Therapeutic drugs using genome-edited pluripotent stem cells

Abstract

Provided is a cell preparation for treating a brain function disorder, a brain disease or a tumor, the cell preparation being characterized by containing a neural stem cell that is differentiated from a pluripotent stem cell having a suicide gene introduced therein.

Description

[Technical field]

[0001] The present invention relates to a cell preparation for treating central nervous system diseases and injuries and a cell preparation for treating tumors. This cell preparation is used to treat central nervous system diseases and injuries, such as brain dysfunction, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and brain tumors and other tumors. When using a cell preparation as a therapeutic cell preparation, especially when used to treat central nervous system diseases and injuries, the tumorigenesis of cells is a problem, but the cell preparation of the present invention is a highly safe cell preparation because it can kill cells by suicide genes. [Background technology]

[0002] Cerebral contusion, a type of traumatic brain injury, is mainly caused by falls and traffic accidents, and still occurs frequently in a wide range of people, from young to old. Physical damage causes necrosis of cells in the brain parenchyma, such as nerve cells and various glial cells, destroying and damaging neural circuits, resulting in severe neurological symptoms and, in the worst case, a disastrous outcome. Treatment for cerebral contusion is performed for early massive edema that occurs within 48 hours of injury, especially the first 24 hours, but it is resistant to conservative treatment such as hyperosmotic diuretics. If consciousness disorder progresses despite conservative treatment, craniotomy is performed to remove the necrotic tissue from the contusion. Although surgery can be expected to improve the life prognosis, the functional prognosis remains poor (Vella MA. Surg Clin North Am. 2017).

[0003] Cerebrovascular disease (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage) is a disease that can be said to be a national disease in Japan, and was once the number one cause of death in Japan. Cerebral infarction causes necrosis of cells in the brain parenchyma, such as nerve cells and various glial cells, due to blood flow disorder. It destroys and damages the neural circuit network, causing severe neurological symptoms and, in the worst case, leading to a tragic outcome (Lindvall O. Stroke. 2011). Due to new treatments such as administration of tissue plasminogen activator (tPA) for the hyperacute phase of cerebral infarction, advances in preventive medicine such as risk factor management, and improvements in public health, the number of deaths from cerebrovascular disease has now fallen to third place, but it remains a disease with a high mortality rate.

[0004] On the other hand, neurodegenerative diseases are diseases that damage higher brain functions, caused by the degeneration and loss of nerve cells due to various reasons. Although much research has been conducted on prevention and treatment methods, there are no drugs that can restore the degenerated and lost nervous system.

[0005] In both cases, current treatments for neurological damage are focused on prevention and prevention of deterioration. Although some functional recovery can be achieved through rehabilitation in response to functional impairment, it is difficult to repair the damage to the neural circuit network, which is a characteristic of the central nervous system, and achieve complete functional recovery with existing treatments. Therefore, a treatment method that can completely repair the neural circuit network is required, aiming at functional recovery, especially the recovery of higher brain functions. Stem cells are considered to be very useful as a carrier for reconstructing damaged neural circuit networks. Methods for this include (1) activation of endogenous neural stem cells present in the subventricular zone of the lateral brain and the subgranular layer of the hippocampus, and (2) stem cell transplantation. However, the survival rate of the newly generated neurons in (1) is extremely low, and reconstruction of the neural circuit network is impossible by this alone. For this reason, (2) is necessary as a means of reconstructing damaged neural circuit networks.

[0006] In stem cell transplantation, it is important to choose the type of transplant donor cells. Various cells have been proposed as candidates for transplant donor cells. To date, functional improvement effects in animal models of cerebral contusion using ES cell-derived neural stem cells (NSCs) and mesenchymal stem cells (MSCs) have been reported (Non-Patent Documents 1-6), and in 2014, bone marrow-derived MSCs (SB623) were transplanted into patients with chronic cerebral contusion (Non-Patent Document 7). Currently, the majority of studies, including ongoing clinical studies, use MSCs (NCT02210624). However, the effects of MSCs are not aimed at reconstructing the circuitry in the strict sense, but are based on the theory that transplanted cells secrete humoral factors that have neurotrophic and protective effects and angiogenesis-promoting effects, thereby indirectly promoting regeneration.

[0007] On the other hand, there is a report that neuroepithelial stem cells are excellent as transplant donor cells due to their high neural differentiation potential (Non-Patent Document 8). There are ethical and practical issues in using neural stem cells as donor cells, but human iPS cell-derived neural stem cells are ideal donor cells that can solve these issues. As with other transplant cell therapies, there is concern about the problem of tumorigenicity in iPS cells, and research is being conducted on production methods that do not use c-Myc, which is one of the causes of this problem. However, this cannot be completely denied, and it has been reported that in an attempt to avoid tumorigenicity, the proliferation ability of the stem cells themselves is reduced and pluripotency is lost (Nakagawa M. Nat Biotechnol 2008; Stadtfeld M. Science. 2008).

[0008] Chemical compounds that target various molecules and have various mechanisms have been developed as therapeutic agents for tumors. However, since these are chemical substances, there is always the problem of side effects, and the development of safe tumor therapeutic agents remains an urgent task. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Riess P. J Neurotrauma. 2007 [Non-Patent Document 2] Tajiri N. Front Syst. Neurosci. 2014 [Non-Patent Document 3] Tian C. Exp Clin Transplant. 2013 [Non-Patent Document 4] Qi L. J Craniofac Surg. 2018 [Non-Patent Document 5] Zanier ER. Crit Care Med. 2011 [Non-Patent Document 6] Turtzo LC. PLoS One. 2015 [Non-Patent Document 7] Steinberg GK. Stroke. 2016 [Non-Patent Document 8] Uchida K. Neurosci Res. 2005 Summary of the Invention [Problem to be solved by the invention]

[0010] Considering clinical application, human iPS cell-derived neural stem cells are ideal donor cells because they can solve ethical and practical problems. However, as mentioned above, iPS cells, like other cell transplant therapies, have the problem of tumorigenicity. The first objective of the present invention is to provide human iPS cell-derived neural stem cells that solve the problem of tumorigenicity.

[0011] The present inventors have also succeeded in producing a cell preparation for treating brain tumors by introducing a suicide gene (a gene encoding an enzyme that converts a prodrug into a substance with cytotoxicity) into human iPS cells and then inducing them to become neural stem cells (WO2018 / 207808 and WO2019 / 098361). The second object of the present invention is to provide a means for constitutively and stably expressing the suicide gene introduced into such human iPS cells.

[0012] Furthermore, if the therapeutic effect of the above-mentioned cell preparation could be predicted in advance, it would be possible to prevent unnecessary treatment and enable efficient treatment. A third object of the present invention is to provide a means for predicting the therapeutic effect of the above-mentioned cell preparation.

[0013] Furthermore, neural stem cells exhibit migration and tropism toward tumors, but it is not clear what molecules cause this. If this can be clarified, it will be possible to produce neural stem cells with enhanced migration and tropism toward tumors and to efficiently select neural stem cells with high migration and tropism. The fourth object of the present invention is to identify molecules that confer such migration and tropism to neural stem cells, and to provide a means for producing neural stem cells with enhanced migration and tropism and a means for efficiently selecting neural stem cells with high migration and tropism.

[0014] Furthermore, neural stem cells used in cell preparations must be highly safe. However, genome editing techniques such as CRISPR / Cas9 used to introduce suicide genes have the problem of off-target effects, in which mutations are introduced into unintended locations. In addition, when producing neural stem cells having a suicide gene, a selection marker gene such as a drug resistance gene is inserted together with the suicide gene to select cells, but from the viewpoint of safety, it is desirable to remove such foreign genes. The fifth object of the present invention is to provide a means for producing neural stem cells that are highly safe. [Means for solving the problem]

[0015] The present inventors conducted extensive research to solve the above problems and as a result, obtained the following findings. 1) When using iPS cell-derived neural stem cells as transplant donor cells, we found that by introducing a suicide gene into iPS cells in advance, if the transplanted cells become tumorigenic, they can be killed by administering a prodrug, solving the problem of tumorigenicity mentioned above. We also found that if a prodrug is administered a certain period of time after transplantation of neural stem cells, undifferentiated cells at risk of tumorigenesis are killed, while the therapeutic effect of the transplanted cells continues.

[0016] 2) We found that by inserting a suicide gene into the 3' side of the β-actin gene translation region of iPS cells, the sensitivity of the suicide gene to prodrugs was increased.

[0017] 3) We found that by measuring the expression levels of the thymidylate synthase gene and dihydropyrimidine dehydrogenase gene in tumor cells, it is possible to estimate the antitumor effect of therapeutic stem cells into which a suicide gene has been introduced against those tumor cells.

[0018] 4) We found that the ligand-receptor pairs of ephrinB and ephrinB receptor (EphB), and the ligand-receptor pairs of CXC motif chemokine 12 (CXCL12) and CXC motif chemokine receptor 4 (CXCR4) are involved in the migration and tropism of neural stem cells toward tumors.

[0019] 5) We found that safe neural stem cells can be produced by using CRISPR / Cas3, which has a low frequency of off-target effects, instead of CRISPR / Cas9 as a genome editing technology, and removing the inserted selection marker gene using the Cre / loxP system, and that the neural stem cells produced in this way have the same therapeutic effect as neural stem cells produced by conventional methods. Furthermore, we found that the safety of neural stem cells can be further improved by replacing the loxP sequence in the Cre / loxP system with a loxP sequence having a mutation, and also found that by combining the Cre / loxP system using this loxP sequence with a homologous recombination vector, not only can the selection marker gene be removed, but a second gene can be inserted into the genome of pluripotent stem cells.

[0020] 6) We found that neural stem cells differentiated from pluripotent stem cells exhibit strong tropism and migration properties toward tumors, including brain tumors, and injured brains.

[0021] The present invention has been completed based on the above findings. That is, the present invention provides the following [1] to

[38] . [1] A cell preparation for treating central nervous system diseases or injuries, comprising neural stem cells differentiated from pluripotent stem cells into which a suicide gene has been introduced.

[0022] [2] The cell preparation for treating central nervous system diseases / injuries described in [1], which is used for treating brain dysfunction.

[0023] [3] The cell preparation for treating central nervous system diseases / injuries described in [1], which is used for treating traumatic brain injury.

[0024] [4] The cell preparation for treating central nervous system diseases / injuries described in [1], which is used for treating spinal cord injuries.

[0025] [5] The cell preparation for treating central nervous system disease / injury described in [1], which is used for treating a neurodegenerative disease.

[0026] [6] The cell preparation for treating central nervous system diseases / injuries described in [1], which is used for treating brain tumors.

[0027] [7] A cell preparation for treating central nervous system diseases or injuries described in [1] to [6], characterized in that a suicide gene is inserted into the 3' side immediately after the translation region of the β-actin gene of the pluripotent stem cell.

[0028] [8] A cell preparation for treating central nervous system diseases or injuries described in [7], characterized in that a sequence encoding a 2A peptide is linked to the 3' side immediately after the translation region of the β-actin gene of a pluripotent stem cell, and a suicide gene is linked to the 3' side of the sequence encoding the 2A peptide.

[0029] [9] A cell preparation for treating a central nervous system disease or injury according to any one of [1] to [8], characterized in that the neural stem cells do not contain a selection marker gene in their genome.

[0030]

[10] The cell preparation for treating central nervous system diseases and injuries according to [9], characterized in that the neural stem cells are obtained by a method comprising the following steps (A) to (D): (A) inserting a gene construct into the genome of a pluripotent stem cell by genome editing, the gene construct comprising a suicide gene, a selection marker gene, and two target sequences of a Cre protein, the selection marker gene being sandwiched between the target sequences of the two Cre proteins; (B) selecting pluripotent stem cells having a suicide gene inserted into their genome from the pluripotent stem cells obtained in step (A) using a selection marker gene; (C) removing a selection marker gene from the genome of the pluripotent stem cells obtained in step (B) using a Cre protein; (D) A step of differentiating the pluripotent stem cells obtained in step (C) into neural stem cells.

[0031]

[11] The cell preparation for treating a central nervous system disease or injury according to

[10] , wherein the target sequence of the Cre protein is a loxP sequence.

[0032]

[12] A cell preparation for treating central nervous system diseases and injuries described in

[10] , characterized in that the target sequences of the two Cre proteins contained in the gene construct are a mutant loxP sequence having a mutation in the upstream repeat sequence and a mutant loxP sequence having a mutation in the downstream repeat sequence, and in the gene construct, the mutant loxP sequence having a mutation in the upstream repeat sequence is located upstream of the selection marker gene, and the mutant loxP sequence having a mutation in the downstream repeat sequence is located downstream of the selection marker gene.

[0033]

[13] The cell preparation for treating a central nervous system disease or injury according to [9], wherein the neural stem cells are obtained by a method comprising the following steps (a) to (d): (a) inserting a gene construct into the genome of a pluripotent stem cell by genome editing, the gene construct comprising a suicide gene, a selection marker gene, a mutant loxP sequence having a mutation in a repeat sequence, and a mutant loxP sequence having a mutation in a spacer sequence, the selection marker gene being sandwiched between the two mutant loxP sequences; (b) selecting pluripotent stem cells having a suicide gene inserted into their genome from the pluripotent stem cells obtained in step (a) using a selection marker gene; (c) removing a selection marker gene from the genome of the pluripotent stem cell obtained in step (b) using a Cre protein and a homologous recombination vector and inserting a second gene into the genome, wherein the homologous recombination vector contains the second gene, a mutant loxP sequence having a mutation in a repeat sequence, and a mutant loxP sequence having a mutation in a spacer sequence, and the second gene is sandwiched between the two mutant loxP sequences; (d) differentiating the pluripotent stem cells obtained in step (c) into neural stem cells.

[0034]

[14] A cell preparation for treating a central nervous system disease or injury according to any one of

[10] to

[13] , characterized in that the genome editing is genome editing using CRISPR / Cas3.

[0035]

[15] A cell preparation for treating a central nervous system disease or injury according to any one of [1] to

[14] , wherein the suicide genes are a cytosine deaminase gene and a uracil phosphoribosyltransferase gene.

[0036]

[16] The cell preparation for treating a central nervous system disease or injury according to

[15] , which is used together with a prodrug that is converted to 5-fluorouracil by cytosine deaminase.

[0037]

[17] A cell preparation for treating a central nervous system disease or injury according to any one of [1] to

[16] , characterized in that the neural stem cells are neural stem cells in which the expression level of at least one selected from ephrin A receptor, ephrin A, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 is increased.

[0038]

[18] The cell preparation for treating central nervous system diseases or injuries described in

[17] , characterized in that the neural stem cells are neural stem cells having increased expression levels of ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif chemokine receptor 4.

[0039]

[19] A cell preparation for treating a central nervous system disease or injury according to any one of [1] to

[16] , characterized in that the neural stem cells are neural stem cells selected using the expression level of at least one selected from ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 as an indicator.

[0040]

[20] The cell preparation for treating central nervous system diseases or injuries described in

[19] , characterized in that the neural stem cells are neural stem cells selected using ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif chemokine receptor 4 as indicators.

[0041]

[21] A method for producing neural stem cells, comprising the following steps (1) and (2): (1) inserting a suicide gene into the 3' side of the translation region of the β-actin gene of a pluripotent stem cell by genome editing; (2) Differentiating the pluripotent stem cells obtained in step (1) into neural stem cells.

[0042]

[22] The method for producing neural stem cells described in

[21] , characterized in that in step (1), a suicide gene is inserted into the 3' side immediately after the translation region of the β-actin gene of the pluripotent stem cell, so that a sequence encoding a 2A peptide is linked to the 3' side immediately after the translation region of the β-actin gene, and the suicide gene is linked to the 3' side of the sequence encoding the 2A peptide.

[0043]

[23] The method for producing neural stem cells according to

[21] or

[22] , wherein step (1) comprises the following steps (1-A) to (1-C): (1-A) a step of inserting a gene construct into the 3' side immediately after the translation region of the β-actin gene of a pluripotent stem cell by genome editing, the gene construct comprising a suicide gene, a selection marker gene, and two target sequences of a Cre protein, the selection marker gene being sandwiched between the target sequences of the two Cre proteins; (1-B) selecting pluripotent stem cells having a suicide gene inserted into their genome from the pluripotent stem cells obtained in step (1-A) using a selection marker gene; (1-C) A step of removing the selection marker gene from the genome of the pluripotent stem cells obtained in step (1-B) using a Cre protein.

[0044]

[24] The method for producing neural stem cells described in

[23] , characterized in that the target sequence of the Cre protein is a loxP sequence.

[0045]

[25] A method for producing neural stem cells described in

[23] , characterized in that the target sequences of the two Cre proteins contained in the gene construct are a mutant loxP sequence having a mutation in the upstream repeat sequence and a mutant loxP sequence having a mutation in the downstream repeat sequence, and in the gene construct, the mutant loxP sequence having a mutation in the upstream repeat sequence is located upstream of the selection marker gene, and the mutant loxP sequence having a mutation in the downstream repeat sequence is located downstream of the selection marker gene.

[0046]

[26] The method for producing neural stem cells according to

[21] or

[22] , wherein step (1) comprises the following steps (1-a) to (1-c): (1-a) inserting a gene construct into the 3' side immediately after the translation region of the β-actin gene of a pluripotent stem cell by genome editing, the gene construct including a suicide gene, a selection marker gene, a mutant loxP sequence having a mutation in a repeat sequence, and a mutant loxP sequence having a mutation in a spacer sequence, the selection marker gene being sandwiched between the two mutant loxP sequences; (1-b) selecting pluripotent stem cells having a suicide gene inserted into their genome from the pluripotent stem cells obtained in step (1-a) using a selection marker gene; (1-c) a step of removing a selection marker gene from the genome of the pluripotent stem cell obtained in step (1-b) using a Cre protein and a homologous recombination vector and inserting a second gene into the genome, wherein the homologous recombination vector contains the second gene, a mutant loxP sequence having a mutation in a repeat sequence, and a mutant loxP sequence having a mutation in a spacer sequence, and the second gene is a homologous recombination vector sandwiched between the two mutant loxP sequences.

[0047]

[27] The method for producing neural stem cells described in

[23] to

[26] , characterized in that the genome editing is genome editing using CRISPR / Cas3.

[0048]

[28] The method for producing neural stem cells according to any one of

[21] to

[27] , wherein the suicide genes are a cytosine deaminase gene and a uracil phosphoribosyltransferase gene.

[0049]

[29] A method for estimating the antitumor effect of a cell preparation for treating central nervous system diseases and injuries described in

[15] on brain tumor cells, the method comprising a step of measuring the expression levels of the thymidylate synthase gene and dihydropyrimidine dehydrogenase gene in the brain tumor cells.

[0050]

[30] A method for selecting neural stem cells with high migratory and / or tropism toward a tumor or damaged site, characterized in that the selection is performed using the expression level of at least one selected from ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 as an indicator.

[0051]

[31] The selection method according to

[30] , characterized in that the expression levels of ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 are used as indicators for selection.

[0052]

[32] A cell preparation for tumor therapy, comprising neural stem cells produced by the method according to any one of

[21] to

[28] , which are differentiated from pluripotent stem cells into which a suicide gene has been introduced.

[0053]

[33] The cell preparation for tumor therapy according to

[32] , characterized in that the neural stem cells are neural stem cells that do not contain a selection marker gene in their genome.

[0054]

[34] The cell preparation for tumor therapy according to

[32] or

[33] , characterized in that the neural stem cells are neural stem cells in which the expression level of at least one selected from ephrin A receptor, ephrin A, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 is increased.

[0055]

[35] The cell preparation for tumor therapy described in

[34] , characterized in that the neural stem cells are neural stem cells having increased expression levels of ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4.

[0056]

[36] The cell preparation for tumor therapy according to

[32] or

[33] , characterized in that the neural stem cells are neural stem cells selected using the expression level of at least one selected from ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 as an indicator.

[0057]

[37] The cell preparation for tumor therapy described in

[34] , characterized in that the neural stem cells are neural stem cells selected using ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif chemokine receptor 4 as indicators.

[0058]

[38] The cell preparation for tumor therapy according to any one of

[32] to

[37] , wherein the tumor is pancreatic cancer.

[0059] This specification includes the contents described in the specifications and / or drawings of Japanese patent applications, which are the basis of the priority of this application, Japanese Patent Application No. 2020-063477, Japanese Patent Application No. 2020-165847, and Japanese Patent Application No. 2020-219455. Effect of the Invention

[0060] The present invention provides a novel tumor treatment preparation and a cell preparation for treating central nervous system diseases and injuries. In particular, when using cell preparations for the treatment of brain dysfunction such as traumatic brain injury including cerebral contusion and cerebral infarction, or for the treatment of neurodegenerative diseases resulting from loss of nerve cells caused by neurodegeneration, the tumorigenesis of cells is a problem, but the cell preparation of the present invention is a highly safe cell preparation because it can kill cells by a suicide gene. [Brief description of the drawings]

[0061] [Figure 1A] Diagram (1) of suicide gene expression and establishment of therapeutic neural stem cells (NSCs). [Figure 1B] Diagram (2) of suicide gene expression and establishment of therapeutic NSCs. [Figure 1C] Diagram (3) of suicide gene expression and establishment of therapeutic NSCs. [Figure 1D] Diagram (4) of suicide gene expression and establishment of therapeutic NSCs. [Figure 2A] Graph showing the migration of therapeutic NSCs (yeast cytosine deaminase (yCD)-NSCs) to malignant gliomas. [Figure 2B] Figure (1) shows the antitumor effect of therapeutic NSCs (yCD-NSCs) on malignant glioma model mice. [Figure 2C] Figure (2) shows the antitumor effect of therapeutic NSCs (yCD-NSCs) on malignant glioma model mice. [Figure 2D] Figure (3) shows the antitumor effect of therapeutic NSCs (yCD-NSCs) on malignant glioma model mice. [Figure 2E] Figure (4) shows the antitumor effect of therapeutic NSCs (yCD-NSCs) on malignant glioma model mice. [Figure 3A] Figure 1: Neuroprotective effect of therapeutic NSCs (yCD-NSCs) against cerebral contusion injury. [Figure 3B] Figure 2: Neuroprotective effect of therapeutic NSCs (yCD-NSCs) against cerebral contusion injury. [Figure 3C] Figure 3: Neuroprotective effect of therapeutic NSCs (yCD-NSCs) against cerebral contusion injury. [Figure 3D] Figure 4: Neuroprotective effect of therapeutic NSCs (yCD-NSCs) against cerebral contusion injury. [Figure 3E] Figure 5: Neuroprotective effect of therapeutic NSCs (yCD-NSCs) against cerebral contusion injury. [Figure 3F] Figure 6: Protective effect of therapeutic NSCs (yCD-NSCs) against cerebral contusion. [Figure 3G] Figure 7: Neuroprotective effect of therapeutic NSCs (yCD-NSCs) against cerebral contusion injury. [Figure 4A] Diagram of the effect of 5-FU release on motor function (1). [Figure 4B] Diagram (2) of the effect of 5-FU release on motor function. [Figure 5A] Diagram (1) regarding the safety of therapeutic NSCs (yCD-NSCs) (differentiated cells). [Figure 5B] Figure (2) regarding the safety of therapeutic NSCs (yCD-NSCs) (differentiated cells). [Figure 5C] Figure (3) regarding the safety of therapeutic NSCs (yCD-NSCs) (differentiated cells). [Figure 6A] Figure (1) Safety of therapeutic NSCs (yCD-NSCs) (mouse model). [Figure 6B] Figure (2) regarding the safety of therapeutic NSCs (yCD-NSCs) (mouse model). [Figure 6C] Figure (3) on the safety of therapeutic NSCs (yCD-NSCs) (mouse model). [Figure 6D] Figure (4) regarding the safety of therapeutic NSCs (yCD-NSCs) (mouse model). [Figure 6E]Figure 5: Safety of therapeutic NSCs (yCD-NSCs) (mouse model). [Figure 7A] Figure 1: Biomarkers of antitumor effects of yCD-NSCs. [Figure 7B] Figure 2: Biomarkers of antitumor effects of yCD-NSCs. [Figure 7C] Figure 3: Biomarkers of antitumor effects of yCD-NSCs. [Figure 7D] Figure 4: Biomarkers of antitumor effects of yCD-NSCs. [Figure 8] Diagram showing an overview of ligand-receptor pairing analysis. [Figure 9] Photograph of neural stem cells (NSCs) and mesenchymal stem cells (MSCs) transplanted into the brain. [Figure 10] Heatmap of autocrine cell-cell interactions. [Figure 11] FIG. 1 shows NSC- and GSC-specific autocrine ligand-receptor pairs suggested by enrichment analysis. [Figure 12] Heatmap of EphB / ephrinB gene expression. [Figure 13] Heatmap of interactions between paracrine cells. [Figure 14] Figure showing t-SNE plot (a method that comprehensively analyzes gene expression in single cells and visualizes cellular similarities on a two-dimensional plot) using single-cell RNA-seq of resected glioblastoma, as previously reported. [Figure 15A] A schematic diagram of a previous method for producing therapeutic stem cells. [Figure 15B] Schematic diagram of the Cre / loxP method for removing a selection marker gene. [Figure 15C] FIG. 1 shows the structures of various vectors used in Example 3. [Figure 16A] FIG. 1 is a schematic diagram showing a method for removing the Puromycin gene and the Venus gene in Example 3. [Figure 16B]FIG. 1 is a schematic diagram showing the cloning procedure in Example 3. [Figure 16C] FIG. 1 shows the results of genomic PCR in Example 3. [Figure 16D] FIG. 2 shows the results of genomic PCR in Example 3. [Figure 16E] FIG. 3 shows the results of genomic PCR in Example 3. [Figure 16F] FIG. 4 shows the results of genomic PCR in Example 3. [Figure 17A] A schematic diagram of a suicide gene therapy for brain tumors. [Figure 17B] Micrographs of iPS cells (top), embryoid bodies (bottom left), and neural stem cells (bottom right) used in Example 3. [Figure 17C] FIG. 1 shows gene expression levels in the cell lines used in Example 3. From the left, each bar graph shows the expression levels of GAPDH, yCD, Puromycin, and Venus. [Figure 18] Micrographs of neural stem cells used in Example 3 in the presence or absence of puromycin. [Figure 19] Microscopic photographs (top row) and a graph showing cell viability (bottom row) of neural stem cells used in Example 3 at various 5-FC concentrations. [Figure 20] Photograph (left) and diagram (right) showing the antitumor effect of neural stem cells used in Example 3 in the presence or absence of 5-FC. [Figure 21] FIG. 1 shows the base sequences of a loxP sequence and a mutant lox sequence. [Figure 22] FIG. 2 is a schematic diagram showing a method for removing an antibiotic resistance gene using the Cre / mutagelox system and a method for inserting a second gene using the Cre / mutagelox system. [Diagram 23] FIG. 1 shows the structure of the vector used in Example 4. [Figure 24] Diagram of drug resistance and fluorescent gene deletion method (1). [Figure 25A] Diagram (2) of the drug resistance and fluorescent gene deletion method. [Figure 25B] Diagram (3) of the drug resistance and fluorescent gene deletion method. [Figure 25C] Diagram (4) of the drug resistance and fluorescent gene deletion method. [Figure 26] Diagram of the second gene insertion method (1). [Figure 27A] Diagram of the second gene insertion method (2). [Figure 27B] Diagram of the second gene insertion method (3). [Figure 27C] Diagram of the second gene insertion method (4). [Figure 28] A diagram showing the expression of various genes in iPS cells and neural stem cells established by the drug resistance / fluorescence gene deletion method. [Figure 29] A diagram showing the response of iPS cells and neural stem cells established by the drug resistance / fluorescence gene deletion method to 5-FC and Puromycin. [Diagram 30] FIG. 13 shows the responses of iPS cells and neural stem cells established by the second gene insertion method to 5-FC and Puromycin. [Diagram 31] FIG. 1 shows the antitumor effect of neural stem cells established by drug resistance / fluorescence gene deletion method or second gene insertion method. [Diagram 32] FIG. 13 is a diagram showing an outline of the experimental method of Example 5. [Figure 33A] Diagram showing the tropism and migration of neural stem cells to pancreatic cancer (1). [Figure 33B] Diagram showing the tropism and migration of neural stem cells to pancreatic cancer (2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The present invention will be described in detail below. (A) Cell preparation The tumor treatment preparation and cell preparation for treating central nervous system diseases and injuries of the present invention are characterized by containing neural stem cells differentiated from pluripotent stem cells into which a suicide gene has been introduced.

[0063] Examples of suicide genes include herpes simplex virus thymidine kinase gene (HSVtk), carboxylesterase, deoxycytidine kinase gene and cytosine arabinoside, cytosine deaminase (CD) gene and uracil phosphoribosyltransferase (UPRT) gene (CD-UPRT gene). In the present invention, the cytosine deaminase (CD) gene and uracil phosphoribosyltransferase (UPRT) gene (CD-UPRT gene) are preferred examples.

[0064] In the present invention, the term "cell preparation for treating central nervous system disease / injury" refers to a cell preparation for treating central nervous system disease and central nervous system injury, and more specifically, refers to a cell preparation used to treat disease or injury in the central nervous system (e.g., brain, spinal cord, etc.). The disease or injury to be treated is not particularly limited, and examples thereof include brain tumor, neurodegenerative disease, brain dysfunction, spinal cord injury, etc., and preferably brain tumor or brain dysfunction. The brain dysfunction to be treated is not particularly limited, and examples thereof include functional disorders caused by physical damage, such as functional disorders caused by cerebral contusion, and functional disorders caused by cerebrovascular disorders (e.g., cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage). Neurodegenerative diseases are diseases caused by loss of nerve cells due to neurodegeneration, and specific examples thereof include Parkinson's disease, Alzheimer's disease, Huntington's disease, and frontotemporal lobar degeneration. Examples of brain tumors include gliomas, medulloblastomas, neuroblastomas, meningiomas, pituitary adenomas, neurilemmomas, primary central nervous system lymphomas, sarcomas, and spinal tumors. In the present invention, any of these brain tumors can be treated, but it is preferable to treat gliomas. When the cell preparation of the present invention is used to treat brain tumors, "treatment" includes not only killing tumor cells, but also reducing tumor cells and inhibiting the proliferation of tumor cells.

[0065] In the present invention, the term "cell preparation for tumor therapy" refers to a cell preparation used in tumor therapy. The cell preparation of the present invention utilizes the characteristics of neural stem cells, namely, migration and tropism to tumor tissue, to allow suicide genes to act on tumor tissue. The "treatment" referred to here includes not only the killing of tumor cells, but also the reduction of tumor cells and the inhibition of tumor cell proliferation. In view of the characteristics of the neural stem cells, the tumors to be treated include not only brain tumors and pancreatic cancer, as mentioned in the examples, but also many other tumors, such as breast cancer, stomach cancer, and lung cancer.

[0066] The term "pluripotent stem cells" in the present invention can be interpreted as the term is usually used by those skilled in the art, and includes, for example, iPS cells and ES cells. ES cells can be used as pluripotent stem cells, but it is preferable to use iPS cells. The iPS cells used are not particularly limited in terms of their origin, the reprogramming factor to be introduced, the method of introducing the reprogramming factor, etc., as long as they can be differentiated into neural stem cells. However, since the cell preparation of the present invention is mainly used to treat diseases and injuries in the human brain, it is preferable to use human-derived iPS cells in such cases. In this case, the iPS cells may be derived from a patient to whom the cell preparation is administered, or may be derived from a human other than the patient. The iPS cells used may be prepared according to a known method, but can also be obtained from research institutes such as the Center for iPS Cell Research and Application (CiRA) of Kyoto University.

[0067] In the present invention, the term "neural stem cells" refers to stem cells that have the ability to supply cells that differentiate into neurons and glial cells. The cell preparation of the present invention includes these neural stem cells, but may also include cells other than neural stem cells as long as they do not have a significant adverse effect on the therapeutic effect of brain damage or brain disease. When neural stem cells are produced from iPS cells according to the method described in Sugai K. Mol Brain 2016, which will be described later, not only neural stem cells but also neural progenitor cells are generated. Therefore, the cell preparation of the present invention may include both neural stem cells and neural progenitor cells.

[0068] The higher the migration and tropism of neural stem cells to tumors, the more neural stem cells can accumulate at disease or damaged sites, and the higher the therapeutic effect can be expected. Therefore, it is preferable to increase the expression levels of those ligand-receptor pairs involved in the above-mentioned migration and tropism in neural stem cells, specifically, ephrin A, ephrin A receptor, ephrin B, ephrin B receptor, and CXC motif chemokine receptor 4. It is preferable to increase the expression levels of all five of ephrin A, ephrin A receptor, ephrin B, ephrin B receptor, and CXC motif chemokine receptor 4, but it is also possible to increase the expression levels of only three of ephrin B, ephrin B receptor, and CXC motif chemokine receptor 4, or to increase the expression level of at least one selected from the three. The expression levels of these ligands and receptors can be increased according to known methods. For example, this can be achieved by introducing the genes for these ligands and receptors into neural stem cells (if necessary, they may be introduced together with a control region that increases the expression level of the genes, such as a strong expression promoter), or by using a substance that has the effect of increasing the expression level of these ligands and receptors.

[0069] Furthermore, since a high therapeutic effect can be expected by using neural stem cells that have inherently high migration and tropism toward tumors, the neural stem cells used may be those selected by the selection method of the present invention described below.

[0070] Carboxylesterase converts CPT-11 to highly toxic 7-ethyl-10-hydroxycamptothecin, which strongly inhibits topoisomerase I. Cytosine arabinoside exerts an antitumor effect when phosphorylated by the deoxycytidine kinase gene. The CD gene and UPRT gene are suicide genes that use 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU) as prodrugs, respectively. Vectors that express these suicide genes are commercially available, and pluripotent stem cells and neural stem cells that express these suicide genes can be produced using such vectors. The CD gene and UPRT gene each have an antitumor effect on their own, but it is known that introducing these two genes into cells produces an antitumor effect that is approximately 100 times greater than that obtained when the CD gene is introduced alone.

[0071] The introduction of the suicide gene into the pluripotent stem cell may be performed using a viral vector, but preferably, it is performed using genome editing. This is because, when the suicide gene is inserted into the genome of the pluripotent stem cell using a viral vector such as a lentivirus vector, the suicide gene is inserted randomly into the chromosome, so there is a concern about gene mutation at the insertion site, activation of surrounding genes, and inactivation of the suicide gene due to position effect. It is believed that such problems can be avoided by using genome editing instead of a viral vector.

[0072] Genome editing can be performed using ZFN, TALEN, CRISPR / Cas9, CRISPR / Cas3, Cre / loxP, etc., and among these, it is preferable to use CRISPR / Cas9 or CRISPR / Cas3, and it is particularly preferable to use CRISPR / Cas3. CRISPR / Cas3 has a longer recognition target sequence than CRISPR / Cas9, so that the frequency of off-target effects is lower and safer neural stem cells can be produced. Genome editing can insert a suicide gene into the housekeeping gene region or safe harbor region AAVS1 of pluripotent stem cells. Examples of housekeeping genes include the β-actin gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, cyclophilin gene, and α-tubulin gene. In the present invention, it is preferable to insert the suicide gene into the 3' side immediately after the translation region of the β-actin gene. In addition, in this case, it is preferable to insert the suicide gene immediately 3' after the translation region of the β-actin gene so that the sequence encoding the 2A peptide is linked to the 3' side immediately after the translation region of the β-actin gene, and the suicide gene is linked to the 3' side of the sequence encoding the 2A peptide.

[0073] When introducing a suicide gene into pluripotent stem cells, a selection marker gene such as an exogenous drug resistance gene or a fluorescent gene is usually introduced together for cell selection. However, it is not preferable from the viewpoint of safety that an exogenous gene such as a selection marker gene remains in the genome of a neural stem cell differentiated from a pluripotent stem cell. For this reason, it is preferable to remove the selection marker gene. There is no particular limitation on the means for removing the selection marker gene, but it is preferable to use a combination of a Cre protein and a target sequence of the Cre protein, such as the Cre / loxP system. Here, the "target sequence of the Cre protein" refers to a loxP sequence (SEQ ID NO: 1) or a loxP sequence having a mutation (mutated loxP sequence). The loxP sequence consists of an upstream repeat sequence, a spacer sequence, and a downstream repeat sequence, and the mutation in the mutant loxP sequence may be present in any of these sequences. A specific example of a mutant loxP sequence having a mutation in the upstream repeat sequence is lox71 (sequence number 2), a specific example of a mutant loxP sequence having a mutation in the spacer sequence is lox2272 (sequence number 4), and a specific example of a mutant loxP sequence having a mutation in the downstream repeat sequence is lox66 (sequence number 3).

[0074] An example of a method for removing a selection marker gene using the Cre / loxP system is to prepare a gene construct containing a suicide gene, a selection marker gene, and two loxP sequences, with the selection marker gene sandwiched between the two loxP sequences, insert this into the genome of a pluripotent stem cell, and then remove the selection marker gene from the genome of the pluripotent stem cell using a Cre protein. However, this method leaves one loxP sequence in the genome of the pluripotent stem cell, which may react with the Cre protein. Therefore, it is preferable to replace the loxP sequence contained in the gene construct with a mutant loxP sequence, and make the loxP sequence remaining in the genome a mutant loxP sequence that is less likely to react with the Cre protein. Specifically, a method can be exemplified in which two loxP sequences contained in a gene construct are replaced with a mutant loxP sequence having a mutation in the upstream repeat sequence and a mutant loxP sequence having a mutation in the downstream repeat sequence, a gene construct is prepared in which the mutant loxP sequence having a mutation in the upstream repeat sequence is located upstream of the selection marker gene and the mutant loxP sequence having a mutation in the downstream repeat sequence is located downstream of the selection marker gene, this is inserted into the genome of a pluripotent stem cell, and then the selection marker gene is removed from the genome of the pluripotent stem cell by Cre protein. In this method, what remains in the genome are mutant loxP sequences having mutations in the upstream repeat sequence and the downstream repeat sequence, which reduces the risk of reacting with Cre protein.

[0075] In addition to the above gene constructs, it is also possible to use a homologous recombination vector to not only remove the selection marker gene from the genome, but also to insert a second gene into the genome at the same time. In this case, the gene construct includes a suicide gene, a selection marker gene, a mutant loxP sequence having a mutation in the repeat sequence, and a mutant loxP sequence having a mutation in the spacer sequence, and the selection marker gene is sandwiched between the two mutant loxP sequences. In addition, the homologous recombination vector includes a second gene, a mutant loxP sequence having a mutation in the repeat sequence, and a mutant loxP sequence having a mutation in the spacer sequence, and the second gene is sandwiched between the two mutant loxP sequences. As the second gene, a gene involved in treatment other than the suicide gene can be used.

[0076] The position of the mutant loxP sequence in the gene construct is not particularly limited, and as shown in FIG. 22C, the mutant loxP sequence having a mutation in the repeat sequence may be located upstream of the selection marker gene, and the mutant loxP sequence having a mutation in the spacer sequence may be located downstream of the selection marker gene, or vice versa. However, when the mutant loxP sequence having a mutation in the repeat sequence is located upstream of the selection marker gene, the mutant loxP sequence preferably has a mutation in the upstream repeat sequence, and conversely, when the mutant loxP sequence having a mutation in the repeat sequence is located downstream of the selection marker gene, the mutant loxP sequence preferably has a mutation in the downstream repeat sequence. This is because the mutant loxP sequence remains in the genome.

[0077] The position of the mutant loxP sequence in the homologous recombination vector is adjusted to be the same as that of the mutant loxP sequence in the gene construct. That is, when the mutant loxP sequence having a mutation in the repeat sequence is located upstream of the selection marker gene in the gene construct and the mutant loxP sequence having a mutation in the spacer sequence is located downstream of the selection marker gene, the mutant loxP sequence having a mutation in the repeat sequence is located upstream of the second gene and the mutant loxP sequence having a mutation in the spacer sequence is located downstream of the second gene in the homologous recombination vector. When the position of the mutant loxP sequence in the gene construct is reversed from the above, the position of the mutant loxP sequence in the homologous recombination vector is reversed from the above.

[0078] When the Cre protein and the homologous recombination vector are applied to the genome into which the gene construct has been inserted, homologous recombination occurs, the selection marker gene is removed from the genome, and a second gene is simultaneously inserted into the genome. A gene encoding the Cre protein may be inserted into the homologous recombination vector, so that the Cre protein and the homologous recombination vector can be applied simultaneously.

[0079] The differentiation of pluripotent stem cells into neural stem cells may be performed according to any known method, including a method using an embryoid body or a method not using an embryoid body. When differentiating from iPS cells, the method can be performed according to, for example, the method described in Stem Cell Reports. 2017 Nov 14;9(5):1675-1691. For example, according to the above method, the formation of embryoid bodies from iPS cells can be performed using a known embryoid body formation medium, and the embryoid body medium contains a TGFβ family inhibitor (e.g., SB431542) and a BMP inhibitor (e.g., LDN-193189). The differentiation of embryoid bodies into neural stem cells can be performed using a known neurosphere medium. For example, the neurosphere medium contains epidermal growth factor, fibroblast growth factor-2, leukemia inhibitory factor, B-27 supplement, and the like. By culturing the embryoid body in the neurosphere medium, a cell mass containing neural stem cells called a neurosphere is formed. The formed neurospheres are collected, dispersed into single cells, and cultured in a neurosphere medium to form neurospheres again. After repeating this procedure several times, the neurospheres can be collected to obtain neural stem cells.

[0080] As mentioned above, in this specification, the "thing" referred to as "neural stem cells" is identified by its manufacturing method rather than by its structure or characteristics. This is because, as cells are part of a living organism, their structure and characteristics are extremely complex, and the task of identifying them would require significantly excessive economic expenditure and time.

[0081] The cell preparation of the present invention may contain, in addition to neural stem cells, other components that are acceptable for the formulation. Such other components include carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc. In addition, dimethyl sulfoxide, serum albumin, etc. may be included to protect the cells during cryopreservation, and antibiotics, etc. may be included to prevent bacterial contamination and proliferation.

[0082] The number of neural stem cells contained in the cell preparation of the present invention can be appropriately determined taking into consideration the type of disease such as tumor, brain disease or brain injury, the subject's sex, age, weight, condition of the affected area, and the condition of the cells used, so as to obtain the desired effect in the treatment of tumors, brain diseases or brain injuries.

[0083] The cell preparation of the present invention may be administered multiple times (e.g., 2 to 10 times) at intervals (e.g., twice a day, once a day, twice a week, once a week, once every two weeks). The dosage can be appropriately determined taking into consideration the type of disease or injury, the subject's sex, age, weight, and condition of the affected area, the condition of the cells used, etc., but is generally 1×10 stem cells per individual (human). 6 pieces~1×10 10 It is preferable to administer 1 to 10 times per person.

[0084] The administration site and administration method of the cell preparation of the present invention are not particularly limited, and examples of the administration method include local administration to the brain or tumor tissue, administration into the carotid artery, and intravenous administration.

[0085] The cell preparation of the present invention may be used together with a prodrug that is activated by a suicide gene. The prodrug can be determined according to the type of suicide gene. For example, when the CD-UPRT gene is used as the suicide gene, a prodrug (e.g., 5-FC) that is converted to 5-fluorouracil by cytosine deaminase can be used. By using such a prodrug, cell death can be induced in cells that have a suicide gene. Whether or not to use a prodrug can be determined according to the disease or damage to be treated. For example, when treating brain dysfunction, the induction of cell death by the use of a prodrug is not directly related to the therapeutic effect, but is intended to kill tumor cells or cells that may become tumorigenic, so it is not necessary to use a prodrug. On the other hand, when treating brain tumors, the induction of cell death by the use of a prodrug is directly related to the therapeutic effect, so the use of a prodrug is essential.

[0086] The administration site and administration method of the prodrug are not particularly limited, and examples of the administration method include oral administration, local administration to the brain or tissue, administration into the carotid artery, intravenous administration, and also intraperitoneal administration.

[0087] The administration time of the prodrug can be determined according to the disease or injury to be treated. For example, when brain injury is the treatment target, the prodrug may be administered at the time when tumor cells are generated, or may be started after a certain time has elapsed since the administration of the cell preparation of the present invention in order to prevent the generation of tumor cells. The start time of such preventive administration is not particularly limited as long as it is a time when the therapeutic effect of neural stem cells is not lost and undifferentiated cells at risk of tumorigenesis can be killed, and may be, for example, 3 to 60 days or 5 to 20 days after the administration of the cell preparation of the present invention. In addition, when brain tumors are the treatment target, the prodrug may be administered before, after, or after the administration of the cell preparation of the present invention, but is usually administered in multiple divided doses after the administration of the cell preparation. The same applies to other tumors.

[0088] The dosage of the prodrug can be determined appropriately taking into consideration the type of prodrug used, the type of disease or injury, the subject's sex, age, weight, and condition of the affected area, but when administering 5-FC, it is preferable to administer 50-200 mg / kg per individual (human) four times a day.

[0089] (B) Method for producing neural stem cells The method for producing neural stem cells of the present invention is characterized by comprising the following steps (1) and (2).

[0090] In step (1), a suicide gene is inserted into the 3' end of the translation region of the β-actin gene in the pluripotent stem cells by genome editing. This step can be performed in the same manner as the introduction of the suicide gene into the pluripotent stem cells described in "(A) Cell preparations."

[0091] In step (2), the pluripotent stem cells obtained in step (1) are differentiated into neural stem cells. This step can be carried out in the same manner as the differentiation of pluripotent stem cells into neural stem cells described in "(A) Cell preparation."

[0092] (C) Method for estimating antitumor effect The method for estimating the antitumor effect of the present invention is a method for estimating the antitumor effect of the above-mentioned cell preparation for treating central nervous system diseases and injuries of the present invention on brain tumor cells, and is characterized by including a step of measuring the expression levels of thymidylate synthase gene and dihydropyrimidine dehydrogenase gene in the brain tumor cells.

[0093] The expression level of a gene can be measured by a known method, for example, RT-qPCR.

[0094] If the measurement shows that the expression levels of the thymidylate synthase gene and the dihydropyrimidine dehydrogenase gene are high, it can be inferred that the anti-tumor effect of the cell preparation for treating central nervous system diseases and injuries of the present invention on the brain tumor cells being measured is low; on the other hand, if the expression levels of the genes are low, it can be inferred that the anti-tumor effect of the cell preparation for treating central nervous system diseases and injuries of the present invention on the brain tumor cells being measured is high.

[0095] (D) Method for selecting neural stem cells with high migratory and homing properties The selection method of the present invention is a method for selecting neural stem cells with high migratory and / or tropism toward tumor or damaged sites, and is characterized in that the selection is performed using the expression level of at least one selected from Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif-type chemokine receptor 4 as an indicator.

[0096] It is preferable to select Ephrin A, Ephrin A receptor, Ephrin B, Ephrin B receptor, and CXC motif-type chemokine receptor 4 using the expression levels of all five of these as indicators, but it is also possible to select using the expression levels of only three of Ephrin B, Ephrin B receptor, and CXC motif-type chemokine receptor 4 as indicators, or to select using the expression level of at least one selected from the above three as an indicator.

[0097] A specific example of the selection method includes the following steps (1) and (2). (1) measuring the expression level of at least one selected from ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4; (2) If the expression level measured in step (1) is higher than a reference value, the neural stem cells are selected as "neural stem cells having high migratory and / or homing ability toward tumor or damaged sites."

[0098] Here, the reference value used for selection is not particularly limited, and for example, the expression levels of ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, or CXC motif-type chemokine receptor 4 in commonly used neural stem cells (e.g., commercially available neural stem cells) may be measured in advance and used as the reference value. EXAMPLES

[0099] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, "NSC" generally means "NS / PCs (Neural stem / Progenitor cells)".

[0100] Example 1 (A) Method <Human iPS cells> The human iPS cells (1210B2) used were obtained from the Center for iPS Cell Research and Application (CiRA), Kyoto University. 1210B2 were established by a method of introducing reprogramming factors into human peripheral blood mononuclear cells using episomal vectors (Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, Goshima N, Yamanaka S. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2013 Mar;31(3):458-66.). Human iPS cells were seeded on plastic culture dishes coated with iMatrix-511 (Nippi Co., Ltd.) and maintained in Stem Fit AK03 or AK03N medium (Ajinomoto Co., Ltd.) using a known feeder-free method (Nakagawa M. Sci Rep. 2014).

[0101] <Induction of differentiation of human iPS cells into neural stem / progenitor cells (NSCs)> Differentiation of human iPS cells into NSCs was induced by a known method (Sugai K. Mol Brain. 2016). First, 10 μM ROCK inhibitor Y276352 (Wako Pure Chemical Industries, Ltd.) was added to the iPS cell culture medium, and after incubation for 1 to 3 hours, the cells were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies, Inc.). The dispersed human iPS cells were suspended in EB (embryoid body) formation medium (Stem Fit medium without C solution, 10 μM SB431542, 100 nM LDN-193189) supplemented with 10 μM ROCK inhibitor Y276352, and plated at 9.0 × 10 cells per well in a low-attachment 96-well culture plate (Prime Surface 96V, Sumitomo Bakelite Co., Ltd.). 3Cells were seeded at a density of 75 μl per well and cultured. One day later, 75 μl of EB formation medium was added, and thereafter, half of the EB formation medium was replaced daily, and the cells were cultured for 13 to 14 days to obtain EBs. The EBs were collected from each well, and suspended in NS (neurosphere) medium (D-MEM / Ham's F-12 medium (containing HEPES), manufactured by Wako Pure Chemical Industries, Ltd.) supplemented with 20 ng / ml Recombinant human epidermal growth factor (manufactured by PeproTech), 20 ng / ml Recombinant human fibroblast growth factor 2 (manufactured by PeproTech), 10 3 units / ml Recombinant human leukemia inhibitory factor (manufactured by Nacalai Tesque), 2% B-27 supplement (manufactured by Thermo Fisher Scientific), and 1 unit / ml heparin sodium (manufactured by AWi Pharma), and cultured for 7 days. The medium was changed once on the 3rd or 4th day of culture. The cell aggregates were collected by centrifugation, dispersed into single cells using TrypLE Select, and suspended in NS medium. 1×10 5 cells / ml were seeded in a low-attachment flask (manufactured by Corning), the medium was changed every 3 to 4 days, and the cells were cultured for 10 days to obtain primary NSCs. Similarly, the primary NSCs were collected by centrifugation, dispersed into single cells using TrypLE Select, suspended in NS medium, and seeded in a low-attachment flask at a density of 1×10 5 cells / ml. The medium was changed every 3 to 4 days, and the cells were cultured for 7 to 10 days to obtain secondary, tertiary, quaternary, and quinary NSCs.

[0102] <Generation of yCD-NSC for therapeutic use by introducing the yCD-UPRT gene into the GAPDH, ACTB, and AAVS1 gene regions using CRISPR / Cas9> Generation of iPS cells transfected with the yCD-UPRT gene in each gene region Using CRISPR / Cas9 genome editing technology, we generated iPS cells with yCD-UPRT fusion suicide gene inserted into the housekeeping gene region GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ACTB (β-actin), and the safe harbor region AAVS1 (PPP1R12C). For genome editing, we created a homologous recombination construct (HR-GAPDH-2A-yCD-UPRT-2A-Bsd) in which GAPDH, yCD-UPRT, and Bsd (blasticidin resistance gene) were inserted in a form connected by a 2A peptide sequence to incorporate yCD-UPRT into the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene region, and a gRNA and Cas9 expression vector construct (U6-GAPDH-gRNA4-Cas9) targeting the vicinity of the GAPDH stop codon. In the case of β-actin, a gRNA expression vector was constructed to target the vicinity of the termination codon of β-actin, as in the case of GAPDH, and the construct for homologous recombination was constructed so that β-actin, yCD-UPRT, and Bsd were inserted in a form connected by the 2A peptide sequence (β-actin-2A-yCD-UPRT-2A-Bsd). The construct for homologous recombination of the AAVS1 region was designed to express yCD-UPRT-2A-Bsd under the EF-1 promoter, and insulator sequences were added to both ends. These genes, yCD-UPRT, and Bsd were inserted in a form connected by the 2A peptide sequence.

[0103] These constructs were introduced into a human iPS cell line (1210B2) by electroporation, and the cells were cultured in the presence of blasticidin S. Homologous recombinant iPS cell lines were confirmed by confirming the base sequence of each clone by genomic PCR and genomic sequencing.

[0104] Generation of yCD-UPRT-expressing NSCs (yCD-NSCs) We induced differentiation of iPS cells with yCD-UPRT in each gene region into NSCs, and obtained secondary to quintuple NSCs (yCD-NSCs). The medium was constantly supplemented with blasticidin S at 1μg / ml. We confirmed that cell death was induced in yCD-UPRT-expressing NSCs by adding 5-FC at 1-5μg / ml to the medium.

[0105] Supplementary Information * Regarding migration and antitumor effects of yCD-NSCs against malignant gliomas yCD-NSCs (labeled with hKO1) were transplanted 1 mm above the tumor mass of the human glioma cell line U87 (ffLuc), and their migration was quantitatively evaluated using brain slice cultures (Tamura R. Mol Brain. 2019). Adipose tissue-derived and bone marrow-derived human mesenchymal stem cell lines (labeled with hKO1) were used as comparison subjects. The migration in the brain was quantitatively evaluated by creating Rose Diagram Maps (Angular histograms) using the direction and migration distance as parameters. The antitumor effect of yCD-NSCs was evaluated by transplanting yCD-NSCs into U87 glioma model mice and administering 5-FC.

[0106] * Regarding the safety of yCD-NSC The right striatum of normal brain was injected with 5 × 10 5 yCD-NSCs (ffLuc) were transplanted, and 5-FC was administered for 2 weeks from 7 days later. A control group was also prepared without 5-FC administration. After 150 days, the mice were decapitated and the brain tissue was evaluated. 5-FU, which is converted from 5-FC, is theoretically thought to have no effect on cells in the brain that are occupied by terminally differentiated cells. However, it may affect neural progenitor cells near the ventricles and cells that undergo slight division, such as vascular endothelial cells. Therefore, the histological evaluation was performed using vascular endothelial cell marker (CD31) and neural stem / progenitor cell marker (Nestin). 5-FC administration was started from 35 days after yCD-NSC transplantation, and it was confirmed by IVIS that the engrafted yCD-NSCs disappeared.

[0107] *Biomarkers for yCD-NSC The sensitivity of U87, U251, SF126, hG008, hG021, GL261, and TSG to 5-FU was evaluated (CCK-8 assay). TMZ was used as a control for comparison. In addition, thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) activities in each cell line and yCD-NSC were quantitatively analyzed by RT-qPCR and Western blotting.

[0108] Differentiation of yCD-UPRT-expressing NSCs into neurons and 5-FU sensitivity of differentiated cells yCD-UPRT-expressing NSCs were differentiated into neurons in vitro. Differentiation was confirmed by the expression and morphology of βIII tubulin and NeuN. The survival of undifferentiated proliferative cells was confirmed by the expression of Ki-67, PCNA, and Nestin. Cell death was evaluated by the expression of cleaved caspase 3. 5-FC was added, and the changes in these differentiated and undifferentiated cells due to 5-FU released by the introduced yCD-UPRT were evaluated.

[0109] Generation of ffLuc-expressing yCD-NSCs The yCD-NSCs thus prepared were infected with a lentiviral vector (CSII-EF-ffLuc) expressing ffLuc (a fusion gene of Venus fluorescent protein and Luc2 firefly luciferase) (Takahashi Y, Tsuji O, Kumagai G, Hara CM, Okano HJ, Miyawaki A, Toyama Y, Okano H, Nakamura M. Comparative study of methods for administering neural stem / progenitor cells to treat spinal cord injury in mice. Cell Transplant. 2011;20(5):727-39.) to obtain yCD-NSCs that stably and highly express ffLuc.

[0110] <Examination of the therapeutic effect of yCD-NSCs in a mouse model of cerebral contusion> A circular craniotomy with a diameter of 2.5 mm was performed on a T cell-deficient mouse (female BALB / c nude mouse, 20 g, 10 w) under general anesthesia, centered 1.25 mm lateral and 1.25 mm rostral to the left of the anterior fontanel. The bone flap was protected to be replaced later. The brain surface was exposed and a cerebral contusion was created by cooling the brain surface using an ACU22XT Amoylus cryosurgery device (Keeler & Weiner) 2.5 mm probe (60°C, 30 sec × 10 sets). Then, 1 × 10 5 yCD-NSCs were transplanted from the anterior fontanel to just below the contusion at the same site (2.5 mm on the left lateral side, 1.25 mm on the rostral side, 2 mm deep). The extracted bone flap was replaced and the skin was sutured to complete the procedure.

[0111] The behavioral evaluation of the mice was performed before surgery, with two sessions of Rota rod (UGO BASILE) and one session of smart rat and mouse grip strength measuring device MK-380Si (Muromachi Kikai) to familiarize the mice with the equipment. The rotation speed of the Rota rod was increased from 2 rpm to 20 rpm over 300 seconds, and the time until the mouse fell off the rod or could no longer walk stably was measured. The Rota rod was performed three times on each measurement day, and the average time was measured. This method is in accordance with a previous report (Tabuse J Clin Neurosci. 2010). Grip strength was measured by placing a plastic cover over the net attached to the measuring device, limiting the area of ​​the net that could be grasped, making it possible to measure the grip strength of only one limb. Grip strength was measured for all four limbs, and when the net was grasped with the five fingers, the tail was pulled horizontally, and the peak value until the limb was released from the net was measured. Each limb was measured seven times, and the average values ​​of the five measurements, excluding the maximum and minimum values, were compared (Alamri FF. Behav Brain Res. 2018).

[0112] After surgery, motor function was evaluated using the above method on days 3, 5, 7, 14, 21, 28, 35, and 42. The time-dependent changes in yCD-NSCs in the same individual were observed every week using the IVIS in vivo imaging system.TM Luciferin (30 mg / ml) (200 μl) was administered intraperitoneally, and images were taken 5 minutes after the intensity reached its peak. In addition, 4 mice were decapitated and perfused to obtain brain tissues on the 7th, 14th, 28th, and 42nd days after the creation of the cerebral contusion. At the time of decapitation, 0.2 ml of 2% Evans blue (Sigma-Aldrich) solution (solvent: sterile saline) was injected into the tail vein of each mouse 2 hours before decapitation, and then the mice were decapitated.

[0113] In addition, aim for about 1 mm below the contusion (3 mm deep from the brain surface) and 5 yCD-NSCs were transplanted on the same day. On day 42, the mice were decapitated and the tissues were evaluated. The transplanted yCD-NSCs were evaluated using the STEM121 human cell-specific antibody.

[0114] <Examination of the safety of yCD-NSCs in a mouse model of cerebral contusion> As described above, contusion was created and yCD-NSCs were transplanted, and 5-FC was administered intraperitoneally at 5 mg / mouse / day for 2 weeks after 7 days. Motor function (grip strength) was compared with both the non-5-FC group and the non-yCD-NSCs transplanted group. Thus, it was confirmed that 5-FU release did not adversely affect brain function.

[0115] (B) Results <Establishment of suicide gene expression and therapeutic NSCs> When a suicide gene (yCD-UPRT) was introduced into human iPS cells using a lentiviral vector, gene silencing occurred during the differentiation induction process into neural stem / progenitor cells (NSCs) (Figure 1A). Therefore, the inventors used the genome editing technology CRISPR / Cas9 to insert the gene into the housekeeping gene region βactin (ACTB), and succeeded in achieving constant stable expression of yCD-UPRT (Figure 1B). yCD-UPRT induces cell death by converting the prodrug antifungal drug 5-FC into the anticancer drug 5-FU (Figure 1C).

[0116] In order to optimize the insertion site of the suicide gene, the inventors also introduced yCD-UPRT into other housekeeping genes, GAPDH and the safe harbor region AAVS1, using CRISPR / Cas9. Silencing occurred in the AAVS1 region in the same way as with the viral vector, and the sensitivity to the prodrug 5-FC in the GAPDH region was less than half that of the βactin region (Figure 1D). Thus, the inventors determined that βactin was the optimal region for gene introduction of yCD-UPRT. Sensitivity to 5-FC was quantitatively analyzed using the CCK-8 assay.

[0117] <Antitumor effect of therapeutic NSCs (yCD-NSCs) on malignant glioma model mice> The inventors have developed a unique method to quantitatively evaluate the homing ability of iPS cell-derived NSCs to tumors in the brain by photographing their migration in real time (see Fig. 2A Rose diagram map). As a result, iPS cell-derived NSCs showed better homing ability and migration ability in the brain than other adipose-derived (AMSC) and bone marrow-derived mesenchymal stem cells (BMSC), which are sometimes used for transplant cell therapy, demonstrating their usefulness as cellular delivery vehicles (Fig. 2A). Therapeutic NSCs (yCD-NSCs) established by the method shown in Fig. 1 were transplanted into a glioma model mouse (U87) (Fig. 2B). This is a treatment that utilizes the tumor migration ability. As a result, administration of 5-FC showed a more effective therapeutic effect than the control (Fig. 2C). Two control groups were prepared: one group in which yCD-NSCs were transplanted but not administered with 5-FC, and one group in which yCD-NSCs were not transplanted but were administered with 5-FC.

[0118] When brain tissue was evaluated 2 weeks after administration of 5-FC, significant tumor shrinkage and complete disappearance were observed compared to the control group (Figure 2D). The tumors were labeled with yellow Venus fluorescent protein. Survival time was also significantly extended compared to both control groups (Figure 2E).

[0119] <Cerebral protective effect of therapeutic NSCs (yCD-NSCs) against cerebral contusion> The inventor has established and reported a cerebral contusion model using a cooling device. By creating a cerebral contusion in the left forebrain, it is possible to stably create a model exhibiting right incomplete lower limb paralysis (Figure 3A). yCD-NSCs were infected with a lentiviral vector expressing ffLuc (a fusion gene of Venus fluorescent protein and Luc2 firefly luciferase) to enable identification of transplanted yCD-NSCs by IVIS (Figure 3B). In the method shown in Figure 2, glioma cell lines were infected and the therapeutic effect was evaluated. yCD-NSCs showed a remarkable migration ability not only to tumor cells but also to the injured brain (cerebral contusion). yCD-NSCs transplanted on the contralateral side of the cerebral contusion began to show signals at the site of cerebral contusion in less than two weeks (Figure 3C).

[0120] When yCD-NSCs were transplanted into the site of cerebral contusion and their engraftment was monitored by IVIS, and 5-FC was administered 35 days later, the signal of yCD-NSCs was completely abolished (Figure 3D). Although tumorigenesis of transplanted cells is always a problem in cell therapy, the yCD-NSCs established by the present inventors are killed by 5-FC administration, which means that they have a safety mechanism against tumorigenesis, making it possible to realize safe regenerative medicine.

[0121] The treatment group that received yCD-NSCs showed a significant improvement in motor function (grip strength) compared to the control group after cerebral contusion (Figure 3E). Furthermore, when the damaged area was visualized using a dye (Evans blue), the damaged area was significantly reduced in the brain tissue of the yCD-NSC-transplanted brain 14 days after the contusion (Figure 3F). This indicates the brain protective effect of yCD-NSCs against acute brain injury. Furthermore, when the brain tissue was evaluated 42 days after transplantation of yCD-NSCs 1 mm below the contusion, migration and accumulation at the contusion site was clearly observed (Figure 3G).

[0122] <Effect of 5-FU release on motor function> The inventors confirmed whether there was any decline in brain function by killing the undifferentiated NSCs (at risk of tumorigenesis) that had exerted a brain protective effect in the acute phase by administering 5-FC. NSCs were transplanted in the hyperacute phase on the day of contusion (Day 0), and brain protective effects were exerted in the acute phase up to Day 7, after which 5-FC was administered for 2 weeks during the subacute phase (Figure 4A). As a result, even when 5-FC was administered from Day 7, motor function did not decline, and functional recovery was achieved similar to that of the 5-FC non-administered group (Figure 4B). In addition, the functional improvement effect was significantly higher than that of the non-NSC transplanted control (Figure 4B).

[0123] <Safety of therapeutic NSCs (yCD-NSCs) (differentiated cells)> To prove that yCD-NSCs do not affect differentiated cells, we differentiated yCD-NSCs into neurons in vitro and administered the prodrug 5-FC (Figure 5A). Neurospheres extended very long processes, which are expected to differentiate into neurons. The cells with such long processes were positive for the neuron marker βIII tubulin (Figure 5B). This indicated that they had differentiated into neurons. However, some Ki-67-positive proliferative cells still remained (Figure 5B). When 5-FC was administered, only the Ki-67-positive proliferative cells died, and the βIII tubulin-positive differentiated cells did not die in response to the released 5-FU. It was shown that cell death occurred in the PCNA-positive proliferative cells, but not in the βIII tubulin-positive differentiated cells (Figure 5B).

[0124] Furthermore, some cells were observed expressing NeuN, a marker for mature neuron, and it was clearly demonstrated that such cells did not undergo cell death due to 5-FU released after administration of 5-FC (Fig. 5C).

[0125] <Safety of therapeutic NSCs (yCD-NSCs) (mouse model)> When yCD-NSC (red) transplanted into glioma stem cell hG008 (green) model mice migrated to tumor cells and were present within the tumor, but as a result of administering 5-FC, although the tumor cells were also killed, they themselves also died and there was no remaining (Figure 6A).

[0126] Theoretically, 5-FC is considered not to affect cells in the brain occupied by terminally differentiated cells, but it may also affect cells that undergo slight division such as neural progenitor cells present near the ventricles and vascular endothelial cells. Therefore, in this study, after transplanting yCD-NSC into the normal brain and administering 5-FC, no cells showing obvious apoptosis were observed in neural progenitor cells (Figure 6B) and vascular endothelial cells (Figure 6C) of the ventricular wall near the transplant site. Since yCD-NSC clusters in tumors and damaged brains, it is suggested that it does not intentionally show directivity near these tissues. The course after transplanting yCD-NSC into the normal brain was tracked by IVIS, but after administering 5-FC, the signal disappeared, indicating its own death (Figure 6D). In addition, A, B, and C in the brain tissue image of Figure 6E correspond to the sites of the fluorescence tissue images in Figures 6A, B, and C, respectively.

[0127] <Biomarkers for the antitumor effect of yCD-NSC> FdUMP, an active metabolite of 5-FU, inhibits the enzymatic activity of the DNA de novo enzyme thymidylate synthase (TS) (Figure 7A). Thus, the de novo synthesis of DNA is suppressed, and DNA damage occurs in a time-dependent manner. In addition, 5-FU taken up into a tumor is usually degraded by the degrading enzyme dihydropyrimidine dehydrogenase (DPD) (Figure 7A). Therefore, the lower the DPD activity, the higher the 5-FU concentration in the tumor, and the lower the TS activity of the target enzyme, the more efficiently FdUMP inhibits TS activity, and the stronger the antitumor effect is expected to be. Therefore, we thought that the expression of TS and DPD would be a biomarker for the antitumor effect of our yCD-NSC. The sensitivity of each glioma cell line to 5-FU was evaluated by CCK-8 assay. As a result, the sensitivity was GL261 (good), TSG, hG008, U87, and U251 (poor) (Figure 7B).

[0128] The gene expression of TS and DPD at the mRNA level was quantitatively analyzed by RT-qPCR. It can be seen that both TS and DPD were high in U251 (Figure 7C). Similar results were obtained by Western blotting. When a graph was created by plotting Figure 7C, the relative gene expression of hG008, TSG, and GL261, which showed a remarkable effect against 5-FU, was 0.5 or less (evaluated with U87 as 1). Furthermore, U251, which was the most resistant to 5-FU, showed a relative gene expression of about 3.0 for both (Figure 7D). From these results, TS and DPD could be biomarkers for this treatment. In addition, ACTB (yCD-NSC) also had particularly high TS activity, and its response to 5-FU was not good (Figure 7D). In other words, it is necessary to determine the suicide gene insertion site with good sensitivity to 5-FC, and the ACTB region evaluated by the present inventor corresponds to that. In addition, the fact that NSCs are not particularly sensitive to 5-FU suggests that it is "safe" and unlikely to have an effect on endogenous neural stem cells.

[0129] Example 2 <Background> Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) have the property of migrating to tumors and damaged sites, and therefore have been used as cellular delivery vehicles (CDVs) for therapeutic genes (References 1 and 2). NSCs can be extracted from fetal tissues, but their low proliferation rate and ethical aspects make their use difficult. The present inventor solved this problem by inducing differentiation of NSCs from human induced pluripotent stem cells (iPS cells). He has been researching a therapeutic strategy that uses NSCs as a CDV for a therapeutic gene (cytosine deaminase-uracil phosphoribosyl transferase: CD-UPRT) for glioblastoma. On the other hand, it is still unclear whether NSCs or MSCs can be a better CDV for glioblastoma.

[0130] The inventors used brain slice cultures (Tamura R. Mol Brain. 2019 [Patent Application 2019-052704]) to evaluate the migration of NSCs and MSCs (derived from adipose tissue and bone marrow) transplanted into the brain toward glioblastoma cells, and found that NSCs showed significantly better migration and tropism toward MSCs. In this study, we performed RNA-seq analysis on NSC lines, MSC lines, and glioblastoma lines owned by the inventors to identify key molecules that cause differences in the migration of NSCs and MSCs.

[0131] <Method> Glioma stem cells (GSCs) have been reported as the root cause of the intractability of malignant gliomas. Unlike glioma cells (GCs), GSCs have the ability to self-renew and form tumors, and are highly invasive and resistant to chemotherapy and radiotherapy, and are considered to be the root cause of recurrence.

[0132] RNA was extracted from human iPS cell-derived NSCs (iPSC-NSCs), human fetal cortex-derived NSCs (FcNSCs), human fetal hippocampus-derived NSCs (FhNSCs), human adipose tissue-derived MSCs (AMSC1, AMSC2), human bone marrow-derived MSCs (BMSC1, BMSC2), human GCs (U87, U251, SF126), and human GSCs (hG008, hG021) (reference 3) using the miRNeasy Serum / Plasma kit (QIAGEN) and the QIAcube (QIAGEN) automation system. RNA-seq analysis was performed by Macrogen Inc.

[0133] In addition, RNA-seq data from previously reported resected glioblastomas was obtained from the NCBI Short Read Archive (SRR359290 and SRR359291) database (Reference 4).

[0134] Differences in gene expression were assessed using the DESeq2 suite of bioinformatics tools with a Benjamini-Hochberg cutoff value of p=0.001 and a log2 fold-change ratio of 10-fold.

[0135] Ligand-receptor pairing analysis The ligand-receptor pairing was performed based on a previously published database (Reference 5). 2552 ligand-receptor pairs were selected (Reference 6), and when both the ligand and the receptor were expressed between cells, they were selected as matched pairs (see Figure 8).

[0136] Single-cell RNA-seq We reviewed previously published single-cell RNA-seq data from resected glioblastoma (reference 7).

[0137] <Result> First, we evaluated autocrine interactions to extract self-repulsive signaling pathways. We focused on self-repulsion because NSCs and MSCs behave differently in the brain even when transplanted alone without tumors. NSCs spread and engraft, while MSCs form cell clusters (Figure 9). In addition, the engraftment behavior is similar to that of GSCs (diffuse engraftment) and GCs (cell cluster formation) [Tamura R. Mol Brain. 2019], so the differences between GSCs and GCs were also used as useful information. As a result, we identified 92 ligand-receptor pairs between NSC-NSC and GSC-GSC (Figure 10). These were not identified in MSCs.

[0138] Enrichment analysis (analysis to determine whether there is a correlation between a specific gene set and expression ratio) suggested that these pairs are related to Eph / ephrin repulsion signaling. In particular, EphB / ehrinB was upregulated in NSCs compared to MSCs. In fact, there are reports that EphB / ephrinB is involved in GSC invasion (Reference 8). Therefore, it was suggested that EphB / ephrinB in particular causes self-repulsion between NSCs and is related to diffuse engraftment.

[0139] Next, we evaluated the paracrine interaction between GSC and NSC or MSC. That is, we considered that there is a ligand released by GSC and a receptor in NSC that senses it, and this pair is involved in the targeting. 27 ligand-receptor pairs were found between NSC and GSC. However, the expression of these was not increased in the inventor's in vitro glioma cell line. This result suggested that the GSC cultured in vitro may not fully reflect the glioblastoma in the brain. Therefore, the inventor performed in silico ligand-receptor pairing analysis using previously reported RNA-seq data of resected glioblastoma. As a result, 8 ligand-receptor pairs were found between the resected glioblastoma and NSC. We focused on the CXCL12 / CXCR4 signal, which has been reported many times so far (Reference 2). As a result, CXCR4 was highly expressed in NSC, but not in MSC. CXCL12 was more highly expressed in resected glioblastomas than in GSCs cultured in vitro.

[0140] Furthermore, the inventors reexamined previously reported single-cell RNA-seq data for glioblastoma. As a result, high expression of CXCL12 was observed in tumor-infiltrating macrophages, which are known to accumulate in the microenvironment surrounding glioblastoma. In vitro cultured GSCs showed low expression of macrophage markers such as CD14 and CD163. These data suggest that CXCL12 in tumor-infiltrating macrophages in the brain may also induce tropism for NSCs expressing CXCR4 (MSCs do not express CXCL12).

[0141] From the above, we concluded that the self-repulsion reaction by EphB-ephrinB and the tropism by CXCL12-CXCR4 are involved, and that these two factors cause the directional migration of NSCs to glioblastoma. There have been no reports to date that point out the difference in migration and tropism between NSCs and MSCs to glioblastoma, or the key molecules that produce this difference. In addition, since high expression of CXCL12 has been reported in other malignant tumors (pancreatic cancer, etc.), it is expected that CXCL12 will also migrate to tumors other than glioblastoma.

[0142] <Cited References> 1. Kim, S.U., Jeung, E.B., Kim, Y.B., Cho, M.H., Choi, K.C. Potential tumor-tropic effect of genetically engineered stem cells expressing suicide enzymes to selectively target invasive cancer in animal models. Anticancer Res. 31:1249-1258 (2011). 2. Vescovi, A.L., Galli, R., Reynolds, B.A. Brain tumour stem cells. Nat Rev Cancer. 6:425-436 (2006). 3. Fukaya, R. et al. MIF Maintains the Tumorigenic Capacity of Brain Tumor-Initiating Cells by Directly Inhibiting p53. Cancer Res. 76,2813-2823 (2016). 4. Chen, L.Y. et al. RNASEQR--a streamlined and accurate RNA-seq sequence analysis program. Nucleic Acids Res. 40, e42 (2012). 5. Camp, J.G. et al. Multilineage communication regulates human liver bud development from pluripotency. Nature. 546, 533-538 (2017). 6. Ramilowski, J.A. et al. A draft network of ligand-receptor-mediated multicellular signalling in human. Nat Commun. 22, 7866 (2015). 7. Neftel, C. et al. An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma. Cell. 178, 835-849 (2019). 8. Nakada, M. et al. The phosphorylation of ephrin-B2 ligand promotes glioma cell migration and invasion. Int J Cancer. 126:1155-1165 (2010).

[0143] Example 3 (A) Background The inventors have devised a treatment method using neural stem / progenitor cells (NSCs) derived from induced pluripotent stem cells (iPS cells) as a cellular delivery vehicle for therapeutic genes, because NSCs have a high migration ability to tumor tissues and damaged brains. A yeast cytosine deaminase (yCD)-uracil phosphoribosyl transferase (UPRT) fusion gene (yCD-UPRT) was used as the therapeutic gene, and it was inserted into the housekeeping locus (ACTB) of iPS cells using genome editing technology, achieving stable and constant expression.

[0144] Until now, after gene transfer into iPS cells, cell selection was performed using drug resistance genes and fluorescent genes, but for clinical application, it is desirable to remove foreign genes such as drug resistance genes as much as possible. In addition, genome editing techniques such as CRISPR-Cas9 have safety concerns due to off-target effects, in which mutations are introduced into untargeted locations. Recently, a new genome editing technique, CRISPR / Cas3, has been developed, and it has been reported that the frequency of off-target effects is lower because the recognition target sequence is longer than that of CRISPR / Cas9.

[0145] (B) Summary of the invention In order to produce safe therapeutic stem cells, we developed a new technology that uses genome editing technology with low off-target effects to introduce therapeutic genes into iPS cells, and then removes the foreign genes used for cell selection.

[0146] Specifically, we use genome editing technology CRISPR / Cas3 to create iPS cells with yCD-UPRT fusion gene, drug resistance gene, and fluorescent gene inserted into the housekeeping locus (ACTB). After fluorescent and drug selection, we use the Cre / loxP system to remove the foreign genes (drug resistance gene and fluorescent gene). Furthermore, cells that have been introduced with a suicide gene (a gene that encodes an enzyme that metabolizes a prodrug and converts it into a tumoricidal substance) will die by themselves when the prodrug is administered [Tamura R. Neurosurgical review. 2019].

[0147] (C) Method <Human iPS cells> The human iPS cells (1210B2) used were obtained from the Center for iPS Cell Research and Application (CiRA), Kyoto University. 1210B2 were established by introducing reprogramming factors into human peripheral blood mononuclear cells using episomal vectors (Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, Goshima N, Yamanaka S. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2013 Mar;31(3):458-66.). Human iPS cells were seeded onto plastic culture dishes coated with iMatrix-511 (Nippon Pharmaceuticals) and maintained in Stem Fit AK03 or AK03N medium (Ajinomoto Co.) using a known feeder-free method (Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, Morizane A, Doi D, Takahashi J, Nishizawa M, Yoshida Y, Toyoda T, Osafune K, Sekiguchi K, Yamanaka S. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep. 2014 Jan 8;4:3594.).

[0148] <Induction of differentiation of human iPS cells into neural stem / progenitor cells (NSCs)> Differentiation of human iPS cells into NSCs was induced by a known method (Sugai K, Fukuzawa R, Shofuda T, Fukusumi H, Kawabata S, Nishiyama Y, Higuchi Y, Kawai K, Isoda M, Kanematsu D, Hashimoto-Tamaoki T, Kohyama J, Iwanami A, Suemizu H, Ikeda E, Matsumoto M, Kanemura Y, Nakamura M, Okano H. Pathological classification of human iPSC-derived neural stem / progenitor cells towards safety assessment of transplantation therapy for CNS diseases. Mol Brain. 2016 Sep 19;9(1):85.). First, 10 μM ROCK inhibitor Y276352 (FUJIFILM Wako Pure Chemical Corporation) was added to the iPS cell medium, and the cells were incubated for 1 to 3 hours, washed with PBS, and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed human iPS cells were suspended in EB (embryoid body) formation medium (Stem Fit medium without solution C, supplemented with 10 μM SB431542 and 100 nM LDN-193189) supplemented with 10 μM ROCK inhibitor Y276352, and plated at 9.0 × 10 cells / well in a low-attachment 96-well culture plate (Prime Surface 96V, Sumitomo Bakelite Co., Ltd.). 3Cells were seeded at a density of 75 μl and cultured. After 1 day, 75 μl of EB formation medium was added, and thereafter, half of the EB formation medium was replaced daily, and EBs were obtained by culturing for 13 to 14 days. EBs were collected from each well and cultured in NS (neurosphere) medium (D-MEM / Ham's F-12 medium (containing HEPES), manufactured by Wako Pure Chemical Industries, Ltd.) supplemented with 20 ng / ml Recombinant human epidermal growth factor (manufactured by PeproTech), 20 ng / ml Recombinant human fibroblast growth factor 2 (manufactured by PeproTech), 1×10 3 units / ml Recombinant human leukemia inhibitory factor (manufactured by Nacalai Tesque), 2% B-27 supplement (manufactured by Thermo Fisher Scientific), and 1 unit / ml heparin sodium (manufactured by Awa Pharma) for 7 days. The medium was changed once on the 3rd or 4th day of culture. Cell aggregates were collected by centrifugation, dispersed into single cells using TrypLE Select, and suspended in NS medium. 1×10 5 cells / ml were seeded in a low-adhesion flask (manufactured by Corning), the medium was changed every 3 to 4 days, and primary NSCs were obtained by culturing for 10 days. Similarly, primary NSCs were collected by centrifugation, dispersed into single cells using TrypLE Select, suspended in NS medium, and seeded in a low-adhesion flask at a density of 1×10 5 cells / ml. The medium was changed every 3 to 4 days, and secondary, tertiary, quaternary, and quinary NSCs were obtained by culturing for 7 to 10 days.

[0149] <Introduction of the yCD-UPRT gene into the ACTB gene region by CRISPR / Cas3 - Production of therapeutic stem cells (Therapeutic stem cell: TSC)> Generation of iPS cells transfected with the yCD-UPRT gene in the ACTB gene region (TiPS1,2) Using CRISPR / Cas3 genome editing technology, we generated iPS cells with a yCD-UPRT fusion suicide gene inserted into the housekeeping gene region ACTB (β-actin). To integrate yCD-UPRT into the ACTB (β-actin) gene region, we created a homologous recombination vector (HR-ACTB-2A-yCD-UPRT-loxP-IRES-Puromycin-2A-Venus-loxP) that contains ACTB and yCD-UPRT linked by a 2A peptide sequence, and an IRES sequence, Puromycin resistance gene, 2A peptide, and fluorescent protein Venus linked downstream of the 2A peptide sequence. We also created a genome cleavage vector (Cas3-crRNA-All-in) that expresses crRNA targeting the vicinity of the stop codon of ACTB and a series of Cas3-related proteins. These constructs were introduced into human iPS cell line (1210B2) by electroporation, cultured in the presence of Puromycin S, and cloned by colony pick-up. Homologous recombinant iPS cell line (TiPS1) was confirmed by confirming the base sequence of each clone by genomic PCR and genomic sequencing. A construct (EF-mCherry-IRES-Cre-hCGpolyA) was prepared for Cre / loxP recombination, in which the fluorescent protein mCherry, IRES sequence, Cre protein, and human growth hormone polyadenylation signal were linked downstream of the EF promoter. This construct was introduced into TiPS1 by electroporation, and cells that emitted mCherry fluorescence and lost Venus fluorescence were cloned by sorting (Figures 15 and 16). Selection marker-removed iPS cell line (TiPS2) was confirmed by confirming the base sequence of each clone by genomic PCR and genomic sequencing.

[0150] Generation of yCD-UPRT expressing NSCs (TSC1, 2) iPS cells (TiPS1,2) with yCD-UPRT integrated into ACTB were induced to differentiate into NSCs, and secondary to quintuple NSCs were obtained. TSC1 is a therapeutic NSC with the selection marker sequence remaining after differentiation from TiPS1, and TSC2 is a therapeutic NSC with the selection marker sequence removed after differentiation from TiPS2 (Figure 17). TSC1 was cultured as a comparison to show that removal of the selection marker (TSC2) did not affect cell proliferation or CD expression. Puromycin S 1μg / ml was constantly added to the TSC1 medium. TSC1 and 2 were confirmed to induce cell death by adding 5-Fluorocytosine (5-FC) 1-7μg / ml to the medium. Cell death was evaluated using the cell proliferation / cytotoxicity assay kit Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Kumamoto, Japan).

[0151] Gene expression analysis of TiPSC1,2, TSC1,2 By real-time qPCR, we compared the mRNA expression of the iPS cell line without genome editing (iPS-nega), TiPSC1, TSC1, TiPSC2, TSC2, iPS-Cas9 with genome editing using CRISPR / Cas9, and NSC-Cas9. TM The mRNA expression levels of GAPDH, ACTB, yCD-UPRT, Puromycin resistance gene, and Venus gene were measured using II (Takara Bio), and the expression level of each gene was normalized by the expression level of ACTB.

[0152] Confirmation of antitumor effects of TSC1 and 2 (TSC1, 2) The glioma cell line U87 (1 × 10 4 ) or glioma stem cell line hG008 (1 × 10 4 pcs) and TSC1 or TSC2 (5 × 10 3(cells) were co-cultured for 1 day, and then 5-FC was added (2 μg / ml). Five days later, cell death was evaluated using Cell Counting Kit-8 (CCK-8), a cell proliferation / cytotoxicity assay kit.

[0153] (D) Results (Induction of differentiation into NSCs) TiPS1 and TiPS2 could be induced to differentiate into NSCs without problems by the above method (TSC1 and TSC2). TSC1 was confirmed to be labeled with Venus fluorescent protein (Figure 17B). Gene expression was confirmed for the expression of yCD-UPRT and the selection marker in TiPS1 and TSC1, and the expression of yCD-UPRT and the non-expression of the selection marker in TiPS2 and TSC2 (Figure 17C). Also, the expression level of yCD-UPRT corrected by the expression level of GAPDH or ACTB was not significantly different from that of iPS-Cas9 and NSC-Cas9. (Figure 17C). Note that the expression level of yCD-UPRT is about 25 - 50% compared to ACTB, which is because it is inserted heterozygously.

[0154] (TSC1,2 resistance to Puromycin) TSC1 was able to be selected without problems by the addition of Puromycin due to the Puromycin resistance gene. TSC2 died immediately upon treatment with Puromycin, and it was confirmed that the Puromycin resistance gene had been removed as expected by the Cre / loxP system (Figure 18).

[0155] (TSC1,2 sensitivity to 5-FC) Both TSC1 and TSC2 had good sensitivity to 5-FC and completely died at a concentration of 1 μg / ml (Figure 19). It was also equivalent to the sensitivity of NSCs prepared by introducing yCD-UPRT into the ACTB locus using CRISPR / Cas9 (Figure 19).

[0156] (Anti-tumor effects of TSC1,2) TSC1,2 were co-cultured with the glioma cell line U87 and the glioma stem cell line hG008, and 5-FC (2μg / ml) was administered. Cell death was evaluated by CCK-8 assay, and both cells showed significant tumor cell death after administration of the prodrug (Figure 20). Administration of 5-FC to U87 and hG008 alone did not result in any particular tumor cell death (Figure 20).

[0157] Example 4 (A) Overview of the mutant loxP sequence As shown in Figure 21A, the lox sequence consists of an upstream repeat sequence, a spacer sequence, and a downstream repeat sequence. Mutant lox sequences with mutations at each site have already been developed (the underlined sites in Figure 21A are the mutation sites).

[0158] When lox71 and lox66 are recombined, one becomes lox71 / 66 and the other becomes loxP (Figure 21B). Since lox71 / 66 has mutations at both ends, it is difficult for Cre to recognize it. Therefore, safety can be increased by leaving lox71 / 66 in the genome. In addition, lox2272, which has a mutation in the spacer sequence, can only recombine with the same lox2272 (Figure 21C).

[0159] (B) Background (1) A method for removing antibiotic resistance genes using the Cre / mutagenesis system In order to enhance the safety of the drug resistance gene / fluorescence gene deletion method described in Example 3, a drug resistance gene / fluorescence gene deletion method was developed using a mutant loxP sequence (arm region mutation) instead of loxP.

[0160] The Cre / loxP system can delete DNA sequences flanked by loxP (e.g., drug resistance genes) by expressing the Cre protein, but one loxP sequence is ultimately left behind, which may potentially react with Cre. To reduce this risk, we developed a method in which the DNA sequence to be deleted (e.g., drug resistance genes) is flanked by loxP (lox71 and lox66) sequences that have mutations in their repeat sequences. The lox71 mutant loxP sequence has a mutation incorporated in the upstream repeat sequence, and the lox66 mutant loxP sequence has a mutation incorporated in the downstream repeat sequence. As a result, the lox71 / 66 fusion sequence that remains after the flanked gene deletion has mutations at both ends, which greatly reduces the risk of reacting with Cre, making safer gene therapy possible (Figure 22B).

[0161] (2) A method for inserting a second gene using the Cre / mutagenesis system We developed a method to insert a second gene and delete a drug resistance gene and a fluorescent gene by using a mutated loxP sequence (spacer sequence mutation).

[0162] Since the mutant lox2272 has a mutation in the spacer sequence, it has the property of reacting with the same mutant lox sequence by Cre. Using this property, it is possible to perform homologous recombination of the target gene using the spacer sequence mutant loxP (in the embodiment, recombination of the region sandwiched by lox71-lox2272 and the region sandwiched by lox66-lox2272). As a result, it is possible to insert another therapeutic gene (second gene) in addition to the suicide gene and delete the drug gene. The Cre / mutation lox system has a higher introduction efficiency and is safer with very few off-target problems than genome editing technologies such as Cas3 (Figure 22C).

[0163] (C) Method (1) Removal of drug resistance marker genes Cas3 expression plasmid (Figure 23(1)) and ACTB recombination plasmid (Figure 23(2)) were introduced into human iPS cells by electroporation, and the ACTB stop codon of the human iPS cell genome was replaced with 2A-yCD / UPRT-lox71-IRES- Puromycin resistance gene-2A-Venus-lox66. Cells in which recombination had occurred were selected by adding 1 μg / ml Puromycin to the medium and culturing. EF-mCherry-IRES-Cre plasmid (Figure 23(4)) was introduced into the same cells by electroporation again, and Venus- / mCherry+ cells were cloned 72 hours later. The genome was extracted from the cloned cells, and the DNA at the insertion site was amplified by PCR to confirm the band length, and the sequence was confirmed by sequencing. The cells were then induced to differentiate into NS / PCs.

[0164] (2) Insertion of a second gene Cas3 expression plasmid (Figure 23(1)) and ACTB recombinant plasmid (Figure 23(3)) were introduced into human iPS cells by electroporation, and the ACTB stop codon of the human iPS cell genome was replaced with 2A-yCD / UPRT-lox71-IRES- Puromycin resistance gene-2A-Venus-lox2272. Cells in which recombination had occurred were selected by adding 1 μg / ml of Puromycin to the medium and culturing. Plasmids containing EF-mCherry-IRES-Cre and lox66-IRES-AmCyan-lox2272 sequences (Figure 23(5)) were introduced into the same cells by electroporation again, and Venus- / AmCyan+ / mCherry+ cells were cloned 72 hours later. Genomes were extracted from the cloned cells, and DNA at the insertion site was amplified by PCR to confirm the band length, and the gene sequence was confirmed. The cells were then induced to differentiate into NS / PCs.

[0165] (3) Confirmation of gene expression The mRNA expression of cells selected in (1) and (2) was compared by real-time qPCR. TMThe mRNA expression levels of GAPDH, ACTB, yCD-UPRT, puromycin resistance gene, Venus, and AmCyan genes were measured using a ELISA kit (Takara Bio), and the expression level of each gene was normalized by the expression level of ACTB or GAPDH.

[0166] (4) Confirmation of antitumor effect The glioma stem cell line hG008 (6 × 10 3 ) and NS / PCs (3 × 10 3 The cells were co-cultured for one day, after which 5-FC was added (5 μg / ml), and the cell proliferation / cytotoxicity assay kit Cell Counting Kit-8 (CCK-8) was performed after five days.

[0167] (D)Result (1) Removal of drug resistance marker genes iPS cells were established in which the suicide gene was inserted and the drug resistance gene and marker were removed (ACTB-2A-yCD / UPRT-lox71 / 66 was incorporated), and they could be induced to differentiate into embryoid bodies (EBs) and NS / PCs (Figure 24). Genomic PCR and gene sequence analysis confirmed that the gene was accurately incorporated into the genome (Figures 25A-C).

[0168] (2) Insertion of a second gene We established iPS cells in which the suicide gene and the second gene were inserted and the drug resistance gene and marker were removed (ACTB-2A-yCD / UPRT-lox71 / 66-IRES-AmCyan-lox2272 was incorporated), and induced differentiation into NS / PCs. We confirmed the fluorescence of AmCyan of the second gene in embryoid bodies (EBs) and NS / PCs (Figure 26). Genomic PCR and gene sequence analysis confirmed accurate integration into the genome (Figures 27A-C).

[0169] (3) Confirmation of gene expression In the iPS cells and NS / PCs established in (1), yCD expression and loss of the Venus and Puromycin resistance genes were confirmed (Figure 28). In the iPS cells and NS / PCs established in (2), yCD and second gene (AmCyan) expression and loss of the Venus and Puromycin resistance genes were confirmed (Figure 28).

[0170] Furthermore, it was confirmed that the iPS cells and NS / PCs cells established in (1) were killed by administration of 5-FC and Puromycin (Figure 29). It was confirmed that the iPS cells and NS / PCs cells established in (2) were killed by administration of 5-FC and Puromycin (Figure 30).

[0171] (4) Confirmation of antitumor effect NS / PCs established in (1) or (2) were co-cultured with the glioma stem cell line hG008 and administered 5-FC (5 μg / ml). Cell death was evaluated by the CCK-8 assay, and both induced significant tumor cell death after administration of the prodrug (Figure 31).

[0172] Example 5: Effect on tumors other than brain tumors Below, the effect on pancreatic cancer will be described as an example of a tumor other than a brain tumor, but the same effect is also observed on other tumors. (A) Background Digestive cancers have a high incidence rate, and pancreatic cancer in particular was the fifth most common cause of cancer deaths in men (17,060 cases) and the third most common cause of cancer deaths in women (16,415 cases) in 2016, surpassing the number of deaths from stomach cancer in women [Reference: National Cancer Center Cancer Information Service]. The 5-year relative survival rate for pancreatic cancer is 7.9% for men and 7.5% for women, making it the most poor prognosis compared to other organ cancers [National Cancer Association 10-year relative survival rate by site and clinical stage]. Multidisciplinary treatment is performed using surgery, chemotherapy, and radiation therapy, but chemotherapy, which is administered systemically, has strong side effects, is prone to distant metastasis, and infiltrates the tumor arteries, so many cases are already considered to be inoperable or difficult to completely resect at the time of diagnosis, and the development of new treatments is desired. 5-FU is used as one of the main chemotherapeutic agents for pancreatic cancer. The NS / PCs derived from suicide gene-transduced iPS cells established by the inventors show tropism and migration toward malignant tumors, making it possible to administer high concentrations of the anticancer drug 5-FU locally to the tumor.

[0173] (B) Method Human pancreatic cancer cell line BxPC3 5×10 6 The iPS-NS / PCs (ffLuc) containing the suicide gene yCD-UPRT were subcutaneously transplanted into the left flank of a NOD / SCID mouse (6W, female) 7 days later. 5 The cells were transplanted subcutaneously into the dorsum of the mouse or administered via the tail vein (Fig. 32). NS / PCs were previously infected with a lentiviral vector (CSII-EF-ffLuc) expressing ffLuc (a fusion gene of Venus fluorescent protein and Luc2 firefly luciferase) (Takahashi Y, Tsuji O, Kumagai G, Hara CM, Okano HJ, Miyawaki A, Toyama Y, Okano H, Nakamura M. Comparative study of methods for administering neural stem / progenitor cells to treat spinal cord injury in mice. Cell Transplant. 2011;20(5):727-39.) to obtain NS / PCs that stably and highly express ffLuc. The time course of changes in NS / PCs in the same individual was observed using the IVIS in vivo imaging system. TM Luciferin (30 mg / ml) (200 μl) was administered intraperitoneally, and images were taken 10 minutes after the intensity reached its peak (FIGS. 33A and B).

[0174] (C) Result In a model mouse in which iPS-NS / PCs (ffLuc) were subcutaneously transplanted on the back, signal accumulation was observed at the site of pancreatic cancer transplantation (left flank) after 18 days using IVIS. In a model mouse in which administration was administered via the tail vein, the cancer was trapped in the lungs on the day of administration, but from 3 days later, signal accumulation gradually began to be observed at the site of pancreatic cancer transplantation (left flank), and after 18 days, very strong accumulation was observed. These results demonstrate that iPS-NS / PCs exhibit strong tropism and migration toward pancreatic cancer, and that iPS-NS / PCs can be used as an effective cellular delivery vehicle for pancreatic cancer (FIGS. 33A and B).

[0175] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Industrial Applicability]

[0176] Since the tumor therapeutic cell preparation and the cell preparation for treating central nervous system diseases and injuries of the present invention can be used as medicines, the present invention can be utilized in industries such as pharmaceutical production.

Claims

1. A cell preparation for treating central nervous system diseases or injuries, comprising neural stem cells differentiated from pluripotent stem cells into which a suicide gene has been introduced, wherein the suicide gene is inserted immediately 3' to the translation region of the β-actin gene of the pluripotent stem cells.

2. 2. The cell preparation for treating central nervous system disease or injury according to claim 1, which is used for treating brain dysfunction.

3. The cell preparation for treating central nervous system diseases or injuries according to claim 1, which is used for treating traumatic brain injury.

4. The cell preparation for treating central nervous system diseases or injuries according to claim 1, which is used for treating spinal cord injuries.

5. The cell preparation for treating central nervous system disease or injury according to claim 1, which is used for treating a neurodegenerative disease.

6. 2. The cell preparation for treating central nervous system diseases or injuries according to claim 1, which is used for treating brain tumors.

7. A cell preparation for treating central nervous system diseases or injuries described in any one of claims 1 to 6, characterized in that a sequence encoding a 2A peptide is linked to the 3' side immediately after the translation region of the β-actin gene of a pluripotent stem cell, and a suicide gene is linked to the 3' side of the sequence encoding the 2A peptide.

8. 8. The cell preparation for treating central nervous system diseases or injuries according to any one of claims 1 to 7, wherein the neural stem cells do not contain a selection marker gene in their genome.

9. The cell preparation for treating a central nervous system disease or injury according to claim 8, wherein the neural stem cells are obtained by a method comprising the following steps (A) to (D): (A) inserting a gene construct into the genome of a pluripotent stem cell by genome editing, the gene construct comprising a suicide gene, a selection marker gene, and two target sequences of a Cre protein, the selection marker gene being sandwiched between the target sequences of the two Cre proteins; (B) selecting pluripotent stem cells having a suicide gene inserted into their genome from the pluripotent stem cells obtained in step (A) using a selection marker gene; (C) removing a selection marker gene from the genome of the pluripotent stem cell obtained in step (B) using a Cre protein; (D) A step of differentiating the pluripotent stem cells obtained in step (C) into neural stem cells.

10. The cell preparation for treating a central nervous system disease or injury according to claim 9, characterized in that the target sequence of the Cre protein is a loxP sequence.

11. The cell preparation for treating central nervous system diseases and injuries described in claim 9, characterized in that the two target sequences of Cre proteins contained in the gene construct are a mutant loxP sequence having a mutation in the upstream repeat sequence and a mutant loxP sequence having a mutation in the downstream repeat sequence, and in the gene construct, the mutant loxP sequence having a mutation in the upstream repeat sequence is located upstream of the selection marker gene, and the mutant loxP sequence having a mutation in the downstream repeat sequence is located downstream of the selection marker gene.

12. The cell preparation for treating a central nervous system disease or injury according to claim 8, wherein the neural stem cells are obtained by a method comprising the following steps (a) to (d): (a) inserting a gene construct into the genome of a pluripotent stem cell by genome editing, the gene construct comprising a suicide gene, a selection marker gene, a mutant loxP sequence having a mutation in a repeat sequence, and a mutant loxP sequence having a mutation in a spacer sequence, the selection marker gene being sandwiched between the two mutant loxP sequences; (b) selecting pluripotent stem cells having a suicide gene inserted into their genome from the pluripotent stem cells obtained in step (a) using a selection marker gene; (c) removing a selection marker gene from the genome of the pluripotent stem cell obtained in step (b) using a Cre protein and a homologous recombination vector and inserting a second gene into the genome, wherein the homologous recombination vector contains the second gene, a mutant loxP sequence having a mutation in a repeat sequence, and a mutant loxP sequence having a mutation in a spacer sequence, and the second gene is sandwiched between the two mutant loxP sequences; (d) differentiating the pluripotent stem cells obtained in step (c) into neural stem cells.

13. The cell preparation for treating central nervous system diseases or injuries described in any one of claims 9 to 12, characterized in that the genome editing is genome editing using CRISPR / Cas3.

14. 14. The cell preparation for treating a central nervous system disease or injury according to claim 1, wherein the suicide genes are a cytosine deaminase gene and a uracil phosphoribosyltransferase gene.

15. The cell preparation for treating a central nervous system disease or injury according to claim 14, characterized in that it is used together with a prodrug that is converted to 5-fluorouracil by cytosine deaminase.

16. The cell preparation for treating central nervous system diseases or injuries according to any one of claims 1 to 15, characterized in that the neural stem cells are neural stem cells in which the expression level of at least one selected from ephrin A receptor, ephrin A, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 is increased.

17. The cell preparation for treating central nervous system diseases and injuries described in claim 16, characterized in that the neural stem cells are neural stem cells in which the expression levels of ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 are increased.

18. The cell preparation for treating a central nervous system disease or injury according to any one of claims 1 to 15, characterized in that the neural stem cells are neural stem cells selected using the expression level of at least one selected from ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 as an indicator.

19. The cell preparation for treating central nervous system diseases or injuries described in claim 18, characterized in that the neural stem cells are neural stem cells selected using ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 as indicators.

20. A method for producing neural stem cells, comprising the steps of: (1) inserting a suicide gene into the 3' side of the translation region of the β-actin gene of a pluripotent stem cell by genome editing; (2) Differentiating the pluripotent stem cells obtained in step (1) into neural stem cells.

21. The method for producing neural stem cells described in claim 20, characterized in that in step (1), a suicide gene is inserted immediately 3' to the translation region of the β-actin gene of the pluripotent stem cell so that a sequence encoding a 2A peptide is linked to the 3' side immediately after the translation region of the β-actin gene, and the suicide gene is linked to the 3' side of the sequence encoding the 2A peptide.

22. The method for producing neural stem cells according to claim 20 or 21, wherein the step (1) comprises the following steps (1-A) to (1-C): (1-A) a step of inserting a gene construct into the 3' side immediately after the translation region of the β-actin gene of a pluripotent stem cell by genome editing, the gene construct comprising a suicide gene, a selection marker gene, and two target sequences of a Cre protein, the selection marker gene being sandwiched between the target sequences of the two Cre proteins; (1-B) selecting pluripotent stem cells having a suicide gene inserted into their genome from the pluripotent stem cells obtained in step (1-A) using a selection marker gene; (1-C) A step of removing the selection marker gene from the genome of the pluripotent stem cells obtained in step (1-B) using a Cre protein.

23. The method for producing neural stem cells according to claim 22, characterized in that the target sequence of the Cre protein is a loxP sequence.

24. The method for producing neural stem cells described in claim 22, characterized in that the two target sequences of Cre protein contained in the gene construct are a mutant loxP sequence having a mutation in the upstream repeat sequence and a mutant loxP sequence having a mutation in the downstream repeat sequence, and in the gene construct, the mutant loxP sequence having a mutation in the upstream repeat sequence is located upstream of the selection marker gene, and the mutant loxP sequence having a mutation in the downstream repeat sequence is located downstream of the selection marker gene.

25. The method for producing neural stem cells according to claim 20 or 21, wherein the step (1) comprises the following steps (1-a) to (1-c): (1-a) inserting a gene construct into the 3' side immediately after the translation region of the β-actin gene of a pluripotent stem cell by genome editing, the gene construct including a suicide gene, a selection marker gene, a mutant loxP sequence having a mutation in a repeat sequence, and a mutant loxP sequence having a mutation in a spacer sequence, the selection marker gene being sandwiched between the two mutant loxP sequences; (1-b) selecting pluripotent stem cells having a suicide gene inserted into their genome from the pluripotent stem cells obtained in step (1-a) using a selection marker gene; (1-c) a step of removing a selection marker gene from the genome of the pluripotent stem cell obtained in step (1-b) using a Cre protein and a homologous recombination vector and inserting a second gene into the genome, wherein the homologous recombination vector comprises the second gene, a mutant loxP sequence having a mutation in a repeat sequence, and a mutant loxP sequence having a mutation in a spacer sequence, and the second gene is a homologous recombination vector sandwiched between the two mutant loxP sequences.

26. The method for producing neural stem cells described in any one of claims 22 to 25, characterized in that the genome editing is genome editing using CRISPR / Cas3.

27. The method for producing neural stem cells according to any one of claims 20 to 26, wherein the suicide gene is a cytosine deaminase gene and a uracil phosphoribosyltransferase gene.

28. A method for estimating the antitumor effect of a cell preparation for treating central nervous system diseases and injuries described in claim 14 on brain tumor cells, the method comprising a step of measuring the expression levels of thymidylate synthase gene and dihydropyrimidine dehydrogenase gene in the brain tumor cells.

29. A method for selecting neural stem cells with high migratory and / or tropism toward tumor or damaged sites, characterized in that the selection is performed using the expression levels of ephrin B receptor, ephrin B, and CXC motif chemokine receptor 4 as indicators.

30. A cell preparation for tumor therapy, comprising neural stem cells produced by the method according to any one of claims 20 to 27, which have been differentiated from pluripotent stem cells into which a suicide gene has been introduced.

31. The cell preparation for tumor therapy according to claim 30, characterized in that the neural stem cells are neural stem cells that do not contain a selection marker gene in their genome.

32. The cell preparation for tumor therapy according to claim 30 or 31, characterized in that the neural stem cells are neural stem cells in which the expression level of at least one selected from ephrin A receptor, ephrin A, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 is increased.

33. The cell preparation for tumor therapy according to claim 32, characterized in that the neural stem cells are neural stem cells in which the expression levels of ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 are increased.

34. The cell preparation for tumor therapy according to claim 30 or 31, characterized in that the neural stem cells are neural stem cells selected using the expression level of at least one selected from ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 as an indicator.

35. The cell preparation for tumor therapy according to claim 34, characterized in that the neural stem cells are neural stem cells selected using ephrin A, ephrin A receptor, ephrin B receptor, ephrin B, and CXC motif-type chemokine receptor 4 as indicators.

36. The cell preparation for tumor therapy according to any one of claims 30 to 35, wherein the tumor is pancreatic cancer.

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