Therapeutic agent using genome-edited pluripotent stem cell

Abstract

To provide means for treating tumors such as breast cancer, gastric cancer, or lung cancer.SOLUTION: There is provided a cell preparation for tumor treatment comprising neural stem cells differentiated from pluripotent stem cells into which a suicide gene has been introduced.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to cell preparations for treating central nervous system diseases and injuries and cell preparations for treating tumors. These cell preparations are used for the treatment of central nervous system disease injuries, such as brain dysfunction, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and brain tumors and other tumors. When using the cell preparation as a therapeutic cell preparation, especially when used for the treatment of central nervous system disease injuries, the tumorigenesis of cells becomes a problem. However, the cell preparation of the present invention can kill cells by a suicide gene, so it is a highly safe cell preparation.

Background Art

[0002] Brain contusion, which is a traumatic brain injury, is mainly caused by falls and traffic accidents, so it still occurs frequently and widely from young people to the elderly. Physical injury causes necrosis of cells in the brain parenchyma such as nerve cells and various glial cells, destroys and damages the nerve circuit network, causes severe nerve symptoms, and in the worst case, follows an unfortunate outcome. The treatment for brain contusion is carried out for the traumatic edema (early massive edema) that occurs within 48 hours after injury, especially within the first 24 hours. However, it is resistant to conservative treatments such as hyperosmotic diuretics. When the disturbance of consciousness progresses even with conservative treatment, craniotomy is performed to remove the contusion necrotic tissue. Although improvement in the prognosis of life can be expected even after surgery, the functional prognosis is still poor (Vella MA. Surg Clin North Am. 2017).

[0003] Cerebrovascular disorders (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage) are diseases that can be said to be the national diseases of our country and were once the leading 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 disorders. It destroys and damages the nerve circuit network, causes severe nerve symptoms, and in the worst case, follows an unfortunate outcome (Lindvall O. Stroke. 2011). With the progress of preventive medicine such as the administration of tissue plasminogen activator (tPA) in the ultra-early stage of cerebral infarction and the management of new treatments and risk factors, and the improvement of public health, the number of deaths from cerebrovascular disorders has now decreased to the third place, but it is still a disease with a high mortality rate.

[0004] On the one hand, neurodegenerative diseases are higher brain function impairment diseases caused by the degeneration and loss of nerve cells due to various causes. Although many studies have been conducted on prevention and treatment methods, there is no therapeutic drug that can restore the degenerated and lost brain nervous system.

[0005] All of them. The current treatment methods for nerve function impairment are prevention and prevention of deterioration. Although some functional recovery can be obtained through rehabilitation for dysfunction, it is difficult to repair the damage of the nerve circuit network, which is a characteristic of the central nervous system, and achieve complete functional recovery with existing treatment methods. Therefore, there is a need for a treatment method that can achieve complete nerve circuit network repair aiming at functional recovery, especially the recovery of higher brain functions. As an agent for reconstructing the damaged nerve circuit network, stem cells are considered to be very useful. The methods include (1) activation of endogenous neural stem cells existing in the subventricular zone of the lateral ventricle and the subgranular layer of the hippocampus, and (2) stem cell transplantation. However, the survival rate of the newly generated nerves in (1) is extremely low, and it is impossible to reconstruct the nerve circuit network only by this. Therefore, (2) is necessary as a means for reconstructing the damaged nerve circuit network.

[0006] In stem cell transplantation, it is important to use what kind of transplanted donor cells. Various cells have been proposed as candidates for transplanted donor cells so far. So far, the functional improvement effects in brain contusion animal models using ES cell-derived neural stem cells (NSCs) and mesenchymal stem cells (MSCs) have been reported (Non-Patent Documents 1 to 6). In 2014, bone marrow-derived MSC (SB623) was transplanted into patients with chronic brain contusion (Non-Patent Document 7). Most of them, including the current ongoing clinical studies, use MSCs (NCT02210624). However, the action of MSCs is not strictly aimed at circuit network reconstruction. It is based on the theory that transplanted cells secrete humoral factors with neurotrophic and protective effects and angiogenesis-promoting effects, and indirectly promote regeneration.

[0007] On the one hand, reports have recognized that neural epithelial stem cells are excellent as transplantation donor cells due to their high neural differentiation ability (Non-Patent Document 8). Although there were ethical and practical problems in using neural stem cells as donor cells, human iPS cell-derived neural stem cells are ideal donor cells that can solve these problems. Since there are concerns about the problem of tumorigenicity in iPS cells as well as in other transplantation cell therapies, production methods that do not use c-Myc, etc., which are one of the causes, have also been studied, but they cannot be completely denied. On the contrary, it has also been reported that in an attempt to avoid tumorigenesis, the proliferation ability of the stem cells themselves decreases and their pluripotency is lost (Nakagawa M. Nat Biotechnol 2008; Stadtfeld M. Science. 2008).

[0008] Although various chemical compounds with various mechanisms against various target molecules have been developed as tumor therapeutic agents, the problem of side effects due to being chemical substances always exists, and a safe tumor therapeutic agent remains an urgent issue.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0010] When considering clinical application, human iPS cell-derived neural stem cells that can solve ethical and practical problems are ideal as donor cells. However, as described above, iPS cells also have the problem of tumorigenicity, similar to other transplant cell therapies. The first object of the present invention is to provide human iPS cell-derived neural stem cells that have solved such tumorigenicity problems.

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

[0012] Furthermore, if the therapeutic effect of the above cell preparation can be predicted in advance, unnecessary treatment can be prevented and efficient treatment becomes possible. The third object of the present invention is to provide a means for predicting the therapeutic effect of the above cell preparation.

[0013] Furthermore, neural stem cells show migratory and tropic properties towards tumors, but it has not been clarified by what molecules this occurs. If this can be clarified, it will be possible to produce neural stem cells with enhanced migratory and tropic properties towards tumors and efficiently select neural stem cells with high migratory and tropic properties. The fourth object of the present invention is to identify molecules that confer such migratory and tropic properties to neural stem cells and provide a means for producing neural stem cells with enhanced migratory and tropic properties and an efficient means for selecting neural stem cells with high migratory and tropic properties.

[0014] Furthermore, the neural stem cells used in the cell preparation must be highly safe. However, genome editing technologies such as CRISPR / Cas9 used in the introduction of suicide genes have a problem of off-target effects where mutations are introduced at 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 for cell selection, but from the viewpoint of safety, it is desirable that such foreign genes be removed. The fifth object of the present invention is to provide a means for producing highly safe neural stem cells.

Means for Solving the Problems

[0015] As a result of intensive studies to solve the above problems, the present inventors obtained the following findings. 1) When using iPS cell-derived neural stem cells as transplantation donor cells, by previously introducing a suicide gene into the iPS cells, when the transplanted cells become tumorigenic, the transplanted cells can be killed by administering a prodrug, and it was found that the above-described problem of tumorigenicity can be solved. In addition, when a prodrug is administered after a certain period has elapsed since the transplantation of neural stem cells, undifferentiated cells at risk of tumorigenesis die while the therapeutic effect by the transplanted cells continues.

[0016] 2) It was found that by inserting a suicide gene into the 3' side immediately after the translation region of the β-actin gene of iPS cells, the sensitivity of the suicide gene to the prodrug increases.

[0017] 3) It was found that by measuring the expression levels of the thymidylate synthase gene and the dihydropyrimidine dehydrogenase gene in tumor cells, the antitumor effect of therapeutic stem cells into which a suicide gene has been introduced against the tumor cells can be estimated.

[0018] 4) It has been found that the ligand and receptor pair of ephrin B and EphB receptor, and the ligand and receptor pair of CXC motif chemokine 12 (CXCL12) and CXC motif chemokine receptor 4 (CXCR4) are involved in the migration and tropism of neural stem cells towards tumors.

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

[0020] 6) It has been found that neural stem cells differentiated from pluripotent stem cells exhibit strong tropism and migration towards tumors including brain tumors and damaged 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 and 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 and injuries according to [1], which is used for treating brain dysfunction.

[0023] The cell preparation for treating central nervous system diseases and injuries according to [1], which is characterized by being used for treating traumatic brain injury.

[0024] The cell preparation for treating central nervous system diseases and injuries according to [1], which is characterized by being used for treating spinal cord injury.

[0025] The cell preparation for treating central nervous system diseases and injuries according to [1], which is characterized by being used for treating neurodegenerative diseases.

[0026] The cell preparation for treating central nervous system diseases and injuries according to [1], which is characterized by being used for treating brain tumors.

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

[0028] The cell preparation for treating central nervous system diseases and injuries according to [7], which is 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 pluripotent stem cells, and a suicide gene is linked to the 3'-side of the sequence encoding the 2A peptide.

[0029] The cell preparation for treating central nervous system diseases and injuries according to [1] to [8], which is characterized in that the neural stem cells are neural stem cells that do not contain a selection marker gene in their genome.

[0030] The cell preparation for treating central nervous system diseases and injuries according to [9], which is characterized in that the neural stem cells are neural stem cells obtained by a method including the following steps (A) to (D). (A) A step of inserting a gene construct into the genome of pluripotent stem cells by genome editing, wherein the gene construct includes a suicide gene, a selection marker gene, and target sequences for two Cre proteins, and the selection marker gene is a gene construct sandwiched between the target sequences for two Cre proteins. (B) Step of selecting pluripotent stem cells in which a suicide gene has been inserted into the genome from among the pluripotent stem cells obtained in step (A) using a selection marker gene (C) Step of removing the selection marker gene from the genome of the pluripotent stem cells obtained in step (B) using Cre protein (D) Step of differentiating the pluripotent stem cells obtained in step (C) into neural stem cells

[0031]

[11] The cell preparation for treating central nervous system diseases and injuries according to

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

[0032]

[12] 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. 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. The cell preparation for treating central nervous system diseases and injuries according to

[10]

[0033]

[13] The cell preparation for treating central nervous system diseases and injuries according to [9], characterized in that the neural stem cells are neural stem cells obtained by a method including the following steps (a) to (d) (a) Step of inserting a gene construct into the genome of pluripotent stem cells by genome editing. 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. The selection marker gene is a gene construct sandwiched between the two mutant loxP sequences (b) Step of selecting pluripotent stem cells in which a suicide gene has been inserted into the genome from among the pluripotent stem cells obtained in step (a) using a selection marker gene (c) A step of removing a selection marker gene from the genome of the pluripotent stem cells obtained in step (b) by Cre protein and a homologous recombination vector, and inserting a second gene into the genome, wherein the homologous recombination vector contains a second gene, a mutant loxP sequence having a mutation in the repetitive sequence, and a mutant loxP sequence having a mutation in the spacer sequence, and the second gene is a homologous recombination vector sandwiched between the two mutant loxP sequences. (d) A step of differentiating the pluripotent stem cells obtained in step (c) into neural stem cells.

[0034]

[14] The cell preparation for treating central nervous system diseases and injuries according to

[10] to

[13] , wherein the genome editing is genome editing using CRISPR / Cas3.

[0035]

[15] The cell preparation for treating central nervous system diseases and injuries according to [1] to

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

[0036]

[16] The cell preparation for treating central nervous system diseases and injuries according to

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

[0037]

[17] The cell preparation for treating central nervous system diseases and injuries according to [1] to

[16] , wherein the neural stem cell is a neural stem cell with increased expression levels of at least one selected from Ephrin A receptor, Ephrin A, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4.

[0038]

[18] The cell preparation for treating central nervous system diseases and injuries according to

[17] , wherein the neural stem cell is a neural stem cell with increased expression levels of Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4.

[0039] 〔19〕The cell preparation for treating central nervous system diseases and injuries according to any one of 〔1〕to 〔16〕, wherein the neural stem cells are selected neural stem cells using the expression level of at least one selected from Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 as an index.

[0040] 〔20〕The cell preparation for treating central nervous system diseases and injuries according to 〔19〕, wherein the neural stem cells are selected neural stem cells using Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 as an index.

[0041] 〔21〕A method for producing neural stem cells, comprising the following steps (1) and (2). (1) A step of inserting a suicide gene into the 3'-side immediately after the translation region of the β-actin gene of pluripotent stem cells by genome editing. (2) A step of differentiating the pluripotent stem cells obtained in step (1) into neural stem cells.

[0042] 〔22〕The method for producing neural stem cells according to 〔21〕, wherein in step (1), a sequence encoding a 2A peptide is ligated to the 3'-side immediately after the translation region of the β-actin gene, and the suicide gene is ligated to the 3'-side of the sequence encoding the 2A peptide, and the suicide gene is inserted into the 3'-side immediately after the translation region of the β-actin gene of pluripotent stem cells.

[0043] 〔23〕The method for producing neural stem cells according to 〔21〕or 〔22〕, wherein step (1) includes 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 pluripotent stem cells by genome editing, wherein the gene construct includes a suicide gene, a selection marker gene, and target sequences for two Cre proteins, and the selection marker gene is sandwiched between the target sequences for two Cre proteins. Step of selecting pluripotent stem cells in which a suicide gene has been inserted into the genome from among the pluripotent stem cells obtained in step (1-A) using a selection marker gene. Step of removing the selection marker gene from the genome of the pluripotent stem cells obtained in step (1-B) using Cre protein.

[0044] The method for producing neural stem cells according to

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

[0045] The method for producing neural stem cells according to

[23] , 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.

[0046] The method for producing neural stem cells according to

[21] or

[22] , characterized in that step (1) includes the following steps (1-a) to (1-c). Step of inserting a gene construct immediately after the translation region of the β-actin gene of pluripotent stem cells by genome editing, wherein the gene construct includes 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, and the selection marker gene is a gene construct sandwiched between the two mutant loxP sequences. Step of selecting pluripotent stem cells in which a suicide gene has been inserted into the genome from among the pluripotent stem cells obtained in step (1-a) using a selection marker gene. Step (1-c) is a step of removing a selection marker gene from the genome of the pluripotent stem cells obtained in step (1-b) by Cre protein and a homologous recombination vector, and inserting a second gene into the genome, wherein the homologous recombination vector includes a second gene, a mutant loxP sequence having a mutation in a repetitive 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 according to 〔23〕~〔26〕, characterized in that the genome editing is genome editing using CRISPR / Cas3.

[0048] 〔28〕The method for producing neural stem cells according to 〔21〕~〔27〕, characterized in that the suicide gene is a cytosine deaminase gene and a uracil phosphoribosyltransferase gene.

[0049] 〔29〕A method for estimating the antitumor effect of the cell preparation for treating central nervous system diseases and injuries according to 〔15〕 against brain tumor cells, comprising the step of measuring the expression levels of the thymidylate synthase gene and the dihydropyrimidine dehydrogenase gene of the brain tumor cells.

[0050] 〔30〕A method for screening neural stem cells with high migratory and / or directional properties against a tumor or injury site, characterized by screening using at least one expression level selected from Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 as an index.

[0051] 〔31〕The screening method according to 〔30〕, characterized by screening using the expression levels of Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 as an index.

[0052] A cell preparation for tumor treatment, which is a neural stem cell produced by the method described in

[32] ,

[21] to

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

[0053] The cell preparation for tumor treatment according to

[32] , wherein the neural stem cell does not contain a selection marker gene in its genome.

[0054] The cell preparation for tumor treatment according to

[32] or

[33] , wherein the neural stem cell is a neural stem cell with an increased expression level of at least one selected from Ephrin A receptor, Ephrin A, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4.

[0055] The cell preparation for tumor treatment according to

[34] , wherein the neural stem cell is a neural stem cell with an increased expression level of Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4.

[0056] The cell preparation for tumor treatment according to

[32] or

[33] , wherein the neural stem cell is a neural stem cell selected using at least one of Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 as an index.

[0057] The cell preparation for tumor treatment according to

[34] , wherein the neural stem cell is a neural stem cell selected using Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 as an index.

[0058] The cell preparation for tumor treatment according to

[32] to

[37] , wherein the tumor is pancreatic cancer.

[0059] The specification includes the content described in the specifications and / or drawings of Japanese Patent Application Nos. 2020-063477, 2020-165847, and 2020-219455, which are the basis of the priority of the present application.

Advantages 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 a cell preparation for the treatment of traumatic brain injury including brain contusion or brain infarction, or neurodegenerative diseases caused by neuronal cell loss resulting from neurodegeneration, the tumorigenesis of cells becomes a problem. However, the cell preparation of the present invention can kill cells by a suicide gene, so it is a highly safe cell preparation.

Brief Description of the Drawings

[0061]

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Mode for Carrying Out the Invention

[0062] Hereinafter, the present invention will be described in detail. (A) Cell preparation The tumor treatment preparation and the 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 the herpes simplex virus thymidine kinase gene (HSVtk), carboxylesterase, deoxycytidine kinase gene, deoxycytidine kinase / cytosine arabinoside, cytosine deaminase (CD) gene, and uracil phosphoribosyltransferase (UPRT) gene (CD-UPRT gene). In the present invention, the cytosine deaminase (CD) gene and the uracil phosphoribosyltransferase (UPRT) gene (CD-UPRT gene) can be cited as preferred examples.

[0064] The "cell preparation for treating central nervous system diseases and injuries" in the present invention means a cell preparation for treating central nervous system diseases and central nervous system injuries. More specifically, it refers to a cell preparation used for treating diseases and injuries in the central nervous system (such as the brain and spinal cord). The diseases and injuries to be treated are not particularly limited, and examples include brain tumors, neurodegenerative diseases, brain dysfunction, spinal cord injuries, etc., and preferably brain tumors or brain dysfunction. The brain dysfunction to be treated is not particularly limited, and examples include dysfunction due to physical injury, such as dysfunction due to brain contusion, dysfunction due to cerebrovascular disorders (such as cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage), etc. Neurodegenerative diseases are diseases caused by the loss of nerve cells due to neurodegeneration, and specific examples include Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal lobar degeneration, etc. Examples of brain tumors include glioma (neuroglioma), medulloblastoma, neuroblastoma, meningioma, pituitary adenoma, schwannoma, primary central nervous system lymphoma, sarcoma, spinal cord tumor, etc. In the present invention, any of these brain tumors can be a treatment target, but it is preferable to treat glioma. When the cell preparation of the present invention is used for the treatment of brain tumors, "treatment" includes not only killing tumor cells but also reducing tumor cells and inhibiting the growth of tumor cells.

[0065] The "cell preparation for tumor treatment" in the present invention means a cell preparation used for the treatment of tumors. The cell preparation of the present invention utilizes the properties of the migratory and directional characteristics of neural stem cells to tumor tissues to cause a suicide gene to act on tumor tissues. Here, the "treatment" includes not only killing tumor cells, but also reducing tumor cells and inhibiting the proliferation of tumor cells. As tumors, due to the characteristics of the neural stem cells mentioned above, not only the brain tumors and pancreatic cancers listed in the examples, but also many tumors, such as breast cancer, gastric cancer, lung cancer, etc., are targeted.

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

[0067] The "neural stem cells" in the present invention refer to stem cells having the ability to supply cells that differentiate into neurons and glial cells. The cell preparation of the present invention contains these neural stem cells, but may contain cells other than neural stem cells as long as it does not have a great adverse effect on the therapeutic effect of brain injury and brain diseases. When neural stem cells are prepared 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 contain both neural stem cells and neural progenitor cells.

[0068] The higher the migration ability and tropism of neural stem cells to tumors, the more neural stem cells can be accumulated at the disease or injury site, and a high therapeutic effect can be expected. Therefore, among the ligand-receptor pairs involved in the above-mentioned migration ability and tropism in neural stem cells, those expressed in neural stem cells, specifically, it is preferable to increase the expression levels of Ephrin A, Ephrin A receptor, Ephrin B, Ephrin B receptor, and CXC motif chemokine receptor 4. Regarding 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 them, but it is also possible to increase only the expression levels of the three of Ephrin B, Ephrin B receptor, and CXC motif chemokine receptor 4, or it is also possible to increase only the expression level of at least one selected from the above-mentioned three. The increase in the expression levels of these ligands and receptors can be carried out according to known methods. For example, a method of introducing the genes of these ligands and receptors into neural stem cells (optionally, it may be introduced together with a control region such as a strong expression promoter that increases the expression level of the gene), a method of using a substance having an effect of increasing the expression levels of these ligands and receptors, etc. can be used.

[0069] In addition, a high therapeutic effect can also be expected by using neural stem cells that originally have high migration ability and tropism to tumors. Therefore, as the neural stem cells, neural stem cells selected by the selection method of the present invention described later may be used.

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

[0071] The introduction of suicide genes into pluripotent stem cells may be carried out using a viral vector, but preferably, it is carried out using genome editing. This is because when a suicide gene is inserted into the genome of pluripotent stem cells using a viral vector such as a lentiviral vector, the suicide gene is randomly inserted into the chromosome, so there are concerns about gene mutations at the insertion site, activation of surrounding genes, and inactivation of the suicide gene due to the position effect. It is considered 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. Among these, it is preferable to perform using CRISPR / Cas9 or CRISPR / Cas3, and it is particularly preferable to perform using CRISPR / Cas3. Since the recognition target sequence of CRISPR / Cas3 is longer than that of CRISPR / Cas9, the frequency of off-target effects is low, and safer neural stem cells can be produced. By genome editing, a suicide gene can be inserted into the region of the housekeeping gene of pluripotent stem cells or the safe harbor region AAVS1. Examples of the housekeeping gene include the β-actin gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, cyclophilin gene, α-tubulin gene, and the like. 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. At this time, it is also preferable to insert the suicide gene into the 3' side immediately after the translation region of the β-actin gene such 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.

[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, the presence of foreign genes such as selection marker genes remaining in the genome of neural stem cells differentiated from pluripotent stem cells is not preferable from the perspective of safety. Therefore, it is preferable to remove the selection marker gene. The means for removing the selection marker gene is not particularly limited, 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 with a mutation (mutated loxP sequence). The loxP sequence consists of an upstream repeat sequence, a spacer sequence, and a downstream repeat sequence, but the mutation in the mutated loxP sequence may be present in any of these sequences. Specific examples of the mutated loxP sequence having a mutation in the upstream repeat sequence include lox71 (SEQ ID NO: 2), specific examples of the mutated loxP sequence having a mutation in the spacer sequence include lox2272 (SEQ ID NO: 4), and specific examples of the mutated loxP sequence having a mutation in the downstream repeat sequence include lox66 (SEQ ID NO: 3).

[0074] As a method for removing a selection marker gene using the Cre / loxP system, a gene construct containing a suicide gene, a selection marker gene, and two loxP sequences, with the selection marker gene flanked by the two loxP sequences, is prepared. This is inserted into the genome of pluripotent stem cells, and then a method of removing the selection marker gene from the genome of pluripotent stem cells by Cre protein can be exemplified. However, in this method, one loxP sequence remains in the genome of pluripotent stem cells, and there is a risk that it will react with Cre protein. Therefore, it is preferable to replace the loxP sequences contained in the gene construct with mutant loxP sequences so that the loxP sequence remaining in the genome is a mutant loxP sequence that is less likely to react with Cre protein. Specifically, the two loxP sequences contained in the 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 such that 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 pluripotent stem cells, and then a method of removing the selection marker gene from the genome of pluripotent stem cells by Cre protein can be exemplified. In this method, what remains in the genome is a mutant loxP sequence having mutations in the upstream repeat sequence and the downstream repeat sequence, and the risk of reacting with Cre protein can be reduced.

[0075] In addition to the above gene construct, it is also possible to use a homologous recombination vector to not only remove the selection marker gene from the genome but also insert a second gene into the genome at the same time. In this case, the gene construct contains 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 a gene construct in which the selection marker gene is sandwiched between the two mutant loxP sequences is used. Further, the homologous recombination vector contains 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 a homologous recombination vector in which the second gene is sandwiched between the two mutant loxP sequences is used. As the second gene, a gene involved in a treatment different from the suicide gene can be used.

[0076] The position of the mutant loxP sequence in the gene construct is not particularly limited. 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. 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 will remain in the genome.

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

[0078] When the Cre protein and the above homologous recombination vector are allowed to act on the genome into which the above gene construct has been inserted, homologous recombination occurs, the selection marker gene is removed from the genome, and at the same time the second gene is inserted into the genome. A gene encoding the Cre protein may be inserted into the homologous recombination vector, whereby the Cre protein and the homologous recombination vector may be allowed to act simultaneously.

[0079] Differentiation of pluripotent stem cells into neural stem cells may be carried out according to any known method, either through embryoid bodies or without embryoid bodies. When differentiating from iPS cells, for example, it can be carried out according to 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 carried out using a known embryoid body formation medium, which contains a TGFβ family inhibitor (e.g., SB431542) and a BMP inhibitor (e.g., LDN-193189). Differentiation of embryoid bodies into neural stem cells can be carried out using a known neurosphere medium. For example, the neurosphere medium contains epidermal growth factor, fibroblast growth factor-2, leukemia inhibitory factor, B-27 supplement, etc. By culturing embryoid bodies in the neurosphere medium, a cell mass containing neural stem cells called neurospheres is formed. The formed neurospheres are collected, dispersed into single cells, and cultured in the neurosphere medium, and neurospheres are formed again. After repeating such operations several times, neural stem cells can be obtained by collecting the neurospheres.

[0080] As described above, in this specification, the "neural stem cells" are specified by the manufacturing method rather than by their structure or properties. This is because since cells are part of a living body, their structure and properties are extremely complex, and the work of specifying them requires extremely excessive economic expenditure and time.

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

[0082] The number of neural stem cells contained in the cell preparation of the present invention can be appropriately determined in consideration of diseases such as tumors, the types of brain diseases and brain injuries, the gender, age, body weight of the subject, the state of the affected part, the state of the cells to be used, etc., so as to obtain the desired effect in the treatment of tumors, brain diseases and brain injuries.

[0083] The cell preparation of the present invention may be administered multiple times (for example, 2 to 10 times) at intervals (for example, twice a day, once a day, twice a week, once a week, once every two weeks). The dosage can be appropriately determined in consideration of the type of disease or injury, the gender, age, body weight of the subject, the state of the affected part, the state of the cells to be used, etc., but 1×10 6 cells to 1×10 10 cells per individual (human), and 1 to 10 administrations are preferred.

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

[0085] The cell preparation of the present invention may be used together with a prodrug 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 (for example, 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 having a suicide gene. Whether to use a prodrug can be determined according to the disease or injury to be treated. For example, when treating brain dysfunction, inducing cell death by using a prodrug is not directly related to the therapeutic effect. It is aimed at killing tumor cells and cells that may become tumorous, so it is not necessarily the case that a prodrug must be used. On the other hand, when treating a brain tumor, inducing cell death by using a prodrug is directly related to the therapeutic effect, so the use of a prodrug is essential.

[0086] The administration site and method of the prodrug are not particularly limited. Examples of the administration method include oral administration, local administration to the brain or tissues, intracarotid administration, intravenous administration, and intraperitoneal administration.

[0087] The administration timing of the prodrug can be determined according to the disease or injury to be treated. For example, when treating a brain injury, it may be administered at the time when tumor cells appear, or in order to prevent the occurrence of tumor cells, administration may be started after a certain period of time has elapsed since the administration of the cell preparation of the present invention. The start timing of such preventive administration is not particularly limited as long as it does not lose the therapeutic effect of neural stem cells and can kill undifferentiated cells at risk of tumorigenesis. For example, it may be 3 to 60 days or 5 to 20 days after the administration of the cell preparation of the present invention. When treating a brain tumor, it may be before, during, or after the administration of the cell preparation of the present invention, but usually, after the administration of the cell preparation, it is administered in multiple divided doses. The same applies to other tumors.

[0088] The dosage of the prodrug can be appropriately determined in consideration of the type of prodrug used, the type of disease or injury, the gender, age, body weight of the subject, the condition of the affected part, etc. In the case of administering 5-FC, it is preferably administered at 50-200 mg / kg per day per individual (human) 4 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 including the following steps (1) and (2).

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

[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 cell preparation for treating central nervous system diseases and injuries of the present invention described above on brain tumor cells, and includes the step of measuring the expression levels of the thymidylate synthase gene and the dihydropyrimidine dehydrogenase gene of the brain tumor cells.

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

[0094] As a result of the measurement, when the expression levels of the thymidylate synthase gene and the dihydropyrimidine dehydrogenase gene are high, it can be estimated that the antitumor effect of the cell preparation for treating central nervous system diseases and injuries of the present invention on the brain tumor cells to be measured is low. On the other hand, when the expression level of the gene is low, it can be estimated that the antitumor effect of the cell preparation for treating central nervous system diseases and injuries of the present invention on the brain tumor cells to be measured is high.

[0095] (D) Method for selecting neural stem cells with high migratory and directional properties The selection method of the present invention is a method for selecting neural stem cells with high migratory and / or directional properties for a tumor or injury site, and is characterized by selecting at least one expression level selected from Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 as an index.

[0096] For Ephrin A, Ephrin A receptor, Ephrin B, Ephrin B receptor, and CXC motif chemokine receptor 4, it is preferable to perform selection using the expression levels of all five of them as an index. However, selection may also be performed using only the expression levels of the three, i.e., Ephrin B, Ephrin B receptor, and CXC motif chemokine receptor 4, or selection may be performed using the expression level of at least one selected from the above three as an index.

[0097] As a specific selection method, for example, a method including the following steps (1) and (2) can be exemplified. (1) A step of measuring the expression level of at least one selected from Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 (2) A step of selecting the neural stem cells as "neural stem cells with high migratory ability and / or directivity to a tumor or damaged site" when the expression level measured in step (1) is higher than a reference value.

[0098] Here, the reference value used for selection is not particularly limited. For example, the expression levels of Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, or CXC motif chemokine receptor 4 of generally used neural stem cells (for example, commercially available neural stem cells, etc.) may be measured in advance and used as the reference value.

Example

[0099] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. In the following examples, "NSC" shall generally mean "NS / PCs (Neural stem / Progenitor cells)".

[0100] 〔Example 1〕 (A) Method <Human iPS cells> The human iPS cells (1210B2) used were obtained from the Institute for Integrated Cell-Material Sciences (CiRA), Kyoto University. 1210B2 was established by a method of introducing reprogramming factors into human peripheral blood mononuclear cells using an episomal vector (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.). The human iPS cells were seeded on plastic culture dishes coated with iMatrix-511 (manufactured by Nippi Inc.) and maintained in culture by a known feeder-free method (Nakagawa M. Sci Rep. 2014) using Stem Fit AK03 or AK03N medium (manufactured by Ajinomoto Co., Inc.).

[0101] <Induction of differentiation from human iPS cells into neural stem / progenitor cells (NSC)> Induction of differentiation from human iPS cells into NSC was performed by a known method (Sugai K. Mol Brain. 2016). First, 10 μM ROCK inhibitor Y276352 (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the medium of iPS cells and incubated for 1 to 3 hours, after which it was washed with PBS and dispersed into single cells using TrypLE Select (manufactured by Life Technologies). The human iPS cells dispersed into single cells were suspended in EB (embryoid body) formation medium (10 μM SB431542 and 100 nM LDN-193189 were added to Stem Fit medium without adding solution C) supplemented with 10 μM ROCK inhibitor Y276352, and 9.0×10 per well of a low-adhesion 96-well culture plate (Prime Surface 96V, manufactured by 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 - 14 days. EBs were collected from each well, and 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)) was added and cultured 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 - 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 - 4 days, and secondary, tertiary, quaternary, and quinary NSCs were obtained by culturing for 7 - 10 days.

[0102] <Generation of yCD-NSC, a therapeutic stem cell, by introducing the yCD-UPRT gene into the GAPDH, ACTB, and AAVS1 gene regions using CRISPR / Cas9> Generation of iPS cells with the yCD-UPRT gene introduced into each gene region Using the CRISPR / Cas9 genome editing technology, iPS cells were generated by inserting the yCD-UPRT fusion suicide gene into the housekeeping gene regions GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ACTB (β-actin), and the safe harbor region AAVS1 (PPP1R12C). For genome editing, the DNA construct for integrating yCD-UPRT into the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene region was inserted in the form of a homologous recombination construct (HR-GAPDH-2A-yCD-UPRT-2A-Bsd) where GAPDH, yCD-UPRT, and Bsd (blasticidin resistance gene) were connected by a 2A peptide sequence, and a gRNA expression vector construct (U6-GAPDH-gRNA4-Cas9) targeting the vicinity of the stop codon of GAPDH and the Cas9 expression vector construct were prepared. In the case of β-actin, similar to GAPDH, a gRNA expression vector targeting the vicinity of the stop codon of β-actin was constructed, and the homologous recombination construct was also constructed in such a way that β-actin, yCD-UPRT, and Bsd were connected by a 2A peptide sequence (β-actin-2A-yCD-UPRT-2A-Bsd). The homologous recombination construct for the AAVS1 region was made 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 a 2A peptide sequence.

[0103] These constructs were introduced into the human iPS cell line (1210B2) by electroporation, cultured in the presence of blasticidin S, and cloning was performed. The confirmation of the homologous recombination iPS cell line was carried out by genomic PCR of each clone and nucleotide sequence confirmation by genomic sequencing.

[0104] Generation of yCD-UPRT-expressing NSCs (yCD-NSCs) From iPS cells with yCD-UPRT integrated into each gene region, differentiation was induced into NSCs to obtain secondary to fifth-generation NSCs (yCD-NSCs). Blasticidin S at 1 μg / ml was constantly added to the medium for culture. It was confirmed that cell death was induced in yCD-UPRT-expressing NSCs by adding 5-FC at 1-5 μg / ml to the medium.

[0105] Supplementary information ※Regarding the migratory and antitumor effects of yCD-NSCs on glioblastoma yCD-NSCs (labeled with hKO1) were transplanted 1 mm above the tumor mass of the human glioma cell line U87 (ffLuc), and their migratory ability was quantitatively evaluated using brain slice culture (Tamura R. Mol Brain. 2019). As comparison controls, human adipose tissue-derived and bone marrow-derived mesenchymal stem cell lines (labeled with hKO1) were used. Using direction and migration distance as parameters, Rose Diagram Maps (Angular histogram) were created to quantitatively evaluate the migratory ability in the brain. 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-NSCs In the right striatum of the normal brain, 5×10 5 individual yCD-NSCs (ffLuc) were transplanted in the same manner as above, and 5-FC was administered for 2 weeks starting from 7 days later. A non-5-FC-administered group was also prepared as a control. Then, after 150 days, the mice were decapitated and the brain tissues were evaluated. 5-FU converted from 5-FC is theoretically considered not to affect the cells in the brain occupied by terminally differentiated cells, but may affect slightly dividing cells such as neural progenitor cells near the ventricle and vascular endothelial cells. Therefore, histological evaluation was performed using vascular endothelial cell marker (CD31) and neural stem cell / progenitor cell marker (Nestin). Also, 5-FC administration was started on the 35th day after yCD-NSC transplantation, and it was confirmed by IVIS that the engrafted yCD-NSCs disappeared.

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

[0108] Differentiation of yCD-UPRT-expressing NSCs into neurons and 5-FU sensitivity of the 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 remaining undifferentiated proliferating cells were 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 to evaluate the changes in these differentiated and undifferentiated cells caused by 5-FU released by the introduced yCD-UPRT.

[0109] Generation of ffLuc-expressing yCD-NSCs The prepared yCD-NSCs were infected with an ffLuc (fusion gene of Venus fluorescent protein and Luc2 firefly luciferase) expression lentiviral vector (CSII-EF-ffLuc) (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 in which ffLuc was stably and highly expressed.

[0110] <Examination of the Therapeutic Effect of yCD-NSC in a Traumatic Brain Injury Model Mouse> Under general anesthesia, a circular craniotomy with a diameter of 2.5 mm centered 1.25 mm lateral and 1.25 mm rostral to the great cerebral vein was performed on a T cell-deficient mouse (Female BALB / c nude mouse, 20 g, 10 w). The bone flap was protected for later replacement. The cerebral surface was exposed, and a traumatic brain injury was created by cooling the cerebral surface using a 2.5 mm probe of the ACU22XT amoyls cryosurgical device (Keeler and Winer) (60 °C for 30 sec × 10 sets). Subsequently, 1 × 10 5 yCD-NSCs were transplanted into the site directly below the injury at the same location (2.5 mm lateral, 1.25 mm rostral, 2 mm depth) from the great cerebral vein. The excised bone flap was replaced, and the skin was sutured to complete the procedure.

[0111] For the behavioral evaluation of the mice, first, the Rota rod (UGO BASILE) was used twice and the grip strength measuring device MK-380Si for smart rats / mice (Muromachi Kikai) was used once before the surgery to familiarize the mice with the instruments. The Rota rod increased the rotation speed from 2 r.p.m to 20 r.p.m 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 was in accordance with the previously reported method (Tabuse J Clin Neurosci. 2010). For grip strength measurement, a plastic cover was placed on the net attached to the measuring instrument to limit the area of the net that could be gripped, enabling the measurement of the grip strength of only one limb. The grip strength was measured for all four limbs. When the net was gripped with five fingers, the tail was pulled horizontally, and the peak value until the limb separated from the net was measured. Each limb was measured seven times, and the average value of five measurements excluding the maximum and minimum values was compared (Alamri FF. Behav Brain Res. 2018).

[0112] After the surgery, the motor function was evaluated using the above method on days 3, 5, 7, 14, 21, 28, 35, and 42. The longitudinal changes of yCD-NSCs in the same individual were observed weekly using an IVIS in vivo imaging system. During IVIS imaging, VivoGlo was used under isoflurane inhalation anesthesiaTM Luciferin at a concentration of 30 mg / ml was intraperitoneally administered at a dose of 200 μl, and imaging was performed 5 minutes after reaching the peak. In addition, on the 7th, 14th, 28th, and 42nd days after the production of brain contusion, four animals were sacrificed by perfusion fixation under decapitation to obtain brain tissues. At the time of decapitation, a 2% solution of Evans blue (Sigma-Aldrich) (the solvent was sterile physiological saline) was injected from the tail vein at a dose of 0.2 ml / animal 2 hours before decapitation, and then decapitation was performed.

[0113] Also, aiming at about 1 mm below the contusion (3 mm in depth from the brain surface), 1×10 5 individuals of yCD-NSCs were transplanted on the same day. Then, on the 42nd day, decapitation was performed to evaluate the tissues. 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 brain contusion> Similar to the above, after making the contusion and transplanting yCD-NSCs, intraperitoneal administration of 5-FC at a dose of 5 mg / animal / day was continuously performed for 2 weeks 7 days later. The motor function (grip strength) was compared with both controls, the 5-FC non-administration group and the yCD-NSC non-transplanted group. Thus, it was confirmed that the release of 5-FU did not have an adverse effect on brain function.

[0115] (B) Results <Establishment of suicide gene-expressing and therapeutic NSCs> In the gene transfer of the suicide gene (yCD-UPRT) into human iPS cells using a lentiviral vector, gene silencing occurred during the process of inducing differentiation into neural stem cells / progenitor cells (NSCs) (Figure 1A). Therefore, the present inventor succeeded in achieving the constitutive and stable expression of yCD-UPRT by inserting it into the housekeeping gene region β-actin (ACTB) using the genome editing technology CRISPR / Cas9 (Figure 1B). yCD-UPRT induces cell death by converting the prodrug antifungal agent 5-FC into the anticancer agent 5-FU (Figure 1C).

[0116] In order to optimize the insertion site of the suicide gene, the present inventors also introduced yCD-UPRT into other housekeeping genes GAPDH and the safe harbor region AAVS1 using CRISPR / Cas9. In the AAVS1 region, silencing occurred similar to that with viral vectors, and in the GAPDH region, the sensitivity to the prodrug 5-FC was less than half that of the β-actin region (Figure 1D). Therefore, the present inventors determined that β-actin is the optimal gene introduction region for yCD-UPRT. The sensitivity to 5-FC was quantitatively analyzed by CCK-8 assay.

[0117] <Antitumor effect of therapeutic NSCs (yCD-NSCs) on glioma model mice> The present inventors have constructed a unique method for quantitatively evaluating the tropism by photographing in real time the behavior of iPS cell-derived NSCs migrating towards tumors in the brain (see Figure 2A Rose diagram map). As a result, iPS cell-derived NSCs showed better tropism and migration in the brain than other adipose-derived (AMSC) and bone marrow-derived mesenchymal stem cells (BMSC) that are sometimes used as transplant cell therapies, indicating their usefulness as a cellular delivery vehicle (Figure 2A). The therapeutic NSCs (yCD-NSCs) established by the method shown in Figure 1 were transplanted into glioma model mice (U87) (Figure 2B). This is a treatment that utilizes the tropism towards tumors. As a result, administration of 5-FC showed an effective therapeutic effect compared to the control (Figure 2C). Note that two control groups were prepared: a group that received transplantation of yCD-NSCs but no 5-FC, and a group that received no transplantation of yCD-NSCs but received 5-FC.

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

[0119] <Brain protective effect of therapeutic NSCs (yCD-NSCs) against brain contusion> The present inventors have established and reported a brain contusion model using a cooling device. By creating a brain contusion in the left forebrain, it is possible to stably create a model presenting right-sided incomplete lower limb paralysis (Figure 3A). The yCD-NSCs were infected with an ffLuc (fusion gene of Venus fluorescent protein and Luc2 firefly luciferase) expression lentiviral vector so that the transplanted yCD-NSCs could be identified by IVIS (Figure 3B). In addition, in the method shown in Figure 2, glioma cell lines were infected to evaluate the therapeutic effect. yCD-NSCs showed remarkable migratory ability not only to tumor cells but also to damaged brains (brain contusions). The yCD-NSCs transplanted contralateral to the brain contusion showed signals at the brain contusion site within less than two weeks (Figure 3C).

[0120] When yCD-NSCs were transplanted into the brain contusion site, engraftment was monitored by IVIS, and 5-FC was administered 35 days later, the signal of yCD-NSCs completely disappeared (Figure 3D). Tumorigenesis of transplanted cells is always a problem in cell therapy, but the yCD-NSCs established by the present inventors are killed by 5-FC administration, which means that they have a safety device against tumorigenesis and enables the realization of safe regenerative medicine.

[0121] In the treatment group transplanted with yCD-NSCs for brain contusion, a significant improvement in motor function (grip strength) was observed compared to the control group (Figure 3E). Furthermore, as a result of visualizing the brain injury site with a dye (Evans blue), in the brain tissue 14 days after brain contusion, a significant reduction in the brain injury site was observed in the brain transplanted with yCD-NSCs (Figure 3F). This means the neuroprotective effect of yCD-NSCs on the acutely injured brain. Also, when yCD-NSCs were transplanted at a point 1 mm below the contusion and the brain tissue was evaluated 42 days later, remarkable migration and clustering were observed at the contusion site (Figure 3G).

[0122] <Effect of 5-FU release on motor function> The present inventors confirmed whether there was a decline in brain function by killing the NSCs that remained in an undifferentiated state (with a risk of tumorigenesis) among those that exerted a brain protection effect during the acute phase, by administering 5-FC. NSCs were transplanted during the ultra-acute phase on the day of contusion production (Day0), exerted a brain protection effect during the acute phase until Day7, and then 5-FC was administered for 2 weeks during the sub-acute phase (Figure 4A). As a result, from Day7, even when 5-FC was administered, the motor function did not decline, and the same functional recovery as the non-5-FC-administered group was obtained (Figure 4B). Also, a significantly improved functional effect was shown compared to the non-NSC-transplanted control (Figure 4B).

[0123] <Safety of therapeutic NSCs (yCD-NSCs) (differentiated cells)> As proof that yCD-NSCs do not affect the differentiated cells, an experiment was conducted in which yCD-NSCs themselves were differentiated into Neurons in vitro and the prodrug 5-FC was administered (Figure 5A). Neurospheres extend very long protrusions, and morphological differentiation into Neurons is expected. The cells that had thus extended long protrusions were positive for the Neuron marker βIII tubulin (Figure 5B). Thus, it was shown that they were differentiating into Neurons. However, on the other hand, some Ki-67-positive proliferating cells still remained (Figure 5B). It was found that when 5-FC was administered, only those Ki-67-positive proliferating cells died, and the βIII tubulin-positive differentiated cells did not die in response to the released 5-FU. It has been shown that cell death occurs in PCNA-positive proliferating cells, but not in βIII tubulin-positive differentiated cells (Figure 5B).

[0124] Also, some cells expressing NeuN, a more mature Neuron marker, were also observed, and it was clearly shown that such cells also did not undergo cell death due to the released 5-FU after 5-FC administration (Figure 5C).

[0125] <Safety of therapeutic NSCs (yCD-NSCs) (model mice)> 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 the cells in the brain occupied by terminally differentiated cells, but it may also affect the neural progenitor cells present near the ventricle and the cells with slight division such as vascular endothelium. Therefore, in this study, after transplanting yCD-NSC into the normal brain and administering 5-FC, no cells with obvious apoptosis were observed in the 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 deliberately 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 of the antitumor effect by yCD-NSC> The active metabolite of 5-FU, FdUMP, inhibits the enzymatic activity of thymidylate synthase (TS), an enzyme in the DNA de novo system (Figure 7A). Thus, the DNA de novo synthesis system is suppressed, and DNA damage occurs in a time-dependent manner. Also, 5-FU taken up into tumors is usually degraded by Dihydropyrimidine dehydrogenase (DPD), a degrading enzyme (Figure 7A). Therefore, it is expected that 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 the TS activity is inhibited by FdUMP, enhancing the antitumor effect. Therefore, the inventors considered that the expression of TS and DPD would be biomarkers for the antitumor effect of their yCD-NSC. The sensitivity of each glioma cell line to 5-FU was evaluated by CCK-8 assay. As a result, the sensitivities were (good) GL261, TSG, hG008, U87, U251 (poor) (Figure 7B).

[0128] The gene expression of the mRNA levels of TS and DPD was quantitatively analyzed by RT-qPCR. In U251, it was found that both TS and DPD were at high levels (Figure 7C). The same result was obtained by Western blotting. When a graph plotting Figure 7C was created, hG008, TSG, and GL261, which showed a significant effect on 5-FU, had a relative gene expression of 0.5 or less (evaluated with U87 as 1) for both. Also, 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. Also, ACTB (yCD-NSC) had a particularly high TS activity and could not be said to have a good response to 5-FU (Figure 7D). That is, it is further required to determine the suicide gene insertion site with good sensitivity to 5-FC, and the ACTB region evaluated by the inventors corresponds to this. Also, the fact that NSC cannot be said to have good sensitivity to 5-FU is also a result suggesting "safety" with little possibility of affecting endogenous neural stem cells.

[0129] 〔Example 2〕 <Background> Since neural stem cells (NSCs) and mesenchymal stem cells (MSCs) have the property of migrating to tumor and damaged sites, they have been used as cellular delivery vehicles (CDVs) for therapeutic genes (Citation Documents 1 and 2). NSCs can be collected from fetal-derived tissues, etc., but their use is not easy due to problems such as their low proliferative ability and ethical aspects. The inventor solved this problem by inducing the differentiation of NSCs from human induced pluripotent stem cells (iPS cells). And the inventor has been researching a therapeutic strategy of using NSCs as a CDV for a therapeutic gene (cytosine deaminase-uracil phosphoribosyl transferase: CD-UPRT) against glioblastoma. On the other hand, regarding glioblastoma, it is not yet clear which of NSCs and MSCs can be a superior CDV.

[0130] The inventor evaluated the migratory ability of NSCs and MSCs (derived from adipose tissue and bone marrow) transplanted into the brain against glioblastoma cells using brain slice culture (Tamura R. Mol Brain. 2019 [Japanese Patent Application No. 2019-052704]). As a result, it became clear that NSCs showed significantly better migration and tropism than MSCs. Therefore, this time, by performing RNA-seq analysis on the NSC strain, MSC strain, and glioblastoma strain held by the inventor, the key molecule that causes differences in the migration of NSCs and MSCs was clarified.

[0131] <Method> Glioma stem cells (GSCs) have been reported as the root cause of the intractability of malignant glioblastoma. Different from glioma cells (GCs), GSCs have the ability of self-renewal and tumor formation, have high invasiveness and chemo- and radiotherapy resistance, and are regarded as 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), two strains of human adipose tissue-derived MSCs (AMSC1, AMSC2), two strains of human bone marrow-derived MSCs (BMSC1, BMSC2), human GC strains (U87, U251, SF126), and human GSC strains (hG008, hG021) (Citation 3) using the miRNeasy Serum / Plasma kit (QIAGEN) with the QIAcube (QIAGEN) automation system. RNA-seq analysis was performed by commissioned analysis by Macrogen Inc.

[0133] In addition, the RNA-seq data of resected glioblastomas that had already been reported were used from the databases of NCBI Short Read Archive (SRR359290 and SRR359291) (Citation 4).

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

[0135] Ligand-receptor pairing analysis Ligand-receptor pairing was referred to the previously reported databases (Citation 5). 2552 ligand-receptor pairs were selected (Citation 6), and when the expression of both the ligand and the receptor was observed in a certain cell type, they were selected as a matched pair (see Figure 8).

[0136] Single-cell RNA-seq The previously reported single-cell RNA-seq data of resected glioblastomas were reexamined (Citation 7).

[0137] <Results> First, to extract the autocrine signaling pathway, autocrine interactions were evaluated. The reason for focusing on autocrine was that NSCs and MSCs behaved differently in the brain even when transplanted alone without tumors. NSCs spread and engrafted, while MSCs formed cell clusters (Figure 9). Also, since the engraftment pattern was similar to that of GSCs (diffuse engraftment) and GCs (cell cluster formation) [Tamura R. Mol Brain. 2019], the differences between GSCs and GCs were also considered important information. As a result, 92 ligand-receptor pairs were identified between NSC-NSC and GSC-GSC (Figure 10). These were not identified in MSCs.

[0138] According to enrichment analysis (an analysis to determine the presence or absence of a correlation between a specific gene set and the expression ratio), these pairs were suggested to be related to Eph / ephrin repulsion signaling. In particular, EphB / ephrinB was upregulated in NSCs compared to MSCs. There is actually a report that EphB / ephrinB is involved in the invasion of GSCs (Citation 8). Therefore, it was suggested that EphB / ephrinB in particular causes autocrine between NSCs and is related to diffuse engraftment.

[0139] Next, the paracrine interaction between GSCs and NSCs or MSCs was evaluated. That is, there are ligands released by GSCs and receptors of NSCs that sense them, and this pair was considered to be involved in directivity. 27 ligand-receptor pairs were identified between NSCs and GSCs. However, their expression was not upregulated in the glioma cell lines of the present inventors in vitro. This result suggested the possibility that GSCs cultured in vitro do not fully reflect glioblastoma in the brain. Therefore, the present inventors performed in silico ligand-receptor pairing analysis using the RNA-seq data of previously reported resected glioblastomas. As a result, 8 ligand-receptor pairs were identified between resected glioblastomas and NSCs. Attention was paid to the CXCL12 / CXCR4 signal, which has been reported many times to date (Citation 2). As a result, CXCR4 was highly expressed in NSCs and not expressed in MSCs. CXCL12 was highly expressed in resected glioblastomas rather than in GSCs cultured in vitro.

[0140] Furthermore, the present inventors reexamined the data of single-cell RNA-seq of previously reported glioblastomas. As a result, high expression of CXCL12 was also observed in tumor-infiltrating macrophages known to cluster in the microenvironment around glioblastomas. GSCs cultured in vitro had low expression of macrophage markers such as CD14 and CD163. These data suggest the possibility that CXCL12 in tumor-infiltrating macrophages in the brain and NSCs (not expressed in MSCs) expressing CXCR4 create directivity.

[0141] From the above, it was concluded that the self-repulsive reaction by EphB-ephrinB and the directivity by CXCL12-CXCR4 are involved, and both of these factors bring about directed migration of NSCs toward glioblastomas. There has been no report pointing out the difference in migration / directivity of NSCs and MSCs toward glioblastomas and the key molecules that generate it. In addition, since CXCL12 has been pointed out to be highly expressed in other malignant tumors (such as pancreatic cancer), migration toward tumors other than glioblastomas is also expected.

[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 Since the present inventors have found that neural stem / progenitor cells (NSC) derived from induced pluripotent stem cells (iPS cells) exhibit high migratory ability to tumor tissues and damaged brains, a treatment method using them as a cellular delivery vehicle for therapeutic genes has been devised. As a therapeutic gene, a yeast cytosine deaminase (yCD)-uracil phosphoribosyl transferase (UPRT) fusion gene (yCD-UPRT) was used and inserted into the housekeeping gene locus (ACTB) of iPS cells by genome editing technology to achieve constitutive and stable expression.

[0144] Previously, after gene introduction into iPS cells, cells were selected using drug resistance genes and fluorescent genes. However, for clinical applications, it is desirable to remove foreign genes such as drug resistance genes as much as possible. In addition, genome editing technologies such as CRISPR-Cas9 have concerns about safety due to off-target effects where mutations occur at unintended locations. Recently, CRISPR / Cas3 has been developed as a new genome editing technology, and it has been reported that the frequency of off-target effects is low because the recognition target sequence is longer than that of CRISPR / Cas9.

[0145] (B) Summary of the Invention To produce highly safe therapeutic stem cells, a new technique was developed to remove foreign genes used for cell selection after introducing a therapeutic gene into iPS cells using a genome editing technique with low off-target effects.

[0146] Specifically, iPS cells were prepared by inserting the yCD-UPRT fusion gene, drug resistance gene, and fluorescent gene into the housekeeping gene locus (ACTB) using the genome editing technique CRISPR / Cas3. After fluorescence and drug selection, the foreign genes (drug resistance gene and fluorescent gene) were removed using the Cre / loxP system. Note that cells into which a suicide gene (a gene encoding an enzyme that metabolizes a prodrug and converts it into an anti-tumor substance) has been introduced will self-destruct upon administration of the prodrug [Tamura R. Neurosurgical review. 2019].

[0147] (C) Method <Human iPS cells> The human iPS cells (1210B2) used were obtained from the Institute for Integrated Cell-Material Sciences (CiRA), Kyoto University. 1210B2 was established by a method of introducing reprogramming factors into human peripheral blood mononuclear cells using an episomal vector (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.). The human iPS cells were seeded on plastic culture dishes coated with iMatrix-511 (manufactured by Nippi Inc.) and maintained in 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.) using Stem Fit AK03 or AK03N medium (manufactured by Ajinomoto Co., Inc.).

[0148] <Induction of differentiation from human iPS cells into neural stem / progenitor cells (NSC)> Differentiation induction from human iPS cells to NSCs was performed 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 (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to the medium of iPS cells, incubated for 1 to 3 hours, washed with PBS, and dispersed into single cells using TrypLE Select (manufactured by Life Technologies). The human iPS cells dispersed into single cells were suspended in an EB (embryoid body) formation medium (10 μM SB431542 and 100 nM LDN-193189 were added to Stem Fit medium without adding solution C) supplemented with 10 μM ROCK inhibitor Y276352, and suspended in a low-adhesion 96-well culture plate (Prime Surface 96V, manufactured by Sumitomo Bakelite Co., Ltd.) at 9.0×10 per well 3The cells were seeded at a density of 75 μl 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 EBs were obtained by culturing for 13 to 14 days. The EBs were collected from each well, and 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 Awi Pharma)) was added, and the cells were 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-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, the primary NSCs were collected by centrifugation, dispersed into single cells using TrypLE Select, suspended in NS medium, and 1×10 5 cells / ml were seeded in a low-adhesion flask, 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] <yCD-UPRT Gene Transfer into the ACTB Gene Region by CRISPR / Cas3 - Generation of Therapeutic Stem Cells (Therapeutic stem cell: TSC)> Generation of iPS cells with the yCD-UPRT gene introduced into the ACTB gene region (TiPS1,2) Using the CRISPR / Cas3 genome editing technology, iPS cells were generated by inserting the yCD-UPRT fusion suicide gene into the housekeeping gene region ACTB (β-actin). The homologous recombination vector for genome editing was constructed such that ACTB and yCD-UPRT were linked by a 2A peptide sequence, and an IRES sequence flanked by loxP sequences, a puromycin resistance gene, a 2A peptide, and a fluorescent protein Venus were inserted downstream in a connected form (HR-ACTB-2A-yCD-UPRT-loxP-IRES-Puromycin-2A-Venus-loxP). The genome cleavage vector was constructed to express a crRNA targeting the vicinity of the stop codon of ACTB and a series of Cas3-related proteins (Cas3-crRNA-All-in). These constructs were introduced into a human iPS cell line (1210B2) by electroporation, cultured in the presence of puromycin S, and cloned by colony picking. The confirmation of the homologous recombination iPS cell line (TiPS1) was performed by genomic PCR and nucleotide sequence confirmation by genomic sequencing of each clone. The Cre / loxP recombination vector was constructed with a fluorescent protein mCherry, an IRES sequence, a Cre protein, and a human growth hormone polyadenylation signal linked downstream of an EF promoter (EF-mCherry-IRES-Cre-hCGpolyA). This construct was introduced into TiPS1 by electroporation, and cells emitting mCherry fluorescence and lacking Venus fluorescence were cloned by sorting (Figs. 15 and 16). The confirmation of the selection marker-removed iPS cell line (TiPS2) was performed by genomic PCR and nucleotide sequence confirmation by genomic sequencing of each clone.

[0150] Generation of yCD-UPRT-expressing NSCs (TSC1, 2) From iPS cells (TiPS1, 2) incorporating yCD-UPRT into ACTB, differentiation induction was performed to NSCs, and secondary to fifth-generation NSCs were obtained. TSC1 is a therapeutic NSC in which the selection marker sequence remaining from the differentiation induction from TiPS1, and TSC2 is a therapeutic NSC in which the selection marker sequence remaining from the differentiation induction from TiPS2 has been removed (Figure 17). In order to show that there are no changes in cell proliferation, CD expression level, etc. even after removing the selection marker (TSC2), TSC1 was cultured as a comparison target. Puromycin S 1 μg / ml was constantly added to the TSC1 medium for culturing. It was confirmed that cell death was induced by adding 1 - 7 μg / ml of 5-Fluorocytosine (5-FC) to the medium for TSC1 and 2. Evaluation of cell death was performed using Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Kumamoto, Japan), which is a cell proliferation / cytotoxicity assay kit.

[0151] Gene expression analysis of TiPSC1,2,TSC1,2 By real-time qPCR, the mRNA expressions of the genome-unedited iPS cell line (iPS-nega), TiPSC1, TSC1, TiPSC2, TSC2, iPS-Cas9 and NSC-Cas9 that had been genome-edited using CRISPR / Cas9 were compared. The mRNA expression levels of GAPDH, ACTB, yCD-UPRT, the puromycin resistance gene, and the Venus gene were measured using TB Green (registered trademark) Premix Ex Taq TM II (Takara Bio), and the expression levels of each gene were normalized by the expression level of ACTB.

[0152] Confirmation of the antitumor effect of TSC1,2 (TSC1, 2) Using a low-adhesion 96-well culture plate (Prime Surface 96V, manufactured by Sumitomo Bakelite Co., Ltd.), the glioma cell line U87 (1×10 4 cells) or the glioma stem cell line hG008 (1×10 4 cells) 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] ><Resistance of TSC1,2 to Puromycin> TSC1 was able to be selected without problems by the addition of Puromycin due to the Puromycin resistance gene. TSC2 immediately died due to Puromycin, and it was confirmed that the Puromycin resistance gene had been removed as expected by the Cre / loxP system (Figure 18).

[0155] ><Sensitivity of TSC1,2 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 with CRISPR / Cas9 (Figure 19).

[0156] ><Antitumor effect of TSC1,2> TSC1 and 2 were co-cultured with glioma cell line U87 and glioma stem cell hG008 cell line, and 5-FC (2 μg / ml) was administered. Cell death was evaluated by CCK-8 assay, and significant tumor cell death occurred in both after prodrug administration (Figure 20). No tumor cell death occurred when 5-FC was administered to U87 and hG008 alone (Figure 20).

[0157] Example 4 (A) Overview of mutant loxP sequences 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 parts 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 to be recognized by Cre. Therefore, safety can be enhanced by leaving lox71 / 66 in the genome. Also, lox2272 with a mutation in the spacer sequence only recombines with the same lox2272 (Figure 21C).

[0159] (B) Background (1) Method for removing antibiotic resistance genes using the Cre / mutant lox system To enhance the safety of the method for removing drug resistance genes and fluorescent genes described in Example 3, a method for removing drug resistance genes and fluorescent genes using a mutant loxP sequence (arm region mutation) instead of loxP was developed.

[0160] The Cre / loxP system can delete DNA sequences (such as drug resistance genes) flanked by loxP by expressing the Cre protein. However, since ultimately one loxP sequence remains, there is a risk of reacting with Cre in case. Therefore, to reduce this risk, a method was developed to sandwich the DNA sequence (such as drug resistance gene) scheduled for deletion with loxP (lox71 and lox66) having mutations in the repetitive sequence. In the mutant sequence of loxP, lox71 has mutations in the upstream repetitive sequence part, and lox66 has mutations in the downstream repetitive sequence part. As a result, the lox71 / 66 fusion sequence remaining after deleting the sandwiched gene has mutations at both ends, so the risk of reacting with Cre is extremely low, enabling safer gene therapy (Figure 22B).

[0161] (2) Second gene insertion method using the Cre / mutant lox system A method was developed to insert a second gene and delete a drug resistance gene and a fluorescent gene using a mutant loxP sequence (spacer sequence mutation).

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

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

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

[0165] (3) Confirmation of gene expression The mRNA expression of the cells selected in (1) and (2) was compared by real-time qPCR. TB Green (registered trademark) Premix Ex Taq TMUsing II (Takara Bio), the mRNA expression levels of the genes of GAPDH, ACTB, yCD-UPRT, Puromycin resistance gene, Venus, and AmCyan were measured, and the expression levels of each gene were normalized by the expression level of ACTB or GAPDH.

[0166] (4) Confirmation of antitumor effect Using a low-adhesion 96-well culture plate (Prime Surface 96V, manufactured by Sumitomo Bakelite Co., Ltd.), glioma stem cell line hG008 (6×10 3 cells) and the NS / PCs established in (1) or (2) (3×10 3 cells) were co-cultured for 1 day, then 5-FC was added (5 μg / ml), and 5 days later, a cell proliferation / cytotoxicity assay kit, Cell Counting Kit-8 (CCK-8) assay was performed.

[0167] (D) Results (1) Removal of drug resistance / marker gene iPS cells in which a suicide gene was inserted and the drug resistance gene / marker was removed (ACTB-2A-yCD / UPRT-lox71 / 66 was integrated) were established and could be induced to differentiate into embryoid bodies (EB) and NS / PCs (Figure 24). It was confirmed by genomic PCR and gene sequence analysis that it was accurately integrated into the genome (Figure 25A - C).

[0168] (2) Insertion of the second gene iPS cells in which a suicide gene and the second gene were inserted and the drug resistance gene / marker was removed (ACTB-2A-yCD / UPRT-lox71 / 66-IRES-AmCyan-lox2272 was integrated) were established and could be induced to differentiate into NS / PCs. Fluorescence of AmCyan of the second gene was confirmed in embryoid bodies (EB) and NS / PCs (Figure 26). It was confirmed by genomic PCR and gene sequence analysis that it was accurately integrated into the genome (Figure 27A - C).

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

[0170] Furthermore, in the iPS cells and NS / PCs cells established in (1), it was confirmed that the cells died upon administration of 5-FC and puromycin (Figure 29). In the iPS cells and NS / PCs cells established in (2), it was confirmed that the cells died upon administration of 5-FC and puromycin (Figure 30).

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

[0172] [Example 5] Effect on tumors other than brain tumors Below, the effect on pancreatic cancer is described as an example of a tumor other than a brain tumor, but other tumors have the same effect. (A) Background Digestive tract cancers have a high incidence rate. In particular, pancreatic cancer ranked fifth (17,060 cases) in the number of cancer deaths among men in 2016 and third (16,415 cases) in the number of cancer deaths among women, exceeding the number of deaths from gastric cancer in women [refer to the Cancer Information Service of the National Cancer Center]. The 5-year relative survival rate is 7.9% in men and 7.5% in women for pancreatic cancer, which has the worst prognosis compared to other organ cancers [10-year relative survival rate by cancer site and clinical stage for all cancers]. Multidisciplinary treatment using surgery, chemotherapy, and radiotherapy is carried out. However, the side effects of chemotherapy, which is administered systemically, are strong, and due to its tendency to cause distant metastasis and infiltration into the tumor artery, etc., in many cases, it is already determined to be unresectable or difficult to perform radical resection at the time of diagnosis, and the development of new treatment methods is desired. 5-FU is used as one of the main chemotherapeutic agents for pancreatic cancer. Since the suicide gene-introduced iPS cell-derived NS / PCs established by the present inventors show tropism and migration to malignant tumors, it becomes possible to administer a high concentration of the anticancer agent 5-FU locally at the tumor.

[0173] (B) Method 5×10 human pancreatic cancer cell line BxPC3 6 cells were transplanted subcutaneously into the left flank of NOD / SCID mice (6 weeks old, female). Seven days later, 5×10 suicide gene yCD-UPRT-introduced iPS-NS / PCs (ffLuc) 5 cells were transplanted subcutaneously into the back or administered via the tail vein (Figure 32). NS / PCs were previously infected with an ffLuc (fusion gene of Venus fluorescent protein and Luc2 firefly luciferase) expression lentiviral vector (CSII-EF-ffLuc) (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 in which ffLuc is stably and highly expressed. The temporal changes of NS / PCs in the same individual were observed using an IVIS in vivo imaging system. During IVIS imaging, 200 μl of VivoGlo TM Luciferin at a concentration of 30 mg / ml was administered intraperitoneally, and imaging was performed 10 minutes after reaching the peak (Figures 33A and B).

[0174] (C) Results In a model mouse with subcutaneous transplantation of iPS-NS / PCs (ffLuc) on the back, signal accumulation was observed at the pancreatic cancer transplantation site (left abdomen) 18 days later using IVIS. In the model mice administered via the tail vein, the signal was trapped in the lungs on the day of administration, but gradually, signal accumulation began to be observed at the pancreatic cancer transplantation site (left abdomen) 3 days later, and very strong accumulation was seen 18 days later. From the above, it was revealed that iPS-NS / PCs also showed strong tropism and migratory ability towards pancreatic cancer, and that iPS-NS / PCs can be used as a good cellular delivery vehicle for pancreatic cancer as well (Figures 33A and B).

[0175] All publications, patents, and patent applications cited in this specification are hereby incorporated by reference in their entirety into this specification.

Industrial Applicability

[0176] Since the tumor treatment cell preparation and the cell preparation for treating central nervous system diseases and injuries of the present invention can be used as pharmaceuticals, the present invention is applicable in industries such as the manufacture of pharmaceuticals.

Claims

1. A cell preparation for tumor treatment, comprising neural stem cells differentiated from pluripotent stem cells into which a suicide gene has been introduced, wherein the neural stem cells are produced by a method comprising the following steps (1) and (2), (1) A step of inserting a suicide gene into the 3'-side immediately after the translation region of the β-actin gene of pluripotent stem cells by genome editing, (2) A step of differentiating the pluripotent stem cells obtained in step (1) into neural stem cells, wherein the tumor is breast cancer, gastric cancer, or lung cancer. A cell preparation for tumor treatment.

2. In step (1), 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, so that the suicide gene is inserted into the 3'-side immediately after the translation region of the β-actin gene of pluripotent stem cells. The cell preparation for tumor treatment according to claim 1.

3. The cell preparation for tumor treatment according to claim 1 or 2, 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 pluripotent stem cells by genome editing, wherein the gene construct comprises a suicide gene, a selection marker gene, and target sequences for two Cre proteins, and the selection marker gene is a gene construct sandwiched between the target sequences for two Cre proteins. (1-B) A step of selecting pluripotent stem cells into which the suicide gene has been inserted into the genome from among the pluripotent stem cells obtained in step (1-A) using the 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 Cre protein.

4. The cell preparation for tumor treatment according to claim 3, wherein the target sequence for Cre protein is a loxP sequence.

5. The target sequences of the two Cre proteins contained in the gene construct are a mutant loxP sequence having a mutation in the upstream repetitive sequence and a mutant loxP sequence having a mutation in the downstream repetitive sequence. In the gene construct, the mutant loxP sequence having a mutation in the upstream repetitive sequence is located upstream of the selection marker gene, and the mutant loxP sequence having a mutation in the downstream repetitive sequence is located downstream of the selection marker gene. The cell preparation for tumor treatment according to claim 3, characterized in that.

6. The cell preparation for tumor treatment according to claim 1 or 2, characterized in that step (1) includes the following steps (1-a) to (1-c). (1-a) A step of inserting a gene construct by genome editing immediately after the translation region of the β-actin gene of pluripotent stem cells. The gene construct includes a suicide gene, a selection marker gene, a mutant loxP sequence having a mutation in the repetitive sequence, and a mutant loxP sequence having a mutation in the spacer sequence. The selection marker gene is a gene construct sandwiched between the two mutant loxP sequences. (1-b) A step of selecting, by means of a selection marker gene, pluripotent stem cells into which a suicide gene has been inserted from among the pluripotent stem cells obtained in step (1-a). (1-c) A step of removing the selection marker gene from the genome of the pluripotent stem cells obtained in step (1-b) by Cre protein and a homologous recombination vector, and inserting a second gene into the genome. The homologous recombination vector includes a second gene, a mutant loxP sequence having a mutation in the repetitive sequence, and a mutant loxP sequence having a mutation in the spacer sequence. The second gene is a homologous recombination vector sandwiched between the two mutant loxP sequences.

7. The cell preparation for tumor treatment according to claims 3 to 6, characterized in that the genome editing is genome editing using CRISPR / Cas3.

8. The cell preparation for tumor treatment according to claims 1 to 7, characterized in that the suicide gene is a cytosine deaminase gene and a uracil phosphoribosyltransferase gene.

9. The cell preparation for tumor treatment according to claims 1 to 8, characterized in that the neural stem cells are neural stem cells that do not contain a selection marker gene in their genome.

10. The cell preparation for tumor treatment according to claims 1 to 9, wherein the neural stem cells are neural stem cells with increased expression levels of at least one selected from Ephrin A receptor, Ephrin A, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4.

11. The cell preparation for tumor treatment according to claim 10, wherein the neural stem cells are neural stem cells with increased expression levels of Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4.

12. The cell preparation for tumor treatment according to claims 1 to 9, wherein the neural stem cells are neural stem cells selected using at least one selected from Ephrin A, Ephrin A receptor, Ephrin B receptor, Ephrin B, and CXC motif chemokine receptor 4 as an index.

13. The cell preparation for tumor treatment according to claim 12, wherein 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 an index.

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