Non-human animal, kit, and method for producing replacement organ

Abstract

A non-human animal having a system for killing a target cell in a living body, the target cell having in a chromosome a cell-specific promoter and an exogenous caspase9 gene inducibly expressed downstream of the cell-specific promoter, or the gene has a high expression promoter in a chromosome and an exogenous caspase9 gene which is subjected to induced expression at the downstream of the high expression promoter.

Description

TECHNICAL FIELD

[0001] The present application relates to non-human animals, kits, and methods of making replacement organs. BACKGROUND

[0002] Systems for inducing death of target cells by a medicament are also widely used for research of intercellular interaction in individuals, research in disease models, research of malignant tumors, and the like. For example, a target cell-induced death system using a herpes virus-derived thymidine kinase gene (HSV-TK) has been developed, and a liver injury model and the like have been produced (see Non-Patent Literature 1).

[0003] However, since the HSV-TK system cannot be applied to non-dividing cells, and since the HSV-TK system has immunogenicity and the like, development of other systems has been explored. Thus, a target cell-induced death system using diphtheria toxin has been developed, and since it has an effect of inducing death of non-dividing cells, its achievements have attracted attention (see Non-Patent Literature 2).

[0004] PRIOR ART DOCUMENTS

[0005] NON-PATENT LITERATURE

[0006] Non-Patent Literature 1: Bonini C, Ferrari G, Verzeletti S, Servida P, Zappone E, Ruggieri L, Ponzoni M, Rossini S, Mavilio F, Traversari C, Bordignon C. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science. 1997 Jun 13;276(5319): 1719-24. doi: 10.1126 / science.276.5319.1719. PMID: 9180086.

[0007] Non-patent literature 2: Saito M, Iwawaki T, Taya C, Yonekawa H, Noda M, Inui Y, Mekada E, Kimata Y, Tsuru A, Kohno K. Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice. Nat Biotechnol. 2001 Aug; 19(8):746-50. doi: 10.1038 / 90795. PMID: 11479567. SUMMARY

[0008] However, since diphtheria toxin used as an inducer is damaging to human cells, it cannot be used for human cells.

[0009] An object of the present application is to provide a non-human animal, a kit, and a method for manufacturing a replacement organ, which do not have an effect of toxicity caused by an inducer on non-target human cells, have a high removal rate of target cells, and have a fast speed of induced death of target cells.

[0010] The present application includes the following embodiments.

[0011] [1] A non-human animal having a system for killing target cells in a living organism, the target cells having a cell-specific promoter and an exogenous caspase 9 gene induced to express downstream of the cell-specific promoter in a chromosome, or having a high-expression promoter and an exogenous caspase 9 gene induced to express downstream of the high-expression promoter in a chromosome.

[0012] [2] The non-human animal according to [1], wherein the exogenous caspase 9 gene is a gene encoding a fusion protein of a dimerization region and caspase 9.

[0013] [3] The non-human animal according to [1], wherein a gene encoding a site-specific recombinase is in a chromosome, and a site-specific recombinase recognition sequence, a transcription termination sequence, and the site-specific recombinase recognition sequence are sequentially included from the 5' side upstream of the exogenous caspase 9 gene.

[0014] [4] A kit having the non-human animal according to [2] and a protein dimer-inducing compound.

[0015] [5] The kit according to [4], further having an apoptosis promoter.

[0016] [6] A method for producing a replacement organ, comprising: an induction step of inducing expression of an exogenous caspase 9 gene in the non-human animal described in any one of [1] to [3]; a killing step of killing a target cell of the non-human animal by the Caspase 9 protein induced to express; and an injection step of injecting a human precursor cell of the same species as the target cell killed into a site where the target cell of the non-human animal is killed.

[0017] [7] The method for producing a replacement organ according to [6], wherein the method further comprises a step of administering an apoptosis promoter.

[0018] According to the present application, it is possible to provide a non-human animal, a kit, and a method for producing a replacement organ, which do not have an influence of toxicity caused by an inducer on non-target human cells, have a high removal rate of target cells, and have a fast speed of induced death of target cells. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 (A) A diagram of the construct of Six2-iCaspase9-tdTomato mouse is shown. (B) is a schematic diagram of the apoptosis mechanism based on Caspase9 activation.

[0020] Figure 2 are bright field images and fluorescence images of each of an embryo and an embryonic kidney of Six2-iCaspase9-tdTomato mouse. The tdTomato expression is confirmed in the Six2 expression region.

[0021] Figure 3 are immunostaining images of kidney organoids made from kidney precursor cells extracted from the embryonic kidney of Six2-iCaspase9 mouse. Non-CID administration (left), CID administration (right) are shown, respectively. The induced death of nephron precursor cells is confirmed in CID administration.

[0022] Figure 4 are fluorescence images after organ culture of the kidney removed from the embryo of Six2-iCaspase9 mouse. The removal of nephron precursor cells by CID administration (lower segment) is confirmed in organ culture.

[0023] Figure 5 are Figure 4 are immunostaining images on day 3. The removal of Six2-positive nephron precursor cells is confirmed in CID administration (right).

[0024] Figure 6An image showing the results of analysis of Six2-iCaspase9-tdTomato mouse neonatal kidney after intraperitoneal administration of AP20187 (CID) to the neonatal pups, 48 hours later. In the upper panel of the CID-administered group, the removal of Six2-positive metanephric precursor cells in the neonatal kidney was confirmed.

[0025] Figure 7 An image of immunostaining in Figure 6 In the upper panel of the CID-administered group, the removal of Six2-positive metanephric precursor cells in the neonatal kidney was confirmed.

[0026] Figure 8 An evaluation result of apoptosis based on TUNEL staining. In the 12th hour after administration (upper left), TUNEL positivity coinciding with Six2-positive metanephric precursor cells was confirmed, and early induction of apoptosis was confirmed.

[0027] Figure 9 is the result of genotyping of the genetically introduced mouse.

[0028] Figure 10 is the result of collecting the kidney of an E13.5 embryo of a double-allele knock-in (homozygote) mouse and culturing after adding CID. (A) is a fluorescence image of tdTomato. (B) is an immunostaining image of metanephric precursor cells. (C) is Figure 10 quantitative results of (B).

[0029] Figure 11 is the result of collecting the kidney of an E13.5 embryo of a single-allele knock-in (heterozygote) mouse and culturing after adding CID and / or AT406. (A) is a fluorescence image of tdTomato. (B) is an immunostaining image of metanephric precursor cells. (C) is Figure 11 quantitative results of (B).

[0030] Figure 12 is the quantitative result of tdTomato in homozygote embryo kidney and heterozygote embryo kidney.

[0031] Figure 13 is the result of subcutaneous administration of CID to a neonatal mouse of a homozygote. (A) is a fluorescence image of tdTomato. (B) is an immunostaining image of metanephric precursor cells.

[0032] Figure 14 is the result of administering CID and AT406 to a neonatal mouse of a heterozygote. (A) is a fluorescence image of tdTomato. (B) is an immunostaining image of metanephric precursor cells.

[0033] Figure 15is a graph showing the protocol of CID administration into the abdominal cavity of a mother carrying a homozygous embryo.

[0034] Figure 16 (A) is the result of confirming the kidney size of neonatal pups to which CID administration was performed at different periods using a fluorescent image of tdtomato. (B) is the result of quantifying the kidney size (long diameter) of (A). Figure 16 (A) is the result of quantifying the kidney size (long diameter) of (A).

[0035] Figure 17 is an immunostaining image of renal progenitor cells with or without CID administration.

[0036] Figure 18 (A) is an immunostaining image of glomeruli to which CID administration was performed at different periods. (B) is the result of quantifying the number of glomeruli of (A). Figure 18 (A) is the result of quantifying the number of glomeruli of (A).

[0037] Figure 19 is the result of evaluating the kidney function of neonatal pups to which CID administration was performed.

[0038] Figure 20 is a graph showing the protocol of the kidney replacement experiment.

[0039] Figure 21 is the result of immunohistostaining images of injecting rat renal progenitor cells (RPCs) into the kidney of a homozygous embryo and culturing for 4 days in a CID-added medium.

[0040] Figure 22 is the result of immunohistostaining images of injecting rat renal progenitor cells into the kidney of a homozygous embryo and culturing for 14 days in a CID-added medium.

[0041] Figure 23 (A) is the result of immunohistostaining images of injecting rat renal progenitor cells into the kidney of a heterozygous embryo and culturing for 4 days in a CID- and AT406-added medium. (B) is the result of immunohistostaining images of injecting rat renal progenitor cells into the kidney of a heterozygous embryo and culturing for 7 days in a CID- and AT406-added medium.

[0042] Figure 24 (A-C) is the result of immunohistostaining images of culturing human NPCs induced from human iPS cells into the kidney of a homozygous embryo.

[0043] Figure 25 (A-B) is the result of immunohistostaining images of injecting human NPCs induced from human iPS cells into the kidney of a homozygous embryo with CID for 1 week. DETAILED DESCRIPTION

[0044] <non-human animal>

[0045] In one embodiment, the present application provides a non-human animal having a system for killing a target cell in a living organism, the target cell having a cell-specific promoter in a chromosome and an exogenous caspase 9 gene downstream of the cell-specific promoter induced to express, or having a high-expression promoter in a chromosome and an exogenous caspase 9 gene downstream of the high-expression promoter induced to express.

[0046] As the non-human animal, for example, cats, dogs, horses, monkeys, cows, sheep, pigs, goats, rabbits, hamsters, guinea pigs, rats, mice, non-human primates (cynomolgus monkeys, marmosets, etc.) and the like can be mentioned. Among them, rodents are preferred. As the rodents, hamsters, guinea pigs, rats, mice and the like can be mentioned, with rats and mice being preferred.

[0047] The exogenous caspase 9 gene is not particularly limited as long as it is a gene capable of inducing cell death in the target cell by high expression, and can be a pro-caspase 9 gene, a caspase 9 gene or a gene fragment of the pro-caspase 9 gene, or a variant. The origin of the exogenous caspase 9 gene is not particularly limited, and is preferably of mammalian origin, more preferably of mouse origin, human origin, particularly preferably of human origin.

[0048] The high-expression promoter is not particularly limited as long as it is a potent promoter, and can be mentioned, for example, CMV promoter, LTR promoter, PGK promoter, SV40 promoter, CK6 promoter, TTR promoter, TK promoter, TRE promoter, HBV promoter, hAAT promoter, LSP promoter, E2F promoter, hTERT promoter, CAG promoter, EF1-α promoter and the like, with the CAG promoter being particularly preferred.

[0049] Further, it is preferred that the target cell has an exogenous caspase 9 gene induced to express in two chromosomes. In the past, there has been no report of a caspase 9-carrying mouse, but by performing expression control of the exogenous caspase 9 gene under the high-expression promoter, or having the introduced gene in two chromosomes, the present inventors have improved the probability of obtaining a caspase 9-carrying mouse.

[0050] In addition, in the case where the single allele has the introduced gene, it is preferred to use a cell apoptosis promoter. The cell apoptosis promoter is not particularly limited, and can be mentioned, for example, IAP inhibitor, BCL2 inhibitor, TRAIL, DNA damaging agent and the like.

[0051] IAP inhibitors, which can include AT406, LCL161, GDC-0917, AEG-35156, TL32711, and the like. BCL2 inhibitors, which can include 4-[4-[[2-(4-chlorophenyl)-5,5-dimethyl-1- cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(4-morpholinyl)-1-[(phenylsulfanyl)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide (ABT-263); tetrachlorodibenzoquinone A; Antimycin; Gossypol ((-)BL-193); Obatoclax; Ethyl-2-amino-6-cyclopentyl-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H chromen-3-carboxylate (HA14-1); Oblimersen (G3139); Bak BH3 peptide; (-)-Gossypol acetic acid (AT-101); 4-[4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylsulfanyl)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-benzamide (ABT-737, CAS 852808-04-9); Navitoclax (ABT-263), and the like.

[0052] DNA damaging agents, which can include alkylating agents, platinum-based agents, intercalating agents, DNA replication inhibitors, and the like. DNA alkylating agents, which can include cyclophosphamide, nitrogen mustard, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, and the like. As platinum-based agents, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, and the like can be included. Intercalating agents, which can include doxorubicin, daunorubicin, idarubicin, mitoxantrone, and the like. DNA replication inhibitors, which can include irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, and the like.

[0053] In the present embodiment, the exogenous caspase9 gene capable of inducing expression can include a gene encoding a fusion protein of a dimerization region and caspase9. The above fusion protein is activated by dimerization by binding with a protein dimer inducer (Chemical Inducers of Dimerization, CIDs, Protein dimerizer), and induces apoptosis to cells.

[0054] FKBP, FK506, and the like can be exemplified. A protein dimer-inducing compound can be exemplified by AP20187, AP1903, and the like. A combination of a multimerization region and a protein dimer-inducing compound can be exemplified by a combination of a fusion protein of FK506 and caspase 9 and AP1903, a combination of a fusion protein of FKBP and caspase 9 and AP20187.

[0055] Further, in the present embodiment, the non-human animal provided with a system for inducing expression of an exogenous caspase 9 gene can be exemplified by a non-human animal having a gene encoding a site-specific recombinase on a chromosome, and having a chromosome containing, in order from the 5' side, a site-specific recombinase recognition sequence, a transcription termination sequence, and the site-specific recombinase recognition sequence upstream of the exogenous caspase 9 gene.

[0056] A site-specific recombinase can be exemplified by Cre, Flpe, Dre, and the like. A site-specific enzyme recognition sequence recognized by a site-specific recombinase can be exemplified by loxP, FRT, rox.

[0057] Further, a fusion protein of a site-specific recombinase and a mutant estrogen receptor (ER) can also be used. For example, a CreERT2 protein is normally present in the cytoplasm, but is transferred to the nucleus by binding to tamoxifen, which is an estrogen derivative, resulting in recombination of a loxP sequence. It is possible to regulate the working time of the Cre-loxP system in a tamoxifen-dependent manner using this.

[0058] Specifically, a system in which a non-human animal expressing an exogenous caspase 9 in a Cre recombinase activity-dependent manner is mated with a Six2-CreERT2 non-human animal into which a CreERT2 gene is introduced downstream of the promoter of Six2, and tamoxifen is brought into contact with the subject organ of the resulting offspring can be exemplified.

[0059] A non-human animal expressing an exogenous caspase 9 in a Cre recombinase activity-dependent manner has a transcription stop sequence sandwiched by loxP sequences upstream of the exogenous caspase 9 gene. Therefore, the exogenous caspase 9 is not expressed in this state. However, when the transcription stop sequence sandwiched by the loxP sequences is removed by the Cre recombinase, the exogenous caspase 9 is expressed.

[0060] In the present embodiment, the exogenous caspase 9 gene can be expressed organ- specifically by appropriately selecting a promoter for expressing the exogenous caspase 9 gene. The organ is not particularly limited, and examples thereof include the liver, the cornea, the skin, the large intestine, the small intestine, the pancreas, the stomach, muscle tissue, the heart, the lung, the esophagus, bone marrow, the kidney, the spleen, the testis, the ovary, and the like, and is preferably an organ to be evaluated in animal experiments. For example, Six2 is a transcription factor that is specifically expressed in metanephric mesenchyme. By using the Six2 promoter, the exogenous caspase 9 gene can be expressed metanephric tissue- specifically.

[0061] For example, when a non-human animal expressing an exogenous caspase 9 is mated with a Six2-CreERT2 non-human animal that specifically expresses CreERT2 in metanephric mesenchyme, a non-human animal in which the exogenous caspase 9 is expressed in metanephric tissue with tissue specificity appears in the resulting offspring.

[0062] The non-human animal of the present embodiment can be used for development of a non-human animal having an organ made of human cells using an organ in a living body as a scaffold. For example, exogenous kidney precursor cells can be injected into the nephrogenic region of a mouse, and after constructing an exogenous kidney (chimeric kidney) integrated into the kidney of a host, the exogenous caspase 9 gene is expressed kidney- specifically, and the kidney of the host is removed, thereby producing a human kidney mouse.

[0063] In the construction of a chimeric kidney, the exogenous cells can include embryonic kidney cells of a mouse (isogenic), a rat (xenogenic), NPCs induced from mouse ES cells, and NPCs induced from human iPS cells. The host mouse is preferably an embryo or a newborn pup. In the case of targeting cells expressing a promoter in an adult, the adult can also be used.

[0064] Further, human stem cells can be transplanted into the brain of a rat to produce a human brain rat, and the non-human animal of the present embodiment can also be utilized in neuroscience. Furthermore, by utilizing the non-human animal of the present embodiment in a human kidney mouse, a model having a higher efficiency of colonization of human cells can be provided.

[0065] In recent years, cancer treatment and the like in cell therapy such as CAR-T therapy are being widely studied, but the onset of GVHD (graft-versus-host disease) in which the transplanted cells attack the self and the possibility of carcinogenesis of the transplanted cells themselves are a concern. A safe and effective induction death system is being explored because it enables reset by inducing death when an adverse event occurs after transplantation of the transplanted cells, which will be a safer cell therapy. In the development of a target cell induction death system, the non-human animal of the present embodiment can be utilized.

[0066] In addition, in recent years, as the number of studies on aging cells as targets increases, the non-human animal of the present embodiment can also be applied to anti-aging studies. The induction death from diphtheria toxin as the prior art is an induction death based on inhibition of protein synthesis, and is close to necrosis. In necrosis, inflammation and the like are induced after cell death. On the other hand, since the present invention activates and utilizes Caspase 9, it is not necrosis but induces apoptosis as programmed death, and it can be considered that the inflammatory response after cell death is weak. Therefore, in aging studies in which even a small amount of inflammatory response becomes an interference, the apoptosis induction death model of the present invention is more suitable than the diphtheria toxin model.

[0067] <Kit>

[0068] In one embodiment, the present invention provides a kit having the above-mentioned non-human animal and the protein dimer-inducing compound.

[0069] The kit of the present embodiment contains the components described in the above-mentioned <<Non-human animal>>. In addition, in the case where the effect of killing target cells differs depending on the type of cells, it is preferable to further contain an apoptosis promoter in addition to the non-human animal and the protein dimer-inducing compound. The details of the apoptosis promoter are the same as the components described in the above-mentioned <<Non-human animal>>.

[0070] <Method for manufacturing a replacement organ>

[0071] In one embodiment, the present invention provides a method for manufacturing a replacement organ, having: an induction step of inducing expression of an exogenous caspase 9 gene in the above-mentioned non-human animal; a killing step of killing target cells of the non-human animal by the Caspase 9 protein induced to be expressed; and an injection step of injecting human precursor cells of the same species as the target cells killed into a site where the target cells of the non-human animal are killed.

[0072] <Exogenous caspase 9 gene expression induction step>

[0073] First, an exogenous caspase 9 gene is induced to be expressed in a non-human animal. As described above, since the non-human animal of the present embodiment has a system for inducing expression of an exogenous caspase 9 gene, an expression inducer is administered to the non-human animal. As described above, the expression inducer can be exemplified by a protein dimer-inducing compound or the like. The method of administration of the expression inducer is not limited, and can be exemplified by oral administration, intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, topical administration, and the like.

[0074] In the case where the subject to be administered is a newborn pup, subcutaneous administration is preferred, and in the case where the subject to be administered is a mother carrying an embryo, intraperitoneal administration is preferred. The number of residual cells (e.g., the number of residual renal cells) can be controlled depending on the timing of administration of the expression inducer.

[0075] [Administration procedure]

[0076] In the case where the effect of killing the target cells is not good depending on the type of cells, an administration procedure of administering an apoptosis promoter is preferred. The method of administration is appropriately adjusted depending on the properties of the apoptosis promoter to be used. The details of the apoptosis promoter are the same as those described in the above-mentioned "non-human animal".

[0077] [Killing procedure]

[0078] By inducing expression of the exogenous caspase9 gene, apoptosis is induced in the cells. In the case where the effect of killing the target cells is not good depending on the type of cells, apoptosis is induced in the cells by administration of an apoptosis promoter.

[0079] [Injection procedure]

[0080] Human precursor cells of the same type as the target cells to be killed are injected into the site in the non-human animal where the target cells are killed. The human precursor cells can also be cells induced from human iPS cells. The injection procedure can also be performed simultaneously with the above-mentioned induction procedure.

[0081] In the case where the replacement ratio is low, a chimeric organ is obtained. In the case where the replacement ratio is high, an organ that can be transplanted into a human is obtained. The method of manufacturing the replacement organ of the present embodiment can also be used for the production of a congenital kidney disease model.

[0082]

EXAMPLE

[0083] Hereinafter, the present application will be described by way of examples, but the present application is not limited to the following examples.

[0084] [Experimental Example 1]

[0085] As shown in Figure 1 , Six2-iCaspase9-tdTomato mice (hereinafter referred to as Six2-iCaspase9-tdTomato mice) in which the Six2 promoter and a gene in which a caspase9 gene and a tdTomato are connected downstream thereof are introduced into the double alleles of mice were produced.

[0086] As shown in Figure 2 , in mouse embryos and mouse embryonic kidneys, expression of tdTomato was confirmed in line with Six2-positive nephron precursor cells.

[0087] [Experimental Example 2]

[0088] Renal precursor cells were isolated from Six2-iCaspase9-tdTomato mice prepared in Experimental Example 1, and kidney organoids were prepared by three-dimensional culture. The kidney organoids were given AP20187 (hereinafter referred to as CID). Figure 3 An immunostaining image of the kidney organoids is shown. It was confirmed that the renal precursor cells underwent apoptosis by CID administration.

[0089] [Experimental Example 3]

[0090] Kidneys were excised from E11 embryos of Six2-iCaspase9-tdTomato mice prepared in Experimental Example 1, and organ culture was performed on a Transwell. In the group in which AP20187 (CID) was administered to the culture medium, it was confirmed that the expression of tdTomato fluorescence of the renal precursor cells was reduced on the second day of administration (refer to Figure 4 ). When CID was also administered in immunostaining, it was confirmed that Six2-positive renal precursor cells were removed (refer to Figure 5 ).

[0091] [Experimental Example 4]

[0092] Kidneys of mouse embryos were recovered 48 hours after AP20187 (CID) was administered intraperitoneally to newborn pups of Six2-iCaspase9-tdTomato mice prepared in Experimental Example 1. Figure 6 Bright field images and fluorescence images of the recovered mouse embryonic kidneys are shown. In the CID-administered group (top), it was confirmed that the renal precursor cells (red) disappeared due to apoptosis.

[0093] Figure 7 Immunostaining images of the same samples are shown. In the immunostaining images, Six2 (white), which is a marker of renal precursor cells, disappeared in the CID-administered group (top), and thus it was confirmed that the renal precursor cells disappeared due to apoptosis.

[0094] [Experimental Example 5]

[0095] Kidneys were excised from E13 embryos of Six2-iCaspase9-tdTomato mice prepared in Experimental Example 1, and organ culture was performed on a Transwell. CID 100 nM was administered to the culture medium, and after 12 hours, apoptosis based on TUNEL staining was evaluated after 18 hours of recovery. At 12 hours of administration, it was confirmed that TUNEL was positive in line with Six2-positive renal precursor cells, and it was confirmed that early apoptosis induction was induced (refer to Figure 8 ).

[0096] [Experimental Example 6]

[0097] In Experimental Example 1, genotyping of the gene-introduced mice was performed when Six2-iCaspase9-tdTomato mice (introducing the Six2 promoter and its downstream caspase9 gene and tdTomato gene into the double alleles of mice) were produced. As shown in Figure 9 , mice into which the gene was introduced into the double alleles (homozygote) and the single allele (heterozygote) were obtained.

[0098] [Experimental Example 7]

[0099] When the kidney of the E13.5 embryo of the double allele knock-in (homozygote) mouse was collected and cultured with the addition of CID, it was confirmed that more than 90% of the NPCs were removed by the addition of CID 100 nM (refer to Figure 10 ). On the other hand, even if the embryonic kidney of the single allele knock-in (heterozygote) individual was added with CID 100 nM, the removal of NPCs was not confirmed (refer to Figure 11 ).

[0100] It is assumed that the difference between the homozygote and the heterozygote is the amount of iCaspase9 expression, and in fact, when the amount of tdtomato protein contained in the knock-in vector was quantified, it was confirmed that the expression amount of the homozygote embryonic kidney was about 2 times that of the heterozygote (refer to Figure 12 ). Again in the culture of the heterozygote embryonic kidney, AT406 as an XIAP inhibitor was added as an apoptosis promoter in addition to CID, and by adding CID 100 nM, AT406 10 μM, the removal of NPCs was confirmed. It was also confirmed that in the administration of AT406 alone, NPCs were not removed, and the removal of NPCs was caused by iCaspase9 (refer to Figure 11 ).

[0101] [Experimental Example 8]

[0102] Further, after subcutaneous administration of CID to the homozygote neonatal mouse, the removal of NPCs was confirmed (refer to Figure 13 ). In the heterozygote neonatal mouse, NPCs were not removed in the administration of CID alone, and NPCs were removed by the administration of AT406 in combination (refer to Figure 14 ), and the same results were confirmed in vivo.

[0103] [Experimental Example 9]

[0104] CID was administered intraperitoneally to the mother who was pregnant with the homozygote embryo (refer to Figure 15). Thus, it was confirmed that the kidney size of the newborn pups (P0) was reduced, and the structure of the cap mesenchyme, which is a nephrogenic microenvironment in which the NPCs form, was destroyed (cf. Figure 16 ). Further, in the immunostaining, it was confirmed that the NPCs disappeared (cf. Figure 17 ). Further, it was also confirmed that the number of glomeruli was reduced (cf. Figure 18 ).

[0105] In the administration at the E11.5 time point, the postnatal individual died of apparent renal failure, but in the administration at the E13.5 time point, it was confirmed that the individual survived. The renal function of the individual one month after birth was evaluated, and the serum urea nitrogen (BUN), urinary albumin / creatinine ratio (ACR) were significantly increased compared to the control group (cf. Figure 19 ).

[0106] [Experimental Example 10]

[0107] By removing the NPCs of the embryonic kidney while transplanting exogenous NPCs, it was verified whether replacement of the renal cells was caused (cf. Figure 20 ). When the renal progenitor cells (RPCs) of the rat were injected into the homozygous embryonic kidney and cultured in the CID-added medium, it was confirmed that the chimeric cap mesenchyme was formed by adhesion to the host's ureteric bud at the 4th day time point (cf. Figure 21 ). In addition, after the cell injection, it was transplanted into an immunodeficient mouse and allowed to develop in vivo, and it was confirmed that the rat-derived cells differentiated into glomeruli or tubules at the 14th day time point, and it was confirmed that about 8 of the glomeruli were replaced with rat-derived glomeruli. It was also confirmed that the rat distal tubule was joined to the collecting duct from the mouse embryonic kidney (cf. Figure 22 ).

[0108] In addition, in the heterozygous embryonic kidney, the rat RPCs were also injected and cultured in the medium to which CID and AT406 were added, and it was confirmed that the chimeric cap mesenchyme was formed at the 4th day time point (cf. Figure 23 (A)), and the rat distal tubule connected to the mouse collecting duct was formed at the 7th day time point (cf. Figure 23 (B)).

[0109] Further, when human NPCs induced from human iPS cells were injected into the kidney of the homozygous embryo and cultured, it was confirmed that the chimeric cap mesenchyme was formed (cf. Figure 24 (A), (B)) and a part was differentiated into renal vesicles (cf. Figure 24 (C)).

[0110] [Experimental Example 11]

[0111] The mouse's embryonic kidney was injected with CID and human NPCs together. One week after injection, human distal renal tubules (GFP+, ECAD+) and GFP-negative embryonic kidney mouse-derived collecting ducts (GFP-, CK8+) were connected (refer to Figure 25 (A)).

[0112] Human-derived glomeruli (GFP+, Nephrin+) were also formed, showing invasion by blood vessels from the immunodeficient mouse (GFP-, CD31+), which facilitated maturation (refer to Figure 25 (B)).

[0113] Industrial applicability

[0114] According to the present application, it is possible to provide a non-human animal in which the removal rate of target cells is high and the speed of induced death of target cells is fast, a kit, and a method for manufacturing a replacement organ.

Claims

1. A non-human animal having a system for killing target cells in a living organism, the target cells having a cell-specific promoter and an exogenous caspase 9 gene downstream of the cell-specific promoter in a chromosome, or having a high-expression promoter and an exogenous caspase 9 gene downstream of the high-expression promoter in a chromosome.

2. The non-human animal of claim 1, wherein, the exogenous caspase 9 gene is a gene encoding a fusion protein of a dimerization region and caspase 9.

3. The non-human animal of claim 1, wherein, a gene encoding a site-specific recombinase in a chromosome; a chromosome having, in order from the 5' side, a site-specific recombinase recognition sequence, a transcription termination sequence, and the site-specific recombinase recognition sequence upstream of the exogenous caspase 9 gene.

4. A kit having the non-human animal of claim 2 and a protein dimer-inducing compound.

5. The kit of claim 4, wherein, the kit further has an apoptosis promoter.

6. A method for producing a replacement organ, comprising: an induction step of inducing expression of an exogenous caspase 9 gene in the non-human animal of any one of claims 1 to 3; a killing step of killing target cells of the non-human animal by a Caspase 9 protein expressed; an injection step of injecting human precursor cells of the same species as the killed target cells into a site where the target cells of the non-human animal are killed.

7. The method for producing a replacement organ according to claim 6, wherein, the method for producing a replacement organ further has a step of administering an apoptosis promoter.

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