WO2010082385A1 - ヒト肝細胞が移植されたマウス - Google Patents

ヒト肝細胞が移植されたマウス Download PDF

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WO2010082385A1
WO2010082385A1 PCT/JP2009/067688 JP2009067688W WO2010082385A1 WO 2010082385 A1 WO2010082385 A1 WO 2010082385A1 JP 2009067688 W JP2009067688 W JP 2009067688W WO 2010082385 A1 WO2010082385 A1 WO 2010082385A1
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mouse
human
hepatocytes
liver
transplanted
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PCT/JP2009/067688
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French (fr)
Japanese (ja)
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末水洋志
川井健司
中村雅登
長谷川雅巳
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財団法人実験動物中央研究所
中外製薬株式会社
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Priority to JP2010546546A priority Critical patent/JP5073836B2/ja
Priority to CN200980154733.8A priority patent/CN102281758B/zh
Priority to KR1020117019068A priority patent/KR101742329B1/ko
Publication of WO2010082385A1 publication Critical patent/WO2010082385A1/ja

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/30—Animal model comprising expression system for selective cell killing, e.g. toxins, enzyme dependent prodrug therapy using ganciclovir
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0368—Animal model for inflammation

Definitions

  • the present invention relates to a mouse transplanted with human hepatocytes, wherein the thymidine kinase gene is retained so that it can be expressed in the liver, and a guanosine analog and hepatocytes isolated from humans are administered. Furthermore, the present invention relates to a mouse transplanted with human hepatocytes, in which the urokinase-type plasminogen activator gene is retained so that it can be expressed in the liver, and hepatocytes isolated from humans are administered. It relates to NOG mice.
  • a method for producing a mouse transplanted with human hepatocytes comprising administering a guanosine analog and a hepatocyte isolated from a human to a mouse in which the thymidine kinase gene is expressed so as to be expressed in the liver.
  • the present invention relates to a manufacturing method including The present invention relates to a method for measuring plasma kinetics of a test substance using these mice, a method for identifying a metabolite of the test substance, and a method for measuring the metabolic rate of the test substance.
  • the present invention relates to a mouse model in which these mice are infected with liver-directed pathogens, and a method for screening drugs and vaccines against liver-directed pathogens using the mouse models.
  • mice that retain human hepatocytes for the purpose of analyzing human-specific hepatitis virus infection, metabolism of administered drugs, or proliferation of the human liver.
  • uPA urokinase type plasminogen activator
  • the uPA transgene activates plasminogen, which regulates the activity of matrix metalloproteases important for hepatocyte proliferation
  • the expression of the uPA transgene itself is sustained and progressive to hepatic stem cells. causes injury. Therefore, the embryonic lethality of the homozygote of the uPA transgene is as high as 30%, and the transplantation time of hepatocytes is limited to 5 to 17 days of age (Patent Document 1).
  • treatment such as transplantation of rat hepatocytes is necessary (Patent Document 2), and maintenance of the strain requires complicated work. The frequency of creating mice with the desired characteristics as well as necessary was also very low.
  • Non-patent Document 4 a chimera mouse having a fumaryl acetoacetate hydrolase (Fah) knockout mouse (Fah ⁇ / ⁇ / Rag2 ⁇ / ⁇ / II2rg ⁇ / ⁇ ) (FRG mouse) as a host has been created (Non-patent Document 4).
  • Fah fumaryl acetoacetate hydrolase
  • the present invention has been made in view of such a situation, and the object thereof is a mouse transplanted with human hepatocytes, wherein the thymidine kinase gene is retained in the liver so that it can be expressed, and a guanosine analog, And a mouse administered with hepatocytes isolated from human, a method for producing the mouse, a mouse transplanted with human hepatocytes, and the urokinase-type plasminogen activating gene can be expressed in the liver.
  • NOG mice to which hepatocytes isolated and administered from humans were administered methods for measuring plasma kinetics of a test substance using these mice, and identifying metabolites of the test substance using the mice
  • the present specification more specifically includes the following inventions; [1] A mouse in which an exogenous thymidine kinase gene is retained so that it can be expressed specifically in the liver, the mouse hepatocytes are replaced with human hepatocytes, and human hepatocytes are transplanted, [2] Mice transplanted with human hepatocytes of [1], wherein the liver has the three-dimensional structure or function of human liver, [3] A mouse into which a human hepatocyte of [1] or [2], wherein the thymidine kinase gene is a human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene, [4] Mice transplanted with human hepatocytes according to any one of [1] to [3], wherein 75% or more of mouse hepatocytes have been replaced with human hepatocytes, [5] Mice transplanted with human hepatocytes according to any one of [1] to [3],
  • a mouse into which a human hepatocyte of [9] is transplanted [11] A mouse into which a human hepatocyte of any one of [1] to [10], wherein hepatocytes isolated from humans are human hepatocyte cell lines, [12] A mouse into which human hepatocytes of [11], wherein the human hepatocyte cell line is HepG2, Hep3B, or HuH-7, [13] A mouse transplanted with any one of the human hepatocytes of [1] to [10], wherein hepatocytes isolated from humans are primary cultured hepatocytes, [14] When the thymidine kinase gene is placed under the control of an albumin promoter, LST-1 promoter, ⁇ -fetoprotein promoter, or ⁇ -TTP promoter, the thymidine kinase gene is retained so that it can be specifically expressed in the liver [ 1] to [13] a mouse transplanted with human hepatocytes of any one of [
  • mice The mouse according to [15], wherein the urokinase type plasminogen activator gene is a mouse urokinase type plasminogen activator gene, [17] Mice transplanted with human hepatocytes according to [15] or [16], wherein 75% or more of mouse hepatocytes are replaced with human hepatocytes, [18] Mice transplanted with human hepatocytes according to [15] or [16], wherein 80% or more of mouse hepatocytes are replaced with human hepatocytes, [19] Mice transplanted with human hepatocytes according to [15] or [16], wherein 90% or more of mouse hepatocytes have been replaced with human hepatocytes, [20] Mice transplanted with human hepatocytes of [15] or [16], wherein 95% or more of mouse hepatocytes are replaced with human hepatocytes, [21] A
  • Human hepatocytes are transplanted, comprising administering to the mouse a suicide substrate and hepatocytes isolated from human, damaging the mouse hepatocytes and replacing the mouse hepatocytes with human hepatocytes.
  • Mouse production method [28] A method for producing a mouse transplanted with human hepatocytes of [27], wherein the thymidine kinase gene is a human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene, [29] A method for producing a mouse transplanted with human hepatocytes of [27] or [28], wherein the suicide substrate is a substance that is metabolized to a toxic substance by thymidine kinase, [30] A method for producing a mouse transplanted with human hepatocytes of [29], wherein the substance to be metabolized is acyclovir or ganciclovir, [31] A method for producing a mouse transplanted with the human hepatocytes of any of [27] to [30], wherein the mouse is an immunodeficient mouse, [32] The human hepatocyte of [31], wherein the immunodeficient mouse is a NOD / SCID mouse, a dKO
  • the thymidine kinase gene is retained under control of an albumin promoter, LST-1 promoter, ⁇ -fetoprotein promoter, or ⁇ -TTP promoter so that the thymidine kinase gene can be specifically expressed in the liver.
  • HCV human hepatitis C virus
  • a reduction in the amount of human HCV infection relative to the amount of infection in a mouse or in a mouse prior to administration of a candidate drug means the anti-HCV activity of the drug;
  • a method for screening a candidate drug having anti-HCV activity [46] The method of [45], wherein the candidate agent is administered before infection with human HCV, [47] The method according to [45] or [46], wherein the candidate drug having anti-HCV activity is an anti-HCV antibody or an HCV-binding fragment thereof, [48] The following steps: (I) the step of reconstituting the immunity of the mouse of any one of [1] to [26] and [37]; (Ii) administering a candidate vaccine to the mouse of (i); and (iii) analyzing the effect of the vaccine on HCV infection, in an untreated mouse or in a mouse before the vaccine administration A decrease in the amount of human HCV infectious relative to the amount of infectious means the anti-HCV activity of the vaccine;
  • mice and [57] dKO mice deficient in RAG-2 and IL-2R ⁇ c are Balb / c dKO mice (RAG-2 KO, IL-2R null ), or NOD dKO mice (RAG-2 KO). IL-2R null ), [56] mice, I will provide a.
  • This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2009-008097 which is the basis of the priority of the present application.
  • FIG. 1 is a diagram showing the structure of a herpes simplex virus type 1 thymidine kinase (UL23 or HSVtk) gene expression unit.
  • FIG. 2 is a diagram showing a restriction map and structure of the transgene of the TK-NOG mouse, and the position of the probe used in the examples.
  • FIG. 3 is a diagram showing the human albumin concentration in the serum of TK-NOG mice transplanted with human hepatocytes.
  • FIG. 4 is a diagram showing proteins in the serum of TK-NOG mice transplanted with human hepatocytes.
  • FIG. 5 is a view showing mRNA expressed in the liver of a TK-NOG mouse transplanted with human hepatocytes.
  • FIG. 1 is a diagram showing the structure of a herpes simplex virus type 1 thymidine kinase (UL23 or HSVtk) gene expression unit.
  • FIG. 2 is a diagram showing a restriction map and structure of the
  • FIG. 6 shows the results of histology and immunohistochemical staining of the liver of TK-NOG mice transplanted with human hepatocytes.
  • FIG. 7 is a diagram showing carboxyfluorescein (CF) excretion into the capillary bile duct (BC) in the liver of TK-NOG mice transplanted with human hepatocytes administered with CFDA.
  • FIG. 8 shows liver h-CK8 / of two different TK-NOG mice obtained 52 days after transplantation of human hepatocytes in the liver of TK-NOG mice transplanted with CFDA and receiving human hepatocytes. It is a figure which shows the result of 18 immunohistochemical staining.
  • FIG. 7 is a diagram showing carboxyfluorescein (CF) excretion into the capillary bile duct (BC) in the liver of TK-NOG mice transplanted with human hepatocytes administered with CFDA.
  • FIG. 8 shows liver h-CK8
  • FIG. 9 is a diagram showing the amount of CF secreted into feces in TK-NOG mice transplanted with human hepatocytes administered with CFDA.
  • FIG. 10 shows the presence of bile CF metabolites in TK-NOG mice transplanted with human hepatocytes administered with CFDA.
  • FIG. 11 is a diagram showing analysis results of bile mice and human transferrin in TK-NOG mice transplanted with human hepatocytes administered with CFDA.
  • FIG. 12 is a diagram showing immunohistochemical staining of H & E and glutamine synthetase (GS) in liver sections obtained from TK-NOG mice transplanted with human hepatocytes.
  • FIG. 13 is a diagram showing the position of the HSVtk expression unit on the chromosome.
  • FIG. 14 is a diagram showing the results of molecular analysis of the TK-NOG mouse transgene.
  • FIG. 15 shows the results of RT-PCR analysis of HSVtk transgene expression.
  • FIG. 16 shows the activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in ganciclovir (GCV) -administered TK-NOG mice (Tg HSVtk) and non-transgenic NOG mice (NTg). .
  • FIG. 17 is a diagram showing the liver of a ganciclovir (GCV) -administered TK-NOG mouse (Tg HSVtk).
  • FIG. 18 is a diagram showing the serum human albumin concentration of TK-NOG mice to which GCV was administered and human hepatocytes were transplanted.
  • FIG. 19 is a diagram showing the correlation between human serum albumin concentration and substitution index (RI) in TK-NOG mice transplanted with human hepatocytes administered with GCV.
  • FIG. 20 is a diagram showing human gene expression in TK-NOG mice to which human hepatocytes were transplanted and repopulated with GCV-administered human hepatocytes.
  • FIG. 21 is a diagram showing the results of renal histopathological analysis in TK-NOG mice to which human hepatocytes were transplanted and repopulated with GCV administered human hepatocytes.
  • FIG. 22 is a diagram showing the results of double immunostaining of mouse albumin and human albumin staining of the liver of a TK-NOG mouse in which human hepatocytes have been transplanted and human hepatocytes have repopulated.
  • FIG. 23 is a diagram showing the results of microdissection of clonally proliferating human hepatocyte colonies in TK-NOG mice in which human hepatocytes were transplanted and human hepatocytes re-proliferated.
  • FIG. 22 is a diagram showing the results of double immunostaining of mouse albumin and human albumin staining of the liver of a TK-NOG mouse in which human hepatocytes have been transplanted and human hepatocytes have repopulated.
  • FIG. 23 is
  • FIG. 24 is a diagram showing the results of PCR analysis for distinguishing between human and mouse hepatocytes.
  • FIG. 25 is a diagram showing the structure of a urokinase-type plasminogen activator (uPA) gene expression unit.
  • FIG. 26 is a diagram showing a restriction map and structure of the transgene of uPA-NOG mouse, and the position of the probe used in the example.
  • FIG. 27 shows the results of RT-PCR analysis of uPA transgene expression.
  • FIG. 28 shows the activity of serum alanine aminotransferase (ALT) in hemizygous (Tg / +) and homozygous (Tg / Tg) transgenic uPA-NOG mice.
  • FIG. ALT serum alanine aminotransferase
  • FIG. 29 is a diagram showing a macroscopic image and a hepatocyte damage image of the liver of a homozygous transgenic uPA-NOG mouse (Tg / Tg).
  • the scale bar in FIG. 29 is 100 ⁇ m.
  • P indicates the portal tract and C indicates the central vein.
  • FIG. 30 is a diagram showing the results of molecular analysis by Southern blotting of a uPA transgene introduced into a uPA-NOG mouse.
  • FIG. 31 is a graph showing liver macroscopic images of hemizygous transgenic uPA-NOG mice (Tg / +) and homozygous transgenic uPA-NOG mice (Tg / Tg1) transplanted with human hepatocytes.
  • FIG. 32 is a graph showing changes in serum human albumin concentration and changes in body weight of uPA-NOG mice.
  • FIG. 33 shows immunoblotting analysis of sera from hemizygous (Tg / +) and homozygous (Tg / Tg1 and 2) transgenic uPA-NOG mice transplanted with mouse serum, human serum, and human hepatocytes. It is a figure which shows the result.
  • FIG. 34 shows the results of histology and immunohistochemical staining of homozygous (Tg / Tg1 and 2) transgenic uPA-NOG mouse liver transplanted with human hepatocytes.
  • FIG. 35 represents the metabolism of Debrisoquin and S-Warfarine in the mouse body with a humanized liver.
  • FIG. 33 shows immunoblotting analysis of sera from hemizygous (Tg / +) and homozygous (Tg / Tg1 and 2) transgenic uPA-NOG mice transplanted with mouse serum, human serum
  • FIG. 36 represents detection of human proliferating hepatocytes expressing Ki-67 antigen in mice with humanized liver.
  • FIG. 37 shows engraftment of human hepatocytes stained with human cytokeratin 8/18 antibody in TK-Balb / c dKO mice and NOD dKO mice.
  • the thymidine kinase disclosed in the present invention originally has an enzyme activity for phosphorylating thymidine to synthesize thymidylate in vivo, but it is a rate-limiting step in normal cells and is hardly detected. Therefore, a suicide substrate that is toxic by being metabolized by thymidine kinase is introduced with a transgene of thymidine kinase and exhibits cytotoxicity only to cells having thymidine kinase activity. Used as a selective therapeutic agent. As such a suicide substrate, a guanosine analog can be preferably used.
  • guanosine analogs include ganciclovir (GCV) and acyclovir, which are widely used as antiviral agents.
  • GCV ganciclovir
  • acyclovir which are widely used as antiviral agents.
  • ganciclovir is phosphorylated by thymidine kinase, an enzyme inherent in the virus, and finally activated ganciclovir-triphosphate by endogenous enzymes guanylate kinase and thymidine kinase in human cells. Is metabolized.
  • Active ganciclovir-triphosphate inhibits DNA polymerase by competitively antagonizing deoxyguanosine-triphosphate (dGTP), a substrate for viral DNA polymerase. Therefore, only viral replication in infected cells infected with the virus is inhibited.
  • dGTP deoxyguanosine-triphosphate
  • thymidine kinase gene is introduced so as to be specifically expressed in the mouse liver.
  • the biological species from which the thymidine kinase gene used in the present invention is derived may be a human or mammal, and may be a prokaryotic cell or a virus, and is not limited to the biological species from which it is derived.
  • human herpes simplex virus type 1-thymidine kinase (HSV-tk) is preferably exemplified as a thymidine kinase used as a specific embodiment of thymidine kinase.
  • HSV-tk human herpes simplex virus type 1-thymidine kinase
  • the HSV-tk gene refers to, for example, a gene represented by the polynucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto.
  • any polynucleotide sequence can be used for the mouse of the present invention as long as it encodes an amino acid sequence encoded by the polynucleotide sequence shown in SEQ ID NO: 1 or an enzyme active portion thereof.
  • amino acid sequence of a polypeptide having an enzymatic activity such as thymidine kinase is slightly changed, that is, one or more amino acids in the amino acid sequence are substituted or deleted, or one or more amino acids are It is a well-known fact that the enzyme activity of the polypeptide may be maintained even when added.
  • a variant may contain conservatively substituted sequences, meaning that certain amino acid residues may be replaced by residues with similar physicochemical characteristics. Yes.
  • conservative substitutions include substitution between aliphatic group-containing amino acid residues such as substitution between Ile, Val, Leu or Ala, or polar group-containing amino acid residues such as substitution between Lys and Arg. Substitution between groups is included. Therefore, for example, a DNA encoding a mutant of thymidine kinase having such a modification in the amino acid sequence encoded by the polynucleotide sequence shown in SEQ ID NO: 1 or an enzyme active portion thereof and having the enzyme activity of thymidine kinase Can also be used for the mouse of the present invention.
  • Mutations resulting from addition, deletion or substitution of amino acids can be performed by, for example, site-directed mutagenesis (for example, Nucleic Acid Research, Vol. 10, No. 20, p. 6487-), which is a well-known technique, in DNA encoding the same. 6500, 1982).
  • site-directed mutagenesis for example, Nucleic Acid Research, Vol. 10, No. 20, p. 6487-
  • “one or more amino acids” refers to a number of amino acids that can be added, deleted or substituted by site-directed mutagenesis, preferably 1 or several, more preferably 1 to 5 More preferably, 1 to 4, more preferably 1 to 3, and still more preferably 1 or 2.
  • Site-directed mutagenesis can be performed, for example, using synthetic oligonucleotide primers complementary to the single-stranded phage DNA to be mutated, as well as the specific mismatch that is the desired mutation, as follows. That is, host cells are transformed with double-stranded DNA synthesized as a complementary strand by phage using the synthetic oligonucleotide as a primer. Transformed bacterial cultures are seeded on agar and plaques are formed from single cells containing phage. Theoretically, 50% of new colonies contain phage with mutations as single strands and the remaining 50% have the original sequence.
  • the obtained plaque is hybridized with a synthetic probe labeled by kinase treatment at a temperature at which it hybridizes with DNA having the desired mutation and does not hybridize with DNA having the original strand.
  • the DNA is then recovered from the plaque that hybridizes to the probe.
  • a gene is treated with a mutagen as a method for substituting, deleting, or inserting one or more amino acids that do not lose its activity in the amino acid sequence of the enzyme.
  • a method and a method of selectively cleaving a gene, then removing, adding or replacing selected nucleotides, and then ligating are also preferable.
  • thymidine kinase that hybridizes under conditions of mild or severe stringency to, for example, a polynucleotide sequence encoding thymidine kinase described in SEQ ID NO: 1 disclosed herein.
  • Isolated DNA and RNA encoding can also be used in the mouse of the present invention.
  • Conditions for hybridization with mild stringency are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989).
  • Mild stringency conditions include 5 ⁇ SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) pre-wash solution and about 55 ° C., 5 ⁇ . SSC, overnight hybridization conditions. Severe stringency conditions include higher temperature hybridization and washing. At that time, the temperature and the salt concentration of the cleaning solution can be appropriately adjusted according to various factors such as the length of the probe. For example, a condition in a range of 5 ⁇ SSC or less and 20 ° C. or more is preferable. Furthermore, when calculated using the polynucleotide sequence encoding thymidine kinase set forth in SEQ ID NO: 1 and BLAST, etc.
  • isolated DNA and RNA that encodes biologically active thymidine kinase having an identity of 95% or more, more preferably 97% or more, and more preferably 97% or more are also used for the mouse of the present invention. can do.
  • “Thymidine kinase gene is retained so that it can be expressed in the liver of a mouse” means that the thymidine kinase gene is contained in a mouse hepatocyte and expressed in the cell. In order to allow the thymidine kinase gene of the present invention to be expressed in the liver of a mouse, a regulatory gene functioning in the liver can be appropriately used.
  • regulatory gene a regulatory gene of a gene encoding a protein functioning in hepatocytes is preferably used.
  • regulatory gene refers to a sequence on DNA that acts to increase or decrease gene transcription efficiency, and includes a promoter, an enhancer, an upstream activation sequence, a silencer, an upstream repression sequence, an attenuator, and the like. It is not limited to these.
  • the biological species from which the regulatory gene is derived is not limited to a specific biological species. In order to allow more appropriate expression in the liver of the mouse, a regulatory gene derived from the mouse can be preferably used.
  • the promoter is not particularly limited as long as the thymidine kinase gene is a promoter expressed in the liver.
  • Examples include albumin promoter, organic anion transporter LST-1 promoter, ⁇ -fetoprotein promoter, ⁇ -tocopherol transport protein ( ⁇ -TTP) gene promoter, and the like.
  • the expression mechanism of genes that are expressed specifically in the liver has been studied very well. That is, cells such as ubiquitous transcription regulatory factor binding sites, liver-specific transcription factor binding sites abundantly present in the liver, and hormones, etc., in the 5 ′ upstream region of the transcription start point of the gene specifically expressed in the liver It is known that upstream activation sequences that respond to external stimuli are packed in a relatively narrow region.
  • Preferred examples of such transcription factors include HNF-1, HNF-3, HNF-4, HNF-6, C / EBP, DBP and the like.
  • the thymidine kinase gene is linked to an upstream activation sequence that functions in the mouse liver. Therefore, for example, a certain region of the above-mentioned albumin promoter, organic anion transporter LST-1 promoter, ⁇ -fetoprotein promoter, ⁇ -tocopherol transport protein ( ⁇ -TTP) gene promoter region is used for the above-mentioned linkage.
  • An upstream activation sequence that binds a liver-specific transcription factor potentially contained by can be used.
  • a liver-specific upstream activation sequence whose sequence has already been identified can be suitably incorporated as a foreign gene by gene recombination techniques.
  • enhancers that increase the level of transcription from a promoter exist not only in the upstream activation sequence but also downstream from the transcription start point.
  • a transgenic mouse having a human angiotensinogen gene having a total length of 14 kb including the 5 ′ upstream 1.3 kb and 3 ′ downstream regions high expression of the gene is observed in the liver.
  • an enhancer was present in a 3.8 kb DNA fragment downstream from the transcription start site of the gene.
  • such an enhancer can also be suitably used to express the thymidine kinase gene in a liver-specific manner.
  • a 3 ′ untranslated sequence containing a poly A addition signal can also be suitably used for expressing the thymidine kinase gene in a liver-specific manner.
  • the thymidine kinase gene is retained so that it can be expressed in the mouse liver, the thymidine kinase gene is linked to an upstream activation sequence that functions in the mouse liver.
  • a 3 ′ untranslated sequence including an enhancer and a poly A addition signal can be suitably linked appropriately.
  • An integrated vector that adjusts the gene expression unit for holding the thymidine kinase gene so that it can be expressed in the liver of the host is inserted by inserting the gene group thus linked into a vector containing a marker gene such as a drug resistance gene.
  • a transgenic mouse into which a gene expression unit prepared from the integration vector has been introduced can be produced.
  • transgenic mice can be suitably produced according to known methods (Proc. Natl. Acad. Sci. USA (1980) 77, 7380-4).
  • a gene expression unit prepared so that the target thymidine kinase gene can be expressed in the liver of a host is introduced into a totipotent cell of a mouse.
  • Suitable examples of totipotent cells into which the gene is introduced include fertilized eggs and early embryos, and cultured cells such as ES cells having multipotency.
  • fertilized eggs into which the gene expression unit can be introduced can be suitably recovered.
  • a gene expression unit is introduced into a mouse fertilized egg by microinjection into the male pronucleus. After in vitro culture of fertilized egg cells, cells that are considered to have successfully introduced the gene expression unit are screened. The screened cells are transplanted into the surrogate mother's oviduct, and then a transgenic chimeric mouse is born.
  • a target transgenic mouse can be produced by selecting an individual into which a transgene has been incorporated into a somatic cell and a germ cell from the obtained individual. More specifically, it can be produced by the method described in the examples described later.
  • human hepatocytes are transplanted into mice, human hepatocytes are rejected due to an immune response to the transplantation of xenogeneic cells. Therefore, in the present invention, xenogeneic cell transplantation is performed as a mouse transplanted with human hepatocytes.
  • An immunodeficient mouse in which the immune response to is inactivated can be preferably used.
  • Such an immunodeficient mouse can also be preferably produced by irradiating the whole body with X-rays.
  • it may be a mouse that is preferably genetically deficient in its immune function.
  • immunodeficient animals include mice obtained by administering asialo GM1 antibody or TM ⁇ 1 to generally available nude mice or SCID mice, and X-irradiated mice.
  • preferred examples of immunodeficient mice whose genetic immune function is deficient include NOD / SCID mice and Rag2 knockout mice that do not have T cells or B cells.
  • dKO mice double knockout mice
  • dKO mice a dKO mouse having a genetic background of Balb / c
  • a dKO mouse having a genetic background of NOD is referred to as a NOD dKO mouse.
  • NOD / SCID / ⁇ c is a mouse derived from NOD / SCID mice and knocking out a common ⁇ chain of IL-2 receptor.
  • null It is also referred to as a NOD / Shi-scid, IL-2R ⁇ KO mouse.
  • NOG mouse is a registered trademark
  • Japanese Patent No. 3753321 Japanese Patent No. 3753321
  • the NOG mouse Since the presence of lymphocytes is not observed in the NOG mouse, the NOG mouse does not show NK activity and is deficient in tree cell function.
  • a gene designed to retain the target thymidine kinase gene so that it can be expressed in the liver of the host is contained in the fertilized egg of the immunodeficient mouse.
  • Preferred examples include transgenic mice resulting from the introduction.
  • an immunodeficient mouse selected to retain the traits of the immunodeficient mouse and to retain the target thymidine kinase gene so that it can be expressed in the liver of the host can also be suitably used.
  • an immunodeficient mouse selected to retain the traits of the immunodeficient mouse and to retain the target thymidine kinase gene so that it can be expressed in the liver of the host can also be suitably used.
  • TK-Balb / cA dKO mice HSV-Tk (+), SCID wild, RAG-2 KO, IL-2R) null
  • TK-NOG mice and NOD dKO mice RAG-2 KO, IL-2R null
  • TK-NOD dKO mice HSV-Tk (+), SCID wild, RAG-2 KO, IL-2R
  • a mouse selected to retain the trait of the NOG mouse and to retain the target thymidine kinase gene so that it can be expressed in the liver of the host is referred to as a TK-NOG mouse.
  • the guanosine analog is metabolized into a toxic substance in the hepatocyte of the mouse. Liver injury occurs in mice.
  • Ganciclovir mentioned as a suitable example of the guanosine analog is metabolized to ganciclovir-triphosphate in mouse hepatocytes and causes liver injury in the mouse.
  • the administration method of the guanosine analog to the mouse can be freely selected.
  • intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, or even application on the skin can be selected as appropriate.
  • it can also be administered into the mouse body by mixing during feeding.
  • the dose of the guanosine analog is not limited, but is 0.1 to 10 mg / Kg body weight, preferably 0.5 to 1.5 mg / Kg body weight.
  • ganciclovir in addition to the above, in addition to the administration of ganciclovir, carbon tetrachloride, D-galactosamine, pyrrolodine can be used as a method for causing liver injury in mice in which the thymidine kinase gene of the present invention is expressed and retained in the liver.
  • a treatment with a known liver injury inducer such as alkaloid or 2-acetylaminofluorene and a surgical treatment such as surgical hepatectomy are also preferred.
  • administration of an anti-mouse Fas antibody to the mouse can cause liver injury in the mouse.
  • the anti-mouse Fas antibody does not bind to Fas antigen expressed in human hepatocytes, but binds to Fas antigen expressed in mouse hepatocytes, thereby specifically causing apoptosis in mouse hepatocytes.
  • a mouse having a NOG mutation in which the urokinase-type plasminogen activator gene is retained in the liver so that it can be expressed in place of the thymidine kinase gene of the present invention is also preferably used.
  • a transgenic mouse resulting from introduction of a gene expression unit prepared to hold the urokinase-type plasminogen activator gene so that it can be expressed in the liver of the host into the totipotent cell of the mouse having the NOG mutation is preferred.
  • the urokinase-type plasminogen activator gene for example, the polynucleotide sequence encoding the mouse urokinase-type plasminogen activator described in SEQ ID NO: 47 can be used.
  • a method similar to the method using the thymidine kinase gene is preferably used.
  • a mouse having a NOG mutation in which both a thymidine kinase gene and a urokinase-type plasminogen activator gene are retained so that they can be expressed in the liver is also preferably used. It is possible to optimize the engraftment of human hepatocytes administered by appropriately selecting when liver injury is caused by the action of thymidine kinase and when liver injury is caused by the action of urokinase-type plasminogen activator it can.
  • the mouse is appropriately combined with a mouse having a thymidine kinase gene retained in an expressible manner in the liver and a mouse having a NOG mutation in which a urokinase-type plasminogen activator gene is retained in the liver. It can be suitably obtained by breeding and selecting a progeny mouse having both a thymidine kinase gene and a urokinase-type plasminogen activator gene that can be bred and expressed in the liver and has a NOG mutation.
  • a gene expression unit prepared because the urokinase-type plasminogen activator gene is retained so that it can be expressed in the liver of the host is used in a mouse in which the thymidine kinase gene is retained so that it can be expressed in the liver of the host. It can also be produced by operations such as introduction into totipotent cells.
  • mice in which the number of human hepatocytes exceeds a certain level.
  • the chimeric mouse 50% or more of the mouse liver, preferably 70% or more, more preferably 75% or more, further preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more of the mouse liver is human.
  • Preferred examples include mice occupied by the hepatocytes.
  • the proportion occupied by human hepatocytes is a method of calculating from a stained image of an antibody recognizing a marker specific to human and mouse hepatocytes, human serum albumin, etc.
  • a method for calculating from a quantitative value of a serum marker is known. Furthermore, a more accurate replacement rate can be confirmed by performing a anatomical examination of the liver of the chimeric mouse after the test by a known method.
  • the cell replacement rate is called RI (Replacement Index).
  • RI Replacement Index
  • the replacement rate of human hepatocytes in the present invention is determined, in the examples of the present invention, it is determined by the ratio occupied by human cytokeratin CK8 / 18 positive hepatocytes in the sections of tissue immunostaining. Embodiments are described.
  • a method for transplanting human hepatocytes into mice in addition to a method for transplanting to the liver via the mouse spleen, a method for transplanting directly from the portal vein can be appropriately employed. It may also be transplanted intraperitoneally or intravenously.
  • the number of human hepatocytes transplanted at one time is 1 to 2,000,000 (2 ⁇ 10 6 ) It can be appropriately selected from cells.
  • the hepatocytes of the present invention include normal hepatocytes, primary hepatocytes, preferably established hepatocytes maintained (including culture, passage, storage) in the presence of serum (eg, fetal bovine serum). It can be suitably used.
  • hepatocytes to be used for transplantation hepatocytes derived from any kind of cells can be suitably used as long as they are human hepatocytes and cells in human liver.
  • hepatocytes obtained from the liver tissue of the subject can be suitably used.
  • a biopsy which is a known method is preferably used.
  • Liver biopsy is a method in which a thin, long needle is inserted directly into the liver from the surface of the skin and the liver tissue is collected. Usually, the site where the needle is punctured is the space between the right lower chest.
  • the puncture part is sterilized after the safety of the puncture part is confirmed using an ultrasonic examination apparatus before surgery. Furthermore, from the skin to the surface of the liver is the target of anesthesia, and the puncture needle is punctured after the skin at the puncture site is made a small incision. Furthermore, commercially available frozen human hepatocytes can also be used. When primary hepatocytes are used, a cell suspension in which hepatocytes from the collected liver or liver tissue are dispersed in ice-cooled Hanks solution using a known technique such as a perfusion method or an infiltration method is used. It is isolated as appropriate by fractionation by centrifugation or the like.
  • the obtained hepatocytes were mixed with 5% CO in a medium such as Williams'E supplemented with bovine serum appropriately. 2 Incubate in the presence at 37 ° C. for 24 hours. Furthermore, cells that have been cultured for about one week by replacing the medium with ASF104 medium (Ajinomoto) etc. every 3 days can be used. Cell culture factors such as HGF and EGF can be added to the medium, and the culture matrix, three-dimensional culture, and the like can be modified as appropriate. When the established hepatocytes are used, the type of hepatocytes is not particularly limited.
  • ATCC American Type Culture Collection
  • Hep3B and HepG2 are registered in the ATCC with registration numbers of HB-8064 and HB-8065, respectively, and HuH-7 is registered in the JCRB cell bank with a registration number of JCRB0403.
  • the guanosine analog and human hepatocytes may be administered to the mouse simultaneously or separately.
  • guanosine analog may be administered into the abdominal cavity of a mouse simultaneously with transplantation of human hepatocytes from the spleen or vein.
  • Replacing mouse hepatocytes with human hepatocytes is called human hepatocytes or liver repopulation, and mice with human hepatocytes replaced with human hepatocytes are human hepatocyte reconstructed chimeric mice.
  • human liver reconstructed chimeric mouse A liver in which hepatocytes are replaced with human hepatocytes is referred to as a humanized liver.
  • the present invention also includes human liver tissue reconstructed in the above mice.
  • the liver tissue has a three-dimensional structure or function of human liver, and 75% or more, preferably 80% or more, more preferably 90% or more of mouse hepatocytes are replaced with human hepatocytes. Have been rebuilt.
  • These chimeric mice having human hepatocytes are used for administration of a test substance about 60 days after transplantation of human hepatocytes, for example.
  • a drug candidate substance is preferably exemplified.
  • Preferred examples of the drug candidate substance include substances that are in the process of drug development and that require prediction of drug interaction in humans. Such a substance can be a main ingredient for the medicinal effect of a pharmaceutical product, or can be a composition containing the main ingredient.
  • the dosage of the drug candidate substance varies depending on the type of the disease targeted by the substance, the type of substance composition, the administration route, etc., but can be appropriately selected from the range of about 0.1 mg / kg body weight to 2000 mg / kg body weight. .
  • the administration route can be appropriately selected from oral, subcutaneous, intravenous or intraperitoneal administration depending on the type of drug candidate substance and its suitable dosage form.
  • the degree of metabolism of a test substance such as a drug candidate substance in the liver of a chimeric mouse, and the metabolite are determined by appropriately measuring and identifying the concentration of the drug candidate substance in mouse plasma by standard methods in the art such as chromatographic methods. Can be done. Further, the measurement can be performed by time-lapse measurement measured at one or a plurality of time points appropriately selected from about 30 minutes to about 24 hours after administration of the candidate substance.
  • Human drug-metabolizing enzymes are CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C10, CYP2C18, CYP2C3, CYP2A3, CYP2C3, CYP2E3, CYP2E3
  • the indicator compounds metabolized by CYP1A2 are caffeine
  • CYP2C9 is tolbutamide
  • CYP2D6 is dextromethorphan
  • CYP2C19 is omeprazole
  • CYP3A4 is erythromycin, and the like.
  • the drug candidate substance After the drug candidate substance is administered to the chimeric mouse of the present invention, a mixture of these indicator compounds is administered, and the drug candidate substance promotes the activity of each enzyme by measuring the plasma concentration of each compound over time. It can be determined whether the substance is a substance that inhibits or a substance that inhibits. For example, when a drug candidate substance is administered, if the caffeine metabolism is promoted and the plasma concentration is decreased, the drug candidate substance can be determined to be a substance that specifically promotes the activity of CYP1A2. That is, how a drug administered to a human using the mouse of the present invention is metabolized in the human liver, and what kind of metabolite is generated, that is, the plasma dynamics of the drug, identification of the metabolite, metabolism Evaluation of speed etc. can be performed.
  • Another object of the present invention is to provide a mouse model that is susceptible to infection with a hepatotropic pathogen such as human HCV by a normal infection route. That is, the present invention shows that the mouse model is susceptible to infection with liver-directed pathogens such as HCV by a normal infection route, and the amount of virus can be equal to that of humans infected with liver-directed pathogens such as HCV over the long term. Provide an animal model that consistently and stably infects.
  • liver-directed pathogens such as HCV
  • HCV liver-directed pathogens
  • the mouse of the present invention can be infected with liver-directed pathogens such as HCV, particularly liver-directed pathogens having a host range limited to primates (eg, humans). Depending on the nature of the pathogen, a chronically infected mouse host can be maintained for weeks to months.
  • the present mouse is chronically infected with HCV (eg, chronic infection), and active HCV infection is at least about 5 weeks, usually at least about 14 to about 20 weeks. Or longer, up to about 35 weeks or longer, or the life of the host mouse.
  • the viral load on the infected host can be similar to the viral load on the infected person (human).
  • the host mouse will have about 10 per ml of serum. 3 ⁇ About 10 4 About 10 pieces 6 Individual virus particles, usually about 10 per ml of serum 3 ⁇ About 10 7 Infection can be maintained at individual virus particle concentrations.
  • the viral load in the infected host is substantially consistent over time, chronic and stable, e.g. the number of virus particles that can be isolated from the serum of an infected untreated host is It does not fluctuate significantly during the sampling period, for example, once a stable infection is established in the host, usually within about 2 to 4 weeks after infection, 1 mL of serum contains a large number of HCV virus particles at the first sampling.
  • the HCV-infected host of the present invention is positive for HCV infection at the next sampling and generally contains a large amount of HCV particles equal to or equivalent to 1 mL of serum.
  • the action of the candidate antiviral substance can be evaluated.
  • the mouse model of the present invention is used to identify agents that ameliorate symptoms caused by viral hepatitis, more specifically HCV infection, and / or more directly, infectious virus Affects the pathogenic mechanism. For example, it suppresses viral infection, reduces viral replication, or otherwise disrupts the viral growth cycle.
  • a candidate agent is administered to a mouse model of the invention and the effect of the candidate agent is compared to a control (eg, an agent with known anti-HCV action (eg, IFN against uninfected mice). -Evaluate to HCV-infected mice administered ⁇ ).
  • the candidate drug can be administered to the HCV-infected mouse of the present invention, and the viral load of the mouse before administration and / or the viral load of the mouse during non-administered HCV infection as a control and the viral load of the administered mouse (for example, Comparison can be made by measuring RT-PCR with serum samples. In general, if a detectable decrease or significant decrease in the amount of virus is observed in an infected mouse after administration of a candidate drug, it means that the target drug has an antiviral effect.
  • Candidate agents can be administered in any of a number of desired ways and / or in a manner suitable for drug delivery.
  • the candidate agent is suitably administered by injection (eg, intravenous injection, intramuscular injection, subcutaneous injection, or direct injection into tissue to achieve the desired effect), oral administration, or any other desirable method.
  • injection eg, intravenous injection, intramuscular injection, subcutaneous injection, or direct injection into tissue to achieve the desired effect
  • oral administration or any other desirable method.
  • In vivo screening methods typically involve several animals and various doses and concentrations of candidate drugs (from non-drug administration to drug doses that can be successfully delivered to animals)
  • the drugs can be administered alone, and can be administered in combination with two or more drugs, especially when the combined administration of the drugs produces a synergistic effect.
  • Candidate agents to be screened include synthetic molecules, natural molecules, or recombinantly produced molecules (eg, low molecular weight molecules; drugs: peptides; antibodies (antigen-binding antibody fragments, eg, those that provide passive immunity) Or other immunotherapeutic agents: endogenous factors (such as polypeptides and plant extracts) contained in eukaryotic cells or prokaryotic cells are preferably used. Of particular importance is a screening assay for drugs with low toxicity to human cells. Candidate drug activity can be assessed in a variety of ways.
  • the action of the drug is related to the presence or absence of the pathogen in the serum sample (eg titer in viral load) or the presence of the pathogen. It can be evaluated by examining a marker to be detected (for example, a protein specific to a pathogen or a nucleic acid encoding the same). Methods that are well known in the art can be adopted as methods for quantitatively and qualitatively detecting and evaluating the presence or absence and severity of viral infection.
  • the activity of a drug against HCV infection can be assessed by examining the presence of virus in serum samples and / or tissue sections (eg, RT-PCR in the case of HCV).
  • RNA of HCV is generated by, for example, detection of ( ⁇ ) strand RNA (HCV replication intermediate) by reverse transcriptase polymerase chain reaction (RT-PCR), competitive RT-PCR, RT-PCR, or treatment. It can be detected by sequencing the viral RNA to detect mutations / shifts (quasispecies evolution) on the viral genome. Or / and any quantitative or qualitative change in the amount of virus particles contained in the tissue section is directly demonstrated by performing the in situ RT-PCR hybridization using the host liver as a biopsy target. Can be done.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • a “vaccine” refers to a substance that, when administered, facilitates the host to initiate an immune response against a target pathogen.
  • the humoral, cellular, or humoral / cellular immune response elicited as a result of vaccine administration can facilitate the suppression of infection by pathogens for vaccine development.
  • liver-directed pathogens eg, bacterial pathogens, viral pathogens, or parasitic pathogens, particularly viral pathogens such as HCV, for example
  • prophylactic vaccines that elicit protective immune responses.
  • therapeutic vaccines that provide protection against infection by liver-directed pathogens by supplying passive immunity or specific active immunity (eg, anti-HCV immunoglobulins) that are rapidly promoted.
  • the immune system of an immunodeficient mouse is, for example, a stem cell, peripheral blood mononuclear cell (PBMC), blood cord cell, hematopoietic cell, or other suitable human origin that provides the animal with a human immune system. It can be reconstituted with cells.
  • Methods for isolating human immune cells, and reconstitution of the immune system of immunodeficient mice are well known in the art (eg, Nature 335: 256-59; Proc. Natl. Acad. Sci. USA 93 (25): 14720-25 may be referred to as appropriate).
  • the human immune cells are obtained from the same donor from which the human hepatocytes used in generating the mice according to the present invention were obtained.
  • human immune cells can be introduced into a host by methods well known in the art (eg, intraperitoneal injection). Effective vaccines can be screened by methods similar to those described above. That is, the candidate vaccine is administered to the chimeric animal before inoculation with the liver-directed pathogen.
  • Candidate vaccines are usually administered by a single bolus administration (eg, intraperitoneal injection, or intramuscular injection, topical or oral administration) followed by one or more boosters. Induction of an immune response can be assessed by examining antigen-specific B and T cell responses in a manner well known in the art.
  • a liver-directed pathogen is administered to the immunized animal.
  • administration is carried out on several immunized mice while increasing the titer of the pathogen.
  • the progression of infection is then observed in immunized and non-immunized control mice, and the severity of infection (eg, assessing the titer of existing pathogens or functioning of human hepatocytes as described above). Evaluated by examining the parameters).
  • Vaccine candidates that result in a significant reduction in pathogen infection that occurs after administration of the candidate vaccine and / or a significant reduction in disease severity are considered effective vaccines.
  • another object of the present invention is to provide a mouse model that can be used for the search, detection, and identification of cells that can become the liver in the future used for cell transplantation treatment, for example, hepatic stem cells.
  • Hepatic stem cells may be differentiated into hepatocytes in the future, and the number of generations necessary for differentiation into hepatocytes is not particularly specified. That is, the present invention performs transsplenic portal vein transplantation of primary isolated cells obtained from human liver tissue to a mouse model, and is used for cell transplantation treatment such as human hepatic stem cells from engrafted liver tissue. Also provided are methods for searching for or further identifying cells that can become liver.
  • a cell population containing cells that can become a future liver used for cell transplantation treatment such as human hepatic stem cells can be collected from the engrafted liver tissue.
  • the present invention also provides a method for collecting a cell population containing cells that can be used as a liver in the future used for cell transplantation treatment such as hepatic stem cells.
  • orthotopic transplantation can also be used as appropriate.
  • Stem cells cultured under appropriate conditions in vitro instead of primary isolated cells and induced to differentiate into hepatocytes can be used as appropriate for this purpose.
  • a method for inducing differentiation into hepatocytes using such stem cells is described in, for example, Gastroenterology (2009) 136, 990-999.
  • tissue immunostaining is performed using antigens that recognize markers for detecting hepatocytes and / or antibodies that recognize proliferating cells. It can be carried out.
  • a marker for detecting hepatocytes can be appropriately selected from albumin, tyrosine aminotransferase, glucose-6-phosphate, coagulation factor (CF) VII, asialoglycoprotein receptor, cytokeratin 8/18, and the like.
  • CF coagulation factor
  • As a cell proliferation marker it can be appropriately selected from Ki-67 antigen, 5-bromo-2′-deoxyuridine uptake ability, PCNA and the like.
  • a cell population containing human hepatic hepatocytes can be obtained by using these markers, and hepatocytes expressing such markers are appropriately isolated using FACSCalibur (Nippon Becton Dickinson Co., Ltd.) Isolated hepatic stem cells can be appropriately used for cell transplantation treatment and the like.
  • TK-NOG mouse and analysis of its function 1.
  • Method This example was performed by the following method.
  • the herpes simplex virus type 1 thymidine kinase (UL23 or HSVtk) gene expression unit was constructed as shown in FIG.
  • a 42 nucleotide polylinker (GATCCAAGCTTATGCAGGTCGACCCGGGCATGCGAATTTCTCGA: SEQ ID NO: 2) was introduced into the Bam HI-Xho I site (pBSII / linker) of pBlueScript II (pBSII; Promega).
  • the HSVtk gene was amplified by PCR at an annealing temperature of 62 ° C.
  • the amplified product was then cloned into the Nhe I-Sal I site (pCI-TK) of the pCI plasmid (Promega).
  • the human growth hormone (hGH) 3 ′ flanking region was amplified by PCR using the following primers at an annealing temperature of 60 ° C.
  • the amplified product was then cloned into the Sma I-EcoR I site (pBSII / linker / hGH) of the pBSII / linker plasmid.
  • the pmAlbEPintUL23GH plasmid DNA was digested with Not I and KpnI, and a 4,4-kb fragment containing no vector portion was prepared as an HSVtk expression unit.
  • This expression unit was microinjected into fertilized eggs of NOD / Shi mice by a known method.
  • Offspring with the transgene were identified by PCR using the HTKF1 forward primer 5'-CACGTCTTTATCCTGGGATACG-3 '(SEQ ID NO: 7) and hGHR1 reverse primer 5'-CACTGGAGTGCAACTTCCA-3' (SEQ ID NO: 8) (annealing temperature 63). ° C).
  • Genomic DNA extracted from the tail tissue was amplified in a 20 ⁇ l reaction mixture at 94 ° C.
  • transgene DNA was identified as an amplification product band of 236 bp on the agarose gel.
  • Female transgenic mice were treated with male NOD.
  • scid and IL2Rg null Gene mutations were genotyped by PCR (Maruyama, C. et al. Exp. Anim. 51, 391-393 (2002): Ito, M.
  • Biotin-labeled DNA was visualized with avidin FITC (Vector Laboratories), and the cells were counterstained with propidium iodide (Sigma-Aldrich Chemie GmbH). Observations were made using a Leica Q550 cytogenic workstation. Chromosomes with a fluorescent signal were identified by the G banding standard. (3) Southern blot analysis Genomic DNA samples were obtained by digesting the kidneys of 12-week-old TK-NOG mice and non-transgenic mice with proteinase K overnight and extracting with phenol: chloroform: ethanol.
  • a 479 bp glyceraldehyde d-phosphagase hydrodealdehyde 3-h phosphatase phosphatase dehydrated with G3PDHF forward primer 5'-TCACCCATCTTCCCGAGAGCGAGA-3 '(SEQ ID NO: 15) and G3PDHR reverse primer 5'-GAAGGCCATGCCCAGTGAGCTT-3' (SEQ ID NO: 16) was used as an internal standard (annealing temperature 65 ° C.).
  • Ganciclovir (GCV) sodium (Denosine-IV; Mitsubishi Tanabe Pharma) or vesicle (saline) was administered intraperitoneally twice daily using a 26 gauge needle.
  • GCV dissolved in water or saline was filter sterilized prior to its administration. All mice were monitored daily. The degree of liver injury was examined by biochemical serologic test values and pathological analysis.
  • peripheral blood was collected from the inferior vena cava for analysis of ALT and AST, and clinical chemistry analysis was performed by FUJI DRI-CHEM7000 (Fujifilm).
  • tissue fixed and paraffin embedded in 4% (v / v) phosphate buffered formalin was stained with hemotoxylin and eosin (H & E).
  • H & E hemotoxylin and eosin
  • the donor's age was 4-7 years. Transplantation was performed in the following general manner. GCV (0.5-1.5 mg / kg) was intraperitoneally administered to 6-8 week old adult TK-NOG recipient mice 5 and 3 days before transplantation. Cell number and viability were measured using a hemocytometer by trypan blue exclusion. 1-2 ⁇ 10 suspended in 40 ⁇ l Hank's Balanced Solution (HBSS) or William's medium E 6 Live liver cells were transplanted into the spleen using a Hamilton syringe with a 26 gauge needle. 1x10 as control 4 Human colon cancer cell line HCT116 (American Type Culture Collection; ATCC) was transplanted into the spleen of non-transgenic NOG mice.
  • HBSS Hank's Balanced Solution
  • HCT116 American Type Culture Collection
  • Human albumin measurement A small amount of blood was collected from the fundus venous plexus every other week using a plastic capillary. The collected blood was diluted 500 to 30000 times with TBS (Tris-buffered saline) containing 1% bovine serum albumin and 0.05% Tween 20 (BSA), and the human albumin concentration was diluted with human albumin ELISA Quantitation Kit (Bethyl Laboratories). Threshold concentration is ⁇ 3x10 3 ng / ml.
  • Table 1 shows the primer information (target gene, sequence) used, annealing temperature, amplification product size, and the like. (10) Histology and immunohistochemical staining Formalin-fixed liver and kidney were embedded in paraffin, and 5 ⁇ m sections thereof were prepared. Some of the sections were immersed in a target retrieval solution (0.1 M citrate buffer, pH 6.0; 1 mM EDTA, pH 9.0), subjected to autoclaving for 10 minutes, and then placed at room temperature for 20 minutes.
  • a target retrieval solution 0.1 M citrate buffer, pH 6.0; 1 mM EDTA, pH 9.0
  • Monoclonal mouse anti-human CK8 / 18 antibody (clone 5D3; Novocastra Laboratories), monoclonal mouse anti-human HLA-class I-A, B, C antibodies (clone EMR8-5; Hokudo), polyclonal goat anti-human albumin antibody (Bethyl Laboratories)
  • a polyclonal rabbit anti-human GLUL antibody (Sigma-Aldrich) was used as the primary antibody.
  • RI which is the percentage of donor human hepatocytes in the recipient liver, was expressed as the percentage of the area occupied by human CK8 / 18 positive hepatocytes in the total area in the immunohistochemically stained section. Livers used to make frozen sections were embedded in OCT compounds (Sakura Finechemicals), frozen in liquid nitrogen and sectioned at a thickness of 5-10 ⁇ m.
  • Genomic DNA is TPMK buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl 2 , And 40 ⁇ g / ml proteinase K).
  • the human and mouse ⁇ -actin genes have the following sequences: Was amplified by PCR using a primer having an annealing temperature of 60 ° C. This primer set amplified not only human ⁇ -actin but also mouse ⁇ -actin gene (245 bp and 271 bp, respectively).
  • a control plasmid (pBSII-hm / ⁇ -ACT) was prepared by inserting corresponding sequences from human and mouse DNA into the Sma I and HindIII sites of the pBSII vector, respectively.
  • (CF) is an organic anion that is excreted from various cells via Mrp2.
  • the ester precursor of this dye, 5-carboxyfluorescein diacetate (5-CFDA, 0.5 nmol; Sigma-Aldrich) was administered by intravenous injection from the tail.
  • the liver was perfused with 50 nmol / L5-CFDA solution for 3 minutes, and the liver was embedded in OCT compound and frozen in liquid nitrogen.
  • This reagent easily penetrates into hepatocytes due to its easy fat solubility, is hydrolyzed by esterase, forms CF (water-soluble fluorescent substrate), and is then excreted in bile.
  • Serial frozen sections 10 ⁇ m thick were prepared and air dried. The fluorescence signal of CF was observed and photographed using an upright microscope Axio Imager (Carl Zeiss) equipped with AxioCam HRm and AxioCam MRc5 CCD cameras (Carl Zeiss).
  • the tissue section was fixed with ethanol, the ethanol concentration of the fixative solution was gradually reduced, and the section was finally immersed in water containing no DNase and RNase. Immunohistochemical staining was then performed using monoclonal mouse anti-human MRP2 antibody (Clone M2 III-6; Millipore), Histofine Simple Stain Mouse MAX PO (M) (Nichirei Bioscience) and DAB substrate. Sections were counterstained with hematoxylin.
  • tissue sections were fixed with 4% paraformaldehyde solution, monoclonal goat anti-human HLA-class I-A, B, C antibodies (clone EMR8-5; Hokudo) and Texas Red labeled streptavidin- (GE Healthcare Bio-Sciences). Was used for immunofluorescence staining. Other tissue sections were stained with hemotoxylin and eosin (H & E). Bile and fecal samples collected from humanized liver NOG mouse # 2-11-5 with a very high degree of substitution and humanized liver NOG # 48-L5 with a very low degree of humanization are 0.1 M Tris-HCl (pH 8.0).
  • Black circles, black triangles, and black squares show the results of assays performed using mice # 12-14, # 6-6-10, and # 2-4-7, respectively.
  • Proteins in the serum of 3 TK-NOG mice were measured by immunoblotting at 70, 44 and 41 days after human hepatocyte transplantation. Mouse and human sera were used as positive and negative controls. The results are shown in FIG. MRNA expressed in the livers of the control and 3 TK-NOG mice was measured by RT-PCR at 70, 44 and 41 days after human hepatocyte transplantation. Mouse and human liver RNA were used as control samples. The results are shown in FIG.
  • FIG. 6 shows # 12-14 (70 days), # 6-6-10 (44 days) and # 2-4-7 (41 days) HE staining (A, C and E, respectively) and anti-human.
  • the results of CK8 / 18 antibody staining (B, D and F, respectively) are shown.
  • G to K indicate serial sections stained with H & E, h-CK8 / 18, HLA, h-albumin and PAS.
  • FIG. 7 shows carboxyfluorescein (CF) excretion into the capillary bile duct (BC).
  • TK-NOG mice A, B, C and D
  • HCT-116, E, F, G and H human colon cancer cell lines
  • 5-CFDA 5-CFDA
  • FIG. 8 shows the results of human CK8 / 18 immunohistochemical staining of two different human liver reconstructed TK-NOG mouse livers obtained 52 days after transplantation of human hepatocytes.
  • FIG. 9 shows the result.
  • FIG. 10 shows a fluorescence image of the metabolite CF in the bile of almost completely humanized # 2-11-5 TK-NOG mice collected 10 hours after administration of 5-CFDA.
  • FIG. 11 shows the analysis result of mouse and human transferrin in bile in humanized TK-NOG mouse # 2-11-5. As shown in FIG.
  • FIG. 12 shows H & E (A and C) and glutamine synthase (GS) of liver sections obtained from TK-NOG mice 6 weeks (A and B) or 16 weeks (C and D) after human hepatocyte transplantation. (B and D) shows immunohistological staining.
  • GS expression was not observed in the central vein region in the human hepatocyte nest 6 weeks after transplantation.
  • GS expression was observed around the central vein in human hepatocyte nests of TK-NOG mouse liver after 16 weeks from transplantation.
  • HSVtk transgenic NOG mice The transgene structure of HSVtk consists of the 3′-UTR of the human growth hormone gene along with the mouse albumin enhancer / promoter (Alb En / Pro), chimeric intron, HSVtk cDNA, and polyadenylation signal (hGHpA).
  • FIG. 1 shows the structure. Arrowheads indicate the position and orientation of the primers used to detect transgene-specific transcripts. The tip of each arrow represents the 3 ′ end of the oligonucleotide.
  • HSVtk-gene transgenic mice were established in the NOD / Shi line (HSVtk-transgenic; line 7-2).
  • the genetic background of NOG mice is the NOD / Shi line itself, scid and IL2Rg null Since the mutation is the only difference, the scid and IL2Rg can then be crossed by crossing with the NOG line.
  • FIG. 13 shows the position on the chromosome of the HSVtk expression unit into which the gene has been introduced. As shown in FIG. 13, TK-NOG mice had an HSVtk expression unit at the chromosome 1G locus.
  • FIG. 14 shows the results of molecular analysis of the HSVtk transgene introduced into TK-NOG mice.
  • Genomic DNA samples from non-transgenic NOG mouse kidney (N) and TK-NOG mouse kidney (T) were analyzed for Bam HI (Ba), Not I and Kpn I (NK), Bgl II (Bg) for Southern blot analysis.
  • Bg Bgl II
  • Bg Bgl II
  • Xba I Xb.
  • FIG. 2 shows a restriction map and structure of the transgene of the TK-NOG mouse.
  • the cloning vector (shaded box indicates the cloning vector) was integrated into the mouse genome in a head-to-tail tandem array structure (black box indicates the transcriptional segment) sandwiched between two HSVtk transgenes.
  • FIG. 15 shows the results of analysis of HSVtk transgene expression by RT-PCR. Detection of the HSVtk transgene was performed using primers that specifically recognize the sequence of the correctly spliced chimeric intron.
  • Non-transgenic NOG mouse liver nLi
  • TK-NOG mouse liver Li
  • kidney Ki
  • spleen Sp
  • lung Lu
  • brain Br
  • skeletal muscle Sm
  • Testicle Te
  • Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal standard.
  • FIG. 16 shows the activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in ganciclovir (GCV) -administered TK-NOG mice (Tg HSVtk) and non-transgenic NOG mice (NTg). .
  • AST serum aspartate aminotransferase
  • ALT alanine aminotransferase
  • Ganciclovir GCV
  • FIG. 17A shows an H & E-stained image of the liver of a TK-NOG mouse administered with PBS as a negative control for ganciclovir administration.
  • FIGS. 17B-D show H & E-stained images of TK-NOG mouse liver after 1.5 mg / kg GCV administration (FIG. 17B), 50 mg / kg GCV administration (FIG. 17C), and 50 mg / kg GCV administration (FIG. 17D).
  • FIG. 17B shows an H & E-stained image of the liver of a TK-NOG mouse administered with PBS as a negative control for ganciclovir administration.
  • FIGS. 17B-D show H & E-stained images of TK-NOG mouse liver after 1.5 mg / kg GCV administration (FIG. 17B), 50 mg / kg GCV administration (FIG. 17C), and 50 mg / kg GCV administration (FIG. 17D).
  • FIG. 17B shows an H & E-stained image of the liver of
  • FIG. 17 shows serum human albumin concentrations in TK-NOG mice to which GCV was administered at various concentrations and human hepatocytes were transplanted. As shown in FIG.
  • FIG. 20 shows human gene expression in TK-NOG mouse liver reconstructed with human hepatocytes. Human and mouse mRNA were used as positive and negative controls, respectively.
  • FIG. 21 shows the results of histopathological analysis of kidneys collected from mice transplanted with human hepatocytes. As shown in FIG. 21, kidney failure was not observed by H & E and PAS staining of kidneys (A and B) of TK-NOG mice with reconstructed liver and kidneys (C and D) of non-transgenic NOG mice. It was. The bar is 100 ⁇ m.
  • Figures 22 to 24 show that the liver of reconstructed TK-NOG mice is not due to cell fusion.
  • FIG. 22 shows the results of double immunofluorescence staining of mouse albumin (green) and human albumin (red). As shown in FIG.
  • FIG. 23 shows the results of capturing clonal proliferating human hepatocyte colonies by the laser microdissection method.
  • the anti-human CK8 / 18-stained frozen section is shown before (A) and after (B) capture by the laser microdissection method.
  • the dashed lines in FIG. 23A indicate the boundaries of the capture region consisting of human and non-human targets (I and II in FIG.
  • FIG. 24 shows the results of PCR analysis performed to distinguish whether the hepatocytes captured by the laser microdissection method are human or mouse.
  • the primer set used amplified not only human ⁇ -actin but also mouse ⁇ -actin gene in the same manner, and products of different sizes were obtained (245 bp and 271 bp, respectively).
  • From the human CK8 / 18 positive colony (FIG. 23A) only the human ⁇ -actin amplicon of the 245-bp band was detected.
  • the mouse ⁇ -actin amplicon of the 271 bp band was detected. Only were detected (FIG. 24, lane “region II”).
  • Mouse and human DNA were used as control samples. A plasmid containing both mouse and human DNA target sequences was also used as a control.
  • GCV ganciclovir
  • human serum albumin was first detected in test recipients within 4 weeks after transplantation and gradually increased to reach a maximum concentration of 11.5 mg / ml several weeks later.
  • FIG. 3 The transplanted human hepatocytes were completely integrated into the liver structure of the recipient mouse (FIG. 6).
  • frozen hepatocytes were used for transplantation only once (the continuous transplantation method in which human hepatocytes were isolated from engrafted TK-NOG mice and transplanted again was not performed. Nevertheless, high levels of human hepatocyte regrowth were observed in the livers of TK-NOG mice.
  • 5-carboxylfocein diacetate 5-CFDA
  • 5-CFDA easily enters hepatocytes due to its lipophilicity, where it is metabolized to water-soluble fluorescent metabolite (CF) by esterase (Mor-Cohen, R. et al. J. Biol. Chem. 276, 36923).
  • Mrp2 an organic anion transporter
  • TK-NOG mice in which more than 90% of the liver is composed of human hepatocytes, excrete 5-CFDA metabolite (water-soluble CF) It was investigated. Although no fluorescence derived from CF was observed in the feces until 6 hours after 5-CFDA administration, the peak reached after 7 hours (FIG.
  • the human liver reconstructed TK-NOG mouse has a biological function in which metabolites such as drugs circulate in the intestinal liver. It was necessary to determine whether the liver reconstructed in TK-NOG mice had the three-dimensional structural characteristics of mature human liver. Therefore, the expression pattern of an enzyme (glutamine synthase (GS)) with different distribution characteristics in the hepatic lobule was examined, and the lobule structure formed in the reconstructed liver showed physiological functions (central vein region and portal vein region). It was evaluated whether it reflects the expression of enzymes induced by nutrient or oxygen concentration gradients. Usually, GS is expressed in hepatocytes in the immediate vicinity of the central venous region (Smith, DDJ et al.
  • TK-NOG TK-NOG platform having a three-dimensional structure such as the biliary network and functional lobule structure (site specific enzyme expression in the lobule), which is a characteristic of the mature liver, is a known uPA transgenic used for transplantation of human liver.
  • mice and Fah knockout mice Overcoming the limitations of mice and Fah knockout mice.
  • the functions of mature liver such as hepatobiliary network and functional lobule structure, can never be reproduced in in vitro experiments such as primary cultured hepatocytes without 3D structure (between cells).
  • Humanized liver reconstructed in TK-NOG mice is a preferred platform for drug metabolism and liver research.
  • mice A mouse urokinase-type plasminogen activator (Plau or uPA) gene expression unit was constructed as shown in FIG.
  • the mouse uPA gene has the following primers: Was amplified by PCR at an annealing temperature of 60 ° C.
  • the herpes simplex virus type 1 thymidine kinase gene portion is removed by double digestion of the herpes simplex virus type 1 thymidine kinase gene expression plasmid (pmAlbEPintUL23GH) described in Example 1 with the restriction enzymes Nhe I and Sal I.
  • pmAlbEPintPlauGH GenBank accession number AB453180
  • mouse uPA gene SEQ ID NO: 47
  • the pmAlbEPintPlauGH plasmid DNA was digested with Not I and Kpn I, and a 4.6-kb fragment without the vector portion was prepared as a uPA expression unit.
  • the expression unit was microinjected into fertilized eggs of NOD / Shi mice by a known method.
  • Offspring with transgenes were identified by PCR using MuPAF1 forward primer 5′-AGTGTATGCAGCCCCCACTACTATG-3 ′ (SEQ ID NO: 50) and hGHR1 reverse primer 5′-CACTGGAGTGCACATCTCCA-3 ′ (SEQ ID NO: 8) (annealing temperature 63 ° C).
  • Genomic DNA extracted from the tail tissue was amplified in a 20 ⁇ l reaction mixture at 94 ° C. for 2 minutes, 94 ° C. for 30 seconds 35 cycles, 63 ° C. for 20 seconds, 68 ° C. for 60 seconds, and 68 ° C. for 3 minutes.
  • the transgene DNA was identified as an amplification product band of 383 bp on the agarose gel.
  • mice Female transgenic mice were treated with male NOD.
  • An individual having a mutation of scid and IL2Rg null was obtained by mating with Cg-Prkdc scid I12rg tm1Sug / ShiJic (NOG).
  • the scid and IL2Rg null gene mutations were genotyped by PCR (Maruyama, C. et al. Exp. Anim. 51, 391-393 (2002): Ito, M. et al. Blood 100, 3175-3182 (2002). )).
  • the official strain name of the produced mouse is NOD.
  • pCIspF forward primer 5′-GAGGCACTGGGGCAGGGTCCC-3 ′ (SEQ ID NO: 13) and MuPAR reverse primer 5′-AGGGCCGACTTTGGTATCAGTG-3 ′ (SEQ ID NO: 51) are used as a transgene of the transgene (mouse uPA (Annealing temperature 65 ° C.).
  • a 479 bp glyceraldehyde d-phosphagase hydrodealdehyde 3-h phosphatase phosphatase dehydrated with G3PDHF forward primer 5'-TCACCCATCTTCCCGAGAGCGAGA-3 '(SEQ ID NO: 15) and G3PDHR reverse primer 5'-GAAGGCCATGCCCAGTGAGCTT-3' (SEQ ID NO: 16) was used as an internal standard (annealing temperature 65 ° C.).
  • (3) Spontaneous liver injury The degree of liver injury was examined by biochemical serum test (variation of alanine aminotransferase aminotransferase (ALT) value) by FUJI DRI-CHEM7000 (Fujifilm).
  • Genomic DNA samples were obtained by digesting the liver and kidney of 8-week-old uPA-NOG mice and non-transgenic mice with proteinase overnight, followed by phenol: chloroform: ethanol extraction.
  • Genomic DNA digested with restriction enzymes XbaI, XhoI, BglII and BamHI was electrophoresed on a 0.6% agarose gel and transferred to a positively charged nylon membrane (F. Hoffmann-La Roche).
  • Hybridization was performed using a DIG-labeled probe prepared using PCR DIG Probe Synthesis Kit (F. Hoffmann-La Roche). The primers used were the following sequences: It was what had. The position recognized by the probe is indicated by * in FIG.
  • transplantation was performed by the following general method. Six-week-old adult hemizygous and homozygous uPA-NOG mice were used as recipients. Cell number and viability were measured using a hemocytometer by trypan blue exclusion. 40 ⁇ l Hank's Balanced Solution (HBSS) or 1-2 ⁇ 10 6 live liver cells suspended in William's medium E were transplanted into the spleen using a Hamilton syringe with a 26 gauge needle. . (6) Measurement of human albumin A small amount of blood was collected every other week from the fundus venous plexus using a plastic capillary.
  • HBSS Hank's Balanced Solution
  • the collected blood was diluted 500 to 30000 times with TBS (Tris-buffered saline) containing 1% bovine serum albumin and 0.05% Tween 20 (BSA), and the human albumin concentration was diluted with human albumin ELISA Quantitation Kit (Bethyl Laboratories). The threshold concentration was ⁇ 3 ⁇ 10 3 ng / ml.
  • a diluted serum sample (dilution ratio: 2000 times) was dissolved in an SDS sample buffer containing 5% ⁇ mercaptoethanol and subjected to SDS-PAGE, it was applied to a Hybond-ECL membrane (GE Healthcare Bio-Sciences). It was transcribed.
  • the transferred membrane was incubated with a biotin-labeled polyclonal goat anti-mouse albumin antibody (A90-234A; Bethyl Laboratories) and a biotin-labeled polyclonal goat anti-human albumin antibody (A80-229A; Bethyl Laboratories). Incubated with horseradish peroxidase (HRP) labeled streptavidin (GE Healthcare Bio-Sciences) for 60 minutes. Biotin labeling was performed by FluoReporter Mini-Biotin-XX Protein Labeling Kit (Invitrogen Corp., CA, USA), and ECL Western Detection System (GE Healthcare Bio-Sciences) and GE Healthcare Bio-Sciences (HEC). Was detected.
  • HRP horseradish peroxidase
  • Goat Ig is an amino acid polymer / peroxidase complex labeled antibody (Histofine Simple Stain Mouse MAX PO (G); Nichirei Bioscience) and diaminobenzidine (DAB) substrate (0.2 mg / ml 3,3′-diaminobenzidine , 0.05 M Tris-HCl, pH 7.6 and 0.005% H 2 O 2 ) and counterstained with hematoxylin. 2. The results were as follows.
  • uPA transgenic NOG mice The transgene structure of uPA, together with mouse albumin enhancer / promoter (Alb En / Pro), chimeric intron, mouse uPA cDNA, and polyadenylation signal (hGHpA) It consists of 3'-UTR.
  • FIG. 25 shows the structure. Arrowheads indicate the position and orientation of the primers used to detect transgene-specific transcripts. The tip of each arrow represents the 3 ′ end of the oligonucleotide. In the NOG strain, the number of offspring obtained by microinjecting a gene fragment is extremely small due to a defect in DNA rearrangement caused by a scid mutation.
  • uPA-gene transgenic mouse was established in the NOD / Shi line (uPA-transgenic; line 11-4). Genetic background of NOG mice were NOD / Shi system itself, since scid the IL2RG null mutation is the only difference, then, by mating with NOG system, to possess scid and IL2RG null mutation, severe immunodeficiency NOG-Tg (Alb-Plau) 11-4 / ShiJic (formally NOD.Cg-Prkdc scid Il2rg tm1Sug Tg (Alb-Plau) 11-4 / ShiJic, abbreviated name: uPA-NOG for short) Called).
  • FIG. 27 shows the results of analysis of uPA transgene expression by RT-PCR.
  • the uPA transgene was detected using primers that specifically recognize the sequence of the correctly spliced chimeric intron.
  • Liver Liver
  • kidney Kidney
  • spleen of non-transgenic NOG mouse Wild
  • hemizygous transgenic uPA-NOG mouse Tg / +
  • homozygous transgenic uPA-NOG mouse Tg / Tg (Splen) was detected and its expression was confirmed in the livers of hemizygous and homozygous transgenic uPA-NOG mice.
  • Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal standard.
  • FIG. 28 is a diagram showing the activity of a liver injury marker / serum alanine aminotransferase (ALT) in hemizygous transgenic uPA-NOG mice (Tg / +) and homozygous transgenic uPA-NOG mice (Tg / Tg). It is.
  • the liver injury marker value in hemizygous transgenic uPA-NOG mice (Tg / +) was not significantly different from the value of non-transgenic NOG mice (Wild) indicated by dotted lines.
  • ALT activity in homozygous transgenic uPA-NOG mice is higher than that of hemizygous transgenic uPA-NOG mice (Tg / +) and non-transgenic NOG mice (Wild) after 6 weeks of age. A significant increase was observed and continued to last up to 14 weeks.
  • Homozygous transgenic uPA-NOG mice (Tg / Tg) liver images (FIG. 29A) and hepatocellular injury images (FIGS. 29B and C) are shown in FIG.
  • FIG. 29B shows an H & E-stained image of the liver of a 6-week-old homozygous mouse (Tg / Tg). Degenerative lesions of hepatocytes and eosin eosinophils were observed mainly in the central venous region.
  • FIG. 30 shows the results of molecular analysis by Southern blotting of the uPA transgene introduced into uPA-NOG mice. Genomic DNA samples from non-transgenic NOG mouse kidney (Kid. (W)) and uPA-NOG mouse kidney (Kid. (Tg)) and uPA-NOG mouse liver (Liv. (Tg)) for Southern blot analysis. Digested with Xba I and Xho I (FIG. 30 left).
  • the 4.6-kb uPA expression unit (Cont.) Used for gene transfer was used as a positive control.
  • the transgene deletion in the liver was reported in the non-patent document 6 of the uPA transgenic mouse, but surprisingly, the transgene deletion in the liver was not observed in the uPA-NOG mouse of the present invention.
  • Genomic DNA samples from non-transgenic NOG mouse kidney (Wild) and uPA-NOG mouse kidney (Tg) were digested with Bgl II and Bam HI for Southern blot analysis (FIG. 30 right). Analysis revealed that uPA-NOG mice possess at least 3 copies of uPA expression units.
  • FIG. 26 shows a restriction map and structure of the transgene of uPA-NOG mice.
  • FIG. 31 shows hemizygous transgenic uPA-NOG mice (Tg / +) transplanted with human hepatocytes (FIG. 31A) and homozygous type.
  • the liver macroscopic image of a transgenic uPA-NOG mouse (Tg / Tg1) (FIG. 31B) is shown.
  • FIG. 32 shows changes in serum human albumin concentration (FIG. 32A) and body weight changes (FIG. 32B) of uPA-NOG mice.
  • Serum human albumin concentration in hemizygous transgenic uPA-NOG mice (Tg / +) is the same level as that of non-transgenic NOG mice (Wild) indicated by dotted lines, and human hepatocyte engraftment was not confirmed It was.
  • the serum human albumin concentration in homozygous transgenic uPA-NOG mice increased from about 4 weeks after transplantation of human hepatocytes, and the serum human albumin concentration was 1 mg in 2 of the 2 transplanted cases. / Ml or more, that is, the replacement rate with human hepatocytes was 10 to 15%.
  • the growth of homozygous transgenic uPA-NOG mice (Tg / Tg1 and 2) transplanted with human hepatocytes was good. The results of analyzing these mouse sera by immunoblotting are shown in FIG. Mouse and human sera were used as positive and negative controls.
  • FIG. 34 shows the results of immunostaining and H & E staining of anti-human albumin antibodies of homozygous transgenic uPA-NOG mice (Tg / Tg1 and 2) 10 weeks after transplantation of human hepatocytes.
  • anti-human albumin antibody staining of Tg / Tg2 homozygous transgenic uPA-NOG mice whose serum human albumin concentration reached 6.5 mg / ml about 80% of the sections were human hepatocytes (FIG. 34B).
  • the anti-human albumin antibody staining positive part FIG.
  • DB 4-hydroxy DB (4-OH DB), WF and 7-hydroxy WF (7-OH WF) by LC-MS / MS
  • Plasma concentrations of DB and 4-OH DB were measured by the following method. It was. Distilled water (150 ⁇ L) was added to mouse plasma (5 ⁇ L), and 20 ⁇ l of an ethanol solution of 20 nM imipramine, which is an internal standard substance, was mixed, followed by solid phase extraction.
  • elution was performed with a 2-propanol / acetonitrile mixture.
  • the eluate was quantified by LC-MS / MS.
  • the observed ions were m / z 176 and 134 for DB, m / z 192 and 132 for 4-OH DB, and m / z 281 and 86 for the internal standard.
  • Plasma concentrations of WF and 7-OH WF were measured by the following method. Distilled water (150 ⁇ L) was added to 5 ⁇ L of mouse plasma, and 20 ⁇ L of 50 nM 2-benzylphenol ethanol solution as an internal standard substance was mixed, followed by solid phase extraction.
  • FIGS. 35A and 35B The blood concentrations of debrisoquin after the lapse of the specified time and its metabolite 4′-hydroxydebrisoquin by CYP2D6 are shown in FIGS. 35A and 35B, respectively. There was no difference in blood concentration of Debrisoquin between the control NOG and hu-Liver (Fig.
  • HBSS Hank's Balanced Solution
  • William's medium E 1-2 ⁇ 10 6 hepatocytes were transplanted into the spleen using a Hamilton syringe with a 26 gauge needle.
  • HBSS Hank's Balanced Solution
  • E 1-2 ⁇ 10 6 hepatocytes were transplanted into the spleen using a Hamilton syringe with a 26 gauge needle.
  • Detection of proliferating human cells Formalin-fixed livers were embedded in paraffin and 5 ⁇ m sections thereof were prepared. The sections were immersed in a target retrieval solution (0.1 M citrate buffer, pH 6.0; 1 mM EDTA, pH 9.0), subjected to autoclaving for 10 minutes, and then placed at room temperature for 20 minutes.
  • Monoclonal mouse anti-human Ki-67antigen antibody (clone MIB-1; Dako) was used as the primary antibody.
  • mouse Ig was treated with amino acid polymer / peroxidase complex-labeled antibody (Histofine Simple Stain Mouse MAX PO (M); Nichirei Bioscience) and diaminobenzidine (DAB; Dojindo Laboratories) substrate (0.2 mg / ml). 3,3′-diaminobenzidine tetrahydrochloride, 0.05M Tris-HCl, pH 7.6, and 0.005% H 2 O 2 ). Sections were counterstained with hematoxylin. 3. Detection of human proliferative hepatocytes in mice with humanized liver FIG.
  • FIG. 36 shows the results of immunostaining human normal hepatocytes engrafted in the liver of TK-NOG and uPA-NOG mice with a cell proliferation marker (MIB-1). It was shown to. Although the Ki-67 antigen has many unclear points in terms of function, it is said that the expression level increases in the S phase and becomes maximum in the M phase. Therefore, the cells in which the expression of Ki-67 antigen was observed indicate that they are in the cell cycle. In FIG. 36A, many cell proliferation marker (anti-human Ki-67 antigen) positive cells were observed in human hepatocyte colonies formed in the TK-NOG mouse liver. In addition, cell proliferation marker positive cells were also observed in human hepatocyte colonies formed in uPA-NOG mouse liver (FIG. 36B).
  • MIB-1 cell proliferation marker
  • human normal hepatocytes include all cell populations in the liver. In both cases where these cells were transplanted as donor cells into TK-NOG and uPA-NOG mice, human hepatocyte colonies were formed in the liver. Furthermore, human-specific cell proliferation marker staining positive cells were observed in human hepatocyte colonies. This indicates that there are hepatocytes or hepatic progenitor cells and hepatic stem cells having the ability to divide and proliferate in donor cells. By using the mouse of the present invention, hepatic progenitor cells and hepatic cells in donor cells can be easily used. The presence of stem cells can be evaluated.
  • TK-Balb / c dKO mice HSV-Tk (+), SCID wild, RAG-2 KO, IL-2R null ) were obtained.
  • Generation of TK-NOD dKO mice TK-dKO mice are crosses between F1 generation mice obtained by mating TK-NOG mice and NOD dKO mice (RAG-2 KO, IL-2R null ) and NOD dKO mice. It was produced by repeating.
  • HSV-Tk, SCID, RAG-2, and IL-2R genes of the offspring mice are examined, and the genetic background is NOD.
  • TK-NOD dKO mice HSV- Tk (+), SCID wild, RAG-2 KO, IL-2R null ) were obtained.
  • the following sequences as primers for HSV-tk genotyping The following sequences as primers for scid genotyping: The following sequences as primers for RAG2 / IL-2R genotyping: The following sequences as primers for IL-2R genotyping: Was used in the PCR reaction according to the method used in the PCR described in Example 1. 3.
  • the mouse of the present invention is a mouse in which liver cells are replaced with human liver cells, and has the structure or function of human liver.
  • the mouse of the present invention can be used to analyze hepatitis virus infection specific to humans, metabolism of administered drugs, or proliferation of human liver.
  • the mouse transplanted with the human hepatocytes of the present invention can exhibit the function of the human liver, and can evaluate the ease of metabolism of the drug in the human liver.

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WO2013162064A1 (ja) * 2012-04-27 2013-10-31 公益財団法人東京都医学総合研究所 ウロキナーゼ型プラスミノーゲンアクチベータートランスジェニックマウス
JP2016082908A (ja) * 2014-10-24 2016-05-19 国立大学法人広島大学 急性重症肝炎モデル非ヒト動物の作製方法、急性重症肝炎モデル非ヒト動物、劇症肝炎治療薬のスクリーニング方法および劇症肝炎治療薬
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WO2013145331A1 (ja) * 2012-03-27 2013-10-03 株式会社トランスジェニック ヒト化マウス
CN104378975A (zh) * 2012-03-27 2015-02-25 转基因股份有限公司 人源化小鼠
JPWO2013145331A1 (ja) * 2012-03-27 2015-12-10 株式会社トランスジェニック ヒト化マウス
WO2013162064A1 (ja) * 2012-04-27 2013-10-31 公益財団法人東京都医学総合研究所 ウロキナーゼ型プラスミノーゲンアクチベータートランスジェニックマウス
JP2013230093A (ja) * 2012-04-27 2013-11-14 Tokyo Metropolitan Institute Of Medical Science ウロキナーゼ型プラスミノーゲンアクチベータートランスジェニックマウス
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JP2016082908A (ja) * 2014-10-24 2016-05-19 国立大学法人広島大学 急性重症肝炎モデル非ヒト動物の作製方法、急性重症肝炎モデル非ヒト動物、劇症肝炎治療薬のスクリーニング方法および劇症肝炎治療薬
WO2021132586A1 (ja) * 2019-12-27 2021-07-01 学校法人高崎健康福祉大学 肝細胞培養膜、それを備えた薬物輸送能評価キット、及び薬物輸送能評価方法
JPWO2021132586A1 (zh) * 2019-12-27 2021-07-01
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CN116103379A (zh) * 2023-01-18 2023-05-12 广东医科大学附属医院 一种动物体内人源细胞的检测方法及用途

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