WO2002011530A1 - Animal transgenique - Google Patents

Animal transgenique Download PDF

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Publication number
WO2002011530A1
WO2002011530A1 PCT/JP2001/006826 JP0106826W WO0211530A1 WO 2002011530 A1 WO2002011530 A1 WO 2002011530A1 JP 0106826 W JP0106826 W JP 0106826W WO 0211530 A1 WO0211530 A1 WO 0211530A1
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Prior art keywords
gene
mmp
extracellular matrix
rat
animal
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PCT/JP2001/006826
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English (en)
Japanese (ja)
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WO2002011530A9 (fr
Inventor
Koji Yoshimura
Atsushi Nishimura
Mayumi Nishida
Kazuhiro Hosono
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Takeda Chemical Industries, Ltd.
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Priority to AU2001277716A priority Critical patent/AU2001277716A1/en
Publication of WO2002011530A1 publication Critical patent/WO2002011530A1/fr
Publication of WO2002011530A9 publication Critical patent/WO2002011530A9/fr

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • 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)
    • 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
    • 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

Definitions

  • the present invention relates to an MMP-19 transgenic non-human mammal.
  • the extracellular matrix is a cell-supporting tissue surrounding cells of the tissue, and is composed of fibrous proteins such as collagen-elastin, glycoproteins such as proteodarican, fibronectin, laminin, and carbohydrates such as hyaluronic acid.
  • Extracellular matrices have a significant effect on cell activities such as cell morphology, metabolism, migration, proliferation, and differentiation, and are associated with many biological phenomena such as development, aging, inflammation, wound healing, immunity, and tumors. It is known. It is also known that extracellular matrix abnormal degradation occurs in various diseases such as rheumatoid arthritis, osteoarthritis, osteoporosis, metastasis and invasion of cancer, arteriosclerosis, and corneal ulcer.
  • MMP matrix metallo rote inase
  • MMP-19 (also called MMP-18 or rasi-1 but the same protein) is a protein consisting of 508 amino acids and, based on its structure, is a new subfamily that does not belong to the above subfamilies. It has been noted as forming MMP (J. Coss ins et al., Biochem. Biophys. Res. Commun. 228: 494-498, 1996, (described as MMP-18)), BA. M. Pendas et al., J. Biol. Clieni. 272: 4281-4286, 1997; I. Massova et al., FASEB J. 12: 1075-1095, 1998). However, only the relationship between MM P—19 physiological function and ovulation has been pointed out (A.
  • New transgenic animals have been thought to enable the development of new medicines that can help prevent and treat joint diseases such as rheumatoid arthritis and osteoarthritis.
  • transgenic non-human animal in which degradation of cartilage matrix observed in joint diseases such as rheumatoid arthritis and osteoarthritis is observed (hereinafter sometimes referred to as a transgenic animal) Therefore, development of a method for mass-producing a joint disease model animal was desired. Disclosure of the invention
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have produced a novel transgenic rat expressing MMP-19 under the control of the type II collagen promoter. Was found to degrade the extracellular matrix and exhibited a phenotype such as shortening of limbs.
  • the exogenous MMP-19 gene has the nucleotide sequence of SEQ ID NO: 12 The animal or part of the living body thereof described in (1) above,
  • Diseases caused by abnormal degradation of extracellular matrix include chondrodysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis, eye disease and The screening method according to the above (13), which is a malignant tumor, (16) A disease caused by abnormal degradation of extracellular matrix containing a substance determined to have a ameliorating effect on a disease caused by abnormal degradation of extracellular matrix by the method described in (13) above Prevention and treatment of medicines,
  • Diseases resulting from abnormal degradation of extracellular matrix include chondrodysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, eye disease and The method for preventing or treating malignant tumor according to the above (18),
  • DNA incorporating the exogenous MMP-19 gene or its mutant gene was introduced into female pseudopregnant rats mated with male rats.
  • FIG. 1 shows a schematic diagram of the plasmid pKS-MMPBcon-19 constructed in Example 2.
  • FIG. 2 shows the results of mRNA detection performed in Example 6.
  • Lanes 1 to 6 show wild-type rats used as controls, and lanes 7 to 12 show organs of transgenic rats.
  • Lanes 1 and 7 are the heart
  • lanes 2 and 8 are the lungs
  • lanes 3 and 9 are the liver
  • lanes 4 and 10 are the spleen
  • lanes 5 and 11 are the kidneys
  • lanes 6 and 12 are the Each shows cartilage.
  • FIG. 3 shows the results of analysis by the Southern hybridization method performed in Example 7. Lanes 1, 3 to 6 and 8 to 13 indicate heterozygous, and lanes 2 and 7 indicate homozygous.
  • the transgenic non-human animal of the present invention is preferably used for embryo development during non-human mammal development (more preferably, for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their primordial cells).
  • the gene transfer method such as calcium phosphate method, electric pulse method, lipofection method, agglutination method, microinjection method, particle gun method, DEAE-dextran method, etc. It is produced by introducing a desired exogenous MMP-19 gene or its mutant gene into a desired cell.
  • the target gene can be introduced into somatic cells, organs of living organisms, tissue cells, and the like by the gene transfer method, and can be used for cell culture, tissue culture, and the like.
  • a transgenic animal can also be produced by fusing the germ cell with a known cell fusion method.
  • a part of the living body of the transgenic animal produced in this manner (for example, 1) cells, tissues, organs, etc. having DNA incorporating the exogenous MMP-19 gene or its mutant gene; The derived cells or tissues are cultured and passaged as necessary. (3) Various proteins or DNA that can be isolated from the transgenic animal) can also be used as the "exogenous MMP-19 gene or DNA" of the present invention.
  • Non-human mammals having a DNA incorporating the sex MMP-19 gene or its mutant gene can be used for the same purpose.
  • the tissue that is a part of the living body of the transgenic animal is preferably a joint tissue or the like.
  • Cells that are part of the living body of the transgenic animal are preferably cells that constitute joint tissues.
  • Non-human mammals that can be the subject of the present invention include red sea lions, bushes, higgins, goats, blue herons, dogs, cats, guinea pigs, hamsters, rats, mice, and the like. Preferred are egrets, dogs, cats, guinea pigs, hamsters, mice or rats, particularly rodents (Rodentia), especially rats (Wistar, SD, etc.), especially Wistar strains. Birds are the most preferred target animals for disease model animals. In addition, birds and the like as bird animals can be used for the same purpose as the “non-human mammal” targeted in the present invention.
  • the exogenous MMP-19 gene to be introduced into the target non-human mammal includes, for example, mammals such as humans, pigs, higgs, goats, rabbits, dogs, cats, guinea pigs, hams, rats, mice, etc. MMP-19 gene derived from animals can be used.
  • the exogenous MMP-19 gene is a gene different from the endogenous gene of the animal into which the gene is introduced, and specifically, the MMP-19 gene isolated or purified from the mammal or the synthesized MMP-19 gene.
  • the exogenous MMP-19 gene is preferably an MMP-19 gene that does not have an intron.
  • mutant gene of the exogenous MMP-19 gene of the present invention those in which a mutation (for example, mutation, site-specific mutation, etc.) has occurred in the DNA of the present invention, specifically, addition of base, deletion, A gene in which substitution with another base or the like has occurred. More specifically, as a result of the addition, deletion, or substitution of another base, 1 to 5 (preferably 1 or 2) amino acids in the amino acid sequence constituting MMP-19 are obtained. It is preferable to carry out mutation so as to cause substitution, addition or deletion, and any mutation may be used as long as it does not lose the function of MMP-119.
  • a mutation for example, mutation, site-specific mutation, etc.
  • the exogenous MMP-19 gene includes, for example, the amino acid sequence represented by SEQ ID NO: 13.
  • a gene encoding a human MMP-19 having an amino acid sequence and having a base sequence represented by SEQ ID NO: 12 is used.
  • the exogenous MMP-19 gene or a mutant gene thereof may be the same or different from the non-human mammal to be introduced or expressed. It may be derived from any mammal.
  • the gene in introducing the gene into a target animal, it is generally advantageous to use the gene as a gene construct (eg, a vector, etc.) linked downstream of a promoter that can be expressed in the cells of the target animal. .
  • various mammals having the MMP- 19 gene highly homologous to the human MMP- 19 gene can be used as targets.
  • Microinjection into a fertilized egg of a non-human mammal eg, a fertilized rat egg
  • a transgenic non-human mammal that highly expresses the target human MMP-19 gene.
  • Examples of expression vectors for the MMP-19 gene include Escherichia coli-derived plasmids, Bacillus subtilis-derived plasmids, yeast-derived plasmids, pacteriophages such as ⁇ phage, retroviruses such as Moroni monoleukemia virus, vaccinia viruses, and baculoviruses. Animal viruses such as viruses are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used, and a plasmid derived from Escherichia coli is particularly preferred.
  • Examples of the promoter that regulates the gene expression of the MM P-19 gene include promoters of genes derived from viruses (such as cytomegalovirus, Moroni leukemia virus, JC virus, and breast cancer virus), and various mammals (human Genes derived from, egrets, dogs, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • viruses such as cytomegalovirus, Moroni leukemia virus, JC virus, and breast cancer virus
  • various mammals human Genes derived from, egrets, dogs, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • EF1-a polypeptide chain elongation factor 1
  • j8 actin and j8 myosin heavy chain
  • myosin light chain 1 and 2 Promoters such as myelin basic protein, serum amyloid P component, renin, etc., and preferably the Moroni leukemia virus promoter 1, human and chicken / 3-actin promoter which can be highly expressed in cartilage tissue. Etc. can be used.
  • the promoter region of the type II collagen gene which is known to be expressed in cartilage, is effective.
  • poly A generally referred to as Yuminata Mineta-1
  • Gene expression can be manipulated using the sequence of each derived gene.
  • the SV40 terminator of simian virus is used.
  • the splicing signal of each gene, the enhancer region, and a part of the intron of the eukaryotic gene 5 'upstream of the promoter overnight region, the promoter overnight region and the translation region
  • the translation region of MMP-19 is derived from the liver, kidney, fibroblasts, etc. of human and various non-human mammals (eg, heron, dog, cat, guinea pig, hamster, rat, mouse, etc.) and commercially available.
  • various non-human mammals eg, heron, dog, cat, guinea pig, hamster, rat, mouse, etc.
  • Using all or part of the genomic DNA derived from a variety of genomic DNA libraries as a raw material, or from RNA derived from liver, kidney, or fibroblasts of human or various non-human mammals Can be obtained using the complementary DNA prepared by the above as a raw material.
  • a translation region mutated by a point mutagenesis method or the like can also be prepared using the translation region of MMP-19 obtained from the above cells or tissues.
  • the above-mentioned translation region is located downstream of the above promoter (preferably at the transcription termination site) as a gene construct (eg, a vector, etc.) that can be expressed in an introduced animal.
  • DNA incorporating the MMP-19 gene can be produced by the usual genetic engineering technique of ligation to the upstream.
  • the presence of the MMP-19 gene in the germ cells of the transgenic animal after gene transfer indicates that the progeny of the transgenic animal has the MMP-19 gene in all of its germ cells and somatic cells Means to do.
  • the offspring of this type of animal that has inherited the gene carry the MMP-19 gene in all of its germinal and somatic cells.
  • the fertilized egg used when introducing the exogenous MMP-19 gene into a target non-human mammal (preferably a rat, particularly preferably a rat of the Wistar strain) or its ancestor fertilized egg is: Male non-human mammals of the same species (preferably male rats, etc., particularly preferably male rats of the Wistar strain) and female non-human mammals (preferably female rats, particularly preferably female rats of the Wistar strain) Are obtained by crossing.
  • Fertilized eggs can also be obtained by natural mating, but after artificially regulating the estrous cycle of female non-human mammals (preferably female rats, particularly preferably female rats of the Wistar strain), A method of crossing with a mammal (preferably a female rat, particularly preferably a female rat of the Wistar strain) is preferable.
  • female non-human mammals preferably female rats, particularly preferably female rats of the Wistar strain
  • a method of crossing with a mammal preferably a female rat, particularly preferably a female rat of the Wistar strain
  • Methods for artificially regulating the estrous cycle of female non-human mammals include, for example, follicle-stimulating hormone (pregnant horse serum gonadotropin, generally abbreviated as PMSG) first, and then luteinizing hormone (human villi) Gonadotropin (generally abbreviated as hCG) is preferably administered by, for example, intraperitoneal injection, but the preferred dose and administration interval of the hormone differ depending on the type of non-human mammal.
  • PMSG horse serum gonadotropin
  • hCG luteinizing hormone
  • Gonadotropin generally abbreviated as hCG
  • the rat be reared for about 1 week under a light condition of about 12 hours (for example, 7: 0 to 19:00) for 8 weeks or older.
  • Non-human mammals are female rats (preferred In the case of a female rat of the Wistar strain, it is usually preferable to administer luteinizing hormone about 48 hours after administration of follicle-stimulating hormone and to mate with male rats to obtain fertilized eggs.
  • the dose of the intense hormone is about 20 to about 50 IU / individual, preferably about 30 IUZ individual, and the dose of luteinizing hormone is about 0 to about 10 IU / individual, preferably about 5 IU / individual. It is an individual.
  • the exogenous MMP-19 gene After the exogenous MMP-19 gene has been introduced into the obtained fertilized egg by the method described above, it is artificially transplanted and implanted into a female non-human mammal, and has a DNA incorporating the exogenous gene. A non-human mammal is obtained.
  • L HRH luteinizing hormone-releasing hormone
  • LHRH or an analog thereof and the timing of mating with a male non-human mammal after its administration differ depending on the type of non-human mammal.
  • the non-human mammal is a female rat (preferably a female rat of the Wistar strain)
  • L HRH or an analog thereof eg, [3,5-DiI-Tyr 5 ] -LH-RH, [Gin 8 ] -LH-RH [D-Ala 6 ]-LH-RH, [des-Gly 1 °]-LH-RH, [D-His (Bzl) 6 ] -LH-RH and their Ethy lamide
  • the dose of L HRH or its analog is usually about 10 to 60 igZ individuals, preferably about 40 g / individual. It is.
  • a method for obtaining a fertilized egg by artificially adjusting the estrous cycle of the female non-human mammal and a method for obtaining the fertilized egg by pseudo-pregnant female non-human mammal in which fertility has been induced are described. It is preferable to use the method in combination with a method of artificially implanting and implanting.
  • the non-human mammal into which the MMP-19 gene of the present invention has been introduced (hereinafter sometimes abbreviated as the non-human mammal of the present invention) has cartilage destruction and has developed joint disease.
  • the non-human mammal of the present invention causes abnormal degradation of the extracellular matrix, and a disease caused by abnormal degradation of the extracellular matrix (eg, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, Rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye disease, malignant tumor, etc.).
  • non-human mammals of the present invention include: 1) shortening and deformation of the limbs, 2) deformation of the skull, 3) malocclusion, 4) excessive maxillary and mandibular incisors, 4) insufficient calcification of the lumbar spine, 4) deformation of the tail vertebra, and And symptoms selected from caudal intervertebral disc defects.
  • non-human mammal of the present invention has the above-mentioned very unique features, it has the following useful uses.
  • the exogenous MMP-19 gene is highly expressed, and cartilage destruction is caused by the enzymatic activity of this MMP-19 gene.
  • Characterized various diseases such as cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye disease, malignant tumor, etc. It can be used to evaluate prophylactic or therapeutic agents for diseases caused by abnormal degradation.
  • the present invention is characterized in that a test substance is applied to the non-human mammal of the present invention or a part of the living body thereof, and the effect of ameliorating a disease caused by abnormal degradation of the extracellular matrix is assayed.
  • Prevention of diseases caused by abnormal degradation of extracellular matrix • Provide a method for screening substances used for treatment.
  • Abnormal degradation of the extracellular matrix includes that caused by an abnormal function of MMP-19.
  • test substance is administered to the non-human mammal of the present invention.
  • the test substance may be a known synthetic compound, peptide, protein, DNA library, or the like, or, for example, a warm-blooded mammal (eg, mouse, rat, pig, pig, sheep, monkey, monkey, etc.). ) Tissue extract and cell culture supernatant are used.
  • the cartilage degradation inhibitor can be evaluated by examining the drug evaluation or therapeutic effect using urinary and blood pyridinoline levels as indicators.
  • Various antigens eg, substrates such as type II collagen and aggrecan
  • bacteria eg, killed Mycobacterium tuberculosis
  • Etc. or a virus (eg, Epstein-Barr virus etc.) is administered systemically or locally, such as joints, once, multiple times or continuously to promote the above disease. These are known to cause rheumatoid arthritis in model animals.
  • the onset can be promoted by administering to the joints cytokines such as IL_1 and TNF ⁇ that induce inflammation and express protease activity. Since the absolute value of the above index is larger in non-human mammals in which the onset is promoted than in non-human mammals in which the onset is not transduced, as well as in non-human mammals in which the onset is not promoted, By performing the evaluation described in (1) using animals, it is possible to evaluate more accurately.
  • cartilage fragments or chondrocytes collected from the non-human mammal of the present invention can be cultured and used for evaluating MMP-19 inhibitors.
  • inhibitors can be evaluated by using these cartilage fragments or chondrocytes as a material for the ⁇ -l9 enzyme and adding a synthetic peptide as a substrate. If necessary, it may be activated with a serine protease such as a mercury compound or trypsin.
  • ⁇ ⁇ —19 promotes cartilage destruction, and by using these evaluation systems, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolism Can be used to evaluate prophylactic and therapeutic agents for osteoarthritis, eye diseases and malignant tumors
  • the non-human mammal of the present invention is pregnant, and the pregnant animal is treated with cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic It can be used to evaluate prophylactic or therapeutic agents for arthropathy, eye diseases and malignancies, and their complications.
  • a test substance is administered to a pregnant non-human mammal of the present invention.
  • the weight, body length and limb length of the offspring after childbirth, morphometry by X-ray photography, various MMPs, TIMP, proteodalican, collagen, keratan sulfate, pyridinoline, hyaluronic acid, osteocalcin, calcium, phosphorus, site Investigate the drug evaluation or therapeutic effect using the biochemistry of cartilage and bone metabolism in blood or urine such as force-in and growth factors as indicators. It is also useful to prepare a pathological specimen as necessary for the experiment, and to examine the degree of cartilage destruction by a method such as safranin O staining.
  • the offspring obtained after mating with non-human mammals were diseases caused by the products (proteins) of these genes as well as MMP-19 (eg, cartilage dysplasia, bone dysplasia, osteoporosis, deformity) Osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye disease and malignancy). Therefore, the offspring can be used for the evaluation of a prophylactic / therapeutic agent for such a disease by a method similar to (1) to (4) or a method analogous thereto.
  • MMPs eg, MMP-1, 8, 13 etc.
  • the test substance when it is determined that administration of a test substance has an effect of ameliorating a disease caused by abnormal degradation of the extracellular matrix, the test substance is extracellular as described above. Prevention of diseases caused by abnormal matrix degradation. Can be selected as a therapeutic drug.
  • Substances selected as the above preventive and therapeutic drugs include, for example, sugar-coated tablets, capsules, elixirs, microcapsules, etc., as needed, orally, or water or other pharmaceutical agents. It can be used parenterally in the form of injections, such as sterile solutions with acceptable solutions, or suspensions.
  • a substance selected as a prophylactic / therapeutic drug may be required to implement a generally accepted formulation with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. Can be prepared by mixing them in unit dosage form. The amount of the active ingredient in such preparations is to be adjusted so that an appropriate dose in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • Solubilizers for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), non-ionic surfactants (eg, Polysorbate 80 TM, HC-50, etc.) ) May be used together.
  • the oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • a buffer eg, phosphate buffer, sodium acetate buffer, etc.
  • a soothing agent eg, benzalkonium chloride, proforce hydrochloride, etc.
  • a stabilizer eg, human serum albumin, polyethylene glycol, etc.
  • a preservative eg, benzyl alcohol, phenol, etc.
  • an antioxidant eg, an antioxidant, and the like may be blended.
  • the prepared injection is usually filled in an appropriate ampoule.
  • the DNA is used alone or after being introduced into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. It can be administered to humans or warm-blooded animals according to conventional means.
  • the DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and can be administered using a gene gun or a catheter such as a hydrogel catheter.
  • the preparations obtained in this way are safe and have low toxicity, for example, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, higgies, bush, horses, cats, cats). , Dogs, monkeys, etc.).
  • the dose of a substance selected as a prophylactic or therapeutic drug may vary depending on the target disease, the subject of administration, the route of administration, and the like. per kg) of the substance is administered per day in an amount of about 0.1 mg to: 00 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose of the substance may vary depending on the subject of administration and the target disease.For example, when administered to an adult (with a body weight of 60 kg) in the form of an injection for the treatment of arthritis It is convenient to administer the substance by injecting about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day into the affected area. . In the case of other animals, the dose can be administered in terms of 60 kg.
  • the non-human mammal of the present invention can be used in an experiment for gene therapy of a patient with an MMP-19 gene abnormality.
  • the above-described transgenic mammal of the present invention can also be used as a cell source for tissue culture.
  • tissue culture For example, by directly analyzing the DNA or RNA in the tissue of the transgenic rat of the present invention or by analyzing the protein expressed by the gene, it is possible to analyze the complex action of the nuclear receptor and the transcription factor. Sex can also be analyzed.
  • culturing cells of a gene-bearing tissue by standard tissue culture techniques and using them to study the function of cells derived from tissues that are generally difficult to culture, such as cells forming cartilage tissue. Can also.
  • the cells for example, it is possible to select a drug that enhances the function of the cells.
  • if there is a high-expressing cell line it is possible to isolate and purify MMP-19 in a large amount and to produce an antibody thereof.
  • sequence numbers in the sequence listing of the present invention indicate the following sequences.
  • [SEQ ID NO: 1] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
  • [SEQ ID NO: 2] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
  • SEQ ID NO: 3 This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
  • SEQ ID NO: 4 This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
  • SEQ ID NO: 5 This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 4 described later.
  • SEQ ID NO: 6 This shows the base sequence of the primer used for PCR (polymerase chain reaction) method performed in Example 4 described later.
  • SEQ ID NO: 7 This shows the base sequence of the primer used for PCR (polymerase chain reaction) method performed in Example 6 described later.
  • SEQ ID NO: 8 This shows the base sequence of the primer used in the PCR (polymerase zetiein reaction) method performed in Example 6 described later.
  • SEQ ID NO: 10 This shows the base sequence of the enhancer region of rat type I collagen gene cloned in Example 1 described later.
  • SEQ ID NO: 11 This shows the base sequence of the DNA fragment containing the splicing site derived from pTB399 used for the construction of pKS-MMPBcon-19 in Example 2 described later.
  • SEQ ID NO: 13 This shows the amino acid sequence of human MMP-19.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the relevant field, and examples thereof are as follows.
  • DNA Deoxylipo nucleic acid
  • RNA Liponucleic acid A adenine
  • Example 1 Cloning of rat type I collagen gene promoter and enhancer
  • the promoter region of the rat type II collagen gene has primers (5, -GTGGTGGTGGAC AACTAGGAAACTCTGG-3 ': SEQ ID NO: 1) designed based on the nucleotide sequence of Kolmo et al. (J. Biol. Chem. 260: 4441, 1985). And (5′-CGAGGCGMTCATGGCTCACCGCG-3 ′: SEQ ID NO: 2) by PCR.
  • the obtained fragment of about 1.2 Kb was cloned into pCRII-TOP0 (referred to as pCRII-promoter2) using a TOPO TA Cloning Kit (manufactured by Invitrogen) according to the attached protocol.
  • the nucleotide sequence of the inserted DNA fragment was confirmed by a conventional method using a DNA sequencer manufactured by ABI, and was found to match the nucleotide sequence of the promoter region of the aforementioned literature (SEQ ID NO: 9).
  • NotI site (5 'side) of the multiple cloning site of pCRII plasmid in pCRI I-Promoter 2 and pCRI I-p A fragment consisting of the SmaI site in the type II collagen gene promoter sequence in the promoter 2 was used in the following experiments.
  • the rat type II enhancer region is derived from a primer (5'-TCCACGCGTTTGGGAAACTTCTTGGCTGCG-) designed to generate an M1uI site based on the nucleotide sequence of Krebsbadi et al. (J. Biol. Chem. 3 ′: SEQ ID NO: 3) and (5′-GCTTCGKGC CGCTACGCGTGGGGCCGGA-3 ′: SEQ ID NO: 4) were obtained by a PCR method. An Eco RI linker was added to the obtained 0.35 Kb M1uI fragment by a conventional method, and inserted into the EcoRI site of pBluescript KSII + (hereinafter referred to as pKS-enhancer 1-4). Call).
  • An expression vector for producing transgenic rats, pKS-MMPBc0n-19, was constructed according to a conventional method.
  • Co 12 A 1 promoter promoter region of rat type II collagen gene, from Not I site in pCRI I multiple cloning site in pCRII-promoter 2 described in Example 1 A 1120 bp fragment in the type II collagen gene promoter in pCR II-promoter 2 up to the Sma I site (the Sma I site was converted to a S a I site by a conventional linker ligation).
  • MMP-19 Approximately 1600 b from the SacI site to the XhoI site in the MMP-19 cDNA of pTB1921 (JP-A-10-080283). P gene fragment. (Sac I site was converted to C 1 a I site and X ho I site was converted to Bg 1 II site by linker ligation).
  • S V40 polyA derived from pTB399 (R. Sasada et al., Cell Structure and Function 12: 205, 1987)! ) Fragment from the Bg1II site to the HindIII site containing the o1yA additional signal.
  • Co 12A1 enhanc er a fragment from Hind II site to Not I site of pKS-enhancer_4 containing the enhancer region of rat type I collagen gene described in Example 1.
  • pKS-MMPBcon-19 was transformed into Escherichia coli JM109 to obtain Escherichia coli JM109 / pKS-MMPBcon-19.
  • Example 3 Creation of transgenic rat
  • the rat SD strain was purchased at 8 weeks of age for egg collection and reared for 1 week at 7:00 to 19:00 12 hours under light conditions. First day, at 11:00, follicle stimulating hormone (pregnant horse serum gonad stimulation) Hormone, PMSG) (30 I UZ individual) was injected intraperitoneally, and at 11:00 on the third day, luteinizing hormone (human chorionic gonadotropin, hCG) (5 IU / individual) was injected intraperitoneally. The strain SD strain lived and bred with individuals after 10 weeks of age at 1: 1 at 15: 0. On the 4th day, the female rats bred at 9:00 were checked for vaginal plugs.
  • follicle stimulating hormone pregnant horse serum gonad stimulation Hormone, PMSG
  • luteinizing hormone human chorionic gonadotropin, hCG
  • Example 2 A pronucleus-forming egg was selected from the fertilized eggs, and the plasmid pKS— MMPB con_19 obtained in Example 2 was cut at 14:30 with Not I, and human MMP prepared to a concentration of 10 g / m1 was prepared.
  • the DNA fragment 1-2 ⁇ 1 containing 19 genes was injected into the male pronucleus of fertilized eggs of the SD strain rat at the single cell stage while observing under a microscope.
  • the egg cells were cultured in a known HER medium (HAM-F12 powder medium (Dainippon Pharmaceutical) 3.180 g, RPMI-1640 powder medium (Dainippon Pharmaceutical) 1.040 g, MEM Eag 1e powder medium Ground (Dainippon Pharmaceutical) 0.95 g, NaHCO s (Wako Pure Chemical) 0.780 g, Penicillin-G (GIBCO BRL) 50000 U and Streptomycin (GIBCO BRL) 5000 0U were dissolved in 500 ml of distilled water At 5:30 pm on the 5th day, confirming the two-cell stage embryos, and then culturing them in Wagner II (Proc. Natl. Acad. Sci. USA 78: 5016, 1981). In accordance with the method described above, it was transplanted into the oviduct of a pseudopregnant female Wistar rat and implanted.
  • Genomic DNA of these two PCR-positive individuals was analyzed by Southern hybridization. That is, 10/1 g of DNA was completely digested with BamHI, electrophoresed on a 2.0% agarose gel, and then transferred to a nylon filter.
  • a DIG fluorescence detection kit manufactured by Roche Diagnostics
  • Example 5 Transgenic rat obtained from heterozygous rat When the B6 OF (first generation (F 0 )) obtained in Example 4 reached the age of 12 weeks, it was bred with the SD line rat and the second generation (F was obtained. At the age of age, heterozygotes were selected by PCR using the method described in Example 4 and used for Example 6.
  • Example 6 Confirmation of human mRNA in cartilage of transgenic rats
  • the transgenic rat obtained in Example 5 was homogenized in ISOGEN (manufactured by Futtsubon Gene) to extract total RNA by a conventional method.
  • ISOGEN manufactured by Futtsubon Gene
  • cDNA was synthesized according to the protocol and used as a template for RT-PCR.
  • the rat and human MMP-19 mRNAs were As a negative control, an individual determined to be a wild type by PCR performed in Example 5 was used. The 538 bp fragment amplified by RT-PCR was incorporated into a human fragment. Completely digested with ApaL I In addition, since the 347 bp and 191 bp fragments were detected, it was confirmed that human MMP-19 mRNA was expressed in the cartilage of the transgenic rat. 19 mRNA was found in heart, lung, liver, spleen, kidney and cartilage ( Figure 2) Example 7 Homozygous Transgenic Rats
  • Example 7 The weight of each of the wild-type, heterozygous and homozygous individuals (B6OF strain, 12-week-old, male) obtained in Example 7 was measured. Furthermore, they found that homozygotes weigh less than heterozygotes.
  • the individual used for weight measurement was anesthetized with Nembutal, and the lengths of forelimbs, hindlimbs and skull were measured with calipers. Heterozygotes did not differ in forelimb, hindlimb, and skull lengths compared to wild-type. Homozygote was found to have shorter forelimbs and hindlimbs than wild-type and heterozygote. It was also found that the skull was flattened in the long axis direction and expanded in the direction perpendicular to the long axis. Table 1 shows the results.
  • the promoter of the integrated gene uses the type I collagen promoter, it was expected that the effect of the transgene (the integrated gene) would be seen at the site where the type I collagen was expressed.
  • the pathology of the right knee joint of the transgenic rat (B6OF strain) was examined and compared between wild type, heterozygous and homozygous.
  • the animals were perfused and fixed from the heart with 4% paraformaldehyde under ether anesthesia, and then the right knee joint was removed. After resection of the muscle tissue and connective tissue attached to the periphery, the joint was divided into two parts in the median direction using a diamond cutter. Each joint was further fixed in 4% paraformaldehyde for 14 hours at room temperature. After the fixation, the tissue pieces were washed with distilled water, and the knee joint was decalcified with 20% EDTA (pH 7.4) for 8 days. Decalcification liquid exchange was performed every day.
  • the 20% EDTA attached to the knee joint was washed with deionized water, and 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol 4 times, and xylene 2 times Each was immersed in order for 1 to 2 hours, followed by dehydration and clearing. Next, it was immersed twice in paraffin at about 60 ° C for 2 hours each to produce a paraffin block based on a tissue force set. The block was cooled rapidly in the refrigerator overnight, and was completely hardened. Slicing was performed by leaving the paraffin block at room temperature and returning it to room temperature, and then using a microtome at 5. The sections were mounted on a slide glass coated with a mass coat while warm bathing.
  • the prepared sections were left overnight in a dryer heated to 37 ° C to dry completely.
  • the following references were used for in- and general staining (hematoxylin eosin double staining method). (Histology Research Method, Yutaka Sano, Nanzando, 1985).
  • staining of sulfated glycosaminoglycan the sections were deparaffinized, dehydrated, washed with hematoxylin for 2 minutes and running water, then washed with a 2% aqueous solution of 1% dextrin for 5 minutes, and immersed in a 1% aqueous solution of acetic acid for 0.1 minutes. Go to Safranin ⁇ ⁇ aqueous solution for 10 minutes.
  • the sections were dehydrated by putting in and out 95% ethanol 100% ethanol three times in each step five times, and finally immersing the sections in 100% ethanol for 5 minutes.
  • the transparency and encapsulation were performed with reference to the following literature. (Histology Research Method, Yutaka Sano, Nanzando, 1985).
  • the sections after staining were examined microscopically in a bright field using an optical microscope. No abnormalities were found in the articular cartilage and growth plate of the femur and tibia of the heterozygote compared to the wild type.
  • the articular cartilage of the femur and tibia of the homozygote had sites where the articular cartilage layer was thinner and thicker than in the wild type, and where the surface layer was replaced by fibrous articular cartilage tissue.
  • the growth plate was found to be ruptured by homozygote.
  • the surface layer of the articular cartilage layer was replaced with fibrous articular cartilage tissue
  • lack of staining at the surface layer indicated the lack of sulfated glycosaminodalican.
  • the direction of the original chondrocyte growth direction was not constant, indicating that normal cartilage differentiation did not occur.
  • Fetuses were double stained with osteochondral cartilage using the Inoue method and the Kimmel method (see Histology Research, Yutaka Sano, Nanzando, 1985) with reference to the following.
  • New transgenic rat (strain name: MMP-B60F) homozygote, shortening of limbs, cranial deformity and caudal vertebra deformity found in adult search were found to occur from embryonic stage . Delayed calcification was observed in the posterior lumbar spine and hind limbs. The incidence of abnormalities in the stomach was about 50% in adults, but in the fetal period, any abnormalities were observed in all cases. From these results, it was inferred that the skeletal abnormalities occurring in the homozygote of the new transgenic rat (strain name: MMP-B60F) were congenital in the fetal period.
  • New transgenic rat (strain name: MMP-B60F) homozygote, skeletal abnormalities are known to occur in adults. There is a thinned part in the articular cartilage of this rat. Whether or not cartilage differentiation was normally occurring at this site was analyzed by examining the expression of various transcription factors by immunostaining.
  • a novel transgenic rat (strain name: MMP-B60F) homozygote, 12-week-old male, section of articular cartilage at the distal end of the femur was prepared in the same manner as in Example 8.
  • the prepared sections were subjected to immunostaining using the following antibodies.
  • FGFR3 Fibroblast Growth Factor Receptor 3
  • IHH Indian hedgehog
  • PTC Patched
  • SMO Smoot end
  • the transgenic non-human mammal of the present invention includes various diseases characterized by cartilage destruction, for example, prophylactic or therapeutic agents for osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis. It can be used for evaluation, experiments for gene therapy of patients with MMP-19 gene abnormality, etc., and culturing cartilage fragments or chondrocytes collected from the transgenic non-human mammal of the present invention. 9 Can be used to evaluate inhibitors.

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Abstract

Cette invention concerne un mammifère non humain transgénique pouvant être utilisé comme cobaye à des fins curatives ou préventives pour des défaillances chondrogènes, des défaillances ostéogénétiques, l'ostéoporose, l'arthrite déformante, la polyarthrite rhumatoïde, l'arthrite, les maladies ophtalmiques, la synovite, les tumeurs malignes et les complications de ces pathologies, et qui permet donc de clarifier les mécanismes pathologique de ces affections, d'étudier des méthodes de traitement pour ces maladies et de procéder à une recherche systématique de remèdes.
PCT/JP2001/006826 2000-08-09 2001-08-08 Animal transgenique WO2002011530A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040157A1 (fr) * 1996-04-25 1997-10-30 Takeda Chemical Industries, Ltd. Metalloprotease matricielle
JPH10309149A (ja) * 1997-03-10 1998-11-24 Takeda Chem Ind Ltd 単球走化性蛋白質類およびその受容体トランスジェニック動物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040157A1 (fr) * 1996-04-25 1997-10-30 Takeda Chemical Industries, Ltd. Metalloprotease matricielle
JPH10309149A (ja) * 1997-03-10 1998-11-24 Takeda Chem Ind Ltd 単球走化性蛋白質類およびその受容体トランスジェニック動物

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
KOHNO ET AL.: "Structure of the promoter of the rat type II procollagen gene", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, no. 7, 1985, pages 4441 - 4447, XP002949721 *
KOLB ET AL.: "The matrix metalloproteinase RASI-1 is expressed in synovial blood vessels of a rheumatoid arthritis patient", IMMUNOLOGY LETTERS, vol. 57, 1997, pages 83 - 88, XP002949728 *
KREBSBACH ET AL.: "Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 8, 1996, pages 4298 - 4303, XP002949722 *
MASSOVA ET AL.: "Matrix metalloproteinases: structures, evolution and diversification", THE FASEB JOURNAL, vol. 12, 1998, pages 1075 - 1095, XP002949725 *
MUELLER ET AL.: "Structure of the human MMP-19 gene", GENE, vol. 252, 11 July 2000 (2000-07-11), pages 27 - 37, XP002949723 *
PENDAS ET AL.: "Identification and characterization of a novel human matrix metalloproteinase with unique structural characteristics, chromosomal location and tissue distribution", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 7, 1997, pages 4281 - 4286, XP002949724 *
STRACKE ET AL.: "Biochemical characterization of the catalytic domain of human matrix metalloproteinase 19", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 20, 19 May 2000 (2000-05-19), pages 14809 - 14816, XP002949726 *
STRACKE ET AL.: "Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matric protein (COMP)", FEBS LETTERS, vol. 478, 2 July 2000 (2000-07-02), pages 52 - 56, XP002949727 *

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