WO2002011530A9 - Animal transgenique - Google Patents

Animal transgenique

Info

Publication number
WO2002011530A9
WO2002011530A9 PCT/JP2001/006826 JP0106826W WO0211530A9 WO 2002011530 A9 WO2002011530 A9 WO 2002011530A9 JP 0106826 W JP0106826 W JP 0106826W WO 0211530 A9 WO0211530 A9 WO 0211530A9
Authority
WO
WIPO (PCT)
Prior art keywords
gene
mmp
rat
extracellular matrix
animal
Prior art date
Application number
PCT/JP2001/006826
Other languages
English (en)
Japanese (ja)
Other versions
WO2002011530A1 (fr
Inventor
Koji Yoshimura
Atsushi Nishimura
Mayumi Nishida
Kazuhiro Hosono
Original Assignee
Takeda Chemical Industries Ltd
Koji Yoshimura
Atsushi Nishimura
Mayumi Nishida
Kazuhiro Hosono
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Chemical Industries Ltd, Koji Yoshimura, Atsushi Nishimura, Mayumi Nishida, Kazuhiro Hosono filed Critical Takeda Chemical Industries Ltd
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 abnormal degradation of extracellular matrix occurs in various diseases such as rheumatoid arthritis, osteoarthritis, osteoporosis, cancer metastasis, invasion, arteriosclerosis, and corneal ulcer.
  • MMP matrix metalloproteinase
  • Human MMP-19 (also called MMP-18 or rasi-1 but the same protein) is a 508 amino acid protein that forms a new subfamily that does not belong to the above subfamily because of its structure. Attention has been drawn (J. Cossins et al., Biochem. Biophys. Res. Co. thigh un. 228: 494-498, 1996, (denoted as MMP-18), BA. M. Pendas et al. , J. Biol. Cheni. 272: 4281-4286, 1997, I. Massova et al., FASEB J. 12: 1075-1095, 1998). However, only the relationship between ovulation and the physiological function of MMP-19 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 animals in which degradation of cartilage matrix observed in joint diseases such as rheumatoid arthritis and osteoarthritis are observed (hereinafter sometimes referred to as transgenic animals) ), And the development of a method for mass-producing animal models of joint disease was desired. Disclosure of the invention
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have produced a novel transgenic rat expressing MMP-19 under the control of type II collagen promoter overnight. As a result, they found that the extracellular matrix of cartilage was degraded and exhibited a phenotype such as shortening of limbs.
  • the exogenous MMP-19 gene has the nucleotide sequence represented by SEQ ID NO: 12.
  • test substance is applied to the animal or a part of the organism described in any of (1) to (6) above, and the effect of ameliorating a disease caused by abnormal degradation of extracellular matrix is assayed. Screening methods for substances used for the prevention and treatment of diseases caused by abnormal degradation of extracellular matrix,
  • 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 an improving 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 containing the exogenous MMP-19 gene or its mutant gene was introduced into female pseudopregnant rats mated with male rats after administration of luteinizing hormone-releasing hormone or its analogs.
  • 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, 8 to 13 indicate heterozygous, and lanes 2 and 7 indicate homozygous. BEST MODE FOR CARRYING OUT THE INVENTION
  • 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).
  • gene transfer methods such as the calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc. It is created by introducing the desired exogenous MMP-19 gene or its mutant gene into the target 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.
  • a part of the living body of a non-human mammal having a DNA incorporating the mutant gene The non-human mammal having 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 targeted in the present invention include porcupines, pigs, sheep, sheep, goats, puppies, dogs, cats, guinea pigs, hampus, rats, mice, and the like. Preferred are egrets, dogs, cats, cats, guinea pigs, hamsters, mice or rats, among which rodents (Rodentia) are preferred, especially rats (Wistar, SD, etc.), especially those of the Wistar strain. Rats 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.
  • exogenous MMP-19 gene to be introduced into the target non-human mammal examples include, for example, human, bush, higgin, goat, rabbit, dog, cat, guinea pig, hamus, rat, mouse
  • a mammalian MMP-19 gene can be used.
  • the exogenous MMP-19 gene is a gene different from the endogenous gene of the animal into which the gene is to be introduced, and specifically, the MMP-19 gene isolated or purified from the mammal or a synthetic MMP-19 gene.
  • the MM P-19 gene is used.
  • 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 a mutation (for example, mutation, site-specific mutation, etc.) in the DNA of the present invention, specifically, the addition of a base Or a gene in which a deletion, 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 are contained in the amino acid sequence constituting MMP-19. It is preferable to mutate so that substitution, addition or deletion occurs in the acid, and any mutation may be used as long as it does not lose the function of MMP-19.
  • the exogenous MMP-19 gene includes, for example, the amino acid sequence represented by SEQ ID NO: 13.
  • a gene encoding the human MMP-19 having the amino acid sequence and having the base sequence represented by SEQ ID NO: 12 is used.
  • the exogenous MMP-19 gene or a mutant gene thereof in the present invention may be of the same or different species as the non-human mammal to be introduced or expressed. It may be derived from any mammal.
  • the gene When 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.
  • a gene construct eg, a vector, etc.
  • various mammals having the MMP-l 19 gene having high homology to the human MMP-l9 gene Hamsters, rats, mice, etc.
  • a vector obtained by ligating the gene downstream of various promoters capable of expressing the human MMP119 gene preferably rats, etc.
  • Microinjection into a fertilized egg of a non-human mammal can produce a transgenic non-human mammal that highly expresses the desired human MMP-19 gene.
  • Examples of the expression vector for the MMP-19 gene include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, pateriophage such as ⁇ phage, retroviruses such as Moroni leukemia virus, vaccinia virus or Animal viruses such as baculovirus are used.
  • 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 MMP-19 gene include, for example, promoters of genes derived from viruses (cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, etc.), various mammals (human, Genes from egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • viruses cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, etc.
  • various mammals human, Genes from egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • AMP-dependent protein kinase 3 I subunit, atrial natriuretic factor, dopamine
  • a Kuching promoter or the like can be used.
  • the promoter region of the type II collagen gene which is known to be expressed in cartilage, is effective.
  • the transgenic mammal has a sequence that terminates the transcription of the target messenger RNA (poly A, generally referred to as “Yuichi Mineta”).
  • poly A generally referred to as “Yuichi Mineta”.
  • it is derived from viruses, various mammals and birds Gene expression can be manipulated using the sequence of each gene.
  • the simian virus SV40 and the like are used.
  • the splicing signal of each gene, the enhancer region, and a part of the intron of the eukaryotic gene are transferred 5 'upstream of the promoter region, between the promoter region and the translation region. Alternatively, it may be linked to the 3 'downstream of the translation region depending on the purpose.
  • the translation region of MMP-19 is derived from DNA derived from the liver, kidney, fibroblasts, etc. of humans and various non-human mammals (eg, herons, dogs, cats, guinea pigs, hamsters, rats, mice, etc.).
  • a known method using all or part of the genomic DNA derived from various commercially available genomic DNA libraries as a raw material, or from RNA derived from liver, kidney, and fibroblasts of human and 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. These are all materials that can be used for transgenic animals.
  • 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 to introduce the exogenous MMP-19 gene into a target non-human mammal preferably a rat or the like, particularly preferably a Wistar strain rat or the like
  • a fertilized egg of its ancestor 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 mammals (preferably female rats, particularly preferably female rats of the Wistar strain) is preferred.
  • 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 chorionic).
  • Gonadotropin generally abbreviated as hCG
  • hCG is preferably administered by, for example, intraperitoneal injection, but the preferred hormone dose and administration interval vary depending on the type of non-human mammal.
  • Non-human mammals are female rats (preferred In the case of female rats of the Wistar strain, it is usually preferable to administer luteinizing hormone about 48 hours after administration of follicle-stimulating hormone and obtain fertilized eggs by mating with male rats.
  • the dose of the hormone is about 20 to about 50 IU / person, preferably about 30 IUZ individuals, and the dose of luteinizing hormone is about 0 to about 10 IUZ individuals, preferably about 5 IUZ individuals. It is.
  • DNA is implanted and implanted artificially into a female non-human mammal, and DNA incorporating the exogenous gene is obtained.
  • a non-human mammal having the above is obtained.
  • L HRH luteinizing hormone-releasing hormone
  • the dose of 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)
  • 111111 is an analog thereof (eg, [3, 5-011]-11-1 ⁇ ).
  • the dose of LHRH or an analog thereof is usually about 10 to 60 individuals, preferably about 40 gZ. It is an individual.
  • a method for artificially regulating the estrous cycle of a female non-human mammal to obtain a fertilized egg and a method for artificially regulating the sexual cycle of the female non-human mammal It is preferable to use the method in combination with the method of transplantation and implantation.
  • 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 diseases, malignant tumors, 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.
  • Characteristic various diseases for example, extracellular matrices such as cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye diseases, malignant tumors It can be used to evaluate prophylactic or therapeutic agents for diseases caused by abnormal degradation of Rix.
  • 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, bush, horsetail, hidge, monkey, human, etc.) Tissue extract, cell culture supernatant, etc. 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.
  • MMP tissue Inhibitor of Met alloproteinase
  • proteodalican collagen
  • keratan Sulfuric acid hyaluronic acid
  • osteocalcin calcium
  • phosphorus cytodynamics
  • blood growth factors etc.
  • drug evaluation or therapeutic effect can be examined using urinary cartilage and bone metabolism biochemical markers as indices. It is also useful to examine the degree of cartilage destruction by methods such as preparation of pathological specimens and staining with safranin O if necessary for experiments.
  • Various antigens eg, substrates such as type II collagen and aggrecan
  • bacteria eg, killed Mycobacterium tuberculosis, etc.
  • the virus eg, Epstein-Barr virus, etc.
  • Epstein-Barr virus is promoted by local, single, multiple or continuous administration of the virus systemically or jointly.
  • MMPs serine proteases such as trypsin, daltathione, active oxygen, nitrogen oxides such as N ⁇ , mercury compounds (eg, 4-aminophenylmercuric acetate, etc.), inflammation
  • the onset can be promoted by administering to the joints cytokines such as IL-1 and TN Fa that induce protease and express protease activity.
  • Non-human mammals in which the onset is promoted are not only non-human mammals in which the gene has not been introduced, but also have a greater absolute value of the above index than non-human mammals in which the onset has not been promoted. By performing the evaluation described in (1) using human animals, the evaluation can be performed with higher accuracy.
  • Cartilage fragments or chondrocytes collected from the non-human mammal of the present invention can be cultured and used for evaluation of MMP-19 inhibitors. Specifically, inhibitors can be evaluated by using these cartilage fragments or chondrocytes as a material for the MMP-19 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.
  • a serine protease such as a mercury compound or trypsin.
  • 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 non-human mammal of the present invention can be transformed with genes such as MMPs (eg, MMP-1, 8, 13 etc.), which are known to degrade cartilage collagen, and aggrecanase, which degrades aggrecan. Introduced non-human mammals are mated and the offspring obtained are diseases caused by the products (proteins) of these genes as well as MMP-19 (eg, cartilage dysplasia, bone dysplasia, osteoporosis, It is expected to develop osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye diseases and malignant tumors. 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. It can be selected as a drug for prevention and treatment of diseases caused by abnormal degradation of matrix.
  • Substances selected as the above preventive and therapeutic drugs include, for example, sugar-coated tablets, forcepsels, elixirs, microforces, etc., orally, or water or other drugs. It can be used parenterally in the form of an injectable preparation such as a sterile solution with a liquid that is acceptable or a suspension.
  • an injectable preparation such as a sterile solution with a liquid that is acceptable or a suspension.
  • 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 solutions 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.), nonionic surfactants (eg, Polysorbate 80 TM, HC 0-50, 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.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, proforce hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • a preservative eg, benzyl alcohol, phenol, etc.
  • the substance selected as a drug for prophylaxis or treatment is DNA
  • insert the DNA alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. After that, 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 low toxic and can be used, for example, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, birds, higgies, bushes, dogs, dogs, 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. ), the substance is administered from about 0.1 mg to about 0.1 mg per day, preferably from about 1.0 to 5 mg, more preferably from about 1.0 to 20 mg.
  • the single dose of the substance may vary depending on the target of administration, target disease, etc., for example, administration to adults (with a body weight of 6 O kg) in the form of injections 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. It is.
  • the dose can be administered in terms of 60 kg.
  • the non-human mammal of the present invention can be used for 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. Further, for example, by directly analyzing DNA or RNA in the tissue of the transgenic rat of the present invention, or by analyzing the protein expressed by the gene, a transcription factor having a complex action of a nuclear receptor can be obtained. It is also possible to analyze the relationship between the two. Alternatively, 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. Further, by using the cells, for example, it is possible to select a drug that enhances the function of the cells. In addition, 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.
  • the 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 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 in the PCR (polymerase chain reaction) method performed in Example 4 described later.
  • SEQ ID NO: 7 This shows the base sequence of the primer used in the 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-MMPB con-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 region of the promoter of the rat type II collagen gene is a primer (5'-GTGGTGGTGGAC AACTAGGAAACTCTGG-3 ': SEQ ID NO: 1) designed based on the nucleotide sequence of Kohno et al. (LBiol. Chem. 260: 4441, 1985). And (5′-CGAGGCGAAKATGGCTCACCGCG-3 ′: SEQ ID NO: 2) by PCR.
  • the obtained fragment of about 1.2 Kb was cloned into pCRII-TOPII using TOPO TA Cloning Kit (manufactured by Invitrogen) according to the attached protocol (referred to as pCRI I-promoter 2).
  • nucleotide sequence of the inserted DNA fragment was confirmed by a conventional method using a DNA sequencer manufactured by ABI, the nucleotide sequence was consistent with the nucleotide sequence of the promoter region in the aforementioned literature (SEQ ID NO: 9).
  • pCRI I Not I site (5 'side) of multiple-cloning site of pCRI I plasmid in promoter 2
  • pCRI I p The fragment consisting of the SmaI site in the type II collagen gene promoter sequence in the robot 2 was used in the following experiments.
  • the rat type II enhancer region was prepared using a primer (5′-TCCACGCGTTTGGGAAACTTCTTGGCTGCG-3) designed based on the nucleotide sequence of Krebsbach et al. (J. Biol. Chem. ': SEQ ID NO: 3) and (5'-GCTTCGTCGC CGCTACGCGTGGGGCCGGA-3': SEQ ID NO: 4).
  • An EcoRI linker was added to the obtained 0.35 Kb MluI fragment by a conventional method, and inserted into the EcoRI site of pBluescript KSII + (hereinafter, referred to as pKS_enhancer1-4).
  • An expression vector for producing a transgenic rat, pKS-MMPBcon- 19, was constructed according to a conventional method.
  • Co 12 A 1 pr omo ter promoter region of rat type II collagen gene, pCR II described in Example 1—from Not I site in the multiple cloning site of pCR II in pr omoter 2 CR II—a 1120 bp fragment to the SmaI site in the type II collagen gene promoter in promoter 2 (the SmaI site was converted to a Sal1 site by conventional linker ligation).
  • SV40 sp1icing a DNA fragment containing a splicing site derived from pTB399 (R. Sasada et al., Cell Structure and Function 12: 205, 1987)
  • MMP-19 Approximately 1600 b from the SacI site to the XhoI site in the MMP-19 cDNA of pTB1921 (Japanese Patent Laid-Open No. 10-080283). P gene fragment. (Sac I site was converted to CI a I site and Xho I site was converted to Bg 1 II site by linker ligation).
  • Co 12 A 1 enh ancer rat type II collagen described in Example 1—enrichment of pKS containing enhancer region of gene—from Hind III site to Not I site of enhancer 1_4 fragment.
  • 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) (301 U / individual) was injected intraperitoneally, and at 11:00 on day 3, male rats were injected intraperitoneally with luteinizing hormone (human chorionic gonadotropin, hCG) (5 IUZ individuals).
  • the SD line lived and bred with individuals after 10 weeks of age at 1: 1 at 15: 0.
  • the female rats bred at 9:00 were checked for vaginal plugs, and at 13:30, the individuals whose vaginal plugs were confirmed were sacrificed and egg collection was started.
  • a pronucleus-forming egg was selected from fertilized eggs, and the plasmid pKS—MMPBc 011-19 obtained in Example 2 was cut from 1:30 at 1:30 with 1 ⁇ 0-se I and adjusted to a concentration of 10 g / m1.
  • DNA fragments 1-21 containing the MMP-19 gene were 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, PMI-1640 powder medium (Dainippon Pharmaceutical) 1.040 g, MEM Eag1e 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 ) And confirmed the two-cell stage embryos at 13:30 on day 5, and then reported to Wagner et al. (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 X g of DNA was completely digested with BamHI, transferred to a nylon filter after 2.0% agarose gel electrophoresis. This filter was combined with a probe obtained by labeling a DNA fragment at the Bg1 II site from the NheI site of MMP-19 (Example 2) with a DIG RNA labeling kit (Roche Diagnostics). Hybridized overnight, washed twice with 2 ⁇ SSC, 0.1% SDS at room temperature, and then twice with 0.1 ⁇ SSC, 0.1% SDS at 68 ° C. For detection, a DIG fluorescence detection kit (manufactured by Roche Diagnostics) was used.
  • Example 5 Acquisition of transgenic rats in heterozygote
  • the B6 OF (first generation (F 0 )) obtained in Example 4 reached the age of 12 weeks, it was bred with the SD strain rat to obtain the second generation (F). The second generation was 4 weeks old.
  • PCR was performed by the method described in Example 4 to select a heterozygote and used in Example 6.
  • Example 6 Confirmation of human mRNA in cartilage of transgenic rat
  • the transgenic rat obtained in Example 5 was homogenized in ISOGEN (manufactured by Futabajin) to extract total RNA by a conventional method.
  • ISOGEN manufactured by Futabajin
  • cDNA was synthesized according to the protocol using First strand cDNA synthesis kit (Amersham Pharmacia Biotech) and used as a template for RT-PCR.
  • rat and human MMP- Simultaneously amplified and detected 19 mRNA, and used as a negative control was an individual determined to be wild-type by PCR performed in Example 5.
  • RT PCR amplified 538 bp fragment was completely digested with ApaLI, which is only present in human fragments. At this time, fragments of 347 bp and 191 bp could be detected, confirming 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 Obtaining homozygous transgenic rats
  • Example 7 The body weight of each of the wild-type, heterozygous and homozygous individuals (B6OF strain, 12-week-old, male) obtained in Example 7 was measured and compared with the wild-type. However, they also found that the weight of homozygotes was smaller than that of 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 animals were port-fixed from the heart with 4% paraformaldehyde under ether anesthesia, and 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 prepared section was left overnight in a drier heated at 37 to dry completely.
  • Deparaffinization 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. Performed in a 1% aqueous solution of safranin for 10 minutes.
  • the sections were dehydrated by taking in and out 95% ethanol and 100% ethanol three times in each step, 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 abnormality was found in the articular cartilage and growth plate of the femur and tibia of the heterozygous 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.
  • New transgenic rat (strain name is MMP—B60F)
  • heterozygous body weight is smaller than wild type
  • homozygous body weight is smaller than heterozygous body
  • homozygous body is wild type.
  • Watashita To determine whether this abnormality was acquired postnatally or innately during fetal life, we examined whether there were abnormalities in fetal skeletal formation.
  • a homozygote of a new transgenic rat (strain name: MMP—B60F) The male of the bird and the female of the homozygote lived together and were mated. After confirmation of mating, pregnant animals were sacrificed on the 18th day of pregnancy, cesarean section was performed, and fetuses were removed. Since all of the removed fetuses were homozygous, we examined whether fetal skeletal abnormalities were seen in all cases.
  • the fetuses were subjected to double staining of osteochondral cartilage with reference to the following (Histology research method, Yutaka Sano, Nanzando, 1985).
  • New transgenic rats (strain name: MMP-B60F) homozygote, shortening of limbs, cranial deformity and caudal vertebra deformity observed in adult searches may have occurred from embryonic stage. all right. Delayed calcification was observed in the posterior lumbar spine and hind limbs. The incidence of abnormalities in the stomach was about 50% in Adalto, but any abnormalities were observed in all cases during the embryonic period. 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 rats (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 Smoothend
  • FGFR3 expression was observed in the proliferating chondrocyte layer at the site where the cartilage layer of the homozygote was thinned, and there was no abnormality.
  • IHH is not The expression of its receptors, PTC and SMO, was not found at this site. Since normal IHH expression is found in pre-hypertrophic chondrocytes, abnormalities occur in the stage where pre-hypertrophic chondrocytes differentiate into hypertrophic chondrocytes at sites where the cartilage layer is thinning Power ⁇ guessed.
  • 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 abnormalities, and the like. Cartilage fragments or chondrocytes collected from the transgenic non-human mammal of the present invention are cultured, and MMP-1 9 Can be used to evaluate inhibitors.
  • 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 abnormalities, and the like.
  • Cartilage fragments or chondrocytes collected from the transgenic non-human mammal of the present invention are cultured, and MMP-1 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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