WO2007083611A1 - Modèle animal d’hypertension pulmonaire spontanée, méthode de production du modèle animal, composition pharmaceutique pour une hypertension pulmonaire et méthode thérapeutique pour une hypertension pulmonaire - Google Patents

Modèle animal d’hypertension pulmonaire spontanée, méthode de production du modèle animal, composition pharmaceutique pour une hypertension pulmonaire et méthode thérapeutique pour une hypertension pulmonaire Download PDF

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WO2007083611A1
WO2007083611A1 PCT/JP2007/050466 JP2007050466W WO2007083611A1 WO 2007083611 A1 WO2007083611 A1 WO 2007083611A1 JP 2007050466 W JP2007050466 W JP 2007050466W WO 2007083611 A1 WO2007083611 A1 WO 2007083611A1
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pulmonary hypertension
human mammal
ox40l
pharmaceutical composition
transgenic non
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Japanese (ja)
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Kazuo Sugamura
Naoto Ishii
Masao Ono
Hiroaki Shimokawa
Kazuko Murata
Kimio Sato
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Japan Science And Technology Agency
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • 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
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

Definitions

  • Pulmonary hypertension model animal, method for producing the same, and pharmaceutical composition and treatment method for pulmonary hypertension Pulmonary hypertension model animal, method for producing the same, and pharmaceutical composition and treatment method for pulmonary hypertension
  • the present invention relates to a pulmonary hypertension spontaneous animal model and a method for producing the same, a pharmaceutical composition and treatment method for pulmonary hypertension, and an activity having a therapeutic effect on pulmonary hypertension using the model animal.
  • the present invention relates to a component screening method and the like.
  • the present inventors have developed a transgene expressing the mouse OX40 ligand (OX40L) gene (Genebank NM 009452) under the control of the mouse lck promoter (Garvinetal, Mo: ellBiol, 8: 3058-3064, 198 8).
  • OX40L mouse OX40 ligand
  • a nick mouse was produced, and it was reported that the mouse developed interstitial pneumonia and inflammatory bowel disease depending on the C57BL / 6 strain (Non-patent Document 1 and Patent Document).
  • these documents do not describe or suggest that the transgenic mouse spontaneously develops pulmonary hypertension and the possibility that the transgenic mouse can be used as a spontaneous animal model of pulmonary hypertension.
  • Non-patent literature l Murata, et al., J. Immunol, 169: 4628-4636, 2002
  • Patent Document 1 Japanese Patent Laid-Open No. 2003-102330
  • OX40L OX40 ligand
  • Non-Patent Document 1 or Patent Document 1 describes that the above-mentioned transgene non-human mammal develops autoimmune diseases such as interstitial pneumonia and inflammatory bowel disease.
  • autoimmune diseases such as interstitial pneumonia and inflammatory bowel disease.
  • primary pulmonary hypertension is a fatal disease of unknown cause, and there is no fact showing a relationship between this disease and autoimmune disease.
  • the knowledge that immune responses are directly involved in the development of pulmonary hypertension is unknown. In these senses, it is completely impossible to conceive of known knowledge that OX40L-Tg mice spontaneously develop pulmonary hypertension.
  • the present invention relates to the following aspects.
  • Aspect 2 The transgenic non-human mammal according to Aspect 1, wherein the right ventricular systolic pressure is increased by 20% or more as compared with control mice.
  • Aspect 3 The transgenic non-human mammal according to Aspect 1 or 2, wherein the OX40L gene consists of the DNA sequence of GenBank accession number NM009452.
  • the OX40L gene is expressed under the control of a T cell-specific lck proximal promoter
  • the transgenic non-human mammal according to any one of aspects 1 to 3, wherein the mammal is introduced.
  • Aspect 5 The transgenic non-human mammal according to any one of Aspects 1 to 4, wherein the non-human mammal is a mouse.
  • Transgenic non-human mammal injects an expression vector containing the OX40L gene into the pronucleus of a fertilized egg of a non-human mammal and introduces the OX40L gene into the non-human mammal
  • the method for producing a transgenic non-human mammal according to any one of embodiments 1 to 6, wherein the method is a non-human mammal.
  • Aspect 8 The production method according to Aspect 7, wherein the transgenic non-human mammal into which the OX40L gene is introduced is produced by purifying the C57BL / 6 strain.
  • Aspect 10 Use of a transgenic non-human mammal produced by the method according to any one of Aspects 7 to 9 as a model animal for spontaneous development of pulmonary hypertension.
  • a method for screening a therapeutically active ingredient for pulmonary hypertension characterized by using the pulmonary hypertension spontaneous non-human mammal according to any one of aspects 1 to 6.
  • Aspect 12 The screening method according to Aspect 11, wherein a test substance is administered to a transgene non-human mammal, and a change in the symptom of pulmonary hypertension is evaluated and measured.
  • a method for treating pulmonary hypertension comprising administering the pharmaceutical composition according to Aspect 16.
  • a method for treating pulmonary hypertension comprising administering the pharmaceutical composition according to any one of aspects 17 to 19.
  • the introduced OX40L gene which can be used as a model animal for spontaneous development of pulmonary hypertension (primary pulmonary hypertension), is constitutively expressed in T cells.
  • a featured transgene non-human mammal is provided.
  • an effective therapeutic method has been established so far, and a therapeutic method for pulmonary hypertension can be provided.
  • FIG.l Lung tissue image of the OX40L-Tg mouse of the present invention (20-week-old OX40L-Tg male mouse (C57BL / 6 strain)) using the lck proximal promoter (upper two photographs and lower left) Photo of). Lung tissue image of the same week-old OX40L-Tg mouse (using j8 actin promoter) bred in the same environment (lower right photo).
  • FIG. 2 is a graph showing an increase in right ventricular systolic pressure in OX40L-Tg mice (left) and littermate wild-type male mice (right) of the present invention.
  • FIG. 3 is a graph showing systematic differences in pulmonary hypertension lesions.
  • score 0.033 + 0.071
  • FIG. 4 is a graph showing that right ventricular systolic pressure is significantly suppressed by administration of anti-OX40L monoclonal antibody.
  • FIG. 5 is a graph showing that the ratio of the right ventricular wall weight to the left ventricular weight is significantly suppressed.
  • FIG. 6 is a photograph of immunostaining of -smooth muscle actin (a-SMA) showing that pulmonary artery thickening is significantly suppressed by administration of anti-OX40L monoclonal antibody.
  • a-SMA smooth muscle actin
  • the pulmonary hypertension spontaneous non-human mammal of the present invention is characterized in that the introduced OX40L gene is constitutively expressed in T cells.
  • the transgenic non-human mammal of the present invention has a right ventricular systolic pressure increased by 20% or more compared to the control mouse.
  • a strong transgene non-human mammal can be produced by any conventional method known to those skilled in the art, for example, as described in Non-Patent Document 1 or Patent Document 1 above. For example, it can be prepared by introducing the mouse OX40L gene into a mouse fertilized egg (F1 fertilized egg (C57B L / 6 X DBA / 2)).
  • OX40 (CD34) is a molecule belonging to the TNF receptor family and is transiently expressed on activated T cells.
  • OX40L is expressed on APCs (antigen-presenting cells) such as activated B cells and activated rod cells.
  • APCs antigen-presenting cells
  • OX40L was originally identified as a human gp34 whose expression is induced by Tax of human T-cell leukemia virus type I (HTLV-I) (Miura et al., 1991, Mol. Cell. Biol, 11,1313). ) And expressed in B cells, rod cells, and endothelial cells (O hshima et al, J Immunol.
  • the OX40L gene (DNA) is a known substance and can be easily obtained by those skilled in the art. I can do it.
  • the mouse OX40L gene DNA deposited under GenBank accession number NM 009452 can be used.
  • the DNA (genomic DNA and cDNA) encoding OX40L in the present invention is complementary to the DNA (or cDNA corresponding thereto) in addition to the DNA having the specific nucleotide sequence described above.
  • the origin of OX40L (gene) is not particularly limited, and examples include those derived from mammals such as rats, mice and humans as described above.
  • hybrida See Chillon, for example, Molecular cloninng third, ed. ( C old Spring Harbor Lab. Press, 2001), or Current 'Protocols' in 'Morekiyura 1 ⁇ ' noisyoron 1 ⁇ (Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987), etc.) In that case, it can be carried out according to the method described in the attached instruction manual.
  • stringent conditions means, for example, a temperature of 60 ° C. to 68 ° C., a sodium concentration of 15 to 900 mM, preferably ⁇ or 15 to 600 mM. The conditions are 15 to 150 mM, pH 6 to 8.
  • DNA that can hybridize under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by the above sequence number
  • the entire base sequence of the DNA examples include DNA containing a base sequence that is about 80% or more, preferably about 95% or more, and more preferably 99% or more on average in terms of the overall degree of homology.
  • OX40L is a protein having the amino acid sequence ability represented by the above SEQ ID No., and in such an amino acid sequence, one or several amino acids are deleted, substituted, or added. A protein consisting of an amino acid sequence (amino acid sequence having sequence homology with the above amino acid sequence) and having at least one action of OX40L is also included.
  • sequence homology between two base or amino acid sequences the sequences are pre-processed to an optimal state for comparison. For example, by inserting a gap in one sequence, the alignment with the other sequence is optimized. The amino acid residue or base at each site is then compared. When the same amino acid residue or base as the corresponding site of the second sequence is present at a site in the first sequence, the sequences are identical at that site. Sequence homology between the two sequences is expressed as a percentage of the total number of sites (all amino acids or all bases) of the number of sites that are identical between the sequences.
  • sequence homology between two base sequences or amino acid sequences can be determined, for example, by the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2 264-2268, 1990 and Proc. Natl. Acad. Sci. USA 90: 5873-5877, 199 3).
  • BLAST programs and FASTA programs that use such algorithms are mainly used to search the database for sequences that show high sequence homology to a given sequence. These can be used, for example, on the Internet website of the National Center for Biotechnology Information.
  • a DNA that exhibits the sequence homology of the base sequence as described above or the sequence homology of the encoded amino acid sequence can also be obtained using hybridization as an index as described above. It can also be found from DNA groups of unknown function obtained by analysis or public databases by publicly used methods by those skilled in the art, for example, by searching using the aforementioned BLAST software.
  • the OX40L gene can be introduced into a fertilized egg by a method known to those skilled in the art using, for example, an expression vector incorporating the OX40L gene. Specifically, microinjection into the pronucleus of a fertilized egg, the obtained egg cells are cultured, and then transplanted into the oviduct of a foster parent, whereby the offspring transgenic non-human mammal of the present invention can be produced.
  • Such an expression vector can be easily constructed based on an appropriate commercially available product (for example, pBlueScript) known to those skilled in the art.
  • Such an expression vector can be in any form known to those skilled in the art, for example, a plasmid, a linear DNA molecule, a protein (peptide), etc., and a complex of the DNA molecule.
  • Such vectors also contain various elements such as promoters, enhancers, silencers, and other gene expression regulatory sequences, cloning sites, drug resistance genes or auxotrophic genes, as known to those skilled in the art. It can be produced by any method.
  • a gene expression regulatory sequence such as a promoter
  • an appropriate one known to those skilled in the art can be used as long as the introduced OX40L gene can be constitutively expressed in T cells.
  • the OX40L gene can be incorporated into an expression vector such that the gene is expressed under the control of a T cell specific promoter, such as a mouse lck proximal promoter and a CD2 promoter.
  • a T cell specific promoter such as a mouse lck proximal promoter and a CD2 promoter.
  • the transgenic non-human mammal introduced with the OX40L gene used in the present invention was prepared in a pure manner to the C57BL / 6 strain, for example, the C57BL / 6 strain by backcrossing. It is preferable that the material is made by purifying the C57BL / 6 pure system. In particular, it is preferable that the backcrossing is performed for at least 12 generations. For example, a person skilled in the art will find a primary mouse having the above-described transgene from among offspring mice produced by transplanting fertilized eggs into which the OX40L gene has been introduced using an appropriate expression vector into the oviduct of a parental mouse.
  • the primary mouse introduced with the OX40L gene is backcrossed with an appropriate kind of mouse, for example, a C57BLZ6 strain mouse at least 12 times by an appropriate means known in the art (for example, PCR or flow cytometry). .
  • transgenic non-human mammal of the present invention include animals usually used as laboratory animals, for example, rodents such as mice and rats.
  • rodents such as mice and rats.
  • C57BL / 6 mice are preferred.
  • the transgenic non-human mammal produced by the above method can be used as a pulmonary hypertension spontaneous model animal.
  • the transgenic non-human mammal of the present invention can be used for screening a therapeutic agent for pulmonary hypertension. It can be used as a model for spontaneous development of blood pressure.
  • Specific procedures of the screening method such as “conditions”, can be appropriately selected and planned by those skilled in the art according to the “amount of test substance” type and the various conditions such as “type of animal” and “condition”.
  • the screening method of the present invention can be a method comprising administering a test substance to a transgenic non-human mammal and evaluating and measuring changes in the symptoms of pulmonary hypertension.
  • a method comprising administering an existing therapeutic agent and test substance for spontaneous development of pulmonary hypertension, and comparing the effect on changes in symptoms of pulmonary hypertension with the existing therapeutic drug and test substance. Is possible.
  • a therapeutic effect on pulmonary hypertension can be obtained by inhibiting or blocking the OX40 signal or OX40LZOX40 interaction. Therefore, based on this finding, a screening method for a therapeutic active ingredient for pulmonary hypertension based on the inhibition of OX40 signal or OX40LZOX40 interaction is provided. Such inhibition / blocking can be measured by any method known to those skilled in the art. For example, whether or not a test substance inhibits or blocks OX40 signal or OX40LZOX 40 interaction can be measured in vitro or in vivo based on the expression level of each gene or the presence or absence of interaction between these proteins. It is.
  • the present invention provides a pharmaceutical composition for pulmonary hypertension comprising the active ingredient obtained by these screening methods, and a method for treating pulmonary hypertension characterized by administering the pharmaceutical composition.
  • a pharmaceutical composition for pulmonary hypertension comprising the active ingredient obtained by these screening methods, and a method for treating pulmonary hypertension characterized by administering the pharmaceutical composition.
  • an active ingredient of such a pharmaceutical composition for example, a substance having an action of inhibiting / blocking OX40 signal or OX40LZOX40 interaction, more specifically, anti-OX40L antibody and soluble OX40 can be mentioned. .
  • anti-OX40L antibody or soluble OX40 By binding the anti-OX40L antibody or soluble OX40 to OX40L, the binding of OX40LZOX40 is inhibited, and their interaction is suppressed / blocked. As a result, pulmonary hypertension can be suppressed.
  • These substances can be easily prepared by those skilled in the art based on, for example, the description of the examples in the
  • the pharmaceutical composition of the present invention can be formulated by a known pharmaceutical method.
  • a pharmacologically acceptable carrier or medium specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, sustained release Agent It is conceivable to formulate and administer it in appropriate combination with the above.
  • the pharmaceutical composition of the present invention may be in the form of an aqueous solution, tablet, capsule, troche, knock tablet, elixir, suspension, syrup, nasal solution, inhalation solution, or the like.
  • the content of the compound of the present invention can be appropriately determined by those skilled in the art according to the purpose of treatment, administration route, treatment target, and the like.
  • administration to the patient may be, for example, percutaneous, intranasal, transtracheal, intramuscular, intraperitoneal, intravenous, intrathecal, intraventricular, or Can be done orally.
  • the pharmaceutical composition of the invention is introduced into the central nervous system by any suitable route including intravenous, intrathecal, intraventricular or intradural injection. Is desirable.
  • a person skilled in the art can appropriately select an appropriate dose according to the patient's age, weight, symptoms, administration method and the like.
  • the dosage and administration method can be appropriately selected by those skilled in the art depending on the tissue transferability of the active ingredient of the pharmaceutical composition of the present invention, the purpose of treatment, the patient's weight, age, symptoms, and the like.
  • the amount of active ingredient can be 0.1 to 1,000 mg / kg body weight, preferably 1 to 100 mg / kg body weight.
  • Example 1 the introduced OX40L gene is constitutively expressed in T cells and is transgeneic.
  • Mice OX40 L-Tg mice
  • a T cell line-specific plasmid vector pl017 a vector in which the poly A signal derived from the human growth hormone gene is incorporated into the lck proximal probe motor
  • Gene donated by Perlmuttaer into which the mouse OX40L-encoding cDNA (DNA sequence of GenBank accession number NM 009452) is inserted, and the gene is constitutively expressed under the control of a T cell-specific lck proximal promoter
  • Such an expression plasmid vector was prepared.
  • This expression plasmid vector was injected into the pronucleus of a mouse fertilized egg (F1 fertilized egg (C57BLZ6 X DBA / 2)) by microinjection.
  • the obtained egg cells were cultured for 1 day and then transplanted into a foster oviduct to produce offspring mice (30 animals).
  • the obtained pups were identified by PCR as to whether or not the OX40L gene was introduced, and the transgenic mice (3 mice) into which the gene was introduced were confirmed.
  • Transgenic mice in which these introductions were confirmed were backcrossed at least 12 times with C57BLZ6 strain mice or BALBZc strain mice, respectively.
  • the OX40L-Tg mouse of the present invention produced by backcrossing to the C57BL / 6 strain of 12 generations or more was reared in an SPF (specific pathogen free) environment until 20 weeks of age, dissected, and lung tissue images Was pathologically searched.
  • Elastica 'Masson staining (upper and lower left) showed marked thickening of the pulmonary artery.
  • OX40L-Tg mice exhibited marked pulmonary artery thickening with thickening of the smooth muscle layer.
  • pulmonary hypertension was scored according to the following criteria.
  • the score of pulmonary artery lesions was as follows. View the accompanying bronchial artery at 10 levels in the EM specimen
  • an anti-OX40L monoclonal antibody (MGP34; rat anti-mouse OX40L antibody IgG) having a property of blocking the binding between OX40 and OX40L is obtained by a known method (Murata et al., Exp. Med. 191: 365, 2000) and examined the ability to suppress the activation of the OX40ZOX40L system and suppress the progression of pulmonary hypertension and pulmonary artery remodeling. Hypoxia-induced pulmonary hypertension was induced based on a known method (Katayose et al, Am J Physiol: 264: 100-106, 1993). Specifically, wild-type mice divided into 4 groups were reared for 5 weeks under the following conditions.
  • the pulmonary artery pressure cannot be measured directly! /
  • the right ventricular systolic pressure which is a general method for evaluating pulmonary artery pressure in mice, was measured. Specifically, the right ventricular systolic pressure was measured by inserting a 25 gauge needle connected to the transducer for pressure measurement directly into the right ventricle from the right edge of the 4th intercostal sternum. Furthermore, the right ventricular wall weight ratio of the right ventricular weight to the left ventricular weight, which is another evaluation standard for evaluating pulmonary hypertension in mice, was measured. As a result, compared with the control antibody administration group, the right ventricular systolic pressure (Fig. 4) and the right ventricular wall weight ratio (Fig. 5) were significantly suppressed in the anti-OX40L monoclonal antibody administration group. .
  • the obtained lung tissue was immunostained with ex-smooth muscle actin (a-SMA) expressed in the smooth muscle layer.
  • a-SMA ex-smooth muscle actin

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Abstract

L’invention concerne un modèle animal d'hypertension pulmonaire primaire humaine, qui peut développer une hypertension pulmonaire spontanément et de façon chronique dans un environnement d’élevage normal et est utile pour l'étude fondamentale de l'hypertension pulmonaire primaire et de ses aspects cliniques et thérapeutiques ; et une méthode thérapeutique efficace pour une hypertension pulmonaire primaire. L’invention concerne donc un mammifère transgénique non humain capable de développer spontanément une hypertension pulmonaire, caractérisé en ce qu'un gène OX40L introduit dans le mammifère est constitutivement exprimé dans un lymphocyte T ; une méthode pour produire un mammifère transgénique non humain ; une méthode pour dépister un agent thérapeutique pour une hypertension pulmonaire, caractérisée en ce qu’on utilise un mammifère transgénique non humain ; une composition pharmaceutique pour une hypertension pulmonaire comprenant un principe actif capable d'inhiber/de bloquer un signal OX40 ou une interaction OX40L/OX40 ; et une méthode pour traiter une hypertension pulmonaire qui consiste à administrer ladite composition pharmaceutique.
PCT/JP2007/050466 2006-01-17 2007-01-16 Modèle animal d’hypertension pulmonaire spontanée, méthode de production du modèle animal, composition pharmaceutique pour une hypertension pulmonaire et méthode thérapeutique pour une hypertension pulmonaire WO2007083611A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003102330A (ja) * 2001-09-28 2003-04-08 Japan Science & Technology Corp Ｏｘ４０ｌ遺伝子を導入した自己免疫疾患モデル非ヒト哺乳動物

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003102330A (ja) * 2001-09-28 2003-04-08 Japan Science & Technology Corp Ｏｘ４０ｌ遺伝子を導入した自己免疫疾患モデル非ヒト哺乳動物

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