WO2012124752A1 - Modèle mammifère non humain du glaucome à tension normale - Google Patents

Modèle mammifère non humain du glaucome à tension normale Download PDF

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WO2012124752A1
WO2012124752A1 PCT/JP2012/056627 JP2012056627W WO2012124752A1 WO 2012124752 A1 WO2012124752 A1 WO 2012124752A1 JP 2012056627 W JP2012056627 W JP 2012056627W WO 2012124752 A1 WO2012124752 A1 WO 2012124752A1
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human mammal
src
cells
human
normal
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柏木　賢治
梧郎 加藤
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国立大学法人山梨大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10002—Non-specific protein-tyrosine kinase (2.7.10.2), i.e. spleen tyrosine kinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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

Definitions

  • the present invention relates to a normal-tension glaucoma model non-human mammal and the like.
  • Glaucoma is a progressive disease that damages the optic nerve for some reason and causes visual field defects. If left untreated, it is likely to eventually lead to blindness.
  • High-intensity glaucoma one of the glaucoma, is caused by the loss of aqueous humor in the anterior chamber and the increase in intraocular pressure (water pressure in the eyeball), which compresses and contracts the optic nerve, thus impairing visual function. This is a disease that narrows the field of vision.
  • Normal-tension glaucoma is a disease that has received particular attention recently because of its high prevalence. There are about 4 million Japanese glaucoma patients, of which about two-thirds are normal-tension glaucoma patients. Normal-tension glaucoma is a pathological condition that presents the same findings (optic nerve atrophy and visual field loss) as high-tension glaucoma despite normal intraocular pressure (usually 10 to 21 mmHg in humans). Are (a) not accompanied by nerve inflammation, (b) the lesion is limited to retinal ganglion cells (RGC), (c) neuropathy progresses over time, (d) It has features such as having a characteristic finding (concave) in the optic nerve head, and (e) maintaining intraocular pressure as normal. Normal-tension glaucoma is difficult to detect early because it progresses slowly and has few subjective symptoms. At present, there is no definitive treatment other than lowering intraocular pressure.
  • Non-patent Document 1 As a normal-tension glaucoma model mouse, the drainage of the aqueous humor was ablated with a laser (Non-patent Document 1), the bead was placed in the anterior chamber (Non-patent Document 2), the drainage vein of the aqueous humor A coagulated product (Non-patent Document 3) is known.
  • All the models have problems such as difficulty in adjusting intraocular pressure, sometimes accompanied by inflammation, and inconsistency in the generation mechanism.
  • Non-patent Document 4 Patent Document 1
  • GLAST is a transporter known as a mechanism for recovering metabotropic glutamate, which is a neurotransmitter.
  • mice those having similar symptoms with the same onset mechanism as actual human normal-tension glaucoma are required, including the gradual progression of symptoms.
  • the present invention provides the following genetically modified non-human mammals and methods for screening agents for preventing and / or treating normal tension glaucoma using the non-human mammals.
  • Ser is the 74th amino acid residue from the N-terminus of the c-Src protein.
  • the non-human mammal has the following conditions: 1) the intraocular pressure is in the normal range, and 2) the number of cells in the retinal ganglion is reduced compared to wild-type non-human mammals, The non-human mammal according to any one of [1] to [4] above, wherein [6] The non-human mammal according to any one of [1] to [5] above, wherein the non-human mammal is a mouse.
  • the method comprises: 1) administering a candidate substance to the non-human mammal and the wild-type non-human mammal according to any one of [1] to [7] above; 2) In each non-human mammal, before the administration, and after a certain period of time after administration, examine the number of optic nerve cells that survive, and 3) compare the test results of each non-human mammal, Evaluating the effectiveness of candidate substances,
  • the method according to [8] above comprising: [9-1] 1) administering a candidate substance to the non-human mammal according to any one of the above [1] to [7] and a control non-human mammal; 2) In each non-human mammal, before the administration, and after a certain period of time after administration, examine the number of optic nerve cells that survive, and 3) compare the test results of each non-human mammal, Evaluating the effectiveness of candidate substances,
  • the method according to [8] above comprising: [10] The method according to [9] above, wherein the examination of the number of
  • the control non-human animal is a wild-type non-human animal and / or a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, is replaced with Ala.
  • [11-1] The method according to [11] above, wherein the Ser in the control non-human animal is the 74th amino acid residue from the N-terminus of the c-Src protein.
  • [11-3] The method according to [11] or [11-1] above, wherein the control gene-modified non-human mammal has the mutant Src gene in a homozygote.
  • [12] A genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Ala is introduced into the Src gene.
  • [12-1] The non-human mammal according to [12] above, wherein the Ser is the 74th amino acid residue from the N-terminus of the c-Src protein.
  • [12-2] The non-human mammal according to [12] or [12-1] above, wherein the non-human mammal has a mutant Src gene in homozygosity.
  • a new non-human mammal that can be used as a normal-tension glaucoma model is provided.
  • the preferred embodiment of the non-human mammal of the present invention exhibits symptoms similar to those of actual human normal-tension glaucoma, such as a slow progression of symptoms.
  • the present invention provides a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Asp or Glu is introduced into the Src gene.
  • the mutant Src gene is expressed to simulate a state in which the Cdk5 phosphorylation site of the c-Src protein is constantly phosphorylated.
  • the non-human mammal of some aspects of the present invention is (a) not accompanied by nerve inflammation (Table 1), (b) the damaged part is a retinal ganglion cell ( RGG: retinal ganglion cell) (Table 1), (c) Neuropathy progresses over time ( Figure 1, Table 1), (d) Intraocular pressure remains normal (FIG. 3, Table 1), etc., showing the same characteristics as the symptoms of normal tension glaucoma in humans. For this reason, the non-human mammal of the preferable aspect of this invention can be used as a normal-tension glaucoma model.
  • the main excitatory neurotransmitter in the neural synapse is glutamate. Glutamate released excessively for some reason binds to NMDA receptors of retinal ganglion cells and promotes the inflow of Ca ions into the cells. As a result, cell death of retinal ganglion cells is induced.
  • Cdk5 proto-oncogene Src phosphorylated by Cdk5
  • a prophylactic and / or therapeutic agent for normal tension glaucoma can be screened.
  • Mutant Src gene c-Src protein is a membrane-bound protein encoded by the proto-oncogene Src gene and functions as a tyrosine kinase in normal cells.
  • the mouse c-Src protein is a protein encoded by a DNA chain having the base sequence shown in SEQ ID NO: 1 (NCBI Accession No. NM_009271.3) and having the amino acid sequence shown in SEQ ID NO: 2 (NCBI Accession No. NP_033297.2).
  • the DNA sequence encoding c-Src protein is shown in NCBI Accession No. NM_031977.1
  • amino acid sequence of c-Src protein is shown in NCBI Accession No. NP_114183.1.
  • the mutant Src gene of the present invention is a gene in which Ser that is a Cdk5 phosphorylation site of the c-Src protein is mutated to Asp or Glu.
  • Ser, which is a Cdk5 phosphorylation site of c-Src protein can be identified by a known method. For example, Shenoy, S. et al., “Purified maturation promoting factor phosphorylates pp60 c-src at the sites phosphorylated during fibroblast mitosis ”Cell 57, pp763-774, Kato, G.
  • Ser which is the Cdk5 phosphorylation site of c-Src protein, is, for example, the 74th amino acid residue from the N-terminal in mice, that is, the 74th amino acid residue from the N-terminal in the amino acid sequence of SEQ ID NO: 2. .
  • This amino acid residue corresponds to codons 604 to 606 from the 5 ′ end in the nucleotide sequence of SEQ ID NO: 1.
  • Ser at the 74th amino acid residue from the N-terminus in the mouse c-Src protein was first revealed to be the Cdk5 phosphorylation site at the corresponding 75th Ser in humans. And is sometimes referred to as “Ser75” in this specification.
  • Ser, the Cdk5 phosphorylation site of the c-Src protein is the 75th amino acid residue from the N-terminus in rats.
  • Ser which is a Cdk5 phosphorylation site of c-Src protein
  • an amino acid that can simulate the phosphorylation state of Ser.
  • an amino acid is Asp or Glu, preferably Asp.
  • the codons “AGC”, “AGT”, “TCA”, “TCC”, “TCG” or “TCT” encoding the Ser of the Src gene encode Asp.
  • the codons “GAC” or “GAT”, or the codons “GAA” or “GAG” encoding Glu In some embodiments of the invention, the codon “TCC” encoding Ser of the Src gene is mutated to codon “GAC” or “GAT” encoding Asp, preferably codon “GAC”.
  • the mutant Src gene of the present invention is further mutated in addition to the amino acid residue of the Cdk5 phosphorylation site within the range in which the function of the mutant Src gene is maintained.
  • 1 to 10 such mutants for example, 1 to 10 sites other than the Cdk5 phosphorylation site in the encoding nucleic acid sequence described above may be subjected to substitution, deletion, addition, or insertion.
  • a mutant in which 1 to 5 bases are substituted, deleted, added, or inserted, or a site other than the Cdk5 phosphorylation site in the amino acid sequence for example, 1 to 10, preferably 1
  • Mutants in which ⁇ 5 amino acids are substituted, deleted, added, or inserted are also included in the mutant Src gene of the present invention.
  • the genetically modified non-human mammal in which a mutation is introduced into the Src gene is heterozygous in which the mutation is introduced into one endogenous Src gene, and the mutation is introduced into two endogenous Src genes.
  • the homozygous type is more preferable.
  • Such introduction of mutations into the endogenous Src gene can be achieved by a known method for producing a genetically modified non-human mammal, such as a gene targeting method.
  • Non-human mammal of the present invention is a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Asp or Glu is introduced into the Src gene. .
  • the non-human mammal of the present invention one or two endogenous Src genes on the homologous chromosome are replaced with the mutant Src gene of the present invention. That is, the non-human mammal of the present invention is a heterozygote or homozygote of the mutant Src gene of the present invention, preferably a homozygote of the mutant Src gene of the present invention.
  • the non-human mammal having the mutant Src gene of the present invention may be any mammal as long as it is a mammal other than a human having the Src gene.
  • Examples of such mammals include cattle, minipigs, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, monkeys, and the like.
  • rodents, particularly mice or rats, which have relatively short ontogeny and biological cycle and are easy to breed, are preferable from the viewpoint of producing a pathological animal model system, and mice are more preferable.
  • the non-human mammal of some embodiments of the present invention has 1) its intraocular pressure in the normal range, and 2) its retinal ganglion cell count is reduced compared to a wild-type non-human mammal.
  • the “wild-type non-human mammal” is, for example, a non-human mammal that does not suffer from glaucoma, and preferably a wild-type (normal) Src gene obtained by mating heterozygous mice of a mutant Src gene A non-human mammal having no glaucoma.
  • the intraocular pressure is in the normal range means that the measured value of the intraocular pressure of the non-human mammal of the present invention falls within the normal value of the intraocular pressure of the wild-type non-human mammal.
  • the normal value of intraocular pressure in wild-type mice is usually 10 to 21 mmHg, and the intraocular pressure is preferably within the normal range in the mouse of the present invention.
  • high intraocular pressure for example, 30 mmHg or more.
  • the intraocular pressure of the non-human mammal can be measured using, for example, an electronic tonometer.
  • the number of neurons in the retinal ganglion is reduced compared to a wild-type non-human mammal, for example, compared to a wild-type non-human mammal, Preferably, it is reduced by at least 20%, more preferably by at least 40%, and even more preferably by 50%.
  • the decrease in the number of neurons in the retinal ganglion can be measured under a microscope by a conventional histochemical method, for example, by hematoxylin / eosin staining using a section.
  • the cultured cell viability of the non-human mammal of the present invention is wild-type.
  • Reduced compared to non-human mammals for example, preferably reduced by at least about 20%, more preferably at least about 40%, and even more preferably about 50% compared to wild-type non-human mammals .
  • glaucoma This decrease in the number of neurons in the retinal ganglion and the decrease in cell viability can be regarded as neurodegeneration or neuronal cell death.
  • glaucoma including normal-tension glaucoma, has atrophy of the optic nerve head and deficits in retinal nerve fibers.
  • the final image of glaucoma is It is thought to be node cell death.
  • the non-human mammal of a preferred embodiment of the present invention has an eye property, that is, that the intraocular pressure is in a normal range, the number of cells in the retinal ganglion is decreased, and the cell viability is cultured. In consideration of the decrease, it can be used as a model non-human mammal of normal pressure glaucoma.
  • the present invention provides a method for producing the non-human mammal of the present invention.
  • the non-human mammal of the present invention can be produced, for example, by a standard method.
  • the method is (a) producing an ES cell in which an endogenous Src gene on a homologous chromosome is mutated to the mutant Src gene of the present invention by gene targeting; (b) producing a chimeric non-human mammal using ES cells; (c) crossing a chimeric non-human mammal with a wild-type non-human mammal to produce a heterozygous non-human mammal; (d) crossing heterozygous non-human mammals to produce homozygous non-human mammals.
  • each said process is demonstrated in detail.
  • a point mutation is introduced into a portion encoding Ser, which is a Cdk phosphorylation site in the Src gene.
  • Point mutations can be introduced by known site-directed mutagenesis methods (eg, Deng, WP et al., “Site-directed mutagenesis of virtually any plasmid by using a unique site”, Anal. Biochem. 200, pp81-88.
  • Point mutations can also be introduced using commercially available kits such as Mutan TM -super Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo).
  • the targeting vector is preferably constructed so that screening of recombinants after homologous recombination is facilitated.
  • a selection marker such as a drug resistance gene or a toxin gene can be linked to the vector for positive-negative selection.
  • Positive-negative selection methods are well known in the art.
  • positive selection utilizes the fact that cells that have not incorporated the selectable marker gene die when they are cultured in a culture medium containing a drug because they do not contain a resistance gene, and the negative selection method uses random integration. In the cells that occurred in the above, the fact that the cells die is used to express the negative selection gene. As a result, only cells that have undergone homologous recombination survive and are selected.
  • selection marker gene for example, neomycin resistance gene (neo), hygromycin B phosphotransferase gene and the like can be used for positive selection, and for example, herpesvirus thymidine kinase gene (HSV-tk), diphtheria toxin A Genes and the like can be used.
  • neomycin resistance gene neo
  • hygromycin B phosphotransferase gene and the like can be used for positive selection
  • herpesvirus thymidine kinase gene (HSV-tk), diphtheria toxin A Genes and the like can be used.
  • Homologous recombination is performed using the targeting vector prepared by the above method.
  • ES cells such as CCE, E14, J1, TT2, D3, BL / 6-III and the like can be appropriately selected and used as ES cells.
  • the targeting vector is introduced into cells.
  • a method for introducing a targeting vector into a cell an electroporation method, a calcium phosphate method, a DEAE-dextran method, a liposome method, or the like can be used.
  • the electroporation method is preferably used in consideration of efficiency, ease of work, and the like. Then, the target genomic DNA sequence in the cell is replaced by homologous recombination of the targeting vector.
  • the selection marker gene may be removed.
  • Selective marker removal is based on Askew GR, Doetschman T, Lingrel JB. Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. Mol Cell Biol. 1993; 13 (7): 4115-24 ., Wu H, Liu X, JeniniseniR. Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells. Proc Natl Acad : 2819-23. Etc. double replacement / tag-and-exchange28procedure; Giese KP, Fedorov NB, Filipkowski RK, Silva AJ.
  • Src gene Whether or not the Src gene is targeted can be confirmed by PCR, Southern blotting or the like.
  • a method for transplanting ES cells into the embryo known methods such as a microinjection method and an aggregation method can be used.
  • mice In the case of mice, first, female mice that have been superovulated with a hormonal agent are mated with male mice. Thereafter, early embryos are collected from the oviduct or uterus on day 2.5 after fertilization when using 8-cell stage embryos, and on day 3.5 after fertilization when using blastocysts. ES cells that have undergone homologous recombination are injected into the collected embryos to produce chimeric embryos.
  • a pseudopregnant female mouse for use as a temporary parent can be obtained by mating a female mouse having a normal cycle with a male mouse castrated by vagina ligation or the like.
  • a chimeric mouse can be produced by transplanting the chimeric embryo produced by the above-mentioned method in utero to the produced pseudopregnant mouse and then giving birth.
  • non-human mammals other than mice
  • chimeric non-human mammals can be produced in the same manner as described above.
  • a male chimeric non-human mammal derived from an ES cell-transplanted embryo is selected from the chimeric non-human mammal obtained as described above.
  • this mouse is mated with a female mouse of a pure mouse strain.
  • ES cells were introduced into the germ line of chimeric mice by the appearance of the coat color of the mouse derived from the ES cell (the mouse that had the genome incorporated into the ES cell) in the born mouse. Can be confirmed.
  • a heterozygous non-human mammal in which the recombinant ES cells transplanted into the embryo are introduced into the germ line is bred.
  • a heterozygous non-human mammal can be obtained by mating the heterozygous non-human mammals obtained as described above.
  • step (c) or (d) can be carried out by extracting chromosomal DNA from the tissue and performing Southern blotting or PCR.
  • RNA can be extracted from the tissue and the gene expression pattern can be analyzed by Northern blot analysis.
  • Western blotting may be performed using an antibody against the protein encoded by the mutant Src gene of the present invention.
  • Phenotypes of heterozygous non-human mammals and homozygous non-human mammals can be analyzed for the established animal lines.
  • Phenotype analysis includes macroscopic observation, internal observation by dissection, tissue section of each organ, observation by X-ray photography, observation of behavior and memory, blood test, serum biochemical test, intraocular pressure test, slit lamp test This is done by examining the fundus.
  • the analysis period may be any period from the embryonic period to the adult period, and is not particularly limited.
  • the intraocular pressure is in the normal range and that the nerve cells of the retinal ganglion are reduced.
  • the non-human mammal of the present invention is a mouse
  • its intraocular pressure is usually about 21 mmHg or less, for example, about 10 to 21 mmHg.
  • This intraocular pressure range may vary somewhat depending on the strain of mouse that is the origin of the ES cells used, or the strain of the mouse that is the origin of the normal embryo that was used to generate the chimeric embryo. Preferably it is less than 30 mmHg.
  • the intraocular pressure can be measured using a known and common method. More specifically, for example, it can be carried out by the method described in the examples described later.
  • the number of nerve cells in the retinal ganglion is reduced as compared to the wild type mouse, for example, at least 20%, more preferably at least 50%.
  • a method for measuring the number of retinal ganglion cells can also be performed using a known and conventional method. More specifically, for example, it can be carried out by the method described in the examples described later.
  • the non-human mammal of the present invention is a optic nerve cell comprising a nerve agent for prevention and / or treatment of normal tension glaucoma, particularly retinal ganglion. It can be used for screening an agent effective for suppressing death or degeneration of cerebral dysfunction, or a decrease in its function, or an agent effective for restoring optic nerve cells or its function.
  • the present invention is a screening method for a preventive and / or therapeutic agent for normal pressure glaucoma, 1) administering a candidate substance to the non-human mammal and wild-type non-human mammal of the present invention; 2) In each non-human mammal, before the administration, and after a certain period of time after administration, examine the number of optic nerve cells that survive, and 3) compare the test results of each non-human mammal, Evaluating the effectiveness of candidate substances,
  • a screening method comprising:
  • Another aspect of the present invention is a screening method for a prophylactic and / or therapeutic agent for normal-tension glaucoma, 1) administering a candidate substance to the non-human mammal of the present invention and a control non-human mammal; 2) In each non-human mammal, before the administration, and after a certain period of time after administration, examine the number of optic nerve cells that survive, and 3) compare the test results of each non-human mammal, Evaluating the effectiveness of candidate substances,
  • a screening method comprising:
  • the control non-human mammal is a wild-type non-human animal and / or a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Ala is introduced into the Src gene.
  • the control non-human mammal is a genetically modified non-human mammal in which a mutation that replaces Ser, which is a Cdk5 phosphorylation site of c-Src protein, with Ala is introduced into the Src gene.
  • a prophylactic and / or therapeutic agent for normal tension glaucoma specific to the Src gene can be screened.
  • Ser which is a Cdk5 phosphorylation site of c-Src protein is the same as described above.
  • Ser, which is the Cdk5 phosphorylation site of c-Src protein is substituted with an amino acid that can mimic the non-phosphorylation state of Ser.
  • Such an amino acid is Ala.
  • the genetically modified non-human mammal used as a control can be produced by a method according to the method for producing the non-human mammal of the present invention.
  • Whether or not the candidate substance is effective for the prevention and / or treatment of normal-tension glaucoma can be determined by examining whether or not the symptoms characteristic of glaucoma can be improved. For example, by counting the number of neurons in the retinal ganglion, administration of the test compound can restore that number by at least 10%, preferably at least 20%, more preferably at least 30% compared to control mice.
  • the test compound may be determined to be pharmaceutically effective.
  • a candidate substance is regularly administered to a group of non-human mammals of the present invention immediately after birth, and another group of non-human mammals of the present invention is administered. Do not administer the candidate substance, 2) measure the number of cells in the retinal ganglion at each age, and 3) select candidate substances that suppress the decrease in the number of retinal ganglion cells over time from the comparison of both You can also
  • Candidate substances include, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, plasma, etc.
  • the compound may be a novel compound or a known compound.
  • These candidate substances may form salts, and the salts of candidate substances include salts with physiologically acceptable acids (for example, organic acids or inorganic acids) and bases (for example, metal acids). And physiologically acceptable acid addition salts are particularly preferred.
  • salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
  • the candidate substance may be a gene vector for expressing a desired protein.
  • the route of administration of the candidate substance various methods may be tried as long as the nature of the candidate substance permits, for example, instillation or oral administration.
  • the administration period and administration mode are also selected so as to maximize the effect of the candidate substance.
  • Such candidate substance types and administration methods may be in accordance with conventional methods in the pharmaceutical or medical fields.
  • non-human mammal of the present invention can be crossed with other types of disease model non-human mammals to produce new disease model non-human mammals.
  • Such use of a disease model non-human mammal is also included in the present invention.
  • Example 1 Production of SD and SA mice (1) Preparation of ES cells (SD) and ES cells (SA) ES cells in which a mutation that replaces Ser75, a Cdk5 phosphorylation site with Asp, was introduced into the Src gene ( ⁇ ES cells (SD) ''), and An ES cell (“ES cell (SA)”) into which a mutation that replaces Ser75 as an oxidation site with Ala was introduced was prepared as follows. In order to eliminate the influence of the selection marker gene and the exogenous site-specific recombination sequence on the expression of endogenous genes and the chromosome structure, a vector for introducing only point mutations was prepared.
  • a partial base sequence of the endogenous Src gene was forwardly connected to both ends of the base sequence in which the HSV-tk gene and the neomycin resistance gene were connected in series.
  • This point mutation-introducing targeting vector was transferred to ES cells by electroporation. From the cells that became resistant to G418 (neomycin), cells in which a point mutation was introduced into one allele by homologous recombination (homologous recombinants) were selected as follows. That is, after selecting cells having a homologous recombination type genome by Southern blotting, cells into which the target point mutation was introduced were selected by PCR-allele-specific probe dot hybridization.
  • FIAU (uracil derivative) resistant and G418 sensitive cells were isolated by deletion of the marker gene by intramolecular homologous recombination, and heterozygotes were obtained by Southern blotting and PCR-allele specific probe dot hybridization. Mutant ES cells were selected.
  • SD mouse (chimera) and SA mouse (chimera) were produced as follows. That is, female C57BL / 6 mice in estrus and male mice were mated, and blastocysts (3.5 day embryos) were collected from the oviduct 4 days later for female mice with vaginal plug formation. A heterozygous mutant ES cell was injected into the blastocyst with a micromanipulator and transplanted into a preparatory uterus prepared in advance to give birth. The contribution rate of ES cells was estimated by the color of the offspring.
  • SD mouse (heterozygote) and SA mouse (heterozygote) were produced as follows. That is, male chimeric mice with a contribution rate of 50% or more were mated with female C57BL / 6 mice, and it was confirmed that ES cells differentiated into germ cells of chimeric mice by the color of the born pups (129 mice) . Furthermore, after extracting genomic DNA from the tail of a pup mouse, it was confirmed by PCR-allele-specific probe dot hybridization that one of the alleles of the Src gene had a point mutation.
  • the resulting germ cell Src gene is crossed with a C57BL / 6 mouse and a chimeric mouse having the desired point mutation, and then the genotype analysis of the born pup is carried out by PCR-allele-specific probe dot hybridization. A body mouse strain was established.
  • SD mouse (homozygote) and SA mouse (homozygote) were prepared as follows. That is, heterozygous mouse strains were mated with each other, and the genotype analysis of the born pups was performed by PCR-allele-specific probe dot hybridization method to establish a homozygous mouse strain into which the target point mutation was introduced.
  • Example 2 Inflammation of optic nerve and damaged part of optic nerve
  • SD mouse (homozygote) and SA mouse (homozygote) obtained in Example 1 inflammation of optic nerve and damaged part of optic nerve are as follows: Observed. The eyeballs are removed from each mouse at 7 months, 11 months, 17 months, and 23 months of age and observed with an optical microscope, and there is no inflammatory findings such as infiltration of inflammatory cells, bleeding, or edema was confirmed histologically.
  • Example 3 Measurement of the number of retinal specimen cells About wild-type normal mice and SD mice (heterozygotes or homozygotes) and SA mice (heterozygotes or homozygotes) obtained in Example 1, retinal specimens The cell number was measured as follows. Eyeballs were removed from each mouse to prepare retinal slice specimens. The examiner who masked the genotype measured the number of retinal ganglion cells with the distance from the optic nerve kept constant for each eye. In order to eliminate measurement variations and errors, multiple measurement points were selected from one eye so that similar positions were selected for all samples.
  • FIGS. 1 and 2 and Table 1 The results are shown in FIGS. 1 and 2 and Table 1 below.
  • FIGS. 1 and 2 and Table 1 As shown in Figure 1 and Table 1, in SD mice (heterozygotes or homozygotes), the number of retinal ganglion cells (RGG) is almost the same as that in wild-type normal mice at 6-7 months of age. However, at 16-23 months of age, the number of RGG was significantly reduced compared to wild type normal mice. When compared by mean, SD mice (heterozygotes) and SD mice (homozygotes) have an average decrease in RGG numbers of about 20% and 20-40%, respectively, compared to wild-type mice. It was. This shows that in SD mice (heterozygotes or homozygotes), the symptoms of glaucoma have progressed over time.
  • Example 4 Measurement of intraocular pressure For wild-type normal mice and SD mice (heterozygotes or homozygotes) and SA mice (heterozygotes or homozygotes) obtained in Example 1, At 18 months of age, intraocular pressure was measured as follows. A rebound tonometer that can measure the intraocular pressure of the mouse most accurately was used. Prior to the measurement, mice were injected with a general anesthetic into the abdominal cavity, and the anesthesia was sufficiently introduced to measure the intraocular pressure measurement. In the measurement, only the value with a good self-reliability judgment result of the measuring instrument was adopted, and after repeating three times, the average value was adopted.
  • the intraocular pressure of SD mice was almost the same as that of wild-type normal mice, and was within the range of normal values.
  • the intraocular pressure of SA mice was also within the range of normal values.
  • Example 5 Survival rate of cultured cells Wild type normal mice, and SD mice (homozygotes) and SA mice (homozygotes) obtained in Example 1, isolated retinal ganglion cells, isolated cells The culture and cell viability were measured as follows. First, retinal ganglion cells were isolated from wild type mice, SD mice (homozygote) and SA mice (homozygote) as follows. Specifically, the immunological technique described in Barres BA, Silverstein BE, Corey DP, Chun LLY. Immunological, morphological, and elecrtrophysiological variation among retinal ganglion calls purified by panning. Neuron. 1988; 1: 791-803. A two-step panning method was used.
  • RGC retinal ganglion cells
  • the survival rate of the cultured cells was measured as follows. Live-dead assay was used to differentiate between live and dead cells (Kashiwagi F, Kashiwagi K, Iizuka Y, Tsukahara S. Effects of brain-derived neurotrophic factor and neurotrophin-4 on isolated cultured: by flow cytometry. Invest Ophthalmol Vis Sci. 2000 Jul; 41 (8): 2373-7., and Kashiwagi K, Iizuka Y, Araie M, Suzuki Y, Tsukahara S. Effects of retinal glial cells on isolated rat sretinal Invest Ophthalmol Vis Sci.2001 Oc; 42 (11): 2686-94.). As a result, viable cells and dead cells show different fluorescent staining. Cells colored by the assay were evaluated by masking the cell conditions under a fluorescence microscope, and live cells and dead cells were evaluated, and the survival rate was quantitatively measured.
  • FIGS. 4 and 5 and Table 1 The results are shown in FIGS. 4 and 5 and Table 1.
  • the survival rate of cultured cells was significantly decreased in SD mice (homozygotes) compared to wild type mice.
  • the SD cell homozygote
  • the survival rate of cultured cells was significantly increased in SA mice (homozygotes) compared to wild-type mice.
  • SD mice are not accompanied by nerve inflammation, the restricted part is limited to retinal ganglion cells, neuropathy progresses over time, normal intraocular pressure It satisfies the conditions such as being maintained as it is, and exhibits symptoms similar to those of human normal-tension glaucoma. Therefore, the SD mouse can be used as a normal-tension glaucoma model. Therefore, it can be seen that the non-human mammal of some aspects of the present invention can be used as a normal-tension glaucoma model non-human mammal.
  • SA mice used as a control model were normal without retinal ganglion cell damage causing normal-tension glaucoma.
  • phosphorylation of the Cdk5 phosphorylation site of the proto-oncogene c-Src is constantly suppressed. Therefore, a drug that suppresses phosphorylation of the Cdk5 phosphorylation site of the proto-oncogene c-Src can suppress damage to retinal ganglion cells that cause normal pressure glaucoma. Therefore, the non-human mammal of some aspects of the present invention can be used in a screening method for a prophylactic and / or therapeutic agent for normal tension glaucoma.
  • [SEQ ID NO: 1] is a nucleotide sequence encoding mouse c-Src protein.
  • SEQ ID NO: 2 is an amino acid sequence of mouse c-Src protein.

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Abstract

La présente invention concerne un mammifère non humain génétiquement modifié, chez lequel une mutation, substituant Asp ou Glu à Ser sur le site de phosphorylation de la Cdk5 de la protéine c-Src, a été introduite dans le gène Src. Ainsi, l'invention concerne un nouveau mammifère non humain et équivalent, qui peut être utilisé comme modèle du glaucome à tension normale.
PCT/JP2012/056627 2011-03-16 2012-03-15 Modèle mammifère non humain du glaucome à tension normale WO2012124752A1 (fr)

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WO2021201170A1 (fr) * 2020-03-31 2021-10-07 スカイファーマ株式会社 Procédé de criblage, procédé de production et procédé de conception de principes actifs de médicaments

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KATO,G. ET AL.: "A functional analysis of the Cdk5-mediated phosphorylation of c-Src using SrcS75D knock-in mice kno.", JOURNAL OF JAPANESE BIOCHEMICAL SOCIETY, 2006, pages A10666 *
KATO,G. ET AL.: "Neuron-specific Cdk5 kinase is responsible for mitosis-independent phosphorylation of c-Src at Ser75 in human Y79 retinoblastoma cells.", J. BIOCHEM., vol. 126, no. 5, November 1999 (1999-11-01), pages 957 - 61 *
KATO,G. ET AL.: "Production of mouse ES cells homozygous for Cdk5-phosphorylated site mutation in c-Src alleles.", J. BIOCHEM., vol. 133, no. 5, May 2003 (2003-05-01), pages 563 - 9 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021201170A1 (fr) * 2020-03-31 2021-10-07 スカイファーマ株式会社 Procédé de criblage, procédé de production et procédé de conception de principes actifs de médicaments

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