WO2023236958A1 - 一种plau和/或plaur基因修饰的非人动物 - Google Patents

一种plau和/或plaur基因修饰的非人动物 Download PDF

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WO2023236958A1
WO2023236958A1 PCT/CN2023/098641 CN2023098641W WO2023236958A1 WO 2023236958 A1 WO2023236958 A1 WO 2023236958A1 CN 2023098641 W CN2023098641 W CN 2023098641W WO 2023236958 A1 WO2023236958 A1 WO 2023236958A1
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human
exon
plaur
plau
animal
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PCT/CN2023/098641
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English (en)
French (fr)
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吕锐利
王琳琳
沈志远
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百奥赛图(北京)医药科技股份有限公司
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Publication of WO2023236958A1 publication Critical patent/WO2023236958A1/zh

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • 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
    • 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

Definitions

  • the invention provides a non-human animal expressing human or chimeric (eg, humanized) PLAU and/or PLAUR proteins and methods of using the same.
  • the present application provides an animal model with human or chimeric PLAU and/or PLAUR proteins.
  • the animal model can express human or chimeric PLAU (eg, humanized PLAU) protein and/or human or chimeric PLAUR (eg, humanized PLAUR) protein. It can be used to study the functions of PLAU and PLAUR genes, and can also be used to screen and evaluate modulators of the PLAU/PLAUR signaling pathway (e.g., anti-human PLAU antibodies or anti-human PLAUR antibodies).
  • the animal model prepared by the method described herein can be used for drug screening, pharmacodynamic research, treatment of immune-related diseases and cancer treatment of human PLAU/PLAUR target sites; the model can also be used to promote the development and design of new drugs, Save time and costs.
  • the present invention provides a powerful tool for studying the function of PLAU/PLAUR protein and a platform for screening anti-cancer drugs.
  • the invention provides a genetically modified non-human animal, one or more cells of the animal expressing a human or chimeric PLAUR protein, and a human PLAUR ligand (PLAU) can bind to the expressed human or chimeric PLAUR protein. , activate downstream signaling pathways.
  • the nucleotide sequence encoding a human or chimeric PLAUR protein is operably linked to an endogenous regulatory element (e.g., endogenous 5'UTR and/or 3'UTR).
  • the nucleotide sequence encoding a human or chimeric PLAUR protein includes 3 homology domains encoding a human PLAUR protein.
  • the nucleotide sequence encoding a human or chimeric PLAUR protein encodes an amino acid sequence that is at least 70%, 75%, or 75% identical to human PLAUR (NP_002650.1, SEQ ID NO: 10). 80%, 85%, 90%, 95%, 99% or 100%. In some embodiments, the nucleotide sequence encoding a human or chimeric PLAUR protein encodes an amino acid sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the nucleotide sequence encoding a human or chimeric PLAUR protein comprises at least 70%, 75%, 80%, 85%, 90% identity to the nucleotide sequence set forth in SEQ ID NO: 15 %, 95%, 99% or 100%.
  • the animal is a mammal, such as a monkey, rodent, mouse, or rat. In some embodiments, the animal is a mouse.
  • one or more cells of the animal express human or chimeric PLAUR protein.
  • one or more cells of the animal express human or chimeric PLAUR protein, and endogenous PLAUR ligand (PLAU) can bind to cells expressing human or chimeric PLAUR protein and activate downstream signaling pathways.
  • one or more cells of the animal express human or chimeric PLAUR protein, and human PLAUR ligand (PLAU) can bind to the expressed human or chimeric PLAUR protein and activate downstream signaling pathways.
  • the invention provides a genetically modified non-human animal, the genome of which comprises a nucleotide sequence encoding an endogenous PLAUR region at the endogenous PLAUR locus substituted by a nucleotide sequence encoding the corresponding region of human PLAUR. replace.
  • the nucleotide sequence encoding a corresponding region of human PLAUR is operably linked to an endogenous regulatory element of an endogenous PLAUR locus, and one or more cells of the animal express human or humanized PLAUR protein.
  • the animal's endogenous PLAUR protein is not expressed or is expressed at a reduced level compared to PLAUR in a wild-type animal.
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and/or part of exon 7.
  • the identity of the nucleotide sequence encoding the corresponding region of human PLAUR to the nucleotide sequence shown in SEQ ID NO: 13 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100%.
  • the nucleotide sequence encoding the endogenous PLAUR region includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and/or part of exon 7.
  • the modified gene in the animal's genome is homozygous or heterozygous for the endogenously replaced locus.
  • the invention provides a non-human animal, the animal comprising at least one cell encoding a nucleotide sequence encoding a human or humanized PLAUR protein, wherein the human or humanized PLAUR protein comprises a protein corresponding to a human
  • the continuous amino acid sequence of the region is identical for at least 50, 100, 150, 200, 250, 300, 310, 311, 312, 320, 330, 331, 332, 333, 334 or 335 consecutive amino acid sequences.
  • the human or humanized PLAUR protein comprises 3 homologous domains of human PLAUR protein.
  • the amino acid sequence of the human or humanized PLAUR protein is at least 70%, 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 10. 99%, or 100%.
  • the nucleotide sequence encoding the corresponding region of human PLAUR is operably linked to an endogenous PLAUR regulatory element.
  • the encoding corresponding region of human PLAUR The nucleotide sequence can be integrated into the animal's endogenous PLAUR locus.
  • the human or humanized PLAUR protein has at least one mouse PLAUR activity and/or human PLAUR activity.
  • the invention provides a method for constructing a genetically modified non-human animal.
  • the nucleotide sequence encoding the endogenous PLAUR region is modified by human Nucleotide sequence replacement of the corresponding region of PLAUR.
  • the animal's endogenous PLAUR protein is not expressed or is expressed at a reduced level compared to PLAUR in a wild-type animal.
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes three homologous domains encoding human PLAUR protein.
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and /or part of exon 7.
  • the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human PLAUR includes at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
  • the nucleotide sequence encoding the endogenous PLAUR region includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and/or part of exon 7.
  • the nucleotide sequence encoding the corresponding region of human PLAUR is operably linked to an endogenous PLAUR regulatory element, such as a promoter.
  • the animal is a mammal, such as a monkey, rodent, mouse or rat.
  • the animal is a mouse.
  • the invention provides a method for constructing a non-human animal expressing human or chimeric PLAUR gene modification, the method comprising at the endogenous mouse PLAUR locus, a nucleotide sequence encoding the endogenous PLAUR region Replacement of the nucleotide sequence with the corresponding region of human PLAUR results in genetically modified non-human animal cells expressing human or chimeric PLAUR proteins.
  • the human or chimeric PLAUR protein comprises 3 homology domains of human PLAUR protein.
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and /or part of exon 7.
  • the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human PLAUR includes at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human PLAUR to the nucleotide sequence shown in SEQ ID NO: 13 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100%.
  • the nucleotide sequence of endogenous PLAUR includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and/or of the mouse PLAUR gene. or part of exon 7.
  • the nucleotide sequence encoding a human or chimeric PLAUR protein is operably linked to a regulatory element of endogenous PLAUR, such as a promoter.
  • the animal is a mammal, such as a monkey, rodent, mouse or rat.
  • the non-human animal is a mouse.
  • the invention provides a construction of genetically modified non-human animal cells expressing human or chimeric PLAUR Method, the method includes replacing the nucleotide sequence encoding the endogenous PLAUR region with the nucleotide sequence of the corresponding region of human PLAUR at the endogenous mouse PLAUR locus to generate genetically modified non-human animal cells, said Animal cells express human or chimeric PLAUR proteins.
  • the human or chimeric PLAUR protein comprises 3 homology domains of human PLAUR protein.
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and /or part of exon 7.
  • the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human PLAUR includes at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human PLAUR to the nucleotide sequence shown in SEQ ID NO: 13 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100%.
  • the nucleotide sequence of endogenous PLAUR includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and/or of the mouse PLAUR gene. or part of exon 7.
  • the nucleotide sequence encoding a human or chimeric PLAUR protein is operably linked to a regulatory element of endogenous PLAUR, such as a promoter.
  • the non-human animal is a mouse.
  • the non-human animal further includes nucleotide sequences encoding human or chimeric proteins selected from the group consisting of PLAUR ligand (PLAU), IL1B, IL6, IL15, At least one of PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4 and TNF- ⁇ .
  • the human or chimeric protein is a human or chimeric PLAU protein.
  • the human or chimeric PLAU protein comprises the full-length protein encoded by the human PLAU gene.
  • the amino acid sequence of the human or chimeric PLAU protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99 identical to the amino acid sequence shown in SEQ ID NO: 2 %, or 100%.
  • the non-human animal further includes nucleotide sequences encoding human or chimeric proteins from other genes, wherein the human or chimeric proteins are selected from the group consisting of PLAUR ligand (PLAU), IL1B, IL6, IL15 , at least one of PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4 and TNF- ⁇ .
  • the human or chimeric protein is a human or chimeric PLAU protein.
  • the human or chimeric PLAU protein comprises the full-length protein encoded by the human PLAU gene.
  • the amino acid sequence of the human or chimeric PLAU protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99 identical to the amino acid sequence shown in SEQ ID NO: 2 %, or 100%.
  • the invention provides a genetically modified non-human animal whose genome comprises at least one chromosome comprising a nucleus encoding a human or chimeric urokinase-type plasminogen activator (PLAU) protein. nucleotide sequence.
  • the nucleotide sequence encoding a human or chimeric PLAU protein is operably linked to a regulatory element of the endogenous PLAU locus of at least one chromosome (e.g., the endogenous 5' UTR and/or 3' UTR).
  • the nucleotide sequence encoding a human or chimeric PLAU protein encodes an amino acid sequence that is at least 70%, 75%, 80%, 85% identical to the amino acid sequence set forth in SEQ ID NO: 2 , 90%, 95%, 99%, or 100%.
  • the animal is a mammal, such as a monkey, a rodent, a mouse or a rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous PLAUR protein or has a reduced expression level compared to PLAU in a wild-type animal. In some embodiments, one or more cells of the animal express human or chimeric PLAU protein.
  • one or more cells of the animal express human or chimeric PLAU protein, and the endogenous PLAU receptor (PLAUR) can bind to the expressed human or chimeric PLAU protein and activate downstream signaling pathways. In some embodiments, one or more cells of the animal express human or chimeric PLAU protein, and human PLAU receptor (PLAUR) can bind to the expressed human or chimeric PLAU protein to activate downstream signaling pathways.
  • the invention provides a genetically modified non-human animal, the genome of the animal comprising at the endogenous PLAU locus, a nucleotide sequence encoding the endogenous PLAU region is replaced by a nucleotide sequence of the corresponding region of human PLAU. Sequence replacement.
  • the nucleotide sequence encoding a corresponding region of human PLAU is operably linked to a regulatory element of an endogenous PLAU locus, and one or more cells of the animal express a human or humanized PLAU protein.
  • the animal does not express endogenous PLAUR protein or has a reduced expression level compared to PLAU in a wild-type animal.
  • the nucleotide sequence encoding the corresponding region of human PLAU includes part of exon 2, exon 3, exon 4, exon 5, exon 6, Portions of exon 7, exon 8, exon 9, exon 10, and/or exon 11. In some embodiments, the nucleotide sequence encoding the corresponding region of human PLAU includes the entire nucleotide sequence of the human PLAU coding region. In some embodiments, the identity of the nucleotide sequence encoding the corresponding region of human PLAU to the nucleotide sequence shown in SEQ ID NO: 7 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human PLAU to the nucleotide sequence shown in SEQ ID NO: 8 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100%.
  • the nucleotide sequence encoding the endogenous PLAU region includes part of exon 2, exon 3, exon 4, exon 5, exon 6, Portions of exon 7, exon 8, exon 9, exon 10, and/or exon 11.
  • the modified gene in the animal's genome is homozygous or heterozygous for the endogenously replaced locus.
  • the invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or humanized PLAU protein, wherein the humanized PLAU protein comprises a region corresponding to a human
  • the continuous amino acid sequences are identical for at least 50, 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, or 431 consecutive amino acids.
  • the amino acid sequence of the humanized PLAU protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the amino acid sequence shown in SEQ ID NO: 2 , or 100%.
  • the nucleotide sequence encoding a human or humanized PLAU protein is operably linked to an endogenous PLAU regulatory element. In some embodiments, the nucleotide sequence encoding a human or humanized PLAU protein can be integrated into the animal's endogenous PLAU locus. In some embodiments, the humanized PLAU protein has at least one mouse PLAU activity and/or human PLAU activity.
  • the invention provides a method for constructing a genetically modified non-human animal.
  • the nucleotide sequence encoding the endogenous PLAU region is modified by human Nucleotide sequence replacement of the corresponding region of PLAU.
  • the endogenous PLAU protein of the non-human animal is not expressed or has a reduced expression level compared to PLAU in a wild-type animal.
  • the nucleotide sequence encoding the corresponding region of human PLAU includes the entire sequence encoding the human PLAU protein.
  • the nucleotide sequence encoding the corresponding region of human PLAU includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and exon of the human PLAU gene. Portions of exon 7, exon 8, exon 9, exon 10, and/or exon 11.
  • the nucleotide sequence encoding the corresponding region of human PLAU comprises an encoded amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% identical to the amino acid sequence shown in SEQ ID NO: 2. %, 95%, 99%, or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human PLAU to the nucleotide sequence shown in SEQ ID NO: 7 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100%.
  • the nucleotide sequence encoding the endogenous PLAU region includes part of exon 2, exon 3, exon 4, exon 5, exon 6, Portions of exon 7, exon 8, exon 9, exon 10, and/or exon 11.
  • the modified gene in the animal's genome is homozygous or heterozygous for the endogenously replaced locus.
  • the invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or humanized PLAU protein, wherein the humanized PLAU protein comprises a region corresponding to a human
  • the continuous amino acid sequences are identical for at least 50, 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, or 431 consecutive amino acids.
  • the amino acid sequence of the humanized PLAU protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the amino acid sequence shown in SEQ ID NO: 2 , or 100%.
  • the nucleotide sequence encoding a human or humanized PLAU protein is operably linked to an endogenous PLAU regulatory element. In some embodiments, the nucleotide sequence encoding a human or humanized PLAU protein can be integrated into the animal's endogenous PLAU locus. In some embodiments, the humanized PLAU protein has at least one mouse PLAU activity and/or human PLAU activity.
  • the invention provides a method for constructing a genetically modified non-human animal.
  • the nucleotide sequence encoding the endogenous PLAU region is modified by human Nucleotide sequence replacement of the corresponding region of PLAU.
  • the endogenous PLAU protein of the non-human animal is not expressed or has a reduced expression level compared to PLAU in a wild-type animal.
  • the nucleotide sequence encoding the corresponding region of human PLAU includes the entire sequence encoding the human PLAU protein.
  • the nucleotide sequence encoding the corresponding region of human PLAU includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and exon of the human PLAU gene. Portions of exon 7, exon 8, exon 9, exon 10, and/or exon 11.
  • the The nucleotide sequence encoding the corresponding region of human PLAU includes the encoded amino acid sequence and the identity of the amino acid sequence shown in SEQ ID NO: 2 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% , or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human PLAU to the nucleotide sequence shown in SEQ ID NO: 7 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100%.
  • the nucleotide sequence encoding the endogenous PLAU region includes part of exon 2, exon 3, exon 4, exon 5, exon 6, Portions of exon 7, exon 8, exon 9, exon 10, and/or exon 11.
  • the nucleotide sequence encoding the corresponding region of human PLAU is operably linked to a regulatory element of endogenous PLAU, such as a promoter.
  • the animal is a mammal, such as a monkey, rodent, mouse, or rat. In some embodiments, the animal is a mouse.
  • the invention provides a method for constructing cells of genetically modified non-human animals that express human or chimeric PLAU proteins, the method comprising using a nucleic acid sequence encoding the corresponding region of human PLAU at the endogenous mouse PLAU locus.
  • the nucleotide sequence encoding the endogenous PLAU region is replaced by nucleotides, resulting in genetically modified non-human animal cells, wherein the animal cells express human or chimeric PLAU proteins.
  • the nucleotide sequence encoding the corresponding region of human PLAU includes the entire sequence of the human PLAU protein.
  • the nucleotide sequence encoding the corresponding region of human PLAU includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and exon of the human PLAU gene. Portions of exon 7, exon 8, exon 9, exon 10, and/or exon 11.
  • the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human PLAU contains at least 70%, 75%, 80%, 85%, 90 identity with the amino acid sequence shown in SEQ ID NO: 2 %, 95%, 99%, or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human PLAU to the nucleotide sequence shown in SEQ ID NO: 7 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99%, or 100%.
  • the nucleotide sequence encoding the endogenous PLAU region includes part of exon 2, exon 3, exon 4, exon 5, exon 6, Portions of exon 7, exon 8, exon 9, exon 10, and/or exon 11.
  • the animal is a mouse.
  • the nucleotide sequence encoding a human or chimeric PLAU protein is operably linked to a regulatory element of endogenous PLAU, such as a promoter.
  • the animal further includes nucleotide sequences encoding human or chimeric proteins from other genes, wherein the human or chimeric proteins are selected from the group consisting of PLAU receptor (PLAUR), IL1B, IL6, IL15, PD -1, at least one of PD-L1, TIGIT, LAG3, CD226, CTLA4 and TNF- ⁇ .
  • PLAU receptor PLAU receptor
  • the human or chimeric protein is a human or chimeric PLAUR protein.
  • the amino acid sequence of the human or chimeric PLAUR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99 identical to the amino acid sequence shown in SEQ ID NO: 14 %, or 100%.
  • the animal further includes nucleotide sequences encoding human or chimeric proteins from other genes, wherein the human or chimeric proteins are selected from the group consisting of PLAU receptor (PLAUR), IL1B, IL6, IL15, PD -1, at least one of PD-L1, TIGIT, LAG3, CD226, CTLA4 and TNF- ⁇ .
  • PLAU receptor PLAU receptor
  • the human or chimeric protein is a human or chimeric PLAUR protein.
  • the amino acid sequence of the human or chimeric PLAUR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99 identical to the amino acid sequence shown in SEQ ID NO: 14 %, or 100%.
  • the invention provides a method for determining the effectiveness of an anti-PLAU and/or PLAUR antibody in treating cancer, the method comprising: 1) administering an anti-PLAU and/or PLAUR antibody to the animal described in the application, wherein said The animals have tumors; 2) Determine the inhibitory effect of anti-PLAU and/or PLAUR antibodies on tumors.
  • the tumor contains one or more tumor cells, wherein the tumor cells are injected into the animal.
  • determining the inhibitory effect of an anti-PLAU/PLAUR antibody on tumors includes measuring tumor volume in the animal.
  • the tumor includes breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor , mesothelial tissue tumors, oropharyngeal tumors, female reproductive system cancers or meningiomas.
  • the invention provides a method for determining the effectiveness of anti-PLAU and/or PLAUR antibodies and other therapeutic agents in treating cancer, the method comprising: 1) administering anti-PLAU and/or PLAUR to an animal described in the application Antibodies, wherein the animals have tumors; 2) determining the inhibitory effect of anti-PLAU and/or PLAUR antibodies on tumors.
  • the animal further includes sequences encoding human or chimeric PD-1, human or chimeric PD-L1, and/or human or chimeric CTLA4.
  • the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and/or an anti-CTLA4 antibody.
  • the tumor contains one or more cells expressing PD-L1 protein. In some embodiments, the tumor contains one or more tumor cells, wherein the tumor cells are injected into the animal. In some embodiments, determining the inhibitory effect of an anti-PLAU/PLAUR antibody on tumors includes measuring tumor volume in the animal.
  • the tumor is breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor , mesothelial tissue tumors, oropharyngeal tumors, female reproductive system cancers or meningiomas.
  • the invention provides a method for determining the effectiveness of anti-PLAU and/or PLAUR antibodies in treating immune diseases, the method comprising: 1) administering anti-PLAU and/or PLAUR antibodies to the non-human animals described in the present application, wherein the non-human animal has an immune disease; 2) determining the effect of anti-PLAU and/or PLAUR antibodies in treating immune diseases.
  • the immune disease is skin ulcer disease, rheumatoid arthritis, stroke.
  • the invention provides a method for determining the effectiveness of an anti-PLAU and/or PLAUR antibody in treating inflammation, the method comprising: 1) administering an anti-PLAU and/or PLAUR antibody to an animal described in the present application, wherein The animal has inflammation; 2) determine the effectiveness of anti-PLAU and/or PLAUR antibodies in treating inflammation.
  • the inflammation is sepsis or an inflammatory disease.
  • the invention provides a method for determining the toxicity of anti-PLAU and/or PLAUR antibodies, said method The method includes: 1) administering anti-PLAU and/or PLAUR antibodies to the animals described in this application; 2) determining the effects of anti-PLAU and/or PLAUR antibodies on the animals. In some embodiments, determining the effect of anti-PLAU and/or PLAUR antibodies on an animal involves measuring the animal's body weight, red blood cell count, hematocrit, and/or hemoglobin.
  • the invention provides a humanized PLAUR protein, which contains three homologous domains of human PLAUR protein.
  • the amino acid sequence of the humanized PLAUR protein has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
  • the amino acid sequence of the humanized PLAUR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the amino acid sequence shown in SEQ ID NO: 15 , or 100%.
  • the present invention provides a humanized PLAUR gene encoding the above-mentioned humanized protein.
  • the humanized PLAUR gene includes part of exon 2, exon 3, exon 4, exon 5, exon 6, and/or part of exon 7 of the human PLAUR gene.
  • the humanized PLAUR gene contains a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90% identical to the nucleotide sequence shown in SEQ ID NO: 14. 95%, 99%, or 100%.
  • the invention provides a humanized PLAU protein comprising all or part of the human PALU protein.
  • the amino acid sequence of the humanized PLAU protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the amino acid sequence shown in SEQ ID NO: 2 , or 100%.
  • the present invention provides a humanized PLAU gene encoding the above-mentioned humanized protein.
  • the humanized PLAU gene includes part of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 2 of the human PLAU gene. Portions of exon 8, exon 9, exon 10, and/or exon 11.
  • the humanized PLAU gene contains a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90% identical to the nucleotide sequence shown in SEQ ID NO: 8. 95%, 99%, or 100%.
  • the invention provides a cell, said cell comprising the above-mentioned humanized PLAUR gene and/or the above-mentioned humanized PLAU gene, and/or said cell expressing any one of the humanized PLAUR genes protein and/or any of the humanized PLAU proteins.
  • the invention provides an animal model, the animal model comprising any of the humanized PLAUR genes and/or any of the humanized PLAU genes, and/or, the animal model Express any one of the humanized PLAUR proteins and/or any one of the humanized PLAU proteins.
  • Figure 1 Schematic diagram comparing mouse PLAU gene and human PLAU locus (not to scale);
  • FIG. 1 Schematic diagram of humanized transformation of mouse PLAU gene (not to scale);
  • FIG. 3 Schematic diagram of PLAU gene targeting strategy and targeting vector design (not to scale);
  • Figure 4 Schematic diagram of the FRT recombination process in PLAU gene humanized mice (not to scale);
  • FIG. 5 Schematic diagram of PLAU gene targeting strategy and targeting vector design (not to scale);
  • Figure 6 PCR identification results of the F1 generation mouse tail of PLAU gene humanized mice, where WT is the wild-type control, H 2 O is the water control, and M is the Marker;
  • FIG. 7 Schematic diagram of Southern Blot detection results, in which WT is the wild-type control
  • Figure 8 RT-PCR detection results of C57BL/6 wild-type mice (+/+) and PLAU gene humanized heterozygous mice (H/+), where H 2 O is water control and GAPDH is glyceraldehyde- 3-phosphate dehydrogenase internal reference;
  • Figure 9 Schematic diagram comparing mouse PLAUR gene and human PLAUR locus (not to scale);
  • Figure 10 Schematic diagram of humanized transformation of mouse PLAUR gene (not to scale);
  • FIG 11 Schematic diagram of PLAUR gene targeting strategy and targeting vector design (not to scale);
  • Figure 12 Schematic diagram of the FRT recombination process in humanized mice with PLAUR gene (not to scale);
  • FIG 13 PCR identification results of the F1 generation of PLAUR gene humanized mice, in which WT is the wild-type control, PC is the positive control, H 2 O is the water control, and M is Marker;
  • Figure 14 RT-PCR detection results of C57BL/6 wild-type mice (+/+) and PLAUR gene humanized heterozygous mice (H/+), where H 2 O is water control and GAPDH is glyceraldehyde- 3-phosphate dehydrogenase internal reference;
  • Figure 15 Western blot detection results of C57BL/6 wild-type mice (+/+) and PLAU gene humanized homozygous mice (H/H), where hPLAU is human PLAU protein and ⁇ -actin is ⁇ -actin. internal reference.
  • Figure 16 ELSA detects the expression of PLAU in serum.
  • Figure 16A shows the detection results of mouse PLAU protein expression;
  • Figure 16B shows the detection results of human PLAU protein expression.
  • Figure 17 A schematic diagram of the tumor volume results of mice after implanting PLAUR humanized colon cancer cell MC38 into PLAU/PLAUR double humanized homozygous mice for anti-tumor efficacy testing;
  • Figure 18 A schematic diagram of the body weight results of mice after implanting PLAUR humanized colon cancer cell MC38 into PLAU/PLAUR double humanized homozygous mice and conducting an anti-tumor efficacy test;
  • Figure 19 Human PLAU amino acid sequence (NP_002649.2; SEQ ID NO: 2) and mouse PLAU amino acid sequence (NP_032899.1; SEQ ID NO: 1);
  • Figure 20 Human PLAU amino acid sequence (NP_002649.2; SEQ ID NO: 2) and rat PLAU amino acid sequence (NP_037217.3; SEQ ID NO: 54);
  • Figure 21 Human PLAUR amino acid sequence (NP_002650.1; SEQ ID NO: 10) and mouse PLAUR amino acid sequence (NP_035243.1; SEQ ID NO: 9);
  • Figure 22 Human PLAUR amino acid sequence (NP_002650.1; SEQ ID NO: 10) and rat PLAUR amino acid sequence (NP_599179.2; SEQ ID NO: 55);
  • UPAS urokinase plasminogen activator system
  • ECM extracellular matrix
  • UPAS urokinase plasminogen activator system
  • PAU prourokinase activator
  • PUAUR prourokinase activator receptor
  • PAI-1 plasminogen activator inhibitor-1
  • PAI-2 plasminogen activator inhibitor-2
  • the regulation of UPAS is strict and cell-specific, involving the regulation of zymogens, enzyme inhibitors and receptors, and plays a role in the reconstruction of the ECM during physiological processes such as embryonic development, wound healing and post-lactation breast tissue development. important role.
  • UPAS In addition to its role in physiological processes, UPAS is active in most tumors, and abnormalities in its regulatory function are related to tumor metastasis phenotype and development. Studies have found that PLAU and PLAUR are closely related to the invasion and metastasis of malignant tumors. They play an important role in extracellular matrix degradation, tissue remodeling, angiogenesis, and cell infiltration and proliferation. They have certain influence on the judgment of tumor malignancy and prognosis. The guiding significance
  • Urokinase-type plasminogen activator (uPA) receptor PAAUR
  • PKAUR Urokinase-type plasminogen activator receptor
  • GPI Glycosylphosphatidylinositol anchor receptor. It contains 3 homologous domains (DI, DII and DIII), which are connected by disulfide bonds. Because of the lack of transmembrane and intracellular regions, PLAUR must cooperate with integrins, protein-coupled receptors (GPCRs), and growth factor receptors to form co-receptors to activate intracellular signals and promote cell movement, invasion, and proliferation. and survive.
  • GPCRs protein-coupled receptors
  • vitronectin is another key ligand of PLAUR, and the binding sites between these two ligands and the receptor are separated.
  • PLAUR simultaneously binds the above two ligands to jointly perform downstream regulation of proteases, cell adhesion and signal transduction.
  • the expression of PLAUR gene is regulated through transcriptional and post-transcriptional binding mechanisms.
  • the upstream region of the PLAUR gene contains cis-acting elements of many transcription factors, including Sp1, AP1, AP2, PEA3, Kruppel-ike factor 4, hypoxia factor 1 ⁇ (HIF-1 ⁇ ), NF- ⁇ B, TCF and LEF, etc. Signaling pathways activate these transcription factors that act on the PLAUR gene, thereby coordinating the expression of PLAUR in tumors.
  • PLAU and PLAUR regulate macrophages, neutrophils, and endothelial cells in the inflamed synovium of patients. Expressed in the synovium of healthy people, PLAU and PLAUR have also received increasing attention in the field of RA treatment.
  • the PLAUR gene (Gene ID: 5329) contains 7 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and exon 7 (Fig. 9).
  • the nucleotide sequence of human PLAUR mRNA is NM_002659.4, and the amino acid sequence of human PLAUR is NP_002650.1 (SEQ ID NO: 10).
  • the corresponding position of each exon is as follows:
  • the human PLAUR gene (NCBI Gene ID: 5329) is located at positions 43646095 to 43670197 of NC_000019.10 on chromosome 19 (GRCh38.p13 (GCF_000001405.39). Based on each nucleotide sequence of the transcript NM_002659.4 The specific positions of the exons are: 5'UTR is located at positions 43670169 to 43670121 of NC_000019.10, exon 1 is located at positions 43670169 to 43670066 of NC_000019.10, intron 1 is located at positions 43670065 to 43667692 of NC_000019.10, exon Exon 2 is located at positions 43667691 to 43667581 of NC_000019.10, intron 2 is located at positions 43667580 to 43665460 of NC_000019.10, exon 3 is located at positions 43665459 to 43665316 of NC_000019.10, and intron 3 is located at NC_000019.1 0th 43665315
  • the 3'UTR is located at positions 43648889 to 43648579 of NC_000019.10. All the above relevant information about the human PLAUR locus can be retrieved on the NCBI website (Gene ID: 5329). The entire contents of which are incorporated herein by reference.
  • the PLAUR gene (Gene ID: 18793) contains 7 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and exon 7 (Fig. 9).
  • the nucleotide sequence of mouse PLAUR mRNA is NM_011113.4
  • the amino acid sequence of mouse PLAUR is NP_035243.1 (SEQ ID NO: 9). Based on the nucleotide sequence and amino acid sequence of transcript NM_011113.4 and its encoded protein NP_035243.1, the corresponding position of each exon is as follows:
  • the mouse PLAUR gene (NCBI Gene ID: 18793) is located at positions 24161857 to 24175393 of NC_000073.7 on chromosome 7 (GRCm39 (GCF_000001635.27)).
  • the specific position of each exon based on the nucleotide sequence of transcript NM_011113.4 is: 5'UTR is located at positions 24161909 to 24161934 of NC_000073.7, exon 1 is located at positions 24161909 to 24161992 of NC_000073.7, within Intron 1 is located at positions 24161993 to 24164555 of NC_000073.7, exon 2 is located at positions 24164556 to 24164666 of NC_000073.7, intron 2 is located at positions 24164667 to 24166099 of NC_000073.7, and exon 3 is located at position NC_000073.7 Positions 24166100 to 24166243, intron 3 is located at positions 24166244 to 24171323 of NC_000073.7, exon 4 is located at positions 2417
  • Figure 21 shows an alignment of the human PLAUR amino acid sequence (NP_002650.1; SEQ ID NO: 10) and the mouse PLAUR amino acid sequence (NP_035243.1; SEQ ID NO: 9). Therefore, the corresponding amino acid residues or regions between human and mouse PLAUR can be found in Figure 21.
  • PLAUR genes, proteins and gene loci for other species are also known in the art.
  • the Gene ID of PLAUR in Rattus norvegicus (rat) is 50692
  • the Gene ID of PLAUR in Macaca mulatta (rhesus monkey) is 710662
  • the gene ID of PLAUR in Canis lupus familiaris (dog) is 476446
  • the gene ID of PLAUR in Sus scrofa (pig) is 476446.
  • the gene ID of PLAUR is 100521017. Relevant information of these genes (eg, intron sequences, exon sequences, and amino acid sequences) can be found in NCBI, the entire content of which is incorporated herein by reference.
  • Figure 22 shows the human PLAUR amino acid sequence (NP_002650.1; SEQ ID NO: 10) and the rat PLAUR amino acid sequence (NP_599179.2; SEQ ID NO: 55). Therefore, the homology region between human and rat PLAUR amino acid sequences can be retrieved in Figure 22.
  • the invention provides a human or chimeric (eg, humanized) PLAUR nucleotide sequence or amino acid sequence.
  • the nucleosides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or exon 7 of the mouse PLAUR gene All or part of the acid sequence is replaced by the corresponding nucleotide sequence of the human PLAUR gene.
  • the "region of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of the mouse PLAUR gene " or "part” is replaced by the corresponding nucleotide sequence or amino acid sequence of the human PLAUR gene.
  • the "area” or “portion” refers to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 930, 940, 950, 1000, 1100, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507 or 1508 bp contiguous nucleotide sequence, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 ,90,100,150,200,220,240,260,280,300,301,302,303,304,305,306,307,308,30
  • the "region" or "portion” is associated with exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or exon
  • the amino acid sequence identity encoded by sub7 is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100%.
  • exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or exon 7 of the mouse PLAUR gene e.g., Part of exon 2, all of exons 3-6 and part of exon 7)
  • the "region", "part” or “whole” sequence is replaced by the corresponding exon 1, exon of the human PLAUR gene 2.
  • Exon 3, exon 4, exon 5, exon 6 and/or exon 7 for example, part of exon 2, all of exons 3-6 and exon 7) "area", "partial” or "whole” sequence replacement.
  • a "region" or "region” of endogenous exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or exon 7 part" is missing.
  • the invention provides a genetically modified non-human animal whose genome includes a human, chimeric or humanized PLAUR nucleotide sequence.
  • the protein encoded by the human, chimeric or humanized PLAUR nucleotide sequence is at least 70%, 80%, 85%, 90% identical to the amino acid sequence shown in SEQ ID NO: 10 , 95% or 100%.
  • the non-human animal genome contains a nucleotide sequence that is at least 70%, 80%, 85%, 90% identical to the nucleotide sequence shown in SEQ ID NO: 11, 12, 13, 14. %, 95% or 100%.
  • the non-human animals described herein comprise a nucleotide sequence encoding a human or humanized PLAUR protein.
  • the human or humanized PLAUR protein comprises the 3 homology domains of the human PLAUR protein, including DI, DII, and DIII.
  • the non-human animals described herein comprise a human or humanized PLAUR gene.
  • the humanized PLAUR gene contains 7 exons.
  • the humanized PLAUR gene comprises humanized exon 2, human exon 3, human exon 4, human exon 5, human exon 6 and/or human Exon 7.
  • the humanized PLAUR gene comprises human intron 2, human intron 3, human intron 4, human intron 5 and/or human intron 6.
  • the humanized PLAU gene comprises a human or humanized 5' UTR.
  • the humanized PLAUR gene comprises a human or humanized 3'UTR.
  • the humanized PLAUR gene comprises an endogenous 5' UTR.
  • the humanized PLAUR gene comprises an endogenous 3'UTR.
  • genetically modified non-human animals can express human PLAUR and/or chimeric (e.g., humanized) PLAUR proteins with endogenous PLAUR gene sequences replaced with human PLAUR gene and/or nucleotide sequences.
  • the amino acid sequence encoding human PLAUR encoded by the human PLAUR gene and/or nucleotide sequence is at least 10%, 20%, 30%, 40%, 50%, 60% identical to the amino acid sequence shown in human PLAUR, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 100%.
  • the PLAUR gene of the endogenous non-human animal is replaced with all or part of the nucleotide sequence encoding the mature PLAUR protein.
  • the genetically modified non-human animal expresses human PLAUR and/or chimeric PLAUR protein (e.g., humanized PLAUR). Replacement of the endogenous mouse locus provides a non-human animal that expresses a human or chimeric PLAUR protein (eg, humanized PLAUR) in the same cell type.
  • the genetically modified mice do not develop the underlying diseases known in the art to be observed in certain other transgenic mice.
  • Human PLAUR or chimeric PLAUR proteins expressed in non-human animals can maintain the function of one or more wild-type or human PLAUR proteins, for example, the expressed PLAUR proteins can bind to human or non-human PLAU proteins. Further, in some embodiments, the genetically modified non-human animal does not express endogenous PLAUR protein.
  • the genome of a non-human animal contains an amino acid sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99 identical to the human PLAUR protein (NP_002650.1; SEQ ID NO:10) % or 100% of the amino acid nucleotide sequence.
  • the genome comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or at least 100% nucleotide sequence.
  • nucleotide sequence encoding the endogenous PLAUR region in the genome of the non-human animal is replaced with the nucleotide sequence encoding the corresponding region of human PLAUR.
  • the nucleotide sequence encoding the endogenous PLAUR region is any sequence of the endogenous PLAUR locus, such as exon 1, exon 2, exon 3, exon 4, Exon 5, exon 6, exon 7, 5'UTR, 3'UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron Sub 6 or any combination thereof.
  • the nucleotide sequence encoding an endogenous PLAUR region is located within an endogenous PLAUR regulatory region.
  • the nucleotide sequence encoding the endogenous PLAUR region is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or or exon 7, or portions thereof.
  • One or more cells of the genetically modified non-human animal express a human or chimeric PLAUR protein (e.g., humanized PLAUR protein).
  • the human or chimeric PLAUR protein comprises at least the amino acid sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 as shown in SEQ ID NO: 10 ,50,60,70,80,90,100,150,200,220,240,260,280,300,310,311,312,313,314,315,316,317,318,319,320,330 , 331, 332, 333, 334 or 335 consecutive amino acid sequences.
  • the genetically modified non-human animal genome includes human PLAUR gene exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and/or All or part of exon 7, or all or part of the nucleotide sequence shown in SEQ ID NO: 13.
  • the genetically modified non-human animal genome includes part of exon 2, all of exons 3-6 and part of exon 7 of the human PLAUR gene.
  • the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 93, 95, 97, 100 of exon 2 of the human PLAUR gene or 111bp continuous nucleotide sequence.
  • the portion of exon 2 includes 97 bp of contiguous nucleotide sequence.
  • the portion of exon 2 includes at least 50 bp or at least 100 bp of nucleotide sequence.
  • the portion of exon 7 comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 251, 252, 253 of exon 7 of the human PLAUR gene , 254, 300, 350, 400, 450, 500, 550, 560 or 565 bp continuous nucleotide sequence.
  • the portion of exon 7 includes 254 bp of contiguous nucleotide sequence.
  • the portion of exon 7 includes at least 50 bp or at least 100 bp of nucleotides.
  • all of exons 3-6 and part of exon 7 of the human PLAUR gene include at least 100-500bp, 500-800bp or 800-900bp continuous nucleotide sequences.
  • the nucleotide sequence encoding the corresponding region of human PLAUR is located at nucleotide sequence 119-1057 of the human PLAUR gene transcript NM_002659.4.
  • the PLAUR gene of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.
  • the humanized PLAUR genome lacks the 5' UTR of the human PLAUR gene.
  • the humanized PLAUR genome comprises an endogenous (e.g., mouse) 5'UTR.
  • the humanized PLAUR genome comprises an endogenous (e.g., mouse) 3'UTR.
  • humanized PLAUR mice comprise replacements of endogenous mouse loci that retain endogenous mouse regulatory elements but include humanized PLAUR coding sequences. Expression of PLAUR in genetically modified heterozygous mice or homozygous mice is completely normal.
  • the invention provides a genetically modified non-human animal, the non-human animal genome comprising a deletion of the endogenous PLAUR gene, wherein the deletion of the endogenous PLAUR gene includes exon 1, exon 2, exon Exon 3, exon 4, exon 5, exon 6 and/or exon 7, or part of the endogenous PLAUR locus.
  • the deletion of the endogenous PLAUR gene includes one or more exons or portions of exons selected from the group consisting of exon 1, exon 2, exon 3, exon Exon 4, exon 5, exon 6 and/or exon 7.
  • the endogenous PLAUR gene deletion further includes one or more introns or portions of introns, the introns being selected from the group consisting of PLAUR gene intron 1, intron 2, intron Intron 3, intron 4, intron 5 and intron 6.
  • the deletions include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507 or 1508 bp continuous nucleotide sequence or more.
  • the deletion of the endogenous PLAUR gene includes at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 910, 911 or 912 bp of contiguous nucleotide sequence or more (for example, at least 50 bp of contiguous nucleotide sequence of exon 2 is deleted, exon All of 3-6, and at least 100 bp of contiguous nucleotide sequence of exon 5).
  • the invention provides a humanized mouse PLAUR genomic DNA sequence, a construct expressing the amino acid sequence of the humanized PLAUR protein; a cell comprising the above construct; and a cell comprising the above cell. organize.
  • the chimeric nucleotide sequence is consistent with the human PLAUR mRNA sequence (e.g., NM_002659.4), the human PLAUR amino acid sequence (e.g., NP_002650.1, SEQ ID NO:10), or a portion thereof (e.g., Part of exon 2, part of exon 3-6 and part of exon 7) has a sequence identity of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the human PLAUR mRNA sequence e.g., NM_002659.4
  • the human PLAUR amino acid sequence e.g., NP_002650.1, SEQ ID NO:10
  • the chimeric nucleic acid sequence described above is operably linked to a promoter or regulatory element, for example, an endogenous mouse PLAUR promoter, an inducible promoter, an enhancer and/or a mouse or human Adjustment element.
  • a promoter or regulatory element for example, an endogenous mouse PLAUR promoter, an inducible promoter, an enhancer and/or a mouse or human Adjustment element.
  • At least a portion of the chimeric nucleic acid sequence described above e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) identical to all or part of the mouse PLAUR nucleotide sequence (e.g., Exon 1, part of exon 2 and part of exon 7 of mouse PLAUR gene transcript NM_011113.4).
  • At least a portion of the chimeric nucleic acid sequence described above e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 cores nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) that differ in whole or in part from the human PLAUR nucleotide sequence (e.g., part of exon 1, exon 2, and exon of human PLAUR gene transcript NM_002659.4 part of exon 7).
  • the human PLAUR nucleotide sequence e.g., part of exon 1, exon 2, and exon of human PLAUR gene transcript NM_002659.4 part of exon 7.
  • At least a portion of the chimeric nucleic acid sequence described above e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) that are identical in whole or in part to the human PLAUR nucleotide sequence (e.g., human PLAUR Gene transcript NM_002659.4 part of exon 2, exons 3-6 and part of exon 7).
  • human PLAUR nucleotide sequence e.g., human PLAUR Gene transcript NM_002659.4 part of exon 2, exons 3-6 and part of exon 7.
  • At least a portion of the amino acid sequence e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30 , 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • amino acid residues e.g., contiguous or non-contiguous amino acid residues
  • human PLAUR protein sequence NP_002650.1 Amino acids 24-335 (SEQ ID NO: 10) amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • the present invention also provides a humanized PLAUR mouse amino acid sequence, wherein the amino acid sequence includes any one of the following groups:
  • the invention also provides a humanized PLAUR nucleotide (e.g., DNA or RNA) sequence, wherein the nucleotide sequence includes any one of the following groups:
  • the encoded amino acid sequence differs from the amino acid sequence shown in SEQ ID NO: 9, 10, 15 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or
  • the encoded amino acid sequence is the same as that shown in SEQ ID NO: 9, 10, 15, including the substitution, deletion and/or insertion of one or more amino acid residues.
  • the present invention further provides a PLAUR genomic DNA sequence of humanized mice.
  • the DNA sequence is obtained by reverse transcription of the transcribed mRNA and is consistent or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 13 or 14.
  • Urokinase type plasminogen activator is a trypsin-like serine endopeptidase that can be synthesized by fibroblasts, neutrophils, monocytes, epithelial cells, and tumor cells .
  • PLAU is initially secreted into the intercellular matrix in the form of enzymatically inactive single-chain zymogen (pro-uPA). Once it leaves the cell, it can be activated by binding to PLAUR on the cell membrane, or by a series of proteases, such as fiber It is activated by the action of proteases, cathepsin B, cathepsin L, kallikrein, trypsin and thermolysin.
  • This activated standard structure contains a growth factor-like domain (EGF-like domain), a kringle domain (the kringle domain) and a A contact reaction domain with serine activity (Peptidase S1domain).
  • This structure has the effect of limiting plasminogen as the primary substrate.
  • Activated uPA can convert plasminogen into plasmin. While plasmin degrades ECM, it can also activate matrix metalloproteinases (MMPs) into active MMPs to enhance the degradation of ECM.
  • MMPs matrix metalloproteinases
  • the human PLAU gene (Gene ID: 5328) contains 11 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6. Exon 7, exon 8, exon 9, exon 10 and exon 11 ( Figure 1).
  • the nucleotide sequence of human PLAU mRNA is NM_002658.6, and the amino acid sequence of human PLAU is NP_002649.2 (SEQ ID NO: 2).
  • SEQ ID NO: 2 Based on the nucleotide sequence and amino acid sequence of transcript NM_002658.6 and its encoded protein NP_002649.2, the corresponding position of each exon is as follows:
  • the human PLAU gene (NCBI Gene ID: 5328) is located at positions 73909182 to 73917497 of NC_000010.11 on chromosome 10 (GRCh38.p13 (GCF_000001405.39)).
  • the specific position of each exon based on the nucleotide sequence of transcript NM_002658.6 is: 5'UTR is located at NC_000010.11 Positions 73911132 to 73911218 and 73911525 to 73911555, exon 1 is located at positions 73911132 to 73911218 of NC_000010.11, intron 1 is located at positions 73911219 to 73911524 of NC_000010.11, and exon 2 is located at NC_0000 10.11 No.
  • intron 2 is located at positions 73911613 to 73912040 of NC_000010.11
  • exon 3 is located at positions 73912041 to 73912068 of NC_000010.11
  • intron 3 is located at positions 73912069 to 73912214 of NC_000010.11
  • exon 4 is located at NC_00001 0 .11 positions 73912215 to 73912322
  • intron 4 is located at positions 73912323 to 73912923 of NC_000010.11
  • exon 5 is located at positions 73912924 to 73913098 of NC_000010.11
  • intron 5 is located at positions 73913099 to NC_000010.11 73913289 bits
  • Exon 6 is located at positions 73913290 to 73913381 of NC_000010.11
  • intron 6 is located at positions 73913382 to 73913538 of NC_000010.11
  • exon 7 is located at positions 73913539 to 73913758 of NC_000010.11
  • intron 7 is located at NC_0000
  • the 3'UTR is located at positions 73916566 to 73917494 of NC_000010.11. All the above relevant information about the human PLAU locus can be retrieved at NCBI (Gene ID: 5328). The entire contents of which are incorporated herein by reference.
  • the mouse PLAU gene (Gene ID: 18792) contains 11 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7. Exon 8, exon 9, exon 10 and exon 11 ( Figure 1)
  • the nucleotide sequence of mouse PLAU mRNA is NM_008873.3
  • the amino acid sequence of mouse PLAU is NP_032899.1 (SEQ ID NO: 1).
  • the corresponding position of each exon is as follows:
  • the mouse PLAU gene (NCBI Gene ID: 18792) is located at positions 20886730 to 20893456 of NC_000080.7 on chromosome 14 (GRCm39 (GCF_000001635.27)).
  • the specific position of each exon in the nucleotide sequence based on transcript NM_008873.3 is: 5'UTR is located at positions 20886728 to 20886802 and 20887117 to 20887146 of NC_000080.7, and exon 1 is located at position 20886728 of NC_000080.7 to position 20886802, intron 1 is located at positions 20886803 to 20887116 of NC_000080.7, exon 2 is located at positions 20887117 to 20887203 of NC_000080.7, intron 2 is located at positions 20887204 to 20887677 of NC_000080.7, exon 3 Located at positions 20887678 to 20887708 of NC_000080.7, intron 3 is located at positions 20887709 to 20887845 of NC_
  • intron 10 is located at positions 20891163 to 20892341 of NC_000080.7
  • exon 11 is located at positions 20892342 to 208934534 of NC_000080.7
  • 3'UTR is located at positions 20892519 to 2089 of NC_000080.7 3453, the above is about rats All relevant information of the PLAU locus can be retrieved at NCBI (Gene ID: 18792). The entire contents of which are incorporated herein by reference.
  • Figure 19 shows the human PLAU amino acid sequence (NP_002649.2; SEQ ID NO: 2) and the mouse PLAU amino acid sequence (NP_032899.1; SEQ ID NO: 1). Therefore, the corresponding amino acid residues or regions between human and mouse PLAU can be found in Figure 19.
  • PLAU genes, proteins and locations for other species are also known in the art.
  • the relevant information of these genes can be found in NCBI and cited in the full text.
  • Figure 20 shows the human PLAU amino acid sequence (NP_002649.2; SEQ ID NO: 2) and the rat PLAU amino acid sequence (NP_037217.3; SEQ ID NO: 54). Therefore, the homology region between human and rat PLAU amino acid sequences can be found in Figure 20 .
  • the invention provides a human or chimeric (eg, humanized) PLAU nucleotide sequence and/or amino acid sequence.
  • exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the mouse PLAU gene the nucleotide sequences of exon 9, exon 10 and exon 11 are all or partially replaced by the corresponding nucleotide sequence or amino acid sequence of the human PLAU gene.
  • exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the mouse PLAU gene the "region" or “part” of exon 9, exon 10 and/or exon 11 is replaced by the corresponding nucleotide or amino acid sequence of the human PLAU gene.
  • the "area” or “portion” refers to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1100, 1200, 1300, 1301, 1302bp, 1350, 1400, 1600, 1800, 2000, 2200, 2300, 2310, 2320, 2330, 2340, 2341, 2342 or 2343bp contiguous nucleotide sequence, or at least 1 ,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,150,200,250,300,350,400,410 , 420, 430 or 431 consecutive amino acid sequences.
  • the "region" or "portion” is the same as exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7 , the amino acid sequence identity encoded by exon 8, exon 9, exon 10 and/or exon 11 is at least 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95% or at least 100%.
  • exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the mouse PLAU gene "region", “portion” of exon 9, exon 10 and/or exon 11 (e.g., part of exon 2, all of exons 3-10, and part of exon 11) ” or “all” sequences are exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon corresponding to the human PLAU gene 8. "Regions”, " Partial or "full” sequence replacement.
  • exogenous exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon A "region” or "portion” of exon 9, exon 10 and/or exon 11 is deleted.
  • the present invention provides a genetically modified non-human animal whose genome includes a human, chimeric or humanized PLAU nucleotide sequence.
  • the protein encoded by the human, chimeric or humanized PLAU nucleotide sequence is at least 70%, 80%, 85%, 90% identical to the amino acid sequence shown in SEQ ID NO: 2 , 95% or 100%.
  • the non-human animal genome contains a nucleotide sequence that has at least 70%, 80%, or 80% identity with the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 85%, 90%, 95% or 100%.
  • the genetically modified non-human animals described herein comprise a nucleotide sequence encoding a human or humanized PLAU protein.
  • the human or humanized PLAU protein comprises all or a portion of a human PLAU protein.
  • the genetically modified non-human animals described herein comprise a human or humanized PLAU gene.
  • the humanized PLAU gene contains 11 exons.
  • the humanized PLAU gene includes humanized exon 2, human exon 3, human exon 4, human exon 5, human exon 6, human exon 7. Human exon 8, human exon 9, human exon 10 and/or humanized exon 11.
  • the humanized PLAU gene comprises human intron 2, human intron 3, human intron 4, human intron 5, human intron 6, human intron 7, human Intron 8, human intron 9 and/or human intron 10.
  • the humanized PLAU gene comprises a human or humanized 5' UTR.
  • the humanized PLAU gene comprises a human or humanized 3'UTR. In some embodiments, the humanized PLAU gene comprises an endogenous 5' UTR. In some embodiments, the humanized PLAU gene comprises an endogenous 3'UTR.
  • genetically modified non-human animals can express human PLAU and/or chimeric (e.g., humanized)
  • the endogenous PLAU gene is replaced by the human PLAU gene and/or nucleotide sequence.
  • the amino acid sequence of the human PLAU gene and/or nucleotide sequence encoding human PLAU is identical to the amino acid sequence shown in human PLAU. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 100%.
  • the endogenous non-human animal's PLAU gene is replaced with all or part of the nucleotide sequence encoding the mature PLAU protein.
  • the genetically modified non-human animal expresses human PLAU and/or chimeric PLAU protein (eg, humanized PLAU) under a mouse endogenous promoter and/or regulatory elements. Replacement of the endogenous mouse locus provides a non-human animal that can express human or chimeric PLAU protein (eg, humanized PLAU) in the same cell type.
  • the genetically modified mice do not develop the underlying diseases known in the art to be observed in certain other transgenic mice.
  • Human PLAU or chimeric PLAU proteins expressed in non-human animals can maintain the function of one or more wild-type or human PLAU proteins, for example, the expressed PLAU protein can bind to human or non-human PLAU proteins.
  • the genetically modified non-human animal does not express endogenous PLAU protein. In some embodiments, the genetically modified non-human animal has reduced expression of endogenous PLAU protein.
  • Endogenous PLAU protein refers to non-human animals (e.g., mice) before genetic modification The endogenous PLAU nucleotide sequence encodes the PLAU protein.
  • the genome of a non-human animal contains an amino acid sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the human PLAU protein (NP_002649.2; SEQ ID NO:2) or 100% amino acid nucleotide sequence.
  • the genome comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or at least 100% nucleotide sequence.
  • nucleotide sequence encoding the endogenous PLAU region in the genome of the non-human animal is replaced with the nucleotide sequence encoding the corresponding region of human PLAU.
  • the nucleotide sequence encoding the endogenous PLAU region is any sequence of the endogenous PLAU locus, such as exon 1, exon 2, exon 3, exon 4, Exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, 5'UTR, 3'UTR, intron 1, intron Intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, or any combination thereof.
  • the nucleotide sequence encoding an endogenous PLAU region is located within an endogenous PLAU regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous PLAU region is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon Exon 7, exon 8, exon 8, exon 9, exon 10 and/or exon 11, or portions thereof, or the entire endogenous PLAU locus thereof.
  • Genetically modified non-human animals have one or more cells expressing human or chimeric PLAU protein (e.g., humanized PLAU protein).
  • the human or chimeric PLAU protein comprises at least the amino acid sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 as shown in SEQ ID NO: 2 , 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 410, 420, 430 or 431 consecutive amino acid sequences.
  • the genetically modified non-human animal genome comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 1 of the human PLAU gene. All or part of exon 7, exon 8, exon 9, exon 10, and/or exon 11, or all or part of the nucleotide sequence shown in SEQ ID NO: 7.
  • the genetically modified non-human animal genome includes part of exon 2, all of exons 3-10, and part of exon 11 of the human PLAU gene.
  • the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 50, 55, 57, 60, 65, 70, 75, 80, 83, 85 of exon 2 of the human PLAU gene , 87 or 88 bp continuous nucleotide sequence.
  • the portion of exon 2 includes 57 bp of contiguous nucleotide sequence.
  • the portion of exon 11 comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 170, 172, 173, 174, 175, 176, 177, 179, 180, 200, 500, 600, 800, 1000, 1100 or 1106 bp contiguous nucleotide sequence. In some implementations, the portion of exon 11 includes 177 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 11 includes at least 50 bp or at least 100 bp of nucleotides.
  • all of exons 3-10 and part of exon 11 of the human PLAU gene contain at least 100-500bp and 500-1000bp continuous nucleotide sequences.
  • the encoded human PLAU phase The nucleotide sequence of the corresponding region is located in the 119th to 1414th nucleotide sequence of the human PLAU gene transcript NM_002658.6.
  • the genetically modified non-human animal PLAU gene is heterozygous or homozygous for the endogenous modified locus.
  • the humanized PLAU genome lacks the 5' UTR of the human PLAU gene.
  • the humanized PLAU genome comprises an endogenous (e.g., mouse) 5'UTR.
  • the humanized PLAU genome comprises an endogenous (e.g., mouse) 3'UTR.
  • humanized PLAU mice comprise replacements of endogenous mouse loci that retain endogenous mouse regulatory elements but include humanized PLAU coding sequences. Expression of PLAU in genetically modified heterozygous mice or homozygous mice is completely normal.
  • the present invention provides a genetically modified non-human animal, the non-human animal genome comprising a deletion of the endogenous PLAU gene, wherein the deletion of the endogenous PLAU gene includes exon 2, exon 3, exon Exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11, or part of the endogenous PLAU locus.
  • the deletion of the endogenous PLAU gene includes one or more exons or portions of exons selected from the group consisting of exon 2, exon 3, exon 4, exon Parts of exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11.
  • the endogenous PLAU gene deletion further includes one or more introns or portions of introns selected from the group consisting of PLAU gene intron 1, intron 2, intron 3. Partial deletion of intron 4, intron 5, intron 6, intron 7, intron 8, intron 9 and/or intron 10.
  • the deletions include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 700, 900, 1000, 1100, 1200, 1250, 1290, 1291, 2192, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1500, 1700, 1800, 2000, 2200, 2300, 2320, 2340, 2341, 2342, 2343bp continuous nucleotide sequence or more nucleotide sequence.
  • the deletion of the endogenous PLAU gene includes exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, At least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1301 or 1302 bp contiguous nucleotide sequence or more (for example, deletion of at least 20 bp contiguous nucleotide sequence of exon 2, all of exon 3-10, exon At least 100 bp of contiguous nucleotide sequence for exon 11).
  • the present invention provides a PLAU genomic DNA sequence of a humanized mouse, and provides a method for expressing humanized PLAU.
  • the invention provides a chimeric (e.g., humanized) PLAU nucleotide sequence and/or amino acid sequence, wherein in some embodiments, the chimeric nucleotide sequence Compatible with mouse endogenous PLAU mRNA (e.g., NM_008873.3), mouse PLAU amino acid sequence (e.g., NP_032899.1, SEQ ID NO:1) or a portion thereof (e.g., 5'UTR, all of exon 1, Part of exon 2, part of exon 11 and 3'UTR) have a sequence identity of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the chimeric nucleotide sequence is consistent with human PLAU mRNA (e.g., NM_002658.6), human PLAU amino acid sequence (e.g., NP_002649.2, SEQ ID NO:2), or a portion thereof (e.g., ect. part of exon 2, exons 3-10 and part of exon 11).
  • the chimeric nucleic acid sequences described above are operably linked to a promoter or regulatory element, such as an endogenous mouse PLAU promoter, an inducible promoter, an enhancer, and/or a mouse or human regulatory element. element.
  • a promoter or regulatory element such as an endogenous mouse PLAU promoter, an inducible promoter, an enhancer, and/or a mouse or human regulatory element. element.
  • At least a portion of the chimeric nucleic acid sequence described above (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) that differ from the mouse PLAU nucleotide sequence in whole or in part (e.g., small Mouse PLAUR gene transcript NM_008873.3 (part of exon 2, exons 3-10 and part of exon 11).
  • At least a portion of the chimeric nucleic acid sequence described above (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) identical to all or part of the mouse PLAU nucleotide sequence (e.g., Mouse PLAU gene transcript NM_008873.3 exon 1, part of exon 2 and part of exon 11).
  • At least a portion of the chimeric nucleic acid sequence described above (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence) that is different from the human PLAU nucleotide sequence in whole or in part (e.g., human PLAUR Gene transcript NM_002658.6 exon 1, part of exon 2 and part of exon 11).
  • At least a portion of the chimeric nucleic acid sequence described above (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) that are identical in whole or in part to the human PLAU nucleotide sequence (e.g., human PLAU part of exon 2, exons 3-10 and exon 11 of gene transcript NM_002658.6).
  • human PLAU nucleotide sequence e.g., human PLAU part of exon 2, exons 3-10 and exon 11 of gene transcript NM_002658.6
  • the chimeric nucleic acid sequence encodes at least a portion of the amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, contiguous or non-contiguous amino acid residues) that differ from all or part of the mouse PLAU protein amino acid sequence (eg, amino acids 1-433 of the mouse PLAU protein sequence NP_032899.1 (SEQ ID NO: 1)).
  • amino acids e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, contiguous or non-contiguous amino acid residues
  • At least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30 , 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) different from all or part of the human PLAU protein amino acid sequence (e.g., human PLAU protein sequence NP_002649.2 Amino acids 1-431 (SEQ ID NO: 2)).
  • the present invention also provides a humanized PLAU mouse amino acid sequence, wherein the amino acid sequence includes any one of the following groups:
  • the invention also provides a humanized PLAU nucleotide (e.g., DNA or RNA) sequence, wherein the nucleotide sequence includes any one of the following groups:
  • the encoded amino acid sequence differs from the amino acid sequence shown in SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or
  • the encoded amino acid sequence is the same as that shown in SEQ ID NO: 2, including one or more substitutions, deletions and/or insertions Amino acid sequence of amino acid residues.
  • chimeric gene or “chimeric nucleic acid” used in the present invention refers to a gene or nucleic acid in which two or more parts of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is identical to that in an animal.
  • the wild-type nucleic acid is different.
  • the chimeric gene or chimeric nucleic acid has at least a portion of the sequence originating from two or more different species, for example, a sequence encoding different proteins or the same (or homologous) sequence encoding two or more different species. ) protein sequence.
  • a chimeric gene or chimeric nucleic acid refers to a humanized gene or humanized nucleic acid.
  • humanized protein or “humanized polypeptide” mentioned in the present invention refers to a protein or polypeptide, wherein at least a part of the protein or polypeptide is derived from a human protein or human polypeptide.
  • humanized protein or humanized protein refers to a human protein or polypeptide.
  • Humanized nucleic acid refers to nucleic acid, wherein at least a portion of the nucleic acid is derived from human nucleic acid. In some embodiments, the nucleic acids in the humanized nucleic acid are entirely of human origin. In some embodiments, the humanized nucleic acid refers to a humanized exon, which may be a human exon or a chimeric exon.
  • the chimeric gene or chimeric nucleic acid is a humanized PLAUR gene or humanized PLAUR nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human PLAUR gene, or at least a portion of the gene or nucleic acid is derived from a non-human PLAUR gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding a PLAUR protein. The encoded PLAUR protein has at least one activity that functions as a human PLAUR or non-human PLAUR protein. For example, interaction with PLAU ligand induces downstream signaling pathways.
  • the chimeric protein or chimeric polypeptide is a humanized PLAUR protein or humanized PLAUR polypeptide.
  • at least a portion of the amino acid sequence of the protein or polypeptide is derived from a human PLAUR protein, or at least a portion of the amino acid sequence of the protein or polypeptide is derived from a non-human PLAUR protein.
  • the humanized PLAUR protein or humanized PLAUR polypeptide has the activity of at least one function of the human PLAUR protein or the non-human PLAUR protein. For example, interaction with PLAUR ligand induces downstream signaling pathways.
  • Such methods include, for example, modifying the genome of a non-ES cell (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transfer to transfer the modified genome to an appropriate cell, such as an oocyte, and under appropriate conditions in Modified cells (eg, modified oocytes) are gestated in non-human animals to form embryos.
  • a non-ES cell e.g., fibroblasts or induced pluripotent stem cells
  • Modified cells eg, modified oocytes
  • the animal is a mammal.
  • the genetically modified non-human animal is a rodent.
  • the rodent may be selected from mice, rats and hamsters.
  • the rodent is selected from the murine family.
  • the genetically modified animal is selected from the group consisting of the family Hamsteridae (e.g., mouse-like hamsters), the family Hamsteridae (e.g., hamsters, New World rats and mice, voles), Muridae (e.g., true mice and rats, gerbils, spiny rats, crested rats), Dormoids (mountain mice, rock mice, tailed rats, Madagascar rats and mice), Dormoids (e.g.
  • the family Hamsteridae e.g., mouse-like hamsters
  • the family Hamsteridae e.g., hamsters, New World rats and mice, voles
  • Muridae e.g., true mice and rats, gerbils, spiny rats
  • the genetically modified rodent is selected from the group consisting of true mice or rats (superfamily Muroidea), gerbils, spiny rats and crested rats.
  • the genetically modified mouse is from a member of the family Muridae.
  • the animal is a rodent.
  • the rodent is selected from the group consisting of mice and rats.
  • the non-human animal is a mouse.
  • the non-human animal is a mouse selected from the group consisting of BALB/c, A, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/ N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/ 6.
  • Genetically modified non-human animals include modifications of endogenous non-human PLAU and/or PLAUR gene loci.
  • the modifications comprise a nucleotide sequence encoding at least a portion of a mature PLAU or PLAUR protein (e.g., at least 10%, 20%, 30%, 40%, 50% identical to the amino acid sequence of a mature PLAU or PLAUR protein , 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% consistency).
  • the present invention provides cells (e.g., ES cells, somatic cells) that may contain genetic modifications as described herein, in many embodiments, genetically modified non-human animals include modification of endogenous PLAU and/or PLAUR in the animal. Modification of genetic loci.
  • the present invention also provides a tumor-bearing non-human mammal, characterized in that the non-human mammal model is obtained by the method described herein.
  • the non-human mammal is a rodent (eg, mouse).
  • the invention also provides a cell or cell line, or primary cell culture, derived from a non-human mammal or its progeny, or a tumor-bearing non-human mammal, or a tissue derived from a non-human mammal or its progeny. , an organ or a culture thereof, or a tumor-bearing non-human mammal, and tumor tissue derived from a non-human mammal or its descendant, when it bears a tumor, or a tumor-bearing tumor of a non-human mammal.
  • non-human mammal produced by any of the methods described herein.
  • non-human mammals genetically modified non-human animals whose genomes comprise DNA of human or humanized PLAU and/or PLAUR are provided.
  • a non-human mammal includes a genetic construct described herein (e.g., Figures 2, 3, 4, 5, 9, Gene constructs shown in 10, 11 and 12).
  • a non-human mammal expressing human or humanized PLAU and/or PLAUR protein is provided.
  • tissue-specific expression of a human or humanized PLAU and/or PLAUR protein is provided.
  • expression of non-human animal human or humanized PLAU and/or PLAUR proteins is controllable. Such as by adding specific inducers or repressors.
  • the specific inducer is selected from Tet-Off System/Tet-On System or Tamoxifen System.
  • the non-human mammal can be any non-human animal known in the art, which can be used in the methods described herein.
  • Preferred non-human mammals are mammals (eg, rodents).
  • the non-human mammal is a mouse.
  • the present invention provides an offspring produced by mating with a non-human mammal of the same genotype or another genotype.
  • the present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny.
  • cell culture-based models can be prepared by the following method.
  • Cell cultures can be obtained by isolation from non-human mammals, or cells can be obtained from cell cultures established using the same constructs and using standard cell transfection techniques. Integration of genetic constructs containing DNA sequences encoding human PLAU and/or PLAUR proteins can be detected by a variety of methods.
  • RT-PCR reverse transcription-polymerase chain reaction
  • protein-level methods including histochemical analysis , Western blot analysis and in vitro binding studies
  • ELSA electroactive polymerase chain reaction
  • RNAdot RNA dot blot assays
  • Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of human or humanized PLAU and/or PLAUR proteins.
  • the genetically modified non-human animals described herein have two or more humanized genes, wherein the humanized genes can be selected from human or humanized genes of PLAU and/or PLAUR.
  • the invention provides a targeting vector targeting the PLAU gene, which includes: a) a DNA fragment homologous to the 5' end of the conversion region to be changed, which is selected from 100-10,000 genomic DNA of the PLAU gene of non-human animals length of nucleotides; b) DNA sequence encoding the donor region; c) DNA fragment homologous to the 3' end of the conversion region to be changed, which is selected from non-human animal PLAU gene genomic DNA, with a length of 100-10,000 Nucleotides.
  • a) the DNA fragment homologous to the 5' end of the conversion region to be changed is selected from a nucleotide sequence that has at least 90% homology with the NCBI accession number NC_000080.7; c) The DNA fragment homologous to the 3' end of the conversion region is selected from a nucleotide sequence that has at least 90% homology with the NCBI accession number NC_000080.7;
  • a) the homologous DNA fragment at the 5' end of the conversion region to be changed is selected from the nucleotide sequence at positions 20883305 to 20887146 with NCBI accession number NC_000080.7; c) the conversion region 3 to be changed The DNA fragment with homology to the 'end is selected from the nucleotide sequence at positions 20893778 to 20896948 with NCBI accession number NC_000080.7;
  • a) the homologous DNA fragment at the 5' end of the conversion region to be changed is selected from the nucleotide sequence at positions 20885866 to 20887146 with NCBI accession number NC_000080.7; c) the conversion region 3 to be changed The DNA fragment homologous to the 'end is selected from the nucleotide sequence at positions 20892519 to 20893251 with NCBI accession number NC_000080.7;
  • the selected genomic nucleotide sequence length of the targeting vector can exceed about 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 5.5kb, 6kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb , 9kb, 9.5kb or 10kb.
  • the conversion region to be changed is located on exons 1 to 11 of the PLAU gene of non-human animals.
  • the conversion region to be changed is located on exons 2 to 11 of the PLAU gene of non-human animals (for example, positions 104-1405 of NM_008873.3).
  • the targeting vector further includes one or more marker genes.
  • a positive selection marker gene or a negative selection marker gene the resistance gene screened for positive clones is the neomycin phosphotransferase coding sequence Neo.
  • the gene encoding the negative selection marker is the gene encoding diphtheria toxin A subunit (DTA).
  • the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 3; the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 5; the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 6.
  • the 5' arm is a nucleotide with at least 90% homology with the NCBI accession number NC_000080.7. Further preferably, the 5' arm sequence includes SEQ ID NO: 3 or 5 The nucleotide sequence is shown. In some embodiments, the 3' arm is a nucleotide with at least 90% homology with the NCBI accession number NC_000080.7. Further preferably, the 3' arm sequence includes SEQ ID NO: 4 or 6 The nucleotide sequence is shown.
  • the targeting vector comprises human sequences (eg, NM_002658.6119-1414).
  • the targeting region in the targeting vector includes: part or all of the nucleotide sequence of the human PLAU gene, preferably exon 2, exon 3, exon 4, exon 5, and exon of the human PLAU gene. Exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11.
  • the nucleotide sequence of the humanized PLAU gene encodes all or part of the nucleotide sequence of the human PLAU protein, and the NCBI protein number is NP_002649.2 (SEQ ID NO: 2).
  • the invention provides a targeting vector targeting the PLAUR gene, including: a) a DNA fragment homologous to the 5' end of the conversion region to be changed, which is selected from 100-10000 lengths of PLAUR genomic DNA of non-human animals nucleotide; b) DNA sequence encoding the donor region; c) DNA fragment homologous to the 3' end of the conversion region to be changed, which is selected from non-human animals PLAU gene genomic DNA, 100-10000 nucleotides in length.
  • a) the DNA fragment homologous to the 5' end of the conversion region to be changed is selected from a nucleotide sequence having at least 90% homology with the NCBI accession number NC_000073.7; c) The DNA fragment homologous to the 3' end of the conversion region is selected from a nucleotide sequence that has at least 90% homology with the NCBI accession number NC_000073.7;
  • a) the homologous DNA fragment at the 5' end of the conversion region to be changed is selected from the nucleotide sequence at positions 24159210 to 24164569 with NCBI accession number NC_000073.7; c) the conversion region 3 to be changed The DNA fragment homologous to the 'end is selected from the nucleotide sequence at positions 24174897 to 24178233 with NCBI accession number NC_000073.7;
  • the selected genomic nucleotide sequence length of the targeting vector can exceed about 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 5.5kb, 6kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb , 9kb, 9.5kb or 10kb.
  • the conversion region to be changed is located on exons 1 to 7 of the PLAUR gene of non-human animals.
  • the conversion region to be changed is located on exons 2 to 7 of the PLAUR gene of non-human animals (for example, positions 100-1011 of NM_011113.4).
  • the targeting vector further includes one or more marker genes.
  • a positive selection marker gene or a negative selection marker gene the resistance gene screened for positive clones is the neomycin phosphotransferase coding sequence Neo.
  • the gene encoding the negative selection marker is the gene encoding diphtheria toxin A subunit (DTA).
  • the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 11; the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 12.
  • the 5' arm is a nucleotide with at least 90% homology with the NCBI accession number NC_000073.7. Further preferably, the 5' arm sequence includes SEQ ID NO: 11 Nucleotide sequence. In some embodiments, the 3' arm is a nucleotide with at least 90% homology with the NCBI accession number NC_000073.7. Further preferably, the 3' arm sequence includes SEQ ID NO: 12 Nucleotide sequence.
  • the targeting vector comprises human sequences (eg, positions 119-1057 of NM_002659.4).
  • the targeting region in the targeting vector includes part or all of the nucleotide sequence of the human PLAUR gene, preferably part of exon 2, all of exons 3-6 and/or exon 7 of the human PLAUR gene. part.
  • the human sequence is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequences set forth in SEQ ID NOs: 7 and 8 % or 100%; in some embodiments, the nucleotide sequence of the humanized PLAUR gene encodes all or part of the nucleotide sequence of the human PLAUR protein, and the NCBI protein number is NP_002650.1 (SEQ ID NO: 10).
  • the present disclosure also relates to cells comprising a targeting vector as described above.
  • the present invention also provides a non-human mammalian cell having any one of the above targeting vectors to and one or more in vitro transcripts of a construct described herein.
  • the cell contains Cas9 mRNA or an in vitro transcript thereof.
  • the genes in the cells are heterozygous. In some embodiments, the genes in the cells are homozygous.
  • the non-human mammalian cells are mouse cells.
  • the cell is a fertilized egg cell.
  • the cells are embryonic stem cells.
  • Genetically modified non-human animals can be prepared through several technologies known in the art, including gene targeting technology using embryonic stem cells, CRISPR/Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, and homing. Nested endonucleases or other molecular biology techniques. In some embodiments, it is preferred to use homologous recombination technology.
  • CRISPR/Cas9 gene editing technology can create genetically modified non-human animals. In some embodiments, CRISPR-Cas9 genome editing is used to create genetically modified non-human animals.
  • the present invention also provides many other methods for genome editing, such as microinjecting a transgenic cell into an enucleated oocyte and fusing the enucleated oocyte with another transgenic cell.
  • a nucleotide sequence encoding the endogenous PLAU region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding the corresponding region of human PLAU.
  • the expression level of the endogenous PLAU protein of the non-human animal is reduced or deleted compared with wild-type PLAU.
  • replacement occurs in germ cells, somatic cells, blastocysts, fibroblasts, or the like. Nuclei from somatic cells or fibroblasts can be inserted into the enucleated oocyte.
  • Figures 3 and 5 show the humanized targeting strategy targeting the mouse PLAU site.
  • the targeting vector contains a 5' homology arm, a human or humanized PLAU gene fragment, and a 3' homology arm. This process involves the use of homologous recombination to replace the endogenous corresponding PLAU sequence with a human or humanized sequence.
  • cleavage upstream and downstream of the target site e.g., by zinc finger nucleases, TALENs, or CRISPR
  • Source PLAU sequence e.g., by zinc finger nucleases, TALENs, or CRISPR
  • the nucleotide sequence encoding the corresponding region of human PLAU includes a nucleotide sequence encoding all or part of the human PLAU protein. Preferably, it includes the entire nucleotide sequence encoding the human PLAU protein.
  • the nucleotide sequence encoding the corresponding region of human PLAU includes at least 50 to at least 431 encoding human PLAU protein, preferably 50, 100, 150, 200, 250, 300, 350, 400, 410, A nucleotide sequence of 420, 425, 428, 429, 430 or 431 consecutive amino acids; further comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2; or, including a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 Indicates that the amino acid sequence identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% nucleotide sequence; Contains a nucleotide sequence encoding a nucleotide sequence that differs from the amino acid sequence shown in SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no
  • the nucleotide sequence encoding the corresponding region of human PLAU includes all or part of exon 1 to exon 11 of the human PLAU gene, and further preferably includes exon 1 to exon 11 of the human PLAU gene. All or part of a combination of one, two or more, or two or more consecutive exons in exon 11, more preferably including all of exon 2 to exon 11 of the human PLAU gene Or part, further preferably includes part of exon 2 of human PLAU gene, all of exons 3-10 and part of exon 11, preferably also includes intron 2 and/or intron 10, wherein, The part of human PLAU gene exon 2 includes at least 20 bp to at least 88 bp of human PLAU gene exon 2, preferably 20, 30, 40, 50, 55, 56, 57, 58, 59, 60, 70, 80 or 88 bp
  • the continuous nucleotide sequence, or the part of human PLAU gene exon 2 includes the nucleotide sequence of the coding region, and the part
  • the nucleotide sequence encoding the corresponding region of human PLAU includes the nucleotide sequence shown in SEQ ID NO: 7; or, the nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 7 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% nucleotide sequence; Contains a nucleotide sequence that differs no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide from the nucleotide sequence shown in SEQ ID NO: 7; or, contains A nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 7, including substitution, deletion and/or insertion of one or more nucleotides.
  • the nucleotide sequence encoding the corresponding region of human PLAU is operably linked to an endogenous regulatory element of the endogenous PLAU gene in at least one chromosome.
  • the endogenous PLAU nucleotide sequence that is replaced in the non-human animal includes all or part of exon 1 to exon 11 of the mouse PLAU gene being replaced, preferably the exon of the non-human animal PLAU gene. All or part of exon 2 to exon 11 are replaced. More preferably, part of exon 2, all of exons 3-10 and part of exon 11 of the non-human animal PLAU gene are replaced.
  • the replaced endogenous PLAU nucleotide sequence encodes the nucleotide sequence of the amino acid sequence shown in SEQ ID NO: 1 is replaced.
  • the human or humanized PLAU gene is regulated in non-human animals through regulatory elements, including but not limited to endogenous promoters.
  • the regulatory elements may be endogenous or exogenous of.
  • the endogenous regulatory element is derived from the PLAU gene of non-human animals.
  • the exogenous regulatory element is derived from the human PLAU gene.
  • the construction method includes replacing all or part of exon 1 to exon 11 of the non-human animal PLAU gene with exon 1 to exon 11 of the human PLAU gene. All or part of, preferably exon 2 to exon 11 of the non-human animal PLAU gene is replaced with all or part of exon 2 to exon 11 of the human PLAU gene.
  • the construction method includes using a gene containing a part of exon 2, all of exons 3-10 and a part of exon 11 of the human PLAU gene, preferably also including introns. 2 and/or intron 9, replacing all or part of exon 2 to exon 11 of the PLAU gene in non-human animals.
  • the construction method includes replacing the non-human animal genome with a nucleotide sequence encoding a human or humanized PLAU protein, or a nucleotide sequence encoding a human or humanized PLAU gene.
  • the construction method includes replacing the nucleotide encoding the amino acid sequence shown in SEQ ID NO: 1 in the non-human animal genome with a genomic DNA sequence, a cDNA sequence or a CDS sequence containing the human PLAU gene. sequence.
  • the construction method includes replacing the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the non-human animal genome with a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2. nucleotide sequence.
  • the construction method includes replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the non-human animal genome with a nucleotide sequence including the nucleotide sequence shown in SEQ ID NO: 7.
  • a nucleotide sequence encoding a region of endogenous PLAUR in the genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding a corresponding region of human PLAUR.
  • the non-human animal has reduced or absent endogenous PLAUR protein expression.
  • replacement occurs in germ cells, somatic cells, blastocysts, fibroblasts, or the like. Nuclei from somatic cells or fibroblasts can be inserted into the enucleated oocyte.
  • FIG 11 shows the humanized targeting strategy for targeting mouse PLAUR.
  • the targeting vector includes a vector composed of a 5' homology arm, a human or humanized PLAUR gene fragment and a 3' homology arm. This process involves the use of homologous recombination to replace the endogenous corresponding PLAUR sequence with a human or humanized sequence.
  • cleavage upstream and downstream of the target site e.g., by zinc finger nucleases, TALENs, or CRISPR
  • homologous recombination is used to replace the human or humanized PLAUR sequence in the mouse.
  • Source PLAUR sequence e.g., by zinc finger nucleases, TALENs, or CRISPR
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes a nucleotide sequence encoding all or part of the human PLAU protein, preferably including nucleotide sequences encoding human PLAUR proteins DI, DII and DIII.
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes a nucleotide sequence encoding all or part of the human PLAUR protein, and further preferably, includes at least 50 to at least 335 nucleotide sequences encoding the human PLAUR protein, Preferably, it is a nucleotide sequence of 50, 100, 150, 200, 250, 300, 310, 311, 312, 313, 314, 320, 330 or 335 consecutive amino acids, and more preferably, it includes the encoding SEQ ID NO: 10
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes all or part of exon 1 to exon 7 of human PLAUR gene, preferably includes exon 1 to exon 7 of human PLAUR gene. All or part of a combination of one, two or more, or two or more consecutive exons, and further preferably includes all or part of exon 1 to exon 7 of the human PLAUR gene, More preferably, it includes part of exon 2, all of exons 3-6 and part of exon 7 of the human PLAUR gene, and preferably also includes introns 2-3 and/or introns 6-7, wherein, The part of human PLAUR gene exon 2 includes at least 20 bp to at least 111 bp of human PLAUR gene exon 2, preferably 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 , 100, 110 or 111bp continuous nucleotide sequence, the part of human PLAUR gene exon 7 includes at least 50bp to at least 565
  • the nucleotide sequence encoding the corresponding region of human PLAUR includes the nucleotide sequence shown in SEQ ID NO: 13; or, includes the nucleotide sequence shown in SEQ ID NO: 13 Identity of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% Nucleotide sequence; alternatively, containing a nucleic acid sequence that differs not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO: 13
  • the nucleotide sequence alternatively, includes a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 13, including substitution, deletion and/or insertion of one or more nucleotides.
  • the nucleotide sequence encoding the corresponding region of human PLAUR is operably linked to an endogenous regulatory element of the endogenous PLAUR gene in at least one chromosome.
  • the endogenous PLAUR nucleotide sequence that is replaced in the non-human animal includes all or part of exon 1 to exon 7 of the mouse PLAUR gene, preferably exon 1 of the non-human animal PLAU gene. Exon 2 to exon All or part of 7 is replaced. More preferably, part of exon 2, all of exons 3 to 6 and part of exon 7 of the non-human animal PLAU gene are replaced. In some embodiments, the replaced endogenous PLAUR nucleotide sequence is replaced with a nucleotide sequence encoding the amino acid sequence shown in positions 25-327 of SEQ ID NO:9.
  • the human or humanized PLAUR gene is regulated in non-human animals through regulatory elements, including but not limited to endogenous promoters.
  • the regulatory elements may be endogenous or exogenous.
  • the endogenous regulatory element is derived from the PLAUR gene of non-human animals.
  • the exogenous regulatory element is derived from the human PLAUR gene.
  • the construction method includes replacing exon 1 to exon 7 of the non-human animal PLAUR gene with all or part of exon 1 to exon 7 of the human PLAUR gene. All or part of exon 2 to exon 7 of the non-human animal PLAUR gene is preferably replaced with all or part of exon 2 to exon 7 of the human PLAUR gene.
  • the construction method includes using a method comprising a part of exon 2, all of exons 3-6 and a part of exon 7 of the human PLAUR gene, preferably also including introns. 2-3 and/or introns 6-7, replacing all or part of exon 2 to exon 7 of the PLAUR gene in non-human animals.
  • the construction method includes replacing the non-human animal genome with a nucleotide sequence encoding a human or humanized PLAUR protein, or a nucleotide sequence encoding a human or humanized PLAUR gene.
  • the construction method includes replacing the nucleotide encoding the amino acid sequence shown in SEQ ID NO: 9 in the non-human animal genome with a genomic DNA sequence, a cDNA sequence or a CDS sequence containing the human PLAUR gene. sequence.
  • the construction method includes replacing the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 9 in the non-human animal genome with a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 10. nucleotide sequence.
  • the construction method includes replacing the amino acid sequence encoding SEQ ID NO: 9 in the non-human animal genome with a nucleotide sequence comprising the amino acid sequence encoding SEQ ID NO: 10.
  • the construction method includes replacing the amino acid sequence shown in positions 25-327 encoding SEQ ID NO: 9 in the non-human animal genome with the nucleotide sequence shown in SEQ ID NO: 13. Nucleotide sequence.
  • the method of constructing a non-human animal includes modifying the coding frame of the PLAU and/or PLAUR gene of the non-human animal to a nucleotide sequence encoding a human or humanized PLAU and/or PLAUR protein or a humanized PLAU and / or
  • the nucleotide sequence of the PLAUR gene is inserted after the endogenous regulatory element of the non-human animal PLAU and/or PLAUR gene, wherein the modification of the coding frame of the non-human animal PLAU and/or PLAUR gene can be achieved by knocking out the non-human animal PLAU and/or
  • the functional region of the PLAUR gene may be inserted into a sequence so that the non-human animal PLAU and/or the PLAUR protein is not expressed or the expression is reduced or the expressed protein has no function.
  • the coding frame of the non-human animal PLAU gene is modified. All or part of the nucleotide sequence of exons 2-11 of the PLAU gene in non-human animals can be knocked out. In some embodiments, the modified non-human animal PLAUR gene coding frame can knock out all or part of the nucleotide sequence of exons 2-7 of the non-human animal PLAUR gene.
  • methods of constructing genetically modified non-human animals include inserting nucleotide sequences encoding human or humanized PLAU and/or PLAUR proteins after endogenous regulatory elements of the non-human animal PLAU and/or PLAUR genes. and/or auxiliary sequences.
  • the auxiliary sequence can be a stop codon, so that the PLAU and/or PLAUR gene humanized animal model expresses human PLAU and/or PLAUR protein in vivo, but does not express non-human animal PLAU and/or PLAUR protein, further , the auxiliary sequence is WPRE, STOP and/or PolyA.
  • the construction method includes constructing non-human animals using the above-mentioned PLAU and/or PLAUR gene targeting vectors and/or PLAU and/or PLAUR gene targeting sgRNA.
  • the construction method includes introducing the above-mentioned targeting vector into non-human animal cells (preferably embryonic stem cells), selecting correct positive clone cells, introducing them into separated blastocysts, and culturing The blastocyst is then transplanted into the fallopian tube of a female non-human animal, allowed to develop, and non-human animals that obtain PLAU and/or PLAUR gene modification are identified and screened.
  • non-human animal cells preferably embryonic stem cells
  • sgRNA targeting the PLAUR gene in order to improve the recombination efficiency, can also be used to construct non-human animals together with the above-mentioned PLAUR gene targeting vector.
  • the construction method includes introducing the above-mentioned PLAU and/or PLAUR gene targeting vector, PLAU and/or PLAUR gene targeting sgRNA and Cas9 into non-human animal cells, and culturing the cells (preferably fertilized eggs), and then transplant the cultured cells into the fallopian tube of a female non-human animal, allow them to develop, and identify and screen non-human animals that obtain PLAU and/or PLAUR gene modification.
  • the non-human animals can be selected from rodents, pigs, rabbits, monkeys, and any other non-human animals that can undergo gene editing to produce gene humanization.
  • the non-human animal is a non-human mammal.
  • the non-human mammal is a rodent. More preferably, the rodent is a rat or mouse.
  • the non-human animal is an immunodeficient non-human mammal.
  • the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey.
  • the immunodeficient rodent is an immunodeficient mouse or rat.
  • the immunodeficient mice are NOD-Prkdc scid IL-2r ⁇ null mice, NOD-Rag 1 -/- -IL2rg -/- mice, Rag 2 -/- -IL2rg -/- mice, NOD /SCID mice or nude mice.
  • the present invention also provides a non-human animal genetically modified by the above-mentioned PLAU and/or PLAUR, and the application of the non-human animal obtained by any of the above-mentioned construction methods.
  • the application includes:
  • the present invention provides a non-human animal expressing human or humanized PLAU and/or PLAUR protein, which can be used for screening of specific modulators of human PLAU and/or PLAUR.
  • the non-human animal is an animal model of human disease.
  • the disease is genetically induced (knock-in or knock-out).
  • the genetically modified non-human animal further comprises a compromised immune system, such as a genetically modified xenograft of human-derived tissue, including human solid tumors (e.g., breast cancer) or hematological tumors (e.g., Lymphocytic neoplasms, B or T cell neoplasms).
  • anti-PLAU and/or PLAUR antibodies block or inhibit PLAU/PLAUR-mediated signaling pathways.
  • the anti-PLAU antibodies described herein can block the interaction between PLAU and the PLAUR complex, thereby inhibiting the PLAU/PLAUR signaling pathway.
  • the PLAUR antibodies described herein can block the interaction between PLAU and the PLAUR complex, thereby inhibiting the PLAU/PLAUR signaling pathway.
  • genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (eg, anti-PLAU antibodies and/or anti-PLAUR antibodies) in treating various immune diseases.
  • the immune diseases include, but are not limited to, GVHD (graft versus host disease), psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic erythema Lupus, rheumatoid arthritis, scleroderma, hyperthyroidism, essential thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, etc.
  • GVHD graft versus host disease
  • psoriasis psoriasis
  • allergies asthma
  • myocarditis nephritis
  • hepatitis preferably non-alcoholic steatohepatitis
  • genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (eg, anti-PLAU antibodies and/or anti-PLAUR antibodies) in treating various inflammatory infections.
  • the inflammation includes acute inflammation and chronic inflammation. Specifically, it includes but is not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinitis, purulent inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation ( Tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).
  • genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (eg, anti-PLAU antibodies and/or anti-PLAUR antibodies) in treating cancer.
  • a therapeutic agent eg, an anti-PLAU antibody and/or an anti-PLAUR antibody
  • the detection includes determining the size and/or proliferation rate of tumor cells.
  • the detection method includes vernier caliper measurement, flow cytometry detection and/or animal live imaging detection.
  • the detection includes assessment of the individual's body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, including changes in food consumption or water consumption.
  • the tumor cells comprise one or more cancer cells that are injected into an animal (eg, the cancer cells are derived from a human or non-human animal).
  • the therapeutic agent inhibits the PLAU/PLAUR signaling pathway. In some embodiments, the therapeutic agent does not inhibit the PLAU/PLAUR signaling pathway.
  • genetically modified non-human animals can be used to detect whether anti-PLAU or anti-PLAUR antibodies are agonists or antagonists.
  • the methods described herein can be used to test the function of a therapeutic agent (e.g., an anti-PLAU antibody or an anti-PLAUR antibody), e.g., whether the therapeutic agent can upregulate an immune response or downregulate an immune response, and/or the Whether the therapeutic is capable of inducing complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC).
  • CMC complement-mediated cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • genetically modified non-human animals can be used to determine effective doses of therapeutic agents to treat a disease (eg, an immune disease) in a subject.
  • the inhibitory effect on tumors can also be determined by methods known in the art, for example, measuring the tumor volume in the animal, and/or determining the tumor (volume) inhibition rate (TGI TV ).
  • therapeutic agents can be used to treat various cancers.
  • Cancer refers to cells with the ability to grow autonomously, that is, an abnormal state or disease characterized by rapid cell growth and proliferation. The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage.
  • Tumor includes but is not limited to lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, Glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocyte tumor, black Myelodysplastic syndromes, myelodysplastic syndromes, and sarcomas.
  • the leukemia is selected from the group consisting of acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia;
  • the lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia;
  • the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma,
  • the tumor is breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer , neuroendocrine tumors, mesothelial tumors, oropharyngeal tumors, female reproductive system cancers, or meningiomas.
  • the invention also provides an assay for determining the toxicity of a therapeutic agent (eg, anti-PLAU antibody or anti-PLAUR antibody).
  • a therapeutic agent eg, anti-PLAU antibody or anti-PLAUR antibody.
  • the method includes administering the antibody to the non-human animal as described above, and assessing the animal's weight change, red blood cell count, hematocrit and/or hemoglobin.
  • the antibody can reduce red blood cells (RBCs), hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%.
  • the animal's body weight is at least 5%, 10%, 20%, 30%, or 40% less than a control group (eg, the average body weight of animals not treated with the antibody).
  • the present invention also provides an animal model constructed by the method described herein for developing products related to human cellular immune processes, manufacturing human antibodies, or a model system for pharmacological, immunological, microbiological and medical research.
  • an animal model generated by the methods described herein is provided for producing and utilizing human cells for animal experimental disease models, studying pathogens, or developing new diagnostic and/or therapeutic strategies for immune processes.
  • the invention also provides for screening, validating, evaluating or studying PLAU and/or PLAUR gene functions, human PLAU and/or PLAUR antibodies, and drugs targeting human PLAU and/or PALUR target sites through animal models generated by the methods described herein. or effectiveness, drugs for immune-related diseases and anti-tumor drugs.
  • the invention also provides a non-human animal with two or more human or chimeric genes, the animal model comprising human or chimeric PLAU and/or PLAUR genes and nucleic acid sequences encoding other human or chimeric proteins.
  • the other genes are non-human animals genetically modified with at least one of IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, and TNF- ⁇ .
  • the above-mentioned non-human animals also express at least one of human or humanized IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4 and TNF- ⁇ protein. kind.
  • the present invention also provides a method for constructing non-human animals with two or more human or chimeric genes.
  • the construction method includes:
  • step (2) Mating the non-human animal provided in step (1) with other genetically modified non-human animals, performing in vitro fertilization or direct gene editing, and conducting screening to obtain multi-gene modified non-human animals.
  • the other genetically modified non-human animals include one or more of the genes IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4 and TNF- ⁇ A combination of humanized non-human animals.
  • PLAU and/or PLAUR are humanized directly with human or chimeric IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4 and/or TNF- ⁇ genetic modifications. on non-human animals.
  • Multigene-modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, e.g., anti-PLAU antibodies or anti-PLAUR antibodies, and for the treatment of cancer or immune diseases (e.g., asthma or idiopathic dermatitis).
  • the methods include administering an anti-PLAU antibody and/or an anti-PLAU antibody and an additional therapeutic agent to an animal, wherein the animal has a tumor or immune disease, and determining the effect of the combination treatment on the immune tumor or immune disease.
  • the additional therapeutic agent is an antibody that specifically binds IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, and/or TNF-alpha.
  • the additional therapeutic agent is an anti-CTLA4 antibody (eg, ipilimumab), an anti-PD-1 antibody (eg, nivolumab), or an anti-PD-L1 antibody.
  • the non-human animals described above also include sequences encoding human or humanized PD-1, sequences encoding human or humanized PD-L1, or sequences encoding human or humanized CTLA-4. .
  • the additional therapeutic agent is an anti-PD-1 antibody (eg, nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.
  • the tumor described above includes one or more tumor cells expressing PD-L1 and/or PD-L2.
  • the combination therapy may also be used to treat various cancers described herein, such as breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, Glioblastoma, prostate cancer, neuroendocrine tumors, mesothelial tumors, oropharyngeal tumors, female reproductive system cancers, or meningiomas.
  • cancers described herein such as breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, Glioblastoma, prostate cancer, neuroendocrine tumors, mesothelial tumors, oropharyngeal tumors, female reproductive system cancers, or meningiomas.
  • the treatment methods described above can be used in combination with conventional cancer chemotherapy agents.
  • methods of treating cancer can be used alone or in combination with methods described herein, including treating the subject with chemotherapy, such as camphorine, doxorubicin, cisplatin, carboplatin, procarbazine, Methylchloride, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorambucil, endosulfan, nitrosula, actinomycin, daunorubicin, bleomycin , primycin, mitomycin, etoposide, verapir, podophyllotoxin, tamoxifen, paclitaxel, transplatinum, 5-fluoruracil, vincristine, vinblastin and/or Triamterin.
  • the method may include performing surgery on the subject to remove at least a portion of the cancer, such as removing a portion or all of a tumor from the patient
  • mice were purchased from the National Rodent Experimental Animal Seed Center, China Institute of Food and Drug Control;
  • BbsI, EcoRI, BamHI, BclI, and AvrII enzymes were purchased from NEB, and the product numbers are; R0539L, R0101L, R0136L, R0160L, and R0174L respectively;
  • Anti-Urokinase antibody [EPR6273], purchased from Abcam, catalog number ab133563;
  • Trizol kit was purchased from TakaRa, catalog number 6110A.
  • Mouse PLAU gene (NCBI Gene ID: 18792, Primary source: MGI: 97611, UniProt ID: P06869, located at positions 20886730 to 20893456 of chromosome 14 NC_000080.7, based on transcript NM_008873.3 and its encoded protein NP_032899.1 (SEQ ID NO: 1)) and human PLAU gene (NCBI Gene ID: 5328, Primary source: HGNC:9052, UniProt ID: P00749, located at positions 73909182 to 73917497 of chromosome 10 NC_000010.11, based on transcript NM_002658. 6 and its encoded protein NP_002649.2 (SEQ ID NO: 2)) are shown in Figure 1.
  • a nucleotide sequence encoding human PLAU protein can be introduced into the endogenous PLAU locus of a mouse, so that the mouse expresses human or humanized PLAU protein.
  • a nucleotide sequence encoding human PLAU protein can be introduced into the endogenous PLAU locus of a mouse, so that the mouse expresses human or humanized PLAU protein.
  • approximately 5.0 kb of the partial sequence of exon 2 to exon 11 of the human PLAU gene was used to replace mouse exon 2.
  • the partial sequence to exon 11 is about 5.3kb, and the schematic diagram of the humanized PLAU locus is shown in Figure 2, realizing humanized transformation of the mouse PLAU gene.
  • FIG. 3 shows the homology arm sequences containing the upstream and downstream mouse PLAU genes on the targeting vector, as well as the A fragment containing the human PLAU DNA fragment.
  • the upstream 5' homology arm sequence (SEQ ID NO: 3) is the same as the nucleotide sequence 20883305 to 20887146 of NCBI accession number NC_000080.7
  • the downstream 3' homology arm sequence (SEQ ID NO: 4) Identical to the nucleotide sequence from 20893778 to 20896948 of NCBI accession number NC_000080.7.
  • the nucleotide sequence of the human PLAU DNA fragment on fragment A (SEQ ID NO: 7) is identical to the nucleotide sequence at positions 73911556 to 73916565 of NCBI accession number NC_000010.11.
  • the targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and is equipped with two site-specific recombination systems FRT arranged in the same direction on both sides of the resistance gene. Recombination site, forming the Neo box (Neo cassette).
  • connection between the 5' end of the Neo box and the mouse gene is designed as: 5'-AAGAAAAGAAAAATCTGATTCAAACAAAGC GATA TCGAATTCCGAAGTTCCTATTCTCTAGAAAGT-3' (SEQ ID NO: 16), where the sequence The last “C” in is the last nucleotide of mouse, and the “G” in the sequence " GATA " is the first nucleotide of the Neo box;
  • the connection between the 3' end of the Neo box and the mouse gene is designed as: 5'-AGTATAGGAACTTCATCAGTCAGGTACATAATGGTGG GAGT CGGTGCTGTCGACGGGGTCAGGTAATGAAGTA-3' (SEQ ID NO: 17), where the sequence The last "C” in the sequence "GAGT” is the last nucleotide of the Neo box, and the first " G “ in the sequence " GAGT " is the first nucleotide of mouse.
  • a coding gene (gene coding for diphtheria toxin A subunit (DTA)) with a negative selection marker was constructed downstream of the 3' homology arm of the targeting vector.
  • DTA diphtheria toxin A subunit
  • the mRNA sequence of the modified humanized mouse PLAU is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 2.
  • Targeting vector construction can be carried out using conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified through enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing was electroporated and transfected into C57BL/6 mouse embryonic stem cells. The resulting cells were screened using positive clone screening marker genes, and PCR and Southern Blot technology were used to detect and confirm the presence of foreign genes. Integrate the situation and screen out the correct positive clone cells. The clones that are positive after PCR identification (primers are shown in Table 5) are then subjected to Southern Blot testing to confirm that there is no random insertion, and then further sequencing is performed to verify that the correct clones are used for the next experiment.
  • the correct positive clone cells (black mice) screened out were introduced into the separated blastocysts (white mice) according to techniques known in the art, and the resulting chimeric blastocysts were transferred to the culture medium for short culture and then transplanted to the recipient.
  • the fallopian tubes of female mice (white mice) can produce F0 generation chimeric mice (black and white).
  • F0 generation chimeric mice and wild-type mice are backcrossed to obtain F1 generation mice, and then F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice.
  • the positive mice and Flp tool mice can also be mated to remove the positive clone screening marker gene (see Figure 4 for a schematic diagram of this process), and then the PLAU gene humanized homozygous mice can be obtained by mating with each other.
  • the CRISPR/Cas9 system can also be introduced for gene editing to design a targeting strategy as shown in Figure 5.
  • the figure shows the homology arm sequences containing the upstream and downstream mouse PLAU genes on the targeting vector, as well as the human PLAU DNA fragment. sequence.
  • the upstream homology arm sequence (5' homology arm, SEQ ID NO: 5) is the same as the nucleotide sequence at positions 20885866 to 20887146 of NCBI accession number NC_000080.7
  • the downstream homology arm sequence (3' homology Arm, SEQ ID NO: 6) is identical to the nucleotide sequence at positions 20892519 to 20893251 of NCBI accession number NC_000080.7
  • the nucleotide sequence of the PLAU DNA fragment (SEQ ID NO: 7) is identical to the nucleotide sequence at positions 73911556 to 73916565 of NCBI accession number NC_000010.11.
  • the mRNA sequence of the modified humanized mouse PLAU is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 2.
  • Targeting vector construction can be carried out using conventional methods, such as enzyme digestion and ligation, direct synthesis, etc. After the constructed targeting vector is initially verified through enzyme digestion, it is sent to a sequencing company for sequencing verification. The correct targeting vector verified by sequencing was used in subsequent experiments.
  • the target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize an sgRNA sequence that recognizes the target site.
  • the target sequence of an exemplary sgRNA on the PLAU gene is as follows:
  • sgRNA1 target site (SEQ ID NO: 20): 5’-GAGGGCTTGTGCACCCAAAGAGG-3’;
  • sgRNA2 target site (SEQ ID NO: 21): 5’-TGAGACCCTCGTGTAGACACCGG-3’;
  • the pT7-sgRNA vector was synthesized by a plasmid synthesis company.
  • the fragment DNA (SEQ ID NO: 30) containing the T7 promoter and sgRNA scaffold was sequentially connected to the scaffold vector (source: Takara, Cat. No. 3299) through enzyme digestion (EcoRI and BamHI). Sequencing verification by a professional sequencing company showed that the target plasmid was obtained.
  • mice such as C57BL/6 mice
  • a microinjector to inject the in vitro transcription products of pT7-PLAU-1 and pT7-PLAU-2 plasmids (use Ambion in vitro transcription kit, follow the instructions) Transcription
  • targeting vector and Cas9mRNA are premixed and injected into the cytoplasm or nucleus of mouse fertilized eggs.
  • Microinjection of fertilized eggs is carried out.
  • mice After injection, the fertilized eggs are transferred to the culture medium for short culture, and then transplanted into the fallopian tube of the recipient female mouse. Development, the obtained mice (F0 generation) will be crossed and selfed to expand the population and establish a stable PLAU gene humanized mouse strain.
  • the genotype of somatic cells of F1 generation mice can be identified by PCR (primers are shown in Table 1).
  • the identification results of exemplary F1 generation mice are shown in Figure 6, in which 9 mice are numbered F1-01 to F1-09. All mice were positive heterozygous mice.
  • mice identified as positive by F1 generation PCR were subjected to Southern blot testing to confirm whether there was random insertion. Cut the mouse tail to extract genomic DNA, use BclI enzyme or AvrII enzyme to digest the genome, transfer to membrane, and hybridize. The lengths of specific probes and target fragments are shown in Table 7. The detection results of the exemplary F1 generation are shown in Figure 7: F1-01 to F1-09 are all positive heterozygous mice. This indicates that this method was used to successfully construct PLAU gene humanized mice that can be stably passaged without random insertion.
  • the probe synthesis primers are as follows:
  • Mouse PLAUR gene (NCBI Gene ID: 18793, Primary source: MGI: 97612, UniProt ID: P35456, located at positions 24161857 to 24175393 of chromosome 7 NC_000073.7, based on transcript NM_011113.4 and its encoded protein NP_035243.1 (SEQ ID NO: 9)) and human PLAUR gene (NCBI Gene ID: 5329, Primary source: HGNC:9053, UniProt ID: Q03405, located at positions 43646095 to 43670197 of chromosome 19 NC_000019.10, based on transcript NM_002659. 4 and its encoded protein NP_002650.1 (SEQ ID NO: 10)) are shown in Figure 9.
  • the nucleotide sequence encoding the human PLAUR protein can be introduced into the endogenous PLAUR locus of the mouse, so that the mouse expresses the human or humanized PLAUR protein.
  • the mouse expresses the human or humanized PLAUR protein.
  • approximately 18.8 kb of the partial sequence of exon 2 to part 7 of the human PLAUR gene was used to replace mouse exon 2.
  • the partial sequence to exon 7 is about 10.3kb, and the schematic diagram of the humanized PLAUR locus is shown in Figure 10, realizing humanized transformation of the mouse PLAUR gene.
  • FIG. 11 shows the homology arm sequences upstream and downstream of the mouse PLAUR gene on the targeting vector, as well as the A1 fragment containing the human PLAUR DNA fragment.
  • the upstream 5' homology arm sequence (SEQ ID NO: 11) is the same as the nucleotide sequence 24159210 to 24164569 of the NCBI accession number NC_000073.7
  • the downstream 3' homology arm sequence (SEQ ID NO: 12) Identical to nucleotide sequence 24174897 to 24178233 of NCBI accession number NC_000073.7.
  • the nucleotide sequence of the human PLAUR DNA fragment on the A1 fragment (SEQ ID NO: 13) is identical to the nucleotide sequence at positions 43648890 to 43667677 of the NCBI accession number NC_000019.10.
  • the targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and is equipped with two site-specific recombination systems Frt arranged in the same direction on both sides of the resistance gene.
  • the recombination site constitutes the Neo cassette.
  • the connection between the 5' end of the Neo box and the human sequence is designed to be 5'-ATTGTACACTTATGAGAGTGAAAAAG ATCA TCTGCGAATCGGACCCACAAGAGCACTGAGGTCGGAAGTTCCTA TTC-3' (SEQ ID NO:41), where the sequence The “T” is the last nucleotide of the human sequence, and the first " A " of the sequence " ATC A” is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human sequence is designed as: 5 '-AAGTATAGGAACTTCATCAGTCCAGGATACATAGATTACCACAACTCCG GTGA AATCACAGATCTCCTGAAAGGGTCTCTGGG-3' (SEQ ID NO: 42), where the sequence The last "C” is the last nucleotide of the Neo box and the first "G” of the sequence " GTGA " is the first nucleotide of the human sequence.
  • a coding gene with a negative selection marker (gene coding for diphtheria toxin A subunit (DTA)) was constructed downstream of the 3' homology arm of the recombinant vector.
  • DTA diphtheria toxin A subunit
  • Targeting vector construction can be carried out using conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified through enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing was electroporated and transfected into C57BL/6 mouse embryonic stem cells. The resulting cells were screened using positive clone screening marker genes, and PCR and Southern Blot technology were used to detect and confirm the presence of foreign genes. Integrate the situation and screen out the correct positive clone cells. The clones that are positive after PCR identification (primers are shown in Table 9) are then subjected to Southern Blot testing to confirm that there is no random insertion, and then further sequencing is performed to verify that the correct clones are used for the next experiment.
  • the correct positive clone cells (black mice) screened out were introduced into the separated blastocysts (white mice) according to techniques known in the art, and the resulting chimeric blastocysts were transferred to the culture medium for short culture and then transplanted to the recipient.
  • the fallopian tubes of female mice (white mice) can produce F0 generation chimeric mice (black and white).
  • F0 generation chimeric mice and wild-type mice are backcrossed to obtain F1 generation mice, and then F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice.
  • the positive mice and Flp tool mice can also be mated to remove the positive clone selection marker gene (see Figure 12 for a schematic diagram of this process), and then the humanized homozygous mice for the PLAUR gene can be obtained by mating with each other.
  • genotype of somatic cells of F1 generation mice can be identified by PCR (primers are shown in Table 4).
  • Exemplary F1 generation mice The identification results are shown in Figure 13, in which mice numbered F1-01, F1-02 and F1-03 are all positive heterozygous mice.
  • Flow cytometry was further used to detect the expression of PLAUR protein in mice with humanized PLAUR gene.
  • One 7-week-old female wild-type C57BL/6 mouse and one 10-week-old female PLAUR gene humanized homozygous mouse were selected.
  • LPS (20ug/200ul) was injected intraperitoneally to stimulate for 2 hours.
  • the peritoneal lavage fluid was collected and used with anti-mouse CD45.
  • Antibody Brilliant Violet 510 TM anti-mouse CD45Antibody (mCD45), anti-mouse CD11b antibody V450Rat Anti-mouse CD11b Antibody (mCD11b), anti-mouse F4/80 antibody FITC anti-mouse F4/80 Antibody (mF4/80), anti-mouse PLAUR Antibody CD87Antibody, anti-mouse, REAfinity TM (mPLAUR), anti-human PLAUR antibody CD87Antibody, anti-human, REAfinity TM (hPLAUR), Zombie NIR TM Fixable Viability Kit and Purified anti-mouse CD16/32 were stained, and the stained The cells were analyzed by flow cytometry, and the characteristics of macrophages in ascitic fluid were: CD45+CD11b+mF4/80+.
  • the macrophages in the ascites of wild-type mice had 99.7% mPLAUR-positive cells (characterized by CD45+CD11b+mF4/80+mPLAUR+) and 2.24% hPLAUR-positive cells (characterized by CD45+CD11b+mF4/80+ hPLAUR+), the macrophages in the ascites of mice homozygous for the humanized PLAUR gene have 0.20% mPLAUR-positive cells (characterized by CD45+CD11b+mF4/80+mPLAUR+), and 99.6% hPLAUR-positive cells (characterized by CD45+CD11b+mF4 /80+hPLAUR+), in summary, it is shown that the PLAUR humanized mice constructed in this example can successfully express the humanized PLAUR protein.
  • PLAU/PLAUR double-gene humanized mice were obtained through screening of positive offspring mice. Conventional methods can be used to confirm the expression of human PLAU protein in positive mice, such as ELSA. Specifically, 3 9-week-old male wild-type C57BL/6 mice (+/+) and 3 male PLAU/PLAUR double-gene humanized homozygous mice (H/H) prepared in this example were selected respectively. Serum was collected after euthanasia by cervical dislocation and tested using mouse uPA ELISA kit and human uPA ELISA kit (URK).
  • ELSA 3 9-week-old male wild-type C57BL/6 mice (+/+) and 3 male PLAU/PLAUR double-gene humanized homozygous mice (H/H) prepared in this example were selected respectively. Serum was collected after euthanasia by cervical dislocation and tested using mouse uPA ELISA kit and human uPA ELISA kit (URK).
  • Flow cytometry can be used to detect hPLAUR protein expression in PLAU/PLAUR double-gene humanized homozygous mice. Specifically, 1 9-week-old male wild-type C57BL/6 mouse (+/+) and 10-week-old male PLAU/PLAUR double-gene humanized homozygous mouse (H/H) prepared in this example were selected.
  • anti-mouse CD45 antibody Brilliant Violet 510 TM anti-mouse CD45 (mCD45), anti-mouse Ly6G antibody Brilliant Violet 650 TM anti-mouse Ly-6G Antibody (mLy6G), and anti-mouse CD11b antibody were used V450 Rat Anti-mouse CD11b Antibody (mCD11b), anti-mouse CD3 antibody Alexa 700 anti-mouse CD3 Antibody (mCD3), anti-mouse F4/80 antibody FITC anti-mouse F4/80Antibody (mF4/80), anti-mouse CD11c antibody Invitrogen CD11c Monoclonal Antibody (N418), PE-Cyanine7, eBioscience (mCD11c), Anti-mouse PLAUR antibody CD87 Antibody, anti-mouse, REAfinity TM (mPLAUR), anti-human PLAUR antibody CD87 Antibody, anti-human
  • flow cytometry can be used to verify whether the PLAU/PLAUR double-gene humanized homozygous mice prepared in this example can bind to the two anti-human PLAUR antibodies Ab1 and Ab2, and 8-week-old male PLAU/PLAUR double-gene humanized mice are selected.
  • One homozygous mouse was homozygous. Injected intraperitoneally with LPS (20ug/200ul) and stimulated for 2 hours to collect peritoneal lavage fluid.
  • Anti-mouse CD45 antibody Brilliant Violet 510 TM anti-mouse CD45 and anti-mouse Ly6G antibody Brilliant Violet 650 TM anti-mouse were used.
  • Ly-6G Antibody anti-mouse CD11b antibody V450Rat Anti-mouse CD11b Antibody, anti-mouse F4/80 antibody FITC anti-mouse F4/80 Antibody, anti-human PLAU antibody Ab1, anti-human PLAUR antibody Ab2, anti-human IgG1 antibody PE Human IgG1 After identification and staining with Isotype Control Recombinant Antibody, anti-mouse IgG1 antibody APC Human IgG1 Isotype Control Recombinant Antibody, Zombie NIR TM Fixable Viability Kit and Purified anti-mouse CD16/32, flow cytometry analysis was performed.
  • PLAU/PLAUR dual-gene human source There were 100% Ab1-positive cells and 98.3% Ab2-positive cells in the mononuclear macrophages in the peritoneal lavage fluid of homozygous mice.
  • LAU/PLAUR double-gene humanized homozygous mice constructed in this example can The anti-human PLAUR antibody was successfully combined and can subsequently be used to evaluate the efficacy of the human PLAUR-targeting antibody.
  • PLAU/PLAUR double-gene humanized homozygous mice were subcutaneously inoculated with MC38 colon cancer cells expressing humanized PLAUR. After the tumor volume grew to about 100 mm, they were divided into a control group or a treatment group according to the tumor volume. The treatment group Antibody drugs targeting human PLAUR were injected, and the control group was injected with an equal volume of PBS. Regularly measure the tumor volume and weigh the mice. By comparing the mouse body weight and tumor volume, the safety and in vivo efficacy of antibody drugs in humanized PLAU/PLAUR mice can be effectively evaluated.
  • mice After vaccination, a single mouse When the mouse tumor volume reaches 3000mm3 , euthanasia should be performed to end the test.
  • the specific grouping, administration, dosage and frequency are shown in Table 12.
  • the tumor volume and body weight of mice during the experiment are shown in Figures 17 and 18 respectively.
  • Table 13 lists the main data and analysis results of the experiment, including tumor volume at the time of grouping and on days 14 and 21 after grouping, mouse survival, and tumor (volume) inhibition rate (Tumor Growth Inhibition Value, TGI TV ) and the statistical difference (P value) between the tumor volumes of mice in the treatment group and the control group.
  • the average tumor volume in the control group was 2237 ⁇ 341mm 3
  • the average tumor volume in the treatment group at the dose levels of Ab1 (10mg/kg) and Ab2 (10mg/kg) was 1926 ⁇ 566mm 3 respectively.
  • 1726 ⁇ 250mm 3 The tumor volumes of mice in all treatment groups were smaller than those of the control group, with TGI TVs of 14.5% and 23.8% respectively, indicating that different doses and types of anti-human PLAUR antibodies have different inhibitory effects on tumors.
  • TGI TVs 14.5% and 23.8%
  • Double-gene modified or multi-gene modified mouse models can also be prepared using this method or the prepared PLAU and PLAUR gene humanized mice.
  • the embryonic stem cells used for blastocyst microinjection can be derived from mice containing other genetic modifications such as PLAUR, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, etc., or, On the basis of humanized PLAU mice, isolation of mouse ES embryonic stem cells and gene recombination targeting technology can also be used to obtain dual-gene or multi-gene modified mouse models of PLAU and other genetic modifications.
  • the homozygous or heterozygous PLAU mice obtained by this method can also be mated with homozygous or heterozygous mice modified by other genes, and their offspring can be screened. According to the laws of Mendelian inheritance, there is a certain probability of obtaining PLAU genes and other genes. Gene-modified double-gene or multi-gene-modified heterozygous mice, and then heterozygotes are mated with each other to obtain double-gene or multi-gene modified homozygotes.
  • PLAUR humanized mice were obtained using the aforementioned Example 2. According to the above method, mice homozygous for PLAUR gene and other genetic modifications can also be obtained. These dual-gene or multi-gene modified mice can be used to verify the in vivo efficacy of targeting human PLAU, PLAUR and other gene regulators.
  • any combination of various embodiments of the present invention can also be carried out. As long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

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Abstract

提供一种表达人或嵌合(例如，人源化)PLAU和/或PLAUR蛋白的非人动物及其使用方法。

Description

一种PLAU和/或PLAUR基因修饰的非人动物
优先权要求
本专利申请要求于2022年6月6日提交的中国专利申请号202210631316.5的优先权，该专利申请的全部内容通过引用并入本文。
技术领域
本发明提供一种表达人或嵌合(例如，人源化)PLAU和/或PLAUR蛋白的非人动物及其使用方法。
背景
传统的药物研发通常使用体外筛选方法，然而这些筛选方法无法提供机体环境(如肿瘤微环境、基质细胞、细胞外基质成分和免疫细胞相互作用等)，导致药物开发失败率较高。此外，鉴于人与动物之间的差异，使用常规实验动物进行体内药理试验获得的试验结果可能无法反映真实的疾病状态和靶向部位的相互作用，导致许多临床试验的结果与动物实验结果存在显著差异。
因此，开发适合人抗体筛选和评价的人源化动物模型将显著提高新药开发效率，降低药物研发成本。
概述
本申请提供一种具有人或嵌合PLAU和/或PLAUR蛋白的动物模型。该动物模型可以表达人或嵌合PLAU(如，人源化PLAU)蛋白和/或人或嵌合PLAUR(如，人源化PLAUR)蛋白。它可用于PLAU和PLAUR基因功能的研究，还可用于PLAU/PLAUR信号通路调节剂(例如，抗人PLAU抗体或抗人PLAUR抗体)的筛选和评估。此外，通过本文所述方法制备的动物模型可用于药物筛选、药效学研究、免疫相关疾病的治疗和人PLAU/PLAUR靶位点的癌症治疗；该模型还可以用于促进新药开发和设计，节省时间和成本。综上所述，本发明为研究PLAU/PLAUR蛋白的功能提供了强有力的工具，为筛选抗癌药物提供了平台。
在一方面，本发明提供了一种基因修饰的非人动物，所述动物的一个或多个细胞表达人或嵌合PLAUR蛋白，人PLAUR配体(PLAU)可以结合表达人或嵌合PLAUR蛋白，激活下游信号通路。在一些实施例中，所述编码人或嵌合PLAUR蛋白的核苷酸序列可操作地连接至至少一条染色体的内源PLAUR基因座的内源调控元件(如，内源5'UTR和/或3'UTR)。在一些实施例中，所述编码人或嵌合PLAUR蛋白的核苷酸序列包含编码人PLAUR蛋白的3个同源结构域。在一些实施例中，所述编码人或嵌合PLAUR蛋白的核苷酸序列编码的氨基酸序列与人PLAUR(NP_002650.1，SEQ ID NO：10)同一性至少为70％、75％、 80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码人或嵌合PLAUR蛋白的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：10第24-335位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码人或嵌合PLAUR蛋白的核苷酸序列包含与SEQ ID NO：15所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述动物是哺乳动物，如猴子、啮齿动物、小鼠或大鼠。在一些实施例中，所述动物是小鼠。在一些实施例中，所述动物的一个或多个细胞表达人或嵌合PLAUR蛋白。在一些实施例中，所述动物的一个或多个细胞表达人或嵌合PLAUR蛋白的细胞，内源PLAUR配体(PLAU)可以结合表达人或嵌合PLAUR蛋白，激活下游信号通路。在一些实施例中，所述动物的一个或多个细胞表达人或嵌合PLAUR蛋白，人PLAUR配体(PLAU)可以结合表达人或嵌合PLAUR蛋白，激活下游信号通路。
在一方面，本发明提供了一种基因修饰的非人动物，所述动物的基因组包含在内源PLAUR基因座处编码内源PLAUR区域的核苷酸序列被人PLAUR相应区域的核苷酸序列替换。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列可操作地连接到内源PLAUR基因座的内源调控元件，并且所述动物的一个或多个细胞表达人或人源化的PLAUR蛋白。在一些实施例中，所述动物的内源PLAUR蛋白不表达或与野生型动物中PLAUR相比表达水平降低。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列包含人PLAUR基因的外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列与SEQ ID NO：13所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码内源PLAUR区域的核苷酸序列包含小鼠PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述动物基因组中修饰的基因对于内源被替换的基因座为纯和或杂合。
在一方面，本发明提供了一种非人动物，所述动物包含至少一个编码人或人源化PLAUR蛋白的核苷酸序列的细胞，其中所述人或人源化PLAUR蛋白包含与人相应区域的连续氨基酸序列至少50、100、150、200、250、300、310、311、312、320、330、331、332、333、334或335个连续氨基酸序列一致。在一些实施例中，所述人或人源化PLAUR蛋白包含人PLAUR蛋白的3个同源结构域。在一些实施例中，所述人或人源化PLAUR蛋白的氨基酸序列与SEQ ID NO：10所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列可操作地连接至内源PLAUR调控元件。在一些实施例中，所述编码人PLAUR相应区域的 核苷酸序列可被整合至所述动物内源PLAUR基因座。在一些实施例中，所述人或人源化PLAUR蛋白具有至少一种小鼠PLAUR活性和/或人PLAUR活性。
在一方面，本发明提供了一种基因修饰的非人动物的构建方法，所述动物的至少一个细胞中，在动物内源PLAUR基因座处，编码内源PLAUR区域的核苷酸序列被人PLAUR相应区域的核苷酸序列替换。在一些实施例中，所述动物的内源PLAUR蛋白不表达或与野生型动物中PLAUR相比表达水平降低。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列包含编码人PLAUR蛋白的3个同源结构域。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列包含人PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：10第24-335所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码内源PLAUR区域的核苷酸序列包含小鼠PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列可操作地连接至内源PLAUR调控元件，如，启动子。在一些实施例中，所述动物为哺乳动物，如，猴子、啮齿动物、小鼠或大鼠。在一些实施例中，所述动物是小鼠。在一方面，本发明提供了一种表达人或嵌合PLAUR基因修饰的非人动物的构建方法，所述方法包括在内源小鼠PLAUR基因座处，编码内源PLAUR区域的核苷酸序列被人PLAUR相应区域的核苷酸序列替换，产生基因修饰的非人动物细胞，所述动物细胞表达人或嵌合PLAUR蛋白。在一些实施例中，所述人或嵌合PLAUR蛋白包含人PLAUR蛋白的3个同源结构域。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列包含人PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：10第24-335所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列与SEQ ID NO：13所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述内源PLAUR的核苷酸序列包含小鼠PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述编码人或嵌合PLAUR蛋白的核苷酸序列可操作地连接至内源PLAUR的调控元件，如，启动子。在一些实施例中，所述动物为哺乳动物，如，猴子、啮齿动物、小鼠或大鼠。在一些实施例中，所述非人动物是小鼠。
在一方面中，本发明提供了一种表达人或嵌合PLAUR的基因修饰非人动物细胞的构建 方法，所述的方法包括在内源小鼠PLAUR基因座处，编码内源PLAUR区域的核苷酸序列被人PLAUR相应区域的核苷酸序列替换，产生基因修饰的非人动物细胞，所述动物细胞表达人或嵌合PLAUR蛋白。在一些实施例中，所述人或嵌合PLAUR蛋白包含人PLAUR蛋白的3个同源结构域。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列包含人PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：10第24-335所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列与SEQ ID NO：13所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述内源PLAUR的核苷酸序列包含小鼠PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述编码人或嵌合PLAUR蛋白的核苷酸序列可操作地连接至内源PLAUR的调控元件，如，启动子。在一些实施例中，所述非人动物是小鼠。在一些实施例中，所述非人动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，所述人或嵌合蛋白选自PLAUR配体(PLAU)、IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种。在一些实施例中，所述人或嵌合蛋白为人或嵌合PLAU蛋白。在一些实施例中，所述人或嵌合PLAU蛋白包含人PLAU基因编码的全长蛋白。在一些实施例中，所述人或嵌合PLAU蛋白的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述非人动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，其中所述人或嵌合蛋白选自PLAUR配体(PLAU)、IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种。在一些实施例中，所述人或嵌合蛋白为人或嵌合PLAU蛋白。在一些实施例中，所述人或嵌合PLAU蛋白包含人PLAU基因编码的全长蛋白。在一些实施例中，所述人或嵌合PLAU蛋白的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
在一方面，本发明提供了一种基因修饰的非人动物，所述动物基因组包含至少一条染色体，所述染色体包含编码人或嵌合尿激酶型纤溶酶原激活剂(PLAU)蛋白的核苷酸序列。在一些实施例中，所述编码人或嵌合PLAU蛋白的核苷酸序列可操作地连接至至少一条染色体的内源PLAU基因座的调控元件(如，内源5'UTR和/或3'UTR)。在一些实施例中，所述编码人或嵌合PLAU蛋白的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例 中，所述动物是哺乳动物，如猴子、啮齿动物、小鼠或大鼠。在一些实施例中，所述动物是小鼠。在一些实施例中，所述动物的内源PLAUR蛋白不表达或与野生型动物中PLAU相比表达水平降低。在一些实施例中，所述动物的一个或多个细胞表达人或嵌合PLAU蛋白。在一些实施例中，所述动物的一个或多个细胞表达人或嵌合PLAU蛋白，内源PLAU受体(PLAUR)可以结合表达人或嵌合PLAU蛋白，激活下游信号通路。在一些实施例中，所述动物的一个或多个细胞表达人或嵌合PLAU蛋白，人PLAU受体(PLAUR)可以结合表达人或嵌合PLAU蛋白，激活下游信号通路。
在一方面，本发明提供了一种基因修饰的非人动物，所述动物的基因组包含在内源PLAU基因座处，编码内源PLAU区域的核苷酸序列被人PLAU相应区域的核苷酸序列替换。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列可操作地连接至内源PLAU基因座的调控元件，所述动物的一个或多个细胞表达人或人源化的PLAU蛋白。在一些实施例中，所述动物的内源PLAUR蛋白不表达或与野生型动物中PLAU相比表达水平降低。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含人PLAU基因的外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含人PLAU编码区的全部核苷酸序列。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：7所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：8所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码内源PLAU区域的核苷酸序列包含小鼠PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所述动物基因组中修饰的基因对于内源被替换的基因座为纯合或杂合。
在一方面，本发明提供了一种非人动物，所述动物包含至少一个编码人或人源化PLAU蛋白的核苷酸序列的细胞，其中所述人源化PLAU蛋白包含与人相应区域的连续氨基酸序列至少50、100、150、200、250、300、350、400、410、420、430、或431个连续氨基酸一致。在一些实施例中，所述人源化PLAU蛋白的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人或人源化PLAU蛋白的核苷酸序列可操作地连接至内源PLAU调控元件。在一些实施例中，所述编码人或人源化PLAU蛋白的核苷酸序列可被整合至所述动物内源PLAU基因座。在一些实施例中，所述人源化PLAU蛋白具有至少一种小鼠的PLAU活性和/或人 PLAU活性。
在一方面，本发明提供了一种基因修饰的非人动物的构建方法，所述动物的至少一个细胞中，在动物内源PLAU基因座处，编码内源PLAU区域的核苷酸序列被人PLAU相应区域的核苷酸序列替换。在一些实施例中，所述非人动物的内源PLAU蛋白不表达或与野生型动物中PLAU相比表达水平降低。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含编码人PLAU蛋白的全部序列。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含人PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含编码的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：7所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码内源PLAU区域的核苷酸序列包含小鼠PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所述动物基因组中修饰的基因对于内源被替换的基因座为纯合或杂合。
在一方面，本发明提供了一种非人动物，所述动物包含至少一个编码人或人源化PLAU蛋白的核苷酸序列的细胞，其中所述人源化PLAU蛋白包含与人相应区域的连续氨基酸序列至少50、100、150、200、250、300、350、400、410、420、430、或431个连续氨基酸一致。在一些实施例中，所述人源化PLAU蛋白的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人或人源化PLAU蛋白的核苷酸序列可操作地连接至内源PLAU调控元件。在一些实施例中，所述编码人或人源化PLAU蛋白的核苷酸序列可被整合至所述动物内源PLAU基因座。在一些实施例中，所述人源化PLAU蛋白具有至少一种小鼠的PLAU活性和/或人PLAU活性。
在一方面，本发明提供了一种基因修饰的非人动物的构建方法，所述动物的至少一个细胞中，在动物内源PLAU基因座处，编码内源PLAU区域的核苷酸序列被人PLAU相应区域的核苷酸序列替换。在一些实施例中，所述非人动物的内源PLAU蛋白不表达或与野生型动物中PLAU相比表达水平降低。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含编码人PLAU蛋白的全部序列。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含人PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所 述编码人PLAU相应区域的核苷酸序列包含编码的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：7所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码内源PLAU区域的核苷酸序列包含小鼠PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列可操作地连接至内源PLAU的调控元件，如，启动子。在一些实施例中，所述动物为哺乳动物，如猴子、啮齿动物、小鼠或大鼠。在一些实施例中，所述动物是小鼠。
在一方面，本发明提供了一种表达人或嵌合PLAU蛋白的基因修饰非人动物的细胞构建方法，所述方法包括在内源小鼠PLAU基因座处，用编码人PLAU相应区域的核苷酸替换编码内源PLAU区域的核苷酸序列，产生基因修饰的非人动物细胞，其中动物细胞表达人或嵌合PLAU蛋白。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含人PLAU蛋白的全部序列。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含人PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：7所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述编码内源PLAU区域的核苷酸序列包含小鼠PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所述动物是小鼠。在一些实施例中，所述编码人或嵌合PLAU蛋白的核苷酸序列可操作地连接至内源PLAU的调控元件，如，启动子。在一些实施例中，所述动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，其中所述人或嵌合蛋白选自PLAU受体(PLAUR)、IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种。在一些实施例中，所述人或嵌合蛋白为人或嵌合PLAUR蛋白。在一些实施例中，所述人或嵌合PLAUR蛋白的氨基酸序列与SEQ ID NO：14所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，其中所述人或嵌合蛋白选自PLAU受体(PLAUR)、IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种。在一 些实施例中，所述人或嵌合蛋白为人或嵌合PLAUR蛋白。在一些实施例中，所述人或嵌合PLAUR蛋白的氨基酸序列与SEQ ID NO：14所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
在一方面，本发明提供了一种测定抗PLAU和/或PLAUR抗体治疗癌症有效性的方法，所述方法包括：1)向本申请所述动物施用抗PLAU和/或PLAUR抗体，其中所述动物具有肿瘤；2)测定抗PLAU和/或PLAUR抗体对肿瘤的抑制作用。在一些实施例中，所述肿瘤包含一个或多个肿瘤细胞，其中肿瘤细胞被注射到动物体内。在一些实施例中，所述测定抗PLAU/PLAUR抗体对肿瘤的抑制作用包含测量动物体内的肿瘤体积。在一些实施例中，所述肿瘤包括乳腺癌、胰腺癌、内分泌癌、头颈癌、胃肠癌、结直肠癌、膀胱癌、非小细胞肺癌、胶质母细胞瘤、前列腺癌、神经内分泌瘤、间皮组织肿瘤、口咽肿瘤、女性生殖系统癌症或脑膜瘤。
在一方面，本发明提供了一种测定抗PLAU和/或PLAUR抗体和其它治疗剂治疗癌症有效性的方法，所述方法包括：1)向本申请所述的动物施用抗PLAU和/或PLAUR抗体，其中所述动物具有肿瘤；2)测定抗PLAU和/或PLAUR抗体对肿瘤的抑制作用。在一些实施例中，所述动物还包括编码人或嵌合PD-1、人或嵌合PD-L1和/或人或嵌合CTLA4的序列。在一些实施例中，所述附加治疗剂是抗PD-1抗体、抗PD-L1抗体和/或抗CTLA4抗体。在一些实施例中，所述肿瘤包含一个或多个细胞表达PD-L1蛋白。在一些实施例中，所述肿瘤包含一个或多个肿瘤细胞，其中肿瘤细胞被注射到动物体内。在一些实施例中，所述测定抗PLAU/PLAUR抗体对肿瘤的抑制作用包含测量动物体内的肿瘤体积。在一些实施例中，所述肿瘤为乳腺癌、胰腺癌、内分泌癌、头颈癌、胃肠癌、结直肠癌、膀胱癌、非小细胞肺癌、胶质母细胞瘤、前列腺癌、神经内分泌瘤、间皮组织肿瘤、口咽肿瘤、女性生殖系统癌症或脑膜瘤。
在一方面，本发明提供了一种测定抗PLAU和/或PLAUR抗体治疗免疫疾病有效性的方法，所述方法包括：1)向本申请所述非人动物施用抗PLAU和/或PLAUR抗体，其中所述非人动物具有免疫疾病；2)测定抗PLAU和/或PLAUR抗体对治疗免疫疾病中的作用。在一些实施例中，所述免疫疾病为皮肤溃疡病、类风湿性关节炎、中风。
在一方面，本发明提供了一种测定抗PLAU和/或PLAUR抗体治疗炎症有效性的方法，所述方法包括：1)向权本申请所述的动物施用抗PLAU和/或PLAUR抗体，其中所述动物具有炎症；2)测定抗PLAU和/或PLAUR抗体对治疗炎症的有效性。在一些实施例中，所述炎症为脓毒症或炎性疾病。
在一方面，本发明提供了一种测定抗PLAU和/或PLAUR抗体毒性的方法，所述方法 包括：1)向本申请所述动物施用抗PLAU和/或PLAUR抗体；2)测定抗PLAU和/或PLAUR抗体对动物的作用。在一些实施例中，所述测定抗PLAU和/或PLAUR抗体对动物的作用涉及测量动物的体重、红细胞计数、血细胞比容和/或血红蛋白。
在一方面，本发明提供了一种人源化PLAUR蛋白，所述人源化蛋白包含人PLAUR蛋白的3个同源结构域。在一些实施例中，所述人源化PLAUR蛋白的氨基酸序列与SEQ ID NO：10第24-335位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。在一些实施例中，所述人源化PLAUR蛋白的氨基酸序列与SEQ ID NO：15所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
在一方面，本发明提供了一种人源化PLAUR基因，所述人源化PLAUR基因编码上述人源化蛋白。所述人源化PLAUR基因包含人PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。在一些实施例中，所述人源化PLAUR基因包含的核苷酸序列与SEQ ID NO：14所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
在一方面，本发明提供了一种人源化PLAU蛋白，所述人源化PLAU蛋白包含人PALU蛋白的全部或部分。在一些实施例中，所述人源化PLAU蛋白的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
在一方面，本发明提供了一种人源化PLAU基因，所述人源化PLAU基因编码上述人源化蛋白。在一些实施例中，所述人源化PLAU基因包含人PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。在一些实施例中，所述人源化PLAU基因包含的核苷酸序列与SEQ ID NO：8所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
在一方面，本发明提供了一种细胞，所述细胞中包含上述人源化PLAUR基因和/或上述人源化PLAU基因，和/或，所述的细胞表达任一所述人源化PLAUR蛋白和/或任一所述人源化PLAU蛋白。
在一方面，本发明提供了一种动物模型，所述的动物模型包含包含任一所述人源化PLAUR基因和/或任一所述人源化PLAU基因，和/或，所述动物模型表达任一所述人源化PLAUR蛋白和/或任一所述人源化PLAU蛋白。
除非另有定义，本文使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。本文描述了用于本发明的方法和材料，也可以使用本领域已知的其他合适的方法和材料。材料、方法和实施例仅是说明性的而不是限制性的。文提及的所有出版物、专利申请、专利、序列、数据库条目和其他参考文献均通过引用整体并入。在冲突的情况下， 以本说明书(包括定义)为准。
附图说明
图1：小鼠PLAU基因和人PLAU基因座对比示意图(非按比例)；
图2：小鼠PLAU基因人源化改造示意图(非按比例)；
图3：PLAU基因打靶策略及靶向载体设计示意图(非按比例)；
图4：PLAU基因人源化小鼠FRT重组过程示意图(非按比例)；
图5：PLAU基因打靶策略及靶向载体设计示意图(非按比例)；
图6：PLAU基因人源化小鼠F1代鼠尾PCR鉴定结果，其中，WT为野生型对照，H2O为水对照，M为Marker；
图7：Southern Blot检测结果示意图，其中WT为野生型对照；
图8：C57BL/6野生型小鼠(+/+)和PLAU基因人源化杂合子小鼠(H/+)RT-PCR检测结果，其中，H2O为水对照，GAPDH为甘油醛-3-磷酸脱氢酶内参；
图9：小鼠PLAUR基因和人PLAUR基因座对比示意图(非按比例)；
图10：小鼠PLAUR基因人源化改造示意图(非按比例)；
图11：PLAUR基因打靶策略及靶向载体设计示意图(非按比例)；
图12：PLAUR基因人源化小鼠FRT重组过程示意图(非按比例)；
图13：PLAUR基因人源化小鼠F1代鼠尾PCR鉴定结果，其中，WT为野生型对照，PC为阳性对照，H2O为水对照，M为Marker；
图14：C57BL/6野生型小鼠(+/+)和PLAUR基因人源化杂合子小鼠(H/+)RT-PCR检测结果，其中，H2O为水对照，GAPDH为甘油醛-3-磷酸脱氢酶内参；
图15：C57BL/6野生型小鼠(+/+)和PLAU基因人源化纯合子小鼠(H/H)Western blot检测结果，其中，hPLAU为人PLAU蛋白，β-actin为β肌动蛋白内参。
图16：ELSA检测血清中PLAU的表达，其中图16A为鼠源PLAU蛋白表达检测结果；图16B为人源PLAU蛋白表达检测结果
图17：将PLAUR人源化结肠癌细胞MC38植入PLAU/PLAUR双人源化纯合子小鼠体内，进行抗肿瘤药效试验后小鼠的肿瘤体积结果示意图；
图18：将PLAUR人源化结肠癌细胞MC38植入PLAU/PLAUR双人源化纯合子小鼠体内，进行抗肿瘤药效试验后小鼠的体重结果示意图；
图19：人PLAU氨基酸序列(NP_002649.2；SEQ ID NO：2)和小鼠PLAU氨基酸序列(NP_032899.1；SEQ ID NO：1)；
图20：人PLAU氨基酸序列(NP_002649.2；SEQ ID NO：2)和大鼠PLAU氨基酸序列(NP_037217.3；SEQ ID NO：54)；
图21：人PLAUR氨基酸序列(NP_002650.1；SEQ ID NO：10)和小鼠PLAUR氨基酸序列(NP_035243.1；SEQ ID NO：9)；
图22：人PLAUR氨基酸序列(NP_002650.1；SEQ ID NO：10)和大鼠PLAUR氨基酸序列(NP_599179.2；SEQ ID NO：55)；
详细说明
在众多重要的生理过程中，尿激酶纤溶酶原激活系统Urokinase plasminogen activator system(UPAS)通过激活无所不在的蛋白纤溶酶来调节纤维蛋白的降解、细胞外基质(ECM)重塑以及细胞迁移等。该系统成员包括尿激酶原激活物(PLAU)、尿激酶原激活物受体(PLAUR)、纤溶酶原激活物抑制剂-1(PAI-1)以及纤溶酶原激活物抑制剂-2(PAI-2)。UPAS的调控是严格的、细胞特异性的，涉及到对酶原、酶抑制剂和受体的调控，在胚胎发育、创面愈合和泌乳后乳腺组织的发育等生理过程中对ECM的重构起重要作用。除了在生理过程中发挥作用外，UPAS在大多数肿瘤中都很活跃，其调节功能的异常与肿瘤转移表型及发展有关。经研究发现，PLAU和PLAUR与恶性肿瘤的侵袭转移关系密切，在对细胞外基质降解，组织重塑，血管生成，细胞浸润增殖中发挥着重要作用，对肿瘤的恶性程度以及预后的判断具有一定的指导意义
PLAUR
尿激酶型纤溶酶原激活物受体(urokinase-type plasminogen activator(uPA)receptor，PLAUR)，是一个富含半胱氨酸的糖基化的单链蛋白，是一种定位于细胞表面的糖基磷脂酰肌醇(GPI)锚受体。它包含3个同源结构域(DI、DII和DIII)，其通过二硫键连接。因为缺少横跨膜和细胞内区域，PLAUR必须与整合素、蛋白偶联受体(GPCRs)和生长因子受体等合作形成共受体，以激活细胞内的信号，促进细胞运动、侵袭、增殖和存活。除了PLAU外，玻连蛋白是PLAUR的另一种关键配体，这两种配体与受体间的结合位点是分开的。PLAUR同时结合以上两种配体来共同进行蛋白酶的下游调节，细胞黏附和信号转导。PLAUR基因的表达通过转录和转录后结合机制进行调控。PLAUR基因的上游区域包含许多转录因子的顺式作用元件，包括Sp1、AP1、AP2、PEA3、Kruppel-ike factor 4、乏氧因子1α(HIF-1α)、NF-κB、TCF和LEF等大量的信号通路激活这些作用在PLAUR基因的转录因子，从而协调PLAUR在肿瘤中的表达。此外，近年来多项研究发现在类风湿性关节炎(RA)患者体内，PLAU和PLAUR在患者发炎的滑膜的巨噬细胞、中性粒细胞和内皮细胞 中表达，在健康人的滑膜中不表达，PLAU和PLAUR在RA治疗领域也受到越来越多的关注。
在人的基因组中，PLAUR基因(Gene ID：5329)包含7个外显子，即外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和外显子7(图9)。人PLAUR mRNA的核苷酸序列为NM_002659.4，人PLAUR的氨基酸序列为NP_002650.1(SEQ ID NO：10)。基于转录本NM_002659.4及其编码蛋白NP_002650.1的核苷酸序列和氨基酸序列中每个外显子对应位置如下：
表1
人PLAUR基因(NCBI Gene ID：5329)位于19号染色体上的NC_000019.10的第43646095至43670197位(GRCh38.p13(GCF_000001405.39)。基于转录本NM_002659.4的核苷酸序列中每个外显子的具体位置为：5'UTR位于NC_000019.10第43670169至43670121位，外显子1位于NC_000019.10第43670169至43670066位，内含子1位于NC_000019.10第43670065至43667692位，外显子2位于NC_000019.10第43667691至43667581位，内含子2位于NC_000019.10第43667580至43665460位，外显子3位于NC_000019.10第43665459至43665316位，内含子3位于NC_000019.10第43665315至43656641位，外显子4位于NC_000019.10第43656640至43656479位，内含子4位于NC_000019.10第43656478至43655574位，外显子5位于NC_000019.10第43655573至43655439位，内含子5位于NC_000019.10第43655438至43652372位，外显子6位于NC_000019.10第43652371至43652225位，内含子6位于NC_000019.10第43652224到 43649144位，外显子7位于NC_000019.10第43649143到43648579位。3'UTR位于NC_000019.10第43648889至43648579位。以上关于人PLAUR基因座的所有相关信息都可以在NCBI网站上(Gene ID：5329)检索到。其全部内容通过引用并入本文。
在鼠的基因组中，PLAUR基因(Gene ID：18793)包含7个外显子，即外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和外显子7(图9)。鼠PLAUR mRNA的核苷酸序列为NM_011113.4，鼠PLAUR的氨基酸序列为NP_035243.1(SEQ ID NO：9)。基于转录本NM_011113.4及其编码蛋白NP_035243.1的核苷酸序列和氨基酸序列中每个外显子对应位置如下：
表2
鼠PLAUR基因(NCBI Gene ID：18793)位于7号染色体上的NC_000073.7的第24161857至24175393位(GRCm39(GCF_000001635.27))。基于转录本NM_011113.4的核苷酸序列中每个外显子的具体位置为：5'UTR位于NC_000073.7第24161909至24161934位，外显子1位于NC_000073.7第24161909至24161992位，内含子1位于NC_000073.7第24161993至24164555位，外显子2位于NC_000073.7第24164556至24164666位，内含子2位于NC_000073.7第24164667至24166099位，外显子3位于NC_000073.7第24166100至24166243位，内含子3位于NC_000073.7第24166244至24171323位，外显子4位于NC_000073.7第24171324至24171479位，内含子4位于NC_000073.7第24171480至24171997位，外显子5位于NC_000073.7第24171998至24172132位，内含子5位于 NC_000073.7第24172133至24173634位，外显子6位于NC_000073.7第24173635至24173781位，内含子6位于NC_000073.7第24173782到24174663位，外显子7位于NC_000073.7第24174664到24175392位，3'UTR位于NC_000073.7第24174897至24175392位。以上关于鼠PLAUR基因座的所有相关信息都可以在NCBI(Gene ID：18793)网站上查找到。其全部内容通过引用并入本文。
图21显示了人PLAUR氨基酸序列(NP_002650.1；SEQ ID NO：10)和小鼠PLAUR氨基酸序列(NP_035243.1；SEQ ID NO：9)比对。因此，在图21中可以找到人与小鼠的PLAUR之间相对应氨基酸残基或区域。
本领域中其他物种的PLAUR基因、蛋白和基因位点也是已知的。例如，Rattus norvegicus(大鼠)的PLAUR的Gene ID:50692、Macaca mulatta(恒河猴)PLAUR的Gene ID:710662、Canis lupus familiaris(狗)中PLAUR的基因ID为476446，Sus scrofa(猪)中PLAUR的基因ID为100521017。这些基因的相关信息(如，内含子序列、外显子序列和氨基酸序列)均可以在NCBI中查找到，其全部内容通过引用并入本文。
图22显示了人PLAUR氨基酸序列(NP_002650.1；SEQ ID NO：10)和大鼠PLAUR氨基酸序列(NP_599179.2；SEQ ID NO：55)。因此，在图22中可以检索到人与大鼠的PLAUR氨基酸序列的同源区域。
本发明提供一种人或嵌合(如，人源化)PLAUR核苷酸序列或氨基酸序列。在一些实施例中，小鼠PLAUR基因的外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7的核苷酸序列的全部或部分被人PLAUR基因相应的核苷酸序列替换。在一些实施例中，小鼠PLAUR基因外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、和/或外显子7的“区域”或“部分”被人PLAUR基因相应核苷酸序列或氨基酸序列替换。所述“区域”或“部分”是指至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、910、911、912、913、914、915、916、917、918、919、920、930、940、950、1000、1100、1200、1250、1300、1350、1400、1450、1500、1501、1502、1503、1504、1505、1506、1507或1508bp连续核苷酸序列，或者至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、220、240、260、280、300、301、302、303、304、305、306、307、308、309、310、320、321、322、323、324、325、326或327个连续氨基酸序列。在一些实施例中，所述“区域”或“部分”与外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7编码的氨基酸序列同一性至少为50％、55％、60％、65％、70％、75％、80％、85％、 90％、95％或至少100％。在一些实施例中，小鼠PLAUR基因的外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7(例如，外显子2的部分、外显子3-6的全部和外显子7的部分)的“区域”、“部分”或“全部”序列被人PLAUR基因相应的外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7(例如，外显子2的部分、外显子3-6的全部和外显子7的部分)的“区域”、“部分”或“全部”序列替换。
在一些实施例中，内源外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7的“区域”或“部分”缺失。
在一些实施例中，本发明提供了一种基因修饰的非人动物，所述非人动物的基因组包括人、嵌合或人源化的PLAUR核苷酸序列。在一些实施例中，所述人、嵌合或人源化的PLAUR核苷酸序列编码的蛋白与SEQ ID NO：10所示氨基酸序列同一性至少为70％、80％、85％、90％、95％或100％。在一些实施例中，所述非人动物基因组包含的核苷酸序列与SEQ ID NO：11、12、13、14所示核苷酸序列同一性至少为70％、80％、85％、90％、95％或100％。
在一些实施例中，本文所述非人动物包含编码人或人源化PLAUR蛋白的核苷酸序列。在一些实施例中，所述人或人源化PLAUR蛋白包含人PLAUR蛋白的3个同源结构域，所述同源结构域包含DI、DII和DIII。
在一些实施例中，本文所述非人动物包含人或人源化PLAUR基因。在一些实施例中，所述人源化PLAUR基因包含7个外显子。在一些实施例中，所述人源化PLAUR基因包含人源化外显子2、人外显子3、人外显子4、人外显子5、人外显子6和/或人源外显子7。在一些实施中，所述人源化PLAUR基因包含人内含子2、人内含子3、人内含子4、人内含子5和/或人内含子6。在一些实施例中，所述人源化PLAU基因包含人或人源化5’UTR。在一些实施中，所述人源化PLAUR基因包含人或人源化3’UTR。在一些实施例中，所述人源化PLAUR基因包含内源5’UTR。在一些实施例中，所述人源化PLAUR基因包含内源3’UTR。
在一些实施例中，基因修饰的非人动物可以表达人PLAUR和/或嵌合(如，人源化)PLAUR蛋白，内源PLAUR基因序列被人PLAUR基因和/或核苷酸序列替换。进一步的，所述人PLAUR基因和/或核苷酸序列编码人PLAUR的氨基酸序列与人PLAUR所示氨基酸序列同一性至少为10％、20％、30％、40％、50％、60％,70％、80％、90％、95％、96％、97％、98％或100％。在各种实施例中，内源非人动物的PLAUR基因被编码成熟PLAUR蛋白的核苷酸序列的全部或部分替换。
在一些实施例中，基因修饰的非人动物在小鼠内源启动子和/或调控元件下表达人 PLAUR和/或嵌合PLAUR蛋白(如，人源化PLAUR)。小鼠内源基因座的替换提供了一种在相同细胞类型中表达人或嵌合PLAUR蛋白(如，人源化PLAUR)的非人动物。经基因修饰的小鼠并未出现本领域已知的在某些其它转基因小鼠中观察到的潜在疾病。在非人动物中表达的人PLAUR或嵌合PLAUR蛋白可以维持一种或多种野生型或人PLAUR蛋白的功能，例如，表达的PLAUR蛋白可以与人或非人PLAU蛋白结合。进一步地，在一些实施例中，基因修饰的非人动物不表达内源PLAUR蛋白。在一些实施例中，基因修饰的非人动物内源PLAUR蛋白表达降低。在本文所述“内源PLAU蛋白”是指基因修饰前的非人动物(如，小鼠)的内源PLAUR核苷酸序列编码的PLAUR蛋白。
非人动物的基因组包含编码与人PLAUR蛋白(NP_002650.1；SEQ ID NO:10)所示氨基酸序列的同一性至少为70％、75％、80％、85％、90％、95％、99％或100％的氨基酸的核苷酸序列。在一些实施例中，所述基因组包含与SEQ ID NO：13和SEQ ID NO：14所示核苷酸序列同一性至少为70％、75％、80％、85％、90％,95％、99％或至少100％的核苷酸序列。
非人动物基因组中编码内源PLAUR区域的核苷酸序列被编码人PLAUR相应区域的核苷酸序列替换。在一些实施例中，所述编码内源PLAUR区域的核苷酸序列是内源PLAUR基因座任一序列，如，外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、5’UTR、3’UTR、内含子1、内含子2、内含子3、内含子4、内含子5、内含子6或其任意组合。在一些实施例中，所述编码内源PLAUR区域的核苷酸序列位于内源PLAUR调控区域内。在一些实施例中，所述编码内源PLAUR区域的核苷酸序列为外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7，或其部分。
基因修饰的非人动物一个或多个细胞表达人或嵌合PLAUR蛋白(如，人源化PLAUR蛋白)。在一些实施例中，人或嵌合PLAUR蛋白至少包含与SEQ ID NO：10所示的氨基酸序列1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、220、240、260、280、300、310、311、312、313、314、315、316、317、318、319、320、330、331、332、333、334或335个连续的氨基酸序列。
在一些实施例中，基因修饰的非人动物基因组中包含人PLAUR基因外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7的全部或部分，或SEQ ID NO：13所示的核苷酸序列的全部或部分。
在一些实施例中，基因修饰的非人动物基因组中包含人PLAUR基因外显子2的部分、外显子3-6的全部和7号外显子的部分。在一些实施例中，外显子2的部分包含人PLAUR基因外显子2至少5、10、20、30、40、50、60、70、80、90、91、93、95、97、100或111bp连续核苷酸序列。在一些实施例中，外显子2的部分包含97bp的连续核苷酸序列。 在一些实施例中，外显子2的部分包括至少50bp或至少100bp的核苷酸序列。在一些实施例中，外显子7的部分包含人PLAUR基因外显子7至少20、30、40、50、60、70、80、90、100、150、200、250、251、252、253、254、300、350、400、450、500、550、560或565bp连续核苷酸序列。在一些实施中，外显子7的部分包含254bp连续核苷酸序列。在一些实施例中，外显子7的部分包括至少50bp或至少100bp的核苷酸。人PLAUR基因外显子2的部分、外显子3-6的全部和外显子7的部分包括至少100-500bp、500-800bp或800-900bp个连续的核苷酸序列。在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列位于人PLAUR基因转录本NM_002659.4的第119-1057位核苷酸序列。
在一些实施例中，基因修饰的非人动物的PLAUR基因对于内源被修饰基因座是杂合的或者是纯合的。
在一些实施例中，所述人源化PLAUR基因组缺少人PLAUR基因的5’UTR。在一些实施例中，所述人源化PLAUR基因组包含内源的(如，小鼠)5’UTR。在一些实施例中，所述人源化PLAUR基因组包含内源的(如，小鼠)3’UTR。在适当的情况下，基于5’侧翼序列的相似性，可以合理地推测小鼠和人PLAUR基因受到相似的调控。如本发明所述，人源化PLAUR小鼠包含内源小鼠基因座的替换，该替换保留小鼠内源调控元件，但包含人源化PLAUR编码序列。基因修饰的杂合子小鼠或纯合子小鼠中PLAUR的表达是完全正常的。
另一方面，本发明提供了一种基因修饰的非人动物，所述非人动物基因组包含内源PLAUR基因的缺失，其中内源PLAUR基因的缺失包含外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7，或内源PLAUR基因座的部分。
在一些实施例中，内源PLAUR基因的缺失包含一个或多个外显子或外显子的部分，所述外显子选自外显子1、外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7。
在一些实施例中，所述内源PLAUR基因缺失进一步包含一个或多个内含子或内含子的部分，所述内含子选自PLAUR基因内含子1、内含子2、内含子3、内含子4、内含子5和内含子6。
在一些实施例中，其中所述缺失包含至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1100、1200、1250、1300、1350、1400、1450、1500、1501、1502、1503、1504、1505、1506、1507或1508bp连续核苷酸序列或更多的核苷酸序列。
在一些实施例中，所述内源PLAUR基因的缺失包含外显子2、外显子3、外显子4、外显子5、外显子6和/或外显子7的至少50、60、70、80、90、100、150、200、250、300、 400、500、600、700、800、900、910、911或912bp连续核苷酸序列或更多的核苷酸序列(例如，缺失外显子2的至少50bp连续核苷酸序列，外显子3-6的全部、和外显子5的至少100bp连续核苷酸序列)。
本发明提供一种人源化小鼠PLAUR基因组DNA序列，提供了一个表达人源化PLAUR蛋白的氨基酸序列的构建体；一种包含上述所述构建体的细胞；一种包含上述所述细胞的组织。
因此，在一些实施例中，本发明提供了一种嵌合的(如，人源化)PLAUR核苷酸序列和/或氨基酸序列，其中在一些实施例中，所述嵌合的核苷酸序列与小鼠内源PLAUR mRNA(如，NM_011113.4)、小鼠PLAUR氨基酸序列(如，NP_035243.1，SEQ ID NO:9)或其部分(如，5’UTR，外显子1的全部、外显子2的部分，外显子7的部分和3’UTR)所示的序列同一性至少为1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％,98％,99％或100％。在一些实施例中，所述嵌合核苷酸序列与人PLAUR mRNA序列(如，NM_002659.4)、人PLAUR氨基酸序列(如，NP_002650.1，SEQ ID NO:10)或其部分(如，外显子2的部分，外显子3-6和外显子7的部分)所示的序列同一性至少为1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。
在一些实施例中，上述所述嵌合的核酸序列可操作地连接到启动子或调节元件上，例如，内源小鼠PLAUR启动子、诱导型启动子、增强子和/或小鼠或人调节元件。
在一些实施方案中，本文所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)不同于小鼠PLAUR核苷酸序列全部或部分(例如，小鼠PLAUR基因转录本NM_011113.4外显子2的部分、外显子3-6以及外显子7的部分)。
在一些实施方案中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)与小鼠PLAUR核苷酸序列的全部或部分相同(例如，小鼠PLAUR基因转录本NM_011113.4的外显子1，外显子2的部分以及外显子7的部分)。
在一些实施方案中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核 苷酸，例如，连续或非连续核苷酸序列)不同于人PLAUR核苷酸序列全部或部分(例如，人PLAUR基因转录本NM_002659.4的外显子1、外显子2的部分以及外显子7的部分)。
在一些实施方案中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)与人PLAUR核苷酸序列全部或部分相同(例如，人PLAUR基因转录本NM_002659.4外显子2的部分、外显子3-6以及外显子7的部分)。
在一些实施方案中，所述嵌合的核酸序列编码的氨基酸至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，如，连续或非连续氨基酸残基)不同于小鼠PLAUR蛋白氨基酸序列的全部或部分(例如，小鼠PLAUR蛋白序列NP_035243.1第25-327位氨基酸(SEQ ID NO：9))。
在一些实施方案中，上述所述氨基酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，例如，连续或非连续氨基酸残基)与小鼠PLAUR蛋白氨基酸序列的全部或部分相同(例如，小鼠PLAUR蛋白序列NP_035243.1第1-24位氨基酸(SEQ ID NO：9))。
在一些实施方案中，上述所述氨基酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，例如，连续或非连续氨基酸残基)不同于人PLAUR蛋白氨基酸序列的全部或部分(例如，人PLAUR蛋白序列NP_002650.1第1-23位氨基酸(SEQ ID NO：10))。
在一些实施方案中，所述氨基酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，例如，连续或非连续氨基酸残基)与人PLAUR蛋白氨基酸序列的全部或部分相同(例如，人PLAUR蛋白序列NP_002650.1第24-335位氨基酸(SEQ ID NO：10))。
本发明还提供一种人源化的PLAUR小鼠氨基酸序列，其中所述氨基酸序列包含下列组中的任一种：
A)SEQ ID NO：9、10、15所示氨基酸序列；
B)与SEQ ID NO：9、10、15所示氨基酸序列同一性至少为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
C)与SEQ ID NO：9、10、15所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；或
D)与SEQ ID NO：9、10、15所示的，包括替换、缺失和/或插入一个或多个氨基酸残 基的氨基酸序列。
本发明还提供一种人源化的PLAUR氨基酸序列，其中所述氨基酸序列包含下列组中的任一种：
A)SEQ ID NO：10第24-335位所示的氨基酸序列；
B)与SEQ ID NO：10第24-33位所示氨基酸序列同一性至少为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
C)与SEQ ID NO：10第24-33位所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；或
D)与SEQ ID NO：10第24-33位所示的，包括替换、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明还提供一种人源化的PLAUR核苷酸(如，DNA或RNA)序列，其中所述核苷酸列包含下列组中的任一种：
A)如SEQ ID NO：13和14所示的核酸序列或编码人源化小鼠PLAUR同源氨基酸序列的核酸序列；
B)能够在低严格条件或严格条件下与SEQ ID NO：13和14所示核苷酸序列杂交的核酸序列；
C)具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或至少90％同源性的核酸序列,与SEQ ID NO：13和14所示核苷酸序列91％、92％、93％、94％、95％、96％、97％、98％或99％相同；
D)其编码的氨基酸序列与SEQ ID NO：9、10、15中所示的氨基酸序列同一性至少为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
E)编码的氨基酸序列与SEQ ID NO：9、10、15所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；或
F)编码的氨基酸序列与SEQ ID NO：9、10、15所示的，包括替换、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明进一步提供了一种人源化小鼠的PLAUR基因组DNA序列。该DNA序列由其转录得到的mRNA逆转录获得,与SEQ ID NO：13或14所示序列同源的DNA序列一致或互补。
PLAU
尿激酶型纤溶酶原激活物(urokinase type plasminogen activator，PLAU)，是一种类似胰酶的丝氨酸内肽酶，可由成纤维细胞、中性粒细胞、单核细胞、上皮细胞、肿瘤细胞合成。 PLAU最初以无酶活性的单链酶原(pro-uPA)的形式分泌到细胞间质中，之后一旦离开细胞，可与细胞膜上的PLAUR结合而被激活，或者被一系列的蛋白酶，如纤维蛋白酶、组织蛋白酶B、组织蛋白酶L、激肽释放酶、胰蛋白酶和嗜热菌蛋白酶作用而被激活。在其158位Lys处裂解成由二硫键连接的双链结构，这个激活后的标准结构，包含1个生长因子样区域(EGF-like domain)、1个kringle区域(the kringle domain)和1个带有丝氨酸活性的接触反应区域(Peptidase S1domain)。这个结构具有把纤维蛋白溶酶原限定为主要底物的作用。被活化的uPA能将纤溶酶原转变成纤溶酶，纤溶酶在降解ECM的同时，可使基质金属蛋白酶(matrix metalloproteinases，MMPs)原活化成有活性的MMPs，加强对ECM的降解。
在人的基因组中人PLAU基因(Gene ID：5328)包含11个外显子，即外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和外显子11(图1)。人PLAU mRNA的核苷酸序列为NM_002658.6，人PLAU的氨基酸序列为NP_002649.2(SEQ ID NO：2)。基于转录本NM_002658.6及其编码蛋白NP_002649.2的核苷酸序列和氨基酸序列中每个外显子对应位置如下：
表3
人PLAU基因(NCBI Gene ID：5328)位于10号染色体上的NC_000010.11的第73909182至73917497位(GRCh38.p13(GCF_000001405.39))。基于转录本NM_002658.6的核苷酸序列中每个外显子的具体位置为：5'UTR位于NC_000010.11第 73911132至73911218位和73911525至73911555位，外显子1位于NC_000010.11第73911132至73911218位，内含子1位于NC_000010.11第73911219至73911524位，外显子2位于NC_000010.11第73911525至73911612位，内含子2位于NC_000010.11第73911613至73912040位，外显子3位于NC_000010.11第73912041至73912068位，内含子3位于NC_000010.11第73912069至73912214位，外显子4位于NC_000010.11第73912215至73912322位，内含子4位于NC_000010.11第73912323至73912923位，外显子5位于NC_000010.11第73912924至73913098位，内含子5位于NC_000010.11第73913099至73913289位，外显子6位于NC_000010.11第73913290至73913381位，内含子6位于NC_000010.11第73913382到73913538位，外显子7位于NC_000010.11第73913539到73913758位，内含子7位于NC_000010.11第73913759到73913979位，外显子8位于NC_000010.11第73913980至73914128位，内含子8位于NC_000010.11第73914129至73914775位，外显子9位于NC_000010.11第73914776至73914916位，内含子9从73914917至73915250位，外显子10位于NC_000010.11第73915251至73915399位，内含子10位于NC_000010.11第73915400至73916388位，外显子11位于NC_000010.11第73916389至73917494位，3'UTR位于NC_000010.11第73916566至73917494位。以上关于人PLAU基因座的所有相关信息都可以在NCBI(Gene ID：5328)检索到。其全部内容通过引用并入本文。
鼠PLAU基因(Gene ID：18792)包含11个外显子，即外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和外显子11(图1)鼠PLAU mRNA的核苷酸序列为NM_008873.3，鼠PLAU的氨基酸序列为NP_032899.1(SEQ ID NO：1)。基于转录本NM_008873.3及其编码蛋白NP_032899.1的核苷酸序列和氨基酸序列中每个外显子对应位置如下：
表4

鼠PLAU基因(NCBI Gene ID：18792)位于14号染色体上的NC_000080.7的第20886730至20893456位(GRCm39(GCF_000001635.27))。基于转录本NM_008873.3的核苷酸序列中每个外显子的具体位置为：5'UTR位于NC_000080.7第20886728至20886802位和20887117至20887146位，外显子1位于NC_000080.7第20886728至20886802位，内含子1位于NC_000080.7第20886803至20887116位，外显子2位于NC_000080.7第20887117至20887203位，内含子2位于NC_000080.7第20887204至20887677位，外显子3位于NC_000080.7第20887678至20887708位，内含子3位于NC_000080.7第20887709至20887845位，外显子4位于NC_000080.7第20887846至20887953位，内含子4位于NC_000080.7第20887954至20888580位，外显子5位于NC_000080.7第20888581至20888755位，内含子5位于NC_000080.7第20888756至20889163位，外显子6位于NC_000080.7第20889164至20889255位，内含子6位于NC_000080.7第20889256到20889399位，外显子7位于NC_000080.7第20889400到20889622位，内含子7位于NC_000080.7第20889623到20889842位，外显子8位于NC_000080.7第20889843至20889991位，内含子8位于NC_000080.7第20889992至20890565位，外显子9位于NC_000080.7第20890566至20890706位，内含子9位于NC_000080.7第20890707至20891013位，外显子10位于NC_000080.7第20891014至20891162位，内含子10位于NC_000080.7第20891163至20892341位，外显子11位于NC_000080.7第20892342至208934534位，3'UTR位于NC_000080.7第20892519至20893453位，以上关于鼠PLAU基因座的所有相关信息均可以在NCBI(Gene ID：18792)检索到。其全部内容通过引用并入本文。
图19显示了人PLAU氨基酸序列(NP_002649.2；SEQ ID NO：2)和小鼠PLAU氨基酸序列(NP_032899.1；SEQ ID NO：1)。因此，在图19中可以找到人与小鼠的PLAU之间相对应氨基酸残基或区域。
本领域中其他物种的PLAU基因、蛋白和位置也是已知的。例如，Rattus norvegicus (大鼠)的PLAU的Gene ID:25619、Macaca mulatta(恒河猴)PLAU的Gene ID:705853、Canis lupus familiaris(狗)中PLAU的基因ID为403426，Sus scrofa(猪)中PLAU的基因ID为396985。这些基因的相关信息(如，内含子序列、外显子序列和氨基酸序列)均可以在NCBI中查找到并且引用至全文。
图20显示了人PLAU氨基酸序列(NP_002649.2；SEQ ID NO：2)和和大鼠PLAU氨基酸序列(NP_037217.3；SEQ ID NO：54)。因此，在图20中可以找到人与大鼠的PLAU氨基酸序列的同源区域。
本发明提供一种人或嵌合(如，人源化)PLAU核苷酸序列和/或氨基酸序列。在一些实施例中，小鼠PLAU基因的外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和外显子11的核苷酸序列全部或部分被人PLAU基因相应的核苷酸序列或氨基酸序列替换。在一些实施例中，小鼠PLAU基因的外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11的“区域”或“部分”被人PLAU基因相应核苷酸或氨基酸序列替换。所述“区域”或“部分”是指至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1100、1100、1200、1300、1301、1302bp、1350、1400、1600、1800、2000、2200、2300、2310、2320、2330、2340、2341、2342或2343bp连续核苷酸序列，或者至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、410、420、430或431个连续氨基酸序列。在一些实施例中，所述“区域”或“部分”与外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11编码的氨基酸序列同一性至少为50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或至少100％。在一些实施例中，小鼠PLAU基因的外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11(例如，外显子2的部分、外显子3-10的全部和外显子11的部分)的“区域”、“部分”或“全部”序列被人PLAU基因相应的外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11(例如，外显子2的部分、外显子3-10的全部和外显子11的部分)的“区域”、“部分”或“全部”序列替换。
在一些实施例中，内源外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11的“区域”或“部分”缺失。
在一些实施例中，本发明提供了一种基因修饰的非人动物，所述非人动物的基因组包括人、嵌合的或人源化的PLAU核苷酸序列。在一些实施例中，所述人、嵌合或人源化的PLAU核苷酸序列编码的蛋白与SEQ ID NO：2所示氨基酸序列同一性至少为70％、80％、85％、90％、95％或100％。在一些实施例中，所述非人动物基因组包含的核苷酸序列与SEQ ID NO：3、4、5、6、7、8所示核苷酸序列同一性至少为70％、80％、85％、90％、95％或100％。
在一些实施例中，本文所述基因修饰的非人动物包含编码人或人源化PLAU蛋白的核苷酸序列。在一些实施例中，所述人或源化PLAU蛋白包含人PLAU蛋白的全部或部分。
在一些实施例中，本文所述基因修饰的非人动物包含人或人源化PLAU基因。在一些实施例中，所述人源化PLAU基因包含11个外显子。在一些实施例中，所述人源化PLAU基因包含人源化外显子2、人外显子3、人外显子4、人外显子5、人外显子6、人外显子7、人外显子8、人外显子9、人外显子10和/或人源化外显子11。在一些实施中，所述人源化PLAU基因包含人内含子2、人内含子3、人内含子4、人内含子5、人内含子6、人内含子7、人内含子8、人内含子9和/或人内含子10。在一些实施例中，所述人源化PLAU基因包含人或人源化的5’UTR。在一些实施中，所述人源化PLAU基因包含人或人源化的3’UTR。在一些实施例中，所述人源化PLAU基因包含内源5’UTR。在一些实施例中，所述人源化PLAU基因包含内源3’UTR。
在一些实施例中，基因修饰的非人动物可以表达人PLAU和/或嵌合(如，人源化)
PLAU蛋白，内源PLAU基因被人PLAU基因和/或核苷酸序列替换，进一步的，所述人PLAU基因和/或核苷酸序列编码人PLAU的氨基酸序列与人PLAU所示氨基酸序列同一性至少为10％、20％、30％、40％、50％、60％,70％、80％、90％、95％、96％、97％、98％或100％。在各种实施例中，内源非人动物的PLAU基因被编码成熟PLAU蛋白的核苷酸序列的全部或部分替换。
在一些实施例中，基因修饰的非人动物在小鼠内源启动子和/或调控元件下表达人PLAU和/或嵌合PLAU蛋白(如，人源化PLAU)。小鼠内源基因座的替换提供了一种可以在相同的细胞类型中表达人或嵌合的PLAU蛋白(如，人源化的PLAU)的非人动物。经基因修饰的小鼠并未出现本领域已知的在某些其它转基因小鼠中观察到的潜在疾病。在非人动物中表达的人PLAU或嵌合PLAU蛋白可以维持一种或多种野生型或人PLAU蛋白的功能，例如，表达的PLAU蛋白可以与人或非人PLAU蛋白结合。进一步地，在一些实施例中，基因修饰的非人动物不表达内源PLAU蛋白。在一些实施例中，基因修饰的非人动物内源PLAU蛋白表达降低。本文所述“内源PLAU蛋白”是指基因修饰前的非人动物(如，小鼠) 的内源PLAU核苷酸序列编码的PLAU蛋白。
非人动物基因组包含编码与人PLAU蛋白(NP_002649.2；SEQ ID NO:2)所示氨基酸序列的同一性至少为70％、75％、80％、85％、90％、95％、99％或100％的氨基酸的核苷酸序列。在一些实施例中，所述基因组包含与SEQ ID NO：7和SEQ ID NO：8所示核苷酸序列同一性至少为70％、75％、80％、85％、90％,95％、99％或至少100％的核苷酸序列。
非人动物基因组中编码内源PLAU区域的核苷酸序列被编码人PLAU相应区域的核苷酸序列替换。在一些实施例中，所述编码内源PLAU区域的核苷酸序列是内源PLAU基因座任一序列，如，外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、外显子11、5’UTR、3’UTR、内含子1、内含子2、内含子3、内含子4、内含子5、内含子6、内含子7、内含子8、内含子9、内含子10或其任意组合。在一些实施例中，所述编码内源PLAU区域的核苷酸序列位于内源PLAU调控区域内。在一些实施例中，所述编码内源PLAU区域的核苷酸序列为外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子8、外显子9、外显子10和/或外显子11，或其部分，或其内源PLAU基因座的全部。
基因修饰的非人动物有一个或多个细胞表达人或嵌合PLAU蛋白(如，人源化PLAU蛋白)。在一些实施例中，人或嵌合PLAU蛋白至少包含与SEQ ID NO：2所示的氨基酸序列1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、410、420、430或431个连续的氨基酸序列。
在一些实施例中，基因修饰的非人动物基因组包含人PLAU基因的外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的全部或部分，或SEQ ID NO：7所示的核苷酸序列的全部或部分。
在一些实施例中，基因修饰的非人动物基因组中包含人PLAU基因外显子2的部分、外显子3-10的全部和外显子11的部分。在一些实施例中，外显子2的部分包含人PLAU基因外显子2至少5、10、20、30、40、50、55、57、60、65、70、75、80、83、85、87或88bp连续核苷酸序列。在一些实施例中，外显子2的部分包含57bp的连续核苷酸序列。在在一些实施例中，外显子11的部分包含人PLAU基因外显子11至少20、30、40、50、60、70、80、90、100、110、120、150、170、172、173、174、175、176、177、179、180、200、500、600、800、1000、1100或1106bp连续核苷酸序列。在一些实施中，11号外显子的部分包含177bp连续核苷酸序列。在一些实施例中，外显子11的部分包含至少50bp或至少100bp的核苷酸。人PLAU基因外显子2的部分、外显子3-10的全部和外显子11的部分包含至少100-500bp、500-1000bp个连续核苷酸序列。在一些实施例中，所述编码人PLAU相 应区域的核苷酸序列位于人PLAU基因转录本NM_002658.6的第119-1414位核苷酸序列。
进一步的，基因修饰的非人动物PLAU基因对于内源被修饰基因座是杂合的或者是纯合的。
在一些实施例中，所述人源化PLAU基因组缺少人PLAU基因的5’UTR。在一些实施例中，所述人源化PLAU基因组包含内源的(如，小鼠)5’UTR。在一些实施例中，所述人源化PLAU基因组包含内源的(如，小鼠)3’UTR。在适当的情况下，基于5’侧翼序列的相似性，可以合理地推测小鼠和人PLAU基因受到相似的调控。如本发明所述，人源化PLAU小鼠包含内源小鼠基因座的替换，该替换保留小鼠内源调控元件，但包含人源化PLAU编码序列。基因修饰的杂合子小鼠或纯合子小鼠中PLAU的表达是完全正常的。
另一方面，本发明提供了一种基因修饰的非人动物，所述非人动物基因组包含内源PLAU基因的缺失，其中内源PLAU基因的缺失包括外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11，或内源PLAU基因座的部分。
在一些实施例中，内源PLAU基因的缺失包含一个或多个外显子或外显子的部分，所述外显子选自外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11部分。
在一些实施例中，内源PLAU基因缺失进一步包含一个或多个或内含子或内含子的部分，所述内含子选自PLAU基因内含子1、内含子2、内含子3、内含子4、内含子5、内含子6、内含子7、内含子8、内含子9和/或内含子10的部分缺失。
在一些实施例中，其中所述缺失包含至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、300、400、500、700、900、1000、1100、1200、1250、1290、1291、2192、1293、1294、1295、1296、1297、1298、1299、1300、1500、1700、1800、2000、2200、2300、2320、2340、2341、2342、2343bp连续核苷酸序列或更多的核苷酸序列。
在一些实施例中，所述内源PLAU基因的缺失包含外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11的至少50、60、70、80、90、100、150、200、250、300、400、500、600、700、800、900、1000、1100、1200、1300、1301或1302bp连续核苷酸序列或更多的核苷酸序列(例如，缺失外显子2至少20bp连续核苷酸序列，外显子3-10的全部、外显子11至少100bp连续核苷酸序列)。
本发明提供一种人源化小鼠的PLAU基因组DNA序列，提供了一个表达人源化PLAU 蛋白氨基酸序列的构建体；一种包含上述所述构建体的细胞；一种包含上述所述细胞的组织。
因此，在一些实施例中，本发明提供了一种嵌合(如，人源化)PLAU核苷酸序列和/或氨基酸序列，其中在一些实施例中，所述嵌合的核苷酸序列与小鼠内源PLAU mRNA(如，NM_008873.3)、小鼠PLAU氨基酸序列(如，NP_032899.1，SEQ ID NO:1)或其一部分(如，5'UTR，外显子1的全部、外显子2的一部分，外显子11的一部分和3'UTR)所示的序列同一性至少为1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％,98％,99％或100％。在一些实施例中，所述嵌合核苷酸序列与人PLAU mRNA(如，NM_002658.6)、人PLAU氨基酸序列(如，NP_002649.2，SEQ ID NO:2)或其一部分(如，外显子2的一部分，外显子3-10和外显子11的一部分)。
在一些实施例中，上述所述嵌合的核酸序列可操作地连接至启动子或调节元件，例如，内源小鼠PLAU启动子、诱导型启动子、增强子和/或小鼠或人调节元件。
在一些实施例中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)不同于小鼠PLAU核苷酸序列全部或部分(例如，小鼠PLAUR基因转录本NM_008873.3外显子2的一部分、外显子3-10以及外显子11的一部分)。
在一些实施例中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)与小鼠PLAU核苷酸序列的全部或部分相同(例如，小鼠PLAU基因转录本NM_008873.3外显子1，外显子2的部分以及外显子11的部分)。
在一些实施例中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)不同于人PLAU核苷酸序列全部或部分(例如，人PLAUR基因转录本NM_002658.6外显子1、外显子2的部分以及外显子11的部分)。
在一些实施例中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)与人PLAU核苷酸序列全部或部分相同(例如，人PLAU基因转录本NM_002658.6外显子2的部分、外显子3-10以及外显子11的部分)。
在一些实施例中，所述嵌合的核酸序列编码的氨基酸至少有一部分(例如，至少1、2、 3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，例如，连续或非连续氨基酸残基)不同于小鼠PLAU蛋白氨基酸序列的全部或部分(例如，小鼠PLAU蛋白序列NP_032899.1第1-433位氨基酸(SEQ ID NO：1))。
在一些实施例中，所述氨基酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，例如，连续或非连续氨基酸残基)不同于人PLAU蛋白氨基酸序列的全部或部分(例如，人PLAU蛋白序列NP_002649.2第1-431位氨基酸(SEQ ID NO：2))。
本发明还提供一种人源化的PLAU小鼠氨基酸序列，其中所述氨基酸序列包含下列组中的任一种：
A)SEQ ID NO：2所示氨基酸序列；
B)与SEQ ID NO：2所示氨基酸序列同一性至少为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
C)与SEQ ID NO：2所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；或
D)与SEQ ID NO：2所示的，包括替换、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明还提供一种人源化的PLAU核苷酸(如，DNA或RNA)序列，其中所述核苷酸列包含下列组中的任一种：
A)如SEQ ID NO：7和8所示的核酸序列或编码人源化小鼠PLAUR同源氨基酸序列的核酸序列；
B)能够在低严格条件或严格条件下与SEQ ID NO：7和8所示核苷酸序列杂交的核酸序列；
C)具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或至少90％同源性的核酸序列,与SEQ ID NO：7和8所示核苷酸序列至少91％、92％、93％、94％、95％、96％、97％、98％或99％相同；
D)其编码的氨基酸序列与SEQ ID NO：2中所示的氨基酸序列同一性至少为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
E)编码的氨基酸序列与SEQ ID NO：2所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；或
F)编码的氨基酸序列与SEQ ID NO：2所示的，包括替换、缺失和/或插入一个或多个 氨基酸残基的氨基酸序列。
本发明进一步提供了一种人源化小鼠的PLAU基因组DNA序列。该DNA序列由其转录得到的mRNA逆转录获得,与SEQ ID NO：7或8所示序列同源的DNA序列一致或互补。
基因修饰的非人动物
本发明所述“基因修饰的非人动物”是指该动物基因组中至少一条染色体具有外源DNA的非人动物。在一些实施例中，至少一个或多个细胞中，例如，基因修饰的非人动物中至少1％、2％、3％、4％、5％、10％、20％、30％、40％、50％的细胞具有外源DNA。具有外源性DNA的细胞可以是各种细胞，例如，内源细胞、体细胞、免疫细胞、T细胞、B细胞、NK细胞、抗原呈递细胞、巨噬细胞、树突状细胞、生殖细胞、囊胚或内源肿瘤细胞。在一些实施例中，提供了一种基因修饰的非人动物，所述动物包含内源PLAU和/或PLAUR基因座和外源PLAU和/或PLAUR基因座(如，人序列)，例如，用一个或多个人源序列替换一个或多个非人序列，或插入一个或多个人源和/或非人序列。动物通常能够通过种系传播将修饰传递给后代。
本发明所述“嵌合基因”或“嵌合核酸”是指基因或核酸，其中所述基因或核酸的两个或多个部分来自不同物种，或者该基因或核酸的至少一个序列与动物中的野生型核酸不同。在一些实施例中，嵌合基因或嵌合核酸具有至少一部分序列具有两个或多个不同的物种来源，例如，编码不同蛋白的序列或编码两个或多个不同物种的相同(或同源)蛋白的序列。在一些实施例中，嵌合基因或嵌合核酸是指人源化基因或人源化核酸。
本发明所述“嵌合蛋白”或“嵌合多肽”是指蛋白或多肽，其中所述多肽或蛋白的两个或多个部分来自不同物种，或者该蛋白或多肽的至少一个序列与动物中的野生型氨基酸序列不同。在一些实施例中，嵌合蛋白或嵌合多肽的至少一部分序列具有两个或多个不同物种来源，例如，不同物种的相同(或同源)蛋白。在一些实施例中，嵌合蛋白或嵌合多肽是指人源化蛋白或人源化多肽。
本发明所述“人源化蛋白”或“人源化多肽”是指蛋白或多肽，其中所述蛋白或多肽的至少一部分来自人蛋白或人多肽。在一些实施例中，人源化蛋白或人源化蛋白是指人蛋白或多肽。
本发明所述“人源化核酸”是指核酸，其中所述核酸的至少一部分来自人核酸。在一些实施例中，人源化核酸中的核酸全部来源于人。在一些实施例中，人源化核酸是指人源化外显子，所述人源化外显子可以是人的外显子或嵌合外显子。
在一些实施例中，嵌合基因或嵌合核酸是人源化PLAU基因或人源化PLAU核酸。在一些实施例中，所述基因或核酸的至少一部分来源于人PLAU基因，或者所述基因或核酸的至少一部分来源于非人PLAU基因。在一些实施例中，所述基因或核酸包含编码PLAU蛋 白的序列。编码的PLAU蛋白具有人PLAU或非人PLAU蛋白功能的至少一种活性。如，与PLAUR受体相互作用诱导下游信号通路。
在一些实施例中，嵌合基因或嵌合核酸是人源化PLAUR基因或人源化PLAUR核酸。在一些实施例中，所述基因或核酸的至少一部分来源于人PLAUR基因，或者所述基因或核酸的至少一部分来源于非人PLAUR基因。在一些实施例中，所述基因或核酸包含编码PLAUR蛋白的序列。编码的PLAUR蛋白具有人PLAUR或非人PLAUR蛋白功能的至少一种活性。如，与PLAU配体相互作用诱导下游信号通路。
在一些实施例中，嵌合蛋白或嵌合多肽是人源化PLAU蛋白或人源化PLAU多肽。在一些实施例中，所述多肽或蛋白的氨基酸序列中的至少一部分来源于人PLAU蛋白，或者所述蛋白或多肽的氨基酸序列的至少一部分来源于非人PLAU蛋白。人源化的PLAU蛋白或人源化的PLAU多肽具有至少一种人PLAU蛋白或非人PLAU蛋白功能的活性。如，与PLAU受体相互作用诱导下游信号通路。
在一些实施例中，嵌合蛋白或嵌合多肽是人源化PLAUR蛋白或人源化PLAUR多肽。在一些实施例中，所述蛋白或多肽的氨基酸序列中的至少一部分来源于人PLAUR蛋白，或者所述蛋白或多肽的氨基酸序列的至少一部分来源于非人PLAUR蛋白。人源化的PLAUR蛋白或人源化的PLAUR多肽具有至少一种人PLAUR蛋白或非人PLAUR蛋白功能的活性。如，与PLAUR配体相互作用诱导下游信号通路。
基因修饰的非人动物可以是各种动物，例如，小鼠、大鼠、兔子、猪、牛(例如，牛、公牛、水牛)、鹿、绵羊、山羊、鸡、猫、狗、雪貂、灵长类动物(例如，狨猴、恒河猴)。对于不容易获得合适的可遗传修饰胚胎干细胞(ES)的非人动物，采用其他方法来构建包含遗传修饰的非人动物。这样的方法包括，例如，修饰非ES细胞基因组(例如，成纤维细胞或诱导多能干细胞)并采用核移植将修饰的基因组转移到合适的细胞，例如卵母细胞，以及在适当的条件下在非人动物中孕育修饰的细胞(例如，修饰的卵母细胞)以形成胚胎。上述所述构建方法在本领域中是已知的，并且在“A.Nagy,et al.,“Manipulating the Mouse Embryo:A Laboratory Manual(Third Edition),”Cold Spring Harbor Laboratory Press,2003”有所描述，其全部内容通过引用并入本文。
在一个方面，所述动物是哺乳动物。在一些实施例中，基因修饰的非人动物是啮齿动物。啮齿动物可以选自小鼠、大鼠和仓鼠。在一个实施方式中，所述啮齿动物选自鼠家族。在一个实施方式中，所述基因修饰的动物来自选自丽仓鼠科(例如小鼠样仓鼠)、仓鼠科(例如仓鼠、新世界大鼠和小鼠、田鼠)、鼠总科(真小鼠和大鼠、沙鼠、刺毛鼠、冠毛大鼠)、马岛鼠科(登山小鼠、岩小鼠、有尾大鼠、马达加斯加大鼠和小鼠)、刺睡鼠科(例如多刺 睡鼠)和鼹形鼠科(例如摩尔大鼠、竹大鼠和鼢鼠)家族。在一个特定实施方式中，所述基因修饰的啮齿动物选自真小鼠或大鼠(鼠总科)、沙鼠、刺毛鼠和冠毛大鼠。在一个实施方式中，所述基因修饰的小鼠来自鼠科家族成员。在一个实施方式中，所述动物是啮齿动物。在一个特定实施方式中，所述啮齿动物选自小鼠和大鼠。在一个实施方式中，所述非人动物是小鼠。
在一个特定实施方式中，所述非人动物是小鼠，其为选自BALB/c、A、A/He、A/J、A/WySN、AKR、AKR/A、AKR/J、AKR/N、TA1、TA2、RF、SWR、C3H、C57BR、SJL、C57L、DBA/2、KM、NIH、ICR、CFW、FACA、C57BL/A、C57BL/An、C57BL/GrFa、C57BL/KaLwN、C57BL/6、C57BL/6J、C57BL/6ByJ、C57BL/6NJ、C57BL/10、C57BL/10ScSn、C57BL/10Cr和C57BL/Ola的C57BL、C58、CBA/Br、CBA/Ca、CBA/J、CBA/st、CBA/H品系的小鼠及NOD、NOD/SCID、NOD-Prkdcscid IL-2rgnull背景的小鼠。
基因修饰的非人动物包括内源非人PLAU和/或PLAUR基因位点的修饰。在一些实施例中，所述修饰包含编码至少一部分成熟PLAU或PLAUR蛋白的核苷酸序列(例如，与成熟的PLAU或PLAUR蛋白氨基酸序列至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％或99％一致性)。虽然在本发明中提供了可包含本文所述基因修饰的细胞(例如，ES细胞、体细胞)，但在许多实施例中，基因修饰的非人动物包括对动物中内源PLAU和/或PLAUR基因位点的修饰。
本发明进一步提供了利用上述方法构建的非人哺乳动物。在一些实施例中，所述非人哺乳动物包含人基因组。在一些实施例中，所述非人哺乳动物为啮齿类动物。进一步优选的，所述啮齿类动物为小鼠。在一些实施例中，所述非人哺乳动物表达由人源化PLAU和/或PLAUR基因编码的蛋白。
此外，本发明还提供了一种携带肿瘤的非人哺乳动物，其特征在于所述非人哺乳动物模型是通过本文所述方法获得的。在一些实施例中，非人哺乳动物是啮齿类动物(如，小鼠)。
本发明还提供了一种细胞或细胞系，或原代细胞培养物，其来源于非人类哺乳动物或其后代、或携带肿瘤的非人类哺乳动物、来源于非人类哺乳动物或其后代的组织、器官或其培养物、或携带肿瘤的非人类哺乳动物、以及来源于非人类哺乳动物或其后代的肿瘤组织，当它携带肿瘤时，或携带非人类哺乳动物的肿瘤。
本发明提供了一种通过本文描述的任一方法产生的非人哺乳动物。在一些实施例中，提供了非人哺乳动物、基因修饰的非人动物，所述基因修饰的非人动物基因组包含人或人源化PLAU和/或PLAUR的DNA。
在一些实施例中，非人哺乳动物包括本文所述遗传构建体(例如，如图2、3、4、5、9、 10、11和12所示的基因构建体)。在一些实施例中，提供了一种表达人或人源化PLAU和/或PLAUR蛋白的非人哺乳动物。在一些实施例中，提供了一种人或人源化PLAU和/或PLAUR蛋白的组织特异性表达。
在一些实施例中，非人动物人或人源化PLAU和/或PLAUR蛋白的表达是可控的。如通过添加特异性诱导物或阻遏物。在一些实施例中，所述特异性诱导物选自四环素系统(Tet-Off System/Tet-On System)或他莫昔芬系统(Tamoxifen System)。
非人哺乳动物可以是本领域已知的任何非人动物，其可用于本文所述方法中。优选的非人哺乳动物是哺乳动物(例如，啮齿类动物)。在一些实施例中，非人哺乳动物是小鼠。
对上述描述的非人哺乳动物进行遗传、分子和行为分析。本发明提供了一种与相同基因型或其他基因型非人哺乳动物交配产生的后代。
本发明提供了一种来源于非人哺乳动物或其后代的细胞系或原代细胞培养物。例如可以通过以下方法制备基于细胞培养的模型。细胞培养物可以通过从非人哺乳动物中分离获得，或者可以使用相同构建体和用标准细胞转染技术建立的细胞培养物中获得细胞。包含编码人PLAU和/或PLAUR蛋白的DNA序列的遗传结构的整合可以通过多种方法检测。
有许多分析方法可用于检测外源性DNA，包括核酸水平的方法(包含使用逆转录-聚合酶链反应(RT-PCR)或Southern Blot以及原位杂交)和蛋白水平的方法(包括组织化学分析，免疫印迹分析和体外结合研究))。此外，目的基因的表达水平可以通过本领域技术人员熟知的ELSA方法进行量化。许多标准的分析方法可用于完成定量检测。例如，可以使用RT-PCR和杂交方法检测转录水平，包括RNA酶保护分析法，Southern Blot，RNA斑点杂交分析(RNAdot)。免疫组织化学染色、流式细胞术、Western blot也可用于检测人源或人源化PLAU和/或PLAUR蛋白的存在。
在一些实施例中，本文所述基因修饰的非人动物具有两个或多个人源化基因，其中人源化基因可以选自PLAU和/或PLAUR的人或人源化基因。
载体
本发明提供了一种靶向PLAU基因的靶向载体，包括：a)为与待改变的转换区5’端同源的DNA片段，其选自非人动物PLAU因基因组DNA的100-10000个长度的核苷酸；b)编码供体区域的DNA序列；c)与待改变的转换区3’端同源的DNA片段，其选自非人动物PLAU基因基因组DNA，长度为100-10000个核苷酸。
在一些实施例中，a)与待改变的转换区5’端同源的DNA片段选自与NCBI登录号为NC_000080.7至少具有90％同源性的核苷酸序列；c)与待改变的转换区3’端同源的DNA片段选自与NCBI登录号为NC_000080.7至少具有90％同源性的核苷酸序列；
在一些实施例中，a)待改变的转换区5’端同源的DNA片段选自于NCBI登录号为NC_000080.7的第20883305至20887146位核苷酸序列；c)待改变的转换区3’端同源的DNA片段选自于NCBI登录号为NC_000080.7的第20893778至20896948位核苷酸序列；
在一些实施例中，a)待改变的转换区5’端同源的DNA片段选自于NCBI登录号为NC_000080.7的第20885866至20887146位核苷酸序列；c)待改变的转换区3’端同源的DNA片段选自于NCBI登录号为NC_000080.7的第20892519至20893251位核苷酸序列；
在一些实施例中，靶向载体所选的基因组核苷酸序列长度可以超过约3kb、3.5kb、4kb、4.5kb、5kb、5.5kb、6kb、6.5kb、7kb、7.5kb、8kb、8.5kb、9kb，9.5kb或10kb。
在一些实施例中，所述待改变的转换区位于非人动物PLAU基因的1号至11号外显子上。
在一些实施例中，所述待改变的转换区位于位于非人动物PLAU基因的2号至11号外显子上(例如NM_008873.3第104-1405位)。
在一些实施例中，所述靶向载体还包含一个或多个标记基因。例如，阳性筛选标记基因或阴性筛选标记基因。在一些实施例中，阳性克隆筛选的抗性基因为新霉素磷酸转移酶编码序列Neo。在一些实施例中，负筛选标记的编码基因为白喉毒素A亚基的编码基因(DTA)。
在一些实施例中，所述5’臂序列如SEQ ID NO：3所示核苷酸序列；所述3’臂序列如SEQ ID NO：4所示核苷酸序列。在一些实施例中，所述5’臂序列如SEQ ID NO：5所示核苷酸序列；所述3’臂序列如SEQ ID NO：6所示核苷酸序列。
在一些实施例中，所述5’臂为与NCBI登录号为NC_000080.7至少具有90％同源性的核苷酸，进一步优选的，所述5’臂序列包含SEQ ID NO：3或5所示核苷酸序列。在一些实施例中，所述3’臂为与NCBI登录号为NC_000080.7至少具有90％同源性的核苷酸，进一步优选的，所述3’臂序列包含SEQ ID NO：4或6所示核苷酸序列。
在一些实施例中，所述靶向载体包含人序列(例如，NM_002658.6119-1414位)。例如，靶向载体中的靶向区域包括：人PLAU基因的部分或全部核苷酸序列，优选人PLAU基因的外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10和/或外显子11。在一些实施例中，人源化PLAU基因的核苷酸序列编码人PLAU蛋白的全部或部分核苷酸序列，NCBI的蛋白号为NP_002649.2(SEQ ID NO：2)。
本发明提供了一种靶向PLAUR基因的靶向载体，包括：a)为与待改变的转换区5’端同源的DNA片段，其选自非人动物PLAUR基因组DNA的100-10000个长度的核苷酸；b)编码供体区域的DNA序列；c)与待改变的转换区3’端同源的DNA片段，其选自非人动物 PLAU基因基因组DNA，长度为100-10000个核苷酸。
在一些实施例中，a)与待改变的转换区5’端同源的DNA片段选自与NCBI登录号为NC_000073.7至少具有90％同源性的核苷酸序列；c)与待改变的转换区3’端同源的DNA片段选自与NCBI登录号为NC_000073.7至少具有90％同源性的核苷酸序列；
在一些实施例中，a)待改变的转换区5’端同源的DNA片段选自于NCBI登录号为NC_000073.7的第24159210至24164569位核苷酸序列；c)待改变的转换区3’端同源的DNA片段选自于NCBI登录号为NC_000073.7的第24174897至24178233位核苷酸序列；
在一些实施例中，靶向载体所选的基因组核苷酸序列长度可以超过约3kb、3.5kb、4kb、4.5kb、5kb、5.5kb、6kb、6.5kb、7kb、7.5kb、8kb、8.5kb、9kb、9.5kb或10kb。
在一些实施例中，所述待改变的转换区位于非人动物PLAUR基因的1号至7号外显子上。
在一些实施例中，所述待改变的转换区位于位于非人动物PLAUR基因的2号至7号外显子上(例如NM_011113.4第100-1011位)。
在一些实施例中，所述靶向载体还包含一个或多个标记基因。例如，阳性筛选标记基因或阴性筛选标记基因。在一些实施例中，阳性克隆筛选的抗性基因为新霉素磷酸转移酶编码序列Neo。在一些实施例中，负筛选标记的编码基因为白喉毒素A亚基的编码基因(DTA)。
在一些实施例中，所述5’臂序列如SEQ ID NO：11所示核苷酸序列；所述3’臂序列如SEQ ID NO：12所示核苷酸序列。
在一些实施例中，所述5’臂为与NCBI登录号为NC_000073.7至少具有90％同源性的核苷酸，进一步优选的，所述5’臂序列包含SEQ ID NO：11所示核苷酸序列。在一些实施例中，所述3’臂为与NCBI登录号为NC_000073.7至少具有90％同源性的核苷酸，进一步优选的，所述3’臂序列包含SEQ ID NO：12所示核苷酸序列。
在一些实施例中，所述靶向载体包含人序列(例如，NM_002659.4的第119-1057位)。例如，靶向载体中的靶向区域包含人PLAUR基因的部分或全部核苷酸序列，优选人PLAUR基因的外显子2的部分、外显子3-6全部和/或外显子7的部分。在一些实施例中，所述人序列与SEQ ID NO：7和8所示核苷酸序列至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％；在一些实施例中，人源化PLAUR基因的核苷酸序列编码人PLAUR蛋白的全部或部分核苷酸序列，NCBI的蛋白号为NP_002650.1(SEQ ID NO：10)。
本公开还涉及包含如上所述的靶向载体的细胞。
此外，本发明还提供了一种非人哺乳动物细胞，其具有上述靶向载体中的任何一种，以 及本文所述构建体的一种或多种体外转录物。在一些实施例中，细胞包含Cas9mRNA或其体外转录物。
在一些实施例中，所述细胞中基因是杂合的。在一些实施例中，所述细胞中的基因是纯合的。
在一些实施例中，所述非人哺乳动物细胞是小鼠细胞。在一些实施例中，所述细胞是受精卵细胞。在一些实施例中，所述细胞是胚胎干细胞。
基因修饰的非人动物的构建方法
基因修饰的非人动物可以通过本领域已知的几种技术制备获得，包括利用胚胎干细胞的基因打靶技术、CRISPR/Cas9技术、锌指核酸酶技术、转录激活子样效应因子核酸酶技术、归巢核酸内切酶或其他分子生物学技术。在一些实施例中，优选使用同源重组技术。在一些实施例中，CRISPR/Cas9基因编辑技术可以构建基因修饰的非人动物。在一些实施例中，CRISPR-Cas9基因组编辑用于产生基因修饰的非人动物。这些基因组编辑技术中的许多技术是本领域已知的，并且在Yin等人的“Delivery technologies for genome editing,”Nature Reviews Drug Discovery 16.6(2017):387-399,中进行了描述，该内容被全部纳入作为引用。本发明还提供了许多其他方法用于基因组编辑，例如，将转基因细胞显微注射到去核卵母细胞中，并将去核卵母细胞与另一个转基因细胞融合。
在一些实施例中，非人动物的至少一个细胞的内源基因组中编码内源PLAU区域的核苷酸序列被编码人PLAU相应区域的核苷酸序列替换。在一些实施例中，所述非人动物内源PLAU蛋白与野生型PLAU相比表达量降低或缺失。在一些实施例中，替换发生在生殖细胞、体细胞、囊胚或成纤维细胞等中。体细胞或成纤维细胞的细胞核可以插入去核卵母细胞中。
图3和图5显示了靶向小鼠PLAU位点的人源化打靶策略。靶向载体包含5'同源臂、人或人源化PLAU基因片段和3'同源臂组成的载体。该过程涉及利用同源重组将人或人源化序列替换内源相应PLAU序列。在一些实施例中，靶位点上游和下游的的切割(例如，通过锌指核酸酶、TALEN或CRISPR)可导致DNA双链断裂，利用同源重组将人或人源化PLAU序列替换鼠内源PLAU序列。
在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含编码人PLAU蛋白的全部或部分的核苷酸序列，优选的，包含编码人PLAU蛋白的全部核苷酸序列。
在一些实施中，所述编码人PLAU相应区域的核苷酸序列包含编码人PLAU蛋白至少50个到至少431个，优选为50、100、150、200、250、300、350、400、410、420、425、428、429、430或431个连续氨基酸的核苷酸序列；更进一步包含编码SEQ ID NO：2所示氨基酸序列的核苷酸序列；或者，包含编码与SEQ ID NO：2所示氨基酸序列同一性至少为 70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％的核苷酸序列；或者，包含编码与SEQ ID NO：2所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸的核苷酸序列；或者，包含编码与SEQ ID NO：2所示氨基酸序列所示的，包括替换、缺失和/或插入一个或多个氨基酸的核苷酸序列。
在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含人PLAU基因的外显子1至外显子11的全部或部分，进一步优选包含人PLAU基因的外显子1至外显子11中的一种、两种或三种以上、连续两种或连续三种以上外显子的组合的全部或部分，更优选包含人PLAU基因外显子2至外显子11的全部或部分，更进一步优选包含人PLAU基因外显子2的部分、外显子3-10的全部和外显子11的部分，优选还包含内含子2和/或内含子10，其中，人PLAU基因外显子2的部分包含人PLAU基因外显子2至少20bp到至少88bp，优选为20、30、40、50、55、56、57、58、59、60、70、80或88bp连续核苷酸序列，或者，人PLAU基因外显子2的部分包含编码区的核苷酸序列，人PLAU基因外显子11的部分包含人PLAU基因外显子11至少50bp到至少1106bp，优选为50、100、150、170、175、176、177、178、179、180、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1050、1100或1106bp连续核苷酸序列，或者，人PLAU基因外显子11的部分包含编码区的核苷酸序列。
在一些实施例中，所述编码人PLAU相应区域的核苷酸序列包含SEQ ID NO：7所示核苷酸序列；或者，包含与SEQ ID NO：7所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％的核苷酸序列；或者，包含与SEQ ID NO：7所示核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸的核苷酸序列；或者，包含具有SEQ ID NO：7所示核苷酸序列所示的，包括替换、缺失和/或插入一个或多个核苷酸的核苷酸序列。
在一些实施例中，所述编码人PLAU相应区域的核苷酸序列可操作的连接到至少一条染色体中内源PLAU基因的内源调控元件。
在一些实施例中，非人动物中被替换的内源PLAU核苷酸序列包括小鼠PLAU基因的外显子1至外显子11的全部或部分被替换，优选非人动物PLAU基因外显子2至外显子11、的全部或部分被替换。更优选的，非人动物PLAU基因的外显子2的部分、外显子3-10的全部和外显子11的部分被替换。在一些实施例中，所述被替换的内源PLAU核苷酸序列编码SEQ ID NO：1所示氨基酸序列的核苷酸序列被替换。
在一些实施例中，所述人或人源化PLAU基因在非人动物体内通过调控元件进行调控，所述调控元件包括但不限于内源启动子。例如，所述调控元件可以是内源或者外源 的。在本发明的一个具体实施方式中，所述内源调控元件来源非人动物PLAU基因。所述外源性调控元件来源于人PLAU基因。
在本发明的一个具体实施例中，所述构建方法包括用包含人PLAU基因外显子1至外显子11的全部或部分替换非人动物PLAU基因的外显子1至外显子11的全部或部分，优选用包含人PLAU基因的外显子2至外显子11的全部或部分替换非人动物PLAU基因的外显子2至外显子11的全部或部分。
在本发明的一个具体实施例中，所述构建方法包括用包含人PLAU基因的外显子2的部分、外显子3-10的全部和外显子11的部分，优选还包含内含子2和/或内含子9，替换非人动物PLAU基因的外显子2至外显子11的全部或部分。
在本发明的一个具体实施例中，所述构建方法包括用包含编码人或人源化PLAU蛋白的核苷酸序列，或，人或人源化PLAU基因的核苷酸序列替换非人动物基因组中编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含人PLAU基因的基因组DNA序列、cDNA序列或CDS序列替换非人动物基因组中编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含编码SEQ ID NO：2所示氨基酸序列的核苷酸序列替换非人动物基因组中编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含SEQ ID NO：7所示核苷酸序列替换非人动物基因组中编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
在一些实施例中，非人动物的至少一个细胞的基因组中编码内源PLAUR区域的核苷酸序列被编码人PLAUR相应区域的核苷酸序列替换。在一些实施例中，所述非人动物内源PLAUR蛋白表达降低或缺失。在一些实施例中，替换发生在生殖细胞、体细胞、囊胚或成纤维细胞等中。体细胞或成纤维细胞的细胞核可以插入去核卵母细胞中。
图11显示了靶向小鼠PLAUR的人源化打靶策略。靶向载体包括5’同源臂、人或人源化PLAUR基因片段和3'同源臂组成的载体。该过程涉及利用同源重组将人或人源化序列序列替换内源相应PLAUR序列。在一些实施例中，靶位点上游和下游的的切割(例如，通过锌指核酸酶、TALEN或CRISPR)可导致DNA双链断裂，利用同源重组将人或人源化PLAUR序列替换鼠内源PLAUR序列。
在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列包含编码人PLAU蛋白的全部或部分的核苷酸序列，优选包含编码人PLAUR蛋白DI、DII和DIII的核苷酸序列。
在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列包含编码人PLAUR蛋白的全部或部分的核苷酸序列，进一步优选的，包含编码人PLAUR蛋白至少50个到至少335个，优选为50、100、150、200、250、300、310、311、312、313、314、320、330或335个连续氨基酸的核苷酸序列，更进一步优选的，包含编码SEQ ID NO：10的24-335位所示氨基酸序列的核苷酸序列；或者，包含编码与SEQ ID NO：10的24-335位所示氨基酸序列同一性至少为65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％的核苷酸序列；或者，包含编码与SEQ ID NO：10的24-335位所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸的核苷酸序列；或者，包含编码具有SEQ ID NO：10的24-335位所示氨基酸序列所示的，包括替换、缺失和/或插入一个或多个氨基酸的核苷酸序列。
在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列包含人PLAUR基因外显子1至外显子7的全部或部分，优选包含人PLAUR基因外显子1至外显子7中的一种、两种或三种以上、连续两种或连续三种以上外显子的组合的全部或部分，进一步优选包含人PLAUR基因外显子1至外显子7的全部或部分，更优选包含人PLAUR基因外显子2的部分、外显子3-6的全部和外显子7的部分，优选还包含内含子2-3和/或内含子6-7，其中，人PLAUR基因外显子2的部分包含人PLAUR基因外显子2至少20bp到至少111bp，优选为20、30、40、50、60、70、80、90、95、96、97、98、99、100、110或111bp连续核苷酸序列，人PLAUR基因外显子7的部分包含人PLAUR基因外显子7至少50bp到至少565bp，优选为50、100、150、200、250、252、253、254、255、256、300、350、400、450、500、550、560或565bp连续核苷酸序列，或者，人PLAUR基因外显子7的部分包含编码区的核苷酸序列。
在本发明的一个具体实施例中，所述编码人PLAUR相应区域的核苷酸序列包含SEQ ID NO：13所示核苷酸序列；或者，包含与SEQ ID NO：13所示核苷酸序列同一性至少为65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％的核苷酸序列；或者，包含与SEQ ID NO：13所示核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸的核苷酸序列；或者，包含具有SEQ ID NO：13所示核苷酸序列所示的，包括替换、缺失和/或插入一个或多个核苷酸的核苷酸序列。
在一些实施例中，所述编码人PLAUR相应区域的核苷酸序列可操作的连接到至少一条染色体中内源PLAUR基因的内源调控元件。
在一些实施例中，非人动物中被替换的内源PLAUR核苷酸序列包括小鼠PLAUR基因的外显子1至外显子7的全部或部分被替换，优选非人动物PLAU基因的外显子2至外显子 7的全部或部分被替换。更优选的，非人动物PLAU基因外显子2的部分、3号至6号外显子的全部和外显子7的部分被替换。在一些实施例中，所述被替换的内源PLAUR核苷酸序列编码SEQ ID NO：9的25-327位所示氨基酸序列的核苷酸序列被替换。
在一些实施例中，所述人或人源化PLAUR基因在非人动物体内通过调控元件进行调控，所述调控元件包括但不限于内源启动子。例如，所述调控元件可以是内源或者外源的。在本发明的一个具体实施方式中，所述内源调控元件来源非人动物PLAUR基因。所述外源性调控元件来源于人PLAUR基因。
在本发明的一个具体实施例中，所述构建方法包括用包含人PLAUR基因的外显子1至外显子7的全部或部分替换非人动物PLAUR基因的外显子1至外显子7的全部或部分，优选用包含人PLAUR基因的外显子2至外显子7的全部或部分替换非人动物PLAUR基因的外显子2至外显子7的全部或部分。
在本发明的一个具体实施例中，所述构建方法包括用包含人PLAUR基因的外显子2的部分、外显子3-6的全部和外显子7的部分，优选还包含内含子2-3和/或内含子6-7，替换非人动物PLAUR基因的外显子2至外显子7的全部或部分。
在本发明的一个具体实施例中，所述构建方法包括用包含编码人或人源化PLAUR蛋白的核苷酸序列，或，人或人源化PLAUR基因的核苷酸序列替换非人动物基因组中编码SEQ ID NO：9所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含人PLAUR基因的基因组DNA序列、cDNA序列或CDS序列替换非人动物基因组中编码SEQ ID NO：9所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含编码SEQ ID NO：10所示氨基酸序列的核苷酸序列替换非人动物基因组中编码SEQ ID NO：9所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含编码SEQ ID NO：10的24-335位所示氨基酸序列的核苷酸序列替换非人动物基因组中编码SEQ ID NO：9的25-327位所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含SEQ ID NO：13所示核苷酸序列替换非人动物基因组中编码SEQ ID NO：9的25-327位所示氨基酸序列的核苷酸序列。
在一些实施例中，构建非人动物的方法包含修饰非人动物PLAU和/或PLAUR基因的编码框，将编码人或人源化PLAU和/或PLAUR蛋白的核苷酸序列或者人源化PLAU和/或 PLAUR基因的核苷酸序列插入非人动物PLAU和/或PLAUR基因内源调控元件之后，其中，所述修饰非人动物PLAU和/或PLAUR基因的编码框可以采用敲除非人动物PLAU和/或PLAUR基因的功能区或者采用插入一段序列，使得非人动物PLAU和/或PLAUR蛋白不表达或表达降低或表达的蛋白无功能，在一些实施例中，所述修饰非人动物PLAU基因的编码框可以敲除非人动物PLAU基因的外显子2-11的全部或部分核苷酸序列。在一些实施例中，所述修饰非人动物PLAUR基因编码框可以敲除非人动物PLAUR基因外显子2-7的全部或部分核苷酸序列。
在一些实施例中，构建基因修饰的非人动物的方法包括在非人动物PLAU和/或PLAUR基因的内源调控元件之后插入编码人或人源化PLAU和/或PLAUR蛋白的核苷酸序列和/或辅助序列。在一些实施例中，辅助序列可以是终止密码子，使得PLAU和/或PLAUR基因人源化动物模型体内表达人PLAU和/或PLAUR蛋白，不表达非人动物PLAU和/或PLAUR蛋白，进一步的，所述辅助序列为WPRE、STOP和/或PolyA。
在一些实施例中，所述构建方法包括使用上述的PLAU和/或PLAUR基因的靶向载体和/或靶向PLAU和/或PLAUR基因的sgRNA进行非人动物的构建。
在本发明的一个具体实施例中，所述构建方法包括将上述靶向载体导入非人动物细胞中(优选为胚胎干细胞)，筛选出正确的阳性克隆细胞导入已分离好的囊胚中，培养该囊胚，然后将培养后的囊胚移植至雌性非人动物输卵管内，允许其发育，鉴定筛选获得PLAU和/或PLAUR基因修饰的非人动物。
在一些实施例中，为提高重组效率，还可以使用靶向PLAUR基因的sgRNA与上述PLAUR基因的靶向载体一起进行非人动物的构建。
在本发明的一个具体实施例中，所述构建方法包括将上述PLAU和/或PLAUR基因的靶向载体、靶向PLAU和/或PLAUR基因的sgRNA及Cas9导入非人动物细胞中，培养该细胞(优选为受精卵)，然后将培养后的细胞移植至雌性非人动物输卵管内，允许其发育，鉴定筛选获得PLAU和/或PLAUR基因修饰的非人动物。
在一些实施例中，所述非人动物可以选自啮齿类动物、猪、兔子、猴子等任何可以进行基因编辑制备基因人源化的非人动物。
优选的，所述非人动物为非人哺乳动物。进一步优选的，所述非人哺乳动物为啮齿类动物。更进一步优选的，所述啮齿类动物为大鼠或小鼠。
优选的，所述非人动物是免疫缺陷的非人哺乳动物。进一步优选的，所述免疫缺陷的非人哺乳动物为免疫缺陷的啮齿类动物、免疫缺陷的猪、免疫缺陷的兔子或免疫缺陷的猴子。更进一步优选的，所述免疫缺陷的啮齿类动物为免疫缺陷的小鼠或大鼠。再进 一步优选的，所述免疫缺陷鼠是NOD-Prkdcscid IL-2rγnull小鼠、NOD-Rag 1-/--IL2rg-/-小鼠、Rag 2-/--IL2rg-/-小鼠、NOD/SCID小鼠或者裸鼠。
基因修饰的非人动物的应用
本发明还提供了一种上述PLAU和/或PLAUR的基因修饰的非人动物、上述任一构建方法获得的非人动物的应用。
在一些实施例中，所述应用包含：
A)涉及人类细胞的与PLAU和/或PLAUR相关的免疫过程的产品开发中的应用；
B)作为药理学、免疫学、微生物学和医学研究的与PLAU和/或PLAUR相关的模型系统中的应用；
C)涉及生产和利用动物实验疾病模型用于与PLAU和/或PLAUR相关的病原学研究和/或用于开发诊断策略和/或用于开发治疗策略中的应用；
D)在体内研究人PLAU和/或PLAUR信号通路调节剂的筛选、药效检测、评估疗效、验证或评价中的应用；或者，
E)研究PLAU和/或PLAUR基因功能，研究针对人PLAU和/或PLAUR靶位点的药物、药效，研究与PLAU和/或PLAUR相关的炎症、免疫相关疾病药物以及抗肿瘤药物方面的应用。
本发明提供了一种表达人或人源化PLAU和/或PLAUR蛋白非人动物，该动物可用于可用于人PLAU和/或PLAUR特异性调节剂的筛选。在一些实施例中，所述非人动物是人疾病动物模型。如，疾病是遗传诱导的(敲入或敲除)。在不同的实施例中，基因修饰的非人动物还包含受损的免疫系统，如，经过基因修饰的人源性组织异种移植，包括人实体瘤(例如，乳腺癌)或血细胞肿瘤(例如，淋巴细胞肿瘤、B或T细胞肿瘤)。
在一些实施方案中，抗PLAU和/或PLAUR抗体阻断或抑制PLAU/PLAUR介导的信号通路。在一些实施例中，本文所描述的抗PLAU抗体可以阻断PLAU与PLAUR复合物之间的相互作用，从而抑制PLAU/PLAUR信号通路。在一些实施例中，本文所描述的PLAUR抗体可以阻断PLAU与PLAUR复合物之间的相互作用，从而抑制PLAU/PLAUR信号通路。
在一些实施例中，基因修饰的非人动物可用于确定治疗剂(如，抗PLAU抗体和/或抗PLAUR抗体)在治疗各种免疫疾病方面的有效性。在一些实施例中，所述免疫疾病包括但不限于GVHD(移植物抗宿主病)、银屑病、过敏、哮喘、心肌炎、肾炎、肝炎(优选为非酒精性脂肪性肝炎)、系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺功能亢进、原发性血小板减少性紫癜、自身免疫性溶血性贫血、溃疡性结肠炎、自身免疫性肝病、糖尿病、疼痛或神经障碍等。
在一些实施例中，基因修饰的非人动物可用于确定治疗剂(如，抗PLAU抗体和/或抗PLAUR抗体)在治疗各种炎症感染方面的有效性。在一些实施例中，所述炎症包括包括急性炎症，也包括慢性炎症。具体的，包括但不限于变质性炎症、渗出性炎症(浆液性炎、纤维素性炎、化脓性炎、出血性炎、坏死性炎、卡他性炎)、增生性炎症、特异性炎症(结核、梅毒、麻疯、淋巴肉芽肿等)。
在一些实施例中，基因修饰的非人动物可用于确定治疗剂(如，抗PLAU抗体和/或抗PLAUR抗体)对治疗癌症的有效性。在一些实施例中，向非人动物施用治疗剂(如，抗PLAU抗体和/或抗PLAUR抗体)，其中所述非人动物具有癌症或肿瘤，检测治疗剂对癌症或肿瘤的抑制作用。在一些实施例中，所述检测包括测定肿瘤细胞的大小和/或增殖速率。在一些实施例中，所述检测方法包括游标卡尺测量、流式细胞检测和/或动物活体成像检测。在一些实施例中，所述检测包括评估个体体重、脂肪量、活化途径、神经保护活性或代谢变化，所述代谢变化包括食物消耗或水消耗的变化。
在一些实施例中，所述肿瘤细胞包括一个或多个被注射到动物体内的癌细胞(如，癌细胞来源于人或非人动物)。在一些实施例中，治疗剂抑制PLAU/PLAUR信号通路。在一些实施例中，治疗剂不抑制PLAU/PLAUR信号通路。
在一些实施例中，基因修饰的非人动物可用于检测抗PLAU或抗PLAUR抗体是激动剂还是拮抗剂。在一些实施例中，本文描述的方法可以用来检测治疗剂(如，抗PLAU抗体或抗PLAUR抗体)的功能，例如，所述治疗剂是否可以上调免疫应答或下调免疫应答，和/或该治疗剂是否能够诱导补体介导的细胞毒性(CMC)或抗体依赖性细胞毒性(ADCC)。在一些实施例中，基因修饰的非人动物可用于确定治疗受试者疾病(例如免疫疾病)的治疗剂的有效剂量。对肿瘤的抑制作用也可以通过本领域已知的方法来确定，例如，测量动物中的肿瘤体积，和/或确定肿瘤(体积)抑制率(TGITV)。肿瘤生长抑制率可以使用公式TGITV、(％)＝(1–TVt/TVc)x 100计算，其中TVt和TVc是治疗组和对照组的平均肿瘤体积(或重量)。
在一些实施例中，治疗剂(如，抗PLAU抗体或抗PLAUR抗体)可以被用于治疗各种癌症。本发明所述“癌症”是指具有自主生长能力的细胞，即以细胞生长迅速增殖为特征的异常状态或病症。该术语旨在包括所有类型的癌性生长或致癌过程、转移性组织或恶性转化的细胞、组织或器官，无论组织病理学类型或侵袭性阶段如何。本发明所述“肿瘤”包括但不限于淋巴瘤、非小细胞肺癌、宫颈癌、白血病、卵巢癌、鼻咽癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、胃癌、膀胱癌、脑胶质瘤、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、肝和胆管癌、食管癌、肾癌、甲状腺癌、头颈部癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色 素瘤、骨髓增生异常综合征、以及肉瘤。其中，所述白血病选自急性淋巴细胞性(成淋巴细胞性)白血病、急性骨髓性白血病、髓性白血病、慢性淋巴细胞性白血病、多发性骨髓瘤、浆细胞白血病、以及慢性骨髓性白血病；所述淋巴瘤选自霍奇金淋巴瘤和非霍奇金淋巴瘤，包括B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、边缘区B细胞淋巴瘤、T细胞淋巴瘤、和瓦尔登斯特伦巨球蛋白血症；所述肉瘤选自骨肉瘤、尤文肉瘤、平滑肌肉瘤、滑膜肉瘤、软组织肉瘤、血管肉瘤、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤、以及软骨肉瘤。在本发明的一个具体实施方式中，所述肿瘤为乳腺癌、胰腺癌、内分泌癌、头颈癌、胃肠癌、结直肠癌、膀胱癌、非小细胞肺癌、胶质母细胞瘤、前列腺癌、神经内分泌瘤、间皮组织肿瘤、口咽肿瘤、女性生殖系统癌症或脑膜瘤。
本发明还提供了一种确定治疗剂(如，抗PLAU抗体或抗PLAUR抗体)毒性的检测方法。所述方法包括向上述所述非人动物施用抗体，评估动物的体重变化、红细胞计数、血细胞比容和/或血红蛋白。在一些实施例中，抗体可使红细胞(RBC)、血细胞比容或血红蛋白降低20％、30％、40％或50％以上。在一些实施例中，动物的体重与对照组(如，未用抗体处理的动物的平均体重)相比至少小5％、10％、20％、30％或40％。
本发明还提供了一种通过本文所述方法构建的动物模型在开发与人类细胞免疫过程相关的产品、制造人抗体、或用于药理学、免疫学、微生物学和医学研究的模型系统。
在一些实施例中，提供了一种通过本文描述的方法生成的动物模型在生产和利用人体细胞的免疫过程的动物实验疾病模型、研究病原体、或制定新的诊断策略和/或治疗策略。
本发明还提供了通过本文所述方法生成的动物模型来筛选、验证、评估或研究PLAU和/或PLAUR基因功能、人PLAU和/或PLAUR抗体、人PLAU和/或PALUR靶向位点的药物或有效性、免疫相关疾病的药物和抗肿瘤药物。
两个或多个人或嵌合基因的非人动物模型
本发明还提供了一种具两个或多个人或嵌合基因的非人动物，所述动物模型包含人或嵌合PLAU和/或PLAUR基因以及编码其他人或嵌合蛋白的核酸序列。在一些实施例中，所述其他基因为IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种基因修饰的非人动物。在一些实施例中，上述所述非人动物还表达人或人源化的IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α蛋白中的至少一种。
本发明还提供了一种两个或多个人或嵌合基因的非人动物的构建方法，所述构建方法包括：
(一)提供上述的构建方法获得非人动物；
(二)将步骤(一)提供的非人动物与其他基因修饰的非人动物交配、体外受精或直接进行基因编辑，并进行筛选，得到多基因修饰的非人动物。
在一些实施例中，所述其他基因修饰的非人动物包括基因IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的一种或两种以上的组合人源化的非人动物。
在一些实施例中，PLAU和/或PLAUR人源化直接在具有人或嵌合IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和/或TNF-α基因修饰的非人动物上进行。
由于这些蛋白可能涉及不同的机制，因此靶向其中两种或多种蛋白的联合疗法可能是一种更有效的治疗方法。事实上，许多相关的临床试验正在进行中，并显示出良好的效果。多基因修饰的非人动物模型可用于确定靶向两种或多种蛋白的联合疗法的有效性，例如，抗PLAU抗体或抗PLAUR抗体，以及用于治疗癌症或免疫疾病(例如，哮喘或特异性皮炎)的附加治疗剂。所述方法包括向动物施用抗PLAU抗体和/或抗PLAU抗体和附加治疗剂，其中动物具有肿瘤或免疫疾病，并确定联合治疗对免疫肿瘤或免疫疾病的影响。在一些实施例中，所述附加治疗剂是特异性结合IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和/或TNF-α的抗体。在一些实施例中，所述附加治疗剂是抗CTLA4抗体(例如，ipilimumab)、抗PD-1抗体(例如，nivolumab)或抗PD-L1抗体。在一些实施例中，上述所述非人动物还包括编码人或人源化PD-1的序列、编码人或人源化PD-L1的序列、或编码人或人源化CTLA-4的序列。在一些实施例中，附加治疗剂是抗PD-1抗体(例如，纳武利尤单抗、帕博利珠单抗)、抗PD-L1抗体或抗CTLA-4抗体。在一些实施例中，上述所述肿瘤包括一个或多个表达PD-L1和/或PD-L2的肿瘤细胞。
在一些实施例中，所述联合疗法还可用于治疗本文所述各种癌症，例如乳腺癌、胰腺癌、内分泌癌、头颈癌、胃肠癌、结直肠癌、膀胱癌、非小细胞肺癌、胶质母细胞瘤、前列腺癌、神经内分泌瘤、间皮组织肿瘤、口咽肿瘤、女性生殖系统癌症或脑膜瘤。
在一些实施例中，上述描述的治疗方法可与常规癌症化疗药联合使用。在一些实施例中，治疗癌症的方法可以单独使用或与本文描述的方法组合使用，包括，用化疗治疗受试者，如樟树碱、多柔比星、顺铂、卡铂、丙卡巴肼、甲氯乙胺、环磷酰胺、阿霉素、异环磷酰胺、美法仑、苯丁酸氮芥、硫丹、硝基苏拉、放线菌素、柔红霉素、博来霉素、普利霉素、丝裂霉素、依托泊苷、维拉皮尔、鬼臼毒素、他莫昔芬、紫杉醇、反铂、5-氟拉嘧啶、长春新碱、长春爆蛋白和/或甲氨蝶呤。所述方法可以包括对受试者进行手术去除至少一部分癌症，如从患者身上切除肿瘤的一部分或全部。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
在下述每一实施例中，设备和材料是从以下所指出的几家公司获得：
C57BL/6小鼠购自中国食品药品检定研究院国家啮齿类实验动物种子中心；
BbsI、EcoRI、BamHI、BclI、AvrII酶购自NEB，货号分别为；R0539L、R0101L、R0136L、R0160L、R0174L；
Anti-Urokinase抗体[EPR6273]，购自Abcam，货号ab133563；
Trizol试剂盒购自TakaRa，货号6110A。
实施例1：PLAU基因人源化小鼠
小鼠PLAU基因(NCBI Gene ID：18792，Primary source：MGI：97611，UniProt ID：P06869，位于14号染色体NC_000080.7的第20886730至20893456位，基于转录本NM_008873.3及其编码蛋白NP_032899.1(SEQ ID NO：1))和人PLAU基因(NCBI Gene ID：5328，Primary source：HGNC:9052，UniProt ID：P00749，位于10号染色体NC_000010.11的第73909182至73917497位，基于转录本NM_002658.6及其编码蛋白NP_002649.2(SEQ ID NO：2))对比示意图如图1所示。
为了达到本发明的目的，可在小鼠内源PLAU基因座引入编码人PLAU蛋白的核苷酸序列，使得该小鼠表达人或人源化PLAU蛋白。具体来说，用基因编辑技术，在小鼠PLAU基因调节元件的控制下，用包含人PLAU基因的2号外显子部分序列至11号外显子部分序列约5.0kb替换小鼠2号外显子的部分序列至11号外显子的部分序列约5.3kb，得到人源化PLAU基因座示意图如图2所示，实现对小鼠PLAU基因的人源化改造。
设计如图3所示的打靶策略，图中显示了靶向载体上含有小鼠PLAU基因上游和下游的同源臂序列，以及包含人PLAU DNA片段的A片段。其中，上游5’同源臂序列(SEQ ID NO：3)与NCBI登录号为NC_000080.7的第20883305至20887146位核苷酸序列相同，下游3’同源臂序列(SEQ ID NO：4)与NCBI登录号为NC_000080.7的第20893778至20896948位核苷酸序列相同。A片段上人PLAU DNA片段的核苷酸序列(SEQ ID NO：7)与NCBI登录号为NC_000010.11的第73911556至73916565位核苷酸序列相同。
靶向载体上还包括用于阳性克隆筛选的抗性基因，即新霉素磷酸转移酶编码序列Neo，并在抗性基因的两侧装上两个同向排列的位点特异性重组系统FRT重组位点，组成Neo盒 (Neo cassette)。其中Neo盒5’端与小鼠基因的连接设计为：5’-AAGAAAAGAAAAAAATCTGATTCAAACAAAGC GATATCGAATTCCGAAGTTCCTATTCTCTAGAAAGT-3’(SEQ ID NO：16)，其中序列中最后一个“C”是小鼠的最后一个核苷酸，序列“GATA”中的“G”是Neo盒的第一个核苷酸；Neo盒3’端与小鼠基因的连接设计为：5’-AGTATAGGAACTTCATCAGTCAGGTACATAATGGTGG GAGTCGGTGCTGTCGACGGGGTCAGGTAATGAAGTA-3’(SEQ ID NO：17)，其中序列中最后一个“C”是Neo盒的最后一个核苷酸，序列“GAGT”中第一个“G”是小鼠的第一个核苷酸。此外，还在靶向载体3’同源臂下游构建了具有负筛选标记的编码基因(白喉毒素A亚基的编码基因(DTA))。改造后的人源化小鼠PLAU的mRNA序列如SEQ ID NO：8所示，表达的蛋白序列如SEQ ID NO：2所示。
靶向载体构建可采用常规方法进行，如酶切连接等。构建好的靶向载体通过酶切进行初步验证后，再送测序公司进行测序验证。将测序验证正确的靶向载体电穿孔转染入C57BL/6小鼠的胚胎干细胞中，利用阳性克隆筛选标记基因对得到的细胞进行筛选，并利用PCR和Southern Blot技术进行检测确认外源基因的整合情况，筛选出正确的阳性克隆细胞。经PCR鉴定(引物如表5所示)为阳性的克隆再进行Southern Blot检测确认无随机插入后，进一步测序验证正确的克隆进行下一步实验。
表5 PCR引物名称及具体序列
将筛选出的正确阳性克隆细胞(黑色鼠)按照本领域已知的技术导入已分离好的囊胚中(白色鼠)，得到的嵌合囊胚转移至培养液中短暂培养后移植至受体母鼠(白色鼠)的输卵管，可生产F0代嵌合体鼠(黑白相间)。将F0代嵌合鼠与野生型鼠回交获得F1代鼠，再将F1代杂合小鼠互相交配即可获得F2代纯合子鼠。还可将阳性鼠与Flp工具鼠交配去除阳性克隆筛选标记基因(该过程示意图见图4)后，再通过互相交配即可得到PLAU基因人源化纯合子小鼠。
此外，还可引入CRISPR/Cas9系统进行基因编辑，设计如图5所示的打靶策略，图中显示了靶向载体上含有小鼠PLAU基因上游和下游的同源臂序列，以及人PLAU DNA片段序列。其中，上游同源臂序列(5’同源臂，SEQ ID NO：5)与NCBI登录号为NC_000080.7的第20885866至20887146位核苷酸序列相同，下游同源臂序列(3’同源臂，SEQ ID NO：6)与NCBI登录号为NC_000080.7的第20892519至20893251位核苷酸序列相同，人 PLAU DNA片段的核苷酸序列(SEQ ID NO：7)与NCBI登录号为NC_000010.11的第73911556至73916565位核苷酸序列相同。改造后的人源化小鼠PLAU的mRNA序列如SEQ ID NO：8所示，表达的蛋白序列如SEQ ID NO：2所示。
靶向载体构建可采用常规方法进行，如酶切连接、直接合成等。构建好的靶向载体通过酶切进行初步验证后，再送测序公司进行测序验证。将测序验证正确的靶向载体用于后续实验。
靶序列决定了sgRNA的靶向特异性和诱导Cas9切割目的基因的效率。因此，高效特异的靶序列选择和设计是构建sgRNA表达载体的前提。设计并合成识别靶位点的sgRNA序列，示例性sgRNA在PLAU基因上的靶序列如下：
sgRNA1靶位点(SEQ ID NO：20)：5’-GAGGGCTTGTGCACCCAAAGAGG-3’；
sgRNA2靶位点(SEQ ID NO：21)：5’-TGAGACCCTCGTGTAGACACCGG-3’；
利用UCA试剂盒检测sgRNA的活性，确定其可介导高效切割效率后，在其5’端及互补链上分别加上酶切位点得到正向寡核苷酸和反向寡核苷酸序列(见表6)，退火后将退火产物连接至pT7-sgRNA质粒(质粒先用BbsI线性化)，获得表达载体pT7-PLAU-1和pT7-PLAU-2。
表6 sgRNA1和sgRNA2序列列表
pT7-sgRNA载体由质粒合成公司合成含有T7启动子及sgRNA scaffold的片段DNA(SEQ ID NO：30)并依次通过酶切(EcoRI及BamHI)连接至骨架载体(来源Takara，货号3299)上，经专业测序公司测序验证，结果表明获得了目的质粒。取小鼠的原核期受精卵，例如C57BL/6小鼠，利用显微注射仪将pT7-PLAU-1和pT7-PLAU-2质粒的体外转录产物(使用Ambion体外转录试剂盒，按照说明书方法进行转录)、靶向载体与Cas9mRNA预混好后注射至小鼠受精卵细胞质或细胞核中。按照《小鼠胚胎操作实验手册(第三版)》 (安德拉斯·纳吉，化学工业出版社，2006)中的方法进行受精卵的显微注射，注射后的受精卵转移至培养液中短暂培养，然后移植至受体母鼠的输卵管中发育，将获得的小鼠(F0代)通过杂交和自交，扩大种群数量，建立稳定的PLAU基因人源化小鼠品系。
可通过PCR鉴定F1代小鼠体细胞的基因型(引物如表1所示)，示例性的F1代小鼠的鉴定结果见图6，其中，编号为F1-01至F1-09的9只小鼠均为阳性杂合子小鼠。
对F1代PCR鉴定为阳性的小鼠进行Southern blot检测，确认是否存在随机插入。剪取鼠尾提取基因组DNA，选用BclI酶或AvrII酶消化基因组，转膜，杂交。具体探针及目的片段的长度见表7，示例性F1代的检测结果如图7所示：F1-01至F1-09均为阳性杂合子小鼠。这表明使用本方法成功构建出可稳定传代且无随机插入的PLAU基因人源化小鼠。
表7具体探针及目的片段的长度
探针合成引物如下：
5’Probe-F(SEQ ID NO：31)：5’-TTACACTCCCTGACGACAAACTTCA-3’，
5’Probe-R(SEQ ID NO：32)：5’-TAACGCAGTCTGTCTGGATCGAGCG-3’；
3’Probe-F(SEQ ID NO：33)：5’-GCTCCAGTCTCCTGAATAGTAGCGC-3’，
3’Probe-R(SEQ ID NO：34)：5’-CTGATGTTTGATTCAAATTATGTGGT-3’；
通过常规检测方法确认阳性小鼠体内人源化PLAU mRNA的表达情况，例如RT-PCR等。具体来说，分别取C57BL/6野生型小鼠和本实施例制得的PLAU基因人源化杂合子小鼠各1只，脱颈安乐死后取小鼠肾脏组织，按照Trizol试剂盒说明书抽提细胞RNA，反转录成cDNA后进行RT-PCR检测(引物见表8)，检测结果如图8所示：在C57BL/6野生型小鼠(+/+)体内中仅检测到鼠PLAU mRNA，没有检测到人PLAU mRNA；仅在PLAU基因人源化杂合子小鼠(H/+)体内检测到人PLAUmRNA。
表8 RT-PCR引物名称及具体序列
进一步地，通过Western blot检测本实施例制备的PLAU基因人源化小鼠体内PLAU蛋白的表达情况。具体来说，分别取8周龄C57BL/6野生型小鼠(+/+)和本实施例制得的PLAU基因人源化纯合子小鼠(H/H)各1只，脱颈安乐死后取小鼠肝脏和肾脏组织，使用Anti-Urokinase抗体进行Western blot检测，结果如图15所示，在C57BL/6野生型小鼠和PLAU基因人源化纯合子小鼠体内均检测出PLAU蛋白，由于Anti-Urokinase抗体具有人鼠PLAU交叉结合活性，综合图8的RT-PCR检测结果可知，在C57BL/6野生型小鼠体内检测到的是鼠PLAU蛋白，在PLAU基因人源化纯合子小鼠体内检测出的是人PLAU蛋白。上述结果表明，本实施例构建的PLAU基因人源化小鼠可成功表达人PLAU蛋白。
实施例2：PLAUR基因人源化小鼠
小鼠PLAUR基因(NCBI Gene ID：18793，Primary source：MGI：97612，UniProt ID：P35456，位于7号染色体NC_000073.7的第24161857至24175393位，基于转录本NM_011113.4及其编码蛋白NP_035243.1(SEQ ID NO：9))和人PLAUR基因(NCBI Gene ID：5329，Primary source：HGNC:9053，UniProt ID：Q03405，位于19号染色体NC_000019.10的第43646095至43670197位，基于转录本NM_002659.4及其编码蛋白NP_002650.1(SEQ ID NO：10))对比示意图如图9所示。
为了达到本发明的目的，可在小鼠内源PLAUR基因座引入编码人PLAUR蛋白的核苷酸序列，使得该小鼠表达人或人源化PLAUR蛋白。具体来说，用基因编辑技术，在小鼠PLAUR基因调节元件的控制下，用包含人PLAUR基因的2号外显子部分序列至7号外显子部分序列约18.8kb替换小鼠2号外显子的部分序列至7号外显子的部分序列约10.3kb，得到人源化PLAUR基因座示意图如图10所示，实现对小鼠PLAUR基因的人源化改造。
设计如图11所示的打靶策略，图中显示了靶向载体上含有小鼠PLAUR基因上游和下游的同源臂序列，以及包含人PLAUR DNA片段的A1片段。其中，上游5’同源臂序列(SEQ ID NO：11)与NCBI登录号为NC_000073.7的第24159210至24164569位核苷酸序列相同，下游3’同源臂序列(SEQ ID NO：12)与NCBI登录号为NC_000073.7的第24174897至24178233位核苷酸序列相同。A1片段上人PLAUR DNA片段的核苷酸序列(SEQ ID NO：13)与NCBI登录号为NC_000019.10的第43648890to 43667677位核苷酸序列相同。
靶向载体上还包括用于阳性克隆筛选的抗性基因，即新霉素磷酸转移酶编码序列Neo，并在抗性基因的两侧装上两个同向排列的位点特异性重组系统Frt重组位点，组成Neo盒(Neo cassette)。其中Neo盒5’端与人序列的连接设计为5’-ATTGTACACTTATGAGAGTGAAAAAG ATCATCTGGCGAATCGGACCCACAAGAGCACTGAGGTCGGAAGTTCCTA TTC-3’(SEQ ID NO:41)，其中序列的“T”是人序列的最后一个核苷酸，序列“ATC A”第一个“A”是Neo盒的第一个核苷酸；Neo盒3’端与人序列的连接设计为：5’-AAGTATAGGAACTTCATCAGTCCAGGATACATAGATTACCACAACTCCG GTGAAATCACAGATCTCCTGAAAGGGTCTCTGGG-3’(SEQ ID NO:42)，其中序列的最后一个“C”是Neo盒的最后一个核苷酸，序列“GTGA”第一个“G”是人序列的第一个核苷酸。此外，还在重组载体3’同源臂下游构建了具有负筛选标记的编码基因(白喉毒素A亚基的编码基因(DTA))。改造后的人源化小鼠PLAUR的mRNA序列如SEQ ID NO：14所示，表达的蛋白序列如SEQ ID NO：15所示。
鉴于人PLAUR具有多种亚型或转录本，本文所述方法可应用于其它亚型或转录本。
靶向载体构建可采用常规方法进行，如酶切连接等。构建好的靶向载体通过酶切进行初步验证后，再送测序公司进行测序验证。将测序验证正确的靶向载体电穿孔转染入C57BL/6小鼠的胚胎干细胞中，利用阳性克隆筛选标记基因对得到的细胞进行筛选，并利用PCR和Southern Blot技术进行检测确认外源基因的整合情况，筛选出正确的阳性克隆细胞。经PCR鉴定(引物如表9所示)为阳性的克隆再进行Southern Blot检测确认无随机插入后，进一步测序验证正确的克隆进行下一步实验。
表9 PCR引物名称及具体序列
将筛选出的正确阳性克隆细胞(黑色鼠)按照本领域已知的技术导入已分离好的囊胚中(白色鼠)，得到的嵌合囊胚转移至培养液中短暂培养后移植至受体母鼠(白色鼠)的输卵管，可生产F0代嵌合体鼠(黑白相间)。将F0代嵌合鼠与野生型鼠回交获得F1代鼠，再将F1代杂合小鼠互相交配即可获得F2代纯合子鼠。还可将阳性鼠与Flp工具鼠交配去除阳性克隆筛选标记基因(该过程示意图见图12)后，再通过互相交配即可得到PLAUR基因人源化纯合子小鼠。
可通过PCR鉴定F1代小鼠体细胞的基因型(引物如表4所示)，示例性的F1代小鼠 的鉴定结果见图13，其中，编号为F1-01、F1-02和F1-03的小鼠均为阳性杂合子小鼠。
通过常规检测方法确认阳性小鼠体内人源化PLAUR mRNA的表达情况，例如RT-PCR等。具体来说，分别取C57BL/6野生型小鼠和本实施例制得的PLAUR基因人源化杂合子小鼠各1只，脱颈安乐死后取小鼠肺部组织，按照Trizol试剂盒说明书抽提细胞RNA，反转录成cDNA后进行RT-PCR检测(引物见表10)，检测结果如图14所示：在C57BL/6野生型小鼠(+/+)体内中仅检测到鼠PLAUR mRNA，没有检测到人PLAUR mRNA；仅在PLAUR基因人源化杂合子小鼠(H/+)体内检测到人PLAURmRNA。
表10 RT-PCR引物名称及具体序列
进一步采用流式细胞术检测PLAUR基因人源化小鼠体内PLAUR蛋白的表达情况。选取7周龄雌性野生型C57BL/6小鼠和10周龄雌性PLAUR基因人源化纯合子小鼠各1只，腹腔注射LPS(20ug/200ul)刺激2h收集腹腔灌洗液，用抗鼠CD45抗体Brilliant Violet 510TM anti-mouse CD45Antibody(mCD45)、抗鼠CD11b抗体V450Rat Anti-mouse CD11b Antibody(mCD11b)、抗鼠F4/80抗体FITC anti-mouse F4/80 Antibody(mF4/80)、抗鼠PLAUR抗体CD87Antibody,anti-mouse,REAfinityTM(mPLAUR)、抗人PLAUR抗体CD87Antibody,anti-human,REAfinityTM(hPLAUR)、Zombie NIRTM Fixable Viability Kit和Purified anti-mouse CD16/32进行染色，将染色后的细胞进行流式检测，腹水中巨噬细胞特征为：CD45+CD11b+mF4/80+。经检测发现，野生型小鼠腹水中巨噬细胞有99.7％mPLAUR阳性细胞(特征为CD45+CD11b+mF4/80+mPLAUR+)，有2.24％hPLAUR阳性细胞(特征为CD45+CD11b+mF4/80+hPLAUR+)，PLAUR基因人源化纯合子小鼠腹水中巨噬细胞有0.20％mPLAUR阳性细胞(特征CD45+CD11b+mF4/80+mPLAUR+)，有99.6％hPLAUR阳性细胞(特征为CD45+CD11b+mF4/80+hPLAUR+)，综上表明本实施例构建的PLAUR人源化小鼠可成功表达人源化PLAUR蛋白。
实施例3：PLAU/PLAUR双基因人源化鼠的制备
使用实施例1制备的PLAU基因人源化小鼠与实施例2制备的PLAUR基因人源化小鼠 交配，通过阳性子代小鼠的筛选，得到PLAU/PLAUR双基因人源化小鼠。可采用常规方法确认阳性小鼠体内人PLAU蛋白的表达情况，如ELSA。具体来说，分别选取9周龄雄性野生型C57BL/6小鼠(+/+)和本实施例制备的雄性PLAU/PLAUR双基因人源化纯合小鼠(H/H)各3只，脱颈安乐死取血清，使用小鼠uPA ELISA试剂盒和人uPA ELISA试剂盒(URK)进行检测，结果显示(图16)鼠的PLAU蛋白仅在野生型C57BL/6小鼠中检测到，人的PLAU蛋白仅在PLAU/PLAUR双基因人源化纯合小鼠中检测到，这表明经人源化改造后小鼠体内可正常表达人的PLAU蛋白。
可采用流式细胞术检测PLAU/PLAUR双基因人源化纯合小鼠体内hPLAUR蛋白表达情况。具体来说，分别选取9周雄性野生型C57BL/6小鼠(+/+)和本实施例制备的10周龄雄性PLAU/PLAUR双基因人源化纯合小鼠(H/H)各1只，脱颈安乐死后取骨髓组织，使用抗鼠CD45抗体Brilliant Violet 510TM anti-mouse CD45(mCD45)、抗鼠Ly6G抗体Brilliant Violet 650TM anti-mouse Ly-6G Antibody(mLy6G)、抗鼠CD11b抗体V450 Rat Anti-mouse CD11b Antibody(mCD11b)、抗鼠CD3抗体Alexa700 anti-mouse CD3 Antibody(mCD3)、抗鼠F4/80抗体FITC anti-mouse F4/80Antibody(mF4/80)、抗鼠CD11c抗体Invitrogen CD11c Monoclonal Antibody(N418),PE-Cyanine7,eBioscience(mCD11c)、抗鼠PLAUR抗体CD87 Antibody,anti-mouse,REAfinityTM(mPLAUR)、抗人PLAUR抗体CD87Antibody,anti-human,REAfinityTM(hPLAUR)、抗人IgG1抗体PE Human IgG1 Isotype Control Recombinant Antibody、抗鼠IgG1抗体APC Human IgG1Isotype Control Recombinant Antibody、Zombie NIRTM Fixable Viability Kit和Purified anti-mouse CD16/32识别染色后，进行流式分析，检测单核巨噬细胞、粒细胞和树突细胞中人或人源化PLAUR蛋白的表达情况，检测结果如表11所示。
表11流式细胞术检测结果
从表11中可以看出在野生型C57BL/6小鼠骨髓的单核巨噬细胞、粒细胞和树突细胞中仅能检测到鼠PLAUR蛋白，而在PLAU/PLAUR双基因人源化纯合子小鼠体内均能检测到人PLAUR蛋白，进一步表明经人源化改造后小鼠体内可正常表达人的PLAU蛋白。
实施例4药效验证
首先可采用流式细胞术验证本实施例制备的PLAU/PLAUR双基因人源化纯合子小鼠与两种抗人PLAUR抗体Ab1和Ab2是否可以结合，选取8周龄雄性PLAU/PLAUR双基因人源化纯合子小鼠1只，腹腔注射LPS(20ug/200ul)刺激2h收集腹腔灌洗液，使用抗鼠CD45抗体Brilliant Violet 510TM anti-mouse CD45、抗鼠Ly6G抗体Brilliant Violet 650TM anti-mouse Ly-6G Antibody、抗鼠CD11b抗体V450Rat Anti-mouse CD11b Antibody、抗鼠F4/80抗体FITC anti-mouse F4/80 Antibody、抗人PLAU抗体Ab1、抗人PLAUR抗体Ab2、抗人IgG1抗体PE Human IgG1 Isotype Control Recombinant Antibody、抗鼠IgG1抗体APC Human IgG1 Isotype Control Recombinant Antibody、Zombie NIRTM Fixable Viability Kit和Purified anti-mouse CD16/32识别染色后，进行流式分析，检测发现，PLAU/PLAUR双基因人源化纯合子小鼠腹腔灌洗液中单核巨噬细胞有100％的Ab1阳性细胞和98.3％的Ab2阳性细胞，综上表明本实例构建的LAU/PLAUR双基因人源化纯合子小鼠可成功结合抗人PLAUR抗体，后续可用于评估靶向人PLAUR抗体的药效。
例如，取PLAU/PLAUR双基因人源化纯合子小鼠皮下接种表达人源化PLAUR结肠癌细胞MC38，待肿瘤体积生长到约100mm3后，根据肿瘤体积分为对照组或治疗组，治疗组注射靶向人PLAUR的抗体药物，对照组注射等体积的PBS。定期测量肿瘤体积并称量小鼠的体重，通过比较小鼠体重和肿瘤体积，可有效评估抗体药物在人源化PLAU/PLAUR小鼠体内安全性和体内药效。
具体来说，取7-9周龄PLAU/PLAUR双基因人源化纯合子小鼠皮下接种表达人源化PLAUR结肠癌细胞MC38(5×105)，待肿瘤体积生长到约100mm3后，将小鼠分为对照组或治疗组(n＝6/组)，治疗组G2和G3注射靶向人PLAUR的IgG1抗体药物Ab1和Ab2，对照组注射等体积的PBS。给药频率为每周给药2次，共给药6次，每周测量肿瘤体积2次并称量小鼠的体重，直至21天(分组后第21天)实验结束，接种后单只小鼠肿瘤体积达到3000mm3时应执行安乐死结束试验。具体的分组、给药、剂量和频率如表12所示，实验过程中小鼠肿瘤体积和体重分别见图17和图18。
表12具体分组、给药、剂量和频率
结果表明，实验过程中各组动物健康状态良好，在实验终点时(分组后21天)，所有治疗组和对照组小鼠体重均出现增长，且在整个实验周期内小鼠体重均无明显区别(图 18)；但从肿瘤体积测量结果上看(图17)，对照组小鼠肿瘤在实验周期内均持续生长，与对照组相比，治疗组G2和G3肿瘤体积增长呈现不同程度的抑制和/或缩小。
表13中列出了实验的主要数据和分析结果，具体包括分组时和分组后14天和第21天的肿瘤体积、小鼠存活情况、肿瘤(体积)抑制率(Tumor Growth Inhibition Value，TGITV)以及治疗组与对照组的小鼠肿瘤体积之间的统计学差异(P值)。
表13肿瘤体积之间的统计学差异
从表13可见，在实验终点时，对照组平均肿瘤体积为2237±341mm3，治疗组在Ab1(10mg/kg)和Ab2(10mg/kg)剂量水平下的平均肿瘤体积分别为1926±566mm3、和1726±250mm3，所有治疗组小鼠的肿瘤体积小于对照组，TGITV分别为14.5％和23.8％，表明不同剂量不同种类的抗人PLAUR抗体对肿瘤抑制作用不同。综上表明，PLAU/PLAUR双基因人源化纯合小鼠可用于候选药物的筛选与评价。
实施例5：PLAU和/或PLAUR多重人源化小鼠的制备
利用本方法或制得的PLAU、PLAUR基因人源化小鼠还可以制备双基因修饰或多基因修饰的小鼠模型。如前述实施例1中，囊胚显微注射使用的胚胎干细胞可选择来源于含有PLAUR、PD-1、PD-L1、CTLA4、OX40、LAG3、TIM3、CD73等其它基因修饰的小鼠，或者，也可在人源化PLAU小鼠的基础上，利用分离小鼠ES胚胎干细胞和基因重组打靶技术，获得PLAU与其它基因修饰的双基因或多基因修饰的小鼠模型。也可将本方法得到的PLAU小鼠纯合子或杂合子与其它基因修饰的纯合或杂合小鼠交配，对其后代进行筛选，根据孟德尔遗传规律，可有一定机率得到PLAU基因与其它基因修饰的双基因或多基因修饰的杂合小鼠，再将杂合子相互交配可以得到双基因或多基因修饰的纯合子。使用前述实施例2获得PLAUR人源化小鼠根据上述方法，同样可以获得PLAUR基因与其它基因修饰的纯合子小鼠。利用这些双基因或多基因修饰的小鼠可以进行靶向人PLAU、PLAUR和其它基因调节剂的体内药效验证等。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单 变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (128)

  1. 一种基因修饰的非人动物，其特征在于，所述动物的基因组包含至少一条染色体，所述染色体包含编码人或嵌合尿激酶型纤溶酶原激活物受体(PLAUR)蛋白的核苷酸序列。
  2. 根据权利要求1所述的动物，其特征在于，所述编码人或嵌合PLAUR蛋白的核苷酸序列可操作地连接至至少一条染色体的内源PLAUR基因座的内源调控元件(如，内源5'UTR和/或3'UTR)。
  3. 根据权利要求1或2所述的动物，其特征在于，所述编码人或嵌合PLAUR蛋白的核苷酸序列包含编码人PLAUR蛋白的3个同源结构域。
  4. 根据权利要求1或2所述的动物，其特征在于，所述编码人或嵌合PLAUR蛋白的核苷酸序列编码的氨基酸序列与人PLAUR(NP_002650.1，SEQ ID NO：10)同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  5. 根据权利要求1或2所述的动物，其特征在于，所述编码人或嵌合PLAUR蛋白的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：10第24-335位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  6. 根据权利要求1或2所述的动物，其特征在于，所述编码人或嵌合PLAUR蛋白的核苷酸序列包含与SEQ ID NO：15所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  7. 根据权利要求1-6任一所述的动物，其特征在于，所述动物是哺乳动物，如猴子、啮齿动物、小鼠或大鼠。
  8. 根据权利要求1-7任一所述的动物，其特征在于，所述动物是小鼠。
  9. 根据权利要求1-8任一所述的动物，其特征在于，所述动物内源PLAUR蛋白不表达或与野生型动物中PLAUR相比表达水平降低。
  10. 根据权利要求1-9任一所述的动物，其特征在于，所述动物的一个或多个细胞表达人或嵌合PLAUR蛋白。
  11. 根据权利要求1-10任一所述的动物，其特征在于，所述动物的一个或多个细胞表达人或嵌合PLAUR蛋白的细胞，内源PLAUR配体(PLAU)可以结合表达人或嵌合PLAUR蛋白，激活下游信号通路。
  12. 根据权利要求1-10任一所述的动物，其特征在于，所述动物的一个或多个细胞表达人或嵌合PLAUR蛋白，人PLAUR配体(PLAU)可以结合表达人或嵌合PLAUR蛋白，激活下游信号通路。
  13. 一种基因修饰的非人动物，其特征在于，所述动物的基因组包含在内源PLAUR基因座处编码内源PLAUR区域的核苷酸序列被人PLAUR相应区域的核苷酸序列替换。
  14. 根据权利要求13所述的动物，其特征在于，所述编码人PLAUR相应区域的核苷酸序列可操作地连接到内源PLAUR基因座的内源调控元件，并且所述动物的一个或多个细胞表达人或人源化的PLAUR蛋白。
  15. 根据权利要求13或14所述的动物，其特征在于，所述动物的内源PLAUR蛋白不表达或与野生型动物中PLAUR相比表达水平降低。
  16. 根据权利要求13-15任一所述的动物，其特征在于，所述编码人PLAUR相应区域的核苷酸序列包含人PLAUR基因的外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。
  17. 根据权利要求13-16任一所述的动物，其特征在于，所述编码人PLAUR相应区域的核苷酸序列与SEQ ID NO：13所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  18. 根据权利要求13-17任一所述的动物，其特征在于，所述编码内源PLAUR区域的核苷酸序列包含小鼠PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。
  19. 根据权利要求13-18任一所述的动物，其特征在于，所述动物基因组中修饰的基因对于内源被替换的基因座为纯和或杂合。
  20. 一种非人动物，其特征在于，所述动物包含至少一个编码人或人源化PLAUR蛋白的核苷酸序列的细胞，其中所述人源化PLAUR蛋白包含与人相应区域的连续氨基酸序列至少50、100、150、200、250、300、310、311、312、320、330、331、332、333、334或335个连续氨基酸序列一致。
  21. 根据权利要求20所述的动物，其特征在于，所述人或人源化PLAUR蛋白包含人PLAUR蛋白的3个同源结构域。
  22. 根据权利要求20或21所述的动物，其特征在于，所述人或人源化PLAUR蛋白的氨基酸序列与SEQ ID NO：10所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  23. 根据权利要求20-22任一所述的动物，其特征在于，所述编码人或人源化PLAUR蛋白的核苷酸序列可操作地连接至内源PLAUR调控元件。
  24. 根据权利要求20-23任一所述的动物，其特征在于，所述编码人PLAUR相应区域的核苷酸序列可被整合至所述动物内源PLAUR基因座。
  25. 根据权利要求20-24任一所述的动物，其特征在于，所述人或人源化PLAUR蛋白具有至少一种小鼠PLAUR活性和/或人PLAUR活性。
  26. 一种基因修饰的非人动物的构建方法，其特征在于，所述动物的至少一个细胞中，在动物内源PLAUR基因座处，编码内源PLAUR区域的核苷酸序列被人PLAUR相应区域的核苷酸序列替换。
  27. 根据权利要求26所述的方法，其特征在在于，所述动物的内源PLAUR蛋白不表达或与野生型动物中PLAUR相比表达水平降低。
  28. 根据权利要求26或27所述的方法，其特征在于，所述编码人PLAUR相应区域的核苷酸序列包含编码人PLAUR蛋白的3个同源结构域。
  29. 根据权利要求26-28任一所述的方法，其特征在于，所述编码人PLAUR相应区域的核苷酸序列包含人PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。
  30. 根据权利要求26-29任一所述的方法，其特征在于，所述编码人PLAUR相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：10第24-335所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  31. 根据权利要求26-30任一所述的方法，其特征在于，所述编码人PLAUR相应区域的核苷酸序列与SEQ ID NO：13所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  32. 根据权利要求26-31任一所述的方法，其特征在于，所述编码内源PLAUR区域的核苷酸序列包含小鼠PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。
  33. 根据权利要求26-32任一所述的方法，其特征在于，所述编码人PLAUR相应区域的核苷酸序列可操作地连接至内源PLAUR调控元件，如，启动子。
  34. 根据权利要求26-33任一所述的方法，其特征在于，所述动物为哺乳动物，如，猴子、啮齿动物、小鼠或大鼠。
  35. 根据权利要求26-34任一所述的方法，其特征在于，所述动物是小鼠。
  36. 一种表达人或嵌合PLAUR的基因修饰非人动物细胞的构建方法，所述方法包括在内源小鼠PLAUR基因座处，编码内源PLAUR区域的核苷酸序列被人PLAUR相应区域的核苷酸序列替换，产生基因修饰的非人动物细胞，所述动物细胞表达人或嵌合PLAUR蛋白。
  37. 根据权利要求36所述的方法，其特征在于，所述人或嵌合PLAUR蛋白包含人PLAUR蛋白的3个同源结构域。
  38. 根据权利要求36或37所述的方法，其特征在于，所述编码人PLAUR相应区域的核苷酸序列包含人PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、 和/或外显子7的部分。
  39. 根据权利要求36-38任一所述的方法，其特征在于，所述编码人PLAUR相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：10第24-335所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  40. 根据权利要求36-39任一所述的方法，其特征在于，所述编码人PLAUR相应区域的核苷酸序列与SEQ ID NO：13所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  41. 根据权利要求36-40任一所述的方法，其特征在于，所述内源PLAUR的核苷酸序列包含小鼠PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。
  42. 根据权利要求36-41任一所述的方法，其特征在于，所述编码人或嵌合PLAUR蛋白的核苷酸序列可操作地连接至内源PLAUR的调控元件，如，启动子。
  43. 根据权利要求36-42任一所述的方法，其特征在于，所述非人动物是小鼠。
  44. 根据权利要求26-34任一所述的方法，其特征在于，所述非人动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，所述人或嵌合蛋白选自PLAUR配体(PLAU)、IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种。
  45. 根据权利要求44所述的方法，其特征在于，所述人或嵌合蛋白为人或嵌合PLAU蛋白。
  46. 根据权利要求44或45所述的方法，其特征在于，所述人或嵌合PLAU蛋白包含人PLAU基因编码的全长蛋白。
  47. 根据权利要求44-46任一所述的方法，其特征在于，所述人或嵌合PLAU蛋白的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  48. 根据权利要求36-43任一所述的方法，其特征在于，所述非人动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，其中所述人或嵌合蛋白选自PLAUR配体(PLAU)、IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种。
  49. 根据权利要求48所述的方法，其特征在于，所述人或嵌合蛋白为人或嵌合PLAU蛋白。
  50. 根据权利要求48或49任一所述的方法，其特征在于，所述人或嵌合PLAU蛋白包含人PLAU基因编码的全长蛋白。
  51. 根据权利要求48-50任一所述的方法，其特征在于，所述人或嵌合PLAU蛋白的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  52. 一种基因修饰的非人动物，其特征在于，所述动物基因组包含至少一条染色体，所述染色体包含编码人或嵌合尿激酶型纤溶酶原激活剂(PLAU)蛋白的核苷酸序列。
  53. 根据权利要求52所述的动物，其特征在于，所述编码人或嵌合PLAU蛋白的核苷酸序列可操作地连接至至少一条染色体的内源PLAU基因座的调控元件(如，内源5'UTR和/或3'UTR)。
  54. 根据权利要求52或53所述的动物，其特征在于，所述编码人或嵌合PLAU蛋白的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  55. 根据权利要求52-54任一所述的动物，其特征在于，所述动物是哺乳动物，如猴子、啮齿动物、小鼠或大鼠。
  56. 根据权利要求52-55任一所述的动物，其特征在于，所述动物是小鼠。
  57. 根据权利要求52-56任一所述的动物，其特征在于，所述动物的内源PLAUR蛋白不表达或与野生型动物中PLAU相比表达水平降低。
  58. 根据权利要求52-57任一所述的动物，其特征在于，所述动物的一个或多个细胞表达人或嵌合PLAU蛋白。
  59. 根据权利要求52-58任一所述的动物，其特征在于，所述动物的一个或多个细胞表达人或嵌合PLAU蛋白，内源PLAU受体(PLAUR)可以结合表达人或嵌合PLAU蛋白，激活下游信号通路。
  60. 根据权利要求52-59任一所述的动物，其特征在于，所述动物的一个或多个细胞表达人或嵌合PLAU蛋白，人PLAU受体(PLAUR)可以结合表达人或嵌合PLAU蛋白，激活下游信号通路。
  61. 一种基因修饰的非人动物，其特征在于，所述动物的基因组包含在内源PLAU基因座处，编码内源PLAU区域的核苷酸序列被人PLAU相应区域的核苷酸序列替换。
  62. 根据权利要求61所述的动物，其特征在于，所述编码人PLAU相应区域的核苷酸序列可操作地连接至内源PLAU基因座的调控元件，所述动物的一个或多个细胞表达人或人源化的PLAU蛋白。
  63. 根据权利要求61或62所述的动物，其特征在于，所述动物的内源PLAUR蛋白不表达或与野生型动物中PLAU相比表达水平降低。
  64. 根据权利要求62-63任一所述的动物，其特征在于，所述编码人PLAU相应区域的核苷酸序列包含人PLAU基因的外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。
  65. 根据权利要求62-64任一所述的动物，其特征在于，所述编码人PLAU相应区域的核苷酸序列包含人PLAU编码区的全部核苷酸序列。
  66. 根据权利要求62-65任一所述的动物，其特征在于，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：7所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  67. 根据权利要求62-66任一所述的动物，其特征在于，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：8所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  68. 根据权利要求62-67任一所述的动物，其特征在于，所述编码内源PLAU区域的核苷酸序列包含小鼠PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。
  69. 根据权利要求62-68任一所述的动物，其特征在于，所述动物基因组中修饰的基因对于内源被替换的基因座为纯合或杂合。
  70. 一种非人动物，其特征在于，所述动物包含至少一个编码人或人源化PLAU蛋白的核苷酸序列的细胞，其中所述人源化PLAU蛋白包含与人相应区域的连续氨基酸序列至少50、100、150、200、250、300、350、400、410、420、430、或431个连续氨基酸一致。
  71. 根据权利要求70所述的动物，其特征在于，所述人源化PLAU蛋白的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  72. 根据权利要求70或71所述的动物，其特征在于，所述编码人或人源化PLAU蛋白的核苷酸序列可操作地连接至内源PLAU调控元件。
  73. 根据权利要求70-72任一所述的动物，其特征在于，所述编码人或人源化PLAU蛋白的核苷酸序列可被整合至所述动物内源PLAU基因座。
  74. 根据权利要求70-73任一所述的动物，其特征在于，所述人源化PLAU蛋白具有至少一种小鼠的PLAU活性和/或人PLAU活性。
  75. 一种基因修饰的非人动物的构建方法，其特征在于，所述动物的至少一个细胞中，在动物内源PLAU基因座处，编码内源PLAU区域的核苷酸序列被人PLAU相应区域的核苷酸序列替换。
  76. 根据权利要求75所述的方法，其特征在于，所述非人动物的内源PLAU蛋白不表达或与野生型动物中PLAU相比表达水平降低。
  77. 根据权利要求75或76所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列包含编码人PLAU蛋白的全部序列。
  78. 根据权利要求75-77任一所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列包含人PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。
  79. 根据权利要求75-78任一所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列包含编码的氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  80. 根据权利要求75-79任一所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：7所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  81. 根据权利要求75-80任一所述的方法，其特征在于，所述编码内源PLAU区域的核苷酸序列包含小鼠PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。
  82. 根据权利要求75-81任一所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列可操作地连接至内源PLAU的调控元件，如，启动子。
  83. 根据权利要求75-82任一所述的方法，其特征在于，所述动物为哺乳动物，如猴子、啮齿动物、小鼠或大鼠。
  84. 根据权利要求75-83任一所述的方法，其特征在于，所述动物是小鼠。
  85. 一种表达人或嵌合PLAU蛋白的基因修饰非人动物的细胞构建方法，所述方法包括在内源小鼠PLAU基因座处，用编码人PLAU相应区域的核苷酸替换编码内源PLAU区域的核苷酸序列，产生基因修饰的非人动物细胞，其中动物细胞表达人或嵌合PLAU蛋白。
  86. 根据权利要求85所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列包含人PLAU蛋白的全部序列。
  87. 根据权利要求85或86所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列包含人PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。
  88. 根据权利要求85-87任一所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：2所示氨基酸序列同一性至少为70％、 75％、80％、85％、90％、95％、99％、或100％。
  89. 根据权利要求85-88任一所述的方法，其特征在于，所述编码人PLAU相应区域的核苷酸序列与SEQ ID NO：7所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  90. 根据权利要求85-89任一所述的方法，其特征在于，所述编码内源PLAU区域的核苷酸序列包含小鼠PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。
  91. 根据权利要求85-90任一所述的方法，其特征在于，所述动物是小鼠。
  92. 根据权利要求86-91任一所述的方法，其特征在于，所述编码人或嵌合PLAU蛋白的核苷酸序列可操作地连接至内源PLAU的调控元件，如，启动子。
  93. 根据权利要求75-84任一所述的方法，其特征在于，所述动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，其中所述人或嵌合蛋白选自PLAU受体(PLAUR)、IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种。
  94. 根据权利要求93所述的方法，其特征在于，所述人或嵌合蛋白为人或嵌合PLAUR蛋白。
  95. 根据权利要求93或94所述的方法，其特征在于，所述人或嵌合PLAUR蛋白的氨基酸序列与SEQ ID NO：14所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  96. 根据权利要求85-92任一所述的方法，其特征在于，所述动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，其中所述人或嵌合蛋白选自PLAU受体(PLAUR)、IL1B、IL6、IL15、PD-1、PD-L1、TIGIT、LAG3、CD226、CTLA4和TNF-α中的至少一种。
  97. 根据权利要求96所述的方法，其特征在于，所述人或嵌合蛋白为人或嵌合PLAUR蛋白。
  98. 根据权利要求96-97所述的方法，其特征在于，所述人或嵌合PLAUR蛋白的氨基酸序列与SEQ ID NO：14所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  99. 一种测定抗PLAU和/或PLAUR治疗剂治疗癌症有效性的方法，其特征在于，所述方法包括：
    1)向权利要求1-25和52-74任一所述的动物施用抗PLAU和/或PLAUR治疗剂，其中所述动物具有肿瘤；
    2)测定抗PLAU和/或PLAUR治疗剂对肿瘤的抑制作用。
  100. 根据权利要求99所述的方法，其特征在于，所述肿瘤包含一个或多个肿瘤细胞，其中肿瘤细胞被注射到动物体内。
  101. 根据权利要求99或100所述的方法，其特征在于，所述测定抗PLAU/PLAUR治疗剂对肿瘤的抑制作用包含测量动物体内的肿瘤体积。
  102. 根据权利要求99-101任一所述的方法，其特征在于，所述肿瘤包括乳腺癌、胰腺癌、内分泌癌、头颈癌、胃肠癌、结直肠癌、膀胱癌、非小细胞肺癌、胶质母细胞瘤、前列腺癌、神经内分泌瘤、间皮组织肿瘤、口咽肿瘤、女性生殖系统癌症或脑膜瘤。
  103. 一种测定抗PLAU和/或PLAUR治疗剂和其它治疗剂治疗癌症有效性的方法，其特征在于，所述方法包括：
    3)向权利要求1-25和52-74任一所述的动物施用抗PLAU和/或PLAUR治疗剂，其中所述动物具有肿瘤；
    4)测定抗PLAU和/或PLAUR治疗剂对肿瘤的抑制作用。
  104. 根据权利要求103所述的方法，其特征在于，所述动物还包括编码人或嵌合PD-1、人或嵌合PD-L1和/或人或嵌合CTLA4的序列。
  105. 根据权利要求103或104所述的方法，其特征在于，所述其它治疗剂是抗PD-1抗体、抗PD-L1抗体和/或抗CTLA4抗体。
  106. 根据权利要求103-105任一所述的方法，其特征在于，所述肿瘤包含一个或多个细胞表达PD-L1蛋白。
  107. 根据权利要求103-106任一所述的方法，其特征在于，所述肿瘤包含一个或多个肿瘤细胞，其中肿瘤细胞被注射到动物体内。
  108. 根据权利要求103-107任一所述的方法，其特征在于，所述测定抗PLAU/PLAUR治疗剂对肿瘤的抑制作用包含测量动物体内的肿瘤体积。
  109. 根据权利要求103-108任一所述的方法，其特征在于，所述肿瘤为乳腺癌、胰腺癌、内分泌癌、头颈癌、胃肠癌、结直肠癌、膀胱癌、非小细胞肺癌、胶质母细胞瘤、前列腺癌、神经内分泌瘤、间皮组织肿瘤、口咽肿瘤、女性生殖系统癌症或脑膜瘤。
  110. 一种测定抗PLAU和/或PLAUR治疗剂治疗免疫疾病有效性的方法，其特征在于，所述方法包括：
    2)向权利要求1-25和52-74任一所述非人动物施用抗PLAU和/或PLAUR治疗剂，其中所述非人动物患有免疫疾病；
    3)测定抗PLAU和/或PLAUR治疗剂对治疗免疫疾病中的作用。
  111. 根据权利要求110所述的方法，其特征在于，所述免疫疾病为皮肤溃疡病、类风湿 性关节炎、中风。
  112. 一种测定抗PLAU和/或PLAUR治疗剂治疗炎症有效性的方法，其特征在于，所述方法包括：
    1)向权利要求1-25和52-74任一所述的动物施用抗PLAU和/或PLAUR治疗剂，其中所述动物具有炎症；
    2)测定抗PLAU和/或PLAUR治疗剂对治疗炎症的有效性。
  113. 根据权利要求112所述的方法，其特征在于，所述炎症为脓毒症或炎性疾病。
  114. 一种测定抗PLAU和/或PLAUR治疗剂毒性的方法，其特征在于，所述方法包括：
    1)向权利要求1-25和52-74任一所述的动物施用抗PLAU和/或PLAUR治疗剂；
    2)测定抗PLAU和/或PLAUR治疗剂对动物的作用。
  115. 根据权利要求114所述的方法，其特征在于，所述测定抗PLAU和/或PLAUR治疗剂对动物的作用涉及测量动物的体重、红细胞计数、血细胞比容和/或血红蛋白。
  116. 一种人源化PLAUR蛋白，其特征在于，所述人源化蛋白包含人PLAUR蛋白的3个同源结构域。
  117. 根据权利要求116所述的人源化PLAUR蛋白，其特征在于，所述人源化PLAUR蛋白的氨基酸序列与SEQ ID NO：10第24-335位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  118. 根据权利要求116或117所述的人源化PLAUR蛋白，其特征在于，所述人源化PLAUR蛋白的氨基酸序列与SEQ ID NO：15所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  119. 一种人源化PLAUR基因，其特征在于，所述人源化PLAUR基因编码权利要求116-118任一所述人源化蛋白。
  120. 根据权利要求119所述的人源化PLAUR基因，其特征在于，所述人源化PLAUR基因包含人PLAUR基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、和/或外显子7的部分。
  121. 根据权利要求119或120所述人源化PLAUR基因，其特征在于，所述人源化PLAUR基因包含的核苷酸序列与SEQ ID NO：14所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  122. 一种人源化PLAU蛋白，其特征在于，所述人源化PLAU蛋白包含人PALU蛋白的全部或部分。
  123. 根据权利要求122所述人源化PLAU蛋白，其特征在于，所述人源化PLAU蛋白的 氨基酸序列与SEQ ID NO：2所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  124. 一种人源化PLAU基因，其特征在于，所述人源化PLAU基因编码权利要求123-124任一所述人源化蛋白。
  125. 根据权利要求124所述的人源化PLAU基因，其特征在于，所述人源化PLAU基因包含人PLAU基因外显子2的部分、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8、外显子9、外显子10、和/或外显子11的部分。
  126. 根据权利要求125或126所述的人源化PLAU基因，其特征在于，所述人源化PLAU基因包含的核苷酸序列与SEQ ID NO：8所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％、或100％。
  127. 一种细胞，其特征在于，所述细胞中包含权利要求119-121任一所述人源化PLAUR基因和/或权利要求124-126任一所述人源化PLAU基因，和/或，所述的细胞表达权利要求116-118任一所述人源化PLAUR蛋白和/或权利要求122-123任一所述人源化PLAU蛋白。
  128. 一种动物模型，其特征在于，所述的动物模型包含包含权利要求119-121任一所述人源化PLAUR基因和/或权利要求124-126任一所述人源化PLAU基因，和/或，所述动物模型表达权利要求116-118任一所述人源化PLAUR蛋白和/或权利要求122-123任一所述人源化PLAU蛋白。
PCT/CN2023/098641 2022-06-06 2023-06-06 一种plau和/或plaur基因修饰的非人动物 WO2023236958A1 (zh)

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