Non-human animal modified by MMP7 gene

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) MMP7 protein and methods of use thereof.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) MMP7 protein and methods of using the same. Background Art

[0002] Traditional drug research and development usually uses in vitro screening methods. However, these screening methods cannot provide the in vivo environment (such as the tumor microenvironment, stromal cells, extracellular matrix components, and immune cell interactions), resulting in a relatively high failure rate of drug development. In addition, due to the differences between humans and animals, the test results obtained from in vivo pharmacological tests using conventional laboratory animals may not reflect the true disease state and the interactions at the target site, resulting in significant differences between the results of many clinical trials and animal experiments.

[0003] Therefore, the development of humanized animal models suitable for human drug screening and evaluation will significantly improve the efficiency of new drug development and reduce the cost of drug research and development. Summary of the Invention

[0004] The present application provides an animal model having a human or chimeric MMP7 protein. The animal model can express a human or chimeric MMP7 (e.g., humanized MMP7) protein. It can be used for the study of the function of the MMP7 gene and also for the screening and evaluation of MMP7 signaling pathway regulators (e.g., therapeutic agents targeting human MMP7, such as antibodies, nucleic acid drugs, and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamic studies, and the treatment of immune diseases, cancers, or inflammations and the treatment of diseases at the human MMP7 target site. The animal model can also be used to facilitate new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of the MMP7 protein and a platform for screening related drugs.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. The genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding a human or chimeric matrix metalloproteinase 7 (MMP7) protein. In some embodiments, the chimeric MMP7 protein is a humanized MMP7 protein. In some embodiments, the nucleotide sequence encoding the human or chimeric MMP7 protein can be a cDNA, CDS or genomic DNA sequence. In some embodiments, the amino acid sequence of the chimeric MMP7 protein comprises an amino acid sequence that is identical to at least 50 to 267 consecutive amino acids of the human MMP7 protein, such as at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 170, 171, 172, 173, 174, 175, 200, 245, 250, 255, 260 or 267 consecutive amino acids, etc. In some embodiments, the nucleotide sequence encoding the human or chimeric MMP7 protein is operably linked to an endogenous regulatory element (such as a promoter and / or UTR, preferably the endogenous 5'UTR) of the endogenous MMP7 locus of at least one chromosome. In some embodiments, the amino acid sequence of the human or chimeric MMP7 protein comprises SEQ ID NO: 2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to human MMP7 (NP_002414.1, SEQ ID NO: 2). In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous MMP7 protein of the non-human animal is not expressed or the expression level is reduced compared to MMP7 in a wild-type animal. In some embodiments, one or more cells of the non-human animal express the human or chimeric MMP7 protein.

[0006] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises a nucleotide sequence of human MMP7 replacing the nucleotide sequence of the corresponding region of endogenous MMP7 at the endogenous MMP7 locus. In some embodiments, the nucleotide sequence of human MMP7 may be a cDNA, CDS or genomic DNA sequence. In some embodiments, the nucleotide sequence encoding a human or chimeric MMP7 protein or the nucleotide sequence of human MMP7 is operably linked to an endogenous regulatory element of the endogenous MMP7 locus. In some embodiments, the endogenous MMP7 protein of the non-human animal is not expressed or has a reduced expression level compared to MMP7 in a wild-type animal. In some embodiments, the modified MMP7 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous locus that has been replaced. In some embodiments, the nucleotide sequence of human MMP7 encodes a human or chimeric MMP7 protein. In some embodiments, the nucleotide sequence of human MMP7 comprises the nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the nucleotide sequence of human MMP7 comprises at least 5 bp to 10,798 bp of contiguous nucleotides of the human MMP7 gene, such as at least 5, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1070, 1071, 1072, 1073, 1080, 1090, 1100, 1118, 1119, 1120, 1500, 2000, 2500, 3000, 4000, 5000, 10000, 10700, 10750, 10751, 10752, 10790 or 10,798 bp of contiguous nucleotides. In some embodiments, the nucleotide sequence of human MMP7 comprises a portion of exon 1 and all of exons 2-6 of the human MMP7 gene, preferably further comprising intron 1. In some embodiments, the nucleotide sequence of human MMP7 comprises a portion of exon 1 to all of exon 6 of the human MMP7 gene. In some embodiments, the nucleotide sequence of human MMP7 further comprises at least 50 bp of contiguous nucleotides downstream of the 3'UTR, such as at least 50, 100, 200, 300, 400, 500, 550, 557, 558, 559, 560 or 1000 bp of contiguous nucleotides. In some embodiments, the nucleotide sequence of human MMP7 further comprises at least 300 bp to 1000 bp of contiguous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human MMP7 comprises a portion of exon 1 of the human MMP7 gene to at least 50 bp of contiguous nucleotides downstream of the 3'UTR.In some embodiments, the portion of exon 1 of the human MMP7 gene comprises a continuous nucleotide sequence of at least 5 bp to 155 bp of exon 1, such as a continuous nucleotide sequence of at least 5, 10, 15, 50, 100, 105, 107, 108, 109, 110, 120, 150 or 155 bp. In some embodiments, the portion of exon 1 of the human MMP7 gene comprises the nucleotide sequence of the coding region. In some embodiments, the portion of exon 1 of the human MMP7 gene comprises the start codon to the last nucleotide of exon 1. In some embodiments, the nucleotide sequence of human MMP7 comprises a continuous nucleotide of at least 50 bp downstream of the 3' UTR from the start codon of the human MMP7 gene. In some embodiments, the nucleotide sequence of human MMP7 comprises a continuous nucleotide of at least 558 bp downstream of the 3' UTR from the start codon of the human MMP7 gene. In some embodiments, the nucleotide sequence of human MMP7 comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the corresponding region of endogenous MMP7 comprises a nucleotide sequence encoding a non-human animal MMP7 protein. In some embodiments, the corresponding region of endogenous MMP7 comprises the nucleotide sequence encoding SEQ ID NO: 1. In some embodiments, the corresponding region of endogenous MMP7 comprises a portion of exon 1 and all of exons 2-6 of the non-human animal MMP7 gene, preferably intron 1. In some embodiments, the corresponding region of endogenous MMP7 comprises a portion of exon 1 to all of exon 6 of the non-human animal MMP7 gene. In some embodiments, the corresponding region of endogenous MMP7 further comprises a continuous nucleotide of at least 50 bp downstream of the 3' UTR, such as a continuous nucleotide of at least 50, 100, 200, 300, 350, 390, 395, 400, 500 or 1000 bp. In some embodiments, the corresponding region of endogenous MMP7 further comprises a continuous nucleotide of at least 50 bp to 500 bp downstream of the 3' UTR. In some embodiments, the corresponding region of endogenous MMP7 comprises a portion of exon 1 of the non-human animal MMP7 gene to a continuous nucleotide of at least 50 bp downstream of the 3' UTR. In some embodiments, the corresponding region of endogenous MMP7 comprises a portion of exon 1 of the non-human animal MMP7 gene to a continuous nucleotide of at least 395 bp downstream of the 3' UTR. In some embodiments, the portion of exon 1 of the non-human animal MMP7 gene comprises a continuous nucleotide sequence of at least 5 bp to 149 bp of exon 1, such as a continuous nucleotide sequence of at least 5, 10, 15, 50, 100, 105, 107, 108, 109, 110, 120, 140 or 149 bp.In some embodiments, the portion of exon 1 of the non-human animal MMP7 gene comprises the nucleotide sequence of the coding region. In some embodiments, the portion of exon 1 of the non-human animal MMP7 gene comprises the start codon to the last nucleotide of exon 1. In some embodiments, the corresponding region of the endogenous MMP7 comprises at least 50 bp of contiguous nucleotides downstream of the 3' UTR starting from the start codon of the non-human animal MMP7 gene. In some embodiments, the corresponding region of the endogenous MMP7 comprises at least 395 bp of contiguous nucleotides downstream of the 3' UTR starting from the start codon of the non-human animal MMP7 gene. In some embodiments, the non-human animal is a mammal, such as a monkey, a rodent; preferably, the non-human animal is a mouse or a rat. In some embodiments, the mRNA transcribed from the modified gene in the genome of the non-human animal comprises SEQ ID NO: 6, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the non-human animal further comprises the nucleotide sequence of a human or chimeric protein encoded by another gene, and the human or chimeric protein includes, but is not limited to, at least one of ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0007] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal contains a nucleotide sequence encoding an endogenous MMP7 region at the endogenous MMP7 locus replaced by a nucleotide sequence encoding the corresponding region of human MMP7. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 can be a cDNA, CDS or genomic DNA sequence. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 is operably linked to an endogenous regulatory element of the endogenous MMP7 locus, and one or more cells of the animal express human or humanized MMP7 protein. In some embodiments, the endogenous MMP7 protein of the non-human animal is not expressed or has a reduced expression level compared to MMP7 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 contains a part of exon 1 and all of exons 2-6 of the human MMP7 gene, preferably intron 1. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 contains a part of exon 1 to all of exon 6 of the human MMP7 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 further contains at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human MMP7 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 contains a part of exon 1 to at least 50 bp of consecutive nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 contains the start codon to at least 50 bp of consecutive nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 contains the start codon to at least 558 bp of consecutive nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 contains a part of human exon 1 to at least 558 bp of consecutive nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 contains SEQ ID NO: 5, or contains a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region contains a part of exon 1 and all of exons 2-6 of the MMP7 gene of a non-human animal (such as a mouse), preferably intron 1. In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region contains a part of exon 1 to all of exon 6 of the MMP7 gene of a non-human animal (such as a mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region further contains at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the MMP7 gene of a non-human animal (such as a mouse).In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises at least 50 consecutive nucleotides downstream of the 3' UTR from the start codon of the MMP7 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises at least 50 consecutive nucleotides downstream of the 3' UTR from a portion of exon 1 of the MMP7 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises 395 consecutive nucleotides downstream of the 3' UTR from a portion of exon 1 of the MMP7 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises 395 consecutive nucleotides downstream of the 3' UTR from the start codon of the MMP7 gene of a non-human animal (e.g., mouse). In some embodiments, the modified MMP7 gene in the non-human animal genome is homozygous or heterozygous for the endogenous locus being replaced.

[0008] In some embodiments, the construction method includes replacing the nucleotide sequence encoding the endogenous MMP7 protein in a non-human animal with a nucleotide sequence encoding a human or chimeric MMP7 protein. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 1 in a non-human animal with the nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the construction method includes replacing the nucleotide sequence of the endogenous MMP7 gene in a non-human animal with the nucleotide sequence of human MMP7. In some embodiments, the construction method includes replacing the nucleotide sequence from a part of exon 1 to the whole of exon 6 of the human MMP7 gene with the nucleotide sequence from a part of exon 1 to the whole of exon 6 of the endogenous MMP7 gene in a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to the whole of exon 6 of the human MMP7 gene with the nucleotide sequence from the start codon to the whole of exon 6 of the endogenous MMP7 gene in a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence from a part of exon 1 to at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human MMP7 gene with the nucleotide sequence from a part of exon 1 to at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the endogenous MMP7 gene in a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human MMP7 gene with the nucleotide sequence from the start codon to at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the endogenous MMP7 gene in a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to at least 558 bp of consecutive nucleotides downstream of the 3'UTR of the human MMP7 gene with the nucleotide sequence from the start codon to at least 395 bp of consecutive nucleotides downstream of the 3'UTR of the endogenous MMP7 gene in a non-human animal. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 1 in a non-human animal with SEQ ID NO: 5 or 6. In some embodiments, the construction method includes replacing the nucleotide sequence from a part of exon 1 to the whole of exon 6 of the endogenous sequence in a non-human animal with SEQ ID NO: 5 or 6. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to the whole of exon 6 of the endogenous sequence in a non-human animal with SEQ ID NO: 5 or 6. In some embodiments, the construction method includes replacing the nucleotide sequence from a part of exon 1 to at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the endogenous sequence in a non-human animal with SEQ ID NO: 5 or 6. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the endogenous sequence in a non-human animal with SEQ ID NO: 5 or 6.

[0009] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or humanized MMP7 protein, wherein the humanized MMP7 protein comprises at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 170, 171, 172, 173, 174, 175, 200, 245, 250, 255, 260 or 267 consecutive amino acid sequences identical to the corresponding region of human, and the non-human animal expresses the human or humanized MMP7 protein. In some embodiments, the nucleotide sequence encoding the human or humanized MMP7 protein is operably linked to an endogenous MMP7 regulatory element. In some embodiments, the nucleotide sequence encoding the human or humanized MMP7 protein is integrated into the endogenous MMP7 locus of the non-human animal. In some embodiments, the human or humanized MMP7 protein has at least one of murine MMP7 activity and / or human MMP7 activity.

[0010] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same. In at least one cell of the non-human animal, at the endogenous MMP7 locus of the non-human animal, the nucleotide sequence encoding the endogenous MMP7 region is replaced by the nucleotide sequence encoding the corresponding region of human MMP7. In some embodiments, the endogenous MMP7 protein of the non-human animal is not expressed or has a reduced expression level compared to MMP7 in a wild-type animal. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises a portion of exon 1 and all of exons 2-6 of the human MMP7 gene, preferably further comprising intron 1. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises a portion of exon 1 to all of exon 6 of the human MMP7 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 further comprises at least 50 bp of continuous nucleotides downstream of the 3' UTR of the human MMP7 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises a portion of exon 1 to at least 50 bp of continuous nucleotides downstream of the 3' UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises the start codon to at least 50 bp of continuous nucleotides downstream of the 3' UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises a portion of exon 1 to at least 558 bp of continuous nucleotides downstream of the 3' UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises the start codon of the human MMP7 gene to at least 558 bp of continuous nucleotides downstream of the 3' UTR. In some embodiments, the amino acid sequence of the corresponding region of human MMP7 comprises SEQ ID NO: 2, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises a portion of exon 1 to all of exon 6 of the MMP7 gene of the non-human animal (such as a mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region further comprises at least 50 bp of continuous nucleotides downstream of the 3' UTR. In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises the start codon of the MMP7 gene of the non-human animal (such as a mouse) to at least 50 bp of continuous nucleotides downstream of the 3' UTR.In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises at least 50 bp of contiguous nucleotides downstream of the 3' UTR from a portion of exon 1 of the MMP7 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises 395 bp of contiguous nucleotides downstream of the 3' UTR from a portion of exon 1 of the MMP7 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises 395 bp of contiguous nucleotides downstream of the 3' UTR from the start codon of the MMP7 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 is operably linked to endogenous MMP7 regulatory elements, such as, a promoter and / or UTR, preferably the 5' UTR.

[0011] In one aspect, the present invention provides a method for constructing a non-human animal cell expressing a human or chimeric MMP7 gene, the construction method comprising replacing, at the endogenous MMP7 locus of a non-human animal (such as a mouse), the nucleotide sequence encoding the endogenous MMP7 region with the nucleotide sequence encoding the corresponding region of human MMP7 to generate a genetically modified non-human animal cell, and the non-human animal cell expressing a human or chimeric MMP7 protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises part of exon 1 to all of exon 6 of the human MMP7 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 further comprises at least 50 bp of contiguous nucleotides downstream of the 3' UTR of the human MMP7 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises part of exon 1 to at least 50 bp of contiguous nucleotides downstream of the 3' UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises the start codon to at least 50 bp of contiguous nucleotides downstream of the 3' UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises part of human exon 1 to at least 558 bp of contiguous nucleotides downstream of the 3' UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises the start codon of the human MMP7 gene to at least 558 bp of contiguous nucleotides downstream of the 3' UTR. In some embodiments, the amino acid sequence of the corresponding region of human MMP7 comprises SEQ ID NO: 2, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the corresponding region of human MMP7 comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises part of exon 1 to all of exon 6 of the MMP7 gene of the non-human animal (such as a mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region further comprises at least 50 bp of contiguous nucleotides downstream of the 3' UTR of the MMP7 gene of the non-human animal (such as a mouse). In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises part of exon 1 of the MMP7 gene of the non-human animal (such as a mouse) to at least 50 bp of contiguous nucleotides downstream of the 3' UTR. In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises the start codon of the MMP7 gene of the non-human animal (such as a mouse) to at least 50 bp of contiguous nucleotides downstream of the 3' UTR.In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises a continuous nucleotide sequence of a portion of exon 1 of the MMP7 gene of a non-human animal (e.g., mouse) to 395 bp downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region comprises a continuous nucleotide sequence from the start codon of the MMP7 gene of a non-human animal (e.g., mouse) to 395 bp downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human or chimeric MMP7 protein is operably linked to regulatory elements of endogenous MMP7, such as, a promoter and / or UTR, preferably the 5'UTR. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding a human or chimeric protein of other genes, and the human or chimeric protein includes, but is not limited to, at least one of ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0012] In one aspect, the present invention provides an application of a non-human animal obtained by the construction method described above, and the application comprises: A) an application in the development of products related to MMP7-related immune processes involving human cells; B) an application as a model system related to MMP7 in pharmacological, immunological, microbiological and medical research; C) an application involving the production and utilization of animal experimental disease models for MMP7-related etiological research and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) an application in the in vivo screening, efficacy detection, evaluation of efficacy, verification or evaluation of human MMP7 signaling pathway regulators; or, E) an application in studying the function of the MMP7 gene, studying the drugs and pharmacodynamics targeting human MMP7 target sites, and studying drugs for cancers, immune diseases or inflammation related to MMP7.

[0013] In one aspect, the present invention provides a method for determining the efficacy or toxicity of a therapeutic agent in treating a disease, the method comprising: 1) administering the therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the disease or the animal. In some embodiments, the therapeutic agent targets MMP7. In some embodiments, the therapeutic agent is an antibody, nucleic acid drug or polypeptide drug targeting MMP7. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent, such as an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody. In some embodiments, the disease includes cancer, immune disease or inflammation. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the solid tumor includes, but is not limited to, digestive tract cancer (such as colorectal cancer), endocrine cancer, pancreatic cancer, head and neck cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma or kidney cancer. In some embodiments, the hematological tumor includes, but is not limited to, lymphocyte tumors, such as B or T cell tumors. In some embodiments, the immune disease includes, but is not limited to, GVHD (graft-versus-host disease), psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty liver disease), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, idiopathic pulmonary fibrosis, multiple sclerosis or neurological disorders, etc. In some embodiments, the immune disease is idiopathic pulmonary fibrosis, asthma, rheumatoid arthritis, psoriasis or multiple sclerosis. In some embodiments, the inflammation includes acute inflammation and also includes chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.). In some embodiments, the inflammation is specific inflammation, arthritis or inflammatory bowel disease (IBD).

[0014] In one aspect, the present invention provides a method for determining the efficacy of a therapeutic agent in treating cancer, the method comprising:

[0015] 1) administering the therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has a tumor; 2) determining the inhibitory effect of the therapeutic agent on the tumor. In some embodiments, the tumor comprises one or more tumor cells injected into the animal. In some embodiments, determining the inhibitory effect of the therapeutic agent on the tumor involves measuring the tumor volume in the animal.

[0016] In one aspect, the present invention provides a method for determining the effectiveness of an anti-MMP7 antibody and an additional therapeutic agent in treating cancer, the method comprising: 1) administering an anti-MMP7 antibody and an additional therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, the non-human animal having a tumor; 2) determining the inhibitory effect on the tumor. In some embodiments, the non-human animal further comprises a sequence encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1. In some embodiments, the tumor comprises one or more tumor cells injected into the animal. In some embodiments, the determining the inhibitory effect on the tumor involves measuring the tumor volume in the animal.

[0017] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating an immune disease, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has an immune disease; 2) determining the therapeutic effect of the therapeutic agent on the immune disease.

[0018] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation, the method comprising:

[0019] 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has inflammation; 2) determining the effectiveness of the therapeutic agent in treating inflammation.

[0020] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the determining the effect of the therapeutic agent on the non-human animal involves measuring the change in body weight of the animal and / or a blood test. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content.

[0021] In one aspect, the present invention provides a humanized MMP7 gene, which comprises any one of the following nucleotide sequences: A) a nucleotide sequence encoding the protein of SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 18 or 19; C) a nucleotide sequence having at least 90% identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 18 or 19; D) a nucleotide sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 18 or 19.

[0022] In one aspect, the present invention provides a cell, tissue or organ, which comprises the humanized MMP7 gene.

[0023] In one aspect, the present invention provides a non-human animal genome, the non-human animal genome comprising at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric MMP7 protein. In some embodiments, the chromosome comprises a human or chimeric MMP7 gene. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human MMP7 protein. In some embodiments, the chromosome comprises a portion of exon 1 to all of exon 6 of the human MMP7 gene. In some embodiments, the chromosome further comprises at least 50 consecutive nucleotides downstream of the 3'UTR of the human MMP7 gene. In some embodiments, the chromosome comprises a portion of exon 1 of the human MMP7 gene to at least 50 consecutive nucleotides downstream of the 3'UTR. In some embodiments, the chromosome comprises the start codon of the human MMP7 gene to at least 50 consecutive nucleotides downstream of the 3'UTR. In some embodiments, the MMP7 in the endogenous genome of the non-human animal is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1 in the endogenous genome of the non-human animal is replaced. In some embodiments, a portion of exon 1 to all of exon 6 of the endogenous MMP7 gene of the non-human animal is replaced. In some embodiments, the replaced endogenous MMP7 gene of the non-human animal further comprises at least 50 consecutive nucleotides downstream of the 3'UTR. In some embodiments, a portion of exon 1 of the endogenous MMP7 gene of the non-human animal to at least 50 consecutive nucleotides downstream of the 3'UTR is replaced. In some embodiments, the start codon of the endogenous MMP7 gene of the non-human animal to at least 50 consecutive nucleotides downstream of the 3'UTR is replaced. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human or chimeric MMP7 protein or the nucleotide sequence of human MMP7 to replace the corresponding portion of the endogenous chromosome of the non-human animal. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 2 is included to replace the nucleotide sequence encoding SEQ ID NO: 1 in the endogenous non-human animal.

[0024] In one aspect, the present invention provides a cell, tissue or organ comprising the above non-human animal genome.

[0025] In one aspect, the present invention provides an animal model, the animal model comprising the humanized MMP7 gene.

[0026] The term "all or part" in the present invention, "all" refers to the whole, "part" refers to the local part in the whole, or the individual parts constituting the whole.

[0027] The term "locus" in the present invention generally represents the position occupied by a gene on a chromosome, and specifically represents a DNA fragment on a certain gene, which can be a gene, a part of a gene, or a gene regulatory region, etc. For example, the described "MMP7 locus" includes any DNA fragment of exons 1-6 of the MMP7 gene and its regulatory region.

[0028] The term "part of exon XX" in the present invention means that the continuous or spaced nucleotide sequence of several, dozens or hundreds of nucleotides is identical to the entire exon nucleotide sequence.

[0029] The term "exon XX to exon XXX" or "exon XX-XXX" or "the whole of exon XX to the whole of exon XXX" or "the whole of exon XX to the whole of exon XXX" in the present invention refers to the exon and the intron between them.

[0030] The term "part of exon x to part of exon xx" or "part of exon x - the whole of exon xx" in the present invention includes the whole or part of the exon and the intron between them. For example, "part of exon 1 to the whole of exon 6" includes part of exon 1, the whole of intron 1, the whole of exon 2, the whole of intron 2, the whole of exon 3, the whole of intron 3, the whole of exon 4, the whole of intron 4, the whole of exon 5, the whole of intron 5 and the whole of exon 6.

[0031] The term "intron xx" in the present application refers to the intron between two exons. For example, intron 3 is the intron between exon 3 and exon 4.

[0032] The term "comprising" or "including" in the present invention is an open-ended writing method, including the specified components or steps described, as well as other specified components or steps that will not be substantially affected. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still has the same or similar activity as the original sequence.

[0033] The term "and / or" in the present invention includes all combinations of the items connected by this term, and should be regarded as each combination having been separately listed in the present application. For example, "A and / or B" includes "A", "A and B" and "B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0034] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This application describes the methods and materials for this invention; other suitable methods and materials known in the art can be used. The materials, methods, and examples are illustrative only and not restrictive. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail.

[0035] Those skilled in the art can easily perceive other conveniences and advantages of this application from the following detailed description.

[0036] MMP7

[0037] In the human genome, the MMP7 gene (NCBI Gene ID: 4316, UniProt ID: P09237, located at positions 102520508 to 102530747 of chromosome 11 NC_000011.10) contains 6 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_002423.5 and its encoded protein NP_002414.1 (SEQ ID NO: 2) are shown in Table 1.

[0038] Table 1

[0039]

[0040] In the mouse genome, the MMP7 gene (NCBI Gene ID: 17393, UniProt ID: Q10738, located at positions 7692095 to 7699587 of chromosome 9 NC_000075.7) contains 6 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_010810.6 and its encoded protein NP_034940.3 (SEQ ID NO: 1) are shown in Table 2.

[0041] Table 2

[0042]

[0043] The MMP7 genes, proteins, and gene loci of other species in the art are also known. For example, Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), and Sus scrofa (pig). The relevant information of these genes (such as intron sequences, exon sequences, and amino acid sequences) can all be found in NCBI, and the entire content is incorporated herein by reference.

[0044] To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). Then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced to achieve the optimal alignment of the two sequences. For example, the comparison of sequences and the determination of the percent identity between two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0045] The percentage of conserved residues with similar physicochemical properties (percent homology), such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percent homology is higher than the percent identity.

[0046] vector

[0047] The present invention provides a targeting vector, comprising: a) a DNA fragment (5' arm or 5' homologous arm) homologous to the 5' end of the region to be modified, which is selected from the genomic DNA of the MMP7 gene and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment (3' arm or 3' homologous arm) homologous to the 3' end of the region to be modified, which is selected from the genomic DNA of the MMP7 gene and has a length of 100 to 10,000 nucleotides.

[0048] In some embodiments, a) the DNA fragment homologous to the 5' end of the region to be modified is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000075.7; c) the DNA fragment homologous to the 3' end of the region to be modified is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000075.7.

[0049] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 0.8 kb, 1 kb,

[0050] 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 18 kb, 19 kb or 20 kb.

[0051] In some embodiments, the region to be modified is located on the MMP7 gene of a non-human animal. In some embodiments, the region to be modified is located on exons 1 to 6 of the MMP7 gene of a non-human animal. In some embodiments, the region to be modified is located on exons 1 to 3'UTR of the MMP7 gene of a non-human animal and at least 50 bp of continuous nucleotides downstream.

[0052] In some embodiments, the 5' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the 3' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 4.

[0053] In some embodiments, the 5' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 18. In some embodiments, the 3' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 19.

[0054] In some embodiments, the targeting vector comprises a human sequence (e.g., positions 102519950 to 102530700 of NC_000011.10). For example, preferably, it is from a part of exon 1 to the whole of exon 6 of the human MMP7 gene. Preferably, it is from the start codon of the human MMP7 gene to at least 50 bp of consecutive nucleotides downstream of the 3'UTR. In some embodiments, the donor region in the targeting vector comprises SEQ ID NO: 5.

[0055] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a positive selection marker gene or a negative selection marker gene. In some embodiments, the resistance gene for positive clone selection encodes the neomycin phosphotransferase sequence Neo or the hygromycin resistance gene sequence HygR. Preferably, the targeting vector further comprises two directly repeated Frt recombination sites flanking the marker gene. In some embodiments, the coding gene for the negative selection marker is the coding gene for the diphtheria toxin A subunit (DTA).

[0056] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the MMP7 gene, and the sgRNA is unique on the target sequence of the gene to be altered and satisfies the sequence arrangement rules of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'. In some embodiments, the targeting site of the sgRNA in the MMP7 gene of a non-human animal (e.g., a mouse) is located in exons 1 to 6. In some embodiments, the targeting site of the sgRNA in the MMP7 gene of a non-human animal (e.g., a mouse) is located in exons 1 to at least 50 bp of consecutive nucleotides downstream of the 3'UTR.

[0057] In some embodiments, the targeting sequences are shown as SEQ ID NO: 21 and / or 22. Thus, the present invention provides an sgRNA sequence for constructing a gene-modified animal model. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 23 and 25. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 24 and 26. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 27 and 16.

[0058] In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 15 and 17.

[0059] In some embodiments, the present application relates to a plasmid construct (e.g., pT7-sgRNA) comprising the sgRNA sequence and / or a cell comprising the construct.

[0060] The present invention also relates to cells comprising the targeting vector or sgRNA vector as described above.

[0061] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned targeting vectors and one or more in vitro transcripts of the construct described in the present application. In some embodiments, the cell comprises Cas9 mRNA or its in vitro transcript.

[0062] In some embodiments, the gene in the cell is heterozygous. In some embodiments, the gene in the cell is homozygous.

[0063] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

[0064] Genetically modified non-human animal

[0065] The "genetically modified non-human animal" as used in the present invention refers to a non-human animal in which at least one chromosome in the genome of the non-human animal has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells having exogenous DNA can be various cells, such as somatic cells, immune cells (such as T cells, B cells, NK cells, antigen-presenting cells, macrophages, dendritic cells or germ cells), blastocysts or tumor cells. In some embodiments, there is provided a genetically modified non-human animal, the non-human animal comprising a modified endogenous MMP7 locus comprising an exogenous sequence (such as a human sequence), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. The non-human animal is generally capable of transmitting the genetic modification to its offspring through the germline.

[0066] The "chimeric (x) gene" or "chimeric (x) nucleic acid" as 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 different from the nucleic acid in the wild-type animal. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid has at least a part of the sequence having two or more different species sources, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid refers to a humanized (x) gene or humanized (x) nucleic acid.

[0067] As used herein, the term "chimeric (x) protein" or "chimeric (x) polypeptide" refers to a protein or polypeptide in which two or more portions of the polypeptide or protein are from different species, or at least one sequence of the protein or polypeptide is different from the amino acid sequence in the wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (x) protein or chimeric (x) polypeptide has two or more different species origins, e.g., the same (or homologous) proteins from different species. In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide refers to a humanized (x) protein or humanized (x) polypeptide.

[0068] As used herein, the term "humanized (x) protein" or "humanized (x) polypeptide" refers to a protein or polypeptide in which at least a portion of the protein or polypeptide is from a human protein or human polypeptide. In some embodiments, the humanized (x) protein or humanized (x) polypeptide refers to a human protein or polypeptide.

[0069] As used herein, the term "humanized (x) nucleic acid" refers to a nucleic acid in which at least a portion of the nucleic acid is from a human. In some embodiments, all of the nucleic acids in the humanized (x) nucleic acid are from a human. In some embodiments, the humanized (x) nucleic acid refers to a humanized exon, which may be a human exon or a chimeric exon.

[0070] In some embodiments, the chimeric MMP7 gene or chimeric MMP7 nucleic acid is a humanized MMP7 gene or humanized MMP7 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is from the human MMP7 gene, and at least a portion of the gene or nucleic acid is from a non-human MMP7 gene. In some embodiments, the gene or nucleic acid contains a sequence encoding an MMP7 protein. The encoded MMP7 protein has at least one activity of a human MMP7 protein or a non-human animal MMP7 protein.

[0071] In some embodiments, the chimeric MMP7 protein or chimeric MMP7 polypeptide is a humanized MMP7 protein or humanized MMP7 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are from a human MMP7 protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are from a non-human animal MMP7 protein. The humanized MMP7 protein or humanized MMP7 polypeptide is functional or has at least one activity of a human MMP7 protein or a non-human animal MMP7 protein.

[0072] The genetically modified non-human animals can be various animals, such as, for example, mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, water buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals for which it is not easy to obtain suitable genetically modifiable embryonic stem cells (ES), other methods are used to construct non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transfer to transfer the modified genome to a suitable cell, such as an oocyte, and gestating the modified cell (e.g., modified oocyte) in a non-human animal under appropriate conditions to form an embryo. The above-described construction methods are known in the art and are described in “A. Nagy, et al.,

[0073] “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2006”, the entire content of which is incorporated herein by reference.

[0074] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent can be selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the Muridae family. In one embodiment, the genetically modified animal is selected from the family Cricetidae (e.g., mouse-like hamsters), Cricetinae (e.g., hamsters, New World rats and mice, voles), Muroidea (mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (mountain mice, rock mice, tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In a particular embodiment, the genetically modified rodent is selected from mice or rats (Muroidea), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0075] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, immunodeficient rodents, immunodeficient rabbits, immunodeficient pigs, immunodeficient monkeys, etc. In some embodiments, the non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse is of the 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant content of the above documents is incorporated herein by reference in its entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is of the BALB strain, such as the BALB / c strain. In some embodiments, the mouse is a hybrid of the BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50% BALB / c - 50% 12954 / Sv; or 50% C57BL / 6 - 50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a strain of BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, or CBA / H. In some embodiments, the non-human animal is a rat. The rat can be selected from Wistar rats, LEA strain, Sprague-Dawley strain, Fischer strain, F344, F6, and Dark Agouti.In some embodiments, the rat strain is a hybrid species of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0076] The genetically modified non-human animal comprises a modification of the endogenous non-human animal MMP7 gene locus. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature MMP7 protein (e.g., having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the mature MMP7 protein). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein are provided in the present invention, in many embodiments, the genetically modified non-human animal comprises a modification of the endogenous MMP7 gene locus in the non-human animal.

[0077] The genetically modified non-human animal may express human MMP7 and / or chimeric (e.g., humanized) MMP7 at the endogenous mouse locus, wherein the endogenous mouse MMP7 gene has been replaced or inserted with the gene of human MMP7 and / or a nucleotide sequence encoding a human MMP7 sequence region or a nucleotide sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the human MMP7 sequence. In some embodiments, the endogenous non-human animal MMP7 gene locus is modified with all or part of the human nucleic acid sequence encoding the mature MMP7 protein.

[0078] In some embodiments, the genetically modified mouse may express human MMP7 and / or chimeric MMP7 (e.g., humanized MMP7) under the control of a mouse promoter and / or mouse regulatory elements. Insertion or replacement at the endogenous mouse locus provides a non-human animal that expresses human MMP7 or chimeric MMP7 (e.g., humanized MMP7) in appropriate cells and in a manner that does not result in the potential pathologies observed in some other transgenic mice known in the art. The human MMP7 or chimeric MMP7 (e.g., humanized MMP7) expressed in the non-human animal may maintain one or more functions of wild-type mouse or human MMP7 in the non-human animal. In addition, in some embodiments, the non-human animal does not express endogenous MMP7. In some embodiments, the endogenous MMP7 expression level in the non-human animal is reduced compared to the MMP7 expression level in the wild-type animal. As used herein, the term "endogenous MMP7" refers to the MMP7 protein expressed from the endogenous MMP7 nucleotide sequence of a non-human animal (e.g., mouse) prior to any genetic modification.

[0079] In some embodiments, the humanized MMP7 locus contains the human 5' UTR. In some embodiments, the humanized MMP7 locus contains the endogenous 5' UTR of a non-human animal (e.g., mouse). In some embodiments, the humanized MMP7 locus contains the human 3' UTR. In some embodiments, the humanized MMP7 locus contains the endogenous 3' UTR of a non-human animal (e.g., mouse). Where appropriate, it can be reasonably assumed that, based on the similarity of the 5' flanking sequences of the mouse and human MMP7 genes, they appear to be regulated similarly. As shown in the present application, in humanized MMP7 mice in which the endogenous mouse MMP7 locus contains an insertion or replacement, which retain mouse regulatory elements but contain a humanized MMP7 coding sequence, do not exhibit pathological phenomena. Both heterozygous and homozygous gene-modified mice of humanized MMP7 are normal.

[0080] The present invention further relates to the genomic DNA sequence of MMP7 of a humanized mouse, the DNA sequence obtained by reverse transcription of mRNA is identical or complementary to this DNA sequence; a construct expressing its amino acid sequence; a cell containing its construct; a tissue including its cell.

[0081] The present invention further relates to a non-human mammal or its offspring produced by the above method. In some embodiments, its genome contains a human gene.

[0082] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0083] In some embodiments, the non-human mammal expresses the protein encoded by the humanized MMP7 gene.

[0084] In addition, the present invention also provides a non-human mammalian model carrying a tumor, and the non-human mammalian model is obtained by the method described in the present application. In some embodiments, the non-human mammal is a rodent (e.g., mouse).

[0085] The present invention also provides a cell or cell line, or a primary cell culture, derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, a tissue, organ or its culture derived from a non-human mammal or its offspring. When carrying a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring or a non-human mammal carrying a tumor.

[0086] The present invention provides a non-human mammal produced by any of the methods described in the present application. In some embodiments, a non-human mammal, a gene-modified non-human animal is provided, and the genome of the gene-modified non-human animal contains the DNA of human or humanized MMP7.

[0087] In some embodiments, the non-human mammal comprises the gene construct described in the present application. In some embodiments, a non-human mammal expressing a human or humanized MMP7 protein is provided. In some embodiments, a tissue specifically expressing a human or humanized MMP7 protein is provided.

[0088] In some embodiments, the expression of the human or humanized MMP7 protein in the non-human animal is controllable. Such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0089] The non-human mammal can be any non-human animal known in the art and can be used in the methods described in the present application. Preferred non-human mammals are mammals (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0090] Genetic, molecular, and behavioral analyses are performed on the non-human mammals described above. The present invention provides an offspring produced by mating a non-human mammal with the same genotype or other genotypes.

[0091] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its offspring. For example, a cell culture-based model can be prepared by the following method. The cell culture can be obtained by isolating from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. The integration of the genetic construct containing the DNA sequence encoding the human MMP7 protein can be detected by various methods.

[0092] There are many analytical methods available for detecting exogenous DNA, including methods at the nucleic acid level (including using reverse transcription-polymerase chain reaction (RT-PCR), Southern Blot, and in situ hybridization) and methods at the protein level (including histochemical analysis, immunoblot analysis, and in vitro binding studies). In addition, the expression level of the target gene can be quantified by ELISA methods well-known to those skilled in the art. Many standard analytical methods can be used to complete quantitative detection. For example, RT-PCR and hybridization methods can be used to detect the transcription level, including ribonuclease protection assay, Southern Blot, RNA dot blot analysis (RNA dot). Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of the human or humanized MMP7 protein.

[0093] In some embodiments, the genetically modified animal described in the present application (e.g., a homozygous humanized MMP7 gene mouse) can express human or humanized MMP7 in one or more liver tissue cells.

[0094] Method for constructing a genetically modified non-human animal

[0095] Genetically modified non-human animals can be prepared by several techniques known in the art, including using homologous recombination techniques, gene targeting techniques for embryonic stem cells, CRISPR / Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases, or other molecular biology techniques. In some embodiments, homologous recombination techniques are preferably used. In some embodiments, CRISPR / Cas9 gene editing techniques can be used to construct genetically modified non-human animals. In some embodiments, CRISPR-Cas9 gene editing techniques are used to generate genetically modified non-human animals. Many of these gene editing techniques are known in the art and are described in Yin et al., "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting gene-edited cells into enucleated oocytes and fusing the enucleated oocytes with another gene-edited cell.

[0096] In some embodiments, the nucleotide sequence encoding the endogenous MMP7 region in the endogenous genome of at least one cell of the non-human animal is replaced with the nucleotide sequence encoding the corresponding region of human MMP7. In some embodiments, the expression level of the endogenous MMP7 protein in the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts, or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0097] The present invention provides a targeting vector. The targeting vector comprises a vector composed of a 5' homologous arm, a human or humanized MMP7 gene fragment, and a 3' homologous arm. This process involves using homologous recombination to replace the endogenous corresponding MMP7 sequence with the human or humanized MMP7 sequence. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nuclease, TALEN, or CRISPR) can result in double-strand breaks in the DNA, and the human or humanized MMP7 sequence is replaced with the murine endogenous MMP7 sequence using homologous recombination.

[0098] Thus, in some embodiments, a method of producing a gene-modified humanized animal includes replacing a nucleic acid sequence encoding an endogenous MMP7 region with a nucleotide sequence encoding a corresponding region of human MMP7 at the endogenous MMP7 locus (or site).

[0099] The present invention also provides a method for establishing a humanized animal model of the MMP7 gene, comprising the following steps:

[0100] (a) Providing a cell (such as a fertilized egg cell) based on the method described in the present application;

[0101] (b) Culturing the cell, preferably culturing the cell in a liquid medium;

[0102] (c) Transplanting the cultured cell into the oviduct or uterus of a recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

[0103] (d) Identifying germline transmission in the offspring of the gene-modified humanized non-human mammal of the pregnant female in step (c).

[0104] In some embodiments, the non-human mammal in the above method is a mouse (such as a C57BL / 6 mouse).

[0105] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0106] In some embodiments, the fertilized egg used in the above method is a C57BL / 6 fertilized egg. Other fertilized eggs that can also be used in the method described in the present application include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0107] The fertilized egg can be from any non-human animal, such as any non-human animal described in the present application. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can introduce DNA into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0108] In some embodiments, a method of producing a genetically modified non-human animal comprises modifying the coding framework of the MMP7 gene of the non-human animal, e.g., by replacing the nucleic acid sequence encoding the endogenous MMP7 region (e.g., CDS, genomic DNA or cDNA sequence) with a nucleotide sequence encoding the corresponding region of human MMP7 under the control of the endogenous regulatory elements of the MMP7 gene of the non-human animal. For example, the sequence of one or more functional regions of the MMP7 gene of the non-human animal can be knocked out or inserted with a sequence such that the endogenous MMP7 protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the MMP7 gene of the modified non-human animal can be all or part of the nucleotide sequence of exons 1 to 6 of the MMP7 gene of the non-human animal.

[0109] In some embodiments, a method of producing a genetically modified non-human animal comprises inserting a nucleotide sequence encoding a human or humanized MMP7 protein and / or an auxiliary sequence after the endogenous regulatory elements of the MMP7 gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon such that the MMP7 gene humanized animal model can express a human or humanized MMP7 protein in vivo but not the MMP7 protein of the non-human animal. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0110] In some embodiments, the construction method comprises obtaining using the above vector. In some embodiments, the vector is a targeting vector and / or an sgRNA vector. In some embodiments, the construction method comprises introducing the targeting vector into the non-human animal or its cells. In some embodiments, the construction method comprises introducing the targeting vector and the sgRNA vector into the non-human animal or its cells.

[0111] In some embodiments, a method for producing a gene-edited non-human animal comprises:

[0112] (1) Providing a plasmid (such as the above vector) containing a human MMP7 gene fragment, the plasmid flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' and 3' homologous arms target endogenous MMP7;

[0113] (2) Providing one or more guide RNAs (sgRNAs) targeting the endogenous MMP7 gene;

[0114] (3) Modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2) and Cas9;

[0115] (4) Transfer the fertilized eggs obtained in step (3) into the oviducts of pseudopregnant (or pregnant) female mice, or transfer the embryonic stem cells obtained in step (3) into blastocysts, and then transfer the blastocysts into the oviducts of pseudopregnant female mice to generate offspring mice that functionally express humanized MMP7 protein;

[0116] (5) Mate the offspring mice obtained in step (4) to obtain homozygous mice.

[0117] In some embodiments, the fertilized eggs are modified by CRISPR with sgRNAs targeting the 5'-terminal targeting site and the 3'-terminal targeting site.

[0118] In some embodiments, the sequence encoding the humanized MMP7 protein is operably linked to endogenous regulatory elements at the endogenous MMP7 locus.

[0119] In some embodiments, the genetically modified non-human animal does not express endogenous MMP7 protein.

[0120] In some embodiments, the method for preparing a gene-edited non-human animal comprises:

[0121] (1) Provide a plasmid (such as the above vector) containing a human or chimeric MMP7 gene fragment, the plasmid flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' and 3' homologous arms target endogenous MMP7;

[0122] (2) Provide one or more guide RNAs (sgRNAs) targeting the endogenous MMP7 gene;

[0123] (3) Modify the genome of fertilized eggs or embryonic stem cells by inserting the human or chimeric MMP7 gene fragment into the genome.

[0124] In some embodiments, the nucleotide sequence encoding the endogenous MMP7 protein in the non-human animal is deleted. In some embodiments, a part of exon 1 to the whole of exon 6 of the endogenous MMP7 gene in the non-human animal is deleted. In some embodiments, a part of exon 1 to at least 50 bp of continuous nucleotides downstream of the 3' UTR of the endogenous MMP7 gene in the non-human animal is deleted. In some embodiments, the start codon of the endogenous MMP7 gene in the non-human animal to at least 50 bp of continuous nucleotides downstream of the 3' UTR is deleted.

[0125] Application of the gene-modified non-human animal

[0126] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence, or inserting a homologous or orthologous human gene or human sequence into a non-human animal at an endogenous non-human animal locus and under the control of an endogenous promoter and / or regulatory element(s), can produce non-human animals having qualities and characteristics that may be significantly different from those of typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or disrupted and a fully human transgene is inserted into the animal's genome and may integrate randomly into the genome. Generally, the location of the integrated transgene is unknown; human protein expression is measured by transcription of the human gene and / or protein assay and / or functional assay. In a human transgene, the upstream and / or downstream of the human sequence provides suitable support for the expression and / or regulation of the transgene.

[0127] Genetically modified animals expressing human or humanized MMP7 protein, for example, in a physiologically appropriate manner, have a variety of uses including, but not limited to, the development of therapeutic methods for human diseases and disorders and the evaluation of the toxicity and / or efficacy of such human therapeutic methods in animal models.

[0128] The present invention also provides an application of the above-mentioned MMP7 gene-modified non-human animal, the non-human animal obtained by any of the above construction methods.

[0129] In some embodiments, the application includes:

[0130] A) Application in the development of products related to MMP7-related immune processes involving human cells;

[0131] B) Application as an MMP7-related model system for pharmacological, immunological, microbiological, and medical research;

[0132] C) Application involving the production and utilization of animal experimental disease models for MMP7-related etiological research and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0133] D) Application in the screening, pharmacodynamic detection, efficacy evaluation, verification, or assessment of human MMP7 signaling pathway modulators in vivo; or,

[0134] E) Application in studying the function of the MMP7 gene, studying the drugs and pharmacodynamics targeting human MMP7 target sites, and studying drugs for cancers, inflammations, and immune diseases related to MMP7.

[0135] The present invention provides a non-human animal expressing a human or humanized MMP7 protein, which can be used for screening human MMP7-specific modulators. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In some embodiments, the genetically modified non-human animal further comprises a damaged immune system, such as a genetically modified human xenograft, including human solid tumors (e.g., breast cancer, gastrointestinal cancer (such as colorectal cancer), endocrine cancer, pancreatic cancer, head and neck cancer, liver cancer, lung cancer, ovarian cancer, endometrial cancer, melanoma or kidney cancer) or hematological tumors (e.g., lymphocyte tumors, preferably B or T cell tumors).

[0136] In some embodiments, the anti-MMP7 antibody blocks or inhibits the MMP7-mediated signaling pathway. In some embodiments, the anti-MMP7 antibody described in the present application can block the interaction between MMP7 complexes, thereby inhibiting the MMP7 signaling pathway.

[0137] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of therapeutic agents (such as therapeutic agents targeting human MMP7, such as antibodies, nucleic acid drugs and / or polypeptide drugs) in treating various immune diseases. In some embodiments, the immune diseases include but are not limited to GVHD (graft-versus-host disease), psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, idiopathic pulmonary fibrosis, multiple sclerosis or neurological disorders, etc. In some embodiments, the immune disease is idiopathic pulmonary fibrosis, asthma, rheumatoid arthritis, psoriasis or multiple sclerosis.

[0138] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of therapeutic agents (such as therapeutic agents targeting human MMP7, such as antibodies, nucleic acid drugs and / or polypeptide drugs) in treating various inflammations. In some embodiments, the inflammation is caused by infection. In some embodiments, the inflammation includes acute inflammation and chronic inflammation. Specifically, it includes but is not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.). In some embodiments, the inflammation is specific inflammation, arthritis or inflammatory bowel disease (IBD).

[0139] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., therapeutic agents targeting human MMP7, such as antibodies, nucleic acid drugs, and / or polypeptide drugs) for treating cancer. In some embodiments, a therapeutic agent (e.g., a therapeutic agent targeting human MMP7, such as an antibody, nucleic acid drug, and / or polypeptide drug) is administered to a non-human animal having cancer or a tumor, and the inhibitory effect of the therapeutic agent on the cancer or tumor is detected. In some embodiments, the detection includes measuring the size and / or proliferation rate of tumor cells. In some embodiments, the detection methods include vernier caliper measurement, flow cytometry, and / or in vivo imaging detection of the animal. In some embodiments, the detection includes evaluating an individual's body weight, fat mass, activation pathway, neuroprotective activity, or metabolic changes, and the metabolic changes include changes in food consumption or water consumption.

[0140] In some embodiments, the tumor cells include one or more cancer cells (e.g., cancer cells derived from a human or non-human animal) injected into the animal. In some embodiments, the therapeutic agent inhibits the MMP7 signaling pathway. In some embodiments, the therapeutic agent does not inhibit the MMP7 signaling pathway.

[0141] In some embodiments, genetically modified non-human animals can be used to detect whether an anti-MMP7 antibody is an agonist or antagonist. In some embodiments, the methods described in the present application can be used to detect the function of a therapeutic agent (e.g., a therapeutic agent targeting human MMP7, such as an antibody, nucleic acid drug, and / or polypeptide drug), for example, whether the therapeutic agent can upregulate or downregulate an immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cell cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a subject's disease (e.g., cancer, inflammation, or immune disease). The inhibitory effect on the tumor 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) growth inhibition rate (TGI TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%) = (1 - T Vt / T Vc ) x 100, where T Vt and T Vc are the average tumor volumes (or weights) of the treatment group and the control group.

[0142] In some embodiments, therapeutic agents (e.g., therapeutic agents targeting human MMP7, such as antibodies, nucleic acid drugs, and / or polypeptide drugs) can be used to treat various cancers. As used herein, "cancer" refers to cells with the ability to grow autonomously, i.e., an abnormal condition or disorder characterized by rapid cell growth and proliferation. This term is intended to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. As used herein, "tumor" or "cancer" 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, glioblastoma, 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 multiforme, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Among them, the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid 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 Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment of the present invention, 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 tumor, oropharyngeal tumor, female reproductive system cancer, or meningioma. In some embodiments, the tumor is a solid tumor or a hematological tumor. In some embodiments, the solid tumor includes, but is not limited to, digestive tract cancers (e.g., colorectal cancer), endocrine cancer, pancreatic cancer, head and neck cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, or kidney cancer. In some embodiments, the hematological tumor includes, but is not limited to, lymphocyte tumors, such as B- or T-cell tumors.

[0143] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (such as a therapeutic agent targeting human MMP7, such as an antibody, a nucleic acid drug, and / or a polypeptide drug). The detection method includes administering the therapeutic agent to the aforementioned non-human animal and evaluating the body weight change and / or blood examination of the non-human animal. In some embodiments, the blood examination includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content. In some embodiments, the therapeutic agent can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the body weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% smaller than that of the control group (such as the average body weight of animals not treated with the therapeutic agent).

[0144] The present invention also provides an animal model constructed by the method described in the present application for developing products related to the human cellular immune process, manufacturing human antibodies, or as a model system for pharmacological, immunological, microbiological, and medical research.

[0145] In some embodiments, provided is an animal model generated by the method described in the present application for producing and utilizing an animal experimental disease model of the immune process of human cells, studying pathogens, or formulating new diagnostic strategies and / or treatment strategies.

[0146] The present invention also provides an animal model generated by the method described in the present application for screening, validating, evaluating, or studying the function of the MMP7 gene, drugs (such as antibodies, nucleic acid drugs, and / or polypeptide drugs) targeting human MMP7 sites, or their effectiveness, drugs for immune diseases, and anti-tumor drugs.

[0147] In some embodiments, the present application provides a method for verifying the in vivo efficacy of TCR-T, CAR-T, and / or other immunotherapies (such as adoptive T cell transfer therapy). For example, the method includes transplanting human tumor cells into the non-human animal described in the present application and applying human CAR-T to the animal with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animal is selected from MMP7 gene humanized non-human animals prepared by the method described in the present application, double or multiple humanized non-human animals (or their offspring) generated by the method described in the present application, non-human animals expressing human or humanized MMP7 protein, or the tumor or inflammation animal model described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat the MMP7-related diseases described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies provide an evaluation method for treating the MMP7-related diseases described in the present application.

[0148] A non-human animal model of two or more human or chimeric genes

[0149] The present invention also provides a method for generating a gene-edited non-human animal model having two or more human or chimeric genes. The non-human animal may comprise a human or chimeric MMP7 gene and a sequence encoding an additional human or chimeric protein.

[0150] In some embodiments, the non-human animal includes, but is not limited to, a non-human animal modified with at least one gene selected from ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the above-mentioned non-human animal also expresses at least one of human or humanized ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 proteins.

[0151] The present invention also provides a method for constructing a non-human animal having two or more human or chimeric genes, the construction method comprising:

[0152] (1) Providing a non-human animal obtained by the above construction method;

[0153] (2) Mating the non-human animal provided in step (1) with other gene-modified non-human animals, performing in vitro fertilization or directly performing gene editing, and screening to obtain a multi-gene-modified non-human animal.

[0154] In some embodiments, the other gene-modified non-human animals include, but are not limited to, non-human animals humanized with one or a combination of two or more genes selected from ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0155] In some embodiments, MMP7 humanization is directly performed on a non-human animal having at least one gene modification selected from human or chimeric ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0156] Because these proteins may be involved in different mechanisms, combination therapies targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are underway and showing good results. A multi-gene modified non-human animal model can be used to determine the effectiveness of combination therapies targeting two or more proteins. For example, a therapeutic agent targeting MMP7 (such as an antibody, nucleic acid drug, and / or polypeptide drug), and an additional therapeutic agent for treating inflammation, cancer, or immune diseases. The method includes administering to the non-human animal a therapeutic agent targeting MMP7 and an additional therapeutic agent, wherein the non-human animal has an inflammatory, tumor, or immune disease, and determining the effect of the combination treatment on the inflammatory, cancer, or immune disease. In some embodiments, the additional therapeutic agent is at least one of antibodies that specifically bind to ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab, nivolumab), or an anti-PD-L1 antibody. In some embodiments, the non-human animal further includes a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the above-mentioned tumor includes one or more tumor cells expressing PD-L1 and / or PD-L2.

[0157] In some embodiments, the combination therapy is used to treat various cancers described in the present application. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the solid tumor is a digestive tract cancer (such as colorectal cancer), an endocrine cancer, a pancreatic cancer, a head and neck cancer, a liver cancer, a lung cancer, a breast cancer, an ovarian cancer, an endometrial cancer, a melanoma, or a kidney cancer. In some embodiments, the hematological tumor includes but is not limited to lymphocyte tumors, such as B or T cell tumors. In some embodiments, the combination therapy is designed to treat the immune diseases described in the present application, such as psoriasis, idiopathic pulmonary fibrosis, asthma, rheumatoid arthritis, or multiple sclerosis. In some embodiments, the methods described in the present application can be used to evaluate combination therapies with some other methods. Methods of treating cancer that can be used alone or in combination with the methods described in the present application include, for example, treating a subject with chemotherapy, such as camptothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, thiotepa, nitrosourea, dacarbazine, daunorubicin, bleomycin, plicamycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, carboplatin, 5-fluorouracil, vincristine, vinblastine, and / or methotrexate. Alternatively, in addition to this, the method can include performing surgery on the subject to remove at least a portion of the cancer, for example, removing a part or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 : Schematic diagram (not to scale) of the comparison between the mouse MMP7 locus and the human MMP7 locus;

[0159] Figure 2 : Schematic diagram (not to scale) of the MMP7 gene targeting strategy and the design of the targeting vector V1;

[0160] Figure 3 : Schematic diagram (not to scale) of the MMP7 gene targeting strategy and the design of the targeting vector V2;

[0161] Figure 4 : Identification results of the F1 generation of MMP7 gene humanized mice by Southern blot, where WT is the wild-type control;

[0162] Figure 5 : ELISA test results, where + / + are wild-type C57BL / 6 mice and H / + are MMP7 gene humanized heterozygous mice;

[0163] Figure 6 : ELISA test results, where + / + are wild-type C57BL / 6 mice and H / H are MMP7 gene humanized homozygous mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0164] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0165] In each of the following embodiments, the equipment and materials were obtained from the several companies indicated below:

[0166] C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Laboratory Animal Seed Center, China National Institute for Food and Drug Control;

[0167] AseI was purchased from NEB, catalog number: R0526S;

[0168] BspHI was purchased from NEB, catalog number: R0517S;

[0169] Human Total MMP-7 Quantikine ELISA Kit was purchased from R&D, catalog number: DMP700;

[0170] Mouse MMP-7 ELISA Kit (Colorimetric) was purchased from NOVUS, catalog number: NBP3-06895.

[0171] Example 1 MMP7 Gene Humanized Mice

[0172] Schematic comparison of the mouse MMP7 gene (Gene ID: 17393, located at positions 7692095 to 7699587 of chromosome 9 NC_000075.7, based on transcript NM_010810.6 and its encoded protein NP_034940.3 (SEQ ID NO: 1)) and the human MMP7 gene (Gene ID: 4316, located at positions 102520508 to 102530747 of chromosome 11 NC_000011.10, based on transcript NM_002423.5 and its encoded protein NP_002414.1 (SEQ ID NO: 2)) is as Figure 1 shown.

[0173] To achieve the object of the present invention, a nucleotide sequence encoding human MMP7 protein can be introduced into the endogenous MMP7 locus of a mouse, such that the mouse expresses human or humanized MMP7 protein. Specifically, using gene editing technology, under the control of the MMP7 gene regulatory elements of the mouse, a sequence of approximately 10.8 kb from a partial sequence of exon 1 to the entire sequence of exon 6 of the human MMP7 gene (such as from the start codon ATG to downstream of the 3' UTR) is used to replace a sequence of approximately 7.9 kb from a partial sequence of exon 1 to the entire sequence of exon 6 of the mouse (such as from the start codon ATG to downstream of the 3' UTR), obtaining a humanized MMP7 locus and achieving humanization of the mouse MMP7 gene.

[0174] To implement the targeting strategy of the present invention, a targeting vector was constructed. The targeting vector V1 ( Figure 2 ) contains homologous arm sequences upstream and downstream of the mouse MMP7 gene, as well as fragment A containing the human MMP7 fragment. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence at positions 7688242 to 7692126 of NCBI accession number NC_000075.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence at positions 7699983 to 7704431 of NCBI accession number NC_000075.7. The nucleotide sequence of the human MMP7 gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence at positions 102519950 to 102530700 of NCBI accession number NC_000011.10; the connection design of the upstream of the human MMP7 fragment sequence with the mouse is:

[0175]

[0176] wherein the last "G" in the sequence " TGTTG " is the last nucleotide of the upstream connection of the mouse and human MMP7 fragment sequences, and the "A" in the sequence is the first nucleotide of the human sequence.

[0177] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two directly repeated site-specific recombination systems, Frt recombination sites, are installed on both sides of the resistance gene to form a Neo cassette. The connection design of the 5' end of the Neo cassette with the human MMP7 gene is:

[0178]

[0179] wherein the sequence "ATGGA The last "A" in "" is the last nucleotide of the human MMP7 gene, and the sequence The first "G" in the sequence is the first nucleotide of the Neo cassette; the connection of the 3'-end of the Neo cassette to the mouse MMP7 gene is designed as:

[0180]

[0181] Among them, the last "C" in the sequence "" ACTTC is the last nucleotide of the Neo cassette, and the "G" in the sequence is the first nucleotide of the connection between the mouse MMP7 gene and the 3'-end of the Neo cassette. The mRNA sequence transcribed from the humanized mouse MMP7 gene after modification is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0182] The construction of the targeting vector can be carried out by conventional methods, such as restriction enzyme ligation, etc. After the constructed targeting vector is preliminarily verified by restriction enzyme digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into the embryonic stem cells of C57BL / 6 mice by electroporation, and the obtained cells are screened using the positive clone screening marker gene to screen out the correct positive clone cells. The correct positive clone cells (black mice) screened out are introduced into the isolated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient female mouse (white mouse) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then homozygous humanized MMP7 gene mice can be obtained by mating with each other.

[0183] In addition, the CRISPR / Cas9 technology can also be used for gene editing, and further design the targeting vector V2 ( Figure 3) The targeting vector V2 contains homologous arm sequences upstream and downstream of the mouse MMP7 gene, as well as the human MMP7 fragment. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 18) is identical to the nucleotide sequence from position 7690623 to 7692126 of NCBI accession number NC_000075.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 19) is identical to the nucleotide sequence from position 7699983 to 7701433 of NCBI accession number NC_000075.7. The nucleotide sequence of the human MMP7 fragment (SEQ ID NO: 5) is identical to the nucleotide sequence from position 102519950 to 102530700 of NCBI accession number NC_000011.10. The connection design between the upstream of the human MMP7 fragment sequence and the mouse is as follows:

[0184]

[0185] Among them, the last "G" in the sequence " TGTTG " is the last nucleotide for the upstream connection between the mouse and human MMP7 fragment sequences, and the "A" in the sequence is the first nucleotide of the human sequence. The connection design between the downstream of the human MMP7 fragment sequence and the mouse is as follows:

[0186]

[0187] Among them, the last "A" in the sequence " ATGGA " is the last nucleotide of the human sequence, and the "G" in the sequence is the first nucleotide for the downstream connection between the mouse sequence and the human MMP7 fragment sequence. The mRNA sequence transcribed from the humanized mouse MMP7 gene after modification is as shown in SEQ ID NO: 6, and the expressed protein sequence is as shown in SEQ ID NO: 2.

[0188] The construction of the targeting vector can be carried out by conventional methods, such as restriction enzyme ligation, direct synthesis, etc. After the constructed targeting vector is preliminarily verified by restriction enzyme digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is used for subsequent experiments.

[0189] The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, the selection and design of a highly efficient and specific target sequence are the prerequisites for constructing the sgRNA expression vector. Design and synthesize the sgRNA sequence that recognizes the target site. Exemplary target sequences of the sgRNA on the MMP7 gene are as follows:

[0190] sgRNA1 target site (SEQ ID NO: 21):

[0191] 5’-TTTGTCCTTCGGTGCTTGCGTGG-3’;

[0192] sgRNA2 target site (SEQ ID NO: 22):

[0193] 5’-CCTCCATTGCATCCCTCAGCTGG-3’;

[0194] After detecting the activity of sgRNA using the UCA kit and determining that it can mediate high cleavage efficiency, restriction enzyme sites were added to the 5' end and complementary strand respectively to obtain the forward and reverse oligonucleotide sequences as shown in Table 3. After annealing, the annealed product was ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain the expression vectors pT7-MMP7-1 and pT7-MMP7-2.

[0195] Table 3 Sequence list of sgRNA1 and sgRNA2

[0196]

[0197]

[0198] The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment DNA (SEQ ID NO: 20) with a T7 promoter and sgRNA scaffold, and was successively ligated to the backbone vector (source: Takara, catalog number 3299) through restriction enzyme digestion (EcoRI and BamHI). After verification by a professional sequencing company, the results showed that the target plasmid was obtained. Take the pronuclear stage fertilized eggs of mice, such as C57BL / 6 mice, and use a microinjector to premix the in vitro transcription products of pT7-MMP7-1 and pT7-MMP7-2 plasmids (using the Ambion in vitro transcription kit and transcribing according to the instructions), the targeting vector and Cas9 mRNA, and then inject them into the cytoplasm or nucleus of mouse fertilized eggs. Microinjection of fertilized eggs was carried out according to the method in "Mouse Embryo Manipulation Experimental Manual (Third Edition)" (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to the culture medium for short-term culture, and then transplanted into the oviduct of the recipient female mouse for development. The obtained mice (F0 generation) were hybridized and self-crossed to expand the population size and establish a stable humanized mouse strain of the MMP7 gene.

[0199] The genotypes of somatic cells of F1 generation mice can be identified by PCR method. For the mice with positive PCR identification in F1 generation, Southern blot detection is carried out to confirm whether there is random insertion. Cut the mouse tail to extract genomic DNA, digest the genome with AseI enzyme or BspHI enzyme respectively, transfer the membrane, and hybridize. The 5’ probe (5’ Probe) and A probe (A Probe) are located upstream of the 5’ homologous arm and on the human MMP7 genomic fragment respectively. The specific probes and the lengths of target fragments are shown in Table 4), and the exemplary results are as Figure 4 shown. Combining the PCR and sequencing results, 4 mice numbered F1-01 to F1-04 are positive mice. This indicates that the humanized MMP7 gene mice that can be stably passed on and have no random insertion can be constructed by using this method.

[0200] Table 4 Specific probes and the lengths of target fragments

[0201] Restriction endonuclease Probe Wild-type fragment size Recombinant sequence fragment size AseI A Probe -- 7.1 kb BspHI 5’ Probe 9.8 kb 12.5 kb

[0202] 5’Probe-F (SEQ ID NO: 11): 5’-GATGTGGGACATTTCTAGAC-3’, 5’Probe-R (SEQ ID NO: 12): 5’-GTGTGGAAATATGACAAATATAG-3’;

[0203] A Probe-F (SEQ ID NO: 13): 5’-CACCACACTATTTTGAGGTCTTCCGCA-3’, A Probe-R (SEQ ID NO: 14): 5’-AACCTAGAGAGTGGCTGCAGCAGGA-3’;

[0204] In addition, the expression of human MMP7 protein in the humanized MMP7 mice can be detected by conventional methods such as ELISA. Specifically, 3 male C57BL / 6 mice ( + / + ) at 8 weeks old and 3 male humanized MMP7 gene heterozygotes (H / +) prepared in this example at 8 weeks old are selected. Take the serum and use Human Total MMP-7 Quantikine ELISA Kit and Mouse MMP-7 ELISA Kit (Colorimetric) for detection. The detection results are as Figure 5 shown.

[0205] From Figure 5As can be seen, when detected using a mouse-specific MMP7 ELISA kit, mouse MMP7 protein was detected in both MMP7 humanized heterozygous mice and C57BL / 6 mice. When detected using a human-specific MMP7 ELISA kit, human MMP7 protein was only detected in MMP7 humanized heterozygous mice.

[0206] In another similar experiment, three 8-week-old female C57BL / 6 mice (+ / +) and three 8-week-old female MMP7 gene humanized homozygotes (H / H) prepared in this example were selected, and their sera were taken for detection. The detection results are as Figure 6 shown. Only mouse MMP7 protein was detected in C57BL / 6 mice, and only human MMP7 protein was detected in MMP7 humanized homozygous mice. It was proved that human MMP7 protein could be successfully expressed in MMP7 gene humanized homozygous mice.

[0207] Example 2 Pharmacodynamic Model

[0208] Using the humanized mice disclosed in the present invention, various human disease models can be induced and prepared, including inflammation, immune diseases, cancer and other models, which can be used to test the in vivo pharmacodynamics of human-specific antibodies. For example, a number of MMP7 humanized mouse homozygotes were taken, and the mice were modeled with idiopathic pulmonary fibrosis. After successful modeling, the mice were divided into a control group or a treatment group. The treatment group randomly selected a drug targeting human MMP7, and the control group was injected with an equal volume of normal saline. The body weights of the mice were weighed regularly, the cytokine levels and lung HE staining were detected, and the in vivo safety and in vivo pharmacodynamics of the compound could be effectively evaluated by comparing the changes in the body weights of the mice and the cytokine indexes.

[0209] Example 3 Preparation of Double-Gene or Multi-Gene Humanized Mice

[0210] The MMP7 gene humanized mice prepared by using this method or obtained can also be used to prepare multi-gene humanized mouse models. For example, in Example 1 above, the embryonic stem cells used for microinjection can be selected from mice modified with at least one gene among ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. Alternatively, on the basis of humanized MMP7 mice, by using the technique of isolating mouse ES embryonic stem cells and gene recombination and targeting, double-gene humanized or multi-gene humanized mouse models can be obtained. The MMP7 mouse homozygotes or heterozygotes obtained by this method can also be mated with other gene-modified mice, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene mice with humanized MMP7 gene and other gene modifications. Then, the heterozygotes can be mated with each other to obtain double-gene or multi-gene modified homozygotes.

[0211] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0212] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

[0213] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric matrix metalloproteinase 7 (MMP7) protein.

2. The construction method according to claim 1, characterized in that: The amino acid sequence of the human or chimeric MMP7 protein comprises SEQ ID NO:2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

2.

3. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises a nucleotide sequence of human MMP7 at the endogenous MMP7 locus replacing the nucleotide sequence of the corresponding region of endogenous MMP7.

4. The construction method according to any one of claims 1 to 3, characterized in that: The nucleotide sequence encoding the human or chimeric MMP7 protein or the nucleotide sequence of the human MMP7 is operably linked to an endogenous regulatory element of an endogenous MMP7 locus; Preferably, the endogenous MMP7 protein of the non-human animal is not expressed or is expressed at a reduced level compared to MMP7 in wild-type animals; Preferably, the modified MMP7 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

5. The construction method according to claim 3 or 4, characterized in that: The nucleotide sequence of human MMP7 encodes human or chimeric MMP7 protein; Preferably, the nucleotide sequence of human MMP7 comprises part of exon 1 to all of exon 6 of the human MMP7 gene, and preferably also includes at least 50 bp of continuous nucleotides downstream of the 3'UTR; Preferably, the nucleotide sequence of human MMP7 comprises a portion of exon 1 of the human MMP7 gene to at least 50 bp of continuous nucleotides downstream of the 3'UTR; Further preferably, the nucleotide sequence of human MMP7 comprises at least 50 bp of continuous nucleotides from the start codon of the human MMP7 gene to the downstream of the 3'UTR; More preferably, the nucleotide sequence of human MMP7 comprises SEQ ID NO: 5, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

5.

6. The construction method according to any one of claims 3 to 5, characterized in that: The endogenous MMP7 corresponding region comprises a nucleotide sequence encoding a non-human animal MMP7 protein; Preferably, the endogenous MMP7 corresponding region includes part of exon 1 to all of exon 6 of the non-human animal MMP7 gene, and preferably also includes at least 50 bp of continuous nucleotides downstream of the 3'UTR; Preferably, the endogenous MMP7 corresponding region includes a portion of exon 1 of the non-human animal MMP7 gene to at least 50 bp of continuous nucleotides downstream of the 3'UTR; Further preferably, the endogenous MMP7 corresponding region comprises at least 50 bp of continuous nucleotides from the start codon of the non-human animal MMP7 gene to the downstream of the 3'UTR.

7. The construction method according to any one of claims 1 to 6, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the non-human animal is a mouse or a rat; Preferably, the mRNA transcribed from the modified gene in the genome of the non-human animal comprises SEQ ID NO: 6, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

6.

8. The construction method according to any one of claims 1 to 7, characterized in that: The non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins include at least one of ICOS, NKP46, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

9. An application of a non-human animal obtained by the construction method according to any one of claims 1 to 8, characterized in that: The application includes: A) Application in product development involving MMP7-related immune processes in human cells; B) Application as a model system related to MMP7 in pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of MMP7-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human MMP7 signaling pathway modulators; or, E) Study the function of MMP7 gene, study the drugs and efficacy targeting human MMP7 target sites, and study the application of MMP7-related cancer, immune disease, neurological disease or inflammatory drug.

10. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 8; 2) Determine the effect of a therapeutic agent on a disease or animal; Preferably, the therapeutic agent is an antibody, nucleic acid drug or polypeptide drug targeting MMP7; further preferably, the therapeutic agent also includes an additional therapeutic agent, such as an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the disease includes cancer, immune disease or inflammation; Further preferably, the cancer is a solid tumor or a blood tumor, such as digestive tract cancer (such as colorectal cancer), endocrine cancer, pancreatic cancer, head and neck cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma or kidney cancer; Further preferably, the immune disease is idiopathic pulmonary fibrosis, asthma, rheumatoid arthritis, psoriasis or multiple sclerosis; Further preferably, the inflammation is specific inflammation, arthritis or inflammatory bowel disease (IBD).

11. A humanized MMP7 gene, characterized in that: The humanized MMP7 gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding the protein of SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 18 or 19; C) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 18 or 19; D) a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 18 or 19.

12. A cell, tissue or organ, characterized in that: The cells, tissues or organs contain the humanized MMP7 gene according to claim 11.

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