SERPINF2 gene modified non-human animal

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) SERPINF2 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) SERPINF2 protein and methods of using the same. Background Art

[0002] Traditional drug development typically 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 in 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, leading to significant differences between the results of many clinical trials and those of 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 SERPINF2 protein. This animal model can express a human or chimeric SERPINF2 (e.g., humanized SERPINF2) protein. It can be used for the study of the function of the SERPINF2 gene and also for the screening and evaluation of SERPINF2 signaling pathway regulators (e.g., therapeutic agents targeting SERPINF2, including antibodies targeting SERPINF2, nucleic acid drugs targeting SERPINF2, 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 research of diseases (including tumors, inflammation, immune diseases, or cerebrovascular diseases) targeting the human SERPINF2 target; this 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 SERPINF2 protein and a platform for screening drugs for the treatment of related diseases.

[0005] On the one hand, 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 serine protease inhibitor family F member 2 (SERPINF2) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric SERPINF2 protein can be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the chimeric SERPINF2 protein is a humanized SERPINF2 protein. In some embodiments, the amino acid sequence of the chimeric SERPINF2 protein comprises an amino acid sequence that is identical to at least 50 to 491, such as 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 490 or 491 consecutive or non-consecutive amino acid sequences of the human SERPINF2 protein. In some embodiments, the nucleotide sequence encoding the human or chimeric SERPINF2 protein is operably linked to an endogenous regulatory element (such as a promoter and / or 5'UTR) of the endogenous SERPINF2 locus of at least one chromosome. In some embodiments, the amino acid sequence of the human or chimeric SERPINF2 protein 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 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 rodent includes a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous SERPINF2 protein of the non-human animal is not expressed or has a reduced expression level compared to SERPINF2 in a wild-type animal. In some embodiments, one or more cells of the non-human animal express the human or chimeric SERPINF2 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 encoding an endogenous SERPINF2 region at the endogenous SERPINF2 locus replaced by a nucleotide sequence encoding the corresponding region of human SERPINF2. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 is operably linked to an endogenous regulatory element of the endogenous SERPINF2 locus. In some embodiments, one or more cells of the non-human animal express human or humanized SERPINF2 protein. In some embodiments, the endogenous SERPINF2 protein of the non-human animal is not expressed or has a reduced expression level compared to SERPINF2 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 comprises a portion of exon 2 and all of exons 3-10 of the human SERPINF2 gene (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 comprises a portion of exon 2, all of exons 3-10, and at least 20 bp of continuous nucleotides downstream of the 3'UTR of the human SERPINF2 gene (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 comprises a portion of exon 2, all of exons 3-10, and at least 300 bp of continuous nucleotides downstream of the 3'UTR of the human SERPINF2 gene (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 comprises from the start codon of the human SERPINF2 gene to at least 300 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 is identical to the nucleotide sequence shown in SEQ ID NO: 5 or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises a portion of exon 2 and all of exons 3-10 of the endogenous SERPINF2 gene of the non-human animal (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises a portion of exon 2, all of exons 3-10, and at least 20 bp of continuous nucleotides downstream of the 3'UTR of the endogenous SERPINF2 gene of the non-human animal (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises a portion of exon 2, all of exons 3-10, and 58 bp of continuous nucleotides downstream of the 3'UTR of the endogenous SERPINF2 gene of the non-human animal (preferably also comprising intron 2).In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises at least 58 consecutive nucleotides from the start codon of the endogenous SERPINF2 gene of a non-human animal to downstream of the 3'UTR. In some embodiments, the modified SERPINF2 gene in the non-human animal genome is homozygous or heterozygous for the endogenous locus being replaced.

[0007] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein at the endogenous SERPINF2 locus of the non-human animal, the nucleotide sequence of the corresponding region of the endogenous SERPINF2 of the non-human animal is replaced with a nucleotide sequence comprising the nucleotide sequence of human SERPINF2. In some embodiments, the nucleotide sequence of human SERPINF2 comprises a nucleotide sequence encoding all or part of a human or chimeric SERPINF2 protein, preferably comprising the nucleotide sequence encoding SEQ ID NO: 2; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the nucleotide sequence of human SERPINF2 can be a genomic DNA sequence, a CDS sequence or a cDNA sequence. In some embodiments, the nucleotide sequence of human SERPINF2 comprises a nucleotide sequence that is continuously or discontinuously identical to the human SERPINF2 gene for at least 50 to at least 12,695 bp, such as at least 50, 100, 500, 1,000, 1,500, 2,249, 2,250, 2,500, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,570, 12,395 or 12,695 bp. In some embodiments, the nucleotide sequence of human SERPINF2 comprises part of exon 2 to all of exon 10 of the human SERPINF2 gene. In some embodiments, the part of exon 2 of the human SERPINF2 gene preferably comprises at least 5 - 67 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 63, 64, 65 or 67 bp of continuous nucleotide sequence, preferably comprising the nucleotide sequence of the coding region. In some embodiments, the nucleotide sequence of human SERPINF2 comprises the nucleotide sequence from the start codon to the stop codon of the human SERPINF2 gene, preferably further comprises the 3'UTR, more preferably further comprises at least 20 bp of continuous nucleotide sequence downstream of the 3'UTR, and even more preferably comprises at least 20 bp to 500 bp of continuous nucleotide sequence, such as at least 20, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of human SERPINF2 comprises the nucleotide sequence from the start codon to all of exon 10 of the human SERPINF2 gene. In some embodiments, the nucleotide sequence of human SERPINF2 comprises the nucleotide sequence from the start codon to at least 20 bp of continuous nucleotide sequence downstream of the 3'UTR.In some embodiments, the nucleotide sequence of human SERPINF2 comprises a continuous nucleotide sequence from the start codon of the human SERPINF2 gene to at least 300 bp downstream of the 3' UTR. In some embodiments, the nucleotide sequence of human SERPINF2 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 of the corresponding region of non-human animal endogenous SERPINF2 comprises a nucleotide sequence encoding all or part of the non-human animal endogenous SERPINF2 protein, preferably comprises the nucleotide sequence encoding SEQ ID NO: 1; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence encoding SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the corresponding region of non-human animal endogenous SERPINF2 comprises part of exon 2 to all of exon 10 of the non-human animal endogenous SERPINF2 gene. In some embodiments, the part of exon 2 of the non-human animal endogenous SERPINF2 gene preferably comprises at least 5 - 67 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 63, 64, 65, 66 or 67 bp of continuous nucleotide sequence, preferably comprises the nucleotide sequence of the coding region. In some embodiments, the nucleotide sequence of the corresponding region of non-human animal endogenous SERPINF2 comprises the nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous SERPINF2 gene, preferably further comprises the 3' UTR, more preferably further comprises at least 20 bp of continuous nucleotide sequence downstream of the 3' UTR, and even more preferably comprises at least 20 bp to 100 bp of continuous nucleotide sequence, such as at least 20, 30, 40, 50, 55, 58, 60, 70, 80, 90 or 100 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of the corresponding region of non-human animal endogenous SERPINF2 comprises the nucleotide sequence from the start codon to all of exon 10 of the non-human animal endogenous SERPINF2 gene. In some embodiments, the nucleotide sequence of the corresponding region of non-human animal endogenous SERPINF2 comprises the nucleotide sequence from the start codon to at least 20 bp of continuous nucleotide sequence downstream of the 3' UTR. In some embodiments, the nucleotide sequence of the corresponding region of non-human animal endogenous SERPINF2 comprises the nucleotide sequence from the start codon to at least 58 bp of continuous nucleotide sequence downstream of the 3' UTR.In some embodiments, the nucleotide sequence encoding the human or chimeric SERPINF2 protein or the nucleotide sequence of human SERPINF2 is operably linked to an endogenous regulatory element of the endogenous SERPINF2 locus. In some embodiments, the regulatory element includes a promoter and / or 5'UTR. In some embodiments, the endogenous SERPINF2 protein of the non-human animal is not expressed or has a reduced expression level compared to SERPINF2 in wild-type animals. In some embodiments, the modified SERPINF2 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous locus being replaced. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the mRNA transcribed from the modified SERPINF2 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 SEQ ID NO: 6. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding another human or chimeric protein, and the other human or chimeric protein preferably comprises at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0008] In one aspect, the present invention provides a non-human animal or a method for constructing the same, the non-human animal comprising at least one cell encoding a nucleotide sequence of a human or humanized SERPINF2 protein, wherein the humanized SERPINF2 protein comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 490 or 491 consecutive amino acid sequences identical to the corresponding region of the human SERPINF2 protein, and the non-human animal expresses the human or humanized SERPINF2 protein. In some embodiments, the nucleotide sequence encoding the human or humanized SERPINF2 protein is operably linked to an endogenous SERPINF2 regulatory element, such as a promoter and / or 5'UTR. In some embodiments, the nucleotide sequence encoding the human or humanized SERPINF2 protein can be integrated into the endogenous SERPINF2 locus of the non-human animal. In some embodiments, the human or humanized SERPINF2 protein has at least one activity, such as endogenous SERPINF2 activity and / or human SERPINF2 activity of the non-human animal.

[0009] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, in which, in at least one cell of the non-human animal, at the endogenous SERPINF2 locus of the non-human animal, the nucleotide sequence encoding the endogenous SERPINF2 region is replaced by the nucleotide sequence encoding the corresponding region of human SERPINF2. In some embodiments, the endogenous SERPINF2 protein of the non-human animal is not expressed or has a reduced expression level compared to SERPINF2 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 comprises a part of exon 2 and all of exons 3-10 of the human SERPINF2 gene (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 comprises a part of exon 2 and all of exons 3-10 of the human SERPINF2 gene and at least 20 bp of continuous nucleotides downstream of the 3'UTR (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 comprises a part of exon 2 and all of exons 3-10 of the human SERPINF2 gene and at least 300 bp of continuous nucleotides downstream of the 3'UTR (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 comprises the start codon of the human SERPINF2 gene to at least 300 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence of the corresponding region of human SERPINF2 is identical to the amino acid sequence shown in SEQ ID NO: 2 or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 is identical to the nucleotide sequence shown in SEQ ID NO: 5 or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises a part of exon 2 and all of exons 3-10 of the endogenous SERPINF2 gene of the non-human animal (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises a part of exon 2 and all of exons 3-10 of the endogenous SERPINF2 gene of the non-human animal and at least 20 bp of continuous nucleotides downstream of the 3'UTR (preferably also comprising intron 2). In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises a part of exon 2 and all of exons 3-10 of the endogenous SERPINF2 gene of the non-human animal and 58 bp of continuous nucleotides downstream of the 3'UTR (preferably also comprising intron 2).In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises at least 58 bp of contiguous nucleotides downstream of the stop codon of the endogenous SERPINF2 gene of a non-human animal to the 3' UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 is operably linked to an endogenous SERPINF2 regulatory element, such as a promoter and / or 5' UTR. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or a rat).

[0010] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric SERPINF2. The construction method includes replacing, at the endogenous SERPINF2 gene locus of the non-human animal, the nucleotide sequence encoding the endogenous SERPINF2 region with the nucleotide sequence encoding the corresponding region of human SERPINF2 to generate a genetically modified non-human animal cell, and the non-human animal cell expresses human or chimeric SERPINF2 protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 includes a part of exon 2 and all of exons 3-10 of the human SERPINF2 gene (preferably also including intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 includes a part of exon 2 and all of exons 3-10 of the human SERPINF2 gene and at least 20 bp of consecutive nucleotides downstream of the 3'UTR (preferably also including intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 includes a part of exon 2 and all of exons 3-10 of the human SERPINF2 gene and at least 300 bp of consecutive nucleotides downstream of the 3'UTR (preferably also including intron 2). In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 includes the start codon of the human SERPINF2 gene to at least 300 bp of consecutive nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence of the corresponding region of human SERPINF2 is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 is identical to or has an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region includes a part of exon 2 and all of exons 3-10 of the endogenous SERPINF2 gene of the non-human animal (preferably also including intron 2). In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region includes a part of exon 2 and all of exons 3-10 of the endogenous SERPINF2 gene of the non-human animal and at least 20 bp of consecutive nucleotides downstream of the 3'UTR (preferably also including intron 2). In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region includes a part of exon 2 and all of exons 3-10 of the endogenous SERPINF2 gene of the non-human animal and 58 bp of consecutive nucleotides downstream of the 3'UTR (preferably also including intron 2).In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 region comprises at least 58 bp of continuous nucleotides downstream of the start codon of the endogenous SERPINF2 gene of a non-human animal to the 3' UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human SERPINF2 is operably linked to regulatory elements of endogenous SERPINF2, such as a promoter and / or 5' UTR. In some embodiments, the non-human animal is a mouse.

[0011] In one aspect, provided is a method for determining the efficacy or toxicity of a therapeutic agent in treating a disease, the method comprising:

[0012] 1) Administering the therapeutic agent to the non-human animal obtained by the above construction method or the non-human animal; 2) Determining the effect of the therapeutic agent on the non-human animal or the disease. In some embodiments, the therapeutic agent includes an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug. In some embodiments, the therapeutic agent further includes an additional therapeutic agent, and the additional therapeutic agent includes one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease includes, but is not limited to, one or more of a tumor, an immune disease, an inflammation, or a cerebrovascular disease.

[0013] In one aspect, the present invention provides a method for determining the efficacy of a therapeutic agent in treating cancer (tumor), the method comprising: 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 therapeutic agent includes an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug. In some embodiments, the cancer comprises injection of one or more tumor cells into the non-human animal. In some embodiments, determining the inhibitory effect of the therapeutic agent on the tumor involves measuring the tumor volume in the non-human animal. In some embodiments, the cancer (tumor) includes a solid tumor or a hematological tumor. In some embodiments, the cancer (tumor) includes one or more of head and neck cancer, lymphocyte tumor, liver cancer, or lung cancer. 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 tumor comprises one or more tumor cells expressing PD-L1.

[0014] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating immune diseases, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the preparation method, wherein the non-human animal suffers from an immune disease; 2) determining the therapeutic effect of the therapeutic agent on the immune disease. In some embodiments, the therapeutic agent comprises an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug. In some embodiments, the immune disease comprises one or more of cirrhosis, asthma, atopic dermatitis, rheumatoid arthritis, psoriasis, or multiple sclerosis.

[0015] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation, 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 inflammation; 2) determining the effectiveness of the therapeutic agent in treating inflammation. In some embodiments, the therapeutic agent comprises an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug. In some embodiments, the inflammation comprises inflammatory bowel disease (IBD).

[0016] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cerebrovascular diseases, 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 a cerebrovascular disease; 2) determining the effectiveness of the therapeutic agent in treating cerebrovascular diseases. In some embodiments, the therapeutic agent comprises an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug. In some embodiments, the cerebrovascular disease comprises thrombosis, ischemic stroke, or stroke.

[0017] 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 therapeutic agent comprises an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug. In some embodiments, determining the effect of the therapeutic agent on the non-human animal comprises measuring the body weight of the non-human animal or performing a blood test. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin content.

[0018] In one aspect, the present invention provides a humanized SERPINF2 gene, which comprises any one of the following nucleotide sequences: A) a nucleotide sequence encoding SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 31 or 32; C) a nucleotide sequence having at least 90% identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 31 or 32; 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, 31 or 32.

[0019] In one aspect, the present invention provides a cell, tissue or organ, the genome of which contains the humanized SERPINF2 gene.

[0020] In one aspect, the present invention provides an animal model, the genome of which contains the humanized SERPINF2 gene.

[0021] In one aspect, the present invention provides an application of the above non-human animal, the non-human animal obtained by the above construction method, the non-human animal cell obtained by the above construction method, the above humanized SERPINF2 gene, the above cell, tissue or organ or the above animal model, and the application comprises: A) an application in the development of products related to the immune process associated with SERPINF2 in human cells; B) an application as a model system related to SERPINF2 in pharmacological, immunological, microbiological and medical research; C) an application involved in the production and utilization of animal experimental disease models for etiological research related to SERPINF2 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 regulators of the human SERPINF2 signaling pathway; or, E) an application in studying the function of the SERPINF2 gene, studying the drugs and drug effects targeting human SERPINF2 target sites, and studying therapeutic drugs for tumors, inflammation, immune diseases or cerebrovascular diseases related to SERPINF2.

[0022] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to the local part in the whole, or the individual parts constituting the whole.

[0023] The term "locus" in the present invention, in a broad sense, represents the position occupied by a gene on a chromosome, and in a narrow sense, represents a DNA fragment on a certain gene, which can be either a gene or a part of a gene or a gene regulatory region, etc. For example, the "SERPINF2 locus" described above includes a DNA fragment arbitrarily selected from exons 1-10 of the SERPINF2 gene.

[0024] The term "part of exon XX" in the present invention means that a continuous or spaced sequence of several, dozens or hundreds of nucleotides is identical to the entire exon nucleotide sequence. For example, a part of exon 2 of the human SERPINF2 gene includes a continuous 5-67 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 63, 64, 65 or 67 bp of continuous nucleotide sequence, preferably including the nucleotide sequence of the coding region.

[0025] The term "exon XX to exon XXX" or "exon XX-XXX" or "the whole of exon XX-XXX" or "the whole of exon XX to the whole of exon XXX" in the present invention refers to including exons and the introns therebetween. For example, the whole of exon 3-10 includes the entire nucleotide sequences of exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9 and exon 10.

[0026] The term "part of exon XX to the whole of exon XXX" in the present invention refers to including the part of exon XX to the whole of exon XXX and the introns therebetween. For example, the part of exon 2 to the whole of exon 10 includes the part 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, the whole of exon 6, the whole of intron 6, the whole of exon 7, the whole of intron 7, the whole of exon 8, the whole of intron 8, the whole of exon 9, the whole of intron 9 and the whole of exon 10 of nucleotide sequences.

[0027] The term "intron xx" in the present invention refers to the intron between two exons. For example, intron 2 refers to the intron between exon 2 and exon 3.

[0028] As used in this invention, the term "comprising" or "including" is an open-ended expression that includes the specified components or steps described, as well as other specified components or steps that do not materially affect. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0029] As used in this invention, the term "and / or" includes all combinations of the items connected by this term, and should be regarded as if each combination has been separately listed in this application. For example, "A and / or B" includes "A", "B", and "A 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".

[0030] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those 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 merely exemplary 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.

[0031] Those skilled in the art can easily perceive other aspects and advantages of this application from the following detailed description.

[0032] SERPINF2

[0033] In the human genome, the SERPINF2 gene (NCBI Gene ID: 5345, UniProt ID: P08697, located at positions 1742871 to 1755265 of chromosome 17 NC_000017.11) contains 10 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, and exon 10. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_000934.4 and its encoded protein NP_000925.2 (SEQ ID NO: 2) are shown in Table 1.

[0034] Table 1

[0035]

[0036] In the mouse genome, the SERPINF2 gene (NCBI Gene ID: 18816, UniProt ID:

[0037] Q61247, located at positions 75322562 to 75330327 of NC_000077.7 on chromosome 11, contains 10 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9 and exon 10. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM_008878.2 and its encoded protein NP_032904.1 (SEQ ID NO: 1) are shown in Table 2.

[0038] Table 2

[0039]

[0040] SERPINF2 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 sequence, exon sequence and amino acid sequence) can all be found in NCBI, and the entire content is incorporated herein by reference.

[0041] 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, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of 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 done 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.

[0042] The percentage of conserved residues (percent homology) with similar physicochemical properties, 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), nonpolar 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.

[0043] vector

[0044] 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 conversion region to be altered, which is selected from the genomic DNA of the SERPINF2 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 conversion region to be altered, which is selected from the genomic DNA of the SERPINF2 gene and has a length of 100 to 10,000 nucleotides.

[0045] In some embodiments, a) the DNA fragment homologous to the 5'-end of the conversion region to be altered is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000077.7; c) the DNA fragment homologous to the 3'-end of the conversion region to be altered is selected from nucleotide sequences having at least 90% homology with NCBI accession number NC_000077.7.

[0046] In some embodiments, a) the DNA fragment homologous to the 5'-end of the conversion region to be altered is selected from the nucleotide sequence at positions 75329281 to 75333189 of NCBI accession number NC_000077.7; c) the DNA fragment homologous to the 3'-end of the conversion region to be altered is selected from the nucleotide sequence at positions 75318585 to 75322503 of NCBI accession number NC_000077.7.

[0047] In some embodiments, a) the DNA fragment homologous to the 5'-end of the conversion region to be altered is selected from the nucleotide sequence at positions 75329281 to 75330558 of NCBI accession number NC_000077.7; c) the DNA fragment homologous to the 3'-end of the conversion region to be altered is selected from the nucleotide sequence at positions 75321512 to 75322503 of NCBI accession number NC_000077.7.

[0048] In some embodiments, the selected genomic nucleotide sequence length of the targeting vector can exceed 0.8 kb, 1 kb,

[0049] 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 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, 17 kb, 18 kb, 19 kb or 20 kb.

[0050] In some embodiments, the conversion region to be altered is located on exons 1 to 10 of the endogenous SERPINF2 gene of a non-human animal, preferably on the nucleotide sequence downstream of exons 1 to the 3'UTR of the endogenous SERPINF2 gene of a non-human animal, and more preferably on the nucleotide sequence downstream of exons 2 to the 3'UTR of the endogenous SERPINF2 gene of a non-human animal.

[0051] In some embodiments, the 5' arm comprises SEQ ID NO: 3 or 31. In some embodiments, the 3' arm comprises SEQ ID NO: 4 or 32.

[0052] In some embodiments, the donor region b) comprises a human sequence, such as the nucleotide sequence at positions 1,744,996 to 1,755,565 of NCBI accession number NC_000017.11 (SEQ ID NO: 5).

[0053] In some embodiments, the targeting vector comprises one or more marker genes (or resistance genes). For example, a resistance gene for positive clone selection or a coding gene for a negative selection marker. In some embodiments, the resistance gene for positive clone selection is the coding sequence Neo of neomycin phosphotransferase. 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 diphtheria toxin A subunit (DTA).

[0054] The present invention also provides vectors for constructing humanized animal models or knockout models. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the SERPINF2 gene. In some embodiments, the sgRNA is unique at the target site in the conversion region to be altered and satisfies the sequence arrangement rules of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'. In some embodiments, the target site of the sgRNA in the non-human animal SERPINF2 gene is located on the exon 2 of the SERPINF2 gene and / or the nucleotide sequence downstream of the 3'UTR. In some embodiments, the target site is shown as SEQ ID NO: 21 and / or 22. Therefore, 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 29. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 28 and 30.

[0055] In some embodiments, the present invention relates to plasmid constructs containing sgRNA (sgRNA vectors, such as pT7-sgRNA) and / or cells comprising the construct.

[0056] The present invention also relates to cells comprising the targeting vector and / or sgRNA vector as described above.

[0057] In some embodiments, the present invention also provides a non-human mammalian cell having any of the above vectors and one or more in vitro transcripts of the plasmid constructs described in the present application. In some embodiments, the non-human mammalian cell contains Cas9 mRNA or its in vitro transcript.

[0058] In some embodiments, the gene in the non-human mammalian cell is heterozygous. In some embodiments, the gene in the non-human mammalian cell is homozygous.

[0059] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the non-human mammalian cell is a fertilized egg cell. In some embodiments, the non-human mammalian cell is an embryonic stem cell. In some embodiments, the non-human mammalian cell is any cell capable of expressing SERPINF2.

[0060] Gene-modified non-human animals

[0061] As used herein, the term "genetically modified non-human animal" or "genetically engineered non-human animal" 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, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in a genetically modified non-human animal or genetically engineered non-human animal, have exogenous DNA. The cells having exogenous DNA can be various types of cells, such as somatic cells, immune cells (e.g., T cells, B cells, NK cells, antigen-presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, there is provided a genetically modified non-human animal, wherein the non-human animal comprises a modified endogenous SERPINF2 locus, comprising an exogenous sequence (e.g., a human sequence), e.g., 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.

[0062] As used herein, the term "chimeric (x) gene" or "chimeric (x) nucleic acid" refers to a gene or nucleic acid in which two or more portions 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 gene or nucleic acid in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (x) gene or chimeric (x) nucleic acid has two or more different origins, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein 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.

[0063] 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 a 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) protein of different species. In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide refers to a humanized (x) protein or humanized (x) polypeptide.

[0064] 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 (x) protein or human (x) polypeptide. In some embodiments, the humanized (x) protein or humanized (x) polypeptide refers to a human (x) protein or (x) polypeptide.

[0065] As used herein, the term "humanized (x) nucleic acid" or "humanized (x) gene" refers to a nucleic acid or a gene. In some embodiments, at least a portion of the nucleic acid or gene is derived from a human (x) gene. In some embodiments, at least a portion of the nucleic acid or gene is derived from a non-human animal (x) gene. In some embodiments, the nucleic acid or gene in the humanized (x) nucleic acid or humanized (x) gene is entirely derived from a human (x) gene. In some embodiments, the humanized (x) nucleic acid or humanized (x) gene refers to a humanized exon, which can be a human exon or a chimeric exon.

[0066] In some embodiments, the chimeric SERPINF2 gene or chimeric SERPINF2 nucleic acid is a humanized SERPINF2 gene or humanized SERPINF2 nucleic acid. In some embodiments, at least a portion of the humanized SERPINF2 gene or humanized SERPINF2 nucleic acid is derived from the human SERPINF2 gene. In some embodiments, at least a portion of the humanized SERPINF2 gene or humanized SERPINF2 nucleic acid is derived from a non-human animal SERPINF2 gene. In some embodiments, the humanized SERPINF2 gene or humanized SERPINF2 nucleic acid contains a sequence encoding the SERPINF2 protein. The encoded SERPINF2 protein has at least one activity, such as the activity of the human SERPINF2 protein and / or the non-human animal SERPINF2 protein.

[0067] In some embodiments, the chimeric SERPINF2 protein or chimeric SERPINF2 polypeptide is a humanized SERPINF2 protein or humanized SERPINF2 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the humanized SERPINF2 protein or humanized SERPINF2 polypeptide are derived from the human SERPINF2 protein. In some embodiments, at least one or more portions of the amino acid sequence of the humanized SERPINF2 protein or humanized SERPINF2 polypeptide are derived from a non-human animal SERPINF2 protein. In some embodiments, the humanized SERPINF2 protein or humanized SERPINF2 polypeptide is functional or has at least one activity, such as the activity of the human SERPINF2 protein or the non-human animal SERPINF2 protein.

[0068] The genetically modified non-human animals can be various non-human 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 into 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 construction methods described above are known in the art and are described in “A. Nagy, et al., “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.

[0069] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. In some embodiments, 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 non-human animal is selected from the families Calomyscidae (e.g., mouse-like hamsters), Cricetidae (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 non-human 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.

[0070] 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 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, for example, in Festing et al., Revised nomenclature for strain129mice, Mammalian Genome 10:836(1999); Auerbach et al., Establishment andChimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem CellLines(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 strain (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 the 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 strain. In some embodiments, the non-human animal is a rat. The rats 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.

[0071] The non-human animal can have one or more other genetic modifications and / or other modifications that are suitable for a particular purpose of preparing a humanized animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) can have one or more modifications that impair, inactivate, or disrupt all or part of the immune system of the non-human animal. Impairment, inactivation, or disruption of the immune system of the non-human animal can include, for example, by chemical means (e.g., administration of a toxin), physical means (e.g., irradiation of the animal), and / or genetic modification (e.g., knockout of one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc null mouse (Ito, M. et al., NOD / SCID / γc null mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can optionally be irradiated or otherwise treated to destroy one or more immune cell types. Thus, in various embodiments, a genetically modified mouse is provided that can include humanization of at least a portion of the non-human animal endogenous SERPINF2 locus and also includes a modification that impairs, inactivates, or partially disrupts the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the type of mouse modification is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null mouse, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scid IL-2Rγ null mouse, NOD Rag 1 - / -IL2rg - / - (NRG) mouse, Rag2 - / - IL2rg - / -(RG) Modifications of mice and their combinations. These transgenic animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mouse may include replacing all or part of the endogenous mature SERPINF2 coding sequence of the mouse with all or part of the human mature SERPINF2 coding sequence, respectively.

[0072] Genetically modified non-human animals include modifications of the endogenous SERPINF2 gene locus (locus) of the non-human animal. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature SERPINF2 protein (e.g., comprising a nucleotide sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence encoding the mature SERPINF2 protein). Although cells (e.g., ES cells, somatic cells) that may contain the gene modification are provided in the present invention, in some embodiments, the genetically modified non-human animal includes a modification of the endogenous SERPINF2 gene locus in the non-human animal.

[0073] The genetically modified non-human animal may express human SERPINF2 and / or chimeric (e.g., humanized) SERPINF2 at the endogenous locus of the non-human animal, wherein the endogenous SERPINF2 gene of the non-human animal has been replaced or inserted with the gene of human SERPINF2 and / or a nucleotide sequence encoding a region of human SERPINF2 or a nucleotide sequence that is identical or has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the human SERPINF2 sequence. In some embodiments, the endogenous SERPINF2 gene locus of the non-human animal is modified with a nucleic acid sequence comprising all or part of the coding sequence of the human mature SERPINF2 protein.

[0074] In some embodiments, a genetically modified non-human animal can express human SERPINF2 and / or chimeric SERPINF2 (e.g., humanized SERPINF2) under the control of a non-human animal endogenous regulatory element. Insertion or replacement at a non-human animal endogenous locus provides a non-human animal that expresses human SERPINF2 or chimeric SERPINF2 (e.g., humanized SERPINF2) 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 SERPINF2 or chimeric SERPINF2 (e.g., humanized SERPINF2) expressed in the non-human animal can maintain one or more functions of a wild-type non-human animal or human SERPINF2 in the non-human animal. Additionally, in some embodiments, the non-human animal does not express endogenous SERPINF2. In some embodiments, the non-human animal endogenous SERPINF2 expression level is reduced compared to the SERPINF2 expression level in a wild-type animal. As used herein, the term "endogenous SERPINF2" refers to the SERPINF2 protein expressed from the endogenous SERPINF2 nucleotide sequence of a non-human animal (e.g., a mouse) prior to any genetic modification.

[0075] In some embodiments, the humanized SERPINF2 gene comprises the 5'UTR of the human SERPINF2 gene. In some embodiments, the humanized SERPINF2 gene comprises the 3'UTR of the human SERPINF2 gene. In some embodiments, the humanized SERPINF2 gene comprises the 5'UTR of a non-human animal endogenous (e.g., mouse endogenous) SERPINF2 gene. In some embodiments, the humanized SERPINF2 gene comprises the 3'UTR of a non-human animal endogenous (e.g., mouse endogenous) SERPINF2 gene. In appropriate cases, it can be reasonably assumed that, based on the similarity of the non-human animal and human SERPINF2 gene sequences, they appear to be regulated similarly. As shown in the present application, humanized SERPINF2 mice containing an insertion or replacement at the non-human animal endogenous SERPINF2 locus, which retain the non-human animal endogenous regulatory elements but contain a humanized SERPINF2 coding sequence, do not exhibit pathologies. Both heterozygous and homozygous gene-modified non-human animals of humanized SERPINF2 are normal.

[0076] The present invention further relates to the genomic DNA sequence of SERPINF2 of a humanized mouse, a DNA sequence obtained by reverse transcription of mRNA that is identical or complementary to this DNA sequence; a construct expressing its amino acid sequence; a cell containing its construct; a tissue or organ including its cell.

[0077] The present invention further relates to non-human mammals produced by the above method. In some embodiments, their genomes contain a part of the human SERPINF2 gene or a nucleotide sequence encoding all or part of the human SERPINF2 protein.

[0078] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0079] In some embodiments, the non-human mammal expresses a protein encoded by the humanized SERPINF2 gene.

[0080] In addition, the present invention also provides a non-human mammal model carrying a tumor, and the non-human mammal model is obtained by the construction method described in the present application. In some embodiments, the non-human mammal is a rodent (such as a mouse).

[0081] 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, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, a tissue, an 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.

[0082] The present invention provides a non-human mammal produced by any of the construction methods described in the present application. In some embodiments, a non-human mammal, a genetically modified non-human animal is provided, and the genome of the genetically modified non-human animal contains DNA of human or humanized SERPINF2.

[0083] In some embodiments, the non-human mammal includes the gene construct described in the present application. In some embodiments, a non-human mammal expressing human or humanized SERPINF2 protein is provided. In some embodiments, a cell, tissue or organ specifically expressing human or humanized SERPINF2 protein is provided.

[0084] In some embodiments, the expression of human or humanized SERPINF2 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).

[0085] The non-human mammal can be any non-human animal known in the art and can be used in the method described in the present application. In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0086] Genetic, molecular, and behavioral analyses are performed on the non-human mammals described above. The present invention provides offspring produced by mating non-human mammals of the same genotype or other genotypes.

[0087] 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 methods. The cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. Integration of a genetic construct containing a DNA sequence encoding the human SERPINF2 protein can be detected by a variety of methods.

[0088] There are many analytical methods available for detecting exogenous DNA, including methods at the nucleic acid level (including the use of 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 transcriptional 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 human or humanized SERPINF2 protein.

[0089] In some embodiments, the genetically modified animals described in the present application (e.g., homozygous humanized mice for the SERPINF2 gene or heterozygous humanized mice for the SERPINF2 gene) can express human or humanized SERPINF2 in one or more cells.

[0090] Method for constructing genetically modified non-human animals

[0091] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting techniques using embryonic stem cells, homologous recombination techniques, 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, the CRISPR / Cas9 gene editing technique can be used to construct genetically modified non-human animals. In some embodiments, the CRISPR / Cas9 gene editing technique is used to generate genetically modified non-human animals. Many of these genome 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 transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0092] In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 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 SERPINF2. In some embodiments, the expression level of the endogenous SERPINF2 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.

[0093] The present invention provides a targeting vector. The targeting vector comprises a vector consisting of a 5' homologous arm, a human or humanized SERPINF2 gene fragment, and a 3' homologous arm. The process involves replacing the corresponding sequence of endogenous SERPINF2 in the non-human animal with the human or humanized SERPINF2 gene fragment using homologous recombination. 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 SERPINF2 gene fragment is used to replace the corresponding sequence of endogenous SERPINF2 in the non-human animal using homologous recombination.

[0094] In some embodiments, the method for preparing a genetically modified humanized animal comprises replacing the nucleotide sequence encoding the endogenous SERPINF2 region in the genome of the non-human animal with the nucleotide sequence encoding the corresponding region of human SERPINF2 at the endogenous SERPINF2 locus (or site).

[0095] The present invention also provides a method for establishing a humanized animal model of the SERPINF2 gene, comprising the following steps:

[0096] (a) Providing cells (such as fertilized egg cells) based on the method described in the present application;

[0097] (b) Culturing the cells (preferably culturing the cells in a liquid medium);

[0098] (c) Transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0099] (d) Identifying germline transmission in the offspring of the genetically modified humanized non-human mammals of the pregnant female in step (c).

[0100] In some embodiments, the non-human mammal in the above method is a mouse (such as a C57BL / 6 mouse).

[0101] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0102] 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.

[0103] 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 cells are derived from rodents. 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.

[0104] In some embodiments, a method of producing a genetically modified non-human animal includes modifying the coding frame of the SERPINF2 gene of the non-human animal, e.g., by replacing the nucleic acid sequence encoding the endogenous SERPINF2 region with a nucleotide sequence encoding the corresponding region of human SERPINF2 (e.g., genomic DNA sequence, CDS sequence, or cDNA sequence) under the control of the endogenous SERPINF2 gene regulatory elements of the non-human animal. For example, one or more functional region sequences of the SERPINF2 gene of the non-human animal can be knocked out or inserted with a sequence such that the endogenous SERPINF2 protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the SERPINF2 gene of the genetically modified non-human animal can be all or part of the nucleotide sequence of exons 1 to 10 of the SERPINF2 gene of the non-human animal, preferably all or part of the nucleotide sequence of exons 2 to 10.

[0105] In some embodiments, a method of producing a genetically modified non-human animal includes inserting a nucleotide sequence encoding a human or humanized SERPINF2 protein and / or an auxiliary sequence after the endogenous regulatory elements of the SERPINF2 gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon such that the SERPINF2 gene humanized animal model can express a human or humanized SERPINF2 protein in vivo but not the SERPINF2 protein of the non-human animal. In some embodiments, the auxiliary sequence includes a stop codon, WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0106] In some embodiments, a method for producing a genetically modified non-human animal includes:

[0107] (1) Providing a plasmid containing a human SERPINF2 gene fragment, the plasmid flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target the endogenous SERPINF2 of the non-human animal;

[0108] (2) Providing one or more guide RNAs (sgRNAs) targeting the endogenous SERPINF2 gene of the non-human animal;

[0109] (3) Modifying the genome of a cell (e.g., a fertilized egg or an embryonic stem cell) by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0110] (4) Transfer the fertilized eggs obtained in step (3) into the oviduct of pseudopregnant female mice, or transfer the embryonic stem cells obtained in step (3) into blastocysts, and then transfer the blastocysts into the oviduct of pseudopregnant female mice to produce offspring mice that functionally express human or humanized SERPINF2 protein. Preferably, the method further comprises:

[0111] (5) Mate the offspring mice obtained in step (4) to obtain homozygous mice.

[0112] In some embodiments, the fertilized eggs are modified by CRISPR with sgRNAs targeting 5'-terminal target sites and 3'-terminal target sites.

[0113] In some embodiments, the human SERPINF2 gene fragment or the sequence encoding human or humanized SERPINF2 protein is operably linked to the endogenous regulatory elements at the endogenous SERPINF2 locus of the non-human animal.

[0114] In some embodiments, the genetically modified non-human animal does not express endogenous SERPINF2 protein.

[0115] In some embodiments, the method for preparing a genetically modified non-human animal comprises:

[0116] (1) Provide a plasmid containing a human or chimeric SERPINF2 gene fragment, flanked by 5' homologous arms and 3' homologous arms, wherein the 5' homologous arms and 3' homologous arms target the endogenous SERPINF2 of the non-human animal;

[0117] (2) Provide one or more guide RNAs (sgRNAs) targeting the endogenous SERPINF2 gene of the non-human animal;

[0118] (3) Modify the genome of a cell (e.g., a fertilized egg or an embryonic stem cell) by inserting the human or chimeric SERPINF2 gene fragment into the genome of the non-human animal.

[0119] In some embodiments, the nucleotide sequence encoding the endogenous SERPINF2 protein in the non-human animal genome is deleted. In some embodiments, all or part of exons 1-10 of the SERPINF2 gene in the non-human animal genome is deleted. In some embodiments, a partial deletion of exon 2 to the entire exon 10 of the SERPINF2 gene in the non-human animal genome is present. In some embodiments, the nucleotide sequence from the start codon to the stop codon of the SERPINF2 gene in the non-human animal genome is deleted. In some embodiments, the deleted nucleotide sequence further comprises a 3'UTR. In some embodiments, the deleted nucleotide sequence further comprises at least 20 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, there is a complete deletion of the SERPINF2 gene from the start codon to exon 10 in the non-human animal genome. In some embodiments, there is a deletion of the nucleotide sequence from the start codon of the SERPINF2 gene to at least 20 bp of continuous nucleotide sequence downstream of the 3'UTR in the non-human animal genome. In some embodiments, there is a deletion of the nucleotide sequence from the start codon of the SERPINF2 gene to at least 58 bp of continuous nucleotide sequence downstream of the 3'UTR in the non-human animal genome.

[0120] In some embodiments, the construction method includes replacing the nucleotide sequence encoding the human SERPINF2 protein with the nucleotide sequence encoding the non-human animal endogenous SERPINF2 protein in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 2 with the nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal genome. In some embodiments, the construction method includes replacing all or part of exons 1-10 of the human SERPINF2 gene with all or part of exons 1-10 of the non-human animal endogenous SERPINF2 gene in the non-human animal genome. In some embodiments, the construction method includes replacing part of exon 2 to all of exon 10 of the human SERPINF2 gene with part of exon 2 to all of exon 10 of the non-human animal endogenous SERPINF2 gene in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to the stop codon of the human SERPINF2 gene with the nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous SERPINF2 gene in the non-human animal genome. In some embodiments, the construction method includes replacing part of exon 2 of the human SERPINF2 gene to at least 20 bp of continuous nucleotide sequence downstream of the 3'UTR with part of exon 2 of the non-human animal endogenous SERPINF2 gene to at least 20 bp of continuous nucleotide sequence downstream of the 3'UTR in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to all of exon 10 of the human SERPINF2 gene with the nucleotide sequence from the start codon to all of exon 10 of the non-human animal endogenous SERPINF2 gene in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to at least 20 bp of continuous nucleotide sequence downstream of the 3'UTR of the human SERPINF2 gene with the nucleotide sequence from the start codon to at least 20 bp of continuous nucleotide sequence downstream of the 3'UTR of the non-human animal endogenous SERPINF2 gene in the non-human animal genome. In some embodiments, the construction method includes replacing the nucleotide sequence from the start codon to at least 300 bp of continuous nucleotide sequence downstream of the 3'UTR of the human SERPINF2 gene with the nucleotide sequence from the start codon to at least 58 bp of continuous nucleotide sequence downstream of the 3'UTR of the non-human animal endogenous SERPINF2 gene in the non-human animal genome. In some embodiments, the construction method includes replacing SEQ ID NO: 5 or 6 with the nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal genome.In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 1 in the genome of a non-human animal with a nucleotide sequence encoding a human or humanized SERPINF2 protein (such as a genomic DNA sequence, a CDS sequence, or a cDNA sequence) or a humanized SERPINF2 gene.

[0121] Use of a gene-modified non-human animal

[0122] 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 gene locus of the non-human animal and under the control of endogenous regulatory elements of the non-human animal (such as a promoter and / or 5'UTR) can produce a non-human animal with qualities and characteristics that may be significantly different from those of a typical knockout plus transgenic animal. In a typical knockout plus transgenic animal, the endogenous gene locus is removed or disrupted, and a fully human transgene is inserted into the genome of the non-human animal and may be randomly integrated into the genome. Generally, the location of the integrated transgene is unknown; the expression of a human protein is measured by transcription of a human gene and / or protein assay and / or functional assay. In a human transgene, the upstream and / or downstream of the human sequence provides appropriate support for the expression and / or regulation of the transgene.

[0123] A gene-modified non-human animal expressing a human or humanized SERPINF2 protein, for example, in a physiologically appropriate manner, provides various uses, including but not limited to developing therapeutic methods for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human therapeutic methods in an animal model.

[0124] The present invention also provides the use of a non-human animal modified with the above SERPINF2 gene and a non-human animal obtained by any of the above construction methods.

[0125] In some embodiments, the use includes:

[0126] A) Use in the development of products related to SERPINF2-related immune processes involving human cells;

[0127] B) Use as a model system related to SERPINF2 in pharmacological, immunological, microbiological, and medical research;

[0128] C) Use in the production and utilization of animal experimental disease models for SERPINF2-related etiological research and / or for developing diagnostic strategies and / or for developing therapeutic strategies;

[0129] D) Use in the screening, pharmacodynamic detection, efficacy evaluation, verification, or assessment of modulators of the human SERPINF2 signaling pathway in vivo; or,

[0130] E) Study the function of the SERPINF2 gene, study the drugs and drug effects targeting human SERPINF2 target sites, and study the applications in the treatment of drugs for tumors, inflammation, immune diseases or cerebrovascular diseases related to SERPINF2.

[0131] The present invention provides a non-human animal expressing human or humanized SERPINF2 protein, which can be used for screening human SERPINF2-specific regulators. 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 different embodiments, the genetically modified non-human animal further comprises a damaged immune system, such as genetically modified human xenograft, including human solid tumors (e.g., breast cancer) or hematological tumors (e.g., lymphocytic tumors (e.g., B or T cell tumors)).

[0132] In some embodiments, the therapeutic agent (e.g., an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug) blocks or inhibits the SERPINF2-mediated signaling pathway. In some embodiments, the therapeutic agent described in the present application (e.g., an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug) can block the interaction between SERPINF2 complexes, thereby inhibiting the SERPINF2 signaling pathway.

[0133] In some embodiments, the genetically modified non-human animal can be used to determine the effectiveness of a therapeutic agent (e.g., an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug) in the treatment of various immune diseases. In some embodiments, the immune diseases include, but are not limited to, GVHD (graft-versus-host disease), psoriasis, cirrhosis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty hepatitis), atopic dermatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, etc. In some embodiments, the immune diseases include asthma, cirrhosis, atopic dermatitis, psoriasis, rheumatoid arthritis or multiple sclerosis.

[0134] In some embodiments, the genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (such as antibodies targeting SERPINF2, nucleic acid drugs targeting SERPINF2, and / or polypeptide drugs) in treating various inflammations (such as infections). In some embodiments, the inflammation includes acute inflammation and also 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 includes inflammatory bowel disease (IBD).

[0135] In some embodiments, the genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (such as antibodies targeting SERPINF2, nucleic acid drugs targeting SERPINF2, and / or polypeptide drugs) in treating cerebrovascular diseases. In some embodiments, a therapeutic agent is administered to a non-human animal that has a cerebrovascular disease, and the effectiveness of the therapeutic agent in treating the cerebrovascular disease is determined. In some embodiments, the cerebrovascular disease is a thrombus, ischemic stroke, or stroke.

[0136] In some embodiments, the genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (such as antibodies targeting SERPINF2, nucleic acid drugs targeting SERPINF2, and / or polypeptide drugs) in treating cancer. In some embodiments, a therapeutic agent (such as an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug) is administered to a non-human animal that has 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 measurement with a vernier caliper, flow cytometry, and / or in vivo animal imaging detection. In some embodiments, the detection includes evaluating the body weight, fat mass, activation pathway, neuroprotective activity, or metabolic changes of an individual, and the metabolic changes include changes in food consumption or water consumption.

[0137] In some embodiments, the tumor cells include one or more cancer cells (such as cancer cells derived from a human or a non-human animal) that are injected into the non-human animal. In some embodiments, the therapeutic agent upregulates or inhibits the SERPINF2 signaling pathway. In some embodiments, the therapeutic agent does not upregulate or inhibit the SERPINF2 signaling pathway.

[0138] In some embodiments, genetically modified non-human animals can be used to detect whether a therapeutic agent (e.g., an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug) is an agonist or an antagonist. In some embodiments, the methods described in the present application can be used to detect the function of a therapeutic agent (e.g., an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug), e.g., 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 an effective dose of a therapeutic agent for treating a subject's disease (e.g., a tumor, an immune disease, an inflammation, or a cerebrovascular disease). In some embodiments, the inhibitory effect on a tumor can also be determined by methods known in the art, e.g., measuring the tumor volume in a non-human 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.

[0139] In some embodiments, therapeutic agents (e.g., antibodies targeting SERPINF2, nucleic acid drugs targeting SERPINF2, and / or polypeptide drugs) can be used to treat various cancers (tumors). As used herein, "cancer" or "tumor" refers to cells with the ability of autonomous growth, i.e., an abnormal state or disease characterized by rapid proliferation of cell growth. 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, "cancer" or "tumor" includes, but is not limited to, lymphoma, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioblastoma, lung cancer (including non-small cell 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 some embodiments, 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 includes solid tumors or hematological tumors. In some embodiments, the tumor includes liver cancer, lymphocyte tumor, head and neck cancer, or lung cancer.

[0140] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (e.g., an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2, and / or a polypeptide drug). The detection method includes administering the therapeutic agent to the non-human animal, evaluating the weight change of the non-human animal or performing a blood test, preferably the blood test 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 weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of the control group (e.g., the average weight of non-human animals not treated with the therapeutic agent).

[0141] 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 for model systems in pharmacological, immunological, microbiological, and medical research.

[0142] In some embodiments, there is provided an animal model generated by the method described in the present application for use in animal experimental disease models of the immune processes of human cells, studying pathogens, or formulating new diagnostic and / or treatment strategies.

[0143] 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 SERPINF2 gene, human SERPINF2 antibodies, or therapeutic drugs or their effectiveness for diseases related to the human SERPINF2 target (such as one or more of tumors, immune diseases, inflammation, or cerebrovascular diseases).

[0144] In some embodiments, the present disclosure 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 animals described in the present application and applying human CAR-T to the non-human animals having human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the animals are selected from SERPINF2 gene humanized non-human animals prepared by the method described in the present application, dual 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 SERPINF2 protein, or the tumor or inflammatory animal models described in the present application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat SERPINF2-related diseases described in the present application (such as one or more of tumors, immune diseases, inflammation, or cerebrovascular diseases). In some embodiments, TCR-T, CAR-T, and / or other immunotherapies provide an evaluation method for treating SERPINF2-related diseases described in the present application (such as one or more of tumors, immune diseases, inflammation, or cerebrovascular diseases).

[0145] Non-human animal models of two or more human or chimeric genes

[0146] The present invention also provides an animal model or non-human animal having two or more human or chimeric genes. The non-human animal or animal model may contain a human or chimeric SERPINF2 gene and a sequence encoding an additional human or chimeric protein.

[0147] In some embodiments, the additional human or chimeric protein comprises at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the non-human animal further expresses at least one of human or humanized LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 proteins.

[0148] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the construction method comprising:

[0149] (i) obtaining a non-human animal by the above construction method;

[0150] (ii) mating the non-human animal provided in step (i) with other genetically modified non-human animals, performing in vitro fertilization or directly performing gene editing, and screening to obtain a multi-gene modified non-human animal.

[0151] In some embodiments, the other genetically modified non-human animals include non-human animals humanized with one or a combination of two or more of the genes LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0152] In some embodiments, the humanization of the SERPINF2 gene is directly performed on a non-human animal having at least one gene modification of human or chimeric LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0153] Since these proteins may be involved in different mechanisms, combination therapies targeting two or more of these proteins may be a more effective treatment. In fact, many related clinical trials are underway and showing good results. Multigene-modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, e.g., therapeutic agents (such as antibodies targeting SERPINF2, nucleic acid drugs targeting SERPINF2, and / or polypeptide drugs), and additional therapeutic agents for treating diseases (such as tumors, immune diseases, inflammation, or cerebrovascular diseases). The method includes administering a therapeutic agent and an additional therapeutic agent to a non-human animal, where the non-human animal has a disease (such as a tumor, immune disease, inflammation, or cerebrovascular disease), and determining the effect of the combination treatment on the disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4, or a nucleic acid drug and / or polypeptide drug targeting 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 or 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 tumor includes one or more tumor cells that express PD-L1 and / or PD-L2.

[0154] In some embodiments, the combination treatment is used to treat the various cancers described in the present application. In some embodiments, the combination treatment is designed to treat the immune diseases described in the present application, such as psoriasis. In some embodiments, the methods described in the present application can be used to evaluate combination treatments 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, e.g., camptothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, thiotepa, nitrosourea, dactinomycin, daunorubicin, bleomycin, plicamycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, carboplatin, 5-fluorouracil, vincristine, vinblastine, and / or methotrexate. Alternatively, in addition, the method can include performing surgery on the subject to remove at least a portion of the cancer, e.g., removing a portion or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1 :Schematic diagram of the comparison between the mouse SERPINF2 locus and the human SERPINF2 locus (not to scale);

[0156] Figure 2 :Schematic diagram of the SERPINF2 gene targeting strategy and the design of targeting vector V1 (not to scale);

[0157] Figure 3 :Schematic diagram of the SERPINF2 gene targeting strategy and the design of targeting vector V2 (not to scale);

[0158] Figure 4 :Identification result of the F1 generation of SERPINF2 gene humanized mice by Southern blot, where WT is the wild-type control;

[0159] Figure 5 :RT-PCR detection result, where + / + is the wild-type C57BL / 6 mouse, H / H is the SERPINF2 gene humanized homozygous mouse, H 2 O is the water control, and GAPDH is the internal reference;

[0160] Figure 6 :ELISA detection result, where + / + is the wild-type C57BL / 6 mouse, and H / H is the SERPINF2 gene humanized homozygous mouse. Detailed implementation manners

[0161] 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 proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0162] In each of the following embodiments, the equipment and materials were obtained from the several companies indicated below:

[0163] C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Laboratory Animal Seed Center, National Institutes for Food and Drug Control, China;

[0164] BclI was purchased from NEB, catalog number R3160S;

[0165] BspHI was purchased from NEB, catalog number R0517S.

[0166] Example 1 SERPINF2 gene humanized mice

[0167] The comparative schematic diagram of the mouse SERPINF2 gene (NCBI Gene ID: 18816, located at positions 75322562 to 75330327 of NC_000077.7 on chromosome 11, based on transcript NM_008878.2 and its encoded protein NP_032904.1 (SEQ ID NO: 1)) and the human SERPINF2 gene (NCBI Gene ID: 5345, located at positions 1742871 to 1755265 of NC_000017.11 on chromosome 17, based on transcript NM_000934.4 and its encoded protein NP_000925.2 (SEQ ID NO: 2)) is as Figure 1 shown.

[0168] To achieve the object of the present invention, a nucleotide sequence encoding the human SERPINF2 protein can be introduced into the endogenous SERPINF2 gene locus of a mouse, such that the mouse expresses the human or humanized SERPINF2 protein. Specifically, using gene editing technology, under the control of the mouse SERPINF2 gene regulatory element, a partial sequence of exon 2 of the human SERPINF2 gene to about 10.6 kb downstream of the 3' UTR is used to replace a partial sequence of exon 2 of the mouse to about 6.8 kb downstream of the 3' UTR, obtaining a humanized SERPINF2 gene locus and realizing the humanization transformation of the mouse SERPINF2 gene.

[0169] To implement the targeting strategy of the present invention, a targeting vector V1 ( Figure 2 ) was constructed. The targeting vector V1 contains homologous arm sequences upstream and downstream of the mouse SERPINF2 gene, and a fragment A containing the human SERPINF2 fragment. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 3) is the same as the nucleotide sequence at positions 75329281 to 75333189 of NCBI accession number NC_000077.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 4) is the same as the nucleotide sequence at positions 75318585 to 75322503 of NCBI accession number NC_000077.7. The nucleotide sequence of the human SERPINF2 gene fragment (SEQ ID NO: 5) is the same as the nucleotide sequence at positions 1744996 to 1755565 of NCBI accession number NC_000017.11; the connection design of the upstream of the human SERPINF2 gene fragment sequence and the mouse sequence is:

[0170] where the sequence " GGAACThe "C" in "" is the last nucleotide at the upstream junction of the mouse sequence and the human SERPINF2 gene fragment sequence, and the sequence The "A" in is the first nucleotide of the human SERPINF2 gene fragment sequence. The connection of the downstream of the human SERPINF2 gene fragment sequence to the mouse sequence is designed as:

[0171]

[0172] where the "A" in the sequence "" CGCCA is the last nucleotide of the human SERPINF2 gene fragment sequence, and the first "G" in the sequence is the first nucleotide at the downstream junction of the mouse sequence and the human SERPINF2 gene fragment sequence.

[0173] The targeting vector also includes a resistance gene for positive clone screening, that is, the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites arranged in the same direction 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 to the human SERPINF2 gene is as follows:

[0174] where the "A" in the sequence "" CGCCA is the last nucleotide at the junction of the human SERPINF2 gene and the 5' end of the Neo cassette, and the first "G" in the sequence is the first nucleotide of the Neo cassette; the connection design of the 3' end of the Neo cassette to the mouse SERPINF2 gene is as follows:

[0175]

[0176] where the last "C" in the sequence "" ACTTC is the last nucleotide of the Neo cassette, and the first "G" in the sequence is the first nucleotide at the junction of the mouse SERPINF2 gene and the 3' end of the Neo cassette. The mRNA sequence transcribed from the humanized mouse SERPINF2 gene after modification is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0177] The construction of the targeting vector can be carried out by conventional methods, such as digestion and ligation. After the constructed targeting vector is preliminarily verified by digestion, it is 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 a positive clone selection marker gene to screen out the correct positive clone cells. The correctly screened positive clone cells (black mice) are introduced into the isolated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to a 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 interbred to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene, and then homozygous humanized mice with the SERPINF2 gene can be obtained by interbreeding.

[0178] In addition, the CRISPR / Cas9 technology can also be used for gene editing to further design the targeting vector V2( Figure 3 ). The targeting vector V2 contains homologous arm sequences upstream and downstream of the mouse SERPINF2 gene, as well as the human SERPINF2 fragment. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 31) is identical to the nucleotide sequence from position 75329281 to 75330558 of NCBI accession number NC_000077.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 32) is identical to the nucleotide sequence from position 75321512 to 75322503 of NCBI accession number NC_000077.7. The nucleotide sequence of the human SERPINF2 fragment (SEQ ID NO: 5) is identical to the nucleotide sequence from position 1744996 to 1755565 of NCBI accession number NC_000017.11. The mRNA sequence transcribed from the SERPINF2 gene in the modified humanized mouse is as shown in SEQ ID NO: 6, and the expressed protein sequence is as shown in SEQ ID NO: 2.

[0179] The construction of the targeting vector can be carried out by conventional methods, such as digestion and ligation, direct synthesis, etc. After the constructed targeting vector is preliminarily verified by digestion, it is sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is used for subsequent experiments.

[0180] The target sequence (target site) 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 premise 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 SERPINF2 gene are as follows:

[0181] sgRNA1 target site (SEQ ID NO: 21): 5'-GTGAGCGCCGTGGATCTCCCGGG-3';

[0182] sgRNA2 target site (SEQ ID NO: 22): 5'-GCTCAGTGCAATCTGCCCCGTGG-3';

[0183] After detecting the activity of sgRNA using the UCA kit and determining its high cleavage efficiency, restriction enzyme sites were added to the 5' end and complementary strand 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 linearized with BbsI first), and the expression vectors pT7-SERPINF2-1 and pT7-SERPINF2-2 were obtained.

[0184] Table 3 sgRNA1 and sgRNA2 sequence list

[0185]

[0186] The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment of DNA (SEQ ID NO: 33) with a T7 promoter and sgRNA scaffold, and was successively ligated to the backbone vector (from 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. Pronuclear stage fertilized eggs of mice, such as C57BL / 6 mice, were taken, and the in vitro transcription products of the pT7-SERPINF2-1 and pT7-SERPINF2-2 plasmids (using the Ambion in vitro transcription kit and transcribed according to the instructions), the targeting vector, and Cas9 mRNA were premixed and then injected 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 SERPINF2 gene.

[0187] 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 genomic DNA with BclI enzyme or BspHI enzyme respectively, transfer the membrane, and hybridize. Probe A (A Probe) and 3' probe (3' Probe) are located on the human SERPINF2 gene fragment and downstream of the 3' homologous arm respectively. The specific probes and the lengths of target fragments are shown in Table 4 below), and the exemplary results are as Figure 4 shown. Combining the PCR and sequencing results, 10 mice numbered from F1-01 to F1-10 are positive mice. This indicates that the method can construct humanized mice with SERPINF2 gene that can be stably passed on and have no random insertion.

[0188] Table 4 Specific probes and the lengths of target fragments

[0189] Restriction endonuclease Probe Wild-type fragment size Recombinant sequence fragment size BclI 3’ Probe 8.5 kb 12.8 kb BspHI A Probe -- 6.2 kb

[0190] A Probe-F (SEQ ID NO: 11): 5'-GAACTGTTTGAACCTGGGAGTTGG-3',

[0191] A Probe-R (SEQ ID NO: 12): 5'-CACCAGGAGGACTTGTAAATAAGCAG-3';

[0192] 3' Probe-F (SEQ ID NO: 13): 5'-CCCTGAACTAGAAGAGATCCTTTAAG-3', 3' Probe-R (SEQ ID NO: 14): 5'-CCACTTCCTCCTCCCCTCTGC-3';

[0193] The expression of mRNA in the humanized mice with SERPINF2 gene can be detected by RT-PCR. Specifically, 1 C57BL / 6 mouse (+ / +) and 1 humanized homozygous SERPINF2 gene mouse (H / H) prepared in this example are selected respectively. After decapitation and euthanasia, liver tissues are taken, and RT-PCR detection is carried out using the primer sequences shown in Table 5 below. The detection results are as Figure 5 shown. As can be seen from Figure 5 , only mouse SERPINF2 mRNA is detected in wild-type C57BL / 6 mice, and human SERPINF2 mRNA is not detected; only human SERPINF2 mRNA is detected in humanized homozygous SERPINF2 gene mice.

[0194] Table 5 RT-PCR primer sequences and the sizes of target fragments

[0195]

[0196] In addition, the expression of human SERPINF2 protein in SERPINF2 humanized mice can be detected by conventional methods such as ELISA. Specifically, three 8-week-old male C57BL / 6 mice (+ / +) and three 12-week-old male SERPINF2 gene homozygous humanized mice (H / H) prepared in this example were selected, plasma was taken, and detected using a human alpha-2 antiplasmin ELISA kit (abcam, ab254502). The detection results are as Figure 6 shown.

[0197] From Figure 6 it can be seen that when detected using a human-specific SERPINF2 ELISA kit, human SERPINF2 protein was detected only in SERPINF2 humanized homozygous mice. Combining the above RT-PCR results, it is proved that human SERPINF2 protein can be successfully expressed in SERPINF2 gene homozygous humanized mice.

[0198] Example 2 Pharmacodynamic model

[0199] The SERPINF2 gene humanized mice prepared in Example 1 of this application can be used to prepare various human disease models, including ischemic stroke, liver cirrhosis, thrombosis and other disease models, and can be used to test the in vivo pharmacodynamics of therapeutic agents (including antibodies targeting SERPINF2, nucleic acid drugs targeting SERPINF2 and / or polypeptide drugs). For example, SERPINF2 gene humanized mice can be used to evaluate the pharmacodynamics, pharmacokinetics of antagonists of the human-specific SERPINF2 signaling pathway and their in vivo therapeutic efficacy in various disease models known in the art.

[0200] For example, take SERPINF2 gene humanized mouse homozygotes, perform thrombosis modeling. After successful modeling, the mice are randomly divided into a control group or a treatment group. The treatment group is given a drug targeting human SERPINF2, and the control group is injected with an equal volume of normal saline. The in vivo safety and in vivo pharmacodynamics of the compound can be effectively evaluated by comparing the changes in mouse body weight and in vivo indicators.

[0201] Example 3 Preparation of double-gene or multi-gene humanized mice

[0202] The humanized mouse of the SERPINF2 gene constructed and obtained by using Example 1 of the present application can also be used to prepare a multi-gene humanized mouse model. For example, in the aforementioned Example 1, the embryonic stem cells used for microinjection can be selected from mice with gene modifications of at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. Alternatively, on the basis of the humanized SERPINF2 mouse, a double-gene humanized or multi-gene humanized mouse model can be obtained by using the techniques of isolating mouse ES embryonic stem cells and gene recombination and targeting. The homozygous or heterozygous humanized mouse of the SERPINF2 gene obtained in the present application 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 a multi-gene mouse with a humanized SERPINF2 gene and other gene modifications. Then, mating the heterozygotes with each other can obtain homozygotes with double-gene or multi-gene modifications.

[0203] 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 belong to the protection scope of the present invention.

[0204] 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 suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0205] In addition, 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 SerpinF2 protein.

2. The construction method according to claim 1, characterized in that: The amino acid sequence of the chimeric SERPINF2 protein comprises an amino acid sequence that is identical to at least 50 to 491 consecutive amino acids of a human SERPINF2 protein; Preferably, the amino acid sequence of the human or chimeric SERPINF2 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: At the non-human animal endogenous SERPINF2 gene locus, the nucleotide sequence of the corresponding region of the non-human animal endogenous SERPINF2 is replaced with a nucleotide sequence comprising human SERPINF2.

4. The construction method according to claim 3, characterized in that: The nucleotide sequence of human SERPINF2 comprises a nucleotide sequence encoding a human or chimeric SERPINF2 protein, preferably comprises a nucleotide sequence encoding SEQ ID NO: 2; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to a nucleotide sequence encoding SEQ ID NO: 2; Preferably, the nucleotide sequence of human SERPINF2 comprises a portion of exon 2 to the entirety of exon 10 of the human SERPINF2 gene, wherein the portion of exon 2 of the human SERPINF2 gene preferably comprises at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of human SERPINF2 comprises the nucleotide sequence from the start codon to the stop codon of the human SERPINF2 gene, preferably further comprises 3'UTR, and more preferably further comprises at least 20 bp of continuous nucleotide sequence downstream of 3'UTR; Preferably, the nucleotide sequence of human SERPINF2 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.

5. The construction method according to claim 3 or 4, characterized in that: The nucleotide sequence of the corresponding region of the endogenous SERPINF2 of a non-human animal comprises a nucleotide sequence encoding an endogenous SERPINF2 protein of a non-human animal, preferably comprises a nucleotide sequence encoding SEQ ID NO: 1; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity with the nucleotide sequence encoding SEQ ID NO: 1; Preferably, the nucleotide sequence of the corresponding region of the endogenous SERPINF2 of the non-human animal includes a portion of exon 2 to the entirety of exon 10 of the endogenous SERPINF2 gene of the non-human animal, wherein the portion of exon 2 of the endogenous SERPINF2 gene of the non-human animal preferably includes at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of the corresponding region of endogenous SERPINF2 of non-human animals includes the nucleotide sequence from the start codon to the stop codon of the endogenous SERPINF2 gene of non-human animals, preferably also includes 3'UTR, and more preferably also includes at least 20bp continuous nucleotide sequence downstream of 3'UTR.

6. The construction method according to any one of claims 1 to 5, characterized in that: The nucleotide sequence encoding the human or chimeric SERPINF2 protein or the nucleotide sequence of human SERPINF2 is operably linked to an endogenous regulatory element of an endogenous SERPINF2 locus; Preferably, the endogenous SERPINF2 protein of the non-human animal is not expressed or is expressed at a reduced level compared to SERPINF2 in wild-type animals; Preferably, the modified SERPINF2 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

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 rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified SERPINF2 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 SEQ ID NO: 6; Preferably, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins, and the other human or chimeric proteins preferably comprise at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

8. 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 7; 2) Determine the effect of therapeutic agents on non-human animals or diseases; Preferably, the therapeutic agent comprises an antibody targeting SERPINF2, a nucleic acid drug targeting SERPINF2 and / or a polypeptide drug; further preferably, the therapeutic agent further comprises an additional therapeutic agent, and the additional therapeutic agent comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the disease includes one or more of immune diseases or cerebrovascular diseases.

9. A humanized SERPINF2 gene, characterized in that: The humanized SERPINF2 gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 31 or 32; C) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 31 or 32; 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, 31 or 32.

10. A cell, tissue or organ, characterized in that: The genome of the cell, tissue or organ comprises the humanized SERPINF2 gene according to claim 9.

11. Use of the non-human animal obtained by the construction method according to any one of claims 1 to 7, the humanized SERPINF2 gene according to claim 9, or the cell, tissue or organ according to claim 10, characterized in that: The application includes: A) Application in product development involving SERPINF2-related immune processes in human cells; B) Application as a model system related to SERPINF2 for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for SERPINF2-related etiological studies 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 SERPINF2 signaling pathway modulators; or, E) To study the function of SERPINF2 gene, the drugs and their efficacy targeting human SERPINF2 target sites, and the application of drugs in the treatment of immune diseases or cerebrovascular diseases related to SERPINF2.

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