INHBE gene modified non-human animal

Abstract

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

Description

Technical Field

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

[0002] Traditional drug development usually uses in vitro screening methods, but these screening methods cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interactions, etc.), resulting in a high failure rate of drug development. In addition, given the differences between humans and animals, the test results obtained from in vivo pharmacology tests using conventional experimental animals may not reflect the actual disease state and the interaction of the target site, resulting in significant differences between the results of many clinical trials and the results of animal experiments.

[0003] Therefore, developing a humanized animal model suitable for screening and evaluation of human antibodies will significantly improve the efficiency of new drug development and reduce the cost of drug development. Summary of the invention

[0004] The present application provides an animal model with human or chimeric INHBE protein. The animal model can express human or chimeric INHBE (e.g., humanized INHBE) protein. It can be used for the study of INHBE gene function, and can also be used for the screening and evaluation of INHBE signaling pathway regulators (e.g., anti-human INHBE antibodies, targeted INHBE nucleic acid drugs and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamics research, treatment of INHBE-related diseases (e.g., tumors, inflammation, immune diseases, metabolic diseases or cardiovascular diseases) and disease treatment of human INHBE target sites; the model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of INHBE protein and provides a platform for screening related drugs.

[0005] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein the genome of the non-human animal comprises at least one chromosome, wherein the chromosome comprises a nucleotide sequence encoding a human or chimeric inhibin subunit βE (INHBE) protein. In some embodiments, the chimeric INHBE protein is a humanized INHBE protein, and the amino acid sequence of the humanized INHBE protein is at least 50 to 350 identical to that of the human INHBE protein, such as 50, 60, 70, 80, 90, 100, 110, 114, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,

[0006] In some embodiments, the amino acid sequence of the humanized INHBE protein is identical to at least 5 to 19, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 consecutive amino acid sequences of the signal peptide of the human INHBE protein. In some embodiments, the amino acid sequence of the humanized INHBE protein is identical to at least 50 to 217, such as 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 217 consecutive amino acid sequences of the leader peptide of the human INHBE protein. In some embodiments, the amino acid sequence of the humanized INHBE protein is identical to at least 50 to 114, such as 50, 60, 70, 80, 90, 100, 110 or 114 consecutive amino acid sequences of the peptide chain of the human INHBE protein. In some embodiments, the amino acid sequence of the human or chimeric INHBE 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. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent comprises a mouse or a rat. In some embodiments, the nucleotide sequence encoding the human or chimeric INHBE protein is operably linked to an endogenous regulatory element of the non-human animal or a human regulatory element, such as a promoter, a 5'UTR and / or a 3'UTR. In some embodiments, the endogenous INHBE protein of the non-human animal is not expressed or is expressed at a reduced level compared to INHBE in a wild-type animal.

[0007] On the one hand, the present invention provides a method for constructing a genetically modified non-human animal, wherein the nucleotide sequence of the corresponding region of the endogenous INHBE of the non-human animal is replaced with a nucleotide sequence comprising human INHBE at the endogenous INHBE locus of the non-human animal. In some embodiments, the nucleotide sequence of human INHBE comprises a nucleotide sequence encoding a human INHBE protein. In some embodiments, the nucleotide sequence of human INHBE comprises a nucleotide sequence encoding a signal peptide of a human INHBE protein. In some embodiments, the nucleotide sequence of human INHBE comprises a nucleotide sequence encoding a leader peptide of a human INHBE protein. In some embodiments, the nucleotide sequence of human INHBE comprises a nucleotide sequence encoding a peptide chain of a human INHBE protein. In some embodiments, the nucleotide sequence of human INHBE comprises a nucleotide sequence encoding 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 with a nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the nucleotide sequence of human INHBE comprises a nucleotide sequence from the start codon to the stop codon of the human INHBE gene, preferably further comprising a nucleotide sequence of 3'UTR. In some embodiments, the nucleotide sequence of human INHBE comprises a nucleotide sequence from part of exon 1 to all of exon 2 of the human INHBE gene, wherein the part of exon 1 of the human INHBE gene comprises at least 5-528 bp, such as at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 295, 296, 297, 298, 299, 300, 350, 400, 450, 500, 520 or 528 bp of continuous nucleotide sequence. In some embodiments, the part of exon 1 of the human INHBE gene comprises a nucleotide sequence of the coding region. In some embodiments, the nucleotide sequence of human INHBE further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR of the human INHBE gene, such as at least 50, 100, 200, 300, 400, 500, 600, 700, 750, 752, 800, 900, 1000, 1200, 1400, 1600, 1800 or 2000 bp, preferably at least 50 bp to 2000 bp of continuous nucleotide sequence, further preferably at least 500 bp to 1000 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of human INHBE comprises at least 50 bp of continuous nucleotide sequence from part of exon 1 of the human INHBE gene to the downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human INHBE comprises at least 50 bp of continuous nucleotide sequence from the start codon of the human INHBE gene to the downstream of the 3'UTR.In some embodiments, the nucleotide sequence of human INHBE 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. In some embodiments, the nucleotide sequence of the corresponding region of endogenous INHBE in a non-human animal comprises a nucleotide sequence from the start codon to the stop codon of the endogenous INHBE gene in a non-human animal, and preferably further comprises a nucleotide sequence of 3'UTR. In some embodiments, the nucleotide sequence of the corresponding region of endogenous INHBE in a non-human animal comprises a nucleotide sequence from part of exon 1 to all of exon 2 of the endogenous INHBE gene in the non-human animal, wherein the part of exon 1 of the endogenous INHBE gene in the non-human animal comprises at least 5-601bp, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 295, 296, 297, 298, 299, 300, 350, 400, 450, 500, 550, 600 or 601bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of the corresponding region of endogenous INHBE in non-human animals further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR of the endogenous INHBE gene in non-human animals, such as at least 50, 100, 200, 300, 400, 500, 600, 700, 730, 736, 750, 800, 900, 1000, 1200, 1400, 1600, 1800 or 2000 bp, preferably at least 50 bp to 2000 bp of continuous nucleotide sequence, further preferably at least 50 bp to 1000 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of human INHBE is operably linked to an endogenous regulatory element of a non-human animal or a human regulatory element, such as a promoter, 5'UTR and / or 3'UTR. In some embodiments, the endogenous INHBE protein of the non-human animal is not expressed or the expression level is reduced compared to INHBE in wild-type animals. In some embodiments, the modified INHBE gene in the genome of the non-human animal is homozygous or heterozygous for the endogenously replaced locus. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent comprises a mouse or a rat. In some embodiments, the mRNA transcribed from the modified INHBE gene in the genome of the non-human animal comprises SEQ ID NO: 6; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 6.In some embodiments, 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 LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0008] On the one hand, 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 at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric inhibin subunit βE (INHBE) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric INHBE protein is operably linked to an endogenous regulatory element or a human regulatory element (e.g., a promoter, 5'UTR and / or 3'UTR) of an endogenous INHBE locus of at least one chromosome. In some embodiments, the amino acid sequence of the human or chimeric INHBE protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to human INHBE (NP_113667.1, SEQ ID NO: 2). In some embodiments, the non-human animal is a mammal, such as a monkey, a rodent (mouse or rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous INHBE protein of the non-human animal is not expressed or the expression level is reduced compared to INHBE in a wild-type animal. In some embodiments, one or more cells of the non-human animal express human or chimeric INHBE protein.

[0009] On the one hand, 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 INHBE region at an endogenous INHBE locus, and the nucleotide sequence encoding the corresponding region of human INHBE is replaced by a nucleotide sequence encoding the corresponding region of human INHBE. In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE is operably linked to an endogenous regulatory element or a human regulatory element of an endogenous INHBE locus. In some embodiments, one or more cells of the non-human animal express human or humanized INHBE protein. In some embodiments, the endogenous INHBE protein of the non-human animal is not expressed or the expression level is reduced compared to INHBE in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises part of exon 1 to all of exon 2 of the human INHBE gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises part of exon 1 of the human INHBE gene to at least 50bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises part of exon 1 of the human INHBE gene to at least 752bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous INHBE region includes a portion of exon 1 to all of exon 2 of the endogenous INHBE gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the endogenous INHBE region includes a portion of exon 1 to at least 50 bp of continuous nucleotides downstream of the 3'UTR of the endogenous INHBE gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the endogenous INHBE region includes a portion of exon 1 to 736 bp of continuous nucleotides downstream of the 3'UTR of the endogenous INHBE gene of a non-human animal. In some embodiments, the modified INHBE gene in the genome of the non-human animal is homozygous or heterozygous for the endogenously replaced locus.

[0010] 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 comprising a nucleotide sequence encoding a human or humanized INHBE protein. In some embodiments, the humanized INHBE protein comprises an amino acid sequence that is consistent with at least 50, 114, 300, 331, or 350 consecutive amino acid sequences of a human INHBE protein. In some embodiments, the non-human animal expresses a human or humanized INHBE protein. In some embodiments, the nucleotide sequence encoding a human or humanized INHBE protein is operably linked to an endogenous INHBE regulatory element or a human INHBE regulatory element. In some embodiments, the nucleotide sequence encoding a human or humanized INHBE protein may be integrated into an endogenous INHBE locus of the non-human animal. In some embodiments, the human or humanized INHBE protein has at least one activity, such as an endogenous INHBE activity of a non-human animal and / or a human INHBE activity.

[0011] On the one hand, the present invention provides a method for constructing a genetically modified non-human animal, the construction method comprising replacing a nucleotide sequence encoding an endogenous INHBE region with a nucleotide sequence encoding a corresponding region of human INHBE at an endogenous INHBE locus of at least one cell of the non-human animal. In some embodiments, the endogenous INHBE protein of the non-human animal is not expressed or the expression level is reduced compared with INHBE in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises part of exon 1 of the human INHBE gene and all of exon 2 (preferably also comprising intron 1). In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises part of exon 1 of the human INHBE gene, all of exon 2, and at least 50bp of continuous nucleotides downstream of the 3'UTR (preferably also comprising intron 1). In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises part of exon 1 of the human INHBE gene, all of exon 2, and at least 752bp of continuous nucleotides downstream of the 3'UTR (preferably also comprising intron 1). In some embodiments, the amino acid sequence of the human INHBE corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human INHBE corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous INHBE region comprises a portion of exon 1 and all of exon 2 of an endogenous INHBE gene of a non-human animal (preferably further comprising intron 1). In some embodiments, the nucleotide sequence encoding the endogenous INHBE region comprises a portion of exon 1, all of exon 2 and at least 50 bp of continuous nucleotides downstream of the 3'UTR of an endogenous INHBE gene of a non-human animal (preferably further comprising intron 1). In some embodiments, the nucleotide sequence encoding the endogenous INHBE region comprises part of exon 1, all of exon 2, and 736 bp of continuous nucleotides downstream of the 3'UTR of the endogenous INHBE gene of a non-human animal (preferably also comprising intron 1). In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE is operably linked to an endogenous INHBE regulatory element or a human INHBE regulatory element, such as a promoter, 5'UTR, or 3'UTR. In some embodiments, the non-human animal is a mammal, such as a monkey, a rodent (mouse or rat).In some embodiments, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins selected from at least one of LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0012] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing a human or chimeric INHBE, the construction method comprising replacing a nucleotide sequence encoding an endogenous INHBE region with a nucleotide sequence encoding a corresponding region of human INHBE at an endogenous INHBE locus of a non-human animal, producing a genetically modified non-human animal cell, the non-human animal cell expressing a human or chimeric INHBE protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises a portion of exon 1 of the human INHBE gene and all of exon 2 (preferably also comprising intron 1). In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises a portion of exon 1 of the human INHBE gene, all of exon 2, and at least 50bp of continuous nucleotides downstream of the 3'UTR (preferably also comprising intron 1). In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE comprises a portion of exon 1 of the human INHBE gene, all of exon 2, and at least 752bp of continuous nucleotides downstream of the 3'UTR (preferably also comprising intron 1). In some embodiments, the amino acid sequence of the human INHBE corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human INHBE corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous INHBE region comprises a portion of exon 1 and all of exon 2 of an endogenous INHBE gene of a non-human animal (preferably further comprising intron 1). In some embodiments, the nucleotide sequence encoding the endogenous INHBE region comprises a portion of exon 1, all of exon 2 and at least 50 bp of continuous nucleotides downstream of the 3'UTR of an endogenous INHBE gene of a non-human animal (preferably further comprising intron 1). In some embodiments, the nucleotide sequence encoding the endogenous INHBE region comprises part of exon 1, all of exon 2, and 736 bp of continuous nucleotides downstream of the 3'UTR of the endogenous INHBE gene of a non-human animal (preferably also comprising intron 1). In some embodiments, the nucleotide sequence encoding the corresponding region of human INHBE is operably linked to a regulatory element of endogenous INHBE or a regulatory element of human INHBE, such as a promoter, 5'UTR, or 3'UTR. In some embodiments, the non-human animal is a mouse.

[0013] On the one hand, a method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease is provided, the method comprising: 1) administering a therapeutic agent to any of the non-human animals described above or to any of the non-human animals obtained by the construction methods described above; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the therapeutic agent comprises an anti-INHBE antibody, a nucleic acid drug targeting INHBE, and / or a polypeptide drug, and preferably further comprises an additional therapeutic agent, such as one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the non-human animal suffers from a disease. In some embodiments, the disease comprises one or more of cancer, immune disease, inflammation, metabolic disease, or cardiovascular disease.

[0014] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer (tumor), the method comprising: 1) administering a therapeutic agent to any of the non-human animals described above or to any of the non-human animals obtained by the construction methods described above, 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 is an anti-INHBE antibody, a nucleic acid drug targeting INHBE, or a polypeptide drug. In some embodiments, the tumor comprises one or more tumor cells injected into an animal. In some embodiments, determining the inhibitory effect of the therapeutic agent on the tumor involves measuring the tumor volume in a non-human animal. In some embodiments, the cancer comprises a solid tumor or a hematological tumor. In some embodiments, the cancer comprises one or more of head and neck cancer, breast cancer, lymphoma, liver cancer, or lung cancer.

[0015] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent (e.g., an anti-INHBE antibody or a nucleic acid drug or polypeptide drug targeting INHBE) and an additional therapeutic agent in treating cancer, the method comprising: 1) administering a therapeutic agent and an additional therapeutic agent to any of the non-human animals described above or to any of the non-human animals obtained by the construction method described above, wherein the non-human animal has a tumor; 2) determining the inhibitory effect on the tumor. In some embodiments, the non-human animal further comprises a sequence encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4. In some embodiments, the additional therapeutic agent is one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1. In some embodiments, the tumor comprises one or more tumor cells injected into a non-human animal. In some embodiments, the determination of the inhibitory effect on the tumor involves measuring the tumor volume in a non-human animal. In some embodiments, the cancer comprises a solid tumor or a hematological tumor. In some implementations, the cancer comprises one or more of head and neck cancer, breast cancer, lymphoma, liver cancer, or lung cancer.

[0016] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent (e.g., an anti-INHBE antibody or a nucleic acid drug or polypeptide drug targeting INHBE) in treating an immune disease, the method comprising: 1) administering a therapeutic agent to any of the non-human animals described above or to a non-human animal obtained by any of the construction methods described above, 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 immune disease comprises one or more of asthma, atopic dermatitis, psoriasis, rheumatoid arthritis, or multiple sclerosis.

[0017] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent (e.g., an anti-INHBE antibody or a nucleic acid drug or polypeptide drug targeting INHBE) in treating inflammation, the method comprising: 1) administering a therapeutic agent to any of the non-human animals described above or to a non-human animal obtained by any of the construction methods described above, wherein the non-human animal has inflammation; 2) determining the therapeutic effect of the therapeutic agent on treating inflammation. In some embodiments, the inflammation includes hepatitis or inflammatory bowel disease (IBD).

[0018] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent (e.g., an anti-INHBE antibody or a nucleic acid drug or polypeptide drug targeting INHBE) in treating a metabolic disease, the method comprising: 1) administering a therapeutic agent to any of the non-human animals described above or to a non-human animal obtained by any of the construction methods described above, wherein the non-human animal has a metabolic disease; 2) determining the therapeutic effect of the therapeutic agent on treating the metabolic disease. In some embodiments, the metabolic disease includes obesity or type II diabetes.

[0019] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent (e.g., an anti-INHBE antibody or a nucleic acid drug or polypeptide drug targeting INHBE) in treating cardiovascular disease, the method comprising: 1) administering a therapeutic agent to any of the non-human animals described above or to a non-human animal obtained by any of the construction methods described above, wherein the non-human animal has a cardiovascular disease; 2) determining the therapeutic effect of the therapeutic agent on the treatment of the cardiovascular disease. In some embodiments, the cardiovascular disease includes coronary heart disease.

[0020] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent (e.g., an anti-INHBE antibody or a nucleic acid drug or polypeptide drug targeting INHBE), the method comprising: 1) administering a therapeutic agent to any of the non-human animals described above or to any of the non-human animals obtained by the construction methods described above; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the determination of the effect of the therapeutic agent on the non-human animal involves measuring the weight of the animal or a blood test. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit and / or hemoglobin content.

[0021] In one aspect, the present invention provides a humanized INHBE gene, comprising 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.

[0022] In one aspect, the present invention provides a cell, tissue or organ, wherein the genome of the cell, tissue or organ comprises the humanized INHBE gene,

[0023] Or, the cells, tissues or organs express human INHBE protein.

[0024] In one aspect, the present invention provides an animal model, comprising the humanized INHBE gene.

[0025] In one aspect, the present invention provides an application of any of the above-mentioned non-human animals, any of the above-mentioned construction methods, any of the above-mentioned non-human animal cells obtained by the construction methods, the humanized INHBE gene, the cells, tissues or organs, and the animal model, the applications comprising: A) application in the development of products involving INHBE-related immune processes in human cells; B) application as a model system related to INHBE in pharmacology, immunology, microbiology and medical research; C) application involving the production and use of animal experimental disease models for INHBE-related pathology research and / or for the development of diagnostic strategies and / or for the development of treatment strategies; D) application in the screening, efficacy testing, efficacy evaluation, verification or evaluation of human INHBE signaling pathway regulators in vivo; or, E) application in studying the function of the INHBE gene, studying drugs and efficacy targeting human INHBE target sites, and studying therapeutic drugs for tumors, inflammation, immune diseases, metabolic diseases or cardiovascular diseases related to INHBE.

[0026] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to a part of the whole, or an individual part that constitutes the whole.

[0027] The term "locus" in the present invention refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a gene in a narrow sense, which can be a gene, a part of a gene, or a regulatory region of a gene, etc. For example, the "INHBE locus" includes a DNA fragment selected from exons 1-2 of the INHBE gene.

[0028] The term "part of exon XX" of the present invention means that a continuous or intermittent nucleotide sequence of several, dozens or hundreds of nucleotide sequences is consistent with the entire exon nucleotide sequence, such as a part of exon 1 of the INHBE gene, including a continuous 5-528 bp, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 295, 296, 297, 298, 299, 300, 350, 400, 450, 500, 520 or 528 bp continuous nucleotide sequence. In some embodiments, the part of exon 1 of the human INHBE gene includes the nucleotide sequence of the coding region.

[0029] The term "exon XX to exon XXX" or "exon XX-XXX" or "exon XX to all of exon XXX" of the present invention refers to the nucleotide sequence including exons and introns in between, for example, exon 1-2 includes all of exon 1, all of intron 1 and all of exon 2.

[0030] The term "part of exon XX to all of exon XXX" in the present invention refers to a nucleotide sequence including part of one exon to all of another exon and introns in between, for example, part of exon 1 to all of exon 2 includes part of exon 1, all of intron 1 and all of exon 2.

[0031] The term "intron xx" in the present invention refers to an intron between two exons, for example, intron 1 is an intron between exon 1 and exon 2.

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

[0033] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0034] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. This application describes methods and materials for use in the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are exemplary and non-limiting only. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0035] Those skilled in the art can easily appreciate other aspects and advantages of the present application from the following detailed description.

[0036] INHBE

[0037] In the human genome, the INHBE gene (NCBI Gene ID: 83729, UniProt ID: P58166, located at positions 57455307 to 57458025 of chromosome 12 NC_000012.12) contains two exons, namely exon 1 and exon 2. The corresponding positions of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_031479.5 and its encoded protein NP_113667.1 (SEQ ID NO: 2) are shown in Table 1:

[0038] Table 1

[0039]

[0040] In the mouse genome, the INHBE gene (NCBI Gene ID: 16326, UniProt ID: O08717, located at positions 127185271 to 127187717 of chromosome 10 NC_000076.7) contains two exons, namely exon 1 and exon 2. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_008382.3 and its encoded protein NP_032408.2 (SEQ ID NO: 1) is shown in Table 2:

[0041] Table 2

[0042]

[0043] INHBE genes, proteins and gene loci of other species are also known in the art. 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 be found in NCBI, the entire contents of which are incorporated into this application by reference.

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

[0045] The percentage (homology percentage) of conservative residues 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 (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0046] Carrier

[0047] The present invention provides a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of the switch region to be changed (5' arm or 5' homology arm), which is selected from the genomic DNA of the INHBE gene and has a length of 100-10000 bp nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the switch region to be changed (3' arm or 3' homology arm), which is selected from the genomic DNA of the INHBE gene and has a length of 100-10000 bp nucleotides.

[0048] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000076.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000076.7.

[0049] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 127187415 to 127191477 of NCBI Accession No. NC_000076.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 127180557 to 127184534 of NCBI Accession No. NC_000076.7.

[0050] In some embodiments, a) a DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 127187415 to 127188991 of NCBI Accession No. NC_000076.7; c) a DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 127183015 to 127184534 of NCBI Accession No. NC_000076.7.

[0051] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.8 kb, 1 kb, 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.

[0052] In some embodiments, the switch region to be altered is located on the nucleotide sequence downstream of exon 1 to 3'UTR of the endogenous INHBE gene of the non-human animal.

[0053] In some embodiments, the 5' homology arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 3 or 31. In some embodiments, the 3' homology arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 4 or 32.

[0054] In some embodiments, the targeting vector comprises a human sequence (eg, nucleotide sequence 57455537 to 57458777 of NC_000012.12). In some embodiments, the nucleotide sequence of the targeting vector encodes all or part of the human INHBE protein.

[0055] In some embodiments, the targeting vector comprises one or more marker genes (resistance genes). For example, the coding gene of the resistance gene for positive clone screening and / or the negative screening marker. In some embodiments, the resistance gene for positive clone screening is the neomycin phosphotransferase coding sequence Neo. Preferably, the targeting vector also includes two Frt recombination sites arranged in the same direction on both sides of the marker gene. In some embodiments, the coding gene of the negative screening marker is the coding gene (DTA) of the diphtheria toxin A subunit.

[0056] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the INHBE gene, and the sgRNA is unique on the target sequence (target site) of the conversion region to be changed, and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG-3' or 5'-CCN-N(20)-3'. In some embodiments, the target site of the sgRNA in the endogenous INHBE gene of a non-human animal is located at the 5'UTR upstream nucleotide sequence, exon 1, intron 1, exon 2 and / or 3'UTR downstream nucleotide sequence. In some embodiments, the target site of the sgRNA in the endogenous INHBE gene of a non-human animal is located at exon 1, and / or, 3'UTR downstream nucleotide sequence.

[0057] In some embodiments, the target site is shown as SEQ ID NO: 21 and / or 22. Therefore, the present invention provides sgRNAs for constructing genetically modified animal models. 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.

[0058] In some embodiments, the present application provides a plasmid construct comprising an sgRNA sequence (ie, an sgRNA vector, such as pT7-sgRNA) and / or a cell comprising the construct.

[0059] The present invention also provides a cell comprising the targeting vector and / or sgRNA vector as described above.

[0060] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned vectors and one or more in vitro transcripts of the construct described in the present application. In some embodiments, the non-human mammalian cell further comprises Cas9 mRNA or its in vitro transcript.

[0061] In some embodiments, the non-human animal cell is a mouse cell. In some embodiments, the non-human animal cell is a fertilized egg cell. In some embodiments, the non-human animal cell is an embryonic stem cell. In some embodiments, the non-human animal cell can be any cell expressing INHBE.

[0062] Genetically modified non-human animals

[0063] The "genetically modified non-human animal" or "genetically modified non-human animal" described in the present invention refers to at least one chromosome in the genome of the non-human animal having exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal or the genetically modified non-human animal have exogenous DNA. Cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (such as T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal or a genetically modified non-human animal is provided, wherein the non-human animal comprises a modified endogenous INHBE locus, comprising an exogenous sequence (such as a human sequence), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. Non-human animals are generally able to pass genetic modifications to offspring through germline transmission.

[0064] The "chimeric gene" or "chimeric nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least a portion of the sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, a chimeric gene or chimeric nucleic acid has at least a portion of the sequence having two or more different species sources, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, a chimeric gene or chimeric nucleic acid refers to a humanized gene or humanized nucleic acid.

[0065] The "chimeric protein" or "chimeric polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more parts of the polypeptide or protein are from different species, or at least a portion of the sequence of the protein or polypeptide is different from the amino acid sequence in the wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric protein or chimeric polypeptide has two or more different species sources, for example, the same (or homologous) protein of different species. In some embodiments, the chimeric protein or chimeric polypeptide refers to a humanized protein or humanized polypeptide.

[0066] The "humanized protein" or "humanized polypeptide" of the present invention refers to a protein or polypeptide. In some embodiments, at least a portion of the protein or polypeptide is derived from a human protein or human polypeptide. In some embodiments, the humanized protein or humanized polypeptide refers to a human protein or polypeptide.

[0067] The "humanized nucleic acid" or "humanized gene" of the present invention refers to a nucleic acid or a gene. In some embodiments, at least a portion of the nucleic acid or gene is from a human nucleic acid or a human gene. In some embodiments, the nucleic acid or gene in the humanized nucleic acid or humanized gene is all derived from humans. In some embodiments, the humanized nucleic acid or humanized gene refers to a humanized exon, and the humanized exon can be a human exon or a chimeric exon.

[0068] In some embodiments, the chimeric gene or chimeric nucleic acid is a humanized INHBE gene or a humanized INHBE nucleic acid. In some embodiments, at least a portion of the humanized INHBE gene or humanized INHBE nucleic acid is derived from a human INHBE gene. In some embodiments, at least a portion of the humanized INHBE gene or humanized INHBE nucleic acid is derived from a non-human animal INHBE gene. In some embodiments, the humanized INHBE gene or humanized INHBE nucleic acid comprises a sequence encoding an INHBE protein. The encoded INHBE protein has at least one activity, such as the activity of a human INHBE protein and / or a non-human animal INHBE protein. In some embodiments, the humanized INHBE gene or humanized INHBE nucleic acid comprises, from the 5' end to the 3' end, a 5'UTR upstream nucleotide sequence of a non-human animal INHBE gene, a portion of exon 1 of a non-human animal INHBE gene, a portion of exon 1 of a human INHBE gene, all of intron 1 of a human INHBE gene, all of exon 2 of a human INHBE gene, and a 3'UTR downstream nucleotide sequence of a human INHBE gene. In some embodiments, the humanized INHBE gene or humanized INHBE nucleic acid comprises, from the 5' end to the 3' end, the 5'UTR upstream nucleotide sequence of the non-human animal INHBE gene, the 5'UTR of the non-human animal INHBE gene, the start codon to the stop codon of the human INHBE gene, the 3'UTR of the human INHBE gene, and the 3'UTR downstream nucleotide sequence of the human INHBE gene.

[0069] In some embodiments, the chimeric protein or chimeric polypeptide is a humanized INHBE protein or a humanized INHBE polypeptide. In some embodiments, at least one or more parts of the amino acid sequence of the humanized INHBE protein or the humanized INHBE polypeptide are from a human INHBE protein. In some embodiments, at least one or more parts of the amino acid sequence of the humanized INHBE protein or the humanized INHBE polypeptide are from a non-human animal INHBE protein. In some embodiments, the humanized INHBE protein or the humanized INHBE polypeptide refers to a human INHBE protein or a human INHBE polypeptide. In some embodiments, the humanized INHBE protein or the humanized INHBE polypeptide is functional, or has at least one activity, such as the activity of a human INHBE protein and / or a non-human animal INHBE protein.

[0070] The non-human animal of genetic modification can be various animals, for example, mouse, rat, rabbit, pig, cattle (for example, cattle, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (for example, marmoset, rhesus monkey).For the non-human animal of suitable genetically modified embryonic stem cell (ES) that is not easy to obtain, adopt alternative methods to construct the non-human animal comprising genetic modification.Such method includes, for example, modifying non-ES cell genome (for example, fibroblast or induced pluripotent stem cell) and adopting nuclear transplantation to transfer the modified genome to suitable cell, for example oocyte, and incubating the modified cell (for example, modified oocyte) in non-human animal under appropriate conditions to form embryo.The above-mentioned construction method is known in the art, and is described in "A.Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), " Cold Spring Harbor Laboratory Press, 2006 ", the entire contents of which are incorporated into the present application by reference.

[0071] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. Rodents can be selected from mice, rats and hamsters. In some embodiments, the rodent is selected from the family of Muridae. In some embodiments, the genetically modified non-human animal is from a family selected from the family of Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (mice and rats, gerbils, spiny mice, crested rats), Isla Muscidae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Spiny Dormouse (e.g., spiny dormouse) and Muridae (e.g., mole rats, bamboo rats and zokors). In some embodiments, the genetically modified rodent is selected from mice or rats (Muroidea), gerbils, spiny mice and crested rats. In some embodiments, the genetically modified mouse is from a member of the Muridae family. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[0072] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, an immunodeficient monkey, 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, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their 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 BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid of a 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 certain embodiments, non-human animals are rodents. In certain embodiments, non-human animals are mice with BALB / c, BALB / cHeAn, BALB / cJ, BALB / cR1, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strains. In certain embodiments, non-human animals are rats. Rats can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6 and Dark Agouti.In some embodiments, the rat strain is a hybrid of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0073] The non-human animal may have one or more other genetic modifications and / or other modifications that are suitable for the specific purpose of preparing a humanized animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) may have one or more modifications that damage, inactivate, or destroy all or part of the immune system of the non-human animal. Damage, inactivation, or destruction of the immune system of the non-human animal may include, for example, chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and / or genetic modification (e.g., knocking out 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 Mice (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 be optionally irradiated or otherwise treated to destroy one or more immune cell types. Therefore, in various embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of the endogenous non-human INHBE locus, and also includes damage, inactivation or partial destruction of the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the mouse modification type is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null Mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scid IL-2Rγ null Mouse, NOD Rag 1 - / - IL - / - (NRG) mouse, Rag2 - / - IL - / -(RG) mice and modifications thereof. These transgenic animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mice may comprise replacement of all or part of the mouse endogenous mature INHBE coding sequence with all or part of the human mature INHBE coding sequence, respectively.

[0074] Genetically modified non-human animals include modifications of endogenous INHBE gene loci in non-human animals. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature INHBE protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a mature INHBE protein amino acid sequence). Although the present invention provides cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein.

[0075] The genetically modified non-human animal can express human INHBE and / or chimeric (e.g., humanized) INHBE at the endogenous locus of the non-human animal, wherein the endogenous INHBE gene of the non-human animal has been replaced or inserted with a nucleotide sequence of a human INHBE gene and / or a region encoding a human INHBE sequence or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% identical to the human INHBE sequence. In some embodiments, the endogenous INHBE locus of the non-human animal is modified by a nucleic acid sequence comprising all or part of the nucleic acid sequence encoding a mature human INHBE protein.

[0076] In some embodiments, the genetically modified non-human animal can express human INHBE and / or chimeric INHBE (e.g., humanized INHBE) under the control of endogenous or human regulatory elements. Insertion or replacement at the endogenous locus of the non-human animal provides a non-human animal that expresses human INHBE or chimeric INHBE (e.g., humanized INHBE) in suitable cells and in a manner that does not cause potential pathology observed in some other transgenic mice known in the art. Human INHBE or chimeric INHBE (e.g., humanized INHBE) expressed in non-human animals can maintain one or more functions of wild-type non-human animals or human INHBE in non-human animals. In some embodiments, non-human animals do not express endogenous INHBE. In some embodiments, the expression level of endogenous INHBE in non-human animals is reduced compared to the expression level of INHBE in wild-type animals. As used herein, the term "endogenous INHBE" refers to an INHBE protein expressed by an endogenous INHBE nucleotide sequence of a non-human animal (e.g., mouse) before any genetic modification.

[0077] In some embodiments, the humanized INHBE gene comprises the 5'UTR of the human INHBE gene. In some embodiments, the humanized INHBE gene comprises the 5'UTR of the endogenous (e.g., mouse endogenous) INHBE gene of a non-human animal. In some embodiments, the humanized INHBE gene comprises the 3'UTR of the endogenous (e.g., mouse endogenous) INHBE gene of a non-human animal. In some embodiments, the humanized INHBE gene comprises the 3'UTR of the INHBE gene of a human. In appropriate cases, it can be reasonably assumed that, based on the similarity of the non-human animal and human INHBE gene sequences, they appear to be similarly regulated. As shown in the present application, the humanized INHBE mouse comprising an insertion or replacement in the endogenous INHBE locus of a non-human animal, which comprises the humanization of the INHBE coding sequence, does not show pathological phenomena. Both genetically modified mice of humanized INHBE heterozygous or homozygous are normal.

[0078] The present invention further relates to the genomic DNA sequence of INHBE gene humanized mice, the DNA sequence obtained by mRNA reverse transcription is consistent with or complementary to the DNA sequence; a construct expressing its amino acid sequence; a cell containing its construct; and a tissue or organ including the cell.

[0079] The present invention further relates to a non-human mammal produced by the above method. In some embodiments, the genome of the non-human mammal comprises all or part of a human INHBE gene or a humanized INHBE gene.

[0080] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0081] In some embodiments, the non-human mammal expresses a protein encoded by a humanized INHBE gene, eg, a human or humanized INHBE protein.

[0082] In addition, the present invention also provides a non-human mammal model carrying a tumor, wherein the non-human mammal model is obtained by the method described in the present application.

[0083] In some embodiments, the non-human mammal is a rodent (eg, a mouse).

[0084] The present invention also provides a cell or cell line, or primary cell culture, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its offspring. When the non-human mammal carries a tumor, the non-human mammal carries a tumor tissue derived from the non-human mammal or its offspring, or a non-human mammal carrying a tumor.

[0085] The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal, a genetically modified non-human animal, wherein the genome of the genetically modified non-human animal comprises DNA of a human or humanized INHBE is provided.

[0086] In some embodiments, a non-human mammal comprises a gene construct described herein. In some embodiments, a non-human mammal expressing a human or humanized INHBE protein is provided. In some embodiments, a cell, tissue or organ that specifically expresses a human or humanized INHBE protein is provided.

[0087] In some embodiments, the expression of human or humanized INHBE protein expressed by non-human animals is controllable. For example, 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).

[0088] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferred non-human mammals are mammals (eg, rodents). In some embodiments, the non-human mammal is a mouse.

[0089] The non-human mammals described above are subjected to genetic, molecular and behavioral analyses. The present invention provides offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0090] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny. For example, a cell culture-based model can be prepared by the following method. 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. The integration of a genetic construct comprising a DNA sequence encoding a human INHBE protein can be detected by a variety of methods.

[0091] There are many analytical methods that can be used to detect exogenous DNA, including nucleic acid level methods (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (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 perform quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcription levels, including RNase protection analysis, Southern Blot, RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of human or humanized INHBE protein.

[0092] In some embodiments, the genetically modified non-human animal described herein (eg, a mouse homozygous for humanized INHBE gene) can express human or humanized INHBE in one or more cells (eg, liver tissue or adipose tissue).

[0093] Methods for constructing genetically modified non-human animals

[0094] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting technology using embryonic stem cells, homologous recombination technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques.

[0095] In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals.

[0096] In some embodiments, CRISPR / Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these genome editing technologies 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.

[0097] In some embodiments, the nucleotide sequence encoding the endogenous INHBE region in the endogenous genome of at least one cell of the genetically modified non-human animal is replaced by a nucleotide sequence encoding the corresponding region of human INHBE. In some embodiments, the expression of endogenous INHBE protein in the genetically modified non-human animal is reduced or absent compared to the wild-type non-human animal. 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.

[0098] The present invention provides a targeting vector. The targeting vector comprises a vector composed of a 5' homology arm, a human or humanized INHBE gene fragment and a 3' homology arm. The process involves using homologous recombination to replace the corresponding sequence of endogenous INHBE in non-human animals with the human or humanized INHBE gene fragment. In some embodiments, cutting upstream and downstream of the target site (for example, by zinc finger nuclease, TALEN or CRISPR) can cause DNA double-strand breaks, and homologous recombination is used to replace the corresponding sequence of endogenous INHBE in non-human animals with the human or humanized INHBE gene fragment.

[0099] Thus, in some embodiments, a method of making a genetically modified humanized non-human animal comprises replacing a nucleotide sequence encoding an endogenous INHBE region at an endogenous INHBE locus (or site) with a nucleotide sequence encoding a corresponding region of human INHBE.

[0100] The present invention also provides a method for establishing an INHBE gene humanized animal model, comprising the following steps:

[0101] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0102] (b) culturing the cells (preferably culturing the cells in a liquid culture medium);

[0103] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

[0104] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0105] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0106] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0107] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0108] The fertilized egg can be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced 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.

[0109] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying the coding frame of the INHBE gene of the non-human animal, for example, by replacing the nucleic acid sequence encoding the endogenous INHBE region with a nucleotide sequence encoding the corresponding region of human INHBE (e.g., a genomic DNA sequence, a CDS sequence, or a cDNA sequence) under the control of the endogenous regulatory element of the INHBE gene of the non-human animal. In some embodiments, one or more functional region sequences of the INHBE gene of the non-human animal can be knocked out or inserted into a sequence so that the endogenous INHBE protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the INHBE gene of the genetically modified non-human animal can be all or part of exon 1 to all or part of exon 2 of the INHBE gene of the non-human animal.

[0110] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or humanized INHBE protein after the endogenous regulatory element of the INHBE gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the INHBE gene humanized animal model can express a human or humanized INHBE protein in vivo, but does not express the INHBE protein of the non-human animal. In some embodiments, the auxiliary sequence also includes WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0111] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0112] (1) providing a plasmid comprising a human INHBE gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target an endogenous INHBE gene of a non-human animal;

[0113] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous INHBE gene of a non-human animal;

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

[0115] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express the humanized INHBE protein. Preferably, the method further comprises:

[0116] (5) The offspring mice obtained in step (4) are mated to obtain homozygous mice.

[0117] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal target site and a 3'-terminal target site.

[0118] In some embodiments, the sequence encoding the humanized INHBE protein is operably linked to an endogenous regulatory element at the endogenous INHBE locus of the non-human animal.

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

[0120] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0121] (1) providing a plasmid comprising a human or chimeric INHBE gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target an endogenous INHBE gene of a non-human animal;

[0122] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous INHBE gene of a non-human animal;

[0123] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric INHBE gene fragment into the genome.

[0124] In some embodiments, the nucleotide sequence encoding the endogenous INHBE protein in the genome of the non-human animal is deleted. In some embodiments, all or part of exons 1-2 of the endogenous INHBE gene of the non-human animal are deleted. In some embodiments, part of exon 1 to all of exon 2 of the endogenous INHBE gene of the non-human animal is deleted. In some embodiments, the nucleotide sequence from the start codon to the stop codon of the endogenous INHBE gene of the non-human animal is deleted, and preferably the deleted sequence also contains 3'UTR. In some embodiments, at least 50bp of continuous nucleotide sequence downstream of the 3'UTR of the endogenous INHBE gene of the non-human animal is deleted. In some embodiments, intron 1 of the endogenous INHBE gene of the non-human animal is deleted.

[0125] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous INHBE protein in the genome of the non-human animal with the nucleotide sequence encoding the human INHBE protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 in the genome of the non-human animal with the nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the construction method comprises replacing the start codon to the stop codon (preferably also including the 3'UTR) of the endogenous INHBE gene of the non-human animal with the nucleotide sequence from the start codon to the stop codon of the human INHBE gene (preferably also including the 3'UTR). In some embodiments, the construction method comprises replacing the nucleotide sequence from part of exon 1 to all of exon 2 of the endogenous INHBE gene of the non-human animal in the genome of the non-human animal with part of exon 1 to all of exon 2 of the human INHBE gene. In some embodiments, the construction method comprises replacing the nucleotide sequence from part of exon 1 to the downstream nucleotide sequence of 3'UTR of the endogenous INHBE gene of the non-human animal in the genome of the non-human animal with part of exon 1 to all of exon 2 of the human INHBE gene. In some embodiments, the construction method comprises replacing the nucleotide sequence from part of exon 1 to the downstream nucleotide sequence of 3'UTR of the endogenous INHBE gene of the non-human animal in the genome of the non-human animal with part of exon 1 to the downstream nucleotide sequence of 3'UTR of the human INHBE gene. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 in the genome of a non-human animal with the genomic DNA sequence, CDS sequence or cDNA sequence of the human INHBE gene. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 in the genome of a non-human animal with the nucleotide sequence shown in SEQ ID NO: 5 or 6.

[0126] Use of genetically modified non-human animals

[0127] 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 the non-human animal endogenous INHBE locus and under the control of endogenous or human regulatory elements, can produce a non-human animal with qualities and characteristics that may be significantly different from typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or destroyed, and the full human transgene is inserted into the genome of the non-human animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; the expression of the human protein is measured by transcription of human gene and / or protein assays and / or functional assays. In human transgenes, the upstream and / or downstream of the human sequence provide suitable support for the expression and / or regulation of the transgene.

[0128] In some embodiments, genetically modified non-human animals or animal models that express human or humanized INHBE proteins, e.g., in a physiologically appropriate manner, provide a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in non-human animals or animal models.

[0129] The present invention also provides an application of any of the above-mentioned INHBE gene-modified non-human animals and any of the above-mentioned construction methods.

[0130] In some embodiments, the application comprises:

[0131] A) Application in the development of products involving human cells for INHBE-related immune processes;

[0132] B) Application as a model system relevant to INHBE for pharmacological, immunological, microbiological and medical research;

[0133] C) applications involving the production and use of animal experimental disease models for the study of the etiology of INHBE and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0134] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of modulators of the human INHBE signaling pathway; or,

[0135] E) Study the function of the INHBE gene, study the drugs and efficacy targeting the human INHBE target site, and study the application of therapeutic drugs for tumors, inflammation, immune diseases, metabolic diseases or cardiovascular diseases related to INHBE.

[0136] The present invention provides a non-human animal expressing human or humanized INHBE protein, which can be used for screening of human INHBE 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 also comprises an impaired immune system, such as a genetically modified human-derived tissue xenograft, including a human solid tumor (e.g., breast cancer) or a blood tumor (e.g., a lymphocyte tumor (e.g., a B or T cell tumor)).

[0137] In some embodiments, anti-INHBE antibodies, INHBE-targeted nucleic acid drugs or polypeptide drugs block or inhibit INHBE-mediated signaling pathways. In some embodiments, anti-INHBE antibodies, INHBE-targeted nucleic acid drugs or polypeptide drugs can block the interaction between INHBE complexes, thereby inhibiting the INHBE signaling pathway.

[0138] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-INHBE antibodies, nucleic acid drugs targeting INHBE, or polypeptide drugs) in treating various metabolic diseases. In some embodiments, the metabolic disease is obesity or type II diabetes.

[0139] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-INHBE antibodies, nucleic acid drugs targeting INHBE, or polypeptide drugs) in treating various cardiovascular diseases. In some embodiments, the cardiovascular disease is coronary heart disease.

[0140] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-INHBE antibodies, nucleic acid drugs targeting INHBE, or polypeptide drugs) in treating various immune diseases. In some embodiments, the immune diseases include, but are not limited to, GVHD (graft versus host disease), psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty hepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, the immune disease is asthma, atopic dermatitis, psoriasis, rheumatoid arthritis, or multiple sclerosis.

[0141] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-INHBE antibodies, INHBE-targeted nucleic acid drugs or polypeptide drugs) in treating various inflammations (e.g., infections). In some embodiments, the inflammation includes acute inflammation and chronic inflammation. Specifically, it includes but is not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.). In some embodiments, inflammation is hepatitis or inflammatory bowel disease (IBD).

[0142] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-INHBE antibodies, nucleic acid drugs targeting INHBE, or polypeptide drugs) for treating cancer. In some embodiments, a therapeutic agent is administered to a non-human animal, wherein the non-human animal 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 determining the size and / or proliferation rate of tumor cells. In some embodiments, the detection method includes vernier caliper measurement, flow cytometry, and / or animal in vivo imaging detection. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, including changes in food consumption or water consumption.

[0143] In some embodiments, the tumor cells include one or more cancer cells injected into a non-human animal (e.g., cancer cells derived from humans or non-human animals). In some embodiments, the therapeutic agent (e.g., anti-INHBE antibody, INHBE-targeted nucleic acid drug, or polypeptide drug) inhibits the INHBE signaling pathway. In some embodiments, the therapeutic agent (e.g., anti-INHBE antibody, INHBE-targeted nucleic acid drug, or polypeptide drug) does not inhibit the INHBE signaling pathway.

[0144] In some embodiments, the genetically modified non-human animals can be used to detect whether a therapeutic agent (e.g., an anti-INHBE antibody, a nucleic acid drug targeting INHBE, or a polypeptide drug) is an agonist or antagonist. In some embodiments, the methods described herein can be used to detect the function of a therapeutic agent (e.g., an anti-INHBE antibody, a nucleic acid drug targeting INHBE, or a polypeptide drug), for example, whether the therapeutic agent can upregulate an immune response or downregulate an immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a disease in a subject (e.g., a tumor, an immune disease, inflammation, a metabolic disease, or a cardiovascular disease). The inhibitory effect on tumors can also be determined by methods known in the art, for example, measuring tumor volume in a non-human animal, and / or determining the tumor growth (volume) 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 is the average tumor volume (or weight) of the treatment group and the control group.

[0145] In some embodiments, therapeutic agents (e.g., anti-INHBE antibodies, INHBE-targeted nucleic acid drugs or polypeptide drugs) can be used to treat various cancers (tumors). "Cancer" as used herein refers to cells with autonomous growth ability, i.e., abnormal states or conditions characterized by rapid proliferation of cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of the histopathological type or invasive stage. "Tumor" as used herein 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, brain glioma, lung cancer (e.g., 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, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein, the leukemia is selected from acute lymphocytic (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's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's 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 tumor, oropharyngeal tumor, female reproductive system cancer or meningioma. In some embodiments, the tumor includes a solid tumor or a hematological tumor. In some embodiments, the tumor includes one or more of head and neck cancer, breast cancer, lymphoma, liver cancer or lung cancer.

[0146] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (e.g., an anti-INHBE antibody, a nucleic acid drug targeting INHBE, or a polypeptide drug). The method comprises administering a therapeutic agent to the above-mentioned non-human animal or a non-human animal obtained by the above-mentioned construction method, assessing the weight change of the non-human animal or performing a blood test, wherein the blood test preferably includes but is not limited to red blood cell count, hematocrit, and / or hemoglobin content. In some embodiments, the therapeutic agent can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the body weight of a non-human animal treated with a therapeutic agent is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average body weight of a non-human animal not treated with a therapeutic agent).

[0147] The present invention also provides an animal model constructed by the method described in the present application for developing products related to human cellular immune processes, producing human antibodies, or a model system for pharmacology, immunology, microbiology and medical research.

[0148] In some embodiments, an animal model generated by the methods described herein is provided for producing and utilizing animal experimental disease models of immune processes of human cells, studying pathogens, or developing new diagnostic strategies and / or therapeutic strategies.

[0149] The present invention also provides an animal model generated by the method described in the present application to screen, verify, evaluate or study INHBE gene function, human INHBE antibodies, drugs or effectiveness of human INHBE target sites, and drugs for INHBE-related diseases (such as tumors, inflammation, immune diseases, metabolic diseases or cardiovascular diseases).

[0150] In some embodiments, the present disclosure provides a method for verifying the in vivo efficacy of TCR-T, CAR-T and / or other immunotherapies (e.g., T cell adoptive transfer therapy). For example, the method includes transplanting human tumor cells into non-human animals described in the present application, and applying human CAR-T to animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animal is selected from INHBE gene humanized non-human animals prepared by the method described in the present application, double-gene or multi-gene humanized non-human animals (or their offspring) produced by the method described in the present application, non-human animals expressing human or humanized INHBE proteins, or tumor-bearing or inflammatory animal models described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat INHBE-related diseases described in the present application. In some embodiments, TCA-T, CAR-T and / or other immunotherapies provide evaluation methods for treating INHBE-related diseases described in the present application.

[0151] Non-human animal models with two or more human or chimeric genes

[0152] The present invention also provides a non-human animal or animal model with two or more human or chimeric gene modifications. The non-human animal or animal model may contain a human or chimeric INHBE gene and a sequence encoding an additional human or chimeric protein.

[0153] In some embodiments, the additional human or chimeric protein comprises at least one of LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the non-human animal also expresses at least one of human or humanized LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 protein.

[0154] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0155] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0156] (ii) mating, in vitro fertilization or direct gene editing of the non-human animals provided in step (i) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0157] 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 LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0158] In some embodiments, INHBE gene humanization is performed directly on a non-human animal having at least one modification of a human or chimeric LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 gene.

[0159] Since these proteins may involve different mechanisms, combination therapy targeting two or more of them may be a more effective treatment method. In fact, many related clinical trials are underway and show good results. Multigene modified non-human animal models can be used to determine the effectiveness of combination therapy targeting two or more proteins, for example, therapeutic agents (anti-INHBE antibodies, nucleic acid drugs targeting INHBE or polypeptide drugs), and additional therapeutic agents for treating diseases (e.g., tumors, inflammation, immune diseases, metabolic diseases or cardiovascular diseases). The method includes administering a therapeutic agent (e.g., an anti-INHBE antibody, a nucleic acid drug targeting INHBE or a polypeptide drug) and an additional therapeutic agent to a non-human animal, wherein the non-human animal suffers from a disease (e.g., a tumor, inflammation, an immune disease, a metabolic disease or a cardiovascular disease), and determining the effect of the combination therapy on the disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and / or CTLA4, or a nucleic acid drug or polypeptide drug that targets one or more of the above targets. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., nivolumab), or an anti-PD-L1 antibody. In some embodiments, the non-human animal further comprises 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 additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0160] In some embodiments, the combination therapy is used to treat various cancers described in the present application, for example, breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer or cancer. In some embodiments, the combination therapy is designed to treat immune diseases described in the present application, such as psoriasis. In some embodiments, the method described in the present application can be used to evaluate the combination therapy with some other methods. The method for treating cancer that can be used alone or in combination with the method described in the present application includes, for example, treating the subject with chemotherapy, for example, camphor alkali, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepate, cyclophosphamide, doxorubicin, ifosamide, melphalan, chlorbutiazine, thiophene, nitrosourea, dacrynic acid, daunorubicin, bleomycin, primycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatinum, 5-fluorouric acid, vincristine, vinblastine and / or methotrexate. Alternatively, or in addition, the method can include performing surgery on the subject to remove at least a portion of the cancer, for example, removing a portion or all of a tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1 : Schematic diagram comparing the mouse INHBE locus and the human INHBE locus (not to scale);

[0162] Figure 2 : Schematic diagram of INHBE gene targeting strategy and targeting vector V1 design (not to scale);

[0163] Figure 3 : Schematic diagram of INHBE gene targeting strategy and targeting vector V2 design (not to scale);

[0164] Figure 4 : Southern blot identification results of F1 generation of INHBE gene humanized mice, where WT is the wild-type control;

[0165] Figure 5 : RT-PCR test results, where + / + is wild-type C57BL / 6 mice, H / + is INHBE gene humanized heterozygous mice, H2O is water control, GAPDH is internal reference;

[0166] Figure 6 : RT-PCR test results, where + / + is wild-type C57BL / 6 mice, H / H is INHBE gene humanized homozygous mice, H2O is water control, GAPDH is internal reference;

[0167] Figure 7:Western Blot test results, where + / + is wild-type C57BL / 6 mice, H / H is INHBE gene humanized homozygous mice, β-actin is the internal reference;

[0168] Figure 8 :qPCR detection results of mRNA in the liver of humanized homozygous INHBE mice under fasting or feeding (not fasting) conditions;

[0169] Fig. 9 : qPCR detection results of mouse INHBE mRNA in the liver after administration of nucleic acid drug A targeting INHBE, where G1 group was injected with PBS and G2 group was injected with nucleic acid drug A targeting human INHBE;

[0170] Fig.10 : qPCR detection results of human INHBE mRNA in the liver after administration of nucleic acid drug A targeting INHBE, where G1 group was injected with PBS and G2 group was injected with nucleic acid drug A targeting human INHBE;

[0171] Fig.11 : qPCR detection results of human INHBE mRNA in the liver after administration of nucleic acid drug B targeting human INHBE, where group G1 was injected with PBS and group G2 was injected with nucleic acid drug B targeting human INHBE;

[0172] Fig. 12A : qPCR detection results of human INHBE mRNA in the liver after administration of nucleic acid drug B targeting human INHBE, where group G1 was not treated, group G2 was injected with normal saline, and group G3 was injected with nucleic acid drug B targeting human INHBE;

[0173] Fig. 12B : Changes in body weight of mice, among which the G1 group was not treated, the G2 group was injected with normal saline, and the G3 group was injected with nucleic acid drug B targeting human INHBE;

[0174] Fig.13 : The weight of subcutaneous fat, inguinal fat and perirenal fat (WAT) after administration of the targeted human INHBE nucleic acid drug B, where the G1 group was not treated, the G2 group was injected with normal saline, and the G3 group was injected with the targeted human INHBE nucleic acid drug B;

[0175] Fig.14A : Relative TG concentration in the liver after administration of nucleic acid drug B targeting human INHBE, where group G1 was not treated, group G2 was injected with normal saline, and group G3 was injected with nucleic acid drug B targeting human INHBE;

[0176] Fig. 14B: Relative TC concentration in the liver after administration of nucleic acid drug B targeting human INHBE, where group G1 was not treated, group G2 was injected with normal saline, and group G3 was injected with nucleic acid drug B targeting human INHBE.

[0177] In each figure, * indicates p<0.05; ** indicates p<0.01; **** indicates p<0.0001. DETAILED DESCRIPTION

[0178] 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. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0179] In each of the following examples, equipment and materials were obtained from the following companies:

[0180] C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Laboratory Animal Seed Center, China Food and Drug Inspection Institutes;

[0181] BsrGI was purchased from NEB, catalog number R3575S;

[0182] BamHI was purchased from NEB, catalog number R3136S;

[0183] Anti-Inhibin beta E chain antibody was purchased from Abcam with the catalog number: ab103167.

[0184] Example 1 INHBE gene humanized mice

[0185] A schematic diagram of the comparison of the mouse INHBE gene (NCBI Gene ID: 16326, located at positions 127185271 to 127187717 of chromosome 10 NC_000076.7, based on transcript NM_008382.3 and its encoded protein NP_032408.2 (SEQ ID NO: 1)) and the human INHBE gene (NCBI Gene ID: 83729, located at positions 57455307 to 57458025 of chromosome 12 NC_000012.12, based on transcript NM_031479.5 and its encoded protein NP_113667.1 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0186] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human INHBE protein can be introduced into the mouse endogenous INHBE locus, so that the mouse expresses a human or humanized INHBE protein. Specifically, using gene editing technology, under the control of the mouse INHBE gene regulatory element, a partial sequence of exon 1 of the human INHBE gene to about 3.2 kb of the nucleotide sequence downstream of the 3'UTR is used to replace a partial sequence of mouse exon 1 to about 2.9 kb of the nucleotide sequence downstream of the 3'UTR, thereby obtaining a humanized INHBE locus and realizing the humanization of the mouse INHBE gene.

[0187] In order to realize the targeting strategy of the present invention, a targeting vector was constructed. The targeting vector V1 ( Figure 2 ) contains upstream and downstream homology arm sequences of the mouse INHBE gene, and an A fragment containing a human INHBE fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 127187415 to 127191477 of NCBI accession number NC_000076.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 127180557 to 127184534 of NCBI accession number NC_000076.7. The nucleotide sequence of the human INHBE gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 57455537 to 57458777 of NCBI accession number NC_000012.12; the connection design of the upstream of the human INHBE fragment sequence and the mouse sequence is:

[0188] The sequence " GGAGC The "C" in the sequence is the last nucleotide of the mouse sequence. The "A" in is the first nucleotide of the human INHBE fragment sequence.

[0189] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human INHBE gene is designed as follows:

[0190]

[0191] The sequence " CTCTG "G" is the last nucleotide of the human INHBE gene, and the sequence The first "G" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the mouse INHBE gene is designed as:

[0192]

[0193] The sequence " ACTTC The last "C" in the sequence is the last nucleotide of the Neo box. The "C" in is the first nucleotide of the mouse INHBE gene. The mRNA sequence transcribed from the INHBE gene in the transformed humanized mouse is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0194] The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using the positive clone screening marker gene to screen the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the technology known in the art, and the obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then INHBE gene humanized homozygous mice can be obtained by mating with each other.

[0195] In addition, CRISPR / Cas9 technology can be used for gene editing to further design the targeting vector V2( Figure 3), the targeting vector V2 contains homology arm sequences upstream and downstream of the mouse INHBE gene, and the human INHBE fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 31) is identical to the nucleotide sequence of positions 127187415 to 127188991 of NCBI accession number NC_000076.7, and the downstream 3' homology arm sequence (SEQ ID NO: 32) is identical to the nucleotide sequence of positions 127183015 to 127184534 of NCBI accession number NC_000076.7. The nucleotide sequence of the human INHBE fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 57455537 to 57458777 of NCBI accession number NC_000012.12. The connection design of the upstream of the human INHBE fragment sequence and the mouse sequence is:

[0196]

[0197] The sequence " GGAGC The "C" in the sequence is the last nucleotide of the mouse sequence. The "A" in is the first nucleotide of the human INHBE fragment sequence. The connection between the human INHBE fragment sequence and the mouse sequence is designed as follows:

[0198]

[0199] The sequence " CTCTG "G" in the sequence is the last nucleotide of the human INHBE fragment sequence. The "C" in is the first nucleotide of the mouse sequence. The mRNA sequence transcribed from the INHBE gene in the transformed humanized mouse is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0200] Conventional methods can be used to construct the targeting vector, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

[0201] The target sequence (target site) determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, the selection and design of efficient and specific target sequence (target site) is a prerequisite for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence (target site) of exemplary sgRNA on the INHBE gene is as follows:

[0202] sgRNA1 target site (SEQ ID NO: 21): 5'-AGGAGAACGCCGCTCTGGTCCTGG-3';

[0203] sgRNA2 target site (SEQ ID NO: 22): 5'-TTCTTAGTTCATAGGTTCGGTGG-3';

[0204] The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 3. After annealing, the annealing products were connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-INHBE-1 and pT7-INHBE-2.

[0205] Table 3 sgRNA1 and sgRNA2 sequence list

[0206]

[0207] The pT7-sgRNA vector was synthesized by a plasmid synthesis company with a fragment DNA containing a T7 promoter and sgRNA scaffold (SEQ ID NO: 33) and connected to a backbone vector (source: Takara, item number 3299) by restriction digestion (EcoRI and BamHI) in sequence. The vector was sequenced and verified by a professional sequencing company, and the results showed that the target plasmid was obtained. The pronuclear fertilized eggs of mice, such as C57BL / 6 mice, were taken, and the in vitro transcription products of pT7-INHBE-1 and pT7-INHBE-2 plasmids (using the Ambion in vitro transcription kit, transcribed according to the instructions), the targeting vector and Cas9 mRNA were premixed and injected into the cytoplasm or nucleus of the mouse fertilized eggs using a microinjector. Microinjection of fertilized eggs was performed according to the method in the "Mouse Embryo Manipulation Experiment Manual (3rd Edition)" (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse for development. The obtained mice (F0 generation) were hybridized and self-fertilized to expand the population and establish a stable INHBE gene humanized mouse strain.

[0208] The PCR method can be used to identify the somatic cell genotype of F1 generation mice, and the F1 generation PCR-positive mice are subjected to Southern blot detection to confirm whether there is random insertion. The mouse tail is cut to extract genomic DNA, and the genome is digested with BsrGI enzyme or BamHI enzyme, respectively, transferred to a membrane, and hybridized. The LR probe (LR Probe) and the 3' probe (3'Probe) are located on the 5' homology arm and downstream of the 3' homology arm, respectively. The specific probes and target fragment lengths are shown in Table 4 below, and the exemplary results are shown in Table 4 below. Figure 4 As shown, combined with the PCR and sequencing results, 11 mice numbered F1-01 to F1-11 were positive mice. This shows that the construction method of the present application can construct INHBE gene humanized mice that can be stably propagated and have no random insertion.

[0209] Table 4 Length of specific probes and target fragments

[0210] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size BnB LR Probe 11.7kb 16.0kb B H 3'Probe 8.1kb 10.2kb

[0211] LR Probe-F (SEQ ID NO: 11): 5'-TCCTATGGACTCCCTCACCTGAATC-3',

[0212] LR Probe-R (SEQ ID NO: 12): 5'-GTCACAGCTACCCTTGACAAAGAGC-3';

[0213] 3'Probe-F (SEQ ID NO: 13): 5'-GACAGGGTACTCCCCTATCACGAAC-3',

[0214] 3'Probe-R (SEQ ID NO: 14): 5'-AAGGAAGAACACGGGGCTAGAGTTC-3';

[0215] The expression of mRNA in humanized INHBE gene mice can be detected by RT-PCR. Specifically, 8-week-old male C57BL / 6 mice (+ / +) and 8-week-old male INHBE gene humanized heterozygotes (H / +) prepared in this example were selected, and liver tissues were obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 5 below. The detection results are shown in Figure 5 As shown. Figure 5 As can be seen, only mouse INHBE mRNA was detected in wild-type C57BL / 6 mice, and human INHBE mRNA was not detected; human INHBE mRNA was only detected in mice with humanized heterozygous INHBE gene.

[0216] Table 5 RT-PCR primer sequences and target fragment sizes

[0217]

[0218] Similar to the above method, the expression of mRNA in humanized homozygous INHBE gene mice can be detected by RT-PCR. 8-week-old male C57BL / 6 mice (+ / +) and 8-week-old male homozygous INHBE gene humanized (H / H) mice prepared in this example were selected, and liver tissue was obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 6. The test results are shown in FIG. Figure 6 As shown. Figure 6 As can be seen, only mouse INHBE mRNA was detected in wild-type C57BL / 6 mice, and human INHBE mRNA was not detected; human INHBE mRNA was only detected in mice homozygous for the humanized INHBE gene.

[0219] Table 6 RT-PCR primer sequences and target fragment sizes

[0220]

[0221] In addition, the expression of human INHBE protein in INHBE gene humanized homozygous mice can be detected by conventional methods such as Western Blot method. Specifically, 8-week-old male C57BL / 6 mice (+ / +) and 8-week-old male INHBE gene humanized homozygous (H / H) prepared in this example were taken, and liver, kidney, lung and spleen tissues were taken after euthanasia by cervical dislocation, and Western Blot detection was performed using human-mouse cross antibody Anti-Inhibin beta E chain antibody. The test results are as follows Figure 7 As shown in the results, it can be seen that the expression of INHBE protein was detected in the liver and kidneys of wild-type C57BL / 6 mice and INHBE gene humanized homozygous mice. Combined with the above RT-PCR results, it shows that the transformed humanized mice can normally express human INHBE protein.

[0222] The specificity of INHBE mRNA expression in the liver of humanized homozygous INHBE mice (H / H) was analyzed by qPCR. Female humanized homozygous INHBE mice aged 8 weeks were randomly divided into a fasting group and a non-fasting group, with 3 mice in each group. After the mice were fasted or fed for 16 hours, liver tissues were collected and the expression level of human INHBE mRNA was detected by qPCR. The results are shown in Figure 8 As shown, the expression level of INHBE was significantly increased in the fasting group of mice, which is consistent with the role of INHBE in preventing lipolysis.

[0223] Example 2 Pharmacodynamic Model

[0224] The humanized mice disclosed in the present invention can be used to induce the preparation of a variety of human disease models, including models of tumors, inflammation, immune diseases, metabolic diseases or cardiovascular diseases, and can be used to test the in vivo efficacy of therapeutic agents targeting humans (such as specific anti-INHBE antibodies, nucleic acid drugs targeting INHBE or polypeptide drugs). For example, INHBE gene humanized mice can be used to evaluate the efficacy, pharmacokinetics and in vivo therapeutic efficacy of antagonists of human-specific INHBE signaling pathways in various disease models known in the art.

[0225] The INHBE gene humanized mice prepared in Example 1 of the present application can be used to evaluate the efficacy of regulators targeting human INHBE in obesity. For example, a number of homozygous INHBE gene humanized mice are taken, and an obese (DIO) mouse model is constructed by feeding a high-fat diet, and then divided into a control group and a drug administration group. The drug administration group is injected with a drug targeting human INHBE, and the control group is injected with an equal volume of normal saline or PBS. The specific experimental steps are as follows:

[0226] In a specific experiment, 9-10 week old homozygous INHBE humanized mice were randomly divided into a control group (G1) and a treatment group (G2) (n=5). On the day of grouping, the treatment group was subcutaneously injected with a nucleic acid drug A targeting human INHBE (1 mpk / mouse), and the control group was injected with an equal volume of PBS. On the 5th day, the mice were euthanized, and liver tissues were taken to detect human and mouse INHBE mRNA. The results showed that mouse INHBE mRNA was not detected in either the control group (G1) or the treatment group (G2). Fig. 9 ); however, the expression of human INHBE mRNA could be detected in both the control group (G1) and the treatment group (G2) ( Fig.10 ), and the expression level of human INHBE mRNA in the drug-treated group (G2) was significantly lower than that in the control group (G1), indicating that the expression of human INHBE in the drug-treated group (G2) can be inhibited by nucleic acid drug A targeting human INHBE.

[0227] In another experiment, 10-week-old INHBE humanized homozygous mice were randomly divided into a control group (G1) and a treatment group (G2) (n=3). On the day of grouping, the treatment group (G2) was subcutaneously injected with another nucleic acid drug B targeting human INHBE (3 mpk / mouse), the sequence of which is shown in Table 7 below, and the control group (G1) was injected with an equal volume of PBS. On the 14th day, the mice were euthanized, and liver tissues were taken to detect human INHBE mRNA. The results are as follows Fig.11 The results also showed that the expression of human INHBE mRNA could be detected in both the control group (G1) and the treatment group (G2) ( Fig.11 ), and the expression level of human INHBE mRNA in the drug-treated group (G2) was significantly lower than that in the control group (G1), which also verified that the expression of human INHBE in the drug-treated group (G2) could be inhibited by nucleic acid drug B targeting human INHBE.

[0228] Table 7 Sequences of nucleic acid drug B targeting INHBE

[0229]

[0230] Among them, m represents 2'-O-methyl, f represents 2'-fluoro, s represents phosphorothioate linkage, Tgn represents thymidine-glycol nucleic acid (GNA) S-isomer, dA represents 2'-deoxy A, and L96 represents GALNAC coupling.

[0231] In another experiment, 7-week-old homozygous INHBE humanized mice were randomly divided into a control group (G1, G2) (n=3) and a treatment group (G3, n=6). The mice in the control group (G2) and the treatment group (G3) were fed a high-fat diet (60% fat calorie feed) for 12 weeks to induce obesity, and the mice in the control group G1 were fed normally. From 12 to 18 weeks, the mice in the treatment group (G3) were subcutaneously injected with the human INHBE-targeted nucleic acid drug B (9 mpk / mouse) once a week. The mice in the control group G1 did not receive any treatment, and the mice in the control group G2 were injected with normal saline. The weight changes were detected on the day of the first drug injection (0 day), 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 49 days ( Fig. 12B ). At 19 weeks, the mice were euthanized and liver and adipose tissues were obtained to detect the expression of human INHBE mRNA in the liver, the total cholesterol TC and triglyceride TG contents, and the fat weight (WAT) of subcutaneous fat, inguinal fat and perirenal fat.

[0232] The results are shown in Figures 12-14. After the mice were injected with the nucleic acid drug B targeting human INHBE, the expression of human INHBE mRNA in the drug group (G3) was significantly reduced compared with the control group (G2). Fig. 12A ), subcutaneous fat, inguinal fat and perirenal fat content decreased ( Fig.13 ), TC and TG concentrations decreased ( Figure 14A-14B ). This indicates that the nucleic acid drug B targeting human INHBE can effectively inhibit weight gain and the increase of TC and TG concentrations. The results show that INHBE gene humanized mice can be used to evaluate the efficacy of drugs that specifically target the human INHBE signaling pathway.

[0233] Example 3 Preparation of double-gene or multi-gene humanized mice

[0234] The INHBE gene humanized mouse prepared by the construction method of the present application can also prepare a double gene or multi-gene humanized mouse model. For example, in the above-mentioned Example 1, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modification of LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4, or, on the basis of the INHBE gene humanized mouse, the double gene humanized or multi-gene humanized mouse model can be obtained by separating mouse ES embryonic stem cells and gene recombination targeting technology. The homozygous or heterozygous INHBE mouse obtained in the present application can also be mated with other gene-modified mice, and the offspring can be screened. According to the Mendel's law of inheritance, there is a certain probability of obtaining a double gene or multi-gene modified mouse modified with a humanized INHBE gene and other genes, and then the heterozygotes can be mated with each other to obtain a homozygous modified with a double gene or multi-gene.

[0235] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0236] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0237] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents 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 inhibin subunit βE (INHBE) protein.

2. The construction method according to claim 1, characterized in that: The chimeric INHBE protein is a humanized INHBE protein, and the amino acid sequence of the humanized INHBE protein is identical to at least 50 to 350 consecutive amino acid sequences of the human INHBE protein; Preferably, the amino acid sequence of the human or chimeric INHBE 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 INHBE gene locus, replacing the nucleotide sequence of the corresponding region of the non-human animal endogenous INHBE with a nucleotide sequence comprising human INHBE; Preferably, the nucleotide sequence of human INHBE comprises a nucleotide sequence from the start codon to the stop codon of the human INHBE gene, and preferably further comprises a nucleotide sequence of 3'UTR; Preferably, the nucleotide sequence of human INHBE comprises the nucleotide sequence from part of exon 1 to the entirety of exon 2 of the human INHBE gene, wherein the part of exon 1 of the human INHBE gene comprises at least 5 bp of continuous nucleotide sequence; Further preferably, the nucleotide sequence of human INHBE further comprises at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR of the human INHBE gene; Preferably, the nucleotide sequence of human INHBE comprises a nucleotide sequence encoding SEQ ID NO: 2; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence encoding SEQ ID No: 2; Preferably, the nucleotide sequence of human INHBE 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; Preferably, the nucleotide sequence of the corresponding region of endogenous INHBE in a non-human animal comprises a nucleotide sequence from the start codon to the stop codon of the endogenous INHBE gene in the non-human animal, and more preferably also comprises a nucleotide sequence of 3'UTR; Preferably, the nucleotide sequence of the corresponding region of the endogenous INHBE of the non-human animal comprises the nucleotide sequence from part of exon 1 to all of exon 2 of the endogenous INHBE gene of the non-human animal, wherein the part of exon 1 of the endogenous INHBE gene of the non-human animal comprises at least 5 bp of continuous nucleotide sequence; Further preferably, the nucleotide sequence of the corresponding region of endogenous INHBE in non-human animals also includes at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR of the endogenous INHBE gene in non-human animals.

4. The construction method according to any one of claims 1 to 3, characterized in that: The nucleotide sequence encoding the human or chimeric INHBE protein or the nucleotide sequence of human INHBE is operably linked to a non-human animal endogenous regulatory element or a human regulatory element, such as a promoter, a 5'UTR and / or a 3'UTR; Preferably, the endogenous INHBE protein of the non-human animal is not expressed or is expressed at a reduced level compared to INHBE in wild-type animals; Preferably, the modified INHBE gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

5. The construction method according to any one of claims 1 to 4, 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 INHBE gene in the genome of the non-human animal comprises SEQ ID NO: 6; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

6.

6. The construction method according to any one of claims 1 to 5, characterized in that: The non-human animal also includes a nucleotide sequence encoding other human or chimeric proteins, which preferably include at least one of LPA, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, Ox40, PD-1, PD-L1 or CTLA4.

7. 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 6; 2) determine the effects of therapeutic agents on non-human animals; Preferably, the therapeutic agent comprises an anti-INHBE antibody, a nucleic acid drug targeting INHBE or a polypeptide drug, and further preferably further comprises an additional therapeutic agent, such as one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the non-human animal suffers from a disease; Preferably, the disease comprises one or more of cancer, immune disease, inflammation, metabolic disease or cardiovascular disease.

8. A humanized INHBE gene, characterized in that: The humanized INHBE 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.

9. A cell, tissue or organ, characterized in that: The genome of the cell, tissue or organ comprises the humanized INHBE gene according to claim 8.

10. Use of the non-human animal obtained by the construction method of any one of claims 1 to 6, the humanized INHBE gene of claim 8, or the cell, tissue or organ of claim 9, characterized in that: The application includes: A) Application in the development of products involving human cells for INHBE-related immune processes; B) Application as a model system relevant to INHBE for pharmacological, immunological, microbiological and medical research; C) applications involving the production and use of animal experimental disease models for the study of the etiology of INHBE 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 INHBE signaling pathway modulators; or, E) Study the function of the INHBE gene, study the drugs and efficacy targeting the human INHBE target site, and study the application of therapeutic drugs for tumors, inflammation, immune diseases, metabolic diseases or cardiovascular diseases related to INHBE.

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