APOC3 gene modified non-human animal

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) APOC3 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) APOC3 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 APOC3 protein. The animal model can express human or chimeric APOC3 (e.g., humanized APOC3) protein. It can be used for the study of APOC3 gene function, and can also be used for the screening and evaluation of APOC3 signaling pathway regulators (e.g., anti-human APOC3 antibodies, nucleic acid drugs and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamics research, treatment of immune diseases, inflammation, cancer, metabolic diseases or cardiovascular diseases, and treatment of diseases at human APOC3 target sites; the animal 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 APOC3 protein and provides a platform for screening related drugs.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric apolipoprotein C3 (APOC3). In some embodiments, the nucleotide sequence encoding a human or chimeric APOC3 protein may be a CDS, a genomic DNA, or a cDNA. In some embodiments, the nucleotide sequence encoding a human or chimeric APOC3 protein is operably linked to an endogenous regulatory element (e.g., a promoter and / or UTR) of an endogenous APOC3 locus of at least one chromosome. In some embodiments, the chimeric APOC3 protein is a humanized APOC3 protein. In some embodiments, the amino acid sequence of the chimeric APOC3 protein comprises at least 5 to 99 consecutive amino acids of a human APOC3 protein, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 consecutive amino acids. In some embodiments, the amino acid sequence of the human or chimeric APOC3 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 non-human animal is a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous APOC3 protein of the non-human animal is not expressed or is expressed at a reduced level compared to APOC3 in wild-type animals. In some embodiments, one or more cells of the non-human animal express the human or chimeric APOC3 protein.

[0006] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the endogenous APOC3 gene is replaced with a nucleotide sequence comprising human APOC3 at the endogenous APOC3 locus of the non-human animal. In some embodiments, the endogenous APOC3 protein of the non-human animal is not expressed or the expression level is reduced compared to APOC3 in wild-type animals. In some embodiments, the nucleotide sequence encoding the human or chimeric APOC3 protein or the nucleotide sequence of human APOC3 is operably linked to an endogenous APOC3 regulatory element, such as a promoter and / or UTR. In some embodiments, the modified APOC3 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the nucleotide sequence of human APOC3 may be a CDS, genomic DNA, or cDNA. In some embodiments, the nucleotide sequence of human APOC3 includes a nucleotide sequence encoding a human or chimeric APOC3 protein. In some embodiments, the nucleotide sequence of human APOC3 comprises at least 5 bp to 3666 bp of continuous nucleotides of the human APOC3 gene, for example, at least 5, 50, 100, 200, 300, 400, 450, 485, 486, 487, 488, 489, 490, 500, 530, 535, 540, 550, 600, 700, 800, 900, 1000, 2000, 2300, 2500, 2600, 2700, 2800, 2900, 2990, 3000, 3166, 3200, 3300, 3400, 3500, 3600 or 3666 bp of continuous nucleotides. In some embodiments, the nucleotide sequence of human APOC3 comprises part of exon 2, all of exon 3 and all of exon 4 of human APOC3 gene, and preferably further comprises intron 2 and / or intron 3. In some embodiments, the nucleotide sequence of human APOC3 comprises part of exon 2 to all of exon 4 of human APOC3 gene. In some embodiments, the part of exon 2 of human APOC3 gene comprises at least 5bp to 68bp continuous nucleotides, for example, at least 5, 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 68bp continuous nucleotides. In some embodiments, the part of exon 2 of human APOC3 gene comprises the nucleotide sequence of coding region. In some embodiments, the part of exon 2 of human APOC3 gene comprises from start codon to the last nucleotide of exon 2.In some embodiments, the nucleotide sequence of human APOC3 further comprises at least 50bp of continuous nucleotides downstream of 3'UTR, for example, at least 50, 100, 200, 300, 400, 450, 490, 500, 510, 520, 600, 700, 800, 900, 1000, 1500 or 2000bp, preferably at least 50bp to 2000bp of continuous nucleotides, further preferably at least 50bp to 1000bp of continuous nucleotides. In some embodiments, the nucleotide sequence of human APOC3 comprises at least 50bp to 2000bp of continuous nucleotides from the start codon to the downstream of 3'UTR. In some embodiments, the nucleotide sequence of human APOC3 comprises at least 500bp of continuous nucleotides from the start codon to the downstream of 3'UTR. In some embodiments, the nucleotide sequence of human APOC3 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 replaced endogenous APOC3 gene comprises part of exon 2, all of exon 3 and all of exon 4 of the endogenous APOC3 gene of a non-human animal. In some embodiments, the nucleotide sequence of the replaced endogenous APOC3 gene further comprises intron 2 and / or intron 3. In some embodiments, the nucleotide sequence of the replaced endogenous APOC3 gene comprises part of exon 2 to all of exon 4 of the endogenous APOC3 gene of a non-human animal. In some embodiments, the portion of exon 2 of the endogenous APOC3 gene of the non-human animal includes at least 5 bp to 68 bp of continuous nucleotides, for example, at least 5, 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 68 bp of continuous nucleotides. In some embodiments, the portion of exon 2 of the endogenous APOC3 gene of the non-human animal includes the nucleotide sequence of the coding region. In some embodiments, the portion of exon 2 of the endogenous APOC3 gene of the non-human animal includes the start codon to the last nucleotide sequence of exon 2. In some embodiments, the nucleotide sequence of the replaced endogenous APOC3 gene further includes at least 50 bp of continuous nucleotides downstream of the 3'UTR, such as at least 50, 100, 200, 300, 310, 320, 321, 322, 330, 340, 350, 400, 500, 600, 700, 800, 900 or 1000 bp, preferably at least 50 bp to 1000 bp of continuous nucleotides, further preferably at least 50 bp to 500 bp of continuous nucleotides. In some embodiments, the nucleotide sequence of the replaced endogenous APOC3 gene comprises at least 50 bp (e.g., at least 50 bp to 1000 bp) of continuous nucleotides from the start codon of the endogenous APOC3 gene of a non-human animal to the downstream of the 3'UTR.In some embodiments, the nucleotide sequence of the replaced endogenous APOC3 gene comprises at least 321bp of continuous nucleotides from the start codon to the 3'UTR downstream of the endogenous APOC3 gene of the non-human animal. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat. In some embodiments, the mRNA transcribed from the modified APOC3 gene in the genome of the non-human animal comprises SEQ ID NO: 6, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes, including but not limited to at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0007] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises a nucleotide sequence encoding an endogenous APOC3 region at an endogenous APOC3 locus, and is replaced by a nucleotide sequence encoding a corresponding region of human APOC3. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 is operably linked to an endogenous regulatory element of the endogenous APOC3 locus, such as a promoter and / or UTR, and one or more cells of the non-human animal express a human or humanized APOC3 protein. In some embodiments, the endogenous APOC3 protein of the non-human animal is not expressed or is expressed at a reduced level compared to APOC3 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 comprises a portion of exon 2 to all of exon 4 of the human APOC3 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 comprises a portion of human exon 2 to all of exon 4 and at least 50 bp (e.g., at least 50 bp to 2000 bp) of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 includes part of human exon 2 to all of exon 4 and at least 500bp of continuous nucleotides downstream of 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 includes SEQ ID NO: 5, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region includes part of exon 2 to all of exon 4 of the APOC3 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region includes part of exon 2 to all of exon 4 of the APOC3 gene of a non-human animal (e.g., mouse) and at least 50bp (e.g., at least 50bp to 1000bp) of continuous nucleotides downstream of 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region includes part of exon 2 to all of exon 4 of the mouse APOC3 gene and 321bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the modified APOC3 gene in the non-human animal genome is homozygous or heterozygous for the endogenous replaced locus.

[0008] In one aspect, the present invention provides a non-human animal or a method for constructing the same, wherein the non-human animal comprises at least one cell encoding a nucleotide sequence of a human or humanized APOC3 protein, wherein the humanized APOC3 protein comprises at least 50, 60, 70, 80, 90 or 99 consecutive amino acid sequences that are consistent with a continuous amino acid sequence of human APOC3, and the non-human animal expresses the human or humanized APOC3 protein. In some embodiments, the nucleotide sequence encoding the human or humanized APOC3 protein is operably linked to an endogenous APOC3 regulatory element. In some embodiments, the nucleotide sequence encoding the human or humanized APOC3 protein can be integrated into the endogenous APOC3 locus of the non-human animal. In some embodiments, the human or humanized APOC3 protein has at least one mouse APOC3 activity and / or human APOC3 activity.

[0009] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein in at least one cell of the non-human animal, at the endogenous APOC3 locus of the non-human animal, the nucleotide sequence encoding the endogenous APOC3 region is replaced by the nucleotide sequence encoding the corresponding region of human APOC3. In some embodiments, the endogenous APOC3 protein of the non-human animal is not expressed or the expression level is reduced compared with APOC3 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 includes part of exon 2 to all of exon 4 of the human APOC3 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 includes part of human exon 2 to all of exon 4 and at least 50bp (e.g., at least 50bp to 2000bp) of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 includes part of human exon 2 to all of exon 4 and at least 500bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence of the human APOC3 corresponding region 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 the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human APOC3 corresponding region 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 encoding the endogenous APOC3 region comprises a portion of exon 2 to all of exon 4 of the APOC3 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region comprises a portion of exon 2 to all of exon 4 of the APOC3 gene of a non-human animal (e.g., mouse) and at least 50 bp (e.g., at least 50 bp to 1000 bp) of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region comprises a portion of exon 2 to all of exon 4 of the APOC3 gene of a non-human animal (e.g., mouse) and 321 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 is operably linked to an endogenous APOC3 regulatory element, such as a promoter and / or UTR.

[0010] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the APOC3 protein endogenously in a non-human animal with a nucleotide sequence encoding a human or chimeric APOC3 protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO:1 endogenously in a non-human animal with a nucleotide sequence encoding a human or chimeric APOC3 protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO:1 endogenously in a non-human animal with a nucleotide sequence encoding SEQ ID NO:2. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO:1 endogenously in a non-human animal with a nucleotide sequence comprising SEQ IDNO:6. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO:1 endogenously in a non-human animal with a nucleotide sequence comprising the human APOC3 gene. In some embodiments, the construction method comprises replacing the nucleotide sequence of the endogenous APOC3 gene of a non-human animal with a nucleotide sequence comprising the human APOC3 gene. In some embodiments, the construction method comprises replacing the exon 2 of the endogenous APOC3 gene of a non-human animal with the exon 2 of the human APOC3 gene. In some embodiments, the construction method includes replacing the entirety of the start codon to exon 4 of the endogenous APOC3 gene of a non-human animal with the entirety of the start codon to exon 4 of the human APOC3 gene. In some embodiments, the construction method includes replacing the entirety of the start codon to exon 4 of the endogenous APOC3 gene of a non-human animal with the entirety of the start codon to exon 4 of the human APOC3 gene. In some embodiments, the construction method includes replacing the entirety of the start codon to exon 4 of the endogenous APOC3 gene of a non-human animal with the entirety of the start codon to exon 4 of the human APOC3 gene. In some embodiments, the construction method includes replacing the entirety of the start codon to exon 4 of the endogenous APOC3 gene of a non-human animal with the entirety of the start codon to exon 4 of the human APOC3 gene. In some embodiments, the construction method includes replacing the entirety of the start codon to exon 4 of the endogenous APOC3 gene of a non-human animal with the entirety of the start codon to exon 4 of the human APOC3 gene. In some embodiments, the construction method includes replacing the entirety of the start codon to exon 4 of the endogenous APOC3 gene of a non-human animal with the entirety of the start codon to exon 4 of the human APOC3 gene. In some embodiments, the construction method comprises replacing the corresponding endogenous region of a non-human animal with a nucleotide sequence comprising SEQ ID NO: 5. In some embodiments, the construction method comprises replacing the endogenous nucleotide sequence encoding SEQ ID NO: 1 of a non-human animal with a nucleotide sequence comprising SEQ ID NO: 5.

[0011] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric APOC3, the construction method comprising replacing a nucleotide sequence encoding an endogenous APOC3 region with a nucleotide sequence of a corresponding region of human APOC3 at an endogenous mouse APOC3 locus, producing a genetically modified non-human animal cell, the non-human animal cell expressing a human or chimeric APOC3 protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 comprises a portion of exon 2 to all of exon 4 of the human APOC3 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 comprises a portion of human exon 2 to all of exon 4 and at least 50bp (preferably at least 50bp to 2000bp) of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 comprises a portion of human exon 2 to all of exon 4 and at least 500bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human APOC3 comprises SEQ ID NO: 2, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region comprises a portion of exon 2 to all of exon 4 of the APOC3 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region comprises a portion of exon 2 to all of exon 4 of the APOC3 gene of a non-human animal (e.g., mouse) and at least 50 bp (e.g., at least 50 bp to 1000 bp) of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region comprises a portion of exon 2 to all of exon 4 of the APOC3 gene of a non-human animal (e.g., mouse) and 321bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOC3 is operably linked to a regulatory element of endogenous APOC3, such as a promoter and / or UTR. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal also includes nucleotide sequences of humans or chimeric proteins encoded by other genes, including but not limited to at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0012] In one aspect, the present invention provides an application of the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method, wherein the application comprises: A) application in the development of products involving immune processes related to APOC3 in human cells; B) application in a model system related to APOC3 for pharmacological, immunological, microbiological and medical research; C) application involving the production and use of animal experimental disease models for pathological research related to APOC3 and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) application in the screening, efficacy detection, efficacy assessment, verification or evaluation of human APOC3 signaling pathway regulators in vivo; or, E) application in studying APOC3 gene function, studying drugs and efficacy targeting human APOC3 target sites, and studying APOC3-related cancer, immune disease, inflammation, metabolic disease or cardiovascular disease drugs.

[0013] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating a disease, the method comprising:

[0014] 1) administering a therapeutic agent to a non-human animal obtained by the construction method or to the non-human animal, wherein the non-human animal has a disease;

[0015] 2) Determine the inhibitory effect of the therapeutic agent on the disease.

[0016] In some embodiments, the therapeutic agent is an anti-APOC3 antibody, a nucleic acid drug or a polypeptide drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent includes but is not limited to an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody. In some embodiments, the disease includes but is not limited to cancer, immune disease, inflammation, metabolic disease or cardiovascular disease. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the cancer is gastrointestinal cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, head and neck cancer, liver cancer or lung cancer. In some embodiments, the immune disease includes but is not limited to GVHD (graft versus host disease), psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty hepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, multiple sclerosis, pain or neurological disorders, etc. In some embodiments, the immune disease is asthma, psoriasis, rheumatoid arthritis, atopic dermatitis or multiple sclerosis. 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, the inflammation is inflammatory bowel disease (IBD). In some embodiments, the disease is cancer, and the cancer comprises one or more tumor cells injected into a non-human animal. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1. In some embodiments, the inhibitory effect of the therapeutic agent on the tumor involves measuring the tumor volume in a non-human animal. In some embodiments, the non-human animal further comprises a sequence encoding a human or chimeric PD-1, a human or chimeric PD-L1 and / or a human or chimeric CTLA4. In certain embodiments, the metabolic disease is a lipid metabolism disease, and the lipid metabolism disease comprises hyperlipidemia, hypertriglyceridemia, dyslipidemia, non-alcoholic fatty liver disease, familial chylomicronemia syndrome (FCS) or lipid metabolism disorder. In certain embodiments, the cardiovascular disease comprises coronary heart disease, cardiomyopathy or atherosclerosis.

[0017] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, the method comprising:

[0018] 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method;

[0019] 2) Determine the effect of therapeutic agents on non-human animals.

[0020] In some embodiments, the therapeutic agent is an anti-APOC3 antibody, a nucleic acid drug, or a polypeptide drug. In some embodiments, the determination of the effect of the therapeutic agent on the non-human animal involves measuring the weight of the non-human animal or a blood test. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit, or hemoglobin content.

[0021] In one aspect, the present invention provides a humanized APOC3 gene, which includes a nucleotide sequence encoding a human APOC3 protein. In some embodiments, the humanized APOC3 gene includes a portion of exon 2 to all of exon 4 of the human APOC3 gene, and preferably also includes at least 50bp (preferably at least 50bp to 2000bp) of continuous nucleotides downstream of the 3'UTR of the human APOC3 gene. In some embodiments, the humanized APOC3 gene includes a portion of human exon 2 to all of exon 4 and at least 500bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the humanized APOC3 gene also includes a portion of a non-human animal APOC3 gene, preferably all of exon 1 of a non-human animal, and further preferably includes a portion of exon 2. In some embodiments, the humanized APOC3 gene is a chimeric APOC3 gene obtained by replacing a portion of exon 2 to all of exon 4 of an endogenous APOC3 gene of a non-human animal with a portion of exon 2 to all of exon 4 of the human APOC3 gene. In some embodiments, the humanized APOC3 gene is a chimeric APOC3 gene obtained by replacing at least 50bp (preferably at least 50bp to 2000bp) of continuous nucleotides from the start codon of the human APOC3 gene to the downstream of the 3'UTR with a non-human animal endogenous APOC3 gene. In some embodiments, the humanized APOC3 gene is a chimeric APOC3 gene obtained by replacing at least 500bp of continuous nucleotides from the start codon of the human APOC3 gene to the downstream of the 3'UTR with a non-human animal endogenous APOC3 gene.

[0022] In a specific embodiment of the present invention, the humanized APOC3 gene comprises any of the following nucleotide sequences:

[0023] A) a nucleotide sequence encoding the protein of SEQ ID NO: 2;

[0024] B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 31 or 32;

[0025] 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;

[0026] 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.

[0027] In one aspect, the present invention provides a non-human animal genome, the non-human animal genome comprising at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric APOC3 protein. In some embodiments, the chromosome comprises a human or chimeric APOC3 gene. In some embodiments, the chromosome comprises a portion of exon 2 to all of exon 4 of the human APOC3 gene. In some embodiments, a portion of exon 2 to all of exon 4 of the APOC3 gene of the endogenous genome of the non-human animal is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1 in the endogenous genome of the non-human animal is replaced. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human or chimeric APOC3 protein or a nucleotide sequence of human APOC3 replaces the corresponding portion of the endogenous chromosome of the non-human animal. In some embodiments, the nucleotide sequence comprising encoding SEQ ID NO: 2 replaces the endogenous nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal.

[0028] In one aspect, the present invention provides a cell, tissue or organ comprising the above-mentioned non-human animal genome.

[0029] In one aspect, the present invention provides a cell, tissue or organ, comprising the humanized APOC3 gene.

[0030] In one aspect, the present invention provides an animal model, comprising the humanized APOC3 gene.

[0031] 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.

[0032] 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 "APOC3 locus" includes exons 1-4 of the APOC3 gene and any DNA fragment in its regulatory region.

[0033] The term "portion of exon XX" of the present invention refers to a continuous or intermittent nucleotide sequence of several, dozens or hundreds of nucleotides that is consistent with the entire exon nucleotide sequence, such as a portion of exon 2 of human APOC3 gene, including continuous 5-68bp, such as at least 5, 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 68bp of continuous nucleotides.

[0034] The term "exon XX to exon XXX" or "exon XX-XXX" or "exon XX to all of exon XXX" or "all of exon XX to all of exon XXX" of the present invention refers to exons and introns in between.

[0035] The term "a portion of exon x to a portion of exon xx" or "a portion of exon x to the entirety of exon xx" of the present invention includes the entirety or portion of an exon and introns therebetween. For example, "a portion of exon 2 to the entirety of exon 4" includes a portion of exon 2, the entirety of intron 2, the entirety of exon 3, the entirety of intron 3, and the entirety of exon 4.

[0036] The term "intron xx" in this application refers to an intron between two exons, for example, intron 2 is an intron between exon 2 and exon 3.

[0037] 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.

[0038] 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".

[0039] 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.

[0040] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description.

[0041] APOC3

[0042] In the human genome, the APOC3 gene (NCBI Gene ID: 345, UniProt ID: P02656, located at positions 116829907 to 116833072 of chromosome 11 NC_000011.10) contains 4 exons, namely exon 1, exon 2, exon 3 and exon 4. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_000040.3 and its encoded protein NP_000031.1 (SEQ ID NO: 2) is shown in Table 1.

[0043] Table 1

[0044]

[0045] In the mouse genome, the APOC3 gene (NCBI Gene ID: 11814, UniProt ID: A0A0R4J1N3, located at positions 46144348 to 46146934 of chromosome 9 NC_000075.7) contains four exons, namely exon 1, exon 2, exon 3 and exon 4. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_023114.4 and its encoded protein NP_075603.1 (SEQ ID NO: 1) is shown in Table 2.

[0046] Table 2

[0047]

[0048]

[0049] APOC3 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.

[0050] 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.

[0051] 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.

[0052] Carrier

[0053] The present invention provides a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of the region to be changed (5' arm or 5' homologous arm), which is selected from the genomic DNA of the APOC3 gene and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the region to be changed (3' arm or 3' homologous arm), which is selected from the genomic DNA of the APOC3 gene and has a length of 100 to 10,000 nucleotides.

[0054] In some embodiments, a) the DNA fragment homologous to the 5' end of the region to be changed is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000075.7; c) the DNA fragment homologous to the 3' end of the region to be changed is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000075.7.

[0055] In some embodiments, a) a DNA fragment homologous to the 5' end of the region to be changed is selected from the nucleotide sequence at positions 46146012 to 46149958 of NCBI Accession No. NC_000075.7; c) a DNA fragment homologous to the 3' end of the region to be changed is selected from the nucleotide sequence at positions 46140309 to 46144026 of NCBI Accession No. NC_000075.7.

[0056] In some embodiments, a) a DNA fragment homologous to the 5' end of the region to be changed is selected from the nucleotide sequence at positions 46146012 to 46147608 of NCBI Accession No. NC_000075.7; c) a DNA fragment homologous to the 3' end of the region to be changed is selected from the nucleotide sequence at positions 46142545 to 46144026 of NCBI Accession No. NC_000075.7.

[0057] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 18 kb, 19 kb or 20 kb.

[0058] In some embodiments, the region to be changed is located at least 50bp to 1000bp downstream of exon 2 to 3'UTR of non-human animal APOC3 gene. In some embodiments, the region to be changed is located at least 50bp to 500bp downstream of exon 2 to 3'UTR of non-human animal APOC3 gene.

[0059] In some embodiments, the 5' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, the 3' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO:4.

[0060] In some embodiments, the 5' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 31. In some embodiments, the 3' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 32.

[0061] In some embodiments, the targeting vector comprises a human sequence (e.g., positions 116830583 to 116833572 of NC_000011.10). For example, the targeting region in the targeting vector includes: a portion or all of the nucleotide sequence of the human APOC3 gene, preferably a portion of exon 2 to all of exon 4 of the human APOC3 gene or at least 50 bp (e.g., at least 50 bp to 2000 bp) from the start codon to the 3'UTR downstream, and further preferably a portion of exon 2 to all of exon 4 of the human APOC3 gene or at least 50 bp to 1000 bp from the start codon to the 3'UTR downstream. In some embodiments, the nucleotide sequence of the targeting vector encodes all or part of the human APOC3 protein, and the protein number of NCBI is NP_000031.1 (SEQ ID NO: 2).

[0062] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a positive screening marker gene or a negative screening marker gene. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo. Preferably, the targeting vector further comprises 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 a coding gene (DTA) of the diphtheria toxin A subunit.

[0063] 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 APOC3 gene, and the sgRNA is unique on the target sequence of the gene to be changed, and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'. In some embodiments, the targeting site of the sgRNA in the mouse APOC3 gene is located at least 50bp to 1000bp downstream of exon 2 to 3'UTR. In some embodiments, the targeting site of the sgRNA in the mouse APOC3 gene is located at least 50bp to 500bp downstream of exon 2 to 3'UTR.

[0064] In some embodiments, the targeting sequence is shown as SEQ ID NO: 21 and / or 22. Therefore, the present invention provides sgRNA sequences for constructing a genetically modified animal model. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 23 and 25. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 24 and 26. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 27 and 29. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 28 and 30.

[0065] In some embodiments, the present application relates to a plasmid construct (e.g., pT7-sgRNA) comprising an sgRNA sequence and / or a cell comprising the construct.

[0066] The present invention also relates to a cell comprising a targeting vector or an sgRNA vector as described above.

[0067] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned targeting vectors and one or more in vitro transcripts of the construct described in the present application. In some embodiments, the cell comprises Cas9mRNA or its in vitro transcript.

[0068] In some embodiments, the cell is heterozygous for the gene. In some embodiments, the cell is homozygous for the gene.

[0069] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

[0070] Genetically modified non-human animals

[0071] The "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome of the non-human animal has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. Cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (e.g., T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal is provided, the non-human animal comprising a modified endogenous APOC3 locus, comprising an exogenous sequence (e.g., 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.

[0072] The "chimeric (x) gene" or "chimeric (x) 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 one sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, a chimeric (x) gene or a chimeric (x) nucleic acid has at least a portion of a 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 (x) gene or a chimeric (x) nucleic acid refers to a humanized (x) gene or a humanized (x) nucleic acid.

[0073] The "chimeric (x) protein" or "chimeric (x) 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 one sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (x) protein or chimeric (x) polypeptide has two or more different species sources, for example, the same (or homologous) proteins of different species. In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide refers to a humanized protein or humanized polypeptide.

[0074] The "humanized (x) protein" or "humanized (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein at least a portion of the protein or polypeptide is derived from a human protein or polypeptide. In some embodiments, the humanized (x) protein or humanized (x) polypeptide refers to a human protein or polypeptide.

[0075] The "humanized (x) nucleic acid" of the present invention refers to a nucleic acid, wherein at least a portion of the nucleic acid is derived from a human. In some embodiments, the nucleic acids in the humanized (x) nucleic acid are all derived from a human. In some embodiments, the humanized (x) nucleic acid refers to a humanized exon, and the humanized exon can be a human exon or a chimeric exon.

[0076] In some embodiments, the chimeric APOC3 gene or chimeric APOC3 nucleic acid is a humanized APOC3 gene or humanized APOC3 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human APOC3 gene, and at least a portion of the gene or nucleic acid is derived from a non-human APOC3 gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding a humanized APOC3 protein. The encoded humanized APOC3 protein has at least one activity of a human APOC3 protein or a non-human animal APOC3 protein.

[0077] In some embodiments, the chimeric APOC3 protein or chimeric APOC3 polypeptide is a humanized APOC3 protein or humanized APOC3 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are from a human APOC3 protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are from a non-human animal APOC3 protein. The humanized APOC3 protein or humanized APOC3 polypeptide is functional, or has at least one activity of a human APOC3 protein or a non-human animal APOC3 protein.

[0078] 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.

[0079] In one aspect, the 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 one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified animal is 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 Mole-shaped Muridae (e.g., mole rats, bamboo rats and zokors). In one embodiment, the genetically modified rodent is selected from mice or rats (Muroidea), gerbils, spiny mice and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0080] 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 non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, 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 C57BL / 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.

[0081] The genetically modified non-human animal includes modification of the endogenous non-human animal APOC3 gene site. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature APOC3 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the mature APOC3 protein amino acid sequence). Although cells (e.g., ES cells, somatic cells) that may include the genetic modifications described herein are provided in the present invention, in many embodiments, the genetically modified non-human animal includes modification of the endogenous APOC3 gene site in the non-human animal.

[0082] The genetically modified non-human animal can express human APOC3 and / or chimeric (e.g., humanized) APOC3 at an endogenous mouse locus, wherein the endogenous mouse APOC3 gene has been replaced or inserted with a human APOC3 gene and / or a nucleotide sequence encoding a region of a human APOC3 sequence or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a human APOC3 sequence. In some embodiments, the endogenous non-human animal APOC3 locus is modified by comprising all or part of a nucleic acid sequence encoding a mature human APOC3 protein.

[0083] In some embodiments, the genetically modified mouse can express human APOC3 and / or chimeric APOC3 (e.g., humanized APOC3) under the control of a mouse promoter and / or mouse regulatory elements. Insertion or replacement at the mouse endogenous locus provides a non-human animal that expresses human APOC3 or chimeric APOC3 (e.g., humanized APOC3) in a suitable cell and in a manner that does not cause potential pathology observed in some other transgenic mice known in the art. Human APOC3 or chimeric APOC3 (e.g., humanized APOC3) expressed in a non-human animal can maintain one or more functions of wild-type mice or human APOC3 in the non-human animal. In addition, in some embodiments, the non-human animal does not express endogenous APOC3. In some embodiments, the expression level of endogenous APOC3 in the non-human animal is reduced compared to the expression level of APOC3 in wild-type animals. As used in the present invention, the term "endogenous APOC3" refers to an APOC3 protein expressed by an endogenous APOC3 nucleotide sequence of a non-human animal (e.g., a mouse) before any genetic modification.

[0084] In some embodiments, the humanized APOC3 locus comprises a human 5'UTR. In some embodiments, the humanized APOC3 locus comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, humanization comprises an endogenous (e.g., mouse) 3'UTR. In some embodiments, humanization comprises a human 3'UTR. Where appropriate, it is reasonable to assume that, based on the similarity of the 5' flanking sequences of the mouse and human APOC3 genes, they appear to be similarly regulated. As shown in the present application, humanized APOC3 mice comprising insertions or substitutions in the endogenous mouse APOC3 locus, which retain mouse regulatory elements but comprise humanization of the APOC3 coding sequence, do not exhibit pathological phenomena. Both genetically modified mice, either heterozygous or homozygous for humanized APOC3, are normal.

[0085] The present invention further relates to the APOC3 genomic DNA sequence of humanized mice, a DNA sequence obtained by reverse transcription of mRNA that is consistent with or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; and a tissue comprising the cell thereof.

[0086] The present invention further relates to a non-human mammal or its offspring produced by the above method. In some embodiments, its genome comprises human genes.

[0087] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0088] In some embodiments, the non-human mammal expresses a protein encoded by a humanized APOC3 gene.

[0089] 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. In some embodiments, the non-human mammal is a rodent (eg, mouse).

[0090] The present invention also provides a cell or cell line derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a 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 it carries a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring, or a non-human mammal carrying a tumor.

[0091] The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal comprises DNA of human or humanized APOC3.

[0092] In some embodiments, a non-human mammal comprises a gene construct as described herein. In some embodiments, a non-human mammal expressing a human or humanized APOC3 protein is provided. In some embodiments, a tissue specifically expressing a human or humanized APOC3 protein is provided.

[0093] In some embodiments, the expression of non-human animal human or humanized APOC3 protein is controllable, such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0094] 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.

[0095] 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.

[0096] 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 APOC3 protein can be detected by a variety of methods.

[0097] There are many analytical methods that can be used to detect exogenous DNA, including methods at the nucleic acid level (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 the ELISA method well known to those skilled in the art. Many standard analytical methods can be used to complete quantitative detection. For example, RT-PCR and hybridization methods can be used to detect 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 APOC3 protein.

[0098] In some embodiments, the genetically modified animals described herein (eg, APOC3 gene homozygous mice) can express human or humanized APOC3 in one or more liver tissue cells.

[0099] Methods for constructing genetically modified non-human animals

[0100] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting technology, homologous recombination technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques using embryonic stem cells. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR-Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these gene 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 genetically modified cells into enucleated oocytes and fusing enucleated oocytes with another genetically modified cell.

[0101] In some embodiments, the nucleotide sequence encoding the endogenous APOC3 region in the endogenous genome of at least one cell of the non-human animal is replaced by the nucleotide sequence encoding the corresponding region of human APOC3. In some embodiments, the expression level of the endogenous APOC3 protein of the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0102] The present invention provides a targeting vector. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized APOC3 gene fragment and a 3' homology arm. The process involves replacing the endogenous corresponding APOC3 sequence with the human or humanized APOC3 sequence by homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nuclease, TALEN or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace the mouse endogenous APOC3 sequence with the human or humanized APOC3 sequence.

[0103] Therefore, in some embodiments, the method of preparing a genetically modified humanized animal comprises replacing a nucleic acid sequence encoding an endogenous APOC3 region with a nucleotide sequence encoding a corresponding region of human APOC3 at the endogenous APOC3 locus (or site).

[0104] The present invention also provides a method for establishing an APOC3 gene humanized animal model, comprising the following steps:

[0105] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0106] (b) culturing the cells, preferably culturing the cells in a liquid culture medium;

[0107] (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;

[0108] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0109] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0110] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0111] 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.

[0112] 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.

[0113] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying the coding frame of the APOC3 gene of the non-human animal, for example, by replacing the nucleic acid sequence encoding the endogenous APOC3 region (e.g., CDS, genomic DNA or cDNA sequence) with a nucleotide sequence encoding the corresponding region of human APOC3 under the control of the endogenous regulatory elements of the APOC3 gene of the non-human animal. For example, one or more functional region sequences of the APOC3 gene of the non-human animal can be knocked out or inserted into a sequence so that the endogenous APOC3 protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the APOC3 gene of the modified non-human animal can be all or part of the nucleotide sequence of exon 2 to exon 4 of the APOC3 gene of the non-human animal.

[0114] 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 APOC3 protein after the endogenous regulatory element of the APOC3 gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the APOC3 gene humanized animal model can express a human or humanized APOC3 protein in vivo, but does not express the APOC3 protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0115] In some embodiments, the construction method includes obtaining using the above-mentioned vector. In some embodiments, the vector is a targeting vector or an sgRNA vector. In some embodiments, the construction method includes introducing the targeting vector into a non-human animal or its cells. In some embodiments, the construction method includes introducing the targeting vector and the sgRNA vector into a non-human animal or its cells.

[0116] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0117] (1) providing a plasmid comprising a human APOC3 gene fragment (e.g., the above-mentioned targeting vector), 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 endogenous APOC3 of a non-human animal;

[0118] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous APOC3 gene;

[0119] (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;

[0120] (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 APOC3 protein;

[0121] (5) The offspring mice obtained in step (4) are mated to obtain homozygous mice.

[0122] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal target site and a 3'-terminal target site.

[0123] In some embodiments, the sequence encoding the humanized APOC3 protein is operably linked to endogenous regulatory elements at the endogenous APOC3 locus.

[0124] In some embodiments, the genetically modified non-human animal does not express endogenous APOC3 protein.

[0125] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0126] (1) providing a plasmid (e.g., the targeting vector described above) comprising a human or chimeric APOC3 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 endogenous APOC3;

[0127] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous APOC3 gene;

[0128] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric APOC3 gene fragment into the genome.

[0129] In some embodiments, the nucleotide sequence encoding the APOC3 protein endogenously in the non-human animal is deleted. In some embodiments, all or part of exons 2-4 of the endogenous APOC3 gene of the non-human animal is deleted. In some embodiments, at least 50bp to 1000bp of continuous nucleotides are deleted from part of exon 2 to the downstream of the 3'UTR of the endogenous APOC3 gene of the non-human animal. In some embodiments, at least 50bp to 500bp of continuous nucleotides are deleted from part of exon 2 to the downstream of the 3'UTR of the endogenous APOC3 gene of the non-human animal. In some embodiments, the start codon of the endogenous APOC3 gene of the non-human animal is deleted to 321bp of continuous nucleotides downstream of the 3'UTR.

[0130] Use of genetically modified non-human animals

[0131] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at an endogenous non-human animal locus and under the control of an endogenous promoter and / or regulatory element can produce a non-human animal with qualities and characteristics that may be significantly different from a typical knockout plus transgenic animal. In a typical knockout plus transgenic animal, the endogenous locus is removed or destroyed, and a full human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of a human gene and / or protein assay and / or functional assay. In a human transgene, the upstream and / or downstream of the human sequence provides suitable support for expression and / or regulation of the transgene.

[0132] In some cases, transgenes with human regulatory elements are expressed in a non-physiological or otherwise unsatisfactory manner, and may actually be harmful to the animal. The present invention demonstrates that replacement or insertion of human sequences at endogenous loci under the control of endogenous regulatory elements to produce humanized animals provides physiologically appropriate expression patterns and levels that are meaningful and appropriate in the context of the physiology of the humanized animal with respect to the physiology of the replaced gene.

[0133] Genetically modified animals that express human or humanized APOC3 protein, 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 animal models.

[0134] The present invention also provides an application of the APOC3 gene-modified non-human animal and the non-human animal obtained by any of the above construction methods.

[0135] In some embodiments, the application comprises:

[0136] A) Application in product development involving APOC3-related immune processes in human cells;

[0137] B) Application as a model system related to APOC3 for pharmacology, immunology, microbiology and medical research;

[0138] C) Applications involving the production and use of animal experimental disease models for the study of APOC3-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0139] D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human APOC3 signaling pathway modulators; or,

[0140] E) To study the function of APOC3 gene, the drugs and efficacy targeting human APOC3 target sites, and the application of drugs related to APOC3 in cancer, inflammation, immune diseases, metabolic diseases or cardiovascular diseases.

[0141] The present invention provides a non-human animal expressing human or humanized APOC3 protein, which can be used for screening of human APOC3-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 cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor).

[0142] In some embodiments, the anti-APOC3 antibody blocks or inhibits the APOC3-mediated signaling pathway. In some embodiments, the anti-APOC3 antibody described herein can block the interaction between APOC3 complexes, thereby inhibiting the APOC3 signaling pathway.

[0143] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-APOC3 antibodies, nucleic acid drugs, 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 liver disease), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, multiple sclerosis, atopic dermatitis, pain or neurological disorders, etc. In some embodiments, the immune disease is asthma, psoriasis, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis.

[0144] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-APOC3 antibodies, nucleic acid drugs, or polypeptide drugs) in treating various inflammatory 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, the inflammation is inflammatory bowel disease (IBD).

[0145] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-APOC3 antibodies, nucleic acid drugs, or polypeptide drugs) in treating various metabolic diseases. In some embodiments, the metabolic disease is a lipid metabolism disease, and the lipid metabolism disease comprises hyperlipidemia, hypertriglyceridemia (HTG), lipid metabolism disorder, dyslipidemia, non-alcoholic fatty liver disease (NAFLD) or familial chylomicronemia syndrome (FCS). In some embodiments, the lipid metabolism disease is hyperlipidemia, hypertriglyceridemia (HTG) or lipid metabolism disorder.

[0146] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-APOC3 antibodies, nucleic acid drugs, or polypeptide drugs) in treating various cardiovascular diseases. In some embodiments, the cardiovascular disease comprises coronary heart disease, atherosclerosis, or cardiomyopathy. In some embodiments, the cardiovascular disease is atherosclerosis.

[0147] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-APOC3 antibodies, nucleic acid drugs, or polypeptide drugs) for treating cancer. In some embodiments, a therapeutic agent (e.g., an anti-APOC3 antibody, a nucleic acid drug, or a polypeptide drug) 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 in vivo animal imaging detection. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, wherein the metabolic changes include changes in food consumption or water consumption.

[0148] In some embodiments, the cancer comprises one or more tumor cells injected into an animal (e.g., tumor cells derived from a human or non-human animal). In some embodiments, the therapeutic agent inhibits the APOC3 signaling pathway. In some embodiments, the therapeutic agent does not inhibit the APOC3 signaling pathway.

[0149] In some embodiments, genetically modified non-human animals can be used to detect whether an anti-APOC3 antibody 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-APOC3 antibody, a nucleic acid drug, 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, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a subject's disease (e.g., cancer, immune disease, inflammation, metabolic disease, or cardiovascular disease). The inhibitory effect on tumors can also be determined by methods known in the art, for example, measuring the tumor volume in the animal, and / or determining the tumor (volume) inhibition rate (TGI). TV ). 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.

[0150] In some embodiments, therapeutic agents (e.g., anti-APOC3 antibodies, nucleic acid drugs, or polypeptide drugs) can be used to treat various cancers. The "cancer" of the present invention refers to cells with autonomous growth ability, that is, an abnormal state or condition characterized by rapid cell growth and proliferation. 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. The "cancer" or "tumor" of the present invention includes, but is not limited to, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, brain glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, 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 a specific embodiment of the present invention, the cancer 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 certain embodiments, the cancer is a solid tumor or a hematological tumor. In certain embodiments, the cancer is gastrointestinal cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, head and neck cancer, liver cancer or lung cancer.

[0151] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (e.g., an anti-APOC3 antibody, a nucleic acid drug, or a polypeptide drug). The method comprises administering an antibody to the non-human animal described above, and evaluating the animal's weight change or blood test. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit, or hemoglobin content. In some embodiments, the antibody can reduce red blood cells (RBC), hematocrit, or hemoglobin content by 20%, 30%, 40%, or more than 50%. In some embodiments, the animal's body weight is at least 5%, 10%, 20%, 30%, or 40% less than a control group (e.g., the average weight of an animal not treated with the antibody).

[0152] 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.

[0153] 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.

[0154] The present invention also provides an animal model generated by the method described in the present application to screen, verify, evaluate or study the safety or effectiveness of APOC3 gene function, human APOC3 antibodies, drugs targeting human APOC3 sites, drugs for immune diseases, metabolic diseases or cardiovascular diseases, and anti-tumor drugs or anti-inflammatory drugs.

[0155] 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 animals described herein, and applying human CAR-T to animals with human tumor cells. The effectiveness of CAR-T therapy can be determined and evaluated. In some embodiments, the animal is selected from APOC3 gene humanized non-human animals prepared by the methods described herein, double or multiple humanized non-human animals (or their offspring) produced by the methods described herein, non-human animals expressing human or humanized APOC3 proteins, or tumor-bearing or inflammatory animal models described herein. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat APOC3-related diseases described herein. In some embodiments, TCA-T, CAR-T and / or other immunotherapies provide evaluation methods for treating APOC3-related diseases described herein.

[0156] Non-human animal models with two or more human or chimeric genes

[0157] The present invention also provides a method for producing a genetically modified non-human animal model having two or more human or chimeric (x) genes. The non-human animal may comprise a human or chimeric APOC3 gene and a sequence encoding an additional human or chimeric (x) protein.

[0158] In some embodiments, the additional human or chimeric (x) genes include but are not limited to at least one genetically modified non-human animal of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the non-human animal described above also expresses at least one of human or humanized LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 proteins.

[0159] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric (x) genes, the method comprising:

[0160] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0161] (ii) mating, in vitro fertilization, or direct gene editing of the non-human animal provided in step (i) with other genetically modified non-human animals, and screening to obtain two or more genetically modified non-human animals.

[0162] In some embodiments, the other genetically modified non-human animals include but are not limited to non-human animals humanized with one or a combination of two or more of the genes LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0163] In some embodiments, APOC3 humanization is performed directly on a non-human animal with a modified human or chimeric LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 gene.

[0164] Since these proteins may be involved in different mechanisms, combination therapy targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are ongoing and have shown good results. Multigene modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, for example, therapeutic agents (e.g., anti-APOC3 antibodies, nucleic acid drugs, or polypeptide drugs), and additional therapeutic agents for the treatment of cancer or immune diseases (e.g., asthma, psoriasis, rheumatoid arthritis, multiple sclerosis, or atopic dermatitis). The method includes administering an anti-APOC3 antibody and an additional therapeutic agent to an animal, wherein the non-human animal has cancer or an immune disease, and determining the effect of the combined therapy on the cancer or immune disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. 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 described above comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0165] In some embodiments, the combination therapy is used to treat various cancers described in the present application, for example, gastrointestinal cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, head and neck cancer, liver cancer or lung cancer. In some embodiments, the combination therapy is designed to treat immune diseases described in the present application, such as asthma, psoriasis, rheumatoid arthritis, atopic dermatitis or multiple sclerosis. 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 a subject with chemotherapy, for example, camphor alkali, doxorubicin, cisplatin, carboplatin, procarbazine, meclorazepam, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chloramphenicol, 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

[0166] Figure 1 : Schematic diagram comparing mouse APOC3 locus and human APOC3 locus (not to scale);

[0167] Figure 2 : Schematic diagram of APOC3 gene targeting strategy and targeting vector V1 design (not to scale);

[0168] Figure 3 : Schematic diagram of APOC3 gene targeting strategy and targeting vector V2 design (not to scale);

[0169] Figure 4 : Southern blot identification results of APOC3 gene humanized mouse F1 generation, where WT is the wild-type control;

[0170] Figure 5 : RT-PCR test results, where + / + is wild-type C57BL / 6 mice, H / H is APOC3 gene humanized homozygous mice, and H2O is water control;

[0171] Figure 6 : ELISA test results, where + / + is wild-type C57BL / 6 mice, and H / H is APOC3 gene humanized homozygous mice;

[0172] Figure 7: Results of blood lipid test in wild-type C57BL / 6 mice and APOC3 humanized mice, where (A) is the concentration of TC in serum, (B) is the concentration of TG in serum, (C) is the concentration of HDL-C in serum, and (D) is the concentration of LD-LC in serum. ** indicates extremely significant difference (P<0.01);

[0173] Figure 8 : Results of blood lipid test in humanized APOC3 mice after administration, where (A) is the concentration of TC in serum, (B) is the concentration of TG in serum, (C) is the concentration of HDL-C in serum, and (D) is the concentration of LDL-C in serum. * indicates significant difference (P<0.05);

[0174] Fig. 9 :qPCR test results in liver tissue of APOC3 humanized mice after administration, ** indicates extremely significant difference (P<0.01);

[0175] Fig.10 : Results of serum ELISA test in APOC3 humanized mice after administration, ** indicates extremely significant difference (P<0.01). DETAILED DESCRIPTION

[0176] 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.

[0177] In each of the following examples, equipment and materials were obtained from the following companies:

[0178] C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Laboratory Animal Seed Center, China Food and Drug Inspection Institutes;

[0179] AseI enzyme was purchased from NEB, catalog number R0526S;

[0180] The AvrII enzyme was purchased from NEB, catalog number R0174S;

[0181] Apolipoprotein C3 Human ELISA Kit was purchased from invitrogen with the catalog number EHAPOC3.

[0182] Example 1 APOC3 gene humanized mice

[0183] A schematic diagram of the comparison of mouse APOC3 gene (NCBI Gene ID: 11814, located at positions 46144348 to 46146934 on chromosome 9 NC_000075.7, based on transcript NM_023114.4 and its encoded protein NP_075603.1 (SEQ ID NO: 1)) and human APOC3 gene (NCBI Gene ID: 345, located at positions 116829907 to 116833072 on chromosome 11 NC_000011.10, based on transcript NM_000040.3 and its encoded protein NP_000031.1 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0184] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human APOC3 protein can be introduced into the mouse endogenous APOC3 locus, so that the mouse expresses a human or humanized APOC3 protein. Specifically, using gene editing technology, under the control of the mouse APOC3 gene regulatory element, a nucleotide sequence from a partial sequence of exon 2 of the human APOC3 gene to a nucleotide sequence downstream of the 3'UTR (e.g., a nucleotide sequence from the start codon ATG to a 500bp nucleotide sequence downstream of the 3'UTR) of about 2.9kb is replaced by a partial sequence of mouse exon 2 to a nucleotide sequence downstream of the 3'UTR (e.g., a nucleotide sequence from the start codon ATG to a 321bp nucleotide sequence downstream of the 3'UTR) of about 1.9kb, to obtain a humanized APOC3 locus, thereby achieving humanization of the mouse APOC3 gene.

[0185] In order to realize the targeting strategy of the present invention, a targeting vector V1 was constructed (see Figure 2 ), the targeting vector contains homology arm sequences upstream and downstream of the mouse APOC3 gene, and fragment A containing the human APOC3 fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 46146012 to 46149958 of NCBI accession number NC_000075.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 46140309 to 46144026 of NCBI accession number NC_000075.7. The nucleotide sequence of the human APOC3 gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 116830583 to 116833572 of NCBI accession number NC_000011.10; the connection design of the upstream of the human APOC3 fragment sequence and the mouse is: 5'-

[0186]

[0187] The sequence " GTGCCThe last "C" in the sequence is the last nucleotide in the mouse. The first "A" in is the first nucleotide of the human sequence. The downstream connection of the human APOC3 fragment sequence and the mouse sequence is designed as: 5'-

[0188] The sequence " CAGTT The last "T" in the sequence is the last nucleotide of the human sequence. The "A" in is the first nucleotide of the mouse 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 APOC3 gene is designed as follows: 5'- The sequence " CAGTT The last of " T " is the last nucleotide of the human APOC3 gene, 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 APOC3 gene is designed as: 5'- The sequence " ACTTC The last "C" in the sequence is the last nucleotide of the Neo box. The "A" in is the first nucleotide of the mouse APOC3 gene. The mRNA sequence transcribed from the modified humanized mouse APOC3 gene is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2. 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 to be correct by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using a positive clone screening marker gene to screen out the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the techniques 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 the 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 mated with each other to obtain APOC3 gene humanized homozygous mice.

[0190] 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 upstream and downstream homology arm sequences of the mouse APOC3 gene, and a human APOC3 fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 31) is identical to the nucleotide sequence of positions 46146012 to 46147608 of NCBI accession number NC_000075.7, and the downstream 3' homology arm sequence (SEQ ID NO: 32) is identical to the nucleotide sequence of positions 46142545 to 46144026 of NCBI accession number NC_000075.7. The nucleotide sequence of the human APOC3 fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 116830583 to 116833572 of NCBI accession number NC_000011.10. The mRNA sequence transcribed from the modified humanized mouse APOC3 gene is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0191] 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.

[0192] The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence of an exemplary sgRNA on the APOC3 gene is as follows:

[0193] sgRNA1 target site (SEQ ID NO: 21): 5'-GGATGCGCTAAGTAGCGTGCAGG-3';

[0194] sgRNA2 target site (SEQ ID NO: 22): 5′-CTGTGCAAACCAGCTGCACGGGG-3′;

[0195] 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-APOC3-1 and pT7-APOC3-2.

[0196] Table 3 Sequence list of sgRNA1 and sgRNA2

[0197]

[0198]

[0199] The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment DNA (SEQ ID NO: 33) of the T7 promoter and sgRNA scaffold and connected to the backbone vector (source: Takara, item number 3299) by restriction digestion (EcoRI and BamHI) in sequence. The sequencing was verified by a professional sequencing company, and the results showed that the target plasmid was obtained. Take the pronuclear fertilized eggs of mice, such as C57BL / 6 mice, and use a microinjector to pre-mix the in vitro transcription products of pT7-APOC3-1 and pT7-APOC3-2 plasmids (using the Ambion in vitro transcription kit, according to the instructions for transcription), the targeting vector and Cas9mRNA and inject them into the cytoplasm or nucleus of the mouse fertilized eggs. 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 a short period of 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 APOC3 gene humanized mouse strain.

[0200] 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 AseI enzyme or AvrII enzyme, respectively, transferred to a membrane, and hybridized. The A probe (A Probe) and the 3' probe (3'Probe) are located on the human APOC3 genome fragment and downstream of the human 3' homology arm, respectively. The specific probes and target fragment lengths are shown in Table 4). The exemplary results are shown in Figure 4 As shown, combined with the PCR and sequencing results, two mice numbered F1-1 and F1-2 were positive mice. This shows that the method can be used to construct APOC3 gene humanized mice that can be stably propagated and have no random insertions.

[0201] Table 4 Length of specific probes and target fragments

[0202] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size AvrII 3'Probe 8.4kb 7.2kb AseI A Probe -- 17.5kb

[0203] A Probe-F (SEQ ID NO: 11): 5'-TCAGATCATCAGGCATTAGATTCTC-3',

[0204] A Probe-R (SEQ ID NO: 12): 5'-CACACTTCTGCTCACACTGGCCC-3';

[0205] 3'Probe-F (SEQ ID NO: 13): 5'-GTGGAAGAGGCTCCCGTCCTGC-3',

[0206] 3'Probe-R (SEQ ID NO: 14): 5'-CGGGCGCTAAGGGGACATCAAG-3';

[0207] The expression of mRNA in APOC3 gene humanized mice can be detected by RT-PCR. Specifically, 7-week-old female C57BL / 6 mice (+ / +) and 8-week-old female APOC3 gene humanized homozygous mice (H / 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. The detection results are shown in FIG. Figure 5 As shown. Figure 5 As can be seen, only mouse APOC3 mRNA was detected in wild-type C57BL / 6 mice, and human APOC3 mRNA was not detected; only human APOC3 mRNA was detected in APOC3 gene humanized homozygous mice.

[0208] Table 5 RT-PCR primer sequences and target fragment sizes

[0209]

[0210]

[0211] In addition, the expression of human APOC3 protein in APOC3 humanized mice can be detected by conventional methods such as ELISA. Specifically, 3 female C57BL / 6 mice (+ / +) aged 7 weeks and 3 female APOC3 homozygous humanized mice (H / H) aged 8 weeks prepared in this example were selected, and serum was collected and tested using a human-specific Apolipoprotein C3 Human ELISA kit. The test results are shown in FIG. Figure 6 shown.

[0212] from Figure 6 As can be seen in the figure, when using the human-specific APOC3 ELISA kit, human APOC3 protein was only detected in APOC3 humanized homozygous mice. Combined with the above RT-PCR results, it is proved that human APOC3 protein can be successfully expressed in APOC3 gene humanized homozygous mice.

[0213] Example 2 Pharmacodynamic Model

[0214] The humanized mice disclosed in the present invention can be used to induce the preparation of various disease models, such as immune diseases, inflammation, cancer, metabolic diseases or cardiovascular diseases. In particular, APOC3 protein can play an important role in regulating lipid abnormalities. The humanized mice disclosed in the present invention can be used to induce lipid metabolic diseases such as hyperlipidemia, hypertriglyceridemia, lipid metabolism disorders, dyslipidemia, non-alcoholic fatty liver disease, familial chylomicronemia syndrome, and disease models of cardiovascular diseases such as coronary heart disease, cardiomyopathy, and atherosclerosis, which can be used to test the in vivo efficacy of human-specific therapeutic drugs.

[0215] For example, 8 5-week-old C57BL / 6 mice and 9 APOC3 humanized homozygous mice were selected and divided into groups to measure the lipid levels in the mice. Group G1 consisted of female C57BL / 6 mice (n=4), group G2 consisted of male C57BL / 6 mice (n=4), group G3 consisted of female APOC3 humanized mice (n=3), and group G4 consisted of male APOC3 humanized mice (n=6). Blood was collected and centrifuged to obtain the supernatant for blood biochemical testing. The main test indicators were TG (triglyceride), TC (total cholesterol), LDL-C (low-density lipoprotein) and HDL-C (high-density lipoprotein) in mice. The results are as follows: Figure 7As shown in the figure, the concentrations of TG, TC, LDL-C and HDL-C in the serum of female APOC3 humanized homozygous mice and male APOC3 humanized homozygous mice were all increased compared with those in the serum of C57BL / 6 wild-type mice of the same sex. In summary, the construction of APOC3 humanized mice does not affect their blood lipid levels.

[0216] APOC3-ASO is a small nucleic acid molecule targeting APOC3. After entering the cell, it can specifically and complementary bind to APOC3 mRNA or Pre-mRNA, inhibit the translation of the mRNA or cause the degradation of APOC3 mRNA, reduce the concentration of triglycerides, and can be used to treat lipid metabolism diseases.

[0217] For another example, six 6-week-old APOC3 humanized homozygous mice were selected and randomly divided into a control group (G1) and a treatment group (G2), with 3 mice in each group. The specific grouping and administration are shown in Table 6. The treatment group was injected with the nucleic acid drug APOC3-ASO analog targeting human APOC3 mRNA, and the control group was injected with an equal volume of PBS. The drugs were administered on day 0 and day 7 after grouping, for a total of 2 times. On the 14th day, the mice were euthanized, and serum was taken for blood biochemical detection and ELISA protein expression detection, among which the serum detection indicators were TG (triglyceride), TC (total cholesterol), LDL-C (low-density lipoprotein) and HDL-C (high-density lipoprotein) of mice; liver tissue was taken, and the expression level of human APOC3 mRNA was detected by qPCR using an RNA extraction kit, and the qPCR primers are shown in Table 7.

[0218] Table 6 Specific groups, administration, dosage and frequency

[0219] Grouping drug Dosage Dosage Dosing frequency G1 PBS — SC QW×2 G2 APOC3-ASO analog 10mg / kg SC QW×2

[0220] Table 7 qPCR primer sequences

[0221]

[0222]

[0223] The results showed that after nucleic acid drug treatment, the levels of TG, TC, HDL-C and LDL-C in the serum of mice in the treatment group G2 were significantly reduced compared with those in the control group G1 ( Figure 8 ) and the expression of human APOC3 protein in the serum of G2 mice was significantly lower than that of G1 mice ( Fig.10 ); The expression level of human APOC3 mRNA in the liver of G2 mice was significantly reduced ( Fig. 9 The inhibition rate was 73.4%, with the content of group G1 as 100%, indicating that APOC3 humanized mice can be used for the in vivo efficacy evaluation of drugs targeting human APOC3.

[0224] Example 3 Preparation of double-gene or multi-gene humanized mice

[0225] The APOC3 gene humanized mouse prepared by the present method can also be used to prepare a 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 modified with at least one gene containing LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4, or, on the basis of humanized APOC3 mice, mouse ES embryonic stem cells can be separated and gene recombination targeting technology can be used to obtain a double-gene humanized or multi-gene humanized mouse model. The homozygous or heterozygous APOC3 mouse obtained by the present method can also be mated with other gene-modified mice, and the offspring can be screened. According to Mendel's genetic law, there is a certain probability of obtaining a multi-gene mouse modified with the humanized APOC3 gene and other genes, and then the heterozygotes can be mated with each other to obtain a homozygous modified with two genes or multiple genes.

[0226] 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.

[0227] 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.

[0228] 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 human or chimeric apolipoprotein C3 (APOC3); Preferably, the amino acid sequence of the human or chimeric APOC3 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.

2. A method for constructing a genetically modified non-human animal, characterized in that: At the endogenous APOC3 gene locus of the non-human animal, the endogenous APOC3 gene is replaced with a nucleotide sequence comprising human APOC3.

3. The construction method according to claim 1 or 2, characterized in that: The endogenous APOC3 protein of the non-human animal is not expressed or is expressed at a reduced level compared to APOC3 in wild-type animals; Preferably, the nucleotide sequence encoding the human or chimeric APOC3 protein or the nucleotide sequence of human APOC3 is operably linked to an endogenous APOC3 regulatory element, such as a promoter; Preferably, the modified APOC3 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

4. The construction method according to claim 2 or 3, characterized in that: The nucleotide sequence of human APOC3 includes part of exon 2 to all of exon 4 of the human APOC3 gene; Preferably, it also contains at least 50 bp of continuous nucleotides downstream of the 3'UTR; Further preferably, the nucleotide sequence of human APOC3 comprises at least 50 bp of continuous nucleotides from the start codon to the downstream of the 3'UTR; More preferably, the nucleotide sequence of human APOC3 comprises SEQ ID NO: 5, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

5.

5. The construction method according to any one of claims 2 to 4, characterized in that: The nucleotide sequence of the replaced endogenous APOC3 gene includes part of exon 2 to all of exon 4 of the endogenous APOC3 gene of a non-human animal; preferably, it also includes at least 50 bp of continuous nucleotides downstream of the 3'UTR; Further preferably, the nucleotide sequence of the replaced endogenous APOC3 gene comprises at least 50 bp of continuous nucleotides from the start codon to the downstream of the 3'UTR of the endogenous APOC3 gene of the non-human animal.

6. The construction method according to any one of claims 1 to 5, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the non-human animal is a mouse or a rat; Preferably, the mRNA transcribed from the modified APOC3 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.

7. The construction method according to any one of claims 1 to 6, characterized in that: The non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

8. An application of a non-human animal obtained by the construction method according to any one of claims 1 to 7, characterized in that: The application includes: A) Application in product development involving APOC3-related immune processes in human cells; B) Application as a model system related to APOC3 for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of APOC3-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human APOC3 signaling pathway modulators; or, E) To study the function of APOC3 gene, the drugs and their efficacy targeting the human APOC3 target site, and the application of APOC3-related drugs in cancer, immune diseases, inflammation, metabolic diseases or cardiovascular diseases.

9. A method for determining the effectiveness of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 7, wherein the non-human animal has a disease; 2) determine the inhibitory effect of the therapeutic agent on the disease; Preferably, the therapeutic agent is an anti-APOC3 antibody, a nucleic acid drug or a polypeptide drug; further preferably, the therapeutic agent further comprises an additional therapeutic agent, such as an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the disease is cancer, immune disease, inflammation, metabolic disease or cardiovascular disease.

10. A method for determining the toxicity of a therapeutic agent, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 7; 2) Determine the effects of therapeutic agents on non-human animals; Preferably, the therapeutic agent is an anti-APOC3 antibody, a nucleic acid drug or a polypeptide drug; preferably, determining the effect of the therapeutic agent on the non-human animal involves measuring the body weight or a blood test of the non-human animal, wherein the blood test includes but is not limited to red blood cell count, hematocrit or hemoglobin content.

11. A humanized APOC3 gene, characterized in that: The humanized APOC3 gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding the protein of SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 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.

12. A cell, tissue or organ, characterized in that: The cell, tissue or organ contains the humanized APOC3 gene according to claim 11.

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