AGT and / or REN gene modified non-human animal

Abstract

The invention belongs to the technical field of biology, and particularly relates to a non-human animal modified by AGT and / or REN genes. The non-human animals can be used in the development of products involving AGT and / or REN-related immune processes of human cells as AGT and / or REN-related model systems for pharmacological, immunological, microbiological and medical studies, relates to production and utilization of animal experiment disease models for etiological research related to AGT and / or REN and / or for development of diagnostic strategies and / or for development of therapeutic strategies, in-vivo research of screening of human AGT and / or REN signaling pathway modulators, drug efficacy detection, evaluation of curative effects, verification or evaluation, research of AGT and / or REN gene functions, and in-vivo research of human AGT and / or REN signaling pathway modulators. The medicine and the medicine effect aiming at the human AGT and / or REN target site are researched, and the medicine for hypertension, kidney diseases and / or cardiovascular diseases related to AGT and / or REN is researched.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a non-human animal modified with AGT and / or REN genes. Background Art

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

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

[0004] The purpose of the first aspect of the present invention is to provide a method for constructing an AGT gene-modified non-human animal.

[0005] The second aspect of the present invention aims to provide an AGT gene-modified non-human animal or its offspring.

[0006] The third aspect of the present invention aims to provide a method for constructing a non-human animal disease model.

[0007] The fourth aspect of the present invention is to provide a non-human animal disease model or its offspring.

[0008] The fifth aspect of the present invention aims to provide a cell, tissue or organ.

[0009] The purpose of the sixth aspect of the present invention is to provide applications of non-human animals or their offspring obtained by the construction method of the first or third aspect of the present invention, and / or cells, tissues or organs of the fifth aspect of the present invention.

[0010] The seventh aspect of the present invention aims to provide a method for determining the effectiveness of an AGT therapeutic agent in treating AGT-related diseases.

[0011] The eighth aspect of the present invention aims to provide a method for determining the toxicity of AGT therapeutic agents.

[0012] The ninth aspect of the present invention aims to provide a method for constructing a non-human animal modified with a REN gene.

[0013] The tenth aspect of the present invention aims to provide a REN gene-modified non-human animal or its offspring.

[0014] The eleventh aspect of the present invention is to provide a method for constructing a non-human animal disease model.

[0015] The twelfth aspect of the present invention is to provide a non-human animal disease model or its offspring.

[0016] The thirteenth aspect of the present invention is to provide a cell, tissue or organ.

[0017] The purpose of the fourteenth aspect of the present invention is to provide the use of the non-human animals or their offspring obtained by the construction method of the ninth or eleventh aspect of the present invention, and / or the cells, tissues or organs of the thirteenth aspect of the present invention.

[0018] The fifteenth aspect of the present invention aims to provide a method for determining the effectiveness of a REN therapeutic agent in treating REN-related diseases.

[0019] The sixteenth aspect of the present invention aims to provide a method for determining the toxicity of REN therapeutic agents.

[0020] The purpose of the seventeenth aspect of the present invention is to provide a method for constructing a non-human animal modified with AGT and REN genes.

[0021] The eighteenth aspect of the present invention aims to provide a non-human animal or its offspring modified with AGT and REN genes.

[0022] The purpose of the nineteenth aspect of the present invention is to provide a method for constructing a non-human animal disease model.

[0023] The twentieth aspect of the present invention aims to provide a non-human animal disease model.

[0024] The twenty-first aspect of the present invention aims to provide a cell, tissue or organ.

[0025] The purpose of the twenty-second aspect of the present invention is to provide applications of non-human animals or their offspring obtained by the construction method of the seventeenth or nineteenth aspect of the present invention, and / or cells, tissues or organs of the twenty-first aspect of the present invention.

[0026] The twenty-third aspect of the present invention aims to provide a method for determining the effectiveness of AGT and / or REN therapeutic agents in treating AGT and / or REN-related diseases.

[0027] The twenty-fourth aspect of the present invention aims to provide a method for determining the toxicity of AGT and / or REN therapeutic agents.

[0028] In a first aspect, the present invention provides a method for constructing a non-human animal modified with an AGT gene, wherein the genome of the non-human animal comprises at least one chromosome comprising all or part of a nucleotide sequence encoding a human AGT protein.

[0029] In some embodiments, the amino acid sequence of the human AGT protein comprises SEQ ID NO: 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0030] A method for constructing an AGT gene-modified non-human animal, comprising introducing a human AGT nucleotide sequence into the endogenous AGT locus of the non-human animal.

[0031] In some embodiments, the introduction is a replacement or insertion; further a substitution.

[0032] In some embodiments, the construction method comprises the following step: replacing the nucleotide sequence of endogenous AGT of a non-human animal with the nucleotide sequence of human AGT.

[0033] In some embodiments, the nucleotide sequence of human AGT comprises all or part of the nucleotide sequence encoding human AGT protein.

[0034] In some embodiments, the amino acid sequence of the human AGT protein comprises SEQ ID NO: 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0035] In some embodiments, the nucleotide sequence of human AGT comprises all or part of exon 1 to exon 5 of the human AGT gene.

[0036] In some embodiments, the nucleotide sequence of human AGT comprises the nucleotide sequence from the start codon to the stop codon of the human AGT gene, preferably further comprises 5'UTR and / or 3'UTR, more preferably further comprises at least 50 bp continuous nucleotide sequence upstream of 5'UTR and / or at least 50 bp continuous nucleotide sequence downstream of 3'UTR.

[0037] In some embodiments, the nucleotide sequence of human AGT comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR.

[0038] In some embodiments, the nucleotide sequence of human AGT comprises nucleotide sequence 230702763-230748399 of NC_000001.11, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto.

[0039] In some embodiments, the nucleotide sequence of human AGT comprises a continuous nucleotide sequence of at least 50 bp downstream of the human AGT gene to the 3'UTR.

[0040] In some embodiments, the nucleotide sequence of human AGT comprises SEQ ID NO: 25, or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto.

[0041] In some embodiments, the human AGT nucleotide sequence is operably linked to an exogenous AGT regulatory element.

[0042] In some embodiments, the exogenous AGT regulatory element is a human AGT regulatory element.

[0043] In some embodiments, the nucleotide sequence of endogenous AGT of a non-human animal comprises all or part of a nucleotide sequence encoding an endogenous AGT protein of a non-human animal.

[0044] In some embodiments, the non-human animal endogenous AGT protein comprises SEQ ID NO: 1, SEQ ID NO: 5, or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and has the same function.

[0045] In some embodiments, the nucleotide sequence of the non-human animal endogenous AGT comprises all or part of exon 1 to exon 5 of the non-human animal endogenous AGT gene.

[0046] In some embodiments, the nucleotide sequence of the endogenous AGT of the non-human animal comprises the nucleotide sequence from the start codon to the stop codon of the endogenous AGT gene of the non-human animal, preferably further comprises 5'UTR and / or 3'UTR, more preferably further comprises at least 50bp continuous nucleotide sequence upstream of the 5'UTR and / or at least 50bp continuous nucleotide sequence downstream of the 3'UTR.

[0047] In some embodiments, the nucleotide sequence of the endogenous AGT of the non-human animal comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR.

[0048] In some embodiments, the nucleotide sequence of the non-human animal endogenous AGT comprises a continuous nucleotide sequence of at least 50 bp downstream of the non-human animal endogenous AGT gene to the 3'UTR.

[0049] In some embodiments, the endogenous AGT protein in the non-human animal is not expressed or is expressed at a reduced level compared to AGT in a wild-type non-human animal.

[0050] In some embodiments, the modified AGT gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

[0051] In some embodiments, gene editing technology is used to introduce the nucleotide sequence of human AGT into the endogenous AGT locus of a non-human animal.

[0052] In some embodiments, the gene editing technology includes gene targeting technology using embryonic stem cells, CRISPR / Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology or homing endonuclease.

[0053] In some embodiments, the sgRNA in the CRISPR / Cas9 comprises sgRNA1 and sgRNA2; the target site sequence of the sgRNA1 comprises SEQ ID NO: 28; and the target site sequence of the sgRNA2 comprises SEQ ID NO: 29.

[0054] In some embodiments, the sequence of the sgRNA1 comprises SEQ ID NO: 30; the sequence of the sgRNA2 comprises SEQ ID NO: 34.

[0055] In some embodiments, the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA1 comprise SEQ ID NO: 30 and SEQ ID NO: 32, respectively; the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA2 comprise SEQ ID NO: 34 and SEQ ID NO: 36, respectively.

[0056] In some embodiments, the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA1 comprise SEQ ID NO: 31 and SEQ ID NO: 33, respectively; the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA2 comprise SEQ ID NO: 35 and SEQ ID NO: 37, respectively.

[0057] In some embodiments, a targeting vector is used to introduce a nucleotide sequence for human AGT at the endogenous AGT locus in a non-human animal.

[0058] In some embodiments, the targeting vector comprises the nucleotide sequence of human AGT; and

[0059] The 5' homology arm of the non-human animal AGT gene and the 3' homology arm of the non-human animal AGT gene.

[0060] In some embodiments, the non-human animal is a mouse or a rat.

[0061] In some embodiments, the 5' homology arm of the non-human animal AGT gene comprises SEQ ID NO: 3, a nucleotide sequence complementary to SEQ ID NO: 3, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0062] In some embodiments, the 3' homology arm of the non-human animal AGT gene comprises SEQ ID NO: 4, a nucleotide sequence complementary to SEQ ID NO: 4, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0063] In some embodiments, the 5' homology arm of the non-human animal AGT gene comprises SEQ ID NO: 23, a nucleotide sequence complementary to SEQ ID NO: 23, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0064] In some embodiments, the 3' homology arm of the non-human animal AGT gene comprises SEQ ID NO: 24, a nucleotide sequence complementary to SEQ ID NO: 24, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0065] In some embodiments, the mRNA transcribed from the modified AGT gene in the genome of the non-human animal comprises SEQ ID NO: 6, SEQ ID NO: 66, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto.

[0066] In some embodiments, the non-human animal further comprises a nucleotide sequence encoding another human or chimeric protein.

[0067] In some embodiments, the other human or chimeric proteins include at least one of REN, APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4; further REN.

[0068] In the present invention, the non-human animals include non-human mammals; further include at least one of non-human primates, rodents, cattle (e.g., cows, bulls, buffaloes), deer, pigs, sheep (e.g., sheep, goats), dogs, rabbits, chickens, cats, ferrets, and horses; and further rodents.

[0069] In some embodiments, the non-human primate comprises at least one of an orangutan, an ape, or a monkey (eg, a marmoset, a rhesus monkey).

[0070] In some embodiments, the rodent comprises at least one of a mouse, a rat, a hamster, and a guinea pig; further comprises at least one of a mouse and a rat.

[0071] In some embodiments, the rodent is selected from the family Muridae; further selected from the families Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny rats, crested rats), Malmyridae (climbing mice, rock mice, tailed rats, Madagascar rats, and mice), Spinellidae (e.g., spiny dormouse), and Mormoriidae (e.g., mole rats, bamboo rats, and zokors).

[0072] In some embodiments, the rodent is a mouse of the C57BL strain selected from the group consisting of 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 the group consisting of 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, and 129 / Sv-ter / +. These mice are described, for example, in 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 mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as the BALB / c strain. In some embodiments, the mouse is a hybrid of the BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having BALB / c, BALB / cHeAn, BALB / cJ, BALB / cR1, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / 01a), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strains and NOD, NOD / SCID, NOD-Prkdc scid IL-2rg null Background of mice.

[0073] The second aspect of the present invention provides an AGT gene-modified non-human animal or its offspring, wherein the AGT gene-modified non-human animal is obtained by the construction method of the first aspect of the present invention.

[0074] The third aspect of the present invention provides a method for constructing a non-human animal disease model, comprising the steps of obtaining a non-human animal or its offspring using the construction method of the first aspect of the present invention, wherein the disease is an AGT-related disease.

[0075] In the present invention, the ATG-related disease comprises at least one of hypertension, kidney disease, and / or cardiovascular disease.

[0076] In the present invention, the ATG-related disease is selected from at least one of familial hyperreninemia / renin deficiency, reninoma, essential hypertension, secondary hypertension, nephropathy, chronic kidney disease, diabetic nephropathy, and heart failure.

[0077] In the present invention, the ATG-related disease includes at least one of tumors, inflammation, and immune diseases.

[0078] In the present invention, the ATG-related disease is selected from hypertension, renal disease, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetes Diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states, including chronic steroid therapy, pheochromocytoma, nephrinoma, secondary aldosteronism, and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR) and fetal growth restriction, eclampsia at least one; further selected from hypertension.

[0079] The fourth aspect of the present invention provides a non-human animal disease model or its progeny, wherein the non-human animal disease model is obtained by the construction method of the third aspect of the present invention.

[0080] The fifth aspect of the present invention provides a cell, tissue or organ derived from a non-human animal or its offspring obtained by the construction method of the first aspect or the third aspect of the present invention.

[0081] In some embodiments, the cells, tissues or organs do not involve reproductive material.

[0082] The sixth aspect of the present invention provides a use of a non-human animal or its progeny obtained by the construction method of the first or third aspect of the present invention, and / or a cell, tissue or organ of the fifth aspect of the present invention, wherein the use comprises:

[0083] A) Application in the development of products involving AGT-related immune processes in human cells;

[0084] B) Application as a model system for AGT-related research in pharmacology, immunology, microbiology, and medicine;

[0085] C) applications involving the production and use of animal experimental disease models for the study of the etiology of AGT and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0086] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human AGT signaling pathway modulators; or

[0087] E) Study the function of the AGT gene, study the drugs and their efficacy targeting the human AGT target site, and study the application of drugs in tumors, inflammation, and / or immune diseases related to AGT.

[0088] In some embodiments, the use does not involve the diagnosis or treatment of a disease.

[0089] The seventh aspect of the present invention provides a method for determining the effectiveness of AGT therapeutic agents in treating AGT-related diseases, comprising the steps of using non-human animals or their offspring obtained by the construction method of the first aspect or the third aspect of the present invention (preferably non-human animals or their offspring obtained by the construction method of the third aspect of the present invention), or cells, tissues or organs of the fifth aspect of the present invention.

[0090] In some embodiments, the method comprises the steps of administering an AGT therapeutic agent to a non-human animal or its offspring obtained by the construction method of the first aspect or the third aspect of the present invention (preferably a non-human animal or its offspring obtained by the construction method of the third aspect of the present invention), and determining the effectiveness of the AGT therapeutic agent in treating AGT-related diseases.

[0091] In some embodiments, the method does not involve the diagnosis or treatment of a disease.

[0092] In the present invention, the AGT therapeutic agent can be a macromolecular drug (eg, an AGT antibody, a polypeptide targeting AGT), a nucleic acid drug, or a small molecule drug.

[0093] The eighth aspect of the present invention provides a method for determining the toxicity of an AGT therapeutic agent, comprising the steps of using a non-human animal or its offspring obtained by the construction method of the first aspect or the third aspect of the present invention (preferably a non-human animal or its offspring obtained by the construction method of the third aspect of the present invention), or cells, tissues or organs of the fifth aspect of the present invention.

[0094] In some embodiments, the method comprises the steps of administering an AGT therapeutic agent to a non-human animal or its offspring obtained by the construction method of the first aspect or the third aspect of the present invention (preferably a non-human animal or its offspring obtained by the construction method of the third aspect of the present invention), and determining the effect of the AGT therapeutic agent on the animal.

[0095] In some embodiments, determining the effect of the AGT therapeutic on the animal comprises measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

[0096] In some embodiments, the method does not involve the diagnosis or treatment of a disease.

[0097] A ninth aspect of the present invention provides a method for constructing a REN gene-modified non-human animal, wherein the genome of the non-human animal comprises at least one chromosome comprising all or part of a nucleotide sequence encoding a human REN protein.

[0098] In some embodiments, the amino acid sequence of the human REN protein comprises SEQ ID NO: 21, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0099] A method for constructing a REN gene-modified non-human animal, comprising introducing a human REN nucleotide sequence into the endogenous REN locus of the non-human animal.

[0100] In some embodiments, the introduction is a replacement or insertion; further a substitution.

[0101] In some embodiments, the construction method comprises the step of replacing the nucleotide sequence of endogenous REN of a non-human animal with the nucleotide sequence of human REN.

[0102] In some embodiments, the human REN nucleotide sequence comprises all or part of a nucleotide sequence encoding a human REN protein.

[0103] In some embodiments, the amino acid sequence of the human REN protein comprises SEQ ID NO: 21, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0104] In some embodiments, the nucleotide sequence of human REN comprises all or part of exon 1 to exon 10 of the human REN gene.

[0105] In some embodiments, the nucleotide sequence of human REN comprises the nucleotide sequence from the start codon to the stop codon of the human REN gene, preferably further comprises 5'UTR and / or 3'UTR, more preferably further comprises at least 50 bp continuous nucleotide sequence upstream of 5'UTR and / or at least 50 bp continuous nucleotide sequence downstream of 3'UTR.

[0106] In some embodiments, the nucleotide sequence of human REN comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR.

[0107] In some embodiments, the nucleotide sequence of human REN comprises nucleotide sequence 204154277-204169793 of NC_000001.11, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto.

[0108] In some embodiments, the human REN nucleotide sequence is operably linked to an exogenous REN regulatory element.

[0109] In some embodiments, the exogenous REN regulatory element is a human REN regulatory element.

[0110] In some embodiments, the nucleotide sequence of the non-human animal endogenous REN comprises all or part of a nucleotide sequence encoding an endogenous REN protein of the non-human animal.

[0111] In some embodiments, the non-human animal endogenous REN protein comprises SEQ ID NO: 20, SEQ ID NO: 22, or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and has the same function.

[0112] In some embodiments, the nucleotide sequence of the non-human animal endogenous REN comprises all or part of exon 1 to exon 9 of the non-human animal endogenous REN gene.

[0113] In some embodiments, the nucleotide sequence of the non-human animal endogenous REN comprises a nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous REN gene, preferably further comprises a 5'UTR and / or a 3'UTR, and more preferably further comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR.

[0114] In some embodiments, the nucleotide sequence of the endogenous REN of the non-human animal comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR.

[0115] In some embodiments, the non-human animal's endogenous REN protein is not expressed or is expressed at a reduced level compared to REN in a wild-type non-human animal.

[0116] In some embodiments, the modified REN gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

[0117] In some embodiments, gene editing technology is used to introduce a human REN nucleotide sequence into the endogenous REN locus of a non-human animal.

[0118] In some embodiments, the gene editing technology includes gene targeting technology using embryonic stem cells, CRISPR / Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology or homing endonuclease.

[0119] In some embodiments, the sgRNA in the CRISPR / Cas9 comprises sgRNA3 and sgRNA4; the target site sequence of the sgRNA3 comprises SEQ ID NO: 56; and the target site sequence of the sgRNA4 comprises SEQ ID NO: 57.

[0120] In some embodiments, the sequence of the sgRNA3 comprises SEQ ID NO: 58; the sequence of the sgRNA4 comprises SEQ ID NO: 62.

[0121] In some embodiments, the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA3 comprise SEQ ID NO: 58 and SEQ ID NO: 60, respectively; the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA4 comprise SEQ ID NO: 62 and SEQ ID NO: 64, respectively.

[0122] In some embodiments, the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA3 comprise SEQ ID NO: 59 and SEQ ID NO: 61, respectively; the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA4 comprise SEQ ID NO: 63 and SEQ ID NO: 65, respectively.

[0123] In some embodiments, a targeting vector is used to introduce a human REN nucleotide sequence at the endogenous REN locus in a non-human animal.

[0124] In some embodiments, the targeting vector comprises the nucleotide sequence of human REN; and

[0125] The 5' homology arm of the non-human animal REN gene and the 3' homology arm of the non-human animal REN gene.

[0126] In some embodiments, the non-human animal is a mouse or a rat.

[0127] In some embodiments, the 5' homology arm of the non-human animal REN gene comprises SEQ ID NO: 42, a nucleotide sequence complementary to SEQ ID NO: 42, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0128] In some embodiments, the 3' homology arm of the non-human animal REN gene comprises SEQ ID NO: 43, a nucleotide sequence complementary to SEQ ID NO: 43, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0129] In some embodiments, the 5' homology arm of the non-human animal REN gene comprises SEQ ID NO: 52, a nucleotide sequence complementary to SEQ ID NO: 52, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0130] In some embodiments, the 3' homology arm of the non-human animal REN gene comprises SEQ ID NO: 53, a nucleotide sequence complementary to SEQ ID NO: 53, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0131] In some embodiments, the mRNA transcribed from the modified REN gene in the genome of the non-human animal comprises SEQ ID NO: 48, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto.

[0132] In some embodiments, the non-human animal further comprises a nucleotide sequence encoding another human or chimeric protein.

[0133] In some embodiments, the other human or chimeric protein includes at least one of AGT, APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4; further being AGT.

[0134] The tenth aspect of the present invention provides a REN gene-modified non-human animal or its offspring, wherein the REN gene-modified non-human animal is obtained by the construction method of the ninth aspect of the present invention.

[0135] The eleventh aspect of the present invention provides a method for constructing a non-human animal disease model, comprising the steps of obtaining a non-human animal or its offspring using the construction method of the ninth aspect of the present invention, wherein the disease is a REN-related disease.

[0136] In the present invention, the REN-related disease comprises at least one of hypertension, kidney disease, and / or cardiovascular disease.

[0137] In the present invention, the REN-related disease is at least one selected from familial hyperreninemia / renin deficiency, reninoma, essential hypertension, secondary hypertension, nephropathy, chronic kidney disease, diabetic nephropathy, and heart failure.

[0138] The twelfth aspect of the present invention provides a non-human animal disease model or its progeny, wherein the non-human animal disease model is obtained by the construction method of the eleventh aspect of the present invention.

[0139] The thirteenth aspect of the present invention provides a cell, tissue or organ derived from a non-human animal or its offspring obtained by the construction method of the ninth or eleventh aspect of the present invention.

[0140] In some embodiments, the cells, tissues or organs do not involve reproductive material.

[0141] The fourteenth aspect of the present invention provides a use of a non-human animal or its progeny obtained by the construction method of the ninth or eleventh aspect of the present invention, and / or a cell, tissue or organ of the thirteenth aspect of the present invention, wherein the use comprises:

[0142] A) Applications in the development of products involving human cells for REN-related immune processes;

[0143] B) Application as a model system for REN-related research in pharmacology, immunology, microbiology, and medicine;

[0144] C) applications involving the production and use of animal experimental disease models for the study of the etiology of REN and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0145] D) in vivo studies for the screening, efficacy testing, efficacy assessment, validation or evaluation of human REN signaling pathway modulators; or

[0146] E) Study the function of the REN gene, study the drugs and their efficacy targeting the human REN target site, and study the application of drugs in hypertension, kidney disease, and / or cardiovascular disease related to REN.

[0147] In some embodiments, the use does not involve the diagnosis or treatment of a disease.

[0148] The fifteenth aspect of the present invention provides a method for determining the effectiveness of REN therapeutic agents in treating REN-related diseases, comprising the steps of using a non-human animal or its offspring obtained by the construction method of the ninth aspect or the eleventh aspect of the present invention (preferably a non-human animal or its offspring obtained by the construction method of the eleventh aspect of the present invention), or a cell, tissue or organ of the thirteenth aspect of the present invention.

[0149] In some embodiments, the method comprises the steps of administering a REN therapeutic agent to a non-human animal or its offspring obtained by the construction method of the ninth aspect or the eleventh aspect of the present invention (preferably a non-human animal or its offspring obtained by the construction method of the eleventh aspect of the present invention), and determining the effectiveness of the REN therapeutic agent in treating REN-related diseases.

[0150] In some embodiments, the method does not involve the diagnosis or treatment of a disease.

[0151] In the present invention, the REN therapeutic agent can be a macromolecular drug (eg, REN antibody, polypeptide targeting REN), a nucleic acid drug or a small molecule drug.

[0152] The sixteenth aspect of the present invention provides a method for determining the toxicity of a REN therapeutic agent, comprising the steps of using a non-human animal or its offspring obtained by the construction method of the ninth aspect or the eleventh aspect of the present invention (preferably a non-human animal or its offspring obtained by the construction method of the eleventh aspect of the present invention), or a cell, tissue or organ of the thirteenth aspect of the present invention.

[0153] In some embodiments, the method comprises the steps of administering a REN therapeutic agent to a non-human animal or its offspring obtained by the construction method of the ninth aspect or the eleventh aspect of the present invention (preferably a non-human animal or its offspring obtained by the construction method of the eleventh aspect of the present invention), and determining the effect of the REN therapeutic agent on the animal.

[0154] In some embodiments, determining the effect of a REN therapeutic on an animal comprises measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

[0155] In some embodiments, the method does not involve the diagnosis or treatment of a disease.

[0156] The seventeenth aspect of the present invention provides a method for constructing a non-human animal modified with AGT and REN genes, wherein the genome of the non-human animal comprises at least two chromosomes, wherein one chromosome comprises all or part of a nucleotide sequence encoding human AGT protein; and the other chromosome comprises all or part of a nucleotide sequence encoding human REN protein.

[0157] In some embodiments, the amino acid sequence of the human AGT protein comprises SEQ ID NO: 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0158] In some embodiments, the amino acid sequence of the human REN protein comprises SEQ ID NO: 21, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0159] A method for constructing a non-human animal modified with AGT and REN genes, wherein a nucleotide sequence of human AGT is introduced into the endogenous AGT locus of the non-human animal; and

[0160] A human REN nucleotide sequence is introduced into the endogenous REN locus of a non-human animal.

[0161] In some embodiments, the introduction is a replacement or insertion; further a substitution.

[0162] In some embodiments, the construction method comprises the following steps:

[0163] Replacing the nucleotide sequence of endogenous AGT of the non-human animal with the nucleotide sequence of human AGT; and

[0164] The nucleotide sequence of the endogenous REN of the non-human animal is replaced with the nucleotide sequence of the human REN.

[0165] In some embodiments, the nucleotide sequence of human AGT is the nucleotide sequence of human AGT in the first aspect of the present invention.

[0166] In some embodiments, the nucleotide sequence of the non-human animal endogenous AGT is the nucleotide sequence of the non-human animal endogenous AGT in the first aspect of the present invention.

[0167] In some embodiments, the endogenous AGT protein in the non-human animal is not expressed or is expressed at a reduced level compared to AGT in a wild-type non-human animal.

[0168] In some embodiments, the modified AGT gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

[0169] In some embodiments, gene editing technology is used to introduce the nucleotide sequence of human AGT into the endogenous AGT locus of a non-human animal.

[0170] In some embodiments, the gene editing technology includes gene targeting technology using embryonic stem cells, CRISPR / Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology or homing endonuclease.

[0171] In some embodiments, the sgRNA in the CRISPR / Cas9 comprises sgRNA1 and sgRNA2; the target site sequence of the sgRNA1 comprises SEQ ID NO: 28; and the target site sequence of the sgRNA2 comprises SEQ ID NO: 29.

[0172] In some embodiments, the sequence of the sgRNA1 comprises SEQ ID NO: 30; the sequence of the sgRNA2 comprises SEQ ID NO: 34.

[0173] In some embodiments, the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA1 comprise SEQ ID NO: 30 and SEQ ID NO: 32, respectively; the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA2 comprise SEQ ID NO: 34 and SEQ ID NO: 36, respectively.

[0174] In some embodiments, the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA1 comprise SEQ ID NO: 31 and SEQ ID NO: 33, respectively; the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA2 comprise SEQ ID NO: 35 and SEQ ID NO: 37, respectively.

[0175] In some embodiments, a targeting vector is used to introduce a nucleotide sequence for human AGT at the endogenous AGT locus in a non-human animal.

[0176] In some embodiments, the targeting vector comprises the nucleotide sequence of human AGT; and

[0177] The 5' homology arm of the non-human animal AGT gene and the 3' homology arm of the non-human animal AGT gene.

[0178] In some embodiments, the non-human animal is a mouse or a rat.

[0179] In some embodiments, the 5' homology arm of the non-human animal AGT gene comprises SEQ ID NO: 3, a nucleotide sequence complementary to SEQ ID NO: 3, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0180] In some embodiments, the 3' homology arm of the non-human animal AGT gene comprises SEQ ID NO: 4, a nucleotide sequence complementary to SEQ ID NO: 4, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0181] In some embodiments, the 5' homology arm of the non-human animal AGT gene comprises SEQ ID NO: 23, a nucleotide sequence complementary to SEQ ID NO: 23, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0182] In some embodiments, the 3' homology arm of the non-human animal AGT gene comprises SEQ ID NO: 24, a nucleotide sequence complementary to SEQ ID NO: 24, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0183] In some embodiments, the mRNA transcribed from the modified AGT gene in the genome of the non-human animal comprises SEQ ID NO: 6, SEQ ID NO: 66, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto.

[0184] In some embodiments, the nucleotide sequence of human REN is the nucleotide sequence of human REN in the ninth aspect of the present invention.

[0185] In some embodiments, the nucleotide sequence of the non-human animal endogenous REN is the nucleotide sequence of the non-human animal endogenous REN in the ninth aspect of the present invention.

[0186] In some embodiments, the non-human animal's endogenous REN protein is not expressed or is expressed at a reduced level compared to REN in a wild-type non-human animal.

[0187] In some embodiments, the modified REN gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

[0188] In some embodiments, gene editing technology is used to introduce a human REN nucleotide sequence into the endogenous REN locus of a non-human animal.

[0189] In some embodiments, the gene editing technology includes gene targeting technology using embryonic stem cells, CRISPR / Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology or homing endonuclease.

[0190] In some embodiments, the sgRNA in the CRISPR / Cas9 comprises sgRNA3 and sgRNA4; the target site sequence of the sgRNA3 comprises SEQ ID NO: 56; and the target site sequence of the sgRNA4 comprises SEQ ID NO: 57.

[0191] In some embodiments, the sequence of the sgRNA3 comprises SEQ ID NO: 58; the sequence of the sgRNA4 comprises SEQ ID NO: 62.

[0192] In some embodiments, the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA3 comprise SEQ ID NO: 58 and SEQ ID NO: 60, respectively; the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA4 comprise SEQ ID NO: 62 and SEQ ID NO: 64, respectively.

[0193] In some embodiments, the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA3 comprise SEQ ID NO: 59 and SEQ ID NO: 61, respectively; the forward oligonucleotide and reverse oligonucleotide sequences for preparing the sgRNA4 comprise SEQ ID NO: 63 and SEQ ID NO: 65, respectively.

[0194] In some embodiments, a targeting vector is used to introduce a human REN nucleotide sequence at the endogenous REN locus in a non-human animal.

[0195] In some embodiments, the targeting vector comprises the nucleotide sequence of human REN; and

[0196] The 5' homology arm of the non-human animal REN gene and the 3' homology arm of the non-human animal REN gene.

[0197] In some embodiments, the non-human animal is a mouse or a rat.

[0198] In some embodiments, the 5' homology arm of the non-human animal REN gene comprises SEQ ID NO: 42, a nucleotide sequence complementary to SEQ ID NO: 42, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0199] In some embodiments, the 3' homology arm of the non-human animal REN gene comprises SEQ ID NO: 43, a nucleotide sequence complementary to SEQ ID NO: 43, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0200] In some embodiments, the 5' homology arm of the non-human animal REN gene comprises SEQ ID NO: 52, a nucleotide sequence complementary to SEQ ID NO: 52, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0201] In some embodiments, the 3' homology arm of the non-human animal REN gene comprises SEQ ID NO: 53, a nucleotide sequence complementary to SEQ ID NO: 53, or a nucleotide sequence having at least 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

[0202] In some embodiments, the mRNA transcribed from the modified REN gene in the genome of the non-human animal comprises SEQ ID NO: 48, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto.

[0203] In some embodiments, the non-human animal further comprises a nucleotide sequence encoding another human or chimeric protein.

[0204] In some embodiments, the additional human or chimeric protein comprises at least one of APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0205] The eighteenth aspect of the present invention provides a non-human animal or its offspring modified by AGT and REN genes, wherein the non-human animal modified by AGT and REN genes is obtained by the construction method of the seventeenth aspect of the present invention.

[0206] The nineteenth aspect of the present invention provides a method for constructing a non-human animal disease model, comprising the steps of obtaining a non-human animal or its offspring using the construction method of the seventeenth aspect of the present invention, wherein the disease is an AGT and / or REN-related disease.

[0207] In the present invention, the ATG and / or REN-related diseases include at least one of hypertension, kidney disease, and / or cardiovascular disease.

[0208] In the present invention, the ATG and / or REN-related diseases are selected from at least one of familial hyperreninemia / renin deficiency, reninoma, essential hypertension, secondary hypertension, kidney disease, chronic kidney disease, diabetic nephropathy, and heart failure.

[0209] In the present invention, AGT and / or REN related diseases include the above-mentioned AGT related diseases and / or REN related diseases of the present invention.

[0210] The twentieth aspect of the present invention provides a non-human animal disease model or its progeny, wherein the non-human animal disease model is obtained by the construction method of the nineteenth aspect of the present invention.

[0211] The twenty-first aspect of the present invention provides a cell, tissue or organ derived from a non-human animal or its offspring obtained by the construction method of the seventeenth or nineteenth aspect of the present invention.

[0212] The twenty-second aspect of the present invention provides a use of a non-human animal or its offspring obtained by the construction method described above (preferably the seventeenth or nineteenth aspect of the present invention), and / or the cells, tissues, or organs described above (preferably the twenty-first aspect of the present invention), the use comprising:

[0213] A) Applications in the development of products involving human cells in immunological processes related to AGT and / or REN;

[0214] B) Use as a model system for pharmacological, immunological, microbiological and medical research related to AGT and / or REN;

[0215] C) applications involving the production and use of animal experimental disease models for the study of the etiology of AGT and / or REN and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0216] D) in vivo studies for the screening, efficacy testing, efficacy assessment, validation or evaluation of modulators of the human AGT and / or REN signaling pathway; or

[0217] E) Study the function of AGT and / or REN genes, study the drugs and drug efficacy targeting human AGT and / or REN target sites, and study the application of drugs in hypertension, kidney disease, and / or cardiovascular disease related to AGT and / or REN.

[0218] In some embodiments, the use does not involve the diagnosis or treatment of a disease.

[0219] The twenty-third aspect of the present invention provides a method for determining the effectiveness of AGT and / or REN therapeutic agents in treating AGT and / or REN-related diseases, comprising the steps of using the non-human animals or their offspring obtained by the construction method described above (preferably the seventeenth aspect of the present invention, or the nineteenth aspect, more preferably the nineteenth aspect of the present invention), or the cells, tissues or organs described above (preferably the twenty-first aspect of the present invention).

[0220] In some embodiments, the method comprises the steps of administering an AGT and / or REN therapeutic agent to a non-human animal or its offspring obtained by the construction method described above (preferably the seventeenth aspect or the nineteenth aspect of the invention, more preferably the nineteenth aspect of the invention), and determining the effectiveness of the AGT and / or REN therapeutic agent in treating AGT and / or REN-related diseases.

[0221] In some embodiments, the method does not involve the diagnosis or treatment of a disease.

[0222] In the present invention, the AGT and / or REN therapeutic agent can be a macromolecular drug (eg, an AGT antibody, a polypeptide targeting AGT, a REN antibody, a polypeptide targeting REN), a nucleic acid drug or a small molecule drug.

[0223] The twenty-fourth aspect of the present invention provides a method for determining the toxicity of AGT and / or REN therapeutic agents, comprising the steps of using the non-human animals or their offspring obtained by the construction method described above (preferably the seventeenth aspect of the present invention, or the nineteenth aspect, more preferably the nineteenth aspect of the present invention), or the cells, tissues or organs described above (preferably the twenty-first aspect of the present invention).

[0224] In some embodiments, the method comprises the steps of administering AGT and / or REN therapeutic agents to the non-human animal or its offspring obtained by the construction method described above (preferably the seventeenth aspect or the nineteenth aspect of the invention, more preferably the nineteenth aspect of the invention), and determining the effects of the AGT and / or REN therapeutic agents on the animal.

[0225] In some embodiments, determining the effect of an AGT and / or REN therapeutic on an animal comprises measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

[0226] In some embodiments, the method does not involve the diagnosis or treatment of a disease.

[0227] The present application provides an animal model with human or chimeric AGT and / or REN protein. The animal model can express human or chimeric AGT and / or REN (e.g., humanized AGT and / or REN) protein. It can be used for the study of AGT and / or REN gene function, and can also be used for the screening and evaluation of AGT and / or REN signaling pathway regulators (e.g., anti-human AGT and / or REN antibodies, oligonucleotide drugs and / or polypeptide drugs). In addition, the animal model prepared by the method described herein can be used for drug screening, pharmacodynamics research, treatment of immune-related diseases and treatment of diseases at human AGT and / or REN target sites; the model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of AGT and / or REN protein and provides a platform for screening related drugs.

[0228] In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric angiotensinogen (AGT) and / or renin (REN) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric AGT and / or REN protein is operably linked to a human regulatory element (e.g., a human 5'UTR) of the endogenous AGT and / or REN locus of at least one chromosome. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric AGT and / or REN protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human AGT (NP_001371408.1, SEQ ID NO: 2) and / or REN (NP_000528.1, SEQ ID NO: 21). In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse or rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous AGT and / or REN protein or expresses it at a reduced level compared to AGT and / or REN in wild-type animals. In some embodiments, one or more cells of the animal express human or chimeric AGT and / or REN protein.

[0229] In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises a nucleotide sequence encoding an endogenous AGT and / or REN region at the endogenous AGT and / or REN locus, replaced with a nucleotide sequence encoding a corresponding region of human AGT and / or REN. In some embodiments, the nucleotide sequence encoding the corresponding region of human AGT and / or REN is operably linked to a human regulatory element, and one or more cells of the animal express human or humanized AGT and / or REN protein. In some embodiments, the endogenous AGT and / or REN protein of the animal is not expressed or is expressed at a reduced level compared to AGT and / or REN in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human AGT comprises all or part of exon 1 to exon 5 of the human AGT gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human REN comprises all or part of exon 1 to exon 10 of the human REN gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human AGT comprises contiguous nucleotides from human exon 1 to exon 5. In some embodiments, the nucleotide sequence encoding the human REN corresponding region comprises contiguous nucleotides from human exon 1 to exon 10. In some embodiments, the nucleotide sequence encoding the human AGT corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to nucleotides 230702763-230748399 of NC_000001.11 or the nucleotide sequence of SEQ ID NO: 25. In some embodiments, the nucleotide sequence encoding the human REN corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to nucleotides 204154277-204169793 of gene accession number NC_000001.11. In some embodiments, the nucleotide sequence encoding the endogenous AGT region comprises all or part of exon 1 to exon 5 of the murine (mouse or rat) AGT gene. In some embodiments, the nucleotide sequence encoding the endogenous REN region comprises all or part of exon 1 to exon 9 of the murine (mouse or rat) REN gene. In some embodiments, the nucleotide sequence encoding the endogenous AGT region comprises contiguous nucleotides from exon 1 to exon 5 of the murine (mouse or rat) AGT gene. In some embodiments, the nucleotide sequence encoding the endogenous REN region comprises contiguous nucleotides from exon 1 to exon 9 of the murine (mouse or rat) REN gene. In some embodiments, the modified AGT gene in the genome of the animal is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the modified REN gene in the genome of the animal is homozygous or heterozygous for the endogenous replaced locus.

[0230] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or humanized AGT and / or REN protein, wherein the humanized AGT and / or REN protein comprises at least 100, 200, 250, 300, 350, 400, 450, 460, 470, or 476 consecutive amino acids identical to a human corresponding region, and the animal expresses the human or humanized AGT and / or REN protein. In some embodiments, the nucleotide sequence encoding the human or humanized AGT and / or REN protein is operably linked to an endogenous AGT and / or REN regulatory element. In some embodiments, the nucleotide sequence encoding the human or humanized AGT and / or REN corresponding region can be integrated into the animal's endogenous AGT and / or REN locus. In some embodiments, the human or humanized AGT and / or REN protein has at least one mouse AGT and / or REN activity and / or human AGT and / or REN activity.

[0231] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein in at least one cell of the animal, at the animal's endogenous AGT and / or REN locus, a nucleotide sequence encoding an endogenous AGT and / or REN region is replaced with a nucleotide sequence encoding a human AGT and / or REN corresponding region. In some embodiments, the animal's endogenous AGT and / or REN protein is not expressed or is expressed at a reduced level compared to AGT and / or REN in a wild-type animal. In some embodiments, the nucleotide sequence encoding the human AGT corresponding region comprises all or part of exon 1 to exon 5 of the human AGT gene. In some embodiments, the nucleotide sequence encoding the human AGT corresponding region comprises contiguous nucleotides from human exon 1 to exon 5. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human AGT corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human AGT corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence at positions 230702763-230748399 of NC_000001.11 or the nucleotide sequence set forth in SEQ ID NO: 25. In some embodiments, the nucleotide sequence encoding the endogenous AGT region comprises all or part of exon 1 to exon 5 of the murine (mouse or rat) AGT gene. In some embodiments, the nucleotide sequence encoding the endogenous AGT region comprises contiguous nucleotides from exon 1 to exon 5 of the murine (mouse or rat) AGT gene. In some embodiments, the nucleotide sequence encoding the human AGT corresponding region is operably linked to a human AGT regulatory element, such as a promoter. In some embodiments, the nucleotide sequence encoding the human REN corresponding region comprises all or part of exon 1 to exon 10 of the human REN gene. In some embodiments, the nucleotide sequence encoding the human REN corresponding region comprises contiguous nucleotides from human exon 1 to exon 10. In some embodiments, the nucleotide sequence encoding the human REN corresponding region encodes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding the human REN corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to nucleotides 204154277-204169793 of gene accession number NC_000001.11. In some embodiments, the nucleotide sequence encoding the endogenous REN region comprises all or part of exon 1 to exon 9 of the murine (mouse or rat) REN gene.In some embodiments, the nucleotide sequence encoding the endogenous REN region comprises contiguous nucleotides from exon 1 to exon 9 of the murine (mouse or rat) REN gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human REN is operably linked to a human REN regulatory element, such as a promoter.

[0232] In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat.

[0233] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell that expresses human or chimeric AGT and / or REN. The method comprises replacing a nucleotide sequence encoding an endogenous AGT and / or REN region at the endogenous mouse AGT and / or REN locus with a nucleotide sequence encoding a corresponding region of human AGT and / or REN, thereby producing a genetically modified non-human animal cell that expresses human or chimeric AGT and / or REN protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human AGT comprises all or part of exon 1 to exon 5 of the human AGT gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human AGT comprises contiguous nucleotides from human exon 1 to exon 5. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human AGT is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the corresponding region of human AGT is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence at positions 230702763-230748399 of NC_000001.11 or the nucleotide sequence set forth in SEQ ID NO: 25. In some embodiments, the nucleotide sequence encoding the endogenous AGT region comprises all or part of exon 1 to exon 5 of the murine (mouse or rat) AGT gene. In some embodiments, the nucleotide sequence encoding the endogenous AGT region comprises contiguous nucleotides from exon 1 to exon 5 of the murine (mouse or rat) AGT gene. In some embodiments, the nucleotide sequence encoding the human or chimeric AGT protein is operably linked to a regulatory element of human AGT, such as a promoter. In some embodiments, the nucleotide sequence encoding the corresponding region of human REN comprises all or part of exon 1 to exon 10 of the human REN gene. In some embodiments, the nucleotide sequence encoding the human REN corresponding region comprises contiguous nucleotides from human exon 1 to exon 10. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human REN corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding the human REN corresponding region is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to nucleotides 204154277-204169793 of gene accession number NC_000001.11. In some embodiments, the nucleotide sequence encoding the endogenous REN region comprises all or part of exon 1 to exon 9 of the murine (mouse or rat) REN gene.In some embodiments, the nucleotide sequence encoding the endogenous REN region comprises consecutive nucleotides from exon 1 to exon 9 of the murine (mouse or rat) REN gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human REN is operably linked to a human REN regulatory element, such as a promoter. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding a human or chimeric protein from other genes, the human or chimeric protein being selected from at least one of APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0234] In some embodiments, the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins are selected from at least one of APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

[0235] In some embodiments, the animal further comprises a sequence encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4.

[0236] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.

[0237] In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1.

[0238] In some embodiments, the tumor comprises one or more tumor cells injected into the animal.

[0239] In some embodiments, said determining the inhibitory effect on a tumor involves measuring tumor volume in the animal.

[0240] In some embodiments, the animal has a NA solid tumor, a hematological tumor, a head and neck cancer, a liver cancer, or a lung cancer.

[0241] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.

[0242] In one aspect, the present invention provides a method for determining the effectiveness of an AGT and / or REN therapeutic agent in treating an immune disease, the method comprising:

[0243] 1) administering an AGT and / or REN therapeutic agent to the non-human animal, wherein the non-human animal suffers from an immune disease;

[0244] 2) Determine the therapeutic effects of AGT and / or REN therapeutic agents on immune diseases.

[0245] In some embodiments, the immune disease is asthma, rheumatoid arthritis, or multiple sclerosis.

[0246] In one aspect, the present invention provides a method for determining the effectiveness of an AGT and / or REN therapeutic agent in treating inflammation, the method comprising:

[0247] 1) administering an AGT and / or REN therapeutic agent to the animal, wherein the animal has inflammation;

[0248] 2) Determine the effectiveness of AGT and / or REN therapeutics for treating inflammation.

[0249] In some embodiments, the inflammatory disease is hypertension, eclampsia, or inflammatory bowel disease (IBD).

[0250] In one aspect, the present invention provides a method for determining the effectiveness of an AGT and / or REN therapeutic agent in treating a disease associated with AGT and / or REN, the method comprising:

[0251] 1) administering an AGT and / or REN therapeutic agent to the animal, wherein the animal has a disease associated with AGT and / or REN;

[0252] 2) Determine the effectiveness of AGT and / or REN therapeutics for treating diseases associated with AGT and / or REN.

[0253] In some embodiments, the disease associated with AGT and / or REN is hypertension or eclampsia.

[0254] In one aspect, the present invention provides a method for determining the effectiveness of an AGT and / or REN therapeutic agent in treating a disease associated with AGT and / or REN, the method comprising:

[0255] 1) administering an AGT and / or REN therapeutic agent to the animal, wherein the animal has a disease associated with AGT and / or REN;

[0256] 2) Determine the effectiveness of AGT and / or REN therapeutics for treating diseases associated with AGT and / or REN.

[0257] In some embodiments, the disease associated with AGT and / or REN is hypertension, kidney disease, and / or cardiovascular disease.

[0258] In one aspect, the present invention provides a method for determining the toxicity of an AGT and / or REN therapeutic agent, the method comprising:

[0259] 1) administering an AGT and / or REN therapeutic agent to the animal;

[0260] 2) Determine the effects of AGT and / or REN treatment on the animals.

[0261] In some embodiments, determining the effect of an AGT and / or REN therapeutic on an animal involves measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

[0262] In some embodiments, the inventions described herein include:

[0263] 1. A genetically modified non-human animal, wherein the genome of the animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric angiotensinogen (AGT) protein.

[0264] 2. The animal described in item 1, wherein the nucleotide sequence encoding the human or chimeric AGT protein is operably linked to a human AGT gene regulatory element (eg, human 5'UTR).

[0265] 3. The animal of item 1 or 2, wherein the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric AGT protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human AGT (NP_001371408.1, SEQ ID NO: 2).

[0266] 4. The animal according to any one of items 1 to 3, wherein the animal is a mammal, such as a monkey, a rodent, a mouse or a rat.

[0267] 5. The animal according to any one of items 1 to 4, wherein the animal is a mouse.

[0268] 6. The animal according to any one of items 1 to 5, wherein the endogenous AGT protein is not expressed in the animal or is expressed at a reduced level compared to AGT in wild-type animals.

[0269] 7. The animal of any one of items 1-6, wherein one or more cells of the animal express human or chimeric AGT protein.

[0270] 8. A genetically modified non-human animal, wherein the genome of the animal comprises a nucleotide sequence encoding an endogenous AGT region at an endogenous AGT locus replaced by a nucleotide sequence corresponding to a human AGT region.

[0271] 9. The animal of item 8, wherein the nucleotide sequence encoding the corresponding region of human AGT is operably linked to an endogenous regulatory element of an endogenous AGT locus, and one or more cells of the animal express human or humanized AGT protein.

[0272] 10. The animal according to item 8 or 9, wherein the endogenous AGT protein of the animal is not expressed or is expressed at a reduced level compared to AGT in wild-type animals.

[0273] 11. The animal according to any one of items 8 to 10, wherein the nucleotide sequence encoding the corresponding region of human AGT comprises all or part of exon 1 to exon 5 of the human AGT gene.

[0274] 12. The animal according to any one of items 8 to 11, wherein the nucleotide sequence encoding the corresponding region of human AGT comprises consecutive nucleotides from human exon 1 to exon 5.

[0275] 13. The animal according to any one of items 8-12, wherein the nucleotide sequence encoding the corresponding region of human AGT is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence at positions 230702763-230748399 of NC_000001.11.

[0276] 14. The animal according to any one of items 8 to 13, wherein the nucleotide sequence encoding the endogenous AGT region comprises all or part of exon 1 to exon 5 of the mouse AGT gene, preferably continuous nucleotides from exon 1 to exon 5 of the mouse AGT gene.

[0277] 15. The animal of any one of items 8-14, wherein the modified AGT gene in the genome of the animal is homozygous or heterozygous for the endogenous replaced locus.

[0278] 16. A non-human animal, wherein the animal comprises at least one cell containing a nucleotide sequence encoding a human or humanized AGT protein, wherein the humanized AGT protein comprises at least 100, 200, 250, 300, 350, 400, 450, 460, 470, 476 consecutive amino acids that are identical to the corresponding region of a human, and the animal expresses the human or humanized AGT protein.

[0279] 17. The animal according to any one of item 16, wherein the nucleotide sequence encoding the human or humanized AGT protein is operably linked to a human AGT regulatory element.

[0280] 18. The animal described in item 16 or 17, wherein the nucleotide sequence encoding the corresponding region of human or humanized AGT can be integrated into the endogenous AGT locus of the animal.

[0281] 19. The animal according to any one of items 16 to 18, wherein the human or humanized AGT protein has at least one of mouse AGT activity and / or human AGT activity.

[0282] 20. A method for constructing a genetically modified non-human animal, wherein in at least one cell of the animal, at the animal's endogenous AGT locus, the nucleotide sequence encoding the endogenous AGT region is replaced by the nucleotide sequence of the corresponding region of human AGT.

[0283] 21. The method of item 20, wherein the endogenous AGT protein of the animal is not expressed or is expressed at a reduced level compared to AGT in wild-type animals.

[0284] 22. The method described in item 20 or 21, wherein the nucleotide sequence encoding the corresponding region of human AGT comprises all or part of exon 1 to exon 5 of the human AGT gene.

[0285] 23. The method described in any one of items 20-22, wherein the nucleotide sequence encoding the corresponding region of human AGT comprises consecutive nucleotides from human exon 1 to exon 5.

[0286] 24. The method described in any one of items 20-23, wherein the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human AGT is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 2.

[0287] 25. The method described in any one of items 20-24, wherein the nucleotide sequence encoding the corresponding region of human AGT is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence at positions 230702763-230748399 of NC_000001.11.

[0288] 26. The method described in any one of items 20-25, wherein the nucleotide sequence encoding the endogenous AGT region comprises all or part of exon 1 to exon 5 of the mouse AGT gene.

[0289] 27. The method described in any one of items 20-26, wherein the nucleotide sequence encoding the endogenous AGT region comprises consecutive nucleotides from exon 1 to exon 5 of the mouse AGT gene.

[0290] 28. The method described in any one of items 20-27, wherein the nucleotide sequence encoding the corresponding region of human AGT is operably linked to a human AGT regulatory element, such as a promoter.

[0291] 29. The method according to any one of items 20 to 28, wherein the animal is a mammal, such as a monkey, a rodent, a mouse or a rat.

[0292] 30. A method for constructing genetically modified non-human animal cells that express human or chimeric AGT, the method comprising replacing the nucleotide sequence encoding the endogenous AGT region at the endogenous mouse AGT locus with the nucleotide sequence of the corresponding region of human AGT, thereby producing genetically modified non-human animal cells that express human or chimeric AGT protein.

[0293] 31. The method described in item 30, wherein the nucleotide sequence encoding the corresponding region of human AGT comprises all or part of exon 1 to exon 5 of the human AGT gene.

[0294] 32. The method described in any one of items 30 or 31, wherein the nucleotide sequence encoding the corresponding region of human AGT comprises consecutive nucleotides from human exon 1 to exon 5.

[0295] 33. The method described in any one of items 30-32, wherein the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human AGT is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 2.

[0296] 34. The method described in any one of items 30-33, wherein the nucleotide sequence encoding the corresponding region of human AGT is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence at positions 230702763-230748399 of NC_000001.11.

[0297] 35. The method described in any one of items 30-34, wherein the nucleotide sequence encoding the endogenous AGT region comprises all or part of exon 1 to exon 5 of the mouse AGT gene.

[0298] 36. The method described in any one of items 30-35, wherein the nucleotide sequence encoding the endogenous AGT region comprises consecutive nucleotides from exon 1 to exon 5 of the mouse AGT gene.

[0299] 37. The method described in any one of items 30-36, wherein the nucleotide sequence encoding the human or chimeric AGT protein is operably linked to a regulatory element of human AGT, such as a promoter.

[0300] 38. The method described in any one of items 30-37, wherein the non-human animal is a mouse.

[0301] 39. The animal of any one of items 1-19, wherein the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from at least one of APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

[0302] 40. The method described in any one of items 20-38, wherein the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from at least one of APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

[0303] 41. A method for determining the effectiveness of an AGT therapeutic agent in treating an AGT-related disease, wherein the method comprises:

[0304] 1) administering an AGT therapeutic agent to the animal of any one of items 1-29 and 39, wherein the animal has an AGT-related disease;

[0305] 2) To determine the effectiveness of AGT therapeutics in treating AGT-related diseases.

[0306] 42. The method described in Item 41, wherein the AGT-related diseases are hypertension and eclampsia.

[0307] 43. A method for determining the toxicity of an AGT therapeutic agent, wherein the method comprises:

[0308] 1) administering an AGT therapeutic agent to the animal of any one of items 1-29 and 39;

[0309] 2) Determine the effect of AGT treatment on animals.

[0310] 44. The method of claim 43, wherein determining the effect of the AGT therapeutic on the animal involves measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

[0311] 45. A humanized AGT gene, wherein the humanized AGT gene comprises any one of the following nucleotide sequences:

[0312] A) nucleotide sequence encoding the protein of SEQ ID NO: 2;

[0313] B) SEQ ID NO: 3, 4, 6, 7, 8, 9 or 10, or nucleotide sequence 230702763-230748399 of NC_000001.11;

[0314] C) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, 4, 6, 7, 8, 9 or 10, or nucleotide sequence 230702763-230748399 of NC_000001.11;

[0315] D) a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to nucleotide sequence 230702763-230748399 of SEQ ID NO: 3, 4, 6, 7, 8, 9 or 10, or NC_000001.11.

[0316] 46. ​​A cell, wherein the cell comprises the humanized AGT gene according to item 45.

[0317] 47. An animal model, wherein the animal model comprises the humanized AGT gene described in item 45.

[0318] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of conflict, the present specification, including definitions, will control.

[0319] Those skilled in the art can easily discern other conveniences and advantages of the present application from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0320] Figure 1 : PCR identification results of the F1 generation of AGT gene humanized mice, where WT is the wild-type control, M is Maker, and H2O is the water control: (A) is the result of PCR identification using primers WT-F and WT-R; (B) is the result of PCR identification using primers WT-F and Mut-R.

[0321] Figure 2 : RT-PCR test results, where + / + represents wild-type C57BL / 6 mice, H / + represents heterozygous mice with humanized AGT gene, and H2O represents water control.

[0322] Figure 3: ELISA test results, where + / + represents wild-type C57BL / 6 mice, and H / + represents heterozygous mice with humanized AGT gene: (A) is the result of ELISA detection of mouse AGT protein; (B) is the result of ELISA detection of human AGT protein.

[0323] Figure 4 : PCR identification results of the F1 generation of humanized AGT gene rats, where WT is the wild-type control, M is Maker, PC is the positive heterozygote control, and H2O is the water control: (A) is the result of PCR identification using primers WT-F1 and WT-R1; (B) is the result of PCR identification using primers WT-F1 and Mut-R1.

[0324] Figure 5 : ELISA test results, where + / + represents wild-type SD rats and H / H represents homozygous rats with humanized AGT gene: (A) is the result of ELISA detection of rat AGT protein; (B) is the result of ELISA detection of human AGT protein.

[0325] Figure 6 : PCR identification results of the F1 generation of humanized REN gene mice, where M is Maker, WT is the wild-type control, and H2O is the water control: (A) is the result of PCR identification using primers WT-F2 and WT-R2; (B) is the result of PCR identification using primers WT-F2 and Mut-R2.

[0326] Figure 7 : ELISA test results, where + / + represents wild-type C57BL / 6 mice and H / + represents heterozygous mice with humanized REN gene: (A) is the result of ELISA detection of mouse REN protein; (B) is the result of ELISA detection of human REN protein. DETAILED DESCRIPTION

[0327] AGT

[0328] In the human genome, the AGT gene (NCBI Gene ID: 183, UniProt ID: P01019, located at positions 230702523 to 230745583 on chromosome 1 NC_000001.11) contains five exons: exon 1, exon 2, exon 3, exon 4, and exon 5. The corresponding positions of each exon based on the nucleotide and amino acid sequences of transcript NM_001384479.1 and its encoded protein NP_001371408.1 (SEQ ID NO: 2) are shown in Table 1.

[0329] Table 1

[0330]

[0331] In the mouse genome, the AGT gene (NCBI Gene ID: 11606, UniProt ID: P11859, located at positions 125283326 to 125296445 on chromosome 8, NC_000074.7) contains five exons: exon 1, exon 2, exon 3, exon 4, and exon 5. The corresponding positions of each exon in the nucleotide and amino acid sequences of transcript NM_007428.4 and its encoded protein NP_031454.4 (SEQ ID NO: 1) are shown in Table 2.

[0332] Table 2

[0333]

[0334]

[0335] In the rat genome, the AGT gene (NCBI Gene ID: 24179, UniProt ID: P01015, located on chromosome 19, NC_086037.1, positions 69426540 to 69447017) contains five exons: exon 1, exon 2, exon 3, exon 4, and exon 5. The corresponding positions of each exon in the nucleotide and amino acid sequences of transcript NM_134432.2 and its encoded protein NP_602308.1 (SEQ ID NO: 5) are shown in Table 3.

[0336] Table 3

[0337]

[0338] REN

[0339] In the human genome, the REN gene (NCBI Gene ID: 5972, UniProt ID: P00797, located on chromosome 1, NC_000001.11, positions 204154819 to 204166337) contains 10 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, and exon 10. The corresponding positions of each exon based on the nucleotide and amino acid sequences of transcript NM_000537.4 and its encoded protein NP_000528.1 (SEQ ID NO: 21) are shown in Table 4.

[0340] Table 4

[0341]

[0342]

[0343] In the mouse genome, the REN gene (NCBI Gene ID: 19701, UniProt ID: P06281, located on chromosome 1, NC_000067.7, positions 133278412 to 133288058) contains nine exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and exon 9. The corresponding positions of each exon in the nucleotide and amino acid sequences of transcript NM_031192.3 and its encoded protein NP_112469.1 (SEQ ID NO: 20) are shown in Table 5.

[0344] Table 5

[0345]

[0346] In the rat genome, the REN gene (NCBI Gene ID: 24715, UniProt ID: P08424, located on chromosome 13, NC_086031.1, positions 47348312 to 47359539) contains nine exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and exon 9. The corresponding positions of each exon in the nucleotide and amino acid sequences of transcript NM_012642.4 and its encoded protein NP_036774.4 (SEQ ID NO: 22) are shown in Table 6.

[0347] Table 6

[0348]

[0349]

[0350] AGT and REN genes, proteins, and gene loci are also known in the art for other species. For example, Macacamulatta (rhesus monkey), Canis lupus familiaris (dog), and Sus scrofa (pig). Information related to these genes (e.g., intron sequences, exon sequences, and amino acid sequences) can be found in NCBI, the entire contents of which are incorporated herein by reference.

[0351] Genetically modified non-human animals

[0352] 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 animal genome has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells with exogenous DNA can be various cells, for example, endogenous cells, somatic cells, immune cells, T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells, germ cells, blastocysts or endogenous tumor cells. In some embodiments, a genetically modified non-human animal is provided, wherein the animal comprises a modified endogenous AGT 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. Animals are generally able to pass genetic modifications to offspring through germline transmission.

[0353] The "chimeric gene" or "chimeric nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is different from the nucleic acid in the wild-type animal. In some embodiments, the chimeric gene or chimeric nucleic acid has at least a portion of the sequence having two or more different species origins, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, the chimeric gene or chimeric nucleic acid refers to a humanized gene or humanized nucleic acid.

[0354] As used herein, a "chimeric protein" or "chimeric polypeptide" refers to a protein or polypeptide in which two or more portions of the polypeptide or protein are derived from different species, or at least one sequence of the protein or polypeptide differs from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of a chimeric protein or chimeric polypeptide is derived from two or more different species, for example, the same (or homologous) proteins from different species. In some embodiments, a chimeric protein or chimeric polypeptide is a humanized protein or polypeptide.

[0355] The "humanized protein" or "humanized 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 protein or polypeptide refers to a human protein or polypeptide.

[0356] As used herein, a "humanized nucleic acid" refers to a nucleic acid wherein at least a portion of the nucleic acid is human. In some embodiments, the nucleic acid in the humanized nucleic acid is entirely human. In some embodiments, the humanized nucleic acid refers to humanized exons, which can be human exons or chimeric exons.

[0357] In some embodiments, the chimeric gene or chimeric nucleic acid is a humanized AGT and / or REN gene or humanized AGT and / or REN nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human AGT and / or REN gene, and at least a portion of the gene or nucleic acid is derived from a non-human AGT and / or REN gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding an AGT and / or REN protein. The encoded AGT and / or REN protein has at least one activity of a human AGT and / or REN protein or a non-human animal AGT and / or REN protein.

[0358] In some embodiments, the chimeric protein or polypeptide is a humanized AGT and / or REN protein or a humanized AGT and / or REN polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a human AGT and / or REN protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human animal AGT and / or REN protein. The humanized AGT and / or REN protein or humanized AGT and / or REN polypeptide is functional or has at least one activity of a human AGT and / or REN protein or a non-human animal AGT and / or REN protein.

[0359] 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) to form embryo in non-human animal under appropriate conditions.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 ", its entire contents are incorporated herein by reference.

[0360] In one aspect, the animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified animal is selected from the families Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Malborinidae (climbing mice, rock mice, tailed rats, Madagascar rats, and mice), Spiny Mouse (e.g., spiny dormouse), and Muridae (e.g., mole rats, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the family Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0361] In some embodiments, the animal is a mouse of the C57BL strain selected from the group consisting of 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 the group consisting of 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as the BALB / c strain. In some embodiments, the mouse is a hybrid of the BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having 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 and a mouse of NOD, NOD / SCID, NOD-Prkdcscid IL-2rgnull background.

[0362] Genetically modified non-human animals include modifications of endogenous non-human animal AGT and / or REN gene loci. In some embodiments, the modifications comprise a nucleotide sequence encoding at least a portion of a mature AGT and / or REN protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a mature AGT and / or REN protein amino acid sequence). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein are provided herein, in many embodiments, the genetically modified non-human animals include modifications of endogenous AGT and / or REN gene loci in the animal.

[0363] The genetically modified animal can express human AGT and / or REN and / or chimeric (e.g., humanized) AGT and / or REN at an endogenous mouse locus, wherein the endogenous mouse AGT and / or REN gene has been replaced or inserted with a human AGT and / or REN gene and / or a nucleotide sequence encoding a region of a human AGT and / or REN sequence, or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% identical to a human AGT and / or REN sequence. In various embodiments, the endogenous non-human animal AGT and / or REN locus is modified with a human nucleic acid sequence comprising all or part of a nucleic acid sequence encoding a mature AGT and / or REN protein.

[0364] In certain embodiments, genetically modified mice can express human AGT and / or REN and / or chimeric AGT and / or REN (e.g., humanized AGT and / or REN) under the control of a mouse promoter and / or mouse regulatory elements. Inserting or replacing the mouse endogenous locus provides a non-human animal that expresses human AGT and / or REN or chimeric AGT and / or REN (e.g., humanized AGT and / or REN) in suitable cells and does not cause the potential pathology observed in some other transgenic mice known in the art. Human AGT and / or REN or chimeric AGT and / or REN (e.g., humanized AGT and / or REN) expressed in animals can maintain one or more functions of wild-type mice or human AGT and / or REN in animals. In addition, in certain embodiments, animals do not express endogenous AGT and / or REN. In certain embodiments, animal endogenous AGT and / or REN expression levels are reduced compared to AGT and / or REN expression levels in wild-type animals. As used herein, the term "endogenous AGT and / or REN" refers to the AGT and / or REN proteins expressed by the endogenous AGT and / or REN nucleotide sequences of a non-human animal (eg, mouse) before any genetic modification.

[0365] In some embodiments, the humanized AGT and / or REN loci comprise a human 5'UTR. In some embodiments, the humanized AGT and / or REN loci comprise an endogenous (e.g., mouse) 5'UTR. In some embodiments, the 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 AGT genes, they appear to be similarly regulated. As shown in the application, humanized AGT and / or REN mice comprising insertions or substitutions in the endogenous mouse AGT and / or REN loci, which retain mouse regulatory elements but comprise humanization of the AGT and / or REN coding sequences, do not exhibit pathological phenomena. Both genetically modified mice, either heterozygous or homozygous for humanized AGT and / or REN, are normal.

[0366] The present invention further relates to the AGT and / or REN genomic DNA sequence of humanized mice, the DNA sequence obtained by mRNA reverse transcription 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 cells thereof.

[0367] The present invention further relates to a non-human mammal produced by the above method. In some embodiments, its genome comprises human genes.

[0368] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0369] In some embodiments, the non-human mammal expresses a protein encoded by a humanized AGT and / or REN gene.

[0370] In addition, the present invention also provides a non-human mammal model bearing a tumor, wherein the non-human mammal model is obtained by the method described herein. In some embodiments, the non-human mammal is a rodent (eg, mouse).

[0371] The present invention also provides a cell or cell line, or primary cell culture derived from a non-human mammal or its progeny, or a non-human mammal carrying a tumor, wherein the cell or cell line is derived from a non-human mammal or its progeny, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its progeny. In the case of a tumor, the cell or cell line is derived from a tumor tissue of a non-human mammal or its progeny, or a non-human mammal carrying a tumor.

[0372] 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 human or humanized AGT and / or REN DNA.

[0373] 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 AGT and / or REN protein is provided. In some embodiments, a tissue specifically expressing a human or humanized AGT and / or REN protein is provided.

[0374] In some embodiments, the expression of non-human animal human or humanized AGT and / or REN protein is controllable. For example, by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0375] 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 (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0376] The non-human mammal described above is subjected to genetic, molecular and behavioral analysis. The present invention provides offspring produced by mating with a non-human mammal of the same genotype or another genotype.

[0377] 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 methods. The cell culture can be obtained by isolation from the non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. Integration of a genetic construct containing a DNA sequence encoding a human AGT protein can be detected by various methods.

[0378] There are many analytical methods that can be used to detect exogenous DNA, including nucleic acid level methods (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, immunoblot analysis and in vitro binding studies). In addition, the expression level of the target gene can be quantified by ELISA methods well known to those skilled in the art. Many standard analytical methods can be used to complete quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcription levels, including RNAse protection assays, Southern Blots, and RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blots can also be used to detect the presence of human or humanized AGT and / or REN proteins.

[0379] In some embodiments, the genetically modified animals described herein (eg, mice homozygous for humanized AGT and / or REN genes) can express human or humanized AGT and / or REN in one or more liver tissue cells.

[0380] Method for constructing genetically modified non-human animals

[0381] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting technology using embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques. 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 genome editing is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6 (2017): 387-399, by Yin et al., which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0382] In some embodiments, the nucleotide sequence encoding the endogenous AGT and / or REN region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding the corresponding region of human AGT and / or REN. In some embodiments, the expression of the endogenous AGT and / or REN protein in the non-human animal is reduced or absent compared to wild-type. In some embodiments, the replacement occurs in a cell such as a germ cell, somatic cell, blastocyst, or fibroblast. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0383] The present invention provides a targeting vector. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized AGT and / or REN gene fragment, and a 3' homology arm. The process involves using homologous recombination to replace the endogenous corresponding AGT and / or REN sequence with the human or humanized AGT and / or REN sequence. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace the endogenous mouse AGT and / or REN sequence with the human or humanized AGT and / or REN sequence.

[0384] Therefore, in some embodiments, the method of preparing a genetically modified humanized animal comprises replacing a nucleic acid sequence encoding an endogenous AGT and / or REN region at the endogenous AGT and / or REN locus (or site) with a nucleotide sequence encoding the corresponding region of human AGT and / or REN.

[0385] The present invention also provides a method for establishing an AGT and / or REN gene humanized animal model, comprising the following steps:

[0386] (a) providing a cell (e.g., a fertilized egg cell) according to the method described herein;

[0387] (b) culturing the cells in a liquid culture medium;

[0388] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0389] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0390] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0391] In some embodiments, the non-human mammal in step (c) is a pseudo-pregnant female.

[0392] 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.

[0393] The fertilized egg can be from any non-human animal, such as any of the non-human animals described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The genetic 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, which can then give birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0394] In some embodiments, the method for preparing a genetically modified animal comprises modifying the coding frame of the AGT and / or REN gene of a non-human animal, for example, replacing the nucleic acid sequence encoding the endogenous AGT and / or REN region (e.g., DNA or cDNA sequence) with a nucleotide sequence encoding the corresponding region of human AGT and / or REN at the endogenous AGT and / or REN locus of the non-human animal. For example, one or more functional region sequences of the AGT and / or REN gene of the non-human animal can be knocked out or inserted into a sequence so that the endogenous AGT and / or REN protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the modified non-human animal's AGT gene can be all or part of the nucleotide sequence of exon 1 to exon 5 of the non-human animal's AGT gene; the coding frame of the modified non-human animal's REN gene can be all or part of the nucleotide sequence of exon 1 to exon 9 of the non-human animal's REN gene.

[0395] In some embodiments, the method for preparing a genetically modified animal comprises inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or humanized AGT and / or REN protein after the endogenous regulatory elements of the AGT and / or REN gene of a non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the AGT and / or REN gene humanized animal model can express the human or humanized AGT and / or REN protein in vivo, but does not express the AGT and / or REN protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0396] In some embodiments, the method for preparing a transgenic animal comprises:

[0397] (1) providing a plasmid comprising a human AGT and / or REN gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous AGT and / or REN;

[0398] (2) providing one or more guide RNAs (sgRNAs) targeting endogenous AGT and / or REN genes;

[0399] (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;

[0400] (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 humanized AGT and / or REN protein;

[0401] (5) The offspring mice obtained in step (4) are mated to obtain homozygous mice.

[0402] In some embodiments, the zygote is modified by CRISPR with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal target site.

[0403] In some embodiments, the sequence encoding the humanized AGT and / or REN protein is operably linked to endogenous regulatory elements at the endogenous AGT and / or REN locus.

[0404] In some embodiments, the genetically modified animal does not express endogenous AGT and / or REN protein.

[0405] In some embodiments, the method for preparing a transgenic animal comprises:

[0406] (1) providing a plasmid comprising a human or chimeric AGT and / or REN gene segment, said plasmid being flanked by 5' homology arms and 3' homology arms, wherein said 5' and 3' homology arms target endogenous AGT;

[0407] (2) providing one or more guide RNAs (sgRNAs) targeting endogenous AGT and / or REN genes;

[0408] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric AGT gene fragment into the genome.

[0409] Use of genetically modified non-human animals

[0410] 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 elements can produce a non-human animal with qualities and characteristics that may be significantly different from those of a typical knockout plus transgenic animal. In a typical knockout plus transgenic animal, the endogenous locus is removed or disrupted and a fully human transgene is inserted into the genome of the animal and may integrate randomly into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and / or protein assays and / or functional assays. In a human transgene, the human sequences upstream and / or downstream provide suitable support for expression and / or regulation of the transgene.

[0411] Genetically modified animals that express human or humanized AGT and / or REN proteins, e.g., in a physiologically appropriate manner, offer 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.

[0412] The present invention also provides the use of the above-mentioned AGT and / or REN gene-modified non-human animals and the non-human animals obtained by any of the above-mentioned construction methods.

[0413] In some embodiments, the application comprises:

[0414] A) Applications in the development of products involving human cells in immunological processes related to AGT and / or REN;

[0415] B) Use as a model system for pharmacological, immunological, microbiological and medical research related to AGT and / or REN;

[0416] C) applications involving the production and use of animal experimental disease models for the study of the etiology of AGT and / or REN and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0417] D) in vivo studies for the screening, efficacy testing, efficacy assessment, validation or evaluation of modulators of the human AGT and / or REN signaling pathway; or

[0418] E) Study the functions of AGT and / or REN genes, study the drugs and drug efficacy targeting human AGT and / or REN target sites, and study the application of drugs in tumors, inflammation, and immune-related diseases related to AGT and / or REN.

[0419] The present invention provides a non-human animal expressing human or humanized AGT and / or REN protein, which can be used to screen for human AGT and / or REN-specific modulators. In some embodiments, the non-human animal is an animal model for human disease. For example, the disease is genetically induced (knock-in or knock-out). In various embodiments, the genetically modified non-human animal further comprises a compromised immune system, such as a genetically modified human tissue xenograft, including a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, B or T cell tumor).

[0420] In some embodiments, anti-AGT and / or REN antibodies block or inhibit AGT and / or REN mediated signaling pathways. In some embodiments, the anti-AGT and / or REN antibodies described herein can block the interaction between AGT and / or REN complexes, thereby inhibiting AGT and / or REN signaling pathways.

[0421] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-AGT antibodies) 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 steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, the immune disease is asthma, rheumatoid arthritis, or multiple sclerosis.

[0422] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-AGT antibodies) 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, inflammation is hypertension, eclampsia, inflammatory bowel disease (IBD).

[0423] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-AGT antibodies) for treating cancer. In some embodiments, a therapeutic agent (e.g., anti-AGT antibody) 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 measuring the size and / or proliferation rate of tumor cells. In some embodiments, the detection method includes vernier caliper measurement, flow cytometry and / or animal in vivo imaging detection. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity or metabolic changes, and the metabolic changes include changes in food consumption or water consumption.

[0424] In some embodiments, the tumor cells include one or more cancer cells injected into an animal (e.g., cancer cells derived from a human or non-human animal). In some embodiments, the therapeutic agent inhibits the AGT signaling pathway. In some embodiments, the therapeutic agent does not inhibit the AGT signaling pathway.

[0425] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-REN antibodies) in treating hypertension, kidney disease, and / or cardiovascular disease. In some embodiments, the hypertension, kidney disease, and / or cardiovascular disease include, but are not limited to, familial hyperreninemia / renin deficiency, reninoma, essential hypertension, secondary hypertension, chronic kidney disease, diabetic nephropathy, and heart failure.

[0426] In some embodiments, genetically modified non-human animals can be used to detect whether anti-AGT antibodies are agonists or antagonists. In some embodiments, the methods described herein can be used to detect the function of therapeutic agents (e.g., anti-AGT antibodies), for example, whether the therapeutic agent can upregulate the immune response or downregulate the 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., an immune 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 (TGITV). The tumor growth inhibition rate can be calculated using the formula TGITV (%) = (1-TVt / TVc) x 100, where TVt and TVc are the average tumor volume (or weight) of the treatment group and the control group.

[0427] In some embodiments, therapeutic agents (such as anti-AGT antibodies) can be used to treat various cancers. "Cancer" as used herein refers to cells with autonomous growth ability, i.e., an abnormal state or condition characterized by rapid proliferation of cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of the histopathological type or invasive stage. "Tumor" as used herein includes but is not limited to lymphoma, 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 one embodiment of the present invention, the tumor is breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tumor, oropharyngeal tumor, female reproductive system cancer or meningioma. In some embodiments, the tumor is a solid tumor, a hematological tumor, head and neck cancer or lung cancer.

[0428] The present invention also provides a method for determining the toxicity of a therapeutic agent (e.g., an anti-AGT and / or REN antibody). The method comprises administering an antibody to a non-human animal as described above and assessing the animal's weight change, red blood cell count, hematocrit, and / or hemoglobin. In some embodiments, the antibody can reduce red blood cells (RBCs), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the animal's 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).

[0429] The present invention also provides an animal model constructed by the method described herein for use in developing products related to human cellular immune processes, producing human antibodies, or as a model system for pharmacology, immunology, microbiology, and medical research.

[0430] In some embodiments, an animal model generated by the methods described herein is provided for producing and utilizing human cell-based immune processes in animal experimental disease models, studying pathogens, or developing new diagnostic and / or therapeutic strategies.

[0431] The present invention also provides animal models generated by the methods described herein for screening, validating, evaluating or studying AGT and / or REN gene functions, human AGT and / or REN antibodies, drugs or effectiveness of human AGT and / or REN target sites, drugs for immune-related diseases, anti-tumor drugs and drugs for preventing and treating hypertension, kidney disease, and / or cardiovascular disease.

[0432] 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 treatment can be determined and evaluated. In some embodiments, the animal is selected from AGT and / or REN 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 AGT and / or REN proteins, or tumor-bearing or inflammatory animal models described herein. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat AGT and / or REN related diseases described herein. In some embodiments, TCR-T, CAR-T and / or other immunotherapies provide evaluation methods for treating AGT and / or REN related diseases described herein.

[0433] Non-human animal models with two or more human or chimeric genes

[0434] The present invention also provides a method for producing a transgenic animal model having two or more human or chimeric genes. The animal may contain a human or chimeric AGT and / or REN gene and a sequence encoding an additional human or chimeric protein.

[0435] In some embodiments, the additional gene is a non-human animal modified with at least one gene of APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. In some embodiments, the non-human animal further expresses at least one of human or humanized APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 proteins.

[0436] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0437] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0438] (2) mating, in vitro fertilization, or direct gene editing of the non-human animal provided in step (1) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0439] In some embodiments, the other genetically modified non-human animals include non-human animals humanized with one or a combination of two or more of the genes APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

[0440] In some embodiments, AGT and / or REN humanization is performed directly on non-human animals with human or chimeric APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 gene modifications.

[0441] Since these proteins may involve different mechanisms, a combination therapy targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are underway and have shown good results. Multigene modified non-human animal models can be used to determine the effectiveness of a combination therapy targeting two or more proteins, for example, anti-AGT and / or REN antibodies, and additional therapeutic agents for treating cancer, immune diseases (e.g., asthma or atopic dermatitis), hypertension, kidney disease, or cardiovascular disease. The method comprises administering an anti-AGT and / or REN antibody and an additional therapeutic agent to an animal, wherein the animal has a tumor or an immune disease, and determining the effect of the combination therapy on the immune tumor or immune disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to APOC3, ANGPTL3, 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 comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0442] In some embodiments, the combination therapy is used to treat various cancers described herein, for example, breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer, or cancer. In some embodiments, the combination therapy is designed to treat immune disorders described herein, such as psoriasis. In some embodiments, the methods described herein can be used to evaluate combination therapy with some other methods. Methods for treating cancer that can be used alone or in combination with the methods described herein include, for example, treating a subject with chemotherapy, for example, camphor, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepate, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chloramphenicol, pyrimethamine, nitrosourea, daktarin, daunorubicin, bleomycin, prinomycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 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, eg, to remove a portion or all of a tumor from the patient.

[0443] The present invention is further described in detail below through specific examples.

[0444] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0445] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the manufacturer. Materials and reagents used in these examples were commercially available unless otherwise specified. Where the manufacturer of the reagent is indicated, similar products from other manufacturers are considered substitutes.

[0446] Equipment and Materials:

[0447] C57BL / 6 mice, Flp transgenic mice, and SD rats were purchased from the National Rodent Laboratory Animal Center of the China Food and Drug Administration.

[0448] Example 1. AGT gene humanized mice

[0449] To achieve the objectives of the present invention, a nucleotide sequence encoding a human AGT protein was introduced into the endogenous mouse AGT locus, enabling the mouse to express a human or humanized AGT protein. Specifically, using gene editing technology, a portion of the approximately 16.4 kb sequence from the 5'UTR upstream to the 3'UTR downstream of the mouse AGT gene was replaced with a sequence encompassing approximately 45.6 kb from the 5'UTR upstream to the 3'UTR downstream of the human AGT gene, resulting in a humanized AGT locus and achieving humanization of the mouse AGT gene.

[0450] To implement the targeting strategy of the present invention, a targeting vector V1 was constructed. The targeting vector V1 contains homology arm sequences upstream and downstream of the mouse AGT gene, and an A segment containing human AGT segment 1. The upstream 5' homology arm sequence is SEQ ID NO: 3, and the downstream 3' homology arm sequence is SEQ ID NO: 4. The nucleotide sequence of human AGT segment 1 is consistent with the sequence at positions 230702763-230748399 of gene accession number NC_000001.11; the upstream connection between the human AGT segment 1 sequence and the mouse AGT segment is designed as follows: (SEQ ID NO: 7), wherein the sequence The last "T" in the sequence is the last nucleotide in the mouse. CCAGCC The first "C" in " is the first nucleotide of the human AGT fragment 1 sequence. The downstream connection between the human AGT fragment 1 sequence and the mouse is designed as follows: (SEQ ID NO: 8), wherein the sequence " TTGGGThe last "G" in " is the last nucleotide of the human AGT fragment 1 sequence. The first "A" in is the first nucleotide of the mouse sequence (ie, the first nucleotide at the downstream junction of the 3' homology arm sequence and human AGT segment 1).

[0451] The targeting vector V1 also contains a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo. Two site-specific recombination system Frt recombination sites are positioned in the same direction on either side of the resistance gene, forming a Neo cassette. The Neo cassette is located in intron 3 of human AGT segment 1, and the connection between the 5' end of the Neo cassette and the human AGT gene is designed as follows:

[0452] (SEQ ID NO: 9), wherein the sequence " CAGCA The last "A" in the sequence is the last nucleotide of the human AGT gene. The "G" in the Neo box is the first nucleotide; the connection between the 3' end of the Neo box and the human AGT gene is designed as follows:

[0453] (SEQ ID NO: 10), wherein the sequence The last "C" in the sequence is the last nucleotide of the Neo box. CACCT The first "C" in " is the first nucleotide of the human AGT gene. The mRNA sequence of the modified humanized mouse AGT is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0454] Targeting vector V1 can be constructed using conventional methods, such as enzyme digestion and ligation. After preliminary verification of the constructed targeting vector V1 by enzyme digestion, it is sent to a sequencing company for sequencing verification. The sequencing-verified targeting vector is electroporated and transfected into embryonic stem cells of C57BL / 6 mice. The resulting cells are screened using a positive clone screening marker gene to identify the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into isolated blastocysts (white mice) according to techniques known in the art. The resulting chimeric blastocysts are transferred to culture medium for a short period of time and then transplanted into the oviducts of recipient female mice (white mice) to produce F0 generation chimeric mice (black and white). F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice. F1 generation heterozygous mice are then mated 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 to obtain AGT gene homozygous mice.

[0455] The genotype of F1 generation mouse somatic cells can be identified by PCR using the primers shown in Table 7. The exemplary results are shown in FIG. Figure 1 As shown, 6 mice numbered F1-1 to F1-6 were positive mice.

[0456] Table 7 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

[0457]

[0458] The expression of mRNA in the humanized AGT gene mice was detected by RT-PCR for the F1 generation PCR-positive mice. Specifically, one 5-week-old C57BL / 6 mouse (+ / +) and one 5-week-old male AGT gene humanized heterozygote (H / +) prepared in this embodiment were selected, and liver tissue was obtained after euthanasia by cervical dislocation. The primer sequences shown in Table 8 were used for RT-PCR detection. The test results are shown in FIG. Figure 2 As shown in the figure, humanized mouse AGT mRNA was not detected in wild-type C57BL / 6 mice, but humanized mouse AGT mRNA was detected in mice with humanized AGT gene heterozygotes.

[0459] Table 8 RT-PCR primer sequences and target fragment sizes

[0460]

[0461] The expression of human AGT protein in AGT gene humanized mice can be detected by conventional methods such as ELISA. Specifically, 3 5-week-old male C57BL / 6 mice (+ / +) and 3 AGT gene humanized heterozygotes (H / +) of the same age prepared in this embodiment were selected, plasma was collected, and the human AGT ELISA kit (Abcam, catalog number ab287170) and mouse AGT ELISA kit (Abcam, catalog number ab245718) were used for detection. The test results are as follows: Figure 3 shown.

[0462] from Figure 3 As can be seen in the results, only mouse AGT protein, not human AGT protein, was detected in wild-type C57BL / 6 mice. When tested using a human-specific AGT ELISA kit, human AGT protein was only detected in AGT humanized heterozygous mice. This demonstrates that human AGT protein can be successfully expressed in mice homozygous for the humanized AGT gene. Furthermore, the plasma level of human AGT in AGT humanized heterozygous mice was approximately 18 μg / mL.

[0463] Example 2 AGT gene humanized rats

[0464] To achieve the objectives of the present invention, a nucleotide sequence encoding a human AGT protein can be introduced into the endogenous rat AGT locus, allowing the rat to express a human or humanized AGT protein. Specifically, using gene editing technology, a portion of the approximately 6.94 kb sequence downstream of the rat AGT to the 3'UTR is replaced with a sequence comprising approximately 8.98 kb downstream of the ATG to the 3'UTR of the human AGT gene, thereby generating a humanized AGT locus and achieving humanization of the rat AGT gene.

[0465] To implement the targeting strategy of the present invention, CRISPR / Cas9 technology was used for gene editing to construct a targeting vector V2, which contained upstream and downstream homology arm sequences of the rat AGT gene and human AGT fragment 2. The upstream 5' homology arm sequence was SEQ ID NO: 23, the downstream 3' homology arm sequence was SEQ ID NO: 24, and the nucleotide sequence of human AGT fragment 2 was SEQ ID NO: 25. The connection between the upstream sequence of human AGT fragment 2 and the rat sequence was designed as follows:

[0466] 5'-

[0467] (SEQ ID NO: 26), wherein the sequence The last "G" in the sequence is the last nucleotide of the upstream junction of rat and human AGT fragment 2 (i.e., the last nucleotide of the 5' homology arm sequence). ATGG The "A" in " is the first nucleotide of human AGT fragment 2. The downstream connection of human AGT fragment 2 sequence and rat is designed as follows:

[0468]

[0469] (SEQ ID NO: 27), wherein the sequence The last "T" in the sequence is the last nucleotide of human AGT fragment 2. GGGC The first "G" in " is the first nucleotide at the downstream junction of rat and human AGT fragment 2 (i.e., the first nucleotide of the 3' homology arm sequence). The mRNA sequence of the modified humanized rat AGT is shown in SEQ ID NO: 66, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0470] Targeting vector V2 can be constructed using conventional methods, such as enzyme digestion, ligation, and direct synthesis. After initial verification of the constructed targeting vector V2 by enzyme digestion, it is sent to a sequencing company for sequencing verification. Targeting vectors that have been verified to be correct by sequencing are used in subsequent experiments.

[0471] The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are prerequisites for constructing sgRNA expression vectors. Design and synthesize the sgRNA sequence that recognizes the target site. The target sequence of the exemplary sgRNA on the AGT gene is as follows:

[0472] sgRNA1 target site (SEQ ID NO: 28): 5'-ATACGCGGTCCCCAGCTGTCAGG-3';

[0473] sgRNA2 target site (SEQ ID NO: 29): 5′-TGTGGATTCTATAGGCACTCTGG-3′;

[0474] After detecting the activity of sgRNA and confirming that it can mediate efficient cleavage efficiency, enzyme cleavage sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 9. After annealing, the annealed products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain expression vectors pT7-AGT-1 and pT7-AGT-2.

[0475] Table 9 Sequence list of sgRNA1 and sgRNA2

[0476]

[0477]

[0478] The pT7-sgRNA vector, synthesized by a plasmid synthesis company, contains a DNA fragment containing the T7 promoter and the sgRNA scaffold (SEQ ID NO: 38). This fragment was subsequently digested with EcoRI and BamHI and ligated to a backbone vector (source: Takara, Cat. No. 3299). Sequencing verification by a professional sequencing company confirmed the acquisition of the desired plasmid. Pronuclear fertilized eggs from rats, such as SD rats, were microinjected with a microinjector to mix the in vitro transcription products of the pT7-AGT-1 and pT7-AGT-2 plasmids (using the Ambion in vitro transcription kit, according to the manufacturer's instructions), the targeting vector V2, and Cas9 mRNA. The injected fertilized eggs were then transferred to culture medium for a short period of time and then transplanted into the oviducts of recipient female mice for development. The resulting rats (F0 generation) were then hybridized and selfed to expand the population and establish a stable AGT gene-humanized rat strain.

[0479] The genotype of F1 rat somatic cells can be identified by PCR using the primers shown in Table 10. The exemplary results are shown in FIG. Figure 4 As shown, the rat numbered F1-1 is a positive rat.

[0480] Table 10 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

[0481]

[0482] The expression of human AGT protein in AGT gene humanized rats can be detected by conventional methods such as ELISA. Specifically, 3 6-week-old female wild-type SD rats (+ / +) and 3 6-week-old AGT gene humanized rat homozygotes (H / H) prepared in this example were selected, blood was collected, and the human AGT ELISA kit (Abcam, catalog number ab287170) and rat AGT ELISA kit (Abcam, catalog number ab288178) were used for detection. The test results are shown in Figure 2. Figure 5 shown.

[0483] from Figure 5 As can be seen in the results, only rat AGT protein, but not human AGT protein, was detected in wild-type SD rats. When tested using a human-specific AGT ELISA kit, human AGT protein was detected only in rats homozygous for the humanized AGT gene, and the serum level of human AGT in these homozygous rats was approximately 28 μg / mL. This demonstrates that human AGT protein can be successfully expressed in rats homozygous for the humanized AGT gene.

[0484] Example 3 REN gene humanized mice

[0485] To achieve the objectives of the present invention, a nucleotide sequence encoding a human REN protein can be introduced into the endogenous REN locus of a mouse, thereby enabling the mouse or rat to express a human or humanized REN protein. Specifically, using gene editing technology, a portion of the approximately 14.07 kb sequence from the 5'UTR upstream to the 3'UTR downstream of the mouse is replaced with a sequence encompassing approximately 15.52 kb from the 5'UTR upstream to the 3'UTR downstream of the human REN gene, thereby generating a humanized REN locus and achieving humanization of the mouse REN gene.

[0486] Taking the construction of a humanized mouse with the REN gene as an example, a targeting vector V3 was constructed to implement the targeting strategy of the present invention. The targeting vector V3 contains upstream and downstream homology arm sequences of the mouse REN gene, as well as an A1 fragment containing a human REN fragment. The upstream 5' homology arm sequence is SEQ ID NO: 42, and the downstream 3' homology arm sequence is SEQ ID NO: 43. The nucleotide sequence of the human REN fragment is consistent with positions 204154277-204169793 of gene accession number NC_000001.11. The connection between the upstream of the human REN fragment and the mouse sequence is designed as follows: (SEQ ID NO: 44), wherein the sequence The "A" in the sequence is the last nucleotide at the upstream junction of the mouse and human REN fragments (i.e., the last nucleotide of the 5' homology arm sequence). CCAG The first "C" in " is the first nucleotide of the human REN fragment. The downstream connection between the human REN fragment and the mouse sequence is designed as follows:

[0487] (SEQ ID NO: 45), wherein the sequence The "A" in the sequence is the last nucleotide of the human REN fragment. GCCT The "G" in " is the first nucleotide at the downstream junction of the mouse and human REN fragments (i.e., the first nucleotide of the 3' homology arm sequence).

[0488] The targeting vector V3 also contains a resistance gene for positive clone screening, the neomycin phosphotransferase coding sequence Neo. Two Frt recombination sites, arranged in the same orientation, are flanked by the resistance gene to form a Neo cassette. The Neo cassette is located within intron 5 of the human REN fragment, and the connection between the 5' end of the Neo cassette and the human REN gene is designed as follows:

[0489] (SEQ ID NO: 46), wherein the sequence " TGGC "C" is the last nucleotide at the junction of the human REN gene and the 5' end of the Neo box. GCGA The first "G" in " is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human REN gene is designed as follows:

[0490] (SEQ ID NO: 47), wherein the sequence " CAACT "T" is the last nucleotide of the Neo box, the sequence " GGCT The first "G" in " is the first nucleotide at the junction of the human REN gene and the 3' end of the Neo box. The mRNA sequence of the modified humanized mouse REN is shown in SEQ ID NO: 48, and the expressed protein sequence is shown in SEQ ID NO: 21.

[0491] Targeting vector V3 can be constructed using conventional methods, such as enzyme digestion and ligation. After preliminary verification of the constructed targeting vector V3 by enzyme digestion, it is sent to a sequencing company for sequencing verification. The sequencing-verified targeting vector is electroporated and transfected into embryonic stem cells of C57BL / 6 mice. The resulting cells are screened using a positive clone screening marker gene to identify the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into isolated blastocysts (white mice) according to techniques known in the art. The resulting chimeric blastocysts are transferred to culture medium for a short period of time and then transplanted into the oviducts of recipient female mice (white mice) to produce F0 generation chimeric mice (black and white). F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice. F1 generation heterozygous mice are then mated 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 to obtain homozygous REN gene humanized mice.

[0492] The genotype of somatic cells of F1 generation mice can be identified by PCR, using the primers shown in Table 11 for detection. The exemplary results are shown in FIG. Figure 6 As shown, three mice numbered F1-1 to F1-3 were positive mice.

[0493] Table 11 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

[0494]

[0495] The expression of human REN protein in REN gene humanized mice can be detected by conventional methods such as ELISA. Specifically, 2 6-week-old male wild-type C57BL / 6 mice (+ / +) and 2 6-week-old heterozygous REN gene humanized mice (H / +) prepared in this example were selected, and serum was collected and tested using human REN ELISA kit (R&D, catalog number DREN00) and mouse REN ELISA kit (Thermo Fisher, catalog number EMREN1). The test results are shown in Figure 2. Figure 7 shown.

[0496] from Figure 7 As can be seen in the figure, only mouse REN protein, but not human REN protein, was detected in wild-type C57BL / 6 mice. When tested using a human-specific REN ELISA kit, both human and mouse REN proteins were detected in heterozygous REN humanized mice, and the level of human REN in the serum of heterozygous REN humanized mice was approximately 2200 pg / mL. This demonstrates that human REN protein can be successfully expressed in humanized REN mice.

[0497] Example 4 REN gene humanized rats

[0498] In order to achieve the purpose of the present invention, a nucleotide sequence encoding human REN protein can be introduced into the endogenous REN locus of rats, so that the rats express human or humanized REN protein. Specifically, using gene editing technology, a nucleotide sequence encoding human REN protein can be introduced into the endogenous REN locus of rats, so that the rats express human or humanized REN protein.

[0499] The approximately 15.52 kb downstream of the UTR replaced the partial sequence of approximately 18.20 kb from the upstream of the rat 5'UTR to the downstream of the 3'UTR) to obtain the humanized REN locus, thereby realizing the humanization transformation of the rat REN gene.

[0500] To implement the targeting strategy of the present invention, CRISPR / Cas9 technology was used for gene editing to design a targeting vector V4 containing upstream and downstream homology arm sequences of the rat AGT gene and a human REN fragment. The upstream 5' homology arm sequence is SEQ ID NO: 52, and the downstream 3' homology arm sequence is SEQ ID NO: 53. The nucleotide sequence of the human REN fragment is consistent with gene accession number NC_000001.11, positions 204154277-204169793. The upstream connection between the human REN fragment and the rat AGT gene is designed as follows:

[0501] (SEQ ID NO: 54), wherein the sequence " CCTCT The last "T" in the sequence " is the last nucleotide of the upstream junction of the rat and human REN fragments (i.e., the last nucleotide of the 5' homology arm sequence). CCAG The first "C" in " is the first nucleotide of the human REN fragment. The downstream connection between the human REN fragment and the rat is designed as follows:

[0502] (SEQ ID NO: 55), wherein the sequence The "A" in the sequence is the last nucleotide of the human REN fragment. CAGAGAC The first "C" in " is the first nucleotide at the downstream junction of the rat and human REN fragments (i.e., the first nucleotide of the 3' homology arm sequence). The mRNA sequence of the modified humanized rat REN is shown in SEQ ID NO: 48, and the expressed protein sequence is shown in SEQ ID NO: 21.

[0503] Targeting vector V4 can be constructed using conventional methods, such as enzyme digestion, ligation, and direct synthesis. After initial verification of the constructed targeting vector V4 by enzyme digestion, it is sent to a sequencing company for sequencing verification. Targeting vectors that have been verified to be correct by sequencing are used in subsequent experiments.

[0504] The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are prerequisites for constructing sgRNA expression vectors. Design and synthesize the sgRNA sequence that recognizes the target site. The target sequence of the exemplary sgRNA on the REN gene is as follows:

[0505] sgRNA3 target site (SEQ ID NO: 56): 5′-CTGCTCTTCCCAAGCCCTAGAGG-3′;

[0506] sgRNA4 target site (SEQ ID NO: 57): 5'-AGAGGGCACGGAAAAACACACAGG-3';

[0507] After testing the activity of sgRNA and confirming that it can mediate efficient cleavage efficiency, enzyme cleavage sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 12. After annealing, the annealed products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain expression vectors pT7-REN-1 and pT7-REN-2.

[0508] Table 12 Sequence list of sgRNA3 and sgRNA4

[0509]

[0510] The pT7-sgRNA vector is synthesized by a plasmid synthesis company. The DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 38) is then ligated to a backbone vector (source: Takara, Catalog No. 3299) using enzyme digestion (EcoRI and BamHI). Sequencing verification by a professional sequencing company confirms the desired plasmid. Pronuclear fertilized eggs from rats, such as Sprague-Dawley rats, are microinjected with a premix of in vitro transcription products of the pT7-REN-1 and pT7-REN-2 plasmids (using the Ambion in vitro transcription kit, according to the manufacturer's instructions), the targeting vector, and Cas9 mRNA. The injected fertilized eggs are transferred to culture medium for a short period of time and then transplanted into the oviducts of recipient female mice for development. The resulting rats (F0 generation) are then hybridized and selfed to expand the population and establish a stable REN gene humanized rat strain.

[0511] Example 5 Pharmacodynamic Model

[0512] The humanized mice (rats or mice) disclosed herein can be used to induce the generation of various human disease models for disorders related to AGT and / or REN, including models of hypertension, heart failure, and eclampsia, which can be used to test the in vivo efficacy of human-specific therapeutic drugs. For example, humanized mice expressing AGT and / or REN genes can be used to evaluate the efficacy, pharmacokinetics, and in vivo therapeutic efficacy of antagonists of the human-specific AGT and / or REN signaling pathways in various disease models known in the art.

[0513] The AGT and / or REN gene humanized mice disclosed in the present invention can be used to detect the inhibitory efficiency of nucleic acid drugs on human AGT and / or REN mRNA. Specifically, 6-8 week-old homozygous AGT gene humanized mice were selected and randomly divided into two groups: a drug administration group and a control group. On the day of grouping (D0), the drug administration group was injected with human AGT targeted nucleic acid drugs (the drug was synthesized according to the WO2021096763 patent), and the control group was injected with an equal volume of PBS. The nucleic acid drug was administered in the form of a PBS aqueous solution. Blood was collected on the 7th day (D7), 14th day (D14), 21st day (D21) and 28th day (D28) after grouping, and the expression of human AGT protein in serum was detected by ELISA. The mice were killed on the 28th day (D28), liver tissue was collected, and the expression level of human AGT mRNA was detected by qPCR.

[0514] Example 6 Preparation of double-gene or multi-gene humanized mice

[0515] Humanized mice with AGT and / or REN genes produced by this method can also be used to create multi-humanized mouse models. For example, in Examples 1-4 above, the embryonic stem cells used for microinjection can be derived from mice modified to contain at least one gene encoding APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. Alternatively, based on humanized AGT and / or REN mice, double-humanized or multi-humanized mouse models can be generated using isolated mouse ES embryonic stem cells and gene recombination targeting techniques. Homozygous or heterozygous AGT and / or REN mice obtained by this method can also be mated with other genetically modified mice, and the offspring screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene mice with humanized AGT and / or REN genes and other genetic modifications. Homozygous mice with double or multiple genetic modifications can then be mated with each other.

[0516] Taking AGT / REN double-gene humanized mice as an example, the homozygous AGT gene humanized mice prepared in Example 1 are mated with the homozygous REN gene humanized mice prepared in Example 3, and their offspring are screened. According to Mendel's law of inheritance, there is a certain probability of obtaining humanized AGT / REN double-gene humanized mice, and then the heterozygotes are mated with each other to obtain homozygotes.

[0517] For another example, taking AGT / REN double-gene humanized rats as an example, the homozygous AGT gene humanized rats prepared in Example 2 are mated with the homozygous REN gene humanized rats prepared in Example 4, and their offspring are screened. According to Mendel's law of inheritance, there is a certain probability of obtaining humanized AGT / REN double-gene humanized rats, and then the heterozygotes are mated with each other to obtain homozygotes.

[0518] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Any construction method a1)-a2): a1) A method for constructing a non-human animal modified with an AGT gene, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising all or part of a nucleotide sequence encoding a human AGT protein; Preferably, the amino acid sequence of the human AGT protein comprises SEQ ID NO: 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function; a2) A method for constructing a non-human animal modified with a REN gene, wherein the genome of the non-human animal comprises at least one chromosome comprising all or part of a nucleotide sequence encoding a human REN protein; Preferably, the amino acid sequence of the human REN protein comprises SEQ ID NO: 21, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

2. Any of the construction methods b1)-b2): b1) A method for constructing an AGT gene-modified non-human animal, characterized in that: introducing a nucleotide sequence of human AGT into the endogenous AGT locus of a non-human animal; b2) A method for constructing a REN gene-modified non-human animal, characterized in that a human REN nucleotide sequence is introduced into the endogenous REN locus of the non-human animal.

3. The construction method according to claim 2, characterized in that The introduction in b1) and b2) is replacement or insertion; further, replacement; Preferably, the construction method b1) comprises the following steps: replacing the nucleotide sequence of endogenous AGT of a non-human animal with the nucleotide sequence of human AGT; Preferably, the nucleotide sequence of human AGT comprises all or part of the nucleotide sequence encoding human AGT protein; Preferably, the amino acid sequence of the human AGT protein comprises SEQ ID NO: 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function; Preferably, the nucleotide sequence of human AGT comprises all or part of exon 1 to exon 5 of the human AGT gene; preferably, the nucleotide sequence of human AGT comprises the nucleotide sequence from the start codon to the stop codon of the human AGT gene, preferably further comprises the 5'UTR and / or 3'UTR, more preferably further comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the nucleotide sequence of human AGT comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR; Preferably, the nucleotide sequence of human AGT comprises nucleotide sequence 230702763-230748399 of NC_000001.11, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto; Preferably, the nucleotide sequence of human AGT comprises a continuous nucleotide sequence of at least 50 bp downstream of the human AGT gene to the 3'UTR; Preferably, the nucleotide sequence of human AGT comprises SEQ ID NO: 25, or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto; Preferably, the human AGT nucleotide sequence is operably linked to an exogenous AGT regulatory element; Preferably, the exogenous AGT regulatory element is a human AGT regulatory element; Preferably, the construction method b2) comprises the following steps: replacing the nucleotide sequence of endogenous REN of a non-human animal with the nucleotide sequence of human REN; Preferably, the nucleotide sequence of human REN comprises all or part of the nucleotide sequence encoding human REN protein; Preferably, the amino acid sequence of the human REN protein comprises SEQ ID NO: 21, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function; Preferably, the nucleotide sequence of human REN comprises all or part of exon 1 to exon 10 of the human REN gene; preferably, the nucleotide sequence of human REN comprises the nucleotide sequence from the start codon to the stop codon of the human REN gene, preferably further comprises a 5'UTR and / or a 3'UTR, more preferably further comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the nucleotide sequence of human REN comprises a continuous nucleotide sequence of at least 50 bp upstream of 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of 3'UTR; Preferably, the nucleotide sequence of human REN comprises nucleotide sequence 204154277-204169793 of NC_000001.11, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto; Preferably, the nucleotide sequence of human REN is operably linked to an exogenous REN regulatory element; Preferably, the exogenous REN regulatory element is a human REN regulatory element.

4. The construction method according to claim 3, characterized in that The nucleotide sequence of the non-human animal endogenous AGT comprises all or part of the nucleotide sequence encoding the non-human animal endogenous AGT protein; Preferably, the non-human animal endogenous AGT protein comprises SEQ ID NO: 1, SEQ ID NO: 5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function; Preferably, the nucleotide sequence of the non-human animal endogenous AGT comprises all or part of exon 1 to exon 5 of the non-human animal endogenous AGT gene; Preferably, the nucleotide sequence of the non-human animal endogenous AGT comprises the nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous AGT gene, preferably further comprises the 5'UTR and / or 3'UTR, more preferably further comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the nucleotide sequence of the non-human animal endogenous AGT comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR; Preferably, the nucleotide sequence of the non-human animal endogenous AGT comprises a continuous nucleotide sequence of at least 50 bp downstream of the non-human animal endogenous AGT gene to the 3'UTR; Preferably, the nucleotide sequence of the non-human animal endogenous REN comprises all or part of the nucleotide sequence encoding the non-human animal endogenous REN protein; Preferably, the non-human animal endogenous REN protein comprises SEQ ID NO: 20, SEQ ID NO: 22, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function; Preferably, the nucleotide sequence of the non-human animal endogenous REN comprises all or part of exon 1 to exon 9 of the non-human animal endogenous REN gene; Preferably, the nucleotide sequence of the non-human animal endogenous REN comprises the nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous REN gene, preferably further comprises a 5'UTR and / or a 3'UTR, more preferably further comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the nucleotide sequence of the non-human animal endogenous REN comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR.

5. The construction method according to any one of claims 2 to 4, characterized in that: b1) The endogenous AGT protein of the non-human animal is not expressed or is expressed at a reduced level compared to AGT in wild-type non-human animals; preferably, the modified AGT gene in the genome of the non-human animal in b1) is homozygous or heterozygous for the endogenous replaced locus; Preferably, the mRNA transcribed from the modified AGT gene in the genome of the non-human animal in b1) comprises SEQ ID NO: 6, SEQ ID NO: 66, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto; Preferably, the non-human animal in b1) further comprises a nucleotide sequence encoding other human or chimeric proteins; Preferably, the other human or chimeric protein comprises at least one of REN, APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4; further REN; Preferably, the endogenous REN protein of the non-human animal in b2) is not expressed or is expressed at a reduced level compared to REN in wild-type non-human animals; Preferably, the modified REN gene in the genome of the non-human animal in b2) is homozygous or heterozygous for the endogenous replaced locus; Preferably, the mRNA transcribed from the modified REN gene in the genome of the non-human animal comprises SEQ ID NO: 48, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto; Preferably, the non-human animal in b2) further comprises a nucleotide sequence encoding other human or chimeric proteins; Preferably, the other human or chimeric proteins include at least one of AGT, APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4; further AGT.

6. Any cell, tissue, or organ from d1) to d2): d1) A cell, tissue, or organ derived from a non-human animal or its progeny obtained by the construction method described in a1) of claim 1 or the construction method described in b1) of any one of claims 2 to 5; d2) A cell, tissue, or organ derived from a non-human animal or its progeny obtained by the construction method described in a2) of claim 1 or the construction method described in b2) of any one of claims 2 to 5.

7. A method for constructing a non-human animal modified with AGT and REN genes, wherein the genome of the non-human animal comprises at least two chromosomes, wherein: One chromosome comprises all or part of a nucleotide sequence encoding a human AGT protein; the other chromosome comprises all or part of a nucleotide sequence encoding a human REN protein; Preferably, the amino acid sequence of the human AGT protein comprises SEQ ID NO: 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function; Preferably, the amino acid sequence of the human REN protein comprises SEQ ID NO: 21, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity thereto and having the same function.

8. A method for constructing a non-human animal modified with AGT and REN genes, comprising introducing a nucleotide sequence of human AGT into the endogenous AGT locus of the non-human animal; and A human REN nucleotide sequence is introduced into the endogenous REN locus of a non-human animal.

9. The construction method according to claim 8, characterized in that: The said introduction is replacement or insertion; further, replacement; Preferably, the construction method comprises the following steps: Replacing the nucleotide sequence of endogenous AGT of the non-human animal with the nucleotide sequence of human AGT; and Replacing the nucleotide sequence of the endogenous REN of the non-human animal with the nucleotide sequence of the human REN; Preferably, the nucleotide sequence of human AGT is the nucleotide sequence of human AGT described in claim 3; Preferably, the nucleotide sequence of the non-human animal endogenous AGT is the nucleotide sequence of the non-human animal endogenous AGT as described in claim 4; Preferably, the endogenous AGT protein in the non-human animal is not expressed or is expressed at a reduced level compared to AGT in a wild-type non-human animal; preferably, the modified AGT gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus; preferably, the mRNA transcribed from the modified AGT gene in the genome of the non-human animal comprises SEQ ID NO: 6, SEQ ID NO: 66, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto; Preferably, the nucleotide sequence of human REN is the nucleotide sequence of human REN as claimed in claim 3; Preferably, the nucleotide sequence of the non-human animal endogenous REN is the nucleotide sequence of the non-human animal endogenous REN as claimed in claim 4; Preferably, the endogenous REN protein in the non-human animal is not expressed or is expressed at a reduced level compared to REN in a wild-type non-human animal; preferably, the modified REN gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus; preferably, mRNA transcribed from the modified REN gene in the genome of the non-human animal comprises SEQ ID NO: 48, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100% identical thereto; Preferably, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins; Preferably, the other human or chimeric proteins include at least one of APOC3, ANGPTL3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

10. A cell, tissue or organ derived from a non-human animal or its progeny obtained by the construction method according to any one of claims 7 to 9.

11. Use of the non-human animal or its progeny obtained by the construction method according to any one of claims 1 to 5 or 7 to 9, and / or the cell, tissue or organ according to claim 6 or 10, comprising: A) Applications in the development of products involving human cells in immunological processes related to AGT and / or REN; B) Use as a model system for pharmacological, immunological, microbiological and medical research related to AGT and / or REN; C) applications involving the production and use of animal experimental disease models for the study of the etiology of AGT and / or REN and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies for the screening, efficacy testing, efficacy assessment, validation or evaluation of modulators of the human AGT and / or REN signaling pathway; or E) Study the function of AGT and / or REN genes, study the drugs and drug efficacy targeting human AGT and / or REN target sites, and study the application of drugs in hypertension, kidney disease, and / or cardiovascular disease related to AGT and / or REN.

12. Either of f1) and f2) f1) A method for determining the effectiveness of an AGT and / or REN therapeutic agent in treating AGT and / or REN-related diseases, comprising the steps of using a non-human animal or its offspring obtained by the construction method of any one of claims 1 to 5, or 7 to 9, and / or a cell, tissue or organ according to claim 6 or 10; Preferably, the method comprises the following steps: administering an AGT and / or REN therapeutic agent to the non-human animal obtained by the construction method according to any one of claims 1 to 5, or 7 to 9, or its offspring, and determining the effectiveness of the AGT and / or REN therapeutic agent in treating an AGT and / or REN-related disease; f2) A method for determining the toxicity of an AGT and / or REN therapeutic agent, comprising the steps of using a non-human animal or its progeny obtained by the construction method of any one of claims 1 to 5 or 7 to 9, and / or a cell, tissue or organ according to claim 6 or 10; Preferably, the method comprises the steps of administering AGT and / or REN therapeutic agents to the non-human animal obtained by the construction method according to any one of claims 1-5 or 7-9 or its offspring, and determining the effects of the AGT and / or REN therapeutic agents on the animal.

13. The construction method, cell, tissue, or organ, use, or method according to any one of claims 1 to 12, characterized in that: The non-human animals include non-human mammals; further include rodents.

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