A gpr75 gene-modified non-human animal and methods of use thereof

Abstract

The present application provides a non-human animal expressing a human or chimeric G protein-coupled receptor 75 (GPR75) protein and methods of using the same. In certain embodiments, the genome of the non-human animal comprises an exogenous polynucleotide sequence encoding a human or chimeric GPR75 protein.

Description

Technical Field

[0001] This invention relates to transgenic nonhuman animals. Specifically, this invention relates to nonhuman animals expressing human or chimeric (e.g., humanized) GPR75 protein and methods of using them. Background Technology

[0002] Traditional drug development typically employs in vitro screening methods. However, these methods cannot provide the in vivo environment (such as the tumor microenvironment, stromal cells, extracellular matrix components, and immune cell interactions), leading to a high failure rate in drug development. Furthermore, given the differences between humans and non-human animals, results obtained from in vivo pharmacological studies using conventional laboratory animals may not reflect the true disease state and target site interactions, resulting in significant discrepancies between clinical trial results and animal experimental results.

[0003] Humanized animals are experimental models created by using techniques such as genetic engineering, cell transplantation, or tissue replacement to imbue animals with human genes, cells, tissues, or immune system components, thereby more closely resembling the human state at physiological, immune, or metabolic levels. Common humanized animals include humanized mice, humanized immune system mice, and transgenic animals carrying specific human organs or metabolic enzymes. These models overcome the limitations of traditional animal models in addressing species differences, providing a more predictive experimental platform for studying human disease mechanisms and drug responses. In drug development, humanized animal models help evaluate the efficacy, safety, and immunogenicity of candidate drugs, improving the success rate of clinical translation.

[0004] Traditional humanized transgenic methods involve randomly inserting humanized genes into the animal's genome. This random insertion often results in poor regulation of humanized gene expression, manifesting in many pathological symptoms in these transgenic animals, thus limiting their application.

[0005] Therefore, it is necessary to develop humanized animal models suitable for drug screening and evaluation, thereby improving the efficiency of new drug development and reducing drug research and development costs. Invention Overview One aspect of this disclosure provides a method for producing genetically modified non-human animals. In some embodiments, the method comprises modifying the genome of the non-human animal to include a foreign polynucleotide sequence encoding a human or chimeric G protein-coupled receptor 75 (GPR75) protein. In some embodiments, the human or chimeric GPR75 protein has at least 70% (75%, 80%, 85%, 90%, 95%, or 99%) identity with the amino acid sequence (SEQ ID NO: 2) of the human GPR75 protein. In some embodiments, the foreign polynucleotide sequence encodes the human GPR75 protein. In some embodiments, the amino acid sequence of the human GPR75 protein is SEQ ID NO: 2. NO: 2. In some embodiments, the exogenous polynucleotide sequence comprises all or part of exons 1 to 2 of the human GPR75 gene; in some embodiments, the exogenous polynucleotide sequence further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human GPR75 gene; in some embodiments, the exogenous polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene and at least 361 bp of continuous nucleotides downstream of the 3'UTR; in some embodiments, the exogenous polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene, at least 361 bp of continuous nucleotides downstream of the 3'UTR and the 5'UTR. The upstream polynucleotide sequence comprises at least 4361 bp of continuous nucleotides; in some embodiments, the exogenous polynucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the polynucleotide sequence shown at positions 53852551-53864328 of the NC_000002.12; in some embodiments, the exogenous polynucleotide sequence is the polynucleotide sequence shown at positions 53852551-53864328 of the NC_000002.12. In some embodiments, the exogenous polynucleotide sequence is regulated by an exogenous regulatory element; in some embodiments, the exogenous regulatory element is the human GPR75 promoter, 5'UTR, and / or 3'UTR.In some embodiments, the exogenous polynucleotide sequence replaces the endogenous GPR75 gene of a non-human animal; in some embodiments, the endogenous GPR75 gene of the non-human animal comprises all or part of exons 1 to 2 of the mouse GPR75 gene; in some embodiments, the endogenous GPR75 gene of the non-human animal further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the mouse GPR75 gene; in some embodiments, the endogenous GPR75 gene of the non-human animal comprises all of exons 1 to 2 of the mouse GPR75 gene and at least 2661 bp of continuous nucleotides downstream of the 3'UTR; in some embodiments, the endogenous GPR75 gene of the non-human animal comprises all of exons 1 to 2 of the mouse GPR75 gene, at least 2661 bp of continuous nucleotides downstream of the 3'UTR and at least 2360 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the genome of the non-human animal further comprises other exogenous polynucleotide sequences encoding human or chimeric proteins; in some embodiments, the human or chimeric protein is selected from at least one of CD98HC, CD3E, CD3D, CD3G, KIT, KITLG, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. In some embodiments, the non-human animal is a mammal; in some embodiments, the mammal is a monkey or a rodent; in some embodiments, the rodent is a mouse or a rat.

[0006] In another aspect, this disclosure provides a non-human animal cell, tissue, or organ whose genome contains a foreign polynucleotide sequence encoding a human or chimeric GPR75 protein. In some embodiments, the human or chimeric GPR75 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence (SEQ ID NO: 2) of the human GPR75 protein. In some embodiments, the foreign polynucleotide sequence encodes the human GPR75 protein. In some embodiments, the amino acid sequence of the human GPR75 protein is SEQ ID NO: 2. In some embodiments, the exogenous polynucleotide sequence comprises all or part of exons 1 to 2 of the human GPR75 gene; in some embodiments, the exogenous polynucleotide sequence further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human GPR75 gene; in some embodiments, the exogenous polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene and at least 361 bp of continuous nucleotides downstream of the 3'UTR; in some embodiments, the exogenous polynucleotide sequence comprises exons 1 to 2 of the human GPR75 gene. The exogenous polynucleotide sequence comprises the entirety of member 2, at least 361 bp of continuous nucleotides downstream of the 3'UTR, and at least 4361 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the exogenous polynucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the polynucleotide sequence shown at positions 53852551-53864328 of the NC_000002.12. In some embodiments, the exogenous polynucleotide sequence is the polynucleotide sequence shown at positions 53852551-53864328 of the NC_000002.12. In some embodiments, the exogenous polynucleotide sequence is regulated by an exogenous regulatory element; in some embodiments, the exogenous regulatory element is the human GPR75 promoter, the 5'UTR, and / or the 3'UTR.

[0007] In another aspect, this disclosure provides a non-human animal genome comprising a foreign polynucleotide sequence encoding a human or chimeric GPR75 protein. In some embodiments, the human or chimeric GPR75 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence (SEQ ID NO: 2) of the human GPR75 protein. In some embodiments, the foreign polynucleotide sequence encodes the human GPR75 protein. In some embodiments, the amino acid sequence of the human GPR75 protein is SEQ ID NO: 2.

[0008] In some embodiments, the exogenous polynucleotide sequence encoding the human or chimeric GPR75 protein comprises all or part of exons 1 to 2 of the human GPR75 gene. In some embodiments, the exogenous polynucleotide sequence further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human GPR75 gene; in some embodiments, the exogenous polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene and at least 361 bp of continuous nucleotides downstream of the 3'UTR; in some embodiments, the exogenous polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene, at least 361 bp of continuous nucleotides downstream of the 3'UTR and at least 4361 bp of continuous nucleotides upstream of the 5'UTR; in some embodiments, the exogenous polynucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the polynucleotide sequence shown at positions 53852551-53864328 of the gene accession number NC_000002.12. In some embodiments, the exogenous polynucleotide sequence is the polynucleotide sequence shown in positions 53852551-53864328 of the gene accession number NC_000002.12.

[0009] In some embodiments, the exogenous polynucleotide sequence encoding the human or chimeric GPR75 protein is regulated by an exogenous regulatory element. In some embodiments, the exogenous regulatory element is the human GPR75 promoter, 5'UTR, and / or 3'UTR. In some embodiments, the exogenous regulatory element comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the exon of the human GPR75 gene. In some embodiments, the exogenous regulatory element comprises at least 361 bp of continuous nucleotides downstream of the 3'UTR of the exon of the human GPR75 gene. In some embodiments, the exogenous regulatory element comprises at least 4361 bp of continuous nucleotides upstream of the 5'UTR of the exon of the human GPR75 gene.

[0010] In some embodiments, the exogenous polynucleotide sequence encoding the human or chimeric GPR75 protein replaces the endogenous GPR75 gene of the non-human animal. In some embodiments, the endogenous GPR75 gene of the non-human animal comprises all or part of exons 1 to 2 of the mouse GPR75 gene; in some embodiments, the endogenous GPR75 gene of the non-human animal further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the mouse GPR75 gene; in some embodiments, the endogenous GPR75 gene of the non-human animal comprises all of exons 1 to 2 of the mouse GPR75 gene and at least 2661 bp of continuous nucleotides downstream of the 3'UTR; in some embodiments, the endogenous GPR75 gene of the non-human animal comprises all of exons 1 to 2 of the mouse GPR75 gene, at least 2661 bp of continuous nucleotides downstream of the 3'UTR and at least 2360 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the exogenous polynucleotide sequence is regulated by endogenous regulatory elements of non-human animals.

[0011] In some embodiments, the genome of the non-human animal further includes other exogenous polynucleotide sequences encoding human or chimeric proteins. In some embodiments, the human or chimeric protein is selected from at least one of CD98HC, CD3E, CD3D, CD3G, KIT, KITLG, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0012] In some embodiments, the non-human animal is a mammal. In some embodiments, the mammal is a monkey or a rodent. In some embodiments, the rodent is a mouse or a rat.

[0013] In another aspect, this disclosure also provides a method for determining the effectiveness of a human GPR75-targeted therapeutic agent in treating a disease. In some embodiments, the method includes: (1) administering the human GPR75-targeted therapeutic agent to a non-human animal provided in this disclosure, wherein the non-human animal has the disease; and (2) evaluating the therapeutic effect of the therapeutic agent on the disease.

[0014] In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the solid tumor is colorectal cancer, bladder cancer, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the hematologic malignancy is chronic lymphocytic leukemia and acute myeloid leukemia.

[0015] In some embodiments, the non-human animal comprises tumor cells injected into the non-human animal. In some embodiments, the evaluation includes measuring the tumor volume in the non-human animal.

[0016] In some embodiments, the disease is an immune disease. In some embodiments, the immune disease is allergy, chronic urticaria, atopic dermatitis, osteoarthritis, asthma, rheumatoid arthritis, multiple sclerosis, psoriasis, chronic obstructive pulmonary disease, or inflammatory bowel disease (IBD).

[0017] In some embodiments, the disease is a metabolic disease. In some embodiments, the metabolic disease is obesity.

[0018] In some embodiments, the human GPR75-targeting therapeutic agent is an antibody, oligonucleotide drug, or peptide drug. In some embodiments, the human GPR75-targeting therapeutic agent is an anti-human GPR75 antibody.

[0019] In some embodiments, the method further includes administering a second therapeutic agent to the non-human animal. In some embodiments, the non-human animal also contains a foreign polynucleotide sequence encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4, and wherein the second therapeutic agent is an anti-human PD-1 antibody, an anti-human PD-L1 antibody, or an anti-human CTLA4 antibody. Attached Figure Description

[0020] Figure 1 The results of PCR identification of humanized mice with the GPR75 gene are shown. Among them, PC is the heterozygous positive control, WT is the wild-type control, and H2O is the water control.

[0021] Figure 2 The results of RT-PCR detection are shown. + / + represent wild-type C57BL / 6 mice, H / H represent humanized homozygous mice of the GPR75 gene, H2O is the water control, and GADPH is the internal control. Invention Details This invention can be implemented in many different forms and is not limited to the embodiments described herein. It should be understood that these embodiments are provided so that a thorough and complete understanding of the disclosure of this invention will be achieved.

[0022] 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 pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Although the numerical ranges and parameter approximations shown in the broad scope of the invention are provided, the values ​​shown in the specific embodiments are described as accurately as possible. However, any value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all scopes disclosed herein should be understood to encompass any and all subscopes contained herein.

[0023] This invention relates to an animal model containing human or chimeric G protein-coupled receptor 75 (GPR75) protein. This animal model can express human or chimeric GPR75 protein (e.g., humanized GPR75). It can be used to study the function of the GPR75 gene and also for screening and evaluating GPR75 signaling pathway regulators (e.g., anti-human GPR75 antibodies, oligonucleotide drugs, and / or peptide drugs). This invention provides a powerful tool for studying the function of the GPR75 protein and a platform for screening related drugs.

[0024] definition As used herein, the terms “non-human animal” and “animal” are used interchangeably throughout the specification and include, but are not limited to, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets), lagomorphs (e.g., rabbits), pigs (e.g., pigs, miniature pigs), equines, canids (e.g., dogs), felines (e.g., dogs), bovines, and other domestic, farm, and zoo animals.

[0025] As used in this article, the term "genome" refers to the sum of genetic information contained in a complete set of monoliths within a cell of an organism, typically composed of DNA. The genome includes all coding genes and non-coding sequences.

[0026] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to an amino acid polymer of any length having at least two amino acids.

[0027] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably to refer to polymers of nucleotides of any length having at least two nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids, and their modified forms.

[0028] As used herein, the terms "chimeric protein" or "chimeric polypeptide" refer to a protein or polypeptide where two or more portions of the polypeptide or protein originate from different species, or where at least one sequence of the protein or polypeptide differs from the amino acid sequence of a wild-type animal. In some embodiments, at least a portion of the sequence of a chimeric protein or chimeric polypeptide has origins from two or more different species, for example, identical (or homologous) proteins from different species. In some embodiments, a chimeric protein or chimeric polypeptide refers to a humanized protein or humanized polypeptide. As used herein, the terms "chimeric gene" or "chimeric nucleic acid" refer to a gene or nucleic acid where two or more portions of the gene or nucleic acid originate from different species, or where at least one sequence of the gene or nucleic acid differs from the nucleic acid of a wild-type animal. In some embodiments, a chimeric gene or chimeric nucleic acid has at least a portion of its sequence having origins from two or more different species, for example, a sequence encoding a different protein or a sequence encoding identical (or homologous) proteins from two or more different species. In some embodiments, a chimeric gene or chimeric nucleic acid refers to a humanized gene or humanized nucleic acid.

[0029] As used herein, the terms "humanized protein" or "humanized polypeptide" refer 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, a humanized protein or humanized polypeptide refers to a human protein or polypeptide. As used herein, the term "humanized nucleic acid" refers to a nucleic acid, wherein at least a portion of the nucleic acid is derived from a human. In some embodiments, all nucleic acids in a humanized nucleic acid are derived from humans. In some embodiments, a humanized nucleic acid refers to a humanized exon, which may be a human exon or a chimeric exon.

[0030] As used herein, the terms “regulatory element” and “gene regulatory element” are used interchangeably to refer to specific regions located on the DNA sequence that precisely regulate the initiation, rate, and efficiency of gene transcription by binding to protein factors. Regulatory elements include, but are not limited to, promoters, enhancers, silencers, and terminators. Depending on their location relative to the gene they regulate, regulatory elements include the 5'UTR (the untranslated region located upstream of the 5' end of a gene) and the 3'UTR (the untranslated region located downstream of the 3' end of a gene).

[0031] As used in this article, the term "cancer" refers to cells with autonomous growth capacity, that is, an abnormal state or condition characterized by rapid cell growth and proliferation. This term is intended to encompass all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignant transformation of cells, tissues, or organs, regardless of histopathological type or stage of invasiveness.

[0032] GPR75 gene The G protein-coupled receptor 75 (GPR75) gene encodes a member of the G protein-coupled receptor family. This gene contains only two exons and is located on human chromosome 2p16. GPR75 consists of 540 amino acids, and its expression level is closely related to the development and progression of various human diseases, including cancer, immune diseases, and metabolic disorders. To date, three ligands have been identified that can bind to GPR75: RANTES, CCL5, and 20-HETE. They participate in pathological processes by activating different downstream signaling pathways. Specifically, 20-HETE, upon binding to GPR75, can activate signaling pathways such as PKC / PLC, C-src, MAPK, NF-κB, and AKT, thereby inducing pro-inflammatory responses, diabetes, vascular dysfunction and remodeling, hypertension, and malignant cell transformation. The binding of CCL5 to GPR75 induces phosphorylation and activation of AKT, glycogen synthase kinase B, and extracellular signal-regulated kinases (ERK1 / 2). Dysregulation of the ERK pathway has been proven to be one of the important mechanisms of cancer metastasis. Furthermore, the binding of RANTES to GPR75 activates the MAPK signaling pathway, a process closely related to cancer occurrence and progression. See: Dashti MR, Gorbanzadeh F, Jafari-Gharabaghlou D, Farhoudi Sefidan Jadid M, Zarghami N. G Protein-Coupled Receptor 75 (GPR75) As a Novel Molecule for Targeted Therapy of Cancer and Metabolic Syndrome. Asian Pac J Cancer Prev. 2023 May 1;24(5):1817-1825, the entire contents of which are incorporated herein by reference.

[0033] In the human genome, the GPR75 gene (NCBI Gene ID: 10936, UniProt ID: O95800, located on chromosome 2 NC_000002.12, vertices 53852912 to 53859967) contains two exons, exon 1 and exon 2. The positions of each exon in the nucleotide and amino acid sequences of transcript NM_006794.4 and its encoded protein NP_006785.1 (SEQ ID NO: 2) are as follows: Table 1. Sequence exon positions of human GPR75 mRNA and corresponding encoded protein positions. In the mouse genome, the GPR75 gene (NCBI Gene ID: 237716, UniProt ID: Q6X632, located on chromosome 11, NC_000077.7, vertices 30834636 to 30846090) contains two exons, exon 1 and exon 2. The positions of each exon in the nucleotide and amino acid sequences of transcript NM_175490.5 and its encoded protein NP_780699.2 (SEQ ID NO: 1) are as follows: Table 2 Mouse GPR75 mRNA sequence exon positions and corresponding encoded protein positions The GPR75 gene, protein, and gene locus are also known in other species in this field. For example, Rattus norvegicus (Rat) Macaca mulatta (Rhesus monkey) Canis lupus familiaris (dog) and Sus scrofa Information about the GPR75 gene in pigs (such as intron sequences, exon sequences, and amino acid sequences) can be found in NCBI, and all of it is incorporated herein by reference.

[0034] Genetically modified non-human animals This disclosure provides, in one aspect, a non-human animal genetically modified with the GPR75 gene. In some embodiments, the genome of the non-human animal contains a foreign polynucleotide sequence encoding a human or chimeric GPR75 protein. In some embodiments, the non-human animal contains at least one genetically modified cell whose genome contains a foreign polynucleotide sequence encoding a human or chimeric GPR75 protein. 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 contain a foreign polynucleotide sequence encoding a human or chimeric GPR75 protein. The cells having the foreign polynucleotide sequence can be various types of cells, such as 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, the non-human animal is capable of passing the genetic modification to offspring through germline propagation.

[0035] The genetically modified non-human animal can be a variety of animals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). In some embodiments, the animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent can be selected from mice, rats, and hamsters. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[0036] In some embodiments, the mice are selected from the C57BL strain. In some embodiments, the C57BL strain is selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mice are selected from strains 129, including 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, MammalianGenome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant content of which is incorporated herein by reference in its entirety. In some embodiments, the mice are BALB strains, such as BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, and BALB / cWt. In some embodiments, the mice are BLD, CM, DX, FX, HZ, JD, KM, PD, QP, SMX, SY, TW, TZ, YP, YX, ZC, ZZ1, or ZZ2 strains, or NOD, NOD / SCID, or NOD-Prkdc strains. scid IL-2rg nullBackground mice. In some embodiments, the mice are derived from hybrid lines (e.g., 50% BALB / c - 50% 129; or 50% C57BL / 6 - 50% 129). In some embodiments, the mice are hybrids of the 129 strain or the C57BL / 6 strain. In some embodiments, the mice are hybrids of the BALB strain and another strain.

[0037] In some embodiments, the genetically modified non-human animals provided in this disclosure express human or chimeric GPR75 protein in at least one cell. In some embodiments, the human or chimeric GPR75 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence (SEQ ID NO: 2) of the human GPR75 protein. In some embodiments, the exogenous polynucleotide sequence encodes the human GPR75 protein. In some embodiments, the amino acid sequence of the human GPR75 protein is SEQ ID NO: 2.

[0038] In some embodiments, the genetically modified non-human animals provided in this disclosure contain a foreign polynucleotide sequence encoding the human or chimeric GPR75 protein in the genome of at least one cell. In some embodiments, the foreign polynucleotide sequence comprises all or part of exons 1 to 2 of the human GPR75 gene. In some embodiments, the foreign polynucleotide sequence further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human GPR75 gene. In some embodiments, the foreign polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene and at least 361 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the foreign polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene, at least 361 bp of continuous nucleotides downstream of the 3'UTR, and at least 4361 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the exogenous polynucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the polynucleotide sequence shown at positions 53852551-53864328 of the NC_000002.12. In some embodiments, the exogenous polynucleotide sequence is the polynucleotide sequence shown at positions 53852551-53864328 of the NC_000002.12.

[0039] In some embodiments, the exogenous polynucleotide sequence encoding the human or chimeric GPR75 protein is regulated by an exogenous regulatory element after insertion into an endogenous safety site in the genome of a non-human animal. In some embodiments, the exogenous regulatory element comprises a human GPR75 promoter, a 5'UTR, and / or a 3'UTR. In some embodiments, the exogenous regulatory element comprises at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 2000 bp, at least 3000 bp, at least 4000 bp, or at least 5000 bp of continuous nucleotides downstream of the 3'UTR of the exon of the human GPR75 gene. In some embodiments, the exogenous regulatory element comprises at least 361 bp of continuous nucleotides downstream of the 3'UTR of the exon of the human GPR75 gene. In some embodiments, the exogenous regulatory element comprises at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 2000 bp, at least 3000 bp, at least 4000 bp, or at least 5000 bp of continuous nucleotides upstream of the 5'UTR of the human GPR75 gene. In some embodiments, the exogenous regulatory element comprises at least 4361 bp of continuous nucleotides upstream of the 5'UTR of the human GPR75 gene. In some embodiments, the exogenous regulatory element is a regulatory system of a specific inducer or repressor. In some embodiments, the regulatory system is a tetracycline system (Tet-Off System / Tet-On System) or a tamoxifen system.

[0040] In some embodiments, the exogenous polynucleotide sequence encoding the human or chimeric GPR75 protein is inserted into or replaces the endogenous GPR75 gene locus of a non-human animal. In some embodiments, the endogenous GPR75 gene of the non-human animal comprises all or part of exons 1 to 2 of the mouse GPR75 gene; in some embodiments, the endogenous GPR75 gene of the non-human animal further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the mouse GPR75 gene; in some embodiments, the endogenous GPR75 gene of the non-human animal comprises all of exons 1 to 2 of the mouse GPR75 gene and at least 2661 bp of continuous nucleotides downstream of the 3'UTR; in some embodiments, the endogenous GPR75 gene of the non-human animal comprises all of exons 1 to 2 of the mouse GPR75 gene, at least 2661 bp of continuous nucleotides downstream of the 3'UTR, and at least 2360 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the exogenous polynucleotide sequence is regulated by endogenous regulatory elements in the nonhuman animal. In some embodiments, the genetically modified nonhuman animal can express human or chimeric GPR75 under the control of endogenous regulatory elements. Insertion or substitution at the endogenous locus provides nonhuman animals that express human or chimeric GPR75 protein in suitable cells without causing potential pathology.

[0041] This disclosure also provides a transgenic animal having two or more human or chimeric genes. The animal may contain a human or chimeric GPR75 gene and a sequence encoding an additional human or chimeric protein. In some embodiments, the additional human or chimeric protein is selected from human or chimeric proteins targeting the CD98HC, CD3E, CD3D, CD3G, KIT, KITLG, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 genes.

[0042] This disclosure also provides a cell line or primary cell culture derived from the genetically modified non-human animal or its offspring. The cell culture can be obtained by isolating cells from the non-human animal, or by using the constructs provided in this disclosure and cell cultures established using standard cell transfection techniques. Therefore, this disclosure, in another aspect, provides a non-human animal cell, tissue, or organ whose genome contains an exogenous polynucleotide sequence encoding a human or chimeric GPR75 protein. In some embodiments, the non-human animal cell does not include embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells (iPS cells), or germ cells.

[0043] This disclosure also provides genomes derived from the genetically modified non-human animals. In some embodiments, the non-human animal genome contains a foreign polynucleotide sequence encoding a human or chimeric GPR75 protein.

[0044] Methods for constructing genetically modified non-human animals The genetically modified nonhuman animals disclosed herein can be prepared using several genome editing techniques known in the art, including homologous recombination using embryonic stem cells, non-homologous end joining (NHEJ) technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, and homing endonuclease technology. In some embodiments, homologous recombination technology is preferred. These genome editing techniques are described in Yin et al., “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6 (2017):387-399, which is incorporated herein by reference.

[0045] In some embodiments, a polynucleotide sequence encoding a human or chimeric GPR75 protein is introduced into the endogenous genome of at least one cell of a non-human animal. In some embodiments, the introduction occurs in embryonic stem cells, germ cells, or blastocysts. In some embodiments, the introduction occurs in somatic cells (e.g., fibroblasts). The nucleus of the somatic cell into which the polynucleotide sequence is introduced can be inserted into an enucleated oocyte to produce a non-human animal.

[0046] In some embodiments, the introduction of the exogenous polynucleotide sequence into the endogenous genome of a non-human animal cell, tissue, or organ can be accomplished using a targeting vector. In some embodiments, the targeting vector comprises a 5' homologous arm, a human or chimeric GPR75 gene fragment, and a 3' homologous arm. In some embodiments, the targeting vector also comprises an auxiliary sequence. In some embodiments, the auxiliary sequence includes a stop codon, an insulator, a WPRE (WHP post-transcriptional response element), a loxP, and / or a polyA.

[0047] In some embodiments, homologous recombination can be used to insert a human or chimeric GPR75 polynucleotide sequence into the genome (e.g., an endogenous safe site) of a nonhuman animal cell, tissue, or organ. In some embodiments, a method for preparing the genetically modified nonhuman animal includes: (1) providing a plasmid containing a fragment of the human GPR75 gene, the plasmid flanked by 5' and 3' homologous arms, wherein the 5' and 3' homologous arms target an endogenous safe site; (2) modifying the genome of a fertilized egg or embryonic stem cell using the plasmid of step (1); (3) transplanting the fertilized egg obtained in step (2) into the oviduct of a pseudopregnant female mouse, or transplanting the embryonic stem cell obtained in step (2) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudopregnant female mouse to produce offspring mice functionally expressing humanized GPR75 protein; and (4) mating the offspring mice obtained in step (3) to obtain homozygous mice.

[0048] In some implementations, DNA double-strand breaks can be caused by cutting upstream and downstream of a target site (e.g., via zinc finger nucleases, TALEN, or CRISPR), and then homologous recombination can be used to insert human or chimeric GPR75 polynucleotide sequences into the genome of non-human animal cells, tissues, or organs.

[0049] For non-human animals from which suitable genetically modified embryonic stem cells (ES) are not readily available, alternative methods can be employed to construct non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent stem cells) and transferring the modified genome to suitable cells, such as oocytes, via nuclear transfer, and gestating the modified cells (e.g., modified oocytes) in a non-human animal under appropriate conditions to form an embryo. The aforementioned methods of construction are known in the art and are described in “A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2006, the entire contents of which are incorporated herein by reference.

[0050] This disclosure also provides a method for generating genetically modified non-human animals having two or more human or chimeric genes. In some embodiments, the method includes: (1) obtaining a non-human animal using the above-described construction method; and (2) mating the non-human animal obtained in step (1) with other genetically modified non-human animals, performing in vitro fertilization or direct gene editing, and screening to obtain a multi-gene-modified non-human animal. In some embodiments, the method includes obtaining fertilized eggs or embryonic stem cells from non-human animals with other gene modifications, and performing gene editing targeting GPR75 on the obtained fertilized eggs or embryonic stem cells to obtain a multi-gene-modified non-human animal.

[0051] Application of genetically modified non-human animals This disclosure also provides, in another aspect, an application of the aforementioned GPR75 gene modification. The applications include: (A) applications in product development involving GPR75-related immune processes in human cells; (B) applications as GPR75-related model systems in pharmacological, immunological, microbiological, and medical research; (C) applications involving the production and use of animal experimental disease models for GPR75-related etiological research and / or for the development of diagnostic and / or therapeutic strategies; (D) applications in the screening, efficacy testing, evaluation, verification, or assessment of human GPR75 signaling pathway modulators in vivo; and (E) applications in studying GPR75 gene function, investigating drugs and their efficacy targeting human GPR75, and studying drugs for GPR75-related tumors, inflammation, and immune-related diseases.

[0052] In some embodiments, this disclosure provides a method for determining the efficacy of a human GPR75-targeted therapeutic agent in treating a disease. In some embodiments, the method includes: (1) administering the human GPR75-targeted therapeutic agent to a non-human animal provided in this disclosure, wherein the non-human animal has the disease; and (2) evaluating the therapeutic effect of the therapeutic agent on the disease.

[0053] In some embodiments, the disease is cancer. The "tumor" includes, but is not limited to, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, 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. The leukemia is selected from acute 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 Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In some embodiments, the tumor is breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tissue tumor, oropharyngeal tumor, female reproductive system cancer, or meningioma.

[0054] In some embodiments, the method includes detecting the inhibitory effect of a therapeutic agent on cancer or tumor. In some embodiments, the detection includes determining the size and / or proliferation rate of tumor cells. In some embodiments, the detection method includes caliper measurement, flow cytometry, and / or in vivo animal imaging. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, including changes in food or water consumption.

[0055] In some embodiments, the disease is an immune disease. In some embodiments, the immune disease includes, but is 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, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, the immune disease is osteoarthritis, asthma, rheumatoid arthritis, or multiple sclerosis.

[0056] In some embodiments, the disease is various types of inflammation. In some embodiments, the inflammation includes both acute and chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, purulent inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, and specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma venereum, etc.). In some embodiments, the inflammation is psoriasis, inflammatory bowel disease, chronic obstructive pulmonary disease, or inflammatory bowel disease (IBD).

[0057] In some embodiments, the disease is a metabolic disease. In some embodiments, the metabolic disease includes metabolic disorders as well as hypermetabolism. Specifically, it includes, but is not limited to, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency and osteoporosis, type 1 diabetes, type 2 diabetes, hyperglycemia, impaired fasting glucose, impaired glucose tolerance, obesity, dyslipidemia, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome or metabolic syndrome, etc. In some embodiments, the metabolic disease is obesity.

[0058] In some embodiments, the human GPR75-targeting therapeutic agent is an antibody, oligonucleotide drug, or peptide drug. In some embodiments, the human GPR75-targeting therapeutic agent is an anti-human GPR75 antibody. In some embodiments, the GPR75-targeting therapeutic agent blocks or inhibits GPR75-mediated signaling pathways. In some embodiments, the GPR75-targeting therapeutic agents described herein can block interactions between GPR75 complexes, thereby inhibiting GPR75 signaling pathways.

[0059] On the other hand, the multi-gene-modified non-human animal models provided in this disclosure can be used to evaluate the effectiveness of combination therapies targeting two or more proteins, such as anti-GPR75 antibodies and adjunctive therapeutic agents for treating cancer or immune diseases (e.g., asthma or atopic dermatitis). In some embodiments, the method includes administering an anti-GPR75 antibody and an adjunctive therapeutic agent to an animal having a tumor or immune disease and determining the effect of the combination therapy on the immunotumor or immune disease. In some embodiments, the adjunctive therapeutic agent is an antibody that specifically binds to CD98HC, CD3E, CD3D, CD3G, KIT, KITLG, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and / or CTLA4.

[0060] In some embodiments, the combination therapy is used to treat various cancers described herein, such as breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, or other cancers. 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 other methods. Methods for treating cancer that can be used alone or in combination with the methods described herein include chemotherapy, such as camphor, doxorubicin, cisplatin, carboplatin, procarbazine, meloratadine, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorpromazine, bismuth subcitrate, nitrosourea, daktarin, daunorubicin, bleomycin, prilocytic erythromycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatinum, 5-fluorouracil, vincristine, vinblastine, and / or methotrexate.

[0061] This disclosure also provides a method for determining the toxicity of a GPR75-targeting therapeutic agent. The method includes administering an antibody to a non-human animal as described above and assessing changes in the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin. In some embodiments, the antibody may reduce red blood cells (RBCs), hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animal's body weight is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average body weight of animals not treated with the antibody).

[0062] In some embodiments, this disclosure provides a method for validating 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 the animals described herein and administering human CAR-T to the animals containing human tumor cells. The effectiveness of CAR-T therapy can be determined and evaluated. In some embodiments, the animals are selected from GPR75 gene-humanized non-human animals prepared by the methods described herein, or dual- or multiple-humanized non-human animals (or their progeny) produced by the methods described herein.

[0063] Example The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0064] In each of the following embodiments, the equipment and materials are obtained from the following companies: C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Experimental Animal Seed Center of the China National Institutes for Food and Drug Control.

[0065] Example 1: GPR75 gene-humanized mice To achieve the objectives of this invention, a nucleotide sequence encoding the human GPR75 protein can be introduced into the mouse endogenous GPR75 locus, enabling the mouse to express the human or humanized GPR75 protein. Specifically, using gene editing technology, approximately 13.34 kb of the sequence from upstream of the 5'UTR to downstream of the 3'UTR in the mouse is replaced with approximately 11.78 kb of the sequence containing the human GPR75 gene upstream of the 5'UTR to downstream of the 3'UTR, resulting in a humanized GPR75 locus and achieving the humanization of the mouse GPR75 gene.

[0066] To implement the targeting strategy of this invention, a targeting vector was constructed. The targeting vector contains upstream and downstream homologous arm sequences of the mouse GPR75 gene, as well as fragment A containing the human GPR75 fragment. Specifically, the upstream 5' homologous arm sequence is SEQ ID NO: 3, the downstream 3' homologous arm sequence is SEQ ID NO: 4, and the nucleotide sequence of the human GPR75 gene fragment is positions 53852551 to 53864328 of NCBI Gene Accession Number NC_000002.12. The linker between the upstream human GPR75 fragment sequence and the mouse fragment is designed as: 5'-AAGATTCTATAATCTCAAAAAGTTCC TTATGGTA TCTTCTGGGTTGAAATAATTCACAAATTG-3' (SEQ ID NO: 6), where the sequence " TTAT The last "T" in the sequence is the last nucleotide at the upstream junction of the mouse sequence and the human GPR75 sequence. GGTA The first "G" in the sequence is the first nucleotide of the human GPR75 sequence.

[0067] The targeting vector also includes an resistance gene for positive clone selection, namely the neomycin phosphotransferase coding sequence Neo. Two co-aligned site-specific recombination system Frt recombination sites are attached to both sides of the resistance gene, forming a Neo cassette. The 5' end of the Neo cassette is linked to the human GPR75 gene as follows: 5'-TTGGGAGGCTGAGGCAGGAGGATCACTTGAGCCCGGGAT TATGGAAG TTCCTATTCTCTAGAAAGTATAG-3' (SEQ ID NO: 7), where the sequence " TATG The "G" in the sequence refers to the last nucleotide of the human GPR75 gene fragment. GAAGThe first "G" in the sequence represents the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the mouse GPR75 gene is designed as: 5'-AAGTTCCTATTCTCTAGAAAGTATAGGAA CTTCCTCA TGGGGAGTGGCACTATTAGGAC-3' (SEQ ID NO: 8), where the sequence " CTTC The last "C" in the sequence "" is the last nucleotide of the Neo box, and the sequence "" CTCA The first "C" in the sequence is the first nucleotide of the mouse GPR75 gene. The mRNA sequence of the modified humanized mouse GPR75 is shown in SEQ ID NO: 5, and the protein sequence it expresses is shown in SEQ ID NO: 2.

[0068] Targeted vector construction can be performed using conventional methods, such as enzyme digestion and ligation. After preliminary validation via enzyme digestion, the constructed targeted vector is sent to a sequencing company for sequencing verification. The sequenced and validated targeted vector is then electroporated into embryonic stem cells of C57BL / 6 mice. Positive clone selection marker genes are used to screen the resulting cells, identifying the correct positive clones. These selected positive clones (black mice) are then introduced into isolated blastocysts (white mice) using techniques known in the art. The resulting chimeric blastocysts are transferred to a culture medium for brief culture before being transplanted into the oviducts of recipient mother 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. F2 generation homozygous mice are then obtained by mating F1 generation heterozygous mice. Alternatively, positive mice can be mated with Flp tool mice to remove the positive clone selection marker genes, followed by mating to obtain GPR75 gene-humanized homozygous mice.

[0069] In addition, gene editing can be performed using CRISPR / Cas9 technology. A targeting vector V2 was designed, containing upstream and downstream homologous arm sequences of the mouse GPR75 gene, as well as a human GPR75 fragment. The upstream 5' homologous arm sequence is SEQ ID NO: 9, the downstream 3' homologous arm sequence is SEQ ID NO: 10, and the nucleotide sequence of the human GPR75 gene fragment is positions 53852551 to 53864328 of NCBI gene accession number NC_000002.12. The downstream linker between the human GPR75 fragment and the mouse GPR75 fragment is designed as: 5'-GGGAGGCTGAGGCAGGAGGATCACTTGAGCCCGGGAT TATGCTCA TGGGGAGTGGCACTATTAGGACCTGT-3' (SEQ ID NO: 31), wherein the sequence " TATGThe "G" in the sequence "" represents the last nucleotide of the human GPR75 fragment sequence. CTCA The first "C" in the sequence is the first nucleotide at the downstream junction of the mouse sequence and the human GPR75 fragment. The mRNA sequence of the modified humanized GPR75 mouse is shown in SEQ ID NO: 5, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0070] Targeting vectors can be constructed using conventional methods, such as enzyme digestion and ligation, or direct synthesis. After initial validation via enzyme digestion, the constructed targeting vector is sent to a sequencing company for sequencing validation. The correctly sequenced targeting vector is then used in subsequent experiments.

[0071] The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 cleavage of the target gene. Therefore, efficient and specific target sequence selection and design are prerequisites for constructing sgRNA expression vectors. An sgRNA sequence recognizing the target site was designed and synthesized. An example sgRNA target sequence on the GPR75 gene is as follows: sgRNA1 target site (SEQ ID NO: 11): 5'-ACTTGATATTAGAGTTACCCAGG-3'; sgRNA2 target site (SEQ ID NO: 12): 5'-CAAGACACCATATTGAAACTTGG-3'; The activity of sgRNA was detected using a UCA kit to confirm that it could mediate high cleavage efficiency. Enzyme sites were added to its 5' end and complementary strand to obtain forward and reverse oligonucleotide sequences, as shown in Table 3. After annealing, the annealed products were ligated into the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain expression vectors pT7-GPR75-1 and pT7-GPR75-2.

[0072] Table 3 Sequence listings of sgRNA1 and sgRNA2 The pT7-sgRNA vector was synthesized by a plasmid synthesis company, containing a fragment DNA (SEQ ID NO: 21) containing the T7 promoter and sgRNA scaffold. This fragment was then sequentially digested with enzymes (EcoRI and BamHI) and ligated into a backbone vector (from Takara, catalog number 3299). Sequencing by a professional sequencing company confirmed the successful acquisition of the target plasmid. Pronuclear zygotes from mice, such as C57BL / 6 mice, were used. The in vitro transcription products of pT7-GPR75-1 and pT7-GPR75-2 plasmids (transcribed using an Ambion in vitro transcription kit according to the manufacturer's instructions), the targeting vector, and Cas9 mRNA were premixed and injected into the cytoplasm or nucleus of mouse zygote cytoplasm using a microinjection apparatus. Following the method described in "Experimental Manual of Mouse Embryo Manipulation (3rd Edition)" (Andras Nagy, Chemical Industry Press, 2006), fertilized eggs were microinjected. After injection, the fertilized eggs were transferred to a culture medium for short-term culture, and then transplanted into the oviduct of the recipient female mouse for development. The resulting mice (F0 generation) were then crossbred and self-crossed to expand the population and establish a stable humanized mouse strain with the GPR75 gene.

[0073] The genotype of F1 generation mouse somatic cells can be identified using PCR. The primers shown in the table below are used for detection. Example results are shown below. Figure 1 As shown, the six mice numbered F1-1 to F1-6 were positive mice.

[0074] Table 4 Primer sequences and recombinant fragment sizes for F1 generation genotype PCR detection The expression of GPR75 gene mRNA in humanized mice can be detected by RT-PCR. Specifically, one 7-week-old C57BL / 6 mouse (+ / +) and one 7-week-old male homozygous humanized GPR75 mouse (H / H) prepared in this study were selected. Brain tissue was collected after euthanasia by cervical dislocation, and RT-PCR was performed using the primer sequences shown in the table below. The detection results are as follows. Figure 2 As shown in the figure, only mouse GPR75 mRNA was detected in wild-type C57BL / 6 mice, and human GPR75 mRNA was not detected; only human GPR75 mRNA was detected in humanized homozygous mice of the GPR75 gene, and mouse GPR75 mRNA was not detected.

[0075] Table 5 RT-PCR primer sequences and target fragment sizes Example 2: Pharmacodynamic Model The humanized mice disclosed in this invention can be used to induce various human disease models, such as obesity models, which can be used to test the in vivo efficacy of human-specific antibodies. For example, GPR75 gene-humanized mice can be used to evaluate the efficacy, pharmacokinetics, and in vivo therapeutic efficacy of antagonists of the human-specific GPR75 signaling pathway in various disease models known in the art.

[0076] Taking the preparation of a disproportionately obese (DIO) model as an example, humanized mice with the GPR75 gene prepared in this invention can be used to induce an obesity model for 12 weeks according to a conventional high-fat diet. These mice are then divided into a control group and a treatment group. The treatment group is randomly assigned to receive a drug targeting human GPR75 (e.g., a GPR75-targeting nucleic acid drug). Depending on the drug, the drug can be administered via various routes such as intraventricular injection, subcutaneous injection, gavage, intraperitoneal injection, or tail vein injection before, during, or after modeling. The control group receives an equal volume of PBS solution. The in vivo efficacy of different human drugs can be evaluated using multiple indicators, including food intake, body weight, changes in body weight, blood levels of insulin, glucagon, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose tolerance curves, pancreatic immunohistochemical staining analysis, and fat percentage. Results showed that compared to the control group, the mice in the treatment group had reduced body weight, changes in body weight, and fat mass (data not shown). This indicates that humanized mice with the GPR75 gene can be used to evaluate the efficacy of human drugs targeting GPR75.

[0077] Example 3: Preparation of dual-gene or multi-gene humanized mice The humanized GPR75 mice obtained using this method can also be used to prepare multi-gene mouse models. For example, in Example 1 above, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modified with CD98HC, CD3E, CD3D, CD3G, KIT, KITLG, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. Alternatively, based on humanized GPR75 mice, mouse ES embryonic stem cells can be isolated and gene recombination targeting technology can be used to obtain dual-humanized or multi-gene mouse models. The homozygous or heterozygous GPR75 mice obtained by this method can also be mated with other gene-modified mice, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene mice with humanized GPR75 gene and other gene modifications. Furthermore, mating heterozygotes with each other can yield homozygotes with dual or multi-gene modifications.

[0078] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention. Furthermore, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. In addition, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, they should also be considered as the content disclosed by the present invention.

Claims

1. A method for producing a genetically modified nonhuman animal, comprising modifying the genome of the nonhuman animal to contain an exogenous polynucleotide sequence encoding a human or chimeric G protein-coupled receptor 75 (GPR75) protein.

2. The method according to claim 1, wherein the human or chimeric GPR75 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence (SEQ ID NO: 2) of the human GPR75 protein; preferably, wherein the exogenous polynucleotide sequence encodes the human GPR75 protein; preferably, wherein the amino acid sequence of the human GPR75 protein is SEQ ID NO:

2.

3. The method according to claim 1 or 2, wherein the exogenous polynucleotide sequence comprises all or part of exons 1 to 2 of the human GPR75 gene; preferably, the exogenous polynucleotide sequence further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human GPR75 gene; preferably, the exogenous polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene and at least 361 bp of continuous nucleotides downstream of the 3'UTR; preferably, the exogenous polynucleotide sequence comprises exons 1 to 2 of the human GPR75 gene and at least 361 bp of continuous nucleotides downstream of the 3'UTR; preferably, the exogenous polynucleotide sequence comprises exons 1 to 2 of the human GPR75 gene and at least 361 bp of continuous nucleotides downstream of the 3'UTR. The exogenous polynucleotide sequence comprises all exon 1 to exon 2, at least 361 bp of continuous nucleotides downstream of the 3'UTR and at least 4361 bp of continuous nucleotides upstream of the 5'UTR; preferably, the exogenous polynucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the polynucleotide sequence shown at positions 53852551-53864328 of the gene accession number NC_000002.12; preferably, the exogenous polynucleotide sequence is the polynucleotide sequence shown at positions 53852551-53864328 of the gene accession number NC_000002.

12.

4. The method according to any one of claims 1-3, wherein the exogenous polynucleotide sequence is regulated by an exogenous regulatory element; preferably, the exogenous regulatory element is a human GPR75 promoter, a 5'UTR, and / or a 3'UTR.

5. The method according to any one of claims 1-4, wherein the exogenous polynucleotide sequence replaces the endogenous GPR75 gene of the non-human animal; preferably, the endogenous GPR75 gene of the non-human animal comprises all or part of exons 1 to 2 of the mouse GPR75 gene; preferably, the endogenous GPR75 gene of the non-human animal further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the exon of the mouse GPR75 gene; preferably, the endogenous GPR75 gene of the non-human animal comprises all of exons 1 to 2 of the mouse GPR75 gene and at least 2661 bp of continuous nucleotides downstream of the 3'UTR; preferably, the endogenous GPR75 gene of the non-human animal comprises all of exons 1 to 2 of the mouse GPR75 gene, at least 2661 bp of continuous nucleotides downstream of the 3'UTR and at least 2360 bp of continuous nucleotides upstream of the 5'UTR.

6. The method according to any one of claims 1-5, wherein the genome of the non-human animal further comprises other exogenous polynucleotide sequences encoding human or chimeric proteins; preferably, wherein the human or chimeric protein is selected from at least one of CD98HC, CD3E, CD3D, CD3G, KIT, KITLG, TFR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

7. The method according to any one of claims 1-6, wherein the non-human animal is a mammal, preferably a monkey or a rodent, and more preferably a mouse or a rat.

8. A non-human animal cell, tissue, or organ, wherein the genome of the non-human animal cell, tissue, or organ contains an exogenous polynucleotide sequence encoding a human or chimeric GPR75 protein.

9. The non-human animal cell, tissue, or organ according to claim 8, wherein the human or chimeric GPR75 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence (SEQ ID NO: 2) of the human GPR75 protein; preferably, wherein the exogenous polynucleotide sequence encodes the human GPR75 protein; more preferably, wherein the amino acid sequence of the human GPR75 protein is SEQ ID NO:

2.

10. The non-human animal cell, tissue, or organ according to claim 8, wherein the exogenous polynucleotide sequence comprises all or part of exons 1 to 2 of the human GPR75 gene; preferably, the exogenous polynucleotide sequence further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human GPR75 gene; preferably, the exogenous polynucleotide sequence comprises all of exons 1 to 2 of the human GPR75 gene and at least 361 bp of continuous nucleotides downstream of the 3'UTR; preferably, the exogenous polynucleotide sequence comprises […]. The exogenous polynucleotide sequence comprises all exons 1 to 2, at least 361 bp of continuous nucleotides downstream of the 3'UTR, and at least 4361 bp of continuous nucleotides upstream of the 5'UTR; preferably, the exogenous polynucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the polynucleotide sequence shown at positions 53852551-53864328 of the gene accession number NC_000002.12; more preferably, the exogenous polynucleotide sequence is the polynucleotide sequence shown at positions 53852551-53864328 of the gene accession number NC_000002.

12.

11. The non-human animal cell, tissue, or organ according to claim 10, wherein the exogenous polynucleotide sequence is regulated by an exogenous regulatory element; preferably, the exogenous regulatory element is a human GPR75 promoter, a 5'UTR, and / or a 3'UTR.

12. A method for determining the efficacy of a human GPR75-targeted therapeutic agent in treating a disease, the method comprising: 1) Administering the targeted human GPR75 therapeutic agent to a non-human animal produced by the method according to any one of claims 1-7, wherein the non-human animal has the disease; and 2) Evaluate the therapeutic effect of the therapeutic agent on the disease.

13. The method according to claim 12, wherein the disease is cancer; preferably, the cancer is a solid tumor or a hematologic malignancy; more preferably, the solid tumor is colorectal cancer, bladder cancer, head and neck cancer, liver cancer or lung cancer; and even more preferably, the hematologic malignancy is chronic lymphocytic leukemia and acute myeloid leukemia.

14. The method of claim 13, wherein the non-human animal comprises tumor cells injected into the non-human animal; preferably, wherein the evaluation includes measuring the tumor volume in the non-human animal.

15. The method of claim 12, wherein the disease is an immune disease; preferably, the immune disease is an allergy, chronic urticaria, atopic dermatitis, osteoarthritis, asthma, rheumatoid arthritis, multiple sclerosis, psoriasis, chronic obstructive pulmonary disease, or inflammatory bowel disease (IBD).

16. The method of claim 12, wherein the disease is a metabolic disease; preferably, the metabolic disease is obesity.

17. The method according to any one of claims 12-16, wherein the human GPR75-targeting therapeutic agent is an antibody, an oligonucleotide drug, or a peptide drug; preferably, the human GPR75-targeting therapeutic agent is an anti-human GPR75 antibody.

18. The method of claim 12, further comprising administering a second therapeutic agent to the non-human animal; preferably, the non-human animal further comprises an exogenous polynucleotide sequence encoding human or chimeric PD-1, human or chimeric PD-L1 and / or human or chimeric CTLA4, and wherein the second therapeutic agent is an anti-human PD-1 antibody, an anti-human PD-L1 antibody or an anti-human CTLA4 antibody.

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