Non-human animal modified by CD300A gene

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) CD300A protein and methods of use thereof.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (eg, humanized) CD300A protein and methods of using the same. Background Art

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

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

[0004] The present application provides an animal model with human or chimeric CD300A protein. The animal model can express human or chimeric CD300A (e.g., humanized CD300A) protein. It can be used for the study of CD300A function, and can also be used for the screening and evaluation of CD300A signaling pathway regulators (e.g., anti-human CD300A antibodies, oligonucleotide drugs and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamics research, treatment of diseases (e.g., cancer, immune diseases or inflammation) and treatment of diseases at human CD300A target sites; the animal model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of CD300A and provides a platform for screening related drugs.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric CD300A molecule (CD300A) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CD300A protein is operably linked to an endogenous regulatory element (e.g., a promoter, 5'UTR and / or 3'UTR) of an endogenous CD300A locus of a non-human animal. In some embodiments, the chimeric CD300A protein is a humanized CD300A protein. In some embodiments, the chimeric CD300A protein comprises a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the chimeric CD300A protein is consistent with at least 50 to 299 consecutive amino acids of the human CD300A protein. In some embodiments, the amino acid sequence of the human or chimeric CD300A protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2, positions 1-196 or SEQ ID NO: 2, positions 1-201; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2, positions 1-196 or SEQ ID NO: 2, positions 1-201. In some embodiments, the amino acid sequence of the chimeric CD300A protein comprises SEQ ID NO: 7; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 7. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or a rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal does not express endogenous CD300A protein or expresses reduced levels of CD300A compared to wild-type animals. In some embodiments, one or more cells of the non-human animal express human or chimeric CD300A protein.

[0006] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing it. In some embodiments, at the endogenous CD300A locus of the non-human animal, the endogenous CD300A gene of the non-human animal is replaced with a nucleotide sequence comprising human CD300A. In some embodiments, the nucleotide sequence of human CD300A is operably linked to an endogenous regulatory element of the endogenous CD300A locus of the non-human animal. In some embodiments, the endogenous CD300A protein of the non-human animal is not expressed or the expression level is reduced compared with CD300A in wild-type animals. In some embodiments, the nucleotide sequence of human CD300A comprises all or part of a nucleotide sequence encoding a human CD300A extracellular region. In some embodiments, the nucleotide sequence of human CD300A comprises all or part of a nucleotide sequence encoding a human CD300A transmembrane region. In some embodiments, the nucleotide sequence of human CD300A comprises all or part of a nucleotide sequence encoding a human CD300A signal peptide. In some embodiments, the nucleotide sequence of human CD300A comprises all or part of a nucleotide sequence encoding a human CD300A cytoplasmic region. In some embodiments, the nucleotide sequence of human CD300A comprises a portion of exon 1 to a portion of exon 4 of the human CD300A gene. In some embodiments, the portion of exon 1 of the human CD300A gene comprises at least 5 bp of continuous nucleotides. In some embodiments, the portion of exon 4 of the human CD300A gene comprises at least 5 bp of continuous nucleotides. In some embodiments, the nucleotide sequence of human CD300A comprises SEQ ID NO: 5; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the replaced endogenous CD300A gene of a non-human animal comprises a portion of exon 1 to a portion of exon 4 of the endogenous CD300A gene of a non-human animal. In some embodiments, the portion of exon 1 of the endogenous CD300A gene of a non-human animal comprises at least 5 bp of continuous nucleotides. In some embodiments, the portion of exon 4 of the endogenous CD300A gene of a non-human animal comprises at least 5 bp of continuous nucleotides. In some embodiments, the modified CD300A gene in the non-human animal genome is homozygous or heterozygous for the endogenously replaced locus. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the non-human animal is a mouse.In some embodiments, the mRNA transcribed from the modified CD300A gene in the genome of the non-human animal comprises SEQ ID NO: 6; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins; preferably, the other human or chimeric proteins comprise at least one of ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0007] In one aspect, the present invention provides a non-human animal or a method for constructing the same. In some embodiments, the non-human animal comprises at least one cell encoding a nucleotide sequence of a human or chimeric CD300A protein. In some embodiments, the amino acid sequence of the human or chimeric CD300A protein is identical to at least 50 to 299, for example, at least 50, 100, 190, 196, 200, 201, 250, 290 or 299 consecutive amino acid sequences of a human CD300A protein. In some embodiments, the chimeric CD300A protein comprises at least 50 to 163, for example, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 or 163 consecutive amino acids of a human CD300A extracellular region. In some embodiments, the chimeric CD300A protein comprises at least 5 to 21, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids of the human CD300A transmembrane region. In some embodiments, the chimeric CD300A protein comprises at least 5 to 17, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 consecutive amino acids of the human CD300A signal peptide. In some embodiments, the nucleotide sequence encoding the human or chimeric CD300A protein is operably linked to an endogenous CD300A regulatory element of a non-human animal. In some embodiments, the nucleotide sequence encoding the human or chimeric CD300A protein can be integrated into an endogenous CD300A locus of a non-human animal. In some embodiments, the human or chimeric CD300A protein has at least one activity, such as endogenous CD300A activity of a non-human animal and / or human CD300A activity.

[0008] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal. In some embodiments, the construction method comprises replacing a nucleotide sequence encoding an endogenous CD300A region of a non-human animal with a nucleotide sequence encoding a corresponding region of human or chimeric CD300A at an endogenous CD300A locus of the non-human animal. In some embodiments, the endogenous CD300A protein of the non-human animal is not expressed or is expressed at a reduced level compared to CD300A in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CD300A comprises at least 20-18426 bp of human CD300A gene, such as 20, 50, 100, 200, 300, 400, 500, 588, 600, 700, 800, 900, 1000, 1500, 1600, 1638, 1700, 1800, 1900, 2000, 5000, 10000, 15000, 18000 or 18426 bp (continuous or non-continuous) of the same nucleotide sequence. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CD300A comprises part of exon 1 to part of exon 4 of human CD300A gene. In some embodiments, the portion of human CD300A gene exon 1 comprises at least 5-110 bp, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 110 bp of continuous nucleotides. In some embodiments, the portion of human CD300A gene exon 4 comprises at least 5-95 bp, such as 5, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90 or 95 bp of continuous nucleotides. In some embodiments, the amino acid sequence of the corresponding region of human or chimeric CD300A comprises SEQ ID NO: 2 or amino acids 1-196 of SEQ ID NO: 2 or amino acids 1-201 of SEQ ID NO: 2; or comprises amino acids that are at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 2 or amino acids 1-196 of SEQ ID NO: 2 or amino acids 1-201 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the corresponding region of chimeric CD300A comprises SEQ ID NO: 7; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CD300A comprises SEQ ID NO:5; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:5.In some embodiments, the nucleotide sequence encoding the endogenous CD300A region of the non-human animal that is replaced includes a nucleotide sequence encoding the endogenous CD300A extracellular region, an endogenous transmembrane region and / or an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the endogenous CD300A region of the non-human animal that is replaced includes a portion of exon 1 to a portion of exon 4 of the endogenous CD300A gene of the non-human animal. In some embodiments, the portion of exon 1 of the endogenous CD300A gene of the non-human animal that is replaced includes at least 5-394bp, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 390 or 394bp of continuous nucleotide sequence. In some embodiments, the portion of exon 4 of the endogenous CD300A gene of a non-human animal that is replaced comprises at least 5-98 bp, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CD300A is operably linked to an endogenous CD300A regulatory element of a non-human animal, such as a promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or a rat).

[0009] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric CD300A, the construction method comprising replacing a nucleotide sequence encoding an endogenous CD300A region with a nucleotide sequence encoding a corresponding region of human CD300A at an endogenous CD300A locus of a non-human animal, producing a genetically modified non-human animal cell, the non-human animal cell expressing a human or chimeric CD300A protein. In some embodiments, the chimeric CD300A protein comprises a human or humanized extracellular region, a human or humanized transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the extracellular region comprises a signal peptide. In some embodiments, the extracellular region does not comprise a signal peptide. In some embodiments, the chimeric CD300A protein comprises or does not comprise a signal peptide. In some embodiments, the signal peptide is preferably endogenous or humanized to a human or non-human animal. In some embodiments, the chimeric CD300A protein comprises a human signal peptide, a human extracellular region, a humanized transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the chimeric CD300A protein comprises a human extracellular region, a humanized transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric CD300A protein comprises a human signal peptide, a human extracellular region, a human transmembrane region and an endogenous cytoplasmic region. In some embodiments, the chimeric CD300A protein comprises a human extracellular region, a human transmembrane region and an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD300A comprises a portion of exon 1 to a portion of exon 4 of the human CD300A gene. Preferably, the nucleotide sequence encoding the corresponding region of human CD300A comprises at least 100-600, 600-1000, 1000-10000 or 10000-15000 nucleotides of a portion of exon 1 to a portion of exon 4 of the human CD300A gene. In some embodiments, the amino acid sequence of the corresponding region of human CD300A comprises SEQ ID NO: 2 or SEQ ID NO: 2 amino acids 1-196 or SEQ ID NO: 2 amino acids 1-201; or comprises amino acids that are at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 2 or SEQ ID NO: 2 amino acids 1-196 or SEQ ID NO: 2 amino acids 1-201. In some embodiments, the nucleotide sequence encoding the endogenous CD300A region to be replaced comprises a portion of exon 1 to a portion of exon 4 of the endogenous CD300A gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD300A is operably linked to a regulatory element of an endogenous CD300A of a non-human animal, such as a promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal is a mouse.

[0010] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent (e.g., an anti-CD300A antibody; or a drug targeting the CD300A signaling pathway) in treating a disease. In some embodiments, the method comprises: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal suffers from a disease; 2) determining the inhibitory effect of the therapeutic agent on the disease. In some embodiments, the therapeutic agent comprises an anti-CD300A antibody or a drug targeting the CD300A signaling pathway; further preferably, an additional therapeutic agent is further included, wherein the additional therapeutic agent comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease comprises one or more of cancer, an immune disease, or inflammation. In some embodiments, the cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the non-human animal. In some embodiments, the cancer comprises injecting one or more cancer cells into the animal. In some embodiments, the cancer is a solid tumor and / or a hematological tumor. In some embodiments, the cancer comprises head and neck cancer, liver cancer, or lung cancer. In some embodiments, the immune disease is urticaria, asthma, atopic dermatitis, allergy, rheumatoid arthritis, psoriasis or multiple sclerosis. In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0011] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent (e.g., an anti-CD300A antibody; or a drug targeting the CD300A signaling pathway). In some embodiments, the method comprises: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the determination of the effect of the therapeutic agent on the non-human animal comprises measuring the weight of the non-human animal or performing a blood test. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit, or hemoglobin content.

[0012] In one aspect, the present invention provides a humanized CD300A protein, in some embodiments, the humanized CD300A protein comprises an extracellular region of a human CD300A protein. In some embodiments, the extracellular region comprises a signal peptide. In some embodiments, the extracellular region does not comprise a signal peptide. In some embodiments, the humanized CD300A protein comprises a signal peptide and an extracellular region of a human CD300A protein. In some embodiments, the humanized CD300A protein further comprises all or part of a transmembrane region of a human CD300A protein. In some embodiments, the humanized CD300A protein comprises a cytoplasmic region of an endogenous CD300A protein of a non-human animal. In some embodiments, the amino acid sequence of the humanized CD300A protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2, positions 1-196 or SEQ ID NO: 2, positions 1-201; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2, positions 1-196 or SEQ ID NO: 2, positions 1-201. In some embodiments, the humanized CD300A protein comprises SEQ ID NO: 7 or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 7.

[0013] In one aspect, the present invention provides a humanized CD300A gene. In some embodiments, the humanized CD300A gene encodes the humanized CD300A protein. In some embodiments, the humanized CD300A gene comprises a portion of exon 1 to a portion of exon 4 of the human CD300A gene. In some embodiments, the humanized CD300A gene comprises a nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 or 33; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 or 33.

[0014] In one aspect, the present invention provides a cell, tissue or organ. In some embodiments, the cell, tissue or organ expresses the above-mentioned humanized CD300A protein. In some embodiments, the genome of the cell, tissue or organ comprises the above-mentioned humanized CD300A gene.

[0015] In one aspect, the present invention provides an animal model. In some embodiments, the animal model expresses the humanized CD300A protein. In some embodiments, the genome of the animal model comprises the humanized CD300A gene.

[0016] In one aspect, the present invention provides an application of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the non-human animal cell obtained by the above-mentioned construction method, the above-mentioned humanized CD300A protein, the above-mentioned humanized CD300A gene, the above-mentioned cell, tissue or organ or the above-mentioned animal model, the application comprising: A) application in the development of products involving CD300A-related immune processes in human cells; B) application as a model system related to CD300A in pharmacology, immunology, microbiology and medical research; C) application involving the production and use of animal experimental disease models for etiology research related to CD300A and / or for the development of diagnostic strategies and / or for the development of treatment strategies; D) application in the screening, efficacy detection, efficacy evaluation, verification or evaluation of human CD300A signaling pathway regulators in vivo; or, E) application in studying CD300A gene function, studying drugs and efficacy targeting human CD300A target sites, and studying CD300A-related tumor, inflammation, and immune disease drugs.

[0017] The term "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a gene in a narrow sense, which can be a gene or a part of a gene. For example, the "CD300A locus" refers to a DNA fragment selected from exons 1-7 of the CD300A gene. In some embodiments, the replaced endogenous CD300A locus of a non-human animal can be a DNA fragment selected from exons 1-7 of the endogenous CD300A gene of a non-human animal.

[0018] The term "exon XX to exon XXX" or "exon XX-XXX" of the present invention refers to exons and introns in between, for example, exon 1 to exon 7 includes the entire nucleotide sequence of exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and exon 7.

[0019] The term "portion of exon XX to portion of exon XXX" of the present invention refers to the portion from portion of one exon to portion of another exon and introns in between, for example, the portion from portion of exon 1 to portion of exon 4 includes the nucleotide sequence of portion of exon 1, all of intron 1, all of exon 2, all of intron 2, all of exon 3, all of intron 3 and part of exon 4.

[0020] The term "intron xx" in the present invention refers to an intron between two exons, for example, intron 1 refers to an intron between exon 1 and exon 2.

[0021] The term "comprising" or "including" in the present invention is an open-ended writing method, which contains the specified components or steps described, as well as other specified components or steps that will not be substantially affected. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0022] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "B" and "A and B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0023] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. This application describes methods and materials for use in the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are exemplary and non-limiting only. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0024] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description.

[0025] CD300A

[0026] In the human genome, the CD300A gene (NCBI Gene ID: 11314, UniProt ID: Q9UGN4-1, located at positions 74466373 to 74484798 of chromosome 17 NC_000017.11) contains 7 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and exon 7. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_007261.4 and its encoded protein NP_009192.2 (SEQ ID NO: 2) is shown in Table 1:

[0027] Table 1

[0028]

[0029] In the mouse genome, the CD300A gene (NCBI Gene ID: 217303, UniProt ID: Q6SJQ0-1, located at positions 114780006 to 114792772 of chromosome 11 NC_000077.7) contains 7 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and exon 7. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_170758.4 and its encoded protein NP_739564.1 (SEQ ID NO: 1) is shown in Table 2:

[0030] Table 2

[0031]

[0032] CD300A genes, proteins and gene loci of other species are also known in the art, for example, Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog) and Sus scrofa (pig), and the relevant information of these genes (such as intron sequence, exon sequence and amino acid sequence) can be found in NCBI, the entire contents of which are incorporated into this application by reference.

[0033] In order to determine the percent identity of two amino acid sequences or two nucleotide sequences, the sequences are compared for the purpose of optimal comparison (e.g., for optimal comparison, gaps can be introduced in one or both of the first and second amino acid or nucleotide sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to achieve the optimal comparison of the two sequences, taking into account the number of gaps and the length of each gap. For example, the comparison of the sequences and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0034] The percentage (homology percentage) of conservative residues with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0035] Carrier

[0036] The present invention provides a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of the switch region to be changed (5' homology arm or 5'

[0037] arm), which is selected from the genomic DNA of the CD300A gene and has a length of 100 to 10,000 bp nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the switch region to be changed (3' homology arm or 3' arm), which is selected from the genomic DNA of the CD300A gene and has a length of 100 to 10,000 bp nucleotides.

[0038] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% identity with NCBI Accession No. NC_000077.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% identity with NCBI Accession No. NC_000077.7.

[0039] In some embodiments, a) a DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 114776853 to 114781190 with NCBI Accession No. NC_000077.7; c) a DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 114788603 to 114792385 with NCBI Accession No. NC_000077.7.

[0040] In some embodiments, a) a DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 114779733 to 114781190 with NCBI Accession No. NC_000077.7; c) a DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 114788603 to 114790138 with NCBI Accession No. NC_000077.7.

[0041] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb or 20 kb.

[0042] In some embodiments, the switch region to be altered is located in exon 1 to exon 7 of the endogenous CD300A gene of a non-human animal, preferably in the region from exon 1 to exon 4.

[0043] In some embodiments, the 5' homology arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 3 or 32. In some embodiments, the 3' homology arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 4 or 33.

[0044] In some embodiments, the donor sequence in the targeting vector comprises a human sequence (eg, nucleotide sequence 74466704 to 74477490 of NCBI accession number NC_000017.11, SEQ ID NO: 5).

[0045] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes), such as resistance genes for positive clone screening or coding genes for negative screening markers. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo, and preferably, the targeting vector further comprises two Frt recombination sites arranged in the same direction on both sides of the marker gene. In some embodiments, the coding gene for the negative screening marker is a coding gene for diphtheria toxin A subunit (DTA).

[0046] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the CD300A gene, and the sgRNA is unique at the target site of the switch region to be changed and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG-3' or 5'-CCN-N(20)-3'.

[0047] In some embodiments, the target site of the sgRNA in the endogenous CD300A gene of the non-human animal is located on exon 1 to exon 7. In some embodiments, the target site of the sgRNA in the endogenous CD300A gene of the non-human animal is located on exon 1 and / or exon 4.

[0048] In some embodiments, the target site (target sequence) is shown as SEQ ID NO: 22 and / or 23. Therefore, the present invention provides sgRNA sequences for constructing a genetically modified animal model. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 24 and 26. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 25 and 27. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 28 and 30. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 29 and 31.

[0049] In some embodiments, the present invention relates to a plasmid construct comprising an sgRNA sequence (ie, an sgRNA vector, such as pT7-sgRNA) and / or a cell comprising the plasmid construct.

[0050] In some embodiments, the present invention also provides a cell comprising the above-mentioned targeting vector and / or sgRNA vector.

[0051] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned vectors and one or more in vitro transcripts of the constructs described in the present application. In some embodiments, the non-human mammalian cell further comprises Cas9 mRNA or its in vitro transcript.

[0052] In some embodiments, the gene in the non-human mammal cell is heterozygous. In some embodiments, the gene in the non-human mammal cell is homozygous.

[0053] In some embodiments, the non-human mammal cell is a mouse cell. In some embodiments, the non-human mammal cell is a fertilized egg cell. In some embodiments, the non-human mammal cell is an embryonic stem cell. In some embodiments, the non-human mammal cell can be any cell capable of expressing CD300A.

[0054] Genetically modified non-human animals

[0055] The "genetically modified non-human animal" or "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome of the non-human animal has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (such as T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal is provided, the non-human animal comprises a modified endogenous CD300A locus, the endogenous CD300A locus comprises an exogenous sequence (such as a human sequence), for example, one or more non-human sequences are replaced with one or more human sequences, or one or more human sequences and / or human / non-human chimeric sequences are inserted into one or more non-human sequences. Non-human animals are often able to pass genetic modifications to their offspring through germline transmission.

[0056] The "chimeric (x) gene" or "chimeric (x) nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least a portion of the sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, a chimeric (x) gene or chimeric (x) nucleic acid has at least a portion of the sequence having two or more different species sources, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, a chimeric (x) gene or chimeric (x) nucleic acid refers to a humanized (x) gene or a humanized (x) nucleic acid.

[0057] The "chimeric (x) protein" or "chimeric (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more parts of the polypeptide or protein are from different species, or at least a portion of the sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of a chimeric (x) protein or a chimeric (x) polypeptide has two or more different species sources, for example, the same (or homologous) proteins of different species. In some embodiments, a chimeric (x) protein or a chimeric (x) polypeptide refers to a humanized (x) protein or a humanized (x) polypeptide.

[0058] The "humanized (x) protein" or "humanized (x) polypeptide" of the present invention refers to a protein or polypeptide. In some embodiments, at least a portion of the protein or polypeptide is derived from a human (x) protein or a human (x) polypeptide. In some implementations, at least a portion of the protein or polypeptide is derived from an endogenous (x) protein or an endogenous (x) polypeptide of a non-human animal. In some embodiments, a humanized (x) protein or a humanized (x) polypeptide refers to a human (x) protein or a human (x) polypeptide.

[0059] The "humanized (x) nucleic acid" or "humanized (x) gene" of the present invention refers to a gene or a nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human (x) gene or a human (x) nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from an endogenous (x) gene of a non-human animal or an endogenous (x) nucleic acid of a non-human animal. In some embodiments, the gene or nucleic acid is entirely derived from a human (x) gene or a human (x) nucleic acid. In some embodiments, a humanized (x) nucleic acid or a humanized (x) gene refers to a humanized exon, which may be a human exon or a chimeric exon.

[0060] In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid is a humanized CD300A gene or a humanized CD300A nucleic acid. In some embodiments, at least a portion of the humanized CD300A gene or the humanized CD300A nucleic acid is derived from a human CD300A gene, and at least a portion of the humanized CD300A gene or the humanized CD300A nucleic acid is derived from an endogenous CD300A gene of a non-human animal. In some embodiments, the humanized CD300A gene or the humanized CD300A nucleic acid comprises a sequence encoding a CD300A protein. The encoded CD300A protein has at least one activity, such as the activity of a human CD300A protein or a non-human animal CD300A protein.

[0061] In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide is a humanized CD300A protein or a humanized CD300A polypeptide. In some embodiments, at least one or more parts of the amino acid sequence of the humanized CD300A protein or humanized CD300A polypeptide are from human CD300A protein. In some embodiments, at least one or more parts of the amino acid sequence of the humanized CD300A protein or humanized CD300A polypeptide are from endogenous CD300A protein of a non-human animal. The humanized CD300A protein or humanized CD300A polypeptide is functional, or has at least one activity, such as the activity of a human CD300A protein or a non-human animal CD300A protein.

[0062] In some embodiments, the humanized CD300A protein comprises an extracellular region, a transmembrane region and / or a cytoplasmic region, preferably, the humanized CD300A protein comprises or does not comprise a signal peptide. In some embodiments, the extracellular region of the humanized CD300A protein is human or humanized. In some embodiments, the signal peptide of the humanized CD300A protein is human or humanized. In some embodiments, the cytoplasmic region of the humanized CD300A protein is endogenous to a human or humanized or non-human animal. In some embodiments, the transmembrane region of the humanized CD300A protein is human or humanized.

[0063] In some embodiments, the genetically modified non-human animal comprises a modification of an endogenous CD300A gene locus in the non-human animal. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature CD300A protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of a mature CD300A protein). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein are provided in the present invention, in some embodiments, the genetically modified non-human animal comprises a modification of an endogenous CD300A gene locus in a non-human animal.

[0064] In some embodiments, the genetically modified non-human animal can express human CD300A and / or chimeric (e.g., humanized) CD300A at the endogenous locus of the non-human animal. In some embodiments, the endogenous CD300A gene of the non-human animal has been replaced or inserted with a human CD300A gene and / or a nucleotide sequence encoding a region of the human CD300A sequence or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% identical to the human CD300A sequence. In some embodiments, the endogenous CD300A locus of the non-human animal is modified by a nucleotide sequence that contains all or part of the nucleotide sequence encoding the mature human CD300A protein.

[0065] In some embodiments, the genetically modified non-human animal can express human CD300A and / or chimeric CD300A (e.g., humanized CD300A) under the control of regulatory elements (e.g., non-human animal endogenous regulatory elements, including promoters, 5'UTRs, and / or 3'UTRs). Insertion or substitution at the endogenous locus of the non-human animal provides a non-human animal that expresses human CD300A or chimeric CD300A (e.g., humanized CD300A) in suitable cells and in a manner that does not cause potential pathologies observed in some other transgenic non-human animals known in the art. Human CD300A or chimeric CD300A (e.g., humanized CD300A) expressed in the non-human animal can maintain one or more functions of wild-type animals or human CD300A in the non-human animal. In some embodiments, the genetically modified non-human animal does not express endogenous CD300A. In some embodiments, the expression level of endogenous CD300A in the genetically modified non-human animal is reduced compared to the expression level of CD300A in wild-type animals. As used in the present invention, the term "endogenous CD300A" refers to the CD300A protein expressed by the endogenous CD300A nucleotide sequence of a non-human animal (eg, mouse) before any genetic modification.

[0066] In some embodiments, the genetically modified non-human animal may have one or more cells expressing human or chimeric CD300A (e.g., humanized CD300A), wherein the human or chimeric CD300A has an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the human or chimeric CD300A comprises or does not comprise a signal peptide. In many cases, the sequences of human CD300A and non-human animal endogenous CD300A (e.g., mouse CD300A) are different, so antibodies that bind to human CD300A or drugs that target human CD300A signaling pathways do not necessarily have the same affinity as non-human animal endogenous CD300A or have the same effect on non-human animal endogenous CD300A. Therefore, non-human animals having a human or humanized extracellular region (preferably also comprising a human or humanized transmembrane region) can be used to better evaluate the effects of therapeutic agents (e.g., anti-CD300A antibodies; or drugs that target CD300A signaling pathways) in animal models.

[0067] In some embodiments, the entire cytoplasmic region of the humanized CD300A is derived from the endogenous CD300A sequence of a non-human animal. In some embodiments, the entire signal peptide and the entire extracellular region of the humanized CD300A are derived from the human CD300A sequence. In some embodiments, the entire transmembrane region of the humanized CD300A is derived from the human CD300A sequence. In some embodiments, the transmembrane region of the humanized CD300A is humanized and comprises the human CD300A sequence and the endogenous CD300A sequence of a non-human animal.

[0068] In some embodiments, the humanized CD300A gene comprises the 5'UTR of the human CD300A gene. In some embodiments, the humanized CD300A gene comprises the 5'UTR of the endogenous CD300A gene of a non-human animal (eg, mouse). In some embodiments, the humanized CD300A gene comprises the 3'UTR of the human CD300A gene. In some embodiments, the humanized CD300A gene comprises the 3'UTR of the endogenous CD300A gene of a non-human animal (eg, mouse). In appropriate cases, it can be reasonably assumed that, based on the similarity of the endogenous CD300A gene of the non-human animal and the human CD300A gene sequence, they appear to be similarly regulated. As shown in the present application, the humanized CD300A comprising an insertion or replacement in the endogenous CD300A locus of a non-human animal retains the endogenous regulatory elements of the non-human animal but comprises the humanization of the CD300A coding sequence, and does not exhibit pathological phenomena. Both genetically modified non-human animals of humanized CD300A heterozygous or homozygous are normal.

[0069] Genetically modified non-human animals can be various animals, for example, mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals that are not easy to obtain suitable genetically modified embryonic stem cells (ES), other methods are used to construct non-human animals containing genetic modifications. Such methods include, for example, modifying the non-ES cell genome (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transplantation to transfer the modified genome to suitable cells, such as oocytes, and incubating the modified cells (e.g., modified oocytes) in non-human animals under appropriate conditions to form embryos. The above-mentioned construction methods are known in the art and are described in "A. Nagy, et al.,

[0070] “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.

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

[0072] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, an immunodeficient monkey, etc. In some embodiments, the non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid strain (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a 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 strain.

[0073] In some embodiments, the non-human animal is a rat. The rat can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a hybrid species of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0074] The non-human animal may have one or more other genetic modifications and / or other modifications that are suitable for the specific purpose of preparing a humanized non-human animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) may have one or more modifications that damage, inactivate, or destroy all or part of the immune system of the non-human animal. Damage, inactivation, or destruction of the immune system of a non-human animal may include, for example, chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and / or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc null Mice (Ito, M. et al., NOD / SCID / γc null mouse:an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can be optionally irradiated or otherwise treated to destroy one or more immune cell types. Therefore, in various embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of the endogenous CD300A locus of a non-human animal, and also includes damage, inactivation or partial destruction of the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the mouse modification type is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null Mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scid IL-2Rγ null Mouse, NOD Rag 1 - / - IL - / - (NRG) mouse, Rag2 - / - IL - / -(RG) mice and modifications thereof. These transgenic animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mice may include replacing all or part of the mouse endogenous mature CD300A coding sequence with all or part of the human mature CD300A coding sequence, respectively.

[0075] The present invention further relates to a non-human mammal or its offspring produced by the above method. In some embodiments, the genome of the non-human mammal or its offspring comprises human genes.

[0076] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0077] In some embodiments, the non-human mammal expresses a protein encoded by a humanized CD300A gene.

[0078] In addition, the present invention also provides a non-human mammal model carrying a tumor, wherein the non-human mammal model is obtained by the method described in the present application. In some embodiments, the non-human mammal is a rodent (eg, mouse).

[0079] The present invention also provides a cell or cell line derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a primary cell culture, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its offspring. When it carries a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring, or a non-human mammal carrying a tumor.

[0080] The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal comprises DNA of human or humanized CD300A.

[0081] 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 CD300A protein is provided. In some embodiments, a cell, tissue or organ specifically expressing a human or humanized CD300A protein is provided.

[0082] In some embodiments, the expression of the human or humanized CD300A protein expressed by the genetically modified non-human animal or the portion of the human CD300A gene or the humanized CD300A gene contained in the genome of the genetically modified non-human animal is controllable, such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0083] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferred non-human mammals are mammals (eg, rodents). In some embodiments, the non-human mammal is a mouse.

[0084] The non-human mammals described above are subjected to genetic, molecular and behavioral analyses. The present invention provides offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0085] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny. For example, a cell culture-based model can be prepared by the following method. The cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. The integration of a genetic construct comprising a DNA sequence encoding a human CD300A protein can be detected by a variety of methods.

[0086] 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 achieve 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 assays (RNAdot). Immunohistochemical staining, flow cytometry, and Western blots can also be used to detect the presence of human or humanized CD300A protein.

[0087] In some embodiments, the genetically modified non-human animals described herein (eg, homozygous or heterozygous mice with humanized CD300A gene) can express human or humanized CD300A in one or more cells.

[0088] Methods for constructing genetically modified non-human animals

[0089] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting technology using embryonic stem cells, homologous recombination technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR / Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in Yin et al. "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0090] In some embodiments, the nucleotide sequence encoding the endogenous CD300A region of the non-human animal in the endogenous genome of at least one cell of the non-human animal is replaced by a nucleotide sequence encoding the corresponding region of human CD300A to form a humanized CD300A gene, and / or the obtained non-human animal expresses human or humanized CD300A protein. In some embodiments, the expression level of the endogenous CD300A protein of the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0091] Preferably, the non-human animal further comprises other gene modifications, more preferably, the other genes include at least one of ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0092] Preferably, the nucleotide sequence of the corresponding region of human CD300A, the humanized CD300A gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) gene locus.

[0093] Preferably, the nucleotide sequence of the corresponding region of human CD300A, the humanized CD300A gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) gene locus.

[0094] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0095] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0096] Therefore, the present invention provides a method for constructing a non-human animal with humanized CD300A gene, wherein the non-human animal expresses human or humanized CD300A protein in vivo, and / or the genome of the non-human animal contains part of the human CD300A gene or the humanized CD300A gene.

[0097] In some embodiments, the construction method allows the non-human animal to express human or humanized CD300A protein in vivo, and / or, the construction method allows the genome of the non-human animal to contain a portion of the human CD300A gene or a humanized CD300A gene.

[0098] In some embodiments, the method of modifying the CD300A locus of a non-human animal to express a human / non-human animal chimeric CD300A polypeptide may include replacing all or part of a nucleotide sequence encoding endogenous CD300A of the non-human animal at the endogenous CD300A locus of the non-human animal with all or part of a nucleotide sequence encoding human CD300A, thereby generating a sequence encoding a chimeric human / non-human animal chimeric CD300A. In some embodiments, the method may include inserting a nucleotide sequence encoding a chimeric human / non-human animal chimeric CD300A at the endogenous CD300A locus of the non-human animal, thereby generating a non-human animal expressing a human / non-human animal chimeric CD300A.

[0099] The present invention provides a targeting vector, which comprises a 5' homology arm, a human or humanized CD300A gene fragment and a 3' homology arm. The process involves using homologous recombination technology to replace the corresponding sequence of endogenous CD300A of non-human animals with the human or humanized CD300A gene fragment. In some embodiments, the cutting upstream and downstream of the target site (for example, by zinc finger nuclease, TALEN or CRISPR) can cause DNA double-strand breaks, and homologous recombination is used to replace the corresponding sequence of endogenous CD300A of non-human animals with the human or humanized CD300A gene fragment.

[0100] Therefore, in some embodiments, the method for preparing a genetically modified humanized non-human animal comprises replacing a nucleotide sequence encoding endogenous CD300A of the non-human animal with a nucleotide sequence of a corresponding region of human CD300A at the endogenous CD300A locus (or site) of the non-human animal.

[0101] The present invention also provides a method for establishing a CD300A gene humanized animal model, comprising the following steps:

[0102] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0103] (b) culturing the cells, preferably culturing the cells in a liquid culture medium;

[0104] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

[0105] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0106] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0107] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0108] 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.

[0109] The fertilized egg can be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0110] In some embodiments, the method for preparing a genetically modified animal includes modifying the coding frame of the CD300A gene of a non-human animal, for example, by replacing the nucleic acid sequence encoding the endogenous CD300A region of the non-human animal (e.g., genomic DNA sequence, CDS sequence or cDNA sequence) with a nucleotide sequence encoding the corresponding region of human CD300A under the control of the endogenous regulatory element of the non-human animal. For example, one or more functional region sequences of the CD300A gene of the non-human animal can be knocked out or inserted into a sequence so that the endogenous CD300A 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 CD300A gene can be all or part of the nucleotide sequence of exon 1 to exon 7 of the CD300A gene of the non-human animal, preferably part of exon 1 to part of exon 4.

[0111] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or humanized CD300A protein after the endogenous regulatory element of the CD300A gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the CD300A gene humanized animal model can express the human or humanized CD300A protein in vivo, but does not express the CD300A protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0112] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0113] (1) providing a plasmid comprising a human CD300A gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target endogenous CD300A of a non-human animal;

[0114] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous CD300A gene of a non-human animal;

[0115] (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;

[0116] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express the humanized CD300A protein. Preferably, the method further comprises:

[0117] (5) The offspring mice obtained in step (4) are mated to obtain homozygous mice.

[0118] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal targeting site.

[0119] In some embodiments, the sequence encoding the humanized CD300A protein is operably linked to endogenous regulatory elements at the endogenous CD300A locus of the non-human animal.

[0120] In some embodiments, the genetically modified non-human animal does not express endogenous CD300A protein.

[0121] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0122] (1) providing a plasmid comprising a human or chimeric CD300A gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target endogenous CD300A of a non-human animal;

[0123] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous CD300A gene of a non-human animal;

[0124] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric CD300A gene fragment into the genome.

[0125] In some embodiments, all or part of the nucleotide sequence encoding the endogenous CD300A protein in the genome of the non-human animal is deleted. In some embodiments, all or part of exon 1 to exon 7 of the endogenous CD300A gene of the non-human animal is deleted. In some embodiments, part of exon 1 to part of exon 4 of the endogenous CD300A gene of the non-human animal is deleted. In some embodiments, the deleted part also includes intron 1, intron 2 and / or intron 3 of the endogenous CD300A gene of the non-human animal.

[0126] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the endogenous CD300A protein in the genome of a non-human animal with the nucleotide sequence encoding the human CD300A protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or ... In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or SEQ ID NO: 1 positions 1-201 or SEQ ID NO: 1 positions 1-206 in the genome of a non-human animal with a nucleotide sequence encoding a human or humanized CD300A protein or a humanized CD300A gene.

[0127] Use of genetically modified non-human animals

[0128] 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 locus of the non-human animal and under the control of endogenous regulatory elements (e.g., promoter, 5'UTR and / or 3'UTR) can produce a non-human animal with qualities and characteristics that may be significantly different from typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or destroyed, and the full human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; the expression of human proteins is measured by transcription of human gene and / or protein assays and / or functional assays. In human transgenes, the upstream and / or downstream of the human sequence provide suitable support for the expression and / or regulation of the transgene.

[0129] In some cases, transgenes with human regulatory elements are expressed in a non-physiologically or otherwise unsatisfactory manner, and may actually be harmful to the non-human animal. The present invention demonstrates that humanized non-human animals are produced by replacing or inserting human sequences at endogenous gene loci in non-human animals under the control of endogenous regulatory elements in the non-human animal, providing physiologically appropriate expression patterns and levels that are meaningful and appropriate in the context of the physiology of the humanized non-human animal with respect to the physiology of the replaced gene.

[0130] Genetically modified non-human animals that express human or humanized CD300A protein, e.g., in a physiologically appropriate manner, provide a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0131] The present invention also provides a use of the CD300A gene-modified non-human animal and a non-human animal obtained by any of the above construction methods.

[0132] In some embodiments, the application comprises:

[0133] A) Application in product development involving CD300A-related immune processes in human cells;

[0134] B) Application as a model system related to CD300A for pharmacology, immunology, microbiology and medical research;

[0135] C) Applications involving the production and use of animal experimental disease models for the study of CD300A-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0136] D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human CD300A signaling pathway modulators; or,

[0137] E) Study the function of CD300A gene, study the drugs and efficacy targeting human CD300A target site, and study the application of CD300A-related cancer, inflammation, and immune disease drugs.

[0138] The present invention provides a non-human animal expressing human or humanized CD300A protein, which can be used for screening of human CD300A-specific therapeutic agents (e.g., anti-CD300A antibodies; or drugs targeting CD300A signaling pathways). The therapeutic agent can reduce or block the interaction between CD300A and the CD300A receptor complex, test whether the therapeutic agent can increase or decrease the immune response, and / or determine whether the therapeutic agent is a CD300A agonist or antagonist. In some embodiments, the non-human animal is an animal model of human disease. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also comprises an impaired immune system, such as a genetically modified human-derived tissue xenograft, including a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor).

[0139] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-CD300A antibodies; or drugs targeting the CD300A signaling pathway) in treating various immune diseases. In some embodiments, the immune diseases include, but are not limited to, GVHD (graft versus host disease), urticaria, multiple sclerosis, psoriasis, allergies, asthma, atopic dermatitis, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty hepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, the immune disease is urticaria, allergies, asthma, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis.

[0140] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-CD300A antibodies; or drugs targeting the CD300A signaling pathway) in treating tumors. In some embodiments, the method involves administering a therapeutic agent to a non-human animal as described herein, wherein the non-human animal suffers from cancer or a tumor; and determining the inhibitory effect of the therapeutic agent on the cancer or tumor. Inhibitory effects that can be determined include, for example, a reduction in tumor size or tumor volume, a reduction in tumor growth, a reduction in the rate of increase of tumor volume in a subject (e.g., compared to the rate of increase of tumor volume in the same subject before treatment or in another subject that has not been treated with such treatment), a reduction in the risk of metastasis or the risk of one or more additional metastases, an increase in survival rate and an increase in life expectancy, etc. The tumor volume of a subject can be determined by various methods, such as by direct measurement, MRI or CT.

[0141] In some embodiments, the tumor comprises one or more cancer cells (e.g., the cancer cells are derived from a human or non-human animal) injected into an animal. In some embodiments, the therapeutic agent activates or inhibits the CD300A signaling pathway. In some embodiments, the therapeutic agent does not activate or inhibit the CD300A signaling pathway.

[0142] In some embodiments, the genetically modified animals can be used to determine whether the antibody is a CD300A agonist or antagonist. In some embodiments, the methods described herein are also designed to determine the effect of a therapeutic agent (e.g., an antibody targeting CD300A; or a drug targeting the CD300A signaling pathway) on CD300A, for example, whether the therapeutic agent can upregulate or downregulate an immune response, and / or whether the 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 to treat a subject's disease (e.g., cancer, inflammation, or immune disease).

[0143] The inhibitory effect on tumors can also be determined by methods known in the art, for example, by measuring tumor volume in animals, and / or determining the tumor (volume) inhibition rate (TGI). TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%)=(1–T Vt / T Vc )×100, where T Vt and T Vc is the average tumor volume (or weight) of the treatment group and the control group.

[0144] In some embodiments, therapeutic agents (e.g., antibodies targeting CD300A; or drugs targeting the CD300A signaling pathway) are designed to treat various cancers. As used in this application, the term "cancer" refers to cells with autonomous growth capacity, that is, an abnormal state or state characterized by rapid proliferating 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. The term "tumor" used in this application refers to cancer cells, such as cancer cell clusters. Cancers that can be treated or diagnosed using the methods described in this application include malignant tumors of various organ systems, such as malignant tumors affecting the lungs, breasts, thyroid, lymph nodes, gastrointestinal and genital urinary tracts, and adenocarcinomas, including malignant tumors such as colon cancer, renal cell carcinoma, prostate cancer, testicular tumors, or non-small cell lung cancer. In some embodiments, the therapeutic agents described in this application are designed to treat or diagnose cancer in a subject. The term "cancer" is generally recognized and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include cancers formed by cervical, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term also includes carcinosarcoma, for example, including malignant tumors composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or tumor cells forming a recognizable glandular structure. The term "sarcoma" is generally recognized and refers to a malignant tumor of mesenchymal origin.

[0145] In certain embodiments, the cancer (tumor) described herein is lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, cancer, bladder cancer, glioma, 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. In certain embodiments, the leukemia is selected from acute lymphocytic (lymphocyte) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia and chronic myelogenous leukemia. In certain embodiments, 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 and T cell lymphoma, and Waldenstrom macroglobulinemia. In certain embodiments, sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma. In a specific embodiment, tumor is solid tumor and / or hematologic tumor. In a specific embodiment, tumor includes head and neck cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer or lung cancer. In certain embodiments, the cancer is solid tumor and / or hematologic tumor.

[0146] In some embodiments, the cancer comprises head and neck cancer, liver cancer, or lung cancer.

[0147] In some embodiments, therapeutic agents (e.g., antibodies targeting CD300A; or drugs targeting the CD300A signaling pathway) are designed to treat various immune diseases, including rheumatoid arthritis, urticaria, allergies, asthma, atopic dermatitis, multiple sclerosis, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease (IBD), ulcerative colitis, or scleroderma. Therefore, the methods described herein can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting CD300A; or drugs targeting the CD300A signaling pathway) in suppressing immune responses. In some embodiments, the immune disease described herein is graft-versus-host disease (GVHD), psoriasis, allergy, asthma, atopic dermatitis, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, urticaria, multiple sclerosis, ulcerative colitis, autoimmune liver disease, diabetes, pain or nervous system disease, etc.

[0148] In some embodiments, therapeutic agents (e.g., antibodies targeting CD300A; or drugs targeting the CD300A signaling pathway) are designed to treat various inflammations, such as viral inflammation. In some embodiments, the inflammation described herein includes acute inflammation and chronic inflammation. Specifically, inflammation includes, but is not limited to, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (e.g., tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, the inflammation described herein includes infection, and infection refers to local tissue and systemic inflammatory responses caused by bacteria, viruses, fungi, parasites, and / or other pathogens that invade the human body. In some embodiments, inflammation is inflammatory bowel disease (IBD).

[0149] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (e.g., an anti-CD300A antibody; or a drug targeting the CD300A signaling pathway). The method comprises administering a therapeutic agent to the above-mentioned non-human animal or a non-human animal obtained by the above-mentioned construction method, and evaluating the weight change or blood test of the non-human animal. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit, or hemoglobin content. In some embodiments, the therapeutic agent can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the body weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average body weight of a non-human animal not treated with an antibody).

[0150] The present invention also provides an animal model constructed by the method described in the present application for developing products related to human cellular immune processes, producing human antibodies, or a model system for pharmacology, immunology, microbiology and medical research.

[0151] In some embodiments, an animal model generated by the methods described herein is provided for producing and utilizing animal experimental disease models of immune processes of human cells, studying pathogens, or developing new diagnostic strategies and / or therapeutic strategies.

[0152] The present invention also provides an animal model generated by the method described in the present application to screen, verify, evaluate or study CD300A gene function, human CD300A antibodies, drugs or effectiveness of human CD300A target sites, drugs for immune diseases and anti-tumor drugs.

[0153] In some embodiments, the present application provides a method for verifying the in vivo efficacy of TCR-T, CAR-T and / or other immunotherapies (e.g., T cell adoptive transfer therapy). For example, the method includes transplanting human tumor cells into non-human animals described in the present application, and applying human CAR-T to animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animal is selected from a CD300A gene humanized non-human animal prepared by the method described in the present application, a CD300A gene humanized non-human animal described in the present application, a double or multiple humanized non-human animal (or its offspring) produced by the method described in the present application, a non-human animal expressing a human or humanized CD300A protein, or a tumor-bearing or inflammatory animal model described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat CD300A-related diseases described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies provide an evaluation method for treating CD300A-related diseases described in the present application.

[0154] Non-human animal models with two or more human or chimeric genes

[0155] The present invention also provides a transgenic animal model or non-human animal having two or more human or chimeric genes. The non-human animal or animal model may contain a human or chimeric CD300A gene and a sequence encoding an additional human or chimeric protein.

[0156] In some embodiments, the additional human or chimeric protein comprises at least one of ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the non-human animal also expresses at least one of human or humanized ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 protein.

[0157] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0158] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0159] (ii) mating, in vitro fertilization, or direct gene editing of the non-human animal provided in step (i) with other genetically modified non-human animals, and screening to obtain two or more genetically modified non-human animals.

[0160] 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 ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0161] In some embodiments, CD300A gene humanization is performed directly on a non-human animal with at least one genetic modification of human or chimeric ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0162] 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 combination therapies targeting two or more proteins, for example, anti-CD300A antibodies, and additional therapeutic agents for treating cancer or immune diseases (e.g., asthma or atopic dermatitis). The method includes administering an anti-CD300A 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 combined therapy on the immune tumor or immune disease. In some embodiments, the additional therapeutic agent includes antibodies that specifically bind to ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and / or CTLA4. In some embodiments, the additional therapeutic agent is one or more of 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 a human or humanized PD-1, a sequence encoding a human or humanized PD-L1, or a sequence encoding a 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.

[0163] In certain embodiments, the combination therapy is used to treat various cancers described herein, for example, solid tumors and / or hematological tumors. In certain embodiments, cancers include head and neck cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer or lung cancer. In certain embodiments, the combination therapy is designed to treat immune diseases described herein, for example, psoriasis.

[0164] In some embodiments, the method described in the present application can be used to evaluate the combined treatment with some other methods. The method for treating cancer that can be used alone or in combination with the method described in the present application includes, for example, treating the subject with chemotherapy, for example, camphor alkali, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepam, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorbutiazine, bithiofen, nitrosourea, dacrynic acid, daunorubicin, bleomycin, plimycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouric acid, vincristine, vinblastine and / or methotrexate. Alternatively, in addition, the method can include surgery on the subject to remove at least a portion of the cancer, for example, removing a portion or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0165] Figure 1 : Schematic diagram comparing mouse CD300A locus and human CD300A locus (not to scale);

[0166] Figure 2 : Schematic diagram of CD300A gene targeting strategy and targeting vector V1 design (not to scale);

[0167] Figure 3 : Schematic diagram of CD300A gene targeting strategy and targeting vector V2 design (not to scale);

[0168] Figure 4 : Southern blot identification results of CD300A gene humanized mouse F1 generation, where WT is the wild-type control;

[0169] Figure 5 : RT-PCR test results, where + / + is wild-type C57BL / 6 mice, H / H is CD300A gene humanized homozygous mice, and H2O is water control. DETAILED DESCRIPTION

[0170] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0171] Materials and methods

[0172] In each of the following examples, equipment and materials were obtained from the following companies:

[0173] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Administration;

[0174] BsrGI was purchased from NEB, catalog number R3575S;

[0175] NcoI was purchased from NEB with the catalog number R3193S;

[0176] Zombie NIR TM Fixable Viability Kit was purchased from Biolegend, catalog number: 423106;

[0177] Purified anti-mouse CD16 / 32 Antibody was purchased from Biolegend, catalog number: 101302;

[0178] Brilliant Violet 510 TM Anti-mouse CD45 Antibody was purchased from Biolegend, catalog number: 103138;

[0179] Brilliant Violet 421 TM Anti-mouse CD3 Antibody was purchased from Biolegend, catalog number: 100228;

[0180] BioLegend Brilliant Violet 711 TM Anti-mouse CD19 Antibody was purchased from Biolegend, catalog number: 115555;

[0181] PE / Cy TM 7 Mouse anti-mouse NK1.1 Antibody was purchased from BD Pharmingen, catalog number: 552878;

[0182] FITC anti-mouse F4 / 80 Antibody was purchased from Biolegend, catalog number: 123108;

[0183] Brilliant Violet 785 TM Anti-mouse / human CD11b Antibody was purchased from Biolegend, catalog number: 101243;

[0184] Alexa 700anti-mouse CD11c Antibody was purchased from Biolegend, catalog number: 117320;

[0185] BD Pharmingen TM Alexa 647 Mouse Anti-Human CLM-8 (CD300a) was purchased from BD Pharmingen, catalog number: 566342.

[0186] Example 1 CD300A gene humanized mice

[0187] A comparison diagram of the mouse CD300A gene (NCBI Gene ID: 217303, located at positions 114780006 to 114792772 on chromosome 11 NC_000077.7, based on transcript NM_170758.4 and its encoded protein NP_739564.1 (SEQ ID NO: 1)) and the human CD300A gene (NCBI Gene ID: 11314, located at positions 74466373 to 74484798 on chromosome 17 NC_000017.11, based on transcript NM_007261.4 and its encoded protein NP_009192.2 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0188] In order to achieve the purpose of the present invention, all or part of the nucleotide sequence encoding the human CD300A protein can be introduced into the mouse endogenous CD300A locus, so that the mouse genome contains a humanized CD300A gene, and / or the mouse expresses a human or humanized CD300A protein. Specifically, using gene editing technology, under the control of the mouse CD300A gene regulatory element, the partial sequence of exon 1 to the partial sequence of exon 4 of the mouse, about 10.8 kb, is replaced with the partial sequence of exon 1 to the partial sequence of exon 4 of the human CD300A gene, about 7.4 kb, to obtain a humanized CD300A locus, thereby achieving humanization of the mouse CD300A gene.

[0189] In order to realize the targeting strategy of the present invention, a targeting vector was constructed. The targeting vector V1 ( Figure 2 ) contains upstream and downstream homology arm sequences of the mouse CD300A gene, and an A fragment containing a human CD300A gene fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 114776853 to 114781190 of NCBI accession number NC_000077.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 114788603 to 114792385 of NCBI accession number NC_000077.7. The nucleotide sequence of the human CD300A gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 74466704 to 74477490 of NCBI accession number NC_000017.11; the connection design of the upstream of the human CD300A fragment sequence and the mouse is: The sequence " GAGAA The last "A" in the sequence is the last nucleotide in the mouse. The "A" in is the first nucleotide of the human sequence. The downstream connection of the human CD300A fragment sequence and the mouse sequence is designed as follows: The sequence " GGGCC The last "C" in the sequence is the last nucleotide of the human sequence. The first "T" in is the first nucleotide of the mouse sequence.

[0190] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human CD300A gene is designed as follows: The sequence " AATCA The last "A" in " is the last nucleotide of the human CD300A gene, the sequence The first "G" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human CD300A gene is designed as: The sequence " ACTTC The last "C" in the sequence is the last nucleotide of the Neo box. The first "C" in is the first nucleotide of the human CD300A gene. The mRNA sequence transcribed from the CD300A gene in the transformed humanized mouse is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 7.

[0191] The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified to be correct by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using the positive clone screening marker gene to screen the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the technology known in the art, and the obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then mated with each other to obtain CD300A gene humanized homozygous mice.

[0192] In addition, CRISPR / Cas9 technology can be used for gene editing to further design the targeting vector V2( Figure 3), the targeting vector V2 contains homology arm sequences upstream and downstream of the mouse CD300A gene, and a human CD300A fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 32) is identical to the nucleotide sequence of positions 114779733 to 114781190 of NCBI accession number NC_000077.7, and the downstream 3' homology arm sequence (SEQ ID NO: 33) is identical to the nucleotide sequence of positions 114788603 to 114790138 of NCBI accession number NC_000077.7. The nucleotide sequence of the human CD300A fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 74466704 to 74477490 of NCBI accession number NC_000017.11. The mRNA sequence transcribed from the CD300A gene in the transformed humanized mouse is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 7.

[0193] Conventional methods can be used to construct the targeting vector, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

[0194] The target sequence (target site) determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence (target site) of the exemplary sgRNA on the CD300A gene is as follows:

[0195] sgRNA1 target site (SEQ ID NO: 22): 5'-CCAAAGAAAGGGCCGTGCTTGGG-3';

[0196] sgRNA2 target site (SEQ ID NO: 23): 5'-AGAGAGGGAGTAATTTAGTCAGG-3';

[0197] The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 3. After annealing, the annealing products were connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-CD300A-1 and pT7-CD300A-2.

[0198] Table 3 Sequence list of sgRNA1 and sgRNA2

[0199]

[0200] The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment DNA (SEQ ID NO: 34) of the T7 promoter and sgRNA scaffold and connected to the backbone vector (source: Takara, item number 3299) by restriction digestion (EcoRI and BamHI) in sequence. The sequencing was verified by a professional sequencing company, and the results showed that the target plasmid was obtained. Take the pronuclear fertilized eggs of mice, such as C57BL / 6 mice, and use a microinjector to pre-mix the in vitro transcription products of pT7-CD300A-1 and pT7-CD300A-2 plasmids (using the Ambion in vitro transcription kit, according to the instructions for transcription), the targeting vector and Cas9 mRNA and inject them into the cytoplasm or nucleus of the mouse fertilized eggs. Microinjection of fertilized eggs was performed according to the method in the Mouse Embryo Manipulation Experiment Manual (3rd Edition) (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to culture medium for a short period of culture and then transplanted into the oviduct of the recipient mother mouse for development. The obtained mice (F0 generation) were hybridized and self-fertilized to expand the population and establish a stable CD300A gene humanized mouse strain.

[0201] The PCR method can be used to identify the somatic cell genotype of F1 generation mice, and the F1 generation PCR-positive mice are subjected to Southern blot detection to confirm whether there is random insertion. The mouse tail is cut to extract genomic DNA, and the genome is digested with NcoI enzyme or BsrGI enzyme, respectively, transferred to a membrane, and hybridized. The 5' probe (A Probe) and the 3' probe (3'Probe) are located on the human CD300A genomic fragment and the 3' homology arm, respectively. The specific probe and target fragment length are shown in Table 4). The exemplary results are shown in Figure 4 As shown, combined with the PCR and sequencing results, 6 mice numbered F1-05, F1-06, F1-10, F1-11, F1-14 and F1-15 were positive mice. This shows that the present method can be used to construct CD300A gene humanized mice that can be stably propagated and have no random insertion.

[0202] Table 4 Length of specific probes and target fragments

[0203] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size BnB 3'Probe 11.8kb 16.2kb NcoI A Probe — 8.8kb

[0204] A Probe-F (SEQ ID NO: 12): 5'-CCTGAGAGTTAATGGGTCAAGATTCTG-3',

[0205] A Probe-R (SEQ ID NO: 13): 5'-CTCTCTCCTGATTAACTTCTCTCTCTG-3';

[0206] 3'Probe-F (SEQ ID NO: 14): 5'-GGTATCAATTCTAGTTAGAGGTGTGTCC-3',

[0207] 3'Probe-R (SEQ ID NO: 15): 5'-ATAGCTTTTGGGTCATCCCGAATGG-3';

[0208] The expression of mRNA in CD300A gene humanized mice can be detected by RT-PCR. Specifically, 6-week-old male C57BL / 6 mice (+ / +) and 6-week-old male CD300A gene humanized homozygous mice (H / H) prepared in this example were selected, and spleen tissue was obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 5. The detection results are shown in FIG. Figure 5 As shown. Figure 5 As can be seen, only mouse CD300A mRNA was detected in wild-type C57BL / 6 mice, and human CD300A mRNA was not detected; only human CD300A mRNA was detected in CD300A gene homozygous mice.

[0209] Table 5 RT-PCR primer sequences and target fragment sizes

[0210]

[0211]

[0212] Flow cytometry was used to confirm the expression of humanized CD300A protein in mice. Specifically, 6-week-old male wild-type C57BL / 6 mice (+ / +) and 6-week-old male homozygous CD300A mice (H / H) were selected, and spleen tissue was taken after euthanasia by cervical dislocation. Splenocytes were collected and analyzed using Zombie NIR. TM Fixable Viability Kit, Purified anti-mouse CD16 / 32 Antibody, Brilliant Violet 510 TM anti-mouse CD45 Antibody, Brilliant Violet 421 TM anti-mouse CD3 Antibody, BioLegend Brilliant Violet711 TM anti-mouse CD19 Antibody, PE / Cy TM7 Mouse anti-mouse NK1.1 Antibody, FITCanti-mouse F4 / 80 Antibody, Brilliant Violet 785 TM anti-mouse / human CD11bAntibody, Alexa 700 anti-mouse CD11c Antibody and specific anti-human antibody BD Pharmingen TM Alexa 647 Mouse Anti-Human CLM-8 (CD300a) and other identification staining were performed for flow cytometry detection.

[0213] The detection results are shown in Table 6. Using specific anti-human antibodies, the expression of humanized CD300A protein was detected only in B cells, NK cells, DC cells, neutrophils, monocytes and macrophages of CD300A gene homozygous mice. Combined with the above RT-PCR results, the CD300A gene homozygous mice constructed using the method of the present application can normally express humanized CD300A protein.

[0214] Table 6 Flow cytometry detection results

[0215] Cell Type Characteristics of CD300A-positive cells C57BL / 6 H / H T cells mCD45+mLy6G-mCD3+mCD19-hCD300A+ 0.24％ 1.31％ B cells mCD45+mLy6G-mCD3-mCD19+hCD300A+ 1.55％ 26.6％ NK cells mCD45+mLy6G-mCD3-mNK1.1+hCD300A+ 2.62％ 12.4％ DC mCD45+mLy6G-mCD3-mCD11c+hCD300A+ 2.30％ 96.3％ Neutrophils mCD45+mLy6G+mCD11b+hCD300A+ 0.54％ 99.6％ Monocytes mCD45+mLy6G-mCD11b+mF4 / 80-hCD300A+ 0.49％ 29.1％ Macrophages mCD45+mLy6G-mCD11b+mF4 / 80+hCD300A+ 0.21％ 99.2％

[0216] Example 2 Pharmacodynamic Model

[0217] The CD300A gene humanized mice prepared by the construction method described in Example 1 of the present application can be used to induce the preparation of a variety of human disease models, including asthma, allergy and other models, and can be used to test the in vivo efficacy of human-specific antibodies. For example, CD300A gene humanized mice can be used to evaluate the efficacy, pharmacokinetics and in vivo therapeutic efficacy of antagonists of the human-specific CD300A signaling pathway in various disease models known in the art.

[0218] For example, CD300A gene humanized mice prepared by the construction method described in Example 1 of the present application can be randomly divided into a control group and a treatment group, and ovalbumin (OVA) is injected for modeling on days 0 and 14. On days 24 and 27, the drug is administered by multiple routes such as intranasal infusion or tail vein injection 30 minutes before triggering an allergic reaction. The in vivo efficacy of the drug can be evaluated by multiple detection indicators such as behavioral scores, serum cytokine detection, and flow cytometry.

[0219] Example 3 Preparation of double-gene or multi-gene humanized mice

[0220] The CD300A gene humanized mouse prepared by the construction method described in Example 1 of the present application can also prepare a double gene or multi-gene humanized mouse model. For example, in the aforementioned Example 1, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modification of ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4, or, on the basis of CD300A humanized mice, the double gene humanized or multi-gene humanized mouse model can be obtained by separating mouse ES embryonic stem cells and gene recombination targeting technology. The homozygous or heterozygous humanized mouse of the CD300A gene obtained in the present application can also be mated with other gene-modified mice, and the offspring can be screened. According to the Mendel's law of inheritance, there is a certain probability of obtaining a multi-gene mouse modified with the humanized CD300A gene and other genes, and then the heterozygotes can be mated with each other to obtain a homozygous modified with double genes or multiple genes.

[0221] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0222] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0223] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric CD300A molecule (CD300A) protein.

2. The construction method according to claim 1, characterized in that: The chimeric CD300A protein comprises a human or humanized extracellular region, a human or humanized transmembrane region and an endogenous cytoplasmic region; Preferably, the amino acid sequence of the chimeric CD300A protein is identical to that of the human CD300A protein in at least 50 to 299 consecutive amino acids; Preferably, the amino acid sequence of the human or chimeric CD300A protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 1-196 or SEQ ID NO: 2 1-201; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 1-196 or SEQ ID NO: 2 1-201; Preferably, the amino acid sequence of the chimeric CD300A protein comprises SEQ ID NO:7; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

7.

3. A method for constructing a genetically modified non-human animal, characterized in that: At the non-human animal endogenous CD300A gene locus, replacing the non-human animal endogenous CD300A gene with a nucleotide sequence comprising human CD300A; Preferably, the nucleotide sequence of human CD300A comprises a portion of exon 1 to a portion of exon 4 of the human CD300A gene; wherein the portion of exon 1 of the human CD300A gene preferably comprises at least 5 bp of continuous nucleotides; the portion of exon 4 of the human CD300A gene preferably comprises at least 5 bp of continuous nucleotides; Further preferably, the nucleotide sequence of human CD300A comprises SEQ ID NO: 5; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity with the nucleotide sequence shown in SEQ ID NO: 5; Preferably, the non-human animal endogenous CD300A gene comprises a portion of exon 1 to a portion of exon 4 of the non-human animal endogenous CD300A gene; wherein, the portion of exon 1 of the non-human animal endogenous CD300A gene preferably comprises at least 5 bp of continuous nucleotides; and the portion of exon 4 of the non-human animal endogenous CD300A gene preferably comprises at least 5 bp of continuous nucleotides.

4. The construction method according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of human CD300A or the nucleotide sequence encoding human or chimeric CD300A protein is operably linked to an endogenous regulatory element of an endogenous CD300A locus of a non-human animal; Preferably, the endogenous CD300A protein of the non-human animal is not expressed or is expressed at a reduced level compared to CD300A in wild-type animals; Preferably, the modified CD300A gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

5. The construction method according to any one of claims 1 to 4, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified CD300A gene in the genome of the non-human animal comprises SEQ ID NO: 6; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

6.

6. The construction method according to any one of claims 1 to 5, characterized in that: The non-human animal also includes a nucleotide sequence encoding other human or chimeric proteins; preferably, the other human or chimeric proteins include at least one of ICOS, NKP46, IL36R, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

7. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 6; 2) Determine the effects of therapeutic agents on non-human animals; Preferably, the non-human animal suffers from a disease; Preferably, the therapeutic agent includes an anti-CD300A antibody or a drug targeting the CD300A signaling pathway; further preferably, it also includes an additional therapeutic agent, and the additional therapeutic agent includes one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the disease includes one or more of cancer, immune disease or inflammation.

8. A humanized CD300A protein, characterized in that: The humanized CD300A protein comprises the extracellular region of the human CD300A protein, and preferably also comprises the whole or part of the transmembrane region of the human CD300A protein; Preferably, the amino acid sequence of the humanized CD300A protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 1-196 or SEQ ID NO: 2 1-201; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 1-196 or SEQ ID NO: 2 1-201; Further preferably, the humanized CD300A protein comprises SEQ ID NO: 7; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO:

7.

9. A humanized CD300A gene, characterized in that: The humanized CD300A gene encodes the humanized CD300A protein according to claim 8; Preferably, the humanized CD300A gene comprises the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 or 33; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 or 33.

10. A cell, tissue or organ, characterized in that: The cell, tissue or organ expresses the humanized CD300A protein according to claim 8; or the genome of the cell, tissue or organ comprises the humanized CD300A gene according to claim 9.

11. Use of the non-human animal obtained by the construction method according to any one of claims 1 to 6, the humanized CD300A protein according to claim 8, the humanized CD300A gene according to claim 9, or the cell, tissue or organ according to claim 10, characterized in that: The applications described include: A) Application in product development involving CD300A-related immune processes in human cells; B) Application as a model system related to CD300A for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of etiology associated with CD300A and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human CD300A signaling pathway modulators; or, E) Study the function of CD300A gene, study the drugs and efficacy targeting human CD300A target site, and study the application of CD300A-related tumor, inflammation, and immune disease drugs.

Images