A mrgrp2 gene-modified non-human animal

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) MRGPRX2 protein and methods of using the same.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing human or chimeric (e.g., humanized) MRGPRX2 protein and a method of using the same. Background Technology

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

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

[0004] This application provides an animal model containing human or chimeric MAS-related GPR family member X2 (MRGPRX2) protein. This animal model can express human or chimeric MRGPRX2 (e.g., humanized MRGPRX2) protein. It can be used to study the function of the MRGPRX2 gene and to screen and evaluate MRGPRX2 signaling pathway regulators (e.g., therapeutics targeting MRGPRX2, including antibodies targeting human MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs). Furthermore, the animal model prepared by the method described in this application can be used for drug screening, pharmacodynamic studies, and research on the treatment of diseases related to the human MRGPRX2 target (e.g., tumors, inflammation, or immune-related diseases); this animal model can also be used to facilitate new drug development and design, saving time and costs. In summary, this invention provides a powerful tool for studying the function of the MRGPRX2 protein and a platform for screening therapeutic drugs for related diseases.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method thereof 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 MAS-associated GPR family member X2 (MRGPRX2) protein. In some embodiments, the chimeric MRGPRX2 protein is a humanized MRGPRX2 protein. In some embodiments, the amino acid sequence of the chimeric MRGPRX2 protein comprises an amino acid sequence consistent with at least 50 to 330, for example, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or 330 consecutive amino acids of the human MRGPRX2 protein. In some embodiments, the nucleotide sequence encoding the human or chimeric MRGPRX2 protein is operatively linked to an endogenous regulatory element (e.g., an endogenous MRGPRB2 promoter) or an exogenous regulatory element (e.g., a CAG promoter) of at least one chromosome. In some embodiments, the nucleotide sequence encoding the human or chimeric MRGPRX2 protein can be a genomic DNA sequence, a CDS sequence, or a cDNA sequence. In some embodiments, the amino acid sequence of the human or chimeric MRGPRX2 protein comprises SEQ ID NO: 2; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to SEQ ID NO: 2. In some embodiments, the non-human animal is a non-human mammal, such as a monkey or rodent (e.g., a mouse or rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous MRGPRB2 locus of the non-human animal is unmodified. In some embodiments, the endogenous MRGPRB2 protein of the non-human animal is not expressed or is expressed at a reduced level compared to MRGPRB2 in wild-type animals. In some embodiments, one or more cells of the non-human animal express the human or chimeric MRGPRX2 protein. In some embodiments, the cells are mast cells. In some embodiments, the cells are characterized by CD45. + CD117 + CD11b - .

[0006] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, the method comprising introducing a nucleotide sequence encoding human or chimeric MRGPRX2 into the genome of a non-human animal. In some embodiments, the introduction is an insertion or substitution. In some embodiments, the introduction into the non-human animal genome includes inserting a safe harbor site. In some embodiments, the introduction into the non-human animal genome includes replacing a corresponding region of the non-human animal's endogenous MRGPRX2 gene. In some embodiments, the safe harbor site is a ROSA26 site or a Hipp11 site. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 is operatively linked to an endogenous regulatory element (e.g., an endogenous MRGPRX2 promoter) or an exogenous regulatory element (e.g., a CAG promoter), and one or more cells of the non-human animal express a human or chimeric MRGPRX2 protein (e.g., a humanized MRGPRX2 protein). In some embodiments, the cells are mast cells. In some embodiments, the cells are characterized by CD45. + CD117 + CD11b -In some embodiments, the nucleotide sequence encoding the human or chimeric MRGPRX2 protein may be a genomic DNA sequence, a CDS sequence, or a cDNA sequence. In some embodiments, the endogenous MRGPRB2 protein in the animal is not expressed or its expression level is reduced compared to MRGPRB2 in wild-type animals. In some embodiments, the nucleotide sequence encoding the human or chimeric MRGPRX2 comprises all or part of exon 3 of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding the human or chimeric MRGPRX2 comprises a portion of exon 3 of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding the human or chimeric MRGPRX2 comprises the coding region of the human MRGPRX2 gene, the 3'UTR, and at least 50 bp of continuous nucleotide sequences downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human or chimeric MRGPRX2 comprises the coding region of the human MRGPRX2 gene, the 3'UTR, and at least 1000 bp of continuous nucleotide sequences downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 39; or comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 39. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 or its transcribed mRNA comprises the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 43; or comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 43. In some embodiments, the modified MRGPRX2 gene in the non-human animal genome is homozygous or heterozygous for the endogenously modified locus.

[0007] In one aspect, a method for constructing a genetically modified non-human animal is provided, comprising introducing a nucleotide sequence of human MRGPRX2 into the genome of a non-human animal. In some embodiments, one or more cells of the non-human animal express human or chimeric MRGPRX2 protein. In some embodiments, the introduction into the non-human animal genome includes the introduction of an endogenous MRGPRX2 or a safe harbor site (e.g., ROSA26 or Hipp11). In some embodiments, the nucleotide sequence encoding the human or chimeric MRGPRX2 protein or the nucleotide sequence of human MRGPRX2 is operatively linked to an endogenous regulatory element (e.g., an endogenous MRGPRX2 promoter) or an exogenous regulatory element (e.g., a CAG promoter). In some embodiments, the endogenous MRGPRX2 protein of the non-human animal is not expressed or is expressed at a reduced level compared to MRGPRX2 in wild-type animals. In some embodiments, the modified MRGPRX2 gene in the non-human animal genome is homozygous or heterozygous for the endogenously modified locus. In some embodiments, one or more cells of the non-human animal express human or chimeric MRGPRX2 protein. In some embodiments, the cells are mast cells. In some embodiments, the cells are characterized by CD45. + CD117 + CD11b -In some embodiments, the nucleotide sequence of the human MRGPRX2 comprises a nucleotide sequence encoding a human or chimeric MRGPRX2 protein. In some embodiments, the nucleotide sequence of the human MRGPRX2 comprises a nucleotide sequence encoding SEQ ID NO: 2; or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the nucleotide sequence of the human MRGPRX2 may be a genomic DNA sequence, a CDS sequence, or a cDNA sequence. In some embodiments, the nucleotide sequence of the human MRGPRX2 comprises at least 50 bp to at least 7263 bp of the human MRGPRX2 gene, for example, a nucleotide sequence of consecutive or discontinuous nucleotides of the following values: 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 950, 990, 1000, 1500, 1900, 1948, 1950, 2000, 2300, 2346, 2500, 2900, 2945, 2948, 2950, ​​3000, 3346, 3500, 4000, 4500, 5000, 5500, 6000, 6263, 6500, 7000, 7200, 7263 bp. In some embodiments, the nucleotide sequence of the human MRGPRX2 comprises all or part of exon 3 of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence of the human MRGPRX2 comprises the nucleotide sequence from the start codon to the stop codon of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence of the human MRGPRX2 further comprises a 3'UTR. In some embodiments, the nucleotide sequence of the human MRGPRX2 further comprises a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR, such as at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 bp downstream of the 3'UTR. In some embodiments, the nucleotide sequence of the human MRGPRX2 comprises a continuous nucleotide sequence of at least 50 bp downstream of the start codon to the 3'UTR of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence of the human MRGPRX2 comprises a continuous nucleotide sequence of at least 1000 bp downstream of the start codon to the 3'UTR of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence of the human MRGPRX2 comprises the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 39; 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 or SEQ ID NO: 39.In some embodiments, the non-human animal genome contains SEQ ID NO: 14, or contains a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 14. In some embodiments, the nucleotide sequence of the non-human animal endogenous MRGPRB2 contains a nucleotide sequence encoding the non-human animal endogenous MRGPRB2 protein. In some embodiments, the nucleotide sequence of the non-human animal endogenous MRGPRB2 contains a nucleotide sequence encoding SEQ ID NO: 1; or contains a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence encoding SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the non-human animal endogenous MRGPRB2 contains all or part of exon 2 of the non-human animal endogenous MRGPRB2 gene. In some embodiments, the nucleotide sequence of the non-human animal endogenous MRGPRB2 contains the nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous MRGPRB2 gene. In some embodiments, the nucleotide sequence of the non-human animal endogenous MRGPRB2 further includes a 3'UTR. In some embodiments, the nucleotide sequence of the non-human animal endogenous MRGPRB2 further includes at least 50 bp downstream of the 3'UTR, for example, at least 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 950, or 963 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of the non-human animal endogenous MRGPRB2 includes at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR from the start codon of the non-human animal endogenous MRGPRB2 gene. In some embodiments, the nucleotide sequence of the non-human animal endogenous MRGPRB2 includes at least 963 bp of continuous nucleotide sequence downstream of the 3'UTR from the start codon of the non-human animal endogenous MRGPRB2 gene. In some embodiments, the sequence of the safe harbor site introduced into the non-human animal includes the nucleotide sequence of human MRGPRX2 and a STOP sequence conditionally controlled by the recombinase. The recombinase is introduced into the non-human animal to obtain non-human animals that specifically express human or chimeric MRGPRX2 in cells expressing the recombinase. In some embodiments, the conditional control of the recombinase is achieved by regulating gene expression through a Cre / Loxp system or a Flp / Frt system. In some embodiments, the cells expressing the recombinase are mast cells. In some embodiments, the expression of the recombinase is regulated by tissue-specific and / or inducible promoters. In some embodiments, the expression of the recombinase is regulated by endogenous MRGPRB2 regulatory elements in the non-human animal (e.g., the MRGPRB2 promoter).In some embodiments, the introduction of recombinase into a non-human animal comprises mating a non-human animal containing the nucleotide sequence of human MRGPRX2 and a conditionally controlled STOP sequence of the recombinase with a non-human animal containing a tissue-specific and / or inducible CRE recombinase. In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human mammal includes monkeys and rodents. In some embodiments, the rodent includes mice or rats. In some embodiments, the mRNA transcribed from the modified MRGPRX2 gene in the genome of the non-human animal comprises SEQ ID NO: 26 or 43; or comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% homology to the nucleotide sequence shown in SEQ ID NO: 26 or 43. In some embodiments, the non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins encoded by other genes are selected from at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0008] In one aspect, the present invention provides a non-human animal or a method thereof for constructing the non-human animal comprising at least one cell containing a nucleotide sequence encoding a human or humanized MRGPRX2 protein. In some embodiments, the humanized MRGPRX2 protein comprises an amino acid sequence identical to a consecutive amino acid sequence of at least 50, 100, 200, 250, 300, or 330 amino acids as shown in the human MRGPRX2 protein (SEQ ID NO: 2). In some embodiments, the non-human animal expresses the human or humanized MRGPRX2 protein. In some embodiments, the nucleotide sequence encoding the human or humanized MRGPRX2 protein is operatively linked to an endogenous regulatory element (e.g., an endogenous MRGPRB2 promoter) or an exogenous regulatory element (e.g., a CAG promoter). In some embodiments, the nucleotide sequence encoding the human or humanized MRGPRX2 can be introduced into the genome of a non-human animal. In some embodiments, the introduction is an insertion or substitution. In some embodiments, the introduction into the non-human animal genome includes the insertion of a safe harbor site. In some embodiments, the safe harbor site is a ROSA26 site or a Hipp11 site. In some embodiments, the introduction of the nonhuman animal genome includes replacing a corresponding region of the nonhuman animal's endogenous MRGPRX2 gene. In some embodiments, the human or humanized MRGPRX2 protein has human MRGPRX2 activity.

[0009] In one aspect, the present invention provides a genetically modified non-human animal or a method thereof for constructing the same, wherein a nucleotide sequence encoding human or chimeric MRGPRX2 is introduced into the genome of at least one cell of the non-human animal. In some embodiments, the endogenous MRGPRX2 protein of the non-human animal is not expressed or its expression level is reduced compared to MRGPRX2 in wild-type animals. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 protein may be a genomic DNA sequence, a CDS sequence, or a cDNA sequence. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises all or part of exon 3 of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises a portion of exon 3 of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises at least 50 bp to 1973 bp of the nucleotide sequence of exon 3 of the human MRGPRX2 gene, for example, at least 50, 100, 500, 900, 950, 990, 993, 1000, 1500, 1900, 1948, 1950, 1970, or 1973 bp of consecutive nucleotides. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the coding region of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the coding region of the human MRGPRX2 gene, the 3'UTR, and at least 50 bp of consecutive amino acids downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the coding region of the human MRGPRX2 gene, the 3'UTR, and at least 1000 bp of consecutive amino acids downstream of the 3'UTR. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the amino acid sequence shown in SEQ ID NO: 2; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 39; 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 or SEQ ID NO: 39.In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 or its transcribed mRAN sequence comprises the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 43; or comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 is operatively linked to an endogenous regulatory element (e.g., an endogenous MRGPRB2 promoter) or an exogenous regulatory element (e.g., a CAG promoter). In some embodiments, the non-human animal is a mammal, such as a monkey or rodent. In some embodiments, the non-human animal is a mouse or rat.

[0010] In one aspect, the present invention provides a method for constructing gene-modified non-human animal cells expressing human or chimeric MRGPRX2, the method comprising introducing a nucleotide sequence encoding human or chimeric MRGPRX2 into the genome of a non-human animal to produce gene-modified non-human animal cells expressing human or chimeric MRGPRX2 protein. In some embodiments, the cells are mast cells. In some embodiments, the cells are characterized by CD45. + CD117 + CD11b -In some embodiments, the non-human animal cells highly express the human or chimeric MRGPRX2 protein. In some embodiments, the importation is an insertion or substitution. In some embodiments, the importation into the non-human animal genome includes inserting a safe harbor site. In some embodiments, the safe harbor site is the ROSA26 site or the Hipp11 site. In some embodiments, the importation into the non-human animal genome includes replacing a corresponding region of the non-human animal's endogenous MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises all or part of exon 3 of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises a portion of exon 3 of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises at least 50 bp to 1973 bp of the nucleotide sequence of exon 3 of the human MRGPRX2 gene, for example, at least 50, 100, 500, 900, 950, 990, 993, 1000, 1500, 1900, 1948, 1950, 1970, or 1973 bp of consecutive nucleotides. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the coding region of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the coding region of the human MRGPRX2 gene, the 3'UTR, and at least 50 bp of consecutive amino acids downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the coding region of the human MRGPRX2 gene, the 3'UTR, and at least 1000 bp of consecutive amino acids downstream of the 3'UTR. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the amino acid sequence shown in SEQ ID NO: 2; or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 comprises the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 39; 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 or SEQ ID NO: 39.In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 or its transcribed mRNA sequence comprises the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 43; 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: 26 or SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding human or chimeric MRGPRX2 protein is operatively linked to an endogenous regulatory element (e.g., an endogenous MRGPRB2 promoter) or an exogenous regulatory element (e.g., a CAG promoter). In some embodiments, the non-human animal is a mouse.

[0011] In one aspect, this application provides a non-human animal specifically expressing human or humanized MRGPRX2, or a method for constructing the same, the method comprising: 1) introducing a nucleotide sequence encoding human or humanized MRGPRX2 into a non-human animal locus, preferably into a safe harbor site (e.g., ROSA26 or Hipp11). In some embodiments, the nucleotide sequence of human or humanized MRGPRX2 is regulated by a conditionally controlled STOP sequence of a recombinase; 2) introducing a recombinase into a non-human animal locus, preferably into a non-human animal endogenous MRGPRB2 locus. In some embodiments, the recombinase is regulated by a tissue-specific expression regulatory element. In some embodiments, the tissue-specific expression regulatory element is a mast cell-specific expression regulatory element, preferably a regulatory element (e.g., a promoter) of non-human animal endogenous MRGPRB2; 3) mating the non-human animal obtained in step 1) and the non-human animal obtained in step 2) to obtain a non-human animal specifically expressing human or humanized MRGPRX2. In some embodiments, the amino acid sequence encoded by the nucleotide sequence of the human or humanized MRGPRX2 protein comprises SEQ ID NO: 2; or comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding human or humanized MRGPRX2 comprises all or part of exon 3 of the human MRGPRX2 gene. In some embodiments, the nucleotide sequence encoding human or humanized MRGPRX2 comprises the coding region of the human MRGPRX2 gene. In some embodiments, the introduction is an insertion or substitution. In some embodiments, the recombinase conditional control is to regulate gene expression via a Cre / Loxp system or a Flp / Frt system. In some embodiments, the nucleotide sequence encoding human or humanized MRGPRX2 is operatively linked to an exogenous regulatory element (e.g., a CAG promoter), and one or more cells of the non-human animal express the human or chimeric MRGPRX2 protein. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal also includes nucleotide sequences of other genes encoding human or chimeric proteins selected from at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0012] In one aspect, a method is provided for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, the method comprising: 1) administering the therapeutic agent to a non-human animal or a non-human animal obtained by the above-described construction method; 2) determining the inhibitory effect of the therapeutic agent on the disease; or determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the therapeutic agent comprises a therapeutic agent targeting MRGPRX2; preferably, it may also comprise other therapeutic agents. In some embodiments, the therapeutic agent targeting MRGPRX2 is an antibody against human MRGPRX2, a nucleic acid drug targeting MRGPRX2, and / or a peptide drug. In some embodiments, the other therapeutic agents include an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease is a disease associated with MRGPRX2 expression, such as a tumor, an immune-related disease, or inflammation. In some embodiments, the tumor is a solid tumor or a hematologic malignancy. In some embodiments, the tumor includes one or more of head and neck cancer, liver cancer, or lung cancer. In some embodiments, the immune-related disease includes one or more of allergies, chronic urticaria, pruritus, asthma, rheumatoid arthritis, or multiple sclerosis. In some embodiments, the inflammation includes inflammatory bowel disease (IBD).

[0013] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating a tumor, the method comprising: 1) administering the therapeutic agent to a non-human animal or a non-human animal obtained by the construction method thereof, the non-human animal having a tumor; and 2) determining the inhibitory effect of the therapeutic agent on the tumor. In some embodiments, the therapeutic agent is an antibody targeting MRGPRX2, a nucleic acid drug targeting MRGPRX2, and / or a peptide drug. In some embodiments, the tumor comprises one or more tumor cells injected into the non-human animal. In some embodiments, determining the inhibitory effect of the therapeutic agent on the tumor involves measuring the tumor volume in the non-human animal. In some embodiments, the tumor is a solid tumor or a hematologic malignancy. In some embodiments, the tumor is head and neck cancer, liver cancer, or lung cancer. In some embodiments, the non-human animal further comprises sequences encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1. In some embodiments, the tumor comprises one or more tumor cells injected into the non-human animal.

[0014] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating an immune-related disease, the method comprising: 1) administering the therapeutic agent to the non-human animal or a non-human animal obtained by the construction method, wherein the non-human animal suffers from an immune disease; and 2) determining the therapeutic effect of the therapeutic agent on the immune-related disease. In some embodiments, the therapeutic agent is an antibody targeting human MRGPRX2, a nucleic acid drug targeting MRGPRX2, and / or a peptide drug. In some embodiments, the immune-related disease is allergy, chronic urticaria, pruritus, asthma, rheumatoid arthritis, or multiple sclerosis.

[0015] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation, the method comprising: 1) administering the therapeutic agent to a non-human animal as described in this application or a non-human animal obtained by the construction method, wherein the non-human animal exhibits inflammation; and 2) determining the effectiveness of the therapeutic agent in treating the inflammation. In some embodiments, the therapeutic agent is an antibody targeting human MRGPRX2, a nucleic acid drug targeting MRGPRX2, and / or a peptide drug. In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0016] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, the method comprising: 1) administering the therapeutic agent to the non-human animal or a non-human animal obtained by the construction method; and 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, determining the effect of the therapeutic agent on the non-human animal involves measuring the animal's weight or performing a blood test. In some embodiments, the blood test includes red blood cell count, hematocrit, and / or hemoglobin.

[0017] In one aspect, the present invention provides a humanized MRGPRX2 gene, said humanized MRGPRX2 gene comprising any of the following nucleotide sequences: A) a nucleotide sequence encoding SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26, 28, 29, 37, 38, 39, 40, 41, 42, 43, 44 or 45; C) a nucleotide sequence having at least 90% identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26, 28, 29, 37, 38, 39, 40, 41, 42, 43, 44 or 45; D) a nucleotide sequence encoding SEQ ID NO: 2. NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26, 28, 29, 37, 38, 39, 40, 41, 42, 43, 44 or 45 nucleotide sequences with identity of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0018] In one aspect, the present invention provides a cell, tissue, or organ containing the aforementioned humanized MRGPRX2 gene or derived from the aforementioned non-human animal or non-human animal obtained by the aforementioned construction method. In some embodiments, the cell, tissue, or organ can develop into an animal individual. In some embodiments, the cell, tissue, or organ cannot develop into an animal individual.

[0019] In one aspect, the present invention provides an animal model comprising the aforementioned humanized MRGPRX2 gene.

[0020] In one aspect, an application is provided for non-human animals or cells, tissues, or organs obtained by the above-described construction method, the applications including: A) applications in product development involving MRGPRX2-related immune processes in human cells; B) applications as MRGPRX2-related model systems in pharmacological, immunological, microbiological, and medical research; C) applications involving the production and utilization of animal experimental disease models for MRGPRX2-related etiological research and / or for the development of diagnostic and / or therapeutic strategies; D) applications in the screening, efficacy testing, efficacy evaluation, validation, or evaluation of human MRGPRX2 signaling pathway modulators in vivo; or, E) applications in studying MRGPRX2 gene function, studying drugs and their efficacy targeting human MRGPRX2, and studying therapeutic drugs for MRGPRX2-related diseases (e.g., tumors, inflammation, immune-related diseases). In some embodiments, the applications are for the diagnostic or therapeutic purposes of diseases. In some embodiments, the applications are for non-disease diagnostic or therapeutic purposes.

[0021] In this application, the term "locus" broadly refers to the position of a gene on a chromosome, and narrowly refers to a segment of DNA on a gene, which can be a gene itself, a part of a gene, or a regulatory region of a gene, etc. For example, the "endogenous MRGPRB2 locus" described in this application includes a DNA segment selected from exons 1-2 of the non-human animal endogenous MRGPRB2 gene.

[0022] The terms “exon XX-exon XXX” or “exon XX to exon XXX” or “all of exon XX to exon XXX” in this application refer to the entire sequence of exons and introns in between, such as the entire sequence of exons 1 to 3, including exon 1, intron 1, exon 2, intron 2 and exon 3.

[0023] The terms "comprising" or "including" in this application are open-ended descriptions, encompassing the specified components or steps described, as well as other specified components or steps that do not materially affect them. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the sequence, but still possess the same or similar activity as the original sequence.

[0024] The term "and / or" in this application includes all combinations of items connected by the term, and should be regarded as each combination being listed separately in this application. For example, "A and / or B" includes "A", "B", and "A and B". As 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".

[0025] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This application describes the methods and materials used in this invention; other suitable methods and materials known in the art may be used. Materials, methods, and embodiments are exemplary and not restrictive. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0026] Other aspects and advantages of this application will be readily apparent to those skilled in the art from the detailed description below.

[0027] MRGPRX2 In the human genome, the MRGPRX2 gene (NCBI Gene ID: 117194, UniProt ID: Q96LB1, located on chromosome 11, NC_000011.10, vertices 19054455 to 19060717) contains three exons: exon 1, exon 2, and exon 3. Table 1 shows the corresponding positions of each exon in the nucleotide and amino acid sequences of transcript NM_001303615.2 and its encoded protein NP_001290544.1 (SEQ ID NO: 2). Table 1

[0028] MRGPRB2 In the mouse genome, the MRGPRB2 gene (NCBI Gene ID: 243979, UniProt ID: Q3KNA1, located on chromosome 7 NC_000073.7, vertices 48200713 to 48207834) contains two exons, exon 1 and exon 2. Table 2 shows the corresponding positions of each exon in the nucleotide and amino acid sequences of transcript NM_175531.4 and its encoded protein NP_780740.2 (SEQ ID NO: 1). Table 2

[0029] Genes, proteins, and gene loci homologous to the human MRGPRX2 gene are also known in other species in this field. Examples include *Rattus norvegicus* (rat), *Macaca mulatta* (rhesus monkey), *Canis lupus familiaris* (dog), and *Sus scrofa* (pig). Information regarding these genes (e.g., intron sequences, exon sequences, and amino acid sequences) is available in NCBI, and its entirety is incorporated herein by reference.

[0030] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position 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 percentage of identity between two sequences is a function of the number of shared positions, taking into account the number of gaps and the length of each gap, which needs to be introduced to achieve optimal alignment of the two sequences. For example, sequence comparison and determination of the percentage of identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5.

[0031] The percentage of conserved residues with similar physicochemical properties (homology percentage), 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), nonpolar 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.

[0032] carrier The present invention provides a targeting vector comprising: a) a DNA fragment (5' homologous arm or 5' arm) homologous to the 5' end of the conversion region to be modified, which is derived from the genomic DNA of the non-human endogenous MRGPRB2 gene and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment (3' homologous arm or 3' arm) homologous to the 3' end of the conversion region to be modified, which is derived from the genomic DNA of the non-human endogenous MRGPRB2 gene and has a length of 100 to 10,000 nucleotides.

[0033] In some embodiments, a) the DNA fragment homologous to the 5' end of the transition region to be modified is derived from a nucleotide sequence with at least 90% homology to the NCBI accession number NC_000073.7; c) the DNA fragment homologous to the 3' end of the transition region to be modified is derived from a nucleotide sequence with at least 90% homology to the NCBI accession number NC_000073.7.

[0034] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.8kb, 1kb, 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 5.5kb, 6kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb, 9kb, 9.5kb, 10kb, 15kb, 16kb, 17kb, 18kb, 19kb, or 20kb.

[0035] In some embodiments, the conversion region to be altered is located on exons 1-2 of the non-human animal endogenous MRGPRB2 gene, preferably on the downstream nucleotide sequence of exons 1 to 3'UTR of the non-human animal endogenous MRGPRB2 gene, and more preferably on the downstream nucleotide sequence of exons 2 to 3'UTR of the non-human animal endogenous MRGPRB2 gene.

[0036] In some embodiments, a) the DNA fragment homologous to the 5' end of the transition region to be altered comprises SEQ ID NO: 37, 44.

[0037] In some embodiments, c) the DNA fragment homologous to the 3' end of the transition region to be altered comprises SEQ ID NO: 38, 45.

[0038] In some embodiments, b) the donor region contains a human sequence. In some embodiments, the donor sequence b) contains SEQ ID NO: 5 or 39.

[0039] In some embodiments, the targeting vector comprises one or more marker genes (or resistance genes). For example, a resistance gene for positive clone selection or a gene encoding a negative selection marker. In some embodiments, the resistance gene for positive clone selection includes the neomycin phosphotransferase coding sequence Neo and / or the hygromycin B phosphotransferase coding sequence HygR. In some embodiments, the targeting vector further includes two co-aligned Frt recombination sites and / or Frt3 recombination sites flanking the marker gene. In some embodiments, the gene encoding the negative selection marker is the gene encoding the diphtheria toxin A subunit (DTA).

[0040] The present invention also provides vectors for constructing humanized animal models or knockout models. In some embodiments, the vectors comprise sgRNA sequences that target the non-human animal endogenous MRGPRB2 gene. In some embodiments, the target site of the sgRNA on the conversion region to be altered is unique and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'.

[0041] Preferably, the target site sequence of the sgRNA is shown in SEQ ID NO: 30 or SEQ ID NO: 46. In some embodiments, the sgRNA is shown in SEQ ID NO: 47 and 49. In some embodiments, the sgRNA is shown in SEQ ID NO: 48 and 50.

[0042] In some embodiments, the present invention relates to plasmid constructs containing sgRNA (sgRNA vectors, such as pT7-sgRNA) and / or cells containing such constructs.

[0043] The present invention also relates to cells comprising the targeting vector and / or sgRNA vector as described above.

[0044] In some embodiments, the present invention provides a non-human mammalian cell having any of the above-described vectors, preferably also having transcripts of one or two of the plasmid constructs. In some embodiments, the non-human mammalian cell further comprises Cas9 mRNA or its in vitro transcript.

[0045] In some embodiments, the genes in the non-human mammalian cells are heterozygous.

[0046] In some embodiments, the genes in the non-human mammalian cells are homozygous.

[0047] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the non-human mammalian cell is a fertilized egg cell. In some embodiments, the non-human mammalian cell is an embryonic stem cell. In some embodiments, the non-human mammalian cell can be any cell capable of expressing MRGPRB2.

[0048] carrier The present invention provides a vector comprising: a) a DNA fragment (5' homologous arm or 5' arm) homologous to the 5' end of the conversion region to be modified, which is derived from genomic DNA of the endogenous Hipp11 or ROSA26 site of a non-human animal and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment (3' homologous arm or 3' arm) homologous to the 3' end of the conversion region to be modified, which is derived from genomic DNA of the endogenous Hipp11 or ROSA26 site of a non-human animal and has a length of 100 to 10,000 nucleotides.

[0049] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.8kb, 1kb, 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 5.5kb, 6kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb, 9kb, 9.5kb, 10kb, 15kb, 16kb, 17kb, 18kb, 19kb, or 20kb.

[0050] In some embodiments, the conversion region to be altered is located at the non-human animal endogenous Hipp11 or ROSA26 site.

[0051] In some embodiments, a) the DNA fragment homologous to the 5' end of the transition region to be altered comprises SEQ ID NO: 3, 12.

[0052] In some embodiments, c) the DNA fragment homologous to the 3' end of the transition region to be altered comprises SEQ ID NO: 4, 13.

[0053] In some embodiments, b) the donor region contains a human sequence. In some embodiments, the donor sequence b) contains SEQ ID NO: 5 or 39.

[0054] In some embodiments, the vector further comprises an auxiliary sequence, which preferably comprises a P2A sequence, a fluorescent group (e.g., EGFP, GFP, mCherry, EmGFP, RFP, tdTomato, YFP, Venus, CFP, Cerulean, mKate2, iRFP and / or Luciferase), a WPRE sequence, or a polyA sequence.

[0055] In some embodiments, the targeting vector comprises one or more marker genes (or resistance genes). For example, a resistance gene for positive clone selection or a gene encoding a negative selection marker. In some embodiments, the resistance gene for positive clone selection includes the neomycin phosphotransferase coding sequence Neo and / or the hygromycin B phosphotransferase coding sequence HygR. In some embodiments, the targeting vector further includes two co-aligned Frt recombination sites and / or Frt3 recombination sites flanking the marker gene. In some embodiments, the gene encoding the negative selection marker is the gene encoding the diphtheria toxin A subunit (DTA).

[0056] In some embodiments, the vector comprises an sgRNA sequence that targets the non-human animal endogenous ROSA26 site. In some embodiments, the target site of the sgRNA on the conversion region to be altered is unique and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'.

[0057] Preferably, the target site sequence of the sgRNA is shown in SEQ ID NO: 15. In some embodiments, the sgRNA is shown in SEQ ID NO: 16 and 18. In some embodiments, the sgRNA is shown in SEQ ID NO: 17 and 19.

[0058] This application also provides a vector for preparing MRGPRB2-iCre in non-human animals, comprising: a) a DNA fragment (5' homologous arm or 5' arm) homologous to the 5' end of the conversion region to be modified, which is derived from the genomic DNA of the endogenous MRGPRB2 gene in non-human animals and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment (3' homologous arm or 3' arm) homologous to the 3' end of the conversion region to be modified, which is derived from the genomic DNA of the endogenous MRGPRB2 gene in non-human animals and has a length of 100 to 10,000 nucleotides.

[0059] In some embodiments, the transition region to be altered is located on exons 1-2 of the non-human animal endogenous MRGPRB2, preferably at exon 2, and more preferably at the start codon.

[0060] In some embodiments, a) the DNA fragment homologous to the 5' end of the transition region to be altered comprises SEQ ID NO: 24, 28.

[0061] In some embodiments, c) the DNA fragment homologous to the 3' end of the transition region to be altered comprises SEQ ID NO: 25, 29.

[0062] In some embodiments, b) the donor region contains a human sequence. In some embodiments, the b) donor sequence contains a coding sequence for a recombinase, such as iCre. In some embodiments, the vector also contains an auxiliary sequence, which preferably contains a P2A sequence, a fluorescent group, a WPRE sequence, or a polyA sequence.

[0063] In some embodiments, the vector for preparing MRGPRB2-iCre nonhuman animals also contains sgRNA, wherein the sgRNA sequence targets the endogenous MRGPRB2 gene in the nonhuman animal. In some embodiments, the target site of the sgRNA on the conversion region to be altered is unique and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'.

[0064] Preferably, the target site sequence of the sgRNA is shown in SEQ ID NO: 30. In some embodiments, the sgRNA is shown in SEQ ID NO: 31 and 33. In some embodiments, the sgRNA is shown in SEQ ID NO: 32 and 34.

[0065] Genetically modified non-human animals The "genetically modified non-human animal" or "genetically modified non-human animal" described in this invention refers to a non-human animal whose genome contains at least one chromosome with 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 or genetically modified non-human animal, contain exogenous DNA. In some embodiments, the cells containing exogenous DNA can be various types of cells, such as somatic cells, immune cells (e.g., T cells, B cells, NK cells, antigen-presenting cells, macrophages, dendritic cells), germ cells, blastocysts, or tumor cells. In some embodiments, a genetically modified non-human animal is provided, which includes a modified endogenous safety site (e.g., ROSA26 or Hipp11) and an exogenous sequence (e.g., a human sequence), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. In some embodiments, a genetically modified non-human animal is provided, the non-human animal containing a modified endogenous MRGPRB2 locus, containing a foreign sequence (e.g., a human sequence), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. Non-human animals are generally able to pass on the genetic modification to their offspring through germline transmission.

[0066] The "chimeric (X) gene" or "chimeric (X) nucleic acid" described in this invention refers to a gene or nucleic acid in which two or more portions of the gene or nucleic acid originate from different species, or where at least one sequence of the gene or nucleic acid differs from that of a nucleic acid in a wild-type animal. In some embodiments, the chimeric (X) gene or chimeric (X) nucleic acid has at least a portion of its sequence originating from two or more different species, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins from two or more different species. In some embodiments, the chimeric (X) gene or chimeric (X) nucleic acid refers to a humanized (X) gene or humanized (X) nucleic acid.

[0067] The "chimeric (X) protein" or "chimeric (X) polypeptide" described in this invention refers to a protein or polypeptide where two or more portions of the polypeptide or protein originate from different species, or where at least one sequence of the protein or polypeptide differs from the amino acid sequence of a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (X) protein or chimeric (X) polypeptide has origins from two or more different species, for example, identical (or homologous) proteins from different species. In some embodiments, the chimeric (X) protein or chimeric (X) polypeptide refers to a humanized (X) protein or humanized (X) polypeptide. In some embodiments, the chimeric (X) protein or chimeric (X) polypeptide refers to a human (X) protein or human (X) polypeptide.

[0068] The "humanized (X) protein" or "humanized (X) polypeptide" as used in this invention refers to a protein or polypeptide, wherein at least a portion of the protein or polypeptide is derived from a human (X) protein or a human (X) polypeptide. In some embodiments, the humanized (X) protein or humanized (X) polypeptide refers to a human (X) protein or a human (X) polypeptide.

[0069] The "humanized (X) nucleic acid" or "humanized (X) gene" described in this invention refers to a nucleic acid or gene, wherein at least a portion of the nucleic acid or gene is derived from a human. In some embodiments, at least a portion of the humanized (X) nucleic acid or humanized (X) gene is derived from a non-human animal. In some embodiments, the humanized (X) nucleic acid or humanized (X) gene refers to a humanized exon, which may be a human exon or a chimeric exon.

[0070] In some embodiments, the chimeric MRGPRX2 gene or chimeric MRGPRX2 nucleic acid is a humanized MRGPRX2 gene or humanized MRGPRX2 nucleic acid. In some embodiments, at least a portion of the humanized MRGPRX2 gene or humanized MRGPRX2 nucleic acid is derived from the human MRGPRX2 gene. In some embodiments, the humanized MRGPRX2 gene or humanized MRGPRX2 nucleic acid contains a sequence encoding the MRGPRX2 protein. The encoded MRGPRX2 protein has at least the activity of the human MRGPRX2 protein.

[0071] In some embodiments, the chimeric MRGPRX2 protein or chimeric MRGPRX2 peptide is a humanized MRGPRX2 protein or a humanized MRGPRX2 peptide. In some embodiments, at least one or more portions of the amino acid sequence of the humanized MRGPRX2 protein or humanized MRGPRX2 peptide are derived from human MRGPRX2 protein. The humanized MRGPRX2 protein or humanized MRGPRX2 peptide is functional, or at least has the activity of human MRGPRX2 protein.

[0072] Genetically modified non-human animals can be various non-human animals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals from which suitable heritably modified embryonic stem cells (ES) are not readily available, other methods are employed to construct genetically modified non-human animals. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent stem cells) and transferring the modified genome to suitable cells, such as oocytes, using nuclear transfer, and gestating the modified cells (e.g., modified oocytes) in a non-human animal under appropriate conditions to form an embryo. The above-described methods of construction are known in the art and are described in “A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2006, the entire contents of which are incorporated herein by reference.

[0073] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. In some embodiments, the rodent may be selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the rat family. In one embodiment, the genetically modified animal is selected from the families Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny rats, crested rats), Madagascar Muridae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Dormicidae (e.g., spiny dormice), and Mole-like Muridae (e.g., mole rats, bamboo rats, and mole rats). In a particular embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny rats, and crested rats. In one embodiment, the genetically modified mouse is a member of the Muridae family. In one embodiment, the non-human animal is a rodent. In a particular embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0074] In some embodiments, the non-human animal may be an immunodeficient non-human mammal. Examples include immunodeficient rodents, immunodeficient rabbits, immunodeficient pigs, and immunodeficient monkeys. In some embodiments, the animal is a C57BL strain mouse, wherein the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, 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, and 129 / Sv-ter / +. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem CellLines (2000), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mice are a cross between the 129 strain and the C57BL / 6 strain. In some embodiments, the mice are a cross between the 129 strain or the C57BL / 6 strain. In some embodiments, the mice are a BALB strain, such as the BALB / c strain. In some embodiments, the mice are a cross between the BALB strain and another strain. In some embodiments, the mice are derived from a hybrid line (e.g., 50% BALB / c - 50% 12954 / Sv; or 50% C57BL / 6 - 50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having the BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, 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. In some embodiments, the non-human animal is a rat. The rat may be selected from Wistar rats, LEA strain, Sprague-Dawley strain, Fischer strain, F344, F6, and Dark Agouti.In some embodiments, the rat strain is a hybrid species selected from two or more strains of Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti. The non-human animal may have one or more other genetic modifications and / or other modifications suitable for the specific purpose of producing a humanized animal. For example, suitable mice for maintaining xenografts (e.g., human cancer or tumors) may have one or more modifications that impair, inactivate, or destroy all or part of the non-human animal's immune system. Impairment, inactivation, or destruction of the non-human animal's immune system can include, for example, by chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and / or genetic modifications (e.g., knockout of one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, and NOD / SCID / γc mice. null Mice (Ito, M. et al., NOD / SCID / γc) null Mouse: an excellent recipient mouse model forengraftment of human cells, Blood 100(9): 3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice may optionally be irradiated or otherwise treated to destroy one or more immune cell types. Thus, in various embodiments, a genetically modified mouse is provided that may include at least a portion of the non-human animal endogenous MRGPRB2 locus, and further includes modifications that impair, inactivate, or partially destroy the non-human animal's immune system (or one or more cell types of the immune system). 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 Mice, NOD Rag 1 - / - IL2rg - / - (NRG) mice, Rag2 - / - IL2rg - / - (RG) Modifications of mice and combinations 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 the replacement of all or part of the coding sequence of the mouse endogenous mature MRGPRB2 gene with all or part of the coding sequence of the human mature MRGPRX2 gene.

[0075] Genetically modified nonhuman animals include modifications to endogenous loci in nonhuman animals (e.g., the endogenous MRGPRB2 locus, ROSA26 site, or Hipp11 site). In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature MRGPRX2 protein (e.g., a nucleotide sequence comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence encoding the mature MRGPRX2 protein). While cells (e.g., ES cells, somatic cells) that may contain the genetic modifications described herein are provided in this invention, in many embodiments, genetically modified nonhuman animals include modifications to endogenous safe harbor sites or the MRGPRB2 locus in nonhuman animals.

[0076] Genetically modified animals can express human MRGPRX2 and / or chimeric (e.g., humanized) MRGPRX2 at non-human endogenous safe harbor sites (e.g., ROSA26 or Hipp11), wherein the endogenous mouse safe harbor site has been replaced or inserted with the human MRGPRX2 gene and / or a nucleotide sequence encoding the human MRGPRX2 sequence region or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% identical to the human MRGPRX2 sequence. In various embodiments, the endogenous non-human animal safe harbor site is modified with a human nucleic acid sequence comprising all or part of the human protein encoding the mature MRGPRX2 protein.

[0077] In some embodiments, genetically modified mice can express human MRGPRX2 and / or chimeric MRGPRX2 (e.g., humanized MRGPRX2) under the control of mouse promoters and / or mouse regulatory elements. Insertion or substitution at the mouse endogenous locus provides non-human animals with expression of human MRGPRX2 or chimeric MRGPRX2 (e.g., humanized MRGPRX2) in suitable cells and in a manner that does not lead to the potential pathology observed in some other transgenic mice known in the art. Human MRGPRX2 or chimeric MRGPRX2 (e.g., humanized MRGPRX2) expressed in non-human animals can maintain one or more functions of human MRGPRX2 in the animal. In some embodiments, the humanized MRGPRX2 locus contains a human 5'UTR. In some embodiments, the humanized MRGPRX2 locus contains an endogenous (e.g., mouse) 5'UTR. In some embodiments, humanization contains an endogenous (e.g., mouse) 3'UTR. Where appropriate, it is reasonable to assume that, based on the similarity of the 5' flanking sequences of the mouse endogenous gene and the human MRGPRX2 gene, they appear to be similarly regulated. As shown in this application, humanized MRGPRX2 mice containing insertions or substitutions at non-human animal endogenous safe harbor sites or endogenous MRGPRX2 loci, which retain non-human animal endogenous regulatory elements but contain the humanized MRGPRX2 coding sequence, do not exhibit pathological phenomena. Both heterozygous and homozygous humanized MRGPRX2 gene-modified mice are normal.

[0078] Genetically modified nonhuman animals include modifications to the endogenous MRGPRB2 gene locus in endogenous nonhuman animals. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature iCre protein (e.g., a nucleotide sequence with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence encoding the mature iCre protein). While cells that may contain the genetic modifications described herein (e.g., ES cells, somatic cells) are provided in this invention, in many embodiments, genetically modified nonhuman animals include modifications to the endogenous MRGPRB2 gene in an animal.

[0079] Genetically modified non-human animals can express the iCre enzyme at the endogenous mouse MRGPRB2 locus, wherein the endogenous mouse MRGPRB2 locus has been replaced or inserted with a nucleotide sequence encoding the iCre enzyme or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% identical to the nucleotide sequence encoding the iCre enzyme. In various embodiments, the endogenous non-human animal MRGPRB2 locus is modified with a nucleic acid sequence containing all or part of the nucleic acid sequence encoding the mature iCre enzyme.

[0080] In some embodiments, genetically modified mice may express the iCre enzyme under the control of the mouse MRGPRB2 promoter and / or mouse MRGPRB2 regulatory elements. Insertion or substitution at the mouse endogenous MRGPRB2 locus provides nonhuman animals with expression of the iCre enzyme in suitable cells and in a manner that does not lead to potential pathologies observed in some other transgenic mice known in the art. The iCre enzyme expressed in nonhuman animals can maintain one or more functions of the iCre enzyme in the animal. Furthermore, in some embodiments, the animal endogenously expresses the iCre enzyme at the MRGPRB2 locus. In some embodiments, the animal does not express endogenous MRGPRB2. In some embodiments, the animal's endogenous MRGPRB2 expression level is reduced compared to the MRGPRB2 expression level in wild-type animals. As used herein, the term "endogenous MRGPRB2" refers to the MRGPRB2 protein expressed by the endogenous MRGPRB2 nucleotide sequence of a nonhuman animal (e.g., a mouse) prior to any genetic modification.

[0081] The present invention further relates to a modified safe harbor site and / or MRGPRB2 genomic DNA sequence of a humanized mouse, wherein the DNA sequence obtained by reverse transcription of mRNA is identical or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell containing the construct thereof; a tissue or organ comprising the cell thereof.

[0082] The present invention further relates to non-human mammals produced by the methods described above. In some embodiments, their genomes contain human genes.

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

[0084] In some embodiments, non-human mammals express a protein encoded by the humanized MRGPRX2 gene.

[0085] In some embodiments, non-human mammals express a protein encoded by a gene sequence that encodes the iCre enzyme.

[0086] Furthermore, the present invention also provides a non-human mammalian model carrying a tumor, which is obtained by the method described in this application. In some embodiments, the non-human mammal is a rodent (e.g., a mouse).

[0087] This invention also provides cells or cell lines, or primary cell cultures, derived from non-human mammals or their offspring, or non-human mammals carrying tumors, that are derived from non-human mammals or their offspring, or non-human mammals carrying tumors, or tissues, organs, or cultures derived from non-human mammals or their offspring. When carrying a tumor, it is derived from tumor tissue of a non-human mammal or its offspring, or from a non-human mammal carrying a tumor.

[0088] This 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, the genome of which contains human or humanized MRGPRX2 DNA.

[0089] In some embodiments, the non-human mammal includes the gene constructs described herein. In some embodiments, a non-human mammal expressing human or humanized MRGPRX2 protein is provided. In some embodiments, a tissue specifically expressing human or humanized MRGPRX2 protein is provided.

[0090] In some embodiments, the expression of the non-human animal human or humanized MRGPRX2 protein is controllable, such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system.

[0091] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferred non-human mammals are mammals (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0092] Genetic, molecular, and behavioral analyses were performed on the non-human mammals described above. This invention provides an offspring produced by mating with a non-human mammal of the same or other genotypes.

[0093] This invention provides a cell line or primary cell culture derived from a non-human mammal or its offspring. For example, cell culture-based models can be prepared by the following methods. Cell cultures can be obtained by isolating cells from non-human mammals, or cells can be obtained using the same construct and a cell culture established with standard cell transfection techniques. Integration of genetic structures containing DNA sequences encoding the human MRGPRX2 protein or Cre enzyme can be detected by various methods.

[0094] Numerous analytical methods are available for detecting exogenous DNA, including nucleic acid-level methods (such as reverse transcription-polymerase chain reaction (RT-PCR) or Southern blotting and in situ hybridization) and protein-level methods (such as histochemical analysis, immunoblotting, and in vitro binding studies). Furthermore, the expression level of the target gene can be quantified using ELISA methods well-known to those skilled in the art. Many standard analytical methods are available for quantitative detection. For example, transcriptional levels can be detected using RT-PCR and hybridization methods, including RNase protection assays, Southern blotting, and RNA dot hybridization (RNAdot). Immunohistochemical staining, flow cytometry, and Western blotting can also be used to detect the presence of human or humanized MRGPRX2 protein.

[0095] In some embodiments, the genetically modified nonhuman animals described herein (e.g., humanized homozygous or heterozygous mice with the MRGPRX2 gene) may express human or humanized MRGPRX2 in one or more cells.

[0096] Methods for constructing genetically modified non-human animals Genetically modified non-human animals can be prepared using several techniques known in the art, including gene targeting techniques using embryonic stem cells, CRISPR / Cas9 technology, homologous recombination technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonucleases, or other molecular biology techniques. In some embodiments, homologous recombination technology is preferred. In some embodiments, CRISPR / Cas9 gene editing technology can be used to construct genetically modified non-human animals. Many of these genome editing techniques are known in the art and 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, such as microinjecting transgenic cells into enucleated oocytes and fusing enucleated oocytes with another transgenic cell.

[0097] In some embodiments, an endogenous gene safe harbor site in at least one cell of a non-human animal is introduced to contain a nucleotide sequence encoding human MRGPRX2. In some embodiments, the substitution occurs in cells such as neurons, mast cells, germ cells, somatic cells, blastocysts, or fibroblasts. The nucleus of a somatic cell or fibroblast may be inserted into an enucleated oocyte.

[0098] To achieve a humanized targeting strategy for endogenous safe sites in mice, a targeting vector comprises a 5' homologous arm, a human or humanized MRGPRX2 gene fragment, and a 3' homologous arm. The process involves introducing a human or humanized MRGPRX2 sequence into the endogenous locus using homologous recombination. In some embodiments, upstream and downstream cleavage of the target site (e.g., via zinc finger nucleases, TALEN, or CRISPR) can lead to DNA double-strand breaks, allowing the introduction of the human or humanized MRGPRX2 sequence into the endogenous locus using homologous recombination.

[0099] Therefore, in some embodiments, the method for preparing genetically modified humanized animals includes introducing a nucleotide sequence encoding human MRGPRX2 at an endogenous safe harbor site.

[0100] This invention also provides a method for establishing a humanized animal model of the MRGPRX2 gene, comprising the following steps: (a) Providing cells (e.g., fertilized egg cells) based on the methods described in this application; (b) Culturing the cells (preferably in a liquid culture medium); (c) Transplanting cultured cells into the fallopian tubes or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal; (d) Identify phylogenetic transmission in the offspring of genetically modified humanized non-human mammals of pregnant females in step (c).

[0101] In some embodiments, the non-human mammal in the above method is a mouse (e.g., a C57BL / 6 mouse).

[0102] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy.

[0103] In some embodiments, the fertilized egg used in the above method is a C57BL / 6 fertilized egg. Other fertilized eggs that can also be used in the method described in this application include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0104] The fertilized egg can originate from any non-human animal, such as any non-human animal described in this application. In some embodiments, the fertilized egg cell originates from a rodent. The gene construct can introduce DNA into the fertilized egg via microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which then gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0105] In some embodiments, the method of preparing a genetically modified nonhuman animal includes modifying the coding framework of the safe harbor site of the nonhuman animal, for example, by introducing a nucleotide sequence encoding human MRGPRX2 (e.g., a DNA, CDS, or cDNA sequence) into a nucleic acid sequence of an endogenous safe harbor site.

[0106] In some embodiments, the method of preparing genetically modified animals includes inserting a nucleotide sequence encoding a human or humanized MRGPRX2 protein and / or an auxiliary sequence at a safe site in a non-human animal. In some embodiments, the auxiliary sequence may be a stop codon, enabling the humanized animal model of the MRGPRX2 gene to express the human or humanized MRGPRX2 protein in vivo. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP, Insulator (isolator), P2A, and / or polyA.

[0107] In some embodiments, the method for preparing genetically modified animals further includes inserting a marker gene at a safe harbor site in a non-human animal. In some embodiments, the marker gene includes EGFP, GFP, mCherry, EmGFP, RFP, tdTomato, YFP, Venus, CFP, Cerulean, mKate2, iRFP, and / or Luciferase. In some embodiments, the marker gene includes EGFP and / or mCherry.

[0108] In some embodiments, the method of preparing genetically modified animals includes modifying the coding framework of the MRGPRB2 gene in a non-human animal, for example, by introducing a nucleotide sequence encoding a Cre enzyme (e.g., a DNA, CDS, or cDNA sequence) into the nucleic acid sequence of the endogenous MRGPRB2 gene locus. For example, one or more functional region sequences of the MRGPRB2 gene in a non-human animal may be knocked out or inserted, such that the endogenous MRGPRB2 protein in the non-human animal is not expressed or its expression level is reduced.

[0109] In some embodiments, the method of preparing a genetically modified animal includes inserting a nucleotide sequence encoding an iCre protein and / or an auxiliary sequence following an endogenous regulatory element of the MRGPRB2 gene in a non-human animal. In some embodiments, the auxiliary sequence may be a stop codon, allowing the non-human animal to express the iCre protein in vivo, but not the non-human MRGPRB2 protein. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0110] In some embodiments, a method for preparing genetically modified non-human animals includes: (1) Provide a plasmid containing a human MRGPRX2 gene fragment, wherein the plasmid is flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' and 3' homologous arms target endogenous safe harbor sites; (2) Provide one or more guide RNAs (sgRNAs) that target endogenous safe harbor sites; (3) Modify the genome of fertilized eggs or embryonic stem cells by using the plasmid from step (1), the sgRNA from step (2), and Cas9; (4) The fertilized eggs obtained in step (3) are transplanted into the fallopian tubes of pseudopregnant female mice, or the embryonic stem cells obtained in step (3) are transplanted into blastocysts, and then the blastocysts are transplanted into the fallopian tubes of pseudopregnant female mice to produce offspring mice that functionally express humanized MRGPRX2 protein. (5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0111] In some embodiments, the fertilized egg is modified by CRISPR with sgRNA targeting both the 5'-end and 3'-end target sites.

[0112] In some embodiments, the sequence encoding the humanized MRGPRX2 protein is operatively linked to an endogenous regulatory element (e.g., the endogenous MRGPRB2 promoter) or an exogenous regulatory element (e.g., the CAG promoter).

[0113] In some embodiments, genetically modified nonhuman animals further express the iCre protein.

[0114] In some embodiments, a method for preparing genetically modified non-human animals includes: (1) Provide a plasmid containing a human or chimeric MRGPRX2 gene fragment, the plasmid having 5' and 3' homologous arms flanking it, wherein the 5' and 3' homologous arms target endogenous safe harbor sites; (2) Provide one or more guide RNAs (sgRNAs) that target endogenous safe harbor sites; (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric MRGPRX2 gene fragment into the genome.

[0115] In some embodiments, a method for preparing genetically modified non-human animals includes: (1) Provide a plasmid containing a gene fragment encoding Cre enzyme, the plasmid having 5' and 3' homologous arms flanking it, wherein the 5' and 3' homologous arms target the endogenous MRGPRB2 locus; (2) Provide one or more guide RNAs (sgRNAs) that target the endogenous MRGPRB2 locus. (3) Modify the genome of fertilized eggs or embryonic stem cells by using the plasmid from step (1), the sgRNA from step (2), and Cas9; (4) The fertilized eggs obtained in step (3) are transplanted into the fallopian tubes of pseudopregnant female mice, or the embryonic stem cells obtained in step (3) are transplanted into blastocysts, and then the blastocysts are transplanted into the fallopian tubes of pseudopregnant female mice to produce offspring mice that functionally express iCre enzymes. (5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0116] In some embodiments, the fertilized egg is modified by CRISPR with sgRNA targeting both the 5'-end and 3'-end target sites.

[0117] In some embodiments, the sequence encoding the iCre enzyme is operatively linked to an endogenous regulatory element at the endogenous MRGPRB2 locus.

[0118] In some embodiments, the genetically modified animals do not express the endogenous MRGPRB2 protein, but express the iCre protein.

[0119] In some embodiments, a method for preparing genetically modified non-human animals includes: (1) Provide a plasmid containing a gene fragment encoding the iCre enzyme, the plasmid having 5' and 3' homologous arms flanking it, wherein the 5' and 3' homologous arms target the endogenous MRGPRB2 locus; (2) Provide one or more guide RNAs (sgRNAs) that target the endogenous MRGPRB2 locus. (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the gene fragment encoding the iCre enzyme into the genome.

[0120] In some embodiments, a method for preparing genetically modified non-human animals includes: (1) Provide a plasmid containing a human MRGPRX2 gene fragment, wherein the plasmid is flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' and 3' homologous arms target the endogenous MRGPRB2 gene locus; (2) Provide one or more guide RNAs (sgRNAs) that target the endogenous MRGPRB2 locus. (3) Modify the genome of fertilized eggs or embryonic stem cells by using the plasmid from step (1), the sgRNA from step (2), and Cas9; (4) The fertilized eggs obtained in step (3) are transplanted into the fallopian tubes of pseudopregnant female mice, or the embryonic stem cells obtained in step (3) are transplanted into blastocysts, and then the blastocysts are transplanted into the fallopian tubes of pseudopregnant female mice to produce offspring mice that functionally express human MRGPRX2. (5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0121] In some embodiments, the nucleotide sequence encoding the MRGPRB2 protein is deleted in the genome of the non-human animal. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1 is deleted in the genome of the non-human animal. In some embodiments, all or part of exons 1-2 of the MRGPRB2 gene are deleted in the genome of the non-human animal. In some embodiments, at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR of the MRGPRB2 gene is deleted in the genome of the non-human animal. In some embodiments, at least 50 bp of continuous nucleotide sequence from the start codon to the 3'UTR of the MRGPRB2 gene is deleted in the genome of the non-human animal. In some embodiments, at least 963 bp of continuous nucleotide sequence downstream of the 3'UTR of the MRGPRB2 gene is deleted in the genome of the non-human animal. In some embodiments, at least 963 bp of continuous nucleotide sequence from the start codon to the 3'UTR of the MRGPRB2 gene is deleted in the genome of the non-human animal.

[0122] In some embodiments, the construction method includes replacing all or part of the nucleotide sequence encoding the endogenous MRGPRB2 protein in a non-human animal genome with the nucleotide sequence encoding the human MRGPRX2 protein. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 1 in a non-human animal genome with the nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the construction method includes replacing all or part of the nucleotide sequence encoding SEQ ID NO: 2 in exons 1-2 of the endogenous MRGPRB2 gene in a non-human animal genome with the nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the construction method includes replacing all or part of the nucleotide sequence encoding exons 1-3 of the human MRGPRX2 gene in exons 1-2 of the endogenous MRGPRB2 gene in a non-human animal genome with the nucleotide sequence encoding the start codon to the 3'UTR of the human MRGPRX2 gene. In some embodiments, the construction method includes replacing at least 50 bp of the start codon to the 3'UTR of the endogenous MRGPRB2 gene in a non-human animal genome with the nucleotide sequence encoding the start codon to the 3'UTR of the endogenous MRGPRB2 gene in a non-human animal genome. In some embodiments, the construction method includes replacing at least 963 bp of the start codon to 3'UTR of the endogenous MRGPRB2 gene in the non-human animal genome with at least 1000 bp of the start codon to 3'UTR of the human MRGPRX2 gene. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal genome with the CDS sequence, cDNA sequence, or genomic DNA sequence of the human MRGPRX2 gene. In some embodiments, the construction method includes replacing the nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal genome with SEQ ID NO: 5, 26, 39, and 43.

[0123] In some embodiments, the construction method includes inserting a nucleotide sequence encoding the human MRGPRX2 protein into a safe harbor site in the endogenous genome of a non-human animal. A non-human animal expressing the recombinase is then constructed. Two non-human animals are then mated to obtain a non-human animal that specifically expresses the human or chimeric MRGPRX2 protein in cells expressing the recombinase. In some embodiments, the sequence inserted into the safe harbor site includes a nucleotide sequence of human MRGPRX2 and a conditionally controlled STOP sequence for the recombinase. In some embodiments, the conditional control of the recombinase is to regulate gene expression via a Cre / Loxp system or a Flp / Frt system. In some embodiments, the conditionally controlled STOP sequence for the recombinase contains recombination sites at both ends of a loxp, the recombination sites being lox sites or frt sites, and the conditionally controlled STOP sequence for the recombinase includes Loxp-STOP-Loxp. In some embodiments, the construction method of the non-human animal expressing the recombinase includes inserting a nucleotide sequence encoding the recombinase into the genome of a non-human animal. In some embodiments, the expression of the recombinase is regulated by an endogenous MRGPRB2 regulatory element in the non-human animal (e.g., the MRGPRB2 promoter). In some embodiments, the nucleotide sequence encoding the recombinase is inserted into the non-human animal endogenous MRGPRB2 gene locus, preferably at the non-human animal endogenous MRGPRB2 start codon. The cells expressing the recombinase include mast cells.

[0124] Application of genetically modified non-human animals Replacing a nonhuman animal gene with a homologous or orthologous human gene or sequence, or inserting a homologous or orthologous human gene or sequence into a nonhuman animal at an endogenous locus and under the control of an endogenous regulatory element (e.g., the nonhuman animal endogenous MRGPRB2 promoter) or an exogenous regulatory element (e.g., the CAG promoter), can produce nonhuman animals 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 a fully human transgene is inserted into the animal's genome, potentially integrating randomly into the genome. Typically, the location of the integrated transgene is unknown; human protein expression is measured by transcriptional assays and / or functional assays of human genes and / or proteins.

[0125] Genetically modified animals expressing human or humanized MRGPRX2 protein, for example in a physiologically suitable manner, provide a variety of uses, including but not limited to developing treatments for human diseases and conditions, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0126] The present invention also provides an application of a non-human animal modified with the above-mentioned MRGPRX2 gene and a non-human animal obtained by any of the above-mentioned construction methods.

[0127] In some embodiments, the application includes: A) Applications in product development involving MRGPRX2-related immune processes in human cells; B) Applications as a model system related to MRGPRX2 in pharmacological, immunological, microbiological and medical research; C) Involving the production and use of animal experimental disease models for etiological studies related to MRGPRX2 and / or for the development of diagnostic and / or therapeutic strategies; D) Application in in vivo studies of the screening, efficacy testing, evaluation, validation, or assessment of modulators of the human MRGPRX2 signaling pathway; or, E) Research the function of the MRGPRX2 gene, study drugs and their efficacy targeting the human MRGPRX2 target site, and study the drug applications for diseases related to MRGPRX2 (including tumors, inflammation, and immune-related diseases).

[0128] This invention provides a non-human animal expressing human or humanized MRGPRX2 protein, which can be used for screening human MRGPRX2-specific modulators (e.g., antibodies targeting MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs). In some embodiments, the non-human animal is a human disease animal model, such as a genetically induced disease (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also includes a compromised immune system, such as a genetically modified human tissue xenograft, including human solid tumors (e.g., breast cancer) or hematologic malignancies (e.g., lymphocytic tumors, including B or T cell tumors)).

[0129] In some embodiments, MRGPRX2 therapeutic agents (e.g., antibodies targeting MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs) block or inhibit MRGPRX2-mediated signaling pathways. In some embodiments, the MRGPRX2 therapeutic agents described herein can block the interactions between MRGPRX2 complexes, thereby inhibiting the MRGPRX2 signaling pathway.

[0130] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting human MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs) in treating various immune diseases. In some embodiments, the immune diseases include, but are not limited to, GVHD (graft-versus-host disease), chronic urticaria, pruritus, psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, immune-related diseases include allergies, chronic urticaria, pruritus, asthma, psoriasis, rheumatoid arthritis, or multiple sclerosis.

[0131] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting human MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs) in treating various inflammatory infections. In some embodiments, the inflammation includes both acute and chronic inflammation. Specifically, this includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, purulent inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, and specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma venereum, etc.). In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0132] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting human MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs) in treating cancer. In some embodiments, therapeutic agents (e.g., anti-human MRGPRX2 antibodies, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs) are administered to non-human animals, wherein the non-human animals have cancer or tumors, and the inhibitory effect of the therapeutic agent on the cancer or tumor is detected. In some embodiments, the detection includes determining the size and / or proliferation rate of tumor cells. In some embodiments, the detection methods include caliper measurement, flow cytometry, and / or in vivo animal imaging. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, wherein the metabolic changes include changes in food consumption or water consumption.

[0133] In some embodiments, the tumor cells comprise one or more cancer cells injected into an animal (e.g., cancer cells derived from humans or non-human animals). In some embodiments, the therapeutic agent inhibits or activates the MRGPRX2 signaling pathway. In some embodiments, the therapeutic agent does not inhibit or activate the MRGPRX2 signaling pathway.

[0134] In some embodiments, genetically modified non-human animals can be used to test whether MRGPRX2 therapeutics (e.g., antibodies targeting MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs) are agonists or antagonists. In some embodiments, the methods described herein can be used to test the function of a therapeutic (e.g., antibodies targeting human MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs), for example, whether the therapeutic can upregulate or downregulate an immune response, and / or whether the therapeutic can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine the effective dose of a therapeutic for treating a subject's disease (e.g., an immune disease). Tumor inhibition can also be determined by methods known in the art, such as measuring tumor volume in an animal and / or determining the tumor (volume) inhibition rate (TGI). TV The tumor growth inhibition rate can be calculated using the formula TGI. TV (%) = (1-T) Vt / T Vc ) x 100 to calculate, where T Vt and T Vc This is the average tumor volume (or weight) in the treatment group and the control group.

[0135] In some embodiments, therapeutic agents (e.g., antibodies targeting human MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs) can be used to treat various cancers. The term "cancer" as used in this invention refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapid cell growth and proliferation. This term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. The term "tumor" as used in this invention includes, but is not limited to, lymphoma, cervical cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioma, lung cancer (e.g., non-small cell lung cancer), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndromes, and sarcoma. The leukemia is selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In some embodiments, the tumor is breast cancer, pancreatic cancer, endocrine carcinoma, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tumor, oropharyngeal tumor, female reproductive system cancer, or meningioma. In some embodiments, the tumor is a non-small cell lung cancer (NSCLC) solid tumor or hematologic malignancy. In some embodiments, the tumor includes head and neck cancer or lung cancer.

[0136] This invention also provides a method for determining the toxicity of a therapeutic agent (e.g., an antibody targeting human MRGPRX2, a nucleic acid drug targeting MRGPRX2, and / or a peptide drug). The method includes administering the therapeutic agent to a non-human animal and assessing changes in the non-human animal's weight or performing blood tests. In some embodiments, the blood tests include red blood cell count, hematocrit, and / or hemoglobin. In some embodiments, the therapeutic agent may reduce red blood cell (RBC), hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animal's weight is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average weight of a non-human animal not treated with the therapeutic agent).

[0137] The present invention also provides an animal model system constructed by the method described in this application for developing products related to human cellular immune processes, manufacturing human antibodies, or for use in pharmacological, immunological, microbiological and medical research.

[0138] In some embodiments, an animal model generated by the method described in this application is provided for use in animal experimental disease models of immune processes of producing and utilizing human cells, for studying pathogens, or for developing new diagnostic and / or therapeutic strategies.

[0139] The present invention also provides animal models generated by the methods described in this application for studying MRGPRX2 gene function, screening, validating or evaluating drugs or efficacy targeting human MRGPRX2-related diseases (e.g., tumors, inflammation, immune-related diseases).

[0140] In some embodiments, this disclosure provides a method for verifying the in vivo efficacy of TCR-T, CAR-T, and / or other immunotherapies (e.g., T-cell adoptive transfer therapy). For example, the method includes transplanting human tumor cells into the non-human animals described in this application and applying human CAR-T to the non-human animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animals are selected from MRGPRX2 gene-humanized non-human animals prepared by the method described in this application, dual-gene or multi-gene humanized non-human animals (or their progeny) generated by the described method, non-human animals expressing human or humanized MRGPRX2 protein, or the tumor- or inflammatory animal models described herein. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat the MRGPRX2-related diseases (e.g., tumors, inflammation, or immune-related diseases). In some embodiments, TCR-T, CAR-T, and / or other immunotherapies provide evaluation methods for treating the MRGPRX2-related diseases (e.g., tumors, inflammation, or immune-related diseases) described in this application.

[0141] Two or more human or chimeric non-human animal models The present invention also provides an 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 MRGPRX2 gene and a sequence encoding an additional human or chimeric protein.

[0142] In some embodiments, the additional human or chimeric protein is a non-human animal genetically modified with at least one of the following: LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. In some embodiments, the aforementioned non-human animal also expresses at least one of the following human or humanized LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 proteins.

[0143] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising: (i) Obtain non-human animals by providing the above construction method; (ii) The non-human animals provided in step (i) are mated with other genetically modified non-human animals, fertilized in vitro, or directly genetically edited, and then screened to obtain multi-gene-modified non-human animals.

[0144] In some embodiments, the other genetically modified non-human animals include humanized non-human animals derived from one or more of the genes LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0145] In some embodiments, the humanization of the MRGPRX2 gene is performed directly in non-human animals with human or chimeric LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4 gene modifications.

[0146] Because these proteins may involve different mechanisms, combination therapies targeting two or more of them may be a more effective treatment. In fact, many relevant clinical trials are underway and showing promising results. Multi-gene modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, such as MRGPRX2 therapeutics (e.g., antibodies targeting MRGPRX2, nucleic acid drugs targeting MRGPRX2, and / or peptide drugs), and adjunctive therapeutics for treating diseases (e.g., tumors, inflammation, or immune-related diseases). The method includes administering the therapeutics and adjunctive therapeutics to non-human animals with tumors or immune diseases and determining the effect of the combination therapy on immunotumor or immune disease. In some embodiments, the adjunctive therapeutic is an antibody that specifically binds to LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4, or a nucleic acid drug and / or peptide drug targeting the aforementioned targets. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), or an anti-PD-L1 antibody. In some embodiments, the non-human animal described above also includes a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0147] In some embodiments, the combination therapy is used to treat various cancers described in this application. In some embodiments, the combination therapy is designed to treat immune-related diseases described herein, such as psoriasis. In some embodiments, the methods described in this application can be used to evaluate combination therapy with some other methods. Methods for treating cancer that can be used alone or in combination with the methods described in this application include, for example, treating a subject with chemotherapy, such as camphor, doxorubicin, cisplatin, carboplatin, procarbazine, meloratadine, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorpromazine, bismuth subcitrate, nitrosourea, daktarin, daunorubicin, bleomycin, priloin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatinum, 5-fluorouracil, vincristine, vinblastine, and / or methotrexate. Alternatively, the methods may include performing surgery on the subject to remove at least a portion of the cancer, for example, removing part or all of a tumor from the patient. Attached Figure Description

[0148] Figure 1 : Schematic diagram of gene targeting strategy and V1 targeting vector design (not to scale); Figure 2 : Schematic diagram of gene targeting strategy and V2 targeting vector design (not to scale); Figure 3 PCR identification results of F1 generation MRGPRX2-flox mice, where PC is the heterozygous positive control; WT is the wild-type control; H2O is the water control; and M is the marker. Figure 4 : Schematic diagram of gene targeting strategy and V3 targeting vector design (not to scale); Figure 5 : Schematic diagram of gene targeting strategy and V4 targeting vector design (not to scale); Figure 6 PCR identification results of F1 generation MRGPRB2-iCre mice, where PC is the positive heterozygous control, WT is the wild-type control, H2O is the water control, and M is the marker; Figure 7 Evans blue assay results: G1 represents wild-type C57BL / 6 mice (+ / +), G3 represents heterozygous MRGPRX2 gene-humanized mice (H / +;H / +), and G5 represents homozygous MRGPRX2 gene-humanized mice (H / H;H / +). ** indicates p < 0.01, and * indicates p < 0.05. Figure 8 The results of mast cell ratio detection show that G2 is a wild-type C57BL / 6 mouse (+ / +), G4 is a heterozygous humanized mouse of the MRGPRX2 gene (H / +;H / +), and G6 is a homozygous humanized mouse of the MRGPRX2 gene (H / H;H / +). Figure 8 (A) represents the ratio of mast cells in the left and right ears of mice in the G2 group. Figure 8 (B) shows the ratio of mast cells in the left and right ears of mice in group G4. Figure 8 (C) represents the ratio of mast cells in the left and right ears of mice in group G6. ** indicates p < 0.01, and * indicates p < 0.05. Figure 9 A schematic diagram comparing the mouse MRGPRB2 locus and the human MRGPRX2 locus (not to scale). Figure 10 : Schematic diagram of gene targeting strategy and V5 targeting vector design (not to scale); Figure 11 : Schematic diagram of gene targeting strategy and V6 targeting vector design (not to scale); Figure 12 PCR identification results of F1 generation of humanized mice V2 with MRGPRX2 gene, where PC is the heterozygous positive control, WT is the wild-type control, H2O is the water control, and M is the marker.

[0149] Figure 13 Changes in body temperature after administration to MRGPRX2 humanized mice. Detailed Implementation

[0150] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0151] In each of the following embodiments, the equipment and materials are obtained from the following companies: C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Experimental Animal Seed Center of the China National Institutes for Food and Drug Control.

[0152] Example 1: Preparation of mice expressing human MRGPRX2 in a tissue-specific manner Preparation of MRGPRX2-flox mice To achieve the objectives of this invention, a nucleotide sequence encoding the human MRGPRX2 protein can be inserted at the Hipp11 or ROSA26 locus in mice. Specifically, using gene editing technology, a nucleotide sequence of approximately 6.23 kb containing the loxP-STOP-loxP element and the CDS sequence of the human MRGPRX2 gene is inserted into the ROSA26 locus in mice, resulting in genetically modified mice with a genome containing the sequence encoding the human MRGPRX2. This locus can undergo Cre-mediated recombination in the presence of Cre, achieving tissue-specific expression of human MRGPRX2 in mice.

[0153] To achieve the targeting strategy of this invention, a target carrier V1 was constructed ( Figure 1 The targeting vector V1 contains upstream and downstream homologous arm sequences, which sequentially include the CAG promoter, the loxP-STOP-loxP element (SEQ ID NO: 55), the human MRGPRX2 gene CDS sequence (SEQ ID NO: 5), P2A, EGFP, WPRE, and the A fragment of polyA. The upstream 5' homologous arm sequence is SEQ ID NO: 3, and the downstream 3' homologous arm sequence is SEQ ID NO: 4. The mouse-to-CAG promoter linkage sequence is as follows: Where the sequence The last "C" in the sequence is the last nucleotide at the junction of the mouse sequence and the CAG promoter. The "C" in the sequence represents the first nucleotide of the CAG promoter. The underlined (curved) sequences are restriction enzyme sites, vector sequences, and other irrelevant sequences. The downstream linker sequence of the CAG promoter to the loxP-STOP-loxP element is as follows: acid, sequence The first "A" in the sequence indicates the first nucleotide of the loxP-STOP-loxP element. The underlined sequences represent restriction enzyme sites, vector sequences, and other irrelevant sequences. The linker sequence upstream of the human MRGPRX2 gene CDS sequence to the loxP-STOP-loxP element is as follows: medium sequence The last "T" in the sequence is the last nucleotide of the loxP-STOP-loxP element. In this sequence, "A" represents the first nucleotide of the human MRGPRX2 gene CDS sequence. The underlined (curved) sequences are unrelated sequences such as restriction enzyme sites and vector sequences. The downstream linker sequence between the human MRGPRX2 gene CDS sequence and EGFP is as follows: The last "G" in the sequence is the last nucleotide of the human MRGPRX2 gene CDS sequence. The “A” in the sequence represents the first nucleotide of EGFP, and the underlined sequence is the P2A sequence.

[0154] The targeting vector also includes an resistance gene for positive clone selection, namely the neomycin phosphotransferase coding sequence Neo, and two co-aligned site-specific recombination system Frt recombination sites flanking the resistance gene, forming a Neo cassette. The connection between the 5' end of the Neo cassette and polyA is designed as follows: acid.

[0155] Targeted vector construction can be performed using conventional methods, such as enzyme digestion and ligation. After preliminary validation via enzyme digestion, the constructed targeted vector is sent to a sequencing company for sequencing verification. The sequenced and validated targeted vector is then electroporated into embryonic stem cells of C57BL / 6 mice. Positive clone selection marker genes are used to screen the resulting cells, identifying the correct positive clones. These selected positive clones (black mice) are then introduced into isolated blastocysts (white mice) using techniques known in the art. The resulting chimeric blastocysts are transferred to culture medium for a short period before being transplanted into the oviducts of recipient mother mice (white mice) to produce F0 generation chimeric mice (black and white). F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice. F2 generation homozygous mice are then obtained by mating F1 generation heterozygous mice. Alternatively, positive mice can be mated with Flp tool mice to remove the positive clone selection marker genes, followed by mating to obtain MRGPRX2-flox homozygous mice.

[0156] In addition, gene editing can be performed using CRISPR / Cas9 technology, and the target vector V2 can be designed. Figure 2 The target vector V2 contains homologous arm sequences upstream and downstream of the mouse ROSA26 site, and an A2 fragment (SEQ ID NO: 14) sequentially comprising the CAG promoter, the loxP-STOP-loxP element (SEQ ID NO: 55), the human MRGPRX2 gene CDS sequence (SEQ ID NO: 5), P2A, EGFP, WPRE, and polyA. The upstream 5' homologous arm sequence is SEQ ID NO: 12, and the downstream 3' homologous arm sequence is SEQ ID NO: 13.

[0157] Targeting vectors can be constructed using conventional methods, such as enzyme digestion and ligation, or direct synthesis. After initial validation via enzyme digestion, the constructed targeting vector is sent to a sequencing company for sequencing validation. The correctly sequenced targeting vector is then used in subsequent experiments.

[0158] The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 cleavage of the target gene. Therefore, efficient and specific target sequence selection and design are prerequisites for constructing sgRNA expression vectors. An sgRNA sequence recognizing the target site was designed and synthesized. An example sgRNA target sequence at the ROSA26 site is as follows: sgRNA1 target site (SEQ ID NO: 15): 5'-AAGGCCGCACCCTTCTCCGGAGG-3'; The activity of sgRNA was detected using a UCA kit to determine that it could mediate high cleavage efficiency. Enzyme sites were added to its 5' end and complementary strand to obtain forward and reverse oligonucleotide sequences as shown in Table 3. After annealing, the annealed products were ligated into the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain the expression vector pT7-ROSA26-1.

[0159] Table 3 sgRNA1 sequence listing

[0160] The pT7-sgRNA vector was synthesized by a plasmid synthesis company, containing a fragment DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 20). This fragment was then sequentially digested with enzymes (EcoRI and BamHI) and ligated into a backbone vector (from Takara, catalog number 3299). Sequencing by a professional sequencing company confirmed the successful acquisition of the target plasmid. Pronuclear zygotes from mice, such as C57BL / 6 mice, were used. The in vitro transcription product of the pT7-ROSA26-1 plasmid (transcribed using an Ambion in vitro transcription kit according to the manufacturer's instructions), the targeting vector, and Cas9 mRNA were premixed and injected into the cytoplasm or nucleus of mouse zygote cytoplasm using a microinjection apparatus. Following the method described in "Experimental Manual of Mouse Embryo Manipulation (3rd Edition)" (Andras Nagy, Chemical Industry Press, 2006), fertilized eggs were microinjected. After injection, the fertilized eggs were transferred to a culture medium for short-term culture, and then transplanted into the oviduct of the recipient female mouse for development. The resulting mice (F0 generation) were then expanded through hybridization and self-fertilization to establish a stable MRGPRX2-flox mouse population.

[0161] The genotype of F1 generation mouse somatic cells can be identified using PCR. Primers shown in Table 4 are used for detection. Example results are shown below. Figure 3 As shown, the six mice numbered F1-01 to F1-06 were positive mice.

[0162] Table 4 Primer sequences and recombinant fragment sizes for F1 generation genotype PCR detection

[0163] Preparation of MRGPRB2-iCre mice To achieve the objectives of this invention, a nucleotide sequence encoding the iCre protein can be introduced into the mouse endogenous MRGPRB2 gene locus, enabling the mouse to express the iCre protein driven by the endogenous MRGPRB2 promoter. Specifically, using gene editing technology, a nucleotide sequence containing iCre, WPRE, and polyA is inserted into the start codon of the mouse MRGPRB2 gene to obtain mice that specifically express iCre.

[0164] To achieve the targeting strategy of this invention, a target carrier V3 was constructed. Figure 4 The targeting vector V3 contains an upstream 5' homologous arm, a downstream 3' homologous arm, and an A3 fragment (SEQ ID NO: 56) containing iCre, WPRE, and polyA in sequence. The upstream 5' homologous arm sequence is SEQ ID NO: 24, and the downstream 3' homologous arm sequence is SEQ ID NO: 25. The A3 fragment of the targeting vector V3 also includes an antibiotic resistance gene for positive clone selection, namely the neomycin phosphotransferase encoding sequence Neo, and two co-aligned site-specific recombination system Frt recombination sites are attached to both sides of the antibiotic resistance gene, forming a Neoassette.

[0165] Targeted vector construction can be performed using conventional methods, such as enzyme digestion and ligation. After preliminary validation via enzyme digestion, the constructed targeted vector is sent to a sequencing company for sequencing verification. The sequenced and validated targeted vector is then electroporated into embryonic stem cells of C57BL / 6 mice. Positive clone selection marker genes are used to screen the resulting cells, identifying the correct positive clones. These selected positive clones (black mice) are then introduced into isolated blastocysts (white mice) using techniques known in the art. The resulting chimeric blastocysts are transferred to culture medium for a short period before being transplanted into the oviducts of recipient mother mice (white mice) to produce F0 generation chimeric mice (black and white). F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice. F2 generation homozygous mice are then obtained by mating F1 generation heterozygous mice. Alternatively, positive mice can be mated with Flp tool mice to remove the positive clone selection marker genes, followed by mating to obtain MRGPRB2-iCre homozygous mice.

[0166] In addition, gene editing can be performed using CRISPR / Cas9 technology, and the target vector V4 can be designed. Figure 5 The targeting vector V4 contains upstream and downstream homologous arm sequences of the mouse MRGPRB2 gene, as well as an A4 fragment containing iCre, WPRE, and polyA in sequence. The upstream 5' homologous arm sequence is SEQ ID NO: 28, and the downstream 3' homologous arm sequence is SEQ ID NO: 29.

[0167] Targeting vectors can be constructed using conventional methods, such as enzyme digestion and ligation, or direct synthesis. After initial validation via enzyme digestion, the constructed targeting vector is sent to a sequencing company for sequencing validation. The correctly sequenced targeting vector is then used in subsequent experiments.

[0168] Design and synthesize sgRNA sequences that recognize target sites. An example sgRNA target sequence on the MRGPRB2 gene is as follows: sgRNA2 target site (SEQ ID NO: 30): 5'-AATTGACATCAACATCCCTTTGG-3'; The activity of sgRNA was detected using a UCA kit to confirm that it could mediate efficient cleavage. Enzyme sites were added to its 5' end and complementary strand to obtain forward and reverse oligonucleotide sequences as shown in Table 5. After annealing, the annealed products were ligated into the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain the expression vector pT7-MRGPRB2-1.

[0169] Table 5 sgRNA2 sequence listing

[0170] Pronuclear zygotes from mice, such as C57BL / 6 mice, were used. The in vitro transcription product of the pT7-MRGPRB2-1 plasmid, the targeting vector, and Cas9 mRNA were premixed and injected into the cytoplasm or nucleus of the mouse zygotes using a microinjection apparatus. Microinjection of the zygotes was performed according to the method described in *The Mouse Embryo Manipulation Manual (Third Edition)*. The injected zygotes were then transferred to culture medium for brief culture before being transplanted into the oviducts of recipient mice for development. The resulting mice (F0 generation) were then multiplied through hybridization and self-fertilization to expand the population and establish a stable MRGPRB2-iCre mouse strain.

[0171] The genotype of F1 generation mouse somatic cells can be identified using PCR. The primers shown in Table 6 were used for detection. Example results are shown below. Figure 6 As shown, the three mice numbered F1-1 to F1-3 were positive mice.

[0172] Table 6 Primer sequences and recombinant fragment sizes for F1 generation genotype PCR detection

[0173] The MRGPRX2-flox mice prepared above were mated with MRGPRB2-iCre mice, and their offspring were screened to obtain heterozygous multi-gene modified mice containing both human or humanized MRGPRX2 genes and iCre genes. The heterozygotes were then mated with each other to obtain homozygous MRGPRX2 gene modified mice.

[0174] This homozygous mouse specifically expresses human MRGPRX2 protein in mast cells and does not express endogenous MRGPRB2. The mRNA sequence transcribed from the modified MRGPRX2 gene in the gene-modified mouse is shown in SEQ ID NO: 26, and the protein sequence it expresses is shown in SEQ ID NO: 2.

[0175] The expression of human MRGPRX2 protein in MRGPRX2 gene-modified mice can be confirmed by flow cytometry. Specifically, one wild-type C57BL / 6 mouse (+ / +), one heterozygous MRGPRX2-modified mouse (MRGPRX2 heterozygote, H / +), and one homozygous MRGPRX2-modified mouse (H / H) were selected. Peritoneal lavage fluid was collected and treated with the following antibodies: Invitrogen CD45 Monoclonal Antibody (30-F11), eFluor™ 506 (purchased from eBioscience, catalog number 69-0451-82), Brilliant Violet 711™ anti-mouse CD117 (c-Kit) Antibody (purchased from BioLegend, catalog number 105835), Brilliant Violet 785™ anti-mouse / human CD11b Antibody (purchased from BioLegend, catalog number 101243), and BioLegend PE anti-human MRGX2. Antibody (purchased from BioLegend, catalog number 359004) is used for flow cytometry detection after staining.

[0176] hMRGPRX2-positive mast cells are characterized by mCD45. + mCD117 + mCD11b - hMRGPRX2 + The results showed that 90.7% of the mast cells in the peritoneal lavage fluid of heterozygous MRGPRX2-modified mice were hMRGPRX2-positive, and 94.3% of the mast cells in the peritoneal lavage fluid of homozygous MRGPRX2-modified mice were hMRGPRX2-positive.

[0177] In another similar experiment, wild-type C57BL / 6 mice (+ / +) and homozygous MRGPRX2 gene-modified mice (H / H) were selected. Spleen tissue and serum were collected, and the samples were stained with leukocyte marker antibodies Invitrogen CD45 Monoclonal Antibody (30-F11), eFluor™ 506, and hMRGPRX2 marker antibody BioLegend PE anti-human MRGX2 Antibody before flow cytometry detection. The results showed that no hMRGPRX2-positive cells were detected in the spleen tissue and serum of wild-type C57BL / 6 mice and homozygous MRGPRX2 gene-modified mice, including T cells, B cells, natural killer cells (NK cells), dendritic cells (DCs), neutrophils (NEs), monocytes (MCs), and macrophages (Mφ). Combined with the above flow cytometry results, this indicates that MRGPRX2 transgenic mice specifically express human MRGPRX2 protein in mast cells.

[0178] The function of human MRGPRX2 protein in MRGPRX2 gene-modified mice can be verified by inducing mast cell degranulation in MRGPRX2 gene-modified mice through neuropeptides (e.g., substance P). Specifically, nine 7-8 week old male wild-type C57BL / 6 mice (+ / +), nine heterozygous male MRGPRX2 gene-modified mice (MRGPRX2 heterozygous (H / +)), and nine homozygous male MRGPRX2 gene-modified mice (MRGPRX2 homozygous (H / H)) were randomly selected and grouped according to Table 7 and administered the drugs. Substance P (purchased from MCE, catalog number HY-P0201) was diluted to 1 nmol with PBS before use, and Evans Blue (purchased from Solarbio, catalog number E8010) was diluted to 5 mg / ml with 0.9% sodium chloride solution before use. On day 0 of the grouping, all mice were injected with 20 μL of substance P in the left ear and an equal volume of PBS solution in the right ear. Immediately afterwards, mice in groups G1, G3, and G5 were injected via the tail vein with 200 μL of Evans blue solution. Twenty minutes after injection, the left and right ears of mice in groups G1, G3, and G5 were harvested. Evans blue dye was extracted using formamide, and the absorbance of the extract was recorded at 620 nm to calculate the Evans blue concentration. The results are shown below. Figure 7 Ear thickness was recorded in mice in groups G2, G4, and G6. Tissue samples from both ears were collected for Wright-Giemza staining. Mast cells in non-degranulated, moderately degranulated, and extensively degranulated groups were counted, and the proportion of mast cells was calculated. Results are shown below. Figure 8 .

[0179] Table 7 Grouping and Dosing Regimen

[0180] When the MRGPRX2 protein functions normally, upon local stimulation by substance P, it mediates mast cell degranulation, releasing histamine and other mediators, thus increasing vascular permeability. Evans blue can bind to plasma albumin; when vascular permeability increases, Evans blue leaks from the blood vessels into the tissue. Therefore, an increased concentration of Evans blue in tissue stimulated by substance P indicates increased vascular permeability and a greater degree of mast cell degranulation at that location. Figure 7 As shown, in wild-type C57BL / 6 mice (+ / +), the Evans blue concentration in the left ear was increased after substance P stimulation compared to the right ear; in heterozygous MRGPRX2-modified mice (H / +), the Evans blue concentration in the left ear was significantly increased after substance P stimulation compared to the right ear (p < 0.01); and in homozygous MRGPRX2-modified mice (H / H), the Evans blue concentration in the left ear was significantly increased after substance P stimulation compared to the right ear (p < 0.05).

[0181] like Figure 8 As shown in (A)-8 (B), in wild-type C57BL / 6 mice (+ / +) and heterozygotes of MRGPRX2 gene-modified mice (H / +), compared with the right ear, the proportion of mast cells with no degranulation and moderate degranulation decreased in the left ear after substance P stimulation, while the proportion of mast cells with extensive degranulation increased significantly (p<0.05), indicating an increased degree of mast cell degranulation. like Figure 8 As shown in (C), in MRGPRX2 gene-modified homozygous mice (H / H), compared with the right ear, after substance P stimulation, the proportion of mast cells without degranulation in the left ear decreased significantly (p<0.01), the proportion of mast cells with moderate degranulation decreased, and the proportion of mast cells with extensive degranulation increased significantly (p<0.05), indicating an increased degree of mast cell degranulation.

[0182] This indicates that in the MRGPRX2 gene-modified mice prepared in this embodiment, the human MRGPRX2 protein can normally mediate mast cell degranulation after stimulation by substance P.

[0183] Example 2: Preparation of MRGPRX2 gene-humanized mice A comparative diagram of the mouse MRGPRB2 gene (NCBI Gene ID: 243979, located on chromosome 7 NC_000073.7, pp. 48200713 to 48207834, based on transcript NM_175531.4 and its encoded protein NP_780740.2 (SEQ ID NO: 1)) and the human MRGPRX2 gene (NCBI Gene ID: 117194, located on chromosome 11 NC_000011.10, pp. 19054455 to 19060717, based on transcript NM_001303615.2 and its encoded protein NP_001290544.1 (SEQ ID NO: 2) is shown below. Figure 9 As shown.

[0184] To achieve the objectives of this invention, a nucleotide sequence encoding the human MRGPRX2 protein can be introduced into the mouse endogenous MRGPRB2 gene locus, enabling the mouse to express the human or humanized MRGPRX2 protein. Specifically, using gene editing technology, approximately 2.97 kb of the sequence downstream of the ATG to 3'UTR of the mouse MRGPRB2 gene is replaced with approximately 2.95 kb of the sequence downstream of the ATG to 3'UTR of the human MRGPRX2 gene, resulting in the humanized MRGPRX2 gene locus and achieving the humanization of the mouse MRGPRB2 gene.

[0185] To achieve the targeting strategy of this invention, a target carrier V5 was constructed. Figure 10 The targeting vector V5 contains upstream and downstream homologous arm sequences of the mouse MRGPRX2 gene, as well as the A5 fragment containing the human MRGPRX2 fragment. Specifically, the upstream 5' homologous arm sequence is SEQ ID NO: 37, the downstream 3' homologous arm sequence is SEQ ID NO: 38, and the nucleotide sequence of the human MRGPRX2 fragment is SEQ ID NO: 39; the human MRGPRX2... The first nucleotide of the MRGPRX2 fragment.

[0186] The targeting vector also includes an resistance gene for positive clone selection, namely the neomycin phosphotransferase coding sequence Neo, and two co-aligned site-specific recombination system Frt recombination sites flanking the resistance gene, forming a Neo cassette. The 5' end of the Neo cassette is linked to the human MRGPRX2 fragment. The first nucleotide linked. The mRNA sequence of the modified humanized mouse MRGPRX2 is shown in SEQ ID NO: 43, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0187] Targeted vector construction can be performed using conventional methods, such as enzyme digestion and ligation. After preliminary validation via enzyme digestion, the constructed targeted vector is sent to a sequencing company for sequencing verification. The sequenced and validated targeted vector is then electroporated into embryonic stem cells of C57BL / 6 mice. Positive clone selection marker genes are used to screen the resulting cells, identifying the correct positive clones. These selected positive clones (black mice) are then introduced into isolated blastocysts (white mice) using techniques known in the art. The resulting chimeric blastocysts are transferred to a culture medium for brief culture before being transplanted into the oviducts of recipient mother mice (white mice) to produce F0 generation chimeric mice (black and white). F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice. F2 generation homozygous mice are then obtained by mating F1 generation heterozygous mice. Alternatively, positive mice can be mated with Flp tool mice to remove the positive clone selection marker genes, and then mated with each other to obtain MRGPRX2 gene-humanized homozygous mice.

[0188] In addition, gene editing can be performed using CRISPR / Cas9 technology, and the target vector V6 can be designed. Figure 11 The targeting vector V6 contains upstream and downstream homologous arm sequences of the mouse MRGPRX2 gene, as well as a human MRGPRX2 fragment. The upstream 5' homologous arm sequence is SEQ ID NO: 44, the downstream 3' homologous arm sequence is SEQ ID NO: 45, and the nucleotide sequence of the human MRGPRX2 fragment is SEQ ID NO: 39. The mRNA sequence transcribed from the modified humanized mouse MRGPRX2 gene is shown in SEQ ID NO: 43, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0189] Targeting vectors can be constructed using conventional methods, such as enzyme digestion and ligation, or direct synthesis. Targeting vectors that have been validated by sequencing are then used in subsequent experiments.

[0190] Design and synthesize sgRNA sequences that recognize target sites. An example sgRNA target sequence on the MRGPRB2 gene is as follows: sgRNA2 target site (SEQ ID NO: 30): 5'-AATTGACATCAACATCCCTTTGG-3'; sgRNA3 target site (SEQ ID NO: 46): 5'-GTGACTGAGTGATGTTACTCAGG-3'; The activity of sgRNA was detected using a UCA kit to confirm that it could mediate efficient cleavage. Enzyme sites were added to its 5' end and complementary strand to obtain forward and reverse oligonucleotide sequences, as shown in Tables 5 and 8, respectively. After annealing, the annealed products were ligated into the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain the expression vectors pT7-MRGPRB2-1 and pT7-MRGPRB2-2.

[0191] Table 8 sgRNA3 sequence listing

[0192] Pronuclear zygotes from mice, such as C57BL / 6 mice, were used. The in vitro transcription products of pT7-MRGPRB2-1 and pT7-MRGPRB2-2 plasmids, the targeting vector, and Cas9 mRNA were premixed and injected into the cytoplasm or nucleus of the mouse zygotes using a microinjection apparatus. Microinjection of the zygotes was performed according to the method described in the "Mouse Embryo Manipulation Experiment Manual (Third Edition)". The injected zygotes were then transferred to culture medium for brief culture before being transplanted into the oviducts of recipient mice for development. The resulting mice (F0 generation) were then multiplied through hybridization and self-fertilization to expand the population and establish a stable MRGPRX2 gene-humanized mouse strain.

[0193] The genotype of F1 generation mouse somatic cells can be identified using PCR. The primers shown in Table 9 are used for detection. Example results are shown below. Figure 12 As shown, the mouse numbered F1-1 is a positive mouse.

[0194] Table 9 Primer sequences and recombinant fragment sizes for F1 generation genotype PCR detection

[0195] Example 3: Preparation of dual-gene or multi-gene humanized mice The method or mice expressing human or humanized MRGPRX2 protein obtained using this method can also be used to prepare multi-gene humanized mouse models. For example, in Examples 1 or 2 above, the embryonic stem cells used for microinjection can be selected from mice modified with at least one gene, including LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. Alternatively, based on mice expressing human or humanized MRGPRX2, dual-gene or multi-gene humanized mouse models can be obtained using isolated mouse ES embryonic stem cells and gene recombination targeting technology. Alternatively, homozygous or heterozygous MRGPRX2 mice obtained by this method can be mated with other gene-modified mice, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene mice with humanized MRGPRX2 gene and other gene modifications. Further mating of heterozygous mice can yield homozygous mice with dual or multi-gene modifications.

[0196] Example 4: Pharmacodynamic Model

[0197] The humanized mice disclosed in this invention can be used to induce various human disease models, including models of allergy, inflammation, chronic urticaria, and pruritus, which can be used to test the in vivo efficacy of drugs targeting human MRGPRX2. For example, mice expressing human or humanized MRGPRX2 protein can be used to evaluate the efficacy, pharmacokinetics, and in vivo therapeutic efficacy of antagonists of the human-specific MRGPRX2 signaling pathway in various disease models known in the art.

[0198] Taking the preparation of an allergy model as an example, mice expressing human or humanized MRGPRX2 protein prepared according to this invention can be used. Mice are weighed and grouped, and drugs are administered via various routes such as gavage, intraperitoneal injection, or tail vein injection. Intradermal sensitization is achieved by injecting DNP-HSA (inducing IgE-dependent inflammation), SP (inducing non-IgE-dependent inflammation), and PBS into the mouse ear or footpads, respectively. After sensitization, ear or footpad tissue is collected, and the in vivo efficacy of different human drugs is evaluated using various detection indicators such as HE pathological examination and toluidine blue pathological examination.

[0199] For example, taking the pruritus model as an example, mice expressing human or humanized MRGPRX2 protein prepared according to this invention can be used. For the first 7 days before grouping, all mice were videotaped for 30 minutes to detect baseline pruritus levels. On day 0, mice were divided into a negative control group, a positive control group, and a drug-treated group according to their baseline pruritus levels. The drug-treated group was administered the drug via various routes, including oral administration, topical application, subcutaneous injection, intradermal injection, gavage, intraperitoneal injection, or tail vein injection. The negative and positive control groups were injected with an equal volume of a mixed solution of 5% DMSO and 95% PEG400. On day 1 of grouping, mice in the positive control group and drug-treated group were intradermally injected in the neck with 10 μL of C48 / 80 solution diluted to 10 μg / mL with PBS to establish the pruritus model. The negative control group was injected with an equal volume of PBS. After the C48 / 80 solution injection, all mice were videotaped for 30 minutes to record the number of pruritus episodes. Subsequently, skin tissue was collected from all mice, and the in vivo efficacy of different human drugs was evaluated using various indicators such as pruritus score, Wright-Gymsa staining pathological examination, and histamine concentration.

[0200] In another experiment, five 6-8 week old female MRGPRX2 gene-humanized homozygous (H / H) mice prepared in Example 2 were randomly divided into three groups: a control group (G1), a modeling group (G2), and a drug-treated group (G3). Groups G2 and G3 were induced to develop an allergic reaction by intravenous injection of Cortistatin-14 (Sigma-Aldrich, catalog number: E0753) at a dose of 1 mg / kg, while group G1 was injected intravenously with an equal volume of PBS. Four hours before modeling, mice in the drug-treated group G3 were intraperitoneally injected with MrgprX2antagonist-1 (MCE, catalog number: 145191) at a dose of 5 mg / kg and a volume of 10 μL / g. Rectal temperature was measured in each group before modeling (0 min), and every 10 minutes from modeling onwards (10, 20, 30, 40, 50, 60, 70, 80, and 90 min), for a total of 10 measurements. The grouping and administration details are shown in Table 10.

[0201] Table 10 Grouping and Dosing Information

[0202] Test results as follows Figure 13As shown, compared to G1, the body temperature of mice in G2 decreased rapidly and then rebounded within 0-90 minutes, indicating successful model establishment. Compared to G2, the decrease in body temperature of mice in G3 within 0-90 minutes was smaller, indicating that the drug MrgprX2antagonist-1 has an inhibitory effect on the temperature decrease induced by Cortistatin-14, and the experimental results are consistent with expectations. The MRGPRX2 gene-humanized mice prepared in this application can be used in preclinical studies to screen and evaluate the in vivo efficacy of drugs targeting MRGPRX2.

[0203] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0204] 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 describe the various possible combinations separately.

[0205] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content 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 contains at least one chromosome containing a nucleotide sequence encoding a human or chimeric MAS-associated GPR family member X2 (MRGPRX2) protein.

2. The construction method according to claim 1, characterized in that, The amino acid sequence of the human or chimeric MRGPRX2 protein comprises SEQ ID NO: 2; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to SEQ ID NO:

2. Preferably, one or more cells of the non-human animal express human or chimeric MRGPRX2 protein; Preferably, the cells are mast cells.

3. A method for constructing a genetically modified non-human animal, characterized in that, The nucleotide sequence of human MRGPRX2 was introduced into the genome of a non-human animal; Preferably, the importation of the non-human animal genome includes the importation of a non-human animal endogenous MRGPRB2 or safe harbor site (e.g., ROSA26 site or Hipp11 site).

4. The construction method according to any one of claims 1-3, characterized in that, The nucleotide sequence encoding the human or chimeric MRGPRX2 protein or the nucleotide sequence of human MRGPRX2 is operatively linked to an endogenous regulatory element (e.g., the endogenous MRGPRB2 promoter) or an exogenous regulatory element (e.g., the CAG promoter). Preferably, the endogenous MRGPRB2 protein in the non-human animal is not expressed or its expression level is reduced compared with that in wild-type animals; Preferably, the modified MRGPRX2 gene in the non-human animal genome is homozygous or heterozygous for the endogenously modified locus; Preferably, one or more cells of the non-human animal express human or chimeric MRGPRX2 protein; Preferably, the cells are mast cells; More preferably, the cells are characterized by CD45. + CD117 + CD11b - .

5. The construction method according to claim 3, characterized in that, The nucleotide sequence of the human MRGPRX2 includes the nucleotide sequence encoding the human or chimeric MRGPRX2 protein; Preferably, the nucleotide sequence of the human MRGPRX2 comprises the nucleotide sequence encoding SEQ ID NO: 2; or comprises a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the nucleotide sequence encoding SEQ ID NO:

2. Preferably, the nucleotide sequence of the human MRGPRX2 can be a genomic DNA sequence, a CDS sequence, or a cDNA sequence; Preferably, the nucleotide sequence of the human MRGPRX2 contains all or part of exon 3 of the human MRGPRX2 gene; Preferably, the nucleotide sequence of the human MRGPRX2 contains the nucleotide sequence from the start codon to the stop codon of the human MRGPRX2 gene; more preferably, it also contains a 3'UTR; more preferably, it also contains a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR; Preferably, the nucleotide sequence of the human MRGPRX2 contains at least 1000 bp of continuous nucleotide sequence downstream of the start codon to the 3'UTR of the human MRGPRX2 gene; Preferably, the nucleotide sequence of the human MRGPRX2 comprises the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 39; or comprises a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO:

39. More preferably, the non-human animal genome contains SEQ ID NO: 14, or contains a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the nucleotide sequence shown in SEQ ID NO:

14.

6. The construction method according to any one of claims 3-5, characterized in that, The nucleotide sequence of the non-human animal endogenous MRGPRB2 contains a nucleotide sequence encoding the non-human animal endogenous MRGPRB2 protein. Preferably, the nucleotide sequence of the non-human animal endogenous MRGPRB2 comprises the nucleotide sequence encoding SEQ ID NO: 1; or comprises a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with the nucleotide sequence encoding SEQ ID NO:

1. Preferably, the nucleotide sequence of the non-human animal endogenous MRGPRB2 contains all or part of exon 2 of the non-human animal endogenous MRGPRB2 gene; Preferably, the nucleotide sequence of the non-human animal endogenous MRGPRB2 contains the nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous MRGPRB2 gene, and more preferably it also contains a 3'UTR, and more preferably it also contains a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR. Preferably, the nucleotide sequence of the non-human animal endogenous MRGPRB2 contains at least 963 bp of continuous nucleotide sequence from the start codon of the non-human animal endogenous MRGPRB2 gene downstream of the 3'UTR.

7. The construction method according to any one of claims 3-6, characterized in that, The sequence of the safe harbor site introduced into the non-human animal includes the nucleotide sequence of human MRGPRX2 and the STOP sequence conditionally controlled by recombinase. The recombinase is introduced into the non-human animal to obtain non-human animals that specifically express human or chimeric MRGPRX2 in cells expressing the recombinase. Preferably, the recombinase is conditionally controlled by regulating gene expression through the Cre / Loxp system or the Flp / Frt system; Preferably, the cells expressing the recombinase are mast cells; Preferably, the expression of the recombinase is regulated by tissue-specific and / or inducible promoters; Preferably, the expression of the recombinase is regulated by a non-human animal endogenous MRGPRB2 regulatory element (e.g., the MRGPRB2 promoter); Preferably, the introduction of recombinase into non-human animals includes mating a non-human animal containing the nucleotide sequence of human MRGPRX2 and a STOP sequence conditionally controlled by the recombinase with a non-human animal containing tissue-specific and / or inducible CRE recombinase.

8. The construction method according to any one of claims 1-7, characterized in that, The non-human animal is a mammal; preferably, the non-human mammal includes monkeys and rodents; more preferably, the rodent includes mice or rats. Preferably, the mRNA transcribed from the modified MRGPRX2 gene in the non-human animal genome contains SEQ ID NO: 26 or 43; or contains a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% uniformity with the nucleotide sequence shown in SEQ ID NO: 26 or 43. Preferably, the non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins encoded by other genes are selected from at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

9. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, characterized in that, The method includes: 1) Administering the therapeutic agent to a non-human animal obtained by any of the construction methods described in claims 1-8; 2) To determine the inhibitory effect of the therapeutic agent on the disease; or to determine the effect of the therapeutic agent on non-human animals; Preferably, the therapeutic agent includes a therapeutic agent that targets MRGPRX2; preferably, it may also include other therapeutic agents; Preferably, the therapeutic agent targeting MRGPRX2 is an antibody against human MRGPRX2, a nucleic acid drug targeting MRGPRX2, and / or a peptide drug. Preferably, the other therapeutic agents include anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies; Preferably, the disease is one associated with MRGPRX2 expression, such as tumors, immune-related diseases, or inflammation; Preferably, the tumor is a solid tumor or a hematologic tumor; more preferably, the tumor includes one or more of head and neck cancer, liver cancer, or lung cancer. Preferably, the immune-related diseases include one or more of the following: allergies, chronic urticaria, pruritus, asthma, psoriasis, rheumatoid arthritis, or multiple sclerosis; Preferably, the inflammation includes inflammatory bowel disease (IBD).

10. An application of a non-human animal obtained by the construction method according to any one of claims 1-8, characterized in that, The applications include: A) Applications in product development involving MRGPRX2-related immune processes in human cells; B) Applications as a model system related to MRGPRX2 in pharmacological, immunological, microbiological and medical research; C) Involving the production and use of animal experimental disease models for etiological studies related to MRGPRX2 and / or for the development of diagnostic and / or therapeutic strategies; D) Application in in vivo studies of the screening, efficacy testing, evaluation, validation, or assessment of modulators of the human MRGPRX2 signaling pathway; or, E) Research the function of the MRGPRX2 gene, study drugs and their efficacy targeting the human MRGPRX2 target site, and study the application of drugs in the treatment of diseases related to MRGPRX2 (such as tumors, inflammation, and immune-related diseases).

11. A humanized MRGPRX2 gene, characterized in that, The humanized MRGPRX2 gene includes: A) The nucleotide sequence encoding SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26, 28, 29, 37, 38, 39, 40, 41, 42, 43, 44 or 45; C) A nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26, 28, 29, 37, 38, 39, 40, 41, 42, 43, 44 or 45; D) A nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26, 28, 29, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

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