A tnfrsf25 gene-modified non-human animal

Abstract

The present application provides a non-human animal expressing a human or chimeric (e.g., humanized) TNFRSF25 protein and methods of using the same.

Description

Technical Field

[0001] This application provides a non-human animal expressing human or chimeric (e.g., humanized) TNFRSF25 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 human drugs 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 tumor necrosis factor receptor superfamily member 25 (TNFRSF25). This animal model can express human or chimeric TNFRSF25 (e.g., humanized TNFRSF25) protein. It can be used to study the function of the TNFRSF25 gene and for screening and evaluating TNFRSF25 signaling pathway regulators (the TNFRSF25 signaling pathway regulators are therapeutic agents targeting TNFRSF25, such as anti-TNFRSF25 antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25). Furthermore, the animal model prepared by the method described in this application can be used for drug screening, pharmacodynamic studies, and the treatment of diseases related to the human TNFRSF25 target (e.g., cancer, immune-related diseases, or inflammation); this animal model can also be used to facilitate new drug development and design, saving time and costs. In summary, this application provides a powerful tool for studying the function of the TNFRSF25 protein and a platform for screening therapeutic drugs for related diseases.

[0005] In one aspect, this application provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises at least one chromosome, the chromosome containing a nucleotide sequence encoding a human or chimeric TNFRSF25 protein. In some embodiments, the amino acid sequence of the chimeric TNFRSF25 protein comprises an amino acid sequence consistent with at least 50 to 417 consecutive amino acid sequences of the human TNFRSF25 protein, such as 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, or 417. In some embodiments, the nucleotide sequence encoding the human or chimeric TNFRSF25 protein is operatively linked to an exogenous regulatory element (e.g., a human TNFRSF25 promoter, 5'UTR, and / or 3'UTR) of at least one chromosome. In some embodiments, the amino acid sequence of the human or chimeric TNFRSF25 protein comprises human TNFRSF25 (NP_003781.1, SEQ ID NO: 2), or comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with human TNFRSF25 (NP_003781.1, SEQ ID NO: 2). In some embodiments, the nucleotide sequence encoding the human or chimeric TNFRSF25 protein may be the genomic DNA, cDNA, or CDS sequence of human TNFRSF25. 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. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous TNFRSF25 protein in the non-human animal is not expressed or its expression level is reduced compared to TNFRSF25 in wild-type animals. In some embodiments, one or more cells of the non-human animal express the human or chimeric TNFRSF25 protein.

[0006] In one aspect, this application provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal contains an endogenous TNFRSF25 locus, and the nucleotide sequence of the endogenous TNFRSF25 is replaced by the nucleotide sequence of human TNFRSF25. In some embodiments, the nucleotide sequence of the human TNFRSF25 comprises at least 5 bp to 10359 bp of continuous nucleotides of the human TNFRSF25 gene, for example, at least 5, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1968, 2000, 3000, 4000, 5000, 5388, 6000, 7000, 8000, 9000, 10000, or 10359 bp of continuous nucleotides. In some embodiments, the nucleotide sequence of the human TNFRSF25 comprises a nucleotide sequence encoding a human or chimeric TNFRSF25 protein. In some embodiments, the nucleotide sequence of the human TNFRSF25 is operatively linked to an exogenous regulatory element (e.g., the human TNFRSF25 promoter, 5'UTR, and / or 3'UTR). In some embodiments, one or more cells of the non-human animal express human or humanized TNFRSF25 protein. In some embodiments, the endogenous TNFRSF25 protein of the non-human animal is not expressed or is expressed at a reduced level compared to TNFRSF25 in wild-type animals. In some embodiments, the modified TNFRSF25 gene in the non-human animal genome is homozygous or heterozygous for the endogenously substituted locus.

[0007] The nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein is a genomic DNA, cDNA, or CDS sequence of human TNFRSF25. In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises all or part of exons 1 to 10 of the human TNFRSF25 gene. In some embodiments, the nucleotide sequence of the human TNFRSF25 or the nucleotide sequence encoding the human or chimeric TNFRSF25 protein comprises at least 50 bp, at least 100 bp, at least 200 bp, at least 500 bp, at least 1000 bp, at least 1500 bp, at least 2000 bp, at least 2500 bp, at least 3000 bp, at least 3500 bp, at least 4000 bp, at least 4500 bp, or at least 5000 bp of continuous nucleotides upstream of the 5'UTR and / or at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 471 bp, or at least 500 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of the human TNFRSF25 or the nucleotide sequence encoding the human or chimeric TNFRSF25 protein comprises at least 50 bp, at least 100 bp, at least 200 bp, at least 500 bp, at least 1000 bp, at least 1500 bp, at least 2000 bp, at least 2500 bp, at least 3000 bp, at least 3500 bp, at least 4000 bp, at least 4500 bp, or at least 5000 bp of continuous nucleotides and all of exons 1 to 10 upstream of the 5'UTR of the human TNFRSF25 gene. In some embodiments, the nucleotide sequence of the human TNFRSF25 or the nucleotide sequence encoding the human or chimeric TNFRSF25 protein comprises at least 50 bp upstream of the 5'UTR of the human TNFRSF25 gene to exon 10. In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises all of exons 1 to 10 of the human TNFRSF25 gene and at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 471 bp, or at least 500 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises at least 50 bp downstream of exons 1 to 3'UTR of the human TNFRSF25 gene.In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, all of exons 1 to 10, and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises all of exons 1 to 10 of the human TNFRSF25 gene and at least 471 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises at least 4500 bp of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, all of exons 1 to 10, and at least 471 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises at least 4500 bp of continuous nucleotides upstream of the 5'UTR to at least 471 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises SEQ ID NO: 5, or comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity to SEQ ID NO: 5. In some embodiments, the nucleotide sequence of endogenous TNFRSF25 comprises a nucleotide sequence encoding a non-human animal TNFRSF25 protein. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises the nucleotide sequence encoding SEQ ID NO: 1, 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: 1. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises all or part of exons 1 to 10 of the non-human animal (e.g., mouse) TNFRSF25 gene. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises all or part of exons 1 to 10 of the non-human animal (e.g., mouse) TNFRSF25 gene.In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises all exons 1 to 9 and a portion of exon 10 of the non-human animal (e.g., mouse) TNFRSF25 gene, preferably also including intron 9. In some embodiments, the portion of exon 10 of the non-human animal TNFRSF25 gene comprises a continuous nucleotide sequence of at least 5 bp to 598 bp of exon 10, for example, a continuous nucleotide sequence of at least 5, 10, 50, 100, 150, 200, 250, 300, 329, 350, 400, 450, 500, 550, or 598 bp. In some embodiments, the portion of exon 10 of the non-human animal TNFRSF25 gene comprises the first nucleotide of exon 10 up to the stop codon. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises exons 1 to the stop codon of the non-human animal (e.g., mouse) TNFRSF25 gene. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises all exons 1 to 9 and the entire coding region of exon 10 of the non-human animal (e.g., mouse) TNFRSF25 gene (preferably also including intron 9). In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 further comprises at least 50 bp, at least 100 bp, at least 150 bp, at least 168 bp, or at least 200 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises all exons 1 to 9, a portion of exon 10 (including intron 9), and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal (e.g., mouse) TNFRSF25. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal (e.g., mouse) TNFRSF25 gene, all exons 1 to 9, and the entire coding region of exon 10 (preferably also including intron 9). In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human TNFRSF25 gene, and exon 1 to the stop codon. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human TNFRSF25 gene to the stop codon. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human TNFRSF25 gene to the entire coding region of exon 10. In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises all exons 1 to 9 of the non-human (e.g., mouse) TNFRSF25 gene, the entire coding region of exon 10, and at least 168 bp of continuous nucleotides upstream of the 5'UTR (preferably also including intron 9).In some embodiments, the nucleotide sequence of the endogenous TNFRSF25 comprises at least 168 bp of continuous nucleotides upstream of the 5'UTR of the nonhuman animal TNFRSF25 gene, and exon 1 to the stop codon.

[0008] In one aspect, this application provides a non-human animal or a method for constructing the same, the non-human animal comprising a cell encoding at least one nucleotide sequence of a human or humanized TNFRSF25 protein, wherein the humanized TNFRSF25 protein comprises at least 50, 100, 200, 300, 400, 410, 415, or 417 consecutive amino acid sequences consistent with the sequence of a human TNFRSF25 protein, and the non-human animal expresses the human or humanized TNFRSF25 protein. In some embodiments, the nucleotide sequence encoding the human or humanized TNFRSF25 protein is operatively linked to an exogenous regulatory element (e.g., a human TNFRSF25 promoter, a 5'UTR, and / or a 3'UTR). In some embodiments, the nucleotide sequence encoding the human or humanized TNFRSF25 protein may be integrated into an endogenous TNFRSF25 locus in the non-human animal. In some embodiments, the human or humanized TNFRSF25 protein has at least one non-human animal (e.g., mouse) TNFRSF25 activity and / or human TNFRSF25 activity.

[0009] In one aspect, this application provides a method for constructing a genetically modified non-human animal, wherein in at least one cell of the non-human animal, at the non-human animal's endogenous TNFRSF25 gene locus, the nucleotide sequence encoding the endogenous TNFRSF25 protein is replaced by a nucleotide sequence encoding the human TNFRSF25 protein. In some embodiments, the endogenous TNFRSF25 protein in the non-human animal is not expressed or its expression level is reduced compared to that in wild-type animals. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all or part of exons 1 to 10 of the human TNFRSF25 gene. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all of exons 1 to 10 of the human TNFRSF25 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all of exons 1 to 10 of the human TNFRSF25 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all exons 1 to 10 of the human TNFRSF25 gene and at least 471 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all exons 1 to 10 of the human TNFRSF25 gene, at least 471 bp of continuous nucleotides downstream of the 3'UTR, and at least 4500 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the amino acid sequence of the human TNFRSF25 protein comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all or part of exons 1 to 10 of the non-human animal (e.g., mouse) TNFRSF25 gene. In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all of exons 1 to 9 and part of exon 10 (preferably also including intron 9) of the non-human animal (e.g., mouse) TNFRSF25 gene. In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all of exons 1 to 9, part of exon 10 (preferably also including intron 9) of the non-human animal (e.g., mouse) TNFRSF25 gene, and at least 50 bp of continuous nucleotides upstream of the 5'UTR.In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all exons 1 to 9 and the entire coding region of exon 10 of the non-human animal (e.g., mouse) TNFRSF25 gene (preferably also including intron 9). In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all exons 1 to 9, the entire coding region of exon 10, and at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal (e.g., mouse) TNFRSF25 gene. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein is operatively linked to an exogenous regulatory element (e.g., the human TNFRSF25 promoter, the 5'UTR, and / or the 3'UTR).

[0010] In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat.

[0011] In one aspect, this application provides a method for constructing gene-modified non-human animal cells expressing human or chimeric TNFRSF25. The method includes replacing the nucleotide sequence encoding the endogenous TNFRSF25 protein at the endogenous TNFRSF25 gene locus in a non-human animal (e.g., a mouse) with the nucleotide sequence encoding the human TNFRSF25 protein, thereby generating gene-modified non-human animal cells expressing human or chimeric TNFRSF25 protein. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all or part of exons 1 to 10 of the human TNFRSF25 gene. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all exons 1 to 10 of the human TNFRSF25 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all exons 1 to 10 of the human TNFRSF25 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all exons 1 to 10 of the human TNFRSF25 gene and at least 471 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises all exons 1 to 10 of the human TNFRSF25 gene, at least 471 bp of continuous nucleotides downstream of the 3'UTR, and at least 4500 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the amino acid sequence of the human TNFRSF25 protein comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human TNFRSF25 protein comprises a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all or part of exons 1 to 10 of the non-human animal (e.g., mouse) TNFRSF25 gene. In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all of exons 1 to 9 and part of exon 10 of the non-human animal (e.g., mouse) TNFRSF25 gene (preferably also including intron 9). In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all of exons 1 to 9, part of exon 10, and at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal (e.g., mouse) TNFRSF25 gene (preferably also including intron 9).In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all exons 1 to 9 and the entire coding region of exon 10 of the non-human animal (e.g., mouse) TNFRSF25 gene, preferably also comprising intron 9. In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein comprises all exons 1 to 9, the entire coding region of exon 10, and at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal (e.g., mouse) TNFRSF25 gene, preferably also comprising intron 9. In some embodiments, the nucleotide sequence encoding the human or chimeric TNFRSF25 protein is operatively linked to an exogenous regulatory element (e.g., the human TNFRSF25 promoter, 5'UTR, and / or 3'UTR). In some embodiments, the non-human animal is a mouse.

[0012] In some embodiments, the construction method includes replacing the nucleotide sequence encoding the human or chimeric TNFRSF25 protein (preferably SEQ ID NO: 2) (preferably genomic DNA, CDS, or cDNA, such as SEQ ID NO: 5 or 7) of the non-human animal endogenous TNFRSF25 protein (preferably SEQ ID NO: 1). In some embodiments, the construction method includes replacing exons 1-10 of the non-human animal endogenous TNFRSF25 gene with the nucleotide sequence encoding the human or chimeric TNFRSF25 protein (preferably SEQ ID NO: 2) (preferably genomic DNA, CDS, or cDNA, such as SEQ ID NO: 5 or 7). In some embodiments, the construction method includes replacing portions of exons 1-9 and exon 10 of the non-human animal endogenous TNFRSF25 gene (preferably also including intron 9) with the nucleotide sequence encoding the human or chimeric TNFRSF25 protein (preferably SEQ ID NO: 2) (preferably genomic DNA, CDS, or cDNA, such as SEQ ID NO: 5 or 7). In some embodiments, the construction method includes replacing exon 1 to the stop codon of the non-human animal endogenous TNFRSF25 gene with a nucleotide sequence (preferably genomic DNA, CDS, or cDNA, such as SEQ ID NO: 5 or 7) encoding a human or chimeric TNFRSF25 protein (preferably SEQ ID NO: 2). In some embodiments, the construction method includes replacing at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene, and exon 1 to the stop codon, with a nucleotide sequence (preferably genomic DNA, CDS, or cDNA, such as SEQ ID NO: 5 or 7) encoding a human or chimeric TNFRSF25 protein (preferably SEQ ID NO: 2). In some embodiments, the construction method includes replacing at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene with a nucleotide sequence (preferably genomic DNA, CDS, or cDNA, such as SEQ ID NO: 5 or 7) encoding a human or chimeric TNFRSF25 protein (preferably SEQ ID NO: 2). In some embodiments, the construction method includes replacing exons 1-10 of the human TNFRSF25 gene with nucleotide sequences encoding the non-human animal endogenous TNFRSF25 protein (preferably SEQ ID NO: 1). In some embodiments, the construction method includes replacing all or part of exons 1-10 of the human TNFRSF25 gene with exons 1-10 of the non-human animal endogenous TNFRSF25 gene. In some embodiments, the construction method includes replacing exons 1-9 and a portion of exon 10 of the non-human animal endogenous TNFRSF25 gene with exons 1-10 of the human TNFRSF25 gene (preferably also including intron 9).In some embodiments, the construction method includes replacing exons 1-10 of the human TNFRSF25 gene with exons 1 to 10 of the non-human animal endogenous TNFRSF25 gene, from exon 1 to 10 ... In some embodiments, the construction method includes replacing all or part of exons 1-10 of the non-human animal endogenous TNFRSF25 gene with at least 50 bp (preferably at least 4500 bp) of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, and at least 50 bp (preferably at least 471 bp) of continuous nucleotides downstream of exons 1-10 and 3'UTR. In some embodiments, the construction method includes replacing a portion of exons 1-9 and exon 10 of the non-human animal endogenous TNFRSF25 gene (preferably also including intron 9) with at least 50 bp (preferably at least 4500 bp) of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, and at least 50 bp (preferably at least 471 bp) of continuous nucleotides downstream of exons 1-10 and 3'UTR. In some embodiments, the construction method includes replacing at least 50 bp (preferably at least 4500 bp) of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, and at least 50 bp (preferably at least 471 bp) of continuous nucleotides downstream of exons 1-10 and the 3'UTR with exons 1 to 471 bp of the non-human animal endogenous TNFRSF25 gene. In some embodiments, the construction method includes replacing at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene, and at least 50 bp (preferably at least 471 bp) of continuous nucleotides downstream of exons 1-10 and the 3'UTR with exons 1 to 471 bp of the human TNFRSF25 gene.In some embodiments, the construction method includes replacing at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene, and at least 50 bp (preferably at least 4500 bp) of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, and at least 50 bp (preferably at least 471 bp) of continuous nucleotides downstream of exons 1-10 and the 3'UTR with exons 1 to the stop codon.

[0013] In some embodiments, the mRNA transcribed from the modified TNFRSF25 gene in the non-human animal genome contains SEQ ID NO: 7, 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: 7.

[0014] In some embodiments, the non-human animal further includes the nucleotide sequence of another human or chimeric protein selected from at least one of TL1A, IL23A, IL12B, A4B7, TNFA, TNFR1, TNFR2, IL6, IL6R, TREM1, PGLYRP1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. In some embodiments, the human or chimeric protein includes a human or chimeric TL1A protein. In some embodiments, the amino acid sequence of the human or chimeric TL1A protein comprises SEQ ID NO: 34 or an amino acid sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% of the identity with SEQ ID NO: 34. In some embodiments, the method of constructing a non-human animal expressing a human or chimeric TL1A protein includes replacing the nucleotide sequence encoding the endogenous TL1A protein (e.g., NP_796345.4) at the endogenous TL1A gene locus of the non-human animal with the nucleotide sequence encoding the human or chimeric TL1A protein (e.g., NP_005109.2 or SEQ ID NO: 34, preferably the genomic DNA, CDS, or cDNA sequence of the human TL1A gene). In some embodiments, the construction method includes replacing all or part of the nucleotide sequence of exons 1-4 of the endogenous TL1A gene of the non-human animal with the nucleotide sequence encoding the human or chimeric TL1A protein (e.g., NP_005109.2 or SEQ ID NO: 34, preferably the genomic DNA, CDS, or cDNA sequence of the human TL1A gene). In some embodiments, the construction method includes replacing a portion of the nucleotide sequence of exon 1 to exon 4 of the non-human animal endogenous TL1A gene with the nucleotide sequence encoding a human or chimeric TL1A protein (e.g., NP_005109.2 or SEQ ID NO: 34, preferably the genomic DNA, CDS, or cDNA sequence of the human TL1A gene). In some embodiments, the construction method includes replacing the nucleotide sequence of the start codon to the stop codon of the non-human animal endogenous TL1A gene with the nucleotide sequence encoding a human or chimeric TL1A protein (e.g., NP_005109.2 or SEQ ID NO: 34, preferably the genomic DNA, CDS, or cDNA sequence of the human TL1A gene). In some embodiments, the construction method includes replacing all or part of the nucleotide sequence of the non-human animal endogenous TL1A protein encoding exons 1-4 with all or part of the exons 1-4 of the human TL1A gene. In some embodiments, the construction method includes replacing all or part of the exons 1-4 of the non-human animal endogenous TL1A gene with all or part of the exons 1-4 of the human TL1A gene.In some embodiments, the construction method includes replacing all or part of exons 1-4 of the human TL1A gene with portions of exons 1 to 4 of the non-human animal endogenous TL1A gene. In some embodiments, the construction method includes replacing all or part of exons 1-4 of the human TL1A gene with the start codon to the stop codon of the non-human animal endogenous TL1A gene. In some embodiments, the construction method includes replacing the nucleotide sequence encoding the TL1A protein in the non-human animal endogenous TL1A gene with the start codon to the stop codon. In some embodiments, the construction method includes replacing all or part of exons 1-4 of the non-human animal endogenous TL1A gene with the start codon to the stop codon. In some embodiments, the construction method includes replacing portions of exons 1 to 4 of the non-human animal endogenous TL1A gene with the start codon to the stop codon. In some embodiments, the construction method includes replacing the start codon to the stop codon of the non-human animal endogenous TL1A gene with the start codon to the stop codon.

[0015] In one aspect, this application provides an application of the non-human animal described herein or a non-human animal obtained by the described construction method, the application comprising: A) applications in product development involving immune processes related to TNFRSF25 and / or TL1A in human cells; B) applications as model systems related to TNFRSF25 and / or TL1A in pharmacological, immunological, microbiological, and medical research; C) applications involving the production and use of animal experimental disease models for etiological research related to TNFRSF25 and / or TL1A and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) applications in the in vivo study of human TNFRSF25 and / or TL1A signaling pathway modulators, including screening, efficacy testing, efficacy assessment, validation, or evaluation; or E) applications in studying TNFRSF25 and / or TL1A gene function, studying drugs and their efficacy targeting human TNFRSF25 and / or TL1A, and studying drugs for cancer, immune-related diseases, or inflammation related to TNFRSF25 and / or TL1A.

[0016] In one aspect, this application provides a method for determining the toxicity of a therapeutic agent or its effectiveness in treating a disease, 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 animal or its inhibitory effect on the disease. In some embodiments, the therapeutic agent comprises a therapeutic agent targeting TNFRSF25 and / or TL1A. In some embodiments, the therapeutic agent further comprises other therapeutic agents. In some embodiments, the therapeutic agent targeting TNFRSF25 and / or TL1A includes, but is not limited to, anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A. In some embodiments, the other therapeutic agents include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies. In some embodiments, the disease includes cancer, immune-related diseases, or inflammation. In some embodiments, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the solid tumor includes head and neck cancer, breast cancer, liver cancer, or lung cancer. In some embodiments, the hematologic malignancy includes lymphocytic tumors (e.g., T-cell tumors or B-cell tumors). In some embodiments, the immune-related disease includes asthma, rheumatoid arthritis, psoriasis, or multiple sclerosis. In some embodiments, the inflammation includes inflammatory bowel disease (IBD) or atopic dermatitis.

[0017] In one aspect, this application provides a method for determining the effectiveness of a therapeutic agent in treating cancer, the method comprising: 1) administering the therapeutic agent to a non-human animal or a non-human animal obtained by the construction method, wherein the non-human animal has a tumor; and 2) determining the inhibitory effect of the therapeutic agent on the tumor. In some embodiments, the therapeutic agent comprises a therapeutic agent targeting TNFRSF25 and / or TL1A. In some embodiments, the therapeutic agent includes, but is not limited to, anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A. In some embodiments, the tumor comprises one or more tumor cells injected into the animal. In some embodiments, determining the inhibitory effect of the therapeutic agent on the tumor involves measuring the tumor volume in the animal. In some embodiments, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the solid tumor includes head and neck cancer, breast cancer, liver cancer, or lung cancer. In some embodiments, the hematologic malignancy includes lymphocytic tumors (e.g., T-cell tumors or B-cell tumors).

[0018] In one aspect, this application provides a method for determining the effectiveness of TNFRSF25 and / or TL1A therapeutic agents and other therapeutic agents in treating cancer, the method comprising: 1) administering TNFRSF25 and / or TL1A therapeutic agents and other therapeutic agents to a non-human animal or a non-human animal obtained by the construction method, the non-human animal having a tumor; 2) determining the inhibitory effect on the tumor. 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 other therapeutic agents include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies. 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 an animal. In some embodiments, determining the inhibitory effect on the tumor involves measuring the tumor volume in the non-human animal. In some embodiments, the non-human animal has a solid tumor or hematologic malignancy. In some embodiments, the non-human animal has breast cancer, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the non-human animal suffers from a hematologic malignancy, including lymphocytic tumors (e.g., T-cell or B-cell tumors). In some embodiments, the other therapeutic agents include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies.

[0019] In one aspect, this application 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 a non-human animal or a non-human animal obtained by the construction method, wherein the non-human animal suffers from an immune-related disease; and 2) determining the therapeutic effect of the therapeutic agent on the immune-related disease. In some embodiments, the therapeutic agent comprises a therapeutic agent targeting TNFRSF25 and / or TL1A. In some embodiments, the therapeutic agent includes, but is not limited to, anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A. In some embodiments, the immune-related disease includes asthma, rheumatoid arthritis, psoriasis, or multiple sclerosis.

[0020] In one aspect, this application 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 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 comprises a therapeutic agent targeting TNFRSF25 and / or TL1A. In some embodiments, the therapeutic agent includes, but is not limited to, anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A. In some embodiments, the inflammation includes inflammatory bowel disease (IBD) or atopic dermatitis.

[0021] In one aspect, this application 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, the therapeutic agent comprises a therapeutic agent targeting TNFRSF25 and / or TL1A. In some embodiments, the therapeutic agent includes, but is not limited to, anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A. In some embodiments, determining the effect of the therapeutic agent on the non-human animal involves measuring changes in the animal's body weight or blood tests. In some embodiments, the blood tests include, but are not limited to, red blood cell count, hematocrit, and / or hemoglobin.

[0022] In one aspect, this application provides a humanized TNFRSF25 gene, which comprises any of the following nucleotide sequences: A) The nucleotide sequence encoding SEQ ID NO: 2; B) The nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 35; C) A nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 35; D) A nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 35.

[0023] In one aspect, this application provides a cell, tissue, or organ that contains the humanized TNFRSF25 gene.

[0024] In one aspect, this application provides an animal model comprising the humanized TNFRSF25 gene.

[0025] In one aspect, this application provides a non-human animal genome comprising at least one chromosome, the chromosome containing a nucleotide sequence encoding a human or chimeric TNFRSF25 protein. In some embodiments, the chromosome contains a human or chimeric TNFRSF25 gene. In some embodiments, the chimeric TNFRSF25 gene is a humanized TNFRSF25 gene. In some embodiments, the chromosome contains a nucleotide sequence encoding a human TNFRSF25 protein. In some embodiments, the chromosome contains a genomic DNA, CDS, or cDNA sequence of a human TNFRSF25 gene. In some embodiments, the chromosome contains all or part of exons 1-10 of the human TNFRSF25 gene. In some embodiments, the chromosome contains at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, exons 1-10, and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the chromosome contains at least 50 bp of continuous nucleotides from upstream of the 5'UTR of the human TNFRSF25 gene to at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the chromosome comprises at least 4500 bp of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, exons 1-10, and at least 471 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the chromosome comprises at least 4500 bp of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene to at least 471 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, TNFRSF25 in the non-human animal endogenous genome is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal endogenous genome is replaced. In some embodiments, all or part of exons 1-10 of the non-human animal endogenous TNFRSF25 gene (preferably exons 1-9 and a portion of exon 10, further including intron 9) is replaced. In some embodiments, exons 1 to the stop codon of the non-human animal endogenous TNFRSF25 gene are replaced. In some embodiments, at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene and all or part of exons 1-10 are replaced. In some embodiments, at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene, and exon 1 to the stop codon, are replaced. In some embodiments, at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene, and the stop codon, are replaced. In some embodiments, the chromosome contains a nucleotide sequence encoding a human or chimeric TNFRSF25 protein, or a nucleotide sequence of human TNFRSF25 replaces the corresponding portion of the non-human animal endogenous chromosome TNFRSF25.In some embodiments, the nucleotide sequence encoding SEQ ID NO: 2 replaces the nucleotide sequence encoding SEQ ID NO: 1 from a non-human animal endogenous source.

[0026] In some embodiments, the cells, tissues, or organs described in this application can develop into an animal individual. In some embodiments, the cells, tissues, or organs described in this application cannot develop into an animal individual.

[0027] In some embodiments, the methods and applications described in this application may be for the purpose of diagnosing or treating a disease, or for the purpose of diagnosing or treating a non-disease.

[0028] In this application, the term "whole or part" means "whole" as the whole and "part" as a part of the whole, or an individual component of the whole.

[0029] 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, a part of a gene, or a regulatory region of a gene. For example, the "TNFRSF25 locus" includes exons 1-10 of the TNFRSF25 gene and a selected segment of DNA from its regulatory region.

[0030] The term "part of exon XX" in this application means that a sequence of several, dozens, or hundreds of nucleotides, either consecutively or at intervals, is identical to the complete exon nucleotide sequence.

[0031] The terms “exon XX to exon XXX”, “exon XX-XXX”, “all exons XX to exon XXX”, or “all exons XX to all exons XXX” in this application refer to exons and introns that include exons and introns between them.

[0032] The term "all or part of exon x to part of exon xx" in this application includes all or part of exon x, part of exon xx, and exons and introns in between. For example, "all of exon 1 to part of exon 10" includes all of exon 1, all of intron 1, all of exon 2, all of intron 2, all of exon 3, all of intron 3, all of exon 4, all of intron 4, all of exon 5, all of intron 5, all of exon 6, all of intron 6, all of exon 7, all of intron 7, all of exon 8, all of intron 8, all of exon 9, all of intron 9, and part of exon 10.

[0033] The term "intron xx" in this application refers to an intron between two exons, for example, intron 9 is an intron between exon 9 and exon 10.

[0034] The terms "comprising" or "including" in this application are open-ended, 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 itself, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the same or similar activity as the original sequence.

[0035] The term "and / or" in this application includes all combinations of the items connected by the term, and should be regarded as each combination having been individually listed in this application. For example, "A and / or B" includes "A", "A and B", 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".

[0036] 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 application pertains. This application describes the methods and materials used in this application; 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.

[0037] Other aspects and advantages of this application will be readily apparent to those skilled in the art from the detailed description below.

[0038] Detailed description TNFRSF25 In the human genome, the TNFRSF25 gene (NCBI Gene ID: 8718, UniProt ID: Q93038, located on chromosome 1 NC_000001.11, vertices 6460786 to 6466173) contains 10 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, and exon 10. Table 1 shows the corresponding positions of each exon based on the nucleotide and amino acid sequences of transcript NM_003790.3 and its encoded protein NP_003781.1 (SEQ ID NO: 2).

[0039] Table 1

[0040] In the mouse genome, the TNFRSF25 gene (NCBI Gene ID: 85030, UniProt ID: B1AWN9, located on chromosome 4 NC_000070.7, vertices 152199985 to 152204568) contains 10 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, and exon 10. The positions of each exon in the nucleotide and amino acid sequences of the transcript NM_001291010.2 and its encoded protein NP_001277939.1 (SEQ ID NO: 1) are shown in Table 2.

[0041] Table 2

[0042] The TNFRSF25 gene, protein, and gene loci of other species in this field are also known. Examples include *Rattus norvegicus* (rat), *Macaca mulatta* (rhesus monkey), *Canis lupus familiaris* (dog), and *Susscrofa* (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.

[0043] 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.

[0044] 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.

[0045] TNFRSF25 gene vector This application provides a targeting vector comprising: a) a DNA fragment (5' arm or 5' homologous arm) homologous to the 5' end of the region to be modified, which is selected from the genomic DNA of the TNFRSF25 gene and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment (3' arm or 3' homologous arm) homologous to the 3' end of the region to be modified, which is selected from the genomic DNA of the TNFRSF25 gene and has a length of 100 to 10,000 nucleotides.

[0046] In some embodiments, a) the DNA fragment homologous to the 5' end of the region to be altered is selected from a nucleotide sequence having at least 90% homology with NCBI accession number NC_000070.7; c) the DNA fragment homologous to the 3' end of the region to be altered is selected from a nucleotide sequence having at least 90% homology with NCBI accession number NC_000070.7.

[0047] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector may exceed approximately 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 18 kb, 19 kb, or 20 kb.

[0048] In some embodiments, the region to be modified is located on the non-human animal TNFRSF25 gene. In some embodiments, the region to be modified is located on exons 1 to 10 of the non-human animal TNFRSF25 gene. In some embodiments, the region to be modified is located on the 5'UTR to exon 10 of the non-human animal TNFRSF25 gene. In some embodiments, the region to be modified is located on at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal TNFRSF25 gene to exon 10. In some embodiments, the region to be modified is located on at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal TNFRSF25 gene to the stop codon of exon 10.

[0049] In some embodiments, the 5' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the 3' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 4.

[0050] In some embodiments, the 5' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 8. In some embodiments, the 3' arm sequence comprises the nucleotide sequence shown in SEQ ID NO: 9.

[0051] In some embodiments, the targeting vector comprises a human sequence (e.g., a donor sequence of a nucleotide sequence of human TNFRSF25, such as the nucleotide sequence encoding SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 7). For example, exons 1 to 10 of the human TNFRSF25 gene are preferred. Preferably, at least 50 bp of continuous nucleotides upstream of the 5'UTR and at least 50 bp of continuous nucleotides downstream of the 3'UTR are present. Preferably, at least 4500 bp of continuous nucleotides upstream of the 5'UTR and at least 471 bp of continuous nucleotides downstream of the 3'UTR are present.

[0052] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a positive selection marker gene or a negative selection marker gene. In some embodiments, the resistance gene for positive clone selection is the neomycin phosphotransferase coding sequence Neo or the hygromycin resistance gene sequence HygR. Preferably, the targeting vector further comprises two co-directed Frt 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).

[0053] In some embodiments, the targeting vector further includes a self-excision cassette. In some embodiments, the self-excision cassette, from 5'-3', includes the following elements: a recombination site loxP for a specific recombination system, a tissue-specific promoter (such as a testis-specific promoter or an ovary-specific promoter), Cre recombinase, a resistance gene (Neo) for positive clone selection, and loxP. In some embodiments, the testis-specific promoter includes, but is not limited to, AMH, STRA8, and SP10.

[0054] This application also provides vectors for constructing humanized animal models or knockout models. In some embodiments, the vector contains an sgRNA sequence that targets the TNFRSF25 gene, and the sgRNA is unique on the target sequence of the gene to be altered and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'. In some embodiments, the target site of the sgRNA in the non-human animal (e.g., mouse) TNFRSF25 gene is located in exons 1 to 10. In some embodiments, the target site of the sgRNA in the non-human animal (e.g., mouse) TNFRSF25 gene is located at least 50 bp upstream of the 5'UTR to exon 10. At least 50 bp upstream of the 5'UTR to the stop codon. At least 168 bp upstream of the 5'UTR to the stop codon.

[0055] In some embodiments, the target site sequence is SEQ ID NO: 10 and / or 11. Therefore, this application provides sgRNAs for constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequence is listed in SEQ ID NO: 12 and 14. In some embodiments, the oligonucleotide sgRNA sequence is listed in SEQ ID NO: 13 and 15. In some embodiments, the oligonucleotide sgRNA sequence is listed in SEQ ID NO: 16 and 18. In some embodiments, the oligonucleotide sgRNA sequence is listed in SEQ ID NO: 17 and 19.

[0056] In some embodiments, this application relates to plasmid constructs (e.g., pT7-sgRNA) comprising sgRNA sequences and / or cells comprising such constructs.

[0057] This application also relates to cells containing the targeting vector or sgRNA vector as described above.

[0058] Furthermore, this application also provides a non-human mammalian cell having any of the aforementioned targeting vectors, and one or more in vitro transcripts of the construct described in this application. In some embodiments, the cell contains Cas9 mRNA or its in vitro transcript.

[0059] In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

[0060] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

[0061] Genetically modified non-human animals The term "genetically modified non-human animal" or "genetically modified non-human animal" as used in this application 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 non-human animal, contain exogenous DNA. Cells with 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, comprising a modified endogenous TNFRSF25 locus (preferably also including a modified endogenous TL1A locus), and containing exogenous sequences (e.g., human sequences), for example, replacing one or more non-human animal endogenous sequences with one or more human sequences, or inserting one or more human and / or non-human animal endogenous sequences. Non-human animals are generally able to pass on genetic modifications to their offspring through germline transmission.

[0062] The term "chimeric (x) gene" or "chimeric (x) nucleic acid" as used in this application 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, at least a portion of the sequence of the chimeric (x) gene or chimeric (x) nucleic acid has two or more different species origins, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid refers to a humanized (x) gene or humanized (x) nucleic acid.

[0063] The term "chimeric (x) protein" or "chimeric (x) polypeptide" as used in this application 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.

[0064] The term "humanized (x) protein" or "humanized (x) polypeptide" as used in this application refers to a protein or polypeptide, wherein at least a portion of the protein or polypeptide is derived from a human protein or human polypeptide. In some embodiments, the humanized (x) protein or humanized (x) polypeptide refers to a human protein or polypeptide.

[0065] The term "humanized (x) nucleic acid" or "humanized (x) gene" as used in this application 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, the nucleic acid or gene in the humanized (x) nucleic acid or "humanized (x) gene" is entirely derived from a human. In some embodiments, the humanized (x) nucleic acid refers to a humanized (x) exon, which may be a human exon or a chimeric exon.

[0066] In some embodiments, the chimeric TNFRSF25 gene or chimeric TNFRSF25 nucleic acid is a humanized TNFRSF25 gene or humanized TNFRSF25 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human TNFRSF25 gene, and at least a portion of the gene or nucleic acid is derived from a non-human TNFRSF25 gene. In some embodiments, the gene or nucleic acid contains a sequence encoding a TNFRSF25 protein. The encoded TNFRSF25 protein has at least the activity of a human TNFRSF25 protein or a non-human animal TNFRSF25 protein.

[0067] In some embodiments, the chimeric TNFRSF25 protein or chimeric TNFRSF25 peptide is a humanized TNFRSF25 protein or a humanized TNFRSF25 peptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or peptide are derived from human TNFRSF25 protein, and at least one or more portions of the amino acid sequence of the protein or peptide are derived from non-human animal TNFRSF25 protein. The humanized TNFRSF25 protein or humanized TNFRSF25 peptide is functional, or has at least the activity of a human TNFRSF25 protein or a non-human animal TNFRSF25 protein.

[0068] In some embodiments, the chimeric TL1A gene or chimeric TL1A nucleic acid is a humanized TL1A gene or humanized TL1A nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human TL1A gene, and at least a portion of the gene or nucleic acid is derived from a non-human TL1A gene. In some embodiments, the gene or nucleic acid contains a sequence encoding a TL1A protein. The encoded TL1A protein has the activity of at least one human TL1A protein or a non-human animal TL1A protein.

[0069] In some embodiments, the chimeric TL1A protein or chimeric TL1A polypeptide is a humanized TL1A protein or a humanized TL1A polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a human TL1A protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human animal TL1A protein. The humanized TL1A protein or humanized TL1A polypeptide is functional, or has at least the activity of a human TL1A protein or a non-human animal TL1A protein.

[0070] 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.

[0071] 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 some embodiments, the rodent is selected from the rat family. In some embodiments, the genetically modified non-human 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 rats and mice, Dormicidae (e.g., spiny dormice), and Mole-like Muridae (e.g., Mole rats, bamboo rats, and mole rats). In some embodiments, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny rats, and crested rats. In some embodiments, the genetically modified mouse is from a member of the Muridae family. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[0072] In some embodiments, the non-human animal may be an immunodeficient non-human mammal, such as an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, or an immunodeficient monkey. In some embodiments, the animal is a mouse of the C57BL strain, 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.

[0073] Non-human animals may have one or more other genetic modifications and / or other modifications suitable for the specific purpose of producing humanized animals. 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, through 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 humanization of the non-human animal endogenous TNFRSF25 and / or TL1A loci, 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 - / -Modifications of (RG) 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 mouse endogenous mature TNFRSF25 gene coding sequence with all or part of the human mature TNFRSF25 coding sequence. In some embodiments, the mice may include the replacement of all or part of the mouse endogenous mature TL1A gene coding sequence with all or part of the human mature TL1A coding sequence. Genetically modified non-human animals include modifications to the non-human animal endogenous TNFRSF25 gene locus. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature TNFRSF25 protein (e.g., containing at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence encoding the mature TNFRSF25 protein). While this application provides for cells that may contain the genetic modifications (e.g., ES cells, somatic cells), in some embodiments, genetically modified non-human animals include modifications to the endogenous TNFRSF25 gene locus in non-human animals.

[0074] Genetically modified non-human animals can express human TNFRSF25 and / or chimeric (e.g., humanized) TNFRSF25 at endogenous loci in non-human animals (e.g., mice), wherein the endogenous TNFRSF25 gene in the non-human animal (e.g., mouse) has been replaced or inserted with the human TNFRSF25 gene and / or the nucleotide sequence encoding the human TNFRSF25 sequence or a nucleotide sequence with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% identity with the human TNFRSF25 sequence. In some embodiments, the endogenous TNFRSF25 locus in the non-human animal is modified with all or part of a nucleic acid sequence encoding the mature human TNFRSF25 protein.

[0075] In some embodiments, genetically modified nonhuman animals (such as mice) may express human TNFRSF25 and / or chimeric TNFRSF25 (e.g., humanized TNFRSF25) under the control of human regulatory elements and / or human regulatory elements. Insertion or substitution at endogenous loci in nonhuman animals (such as mice) provides nonhuman animals that express human TNFRSF25 or chimeric TNFRSF25 (e.g., humanized TNFRSF25) in suitable cells in a manner that does not lead to potential pathologies observed in some other transgenic nonhuman animals (such as mice) known in the art. Expression of human TNFRSF25 or chimeric TNFRSF25 (e.g., humanized TNFRSF25) in nonhuman animals can maintain one or more functions of wild-type nonhuman animals (such as mice) or human TNFRSF25 in nonhuman animals. Furthermore, in some embodiments, nonhuman animals do not express endogenous TNFRSF25. In some embodiments, the expression level of endogenous TNFRSF25 in nonhuman animals is reduced compared to the TNFRSF25 expression level in wild-type animals. As used in this application, "endogenous TNFRSF25" refers to the TNFRSF25 protein expressed by the endogenous TNFRSF25 nucleotide sequence of a non-human animal (such as a mouse) prior to any genetic modification.

[0076] In some embodiments, the humanized TNFRSF25 gene comprises the 5' UTR of the human TNFRSF25 gene. In some embodiments, the humanized TNFRSF25 gene comprises the 5' UTR of the endogenous TNFRSF25 gene from a non-human animal (e.g., mouse). In some embodiments, the humanized TNFRSF25 locus comprises the 3' UTR of the human TNFRSF25 gene. In some embodiments, the humanized TNFRSF25 locus comprises the 3' UTR of the TNFRSF25 gene from a non-human animal (e.g., mouse).

[0077] This application further relates to the TNFRSF25 genomic DNA sequence of humanized non-human animals (such as mice), the DNA sequence obtained by reverse transcription of mRNA being identical or complementary to the DNA sequence; a construct expressing the amino acid sequence; a cell containing the construct; and a tissue or organ containing the cell.

[0078] This application further relates to non-human mammals produced by the methods described above. In some embodiments, their genomes contain human genes.

[0079] In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0080] In some embodiments, non-human mammals express a protein encoded by the humanized TNFRSF25 gene.

[0081] Furthermore, this application also provides a non-human mammalian model carrying a tumor, which is obtained by the construction method described in this application. In some embodiments, the non-human mammal is a rodent (e.g., a mouse).

[0082] This application 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.

[0083] This application provides a non-human mammal produced by any of the construction methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal contains human or humanized TNFRSF25 DNA.

[0084] In some embodiments, the non-human mammal includes the gene constructs described in this application (e.g., plasmid vectors). In some embodiments, a non-human mammal expressing human or humanized TNFRSF25 protein is provided. In some embodiments, a tissue specifically expressing human or humanized TNFRSF25 protein is provided. In some embodiments, an organ specifically expressing human or humanized TNFRSF25 protein is provided.

[0085] In some embodiments, the expression of human or humanized TNFRSF25 protein in non-human animals 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.

[0086] The non-human mammal can be any non-human animal known in the art, which can be used in the methods described in this application. Preferred non-human mammals are rodents. In some embodiments, the non-human mammal is a mouse.

[0087] Genetic, molecular, and behavioral analyses were performed on the non-human mammals described above. This application provides a method for providing offspring produced by mating with a non-human mammal of the same or other genotypes.

[0088] This application provides a cell line or primary cell culture derived from a non-human mammal or its offspring. For example, a cell culture-based model can be prepared using the following methods. The cell culture can be obtained by isolating cells from a non-human mammal, or cells can be obtained using the same construct and a cell culture established using standard cell transfection techniques. Integration of the genetic structure containing the DNA sequence encoding the human TNFRSF25 protein can be detected using various methods.

[0089] 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 TNFRSF25 protein.

[0090] In some embodiments, the genetically modified non-human animals described in this application (e.g., homozygous or heterozygous mice with humanized TNFRSF25 gene) can express human or humanized TNFRSF25 in one or more liver tissue cells.

[0091] 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 genome editing is used to produce 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. This application also provides many other methods for genome editing, such as microinjecting genetically modified cells into enucleated oocytes and fusing enucleated oocytes with another genetically modified cell.

[0092] In some embodiments, the nucleotide sequence encoding the endogenous TNFRSF25 protein in the endogenous genome of at least one cell of a non-human animal is replaced by the nucleotide sequence encoding the human TNFRSF25 protein. In some embodiments, the expression level of the endogenous TNFRSF25 protein in the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts, or fibroblasts. The nucleus of the somatic cell or fibroblast may be inserted into an enucleated oocyte.

[0093] This application provides a targeting vector. The targeting vector comprises a 5' homologous arm, a human or humanized TNFRSF25 gene fragment, and a 3' homologous arm. The process involves replacing the corresponding endogenous TNFRSF25 sequence with a human or humanized TNFRSF25 sequence using homologous recombination. In some embodiments, upstream and downstream cleavage at the target site (e.g., via zinc finger nucleases, TALEN, or CRISPR) can lead to DNA double-strand breaks, and the endogenous TNFRSF25 sequence in a non-human animal (e.g., mouse) is replaced with a human or humanized TNFRSF25 sequence using homologous recombination.

[0094] Therefore, in some embodiments, the method of preparing genetically modified humanized animals includes replacing the nucleic acid sequence encoding the endogenous TNFRSF25 protein at the endogenous TNFRSF25 locus (or site) with the nucleotide sequence encoding the human TNFRSF25 protein.

[0095] This application also provides a method for establishing a humanized animal model of the TNFRSF25 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).

[0096] In some embodiments, the non-human mammal in the above method is a mouse (e.g., a C57BL / 6 mouse).

[0097] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy.

[0098] 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.

[0099] 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.

[0100] In some embodiments, the method for constructing genetically modified non-human animals includes modifying the coding frame of the non-human animal's TNFRSF25 gene, for example, by replacing the nucleic acid sequence encoding the endogenous TNFRSF25 protein with a nucleotide sequence encoding the human TNFRSF25 protein (e.g., genomic DNA, CDS, or cDNA sequence) under the control of exogenous regulatory elements (e.g., the human TNFRSF25 promoter, 5'UTR, and / or 3'UTR). For example, one or more functional region sequences of the non-human animal's TNFRSF25 gene may be knocked out or inserted, causing the non-human animal's endogenous TNFRSF25 protein to be unexpressed or its expression level to be reduced. In some embodiments, the coding frame of the genetically modified non-human animal's TNFRSF25 gene may be all or part of the nucleotide sequence of exons 1 to 10 of the non-human animal's TNFRSF25 gene.

[0101] In some embodiments, the method for constructing genetically modified nonhuman animals includes inserting a nucleotide sequence encoding a human or humanized TNFRSF25 protein and / or an auxiliary sequence into the TNFRSF25 locus of a nonhuman animal. In some embodiments, the auxiliary sequence may be a stop codon, enabling the humanized animal model of the TNFRSF25 gene to express the human or humanized TNFRSF25 protein in vivo, but not the nonhuman TNFRSF25 protein. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0102] In some embodiments, the construction method includes obtaining the vector described above. In some embodiments, the vector is a targeting vector and / or an sgRNA vector. In some embodiments, the construction method includes introducing the targeting vector into a non-human animal or its cells. In some embodiments, the construction method includes introducing the targeting vector and the sgRNA vector into a non-human animal or its cells.

[0103] In some embodiments, a method for preparing genetically modified non-human animals includes: (1) Provide a plasmid containing a human TNFRSF25 gene fragment, wherein the plasmid is flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' homologous arm and the 3' homologous arm target endogenous TNFRSF25 in non-human animals; (2) Provide one or more guide RNAs (sgRNAs) that target the endogenous TNFRSF25 gene in non-human animals; (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 non-human animals (e.g., 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 non-human animals (e.g., mice) to produce offspring non-human animals (e.g., mice) that functionally express humanized TNFRSF25 protein. (5) Mating the offspring non-human animals (such as mice) obtained in step (4) to obtain homozygous non-human animals (such as mice).

[0104] In some embodiments, the fertilized egg is modified by CRISPR with sgRNA targeting both the 5'-end and 3'-end target sites.

[0105] In some embodiments, the sequence encoding the humanized TNFRSF25 protein is operatively linked to exogenous regulatory elements (e.g., the human TNFRSF25 promoter, 5'UTR, and / or 3'UTR).

[0106] In some embodiments, genetically modified nonhuman animals do not express endogenous TNFRSF25 protein.

[0107] In some embodiments, the method for preparing genetically modified non-human animals includes: (1) Provide a plasmid containing a human or chimeric TNFRSF25 gene fragment, wherein the plasmid is flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' homologous arm and the 3' homologous arm target endogenous TNFRSF25; (2) Provide one or more guide RNAs (sgRNAs) that target the endogenous TNFRSF25 gene; (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric TNFRSF25 gene fragment into the genome.

[0108] In some embodiments, all or part of the nucleotide sequence encoding the TNFRSF25 protein in the non-human animal endogenous gene is deleted. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1 in the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% nucleotide sequence identity with SEQ ID NO: 1 is deleted in the non-human animal. In some embodiments, all or part of exons 1-10 of the non-human animal endogenous TNFRSF25 gene is deleted. In some embodiments, all of exon 1 to part of exon 10 of the non-human animal endogenous TNFRSF25 gene is deleted. In some embodiments, the 5'UTR of the non-human animal endogenous TNFRSF25 gene is deleted. In some embodiments, the 5'UTR and at least 50 bp upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene are deleted. In some embodiments, at least 50 bp upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene to part of exon 10 is deleted. In some embodiments, at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene is deleted up to the stop codon. In some embodiments, at least 168 bp of continuous nucleotide sequence upstream of the 5'UTR of the non-human animal endogenous TNFRSF25 gene is deleted up to the stop codon.

[0109] Two or more human or chimeric non-human animal models This application also provides a method for generating a genetically modified animal model having two or more human or chimeric genes. The animal model may contain a human or chimeric TNFRSF25 gene and a sequence encoding an additional human or chimeric protein.

[0110] In some embodiments, the additional human or chimeric protein is a non-human animal genetically modified with at least one of the following: TL1A, IL23A, IL12B, A4B7, TNFA, TNFR1, TNFR2, IL6, IL6R, TREM1, PGLYRP1, 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 TL1A, IL23A, IL12B, A4B7, TNFA, TNFR1, TNFR2, IL6, IL6R, TREM1, PGLYRP1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 proteins.

[0111] This application 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.

[0112] In some embodiments, the other genetically modified non-human animals include humanized non-human animals containing one or more of the genes TL1A, IL23A, IL12B, A4B7, TNFA, TNFR1, TNFR2, IL6, IL6R, TREM1, PGLYRP1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0113] In some embodiments, TNFRSF25 humanization is performed directly on non-human animals with human or chimeric TL1A, IL23A, IL12B, A4B7, TNFA, TNFR1, TNFR2, IL6, IL6R, TREM1, PGLYRP1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 gene modifications.

[0114] In some embodiments, a method for constructing an animal model is provided, the method comprising obtaining immune cells from a non-human animal modified with the TNFRSF25 gene, or from two or more non-human animals with human or chimeric genes, and introducing them into another non-human animal. In some embodiments, the immune cells include lymphocytes, such as T cells, preferably CD4+CD45RB. high T cells. In some embodiments, the other non-human animal may be a wild-type non-human animal or a non-human animal with the TL1A gene knocked out.

[0115] 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 have shown 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 therapeutics targeting TNFRSF25 and / or TL1A, including but not limited to anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A, and adjunctive therapeutics for treating diseases (e.g., tumors, inflammation, or immune-related diseases). The method involves administering therapeutics targeting TNFRSF25 and / or TL1A and adjunctive therapeutics to non-human animals with disease and determining the effect of the combination therapy on the disease. In some embodiments, the additional therapeutic agent includes, but is not limited to, antibodies that specifically bind to TL1A, IL23A, IL12B, A4B7, TNFA, TNFR1, TNFR2, IL6, IL6R, TREM1, PGLYRP1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. In some embodiments, the additional therapeutic agent includes, but is not limited to, anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD-1 antibodies (e.g., pembrolizumab or nivolumab), or anti-PD-L1 antibodies. In some embodiments, the non-human animal further 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 described above comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0116] 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 in this application, such as asthma, rheumatoid arthritis, psoriasis, or multiple sclerosis. 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 a subject to remove at least a portion of the cancer, for example, removing part or all of a tumor from a patient.

[0117] Application of genetically modified non-human animals Replacing nonhuman animal genes with homologous or orthologous human genes or sequences at endogenous loci in nonhuman animals, or inserting homologous or orthologous human genes or sequences into nonhuman animals, 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. Typically, the location of the integrated transgene is unknown; human protein expression is measured through transcriptional assays and / or functional assays of human genes and / or proteins.

[0118] Genetically modified non-human animals expressing human or humanized TNFRSF25 and / or TL1A proteins, 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.

[0119] This application also provides the application of non-human animals modified with the above-mentioned TNFRSF25 and / or TL1A genes, and non-human animals obtained by any of the above-mentioned construction methods.

[0120] In some embodiments, the application includes: A) Applications in product development involving immune processes in human cells related to TNFRSF25 and / or TL1A; B) Application as a model system related to TNFRSF25 and / or TL1A in pharmacological, immunological, microbiological and medical research; C) Involving the production and use of animal experimental disease models for etiological studies related to TNFRSF25 and / or TL1A and / or for the development of diagnostic and / or therapeutic strategies; D) Its application in the screening, efficacy testing, efficacy assessment, validation, or evaluation of human TNFRSF25 and / or TL1A signaling pathway modulators in vivo; or, E) Investigate the function of TNFRSF25 and / or TL1A genes, study drugs and their efficacy targeting human TNFRSF25 and / or TL1A, and study the drug applications for diseases related to TNFRSF25 and / or TL1A (such as tumors, inflammation, and immune-related diseases).

[0121] This application provides a non-human animal expressing human or humanized TNFRSF25 and / or TL1A proteins, which can be used for screening human TNFRSF25 and / or TL1A-specific regulators. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also includes a compromised immune system, such as a genetically modified human tissue xenograft, including human solid tumors (e.g., breast cancer, lung cancer, head and neck cancer, liver cancer) or hematologic malignancies (e.g., lymphocytic tumors, including B-cell or T-cell tumors).

[0122] In some embodiments, therapeutic agents targeting TNFRSF25 and / or TL1A block or inhibit TNFRSF25 and / or TL1A-mediated signaling pathways. In some embodiments, the therapeutic agents targeting TNFRSF25 and / or TL1A described herein can block the interaction between the TNFRSF25 and / or TL1A complex, thereby inhibiting the TNFRSF25 and / or TL1A-targeting signaling pathways.

[0123] In some embodiments, genetically modified nonhuman animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A) in treating various immune-related diseases. In some embodiments, the immune-related diseases include, but are not limited to, GVHD (graft-versus-host disease), psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, immune-related diseases include asthma, rheumatoid arthritis, psoriasis, or multiple sclerosis.

[0124] In some embodiments, genetically modified nonhuman animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A) in treating various types of inflammation, such as infectious inflammation. 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 includes inflammatory bowel disease (IBD) or atopic dermatitis.

[0125] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A) in treating cancer. In some embodiments, therapeutic agents (e.g., anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A) 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.

[0126] In some embodiments, the tumor cells comprise one or more cancer cells injected into a non-human animal (e.g., cancer cells derived from humans or non-human animals). In some embodiments, the therapeutic agent inhibits or activates the TNFRSF25 and / or TL1A signaling pathways. In some embodiments, the therapeutic agent does not inhibit or activate the TNFRSF25 and / or TL1A signaling pathways.

[0127] In some embodiments, genetically modified non-human animals can be used to test whether a therapeutic agent (e.g., an anti-TNFRSF25 and / or TL1A antibody, a nucleic acid drug and / or peptide drug targeting TNFRSF25 and / or TL1A) is an agonist or antagonist. In some embodiments, the methods described herein can be used to test the function of a therapeutic agent (e.g., an anti-TNFRSF25 and / or TL1A antibody, a nucleic acid drug and / or peptide drug targeting TNFRSF25 and / or TL1A), for example, whether the therapeutic agent upregulates or downregulates the immune response, and / or whether the therapeutic agent 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 agent for treating a subject's disease (e.g., an immune-related disease, tumor, or inflammation). Tumor inhibition can also be determined by methods known in the art, such as measuring tumor volume in a non-human animal, and / or determining the tumor (volume) growth 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.

[0128] In some embodiments, therapeutic agents (e.g., anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A) can be used to treat various cancers. As used in this application, "cancer" 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. As used in this application, "cancer" and "tumor" are used interchangeably. The term "tumor" as used in this application 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 (such as 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 syndrome, and sarcoma. The leukemia is selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In some embodiments, the tumor is breast cancer, pancreatic cancer, endocrine carcinoma, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, lung cancer (such as non-small cell lung cancer), glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tissue tumor, oropharyngeal tumor, female reproductive system cancer, or meningioma. In some embodiments, the tumor is a solid tumor or a hematologic malignancy. In some embodiments, the solid tumor includes head and neck cancer, breast cancer, liver cancer, or lung cancer. In some embodiments, the hematologic malignancy includes lymphocytic tumors (e.g., T-cell tumors or B-cell tumors).

[0129] This application also provides a method for determining the toxicity of a therapeutic agent (e.g., an anti-TNFRSF25 and / or TL1A antibody, a nucleic acid drug and / or peptide drug targeting TNFRSF25 and / or TL1A). The method includes administering the therapeutic agent to a non-human animal and assessing changes in the non-human animal's weight or blood tests. In some embodiments, the blood tests include, but are not limited to, red blood cell count, hematocrit, and / or hemoglobin. In some embodiments, the antibody may reduce red blood cells (RBCs), hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average weight of non-human animals not treated with the therapeutic agent).

[0130] This application also provides a model system for developing animal models constructed by the methods described herein, for use in developing products related to human cellular immune processes, manufacturing human drugs (such as antibodies), or for use in pharmacological, immunological, microbiological, and medical research.

[0131] 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.

[0132] This application also provides animal models generated by the methods described in this application for screening, validating, evaluating or studying the function of TNFRSF25 and / or TL1A genes, human TNFRSF25 and / or TL1A antibodies, and therapeutic agents or efficacy for diseases (tumors, inflammation or immune-related diseases) related to human TNFRSF25 and / or TL1A targets.

[0133] In some embodiments, this application provides a method for verifying the in vivo efficacy of TCR-T, CAR-T, and / or other immunotherapies (e.g., T-cell adoptive transfer therapy). For example, the method includes transplanting human tumor cells into a non-human animal as described in this application and applying human CAR-T to the non-human animal with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animal is selected from humanized non-human animals with TNFRSF25 and / or TL1A genes prepared by the method described in this application, dual-gene or multi-gene humanized non-human animals (or their progeny) generated by the method described in this application, non-human animals expressing human or humanized TNFRSF25 and / or TL1A proteins, or animal models carrying tumors or inflammation as described in this application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat TNFRSF25 and / or TL1A-related diseases as described in this application. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies provide evaluation methods for treating TNFRSF25 and / or TL1A-related diseases as described in this application. Attached Figure Description

[0134] 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 humanized mice with TNFRSF25 gene, where PC is the positive control, WT is the wild-type control, H2O is the water control, and M is the marker; Figure 4 RT-PCR results: + / + indicates wild-type C57BL / 6 mice; H / H; H / H indicates homozygous TL1A / TNFRSF25 dual-gene humanized mice; GAPDH is an internal control. Figure 4 (A) shows the TL1A detection results. Figure 4 (B) shows the results of TNFRSF25 detection; Figure 5 ELISA results: + / + indicates wild-type C57BL / 6 mice, H / H; H / H indicates homozygous TL1A / TNFRSF25 dual-gene humanized mice.

[0135] Figure 6 : Changes in body weight (A) and DAI score (B) in TL1A / TNFRSF25 dual-gene humanized mouse model after drug administration.

[0136] Figure 7Changes in colon length (A), weight (B), and density (C) after administration to TL1A / TNFRSF25 dual-gene humanized mice model.

[0137] Figure 8 The results of body weight change (A) and DAI score (B) of the test mice after drug administration to humanized mouse T cells with TL1A / TNFRSF25 dual genes.

[0138] Figure 9 Changes in colon length (A), weight (B), and density (C) in test mice after administration of TL1A / TNFRSF25 dual-gene humanized mouse T-cell model. Detailed Implementation

[0139] The present application will be further described below with reference to specific embodiments, and the advantages and features of the present application will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present application. 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 application without departing from the spirit and scope of the present application, but such modifications and substitutions all fall within the protection scope of the present application.

[0140] In each of the following embodiments, the equipment and materials are obtained from the following companies: C57BL / 6 mice were purchased from the National Rodent Experimental Animal Seed Center of the National Institutes for Food and Drug Control, China. The Human TL1A / TNFSF15 DuoSet ELISA was purchased from R&D, catalog number DY1319-05.

[0141] Example 1: Humanized TNFRSF25 gene mice The TNFRSF25 gene-humanized mouse in this embodiment was designed based on the mouse TNFRSF25 gene (NCBI Gene ID: 85030, located on chromosome 4 NC_000070.7, pp. 152199985 to 152204568, based on transcript NM_001291010.2 and its encoded protein NP_001277939.1 (SEQ ID NO: 1)) and the human TNFRSF25 gene (Gene ID: 8718, located on chromosome 1 NC_000001.11, pp. 6460786 to 6466173), based on transcript NM_003790.3 and its encoded protein NP_003781.1 (SEQ ID NO: 2).

[0142] To achieve the objectives of this application, a nucleotide sequence encoding the human TNFRSF25 protein can be introduced into the mouse endogenous TNFRSF25 gene locus, enabling the mouse to express the human or humanized TNFRSF25 protein. Specifically, using gene editing technology, approximately 3.99 kb of the sequence upstream of the mouse 5'UTR to the stop codon (TGA) is replaced with approximately 10.36 kb upstream of the 5'UTR and downstream of the 3'UTR containing the human TNFRSF25 gene, resulting in a humanized TNFRSF25 gene locus, thus achieving the humanization of the mouse TNFRSF25 gene.

[0143] To achieve the targeting strategy of this application, a targeting vector V1 was constructed ( Figure 1 The targeting vector V1 contains upstream and downstream homologous arm sequences of the mouse TNFRSF25 gene, as well as fragment A containing the human TNFRSF25 fragment. Specifically, the upstream 5' homologous arm sequence is SEQ ID NO: 3, the downstream 3' homologous arm sequence is SEQ ID NO: 4, and the nucleotide sequence of the human TNFRSF25 fragment is SEQ ID NO: 5. The connection between the upstream human TNFRSF25 fragment and the mouse gene is designed as follows:

[0144] Among them, the sequence " The last one in " "This is the last nucleotide linked to the 5' end of the mouse-human TNFRSF25 fragment, sequence..." “ in "It is the first nucleotide of the human TNFRSF25 fragment."

[0145] The targeting vector also includes a self-excision cassette, located between the human TNFRSF25 fragment and its downstream 3' homologous arm. From 5' to 3', the cassette contains the following elements: a loxP recombination site for a specific recombination system, a tissue-specific promoter (testis-specific promoters such as AMH, ovarian-specific promoters), Cre recombinase, the resistance gene (Neo) for positive clone selection, and loxP. During germline transfer, the Cre recombinase is expressed under the regulation of the specific promoter and is removed from the mouse cell genome along with Neo. The 5' linking sequence of the self-excision cassette to the human TNFRSF25 fragment is as follows:

[0146] The sequence " The last one in " "This is the last nucleotide of the human TNFRSF25 fragment, sequence..." The first " in" " is the first nucleotide of the self-excision box; The 3' end of the self-excision box and the mouse connection sequence are as follows:

[0147] The sequence " The last one in " "This is the last nucleotide of the self-excision cassette fragment, sequence number." “ in "This is the first nucleotide at the junction of the mouse and the 3' end of the self-excision box."

[0148] The mRNA sequence transcribed from the TNFRSF25 gene of the modified humanized mouse is shown in SEQ ID NO: 7, and the protein sequence expressed is shown in SEQ ID NO: 2.

[0149] 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 female mice (white mice) to produce F0 generation chimeric mice (black and white). Backcrossing F0 generation chimeric mice (males) with wild-type mice (females) yields F1 generation mice. Mating F1 generation heterozygous mice with each other yields F2 generation homozygous mice.

[0150] In addition, gene editing can be performed using CRISPR / Cas9 technology, and the target vector V2 can be designed. Figure 2 The targeting vector V2 contains upstream and downstream homologous arm sequences of the mouse TNFRSF25 gene, as well as a human TNFRSF25 fragment. Specifically, the upstream 5' homologous arm sequence is SEQ ID NO: 8, the downstream 3' homologous arm sequence is SEQ ID NO: 9, and the nucleotide sequence of the human TNFRSF25 fragment is SEQ ID NO: 5. The connection between the upstream human TNFRSF25 fragment and the mouse gene is designed as follows:

[0151] The sequence " The last one in " "This is the last nucleotide linked to the 5' end of the mouse-human TNFRSF25 fragment, sequence..." “ in " is the first nucleotide of the human TNFRSF25 fragment. The downstream linker of the human TNFRSF25 fragment to the mouse fragment is designed as follows:

[0152] The sequence " The last one in " "This is the last nucleotide of the human TNFRSF25 fragment, sequence..." The first " in" " is the first nucleotide linked to the 3' end of the mouse-human TNFRSF25 fragment. The mRNA sequence transcribed from the modified humanized mouse TNFRSF25 gene is shown in SEQ ID NO: 7, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0153] 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.

[0154] The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 cleavage of the target gene. Therefore, efficient and specific target sequence selection and design are prerequisites for constructing sgRNA expression vectors. An sgRNA sequence recognizing the target site was designed and synthesized. An example sgRNA target sequence on the TNFRSF25 gene is as follows: sgRNA1 target site (SEQ ID NO: 10): 5'-TCCAGAACAACTCCCCTCGGGGG-3'; sgRNA2 target site (SEQ ID NO: 11): 5'-CGGTGCCATCTATGCGGCTCTGG-3'; The activity of sgRNA was detected using a UCA kit to confirm that it could mediate high cleavage efficiency. Enzyme sites were added to its 5' end and complementary strand to obtain forward and reverse oligonucleotide sequences as shown in Table 3. After annealing, the annealed products were ligated into the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain the expression vectors pT7-TNFRSF25-1 and pT7-TNFRSF25-2.

[0155] Table 3 Sequence listings of sgRNA1 and sgRNA2

[0156] The pT7-sgRNA vector was synthesized by a plasmid synthesis company, containing a fragment DNA (SEQ ID NO: 20) containing the T7 promoter and sgRNA scaffold. This fragment was then sequentially digested with enzymes (EcoRI and BamHI) and ligated into a backbone vector (from Takara, catalog number 3299). Sequencing by a professional sequencing company confirmed the successful acquisition of the target plasmid. Pronuclear zygotes from mice, such as C57BL / 6 mice, were used. The in vitro transcription products of pT7-TNFRSF25-1 and pT7-TNFRSF25-2 plasmids (transcribed using an Ambion in vitro transcription kit according to the manufacturer's instructions), the targeting vector, and Cas9 mRNA were premixed and injected into the cytoplasm or nucleus of mouse zygote cytoplasm using a microinjection apparatus. Following the method described in "Experimental Manual of Mouse Embryo Manipulation (3rd Edition)" (Andras Nagy, Chemical Industry Press, 2006), fertilized eggs were microinjected. After injection, the fertilized eggs were transferred to a culture medium for short-term culture, and then transplanted into the oviduct of the recipient female mouse for development. The resulting mice (F0 generation) were then expanded through hybridization and self-fertilization to establish a stable humanized mouse strain of the TNFRSF25 gene.

[0157] The genotype of F1 generation mouse somatic cells can be identified using PCR. The primers shown in Table 4 were used for detection. Example results are shown below. Figure 3 As shown, six mice numbered F1-1 to F1-6 were positive mice. Heterozygous mice from the humanized TNFRSF25 locus were mated to obtain homozygotes.

[0158] Table 4 Primer sequences and recombinant fragment sizes for F1 generation genotype PCR detection

[0159] Example 2: Preparation of dual-gene or multi-gene humanized mice The TNFRSF25 gene-humanized mice obtained using this method can also be used to prepare dual-gene or multi-gene humanized mouse models. For example, in Example 1 above, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modified with TL1A, IL23A, IL12B, A4B7, TNFA, TNFR1, TNFR2, IL6, IL6R, TREM1, PGLYRP1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. Alternatively, dual-gene or multi-gene humanized mouse models can be obtained by using isolated mouse ES embryonic stem cells and gene recombination targeting technology based on humanized TNFRSF25 mice. Alternatively, the humanized mice containing the TNFRSF25 gene obtained according to the method in Example 1 above can be mated with mice with other gene modifications, and their offspring can be screened. According to Mendelian inheritance laws, there is a certain probability of obtaining humanized TNFRSF25 and other gene-modified dual-gene / multi-gene mice. Then, the heterozygotes can be mated with each other to obtain homozygotes with dual-gene / multi-gene modifications.

[0160] In one specific embodiment, the embryonic stem cells or fertilized eggs used in Example 1 above are derived from TL1A gene-humanized mice to obtain TL1A / TNFRSF25 dual-gene humanized mice. TL1A gene-humanized mice are created by introducing a nucleotide sequence encoding the human TL1A protein into the mouse's endogenous TL1A gene locus, thereby enabling the mouse to express the human or humanized TL1A protein. Among them, the TL1A gene humanized mouse was designed based on the transcript NM_177371.4 of the mouse TL1A gene (NCBI Gene ID: 326623, UniProt ID: Q5UBV8, located at positions 63642837 to 63663296 of chromosome 4 NC_000070.7) and its encoded protein NP_796345.4, and the transcript NM_005118.4 of the human TL1A gene (NCBI Gene ID: 9966, UniProt ID: O95150, located at positions 114784635 to 114806039 of chromosome 9 NC_000009.12) and its encoded protein NP_005109.2.

[0161] Specifically, the TL1A gene-humanized mouse was created by using gene editing technology. Under the control of the mouse TL1A gene regulatory elements, approximately 15.4 kb of the sequence from exon 1 to exon 4 of the human TL1A gene was replaced with approximately 15.2 kb of the sequence from exon 1 to exon 4, thus achieving the humanization of the mouse TL1A gene. The protein sequence expressed by the TL1A gene in the modified humanized mouse is shown in SEQ ID NO: 34.

[0162] The expression of TL1A / TNFRSF25 dual-gene humanized mice could be detected by RT-PCR. Specifically, one 9-week-old wild-type C57BL / 6 mouse (+ / +) and one 9-week-old male homozygous TL1A / TNFRSF25 dual-gene humanized mouse (H / H; H / H) prepared in this example were selected. After euthanasia, spleen, lung, and colon tissues were collected. RT-PCR was performed using the primer sequences shown in Table 5. The detection results are as follows: Figure 4 As shown. From Figure 4 It can be seen that only mouse TL1A and TNFRSF25 mRNA were detected in wild-type C57BL / 6 mice (+ / +), and human TL1A or TNFRSF25 mRNA was not detected; human TL1A and TNFRSF25 mRNA were detected in homozygous humanized mice with TL1A / TNFRSF25 dual genes (H / H; H / H).

[0163] Table 5 RT-PCR primer sequences and target fragment sizes

[0164] The expression of soluble humanized TL1A in TL1A / TNFRSF25 dual-gene humanized mice can be detected by ELISA. Specifically, three 10-week-old female wild-type C57BL / 6 mice (+ / +) and three 10-week-old female homozygous TL1A / TNFRSF25 dual-gene humanized mice (H / H; H / H) were selected. Dendritic cells were isolated from bone marrow, stimulated with 1 μg / mL LPS for 24 h, and the supernatant was collected. The level of soluble humanized TL1A was detected using the Human TL1A / TNFSF15 DuoSet ELISA kit (purchased from R&D, catalog number DY1319-05). The results are as follows. Figure 5 .like Figure 5 As shown, soluble humanized TL1A was detected only in homozygous TL1A / TNFRSF25 dual-gene humanized mice (H / H; H / H), indicating that humanized TL1A can be normally expressed and function normally in TL1A / TNFRSF25 dual-gene humanized mice.

[0165] The expression of human TNFRSF25 protein in TL1A / TNFRSF25 dual-gene humanized mice can be confirmed by flow cytometry. Specifically, peripheral blood and spleen tissues from 8-week-old male wild-type C57BL / 6 mice and homozygous TL1A / TNFRSF25 dual-gene humanized mice (H / H;H / H) were used. The following antibodies were used: BioLegend PerCP anti-mouse CD45 Antibody (mCD45), Alexa Fluor® 700 anti-mouse CD3 Antibody (mCD3), Brilliant Violet 510™ anti-mouse CD4 Antibody (mCD4), Brilliant Violet 711™ anti-mouse CD8a (mCD8a), PE / Cyanine7 anti-mouse CD25 Antibody (mCD25), and FOXP3 Monoclonal Antibody (FJK-16s). PerCP-eFluor™ 710... eBioscience™ (mFOXP3), human-mouse cross-anti-TNFRSF25 antibody APC anti-mouse DR3(TNFRSF25) (mTNFRSF25), and anti-human TNFRSF25 antibody PE anti-human DR3(TNFRSF25) Antibody (hTNFRSF25) were identified and stained before flow cytometry detection.

[0166] In the spleen of wild-type C57BL / 6 mice, 14.0% of mTNFRSF25-positive cells (characterized by mCD45+mCD3+mCD4+mCD8a-mCD25+mFOXP3+mTNFRSF25+) and 2.11% of hTNFRSF25-positive cells (characterized by mCD45+mCD3+mCD4+mCD8a-mCD25+mFOXP3+hTNFRSF25+) were observed; in the spleen of homozygous TL1A / TNFRSF25 dual-gene humanized mice, 3.64% of mTNFRSF25-positive cells (characterized by mCD45+mCD3+mCD4+mCD8a-mCD25+mFOXP3+mTNFRSF25+) and 7.39% of hTNFRSF25-positive cells (characterized by mCD45+mCD3+) were observed. mCD4+mCD8a-mCD25+mFOXP3+hTNFRSF25+).

[0167] In the peripheral blood of wild-type C57BL / 6 mice, 12.7% of mTNFRSF25-positive cells (characterized by mCD45+mCD3+mCD4+mCD8a-mCD25+mFOXP3+mTNFRSF25+) and 1.03% of hTNFRSF25-positive cells (characterized by mCD45+mCD3+mCD4+mCD8a-mCD25+mFOXP3+hTNFRSF25+) were observed; in the peripheral blood of homozygous TL1A / TNFRSF25 dual-gene humanized mice, 4.26% of mTNFRSF25-positive cells (characterized by mCD45+mCD3+mCD4+mCD8a-mCD25+mFOXP3+mTNFRSF25+) and 4.52% of hTNFRSF25-positive cells were observed. hTNFRSF25 positive cells (characterized by mCD45+mCD3+mCD4+mCD8a-mCD25+mFOXP3+hTNFRSF25+).

[0168] The results showed that human TNFRSF25 protein expression was detected in the spleen and peripheral blood of homozygous TL1A / TNFRSF25 dual-gene humanized mice. Combined with RT-PCR results, human TNFRSF25 protein was normally expressed in TL1A / TNFRSF25 dual-gene humanized mice.

[0169] Example 3: Pharmacodynamic Model

[0170] The humanized mice disclosed in this application can be used to induce and prepare various human disease models, such as inflammatory bowel disease, psoriasis, and rheumatoid arthritis, which can be used to test the in vivo efficacy of human-specific drugs. For example, humanized mice with TNFRSF25 and / or TL1A genes can be used to evaluate the efficacy, pharmacokinetics, and in vivo therapeutic efficacy of antagonists of the human-specific TNFRSF25 and / or TL1A signaling pathways in various disease models known in the art.

[0171] Taking a colitis model as an example, TL1A / TNFRSF25 dual-gene humanized mice were randomly divided into a negative control group, a positive control group, or a treatment group. The positive control group and the treatment group were orally administered 100 mL of 2%-3% dextran sulfate (DSS) to induce colitis, while the negative control group was orally administered an equal volume of drinking water. After successful modeling, the treatment group was randomly assigned to receive a drug targeting human TNFRSF25 and / or TL1A. Depending on the drug, administration could be done before, during, or after modeling via various routes such as intradermal injection, subcutaneous injection, gavage, intraperitoneal injection, or tail vein injection. The negative control group and the positive control group were injected with an equal volume of physiological saline. Mouse weight and fecal consistency were measured periodically. The in vivo efficacy of different human drugs was evaluated by comparing multiple indicators, including mouse weight, fecal consistency, fecal bleeding, and colonic hematologic malignancy (HE) scores.

[0172] In some embodiments of the present invention, homozygous 8-10 week old female TL1A / TNFRSF25 dual-gene humanized mice prepared in Example 2 were randomly divided into 6 groups (n=10 / group). On the day of grouping (D-1), the mice in each group were weighed and administered the drugs. Among them, groups G3 and G4 were intraperitoneally injected with 25 mg / kg of anti-TL1A antibody RVT-3101 (heavy chain sequence as shown in SEQ ID NO.38, light chain sequence as shown in SEQ ID NO.39) and Tulisokibart (heavy chain sequence as shown in SEQ ID NO.40, light chain sequence as shown in SEQ ID NO.41), respectively; groups G5 and G6 were intraperitoneally injected with 25 mg / kg of anti-human TNFRSF25 antibody Ab-1 and Ab-2, respectively; group G2 was intraperitoneally injected with an equal volume of PBS solution; each group was administered the drugs once every 2 days for a total of 3 times. Group G1 was not administered the drugs. On the second day after grouping (D0), mice in groups G2-G6 were perfused with 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution into the colon to establish an acute colitis model, while mice in group G1 were perfused with an equal volume of 50% ethanol solution into the colon. Specific groupings and drug administration details are shown in Table 6.

[0173] During the experiment, the weight changes and fecal condition of mice in each group were recorded daily, and the Disease Activity Index (DAI) was scored (see Table 7 for the indicators). On day 6 (D5) after grouping, mice in each group were euthanized, and colon tissue was collected for photography. The colon length and the weight of the colon after removing the contents were measured, and the colon density was calculated based on "colon weight / colon length".

[0174] Table 6 Grouping and Dosage

[0175] Table 7 DAI Scoring Indicators

[0176] The experimental results showed that on day 2 after grouping (D1), compared with group G1, the changes in body weight of mice in groups G2-G6 were significantly reduced. Figure 6 A), DAI scores both increased significantly ( Figure 6 B), indicating that the TNBS-induced acute colitis model was successfully established. At each experimental endpoint, compared with the modeling control group G2, the body weight changes of mice in the treatment groups G3-G6 were all significantly increased (B). Figure 6 A), DAI score decreased significantly ( Figure 6 B), the length of the colon showed a significant increase to varying degrees. Figure 7 A), mouse colon weight and density were significantly reduced ( Figure 7 B. Figure 7 C). This indicates that the use of TL1A-targeting antibody drugs RVT-3101 and Tulisokibart, and TNFRSF25-targeting antibody drugs Ab-1 and Ab-2, have good therapeutic effects on TNBS-induced acute colitis. These results demonstrate that the TL1A / TNFRSF25 dual-gene humanized mice prepared in this invention can be used to construct colitis models and for in vivo efficacy evaluation of human TL1A and / or TNFRSF25-targeting antibody drugs.

[0177] In another experiment, TL1A / TNFRSF25 dual-gene humanized mice were euthanized, and spleen tissue was collected in pre-cooled DPBS solution. After grinding, the cell suspension was filtered through a 70μm cell filter, centrifuged, and the cells were collected and counted. CD4+ T cells were isolated using the EasySep™ Mouse CD4+ T Cell Sorting Kit (Stemcell, catalog number: 19852), and labeled with CD4 and CD45RB flow cytometry antibodies. CD4+CD45RB cells were then sorted using flow cytometry. high T cells and CD4+CD45RB low T cells were used. B-hTL1A, Rag2 KO mice (Biocytogen, catalog number: 114011) were randomly divided into 4 groups (n=7 / group). On the day of grouping (D0), mice in groups G2-G4 were intraperitoneally injected with 5×10⁻⁵ T cells. 5 One CD4+CD45RB high An acute colitis model was established using T cells. G1 group mice were intraperitoneally injected with 5×10⁵ T cells. 5 One CD4+CD45RB lowT cells. Each group was administered the following regimen starting on the day of grouping (D0) or 16 days after grouping (D16) (see Table 8 for details): Group G3 received intraperitoneal injections of 25 mg / kg anti-TL1A antibody Tulisokibart every two days starting from D0, for a total of 22 injections; Group G4 received intraperitoneal injections of 25 mg / kg Tulisokibart every two days starting from D16, for a total of 14 injections; Group G2 (modeling control group) received intraperitoneal injections of an equal volume of PBS solution every two days starting from D0, for a total of 22 injections.

[0178] During the experiment, mouse weight and DAI scores were recorded every three days (scoring criteria are shown in Table 9). At the experimental endpoint (D42), all mice were euthanized, colon tissue from each group was collected and photographed, colon length and weight (after removing contents) were measured, and colon density was calculated based on colon / weight.

[0179] Table 8 Grouping and Dosing Information

[0180] Table 9 DAI Scoring Indicators

[0181] Experimental results showed that, compared with the model control group G2, the body weight changes of mice in the drug-treated groups G3 and G4 were significantly increased. Figure 8 A), DAI score decreased significantly ( Figure 8 B), significantly increased colon length ( Figure 9 A), colon density was significantly reduced ( Figure 9 C), colon weight decreased to varying degrees ( Figure 9 B). This indicates that both prophylactic and therapeutic administration methods have a therapeutic effect on T-cell-induced colitis.

[0182] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application. It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this application will not further describe the various possible combinations.

[0183] Furthermore, various different implementations of this application can be combined in any way, as long as they do not violate the spirit of this application, they should also be regarded as the content disclosed in this application.

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 tumor necrosis factor receptor superfamily member 25 (TNFRSF25) protein.

2. The construction method according to claim 1, characterized in that, The amino acid sequence of the human or chimeric TNFRSF25 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.

3. A method for constructing a genetically modified non-human animal, characterized in that, The genome of the non-human animal contains the endogenous TNFRSF25 locus, where the nucleotide sequence of the endogenous TNFRSF25 is replaced by the nucleotide sequence of the human TNFRSF25.

4. The construction method according to any one of claims 1-3, characterized in that, The nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein can be operatively linked to exogenous regulatory elements (e.g., human TNFRSF25 promoter, 5'UTR and / or 3'UTR). Preferably, the endogenous TNFRSF25 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 TNFRSF25 gene in the non-human animal genome is homozygous or heterozygous for the endogenously replaced locus; Preferably, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein is the genomic DNA, cDNA or CDS sequence of human TNFRSF25.

5. The construction method according to any one of claims 1-4, characterized in that, The nucleotide sequence of the human TNFRSF25 contains a nucleotide sequence encoding the human or chimeric TNFRSF25 protein; Preferably, the nucleotide sequence of the human TNFRSF25 or the nucleotide sequence encoding the human or chimeric TNFRSF25 protein includes all or part of exons 1 to 10 of the human TNFRSF25 gene, and preferably also includes at least 50 bp of continuous nucleotides upstream of the 5'UTR and / or at least 50 bp of continuous nucleotides downstream of the 3'UTR; Preferably, the nucleotide sequence of the human TNFRSF25 or the nucleotide sequence encoding the human or chimeric TNFRSF25 protein contains at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human TNFRSF25 gene, all of exons 1 to 10, and at least 50 bp of continuous nucleotides downstream of the 3'UTR. Preferably, the nucleotide sequence of human TNFRSF25 or the nucleotide sequence encoding human or chimeric TNFRSF25 protein comprises at least 4500 bp of continuous nucleotides upstream of the 5'UTR, all exons 1 to 10 of the human TNFRSF25 gene, and at least 471 bp of continuous nucleotides downstream of the 3'UTR. Preferably, the nucleotide sequence of the human TNFRSF25 or the nucleotide sequence encoding the human or chimeric TNFRSF25 protein comprises at least 4500 bp of continuous nucleotides upstream of the 5'UTR to at least 471 bp of continuous nucleotides downstream of the 3'UTR; More preferably, the nucleotide sequence of the human TNFRSF25 or the nucleotide sequence encoding the human or chimeric TNFRSF25 protein contains SEQ ID NO: 5, or contains a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identity with SEQ ID NO:

5.

6. The construction method according to any one of claims 3-5, characterized in that, The nucleotide sequence of the endogenous TNFRSF25 contains a nucleotide sequence encoding the non-human TNFRSF25 protein; Preferably, the nucleotide sequence of the endogenous TNFRSF25 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 endogenous TNFRSF25 comprises all or part of exons 1 to 10 of the non-human animal TNFRSF25 gene, for example, all of exon 1 to part of exon 10; more preferably, it also includes at least 50 bp of continuous nucleotides upstream of the 5'UTR; Preferably, the nucleotide sequence of the endogenous TNFRSF25 contains exon 1 to the stop codon of the non-human animal TNFRSF25 gene; Preferably, the nucleotide sequence of the endogenous TNFRSF25 contains at least 50 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal TNFRSF25 gene, and exon 1 to the stop codon; Preferably, the nucleotide sequence of the endogenous TNFRSF25 contains at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal TNFRSF25 gene, and exon 1 to the stop codon; More preferably, the nucleotide sequence of the endogenous TNFRSF25 contains at least 168 bp of continuous nucleotides upstream of the 5'UTR of the non-human animal TNFRSF25 gene up to the stop codon.

7. The construction method according to any one of claims 1-6, characterized in that, The non-human animal is a mammal, such as a monkey or a rodent; the rodent is, for example, a mouse or a rat. Preferably, the mRNA transcribed from the modified TNFRSF25 gene in the non-human animal genome contains SEQ ID NO: 7, 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:

7.

8. The construction method according to any one of claims 1-7, characterized in that, The non-human animals also include nucleotide sequences of other human or chimeric proteins, wherein the human or chimeric protein is selected from at least one of TL1A, IL23A, IL12B, A4B7, TNFA, TNFR1, TNFR2, IL6, IL6R, TREM1, PGLYRP1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4; Preferably, the human or chimeric protein includes the human or chimeric TL1A protein; Preferably, the amino acid sequence of the human or chimeric TL1A protein comprises SEQ ID NO: 34 or contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to SEQ ID NO:

34.

9. An application of a non-human animal obtained by the construction method according to any one of claims 1-8, characterized in that, The application includes: A) Applications in product development involving immune processes in human cells related to TNFRSF25 and / or TL1A; B) Application as a model system related to TNFRSF25 and / or TL1A in pharmacological, immunological, microbiological and medical research; C) Involving the production and use of animal experimental disease models for etiological studies related to TNFRSF25 and / or TL1A and / or for the development of diagnostic and / or therapeutic strategies; D) Its application in the screening, efficacy testing, efficacy assessment, validation, or evaluation of human TNFRSF25 and / or TL1A signaling pathway modulators in vivo; or, E) Investigate the function of TNFRSF25 and / or TL1A genes, investigate drugs and their efficacy targeting human TNFRSF25 and / or TL1A, and investigate the application of drugs related to cancer, immune-related diseases, or inflammation associated with TNFRSF25 and / or TL1A.

10. A method for determining the toxicity of a therapeutic agent or its effectiveness 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) Determine the effects of the therapeutic agent on animals or its inhibitory effect on the disease; Preferably, the therapeutic agent includes a therapeutic agent targeting TNFRSF25 and / or TL1A, and preferably also includes other therapeutic agents; Preferably, the therapeutic agents targeting TNFRSF25 and / or TL1A include anti-TNFRSF25 and / or TL1A antibodies, nucleic acid drugs and / or peptide drugs targeting TNFRSF25 and / or TL1A; Preferably, the other therapeutic agents include anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies; Preferably, the disease includes cancer, immune-related diseases, or inflammation; More preferably, the cancer is a solid tumor or a hematologic tumor; the solid tumor is, for example, head and neck cancer, breast cancer, liver cancer, or lung cancer; the hematologic tumor is, for example, a lymphocytic tumor (e.g., a T-cell tumor or a B-cell tumor). More preferably, the immune-related diseases include asthma, rheumatoid arthritis, psoriasis, or multiple sclerosis; More preferably, the inflammation includes inflammatory bowel disease (IBD) or atopic dermatitis.

11. A humanized TNFRSF25 gene, characterized in that, The humanized TNFRSF25 gene contains any of the following nucleotide sequences: A) The nucleotide sequence encoding SEQ ID NO: 2; B) The nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 35; C) A nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 35; D) A nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 35.

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