A non-human animal with humanized TREM1 gene, its construction method and application

Abstract

The present invention provides a non-human animal with humanized TREM1 gene, a method for constructing the same, a humanized TREM1 protein, a humanized TREM1 gene, a targeting vector for TREM1 gene and its application in the field of biomedicine. By means of homologous recombination, the nucleotide sequence encoding human TREM1 protein is introduced into the genome of the non-human animal, and the human or humanized TREM1 protein can be normally expressed in the animal body. It can be used as an animal model for studying the signal mechanism of human TREM1, screening drugs for inflammation, tumors or immune-related diseases, and has important application value for the research and development of new drugs for immune targets.

Description

Technical Field

[0001] The present invention belongs to the fields of animal genetic engineering and genetic modification, and particularly relates to a method for constructing a non-human animal with humanized TREM1 gene and its application in biomedicine. Background Art

[0002] TREM1 (Triggering receptor expressed on myeloid cells-1) is a receptor expressed on the surface of neutrophils, monocytes, and macrophages. It cooperates with classical pattern recognition receptors (PRRs), such as the Toll-like receptor (TLR) family and the Nod-like receptor (NLR) family, to amplify the inflammatory response mediated by microbial components in host defense.

[0003] TREM1 lacks a signal transduction motif. Membrane-bound TREM1 can interact with the adaptor protein DAP12 and transduce signals, leading to cytoskeletal rearrangement and activation of transcription factors, as well as the expression of pro-inflammatory cytokines, chemokines, and cell surface molecules. In addition, activation of TLRs can lead to upregulation of TREM1 expression. TREM1 and TLR4 can synergistically promote the production of pro-inflammatory mediators by activating common signaling pathways (including activation of PI3K, ERK1 / 2, IRAK1, and Nf-κB). Soluble sTREM1 is upregulated in inflammation and is one of the important markers in the diagnosis and treatment of inflammatory diseases.

[0004] TREM1 is a myeloid cell surface receptor that is expressed in a variety of tumor microenvironments, especially concentrated on MDSC cells. PY159 is an activating monoclonal antibody with ADCC effect, which can lead to the remodeling of the immune response phenotype of the TME, generally upregulating multiple immune pathways in the TME, including T cells, NK cells, and myeloid cells, and having good pharmacodynamic results and potential for treating a wide range of tumor types. In addition, TREM1, as an amplifier of the inflammatory response, implies its anti-tumor effect. However, TREM1 activation may activate the systemic immune system, and specific targeting of the tumor microenvironment can be considered.

[0005] With the continuous development and maturity of genetic engineering technology, the replacement or substitution of homologous genes in animals with human genes has been achieved. Developing humanized experimental animal models in this way is the future development direction of animal models. Among them, the gene humanized animal model, that is, using gene editing technology to replace the homologous gene in the animal genome with a humanized normal or mutant gene, can establish normal or mutant gene animal models that are closer to human physiological or disease characteristics. Gene humanized animals not only have important application values themselves, such as improving and enhancing the humanization of cell or tissue transplantation through gene humanization, but more importantly, due to the insertion of human gene fragments, human proteins can be expressed or partially expressed in the animal body, which can be used as the target of drugs that can only recognize human protein sequences, providing the possibility for screening anti-human antibodies and other drugs at the animal level. However, due to the differences between animals and humans in physiology and pathology, coupled with the complexity of genes, for example, the identity of human and mouse TREM1 proteins is 46.1%, how to construct an "effective" gene humanized animal model for new drug research and development remains the biggest challenge.

[0006] In view of the complex mechanism of action of TREM1 and its great application value in the field of tumor treatment, in order to further explore its related biological characteristics, improve the effectiveness of preclinical pharmacodynamic tests, increase the success rate of research and development, make preclinical tests more effective and minimize research and development failures, there is an urgent need in this field to develop non-human animal models of the TREM1-related signaling pathway. In addition, the non-human animals obtained by this method can also be mated with other gene humanized non-human animals to obtain multi-gene humanized animal models for screening and evaluating the pharmacodynamic studies of human drugs and combination drugs targeting this signaling pathway. The present invention has broad application prospects in academic and clinical research. Summary of the Invention

[0007] In the first aspect of the present invention, a method for constructing a non-human animal humanized with the TREM1 gene is provided, and human or humanized TREM1 protein is expressed in the non-human animal.

[0008] Preferably, the expression of the endogenous TREM1 protein in the non-human animal is reduced or absent.

[0009] Preferably, the humanized TREM1 protein comprises all or part of the human TREM1 protein.

[0010] Preferably, the humanized TREM1 protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human TREM1 protein.

[0011] Preferably, the humanized TREM1 protein comprises all or part of the amino acid sequence encoded by exons 1 to 4 of the human TREM1 gene. More preferably, it comprises all or part of the amino acid sequence encoded by any one, two, three or more, two consecutive or three or more consecutive combinations of exons 1 to 4. Even more preferably, it comprises the amino acid sequence encoded by part of exon 1, all of exons 2 to 3, and part of exon 4, wherein the part of exon 1 comprises at least 20 bp of nucleotide sequence, for example, at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the part of exon 1 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 1, and the part of exon 4 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the part of exon 4 comprises the nucleotide sequence encoding the extracellular region.

[0012] In a specific embodiment of the present invention, the humanized TREM1 protein comprises all or part of the extracellular region of the human TREM1 protein. Preferably, it comprises the extracellular region of the human TREM1 protein with at least 50 consecutive amino acids, for example, the extracellular region of the human TREM1 protein with at least 50, 70, 90, 100, 150, 180, 181, 182, 183, 184, 185 consecutive amino acids; the extracellular region of the humanized TREM1 protein comprises the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference from the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence including substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 21-205 of SEQ ID NO: 2.

[0013] In a specific embodiment of the present invention, the humanized TREM1 protein further comprises all or part of the signal peptide of human TREM1 protein. Preferably, it comprises the signal peptide of human TREM1 protein with at least 10 consecutive amino acids, such as the signal peptide of human TREM1 protein with at least 10, 15, 16, 17, 18, 19, 20 consecutive amino acids; the signal peptide of the humanized TREM1 protein comprises the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2; or, it comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence that includes substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 1-20 of SEQ ID NO: 2.

[0014] Preferably, the humanized TREM1 protein comprises the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence that includes substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 1-205 of SEQ ID NO: 2.

[0015] Preferably, the humanized TREM1 protein further comprises a part of the TREM1 protein of a non-human animal, preferably the transmembrane region and / or cytoplasmic region of the TREM1 protein of a non-human animal.

[0016] In a specific embodiment of the present invention, the humanized TREM1 protein further comprises all or part of a non-human animal TREM1 protein. Preferably, it comprises the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1; or, it comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence shown in positions 203-230 of SEQ ID NO: 1, including substitution, deletion and / or insertion of one or more amino acid residues.

[0017] In a specific embodiment of the present invention, the amino acid sequence of the humanized TREM1 protein comprises any one of the following groups:

[0018] A) The amino acid sequence shown in SEQ ID NO: 7;

[0019] B) Having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 7;

[0020] C) Differing from the amino acid sequence shown in SEQ ID NO: 7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0021] D) An amino acid sequence shown in SEQ ID NO: 7, including substitution, deletion and / or insertion of one or more amino acid residues.

[0022] Preferably, the genome of the non-human animal comprises a human or humanized TREM1 gene, and the humanized TREM1 gene comprises a part of the human TREM1 gene.

[0023] Preferably, the humanized TREM1 gene comprises all or part of exons 1 to 4 of the human TREM1 gene. More preferably, the humanized TREM1 gene comprises all or part of any one, two, three or more, two consecutive or three or more consecutive combinations of exons 1 to 4 of the human TREM1 gene. Even more preferably, the humanized TREM1 gene comprises a part of exon 1, all of exons 2 to 3 and a part of exon 4 of the human TREM1 gene, preferably further comprises introns 1-2 and / or introns 3-4, and more preferably comprises any intron between exons 1-4. Among them, the part of exon 1 comprises at least 20 bp of nucleotide sequence, for example, at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the part of exon 1 comprises from the start codon to the last nucleotide of exon 1, and the part of exon 4 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the part of exon 4 comprises the nucleotide sequence encoding the extracellular region.

[0024] In a specific embodiment of the present invention, the humanized TREM1 gene comprises the nucleotide sequence shown in SEQ ID NO: 5; or, has an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% with the nucleotide sequence shown in SEQ ID NO: 5; or, differs from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5, including substitution, deletion and / or insertion of one or more nucleotides.

[0025] Preferably, the humanized TREM1 gene comprises all or part of the nucleotide sequence encoding human TREM1 protein. More preferably, it comprises all or part of the nucleotide sequence encoding the extracellular region of human TREM1 protein, and even more preferably, it comprises all or part of the nucleotide sequence encoding the signal peptide of human TREM1 protein, wherein the humanized TREM1 gene comprises the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more nucleotides.

[0026] Preferably, the humanized TREM1 gene further comprises all or part of the non-human animal TREM1 gene. More preferably, it comprises all or part of exons 1 to 4 of the non-human animal TREM1 gene, and even more preferably, it comprises all or part of exon 1 and / or exon 4 of the non-human animal TREM1 gene.

[0027] In a specific embodiment of the present invention, the humanized TREM1 gene further comprises the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1, including substitution, deletion and / or insertion of one or more nucleotides.

[0028] In a specific embodiment of the present invention, the nucleotide sequence of the humanized TREM1 gene comprises any one of the following groups:

[0029] A) The transcribed mRNA is the nucleotide sequence shown in SEQ ID NO: 6;

[0030] B) The transcribed mRNA has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 6;

[0031] C) The transcribed mRNA differs from the nucleotide sequence shown in SEQ ID NO: 6 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide;

[0032] D) The transcribed mRNA has a nucleotide sequence shown in SEQ ID NO: 6, including a nucleotide sequence with substitution, deletion and / or insertion of one or more nucleotides; or,

[0033] E) Comprising the nucleotide sequence shown in SEQ ID NO: 10 and / or 11.

[0034] Preferably, the humanized TREM1 gene further comprises a specific inducer or repressor. More preferably, the specific inducer or repressor can be a substance that can be induced or repressed conventionally. In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0035] Preferably, the construction method includes introducing a donor nucleotide sequence into the TREM1 gene locus of a non-human animal.

[0036] Preferably, the donor nucleotide sequence comprises one of the following groups:

[0037] A) A nucleotide sequence encoding a human or humanized TREM1 protein;

[0038] B) All or part of a nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of a human TREM1 protein. Preferably, all or part of the nucleotide sequence encoding the extracellular region of a human TREM1 protein. More preferably, a nucleotide sequence encoding at least 50 consecutive amino acids of the extracellular region of a human TREM1 protein. Even more preferably, it further comprises all or part of the nucleotide sequence encoding the signal peptide of a human TREM1 protein. Even more preferably, a nucleotide sequence encoding at least 10 consecutive amino acids of the signal peptide of a human TREM1 protein. Even further preferably, a nucleotide sequence encoding the amino acids at positions 1-205 of SEQ ID NO: 2.

[0039] C) A nucleotide sequence of a human or humanized TREM1 gene; or,

[0040] D) A portion of the human TREM1 gene, preferably all or part of exons 1 to 4 of the human TREM1 gene. More preferably, it includes all or part of a combination of any one, two, three or more, two consecutive or three or more consecutive exons among exons 1 to 4. Even more preferably, it includes a part of exon 1, all of exons 2 to 3, and a part of exon 4, wherein the part of exon 1 contains at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the part of exon 1 contains the nucleotide sequence from the start codon to the last nucleotide of exon 1, and the part of exon 4 contains at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the part of exon 4 contains the nucleotide sequence encoding the extracellular region. More preferably, it contains the nucleotide sequence shown in SEQ ID NO: 5; or, contains a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5; or, contains a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, contains a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5, including substitution, deletion and / or insertion of one or more nucleotides.

[0041] Preferably, the introduction described in the present application includes, but is not limited to, insertion, replacement or transgenesis, and the replacement is preferably in situ replacement or insertion.

[0042] Preferably, the human or humanized TREM1 gene is operably linked to at least one endogenous TREM1 endogenous regulatory element on the chromosome.

[0043] Preferably, the human or humanized TREM1 gene is regulated by regulatory elements in non-human animals. More preferably, the regulatory elements can be endogenous or exogenous.

[0044] Preferably, the regulatory elements include, but are not limited to, endogenous promoters.

[0045] In a specific embodiment of the present invention, the endogenous regulatory element is from the TREM1 gene of non-human animals. The exogenous regulatory element is from the human TREM1 gene.

[0046] Preferably, the position of the introduction is located after the endogenous regulatory element of the TREM1 gene.

[0047] Preferably, the introduction is a replacement or insertion. Optionally, the introduction into the non-human animal TREM1 locus is to replace the corresponding region of the non-human animal. Further preferably, all or part of exons 1 to 4 of the non-human animal TREM1 gene are replaced. More preferably, part of exon 1, all of exons 2 to 3, and part of exon 4 of the non-human animal are replaced.

[0048] Preferably, the nucleotide sequence encoding the amino acids shown at positions 1-202 of SEQ ID NO:1 is replaced.

[0049] Preferably, a non-human animal with humanized TREM1 gene is constructed using gene editing technology, and the gene editing technology includes gene targeting technology using embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques.

[0050] In a specific embodiment of the present invention, the construction method includes modifying the coding frame of the non-human animal TREM1 gene, and inserting the nucleotide sequence encoding human or humanized TREM1 protein or the nucleotide sequence of the humanized TREM1 gene after the endogenous regulatory element of the non-human animal TREM1 gene. Among them, the modification of the coding frame of the non-human animal TREM1 gene can be achieved by knocking out the functional region of the non-human animal TREM1 gene or by inserting a sequence, so that the non-human animal TREM1 protein is not expressed, or the expression is reduced, or the expressed protein is non-functional. Further preferably, the modification of the coding frame of the non-human animal TREM1 gene can be knocking out all or part of exons 1 to 4 of the non-human animal TREM1 gene, preferably part of exon 1, all of exons 2 to 3, and part of exon 4.

[0051] Preferably, a non-human animal is constructed using a targeting vector.

[0052] Preferably, the targeting vector contains a donor nucleotide sequence, and the donor nucleotide sequence contains one of the following groups:

[0053] A) The nucleotide sequence encoding human or humanized TREM1 protein;

[0054] B) all or part of the nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of human TREM1 protein, preferably all or part of the nucleotide sequence encoding the extracellular region of human TREM1 protein, more preferably the nucleotide sequence encoding at least 50 consecutive amino acids in the extracellular region of human TREM1 protein, more preferably, further comprising all or part of the nucleotide sequence encoding the signal peptide of human TREM1 protein, still more preferably, the nucleotide sequence encoding at least 10 consecutive amino acids in the signal peptide of human TREM1 protein, still further preferably, the nucleotide sequence encoding the amino acids at positions 1-205 of SEQ ID NO: 2.

[0055] C) the nucleotide sequence of human or humanized TREM1 gene; or,

[0056] D) a part of the human TREM1 gene, preferably all or part of exons 1 to 4 of the human TREM1 gene. More preferably, it comprises all or part of a combination of any one, two, three or more, two consecutive or three or more consecutive exons among exons 1 to 4. Still more preferably, it comprises a part of exon 1, all of exons 2 to 3 and a part of exon 4, wherein the part of exon 1 comprises at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the part of exon 1 comprises the nucleotides from the start codon to the last nucleotide of exon 1, the part of exon 4 comprises at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the part of exon 4 comprises the nucleotide sequence encoding the extracellular region. Further preferably, the targeting vector comprises the nucleotide sequence shown in SEQ ID NO: 5; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5, including substitution, deletion and / or insertion of one or more nucleotides.

[0057] Preferably, the targeting vector further comprises a 5' arm (5' homologous arm) and / or a 3' arm (3' homologous arm).

[0058] The 5'-arm is a DNA fragment homologous to the 5'-end of the conversion region to be modified, and it is selected from nucleotides with a length of 100 - 10,000 of the genomic DNA of the non-human animal TREM1 gene. Preferably, the 5'-arm is a nucleotide having at least 90% homology with NCBI accession number NC_000083.7. Further preferably, the 5'-arm sequence is as shown in SEQ ID NO: 3 or 8.

[0059] The 3'-arm is a second DNA fragment homologous to the 3'-end of the conversion region to be modified, and it is selected from nucleotides with a length of 100 - 10,000 of the genomic DNA of the non-human animal TREM1 gene; preferably, the 3'-arm is a nucleotide having at least 90% homology with NCBI accession number NC_000083.7; further preferably, the 3'-arm sequence is as shown in SEQ ID NO: 4 or 9.

[0060] Preferably, the targeting vector further comprises SEQ ID NO: 10, 11, 12, and / or 13.

[0061] Preferably, the conversion region to be modified is located on exons 1 to 4 of the non-human animal TREM1 gene.

[0062] Preferably, the targeting vector further comprises a marker gene. Further preferably, the marker gene is a coding gene for a negative selection marker. Even more preferably, the coding gene for the negative selection marker is the coding gene for diphtheria toxin A subunit (DTA).

[0063] In a specific embodiment of the present invention, the targeting vector further includes a resistance gene for positive clone screening. Further preferably, the resistance gene for positive clone screening is the coding sequence Neo of neomycin phosphotransferase.

[0064] In a specific embodiment of the present invention, the targeting vector further includes a specific recombination system. Further preferably, the specific recombination system is the Frt recombination site (the conventional LoxP recombination system can also be selected). There are 2 specific recombination systems, which are respectively installed on both sides of the resistance gene.

[0065] Preferably, to improve the recombination efficiency, an sgRNA targeting the TREM1 gene can also be used together with the above targeting vector for the construction of non-human animals. Among them, the sgRNA targets the non-human animal TREM1 gene, and at the same time, the sequence of the sgRNA is on the target sequence of the TREM1 gene to be modified.

[0066] Preferably, the sgRNA target site is located on the exon 1 to exon 4 sequences of the TREM1 gene.

[0067] Preferably, the target site of the sgRNA is located on the exon 1 and / or exon 4 sequence of the TREM1 gene.

[0068] Preferably, the target sequence of the sgRNA on the TREM1 gene is as shown in SEQ ID NO: 16 or 17.

[0069] In a specific embodiment of the present invention, the construction method includes introducing the above-mentioned targeting vector, the sgRNA targeting the TREM1 gene, and Cas9 into non-human animal cells, culturing the cells (preferably fertilized eggs), and then transplanting the cultured cells into the fallopian tube of a female non-human animal to allow it to develop, and identifying and screening to obtain a non-human animal with humanized TREM1 gene.

[0070] According to some embodiments of the present invention, the construction method further includes: mating a non-human animal with humanized TREM1 gene with other gene-modified non-human animals, in vitro fertilization, or directly performing gene editing, and screening to obtain a multi-gene-modified non-human animal.

[0071] Preferably, the other gene is a non-human animal modified by at least one gene selected from IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, and TNF-α.

[0072] Preferably, the non-human animal also expresses at least one of human or humanized IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, and TNF-α proteins.

[0073] Preferably, the human or humanized TREM1 gene and / or other genes are homozygous for the endogenous modified locus.

[0074] Preferably, the human or humanized TREM1 gene and / or other genes are heterozygous for the endogenous modified locus.

[0075] Preferably, the non-human animal can be selected from any non-human animal that can be genetically edited to prepare a gene-humanized non-human animal, such as rodents, pigs, rabbits, monkeys, etc.

[0076] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0077] Preferably, the non-human animal is an immunodeficient non-human mammal. More preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc scid IL-2rγ null mice, NOD-Rag 1 - / - -IL2rg - / - (NRG) mice, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0078] In a second aspect of the present invention, there is provided a non-human animal with humanized TREM1 gene, and the non-human animal expresses human or humanized TREM1 protein in vivo.

[0079] Preferably, the expression of endogenous TREM1 protein in the non-human animal is reduced or absent.

[0080] Preferably, the humanized TREM1 protein comprises all or part of the human TREM1 protein.

[0081] Preferably, the humanized TREM1 protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human TREM1 protein.

[0082] Preferably, the humanized TREM1 protein comprises the amino acid sequence encoded by all or part of exons 1 to 4 of the human TREM1 gene. More preferably, it comprises all or part of the amino acid sequence encoded by any one, two, three or more, two consecutive or three or more consecutive combinations of exons 1 to 4. Even more preferably, it comprises the partial amino acid sequence of exon 1, the entire amino acid sequence of exons 2 to 3 and the partial amino acid sequence of exon 4, wherein the partial exon 1 sequence comprises at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the partial exon 1 sequence comprises the nucleotide sequence from the start codon to the last nucleotide of exon 1, the partial exon 4 sequence comprises at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the partial exon 4 sequence comprises the nucleotide sequence encoding the extracellular region.

[0083] In a specific embodiment of the present invention, the humanized TREM1 protein comprises all or part of the extracellular region of the human TREM1 protein. Preferably, it comprises the extracellular region of the human TREM1 protein having at least 50 consecutive amino acids, such as the extracellular region of the human TREM1 protein having at least 50, 70, 90, 100, 150, 180, 181, 182, 183, 184, 185 consecutive amino acids; the extracellular region of the humanized TREM1 protein comprises the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence shown in positions 21-205 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more amino acid residues.

[0084] In a specific embodiment of the present invention, the humanized TREM1 protein further comprises all or part of the signal peptide of the human TREM1 protein. Preferably, it comprises the signal peptide of the human TREM1 protein having at least 10 consecutive amino acids, such as the signal peptide of the human TREM1 protein having at least 10, 15, 16, 17, 18, 19, 20 consecutive amino acids; the signal peptide of the humanized TREM1 protein comprises the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2; or, it comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence shown in positions 1-20 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more amino acid residues.

[0085] Preferably, the humanized TREM1 protein comprises the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2; or, comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence that includes substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 1-205 of SEQ ID NO: 2.

[0086] Preferably, the humanized TREM1 protein further comprises a portion of a non-human animal TREM1 protein, preferably the transmembrane region and / or cytoplasmic region of a non-human animal TREM1 protein.

[0087] In a specific embodiment of the present invention, the humanized TREM1 protein further comprises all or a portion of a non-human animal TREM1 protein, preferably, comprises the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1; or, comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1; or, comprises an amino acid sequence that differs from the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence that includes substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 203-230 of SEQ ID NO: 1.

[0088] In a specific embodiment of the present invention, the amino acid sequence of the humanized TREM1 protein comprises any one of the following groups:

[0089] A) The amino acid sequence shown in SEQ ID NO: 7;

[0090] B) Having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 7;

[0091] C) Differing from the amino acid sequence shown in SEQ ID NO: 7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0092] D) an amino acid sequence as shown in SEQ ID NO: 7, including substitution, deletion and / or insertion of one or more amino acid residues.

[0093] Preferably, the non-human animal genome contains a human or humanized TREM1 gene, and the humanized TREM1 gene contains a part of the human TREM1 gene.

[0094] Preferably, the humanized TREM1 gene contains all or part of exons 1 to 4 of the human TREM1 gene. More preferably, the humanized TREM1 gene contains all or part of any one, two, three or more, two consecutive or three or more consecutive combinations of exons 1 to 4 of the human TREM1 gene. Even more preferably, the humanized TREM1 gene contains a part of exon 1, all of exons 2 to 3 and a part of exon 4 of the human TREM1 gene, preferably also contains introns 1-2 and / or introns 3-4, and more preferably contains any intron between exons 1-4, wherein the part of exon 1 contains at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the part of exon 1 contains the nucleotide sequence from the start codon to the last nucleotide of exon 1, the part of exon 4 contains at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the part of exon 4 contains the nucleotide sequence encoding the extracellular region.

[0095] In a specific embodiment of the present invention, the humanized TREM1 gene contains the nucleotide sequence shown in SEQ ID NO: 5; or, contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5; or, contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 5; or, contains a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5, including substitution, deletion and / or insertion of one or more nucleotides.

[0096] Preferably, the humanized TREM1 gene comprises all or part of the nucleotide sequence encoding the human TREM1 protein. More preferably, it comprises all or part of the nucleotide sequence encoding the extracellular region of the human TREM1 protein, and even more preferably, it comprises all or part of the nucleotide sequence encoding the signal peptide of the human TREM1 protein, wherein the humanized TREM1 gene comprises the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more nucleotides.

[0097] Preferably, the humanized TREM1 gene further comprises all or part of the non-human animal TREM1 gene. More preferably, it comprises all or part of exons 1 to 4 of the non-human animal TREM1 gene, and even more preferably, it comprises all or part of exon 1 and / or exon 4 of the non-human animal TREM1 gene.

[0098] In a specific embodiment of the present invention, the humanized TREM1 gene further comprises the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1, including substitution, deletion and / or insertion of one or more nucleotides.

[0099] In a specific embodiment of the present invention, the nucleotide sequence of the humanized TREM1 gene comprises any one of the following groups:

[0100] A) The transcribed mRNA is the nucleotide sequence shown in SEQ ID NO: 6;

[0101] B) The transcribed mRNA has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 6;

[0102] C) The transcribed mRNA differs from the nucleotide sequence shown in SEQ ID NO: 6 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide;

[0103] D) The transcribed mRNA has a nucleotide sequence shown in SEQ ID NO: 6, including nucleotide sequences with substitutions, deletions and / or insertions of one or more nucleotides; or,

[0104] E) It contains the nucleotide sequences shown in SEQ ID NO: 10 and / or 11.

[0105] Preferably, the nucleotide sequence of the human or humanized TREM1 gene is operably linked to an endogenous regulatory element at the endogenous TREM1 locus in at least one chromosome.

[0106] Preferably, the human TREM1 gene is introduced into the TREM1 locus of a non-human animal.

[0107] Preferably, the human TREM1 gene contains all or part of exons 1 to 4 of the human TREM1 gene. More preferably, it contains all or part of any one, two, three or more, two consecutive or three or more consecutive combinations of exons 1 to 4. Even more preferably, it contains a part of exon 1, all of exons 2 to 3 and a part of exon 4, preferably also contains introns 1-2 and / or introns 3-4, and more preferably contains any intron between exons 1-4, wherein the part of exon 1 contains at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the part of exon 1 contains the nucleotide sequence from the start codon to the last nucleotide of exon 1, the part of exon 4 contains at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the part of exon 4 contains the nucleotide sequence encoding the extracellular region.

[0108] Preferably, a nucleotide sequence comprising SEQ ID NO: 5; or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 5; or a nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 5; or a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5, including substitution, deletion and / or insertion of one or more nucleotides, is introduced into the TREM1 locus of a non-human animal.

[0109] Preferably, a nucleotide sequence encoding all or part of the human TREM1 protein is introduced into the TREM1 locus of a non-human animal, preferably all or part of the nucleotide sequence encoding the extracellular region of the human TREM1 protein, more preferably all or part of the nucleotide sequence encoding the signal peptide of the human TREM1 protein.

[0110] Preferably, a nucleotide sequence comprising the nucleotide sequence of positions 1-205 of SEQ ID NO: 2; or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2; or a nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2; or a nucleotide sequence having the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more nucleotides, is introduced into the TREM1 locus of a non-human animal.

[0111] According to some embodiments of the present invention, the non-human animal further comprises other gene modifications, and the other genes are selected from at least one of IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4 and TNF-α.

[0112] Preferably, the human or humanized TREM1 gene and / or other genes are homozygous for the endogenous modified locus.

[0113] Preferably, the human or humanized TREM1 gene and / or other genes are heterozygous for the endogenous modified locus.

[0114] Preferably, the humanized TREM1 gene further includes a specific inducer or repressor. More preferably, the specific inducer or repressor can be a substance that can be induced or repressed conventionally.

[0115] In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0116] Preferably, the non-human animal can be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc. that can be genetically edited to prepare gene humanization.

[0117] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0118] Preferably, the non-human animal is an immunodeficient non-human mammal. More preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or a rat. Most preferably, the immunodeficient mouse is NOD-Prkdc scid IL-2rγ null mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mouse, NOD / SCID mouse or nude mouse.

[0119] In a third aspect of the present invention, a targeting vector is provided. The targeting vector contains a donor nucleotide sequence, and the donor nucleotide sequence contains one of the following groups:

[0120] A) a nucleotide sequence encoding a human or humanized TREM1 protein;

[0121] B) all or part of the nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of human TREM1 protein. Preferably, all or part of the nucleotide sequence encoding the extracellular region of human TREM1 protein. More preferably, the nucleotide sequence encoding at least 50 consecutive amino acids in the extracellular region of human TREM1 protein. Even more preferably, it further includes all or part of the nucleotide sequence encoding the signal peptide of human TREM1 protein. Even more preferably, the nucleotide sequence encoding at least 10 consecutive amino acids in the signal peptide of human TREM1 protein. Still more preferably, the nucleotide sequence encoding the amino acids at positions 1-205 of SEQ ID NO: 2;

[0122] C) the nucleotide sequence of human or humanized TREM1 gene; or,

[0123] D) a part of the human TREM1 gene. Preferably, all or part of exons 1 to 4 of the human TREM1 gene. More preferably, all or part of a combination of any one, two, three or more, two consecutive or three or more consecutive exons among exons 1 to 4. Even more preferably, it includes a part of exon 1, all of exons 2 to 3, and a part of exon 4, wherein the part of exon 1 contains at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the part of exon 1 contains the nucleotide sequence from the start codon to the last nucleotide of exon 1, and the part of exon 4 contains at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the part of exon 4 contains the nucleotide sequence encoding the extracellular region. More preferably, the targeting vector contains the nucleotide sequence shown in SEQ ID NO: 5; or, contains a nucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 5; or, contains a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, contains a nucleotide sequence with substitutions, deletions and / or insertions of one or more nucleotides as shown in the nucleotide sequence shown in SEQ ID NO: 5.

[0124] Preferably, the targeting vector further includes a 5' arm (5' homologous arm) and / or a 3' arm (3' homologous arm).

[0125] The 5'-arm is a DNA fragment homologous to the 5'-end of the conversion region to be altered, which is selected from nucleotides with a length of 100 to 10,000 of the genomic DNA of the TREM1 gene of a non-human animal. Preferably, the 5'-arm is a nucleotide having at least 90% homology with the NCBI accession number NC_000083.7. More preferably, the 5'-arm sequence is as shown in SEQ ID NO: 3 or 8.

[0126] The 3'-arm is a second DNA fragment homologous to the 3'-end of the conversion region to be altered, which is selected from nucleotides with a length of 100 to 10,000 of the genomic DNA of the TREM1 gene of a non-human animal; preferably, the 3'-arm is a nucleotide having at least 90% homology with the NCBI accession number NC_000083.7; more preferably, the 3'-arm sequence is as shown in SEQ ID NO: 4 or 9.

[0127] Preferably, the targeting vector further comprises SEQ ID NO: 10, 11, 12, and / or 13.

[0128] Preferably, the conversion region to be altered is located on exons 1 to 4 of the TREM1 gene of a non-human animal.

[0129] Preferably, the targeting vector further comprises a marker gene. More preferably, the marker gene is a coding gene for a negative selection marker. Even more preferably, the coding gene for the negative selection marker is the coding gene for diphtheria toxin A subunit (DTA).

[0130] In a specific embodiment of the present invention, the targeting vector further includes a resistance gene for positive clone screening. More preferably, the resistance gene for positive clone screening is the coding sequence Neo of neomycin phosphotransferase.

[0131] In a specific embodiment of the present invention, the targeting vector further includes a specific recombination system. More preferably, the specific recombination system is the Frt recombination site (the conventional LoxP recombination system can also be selected). There are 2 specific recombination systems, which are respectively installed on both sides of the resistance gene.

[0132] In the fourth aspect of the present invention, an sgRNA is provided, and the sgRNA targets the TREM1 gene of a non-human animal, and its target site is located on the exon 1 to exon 4 sequences of the TREM1 gene.

[0133] Preferably, the target site of the sgRNA is located on the exon 1 and / or exon 4 sequences of the TREM1 gene.

[0134] Preferably, the target sequence of the sgRNA on the TREM1 gene is as shown in SEQ ID NO: 16 or 17.

[0135] In the fifth aspect of the present invention, there is provided a DNA molecule encoding the above sgRNA. Preferably, the double strand of the DNA molecule is the upstream and downstream sequences of the sgRNA, or the forward oligonucleotide sequence or the reverse oligonucleotide sequence after adding restriction enzyme sites.

[0136] In a specific embodiment of the present invention, the nucleotide sequences of the double strands of the DNA molecule are as shown in SEQ ID NO: 16 and SEQ ID NO: 19, SEQ ID NO: 18 and SEQ ID NO: 20, SEQ ID NO: 17 and SEQ ID NO: 22, or SEQ ID NO: 21 and SEQ ID NO: 23.

[0137] In the sixth aspect of the present invention, there is provided a vector containing the above sgRNA.

[0138] In the seventh aspect of the present invention, there is provided a cell containing the above targeting vector, the above sgRNA, the above DNA molecule and / or the above vector.

[0139] In the eighth aspect of the present invention, there is provided an application of the above targeting vector, the above sgRNA, the above DNA molecule, the above vector and / or the above cell in TREM1 gene editing. Preferably, the application includes, but is not limited to, knockout, insertion or replacement.

[0140] In the ninth aspect of the present invention, there is provided a humanized TREM1 protein, which contains all or part of the human TREM1 protein.

[0141] Preferably, the humanized TREM1 protein contains all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human TREM1 protein.

[0142] Preferably, the humanized TREM1 protein comprises all or part of the amino acid sequence encoded by exons 1 to 4 of the human TREM1 gene. More preferably, it comprises all or part of the amino acid sequence encoded by a combination of any one, two, three or more, two consecutive or three or more consecutive exons among exons 1 to 4. Even more preferably, it comprises the amino acid sequence encoded by a partial sequence of exon 1, the whole sequences of exons 2 to 3, and a partial sequence of exon 4, wherein the partial sequence of exon 1 comprises at least 20 bp of nucleotide sequence, for example, at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the partial sequence of exon 1 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 1, and the partial sequence of exon 4 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the partial sequence of exon 4 comprises the nucleotide sequence encoding the extracellular region.

[0143] In a specific embodiment of the present invention, the humanized TREM1 protein comprises all or part of the extracellular region of the human TREM1 protein. Preferably, it comprises the extracellular region of the human TREM1 protein with at least 50 consecutive amino acids, for example, the extracellular region of the human TREM1 protein with at least 50, 70, 90, 100, 150, 180, 181, 182, 183, 184, 185 consecutive amino acids; the extracellular region of the humanized TREM1 protein comprises the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 21-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence including substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 21-205 of SEQ ID NO: 2.

[0144] In a specific embodiment of the present invention, the humanized TREM1 protein further comprises all or part of the signal peptide of the human TREM1 protein. Preferably, it comprises the signal peptide of the human TREM1 protein with at least 10 consecutive amino acids, such as the signal peptide of the human TREM1 protein comprising at least 10, 15, 16, 17, 18, 19, 20 consecutive amino acids; the signal peptide of the humanized TREM1 protein comprises the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2; or, it comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-20 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence shown in positions 1-20 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more amino acid residues.

[0145] Preferably, the partial humanized TREM1 protein comprises the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence shown in positions 1-205 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more amino acid residues.

[0146] Preferably, the humanized TREM1 protein further comprises a part of the TREM1 protein of a non-human animal, preferably the transmembrane region and / or cytoplasmic region of the TREM1 protein of a non-human animal.

[0147] In a specific embodiment of the present invention, the humanized TREM1 protein further comprises all or part of the non-human animal TREM1 protein. Preferably, it comprises the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1; or, it comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1; or, it comprises an amino acid sequence that differs from the amino acid sequence shown in positions 203-230 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, it comprises an amino acid sequence that includes substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 203-230 of SEQ ID NO: 1.

[0148] In a specific embodiment of the present invention, the amino acid sequence of the humanized TREM1 protein comprises any one of the following groups:

[0149] A) The amino acid sequence shown in SEQ ID NO: 7;

[0150] B) Having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 7;

[0151] C) Differing from the amino acid sequence shown in SEQ ID NO: 7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0152] D) An amino acid sequence that includes substitution, deletion and / or insertion of one or more amino acid residues as shown in SEQ ID NO: 7.

[0153] Preferably, the non-human animal can be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc. that can be genetically edited to prepare gene humanization.

[0154] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0155] Preferably, the non-human animal is an immunodeficient non-human mammal. More preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or a rat. Most preferably, the immunodeficient mouse is NOD-Prkdcscid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) Mouse, Rag 2 - / - -IL2rg - / - (RG) Mouse, NOD / SCID mouse or nude mouse.

[0156] In the tenth aspect of the present invention, there is provided a humanized TREM1 gene, and the humanized TREM1 gene contains a part of the human TREM1 gene.

[0157] Preferably, the humanized TREM1 gene encodes the above-mentioned humanized TREM1 protein.

[0158] Preferably, the humanized TREM1 gene contains all or part of exons 1 to 4 of the human TREM1 gene. Further preferably, the humanized TREM1 gene contains any one, two, three or more, two consecutive or three or more consecutive combinations of exons 1 to 4 of the human TREM1 gene in whole or in part. Even more preferably, the humanized TREM1 gene contains a part of exon 1, all of exons 2 to 3, and a part of exon 4 of the human TREM1 gene, preferably also contains introns 1-2 and / or introns 3-4, and more preferably contains any intron between exons 1-4, wherein the part of exon 1 contains at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 45, 46, 47, 48, 49, 50, 70, 76 bp of nucleotide sequence; preferably, the part of exon 1 contains the nucleotide sequence from the start codon to the last nucleotide of exon 1, and the part of exon 4 contains at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 2600, 2626 bp of nucleotide sequence; preferably, the part of exon 4 contains the nucleotide sequence encoding the extracellular region.

[0159] In a specific embodiment of the present invention, the humanized TREM1 gene comprises the nucleotide sequence shown in SEQ ID NO: 5; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 5; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5, including substitution, deletion and / or insertion of one or more nucleotides.

[0160] Preferably, the humanized TREM1 gene comprises all or part of the nucleotide sequence encoding the human TREM1 protein. More preferably, it comprises all or part of the nucleotide sequence encoding the extracellular region of the human TREM1 protein, and even more preferably, it comprises all or part of the nucleotide sequence encoding the signal peptide of the human TREM1 protein. Among them, the humanized TREM1 gene comprises the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more nucleotides.

[0161] Preferably, the humanized TREM1 gene further comprises all or part of the non-human animal TREM1 gene. More preferably, it comprises all or part of exons 1 to 4 of the non-human animal TREM1 gene, and even more preferably, it comprises all or part of exon 1 and / or exon 4 of the non-human animal TREM1 gene.

[0162] In a specific embodiment of the present invention, the humanized TREM1 gene further comprises a nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1; or, comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having the nucleotide sequence encoding positions 203-230 of SEQ ID NO: 1, including substitution, deletion and / or insertion of one or more nucleotides.

[0163] In a specific embodiment of the present invention, the nucleotide sequence of the humanized TREM1 gene comprises any one of the following groups:

[0164] A) The transcribed mRNA is the nucleotide sequence shown in SEQ ID NO: 6;

[0165] B) The transcribed mRNA has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 6;

[0166] C) The transcribed mRNA differs from the nucleotide sequence shown in SEQ ID NO: 6 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide;

[0167] D) The transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 6, including substitution, deletion and / or insertion of one or more nucleotides; or,

[0168] E) Comprises the nucleotide sequence shown in SEQ ID NO: 10 and / or 11.

[0169] Preferably, the humanized TREM1 gene further comprises a specific inducer or repressor. More preferably, the specific inducer or repressor can be a substance that can be induced or repressed conventionally. In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0170] Preferably, the non-human animal can be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc. that can be genetically edited to prepare a gene humanized animal.

[0171] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0172] Preferably, the non-human animal is an immunodeficient non-human mammal. More preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or a rat. Most preferably, the immunodeficient mouse is NOD-Prkdc scid IL-2rγ null mice, NOD-Rag 1 - / - -IL2rg - / - (NRG) mice, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0173] In the eleventh aspect of the present invention, there is provided a non-human animal with a deletion of the TREM1 gene, wherein the non-human animal lacks all or part of exon 1 to exon 4 of the endogenous TREM1 gene, preferably part of exon 1, all of exon 2 to exon 3, and part of exon 4.

[0174] In the twelfth aspect of the present invention, there is provided a method for constructing a non-human animal with a deletion of the TREM1 gene, the construction method comprising preparing a non-human animal using the above-mentioned targeting vector or the above-mentioned sgRNA.

[0175] In the thirteenth aspect of the present invention, there is provided a cell with a deletion of the TREM1 gene, wherein the cell lacks all or part of exon 1 to exon 4 of the TREM1 gene, preferably part of exon 1, all of exon 2 to exon 3, and part of exon 4.

[0176] In the fourteenth aspect of the present invention, there is provided a method for constructing a cell with a deletion of the TREM1 gene, comprising constructing a cell with a deletion of the TREM1 gene using the above-mentioned targeting vector and / or the above-mentioned sgRNA.

[0177] In the fifteenth aspect of the present invention, there is provided a cell, tissue or organ, wherein the cell, tissue or organ expresses the above-mentioned humanized TREM1 protein, and the genome of the cell, tissue or organ contains the above-mentioned humanized TREM1 gene. Alternatively, the cell, tissue or organ is derived from the above-mentioned non-human animal, or the non-human animal obtained by the above-mentioned construction method.

[0178] In the sixteenth aspect of the present invention, a tumor tissue after tumor-bearing is provided, and the tumor tissue expresses the above-mentioned humanized TREM1 protein. Alternatively, the tumor tissue after tumor-bearing is derived from the above-mentioned non-human animal, or the non-human animal obtained by the above-mentioned construction method.

[0179] In the seventeenth aspect of the present invention, an animal model is provided, and the animal model is derived from the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

[0180] In the eighteenth aspect of the present invention, a method for constructing an animal model is provided, and the method is carried out by using the above-mentioned constructed non-human animal, non-human animal or its offspring, and non-human animal with gene deletion. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

[0181] In the nineteenth aspect of the present invention, an application of the above-mentioned non-human animal and the non-human animal obtained by the above-mentioned construction method in constructing an animal model is provided. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

[0182] In the twentieth aspect of the present invention, an application of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method or the above-mentioned animal model in preparing a drug for treating tumors, inflammation or immune-related diseases is provided.

[0183] In the twenty-first aspect of the present invention, a cell with humanized TREM1 gene is provided, and the cell expresses human or humanized TREM1 protein.

[0184] Preferably, the cell expresses the above-mentioned humanized TREM1 protein.

[0185] Preferably, a part of the human TREM1 gene is included in the genome of the cell. More preferably, the cell contains the above-mentioned humanized TREM1 gene.

[0186] In the twenty-second aspect of the present invention, a construct containing the above-mentioned humanized TREM1 gene or a construct expressing the above-mentioned humanized TREM1 protein is provided. Preferably, the construct can be a plasmid.

[0187] In the twenty-third aspect of the present invention, a cell containing the above-mentioned construct is provided.

[0188] In the twenty-fourth aspect of the present invention, a tissue containing the above-mentioned cell is provided.

[0189] In the twenty-fifth aspect of the present invention, a genome of a non-human animal with humanized TREM1 gene is provided.

[0190] Preferably, the genome contains all or part of the human or humanized TREM1 gene, and / or contains all or part of the nucleotide sequence encoding the human or humanized TREM1 protein.

[0191] Preferably, the humanized TREM1 gene is the above-mentioned humanized TREM1 gene.

[0192] Preferably, the humanized TREM1 protein is the above-mentioned humanized TREM1 protein.

[0193] Preferably, the genome contains, at the endogenous TREM1 locus of a non-human animal, a genomic fragment of the human TREM1 gene (preferably all or part of the sequence encoding the signal peptide and / or extracellular region of human TREM1), and / or a genomic fragment of the non-human animal TREM1 gene (preferably all or part of the sequence encoding the transmembrane region and / or cytoplasmic region of non-human animal TREM1), and the genomic fragment of the non-human animal TREM1 gene is introduced to form a modified TREM1 gene.

[0194] Preferably, the genome contains, at the endogenous TREM1 locus of a non-human animal, the humanized TREM1 gene introduced into the genomic fragment of the non-human animal TREM1 gene to form a modified TREM1 gene.

[0195] The modified TREM1 gene encodes a humanized TREM1 protein.

[0196] Preferably, the introduction is insertion or replacement.

[0197] Preferably, the replacement is to replace all or part of exons 1 to 4 of the non-human animal TREM1 gene. Further preferably, part of exon 1, all of exons 2 to 3, and part of exon 4 of the non-human animal are replaced.

[0198] Preferably, the expression of the modified TREM1 gene is controlled by the endogenous regulatory elements of the non-human animal.

[0199] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare gene humanization, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0200] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. More preferably, the rodent is a rat or a mouse.

[0201] In the twenty-sixth aspect of the present invention, there is provided a cell, tissue or organ comprising the genome of the above-mentioned non-human animal with humanized TREM1 gene.

[0202] Preferably, any of the above cells, tissues or organs or tumor tissues after tumor-bearing include cells, tissues or organs or tumor tissues after tumor-bearing that can develop into an animal individual or cannot develop into an animal individual.

[0203] Preferably, any of the above cells, tissues or organs or tumor tissues after tumor-bearing include cells, tissues or organs or tumor tissues after tumor-bearing that can develop into an animal individual or cannot develop into an animal individual.

[0204] In the twenty-seventh aspect of the present invention, there is provided an application of the above-mentioned humanized TREM1 protein, the above-mentioned humanized TREM1 gene, the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method, any of the above cells, tissues or organs, or tumor tissue, the above animal model, and the application includes:

[0205] A) Application in the development of products related to TREM1-related immune processes involving human cells;

[0206] B) Application as a model system related to TREM1 in pharmacological, immunological, microbiological and medical research;

[0207] C) Application related to the production and utilization of animal experimental disease models for etiological research related to TREM1 and / or for developing diagnostic strategies and / or for developing therapeutic strategies;

[0208] D) Application in the screening, efficacy detection, evaluation of efficacy, verification or evaluation of human TREM1 signaling pathway regulators in vivo; or,

[0209] E) Application in studying the function of the TREM1 gene, studying the drugs and drug effects targeting human TREM1 target sites, studying drugs for immune-related diseases related to TREM1 and anti-tumor drugs.

[0210] Preferably, the application includes a method for treating and / or diagnosing a disease, or a method for treating and / or diagnosing a non-disease.

[0211] In the twenty-eighth aspect of the present invention, there is provided a method for screening a human TREM1-specific regulator from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method or the above-mentioned tumor-bearing or inflammatory animal model.

[0212] In the twenty-ninth aspect of the present invention, there is provided a method for screening a human TREM1-specific regulator, and the screening method includes applying a regulator to an individual implanted with tumor cells and detecting tumor inhibition; wherein, the individual is selected from the above-mentioned non-human animal or the non-human animal constructed by the above method or the above-mentioned tumor-bearing animal model.

[0213] Preferably, the modulator is selected from CAR-T and drugs. More preferably, the drug is an antibody. Specifically, the drug can be an anti-TREM1 antibody.

[0214] Preferably, the modulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0215] Preferably, the detection includes measuring the size and / or proliferation rate of tumor cells.

[0216] Preferably, the detection methods include measurement with vernier calipers, flow cytometry detection, and / or in vivo imaging detection of animals.

[0217] Preferably, the detection includes evaluating the body weight, fat mass, activation pathway, neuroprotective activity, or metabolic changes of an individual, and the metabolic changes include changes in food consumption or water consumption.

[0218] Preferably, the tumor cells are derived from humans or non-human animals.

[0219] Preferably, the screening method includes treatment methods and non-treatment methods.

[0220] In a specific embodiment, the screening method detects and evaluates the effect of the modulator to determine whether the modulator has a therapeutic effect, that is, the therapeutic effect is not inevitable, but only a possibility.

[0221] In the thirtieth aspect of the present invention, a method for screening or evaluating drugs for human use is provided. The method includes constructing an individual of a disease animal model, administering a candidate drug to the individual of the disease animal model, and performing a pharmacodynamic detection and / or comparison on the individual administered with the candidate drug. Among them, the individual is selected from the non-human animals obtained by the above construction method, the above non-human animals or their offspring, or the above tumor-bearing or inflammatory animal models.

[0222] Preferably, the drug screening or evaluation method includes treatment methods and non-treatment methods.

[0223] In a specific embodiment, this method is used to screen or evaluate drugs, detect and compare the pharmacodynamic effects of candidate drugs to determine which candidate drugs can be used as drugs and which cannot, or compare the pharmacodynamic sensitivity of different drugs, that is, the therapeutic effect is not inevitable, but only a possibility.

[0224] Preferably, the candidate drugs include targeted drugs. More preferably, the targeted drug is an antigen-binding protein. In a specific embodiment of the present invention, the antigen-binding protein is an antibody.

[0225] Preferably, the candidate drug is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0226] Preferably, the detection includes measuring the size and / or proliferation rate of tumor cells; preferably, the methods for detection include measurement with a vernier caliper, flow cytometry, and / or in vivo imaging of animals.

[0227] Preferably, the detection includes evaluating the body weight, fat mass, activation pathway, neuroprotective activity, or metabolic changes of an individual, and the metabolic changes include changes in food consumption or water consumption.

[0228] Preferably, any of the above non-human animals is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0229] Preferably, any of the above non-human animals can also be selected from pigs, rabbits, monkeys, and any other non-human animals that can be genetically edited to prepare humanized genes.

[0230] The "immune-related diseases" described in the present invention include, but are not limited to, allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or nerve disorders, etc.

[0231] The "tumors" described in the present invention include, but are not limited to, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioblastoma, 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 multiforme, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Among them, the leukemia is selected from acute lymphoblastic (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 lymphoma and non-Hodgkin 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 sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment of the present invention, the tumors are breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, and liver cancer.

[0232] As used herein, "treating" (or "treat" or "treatment") means slowing, interrupting, arresting, controlling, halting, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve complete elimination of all disease-related signs, symptoms, conditions, or disorders. The terms "treating" and the like refer to therapeutic interventions that improve the signs, symptoms, etc. of a disease or pathological condition after the disease has begun to develop.

[0233] As used herein, "locus" broadly represents the position occupied by a gene on a chromosome, and narrowly represents a DNA fragment on a certain gene, that is, it can be a gene or a part of a gene. For example, the "TREM1 locus" refers to an optional DNA fragment on exons 1 to 4 of the TREM1 gene. In a specific embodiment of the present invention, the replaced TREM1 locus can be an optional DNA fragment on exons 1 to 4 of the TREM1 gene.

[0234] As used herein, "nucleotide sequence" includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences. Preferably, it is DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, tRNA.

[0235] As used herein, "all or part" means "all" is the whole, and "part" is a local part of the whole or an individual constituting the whole.

[0236] As used herein, "humanized TREM1 protein" contains a part derived from human TREM1 protein. Among them, the "human TREM1 protein" is the same as "the whole of human TREM1 protein", that is, its amino acid sequence is identical to the full-length amino acid sequence of human TREM1 protein. The "part of human TREM1 protein" is a continuous or spaced 5-234 (preferably 10-205) amino acid sequence that is identical to the amino acid sequence of human TREM1 protein or has more than 70% homology with the amino acid sequence of human TREM1 protein.

[0237] The "humanized TREM1 gene" described in the present invention comprises a part derived from the human TREM1 gene and a part of a non-human TREM1 gene. Among them, the "human TREM1 gene" is the same as the "entire human TREM1 gene", that is, its nucleotide sequence is identical to the full-length nucleotide sequence of the human TREM1 gene. The "part of the human TREM1 gene" is a continuous or discontinuous nucleotide sequence of 20 - 19308 bp (preferably 20 - 10441 bp, 20 - 3252 bp, or 20 - 615 bp) that is identical to the human TREM1 nucleotide sequence or has more than 70% homology with the human TREM1 nucleotide sequence.

[0238] The "exon No. xx to xxx" or the "entire exon No. xx to xxx" described in the present invention comprises the nucleotide sequence of the exon and the intron therebetween. For example, the "exon No. 1 to 2" comprises the entire nucleotide sequence of exon No. 1, intron No. 1 - 2, and exon No. 2.

[0239] The "intron No. x - xx" described in the present invention represents the intron between exon No. x and exon No. xx. For example, "intron No. 1 - 2" represents the intron between exon No. 1 and exon No. 2.

[0240] The "part of the exon" described in the present invention means that a continuous or discontinuous nucleotide sequence of several, dozens, or hundreds of nucleotides is identical to the entire exon nucleotide sequence. For example, a part of exon No. 1 of the human TREM1 gene comprises a continuous or discontinuous nucleotide sequence of 5 - 76 bp, preferably 10 - 49 bp, that is identical to the nucleotide sequence of exon No. 1 of the human TREM1 gene. In a specific embodiment of the present invention, the "part of exon No. 1" comprised in the "humanized TREM1 gene" includes at least the nucleotides from the start codon to the last nucleotide of exon No. 1.

[0241] The "cell" described in the present invention can be a fertilized egg cell or other somatic cells, preferably including but not limited to platelets, monocytes, microglial cells, endothelial cells, neutrophils, activated macrophages, B cell precursors, dendritic cells, natural killer cells, late B cells, or plasma cells, etc. Therefore, depending on the different cell sources, some of the cells described in the present application can develop into animal individuals, while some cannot.

[0242] The "TREM1 protein" described in the present invention, such as "human TREM1 protein", "non-human animal TREM1 protein", or "humanized TREM1 protein", all comprise a signal peptide, an extracellular region, an intracellular region, and / or a transmembrane region.

[0243] As used herein, "comprising" or "including" are open-ended descriptions that include the specified components or steps described, as well as other specified components or steps that do not materially affect. However, when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein.

[0244] As used herein, "homology" means that, in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, so that the sequences used have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity compared to the sequences obtained from the prior art.

[0245] Those skilled in the art can determine and compare sequence elements or degrees of identity to distinguish additional mouse and human sequences.

[0246] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following references. For example: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes V (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0247] The above only summarizes some aspects of the present invention and should not be considered, nor is it intended to be, a limitation of the present invention in any respect.

[0248] All patents and publications mentioned in this specification are incorporated into the present invention by reference in their entirety. Those skilled in the art should recognize that certain changes can be made to the present invention without departing from the concept or scope of the present invention. The following examples further illustrate the present invention in detail and should not be considered as limiting the present invention or the scope of the specific methods described in the present invention.

[0249] Advantages of the present invention:

[0250] By using gene editing technology to replace homologous genes in the animal genome with human normal or mutant genes, a gene humanized animal model closer to human physiological or disease characteristics is established, enabling the expression of human proteins in its body, which serves as a target for drugs that can only recognize human protein sequences, providing the possibility for screening anti-human antibodies and other drugs at the animal level.

[0251] Using the gene humanized animal model to establish various disease models can be used for the pharmacological and efficacy evaluation of anti-human antibody drugs.

[0252] Furthermore, the technical solution of humanizing the TREM1 gene designed in the present invention enables non-human animals containing the above humanized TREM1 gene to express humanized or corresponding human proteins, which can better simulate the signal pathway in the human body, provide a more humanized microenvironment for screening reagents suitable for humans, and thus the screened reagents have better efficacy.

[0253] Furthermore, for the convenience of expressing the humanized TREM1 gene, the present invention optimizes the selection of the fragment of the imported human TREM1 gene and the selection of the import position. The human TREM1 gene can be imported in a non-random manner and correctly express humanized or corresponding human proteins, can be stably passed on, and does not affect other functions of non-human animals at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0254] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0255] Figure 1 : Schematic diagram of the comparison between mouse TREM1 gene and human TREM1 gene locus (not to scale);

[0256] Figure 2 : Schematic diagram of the humanization modification of mouse TREM1 gene (not to scale);

[0257] Figure 3 : Schematic illustration of the TREM1 gene targeting strategy and the design of the targeting vector Figure 1 (not to scale);

[0258] Figure 4 : Schematic diagram of the FRT recombination process of TREM1 gene humanized mice (not to scale);

[0259] Figure 5 : Schematic illustration of the TREM1 gene targeting strategy and the design of the targeting vector Figure 2 (not to scale);

[0260] Figure 6 : Results of tail PCR identification of F1 generation of TREM1 gene humanized mice, where WT is wild type, H2O is water control, and M is Marker;

[0261] Figure 7 : Schematic diagram of the results of Southern Blot detection of positive clones, WT is wild type;

[0262] Figure 8 : Results of flow cytometry detection of the proportion of white blood cell subsets in the spleen;

[0263] Figure 9 : Results of flow cytometry detection of the proportion of T cell subsets in the spleen;

[0264] Figure 10 : Results of flow cytometry detection of the proportion of white blood cell subsets in peripheral blood;

[0265] Figure 11 : Results of flow cytometry detection of the proportion of T cell subsets in peripheral blood;

[0266] Figure 12 : Results of flow cytometry detection of the proportion of white blood cell subsets in lymph nodes;

[0267] Figure 13 : Results of flow cytometry detection of the proportion of T cell subsets in lymph nodes;

[0268] Figure 14 : Detection of the expression of sTREM1 in serum by ELISA. Specific implementation manners

[0269] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not impose any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0270] In each of the following embodiments, the equipment and materials were obtained from the several companies indicated below:

[0271] The NdeI and BsrGI enzymes were purchased from NEB, with catalog numbers R0111S and R0575S respectively;

[0272] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center, China National Institute for Food and Drug Control;

[0273] Brilliant Violet 510 TM The anti-mouse CD45 Antibody was purchased from Biolegend, catalog number 103138;

[0274] V450 Rat Anti-mouse CD11b was purchased from BD Horizon, catalog number 560455;

[0275] The PerCP anti-mouse Ly-6G / Ly-6C(Gr-1)Antibody was purchased from Biolegend, catalog number 108426;

[0276] The FITC anti-mouse F4 / 80 was purchased from Biolegend, catalog number 123108;

[0277] The PerCP / Cyanine5.5 anti-mouse TCRβchain Antibody was purchased from Biolegend, catalog number 109228;

[0278] The FITC anti-mouse CD19 Antibody was purchased from Biolegend, catalog number 115506;

[0279] The Mouse TREM-1PE-conjugated Antibody was purchased from RD, catalog number FAB1187P;

[0280] The APC anti-human CD354(TREM-1)Antibody was purchased from Biolegend, catalog number 314909;

[0281] The APC Mouse IgG1,κ Isotype Ctrl(FC) Antibody was purchased from Biolegend, catalog number 400122;

[0282] The PE Rat IgG2a,κ Isotype Ctrl Antibody was purchased from Biolegend, catalog number 400508;

[0283] Zombie NIR TM The Fixable Viability Kit was purchased from Biolegend, catalog number 423106;

[0284] The Purified anti-mouse CD16 / 32 was purchased from Biolegend, catalog number 101302;

[0285] The TREM1-PI64062-hIgG1-SI was purchased from Biolegend, catalog number 20210910.

[0286] The Mouse / Rat TREM-1 Quantikine ELISA Kit was purchased from RD, catalog number MTRM10;

[0287] The Human TREM-1 Quantikine ELISA Kit was purchased from RD, catalog number DTRM10C.

[0288] Example 1: Humanized mice with the TREM1 gene

[0289] The comparison schematic diagram of the mouse TREM1 gene (NCBI Gene ID: 58217, Primary source: MGI: 1930005, UniProt ID: Q9JKE2, located at positions 48539763 to 48553955 of NC_000083.7 on chromosome 17, based on transcript NM_021406.5 and its encoded protein NP_067381.1 (SEQ ID NO: 1)) and the human TREM1 gene (NCBI Gene ID: 54210, Primary source: HGNC:17760, UniProt ID: Q9NP99-1, located at positions 41267385 to 41286692 of NC_000006.12 on chromosome 6, based on transcript NM_018643.5 and its encoded protein NP_061113.1 (SEQ ID NO: 2)) is as shown Figure 1 as follows.

[0290] To achieve the object of the present invention, a nucleotide sequence encoding human TREM1 protein can be introduced into the endogenous TREM1 gene locus of a mouse, such that the mouse expresses human or humanized TREM1 protein. Specifically, using gene editing technology, under the control of the mouse TREM1 gene regulatory element, a partial sequence of exon 1 to a partial sequence of exon 4 of the human TREM1 gene, approximately 10.4 kb, is used to replace a partial sequence of exon 1 to a partial sequence of exon 4 of the mouse, approximately 11.8 kb, to obtain a schematic diagram of the humanized TREM1 gene locus as shown in Figure 2 shown, to achieve humanization of the mouse TREM1 gene.

[0291] Design a targeting strategy as shown in Figure 3 shown. The figure shows that the targeting vector contains homologous arm sequences upstream and downstream of the mouse TREM1 gene, and fragment A containing the human TREM1 sequence (SEQ ID NO: 5). Among them, the upstream homologous arm sequence (5' homologous arm, SEQ ID NO: 3) is identical to the nucleotide sequence at positions 48535775 to 48539822 of NCBI accession number NC_000083.7, and the downstream homologous arm sequence (3' homologous arm, SEQ ID NO: 4) is identical to the nucleotide sequence at positions 48551590 to 48555472 of NCBI accession number NC_000083.7. The nucleotide sequence of human TREM1 on fragment A (SEQ ID NO: 5) is identical to the nucleotide sequence at positions 41276215 to 41286655 of NCBI accession number NC_000006.12; the connection design of the upstream of the human TREM1 sequence to the mouse is: Among them, the last "G" in the sequence "AAGG" is the last nucleotide of the mouse, and the first "A" in the sequence "ATGA" is the first nucleotide of the human. The connection design of the downstream of the human TREM1 sequence to the mouse is Among them, the last "C" in the sequence "CAAC" is the last nucleotide of the human, and the "G" in the sequence "GTTA" is the first nucleotide of the mouse sequence.

[0292] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection design of the 5' end of the Neo cassette to the human gene is: Among them, the A in the sequence "GGCA" is the last nucleotide of the human, and the first G in the sequence "GTCG" is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo cassette and the human gene is designed as: Among them, the last C in the sequence "ATCC" is the last nucleotide of the Neo cassette, and the first "G" in the sequence "GGGT" is the first nucleotide of the human. In addition, a coding gene with a negative selection marker (the coding gene of diphtheria toxin subunit A (DTA)) was constructed downstream of the 3' homologous arm of the targeting vector. The mRNA sequence of the humanized mouse TREM1 after modification is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 7.

[0293] In view of the fact that human TREM1 has multiple subtypes or transcripts, the methods described herein can be applied to other subtypes or transcripts. Vector construction can be carried out by conventional methods, such as digestion and ligation. After the constructed targeting vector is preliminarily verified by digestion, it is sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into the embryonic stem cells of C57BL / 6 mice by electroporation, and the obtained cells are screened using the positive clone selection marker gene, and the integration of foreign genes is detected and confirmed using PCR and Southern Blot techniques. The correct positive clone cells are screened out, and the clones identified as positive by PCR (the primers are shown in Table 1) are further subjected to Southern Blot detection. The clones positive in the Southern detection and further verified as positive by sequencing and without random insertion are used for the next experiment.

[0294] Table 1 Names and specific sequences of PCR primers

[0295]

[0296] The correct positive clone cells (black mice) screened out are introduced into the separated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient female mouse (white mouse) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene (the schematic diagram of this process is shown in Figure 4 ) and then, by mating with each other, homozygous mice with humanized TREM1 gene can be obtained.

[0297] In addition, the CRISPR / Cas system can also be introduced for gene editing, and the design is as Figure 5The shown targeting strategy, in the figure, the homologous arm sequences upstream and downstream of the mouse TREM1 gene and the human TREM1 fragment sequence are shown on the targeting vector. Among them, the upstream homologous arm sequence (5' homologous arm, SEQ ID NO: 8) is identical to the nucleotide sequence from position 48538483 to 48539822 of NCBI accession number NC_000083.7, the downstream homologous arm sequence (3' homologous arm, SEQ ID NO: 9) is identical to the nucleotide sequence from position 48551590 to 48552918 of NCBI accession number NC_000083.7, and the nucleotide sequence of the human TREM1 fragment is identical to the nucleotide sequence from position 41276215 to 41286655 of NCBI accession number NC_000006.12 (SEQ ID NO: 5).

[0298] The construction of the targeting vector can be carried out by conventional methods, such as restriction enzyme digestion and ligation, direct synthesis, etc. After the constructed targeting vector is preliminarily verified by restriction enzyme digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is used for subsequent experiments.

[0299] The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, the selection and design of highly efficient and specific target sequences are the premise for constructing the sgRNA expression vector. Design and synthesize the sgRNA sequences that recognize the target sites. The target sequences of each sgRNA on the TREM1 gene are as follows:

[0300] sgRNA1 target site (SEQ ID NO: 16): 5'-CAGTATGTAGCCTGTATGGCTGG-3';

[0301] sgRNA2 target site (SEQ ID NO: 17): 5'-GGGAAGAATTTTCTACTCCATGG-3';

[0302] Use the UCA kit to detect and confirm the activity of the sgRNA. Subsequently, add restriction enzyme sites to the 5' end and its complementary strand respectively to obtain the forward oligonucleotide and reverse oligonucleotide sequences (see Table 2). After annealing, the annealed product is ligated to the pT7-sgRNA plasmid (the plasmid is first linearized with BbsI) to obtain the expression vectors pT7-TREM1-1 and pT7-TREM1-2.

[0303] Table 2 List of sgRNA1 and sgRNA2 sequences

[0304]

[0305]

[0306] The pT7-sgRNA vector was synthesized by a plasmid synthesis company. A DNA fragment (SEQ ID NO: 24) containing the T7 promoter and the sgRNA scaffold was ligated to the backbone vector (source: Takara, catalog number 3299) through enzymatic digestion (EcoRI and BamHI) in sequence. After verification by a professional sequencing company, the results showed that the target plasmid was obtained. Take the pronuclear stage fertilized eggs of mice, such as C57BL / 6 mice. Using a microinjector, the in vitro transcription products of pT7-TREM1-1 and pT7-TREM1-2 plasmids (transcribed using the Ambion in vitro transcription kit according to the instructions), the targeting vector and Cas9 mRNA were premixed and then injected into the cytoplasm or nucleus of mouse fertilized eggs. According to the method in "Mouse Embryo Manipulation Experimental Manual (Third Edition)" (Andras Nagy, Chemical Industry Press, 2006), the fertilized eggs were microinjected. The injected fertilized eggs were transferred to the culture medium for short-term culture, and then transplanted into the oviduct of the recipient female mouse for development. The obtained mice (F0 generation) were hybridized and self-crossed to expand the population size and establish a stable humanized mouse strain of the TREM1 gene. The genotype of somatic cells of F0 generation mice can be identified by PCR (the primers are shown in Table 3). For mice identified as positive by PCR, Southern Blot detection was performed. For mice positive in Southern detection, further sequencing verification was carried out to confirm whether there was random insertion.

[0307] Table 3 Names and specific sequences of F0 generation PCR primers

[0308]

[0309] The success of the mice prepared by the above method can be identified by various methods. For example, the genotype of somatic cells of F1 generation mice can be identified by PCR (the primers are shown in Table 4). The identification results of exemplary F1 generation mice are shown in Figure 6 , among which, the mice numbered F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, F1-08, F1-09, F1-10, F1-11, F1-12, F1-13 and F1-14 are positive heterozygous mice.

[0310] Table 4 Names and specific sequences of F1 generation PCR primers

[0311]

[0312] The mice with positive PCR identification in the F1 generation were subjected to Southern blot detection to confirm the presence of random insertion. The genomic DNA was extracted by cutting the mouse tails, and the genome was digested with BsrGI enzyme or NdeI enzyme, transferred to the membrane, and hybridized. The specific probes and the lengths of the target fragments are shown in Table 5, and the detection results are as Figure 7 shown, among which the mice numbered F1-05, F1-06, F1-07, F1-08, F1-09, F1-10, F1-11, F1-12, F1-13, and F1-14 were all positive and had no random insertion.

[0313] Table 5 Specific probes and the lengths of the target fragments

[0314]

[0315]

[0316] The primers for probe synthesis are as follows:

[0317] A Probe(5’)-F(SEQ ID NO: 29): 5’-TTCCGAGGTTTCCCTAGAAACTGGA-3’, A Probe(5’)-R(SEQ ID NO: 30): 5’-TGGAGTCCGAACTGGGTTAACC-3’;

[0318] 3’Probe-F(SEQ ID NO: 31): 5’-GTCAGTTTCTATGTGGGGAAGC-3’,

[0319] 3’Probe-R(SEQ ID NO: 32): 5’-GTTGATTGTTCCTTTAAGGGAG-3’;

[0320] The expression of human or humanized TREM1 protein in the positive mice can be confirmed by conventional detection methods, such as flow cytometry, etc. Specifically, one 6-week-old male C57BL / 6 wild-type mouse and one 6-week-old male TREM1 gene humanized heterozygous mouse were taken respectively. After cervical dislocation and euthanasia, peripheral blood was taken, and anti-mouse CD45 antibody Brilliant Violet510 TMAnti-mouse CD45 (mCD45), anti-mouse CD11b antibody V450 Rat Anti-mouse CD11b (mCD11b), anti-mouse Ly-6G / Ly-6C (Gr-1) antibody PerCP anti-mouse Ly-6G / Ly-6C (Gr-1) Antibody (mGr-1), anti-mouse F4 / 80 antibody FITC anti-mouse F4 / 80 (mF4 / 80), anti-mouse TCRβ antibody PerCP / Cyanine5.5 anti-mouse TCRβ chain Antibody (mTCRβ), anti-mouse CD19 antibody FITC anti-mouse CD19 Antibody (mCD19), anti-mouse TREM1 antibody Mouse TREM-1 PE-conjugated Antibody (mTREM1), anti-human TREM1 antibody APC anti-human CD354 (TREM-1) Antibody (hTREM1), anti-mouse IgG2a antibody PE Rat IgG2a,κ Isotype Ctrl Antibody, anti-mouse CD16 / 32 antibody Purified anti-mouse CD16 / 32, etc. After identification and staining, flow cytometry was performed to detect the expression of human or humanized TREM1 protein.

[0321] The results showed that 0.57% of the granulocytes in the peripheral blood of C57BL / 6 mice (characterized by mCD45+mGr-1+) were hTREM1-positive cells (characterized by mCD45+mGr-1+hTREM1), and 89.3% were mTREM1-positive cells (characterized by mCD45+mGr-1+mTREM1). In the peripheral blood granulocytes of TREM1 heterozygous mice (characterized by mCD45+mGr-1+), 16.3% were hTREM1-positive cells (characterized by mCD45+mGr-1+hTREM1+), and 92.7% were mTREM1-positive cells (characterized by mCD45+mGr-1+mTREM1+). This indicated that humanized TREM1 protein could only be detected in the TREM1 humanized heterozygous mice.

[0322] The heterozygous mice identified as positive in the F1 generation were mated with each other to obtain TREM1 gene humanized homozygous mice. Three 6-week-old C57BL / 6 wild-type mice and three TREM1 gene humanized homozygous mice (H / H) were subcutaneously inoculated with mouse colon cancer cells MC38. When the tumor volume grew to approximately 500 mm 3At that time, after decapitation for euthanasia, tumor tissues and peripheral blood were taken, and the expression of human or humanized TREM1 protein in macrophages, monocytic MDSCs (M-MDSC), granulocytic MDSCs (G-MDSC), T cells, and B cells was detected by flow cytometry similar to the above. The detection results are shown in Table 6.

[0323] Table 6 Detection Results by Flow Cytometry

[0324]

[0325] As can be seen from Table 6, murine TREM1 protein could be detected in macrophages, monocytic MDSCs, and granulocytic MDSCs in the tumors and peripheral blood of wild-type C57BL / 6 mice; while only humanized TREM1 protein could be detected in TREM1 humanized homozygous mice, and murine TREM1 protein could not be detected. In addition, murine TREM1 protein, human or humanized TREM1 protein were not detected in T cells and B cells in the tumors and peripheral blood of wild-type C57BL / 6 mice and TREM1 humanized homozygous mice.

[0326] Furthermore, flow cytometry was used to perform immunophenotyping detection on the spleen, peripheral blood, and lymph node tissues of C57BL / 6 wild-type mice and TREM1 gene humanized homozygous mice (H / H). The detection results of leukocyte subtypes and T cell subtypes in the spleen are shown in Figure 8 and Figure 9 respectively. The detection results of leukocyte subtypes and T cell subtypes in peripheral blood are shown in Figure 10 and Figure 11 respectively. As can be seen from the figures, the leukocyte subtypes such as B cells (BCells), T cells, NK cells, CD4+ T cells, CD8+ T cells, granulocytes, DC cells, macrophages, and monocytes in the spleen and peripheral blood of TREM1 gene humanized homozygous mice were basically the same as those of C57BL / 6 wild-type mice ( Figure 8 and Figure 10 ), and the percentages of T cell subtypes such as CD4+ T cells, CD8+ T cells, and Tregs cells were basically the same as those of C57BL / 6 wild-type mice ( Figure 9 and Figure 11 ).

[0327] The detection results of white blood cell subtypes and T cell subtypes in lymph nodes are shown respectively as Figure 12 and Figure 13 shown. It can be seen from the figure that the white blood cell subtypes such as T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells, etc. in the lymph nodes of TREM1 gene humanized homozygous mice are basically the same as those of C57BL / 6 wild-type mice( Figure 12 ), and the percentages of T cell subtypes such as CD4+ T cells, CD8+ T cells and Tregs cells are basically the same as those of C57BL / 6 wild-type mice( Figure 13 ).

[0328] It indicates that the humanized modification of the TREM1 gene does not affect the differentiation, development and distribution of white blood cells and T cells in mice.

[0329] Furthermore, flow cytometry was used to detect the phosphorylation of spleen tyrosine kinase (p-syk+) in TREM1 gene humanized homozygous mice, and ELISA was used to detect the expression of soluble TREM1 (sTREM1) in serum to verify whether the signaling pathway of humanized mice is normal. Specifically, 3 six-week-old female wild-type C57BL / 6 mice (+ / +) and 3 nine-week-old female TREM1 gene humanized homozygous mice (H / H) were randomly divided into a control group and an LPS group. The LPS group was intraperitoneally injected with LPS (500 μg / mouse), and the control group was injected with an equal volume of PBS or normal saline. After 12 h, peritoneal lavage fluid and serum were collected, and anti-mouse CD45 antibody Brilliant Violet 510 TM anti-mouse CD45 (mCD45), anti-mouse CD11b antibody V450 Rat Anti-mouseCD11b (mCD11b), anti-mouse Ly-6G / Ly-6C (Gr-1) antibody PerCP anti-mouse Ly-6G / Ly-6C (Gr-1)Antibody (mGr-1), anti-mouse F4 / 80 antibody FITC anti-mouse F4 / 80 (mF4 / 80), anti-mouse TREM1 antibody MouseTREM-1PE-conjugated Antibody (mTREM1), anti-human TREM1 antibody APC anti-human CD354 (TREM-1)Antibody (hTREM1), anti-mouse FC antibody APC Mouse IgG1,κIsotype Ctrl (FC)Antibody (mFC), anti-mouse IgG2a antibody PE Rat IgG2a,κIsotype Ctrl Antibody, Zombie NIR TMThe Fixable ViabilityKit, Purified anti-mouse CD16 / 32, and TREM1-PI64062-hIgG1-SI were used to detect the phosphorylation of spleen tyrosine kinase in peritoneal lavage macrophages. Flow cytometry results showed that in the absence of LPS stimulation, the percentages of p-syk+ in wild-type C57BL / 6 mice and TREM1 gene humanized homozygous mice were 0.84% and 0.64% respectively. After 12 hours of LPS stimulation, the percentages of p-syk+ in C57BL / 6 mice and TREM1 gene humanized homozygous mice were 16% and 19.4% respectively.

[0330] ELISA was used to detect the expression of sTREM1 in serum using the Mouse / Rat TREM-1 Quantikine ELISA Kit and the Human TREM-1 Quantikine ELISA Kit. As Figure 14 shown, mouse sTREM1 was detected in wild-type C57BL / 6 mice, and human sTREM1 was not detected. Only human sTREM1 was detected in TREM1 gene humanized homozygous mice. In the absence of LPS stimulation, the expression levels of sTREM1 in the sera of wild-type C57BL / 6 mice and TREM1 gene humanized homozygous mice were low. After 12 hours of LPS stimulation, the expression levels of sTREM1 in both sera increased significantly, and sTREM1 was upregulated in the sera of TREM1 gene humanized homozygous mice, indicating that TREM1 can be activated.

[0331] The above results indicate that under LPS stimulation, the TREM1 receptor is activated, MMP induces the release of sTREM1 into the supernatant, and the activation of TREM1 induces the rapid phosphorylation of syk, indicating that the TREM1 signaling function of the TREM1 gene humanized homozygous mice after modification is normal.

[0332] Example 2 Identification of gene knockout mice

[0333] Since the cleavage of Cas9 causes double-strand breaks in DNA, and the repair by chromosomal homologous recombination will randomly generate insertion / deletion mutations, there is a chance to obtain gene knockout mice with loss of TREM1 protein function. For this purpose, two pairs of primers with the sequences shown in Table 7 were designed for PCR detection. The positive detection result indicated that gene knockout mice of TREM1 were successfully obtained.

[0334] Table 7 Names and specific sequences of PCR primers for gene knockout mice

[0335]

[0336] Example 3 Efficacy verification

[0337] The TREM1 humanized mice prepared by this method can be used to evaluate the efficacy of drugs targeting human TREM1. For example, homozygous TREM1 humanized mice are subcutaneously inoculated with mouse colon cancer cells MC38. When the tumor volume grows to about 100 mm 3 3, they are divided into a control group or a treatment group according to the tumor volume. The treatment group randomly selects a drug targeting human TREM1, and the control group is injected with an equal volume of physiological saline. The tumor volume is measured regularly and the body weight of the mice is weighed. The in vivo safety and in vivo efficacy of the drug can be effectively evaluated by comparing the changes in the body weight of the mice and the size of the tumors.

[0338] Example 4 Preparation of double humanized or multiple double humanized mice

[0339] The TREM1 mice prepared by this method can also be used to prepare double humanized or multiple humanized mouse models. For example, in Example 1 above, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing other gene modifications such as IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4 or TNF-α. Alternatively, on the basis of humanized TREM1 mice, by separating mouse ES embryonic stem cells and gene recombination targeting technology, a double-gene or multi-gene modified mouse model of TREM1 and other gene modifications can be obtained. The homozygous or heterozygous TREM1 mice obtained by this method can also be mated with homozygous or heterozygous mice with other gene modifications, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining heterozygous mice with double-gene or multi-gene modifications of humanized TREM1 and other gene modifications. Then, mating the heterozygotes with each other can obtain homozygotes with double-gene or multi-gene modifications. These double-gene or multi-gene modified mice can be used to verify the in vivo efficacy of drugs targeting human TREM1 and other gene regulators, etc.

[0340] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0341] In addition, it should be noted that, in the various specific technical features described in the above specific embodiments, they can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0342] In addition, any combination can be made between different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for constructing a humanized non-human animal with TREM1 gene, characterized in that, The humanized TREM1 protein is expressed in the non-human animal, the genome of the non-human animal contains the humanized TREM1 gene, the humanized TREM1 protein contains a part of the human TREM1 protein and a part of the non-human animal TREM1 protein, the part of the human TREM1 protein is the signal peptide and extracellular region of the human TREM1 protein, the part of the non-human animal TREM1 protein is the transmembrane region and cytoplasmic region of the non-human animal TREM1 protein, the humanized TREM1 gene contains a part of the human TREM1 gene, the part of the human TREM1 gene encodes the part of the human TREM1 protein, the part of the human TREM1 gene is the nucleotide sequence encoding positions 1-205 of SEQ ID NO: 2, and the construction method includes introducing a donor nucleotide sequence into the non-human animal TREM1 locus, and the donor nucleotide sequence contains one of the following groups: A) The nucleotide sequence encoding the part of the human TREM1 protein; or, B) The nucleotide sequence of the part of the human TREM1 gene; The introduction of the non-human animal TREM1 locus is to replace the corresponding region of the non-human animal. The part of the human TREM1 gene or the humanized TREM1 gene is regulated by endogenous regulatory elements in the non-human animal. The expression of the endogenous TREM1 protein in the non-human animal is reduced or absent, and the non-human animal is a mouse.

2. The construction method according to claim 1, wherein The amino acid sequence of the humanized TREM1 protein is the amino acid sequence shown in SEQ ID NO:

7.

3. The construction method according to claim 1, characterized in that The part of the human TREM1 gene is the nucleotide sequence shown in SEQ ID NO:

5.

4. The construction method according to claim 1, wherein The humanized TREM1 gene further contains a part of the non-human animal TREM1 gene.

5. The construction method according to claim 4, characterized in that, The humanized TREM1 gene contains a part of exon 1 and a part of exon 4 of the non-human animal TREM1 gene.

6. The construction method according to claim 1, wherein The nucleotide sequence of the humanized TREM1 gene contains: A) The transcribed mRNA is the nucleotide sequence shown in SEQ ID NO: 6; B) Containing the nucleotide sequence shown in SEQ ID NO: 10 and / or 11.

7. The construction method according to claim 1, wherein A targeting vector is used for the construction of the non-human animal, and the targeting vector contains the donor nucleotide sequence.

8. The construction method according to claim 7, wherein The targeting vector further contains a 5' arm and a 3' arm. The 5' arm sequence is as shown in SEQ ID NO: 3 or 8; the 3' arm sequence is as shown in SEQ ID NO: 4 or 9.

9. The construction method according to any one of claims 1-8, characterized in that, The construction method further includes mating the non-human animal with humanized TREM1 gene with other genetically modified non-human animals, in vitro fertilization or direct gene editing, and screening to obtain a multi-genetically modified non-human animal.

10. The construction method according to claim 9, characterized in that, The other genes are selected from at least one of IL1A, IL1B, IL6, IL15, PD-1, PD-L1, TIGIT, LAG3, CD226, CTLA4, and TNF-α.

11. The construction method according to claim 10, wherein The humanized TREM1 gene and / or other genes are homozygous for the endogenous modified locus.

12. The construction method according to claim 10, characterized in that, The humanized TREM1 gene and / or other genes are heterozygous for the endogenous modified locus.

13. A targeting vector, characterized in that, The targeting vector contains a donor nucleotide sequence. The targeting vector includes introducing the donor nucleotide sequence into the TREM1 locus of a non-human animal to construct a non-human animal with a humanized TREM1 gene. The non-human animal expresses a humanized TREM1 protein in vivo. The humanized TREM1 protein contains a portion of the human TREM1 protein and a portion of the non-human animal TREM1 protein. The portion of the human TREM1 protein is the signal peptide and extracellular region of the human TREM1 protein. The portion of the non-human animal TREM1 protein is the transmembrane region and cytoplasmic region of the non-human animal TREM1 protein. The donor nucleotide sequence contains a nucleotide sequence encoding the portion of the human TREM1 protein. The donor nucleotide sequence is the nucleotide sequence encoding positions 1-205 of SEQ ID NO:

2. The non-human animal is a mouse.

14. The targeting vector according to claim 13, wherein The targeting vector contains the nucleotide sequence shown in SEQ ID NO:

5.

15. The targeting vector according to any one of claims 13-14, characterized in that, The targeting vector further contains a 5' arm and a 3' arm. The 5' arm sequence is as shown in SEQ ID NO: 3 or 8. The 3' arm sequence is as shown in SEQ ID NO: 4 or 9.

16. A humanized TREM1 protein, characterized in that, The humanized TREM1 protein contains a portion of the human TREM1 protein and a portion of the non-human animal TREM1 protein. The portion of the human TREM1 protein is the signal peptide and extracellular region of the human TREM1 protein. The portion of the non-human animal TREM1 protein is the transmembrane region and cytoplasmic region of the non-human animal TREM1 protein. The non-human animal is a mouse.

17. The humanized TREM1 protein according to claim 16, characterized in that, The portion of the human TREM1 protein is the amino acid sequence shown in positions 1-205 of SEQ ID NO:

2.

18. The humanized TREM1 protein according to any one of claims 16-17, characterized in that, The amino acid sequence of the humanized TREM1 protein is the amino acid sequence shown in SEQ ID NO:

7.

19. A humanized TREM1 gene, characterized in that, The humanized TREM1 gene contains a portion of the human TREM1 gene. The humanized TREM1 gene encodes the humanized TREM1 protein according to any one of claims 16-18.

20. The humanized TREM1 gene according to claim 19, characterized in that, The portion of the human TREM1 gene is the nucleotide sequence shown in SEQ ID NO:

5.

21. The humanized TREM1 gene according to claim 19, wherein, The humanized TREM1 gene further contains a portion of the non-human animal TREM1 gene.

22. The humanized TREM1 gene according to claim 21, wherein The humanized TREM1 gene contains a portion of exon 1 and a portion of exon 4 of the non-human animal TREM1 gene.

23. The humanized TREM1 gene according to any one of claims 19-22, characterized in that, The nucleotide sequence of the humanized TREM1 gene contains any one of the following groups: A) The transcribed mRNA is the nucleotide sequence shown in SEQ ID NO:

6. B) It contains the nucleotide sequence shown in SEQ ID NO: 10 and / or 11.

24. A cell, tissue or organ, characterized in that, The genome of the cell, tissue or organ as described above contains the humanized TREM1 protein as described in any one of claims 16-18, or the cell, tissue or organ as described above expresses the humanized TREM1 gene as described in any one of claims 19-23, or the non-human animal obtained by the construction method as described in any one of claims 1-12, and the cell, tissue or organ cannot develop into an individual animal.

25. The cell, tissue or organ according to claim 24, characterized in that, The tissue as described above includes tumor tissue.

26. Use of the humanized TREM1 protein according to any one of claims 16-18, the humanized TREM1 gene according to any one of claims 19-23, the non-human animal obtained by the construction method according to any one of claims 1-12, or the cell, tissue or organ according to any one of claims 24-25, characterized in that, The application as described above includes: A) Application in the development of products related to the TREM1-related immune process involving human cells; B) Application as a model system related to TREM1 in pharmacological, immunological, microbiological and medical research; C) Application in the production and utilization of animal experimental disease models for TREM1-related etiological research and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) Application in the screening, efficacy detection, evaluation of efficacy, verification or evaluation of human TREM1 signaling pathway regulators in vivo; or, E) Application in studying the function of the TREM1 gene, studying the drugs and drug effects targeting human TREM1 target sites, studying drugs for immune-related diseases related to TREM1 and anti-tumor drugs. The application as described above is a non-disease treatment and / or diagnosis method.

Images