A non-human animal modified with CLEC10A gene

Abstract

The present invention provides a non-human animal expressing a human or chimeric (eg, humanized) CLEC10A protein and methods of using the same.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (eg, humanized) CLEC10A protein and methods of using the same. Background Art

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

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

[0004] The present application provides an animal model with human or chimeric CLEC10A protein. The animal model can express human or chimeric CLEC10A (e.g., humanized CLEC10A) protein. It can be used for the study of CLEC10A gene function, and can also be used for the screening and evaluation of CLEC10A signaling pathway regulators (e.g., anti-human CLEC10A antibodies, nucleic acid drugs targeting CLEC10A, and / or polypeptide drugs targeting CLEC10A). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamics research, treatment of inflammation, cancer or immune diseases, and treatment of diseases at human CLEC10A target sites; the model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of CLEC10A protein and provides a platform for screening related drugs.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric C-type lectin-like domain 10A (CLEC10A) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein can be a cDNA, CDS or genomic DNA sequence. In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein is operably linked to an endogenous regulatory element (e.g., an endogenous promoter and / or UTR, such as a 5'UTR and / or a 3'UTR) of an endogenous MGL locus of at least one chromosome. In some embodiments, the chimeric CLEC10A protein is a humanized CLEC10A protein. In some embodiments, the amino acid sequence of the chimeric CLEC10A protein comprises at least 5 to 316 consecutive amino acids identical to a human CLEC10A protein, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 255, 256, 257, 260, or 316 consecutive amino acids identical to the human CLEC10A protein, etc. In some embodiments, the chimeric CLEC10A protein comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the human or chimeric CLEC10A protein is identical or has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence of human CLEC10A (NP_878910.1, SEQ ID NO: 2). In some embodiments, the amino acid sequence of the human or chimeric CLEC10A protein comprises amino acids 61-316 of SEQ ID NO: 2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 61-316 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the human or chimeric CLEC10A protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 7. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat. In some embodiments, the non-human animal does not express endogenous MGL protein or the expression level is reduced compared to MGL in wild-type animals. In some embodiments, one or more cells of the non-human animal express human or chimeric CLEC10A protein.

[0006] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein the endogenous MGL gene is replaced with a nucleotide sequence of human CLEC10A at the endogenous MGL locus of the non-human animal. In some embodiments, the nucleotide sequence of human CLEC10A can be a cDNA, CDS or genomic DNA sequence. In some embodiments, the endogenous MGL protein of the non-human animal is not expressed or the expression level is reduced compared with MGL in wild-type animals. In some embodiments, the nucleotide sequence of human CLEC10A or the nucleotide sequence encoding the human or chimeric CLEC10A protein is operably linked to the regulatory elements of endogenous MGL, such as a promoter and / or UTR. In some embodiments, the modified CLEC10A gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the nucleotide sequence of human CLEC10A comprises a nucleotide sequence encoding the extracellular region of human CLEC10A. In some embodiments, the nucleotide sequence of human CLEC10A comprises a nucleotide sequence encoding amino acids 61-316 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence of human CLEC10A comprises at least 5 bp to 5715 bp of continuous nucleotides of human CLEC10A gene, for example, at least 5, 50, 100, 200, 300, 400, 450, 485, 486, 487, 488, 489, 490, 500, 530, 535, 540, 550, 600, 700, 800, 900, 1000, 1200, 1250, 1260, 1270, 1280, 1290, 1310, 1321, 1331, 1341, 1350, 1360, 1371, 1381, 1391, 1402, 1413, 1423, 1433, 1444, 1455, 1460, 1471, 1482, 1493, 1500, 1511, 1521, 1533, 1545, 1556, 1566, 1571, 1581, 1591, 1601, 1613, 1624, 1635, 1640, 1650, 1661, 1671, 1681, 1691, 1701, 171 500, 5500, 5700 or 5715 bp of consecutive nucleotides. In some embodiments, the nucleotide sequence of human CLEC10A comprises part of exon 3 to all of exon 9 of human CLEC10A gene, preferably further comprises at least 50 bp of continuous nucleotides downstream of 3'UTR, for example, at least 50, 100, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 bp of continuous nucleotides, preferably at least 50 bp to 1000 bp of continuous nucleotides downstream of 3'UTR. In some embodiments, the nucleotide sequence of human CLEC10A comprises part of exon 3 to at least 50 bp of continuous nucleotides downstream of 3'UTR of human CLEC10A gene.In some embodiments, the portion of exon 3 of the human CLEC10A gene comprises at least 1 bp of continuous nucleotides, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 110 or 117 bp, preferably at least 1 to 10 bp of continuous nucleotides. In some embodiments, the nucleotide sequence of human CLEC10A comprises SEQ ID NO: 5, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the replaced endogenous MGL gene comprises a nucleotide sequence encoding an MGL1 protein. In some embodiments, the replaced endogenous MGL gene comprises a nucleotide sequence encoding an extracellular region of MGL2. In some embodiments, the replaced endogenous MGL gene comprises a nucleotide sequence encoding amino acids 73-332 of SEQ ID NO: 35. In some embodiments, the replaced endogenous MGL gene comprises a nucleotide sequence encoding an MGL1 protein and an extracellular region of MGL2. In some embodiments, the replaced endogenous MGL gene comprises a nucleotide sequence encoding amino acids 73-332 of SEQ ID NO: 1 and SEQ ID NO: 35. In some embodiments, the replaced endogenous MGL gene comprises all or part of the MGL1 gene and / or the MGL2 gene. In some embodiments, the replaced endogenous MGL gene comprises all or part of the MGL1 gene and the MGL2 gene. In some embodiments, the replaced MGL1 gene comprises all of exon 1 to exon 10 of the endogenous MGL1 gene, preferably also comprising at least 50 bp of continuous nucleotides downstream of the 3'UTR, such as at least 50, 100, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 bp of continuous nucleotides, preferably at least 50 bp to 1000 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the replaced MGL1 gene comprises at least 50 bp of continuous nucleotides downstream of the exon 1 to the 3'UTR of the endogenous MGL1 gene. In some embodiments, the replaced MGL2 gene includes all of exon 3 to exon 10 of the endogenous MGL2 gene. In some embodiments, the replaced portion of exon 3 of the endogenous MGL2 gene comprises at least 1 bp of continuous nucleotides, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 110 or 111 bp of continuous nucleotides, preferably at least 1 to 10 bp of continuous nucleotides. In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is a monkey or a rodent (e.g., a mouse or a rat).In some embodiments, the mRNA transcribed from the modified gene in the genome of the non-human animal comprises SEQ ID NO: 6, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the non-human animal further comprises other human or chimeric proteins, and the human or chimeric proteins comprise at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0007] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the MGL protein endogenously in a non-human animal with a nucleotide sequence encoding a human or chimeric CLEC10A protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the MGL1 and MGL2 proteins endogenously in a non-human animal with a nucleotide sequence encoding a human or chimeric CLEC10A protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the extracellular region of MGL2 endogenously in a non-human animal with a nucleotide sequence encoding the extracellular region of human CLEC10A. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the extracellular region of MGL1 and MGL2 endogenously in a non-human animal with a nucleotide sequence encoding the extracellular region of human CLEC10A. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the extracellular region of MGL1 and MGL2 endogenously in a non-human animal with a nucleotide sequence encoding the extracellular region of human CLEC10A. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 and SEQ ID NO: 35 endogenously in a non-human animal with a nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the construction method comprises replacing the nucleotide sequence of positions 73-332 of the non-human animal endogenous encoding SEQ ID NO: 1 and SEQ ID NO: 35 with the nucleotide sequence comprising positions 61-316 encoding SEQ ID NO: 2. In some embodiments, the construction method comprises replacing the nucleotide sequence of the non-human animal endogenous encoding SEQ ID NO: 1 and SEQ ID NO: 35 with the nucleotide sequence comprising SEQ ID NO: 6. In some embodiments, the construction method comprises replacing the nucleotide sequence of the non-human animal endogenous MGL gene with the nucleotide sequence comprising the human CLEC10A gene. In some embodiments, the construction method comprises replacing the nucleotide sequence of the non-human animal endogenous MGL1 and MGL2 genes with the nucleotide sequence comprising the human CLEC10A gene. In some embodiments, the construction method comprises replacing the part of exon 3 to all of exon 10 of the non-human animal endogenous MGL2 gene with the part of exon 3 to all of exon 9 of the human CLEC10A gene. In some embodiments, the construction method comprises replacing exons 1-10 of endogenous MGL1 of non-human animals and part of exon 3 to all of exon 10 of endogenous MGL2 gene with part of exon 3 to all of exon 9 of human CLEC10A gene. In some embodiments, the construction method comprises replacing part of exon 3 to all of exon 10 of endogenous MGL2 gene of non-human animals with part of exon 3 to at least 50 bp downstream of 3'UTR of human CLEC10A gene.In some embodiments, the construction method comprises replacing exon 1 to at least 50 bp downstream of 3'UTR of endogenous MGL1 of non-human animals and all of exon 10 of exon 3 of endogenous MGL2 gene with a portion of exon 3 to at least 50 bp downstream of 3'UTR of human CLEC10A gene. In some embodiments, the construction method comprises replacing exon 1 to at least 300 bp downstream of 3'UTR of endogenous MGL1 of non-human animals and all of exon 10 of exon 3 of endogenous MGL2 gene with a portion of exon 3 to at least 300 bp downstream of 3'UTR of human CLEC10A gene. In some embodiments, the construction method comprises replacing the corresponding endogenous region of non-human animals with a nucleotide sequence comprising SEQ ID NO: 5. In some embodiments, the construction method comprises replacing the nucleotide sequence of positions 73-332 of endogenous non-human animal encoding SEQ ID NO: 35 with a nucleotide sequence comprising SEQ ID NO: 5. In some embodiments, the construction method comprises replacing the nucleotide sequence at positions 73-332 of SEQ ID NO: 1 and SEQ ID NO: 35 endogenously encoded by a non-human animal with the nucleotide sequence shown in SEQ ID NO: 5.

[0008] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises a nucleotide sequence encoding a human or chimeric CLEC10A protein at an endogenous MGL locus replacing a nucleotide sequence encoding a corresponding region of endogenous MGL. In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein is operably linked to an endogenous regulatory element of an endogenous MGL locus. In some embodiments, the chimeric CLEC10A protein is a humanized CLEC10A protein. In some embodiments, one or more cells of the non-human animal express a human or humanized CLEC10A protein. In some embodiments, the endogenous MGL protein of the non-human animal is not expressed or is expressed at a reduced level compared to MGL in wild-type animals. In some embodiments, the corresponding region encoding the endogenous MGL is the entire endogenous MGL1 protein and / or the extracellular region of the MGL2 protein. In some embodiments, the corresponding region encoding the endogenous MGL is the entire endogenous MGL1 protein and the extracellular region of the MGL2 protein. In some embodiments, one or more cells of the non-human animal express a chimeric CLEC10A protein. In some embodiments, the chimeric CLEC10A protein comprises an extracellular region, a transmembrane region and a cytoplasmic region, wherein the extracellular region is identical to the extracellular region of human CLEC10A or is at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% identical. In some embodiments, the extracellular region is identical to the extracellular region of human CLEC10A for at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 or 256 consecutive amino acids (e.g., amino acids 61-316 of SEQ ID NO: 2). In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein comprises all or part of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and / or exon 9 of the human CLEC10A gene, preferably also includes intron 3, intron 4, intron 5, intron 6, intron 7 and / or intron 8. In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein comprises part of exon 3 to all of exon 9 of the human CLEC10A gene, preferably also includes at least 50 bp (preferably at least 50 bp to 1000 bp) of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein comprises part of exon 3 of the human CLEC10A gene to at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein comprises a portion of exon 3 of the human CLEC10A gene to at least 300 bp of continuous nucleotides downstream of the 3'UTR.In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein comprises SEQ ID NO: 5, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous MGL comprises all or part of the MGL1 gene and / or the MGL2 gene of a non-human animal (e.g., mouse) ... In some embodiments, the modified CLEC10A gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

[0009] In one aspect, the present invention provides a non-human animal or a method for constructing the same, wherein the non-human animal comprises at least one cell encoding a nucleotide sequence of a human or chimeric CLEC10A protein, wherein the human or chimeric CLEC10A protein comprises at least 50, 60, 70, 80, 90, 100, 200 or 256 consecutive amino acids of a continuous amino acid sequence of a human CLEC10A protein, and the non-human animal expresses the human or chimeric CLEC10A protein. In some embodiments, the chimeric CLEC10A protein comprises at least 50 consecutive amino acids of the extracellular region of human CLEC10A. In some embodiments, the human or chimeric CLEC10A protein comprises amino acids 61-316 of SEQ ID NO: 2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 61-316 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein is operably linked to an endogenous MGL regulatory element. In some embodiments, the nucleotide sequence encoding the human or chimeric CLEC10A protein can be integrated into the non-human animal endogenous MGL locus. In some embodiments, the human or chimeric CLEC10A protein has at least one non-human animal (e.g., mouse) MGL activity and / or human CLEC10A activity.

[0010] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal. In some embodiments, in at least one cell of the non-human animal, at the non-human animal endogenous MGL locus, the nucleotide sequence encoding the endogenous MGL region is replaced by a nucleotide sequence encoding the corresponding region of human or chimeric CLEC10A. In some embodiments, the endogenous MGL protein of the non-human animal is not expressed or the expression level is reduced compared with MGL in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CLEC10A comprises all or part of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and / or exon 9 of the human CLEC10A gene, preferably further comprising intron 3, intron 4, intron 5, intron 6, intron 7 and / or intron 8. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CLEC10A comprises part of exon 3 to all of exon 9 of the human CLEC10A gene, preferably also comprising at least 50bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CLEC10A comprises a portion of exon 3 of the human CLEC10A gene to at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence of the corresponding region of human or chimeric CLEC10A comprises amino acids 61-316 of SEQ ID NO: 2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 61-316 of SEQ ID NO: 2. In some embodiments, the endogenous MGL region is the extracellular region of the endogenous MGL2 protein. In some embodiments, the endogenous MGL region comprises the entire endogenous MGL1 protein and the extracellular region of the MGL2 protein. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human or chimeric CLEC10A comprises SEQ ID NO: 2 or 7, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 2 or 7. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CLEC10A comprises SEQ ID NO: 5, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous MGL region comprises all or part of the MGL2 gene of a non-human animal (e.g., mouse), preferably, the nucleotide sequence encoding the endogenous MGL region comprises all or part of the MGL1 gene of a non-human animal (e.g., mouse) and the MGL2 gene of a non-human animal (e.g., mouse).In some embodiments, the nucleotide sequence encoding the endogenous MGL region comprises a portion of exon 3 and all of exons 4-10 of the MGL2 gene of a non-human animal (e.g., a mouse). In some embodiments, the nucleotide sequence encoding the endogenous MGL region comprises a portion of exon 3 to all of exon 10 of the MGL2 gene of a non-human animal (e.g., a mouse). In some embodiments, the nucleotide sequence encoding the endogenous MGL region comprises all of exons 1-10 of the MGL1 gene of a non-human animal (e.g., a mouse) and a portion of exon 3 to all of exon 10 of the MGL2 gene of a non-human animal (e.g., a mouse). In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric CLEC10A is operably linked to an endogenous MGL regulatory element, such as a promoter and / or UTR. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the non-human animal is a mouse or a rat.

[0011] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric CLEC10A, the method comprising replacing a nucleotide sequence encoding an endogenous MGL region with a nucleotide sequence encoding a corresponding region of human CLEC10A at an endogenous non-human animal (e.g., mouse) MGL locus to produce a genetically modified non-human animal cell. In some embodiments, the non-human animal cell expresses a human or chimeric CLEC10A protein. In some embodiments, the chimeric CLEC10A protein comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLEC10A comprises all or part of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and / or exon 9 of the human CLEC10A gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLEC10A comprises all of exon 9 from part of exon 3 of the human CLEC10A gene, preferably further comprising at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLEC10A comprises a portion of exon 3 of the human CLEC10A gene to at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human CLEC10A comprises a portion of exon 3 of the human CLEC10A gene to at least 300 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human CLEC10A comprises SEQ ID NO: 2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human CLEC10A comprises amino acids 61-316 of SEQ ID NO: 2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence 61-316 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the endogenous MGL region comprises all or part of the MGL2 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous MGL region comprises all or part of the MGL1 gene of a non-human animal (e.g., mouse) and the MGL2 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous MGL region comprises part of exon 3 to all of exon 10 of the MGL2 gene of a non-human animal (e.g., mouse).In some embodiments, the nucleotide sequence encoding the endogenous MGL region includes all of exons 1-10 of the MGL1 gene of a non-human animal (e.g., mouse) and part of exon 3 to all of exon 10 of the MGL2 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the endogenous MGL region includes all of exon 1 of the MGL1 gene of a non-human animal (e.g., mouse) to at least 50 bp of continuous nucleotides downstream of the 3'UTR and part of exon 3 to all of exon 10 of the MGL2 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the corresponding region of human CLEC10A is operably linked to a regulatory element of endogenous MGL, such as a promoter and / or UTR. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins include but are not limited to at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0012] In one aspect, the present invention provides an application of the non-human animal or the non-human animal obtained by the construction method, the application comprising: A) application in the development of products involving immune processes related to CLEC10A in human cells; B) application as a model system related to CLEC10A in pharmacology, immunology, microbiology and medical research; C) application involving the production and use of animal experimental disease models for pathological research related to CLEC10A and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) application in in vivo research on the screening, efficacy detection, efficacy assessment, verification or evaluation of human CLEC10A signaling pathway regulators; or, E) application in studying the function of the CLEC10A gene, studying drugs and efficacy targeting human CLEC10A target sites, and studying cancer, immune diseases or inflammatory drugs related to CLEC10A.

[0013] In one aspect, the present invention provides a method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the disease. In some embodiments, the therapeutic agent comprises an anti-CLEC10A antibody, a nucleic acid drug targeting CLEC10A, and / or a polypeptide drug targeting CLEC10A. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the non-human animal suffers from a disease. In some embodiments, the determination of the effect of the therapeutic agent on the animal includes but is not limited to measuring the weight of the animal or a blood test. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit, or hemoglobin content. In some embodiments, the effect is an inhibitory effect. In some embodiments, the disease is cancer, an immune disease, or inflammation. In some embodiments, the cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the non-human animal. In some embodiments, the cancer comprises injecting one or more cancer cells into the animal. In some embodiments, the cancer is a solid tumor and / or a blood tumor. In some embodiments, the cancer is ovarian cancer, head and neck cancer, liver cancer or lung cancer. In some embodiments, the immune disease includes but is not limited to GVHD (graft versus host disease), Crohn's disease, ankylosing spondylitis, atopic dermatitis, psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty hepatitis), systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, hyperthyroidism, thrombocytopenic purpura (e.g., primary or idiopathic), autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, etc. In some embodiments, the immune disease is asthma, atopic dermatitis, rheumatoid arthritis or multiple sclerosis. In some embodiments, inflammation includes but is not limited to degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (e.g., tuberculosis, syphilis, leprosy or lymphogranuloma). In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0014] In one aspect, the present invention provides a humanized CLEC10A protein. In some embodiments, the humanized CLEC10A protein comprises all or part of a human CLEC10A protein. In some embodiments, the humanized CLEC10A protein comprises the extracellular region of a human CLEC10A protein, a non-human animal endogenous transmembrane region, and a non-human animal endogenous cytoplasmic region. In some embodiments, the humanized CLEC10A protein comprises the extracellular region of a human CLEC10A protein, a non-human animal endogenous MGL2 transmembrane region, and a non-human animal endogenous MGL2 cytoplasmic region. In some embodiments, the extracellular region of the human CLEC10A protein comprises SEQ ID NO: 2 or its 61-316 amino acid sequence. In some embodiments, the non-human animal endogenous MGL2 cytoplasmic region comprises SEQ ID NO: 35 or its 1-45 amino acid sequence. In some embodiments, the non-human animal endogenous MGL2 cytoplasmic region comprises SEQ ID NO: 35 or its 46-70 amino acid sequence. In some embodiments, the humanized CLEC10A protein comprises SEQ ID NO: 2 or amino acid sequence 61-316 thereof, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 2 or amino acid sequence 61-316 thereof. In some embodiments, the humanized CLEC10A protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0015] In one aspect, the present invention provides a humanized CLEC10A gene. In some embodiments, the humanized CLEC10A gene encodes the humanized CLEC10A protein. In some embodiments, the humanized CLEC10A gene comprises a nucleotide sequence encoding a human CLEC10A extracellular region. In some embodiments, the humanized CLEC10A gene comprises a portion of exon 3 of the human CLEC10A gene to all of exon 9. In some embodiments, the humanized CLEC10A gene further comprises at least 50bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the humanized CLEC10A gene comprises a portion of human exon 3 to at least 50bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the humanized CLEC10A gene further comprises a nucleotide sequence encoding a non-human animal MGL cytoplasmic region and / or a transmembrane region. In some embodiments, the humanized CLEC10A gene further comprises a nucleotide sequence encoding a non-human animal MGL2 cytoplasmic region and / or a transmembrane region. In some embodiments, the humanized CLEC10A gene further comprises all of exons 1-2 and part of exon 3 of the non-human animal MGL2 gene, and preferably further comprises intron 2. In some embodiments, the humanized CLEC10A gene further comprises all of exon 1 to part of exon 3 of the non-human animal MGL2 gene. In some embodiments, the part of exon 3 of the MGL2 gene comprised by the humanized CLEC10A gene comprises at least 5 bp of continuous nucleotides, for example, at least 5, 10, 20, 50, 80, 100, 105, 106, 107, 108, 109, 110, 111 bp of continuous nucleotides, preferably at least 100 bp to 110 bp of continuous nucleotides. In some embodiments, the humanized CLEC10A gene comprises the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 22 or 23, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 22 or 23.

[0016] In one aspect, the present invention provides a cell, tissue or organ, comprising the humanized CLEC10A protein and / or the humanized CLEC10A gene.

[0017] In one aspect, the present invention provides a non-human animal genome, the non-human animal genome comprising at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric CLEC10A protein. In some embodiments, the chromosome comprises a human or chimeric CLEC10A gene. In some embodiments, the chromosome comprises a portion of exon 3 to all of exon 9 of the human CLEC10A gene. In some embodiments, the MGL gene of the endogenous genome of the non-human animal is replaced. In some embodiments, the MGL1 of the endogenous genome of the non-human animal is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1 in the endogenous genome of the non-human animal is replaced. In some embodiments, a portion of exon 3 to all of exon 10 of the endogenous MGL2 gene of the non-human animal is replaced. In some embodiments, a portion of exon 3 to at least 50 bp of continuous nucleotides downstream of the 3'UTR of the endogenous MGL2 gene of the non-human animal is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 35 at positions 73-332 in the endogenous genome of the non-human animal is replaced. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 1 and the nucleotide sequence encoding SEQ ID NO: 35 at positions 73-332 are replaced in the endogenous genome of the non-human animal. In some embodiments, the chromosome comprises a nucleotide sequence encoding a human or chimeric CLEC10A protein or a nucleotide sequence of human CLEC10A replaces the corresponding portion of the endogenous chromosome of the non-human animal. In some embodiments, the nucleotide sequence encoding SEQ ID NO: 2 at positions 61-316 replaces the endogenous nucleotide sequence encoding SEQ ID NO: 1 and SEQ ID NO: 35 at positions 73-332 in the non-human animal.

[0018] In one aspect, the present invention provides a cell, tissue or organ comprising the above-mentioned non-human animal genome.

[0019] In one aspect, the present invention provides an animal model, wherein the animal model comprises the humanized CLEC10A protein and the humanized CLEC10A gene.

[0020] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to a part of the whole, or an individual part that constitutes the whole.

[0021] The term "locus" in the present invention refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a gene in a narrow sense, which can be a gene, a part of a gene, or a regulatory region of a gene, etc. For example, the "MGL1 locus" includes exons 1-10 of the MGL1 gene and a DNA fragment optionally in its regulatory region. For example, the "MGL2 locus" includes exons 1-10 of the MGL1 gene and a DNA fragment optionally in its regulatory region.

[0022] The term "portion of exon XX" of the present invention means a continuous or intermittent nucleotide sequence of several, dozens or hundreds of nucleotides that is consistent with the entire exon nucleotide sequence, such as a portion of exon 3 of human CLEC10A gene, including continuous 1-117bp, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 100, 110 or 117bp of continuous nucleotides.

[0023] The term "exon XX to exon XXX" or "exon XX-XXX" or "exon XX to all of exon XXX" or "all of exon XX to all of exon XXX" of the present invention refers to exons and introns in between.

[0024] The term "a portion of exon x to a portion of exon xx" or "a portion of exon x to the entirety of exon xx" of the present invention includes all or a portion of an exon and introns therebetween. For example, "a portion of exon 3 to the entirety of exon 9" includes a portion of exon 3, the entirety of intron 3, the entirety of exon 4, the entirety of intron 4, the entirety of exon 5, the entirety of intron 5, the entirety of exon 6, the entirety of intron 6, the entirety of exon 7, the entirety of intron 7, the entirety of exon 8, the entirety of intron 8, and the entirety of exon 9.

[0025] The term "intron xx" in this application refers to an intron between two exons, for example, intron 3 is the intron between exon 3 and exon 4.

[0026] The term "comprising" or "including" in the present invention is an open-ended writing method, which contains the specified components or steps described, as well as other specified components or steps that will not be substantially affected. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0027] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0028] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. This application describes methods and materials for use in the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are exemplary and non-limiting only. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0029] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description.

[0030] CLEC10A

[0031] CLEC10A, also known as CD301, is also known as macrophage galactose-type lectin MGL, and is a member of C-type lectinreceptors (CLRs). It is a type II transmembrane receptor protein that is mainly expressed on DC cells (immature dendritic cells iDC) and activated macrophages. This molecule is a member of the C-type lectin family. This molecule is Ca 2+ MGL is a lectin receptor that specifically recognizes terminal α- or β-N-acetylgalactosamine (GalNAc or Tn) residues within the CLR family of molecules. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds to terminal N-acetylgalactosamine glycan residues, such as Tn and sialyl Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites, mediating recognition and clearance of various pathogens such as spirochetes, bacteria, and filoviruses, completing antigen presentation and mediating endocytosis. MGL recognizes Tn structures on tumor-associated MUC1 glycoproteins and self-glycoproteins CD43 and CD45, which can induce inhibitory antitumor immune responses characterized by increased IL-10 production and induction of regulatory and effector T cells. Therefore, specific antibodies can be developed against the CDR region to block tumor cells from escaping immune clearance by specifically recognizing tumor-associated glycan motifs that are not present in healthy tissues and targeting MGL.

[0032] In mice, there are two MGL homologues, MGL1 and MGL2, which are expressed by dermal DCs and alternatively activated macrophages. Although there is a high degree of sequence homology between MGL1 and MGL2 (91.5%), the sequences corresponding to the cytoplasm and CRD show differences (86% homology), and MGL1 is not expressed in Lewis X and Lewis A The structure is highly specific, and MGL2 is closer to human MGL in function, recognizing N-galactosamine and galactose, including O-linked Tn antigen, TF antigen and core 2 structure. MGL participates in signal transduction of DC and T cells as a signal receptor, and plays a very important role in pathogen immunity and tumor immunity.

[0033] In the human genome, the CLEC10A gene (Gene ID: 10462, located at positions 7074537 to 7080251 of chromosome 17 NC_000017.11) contains 9 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and exon 9. The nucleotide sequence of human CLEC10A mRNA is NM_182906.4, and the amino acid sequence of human CLEC10A is NP_878910.1 (SEQ ID NO: 2). The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_182906.4 and its encoded protein NP_878910.1 is shown in Table 1.

[0034] Table 1

[0035]

[0036] In the mouse MGL genome, the MGL1 gene (Gene ID: 17312, located at positions 70057449 to 70061662 of chromosome 11 NC_000077.7) contains 10 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9 and exon 10. The nucleotide sequence of mouse MGL1 mRNA is NM_010796.3, and the amino acid sequence of mouse MGL1 is NP_034926.1 (SEQ ID NO: 1). The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_010796.3 and its encoded protein NP_034926.1 is shown in Table 2.

[0037] Table 2

[0038]

[0039]

[0040] In the mouse MGL genome, the MGL2 gene (Gene ID: 216864, located at positions 70020953 to 70028368 of chromosome 11 NC_000077.7) contains 10 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9 and exon 10. The nucleotide sequence of mouse MGL2 mRNA is NM_145137.2, and the amino acid sequence of mouse MGL2 is NP_660119.1 (SEQ ID NO: 35). The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_145137.2 and its encoded protein NP_660119.1 is shown in Table 3.

[0041] Table 3

[0042]

[0043] CLEC10A genes, proteins and gene loci of other species are also known in the art, for example, Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog) and Sus scrofa (pig), and the relevant information of these genes (e.g., intron sequences, exon sequences and amino acid sequences) can be found in NCBI, the entire contents of which are incorporated into this application by reference.

[0044] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are compared for the purpose of optimal comparison (e.g., for optimal comparison, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides on the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to achieve the optimal comparison of the two sequences, taking into account the number of gaps and the length of each gap. For example, the comparison of the sequences and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0045] The percentage (homology percentage) of conservative residues with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0046] Carrier

[0047] The present invention provides a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of the region to be changed (5' arm or 5' homologous arm), which is selected from the genomic DNA of the MGL gene and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the region to be changed (3' arm or 3' homologous arm), which is selected from the genomic DNA of the MGL gene and has a length of 100 to 10,000 nucleotides.

[0048] In some embodiments, a) the DNA fragment homologous to the 5' end of the region to be changed is selected from a nucleotide sequence having at least 90% homology with NCBI Accession No. NC_000077.7; c) the DNA fragment homologous to the 3' end of the region to be changed is selected from a nucleotide sequence having at least 90% homology with NCBI Accession No. NC_000077.7.

[0049] In some embodiments, a) a DNA fragment homologous to the 5' end of the region to be changed is selected from the nucleotide sequence at positions 70021924 to 70025984 of NCBI Accession No. NC_000077.7; c) a DNA fragment homologous to the 3' end of the region to be changed is selected from the nucleotide sequence at positions 70061963 to 70065620 of NCBI Accession No. NC_000077.7.

[0050] In some embodiments, a) a DNA fragment homologous to the 5' end of the region to be changed is selected from the nucleotide sequence at positions 70024418 to 70025984 of NCBI Accession No. NC_000077.7; c) a DNA fragment homologous to the 3' end of the region to be changed is selected from the nucleotide sequence at positions 70061963 to 70063540 of NCBI Accession No. NC_000077.7.

[0051] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 18 kb, 19 kb or 20 kb.

[0052] In some embodiments, the region to be changed is located on the non-human animal MGL1 gene and / or MGL2 gene. In some embodiments, the region to be changed is located at least 50bp downstream of exon 3 to 3'UTR of the non-human animal MGL2 gene. In some embodiments, the region to be changed is located at least 50bp downstream of exon 3 to 3'UTR of the non-human animal MGL1 gene and MGL2 gene.

[0053] In some embodiments, the 5' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, the 3' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO:4.

[0054] In some embodiments, the 5' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 22. In some embodiments, the 3' arm sequence comprises the nucleotide sequence set forth in SEQ ID NO: 23.

[0055] In some embodiments, the targeting vector comprises a human sequence (e.g., positions 7074237 to 7078000 of NC_000017.11). For example, the donor region in the targeting vector comprises a nucleotide sequence encoding the extracellular region of human CLEC10A. Preferably, the portion of exon 3 to the entirety of exon 9 of the human CLEC10A gene, and more preferably, the portion of exon 3 to at least 50 bp downstream of the 3'UTR. In some embodiments, the donor region in the targeting vector comprises SEQ ID NO: 5.

[0056] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a positive screening marker gene or a negative screening marker gene. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo. Preferably, the targeting vector further comprises two Frt recombination sites arranged in the same direction on both sides of the marker gene. In some embodiments, the coding gene of the negative screening marker is a coding gene (DTA) of the diphtheria toxin A subunit.

[0057] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the MGL gene, and the sgRNA is unique on the target sequence of the gene to be changed, and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'. In some embodiments, the targeting site of the sgRNA in the MGL2 gene of a non-human animal (e.g., mouse) is located at exon 3 to exon 9.

[0058] In some embodiments, the targeting sequence is shown as SEQ ID NO: 25 and / or 26. Therefore, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 27 and 29. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 28 and 30. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 31 and 33. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 32 and 34.

[0059] In some embodiments, the present application relates to a plasmid construct (e.g., pT7-sgRNA) comprising an sgRNA sequence and / or a cell comprising the construct.

[0060] The present invention also relates to a cell comprising a targeting vector or an sgRNA vector as described above.

[0061] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned targeting vectors and one or more in vitro transcripts of the construct described in the present application. In some embodiments, the cell comprises Cas9mRNA or its in vitro transcript.

[0062] In some embodiments, the cell is heterozygous for the gene. In some embodiments, the cell is homozygous for the gene.

[0063] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

[0064] Genetically modified non-human animals

[0065] The "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome of the non-human animal has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (such as T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells) germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal is provided, the non-human animal comprises a modified endogenous MGL locus, comprising an exogenous sequence (such as a human sequence), for example, one or more non-human sequences are replaced with one or more human sequences, or one or more human and / or non-human sequences are inserted. Non-human animals are generally able to pass genetic modifications to offspring through germline transmission.

[0066] The "chimeric (x) gene" or "chimeric (x) nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, a chimeric (x) gene or a chimeric (x) nucleic acid has at least a portion of a sequence having two or more different species sources, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, a chimeric (x) gene or a chimeric (x) nucleic acid refers to a humanized (x) gene or a humanized (x) nucleic acid.

[0067] The "chimeric (x) protein" or "chimeric (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more parts of the polypeptide or protein are from different species, or at least one sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (x) protein or chimeric (x) polypeptide has two or more different species sources, for example, the same (or homologous) proteins of different species. In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide refers to a humanized (x) protein or humanized (x) polypeptide.

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

[0069] The "humanized (x) nucleic acid" of the present invention refers to a nucleic acid, wherein at least a portion of the nucleic acid is derived from a human. In some embodiments, the nucleic acids in the humanized (x) nucleic acid are all derived from a human. In some embodiments, the humanized (x) nucleic acid refers to a humanized exon, and the humanized exon can be a human exon or a chimeric exon.

[0070] In some embodiments, the chimeric CLEC10A gene or chimeric CLEC10A nucleic acid is a humanized CLEC10A gene or a humanized CLEC10A nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human CLEC10A gene, and at least a portion of the gene or nucleic acid is derived from a non-human MGL gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding a CLEC10A protein. The encoded CLEC10A protein has at least one activity of a human CLEC10A protein or a non-human animal MGL protein.

[0071] In some embodiments, the chimeric CLEC10A protein or chimeric CLEC10A polypeptide is a humanized CLEC10A protein or humanized CLEC10A polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are from human CLEC10A protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are from non-human animal MGL protein. The humanized CLEC10A protein or humanized CLEC10A polypeptide is functional, or has at least one activity of a human CLEC10A protein or non-human animal MGL protein.

[0072] In some embodiments, the CLEC10A extracellular region is human or humanized. In some embodiments, the CLEC10A cytoplasmic region is human or humanized. In some embodiments, the CLEC10A transmembrane region is human or humanized.

[0073] The genetically modified non-human animal comprises a modification of an endogenous non-human animal MGL gene locus. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature CLEC10A protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a mature CLEC10A protein amino acid sequence). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein are provided in the present invention, in many embodiments, the genetically modified non-human animal comprises a modification of an endogenous MGL gene locus in a non-human animal.

[0074] The genetically modified non-human animal can express human CLEC10A and / or a chimeric (e.g., humanized) CLEC10A protein at an endogenous mouse locus, wherein the endogenous mouse MGL gene has been replaced or inserted with a gene for human CLEC10A and / or a nucleotide sequence encoding a human CLEC10A protein or a nucleotide sequence at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 100% identical to a human CLEC10A sequence. In various embodiments, the endogenous non-human animal MGL locus is modified with a human full or partial nucleic acid sequence encoding a mature CLEC10A protein.

[0075] In some embodiments, the genetically modified mouse can express human CLEC10A and / or chimeric CLEC10A (e.g., humanized CLEC10A) proteins under the control of a mouse promoter and / or mouse regulatory elements. Insertion or replacement at the mouse endogenous locus provides a non-human animal that expresses human CLEC10A or chimeric CLEC10A (e.g., humanized CLEC10A) proteins in suitable cells and in a manner that does not cause potential pathology observed in some other transgenic mice known in the art. Human CLEC10A or chimeric CLEC10A (e.g., humanized CLEC10A) proteins expressed in non-human animals can maintain one or more functions of wild-type mice or human CLEC10A in non-human animals. In addition, in some embodiments, the non-human animal does not express endogenous MGL. In some embodiments, the endogenous MGL expression level of the non-human animal is reduced compared to the MGL expression level in the wild-type animal. As used herein, the term "endogenous MGL" refers to the MGL protein expressed by the endogenous MGL nucleotide sequence of a non-human animal (eg, mouse) before any genetic modification.

[0076] The genetically modified non-human animal may have one or more cells expressing a human or chimeric CLEC10A (e.g., humanized CLEC10A) protein having, from the N-terminus to the C-terminus, a cytoplasmic region, a transmembrane region, and an extracellular region. In many cases, human CLEC10A and non-human MGL (e.g., mouse MGL) sequences are different, so antibodies that bind to human CLEC10A do not necessarily have the same affinity as non-human MGL or have the same effect on non-human MGL. Therefore, genetically modified non-human animals with human or humanized extracellular regions can be used to better evaluate the effects of anti-human CLEC10A antibodies in animal models.

[0077] In some embodiments, the entire cytoplasmic region and transmembrane region of the humanized CLEC10A protein described herein are derived from an endogenous MGL (preferably MGL2) sequence.

[0078] In some embodiments, the humanized CLEC10A locus comprises a human 5'UTR. In some embodiments, the humanized CLEC10A locus comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'UTR. In some embodiments, the humanized CLEC10A locus comprises a human 3'UTR. Where appropriate, it is reasonable to assume that, based on the similarity of the 5' flanking sequences of the mouse and human CLEC10A genes, they appear to be similarly regulated. As shown in the present application, humanized CLEC10A mice containing insertions or substitutions in the endogenous mouse MGL locus, which retain mouse regulatory elements but contain humanization of the CLEC10A coding sequence, do not exhibit pathological phenomena. Both genetically modified mice, either heterozygous or homozygous for humanized CLEC10A, are normal.

[0079] The non-human animal of genetic modification can be various animals, for example, mouse, rat, rabbit, pig, cattle (for example, cattle, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (for example, marmoset, rhesus monkey).For the non-human animal of suitable genetically modified embryonic stem cell (ES) that is not easy to obtain, adopt alternative methods to construct the non-human animal comprising genetic modification.Such method includes, for example, modifying non-ES cell genome (for example, fibroblast or induced pluripotent stem cell) and adopting nuclear transplantation to transfer the modified genome to suitable cell, for example oocyte, and incubating the modified cell (for example, modified oocyte) in non-human animal under appropriate conditions to form embryo.The above-mentioned construction method is known in the art, and is described in "A.Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), " Cold Spring Harbor Laboratory Press, 2006 ", the entire contents of which are incorporated into the present application by reference.

[0080] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. Rodents can be selected from mice, rats and hamsters. In one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified non-human animal is selected from the family of Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Malboridae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Spiny Dormouse (e.g., spiny dormouse) and Muridae (e.g., mole rats, bamboo rats and zokors). In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny mice and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0081] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, an immunodeficient monkey, etc. In some embodiments, the non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid strain (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In certain embodiments, non-human animals are rodents. In certain embodiments, non-human animals are mice with BALB / c, BALB / cHeAn, BALB / cJ, BALB / cR1, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strains. In certain embodiments, non-human animals are rats. Rats can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6 and Dark Agouti.In some embodiments, the rat strain is a hybrid of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0082] The non-human animal may have one or more other genetic modifications and / or other modifications that are suitable for the specific purpose of preparing a humanized animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) may have one or more modifications that damage, inactivate, or destroy all or part of the immune system of the non-human animal. Damage, inactivation, or destruction of the immune system of the non-human animal may include, for example, chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and / or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc null Mice (Ito, M. et al., NOD / SCID / γc null mouse:an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can be optionally irradiated, or otherwise treated to destroy one or more immune cell types. Therefore, in various embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of endogenous non-human MGL1 and / or MGL2 loci, and also includes damage, inactivation or partial destruction of the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the mouse modification type is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null Mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scid IL-2Rγ null Mouse, NOD Rag 1 - / - IL - / - (NRG) mouse, Rag2 - / - IL - / -(RG) mice and modifications of combinations thereof. These transgenic animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mice may include replacing all or part of the mouse endogenous mature MGL1 and / or MGL2 coding sequences with all or part of the human mature CLEC10A coding sequence, respectively.

[0083] The present invention further relates to a non-human mammal or its offspring produced by the above method. In some embodiments, its genome comprises human genes.

[0084] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0085] In some embodiments, the non-human mammal expresses a protein encoded by a humanized CLEC10A gene.

[0086] In addition, the present invention also provides a non-human mammal model carrying a tumor, wherein the non-human mammal model is obtained by the method described in the present application. In some embodiments, the non-human mammal is a rodent (eg, mouse).

[0087] The present invention also provides a cell or cell line derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a primary cell culture, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its offspring. When it carries a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring, or a non-human mammal carrying a tumor.

[0088] The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal comprises DNA of human or humanized CLEC10A.

[0089] In some embodiments, a non-human mammal comprises a gene construct described herein. In some embodiments, a non-human mammal expressing a human or humanized CLEC10A protein is provided. In some embodiments, a tissue specifically expressing a human or humanized CLEC10A protein is provided.

[0090] In some embodiments, the expression of non-human animal human or humanized CLEC10A protein is controllable, such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

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

[0092] The non-human mammals described above are subjected to genetic, molecular and behavioral analyses. The present invention provides offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0093] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny. For example, a cell culture-based model can be prepared by the following method. The cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. The integration of a genetic construct comprising a DNA sequence encoding a human CLEC10A protein can be detected by a variety of methods.

[0094] There are many analytical methods that can be used to detect exogenous DNA, including methods at the nucleic acid level (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, immunoblot analysis and in vitro binding studies). In addition, the expression level of the target gene can be quantified by the ELSA method well known to those skilled in the art. Many standard analytical methods can be used to perform quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcription levels, including RNAse protection analysis, Southern Blot, RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of human or humanized CLEC10A protein.

[0095] Methods for constructing genetically modified non-human animals

[0096] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting technology, homologous recombination technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques using embryonic stem cells. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR-Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these gene editing technologies are known in the art and are described in Yin et al. "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting genetically modified cells into enucleated oocytes and fusing enucleated oocytes with another genetically modified cell.

[0097] In some embodiments, the nucleotide sequence encoding the endogenous MGL region in the endogenous genome of at least one cell of the non-human animal is replaced by the nucleotide sequence encoding the corresponding region of human CLEC10A. In some embodiments, the expression level of the endogenous MGL protein of the non-human animal is reduced or absent compared with the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0098] Preferably, the non-human animal further comprises other genetic modifications. More preferably, the other genes include but are not limited to at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0099] Preferably, the human or humanized CLEC10A gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) locus.

[0100] Preferably, the human or humanized CLEC10A gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

[0101] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0102] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0103] Therefore, the present invention provides a method for constructing a non-human animal with a humanized CLEC10A gene, wherein the non-human animal expresses human or humanized CLEC10A protein in vivo, and / or the genome of the non-human animal contains a portion of the human CLEC10A gene or a humanized CLEC10A gene.

[0104] In some embodiments, a method of modifying the MGL locus of a mouse to express a chimeric human / mouse CLEC10A polypeptide may comprise replacing a nucleotide sequence encoding mouse MGL1 and MGL2 at an endogenous mouse MGL locus with a nucleotide sequence encoding human CLEC10A, thereby generating a sequence encoding a chimeric human / mouse CLEC10A. In some embodiments, the method may comprise inserting a nucleotide sequence encoding a chimeric human / mouse CLEC10A at an endogenous mouse MGL locus, thereby generating a sequence encoding a chimeric human / mouse CLEC10A.

[0105] The present invention also provides a method for establishing a CLEC10A gene humanized animal model, comprising the following steps:

[0106] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0107] (b) culturing the cells, preferably culturing the cells in a liquid culture medium;

[0108] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

[0109] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0110] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0111] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0112] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0113] The fertilized egg can be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0114] In some embodiments, the method for preparing a genetically modified non-human animal comprises modifying the coding framework of the MGL gene of the non-human animal, for example, by replacing the nucleic acid sequence encoding the endogenous MGL region (e.g., CDS, genomic DNA or cDNA sequence) with a nucleotide sequence encoding the corresponding region of human CLEC10A under the control of the endogenous regulatory elements of the MGL gene of the non-human animal, so that the endogenous MGL protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, one or more functional region sequences of the MGL1 and / or MGL2 genes of the non-human animal can be knocked out or inserted with sequences, so that the endogenous MGL1 and / or MGL2 proteins of the non-human animal cannot be expressed or the expression level is reduced.

[0115] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence encoding a human or humanized CLEC10A protein and / or an auxiliary sequence after the endogenous regulatory element of the MGL gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the CLEC10A gene humanized animal model can express the human or humanized CLEC10A protein in vivo, but does not express the MGL protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0116] In some embodiments, the construction method includes obtaining using the above-mentioned vector. In some embodiments, the vector is a targeting vector and / or an sgRNA vector. In some embodiments, the construction method includes introducing the targeting vector into a non-human animal or its cells. In some embodiments, the construction method includes introducing the targeting vector and the sgRNA vector into a non-human animal or its cells.

[0117] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0118] (1) providing a plasmid (such as the above-mentioned targeting vector) comprising a human CLEC10A gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous MGL;

[0119] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous MGL gene;

[0120] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2) and Cas9;

[0121] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express the humanized CLEC10A protein;

[0122] (5) The offspring mice obtained in step (4) are mated to obtain homozygous mice.

[0123] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal targeting site.

[0124] In some embodiments, the sequence encoding the humanized CLEC10A protein is operably linked to endogenous regulatory elements at the endogenous MGL locus.

[0125] In some embodiments, the genetically modified non-human animal does not express endogenous MGL protein.

[0126] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0127] (1) providing a plasmid (e.g., the above-mentioned targeting vector) comprising a human or chimeric CLEC10A gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target endogenous MGL of a non-human animal;

[0128] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous MGL gene;

[0129] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric CLEC10A gene fragment into the genome.

[0130] In some embodiments, the nucleotide sequence encoding the endogenous MGL protein of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding the endogenous MGL1 and / or MGL2 protein of the non-human animal is deleted. In some embodiments, all or part of exons 1-10 of the endogenous MGL1 gene of the non-human animal is deleted. In some embodiments, all or part of exon 1 to at least 50bp downstream of the 3'UTR of the endogenous MGL1 gene of the non-human animal is deleted. In some embodiments, part of exon 3 to all of exon 10 of the endogenous MGL2 gene of the non-human animal is deleted. In some embodiments, the nucleotide sequence encoding the extracellular region in the endogenous MGL2 gene of the non-human animal is deleted.

[0131] Use of genetically modified non-human animals

[0132] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at an endogenous non-human animal locus and under the control of an endogenous promoter and / or regulatory element can produce a non-human animal with qualities and characteristics that may be significantly different from a typical knockout plus transgenic animal. In a typical knockout plus transgenic animal, the endogenous locus is removed or destroyed, and a full human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of a human gene and / or protein assay and / or functional assay. In a human transgene, the upstream and / or downstream of the human sequence provides suitable support for expression and / or regulation of the transgene.

[0133] In some cases, transgenes with human regulatory elements are expressed in a non-physiological or otherwise unsatisfactory manner, and may actually be harmful to the animal. The present invention demonstrates that replacement or insertion of human sequences at endogenous loci under the control of endogenous regulatory elements to produce humanized animals provides physiologically appropriate expression patterns and levels that are meaningful and appropriate in the context of the physiology of the humanized animal with respect to the physiology of the replaced gene.

[0134] Genetically modified animals that express human or humanized CLEC10A protein, e.g., in a physiologically appropriate manner, provide a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0135] The present invention also provides a use of the CLEC10A gene-modified non-human animal and a non-human animal obtained by any of the above construction methods.

[0136] In some embodiments, the application comprises:

[0137] A) Application in product development involving CLEC10A-related immune processes in human cells;

[0138] B) Application as a model system related to CLEC10A for pharmacological, immunological, microbiological and medical research;

[0139] C) Applications involving the production and use of animal experimental disease models for the study of the etiology of CLEC10A and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0140] D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human CLEC10A signaling pathway modulators; or

[0141] E) Study the function of CLEC10A gene, study the drugs and efficacy targeting human CLEC10A target sites, and study the application of CLEC10A-related drugs in cancer, inflammation, and immune diseases.

[0142] The present invention provides a non-human animal expressing a human or humanized CLEC10A protein, which can be used for screening of human CLEC10A specific therapeutic agents. The therapeutic agent can reduce or block the interaction between CLEC10A and the CLEC10A receptor complex, test whether the therapeutic agent can increase or decrease the immune response, and / or determine whether the therapeutic agent is a CLEC10A agonist or antagonist. In some embodiments, the non-human animal is an animal model of human disease. For example, the disease is genetically induced (knock-in or knock-out). In various embodiments, the genetically modified non-human animal also comprises an impaired immune system, such as a genetically modified human-derived tissue xenograft, including a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor).

[0143] In some embodiments, the genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-CLEC10A antibodies, nucleic acid drugs targeting CLEC10A, and / or polypeptide drugs targeting CLEC10A) in treating various immune diseases. In some embodiments, the immune diseases include, but are not limited to, GVHD (graft versus host disease), Crohn's disease, ankylosing spondylitis, atopic dermatitis, psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, hyperthyroidism, thrombocytopenic purpura (e.g., primary or idiopathic), autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, the immune disease is asthma, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis.

[0144] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-CLEC10A antibodies, nucleic acid drugs targeting CLEC10A, and / or polypeptide drugs targeting CLEC10A) in treating various inflammations. In some embodiments, inflammation includes, but is not limited to, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (e.g., tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, inflammation is inflammatory bowel disease (IBD).

[0145] In some embodiments, the genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-CLEC10A antibodies, nucleic acid drugs targeting CLEC10A, and / or polypeptide drugs targeting CLEC10A; or drugs targeting the CLEC10A signaling pathway) in treating tumors or cancer. In some embodiments, the method involves administering a therapeutic agent to a non-human animal as described herein, wherein the animal suffers from cancer or a tumor; and determining the inhibitory effect of the therapeutic agent on the cancer or tumor. The inhibitory effects that can be determined include, for example, a reduction in tumor size or tumor volume, a reduction in tumor growth, a reduction in the rate of increase of tumor volume in a subject (e.g., compared to the rate of increase of tumor volume in the same subject before treatment or in another subject that has not been treated with such treatment), a reduction in the risk of metastasis or the risk of one or more additional metastases, an increase in survival rate and an increase in life expectancy, etc. The tumor volume of a subject can be determined by various methods, such as by direct measurement, MRI, or CT. In some embodiments, the antibody can directly target the CLEC10A protein.

[0146] In some embodiments, the tumor comprises one or more cancer cells (eg, human or mouse cancer cells) injected into the animal. In some embodiments, the antibody activates the CLEC10A signaling pathway. In some embodiments, the antibody does not activate the CLEC10A signaling pathway.

[0147] In some embodiments, the genetically modified non-human animals can be used to determine whether an antibody is a CLEC10A agonist or antagonist. In some embodiments, the methods described herein are also designed to determine the effect of a therapeutic agent (e.g., an antibody targeting CLEC10A, a nucleic acid drug and / or a polypeptide drug targeting CLEC10A; or a drug targeting the CLEC10A signaling pathway) on CLEC10A, for example, whether the therapeutic agent can upregulate an immune response or downregulate an immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent to treat a disease in a subject, such as cancer or a tumor.

[0148] The inhibitory effect on tumors can also be determined by methods known in the art, for example, by measuring tumor volume in animals, and / or determining the tumor (volume) inhibition rate (TGI). TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%)=(1–T Vt / T Vc )×100, where T Vt and T Vc is the average tumor volume (or weight) of the treatment group and the control group.

[0149] In some embodiments, therapeutic agents (e.g., antibodies targeting CLEC10A, nucleic acid drugs targeting CLEC10A, and / or polypeptide drugs targeting CLEC10A; or drugs targeting the CLEC10A signaling pathway) are designed to treat various cancers or tumors. As used in this application, the term "cancer" refers to cells with autonomous growth capacity, i.e., an abnormal state or state characterized by rapid proliferating cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. The term "tumor" used in this application refers to cancer cells, such as cancer cell clusters. Cancers that can be treated or diagnosed using the methods described in this application include malignant tumors of various organ systems, such as malignant tumors affecting the lungs, breasts, thyroid, lymph, gastrointestinal and genital urinary tracts, and adenocarcinomas, including malignant tumors such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancer. In some embodiments, the therapeutic agents described in this application are designed to treat or diagnose cancer in a subject. The term "cancer" is generally recognized and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, urogenital cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include cancers formed by cervical, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term also includes carcinosarcoma, for example, including malignant tumors composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or tumor cells forming a recognizable glandular structure. The term "sarcoma" is generally recognized and refers to a malignant tumor of mesenchymal origin.

[0150] In some embodiments, the cancer described herein is lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, cancer, bladder cancer, glioma, cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome and sarcoma. In some embodiments, the leukemia is selected from acute lymphocytic (lymphocytic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia and chronic myelogenous leukemia. In certain embodiments, lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma and T cell lymphoma, and Waldenstrom macroglobulinemia. In certain embodiments, sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma. In certain specific embodiments, tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer or cancer. In certain embodiments, described cancer is solid tumor and / or hematologic tumor. In certain embodiments, described cancer is ovarian cancer, head and neck cancer, liver cancer or lung cancer.

[0151] In some embodiments, therapeutic agents (e.g., antibodies targeting CLEC10A, nucleic acid drugs targeting CLEC10A, and / or polypeptide drugs targeting CLEC10A; or drugs targeting the CLEC10A signaling pathway) are designed to treat various immune diseases. Therefore, the methods described in the present application can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting CLEC10A, nucleic acid drugs targeting CLEC10A, and / or polypeptide drugs targeting CLEC10A; or drugs targeting the CLEC10A signaling pathway) in inhibiting immune responses. In some embodiments, the immune disease described herein is graft-versus-host disease (GVHD), Crohn's disease, ankylosing spondylitis, atopic dermatitis, psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty liver disease), systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, hyperthyroidism, thrombocytopenic purpura (e.g., primary or idiopathic), autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or nervous system disease, etc. In some embodiments, the therapeutic agent (e.g., an antibody targeting CLEC10A, a nucleic acid drug targeting CLEC10A and / or a polypeptide drug targeting CLEC10A; or a drug targeting the CLEC10A signaling pathway) is designed to treat various inflammations, such as viral inflammation. In some embodiments, the inflammation described herein includes acute inflammation and chronic inflammation. Specifically, inflammation includes, but is not limited to, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (e.g., tuberculosis, syphilis, leprosy, or lymphogranuloma). In certain embodiments, inflammation described herein includes infection, and infection refers to local tissue and systemic inflammatory responses caused by bacteria, viruses, fungi, parasites, and / or other pathogens that invade the human body. In certain embodiments, inflammation is inflammatory bowel disease (IBD).

[0152] The present invention also provides a method for determining the toxicity of a therapeutic agent (e.g., an anti-CLEC10A antibody, a nucleic acid drug targeting CLEC10A, and / or a polypeptide drug targeting CLEC10A). The method comprises administering a therapeutic agent to the non-human animal described above, and evaluating the animal's weight change or blood test. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit, or hemoglobin content. In some embodiments, the therapeutic agent can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the weight of the animal is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average weight of an animal not treated with the antibody).

[0153] The present invention also provides an animal model constructed by the method described in the present application for developing products related to human cellular immune processes, producing human antibodies, or a model system for pharmacology, immunology, microbiology and medical research.

[0154] In some embodiments, an animal model generated by the methods described herein is provided for producing and utilizing animal experimental disease models of immune processes of human cells, studying pathogens, or developing new diagnostic strategies and / or therapeutic strategies.

[0155] The present invention also provides an animal model generated by the method described in the present application to screen, verify, evaluate or study the safety or effectiveness of CLEC10A gene function, human CLEC10A antibodies, drugs targeting human CLEC10A sites, drugs for immune diseases, anti-inflammatory drugs and anti-tumor drugs.

[0156] In some embodiments, the present disclosure provides a method for verifying the in vivo efficacy of TCR-T, CAR-T and / or other immunotherapies (e.g., T cell adoptive transfer therapy). For example, the method includes transplanting human tumor cells into non-human animals described herein, and applying human CAR-T to animals with human tumor cells. The effectiveness of CAR-T therapy can be determined and evaluated. In some embodiments, the non-human animal is selected from a CLEC10A gene humanized non-human animal prepared by the method described herein, a CLEC10A gene humanized non-human animal described herein, a double or multiple gene humanized non-human animal (or its offspring) produced by the method described herein, a non-human animal expressing a human or humanized CLEC10A protein, or a tumor-bearing or inflammatory animal model described herein. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat CLEC10A-related diseases described herein. In some embodiments, TCA-T, CAR-T and / or other immunotherapies provide an evaluation method for treating CLEC10A-related diseases described herein.

[0157] Non-human animal models with two or more human or chimeric genes

[0158] The present invention also provides a method for generating a genetically modified animal model having two or more human or chimeric genes. The animal model may comprise a human or chimeric CLEC10A gene and a sequence encoding an additional human or chimeric protein. In some embodiments, the animal model comprises a human or humanized CLEC10A gene.

[0159] In some embodiments, the additional human or chimeric proteins include, but are not limited to, at least one genetically modified non-human animal of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the non-human animal described above also expresses at least one of human or humanized ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 proteins.

[0160] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0161] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0162] (ii) mating, in vitro fertilization, or direct gene editing of the non-human animal provided in step (i) with other genetically modified non-human animals, and screening to obtain two or more genetically modified non-human animals.

[0163] In some embodiments, the other genetically modified non-human animals include but are not limited to non-human animals humanized with one or a combination of two or more of the genes ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

[0164] In some embodiments, humanization is performed directly on a non-human animal with a modified human or chimeric ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 gene.

[0165] Since these proteins may be involved in different mechanisms, combination therapy targeting two or more of them may be a more effective treatment method. In fact, many related clinical trials are underway and have shown good results. Multigene modified non-human animal models can be used to determine the effectiveness of combination therapy targeting two or more proteins, for example, anti-CLEC10A antibodies, nucleic acid drugs targeting CLEC10A and / or polypeptide drugs targeting CLEC10A, and additional therapeutic agents for treating inflammation, cancer or immune diseases (e.g., asthma, rheumatoid arthritis, multiple sclerosis or atopic dermatitis). The method includes administering an anti-CLEC10A antibody (or a nucleic acid drug targeting CLEC10A and / or a polypeptide drug targeting CLEC10A) and an additional therapeutic agent to a non-human animal, wherein the non-human animal has inflammation, tumor or immune disease, and determining the effect of the combination therapy on inflammation, tumor or immune disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the additional therapeutic agent includes but is not limited to anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab) or anti-PD-L1 antibodies. In some embodiments, the non-human animal further includes a sequence encoding a human or humanized PD-1, a sequence encoding a human or humanized PD-L1, or a sequence encoding a human or humanized CTLA-4. In some embodiments, the tumor includes one or more tumor cells expressing PD-L1 and / or PD-L2.

[0166] In some embodiments, the combination therapy is used to treat various cancers described herein, for example, solid tumors and / or blood tumors, such as ovarian cancer, head and neck cancer, liver cancer or lung cancer. In some embodiments, the combination therapy is designed to treat immune diseases described herein, such as asthma, atopic dermatitis, rheumatoid arthritis or multiple sclerosis.

[0167] In some embodiments, the method described in the present application can be used to evaluate the combined treatment with some other methods. The method for treating cancer that can be used alone or in combination with the method described in the present application includes, for example, treating the subject with chemotherapy, for example, camphor alkali, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepam, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorbutiazine, bithiofen, nitrosourea, dacrynic acid, daunorubicin, bleomycin, plimycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouric acid, vincristine, vinblastine and / or methotrexate. Alternatively, in addition, the method can include surgery on the subject to remove at least a portion of the cancer, for example, removing a portion or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0168] Figure 1 : Schematic diagram comparing the mouse MGL locus and the human CLEC10A locus (not to scale);

[0169] Figure 2 : Schematic diagram of CLEC10A gene targeting strategy and targeting vector V1 design (not to scale);

[0170] Figure 3 : Schematic diagram of CLEC10A gene targeting strategy and targeting vector V2 design (not to scale);

[0171] Figure 4 : Southern blot identification results of the F1 generation of CLEC10A gene humanized mice, where WT is the wild-type control;

[0172] Figure 5 : RT-PCR test results, where + / + is wild-type C57BL / 6 mice, H / H is CLEC10A gene humanized homozygous mice, H 2 O is water control. DETAILED DESCRIPTION

[0173] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0174] Materials and methods

[0175] In each of the following examples, equipment and materials were obtained from the following companies:

[0176] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Administration;

[0177] BstEII enzyme was purchased from NEB, catalog number R3162S;

[0178] StuI enzyme was purchased from NEB, catalog number R0187S;

[0179] BioLegend PerCP anti-mouse CD45 Antibody was purchased from Biolegend, catalog number 103130;

[0180] Brilliant Violet 421 TM anti-mouse CD3 Antibody was purchased from Biolegend, catalog number 100228;

[0181] Alexa 700anti-mouse CD11c Antibody was purchased from Biolegend, catalog number 117320;

[0182] Human CLEC 10A / CD301 Alexa 488-conjugated Antibody was purchased from R&D, catalog number FAB48881G;

[0183] APC anti-mouse CD301 (MGL1 / MGL2) Antibody was purchased from Biolegend, catalog number 145707;

[0184] BioLegend PE anti-human CD301 (CLEC10A) Antibody was purchased from Biolegend, catalog number 354703;

[0185] PE Mouse IgG2a, κIsotype Ctrl (FC) Antibody was purchased from Biolegend, catalog number 400214;

[0186] APC Rat IgG2b, κIsotype Ctrl Antibody was purchased from Biolegend, catalog number 400612;

[0187] Zombie NIR TM Fixable Viability Kit was purchased from Biolegend, catalog number 423106;

[0188] Purified anti-mouse CD16 / 32 was purchased from Biolegend, catalog number 101302.

[0189] Example 1 CLEC10A gene humanized mice

[0190] Mouse MGL1 gene (Gene ID: 17312, located at positions 70057449 to 70061662 of chromosome 11 NC_000077.7, based on transcript NM_010796.3 and its encoded protein NP_034926.1 (SEQ ID NO: 1)), mouse MGL2 gene (Gene ID: 216864, located at positions 70020953 to 70028368 of chromosome 11 NC_000077.7, based on transcript NM_145137.2 and its encoded protein NP_660119.1 (SEQ ID NO: 35)) and human CLEC10A gene (GeneID: 10462, located at positions 7074537 to 7080251 of chromosome 17 NC_000017.11, based on transcript NM_182906.4 and its encoded protein NP_878910.1 (SEQ ID NO: 2)) are shown in the schematic diagram of the comparison. Figure 1 shown.

[0191] In order to achieve the purpose of the present invention, a nucleotide sequence encoding human CLEC10A protein can be introduced into the mouse endogenous MGL locus, so that the mouse expresses human or humanized CLEC10A protein. Specifically, using gene editing technology, under the control of the mouse MGL gene regulatory element, at least the partial sequence of exon 3 of the mouse MGL2 gene to the partial nucleotide sequence of about 3.7 kb downstream of 3'UTR is used to replace the entire nucleotide sequence of about 2.3 kb from the partial sequence of exon 3 to exon 10; further, the partial sequence of exon 3 of the human CLEC10A gene to the partial nucleotide sequence of about 3.7 kb downstream of 3'UTR can be used to replace the partial genomic sequence of about 35.9 kb of the mouse MGL, specifically including the nucleotide sequence of exon 1 to 300 bp downstream of 3'UTR of the mouse MGL1 gene and the partial nucleotide sequence of exon 3 to exon 10 of the mouse MGL2 gene, to obtain a humanized CLEC10A locus, and realize the humanization transformation of the mouse MGL gene.

[0192] In order to realize the present invention, a targeting vector was constructed. The targeting vector V1 ( Figure 2) contains upstream and downstream homology arm sequences of the mouse MGL gene, and an A fragment containing a human CLEC10A gene fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 70021924 to 70025984 of NCBI accession number NC_000077.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 70061963 to 70065620 of NCBI accession number NC_000077.7. The nucleotide sequence of the human CLEC10A gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 7074237 to 7078000 of NCBI accession number NC_000017.11; the connection design of the upstream of the human CLEC10A fragment sequence and the mouse is: The sequence " GATCC The last "C" in the sequence is the last nucleotide in the mouse. The "C" in is the first nucleotide of the human sequence.

[0193] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human CLEC10A gene is designed as follows: The sequence " AGCAT "T" in the sequence is the last nucleotide of the human CLEC10A gene. The first "G" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the mouse MGL gene is designed as: The sequence " ACTTC The last "C" in the sequence is the last nucleotide of the Neo box. The "T" in is the first nucleotide of the mouse MGL gene. The mRNA sequence transcribed from the CLEC10A gene in the transformed humanized mouse is shown in SEQ ID NO:6, and the expressed protein sequence is shown in SEQ ID NO:7.

[0194] The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using the positive clone screening marker gene to screen the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the technology known in the art, and the obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then mated with each other to obtain CLEC10A gene humanized homozygous mice.

[0195] In addition, CRISPR / Cas9 technology can be used to edit the endogenous loci of mice and further design the targeting vector V2( Figure 3 ), the targeting vector V2 contains homology arm sequences upstream and downstream of the mouse MGL gene, and the human CLEC10A fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 22) is identical to the nucleotide sequence of positions 70024418 to 70025984 of NCBI accession number NC_000077.7, and the downstream 3' homology arm sequence (SEQ ID NO: 23) is identical to the nucleotide sequence of positions 70061963 to 70063540 of NCBI accession number NC_000077.7. The nucleotide sequence of the human CLEC10A fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 7074237 to 7078000 of NCBI accession number NC_000017.11. The connection design of the upstream of the human CLEC10A fragment sequence and the mouse is: The sequence " GATCC The last "C" in the sequence is the last nucleotide in the mouse. The "C" in is the first nucleotide of the human sequence. The downstream connection of the human CLEC10A fragment sequence and the mouse sequence is designed as follows: The sequence " AGCAT "T" is the last nucleotide in the human sequence. The "T" in is the first nucleotide of the mouse sequence. The mRNA sequence transcribed from the modified humanized mouse CLEC10A gene is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 7.

[0196] Conventional methods can be used to construct the targeting vector, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

[0197] The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence of the exemplary sgRNA on the CLEC10A gene is as follows:

[0198] sgRNA1 target site (SEQ ID NO: 25): 5'-GCAAGAGCCCTACCTAGTACTGG-3';

[0199] sgRNA2 target site (SEQ ID NO: 26): 5'-TTCAAGTTCAGGAACGGTGTGGG-3';

[0200] The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 4. After annealing, the annealing products were connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-CLEC10A-1 and pT7-CLEC10A-2.

[0201] Table 4 Sequence list of sgRNA1 and sgRNA2

[0202]

[0203] The pT7-sgRNA vector was synthesized by a plasmid synthesis company with a fragment DNA containing a T7 promoter and sgRNA scaffold (SEQ ID NO: 24) and connected to a backbone vector (source: Takara, item number 3299) by restriction digestion (EcoRI and BamHI) in sequence. The vector was sequenced and verified by a professional sequencing company, and the results showed that the target plasmid was obtained. The pronuclear fertilized eggs of mice, such as C57BL / 6 mice or BALB / c mice, were taken, and the in vitro transcription products of pT7-CLEC10A-1 and pT7-CLEC10A-2 plasmids (using the Ambion in vitro transcription kit, transcribed according to the instructions), the targeting vector and Cas9mRNA were premixed and injected into the cytoplasm or nucleus of the mouse fertilized eggs using a microinjector. Microinjection of fertilized eggs was performed according to the method in the Mouse Embryo Manipulation Experiment Manual (3rd Edition) (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to culture medium for a short period of culture and then transplanted into the oviduct of the recipient mother mouse for development. The obtained mice (F0 generation) were hybridized and self-fertilized to expand the population and establish a stable CLEC10A gene humanized mouse strain.

[0204] The PCR method can be used to identify the somatic cell genotype of F1 generation mice, and the F1 generation PCR-positive mice are subjected to Southern blot detection to confirm whether there is random insertion. The mouse tail is cut to extract genomic DNA, and the genome is digested with BstEII enzyme or StuI enzyme, respectively, transferred to a membrane, and hybridized. The 5' probe (5'Probe) and the A probe (A Probe) are located upstream of the 5' homology arm and on the human CLEC10A genome fragment, respectively. The specific probes and target fragment lengths are shown in Table 5, and the exemplary results are shown in Table 5. Figure 4 As shown, combined with the PCR and sequencing results, three mice numbered F1-12 to F1-14 were positive mice. This indicates that the present method can be used to construct CLEC10A gene humanized mice that can be stably propagated and have no random insertions.

[0205] Table 5 Length of specific probes and target fragments

[0206] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size BsE A'Probe -- 4.9kb StuI 5'Probe 5.8kb 7.0kb

[0207] 5'Probe-F (SEQ ID NO: 12): 5'-GCCTGGCAAGTACCTTATTGACTG-3',

[0208] 5'Probe-R (SEQ ID NO: 13): 5'-AACACTGTAAATGAACAAAGTCACA-3';

[0209] A Probe-F (SEQ ID NO: 14): 5'-AGTAGAGATGACTTTGACAAATATG-3',

[0210] A Probe-R (SEQ ID NO: 15): 5'-CTCAATCACATAGGACTTTCGAAGGC-3';

[0211] The expression of mRNA in humanized CLEC10A mice can be detected by RT-PCR. Specifically, 8-week-old male C57BL / 6 mice (+ / +) and 8-week-old male homozygous CLEC10A mice (H / H) prepared in this example were selected, and spleen tissue was obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 6. The detection results are shown in FIG. Figure 5 As shown. Figure 5 As can be seen, only mouse MGL mRNA was detected in wild-type C57BL / 6 mice, and human CLEC10A mRNA was not detected; only human CLEC10A mRNA was detected in CLEC10A gene homozygous mice.

[0212] Table 6 RT-PCR primer sequences and target fragment sizes

[0213]

[0214] The expression of humanized CLEC10A protein in positive mice can be confirmed by conventional detection methods, such as flow cytometry. Specifically, 8-week-old female C57BL / 6 wild-type mice and 8-week-old female CLEC10A homozygous mice (H / H) were taken, euthanized by cervical dislocation, and bone marrow tissue was obtained. The anti-mouse CD45 antibody BioLegendPerCP anti-mouse CD45 Antibody (mCD45) and the anti-mouse CD11c antibody Alexa 700anti-mouseCD11c Antibody(mCD11c), anti-mouse MGL antibody APC anti-mouse CD301(MGL1 / MGL2)Antibody(mMGL), anti-human CLEC10A antibody BioLegend PE anti-human CD301(CLEC10A)Antibody(hCLEC10APE), anti-human CLEC10A antibody Human CLEC10A / CD301 Alexa 488-conjugated Antibody (hCLEC10A AF488), Anti-rat IgG2A Antibody PE Mouse IgG2a, κIsotype Ctrl (FC) Antibody, Anti-rat IgG2b Antibody APC Rat IgG2b, κIsotype Ctrl Antibody, Zombie NIR TM The expression of humanized CLEC10A protein in undifferentiated dendritic cells (BMDC) in the bone marrow was detected by flow cytometry after staining with Fixable Viability Kit and Purified anti-mouse CD16 / 32. The detection results are shown in Table 7.

[0215] Table 7 Flow cytometry detection results

[0216]

[0217]

[0218] As can be seen from Table 7, only the expression of mouse MGL protein can be detected in immature BMDC cells of wild-type C57BL / 6 mice, and only the expression of humanized CLEC10A protein can be detected in immature BMDC cells of CLEC10A humanized homozygous mice. In summary, the construction of CLEC10A humanized mice was successful.

[0219] Example 2 Pharmacodynamic Model

[0220] The CLEC10A gene humanized mice prepared by this method can be used to evaluate the efficacy of regulators targeting human CLEC10A in tumors, inflammation or immune diseases. Taking tumors as an example, MC38 cells were subcutaneously inoculated in homozygous CLEC10A humanized mice, and the tumor volume grew to about 100 mm. 3 The mice were then divided into a control group or a treatment group according to the tumor volume. The treatment group randomly selected drugs targeting human CLEC10A, and the control group was injected with an equal volume of saline. The tumor volume was measured regularly and the mice were weighed. By comparing the weight changes of the mice and the tumor size, the in vivo safety and in vivo efficacy of the compound can be effectively evaluated.

[0221] Example 3 Preparation of double-gene or multi-gene humanized mice

[0222] The CLEC10A gene humanized mouse prepared by the present method can also be used to prepare a multi-gene humanized mouse model. For example, in the above-mentioned Example 1, the embryonic stem cells used for microinjection can be selected from mice modified with at least one gene containing ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4, or, on the basis of humanized CLEC10A mice, mouse ES embryonic stem cells can be separated and gene recombination targeting technology can be used to obtain a double gene humanized or multi-gene humanized mouse model. The homozygous or heterozygous CLEC10A mouse obtained by the present method can also be mated with other gene-modified mice, and the offspring can be screened. According to the Mendel's law of inheritance, there is a certain probability of obtaining a multi-gene mouse modified with the humanized CLEC10A gene and other genes, and then the heterozygotes can be mated with each other to obtain a homozygous modified with two genes or multiple genes.

[0223] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0224] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0225] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric C-type lectin-like domain 10A (CLEC10A) protein.

2. The construction method according to claim 1, characterized in that: The chimeric CLEC10A protein comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region; Preferably, the amino acid sequence of the human or chimeric CLEC10A protein comprises amino acids 61-316 of SEQ ID NO:2, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 61-316 of SEQ ID NO:2; Further preferably, the amino acid sequence of the chimeric CLEC10A protein comprises SEQ ID NO:7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

7.

3. A method for constructing a genetically modified non-human animal, characterized in that: At the endogenous MGL gene locus of the non-human animal, the endogenous MGL gene is replaced with the nucleotide sequence of human CLEC10A.

4. The construction method according to any one of claims 1 to 3, characterized in that: The endogenous MGL protein of the non-human animal is not expressed or is expressed at a reduced level compared to MGL in wild-type animals; Preferably, the nucleotide sequence of human CLEC10A or the nucleotide sequence encoding human or chimeric CLEC10A protein is operably linked to a regulatory element of endogenous MGL, such as a promoter and / or UTR; Preferably, the modified CLEC10A gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

5. The construction method according to claim 3 or 4, characterized in that: The nucleotide sequence of human CLEC10A includes part of exon 3 to all of exon 9 of the human CLEC10A gene, and preferably also includes at least 50 bp of continuous nucleotides downstream of the 3'UTR; Preferably, the nucleotide sequence of human CLEC10A comprises a portion of exon 3 to at least 50 bp of continuous nucleotides downstream of the 3'UTR; Further preferably, the nucleotide sequence of human CLEC10A comprises SEQ ID NO: 5, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:

5.

6. The construction method according to any one of claims 3 to 5, characterized in that: The replaced endogenous MGL gene includes all or part of the MGL1 gene and the MGL2 gene; Preferably, the replaced MGL1 gene includes all of exon 1 to exon 10 of the endogenous MGL1 gene, and further preferably, includes at least 50 bp of continuous nucleotides from exon 1 to the downstream of the 3'UTR of the endogenous MGL1 gene; Preferably, the replaced MGL2 gene includes part of exon 3 to the entirety of exon 10 of the MGL2 gene.

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

6.

8. The construction method according to any one of claims 1 to 7, characterized in that: The non-human animal also includes other human or chimeric proteins, and the human or chimeric proteins include at least one of ICOS, NKP46, HER2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

9. An application of a non-human animal obtained by the construction method according to any one of claims 1 to 8, characterized in that: The application includes: A) Application in product development involving CLEC10A-related immune processes in human cells; B) Application as a model system related to CLEC10A for pharmacological, immunological, microbiological and medical research; C) Applications involving the production and use of animal experimental disease models for the study of the etiology of CLEC10A and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human CLEC10A signaling pathway modulators; or E) Study the function of CLEC10A gene, study the drugs and efficacy targeting human CLEC10A target sites, and study the application of CLEC10A-related cancer, immune disease or inflammatory drugs.

10. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 8; 2) Determine the effect of a therapeutic agent on a disease; Preferably, the therapeutic agent comprises an anti-CLEC10A antibody, a nucleic acid drug targeting CLEC10A and / or a polypeptide drug targeting CLEC10A; further preferably, the therapeutic agent further comprises an additional therapeutic agent, more preferably, the additional therapeutic agent comprises an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; Preferably, the disease is cancer, immune disease or inflammation.

11. A humanized CLEC10A protein, characterized in that: The humanized CLEC10A protein comprises SEQ ID NO:7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO:

7.

12. A humanized CLEC10A gene, characterized in that: The humanized CLEC10A gene encodes the humanized CLEC10A protein according to claim 11; Preferably, the humanized CLEC10A gene comprises the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 22 or 23, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 22 or 23.

13. A cell, tissue or organ, characterized in that: The cells, tissues or organs contain the humanized CLEC10A protein according to claim 11 and / or the humanized CLEC10A gene according to claim 12.

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