CD209 gene modified non-human animal

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) CD209 protein and methods of use thereof.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (eg, humanized) CD209 protein and methods of using the same. Background Art

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

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

[0004] However, the construction of humanized animal models is very difficult. For example, the non-patent literature: Generation and utility of genetically humanized mouse models (Scheer N, Snaith M, Wolf CR, Seibler J., Drug Discovery Today; 18(23-24): 1200-11, 2013) discloses that the humanization of any gene carries certain risks, and even with carefully designed strategies, it is impossible to ensure the expression and function of human genes in non-human animals. Summary of the invention

[0005] The present application provides a non-human animal or animal model with human or chimeric CD209 protein or a method for constructing it. The non-human animal or animal model can express human or chimeric CD209 (e.g., humanized CD209) protein. It can be used for the study of CD209 gene function, and can also be used for the screening and evaluation of CD209 signaling pathway regulators (e.g., anti-human CD209 antibodies, oligonucleotide drugs and / or polypeptide drugs). In addition, the non-human animal or animal model prepared by the construction method can be used for drug screening, pharmacodynamics research, treatment of CD209-related diseases (e.g., tumors, immune diseases or viral infections) and disease treatment of human CD209 target sites; the non-human animal or animal model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of CD209 gene / protein and provides a platform for screening CD209-related disease therapeutic drugs.

[0006] On the one hand, 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 CD209 molecule (CD209) protein. In some embodiments, the nucleotide sequence encoding a human or chimeric CD209 protein is operably linked to an endogenous regulatory element (e.g., promoter, 5'UTR and / or 3'UTR) of an endogenous CD209 locus of at least one chromosome. In some embodiments, the chimeric CD209 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region, and a non-human animal endogenous cytoplasmic region. In some embodiments, the amino acid sequence of the human or chimeric CD209 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 38-404 or SEQ ID NO: 2 positions 39-404, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 2 or SEQ ID NO: 2 positions 38-404 or SEQ ID NO: 2 positions 39-404. In some embodiments, the amino acid sequence of the chimeric CD209 protein comprises SEQ ID NO: 1 or SEQ ID NO: 1 positions 1-47 or SEQ ID NO: 1 positions 1-46, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 1 or SEQ ID NO: 1 positions 1-47 or SEQ ID NO: 1 positions 1-46. In some embodiments, the amino acid sequence of the chimeric CD209 protein comprises SEQ ID NO: 7, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 7. In some embodiments, the non-human animal comprises a mammal, such as a monkey or a rodent. In some embodiments, the rodent comprises a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous CD209 protein of the non-human animal is not expressed or is expressed at a reduced level compared to CD209 in wild-type animals. In some embodiments, one or more cells of the non-human animal express a human or chimeric CD209 protein.

[0007] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the endogenous CD209 gene of the non-human animal is replaced with a nucleotide sequence comprising human CD209 at the endogenous CD209 locus of the non-human animal. In some embodiments, the replacement of the endogenous CD209 gene of the non-human animal comprises a nucleotide sequence that replaces the extracellular region and / or transmembrane region encoding the endogenous CD209 protein of the non-human animal. In some embodiments, the replaced endogenous CD209 gene of the non-human animal also comprises a nucleotide sequence that encodes a portion of the cytoplasmic region of the endogenous CD209 protein. In some embodiments, one or more cells of the non-human animal express a chimeric CD209 protein, wherein the chimeric CD209 comprises a cytoplasmic region, a transmembrane region and / or an extracellular region, wherein the transmembrane region and / or the extracellular region comprises a human CD209 transmembrane region and / or a human CD209 extracellular region, or comprises a transmembrane region and / or a human CD209 extracellular region that is at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% identical to a human CD209 transmembrane region and / or a human CD209 extracellular region. In some embodiments, the extracellular region and / or transmembrane region is identical to the extracellular region of human CD209 protein and / or the transmembrane region of human CD209 for at least 10, 20, 21, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 345, 346, 347, 348, 349, 350, 360, 365, 366 or 367 consecutive amino acids. In some embodiments, the transmembrane region is identical to the transmembrane region of human CD209 for at least 5, 6, 7, 8, 9, 10, 15, 20 or 21 consecutive amino acids. In some embodiments, the extracellular region is identical to the extracellular region of human CD209 for at least 5, 10, 50, 100, 150, 200, 250, 300, 310, 320, 330, 340, 345 or 346 consecutive amino acids. In some embodiments, the nucleotide sequence of human CD209 includes a portion of exon 3 of the human CD209 gene to all of exon 7. In some embodiments, the portion of exon 3 of the human CD209 gene includes at least 5bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of human CD209 includes at least 50bp of continuous nucleotides downstream of the 3'UTR of the human CD209 gene. In some embodiments, the nucleotide sequence of human CD209 includes a portion of exon 3 of the human CD209 gene, all of exons 4-7, and at least 50bp of continuous nucleotides downstream of the 3'UTR (e.g., at least 300bp). In some embodiments, the nucleotide sequence of human CD209 includes a portion of exon 3 of the human CD209 gene to at least 50bp of continuous nucleotides downstream of the 3'UTR (e.g., at least 300bp).In some embodiments, the nucleotide sequence of human CD209 comprises SEQ ID NO: 5, or comprises a nucleotide sequence with an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, part of exon 3 to all of exon 7 of the endogenous CD209 gene of the non-human animal is replaced. In some embodiments, part of exon 3 of the endogenous CD209 gene of the non-human animal includes at least 5bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence encoding the human or chimeric CD209 protein or the nucleotide sequence of human CD209 is operably linked to the endogenous regulatory elements of the endogenous CD209 locus of the non-human animal, such as a promoter and / or 5'UTR. In some embodiments, the endogenous CD209 protein of the non-human animal is not expressed or the expression level is reduced compared to CD209 in wild-type animals. In some embodiments, the modified CD209 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenously replaced locus. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a mouse or a rat. In some embodiments, the mRNA transcribed from the modified CD209 gene in the genome of the non-human animal comprises SEQ ID NO: 6, or comprises 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 also includes nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins preferably include at least one of CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0008] 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 CD209 protein, wherein the human or chimeric CD209 protein comprises at least 50, 60, 70, 80, 90, 100, 200, 300, 360, 365, 366, 367, 368, 369, 370, 380, 390, 400 or 404 identical contiguous amino acid sequences of a human CD209 protein. In some embodiments, the non-human animal expresses a human or chimeric CD209 protein. In some embodiments, the human or chimeric CD209 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 39-404 or SEQ ID NO: 2 38-404, or comprises an amino acid sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with SEQ ID NO: 2 or SEQ ID NO: 2 39-404 or SEQ ID NO: 2 38-404. In some embodiments, the nucleotide sequence encoding the human or chimeric CD209 protein is operably linked to a non-human animal endogenous CD209 regulatory element. In some embodiments, the nucleotide sequence encoding the human or chimeric CD209 protein can be integrated into the non-human animal endogenous CD209 locus. In some embodiments, the human or chimeric CD209 protein has at least one CD209 activity, such as a non-human animal endogenous CD209 activity and / or a human CD209 activity.

[0009] On the one hand, the present invention provides a method for constructing a genetically modified non-human animal, in at least one cell of the non-human animal, at the endogenous CD209 locus of the non-human animal, the nucleotide sequence encoding the endogenous CD209 region is replaced by a nucleotide sequence encoding the corresponding region of human CD209 or a chimeric CD209 protein. In some embodiments, the endogenous CD209 protein of the non-human animal is not expressed or the expression level is reduced compared with CD209 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD209 or the chimeric CD209 protein includes a portion of exon 3 of the human CD209 gene to all of exon 7. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD209 or the chimeric CD209 protein also includes a nucleotide sequence downstream of the 3'UTR of the human CD209 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human CD209 or the chimeric CD209 protein includes a portion of exon 3 of the human CD209 gene, all of exons 4-7, and a nucleotide sequence downstream of the 3'UTR. In some embodiments, the portion of human CD209 gene exon 3 includes at least 5-72bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 71 or 72bp continuous nucleotide sequence. In some embodiments, the 3'UTR downstream nucleotide sequence of the human CD209 gene includes at least 50-1000bp, such as at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000bp continuous nucleotide sequence. In some embodiments, the 3'UTR downstream nucleotide sequence of the human CD209 gene includes at least 200-400bp continuous nucleotides downstream of the 3'UTR of the human CD209 gene. In some embodiments, the nucleotide sequence encoding the human CD209 corresponding region or chimeric CD209 protein comprises a portion of exon 3 of the human CD209 gene to at least 300 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the amino acid sequence of the human CD209 corresponding region or chimeric CD209 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 39-404 or SEQ ID NO: 2 38-404, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity with SEQ ID NO: 2 or SEQ ID NO: 2 39-404 or SEQ ID NO: 2 38-404 amino acid sequence. In some embodiments, the endogenous CD209 region is an endogenous CD209 cytoplasmic region, an extracellular region and / or a transmembrane region.In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human CD209 or the chimeric CD209 protein comprises SEQ ID NO: 2 or 7, or comprises 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 CD209 or the chimeric CD209 protein comprises SEQ ID NO: 5, or comprises 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 CD209 region comprises a portion of exon 3 to all of exon 7 of the CD209 gene of a non-human animal (e.g., mouse). In some embodiments, the nucleotide sequence encoding the corresponding region of human CD209 is operably linked to an endogenous CD209 regulatory element of a non-human animal, such as a 5'UTR and / or a promoter. In some embodiments, the non-human animal comprises a mammal, such as a monkey or a rodent. In some embodiments, the rodent comprises a mouse or a rat. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, wherein the other genes comprise at least one of CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0010] On the one hand, the present invention provides a method for constructing a non-human animal cell that expresses a human or chimeric CD209 gene modification, the method comprising replacing the non-human animal endogenous CD209 gene with a nucleotide sequence comprising human CD209 at the non-human animal endogenous CD209 locus to produce a genetically modified non-human animal cell. In some embodiments, the genetically modified non-human animal cell expresses a human or chimeric CD209 protein. In some embodiments, the chimeric CD209 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region, and a non-human animal endogenous cytoplasmic region. In some embodiments, the nucleotide sequence of the human CD209 comprises a portion of exon 3 of the human CD209 gene to all of exon 7, preferably, a portion of exon 3 of the human CD209 gene comprises at least 5bp continuous nucleotide sequence. In some embodiments, the nucleotide sequence of the human CD209 also comprises at least 50bp continuous nucleotides downstream of the 3'UTR of the human CD209 gene. In some embodiments, the nucleotide sequence of human CD209 comprises part of exon 3 of human CD209 gene, all of exons 4-7 and at least 50bp of continuous nucleotide sequence downstream of 3'UTR. In some embodiments, the nucleotide sequence of human CD209 comprises part of exon 3 of human CD209 gene to at least 50bp of continuous nucleotide sequence downstream of 3'UTR (e.g., 300bp of continuous nucleotide sequence). In some embodiments, the amino acid sequence of the chimeric CD209 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 38-404 or SEQ ID NO: 2 39-404 or SEQ ID NO: 7, or comprises an amino acid sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% with SEQ ID NO: 2 or SEQ ID NO: 2 38-404 or SEQ ID NO: 2 39-404 or SEQ ID NO: 7. In some embodiments, part of exon 3 to all of exon 7 of the endogenous CD209 gene of a non-human animal is replaced, preferably, part of exon 3 of the endogenous CD209 gene of a non-human animal includes at least 5 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of human CD209 is operably linked to a regulatory element of endogenous CD209 of a non-human animal, such as 5'UTR and / or a promoter. In some embodiments, the non-human animal is a mouse.

[0011] In one aspect, the present invention provides a method for determining the effectiveness 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, wherein the non-human animal suffers from a disease; 2) determining the inhibitory effect of the therapeutic agent on the disease. In some embodiments, the therapeutic agent comprises an anti-CD209 antibody, an oligonucleotide drug, or a polypeptide drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease comprises a tumor, an immune disease, or a viral infection. In some embodiments, the tumor comprises one or more of breast cancer, head and neck cancer, colon cancer, liver cancer, or lung cancer. In some embodiments, the viral infection comprises one or more of AIDS (HIV), Ebola virus, or acute respiratory syndrome (SARS). In some embodiments, the immune disease comprises one or more of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, cerebellar atrophy, multiple sclerosis, or Parkinson's disease. In some embodiments, 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 tumor comprises one or more cancer cells injected into a non-human animal.

[0012] In one aspect, the present invention provides a method for determining the toxicity of a CD209 therapeutic agent, the method comprising: 1) administering a CD209 therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the CD209 therapeutic agent on the non-human animal. In some embodiments, the determination of the effect of the CD209 therapeutic agent on the non-human animal comprises measuring the weight of the non-human animal or a blood test. In some embodiments, the blood test comprises one or more of red blood cell count, hematocrit, or hemoglobin content.

[0013] In one aspect, the present invention provides a humanized CD209 protein, comprising all or part of a human CD209 protein. In some embodiments, the humanized CD209 protein comprises all or part of a non-human animal CD209 protein. In some embodiments, the humanized CD209 protein comprises a portion of a human CD209 protein and a portion of a non-human animal CD209 protein. In some embodiments, the humanized CD209 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 38-404 or SEQ ID NO: 2 positions 39-404, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity with SEQ ID NO: 2 or SEQ ID NO: 2 positions 38-404 or SEQ ID NO: 2 positions 39-404. In some embodiments, the humanized CD209 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.

[0014] In one aspect, the present invention provides a humanized CD209 gene, which encodes the above-mentioned humanized CD209 protein. In some embodiments, the humanized CD209 gene comprises part of exon 3 of the human CD209 gene, all of exons 4-7, and 3'UTR downstream nucleotide sequence. In some embodiments, the humanized CD209 gene comprises part of exon 3 of the human CD209 gene to 3'UTR downstream nucleotide sequence. In some embodiments, the nucleotide sequence contained in the humanized CD209 gene comprises SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 or 33, or comprises a nucleotide sequence identical to SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 or 33, which is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

[0015] In one aspect, the present invention provides a cell, tissue or organ, wherein the cell, tissue or organ expresses the above-mentioned humanized CD209 protein, and / or the cell, tissue or organ comprises the above-mentioned humanized CD209 gene.

[0016] In one aspect, the present invention provides an animal model, wherein the animal model expresses the above-mentioned humanized CD209 protein and / or the animal model comprises the above-mentioned humanized CD209 gene.

[0017] In one aspect, the present invention provides an application of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the cell obtained by the above-mentioned construction method, the above-mentioned humanized CD209 protein, the above-mentioned humanized CD209 gene, the above-mentioned cell, tissue or organ or the above-mentioned animal model. In some embodiments, the application includes: A) application in product development involving CD209-related immune processes in human cells; B) application in a model system related to CD209 for pharmacological, immunological, microbiological and medical research; C) application involving the production and use of animal experimental disease models for etiological research related to CD209 and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) application in the screening, efficacy detection, evaluation of efficacy, verification or evaluation of human CD209 signaling pathway regulators in vivo; or, E) application in studying CD209 gene function, studying drugs and efficacy targeting human CD209 target sites, and studying drugs for the treatment of diseases related to CD209.

[0018] 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.

[0019] The term "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a gene in a narrow sense, which can be a gene or a part of a gene. For example, the "CD209 locus" refers to a DNA fragment of any one of the CD209 gene. In some embodiments, the replaced non-human animal endogenous CD209 locus can be a DNA fragment of any one of exons 1-7 of the non-human animal endogenous CD209 gene.

[0020] The term "portion of exon XX" of the present invention means that continuous or intermittent several, dozens or hundreds of nucleotide sequences are consistent with the entire exon nucleotide sequence, for example, a portion of exon 3 of the human CD209 gene, including continuous or intermittent 5-72bp, for example 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72bp nucleotide sequence is consistent with the nucleotide sequence of exon 3 of the human CD209 gene.

[0021] The term "exon XX-XXX" or "exon XX to exon XXX" of the present invention refers to the nucleotide sequence from one exon to another exon and the introns in between, for example, exon 1 to exon 7 or exons 1-7 include all nucleotide sequences between exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and exon 7.

[0022] The term "part of exon XX to all of exon XXX" in the present invention refers to the nucleotide sequence from part of one exon to all of another exon and the introns in between, for example, part of exon 3 to all of exon 7 includes part of exon 3, all of intron 3, all of exon 4, all of intron 4, all of exon 5, all of intron 5, all of exon 6, all of intron 6 and all of exon 7.

[0023] The term "intron xx" in the present invention refers to an intron between two exons, for example, intron 3 refers to an intron between exon 3 and exon 4.

[0024] 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 nucleotide sequence, the protein or nucleotide 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.

[0025] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "B" and "A and B"; for another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0026] 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.

[0027] Those skilled in the art can easily appreciate other aspects and advantages of the present application from the following detailed description.

[0028] Detailed description

[0029] CD209

[0030] In the human genome, the CD209 gene (Gene ID: 30835) contains 7 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and exon 7 ( Figure 1 ). The nucleotide sequence of human CD209 mRNA is NM_021155.4, and the amino acid sequence of human CD209 is NP_066978.1 (SEQ ID NO: 2). The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_021155.4 and its encoded protein NP_066978.1 is shown in Table 1:

[0031] Table 1

[0032]

[0033] In the mouse genome, the CD209 gene (Gene ID: 170786) contains 7 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and exon 7 ( Figure 1 ). The nucleotide sequence of mouse CD209 mRNA is NM_133238.5, and the amino acid sequence of mouse CD209 is NP_573501.1 (SEQ ID NO: 1). The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_133238.5 and its encoded protein NP_573501.1 is shown in Table 2:

[0034] Table 2

[0035]

[0036]

[0037] CD209 genes, proteins and gene loci of other species are also known in the art. For example, the relevant information (e.g., intron sequences, exon sequences and amino acid sequences) of Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), Sus scrofa (pig) genes can be found in NCBI, the entire contents of which are incorporated into this application by reference.

[0038] The present invention provides a human or chimeric (e.g., humanized) CD209 nucleotide sequence or a human or chimeric (e.g., humanized) CD209 amino acid sequence. In some embodiments, all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and / or exon 7 of the endogenous CD209 gene of a non-human animal, or all or part of the nucleotide sequence encoding the extracellular region, transmembrane region and / or cytoplasmic region of the endogenous CD209 protein of a non-human animal is replaced by the nucleotide sequence of human CD209. The above “part” refers to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, 1400, 1480, 1484, 1500, 2000, 2500, 3000, 3500, 4000, 4500 194, 195, 200, 210, 220, 230 or 238 consecutive amino acid sequences. In some embodiments, the "portion" is identical or identical to exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7 of an endogenous CD209 gene of a non-human animal, or to a nucleotide sequence encoding an extracellular region, a transmembrane region and / or a cytoplasmic region of an endogenous CD209 protein of a non-human animal, and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99.5%. In some embodiments, "partial" or "complete" sequences of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous CD209 gene of a non-human animal (e.g., part of exon 3 to all of exon 7 of the endogenous CD209 gene of a non-human animal) are replaced by "partial" or "complete" sequences of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 of the human CD209 gene (e.g., part of exon 3 to the 3'UTR downstream nucleotide sequence of the human CD209 gene). In some embodiments, the extracellular region comprises a signal peptide. In some embodiments, the extracellular region does not comprise a signal peptide.In some embodiments, the nucleotide sequence of the human CD209 gene or the amino acid sequence encoded by it comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 4 200, 4284, 5000, 6000, 6500, 6531, 6800 or 6831 bp of continuous nucleotide sequence, or, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 250, 300, 350, 360, 365, 366, 367, 368, 369, 370, 400 or 404 continuous amino acid sequence.

[0039] In some embodiments, "all or part" of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and / or exon 7 of the endogenous CD209 gene of a non-human animal is deleted. In some embodiments, the deleted endogenous CD209 gene of a non-human animal includes part of exon 3 to all of exon 7 of the endogenous CD209 gene of the non-human animal. Among them, the deleted part of exon 3 of the endogenous CD209 gene of the non-human animal includes at least 5-90bp, for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85 or 90bp of continuous nucleotide sequence.

[0040] In some embodiments, the "partial" deletion of the extracellular region, transmembrane region and / or cytoplasmic region of the endogenous CD209 protein of a non-human animal. In some embodiments, the domain of the deleted endogenous CD209 protein of a non-human animal includes part of the cytoplasmic region, all of the transmembrane region and / or all of the extracellular region.

[0041] In some embodiments, the present invention provides a genetically modified non-human animal, the genome of which comprises a chimeric CD209 (e.g., humanized CD209) nucleotide sequence. In some embodiments, the protein encoded by the chimeric CD209 nucleotide sequence comprises a human or humanized extracellular region, a human or humanized transmembrane region, and a non-human animal endogenous cytoplasmic region. In some embodiments, the encoded protein is identical or identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-47, or SEQ ID NO: 1, positions 1-46, or SEQ ID NO: 2, positions 38-404, or SEQ ID NO: 2, positions 39-404, or SEQ ID NO: 7, for at least 70%, 80%, 85%, 90%, 95%, or 99.5%. In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence that is identical or at least 70%, 80%, 85%, 90%, 95% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 or 33.

[0042] In some embodiments, the genetically modified non-human animal expresses a human or humanized CD209 protein, or comprises a nucleotide sequence encoding a human or humanized CD209 protein. In some embodiments, the humanized CD209 protein comprises a cytoplasmic region, a transmembrane region, and an extracellular region from the N-terminus to the C-terminus. In some embodiments, the humanized CD209 protein comprises a human or humanized transmembrane region, for example, a sequence comprising a human or humanized transmembrane region is consistent or identical to SEQ ID NO: 2, 39-58 or SEQ ID NO: 2, 38-58 amino acids, or at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the human or humanized CD209 protein comprises a human or humanized extracellular region, for example, a sequence comprising a human or humanized extracellular region is consistent or identical to SEQ ID NO: 2, 59-404 amino acids, at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the sequence contained in the human or humanized CD209 protein is at least 70%, 80%, 85%, 90%, 95% or 99.5% identical to SEQ ID NO: 2, amino acids 39-404 or SEQ ID NO: 2, amino acids 38-404. In some embodiments, the humanized CD209 protein comprises a cytoplasmic region endogenous to a non-human animal. For example, the sequence contained in the cytoplasmic region of endogenous CD209 of a non-human animal is at least 70%, 80%, 85%, 90%, 95% or 99.5% identical to SEQ ID NO: 1, amino acids 1-47 or SEQ ID NO: 1, amino acids 1-46. In some embodiments, all or part of the nucleotide sequence encoding the cytoplasmic region, extracellular region and transmembrane region of non-human animal CD209 (SEQ ID NO: 1) is replaced or inactivated. In some embodiments, the nucleotide sequence encoding amino acids 48-238 of SEQ ID NO: 1 or 47-238 of SEQ ID NO: 1 is replaced.

[0043] In some embodiments, the genetically modified non-human animal comprises a human or humanized CD209 gene. In some embodiments, the humanized CD209 gene comprises, from 5' to 3', in sequence: all of exons 1-2 of an endogenous CD209 gene of a non-human animal (such as NM_133238.5 1-211), intron 2 of an endogenous CD209 gene of a non-human animal, part of exon 3 of an endogenous CD209 gene of a non-human animal (such as NM_133238.5 212-219), part of exon 3 of a human CD209 gene (such as NM_021155.4 138-201 nucleotide sequence), intron 3 of a human CD209 gene, all of exons 4-7 of a human CD209 gene (such as NM_021155.4 202-4284 nucleotide sequence), and at least 50 bp of continuous nucleotide sequence (e.g., 300 bp of continuous nucleotide sequence) downstream of the 3'UTR of the human CD209 gene.

[0044] In some embodiments, the humanized CD209 gene comprises 7 exons. In some embodiments, the humanized CD209 gene comprises non-human animal endogenous CD209 gene exon 1, non-human animal endogenous CD209 gene exon 2, humanized exon 3, human CD209 gene exon 4, human CD209 gene exon 5, human CD209 gene exon 6 and / or human CD209 gene exon 7.

[0045] In some embodiments, the endogenous CD209 gene sequence in the genetically modified non-human animal is replaced by a human CD209 gene and / or nucleotide sequence, so that the non-human animal can express human CD209 and / or chimeric (e.g., humanized) CD209 protein. In some embodiments, the amino acid sequence encoded by the human CD209 gene and / or nucleotide sequence is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 2 positions 38-404 or SEQ ID NO: 2 positions 39-404 or SEQ ID NO: 7. In some embodiments, the nucleotide sequence of the endogenous CD209 gene of the non-human animal is replaced by a nucleotide sequence encoding all or part of the nucleotide sequence of the mature human CD209 protein.

[0046] In some embodiments, the genetically modified non-human animal expresses human CD209 and / or chimeric CD209 protein (e.g., humanized CD209) under the control of endogenous regulatory elements (e.g., promoters and / or 5'UTR). The replacement of the non-human animal endogenous CD209 locus provides a non-human animal expressing human or chimeric CD209 protein (e.g., humanized CD209) in the same type of cells. The genetically modified non-human animal does not have potential diseases observed in some other transgenic non-human animals known in the art. The chimeric CD209 protein expressed in the non-human animal can maintain the function of one or more wild-type or human CD209 proteins. In some embodiments, the genetically modified non-human animal does not express endogenous CD209 protein. In some embodiments, the endogenous CD209 protein expression of the genetically modified non-human animal is reduced. "Endogenous CD209 protein" described in the present application refers to the CD209 protein encoded by the endogenous CD209 nucleotide sequence of the non-human animal (e.g., mouse) before genetic modification.

[0047] In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence encoding 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 SEQ ID NO: 2, positions 38-404 or SEQ ID NO: 2, positions 39-404 or SEQ ID NO: 7. In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence set forth in SEQ ID NO: 5.

[0048] In some embodiments, the endogenous CD209 gene in the genetically modified non-human animal genome is replaced by the nucleotide sequence of human CD209. In some embodiments, the replaced endogenous CD209 gene includes any sequence of the endogenous CD209 gene of the non-human animal, such as exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6 or any combination thereof. In some embodiments, the endogenous CD209 gene is located in the non-human animal endogenous CD209 regulatory region. In some embodiments, the replaced non-human animal endogenous CD209 gene includes part of exon 3 to all of exon 7. In some embodiments, the portion of exon 3 of the endogenous CD209 gene of the non-human animal that is replaced includes at least 5-90bp, for example at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85 or 90bp of continuous nucleotide sequence.

[0049] In some embodiments, one or more cells of the genetically modified non-human animal express a human or chimeric CD209 protein (e.g., a humanized CD209 protein). In some embodiments, the human or chimeric CD209 protein comprises at least 50, 60, 70, 80, 90, 100, 200, 300, 360, 366, 367, 368, 369, 370, 380, 390, 400, or 404 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2 38-404 or SEQ ID NO: 2 39-404.

[0050] In some embodiments, the genome of the genetically modified non-human animal contains all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and / or exon 7 of the human CD209 gene, or all or part of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 38-404 or SEQ ID NO: 2 positions 39-404.

[0051] In some embodiments, the genetically modified non-human animal genome comprises a portion of human CD209 gene exon 3 to 3'UTR downstream nucleotide sequence. In some embodiments, the nucleotide sequence downstream of the 3'UTR of the human CD209 gene comprises at least 50-1000bp, for example, at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000bp continuous nucleotide sequence. In some embodiments, the nucleotide sequence downstream of the 3'UTR of the human CD209 gene comprises at least 200-400bp continuous nucleotide sequence. In some embodiments, the genome of the genetically modified non-human animal comprises the nucleotide sequence of position 138-4284 of the human CD209 gene transcript NM_021155.4.

[0052] In some embodiments, the chimeric (eg, humanized) CD209 gene, or the nucleotide sequence encoding the chimeric (eg, humanized) CD209 protein, is heterozygous or homozygous for the endogenous modified locus.

[0053] In some embodiments, the humanized CD209 gene comprises the 5'UTR of the human CD209 gene. In some embodiments, the humanized CD209 gene comprises the (e.g., mouse) 5'UTR of the endogenous CD209 gene of a non-human animal. In some embodiments, the humanized CD209 gene comprises the 3'UTR of the human CD209 gene. In some embodiments, the humanized CD209 gene comprises the (e.g., mouse) 3'UTR of the endogenous CD209 gene of a non-human animal. In appropriate cases, based on the similarity of the sequence, it can be reasonably inferred that the non-human animal and human CD209 genes are subject to similar regulation. As described in the present invention, the non-human animal humanized CD209 gene comprises the replacement of the endogenous gene locus of the non-human animal, which retains the endogenous regulatory elements of the non-human animal, but comprises the humanization of the CD209 coding sequence. The expression of CD209 in the heterozygous non-human animal or homozygous non-human animal humanized CD209 gene is completely normal.

[0054] In some embodiments, the present invention provides a genetically modified non-human animal, the genome of the genetically modified non-human animal comprising a deletion of an endogenous CD209 gene of the non-human animal, the deletion comprising one or more exons or portions of exons. In some embodiments, the deletion of the endogenous CD209 gene of the non-human animal comprises all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and / or exon 7. In some embodiments, the deleted exons include all of exon 7 from part of exon 3. In some embodiments, the deletion of the endogenous CD209 gene of the non-human animal further comprises one or more introns or portions of introns, the introns comprising intron 1, intron 2, intron 3, intron 4, intron 5 and / or intron 6. In some embodiments, the deleted introns comprise intron 3, intron 4, intron 5 and / or intron 6.

[0055] The present invention provides a humanized mouse CD209 genomic DNA sequence; or a construct expressing the amino acid sequence of the humanized CD209 protein; or a cell comprising the construct; or a tissue or organ comprising the cell.

[0056] In some embodiments, the present invention provides a chimeric (eg, humanized) CD209 nucleotide sequence and / or amino acid sequence. In some embodiments, the chimeric CD209 nucleotide sequence and / or amino acid sequence is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to an endogenous CD209 mRNA of a non-human animal (e.g., NM_133238.5), an endogenous CD209 amino acid sequence of a non-human animal (e.g., NP_573501.1, SEQ ID NO: 1), or a portion thereof (e.g., all of exon 1 to exon 3 of an endogenous CD209 gene of a non-human animal). In some embodiments, the chimeric CD209 nucleotide sequence and / or amino acid sequence is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to a human CD209 mRNA sequence (e.g., NM_021155.4), a human CD209 amino acid sequence (e.g., NP_066978.1, SEQ ID NO: 2), or a portion thereof (e.g., a portion of exon 3 of a human CD209 gene to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR).

[0057] In some embodiments, the nucleotide sequence encoding amino acids 48-238 or 47-238 of non-human CD209 (SEQ ID NO: 1) is replaced by a nucleotide sequence encoding amino acids 39-404 or 38-404 of human CD209 (SEQ ID NO: 2).

[0058] In some embodiments, the human CD209 nucleotide sequence or chimeric CD209 nucleotide sequence is operably linked to a regulatory element, such as a promoter of an endogenous CD209 gene of a non-human animal, a 5'UTR of an endogenous CD209 gene of a non-human animal, an inducible promoter, or an enhancer.

[0059] In some embodiments, at least a portion of the chimeric CD209 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 bp of nucleotides, e.g., a continuous or non-contiguous nucleotide sequence) is different from all or part of the endogenous CD209 nucleotide sequence of a non-human animal (e.g., part of exon 3 to all of exon 7 of the mouse CD209 gene transcript NM_133238.5).

[0060] In some embodiments, at least a portion of the chimeric CD209 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 bp of nucleotides, e.g., a continuous or non-contiguous nucleotide sequence) is identical to all or part of the non-human animal CD209 nucleotide sequence (e.g., all of exon 1 to part of exon 3 of the mouse CD209 gene transcript NM_133238.5).

[0061] In some embodiments, at least a portion of the chimeric CD209 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 bp of nucleotides, e.g., a continuous or non-contiguous nucleotide sequence) is different from all or part of the human CD209 nucleotide sequence (e.g., all of exon 1 to part of exon 3 of the human CD209 gene transcript NM_021155.4).

[0062] In some embodiments, at least a portion of the chimeric CD209 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 bp of nucleotides, e.g., a continuous or non-continuous nucleotide sequence) is identical to all or part of the human CD209 nucleotide sequence (e.g., a portion of exon 3 of the human CD209 gene transcript NM_021155.4 to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR).

[0063] In some embodiments, at least a portion of the amino acids encoded by the chimeric CD209 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, such as consecutive or non-consecutive amino acid residues) is different from all or part of the amino acid sequence of the non-human animal CD209 protein (e.g., amino acids 48-238 or 47-238 of the mouse CD209 protein sequence NP_573501.1 (SEQ ID NO: 1)).

[0064] In some embodiments, at least a portion of the amino acid sequence encoded by the chimeric CD209 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is identical to all or part of the amino acid sequence of the non-human animal CD209 protein (e.g., amino acids 1-47 or 1-46 of the mouse CD209 protein sequence NP_573501.1 (SEQ ID NO: 1)).

[0065] In some embodiments, at least a portion of the amino acid sequence encoded by the chimeric CD209 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is identical to all or part of the amino acid sequence of the human CD209 protein (e.g., amino acids 39-404 or 38-404 of the human CD209 protein sequence NP_066978.1 (SEQ ID NO: 2)).

[0066] The present invention also provides a humanized CD209 amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0067] A) the amino acid sequence shown in SEQ ID NO: 1, positions 1 to 47, SEQ ID NO: 1, positions 1 to 46, SEQ ID NO: 2, positions 39 to 404, or positions 38 to 404, or SEQ ID NO: 7;

[0068] B) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1, positions 1-47, or SEQ ID NO: 1, positions 1-46, or SEQ ID NO: 2, positions 39-404, or positions 38-404, or SEQ ID NO: 7;

[0069] C) differs from the amino acid sequence of SEQ ID NO: 1 at positions 1-47, or SEQ ID NO: 1 at positions 1-46, or SEQ ID NO: 2 at positions 39-404, or 38-404, or SEQ ID NO: 7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0070] D) an amino acid sequence comprising a substitution, deletion and / or insertion of one or more amino acid residues compared to positions 1 to 47 of SEQ ID NO: 1 or positions 1 to 46 of SEQ ID NO: 1 or positions 39 to 404 of SEQ ID NO: 2 or positions 38 to 404 of SEQ ID NO: 7.

[0071] The present invention also provides a humanized CD209 nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

[0072] A) a nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 and / or 33, or all or part of a nucleotide sequence encoding a homologous amino acid sequence of human CD209;

[0073] B) a nucleotide sequence that can hybridize to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 and / or 33 under low stringency conditions or stringent conditions;

[0074] C) a nucleotide sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 90% homology to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 8, 9, 10, 11, 32 and / or 33;

[0075] D) the amino acid sequence encoded by it is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in any one or more of SEQ ID NO:1, positions 1-47, SEQ ID NO:1, positions 38-404, SEQ ID NO:2, positions 39-404, or SEQ ID NO:7;

[0076] E) the encoded amino acid sequence differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid from the amino acid sequence set forth in any one or more of SEQ ID NO: 1 at positions 1-47, SEQ ID NO: 1 at positions 1-46, SEQ ID NO: 2 at positions 38-404, SEQ ID NO: 2 at positions 39-404 and / or SEQ ID NO: 7; or

[0077] F) The amino acid sequence encoded by the amino acid sequence is the same as that shown in any one or more of SEQ ID NO: 1-47, SEQ ID NO: 1-46, SEQ ID NO: 2 38-404, SEQ ID NO: 2 39-404 and / or SEQ ID NO: 7, including the amino acid sequence in which one or more amino acid residues are replaced, deleted and / or inserted.

[0078] The present invention provides a humanized CD209 genomic DNA sequence, which is obtained by reverse transcription of mRNA transcribed from the humanized CD209 genomic DNA sequence and is consistent with or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6.

[0079] In order to determine the percent identity of two amino acid sequences or two nucleotide sequences, the sequences are compared for the purpose of optimal comparison (e.g., for optimal comparison, gaps can be introduced in one or both of the first and second amino acid or nucleotide sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to achieve the optimal comparison of the two sequences, taking into account the number of gaps and the length of each gap. For example, the comparison of the sequences and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0080] 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.

[0081] The present invention also provides cells, tissues and non-human animals (e.g., mice) comprising nucleotide sequences of human CD209, as well as cells, tissues and non-human animals (e.g., mice) expressing human or chimeric (e.g., humanized) CD209 at the endogenous CD209 locus of the non-human animal.

[0082] Genetically modified non-human animals

[0083] The "genetically modified non-human animal" or "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome 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 or the genetically modified non-human animal have exogenous DNA. 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, wherein the genetically modified non-human animal comprises a modified endogenous CD209 locus, and the modified endogenous CD209 locus comprises an exogenous sequence (such as a human sequence). For example, one or more non-human sequences are replaced with one or more human sequences, or one or more human and / or non-human sequences are inserted. Non-human animals are generally able to pass genetic modifications to offspring through germline transmission.

[0084] 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 the nucleotide sequence of at least a part of the gene or nucleic acid is different from the nucleotide sequence in a wild-type non-human animal. In some embodiments, the nucleotide sequence of at least a part of the chimeric (x) gene or chimeric (x) nucleic acid has 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, the chimeric (x) gene or chimeric (x) nucleic acid refers to a humanized (x) gene or a humanized (x) nucleic acid.

[0085] The "chimeric (x) protein" or "chimeric (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more parts of the polypeptide or protein are from different species, or at least a portion of the amino acid sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, the amino acid sequence of at least a portion 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.

[0086] 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 (x) protein or human (x) polypeptide. In some embodiments, the humanized protein or humanized polypeptide refers to a human (x) protein or human (x) polypeptide.

[0087] The "humanized (x) nucleic acid" or "humanized (x) gene" 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, at least a portion of the nucleic acid is derived from a non-human animal. In some embodiments, the humanized (x) nucleic acid or humanized (x) gene refers to a humanized exon, and the humanized exon can be a human exon or a chimeric exon.

[0088] In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid is a humanized CD209 gene or a humanized CD209 nucleic acid. In some embodiments, at least a portion of the humanized CD209 gene or the humanized CD209 nucleic acid is derived from a human CD209 gene, and at least a portion of the humanized CD209 gene or the humanized CD209 nucleic acid is derived from an endogenous CD209 gene of a non-human animal. In some embodiments, the humanized CD209 gene or the humanized CD209 nucleic acid comprises a sequence encoding a CD209 protein. The encoded CD209 protein has at least one activity of a human CD209 protein or an endogenous CD209 protein of a non-human animal.

[0089] In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide is a humanized CD209 protein or a humanized CD209 polypeptide. In some embodiments, at least one or more parts of the amino acid sequence of the humanized CD209 protein or humanized CD209 polypeptide are from human CD209 protein. In some embodiments, at least one or more parts of the amino acid sequence of the humanized CD209 protein or humanized CD209 polypeptide are from endogenous CD209 protein of non-human animals. In some embodiments, the humanized CD209 protein or humanized CD209 polypeptide is functional, or has at least one activity of human CD209 protein and / or endogenous CD209 protein of non-human animals.

[0090] In some embodiments, the humanized CD209 protein includes an extracellular region, a cytoplasmic region and a transmembrane region. In some embodiments, the extracellular region includes the extracellular region of human CD209 protein. In some embodiments, the cytoplasmic region includes the cytoplasmic region of human CD209 protein. In some embodiments, the transmembrane region includes the transmembrane region of human CD209 protein. In some embodiments, the cytoplasmic region includes the cytoplasmic region of non-human animal endogenous CD209 protein.

[0091] In some embodiments, the genetically modified non-human animal includes modification of the endogenous CD209 gene site (locus) of the non-human animal. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature human CD209 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a mature human CD209 protein amino acid sequence). Although cells (e.g., ES cells, somatic cells) that may include the genetic modifications described herein are provided in the present invention, in many embodiments, the genetically modified non-human animal includes modification of the endogenous CD209 gene site in the non-human animal.

[0092] In some embodiments, the genetically modified non-human animal can express human CD209 and / or chimeric (e.g., humanized) CD209 proteins at the non-human animal endogenous CD209 locus, and the non-human animal endogenous CD209 gene has been replaced or inserted with a human CD209 gene and / or a nucleotide sequence encoding a human CD209 sequence or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to or identical to the human CD209 sequence. In some embodiments, the non-human animal endogenous CD209 locus is modified by all or part of a nucleotide sequence encoding a mature human CD209 protein.

[0093] In some embodiments, genetically modified non-human animals can express human CD209 and / or chimeric CD209 (e.g., humanized CD209) under the control of endogenous regulatory elements (e.g., promoters and / or 5'UTR) of non-human animals. Insertion or replacement at the endogenous CD209 locus of non-human animals provides non-human animals that express human CD209 or chimeric CD209 (e.g., humanized CD209) in suitable cells and do not cause potential pathological changes observed in some other transgenic non-human animals known in the art. In some embodiments, human CD209 or chimeric CD209 (e.g., humanized CD209) expressed in non-human animals can maintain one or more functions of wild-type non-human animals and / or human CD209 in non-human animals. In addition, in some embodiments, genetically modified non-human animals do not express endogenous CD209. In some embodiments, compared with the CD209 expression level in wild-type animals, the expression level of endogenous CD209 in genetically modified non-human animals is reduced. As used herein, the term "endogenous CD209" refers to the CD209 protein expressed by the endogenous CD209 nucleotide sequence of a non-human animal (eg, mouse) before any genetic modification.

[0094] In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to human CD209 (SEQ ID NO: 2 or SEQ ID NO: 2 positions 38-404 or SEQ ID NO: 2 positions 39-404). In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 5 or 6. In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to NM_021155.4 positions 138-4284.

[0095] In some embodiments, the genome of the genetically modified non-human animal may include replacing the non-human animal endogenous CD209 gene with a nucleotide sequence of human CD209 at the non-human animal endogenous CD209 locus. In some embodiments, the replaced non-human animal endogenous CD209 gene can be any sequence of the non-human animal endogenous CD209 gene, such as exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, or any combination thereof. In some embodiments, the replaced non-human animal endogenous CD209 gene is within the regulatory region of the non-human animal endogenous CD209 gene. In some embodiments, the replaced non-human animal endogenous CD209 gene includes part of exon 3 to all of exon 7 of the non-human animal endogenous CD209 gene.

[0096] In some embodiments, the genetically modified non-human animal may have one or more cells expressing human or chimeric CD209 protein (e.g., humanized CD209), wherein the humanized CD209 includes a cytoplasmic region, a transmembrane region, and an extracellular region from the N-terminus to the C-terminus. In some embodiments, the extracellular region comprises a sequence that is consistent or at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the extracellular region of human CD209 (e.g., SEQ ID NO: 2 amino acids 59-404). In some embodiments, the extracellular region has a sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 340, or 346 amino acids (continuous or non-continuous) that is consistent with the extracellular region of the human CD209 protein. In some embodiments, the extracellular region described in the present application does not include a signal peptide. In some embodiments, the transmembrane region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the transmembrane region of human CD209 (e.g., amino acids 39-58 or 38-58 of SEQ ID NO: 2). In some embodiments, the transmembrane region has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids (continuous or non-continuous) that are identical to the transmembrane region of human CD209 protein. In some embodiments, the cytoplasmic region comprises an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the cytoplasmic region of endogenous CD209 protein of non-human animals (e.g., amino acids 1-47 of SEQ ID NO: 1 or amino acids 1-46 of SEQ ID NO: 1). In some embodiments, the cytoplasmic region has at least 5, 10, 20, 30, 40, 47, 48, 50 or 51 amino acids (contiguous or non-contiguous) that are identical to the cytoplasmic region of an endogenous CD209 protein of a non-human animal (eg, a mouse CD209 protein).

[0097] In some embodiments, the entire extracellular region and the entire transmembrane region of the humanized CD209 described herein are derived from the human CD209 sequence.

[0098] Because in many cases, human CD209 and non-human animal CD209 (e.g., mouse CD209) sequences are different, antibodies that bind to human CD209 do not necessarily have the same affinity or effect on non-human animal CD209 as when binding to human CD209. Therefore, non-human animals with human or humanized sequences can be used to better evaluate the effects of anti-human CD209 antibodies in animal models.

[0099] In some embodiments, the genome of the genetically modified non-human animal includes: a portion of exon 3 of the human CD209 gene to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; or a nucleotide sequence encoding amino acids 39-404 of SEQ ID NO: 2 or 38-404 of SEQ ID NO: 2.

[0100] In some embodiments, the genome of the genetically modified non-human animal includes at least 50-1000bp of continuous nucleotide sequence downstream of the 3'UTR of the human CD209 gene, for example, at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000bp of continuous nucleotide sequence. In some embodiments, the genome of the genetically modified non-human animal includes at least 200-400bp of continuous nucleotide sequence downstream of the 3'UTR of the human CD209 gene.

[0101] In some embodiments, there is a nucleotide sequence encoding a chimeric human / non-human CD209 polypeptide at the endogenous CD209 locus of a genetically modified non-human animal, and the cell surface of the genetically modified non-human animal expresses functional CD209. In some embodiments, the human portion of the chimeric human / non-human CD209 polypeptide may include the amino acid sequence encoded by part of exon 3 to all of exon 7 of the human CD209 gene. In some embodiments, the human portion of the chimeric human / non-human CD209 polypeptide comprises at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 2, positions 39-404 or SEQ ID NO: 2, amino acids 38-404.

[0102] In some embodiments, the modified gene in the genome of the genetically modified non-human animal is homozygous or heterozygous for the endogenously replaced locus. In a specific embodiment, the modified CD209 gene in the genome of the genetically modified non-human animal is homozygous or heterozygous for the endogenously replaced locus.

[0103] In some embodiments, the humanized CD209 gene comprises the 5'UTR of the human CD209 gene. In some embodiments, the humanized CD209 gene comprises the 3'UTR of the human CD209 gene. In some embodiments, the humanized CD209 gene comprises the 5'UTR of the endogenous (eg mouse) CD209 gene of a non-human animal. In some embodiments, the humanized CD209 gene comprises the 3'UTR of the endogenous (eg mouse) CD209 gene of a non-human animal. In appropriate cases, it can be reasonably assumed that, based on the similarity of the non-human animal and human CD209 gene sequences, they may be subject to similar regulation. As shown in the present application, the humanized CD209 gene is included in the endogenous CD209 locus of a non-human animal, which retains the endogenous regulatory elements of non-human animals but comprises the humanization of the CD209 coding sequence, and does not show pathological phenomena. Both heterozygous or homozygous genetically modified mice of the CD209 gene humanization are normal.

[0104] In some embodiments, the present invention also provides a genetically modified non-human animal, whose genome comprises a disruption of the endogenous CD209 gene of the non-human animal, wherein the disruption of the endogenous CD209 gene of the non-human animal comprises a deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and / or exon 7, or a partial deletion thereof.

[0105] In some embodiments, the disruption of the endogenous CD209 gene of the non-human animal further comprises the deletion of one or more introns among intron 1, intron 2, intron 3, intron 4, intron 5, and intron 6.

[0106] In some embodiments, the disruption of the endogenous CD209 gene of the non-human animal comprises a deletion of at least 50 bp of nucleotides from part of exon 3 to all of exon 7 of the endogenous CD209 gene of the non-human animal.

[0107] 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.

[0108] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. Rodents can be selected from mice, rats and hamsters. In one embodiment, the rodent is selected from the family of Muridae. In one embodiment, the genetically modified non-human animal is from a family selected from the family of Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (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.

[0109] 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 animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid strain (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a BALB / c, BALB / cHeAn, BALB / cJ, BALB / cR1, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strain.

[0110] In some embodiments, the non-human animal is a rat. The rat can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a hybrid species of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0111] In some embodiments, the non-human animal may have one or more other genetic modifications and / or other modifications that are suitable for the specific purpose of preparing a humanized non-human animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human tumor) may have one or more modifications that damage, inactivate, or destroy all or part of the immune system of the non-human animal. Damage, inactivation, or destruction of the immune system of a non-human animal may include, for example, chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and / or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc null Mice (Ito, M. et al., NOD / SCID / γc null mouse:an excellent recipient mouse modelfor 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 some embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of the endogenous CD209 locus of a non-human animal, and also includes damage, inactivation or partial destruction of the immune system (or one or more cell types of the immune system) of a 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 non-human animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, all or part of the mouse endogenous mature CD209 coding sequence is replaced with all or part of the human mature CD209 coding sequence.

[0112] The present invention further relates to a non-human mammal produced by the above construction method. In some embodiments, its genome comprises all or part of the human CD209 gene.

[0113] In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0114] In some embodiments, the non-human mammal expresses a protein encoded by a humanized CD209 gene.

[0115] 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).

[0116] 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.

[0117] The present invention provides a non-human mammal produced by any of the methods or construction methods described herein. In some embodiments, a non-human mammal, a genetically modified non-human animal, the genome of which comprises human or humanized CD209 DNA is provided.

[0118] In some embodiments, the non-human mammal or genetically modified non-human animal comprises a genetic construct (e.g., Figure 2 and Figure 5 In some embodiments, a non-human mammal expressing a human or humanized CD209 protein is provided. In some embodiments, a cell, tissue or organ that specifically expresses a human or humanized CD209 protein is provided.

[0119] In some embodiments, the expression of human or humanized CD209 protein in genetically modified non-human animals is controllable. Such as by adding specific inducers or repressors. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0120] The non-human mammal can be any non-human animal known in the art that can be used in the methods. In some embodiments, the non-human mammal includes a rodent. In some embodiments, the non-human mammal is a mouse.

[0121] The non-human mammals are subjected to genetic, molecular and behavioral analysis. The present invention provides offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0122] 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 CD209 protein can be detected by a variety of methods.

[0123] 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 ELISA methods well known to those skilled in the art. Many standard analytical methods can be used to complete 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 CD209 protein.

[0124] Carrier

[0125] The present invention provides a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of the switch region to be changed (5' homology arm or 5' arm), which is selected from the genomic DNA of the CD209 gene and has a length of 100 to 10,000 nucleotides; b) a donor sequence; and c) a DNA fragment homologous to the 3' end of the switch region to be changed (3' homology arm or 3' arm), which is selected from the genomic DNA of the CD209 gene and has a length of 100 to 10,000 nucleotides.

[0126] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence with at least 90% identity to NCBI Accession No. NC_000074.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence with at least 90% identity to NCBI Accession No. NC_000074.7.

[0127] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 3798406 to 3802838 of NCBI Accession No. NC_000074.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 3789138 to 3793394 of NCBI Accession No. NC_000074.7.

[0128] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 3798406 to 3799944 of NCBI Accession No. NC_000074.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 3791988 to 3793394 of NCBI Accession No. NC_000074.7.

[0129] In some embodiments, the length of the genomic nucleotide sequence selected by 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.

[0130] In some embodiments, the switch region to be changed is located on exon 1 to exon 7 of the CD209 gene of a non-human animal. In some embodiments, the switch region to be changed is located on exon 3 to exon 7 of the CD209 gene of a non-human animal.

[0131] In some embodiments, the targeting vector further comprises one or more marker genes. For example, a resistance gene for positive clone screening or a coding gene for a negative screening marker. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo. In some embodiments, the coding gene for a negative screening marker is a coding gene for a diphtheria toxin A subunit (DTA).

[0132] In some embodiments, the 5' arm is a nucleotide having at least 90% identity with NCBI Accession No. NC_000074.7, and more preferably, the 5' arm sequence comprises the nucleotide sequence shown in SEQ ID NOs: 3 and 32. In some embodiments, the 3' arm is a nucleotide having at least 90% identity with NCBI Accession No. NC_000074.7, and more preferably, the 3' arm sequence comprises the nucleotide sequence shown in SEQ ID NOs: 4 and 33.

[0133] In some embodiments, the targeting vector comprises a donor sequence. In some embodiments, the donor sequence comprises a human sequence (e.g., positions 7739693 to 7746523 of NC_000019.10). For example, the human sequence in the targeting vector includes: part or all of the nucleotide sequence of the human CD209 gene, preferably all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and / or exon 7 of the human CD209 gene, preferably also including at least 50bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the human sequence in the targeting vector includes: part of exon 3 of the human CD209 gene to at least 50bp of continuous nucleotide sequence downstream of the 3'UTR (e.g., at least 300bp). In some embodiments, the human sequence encodes all or part of the human CD209 protein, such as SEQ ID NO: 2 or SEQ ID NO: 2 positions 39-404 or SEQ ID NO: 2 positions 38-404. In some embodiments, the protein encoded by the donor sequence is SEQ ID NO: 7.

[0134] In some embodiments, the targeting vector comprises, from the 5' end to the 3' end, a 5' arm, a portion of exon 3 of the human CD209 gene to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR, and a 3' arm.

[0135] 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 a non-human animal endogenous CD209 gene, and the sgRNA is unique on the target sequence (target site) on the conversion region to be changed, and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the target site of the sgRNA in the non-human animal endogenous CD209 gene is located at exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7 and / or 3'UTR downstream nucleotide sequence. In some embodiments, the target site is located on exon 3 of the non-human animal endogenous CD209 gene, and / or on exon 7.

[0136] In some embodiments, the target sequence is shown as SEQ ID NO: 22 and / or 23. Therefore, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some embodiments, the sgRNA sequences are listed in SEQ ID NO: 24 and 26. In some embodiments, the sgRNA sequences are listed in SEQ ID NO: 25 and 27. In some embodiments, the sgRNA sequences are listed in SEQ ID NO: 28 and 30. In some embodiments, the sgRNA sequences are listed in SEQ ID NO: 29 and 31.

[0137] In some embodiments, the present application also includes a plasmid construct of an sgRNA sequence (sgRNA vector, such as pT7-sgRNA) and / or a cell comprising the sgRNA vector.

[0138] The present invention also relates to a cell comprising a targeting vector as described above.

[0139] 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 constructs described herein (e.g., gene constructs or sgRNA vectors). In some embodiments, the cell comprises Cas9mRNA or its in vitro transcript.

[0140] In some embodiments, the cell is heterozygous for the gene. In some embodiments, the cell is homozygous for the gene.

[0141] 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. In some embodiments, the cell can be any cell expressing the CD209 protein.

[0142] Methods for constructing genetically modified non-human animals

[0143] Genetically modified non-human animals can be prepared by several gene editing techniques known in the art, including homologous recombination technology using embryonic stem cells, gene targeting technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR / Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in Yin et al. "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0144] In some embodiments, the nucleotide sequence encoding the endogenous CD209 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 CD209. In some embodiments, the expression of the endogenous CD209 protein of the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0145] Figure 2 and Figure 5 The humanization targeting strategy of the mouse CD209 locus is shown. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized CD209 gene fragment and a 3' homology arm. The process involves replacing the endogenous CD209 sequence of a non-human animal with a human or humanized CD209 gene fragment using homologous recombination. In some embodiments, cutting upstream and downstream of the target site (e.g., by zinc finger nuclease, TALEN or CRISPR) can result in a double-stranded DNA break, and homologous recombination is used to replace the endogenous CD209 sequence of a non-human animal with a human or humanized CD209 sequence.

[0146] In some embodiments, the non-human animal is constructed by introducing any of the following nucleotide sequences into the CD209 locus of the non-human animal:

[0147] A) a portion of the human CD209 gene, preferably comprising all or part of exons 1-7 of the human CD209 gene, and further preferably comprising part of exon 3 to all of exon 7 of the human CD209 gene, wherein the portion of exon 3 of the human CD209 gene includes at least 5-72 bp, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 71 or 72 consecutive nucleotide sequences; preferably, the portion of the human CD209 gene further comprises at least 50-1000 bp downstream of the 3'UTR of the human CD209 gene, such as at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 bp of consecutive nucleotide sequences. In some embodiments, the portion of the human CD209 gene further comprises at least 200-400 bp of continuous nucleotide sequence downstream of the 3'UTR of the human CD209 gene. In some embodiments, the portion of the human CD209 gene comprises a portion of exon 3 of the human CD209 gene to a nucleotide sequence downstream of the 3'UTR. In some embodiments, the portion of the human CD209 gene comprises the nucleotide sequence shown in SEQ ID NO: 5; or, comprises at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the nucleotide sequence shown in SEQ ID NO: 5; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 5; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO: 5, including substitution, deletion and / or insertion of one or more nucleotides;

[0148] B) all or part of the nucleotide sequence encoding human CD209 protein, preferably comprising all or part of the nucleotide sequence encoding the extracellular region, cytoplasmic region and / or transmembrane region of human CD209 protein, further preferably comprising all or part of the nucleotide sequence encoding the extracellular region of human CD209 protein, and further preferably comprising the nucleotide sequence encoding the amino acids as shown at positions 59-404 of SEQ ID NO:2; or, comprising a nucleotide sequence encoding amino acids having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the amino acid sequence as shown at positions 59-404 of SEQ ID NO:2; or, comprising a nucleotide sequence encoding amino acids having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference with the amino acid sequence as shown at positions 59-404 of SEQ ID NO:2; or, comprising a nucleotide sequence encoding a nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference with the amino acid sequence as shown at positions 59-404 of SEQ ID NO:2 NO:2, positions 59-404, comprising substitution, deletion and / or insertion of one or more amino acid residues; preferably further comprising the whole or part of the nucleotide sequence encoding the transmembrane region of human CD209 protein, and further preferably comprising the nucleotide sequence encoding the amino acids as shown in SEQ ID NO:2, positions 39-58 or positions 38-58; or, comprising a nucleotide sequence encoding an amino acid having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with the amino acid sequence as shown in SEQ ID NO:2, positions 39-58 or positions 38-58; or, comprising a nucleotide sequence encoding an amino acid having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference with the amino acid sequence as shown in SEQ ID NO:2, positions 39-58 or positions 38-58; or, comprising a nucleotide sequence encoding an amino acid having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference with the amino acid sequence as shown in SEQ ID NO:2, positions 39-58 or positions 38-58; NO:2 The nucleotide sequence shown at positions 39-58 or 38-58, including substitution, deletion and / or insertion of one or more amino acid residues. ;

[0149] C) the whole or part of the nucleotide sequence encoding human CD209 protein comprises a nucleotide sequence encoding the amino acids set forth in SEQ ID NO:7; or, comprises a nucleotide sequence encoding an amino acid having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to the amino acid sequence set forth in SEQ ID NO:7; or, comprises a nucleotide sequence encoding an amino acid having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference from the amino acid sequence set forth in SEQ ID NO:7; or, comprises a nucleotide sequence encoding a nucleotide sequence encoding a nucleotide sequence having at least 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference from the amino acid sequence set forth in SEQ ID NO:7;

[0150] D) a nucleotide sequence encoding a human or humanized CD209 protein; or,

[0151] E) Nucleotide sequence of human or humanized CD209 gene.

[0152] Preferably, the non-human animal is constructed using the above-mentioned targeting vector and / or sgRNA vector.

[0153] Preferably, the non-human animal further comprises other gene modifications, more preferably, the other genes are selected from at least one of CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

[0154] Preferably, the human or humanized CD209 gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) locus.

[0155] Preferably, the human or humanized CD209 gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

[0156] 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.

[0157] 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.

[0158] Therefore, the present invention provides a method for constructing a non-human animal with a humanized CD209 gene, wherein the non-human animal expresses human or humanized CD209 protein in vivo, and / or the genome of the non-human animal contains a portion of the human CD209 gene or the humanized CD209 gene.

[0159] In some embodiments, the method for preparing a genetically modified humanized non-human animal includes replacing the endogenous CD209 gene of the non-human animal with a nucleotide sequence encoding human CD209 at the endogenous CD209 locus (or site) of the non-human animal. The replaced sequence may include regions (e.g., part or all of regions) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and / or exon 7 of the human CD209 gene. In some embodiments, the replaced sequence includes a portion of exon 3 of the human CD209 gene to a nucleotide sequence downstream of the 3'UTR (e.g., a nucleotide sequence of positions 138-4284 of NM_021155.4).

[0160] In some embodiments, the method of modifying the CD209 locus of a mouse to express a chimeric human / mouse CD209 polypeptide may include replacing a nucleotide sequence encoding mouse CD209 at the mouse endogenous CD209 locus with a nucleotide sequence encoding human CD209, thereby generating a sequence encoding chimeric human / mouse CD209. In some embodiments, the method may include inserting a nucleotide sequence encoding chimeric human / mouse CD209 at the mouse endogenous CD209 locus, thereby generating a sequence encoding chimeric human / mouse CD209.

[0161] The present invention also provides a method for establishing a CD209 gene humanized animal model, comprising the following steps:

[0162] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0163] (b) culturing the cells (preferably in a liquid culture medium);

[0164] (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;

[0165] (d) identifying germline transmission in the offspring of the non-human mammal with humanized CD209 gene of the pregnant female in step (c).

[0166] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0167] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0168] 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.

[0169] 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.

[0170] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying the coding frame of the CD209 gene of the non-human animal, for example, by replacing the nucleotide sequence (e.g., locus DNA sequence, CDS sequence or cDNA sequence) encoding the endogenous CD209 region with the nucleotide sequence encoding the corresponding region of human CD209 under the control of the endogenous regulatory element of the CD209 gene of the non-human animal. For example, one or more functional region sequences of the CD209 gene of the non-human animal can be knocked out or inserted into a sequence so that the endogenous CD209 protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the CD209 gene of the genetically modified non-human animal can be all or part of the nucleotide sequence of exon 1 to exon 7 of the endogenous CD209 gene of the non-human animal.

[0171] In some embodiments, the method for preparing a genetically modified non-human animal includes inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or humanized CD209 protein after the endogenous regulatory element of the CD209 gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon so that the CD209 gene humanized animal model can express a human or humanized CD209 protein in vivo, but does not express the CD209 protein of the non-human animal. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0172] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0173] (1) providing a plasmid comprising a human CD209 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 CD209 of a non-human animal;

[0174] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous CD209 gene of a non-human animal;

[0175] (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;

[0176] (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 CD209 protein.

[0177] In some embodiments, the method further comprises (5) mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0178] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal target site and a 3'-terminal target site.

[0179] In some embodiments, the nucleotide sequence encoding the human or humanized CD209 protein is operably linked to endogenous regulatory elements at the endogenous CD209 locus of the non-human animal.

[0180] In some embodiments, the genetically modified non-human animal does not express endogenous CD209 protein.

[0181] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0182] (1) providing a plasmid comprising a human or chimeric CD209 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 CD209 of a non-human animal;

[0183] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous CD209 gene of a non-human animal;

[0184] (3) Modifying the genome of fertilized eggs or embryonic stem cells by inserting the human or chimeric CD209 gene fragment into the endogenous CD209 gene of a non-human animal.

[0185] In some embodiments, the method comprises replacing part of exon 3 to all of exon 7 of the endogenous CD209 gene of a non-human animal with part of exon 3 to the nucleotide sequence downstream of 3'UTR of the human CD209 gene.

[0186] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 1 positions 48-238 or SEQ ID NO: 1 positions 47-238 in the genome of a non-human animal with a nucleotide sequence encoding a human or humanized CD209 protein or a human or humanized CD209 gene.

[0187] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 1 48-238 or SEQ ID NO: 1 47-238 in the genome of a non-human animal with the genomic DNA sequence, cDNA sequence or CDS sequence of human CD209.

[0188] In some embodiments, the construction method includes replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 1 48-238 or SEQ ID NO: 1 47-238 in the genome of a non-human animal with a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 2 39-404 or SEQ ID NO: 2 38-404.

[0189] In some embodiments, the construction method includes replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 1 positions 48-238 or SEQ ID NO: 1 positions 47-238 in the genome of a non-human animal with SEQ ID NO: 5.

[0190] Use of genetically modified non-human animals

[0191] 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 the non-human animal endogenous CD209 locus and under the control of endogenous regulatory elements (e.g., promoter and / or 5'UTR) can produce a non-human animal with qualities and characteristics that may be significantly different from typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or destroyed, and the full human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; the expression of human proteins is measured by transcription of human gene and / or protein assays and / or functional assays. In human transgenes, the upstream and / or downstream of the human sequence provide suitable support for the expression and / or regulation of the transgene.

[0192] 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 non-human animal. The present invention demonstrates that the replacement or insertion of human sequences at endogenous loci under the control of endogenous regulatory elements produces humanized non-human animals, providing 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.

[0193] Genetically modified non-human animals that express human or humanized CD209 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.

[0194] The present invention also provides the use of the CD209 gene-modified non-human animal and the non-human animal obtained by any of the above construction methods.

[0195] In some embodiments, the application comprises:

[0196] A) Application in product development involving CD209-related immune processes in human cells;

[0197] B) Application as a model system related to CD209 for pharmacology, immunology, microbiology and medical research;

[0198] C) Applications involving the production and use of animal experimental disease models for the study of CD209-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0199] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human CD209 signaling pathway modulators; or,

[0200] E) Study the function of CD209 gene, study the drugs and efficacy targeting human CD209 target site, and study the application of drugs in treating diseases related to CD209.

[0201] The present invention provides a non-human animal expressing human or humanized CD209 protein, which can be used for screening of human CD209 specific therapeutic agents. Whether the test therapeutic agent can increase or decrease the immune response, and / or determine whether the therapeutic agent is a CD209 agonist or antagonist. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In some embodiments, the genetically modified non-human animal also includes an impaired immune system, such as genetically modified human-derived tissue xenografts, including human solid tumors (e.g., breast cancer, head and neck cancer, colon cancer, liver cancer or lung cancer) or blood cell tumors (e.g., lymphocyte tumors, B or T cell tumors).

[0202] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-CD209 antibodies, oligonucleotide drugs, or polypeptide drugs; or drugs targeting the CD209 signaling pathway) in treating tumors. In some embodiments, a therapeutic agent is administered to a non-human animal or a non-human animal obtained by the above-mentioned construction method, wherein the non-human animal suffers from a tumor; and the inhibitory effect of the therapeutic agent on the tumor is determined. 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 without 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, anti-CD209 antibodies can directly target cells expressing CD209.

[0203] In some embodiments, the tumor comprises one or more tumor cells (e.g., human or mouse tumor cells) injected into an animal. In some embodiments, the antibody activates the CD209 signaling pathway. In some embodiments, the antibody does not activate the CD209 signaling pathway.

[0204] In some embodiments, genetically modified non-human animals can be used to determine whether an antibody is a CD209 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, oligonucleotide drug, or polypeptide drug targeting CD209; or a drug targeting a CD209 signaling pathway) on CD209, for example, whether the therapeutic agent can upregulate 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, transgenic non-human animals can be used to determine the effective dose of a therapeutic agent to treat a disease in a subject, such as a tumor.

[0205] The inhibitory effect on tumors can also be determined by methods known in the art, for example, by measuring tumor volume in non-human 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 The mean tumor volume (or weight) of the treatment group and the control group, respectively.

[0206] In some embodiments, therapeutic agents (e.g., antibodies, oligonucleotide drugs, or polypeptide drugs targeting CD209; or drugs targeting CD209 signaling pathways) are designed to treat various cancers (tumors). As used in this application, the term "cancer" refers to cells with autonomous growth ability, i.e., an abnormal state or state characterized by rapid proliferation of cell growth. The term is intended to include all types of cancer 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, testicular tumors, and non-small cell lung cancers. 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.

[0207] In certain embodiments, the cancer (tumor) described herein is lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, cancer, bladder cancer, glioma, cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome and sarcoma. In certain embodiments, the leukemia is selected from acute lymphocytic (lymphocyte) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma and T-cell lymphoma, and Waldenstrom's macroglobulinemia. In some embodiments, the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma. In some embodiments, the tumor comprises breast cancer, head and neck cancer, colon cancer, liver cancer or lung cancer.

[0208] In some embodiments, therapeutic agents (e.g., antibodies targeting CD209; or drugs targeting CD209 signaling pathways) are designed to treat various immune diseases. Therefore, the methods described herein can be used to determine the effectiveness of therapeutic agents (e.g., antibodies, oligonucleotide drugs, or polypeptide drugs targeting CD209; or drugs targeting CD209 signaling pathways) in suppressing immune responses. In some embodiments, the immune diseases described herein include one or more of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, cerebellar atrophy, multiple sclerosis, and Parkinson's disease.

[0209] The present invention also provides a method for determining the effectiveness of a therapeutic agent (e.g., an anti-CD209 antibody, an oligonucleotide drug, or a polypeptide drug) in treating a viral infection, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the above construction method, wherein the non-human animal suffers from a viral infection; 2) determining the therapeutic effect of the therapeutic agent on the viral infection. In some embodiments, the viral infection includes one or more of AIDS (HIV), Ebola virus, or acute respiratory syndrome (SARS).

[0210] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (e.g., an anti-CD209 antibody, an oligonucleotide drug, or a polypeptide drug). The method comprises administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, and measuring the weight change or blood test of the non-human animal; preferably, the blood test comprises one or more of red blood cell count, hematocrit, or hemoglobin content. In some embodiments, the therapeutic agent can reduce the red blood cell (RBC), hematocrit, or hemoglobin content by 20%, 30%, 40%, or 50% or more. In some embodiments, the body weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of the control group (e.g., the average body weight of non-human animals not treated with the therapeutic agent).

[0211] In some embodiments, the present invention also provides an animal model obtained by any construction method for developing products related to human cellular immune processes, producing human antibodies, or a model system for pharmacology, immunology, microbiology and medical research.

[0212] In some embodiments, the present invention also provides an animal model obtained by any of the above-mentioned construction methods for producing and utilizing animal experimental disease models of human cell immune processes, studying pathogens, or formulating new diagnostic strategies and / or treatment strategies.

[0213] In some embodiments, the present invention also provides an animal model obtained by any of the above-mentioned construction methods for screening, verifying, evaluating or studying CD209 gene function, human CD209 antibodies, therapeutic drugs or effectiveness of human CD209-related diseases, such as tumors, immune diseases or viral infections.

[0214] 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 in the present application, and applying human CAR-T to non-human animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animal is selected from a CD209 gene humanized non-human animal prepared by the method described in the present application, a CD209 gene humanized non-human animal described in the present application, a double gene or multi-gene humanized non-human animal (or its offspring) produced by the method described in the present application, a non-human animal expressing a human or humanized CD209 protein, or a tumor-bearing or inflammatory animal model described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat CD209-related diseases (e.g., tumors, immune diseases or viral infections). In some embodiments, TCA-T, CAR-T and / or other immunotherapies provide an evaluation method for treating CD209-related diseases of the present application.

[0215] Non-human animal models with two or more human or chimeric genes

[0216] The present invention also provides a transgenic animal model or non-human animal having two or more human or chimeric genes. The animal model or non-human animal may comprise a human or chimeric (eg, humanized) CD209 gene and a sequence encoding an additional human or chimeric protein.

[0217] In some embodiments, the additional human or chimeric protein comprises at least one of CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the animal model or non-human animal also expresses at least one of human or humanized CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 proteins.

[0218] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0219] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0220] (ii) mating, in vitro fertilization or direct gene editing of the non-human animals provided in step (i) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0221] In some embodiments, the other genetically modified non-human animals include non-human animals modified with one or a combination of two or more of the genes CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

[0222] In some embodiments, humanization of the CD209 gene is performed directly on a non-human animal with a modified human or chimeric CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 gene.

[0223] Since these proteins may involve different mechanisms, a combined therapy targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are underway and show good results. Multigene modified non-human animal models can be used to determine the effectiveness of combined therapies targeting two or more proteins, for example, anti-CD209 antibodies, and additional therapeutic agents for treating tumors or immune diseases (e.g., Alzheimer's disease). Including administering anti-CD209 antibodies and additional therapeutic agents to non-human animals, wherein the non-human animals have tumors or immune diseases, and determining the effects of combined therapy on immune tumors or immune diseases. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the non-human animal described above further comprises a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody (e.g., ipilimumab). In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0224] In some embodiments, the combination therapy is used to treat tumors, such as breast cancer, head and neck cancer, colon cancer, liver cancer, or lung cancer. In some embodiments, the combination therapy is used to treat viral infections, such as one or more of AIDS (HIV), Ebola virus, or acute respiratory syndrome (SARS). In some embodiments, the combination therapy is used to treat immune diseases, such as one or more of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease.

[0225] 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 performing surgery on the subject to remove at least a portion of the tumor, for example, removing a portion or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0226] Figure 1 : Schematic diagram comparing mouse CD209 locus and human CD209 locus (not to scale);

[0227] Figure 2 : Schematic diagram of CD209 gene targeting strategy and targeting vector V1 design (not to scale);

[0228] Figure 3 : Southern blot identification results of CD209 gene humanized mouse F1 generation, where WT is the wild-type control;

[0229] Figure 4 : RT-PCR test results, where + / + is wild-type C57BL / 6 mice, H / H is CD209 gene humanized homozygous mice, and H2O is water control;

[0230] Figure 5 : Schematic diagram of CD209 gene targeting strategy and targeting vector V2 design (not to scale). DETAILED DESCRIPTION

[0231] 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.

[0232] Materials and methods

[0233] In each of the following examples, equipment and materials were obtained from the following companies:

[0234] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Administration;

[0235] StuI and BclI enzymes were purchased from NEB with catalog numbers R0187S and R0160S;

[0236] Brilliant Violet 510 TM Anti-mouse CD45Antibody was purchased from Biolegend, catalog number 103138;

[0237] Alexa 700anti-mouse CD11c Antibody was purchased from Biolegend, catalog number 117320;

[0238] Brilliant Violet 650 TM Anti-mouse Ly-6G Antibody was purchased from Biolegend, catalog number 127641;

[0239] PE / Cyanine7 anti-mouse F4 / 80Antibody was purchased from Biolegend, catalog number 123114;

[0240] Brilliant Violet 785 TM anti-mouse / human CD11b Antibody was purchased from Biolegend, catalog number 101243;

[0241] APC anti-human CD209 (DC-SIGN) Antibody was purchased from Biolegend, catalog number 330107;

[0242] Zombie NIR TMFixable Viability Kit was purchased from Biolegend, catalog number 423106;

[0243] Purified anti-mouse CD16 / 32 was purchased from Biolegend, catalog number 101302.

[0244] Example 1 CD209 gene humanized mice

[0245] Mouse CD209 gene (NCBI Gene ID: 170786, UniProt ID: Q91ZX1-1, located at positions 3793395 to 3798984 of chromosome 8 NC_000074.7, based on transcript NM_133238.5 and its encoded protein NP_573501.1 (SEQ ID NO: 1)) and human CD209 gene (NCBI Gene ID: 30835, UniProt ID: Q9NNX6-1, located at positions 7739993 to 7747534 of chromosome 19 NC_000019.10, based on transcript NM_021155.4 and its encoded protein NP_066978.1 (SEQ ID NO: 2)), the comparison diagram is shown in the following figure. Figure 1 shown.

[0246] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human CD209 protein can be introduced into the mouse endogenous CD209 locus, so that the mouse expresses a human or humanized CD209 protein. Specifically, using gene editing technology, under the control of the mouse CD209 gene regulatory element, the partial sequence of exon 3 of the human CD209 gene to the nucleotide sequence downstream of the 3'UTR of about 6.8kb is used to replace the entire sequence of about 5.0kb of mouse exon 3 to exon 7, to obtain a humanized CD209 locus, thereby achieving humanization of the mouse CD209 gene.

[0247] Design as Figure 2 The targeting strategy shown, Figure 2The targeting vector V1 contains homology arm sequences upstream and downstream of the mouse CD209 gene, as well as an A fragment containing a human CD209 gene fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 3798406 to 3802838 of NCBI accession number NC_000074.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 3789138 to 3793394 of NCBI accession number NC_000074.7. The nucleotide sequence of the human CD209 gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 7739693 to 7746523 of NCBI accession number NC_000019.10; the connection design of the upstream of the human CD209 fragment sequence and the mouse is: The sequence " GCCTG The last "G" in the sequence is the last nucleotide in the mouse. The first "G" in is the first nucleotide of the human sequence.

[0249] 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 CD209 gene is designed as follows: The sequence The "T" in the sequence is the last nucleotide of the human CD209 gene. AATATT The first "A" in " is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the mouse CD209 gene is designed as: The sequence " GGATCC The last "C" in the sequence is the last nucleotide of the Neo box. The first "T" in is the first nucleotide of the mouse CD209 gene. The mRNA sequence transcribed from the modified humanized CD209 gene is shown in SEQ ID NO: 6, and the expressed humanized CD209 protein sequence is shown in SEQ ID NO: 7.

[0252] 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 CD209 gene humanized homozygous mice.

[0253] In addition, CRISPR / Cas9 technology can be used for gene editing to further design Figure 5 The targeting strategy shown, Figure 5 The homology arm sequences upstream and downstream of the mouse CD209 gene and the human CD209 fragment are shown in the targeting vector V2. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 32) is identical to the nucleotide sequence of positions 3798406 to 3799944 of NCBI accession number NC_000074.7, and the downstream 3' homology arm sequence (SEQ ID NO: 33) is identical to the nucleotide sequence of positions 3791988 to 3793394 of NCBI accession number NC_000074.7. The nucleotide sequence of the human CD209 fragment (SEQID NO: 5) is identical to the nucleotide sequence of positions 7739693 to 7746523 of NCBI accession number NC_000019.10. The connection design of the upstream of the human CD209 fragment sequence and the mouse is:

[0254] The sequence " GCCTG The last "G" in the sequence is the last nucleotide in the mouse. The first "G" in is the first nucleotide of the human sequence. The downstream connection of the human CD209 fragment sequence and the mouse sequence is designed as: The sequence " AGAAT "T" is the last nucleotide in the human sequence. The first "T" in is the first nucleotide of the mouse sequence. The mRNA sequence transcribed from the modified humanized CD209 gene is shown in SEQ ID NO: 6, and the expressed humanized CD209 protein sequence is shown in SEQ ID NO: 7.

[0256] 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.

[0257] 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 CD209 gene is as follows:

[0258] sgRNA1 target site (SEQ ID NO: 22): 5′-AGACTATCTAAGAGAGACGGGGG-3′;

[0259] sgRNA2 target site (SEQ ID NO: 23): 5'-ATGACTAATAGTTAAGGAATTGG-3';

[0260] The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 3. After annealing, the annealing products were connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-CD209-1 and pT7-CD209-2.

[0261] Table 3 sgRNA1 and sgRNA2 sequence list

[0262]

[0263] The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment DNA (SEQ ID NO: 34) of the T7 promoter and sgRNA scaffold and connected to the backbone vector (source: Takara, item number 3299) by restriction digestion (EcoRI and BamHI) in sequence. The sequencing verification by a professional sequencing company showed that the target plasmid was obtained. The pronuclear fertilized eggs of mice, such as C57BL / 6 mice, were taken, and the in vitro transcription products of pT7-CD209-1 and pT7-CD209-2 plasmids (using the Ambion in vitro transcription kit, transcribed according to the instructions), the targeting vector and Cas9 mRNA 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 CD209 gene humanized mouse strain.

[0264] The somatic cell genotype of the F1 generation mice was identified by PCR, and the mice identified as positive by PCR were then subjected to Southern blot detection (cell DNA was digested with StuI and BclI respectively and hybridized with two probes, and the probes and target fragment lengths are shown in Table 4 below) to confirm whether there was random insertion. Figure 3 As shown, combined with the PCR and sequencing results, none of the 11 mice numbered F1-1 to F1-11 had random insertions. This indicates that the present method can be used to construct CD209 gene humanized mice that can be stably propagated and have no random insertions.

[0265] Table 4 Length of specific probes and target fragments

[0266] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size StuI 5'Probe 6.1kb 7.6kb BclI 3'Probe 6.6kb 12.1kb

[0267] 5'Probe-F: 5'-GGTGTCTCTGACAAGATATGGTGTTG-3' (SEQ ID NO: 12);

[0268] 5'Probe-R: 5'-TGCCACCTCCTTGAATCTTACTTAG-3' (SEQ ID NO: 13);

[0269] 3'Probe-F: 5'-CTCCCTGTTTTCCACTCTGCCTTG-3' (SEQ ID NO: 14);

[0270] 3'Probe-R: 5'-GTGATGGGCTCAAATGGTCACAGA-3' (SEQ ID NO: 15);

[0271] The expression of mRNA in CD209 gene humanized mice can be detected by RT-PCR. Specifically, 8-week-old C57BL / 6 mice (+ / +) and 7-week-old female CD209 gene humanized homozygous mice (H / H) prepared in this example were selected, and the spleens were taken after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 5 below. The detection results are shown in FIG. Figure 4 As shown in the figure, it can be seen that only mouse CD209 mRNA was detected in wild-type C57BL / 6 mice, and human CD209 mRNA was not detected; only human CD209 mRNA was detected in CD209 gene homozygous mice.

[0272] Table 5 RT-PCR primer sequences and target fragment sizes

[0273]

[0274]

[0275] Flow cytometry was used to confirm the expression of humanized CD209 protein in mice. Specifically, peripheral blood and spleen tissues of 8-week-old male wild-type C57BL / 6 mice and CD209 homozygous mice were selected and analyzed with the leukocyte marker antibody Brilliant Violet510. TM anti-mouse CD45Antibody(mCD45), anti-mouse CD11c antibody 700anti-mouse CD11c Antibody (mCD11c), anti-mouse Ly-6G antibody Brilliant Violet650 TM anti-mouse Ly-6G Antibody(mLy-6G), anti-mouse F4 / 80 antibody PE / Cyanine7 anti-mouse F4 / 80Antibody(mF4 / 80), anti-mouse CD11b antibody Brilliant Violet 785 TMAfter staining with anti-mouse / human CD11bAntibody (mCD11b), anti-human CD209 specific antibody APC anti-human CD209 (DC-SIGN)Antibody (hCD209), etc., flow cytometry was performed to detect the expression of humanized CD209 protein in dendritic cells and macrophages in peripheral blood and spleen. The test results are shown in Table 6.

[0276] Table 6 Flow cytometry detection results

[0277]

[0278] The results showed that the expression of humanized CD209 protein could be detected in the dendritic cells and macrophages in the peripheral blood and spleen of CD209 gene homozygous humanized mice, but the expression of humanized CD209 protein was not detected in the dendritic cells and macrophages in the peripheral blood and spleen of C57BL / 6 wild-type mice, indicating that CD209 gene homozygous humanized mice can normally express humanized CD209 protein. In summary, CD209 gene humanized mice were successfully constructed.

[0279] Example 2 Model and efficacy verification

[0280] The CD209 humanized mice prepared by the method described in Example 1 can be used to evaluate the efficacy of modulators targeting human CD209. For example, CD209 humanized homozygous mice are subcutaneously inoculated with colon cancer cells MC38, and the tumor volume grows 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 was injected with an antibody drug targeting human CD209, 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 and tumor volume of the mice, the safety and in vivo efficacy of the antibody drug in humanized CD209 mice can be effectively evaluated.

[0281] Example 3 Preparation of double-gene or multi-gene humanized mice

[0282] The CD209 gene humanized mouse prepared by the present application 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 genes such as CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4, or, on the basis of humanized CD209 mice, separate mouse ES embryonic stem cells and gene recombination targeting technology can be used to obtain a double gene humanized or multi-gene humanized mouse model. The homozygous or heterozygous humanized mouse of the CD209 gene obtained in the present application can also be mated with other gene-modified mice, and the offspring can be screened. According to the Mendel's law of inheritance, there is a certain probability of obtaining a multi-gene mouse modified with the humanized CD209 gene and other genes, and then the heterozygotes can be mated with each other to obtain a homozygous modified with double genes or multiple genes.

[0283] 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.

[0284] 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.

[0285] 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 CD209 protein.

2. The construction method according to claim 1, characterized in that: The chimeric CD209 protein comprises a human or humanized extracellular region, a human or humanized transmembrane region and a non-human animal endogenous cytoplasmic region.

3. The construction method according to claim 1 or 2, characterized in that: The amino acid sequence of the human or chimeric CD209 protein comprises SEQ ID NO: 2 or positions 38-404 of SEQ ID NO: 2 or positions 39-404 of SEQ ID NO: 2, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO: 2 or positions 38-404 of SEQ ID NO: 2 or positions 39-404 of SEQ ID NO:

2.

4. The construction method according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the chimeric CD209 protein comprises SEQ ID NO:7, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to SEQ ID NO:

7.

5. A method for constructing a genetically modified non-human animal, characterized in that: At the non-human animal endogenous CD209 gene locus, the non-human animal endogenous CD209 gene is replaced with a nucleotide sequence comprising human CD209.

6. The construction method according to claim 5, characterized in that: The nucleotide sequence of human CD209 includes a portion of exon 3 to the entirety of exon 7 of the human CD209 gene; preferably, the portion of exon 3 of the human CD209 gene includes at least 5 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of human CD209 comprises a portion of exon 3 of the human CD209 gene to at least 50 bp of continuous nucleotides downstream of the 3'UTR; Preferably, the nucleotide sequence of human CD209 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.

7. The construction method according to any one of claims 5-6, characterized in that: Part of exon 3 to all of exon 7 of the endogenous CD209 gene of the non-human animal are replaced. Preferably, the part of exon 3 of the endogenous CD209 gene of the non-human animal includes at least 5 bp of continuous nucleotide sequence.

8. The construction method according to any one of claims 1 to 7, characterized in that: The nucleotide sequence encoding the human or chimeric CD209 protein or the nucleotide sequence of human CD209 is operably linked to an endogenous regulatory element of an endogenous CD209 locus of a non-human animal, such as a promoter and / or 5'UTR; Preferably, the endogenous CD209 protein of the non-human animal is not expressed or is expressed at a reduced level compared to CD209 in wild-type animals; Preferably, the modified CD209 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

9. The construction method according to any one of claims 1 to 8, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent includes a mouse or a rat.

10. The construction method according to any one of claims 1 to 9, characterized in that: The mRNA transcribed from the modified CD209 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.

11. The construction method according to any one of claims 1 to 10, characterized in that: The non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins preferably include at least one of CFD, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

12. An application of a non-human animal obtained by the construction method according to any one of claims 1 to 11, characterized in that: The applications include: A) Application in product development involving CD209-related immune processes in human cells; B) Application as a model system related to CD209 for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of CD209-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) Application in the screening, efficacy testing, efficacy assessment, validation or evaluation of human CD209 signaling pathway modulators in vivo; or, E) Study the function of CD209 gene, study the drugs and efficacy targeting human CD209 target site, and study the application of drugs in treating diseases related to CD209.

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