Non-human animals with humanized clec9a gene

Abstract

Disclosed herein is a genetically modified rodent, the genetically modified rodent comprising a humanized Clec9a gene in its genome. The invention also discloses an isolated rodent tissue or cell. The isolated rodent tissue or cell comprises a rodent embryonic stem cell and a rodent embryo; a method of making the genetically modified rodent; and methods of using the genetically modified rodents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 355,948, filed on June 27, 2022, the entire contents of which are incorporated herein by reference.

[0003] Incorporation by Reference into the Sequence Listing

[0004] The sequence listing is an XML format file named 39341WO_10900WO01_SequenceListing.xml, created on June 13, 2023, with a size of 53KB, which was submitted to the United States Patent and Trademark Office via the Patent Center and is incorporated herein by reference in its entirety. Background Art

[0005] C-type lectin domain family 9 member A (Clec9a) is a C-type lectin-like receptor expressed as a glycosylated dimer on the cell surface of dendritic cells and a small subset of monocytes. Clec9a can mediate endocytosis but not phagocytosis. Clec9a has a cytoplasmic immunoreceptor tyrosine-based activation-like motif ("ITAM"), which has been reported to recruit Syk kinase and induce the production of proinflammatory cytokines. Summary of the invention

[0006] In some embodiments, disclosed herein is a genetically modified rodent comprising a humanized Clec9a gene in its genome, wherein the humanized Clec9a gene comprises a rodent Clec9a nucleic acid sequence and a human CLEC9A nucleic acid sequence, wherein the humanized Clec9a gene encodes a humanized Clec9a polypeptide, and the humanized Clec9a polypeptide comprises an extracellular domain substantially identical to the extracellular domain of the human CLEC9A protein.

[0007] In some embodiments, the humanized Clec9a protein comprises a cytoplasmic-transmembrane sequence that is substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein (e.g., an endogenous rodent Clec9a protein). In some embodiments, the humanized Clec9a protein comprises a cytoplasmic-transmembrane sequence that is substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein (e.g., an endogenous rodent Clec9a protein).

[0008] In some embodiments, the human CLEC9A nucleic acid sequence in the humanized Clec9a gene encodes at least a majority of the extracellular domain of the human CLEC9A protein. In some embodiments, the human CLEC9A nucleic acid sequence in the humanized Clec9a gene encodes amino acids 57-241 of human CLEC9A (e.g., human CLEC9A as shown in SEQ ID NO: 4). In some embodiments, the human CLEC9A nucleic acid sequence comprises a stop codon in exon 3 to exon 6 of the human CLEC9A gene. In some embodiments, the human CLEC9A nucleic acid sequence comprises exon 3 to exon 6 of the human CLEC9A gene (i.e., to the 3' end of exon 6).

[0009] In some embodiments, the rodent Clec9a nucleic acid sequence in the humanized Clec9a gene comprises a nucleotide sequence of a rodent Clec9a gene encoding at least a majority of a cytoplasmic-transmembrane sequence of a rodent Clec9a protein (e.g., an endogenous rodent Clec9a protein). In some embodiments, the rodent Clec9a nucleic acid sequence comprises exon 1 and exon 2 of a rodent Clec9a gene (e.g., an endogenous rodent Clec9a gene).

[0010] In some embodiments, the humanized Clec9a gene comprises (i) exon 1 and exon 2 of a rodent Clec9a gene (e.g., an endogenous rodent Clec9a gene), and (ii) a stop codon in exon 3 to exon 6 of a human CLEC9A gene, optionally exon 3 to exon 6. In some embodiments, the humanized Clec9a gene comprises the 3'UTR of a rodent Clec9a gene. In some embodiments, the humanized Clec9a gene comprises (i) exon 1 and exon 2 of a rodent Clec9a gene (e.g., an endogenous rodent Clec9a gene), (ii) exon 3 to exon 6 of a human CLEC9A gene, and (iii) the 3'UTR of a rodent Clec9a gene (e.g., an endogenous rodent Clec9a gene).

[0011] In some embodiments, the humanized Clec9a gene is operably linked to a rodent Clec9a promoter, such as an endogenous rodent Clec9a promoter.

[0012] In some embodiments, the humanized Clec9a gene is located at a locus other than the endogenous rodent Clec9a locus. In some embodiments, the humanized Clec9a gene is located at the endogenous rodent Clec9a locus.

[0013] In some of the embodiments in which the humanized Clec9a gene is located at the endogenous rodent Clec9a locus, the humanized Clec9a gene is formed by replacing the rodent Clec9a genomic DNA at the endogenous rodent Clec9a locus with a human CLEC9A nucleic acid. In some embodiments, the humanized Clec9a gene is formed by replacing the rodent genomic DNA comprising at least a majority of the nucleotide sequence encoding the extracellular domain of the endogenous rodent Clec9a protein with a human CLEC9A nucleic acid encoding at least a majority of the extracellular domain of the human CLEC9A protein. In some embodiments, the rodent is a mouse, and wherein the replaced mouse genomic DNA comprises a stop codon in exon 3 to exon 6 of the endogenous mouse Clec9a gene, and the human genomic DNA comprises a stop codon in exon 3 to exon 6 of the human CLEC9A gene (optionally exon 3 to exon 6).

[0014] In some embodiments, the rodent is heterozygous for the humanized Clec9a gene.

[0015] In some embodiments, the rodent is homozygous for the humanized Clec9a gene.

[0016] In some embodiments, the humanized Clec9a polypeptide is expressed from a humanized Clec9a gene on dendritic cells in a rodent.

[0017] In some embodiments, the rodent is a mouse or a rat.

[0018] In some embodiments, disclosed herein is a rodent tissue or cell separated, the genome of the rodent tissue or cell separated comprising the humanized Clec9a gene described herein. In some embodiments, the rodent cell is a rodent embryonic stem cell. In some embodiments, the rodent cell is an egg or sperm. In some embodiments, the rodent tissue or cell separated is a mouse tissue or mouse cell, or a rat tissue or rat cell.

[0019] In some embodiments, disclosed herein is a rodent embryo comprising a rodent embryonic stem cell comprising a humanized Clec9a gene described herein.

[0020] In some embodiments, disclosed herein is a method for preparing a genetically modified rodent. In some embodiments, the method comprises modifying the rodent genome to include a humanized Clec9a gene, wherein the humanized Clec9a gene comprises a rodent Clec9a nucleic acid sequence and a human CLEC9A nucleic acid sequence, and encodes a humanized Clec9a polypeptide comprising an extracellular domain substantially identical to the extracellular domain of a human CLEC9A protein; and preparing a rodent comprising the modified rodent genome.

[0021] In some embodiments, modifying the rodent genome comprises the following steps: introducing a nucleic acid molecule comprising a human CLEC9A nucleic acid sequence into the genome of a rodent embryonic stem (ES) cell, obtaining a rodent ES cell in which the human CLEC9A nucleic acid sequence has been integrated into the endogenous Clec9a locus to replace the rodent Clec9a genomic DNA to form a humanized Clec9a gene, and producing a rodent from the obtained rodent ES cell. In some embodiments, the human CLEC9A nucleic acid sequence encodes at least a majority of the extracellular domain of the human CLEC9A protein. In some embodiments, the nucleic acid molecule introduced into the ES cell further comprises a 5' homology arm and a 3' homology arm flanking the human CLEC9A nucleic acid sequence, wherein the 5' homology arm and the 3' homology arm are homologous to the nucleic acid sequence at the endogenous rodent locus flanking the rodent Clec9a genomic DNA to be replaced. In some embodiments, the humanized Clec9a gene is operably linked to a rodent Clec9a promoter, eg, an endogenous rodent Clec9a promoter at an endogenous rodent Clec9a locus.

[0022] In some embodiments of the methods, the rodent is a mouse or a rat.

[0023] In some embodiments, disclosed herein is a targeting nucleic acid construct comprising a human CLEC9A nucleic acid sequence to be integrated into a rodent Clec9a gene at an endogenous rodent Clec9a locus, the human CLEC9A nucleic acid sequence flanked by a 5' nucleotide sequence and a 3' nucleotide sequence homologous to the nucleotide sequence at the rodent Clec9a locus, wherein integration of the human CLEC9A nucleic acid sequence into the rodent Clec9a gene replaces the rodent Clec9a genomic DNA with the human CLEC9A nucleic acid sequence, thereby forming a humanized Clec9a gene, and wherein the human CLEC9A nucleic acid sequence encodes at least a majority of the extracellular domain of the human CLEC9A protein. In some embodiments of the targeting nucleic acid, the rodent is a mouse or a rat.

[0024] In some embodiments, disclosed herein is an in vitro method for producing a genetically modified rodent cell, the in vitro method comprising introducing a targeting vector into a rodent cell, the targeting vector comprising a human CLEC9A nucleic acid sequence encoding at least a majority of the extracellular domain of a human CLEC9A protein, the human CLEC9A nucleic acid sequence flanking a rodent homology arm, the rodent homology arm mediating the integration of the human CLEC9A nucleotide sequence into the endogenous rodent Clec9a locus, the integration causing the rodent Clec9a genomic DNA to be replaced by the human CLEC9A nucleic acid sequence to form a humanized Clec9a gene as described herein, thereby producing a genetically modified rodent cell. In some embodiments, the rodent cell is a mouse cell or a rat cell. In some embodiments, the rodent cell is a rodent ES cell, and the method produces a genetically modified rodent ES cell.

[0025] In some embodiments, disclosed herein is a method for evaluating the pharmacokinetic properties of a candidate drug, the method comprising administering the candidate drug to a rodent as described herein, and performing one or more assays to determine the pharmacokinetic properties of the candidate drug in the rodent. In some embodiments, the candidate drug is an antibody that binds to human CLEC9A. In some embodiments, the candidate drug is an antibody that can bind to human CLEC9A.

[0026] In some embodiments, disclosed herein is a method for screening or evaluating a candidate drug targeting human CLEC9A, the method comprising administering the candidate drug to a rodent as described herein, and performing one or more assays to determine whether the candidate drug has an effect on the rodent (e.g., inducing activation of immune cells (such as T cells)). In some embodiments, one or more assays include an assay for measuring T cell proliferation in rodents. In some embodiments, one or more assays include an assay for measuring CD4+T cell proliferation in rodents. In some embodiments, one or more assays include an assay for measuring CD8+T cell proliferation in rodents. In some embodiments, the candidate drug includes an antibody that binds to human CLEC9A. In some embodiments, the candidate drug includes an antibody that binds to human CLEC9A, and the human CLEC9A is fused to one or more peptides recognized by MHC molecules on dendritic cells of rodents. In some embodiments, one or more peptides include OVA peptide I ("OTI", amino acids 257-264 of ovalbumin) and / or OVA peptide II ("OTII", amino acids 323-339 of ovalbumin). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1AAn exemplary embodiment of the humanization strategy of the mouse Clec9a gene is depicted. The mouse Clec9a gene and the human CLEC9A gene are represented by horizontal lines, and their exons are represented by boxes located on the horizontal lines. A 7138bp continuous mouse Clec9a genomic fragment at the endogenous mouse Clec9a locus (including the stop codon in exon 3 to exon 6) is replaced by a 4840bp human CLEC9A genomic fragment containing exon 3 to exon 6. As a result, the entire extracellular domain of mouse Clec9a (including the "CLECT-NK receptor-like" domain) (amino acids 57-238) is replaced by the extracellular domain of human CLEC9A (amino acids 57-241), while the N-terminal 31 amino acids of mouse Clec9a (including the "ITAM-like" motif) and the transmembrane domain (amino acids 32-56) are retained. See also Figure 1D Also indicated are the positions of primers and probes used to confirm correct targeting in human allele gain and mouse allele loss assays (represented by 7764hTU, 7764hTD, 7764mTU, and 7764mTD). The sequences of the primers and probes are shown in Table 2.

[0028] Figure 1B Depicted is an exemplary embodiment (not drawn to scale) of a targeting nucleic acid construct comprising a humanized fragment containing human CLEC9A exons 3-6, followed by a neomycin cassette with LoxP sites, flanked by 45.6 kb of mouse 5' homology arms (including exon 1 and exon 2 of mouse Clec9a) and 112.6 kb of mouse 3' homology arms (including 3'UTR of exon 6 and downstream sequences of mouse Clec9a) from BAC clone Rp23-248K14. A targeting nucleic acid construct comprising a humanized Clec9a gene can be introduced into mouse embryonic stem cells for targeted insertion into the mouse genome. Connections ("A", "B" and "C") indicate their sequences as provided.

[0029] Figure 1C Depicted are the Clec9a locus for mouse (i.e., removal of Figure 1B An exemplary embodiment of a humanization strategy for a humanized Clec9a locus obtained by targeting a neomycin cassette to a humanized Clec9a locus generated by the described targeting construct (not drawn to scale). Connections ("A" and "D") also indicate their sequences as provided.

[0030] Figure 1DAn alignment of exemplary protein sequences of mouse Clec9a (SEQ ID NO: 2), human CLEC9A (SEQ ID NO: 4), and humanized ("7765 mutant") Clec9a (SEQ ID NO: 7) is shown. The transmembrane segment ("TM") is underlined; the extracellular domain is identified by a solid box; the CLECT-NK receptor-like domain is indicated by a dotted line.

[0031] Figures 2A-2D Detection of Clec9a mRNA in wild-type ("WT") mice (endogenous Clec9a gene is not humanized), mice heterozygous for the humanized Clec9a gene ("Het"), and mice homozygous for the humanized Clec9a gene ("HumIn") is shown. RT-PCR was performed on bulk RNA derived from spleen cells using probes for mouse or human Clec9a nucleotide sequences. 2A-2B: Analysis was performed using probes targeting exons 1-2 or exons 3-4 of WT, Het, or HumIn mice. 2C-2D: Analysis was performed using probes targeting exons 2-3 or exons 5-6 of WT, Het, or HumIn mice. n=3 for each mouse genotype; Gapdh was used as an internal control.

[0032] Figure 3A-3B mClec9a expression is shown to be restricted to circulating dendritic cells (also called "cDC1") in wild-type ("WT") mice and mice heterozygous for the humanized Clec9a gene ("Het"). 3A: Gating strategy for mouse splenocytes. Cells were gated based on size, single cells, and live cells. Dendritic cells were gated for CD11c + MHC-II + Set the gate and + and CD11b + Further sub-gating was performed. 3B: The figure depicts the expression of mouse Clec9a ("mClec9a") in WT, Het or mice homozygous for the humanized Clec9a gene ("HumIn"), respectively. FMO; fluorescence minus one.

[0033] Figure 4A-4B Humanized Clec9a expression is shown to be restricted to cDC1 in mice heterozygous for the humanized Clec9a gene ("Het") and mice homozygous for the humanized Clec9a gene ("HumIn"). 4A: Gating strategy for mouse splenocytes. Cells were gated based on size, single cells, and live cells. Dendritic cells were gated for CD11c + MHC-II + Set the gate and + and CD11b +Further sub-gating was performed. 4B: The figure depicts the expression of humanized Clec9a (hClec9a) in wild-type ("WT"), Het or HumIn mice, respectively. FMO; fluorescence minus one.

[0034] Figure 5 Humanized Clec9a expression is shown to be restricted to cDC1 in mice heterozygous for the humanized Clec9a gene ("Het") and mice homozygous for the humanized Clec9a gene ("HumIn"). The left panel shows a dot plot of humanized Clec9a expression on Xcr1+ cells. The right panel shows a histogram depicting the expression of humanized Clec9a ("hClec9a") in wild-type ("WT"), Het or HumIn mice. FMO; fluorescence minus one.

[0035] Figure 6A-6B Humanized Clec9a expression was shown to be restricted to cDC1. + Gating of B cells (upper panel) shows the lack of mouse or human CLEC9A expression, as shown in the histograms corresponding to wild-type ("WT") mice, mice heterozygous for the humanized Clec9a gene ("Het"), and mice homozygous for the humanized Clec9a gene ("HumIn"). FMO; fluorescence minus one. 6B: Splenic CD4 + and CD8 + Gating of T cells (upper panel) demonstrated lack of expression of mouse or humanized Clec9a as shown in the histograms corresponding to WT, Het, or HumIn mice. FMO; fluorescence minus one.

[0036] Figure 7A-7B Antibody-antigen fusion targeting Clec9a can elicit strong CD4+ T cell responses in vivo. 7A: Gating of Ki67+OT-II tetramer+ T cells in mice injected with anti-human CLEC9 antibody fused to OVA peptide, isotype antibody fused to OVA peptide, PBS, full-length OVA, or OVA peptide containing both OT-I and OT-II antigens. 2×10 6 CFSE-labeled OT-II T cells were transferred to mice and one day later PBS, full-length OVA or OVA peptide and 15μg, 7.5μg and 3.75μg of antibody were given. Spleens were harvested 72 hours after antibody injection and analyzed by flow cytometry. 7B: Fig. 7A Shown is a depiction of Ki67 + OT-II tetramer +Bar graph of absolute counts of T cells. #, &, and @ indicate statistical significance with 15 μg, 7.5 μg, and 3.75 μg of isotype-OVA peptide fusion per mouse, respectively. Error bars indicate standard deviation; one-way ANOVA.

[0037] Figures 8A-8B Antibody-antigen fusion targeting Clec9a can elicit strong CD8 T cell responses in vivo. (A) Gating of Ki67+OT-I tetramer+T cells in mice injected with anti-human CLEC9 fused to OVA peptide, isotype antibody fused to OVA peptide, PBS, full-length OVA, or OVA peptide containing both OT-I and OT-II antigens. 6 CFSE-labeled OT-I T cells were transferred into mice and one day later, PBS, full-length OVA or OVA peptide and 15 μg, 7.5 μg and 3.75 μg of antibody were administered. Spleens were harvested 72 hours after antibody injection and analyzed by flow cytometry. (B) Fig. 8A Shown is a depiction of Ki67 + OT-I tetramer + Bar graph of absolute counts of T cells. # indicates statistical significance with 15 μg of isotype-OVA peptide per mouse. Error bars indicate standard deviation. One-way ANOVA. DETAILED DESCRIPTION

[0038] Disclosed herein are rodents (such as, but not limited to, mice and rats) that are genetically modified to contain humanized Clec9a genes. Also disclosed herein are compositions (e.g., targeting vectors) and methods for preparing such genetically modified rodents. The rodents disclosed herein can be used, for example, but not limited to, as an in vivo system for evaluating candidate compounds for human CLEC9A, such as anti-human Clec9A antibodies, which target dendritic cells to treat a wide range of immune-related indications, including but not limited to autoimmune infectious diseases and cancer, etc. Therefore, also disclosed herein are methods for evaluating candidate compounds for human CLEC9A using genetically modified rodents.

[0039] Humanization of rodent Clec9a

[0040] Clec9a is a C-type lectin-like receptor expressed as a glycosylated dimer on the cell surface of dendritic cells and a small subset of monocytes. Clec9a can mediate endocytosis, but not phagocytosis. Clec9a has a cytoplasmic immunoreceptor tyrosine-based activation-like motif ("ITAM") at its N-terminal portion, which is a conserved sequence of four amino acids repeated twice and is believed to recruit Syk kinases and induce the production of proinflammatory cytokines. See, e.g., Huysamen et al., J Biol Chem 283 (24), 16693-16701 (2008), which is incorporated herein by reference in its entirety.

[0041] Exemplary Clec9a sequences, including nucleic acid and protein sequences of human, mouse, rat, and humanized (mouse-human hybrid) Clec9a, are disclosed in the Sequence Listing and summarized in Table 1. Figure 1D An alignment of human CLEC9A, mouse Clec9a, and humanized (mouse-human hybrid) Clec9a protein sequences is provided.

[0042] For simplicity, the numbering of exons herein refers to the coding exons of the Clec9a gene. For example, exon 1 of the Clec9a gene herein refers to the first coding exon of the Clec9a gene.

[0043] Table 1. Sequence summary

[0044]

[0045]

[0046]

[0047] In some embodiments, the rodents disclosed herein comprise a humanized Clec9a gene in the germline.

[0048] In some embodiments, the rodent disclosed herein comprises a humanized Clec9a gene in its genome, which comprises a nucleotide sequence of a rodent Clec9a gene (e.g., an endogenous rodent Clec9a gene) and a nucleotide sequence of a human CLEC9A gene. As used herein, a "nucleotide sequence of a gene" comprises all or part of a genomic sequence, mRNA or cDNA sequence of a gene. As a non-limiting example, a nucleotide sequence of a human CLEC9A gene comprises all or part of a genomic sequence, mRNA or cDNA sequence of a human CLEC9A gene. The nucleotide sequence of the rodent Clec9a gene and the nucleotide sequence of the human CLEC9A gene are operably linked to each other so that the humanized Clec9a gene in the rodent genome encodes a humanized Clec9a protein, which maintains the protein structure of Clec9a (including an ITAM-containing cytoplasmic domain, a transmembrane domain, and an extracellular domain) and exerts the function of the Clec9a protein (e.g., recruiting Syk kinase and inducing the production of proinflammatory cytokines).

[0049] As used herein, "human CLEC9A" genes and proteins refer to CLEC9A genes and proteins of human origin. In some embodiments, the human CLEC9A protein comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the human CLEC9A protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the human CLEC9A protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the human CLEC9A protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0050] As used herein, "rodent Clec9a" genes and proteins refer to Clec9a genes and proteins of rodent (e.g., mouse or rat) origin. In some embodiments, the mouse Clec9a protein comprises an amino acid sequence of SEQ ID NO:2. In some embodiments, the mouse Clec9a protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the mouse Clec9a protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the mouse Clec9a protein comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQID NO:2. In some embodiments, the rat Clec9a protein comprises an amino acid sequence of SEQ ID NO:25. In some embodiments, the rat Clec9a protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, the rat Clec9a protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, the rat Clec9a protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:25.

[0051] In some embodiments, the genetically modified rodent comprises a humanized Clec9a gene in its genome, wherein the humanized Clec9a gene encodes a humanized Clec9a protein containing an extracellular domain substantially identical to the extracellular domain of a human CLEC9A protein. In some embodiments, an extracellular domain substantially identical to the extracellular domain of a human CLEC9A protein exhibits the same functionality (e.g., ligand binding properties) as the extracellular domain of a human CLEC9A protein. An extracellular domain or polypeptide that is "substantially identical to the extracellular domain of a human CLEC9A protein" may be a polypeptide that has at least 95% identity in sequence to the extracellular domain of a human CLEC9A protein, a polypeptide that has at least 98% identity in sequence to the extracellular domain of a human CLEC9A protein, or a polypeptide that has at least 99% identity in sequence to the extracellular domain of a human CLEC9A protein. The extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human CLEC9A protein" can be a polypeptide that has 100% sequence identity with the extracellular domain of the human CLEC9A protein. Alternatively or additionally, the extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human CLEC9A protein" can be a polypeptide that differs from the extracellular domain of the human CLEC9A protein by no more than 5 amino acids, a polypeptide that differs from the extracellular domain of the human CLEC9A protein by no more than 4 amino acids, a polypeptide that differs from the extracellular domain of the human CLEC9A protein by no more than 3 amino acids, a polypeptide that differs from the extracellular domain of the human CLEC9A protein by no more than 2 amino acids, or a polypeptide that differs from the extracellular domain of the human CLEC9A protein by no more than 1 amino acid. Alternatively or additionally, the extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human CLEC9A protein" may be a polypeptide that differs from the extracellular domain of the human CLEC9A protein only at the N-terminal or C-terminal portion of the extracellular domain, for example, by adding, deleting and / or replacing amino acids at the N-terminal and / or C-terminal portion of the extracellular domain (i.e., the difference from the N-terminal or C-terminal end of the extracellular domain is within 5-10 amino acids). Alternatively or additionally, the extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human CLEC9A protein" may be a polypeptide having one or more of the characteristics described above, for example, a polypeptide that has at least 95% identity in sequence to the extracellular domain of the human CLEC9A protein and differs from the extracellular domain of the human CLEC9A protein by no more than 5 amino acids only at the N-terminal or C-terminal portion of the extracellular domain, or a polypeptide that has at least 98% identity in sequence to the extracellular domain of the human CLEC9A protein and differs from the extracellular domain of the human CLEC9A protein by no more than 3 amino acids only at the N-terminal or C-terminal portion of the extracellular domain.In some embodiments, the human CLEC9A protein comprises an amino acid sequence as shown in SEQ ID NO: 4, and its extracellular domain consists of amino acids 57-241 of SEQ ID NO: 4. In some embodiments, the extracellular domain of the humanized Clec9a protein encoding the humanized Clec9a gene is substantially the same as the extracellular domain of the human CLEC9A protein shown in SEQ ID NO: 4, i.e., substantially the same as amino acids 57-241 of SEQ ID NO: 4. For example, the humanized Clec9a gene encoding the humanized Clec9a protein has an extracellular domain comprising amino acids 57-241, 58-241, 59-241, 60-241, 57-240, 57-239, 57-238, or 57-237 of SEQ ID NO: 4. In some embodiments, the humanized Clec9a gene encodes a humanized Clec9a protein having an extracellular domain comprising amino acids 57-241 of SEQ ID NO: 4. In some embodiments, the humanized Clec9a gene encodes a humanized Clec9a protein having an extracellular domain comprising amino acids 58-241 of SEQ ID NO: 4. In some embodiments, the humanized Clec9a gene encodes a humanized Clec9a protein having an extracellular domain comprising amino acids 59-241 of SEQ ID NO: 4. In some embodiments, the humanized Clec9a gene encodes a humanized Clec9a protein having an extracellular domain comprising amino acids 57-240 of SEQ ID NO: 4. In some embodiments, the humanized Clec9a gene encodes a humanized Clec9a protein having an extracellular domain comprising amino acids 57-239 of SEQ ID NO: 4.

[0052] In some embodiments, the humanized Clec9a protein encoded by the humanized Clec9a gene contains a cytoplasmic-transmembrane sequence substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein (e.g., an endogenous rodent Clec9a protein) (i.e., a sequence comprising both a transmembrane domain and a cytoplasmic domain). In some embodiments, the cytoplasmic-transmembrane sequence substantially identical to the cytoplasmic-transmembrane sequence of an endogenous rodent Clec9a protein exhibits the same function (e.g., signal transduction and / or interaction with intracellular molecules) as the cytoplasmic-transmembrane sequence of a rodent Clec9a protein (such as an endogenous rodent Clec9a protein). The cytoplasmic-transmembrane sequence or polypeptide that is "substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein" can be a polypeptide that has at least 95% identity in sequence to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein or a polypeptide that has at least 98% identity in sequence to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein. The cytoplasmic-transmembrane sequence or polypeptide that is "substantially identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein" may be a polypeptide that is identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein. Alternatively or additionally, the cytoplasmic-transmembrane sequence or polypeptide that is "substantially identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein" may be a polypeptide that differs from the cytoplasmic-transmembrane sequence of the rodent Clec9a protein by no more than 3 amino acids, a polypeptide that differs from the cytoplasmic-transmembrane sequence of the rodent Clec9a protein by no more than 2 amino acids, or a polypeptide that differs from the cytoplasmic-transmembrane sequence of the rodent Clec9a protein by no more than 1 amino acid. Alternatively or additionally, a cytoplasmic-transmembrane sequence or polypeptide that is "substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein" may be a polypeptide that differs from the cytoplasmic-transmembrane sequence of a rodent Clec9a protein only at the N-terminus or C-terminus, for example, by adding, deleting or substituting amino acids at the N-terminal or C-terminal portion of the transmembrane-cytoplasmic sequence. Alternatively or additionally, the cytoplasmic-transmembrane sequence or polypeptide that is "substantially identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein" can be a polypeptide having one or more of the characteristics described above, for example, a polypeptide that has at least 95% identity in sequence to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein and differs from the cytoplasmic-transmembrane sequence of the rodent Clec9a protein by no more than 3 amino acids at the N-terminus or C-terminus; or a polypeptide that has at least 95% identity in sequence to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein and differs from the cytoplasmic-transmembrane sequence of the rodent Clec9a protein by no more than 2 amino acids at the N-terminus or C-terminus.By "the N-terminal or C-terminal portion of the cytoplasmic-transmembrane sequence" is meant a portion that differs from the N-terminus of the cytoplasmic domain or from the C-terminus of the transmembrane domain within 3-5 amino acids. In some embodiments, the humanized Clec9a protein contains a cytoplasmic-transmembrane sequence that is substantially identical to the cytoplasmic-transmembrane sequence of a mouse Clec9a protein (such as an endogenous mouse Clec9a protein). In some embodiments, the humanized Clec9a protein contains a cytoplasmic-transmembrane sequence that is substantially identical to the cytoplasmic-transmembrane sequence of a rat Clec9a protein (such as an endogenous rat Clec9a protein).

[0053] In some embodiments, the humanized Clec9a gene in the genome of the genetically modified rodent comprises a nucleotide sequence of a human CLEC9A gene ("human CLEC9A nucleotide sequence") and a nucleotide sequence of a rodent Clec9a gene ("rodent Clec9a nucleotide sequence", such as an endogenous rodent Clec9a nucleotide sequence), wherein the human CLEC9A nucleotide sequence encodes at least a majority of the extracellular domain of the human CLEC9A protein. Examples of a majority of the extracellular domain of human CLEC9A can include amino acids 57-241, 57-240, 57-239, 58-241, or 59-241 of SEQ ID NO:4. In some embodiments, a majority of the extracellular domain comprises amino acids 57-241 of SEQ ID NO:4. In some embodiments, a majority of the extracellular domain comprises amino acids 57-240 of SEQ ID NO:4. In some embodiments, a majority of the extracellular domain comprises amino acids 57-239 of SEQ ID NO:4. In some embodiments, a majority of the extracellular domain comprises amino acids 58-241 of SEQ ID NO:4. In some embodiments, a majority of the extracellular domain comprises amino acids 59-241 of SEQ ID NO:4. In some embodiments, the human CLEC9A nucleotide sequence is a cDNA sequence. In some embodiments, the human CLEC9A nucleotide sequence in the humanized Clec9a gene encodes the extracellular domain of a human CLEC9A protein (e.g., a human CLEC9A protein as defined by SEQ ID NO:4). In some embodiments, the human CLEC9A nucleotide sequence is a genomic fragment of a human CLEC9A gene. In some embodiments, the human CLEC9A nucleotide sequence is a genomic fragment of a human CLEC9A gene comprising a stop codon in exon 3 to exon 6. In some embodiments, the human CLEC9A nucleotide sequence is a genomic fragment of a human CLEC9A gene comprising exon 3 to exon 6 (i.e., to the 3' untranslated region in exon 6).

[0054] In some embodiments, the humanized Clec9a gene in the genome of the genetically modified rodent comprises a rodent Clec9a nucleotide sequence and a human CLEC9A nucleotide sequence, wherein the rodent Clec9a nucleotide sequence encodes a polypeptide substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein (e.g., an endogenous rodent Clec9a protein). In some embodiments, the rodent Clec9a nucleotide sequence present in the humanized Clec9a gene encodes the cytoplasmic-transmembrane sequence of an endogenous rodent Clec9a protein. In some embodiments, the rodent Clec9a nucleotide sequence present in the humanized Clec9a gene comprises exon 1 (all or part, e.g., a coding portion) and exon 2 of a rodent (e.g., an endogenous rodent) Clec9a gene. In some embodiments, the rodent Clec9a nucleotide sequence present in the humanized Clec9a gene is a mouse Clec9a nucleotide sequence; and in some such embodiments, the mouse Clec9a nucleotide sequence comprises the coding portion of the mouse Clec9a gene (e.g., an endogenous mouse Clec9a gene), exon 1 (and optionally also the 5'UTR of exon 1) and exon 2. In some embodiments, the rodent Clec9a nucleotide sequence present in the humanized Clec9a gene also comprises the 3'UTR of the rodent Clec9a gene. In some embodiments, the 3'UTR of the rodent Clec9a gene is located downstream of the 3'UTR of the human CLEC9A gene.

[0055] In some embodiments, the humanized Clec9a gene is operably linked to a rodent Clec9a 5' regulatory sequence, such as an endogenous rodent Clec9a regulatory sequence, e.g., a 5' transcriptional regulatory sequence such as a promoter and / or enhancer, such that expression of the humanized Clec9a gene is under the control of the rodent Clec9a 5' regulatory sequence.

[0056] In some embodiments, the humanized Clec9a gene is located at the endogenous rodent Clec9a locus. In some embodiments, the humanized Clec9a gene is located at a locus other than the endogenous rodent Clec9a locus; for example, as a result of random integration. In some embodiments, the humanized Clec9a gene is located at the ROSA26 locus (the locus is as described in Zambrowicz et al., 1997, PNAS USA 94:3789-3794, which is incorporated herein by reference). In some embodiments where the humanized Clec9a gene is located at a locus other than the endogenous rodent Clec9a locus, the rodent cannot express rodent Clec9a protein, for example, as a result of inactivation (e.g., total or partial deletion) of the endogenous rodent Clec9a gene.

[0057] In some embodiments where the humanized Clec9a gene is located at an endogenous rodent Clec9a locus, the humanized Clec9a gene is generated by replacing the nucleotide sequence of the endogenous rodent Clec9a gene at the endogenous rodent Clec9a locus with the nucleotide sequence of the human CLEC9A gene.

[0058] In some embodiments, the nucleotide sequence of the endogenous rodent Clec9a gene at the replaced endogenous rodent Clec9a locus is a genomic fragment of an endogenous rodent Clec9a gene encoding at least a majority of the extracellular domain of the rodent Clec9a protein. In some embodiments, the rodent is a mouse, and the replaced mouse Clec9a genomic fragment encodes at least a majority of the extracellular domain of the endogenous mouse Clec9a protein. For example, the extracellular domain of the mouse Clec9a of SEQ ID NO:2 is defined by amino acids 57-238, and examples of most of the extracellular domain can include amino acids 57-238, 57-237, 57-236, 58-238, or 59-238 of SEQ ID NO:2. In some embodiments, most of the extracellular domain of the mouse Clec9a protein includes amino acids 57-238 of SEQ ID:2. In some embodiments, a majority of the extracellular domain of the mouse Clec9a protein comprises amino acids 57-237 of SEQ ID: 2. In some embodiments, a majority of the extracellular domain of the mouse Clec9a protein comprises amino acids 57-236 of SEQ ID: 2. In some embodiments, a majority of the extracellular domain of the mouse Clec9a protein comprises amino acids 58-238 of SEQ ID: 2. In some embodiments, a majority of the extracellular domain of the mouse Clec9a protein comprises amino acids 59-238 of SEQ ID: 2. In some embodiments, the replaced mouse Clec9a genomic fragment comprises a stop codon in exon 3 to exon 6.

[0059] In some embodiments, the nucleotide sequence of the human CLEC9A gene that replaces the genomic fragment of the rodent Clec9a gene at the endogenous rodent Clec9a locus is a cDNA sequence. In some embodiments, the human CLEC9A nucleotide sequence that replaces the genomic fragment of the rodent Clec9a gene at the endogenous rodent Clec9a locus is a genomic fragment of the human CLEC9A gene. In some embodiments, the genomic fragment of the human CLEC9A gene that replaces the genomic fragment of the rodent Clec9a gene at the endogenous rodent Clec9a locus includes all or part of the exons of the human CLEC9A gene that encodes at least a majority of the extracellular domain of the human CLEC9A protein. Examples of a majority of the extracellular domain of human CLEC9A, for example, amino acids 57-241, 57-240, 57-239, 58-241, or 59-241 of SEQ ID NO: 4 are described above. In some embodiments, a majority of the extracellular domain of human CLEC9A comprises amino acids 57-241 of SEQ ID NO: 4. In some embodiments, a majority of the extracellular domain of human CLEC9A comprises amino acids 57-240 of SEQ ID NO: 4. In some embodiments, a majority of the extracellular domain of human CLEC9A comprises amino acids 57-239 of SEQ ID NO: 4. In some embodiments, a majority of the extracellular domain of human CLEC9A comprises amino acids 58-241 of SEQ ID NO: 4. In some embodiments, a majority of the extracellular domain of human CLEC9A comprises amino acids 59-241 of SEQ ID NO: 4. In some embodiments, the human genomic fragment comprises exon 3 to the stop codon in exon 6 of human CLEC9A. In some embodiments, the human genomic fragment comprises exon 3 to the 3' end of exon 6 of human CLEC9A (i.e., comprises the 3'UTR of human CLEC9A).

[0060] In some embodiments, the human CLEC9A nucleotide sequence inserted into the endogenous rodent Clec9a locus is operably linked to a genomic sequence of a rodent Clec9a gene encoding a polypeptide substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein (such as an endogenous rodent, e.g., mouse or rat, Clec9a protein). In some embodiments, the genomic sequence of the rodent Clec9a gene comprises exon 1 and exon 2 of a rodent Clec9a gene (e.g., an endogenous mouse or rat Clec9a gene). In some embodiments, the genomic sequence of the rodent Clec9a gene further comprises the 3'UTR of the rodent Clec9a gene.

[0061] In some embodiments, the rodent is a mouse, and a genomic fragment of an endogenous mouse Clec9a gene at an endogenous mouse Clec9a locus comprising a stop codon in exon 3 to exon 6 of a mouse Clec9a gene (encoding a mouse Clec9a extracellular domain) has been replaced by a genomic fragment of a human CLEC9A gene comprising a stop codon in exon 3 to exon 6 encoding a human CLEC9A extracellular domain. In some embodiments, a humanized Clec9a gene is formed at an endogenous mouse Clec9a locus and comprises exons 1-2 of a mouse Clec9a gene, exons 3 to exon 6 of a human CLEC9A gene, optionally followed by the 3'UTR of the mouse Clec9a gene.

[0062] In some embodiments, the rodents provided herein are heterozygous for the humanized Clec9a gene in their genome. In some embodiments, the rodents provided herein are homozygous for the humanized Clec9a gene in their genome.

[0063] In some embodiments, the humanized Clec9a gene results in expression of the encoded humanized Clec9a protein in a rodent. In some embodiments, the humanized Clec9a protein is expressed in cells and tissues, wherein the corresponding rodent Clec9a protein in a control rodent (e.g., a rodent without a humanized Clec9a gene) is typically expressed, for example, on dendritic cells.

[0064] In some embodiments, a rodent disclosed herein is unable to express a rodent Clec9a protein, for example, as a result of inactivation (eg, complete or partial deletion) or replacement (complete or partial) of an endogenous rodent Clec9a gene.

[0065] Rodent species and strains

[0066] In some embodiments, as non-limiting examples, rodents of the present disclosure include mice, rats and hamsters. In some embodiments, rodents are selected from superfamily Muroidea. In some embodiments, rodents of the present disclosure are from the family selected from Calomyscidae (e.g., mouse-like hamster), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (Muridae) (true mice and rats, gerbils, spiny rats, crowned rats), Nesomyidae (climbing rats, rock rats, rats with tails, Madagascar rats and mice), Platacanthomyidae (e.g., spiny rod dormice) and Spalacidae (e.g., moles, bamboo rats and zokors). In some embodiments, rodents of the present disclosure are selected from true mice or rats (Muridae), gerbils, spiny rats and crowned rats. In some embodiments, the mice of the present disclosure are from a member of the family Muridae.

[0067] In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a mouse selected from the following C57BL strains: C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the rodent is a 129 strain selected from the group consisting of: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach et al., 2000, Biotechniques 29(5):1024-1028, 1030, 1032; both of which are incorporated herein by reference in their entirety). In some embodiments, the rodent is a mouse of a mixture of 129 strains and C57BL / 6 strains. In some embodiments, the rodent is a mouse of a mixture of the aforementioned 129 strains or a mixture of the aforementioned BL / 6 strains. In some embodiments, the rodent is a mouse of a BALB strain (e.g., BALB / c strain). In some embodiments, the rodent is a mouse of a mixture of the BALB strain and another aforementioned strain.

[0068] In some embodiments, the rodent is a rat. In some certain embodiments, the rat is selected from Wistar rat, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6 and Dark Agouti. In some embodiments, the rat strain as described herein is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6 and Dark Agouti.

[0069] Genetically modified rodent tissues and cells

[0070] In some embodiments, disclosed herein is an isolated rodent cell or tissue, the genome of which comprises a humanized Clec9a gene.

[0071] In some embodiments, the tissue is selected from adipose, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, ovum, and combinations thereof.

[0072] In some embodiments, the cell is selected from a dendritic cell or a monocyte.

[0073] In some embodiments, the isolated rodent cell is a rodent embryonic stem cell. In some embodiments, the isolated rodent cell is a rodent egg or a rodent sperm.

[0074] Compositions and methods for preparing humanized rodents

[0075] Disclosed herein is a targeting vector (or nucleic acid construct) comprising a human CLEC9A nucleotide sequence that is desired to be integrated into a rodent locus to form a humanized Clec9a gene as described herein.

[0076] In some embodiments, the targeting vector comprises a human CLEC9A nucleotide sequence encoding at least a majority of the extracellular domain of a human CLEC9A protein as described above. In some embodiments, the human CLEC9A nucleotide sequence encodes a polypeptide comprising amino acids 57-241 of SEQ ID NO: 4. In some embodiments, the human CLEC9A nucleotide sequence comprises a stop codon in exon 3 to exon 6 encoding the extracellular domain of a human CLEC9A protein.

[0077] The targeting vector also comprises 5' and 3' rodent sequences flanking the human nucleotide sequence to be integrated, also referred to as 5' and 3' homology arms, which mediate homologous recombination and integration of the human nucleotide sequence to the target rodent locus (e.g., endogenous rodent Clec9a locus), thereby forming a humanized gene as described above. Typically, the 5' and 3' flanking rodent sequences in the targeting vector are identical or substantially identical (e.g., having at least 98% or at least 99% identity) to the nucleotide sequences of the rodent nucleotide sequences corresponding to the target rodent locus to be replaced by the human nucleotide sequence. In some embodiments, the targeting vector comprises a humanized gene as described above. In some embodiments, the targeting vector comprises a humanized Clec9a gene comprising a human CLEC9A nucleotide sequence and a rodent Clec9a nucleotide sequence, as described above. In some embodiments, the targeting vector comprises a humanized Clec9a gene, wherein the humanized Clec9a gene comprises exons 1-2 of a rodent Clec9a gene and exons 3-6 of a human CLEC9A gene, and optionally the humanized Clec9a gene is flanked by 5' and 3' rodent homology arms.

[0078] In some embodiments, the targeting vector includes a selectable marker gene. The selectable marker gene can be inserted into the intron of the human genome sequence to be integrated. In some embodiments, the selectable marker gene is provided as a self-deletion box, which can be deleted after successfully integrating the human nucleotide sequence.

[0079] In an exemplary embodiment, bacterial homologous recombination and Technology, from the bacterial artificial chromosome (BAC) clone carrying rodent Clec9a genomic DNA to produce targeting vector (see, for example, US6,586,251 and Valenzuela et al. (2003) Nature Biotech.21 (6): 652-659, which is incorporated herein by reference in its entirety). Due to bacterial homologous recombination, the rodent genomic sequence is deleted from the BAC clone, and the human nucleotide sequence is inserted, thereby producing a modified BAC clone carrying the human nucleotide sequence, the human nucleotide sequence is flanked by 5' and 3' rodent homology arms. In some embodiments, the human nucleotide sequence can be a cDNA sequence or human genomic DNA. Once linearized, the modified BAC clone can be introduced into rodent embryonic stem (ES) cells.

[0080] In some embodiments, the invention provides the use of a targeting vector as described herein for preparing modified rodent embryonic stem (ES) cells. The targeting vector can be introduced into rodent ES cells by, for example, electroporation. Both mouse ES cells and rat ES cells have been described in the art. See, for example, US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000A1 (all of which are incorporated herein by reference in their entirety), which describe mouse ES cells and methods for preparing genetically modified mice. Method; US2014 / 0235933A1 (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828A1 (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) Nat Protoc.6(6):doi:10.1038 / nprot.2011.338 (all of which are incorporated herein by reference in their entirety), which describe rat ES cells and methods for preparing genetically modified rats, which can be used to prepare modified rodent embryos, which can then be used to prepare rodents.

[0081] In some embodiments, ES cells can be selected that have a desired human nucleotide sequence (e.g., a human CLEC9A nucleotide sequence) integrated into the genome. In some embodiments, ES cells are selected based on the loss of the rodent allele and / or the gain of the human allele assay. In some embodiments, the selected ES cells are then used as donor ES cells to be used in the transplantation of a human ES cell by using Method (see, for example, US 7,576,259, US 7,659,442, US 7,294,754 and US2008-0078000 A1, all of which are incorporated by reference in their entirety) or the method described in US 2014 / 0235933A1 and US2014 / 0310828 A1 (the documents are all incorporated by reference in their entirety) is injected into pre-morula embryos (e.g., 8-cell stage embryos). In some embodiments, the embryo comprising the donor ES cell is incubated and implanted into a surrogate mother to produce F0 rodents. The rodent pups carrying human nucleotide sequences can be identified by genotyping DNA separated from tails using the loss of rodent alleles and / or the gain of human allele determination.

[0082] In some embodiments, rodents heterozygous for a humanized gene can be crossed to produce homozygous rodents.

[0083] A humanized rodent as described herein (ie, a rodent comprising a humanized Clec9a gene) can be mated or crossed with another rodent. Thus, breeding methods and offspring obtained from such breeding are also embodiments of the present disclosure.

[0084] In some embodiments, a method is provided, the method comprising mating a first rodent as described above (e.g., a rodent whose genome comprises a humanized Clec9a gene) with a second rodent to produce an offspring rodent whose genome comprises a humanized Clec9a gene. The offspring can have other desired phenotypes or genetic modifications inherited from the second rodent used in breeding. In some embodiments, the offspring rodent is heterozygous for one or more humanized genes from the first rodent. In some embodiments, the offspring rodent is homozygous for the humanized gene from the first rodent.

[0085] In some embodiments, a progeny rodent having a genome comprising a humanized Clec9a gene is provided, wherein the progeny rodent is produced by a method comprising mating a first rodent having a genome comprising a humanized Clec9a gene with a second rodent. In some embodiments, the progeny rodent is heterozygous for the humanized Clec9a gene from the first rodent. In some embodiments, the progeny rodent is homozygous for the humanized Clec9a gene from the first rodent.

[0086] In some embodiments, disclosed herein is an in vitro method for producing a genetically modified rodent cell, the in vitro method comprising introducing a targeting vector into a rodent cell, the targeting vector comprising a human CLEC9A nucleic acid sequence encoding at least a majority of the extracellular domain of a human CLEC9A protein, the human CLEC9A nucleic acid sequence flanking a rodent homology arm, the rodent homology arm mediating the integration of the human CLEC9A nucleotide sequence into the endogenous rodent Clec9a locus, the integration causing the rodent Clec9a genomic DNA to be replaced by the human CLEC9A nucleic acid sequence to form a humanized Clec9a gene as described herein, thereby producing a genetically modified rodent cell. In some embodiments, the rodent cell is a mouse cell or a rat cell. In some embodiments, the rodent cell is a rodent ES cell, and the method produces a genetically modified rodent ES cell.

[0087] Humanized rodent approach

[0088] The rodents disclosed herein provide a useful in vivo system and source of biological material for identifying and testing compounds for their potential to treat human diseases in which CLEC9A may play a role, including immune-related indications, i.e., conditions that may involve or result from immune dysregulation, such as autoimmune infectious diseases and cancer, among others.

[0089] In some embodiments, the genetically modified rodents disclosed herein are used to evaluate agents targeting human CLEC9A. In some embodiments, the agent is an antibody that specifically binds to human CLEC9A. Candidate agents such as anti-human CLEC9A antibodies can be administered to rodents disclosed herein at multiple doses (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / mg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg or higher). The agent can be administered via any desired route of administration (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.).

[0090] In some embodiments, the genetically modified rodents disclosed herein are used to evaluate the pharmacokinetic properties of candidate agents (such as anti-human CLEC9A antibodies). The agent is administered to a genetically modified rodent. Blood is isolated from the animal at multiple time points (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more). The various assays that can be performed to determine pharmacokinetic properties may include, but are not limited to, the manner in which the animal processes the drug into various metabolites (or one or more drug metabolites, including the detection of the presence or absence of toxic metabolites), drug half-life, circulating levels of the drug after administration (e.g., serum concentrations of the drug), anti-drug reactions (e.g., anti-drug antibodies), drug absorption and distribution, routes of administration, excretion pathways, and / or drug clearance.

[0091] In some embodiments, the genetically modified rodents disclosed herein are used to evaluate agents (e.g., antibodies) targeting human CLEC9A to determine whether the agent has an effect on the rodent, for example, whether the agent triggers the activation of dendritic cell-dependent immune cells (such as T cells). This evaluation can be performed by the following steps: administering the agent disclosed herein to the rodent and evaluating the immune cells in the rodent to determine whether the immune cells are activated, for example, the proliferation of T cells and / or the production of cytokines. It can be compared with a control rodent to which the agent is not administered or a control rodent to which a control agent (e.g., an isotype antibody) is administered. In some embodiments, an antibody-peptide fusion is administered to a rodent disclosed herein (e.g., a mouse or rat expressing a humanized Clec9a protein on dendritic cells), wherein the antibody binds to the extracellular domain of human CLEC9A, and wherein the peptide includes one or more antigens recognized by the major histocompatibility complex (MHC) molecule on the dendritic cell. In some embodiments, the peptides include antigens recognized by MHC class I molecules (e.g., amino acids 257-264 of ovalbumin, also known as OTI peptides) and antigens recognized by MHC class II molecules (e.g., amino acids 323-339 of ovalbumin, also known as OTII peptides). The rodents are then examined to determine whether exposure to the antibody-peptide fusion will induce a T cell response (e.g., T cell proliferation). In some embodiments, the rodents are examined to measure whether there is CD4+T cell proliferation. In some embodiments, the rodents are examined to measure whether there is CD8+T cell proliferation. Methods and assays for evaluating T cell responses in animals are well known in the art and are also described in the Examples section below. Comparisons can be made with control rodents not administered antibody-peptide fusions or control rodents administered isotype antibody-peptide fusions, isotype antibodies, or peptides alone. Any observed T cell response and the extent of the response can be related to the efficacy of the antibody being tested.

[0092] This specification is further illustrated by the following examples, which should not be construed as limiting in any way.The contents of all cited documents (including references, issued patents, and published patent applications cited throughout this application) are hereby expressly incorporated by reference in their entirety.

[0093] Example 1. Humanization of the endogenous mouse Clec9a gene

[0094] Using bacterial artificial chromosome (BAC) cloning and Technology was used to construct a targeting vector for humanization of the endogenous Clec9a gene (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotechnology 21(6):652-659; all of these documents are incorporated herein by reference).

[0095] The BAC clone RP23-248K14 containing the mouse Clec9a gene was used and modified as follows. In short, a DNA fragment containing a mouse 5' homologous nucleotide sequence (100 bp), 4,840 bp of human CLEC9A genomic DNA (containing exons 3, 4, 5, 6 of the human CLEC9A gene and their respective introns 3, 4, 5 (including complete human 3'UTR)), 4,809 bp of self-deletion neomycin box and 3' mouse homologous sequence (100 bp) was generated. This DNA fragment was used to modify the BAC clone RP23-248K14 by homologous recombination in bacterial cells. As a result, the 7,138 bp mouse Clec9a genomic fragment (encoding the extracellular domain of the mouse Clec9a protein) in the BAC clone was replaced by a 4,840 bp human CLEC9A genomic fragment and a subsequent 4,809 bp of self-deletion neomycin box. Specifically, the replaced mouse Clec9a genomic fragment contains the entire exon 3 to the stop codon of the last coding exon (exon 6) of the mouse Clec9a gene ( Figure 1A-1B The inserted human CLEC9A genomic fragment includes exons 3, 4, 5 and their respective introns to the stop codon of the last coding exon (exon 6), including the complete 3'UTR of human CLEC9A ( Figure 1A-1B The resulting modified BAC clone contained, from 5' to 3': (i) a 5' mouse homology arm containing approximately 45.6 kb of mouse genomic DNA, including mouse Clec9a 5'UTR, mouse Clec9a exons 1 and 2; (ii) an approximately 4,840 bp human CLEC9A genomic fragment containing exons 3, 4, 5, and 6 (including the 3'UTR of human CLEC9A); (iii) an approximately 4,809 bp self-deleting neomycin cassette, followed by (iv) a 112.6 kb 3' mouse homology arm containing mouse Clec9a 3'UTR and the rest of the mouse genomic DNA in the original BAC clone ( Figure 1A-1B ). Figure 1BThe junction sequence is also listed. A portion of the modified BAC clone containing the human CLEC9A genomic fragment and the self-deleted neomycin cassette, as well as the upstream and downstream of the insertion junction are shown in SEQ ID NO: 5. The amino acid sequence of the protein encoded by the humanized Clec9a gene is shown in SEQ ID NO: 7 and Figure 1D shown. Figure 1D An alignment of the humanized Clec9a protein ("7765 mutant protein"), mouse Clec9a protein (SEQ ID NO: 2), and human CLEC9A protein (SEQ ID NO: 4) is provided.

[0096] As described above, a modified BAC clone containing a humanized Clec9a gene is used to electroporate mouse embryonic stem (ES) cells to generate modified ES cells containing a humanized Clec9a gene. ES cells containing a forward targeting of a humanized Clec9a gene are identified by detecting the presence of a human CLEC9A sequence (e.g., exons 3-6 of human CLEC9A) and confirming the absence and / or retention of a mouse Clec9a sequence (e.g., absence of exons 3-6 of mouse Clec9a) (Valenzuela et al., see above). Table 1 shows primers and probes used to confirm the humanization of an endogenous Clec9a gene as described above ( Figure 1A-1B ). Select the forward-targeted ES cell clone as the donor cell and use Method (see, e.g., Poueymirou et al., 2007, Nature Biotech. 25 (1): 91-99, US 7,576,259, US 7,659,442, US 7,294,754 and US2008-0078000 A1, all of which are incorporated herein by reference in their entirety) or the method described in US2014 / 0235933 A1 and US2014 / 0310828 A1 injects donor cells into pre-morula embryos (e.g., 8-cell embryos). Embryos containing donor ES cells are incubated until the blastocyst stage and then implanted into surrogate mothers to produce F0 rodents derived entirely from donor ES cells. Using an allele assay that detects the presence of human CLEC9A gene sequences (Valenzuela et al., supra), mice carrying the humanized Clec9a allele were confirmed and identified by genotyping of DNA isolated from tail snips. The neomycin selection cassette was removed by crossing offspring generated by ES clones with a deleter rodent strain expressing Cre recombinase. Figure 1C The humanized Clec9a locus after the deletion cassette is depicted in and the junction sequence is shown in Figure 1CLower panel. Mice heterozygous for the humanized Clec9a locus were crossed to obtain homozygous.

[0097] Table 2

[0098]

[0099]

[0100] Example 2. Characterization of genetically modified mice containing a humanized Clec9a gene.

[0101] To determine whether the genetically modified mice described in Example 1 express humanized Clec9a, mRNA expression was examined. RNA was extracted from spleen cells of wild-type (WT) mice (endogenous Clec9a gene is not humanized), mice heterozygous for the humanized Clec9a gene (Het), or mice homozygous for the humanized Clec9a gene (HumIn), and RT-PCR was performed using probes that detect mouse or human Clec9a. This series of experiments showed that WT and Het mice express mouse Clec9a ( Figure 2A and 2B These results also showed that Het and HumIn mice expressed humanized Clec9a ( Figure 2C-2D ).

[0102] To ensure the expression of humanized Clec9a, mice were injected with Flt3L to expand the dendritic cell population. Splenocytes were then isolated from wild-type (WT), heterozygous (Het), or homozygous (HumIn) mice and analyzed using FACS. After gating on live cells and CD45+ cells, the expression of dendritic cells was analyzed based on MHC-II + CD11c + Dendritic cells were gated and cDC1 (Xcr1 + ) cells to set gates (Fig. 3A) Then, cells expressing mouse Clec9a were sorted ( Figure 3B ) and determined that mouse Clec9a was expressed only in wild-type and heterozygous mice, the latter carrying one copy of mouse Clec9a and one copy of humanized Clec9a. As expected, HumIn mice homozygous for humanized Clec9a did not express the mouse protein. In contrast, Xcr1 derived from HumIn mice as well as Het mice + Humanized Clec9a was detected on DCs ( Figure 4B and Figure 5 ). As expected, wild-type mice did not express humanized Clec9a protein.

[0103] We then determined whether the genetic modification (humanization of the endogenous Clec9a gene in mice) affects expression in addition to dendritic cells. To this end, we stained B cells (CD19 + )( Fig. 6A ) or CD4 + and CD8 + T cells ( Figure 6B ) were gated and the expression of mouse or humanized Clec9a was detected. No expression of humanized Clec9a was detected on the aforementioned cell populations, indicating that expression was limited to dendritic cells.

[0104] In vivo proliferation assays were performed to determine whether humanized Clec9a exerted the expected function in genetically modified mice. Figure 7A-7B and Figures 8A-8B We showed that targeting humanized Clec9a expressed on dendritic cells by using an anti-human CLEC9a extracellular domain antibody and a fusion of peptides containing OT-I and OT-II antigens could elicit a strong in vivo T cell response, indicating that humanized Clec9a expressed on dendritic cells in genetically modified mice was fully functional.

[0105] Materials and Methods

[0106] Flt3L injections - Mice were injected intraperitoneally daily for 5 days with 10 μg of human Flt3L (purified in-house) in 100 μL PBS.

[0107] Flow cytometry-spleen is placed in serum-free cold RPMI 1640 for processing, and then gently homogenized using MACS dissociator (Miltenyi, catalog #130-093-235). Mechanical fragmentation of spleen cell suspension is performed by 70 μm nylon cell strainer (Miltenyi, catalog #130-110-916) with syringe plunger on the back. Red blood cells (RBC) are removed from spleen single cell suspension using ACK lysis buffer (Gibco, catalog #A10492-01). At room temperature, cells are stained for 10 minutes with live / dead Blue-Fixable blue dye (eBioscience; catalog #L23105) and mouse Fc blocker (Biolegend, catalog #101320). Cells are washed twice in cell staining buffer (Biolegend, product #420201).Cells were stained in Brilliant staining buffer (BD Biosciences catalog #566349) for thirty minutes on ice with the following monoclonal antibodies: CD45 BV510 (clone 30-F11, Biolegend, catalog #10138), CD11b BUV395 (clone M1 / 70, BD Bioscience, catalog #563553), MHC-II Alexa Fluor 700 (clone M5 / 114.152, Biolegend, catalog #107622), CD11c Pe-Cy7 (clone N418, Biolegend, catalog #117318), F4 / 80 BV605 (clone BM8, catalog #123133), CD8a BUV805 (clone 53-6.7, BD Bioscience, catalog #612898), Xcr1 BV421 (clone ZET, Biolegend, catalog #148216), human Clec9a APC (clone 8F9, Biolegend, catalog #353806, against the extracellular domain of human CLEC9a protein), CD86 PerCP-Cy-5.5 (clone GL-1, Biolegend, catalog #105028), PDL1 BV711 (clone 10.F.9G2, catalog #124319), mouse Clec9aPE (clone 42D2, eBioscience, catalog #L2129905, against the extracellular domain of mouse Clec9a), Gr-1FITC (clone RB6-8C5, Biolegend, catalog #108406, against Gr-1, a protein expressed on monocytes, granulocytes and neutrophils), CD19 APC-Cy7 (clone 6D5, Biolegend, catalog #115530), CD4 BV786 (clone RM4-5, BD Bioscience, catalog #563727), and NK1.1 BV650 (clone PKI36, BD Bioscience, catalog #564143).

[0108] RNA extraction and RT-PCR - MagMAX was used according to the manufacturer's instructions. TM Total RNA was purified using the MagMAX-96 Microarray Total RNA Isolation Kit catalog #AM1839 (Ambion by Life Technologies). TM Turbo TM DNase buffer and TURBO DNase were used to remove genomic DNA. VILO TM mRNA (up to 2.5 μg) was reverse transcribed into cDNA using Master Mix Catalog #11755500 (Invitrogen by Life Technologies). cDNA was diluted to 0.5-5 ng / μL. 2.5-25 ng cDNA input was amplified using the ABI7900HT Sequence Detection System (Applied Biosystems) using SensiFAST Hi-ROX Master Mix (1×100 mL) Catalog #CSA-01113 (BIOLINE).

[0109] In vivo proliferation assay - CD4+ T cells or CD8+ T cells were purified from B6.Cg-Tg(TcraTcrb)425Cbn / mouse spleen cells using EasySep Mouse CD4+ or CD8+ T Cell Isolation Kit (StemCell Tech; Catalog #19852 for CD4+ T cell isolation, and Catalog #19853 for CD8+ T cell isolation). These purified T cells were then labeled with CFSE cell trace dye (Invitrogen; Catalog #C34554A). 2E+06 cells were injected retro-orbitally into each HumIn mouse expressing humanized Clec9a. Twenty-four hours later, mice received the following treatment groups subcutaneously: 15 μg anti-hCLEC9a peptide (n=3), 7.5 μg anti-hCLEC9a peptide (n=3), 3.75 μg anti-hCLEC9a peptide (n=3). The anti-hCLEC9a peptide is an antibody-peptide fusion, wherein the antibody is directed against the extracellular domain of human CLEC9a, and the peptide comprises the OT-I (amino acids 257-264 of OVA) and OT-II (amino acids 323-339 of OVA) antigens. Isotype-peptide fusions (i.e., fusions between isotype antibodies and peptides comprising OT-I and OT-II antigens) were administered in a similar manner. Another group of mice received 5 mg / kg ovalbumin EndoFit ((n=3) InvivoGen; catalog #vac-pova) and 0.03366 mg / kg of the peptide alone (n=3). After 72 hours, the mice were euthanized, and the spleens were harvested and purified for flow cytometry.

Claims

1. A genetically modified rodent comprising in its genome: A humanized Clec9a gene, comprising: Rodent Clec9a nucleic acid sequence, and Human CLEC9A nucleic acid sequence, The humanized Clec9a gene encodes a humanized Clec9a polypeptide, and the humanized Clec9a polypeptide comprises an extracellular domain that is substantially identical to the extracellular domain of the human CLEC9A protein.

2. The genetically modified rodent of claim 1, wherein the extracellular domain of the humanized Clec9a polypeptide has at least 90% sequence identity with the extracellular domain of the human CLEC9A protein.

3. The genetically modified rodent of claim 1 or claim 2, wherein the human CLEC9A protein comprises the amino acid sequence shown in SEQ ID NO:

4.

4. The genetically modified rodent of any one of claims 1 to 3, wherein the humanized Clec9a protein comprises a cytoplasmic-transmembrane sequence that is substantially identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein.

5. The genetically modified rodent of claim 4, wherein the cytoplasmic-transmembrane sequence of the humanized Clec9a protein is at least 90% identical in sequence to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein.

6. The genetically modified rodent of claim 4 or claim 5, wherein the rodent Clec9a protein is an endogenous rodent Clec9a protein.

7. The genetically modified rodent of any one of claims 1 to 6, wherein the human CLEC9A nucleic acid sequence comprises exon 3 to exon 6 of the human CLEC9A gene.

8. The genetically modified rodent of any one of claims 1 to 7, wherein the rodent Clec9a nucleic acid sequence comprises exon 1 and exon 2 of the rodent Clec9a gene.

9. The genetically modified rodent of any one of claims 1 to 8, wherein the rodent Clec9a nucleic acid sequence comprises the 3' untranslated region of exon 6 of the rodent Clec9a gene.

10. The genetically modified rodent of claim 8 or claim 9, wherein the rodent Clec9a gene is an endogenous rodent Clec9a gene.

11. The genetically modified rodent of claim 1, wherein the humanized Clec9a protein comprises the amino acid sequence of SEQ ID NO:

7.

12. The genetically modified rodent of any one of claims 1 to 11, wherein the humanized Clec9a gene is operably linked to a rodent Clec9a promoter.

13. The genetically modified rodent of claim 12, wherein the rodent Clec9a promoter is an endogenous rodent Clec9a promoter.

14. The genetically modified rodent of any one of claims 1 to 13, wherein the humanized Clec9a gene is located at an endogenous rodent Clec9a locus.

15. The genetically modified rodent of claim 14, wherein the humanized Clec9a gene is formed by replacing rodent Clec9a genomic DNA at the endogenous rodent Clec9a locus with the human CLEC9A nucleic acid.

16. The genetically modified rodent of claim 15, wherein the humanized Clec9a gene is formed by replacing rodent genomic DNA comprising a stop codon in exon 3 to exon 6 of an endogenous rodent Clec9a gene with the human CLEC9A nucleic acid, and wherein the human CLEC9A nucleic acid comprises exon 3 to exon 6 of the human CLEC9A gene.

17. The genetically modified rodent of any one of claims 1 to 16, wherein the rodent is heterozygous for the humanized Clec9a gene.

18. The genetically modified rodent of any one of claims 1 to 16, wherein the rodent is homozygous for the humanized Clec9a gene.

19. The genetically modified rodent of any one of claims 1 to 18, wherein the rodent expresses the humanized Clec9a polypeptide on dendritic cells.

20. The genetically modified rodent of any one of claims 1 to 19, wherein the rodent is a mouse or a rat.

21. The genetically modified rodent of claim 20, wherein the rodent is a mouse and the humanized Clec9a polypeptide comprises the amino acid sequence shown in SEQ ID NO:

7.

22. An isolated rodent tissue or cell, the genome of which comprises a humanized Clec9a gene, the humanized Clec9a gene comprising a rodent Clec9a nucleic acid sequence and a human CLEC9A nucleic acid sequence, wherein the humanized Clec9a gene encodes a humanized Clec9a polypeptide, the humanized Clec9a polypeptide comprising an extracellular domain substantially identical to the extracellular domain of a human CLEC9A protein.

23. The isolated rodent tissue or cell of claim 22, wherein the rodent cell is a rodent embryonic stem cell.

24. The isolated rodent tissue or cell of claim 22, wherein the rodent cell is a rodent germ cell.

25. The isolated rodent tissue or cell of any one of claims 22 to 24, wherein the extracellular domain of the humanized Clec9a polypeptide is at least 90% identical in sequence to the extracellular domain of the human CLEC9A protein.

26. The isolated rodent tissue or cell of claim 25, wherein the human CLEC9A protein comprises the amino acid sequence shown in SEQ ID NO:

4.

27. The isolated rodent tissue or cell of any one of claims 22 to 26, wherein the humanized Clec9a protein comprises a cytoplasmic-transmembrane sequence that is substantially identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein.

28. The isolated rodent tissue or cell of claim 27, wherein the cytoplasmic-transmembrane sequence of the humanized Clec9a protein is at least 90% identical in sequence to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein.

29. The isolated rodent tissue or cell of any one of claims 22 to 28, wherein the rodent tissue or cell is a mouse tissue or cell, or a rat tissue or cell.

30. The isolated rodent tissue or cell of claim 29, wherein the rodent tissue or cell is a mouse tissue or cell, and the humanized Clec9a protein comprises the amino acid sequence of SEQ ID NO:

7.

31. A rodent embryo comprising the rodent embryonic stem cell of claim 23.

32. A method for preparing a genetically modified rodent, the method comprising: Modifying the rodent genome to include a humanized Clec9a gene, wherein the humanized Clec9a gene includes a rodent Clec9a nucleic acid sequence and a human CLEC9A nucleic acid sequence and encodes a humanized Clec9a polypeptide including an extracellular domain that is substantially identical to an extracellular domain of a human CLEC9A protein; and Producing the rodent comprising a modified rodent genome.

33. The method of claim 32, wherein the modification comprises Introducing a nucleic acid molecule comprising the human CLEC9A nucleic acid sequence into the genome of a rodent embryonic stem (ES) cell, obtaining a rodent ES cell in which the human CLEC9A nucleic acid sequence has been integrated into the endogenous Clec9a locus to replace the rodent Clec9a genomic DNA to form the humanized Clec9a gene, and Rodents are generated from the obtained rodent ES cells.

34. The method of claim 32 or claim 33, wherein the humanized Clec9a polypeptide comprises an extracellular domain that is at least 90% identical in sequence to the extracellular domain of the human CLEC9A protein.

35. The method of any one of claims 32 to 34, wherein the human CLEC9A protein comprises the amino acid sequence shown in SEQ ID NO:

4.

36. The method of any one of claims 32 to 35, wherein the humanized Clec9a protein comprises a cytoplasmic-transmembrane sequence that is substantially identical to a cytoplasmic-transmembrane sequence of a rodent Clec9a protein.

37. The method of claim 36, wherein the humanized Clec9a protein comprises the cytoplasmic-transmembrane sequence that is at least 90% identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein.

38. The method of claim 36 or claim 37, wherein the rodent Clec9a protein is an endogenous rodent Clec9a protein.

39. The method of any one of claims 32 to 38, wherein the human CLEC9A nucleic acid sequence comprises exon 3 to exon 6 of the human CLEC9A gene.

40. The method of any one of claims 32 to 39, wherein the rodent Clec9a nucleic acid sequence comprises exon 1 and exon 2 of the rodent Clec9a gene.

41. The method of any one of claims 32 to 40, wherein the rodent Clec9a nucleic acid sequence comprises the 3' untranslated region of exon 6 of the rodent Clec9a gene.

42. The method of any one of claims 32 to 41, wherein the humanized Clec9a gene is operably linked to the endogenous rodent Clec9a promoter at the endogenous rodent Clec9a locus.

43. The method of any one of claims 32 to 42, wherein the rodent is heterozygous for the humanized Clec9a gene.

44. The method of any one of claims 32 to 42, wherein the rodent is homozygous for the humanized Clec9a gene.

45. The rodent of any one of claims 32 to 44, wherein the rodent is a mouse or a rat.

46. ​​A targeting nucleic acid construct, comprising a human CLEC9A nucleic acid sequence to be integrated into a rodent Clec9a gene at an endogenous rodent Clec9a locus, flanked by a 5' nucleotide sequence and a 3' nucleotide sequence homologous to the nucleotide sequence at the rodent Clec9a locus, wherein the human CLEC9A nucleic acid sequence is integrated into the rodent Clec9a gene so that the rodent Clec9a genomic DNA is replaced by the human CLEC9A nucleic acid sequence, thereby forming a humanized Clec9a gene, and The humanized Clec9a gene encodes a humanized Clec9a polypeptide, and the humanized Clec9a polypeptide comprises an extracellular domain that is substantially identical to the extracellular domain of the human CLEC9A protein.

47. The targeting nucleic acid of claim 46, wherein the human CLEC9A nucleic acid sequence comprises exon 3 to exon 6 of the human CLEC9A gene.

48. The targeting nucleic acid of claim 46 or claim 47, wherein the rodent is a mouse or a rat.

49. An in vitro method for producing genetically modified rodent cells, the method comprising The targeting nucleic acid construct described in any one of claims 46 to 48 is introduced into rodent cells, thereby integrating the human CLEC9A nucleic acid sequence into the endogenous rodent Clec9a gene locus, and replacing the rodent Clec9a genomic DNA with the human CLEC9A nucleic acid sequence to form a humanized Clec9a gene, thereby producing the genetically modified rodent cells.

50. The method of claim 49, wherein the rodent cell is a rodent ES cell.

51. A method for evaluating the pharmacokinetic properties of a candidate drug, the method comprising: administering the candidate drug to a rodent as claimed in any one of claims 1 to 21; One or more assays are performed to determine the pharmacokinetic properties of the candidate drug in the rodent.

52. A method for screening a candidate drug targeting human CLEC9A, the method comprising: administering the candidate drug to a rodent as claimed in any one of claims 1 to 21; as well as One or more assays are performed to determine whether the candidate drug has an effect on the rodent.

53. The method of claim 52, wherein the one or more assays comprise an assay to measure proliferation of T cells.

54. The method of claim 53, wherein the assay measures proliferation of CD4+ T cells.

55. The method of claim 53, wherein the assay measures proliferation of CD8+ T cells.

56. The method of any one of claims 51 to 55, wherein the candidate drug is an antibody that binds to human CLEC9A.

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