Non-human animals having a humanized CLEC9A gene
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- REGENERON PHARMACEUTICALS INC
- Filing Date
- 2023-06-26
- Publication Date
- 2026-05-25
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Abstract
Description
Background Art
[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 355,948, filed Jun. 27, 2022, the entire contents of which are incorporated herein by reference.
[0002] Incorporation by Reference of Sequence Listing A 53 KB XML - formatted sequence listing named 39341WO_10900WO01_SequenceListing.xml, created on Jun. 13, 2023 and filed with the United States Patent and Trademark Office via Patent Center, is incorporated herein by reference in its entirety.
[0003] C - type lectin domain family 9 member A (Clec9a) is a C - type lectin - like receptor that is expressed as a glycosylated dimer on the cell surface of dendritic cells and a small subset of monocytes. Clec9a can mediate endocytosis rather than phagocytosis. Clec9a has a cytoplasmic immunoreceptor tyrosine - based activation - like motif (ITAM) that has been reported to mobilize the Syk kinase and induce pro - inflammatory cytokine production.
Summary of the Invention
Means for Solving the Problems
[0004] In some embodiments, disclosed herein is a genetically modified rodent that contains a humanized Clec9a gene in its genome, the humanized Clec9a gene comprising a rodent Clec9a nucleic acid sequence and a human CLEC9A nucleic acid sequence, the humanized Clec9a gene encoding a humanized Clec9a polypeptide that contains an extracellular domain substantially identical to the extracellular domain of the human CLEC9A protein.
[0005] 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 identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein (e.g., an endogenous rodent Clec9a protein).
[0006] In some embodiments, the human CLEC9A nucleic acid sequence in the humanized Clec9a gene encodes at least a substantial portion 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 set forth in SEQ ID NO: 4). In some embodiments, the human CLEC9A nucleic acid sequence comprises from exon 3 to the stop codon of exon 6 of the human CLEC9A gene. In some embodiments, the human CLEC9A nucleic acid sequence comprises from exon 3 to exon 6 of the human CLEC9A gene (i.e., to the 3' end of exon 6).
[0007] In some embodiments, the rodent Clec9a nucleic acid sequence in the humanized Clec9a gene comprises the nucleotide sequence of a rodent Clec9a gene that encodes at least a substantial portion of the 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 exons 1 and 2 of a rodent Clec9a gene (e.g., an endogenous rodent Clec9a gene).
[0008] In some embodiments, the humanized Clec9a gene comprises (i) exons 1 and 2 of a rodent Clec9a gene (e.g., the endogenous rodent Clec9a gene), and (ii) from exon 3 to the stop codon of exon 6 of the human CLEC9A gene, optionally from exon 3 to exon 6. In some embodiments, the humanized Clec9a gene comprises the 3’UTR of the rodent Clec9a gene. In some embodiments, the humanized Clec9a gene comprises (i) exons 1 and 2 of a rodent Clec9a gene (e.g., the endogenous rodent Clec9a gene), (ii) from exon 3 to exon 6 of the human CLEC9A gene, and (iii) the 3’UTR of the rodent Clec9a gene (e.g., the endogenous rodent Clec9a gene).
[0009] In some embodiments, the humanized Clec9a gene is operably linked to a rodent Clec9a promoter, such as the endogenous rodent Clec9a promoter.
[0010] 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.
[0011] In some embodiments where the humanized Clec9a gene is located at the endogenous rodent Clec9a locus, the humanized Clec9a gene is formed as a result of replacing rodent Clec9a genomic DNA with human CLEC9A nucleic acid at the endogenous rodent Clec9a locus. In some embodiments, the humanized Clec9a gene is formed as a result of replacing rodent genomic DNA containing a nucleotide sequence encoding at least a substantial portion of the extracellular domain of the endogenous rodent Clec9a protein with human CLEC9A nucleic acid encoding at least a substantial portion of the extracellular domain of the human CLEC9A protein. In some embodiments, the rodent is a mouse, and the replaced mouse genomic DNA includes from exon 3 to the stop codon of exon 6 of the endogenous mouse Clec9a gene, and the human genomic DNA includes from exon 3 to the stop codon of exon 6 of the human CLEC9A gene (optionally, from exon 3 to exon 6).
[0012] In some embodiments, the rodent is heterozygous for the humanized Clec9a gene.
[0013] In some embodiments, the rodent is homozygous for the humanized Clec9a gene.
[0014] In some embodiments, the humanized Clec9a polypeptide is expressed on dendritic cells from the humanized Clec9a gene in a rodent.
[0015] In some embodiments, the rodent is a mouse or a rat.
[0016] In some embodiments, disclosed herein is an isolated rodent tissue or cell comprising the humanized Clec9a gene described herein in its genome. In some embodiments, the rodent cell is a rodent embryonic stem cell. In some embodiments, the rodent cell is an egg or a sperm. In some embodiments, the isolated rodent tissue or cell is a mouse tissue or mouse cell, or a rat tissue or rat cell.
[0017] In some embodiments, disclosed herein is a rodent embryo comprising rodent embryonic stem cells comprising a humanized Clec9a gene as described herein.
[0018] In some embodiments, disclosed herein is a method of generating a genetically modified rodent. In some embodiments, the method comprises modifying a rodent genome to comprise 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 the human CLEC9A protein, and generating a rodent comprising the modified rodent genome.
[0019] In some embodiments, modifying the rodent genome comprises introducing a nucleic acid molecule comprising a human CLEC9A nucleic acid sequence into the genome of rodent embryonic stem (ES) cells, obtaining rodent ES cells in which the human CLEC9A nucleic acid sequence has been integrated into the endogenous Clec9a locus to replace rodent Clec9a genomic DNA, thereby forming a humanized Clec9a gene, and generating a rodent animal from the obtained rodent ES cells. In some embodiments, the human CLEC9A nucleic acid sequence encodes at least a substantial portion of the extracellular domain of the human CLEC9A protein. In some embodiments, the nucleic acid molecule introduced into the ES cells further comprises a 5' homology arm and a 3' homology arm flanking the human CLEC9A nucleic acid sequence, and the 5' homology arm and the 3' homology arm are homologous to the nucleic acid sequences of 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, such as the endogenous rodent Clec9a promoter at the endogenous rodent Clec9a locus.
[0020] In some embodiments of the method, the rodent is a mouse or a rat.
[0021] In some embodiments, disclosed herein is a targeted nucleic acid construct comprising a human CLEC9A nucleic acid sequence integrated into a rodent Clec9a gene at the endogenous rodent Clec9a locus, wherein the human CLEC9A nucleic acid sequence is flanked by a 5' nucleotide sequence and a 3' nucleotide sequence that are homologous to the nucleotide sequence at the rodent Clec9a locus, and as a result of the integration of the human CLEC9A nucleic acid sequence into the rodent Clec9a gene, the rodent Clec9a genomic DNA is replaced by the human CLEC9A nucleic acid sequence, thereby forming a humanized Clec9a gene, and the human CLEC9A nucleic acid sequence encodes at least a substantial portion of the extracellular domain of the human CLEC9A protein. In some embodiments of the targeted nucleic acid, the rodent is a rat or a mouse.
[0022] In some embodiments, disclosed herein is an in vitro method for generating a genetically modified rodent cell, the method comprising introducing into a rodent cell a targeting vector comprising a human CLEC9A nucleic acid sequence encoding at least a substantial portion of the extracellular domain of the human CLEC9A protein, flanked by rodent homology arms that mediate the integration of the human CLEC9A nucleic acid sequence into the endogenous rodent Clec9a locus, such that the rodent Clec9a genomic DNA is replaced by the human CLEC9A nucleic acid sequence, thereby forming the humanized Clec9a gene described herein, and thereby generating 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 generates a genetically modified rodent ES cell.
[0023] In some embodiments, disclosed herein is a method for evaluating the pharmacokinetic properties of a candidate agent, the method comprising administering the candidate agent to a rodent described herein and performing one or more assays to determine the pharmacokinetic properties of the candidate agent in the rodent. In some embodiments, the candidate agent is an antibody that binds to human CLEC9A. In some embodiments, the candidate agent is an antibody capable of binding to human CLEC9A.
[0024] In some embodiments, disclosed herein is a method for screening or evaluating a candidate agent that targets human CLEC9A, the method comprising administering the candidate agent to a rodent described herein and performing one or more assays to determine whether the candidate agent has an effect on the rodent, e.g., whether it induces activation of immune cells such as T cells. In some embodiments, the one or more assays include an assay for measuring T cell proliferation in the rodent. In some embodiments, the one or more assays include an assay for measuring the proliferation of CD4+ T cells in the rodent. In some embodiments, the one or more assays include an assay for measuring the proliferation of CD8+ T cells in the rodent. In some embodiments, the candidate agent includes an antibody that binds to human CLEC9A. In some embodiments, the candidate agent includes an antibody that binds to human CLEC9A and is fused to one or more peptides recognized by MHC molecules on dendritic cells of the rodent. In some embodiments, the 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
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DETAILED DESCRIPTION OF THE INVENTION
[0036] Rodents (such as, but not limited to, mice and rats) genetically modified to contain a humanized Clec9a gene are disclosed herein. Also disclosed herein are compositions (e.g., targeting vectors) and methods for producing such genetically modified rodents. The rodents disclosed herein are used, for example, but not limited to, as an in vivo system for evaluating candidate compounds directed against human CLEC9A, such as anti-human Clec9A antibodies, and can target dendritic cells for a wide range of immune-related indications including, but not limited to, autoimmune, infectious diseases, and cancer. Accordingly, methods of using genetically modified rodents for evaluating candidate compounds directed against human CLEC9A are also disclosed herein.
[0037] Humanization of Rodent Clec9a Clec9a is a C-type lectin-like receptor that is expressed as a glycosylated dimer on the cell surface of dendritic cells and a small subset of monocytes. Clec9a can mediate endocytosis rather than phagocytosis. Clec9a has a cytoplasmic immunoreceptor tyrosine-based activation-like motif (the "ITAM") in its N-terminal portion. The ITAM is a conserved sequence in which 4 amino acids are repeated twice and is thought to recruit the Syk kinase and induce the production of pro-inflammatory cytokines. See, for example, Huysamen et al., J Biol Chem 283(24), 16693-16701(2008), which is incorporated herein by reference in its entirety.
[0038] Exemplary Clec9a sequences, including the 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. An alignment of the protein sequences of human CLEC9A, mouse Clec9a, and humanized (mouse-human hybrid) Clec9a is shown in FIG. 1D.
[0039] For the sake of simplicity, the exon numbering in this specification refers to the coding exons of the Clec9a gene. For example, exon 1 of the Clec9a gene refers to the first coding exon of the Clec9a gene in this specification. [Table 1]
[0040] In some embodiments, the rodents disclosed herein contain a humanized Clec9a gene in the germline.
[0041] In some embodiments, the rodents disclosed herein contain a humanized Clec9a gene that contains the nucleotide sequence of the rodent Clec9a gene (e.g., the endogenous rodent Clec9a gene) and the nucleotide sequence of the human CLEC9A gene in its genome. As used herein, "the nucleotide sequence of a gene" includes the genomic sequence, mRNA sequence, or cDNA sequence of all or part of that gene. By way of non-limiting example, the nucleotide sequence of the human CLEC9A gene includes the genomic sequence, mRNA sequence, or cDNA sequence of all or part of the 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 such that the humanized Clec9a gene in the rodent genome encodes a humanized Clec9a protein that maintains the protein structure of Clec9a (including the ITAM-containing cytoplasmic domain, transmembrane domain, and extracellular domain) and performs the functions of the Clec9a protein (e.g., mobilizes the Syk kinase and induces pro-inflammatory cytokines).
[0042] As used herein, the "human CLEC9A" gene and protein refer to the CLEC9A gene and protein derived from humans. 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 having at least 95% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the human CLEC9A protein comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the human CLEC9A protein comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 4.
[0043] As used herein, the "rodent Clec9a" gene and protein refer to the Clec9a gene and protein derived from rodents (e.g., mouse or rat). In some embodiments, the mouse Clec9a protein comprises the 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 with 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 with 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 with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the rat Clec9a protein comprises the 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 with 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 with the amino acid sequence of SEQ ID NO: 25. In some embodiments, the rat Clec9a protein comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 25.
[0044] In some embodiments, the genetically modified rodent contains a humanized Clec9a gene in its genome, and this humanized Clec9a gene encodes a humanized Clec9a protein containing an extracellular domain that is substantially identical to the extracellular domain of the human CLEC9A protein. In some embodiments, the extracellular domain that is substantially identical to the extracellular domain of the human CLEC9A protein exhibits the same functionality (e.g., ligand binding properties) as the extracellular domain of the human CLEC9A protein. An extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human CLEC9A protein" can be a polypeptide having at least 95% sequence identity with the extracellular domain of the human CLEC9A protein, a polypeptide having at least 98% sequence identity with the extracellular domain of the human CLEC9A protein, or a polypeptide having at least 99% sequence identity with the extracellular domain of the human CLEC9A protein. An extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human CLEC9A protein" can be a polypeptide having 100% sequence identity with the extracellular domain of the human CLEC9A protein. Alternatively or additionally, an 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, an extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human CLEC9A protein" can be a polypeptide that is different from the extracellular domain of the human CLEC9A protein only at the N-terminal or C-terminal portion of the extracellular domain (i.e., within 5 to 10 amino acids from the N-terminal or C-terminal of the extracellular domain) by having an addition, deletion, and / or substitution of amino acids. Alternatively or additionally, an extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human CLEC9A protein" can be a polypeptide having one or more of the features detailed above, for example, a polypeptide having at least 95% sequence identity with the extracellular domain of the human CLEC9A protein and differing from the extracellular domain of the human CLEC9A protein by no more than 5 amino acids only at the N-terminal portion or C-terminal portion of the extracellular domain, or a polypeptide having at least 98% sequence identity with the extracellular domain of the human CLEC9A protein and differing from the extracellular domain of the human CLEC9A protein by no more than 3 amino acids only at the N-terminal portion or C-terminal portion of the extracellular domain. In certain embodiments, the human CLEC9A protein comprises the amino acid sequence set forth in SEQ ID NO: 4, and its extracellular domain is composed of amino acids 57-241 of SEQ ID NO: 4. In some embodiments, the humanized Clec9a gene encodes a humanized Clec9a protein whose extracellular domain is substantially identical to the extracellular domain of the human CLEC9A protein set forth in SEQ ID NO: 4, i.e., substantially identical to amino acids 57-241 of SEQ ID NO: 4. For example, the humanized Clec9a gene encodes a humanized Clec9a protein having 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 to 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 to 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 to 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 to 239 of SEQ ID NO: 4.
[0045] In some embodiments, the humanized Clec9a gene encodes a humanized Clec9a protein that contains a cytoplasmic-transmembrane sequence that is substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein, such as an endogenous rodent Clec9a protein (i.e., a sequence that includes both a transmembrane domain and a cytoplasmic domain). In some embodiments, the cytoplasmic-transmembrane sequence that is substantially identical to the cytoplasmic-transmembrane sequence of the endogenous rodent Clec9a protein exhibits the same functionality (e.g., signal transduction and / or interaction with intracellular molecules) as the cytoplasmic-transmembrane sequence of a rodent Clec9a protein, such as the endogenous rodent Clec9a protein. A 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% sequence identity to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein, or a polypeptide that has at least 98% sequence identity to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein. A cytoplasmic-transmembrane sequence or polypeptide that is "substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein" can be a polypeptide that is identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein. Alternatively or additionally, a cytoplasmic-transmembrane sequence or polypeptide that is "substantially identical to the cytoplasmic-transmembrane sequence of a rodent Clec9a protein" can be a polypeptide that differs from the cytoplasmic-transmembrane sequence of a rodent Clec9a protein by no more than 3 amino acids, a polypeptide that differs from the cytoplasmic-transmembrane sequence of a rodent Clec9a protein by no more than 2 amino acids, or a polypeptide that differs from the cytoplasmic-transmembrane sequence of a 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" can 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 having an addition, deletion, or substitution of an amino acid in the N-terminal portion or C-terminal portion of the cytoplasmic-transmembrane sequence.Alternatively or additionally, a 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 features detailed above. For example, a polypeptide having at least 95% sequence identity with the cytoplasmic-transmembrane sequence of the rodent Clec9a protein and differing 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 having at least 95% sequence identity with the cytoplasmic-transmembrane sequence of the rodent Clec9a protein and differing from the cytoplasmic-transmembrane sequence of the rodent Clec9a protein by no more than 2 amino acids at the N-terminus or C-terminus. "The N-terminal portion or C-terminal portion of the cytoplasmic-transmembrane sequence" means within 3 to 5 amino acids from the N-terminus of the cytoplasmic domain or from the C-terminus of the transmembrane domain. 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 the 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 the endogenous rat Clec9a protein).
[0046] In some embodiments, the humanized Clec9a gene in the genome of the genetically modified rodent comprises the nucleotide sequence of the human CLEC9A gene ("human CLEC9A nucleotide sequence") and the nucleotide sequence of the rodent Clec9a gene ("rodent Clec9a nucleotide sequence", e.g., the endogenous rodent Clec9a nucleotide sequence), and the human CLEC9A nucleotide sequence encodes at least a substantial portion of the extracellular domain of the human CLEC9A protein. Examples of a substantial portion 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, the substantial portion of the extracellular domain comprises amino acids 57 - 241 of SEQ ID NO: 4. In some embodiments, the substantial portion of the extracellular domain comprises amino acids 57 - 240 of SEQ ID NO: 4. In some embodiments, the substantial portion of the extracellular domain comprises amino acids 57 - 239 of SEQ ID NO: 4. In some embodiments, the substantial portion of the extracellular domain comprises amino acids 58 - 241 of SEQ ID NO: 4. In some embodiments, the substantial portion 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 the human CLEC9A protein (e.g., the human CLEC9A protein defined in SEQ ID NO: 4). In some embodiments, the human CLEC9A nucleotide sequence is a genomic fragment of the human CLEC9A gene. In some embodiments, the human CLEC9A nucleotide sequence is a genomic fragment of the human CLEC9A gene that includes from exon 3 to the stop codon of exon 6. In some embodiments, the human CLEC9A nucleotide sequence is a genomic fragment of the human CLEC9A gene that includes from exon 3 to exon 6, i.e., up to the 3' untranslated region of exon 6.
[0047] 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, and the rodent Clec9a nucleotide sequence encodes a polypeptide 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 rodent Clec9a nucleotide sequence present in the humanized Clec9a gene encodes the cytoplasmic-transmembrane sequence of the 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., the coding portion) and exon 2 of a rodent (e.g., 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 coding exon 1 (and optionally also the 5’UTR of exon 1) and exon 2 of a mouse Clec9a gene (e.g., an endogenous mouse Clec9a gene). 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.
[0048] In some embodiments, the humanized Clec9a gene is operably linked to a 5’ regulatory sequence of rodent Clec9a, 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 5’ regulatory sequence of rodent Clec9a.
[0049] In some embodiments, the humanized Clec9a gene is at the endogenous murine Clec9a locus. In some embodiments, the humanized Clec9a gene is at a locus other than the endogenous murine Clec9a locus, for example, as a result of random integration. In some embodiments, the humanized Clec9a gene is 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 at a locus other than the endogenous murine Clec9a locus, the mouse is unable to express murine Clec9a protein, for example, as a result of inactivation (e.g., deletion in whole or in part) of the endogenous murine Clec9a gene.
[0050] In some embodiments where the humanized Clec9a gene is at the endogenous murine Clec9a locus, the humanized Clec9a gene may result from replacement of the nucleotide sequence of the endogenous murine Clec9a gene with the nucleotide sequence of the human CLEC9A gene at the endogenous murine Clec9a locus.
[0051] In some embodiments, the nucleotide sequence of the endogenous rodent Clec9a gene at the replaced endogenous rodent Clec9a locus is a genomic fragment of the endogenous rodent Clec9a gene encoding at least a substantial portion 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 substantial portion of the extracellular domain of the endogenous mouse Clec9a protein. For example, the extracellular domain of mouse Clec9a of SEQ ID NO: 2 is defined by amino acids 57-238, and examples of substantial portions 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, the substantial portion of the extracellular domain of the mouse Clec9a protein includes amino acids 57-238 of SEQ ID NO: 2. In some embodiments, the substantial portion of the extracellular domain of the mouse Clec9a protein includes amino acids 57-237 of SEQ ID NO: 2. In some embodiments, the substantial portion of the extracellular domain of the mouse Clec9a protein includes amino acids 57-236 of SEQ ID NO: 2. In some embodiments, the substantial portion of the extracellular domain of the mouse Clec9a protein includes amino acids 58-238 of SEQ ID NO: 2. In some embodiments, the substantial portion of the extracellular domain of the mouse Clec9a protein includes amino acids 59-238 of SEQ ID NO: 2. In some embodiments, the replaced mouse Clec9a genomic fragment includes from exon 3 to the stop codon of exon 6.
[0052] 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 comprises all or part of the exons of the human CLEC9A gene that encode at least a substantial portion of the extracellular domain of the human CLEC9A protein. Examples of substantial portions of the extracellular domain of human CLEC9A are described above, such as amino acids 57-241, 57-240, 57-239, 58-241, or 59-241 of SEQ ID NO: 4. In some embodiments, the substantial portion of the extracellular domain of human CLEC9A comprises amino acids 57-241 of SEQ ID NO: 4. In some embodiments, the substantial portion of the extracellular domain of human CLEC9A comprises amino acids 57-240 of SEQ ID NO: 4. In some embodiments, the substantial portion of the extracellular domain of human CLEC9A comprises amino acids 57-239 of SEQ ID NO: 4. In some embodiments, the substantial portion of the extracellular domain of human CLEC9A comprises amino acids 58-241 of SEQ ID NO: 4. In some embodiments, the substantial portion 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 from exon 3 to the stop codon of exon 6 of human CLEC9A. In some embodiments, the human genomic fragment comprises from exon 3 to the 3' end of exon 6 of human CLEC9A (i.e., including the 3' UTR of human CLEC9A).
[0053] In some embodiments, the human CLEC9A nucleotide sequence inserted within the endogenous rodent Clec9a locus is operably linked to a genomic sequence of a rodent Clec9a gene encoding a polypeptide that is substantially identical to the cytoplasmic-membrane spanning sequence of the rodent Clec9a protein (endogenous rodent, such as a mouse or rat Clec9a protein). In some embodiments, the genomic sequence of the rodent Clec9a gene includes exon 1 and exon 2 of the rodent Clec9a gene (e.g., the endogenous mouse or rat Clec9a gene). In some embodiments, the genomic sequence of the rodent Clec9a gene also includes the 3’UTR of the rodent Clec9a gene.
[0054] In some embodiments, the rodent is a mouse, and the genomic fragment of endogenous mouse Clec9a in the endogenous mouse Clec9a locus including from exon 3 to the stop codon of exon 6 of the mouse Clec9a gene (encoding the mouse Clec9a extracellular domain) is replaced with a genomic fragment of the human CLEC9A gene including from exon 3 to the stop codon of exon 6 encoding the human CLEC9A extracellular domain. In some embodiments, the humanized Clec9a gene is formed at the endogenous mouse Clec9a locus and includes exon 1-2 of the mouse Clec9a gene, exons 3 to 6 of the human CLEC9A gene, and subsequently optionally the 3’UTR of the mouse Clec9a gene.
[0055] 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.
[0056] In some embodiments, the humanized Clec9a gene results in the expression of a humanized Clec9a protein encoded in rodents. In some embodiments, the humanized Clec9a protein is expressed in cells and tissues where the counterpart rodent Clec9a protein in control rodents (e.g., rodents without the humanized Clec9a gene) is typically expressed, e.g., on dendritic cells.
[0057] In some embodiments, the rodents disclosed herein are unable to express rodent Clec9a protein as a result of, for example, inactivation (e.g., complete or partial deletion) or replacement (in whole or in part) of the endogenous rodent Clec9a gene.
[0058] Rodent species and strains In some embodiments, the rodents of the disclosure include, by way of non-limiting example, mice, rats, and hamsters. In some embodiments, the rodent is selected from the superfamily Muroidea. In some embodiments, the rodents of the disclosure are from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, vole), Muridae (purebred mice and rats, jerboas, spiny mice, tufted mice), Nesomyidae (climbing mice, rock mice, white-tailed rat, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormouse), and Spalacidae (e.g., blind mole rats, bamboo rats, and zokors). In some embodiments, the rodents of the disclosure are selected from purebred mice or rats (Muridae), jerboas, spiny mice, and tufted mice. In some embodiments, the mice of the disclosure are from a member of the family Muridae.
[0059] In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a mouse of the C57BL strain selected from 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 mouse of the 129 strain selected from the group consisting of the strains 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 entireties). In some embodiments, the rodent is a mouse that is a mixture of the 129 strain and the C57BL / 6 strain. In some embodiments, the rodent is a mouse that is a mixture of the aforementioned 129 strains or the aforementioned BL / 6 strains. In some embodiments, the rodent is a mouse of the BALB strain, e.g., a BALB / c strain mouse. In some embodiments, the rodent is a mouse that is a mixture of the BALB strain and another aforementioned strain.
[0060] In some embodiments, the rodent is a rat. In some specific embodiments, the rat is selected from Wistar rats, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strains described herein are a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
[0061] Tissues and Cells of Genetically Modified Rodents In some embodiments, isolated rodent cells or tissues comprising a humanized Clec9a gene in their genome are disclosed herein.
[0062] In some embodiments, the tissue is selected from fat, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, egg, and combinations thereof.
[0063] In some embodiments, the cell is selected from dendritic cells or monocytes.
[0064] 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.
[0065] Compositions and Methods for Producing Humanized Rodents 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.
[0066] In some embodiments, the targeting vector comprises a human CLEC9A nucleotide sequence encoding at least a substantial portion of the extracellular domain of the human CLEC9A protein described hereinabove. In some embodiments, the human CLEC9A nucleotide sequence encodes a polypeptide comprising amino acids 57 to 241 of SEQ ID NO: 4. In some embodiments, the human CLEC9A nucleotide sequence comprises from exon 3 to the stop codon of exon 6 encoding the extracellular domain of the human CLEC9A protein.
[0067] The targeting vector also comprises 5' rodent sequences and 3' rodent sequences located flanking the human nucleotide sequence to be integrated, which are also known as 5' homology arms and 3' homology arms and mediate homologous recombination and integration of the human nucleotide sequence into a target rodent locus (e.g., the endogenous rodent Clec9a locus) to form the humanized gene described hereinabove. Typically, the 5' and 3' flanking rodent sequences are identical or substantially identical (e.g., at least 98% or at least 99% identical) to the nucleotide sequences flanking the corresponding rodent nucleotide sequence at the target rodent locus to be replaced by the human nucleotide sequence. In some embodiments, the targeting vector comprises the humanized gene described hereinabove. In some embodiments, the targeting vector comprises a humanized Clec9a gene comprising the human CLEC9A nucleotide sequence and the rodent Clec9a nucleotide sequence described hereinabove. In some embodiments, the targeting vector comprises a humanized Clec9a gene, the humanized Clec9a gene comprising exons 1-2 of the rodent Clec9a gene and exons 3-6 of the human CLEC9A gene, and optionally the humanized Clec9a gene is flanked by 5' rodent homology arms and 3' rodent homology arms.
[0068] In some embodiments, the targeting vector includes a selectable marker gene. The selectable marker gene can be inserted into an intron of the human genomic sequence to be integrated. In some embodiments, the selectable marker gene is provided as a self-deleting cassette that can be deleted after successful integration of the human nucleotide sequence.
[0069] In an exemplary embodiment, the targeting vector is generated from a bacterial artificial chromosome (BAC) clone carrying rodent Clec9a genomic DNA using bacterial homologous recombination and VELOCIGENE® technology (see, e.g., U.S. Patent No. 6,586,251, which is hereby incorporated by reference in its entirety, and Valenzuela et al. (2003) Nature Biotech. 21(6):652-659). As a result of bacterial homologous recombination, the rodent genomic sequence is deleted from the BAC clone, the human nucleotide sequence is inserted, and a modified BAC clone is generated that retains the human nucleotide sequence flanked by 5' and 3' rodent homology arms. In some embodiments, the human nucleotide sequence can be a cDNA sequence or human genomic DNA. The modified BAC clone can be introduced into rodent embryonic stem (ES) cells when linearized.
[0070] In some embodiments, the invention provides for the use of the targeting vectors described herein for generating modified rodent embryonic stem (ES) cells. The targeting vectors can be introduced into rodent ES cells, for example, by electroporation. Both mouse ES cells and rat ES cells are described in the art. For example, U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 7,294,754, and U.S. Patent Application Publication No. 20080078000 A1, which describe mouse ES cells and the VELOCIMOUSE® method for generating genetically modified mice (all of which are incorporated herein by reference); U.S. Patent Application Publication No. 2014 / 0235933 A1 (Regeneron Pharmaceuticals, Inc.), U.S. Patent Application Publication No. 2014 / 0310828 A1 (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, which describe rat ES cells and methods for generating genetically modified rats (all of which are incorporated herein by reference). The foregoing documents can be used to generate modified rodent embryos and, in turn, rodents.
[0071] In some embodiments, ES cells having a desired human nucleotide sequence (e.g., a human CLEC9A nucleotide sequence) integrated into the genome can be selected. In some embodiments, the ES cells are selected based on a rodent allele loss assay and / or a human allele gain assay. In some embodiments, the selected ES cells are then used as donor ES cells for injection into a pre-morula stage embryo (e.g., an 8-cell stage embryo) by the VELOCIMOUSE® method (see, e.g., U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 7,294,754, and U.S. Patent Application Publication No. 2008-0078000 A1, which are all incorporated by reference in their entirety), or by the methods described in U.S. Patent Application Publication No. 2014 / 0235933 A1 and U.S. Patent Application Publication No. 2014 / 0310828 A1, which are both incorporated by reference in their entirety. In some embodiments, the embryo containing the donor ES cells is incubated and transferred to a surrogate mother to produce F0 rodents. Rodent pups carrying the human nucleotide sequence can be identified by genotyping DNA isolated from a tail fragment using a rodent allele loss assay and / or a human allele gain assay.
[0072] In some embodiments, rodents heterozygous for the humanized gene can be mated to produce homozygous rodents.
[0073] The humanized rodents described herein (i.e., rodents containing the humanized Clec9a gene) can breed or mate with another rodent. Accordingly, methods of breeding and progeny obtained from such breeding are also embodiments of the present disclosure.
[0074] In some embodiments, provided is a method comprising breeding a first rodent described above herein, e.g., a rodent having a humanized Clec9a gene in its genome, with a second rodent, resulting in progeny rodents having a humanized Clec9a gene in their genomes. The progeny may have other desirable phenotypes or genetic modifications inherited from the second rodent used in the breeding. In some embodiments, the progeny rodents are heterozygous for one or more humanized genes derived from the first rodent. In some embodiments, the progeny rodents are homozygous for the humanized gene derived from the first rodent.
[0075] In some embodiments, provided are progeny rodents having a humanized Clec9a gene in their genomes, which are generated by a method comprising breeding a first rodent having a humanized Clec9a gene in its genome with a second rodent. In some embodiments, the progeny rodents are heterozygous for the humanized Clec9a gene derived from the first rodent. In some embodiments, the progeny rodents are homozygous for the humanized Clec9a gene derived from the first rodent.
[0076] In some embodiments, disclosed herein is an in vitro method for generating genetically modified rodent cells, the method comprising introducing into a rodent cell a targeting vector comprising a human CLEC9A nucleic acid sequence encoding at least a substantial portion of the extracellular domain of the human CLEC9A protein, flanked by rodent homology arms that mediate the integration of the human CLEC9A nucleic acid sequence into the endogenous rodent Clec9a locus, such that the rodent Clec9a genomic DNA is replaced by the human CLEC9A nucleic acid sequence, thereby forming the humanized Clec9a gene described herein and generating genetically modified rodent cells. In some embodiments, the rodent cells are mouse cells or rat cells. In some embodiments, the rodent cells are rodent ES cells and the method generates genetically modified rodent ES cells.
[0077] Methods of Using Humanized Rodents The rodents disclosed herein provide a useful in vivo system and source of biological materials for identifying and testing compounds for the potential to treat human diseases, including immune-related indications where CLEC9A can play a role, i.e., conditions that can be involved in or result from dysregulated immune function such as autoimmunity, infectious diseases, and cancer.
[0078] In some embodiments, the genetically modified rodents disclosed herein are used to evaluate agents that target 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 the rodents disclosed herein at various 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 more). The agent may be administered via any desired route of administration (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.).
[0079] 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 the genetically modified rodent. Blood is isolated from the animal at various 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). Various assays can be performed to determine the pharmacokinetic properties. Such pharmacokinetic properties include, but are not limited to, how the animal processes the agent into various metabolites (or detection of the presence or absence of one or more drug metabolites, including toxic metabolites), drug half-life, the circulating levels of the agent after administration (e.g., the serum concentration of the agent), anti-drug response (e.g., anti-drug antibodies), absorption and distribution of the agent, route of administration, and excretion and / or clearance pathways of the agent.
[0080] In some embodiments, to determine whether a drug has an effect on a rodent, e.g., whether the drug induces dendritic cell-dependent activation of immune cells (such as T cells), the genetically modified rodents disclosed herein are used to evaluate a drug that targets human CLEC9A (such as an antibody). Such an evaluation can be achieved by administering the drug to the rodents disclosed herein and evaluating the immune cells in the rodents to determine whether there is activation of immune cells, e.g., proliferation of T cells and / or production of cytokines. Comparison can be made with control rodents that are not administered the drug or are administered a control drug (such as an isotype antibody). In some embodiments, the rodents disclosed herein, e.g., mice or rats that express a humanized Cle9a protein on dendritic cells, are administered an antibody-peptide fusion, wherein the antibody binds to the extracellular domain of human CLEC9A and the peptide comprises one or more antigens recognized by major histocompatibility complex (MHC) molecules on dendritic cells. In some embodiments, the peptide comprises an antigen recognized by MHC class I molecules (e.g., amino acids 257-264 of ovalbumin, also known as the OTI peptide) and an antigen recognized by MHC class II molecules (e.g., amino acids 323-339 of ovalbumin, also known as the OTII peptide). The rodents are then examined to determine whether exposure to the antibody-peptide fusion induces a T cell response (such as T cell proliferation). In some embodiments, the rodents are examined to measure whether there is proliferation of CD4+ T cells. In some embodiments, the rodents are examined to measure whether there is proliferation of CD8+ T cells. Methods and assays for evaluating T cell responses in animals are well known in the art and are also described in the following example sections of this specification. Comparison can be made with control rodents that are not administered the antibody-peptide fusion or are administered an isotype antibody-peptide fusion, an isotype antibody, or the peptide alone. Any observed T cell response and the degree of the response can be correlated with the effectiveness of the antibody being tested.
Example
[0081] This description is further illustrated by the following examples, which should in no way be construed as limiting. The content of all cited references (including references cited throughout this application, issued patents, and published patent applications) is hereby incorporated by reference in its entirety into this specification.
[0082] Example 1. Humanization of the endogenous mouse Clec9a gene A targeting vector for humanizing the endogenous Clec9a gene was constructed using bacterial artificial chromosome (BAC) clones and VELOCIGENE® technology (see, for example, U.S. Patent No. 6,586,251, the content of which is incorporated herein by reference, and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659).
[0083] The BAC clone RP23-248K14 containing the mouse Clec9a gene was used and modified as follows. Briefly, a DNA fragment was generated 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 the complete human 3' UTR), a 4,809 bp self-deleting neomycin cassette, and a 3' mouse homologous sequence (100 bp). This DNA fragment was used to modify the BAC clone RP23-248K14 via homologous recombination in bacterial cells. As a result, a 7,138 bp mouse Clec9a genomic fragment (encoding the extracellular domain of mouse Clec9a) in the BAC clone was replaced by approximately 4,840 bp of the human CLEC9A genomic fragment followed by approximately 4,809 bp of the self-deleting neomycin cassette. Specifically, the replaced mouse Clec9a genomic fragment contained all exons 3 to the stop codon of the last coding exon (exon 6) of the mouse Clec9a gene (Figs. 1A - 1B). The inserted human CLEC9A genomic fragment contained exons 3, 4, 5 of human CLEC9A and their respective introns up to the stop codon of the last coding exon (exon 6) and included the full-length 3' UTR (Figs. 1A - 1B). The resulting modified BAC clone contained, in the 5' to 3' direction, (i) a 5' mouse homologous arm containing approximately 45.6 kb of mouse genomic DNA including the 5' UTR of mouse Clec9a and exons 1 and 2 of mouse Clec9a, (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) an 112.6 kb 3' mouse homologous arm containing the 3' UTR of mouse Clec9a and the remaining mouse genomic DNA in the original BAC clone (Figs. 1A - 1B). The junction sequences are also described at the bottom of Fig. 1B. A part of the modified BAC clone containing the human CLEC9A genomic fragment, the self-deleting neomycin cassette, and the upstream and downstream of the insertion junction is specified in SEQ ID NO: 5.The amino acid sequence of the protein encoded by the humanized Clec9a gene is specified in SEQ ID NO: 7 and FIG. 1D. An alignment of this humanized Clec9a protein (“7765 mutant protein”), mouse Clec9a protein (SEQ ID NO: 2), and human Clec9a protein (SEQ ID NO: 4) is shown in FIG. 1D.
[0084] Using the above-described modified BAC clone containing the humanized Clec9a gene, mouse embryonic stem (ES) cells were electroporated to generate modified ES cells containing the humanized Clec9a gene. Targeted positive ES cells containing the humanized Clec9a gene were identified by an assay (Valenzuela et al., supra) that detects the presence of the human CLEC9A sequence (e.g., exons 3-6 of human CLEC9A), and the loss and / or retention of the mouse Clec9a sequence (e.g., exons 3-6 of mouse Clec9a) was determined. Table 1 specifies the primers and probes used to confirm the humanization of the endogenous Clec9a gene described above (FIGS. 1A-1B). Properly targeted ES cells were selected as donor ES cells and injected into pre-morula stage embryos (e.g., 8-cell stage embryos) by the VELOCIMOUSE® method (e.g., Poueymirou et al., 2007, Nature Biotech. 25(1):91-99, which is hereby incorporated by reference in its entirety, U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 7,294,754, and U.S. Patent Application Publication No. 20080078000 A1) or the methods described in U.S. Patent Application Publication No. 2014 / 0235933 A1 and U.S. Patent Application Publication No. 2014 / 0310828 A1 (both of which are hereby incorporated by reference in their entirety). Embryos containing donor ES cells were incubated to the blastocyst stage and then transferred to surrogate mothers to produce F0 rodents that were entirely derived from the donor ES cells. Mouse carrying the humanized Clec9a allele was confirmed and identified by genotyping of DNA isolated from tail fragments using an allelic modification assay (Valenzuela et al., supra) that detects the presence of the human CLEC9A gene sequence. The progeny generated from the ES clone were mated with a deletor rodent strain expressing Cre recombinase to remove the neomycin selection cassette. The humanized Clec9a locus after cassette deletion is illustrated in FIG. 1C, and the junction sequence is shown at the bottom of FIG. 1C. Heterozygous mice for the humanized Clec9a locus were mated to obtain homozygotes.
Table 2
[0085] Example 2. Characterization of Genetically Modified Mice Containing Humanized Clec9a Gene To determine whether the genetically modified mice described in Example 1 expressed humanized Clec9a, mRNA expression was examined. RNA was extracted from splenocytes obtained from wild-type (WT) mice (without humanization of the endogenous Clec9a gene), mice heterozygous for the humanized Clec9a gene (Het), or mice homozygous for the humanized Clec9a gene (HumIn), and subjected to RT-PCR using a probe that detects mouse or human Clec9a. This experimental line showed that WT mice and Het mice expressed mouse Clec9a (Figures 2A and 2B). These results also showed that Het mice and HumIn mice expressed humanized Clec9a (Figures 2C - 2D).
[0086] To ensure that humanized Clec9a was expressed, mice were injected with Flt3L to expand the dendritic cell population. Subsequently, splenocytes were isolated from wild-type (WT) mice, heterozygous (Het) mice, or homozygous (HumIn) mice and analyzed using FACS. Dendritic cells were gated on live cells and CD45 + cells and then gated based on MHC-II + CD11c + and subsequently gated on cDC1 (Xcr1 + ) cells (Figure 3A). Next, cells expressing mouse Clec9a were sorted (Figure 3B), and it was determined that mouse Clec9a was expressed only in wild-type mice and heterozygous mice, and the latter carried 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. Conversely, humanized Clec9a was detected in Xcr1 +Detected in DCs (Figure 4B and Figure 5). As expected, wild-type mice did not express the humanized Clec9a protein.
[0087] Next, it was determined whether genetic modification (humanization of the endogenous Clec9a gene in mice) affected expression beyond dendritic cells. For this purpose, B cells (CD19 + )(Figure 6A) or CD4 + and CD8 + T cells (Figure 6B) were gated, and expression of either mouse Clec9a or humanized Clec9a was probed. Expression of humanized Clec9a was not detected on the aforementioned cell populations, indicating that expression was limited to dendritic cells.
[0088] To determine whether humanized Clec9a functions as intended in genetically modified mice, an in vivo proliferation assay was performed. In Figures 7A - 7B and Figures 8A - 8B, it has been demonstrated that by using a fusion of an anti-human CLEC9a extracellular domain antibody with peptides containing the OT-I antigen and the OT-II antigen to target humanized Clec9a expressed on dendritic cells, a strong T cell response was induced in vivo, indicating that humanized Clec9a expressed on dendritic cells in genetically modified mice is fully functional.
[0089] Materials and Methods Flt3L injection - Mice were intraperitoneally injected daily for 5 days with 10 μg of human Flt3L (purified in-house) in 100 μL of PBS.
[0090] Flow cytometry - The spleen was processed in cold serum-free RPMI 1640 and homogenized using a gentle MACS dissociator (Miltenyi, catalog number 130-093-235). The spleen cell suspension was mechanically disrupted by passing it through a 70-μm nylon cell strainer (Miltenyi, catalog number 130-110-916) attached to the rear of a syringe plunger. Red blood cells (RBCs) were removed from the spleen single-cell suspension using ACK lysis buffer (Gibco, catalog number A10492-01). Cells were washed with Live / Dead Blue-Fixable Blue Dye (eBioscience, catalog number L23105) and mouse Fc block (BioLegend, catalog number 101320) for 10 minutes at room temperature. Cells were washed twice in cell staining buffer (BioLegend, catalog number 420201). Cells were stained in Brilliant Stain Buffer (BD Biosciences, catalog number 566349) on ice for 30 minutes using the following monoclonal antibodies.CD45 BV510 (clone 30-F11, BioLegend, catalog number 10138), CD11b BUV395 (clone M1 / 70, BD Biosciences, catalog number 563553), MHC-II Alexa Fluor 700 (clone M5 / 114.152, BioLegend, catalog number 107622), CD11c Pe-Cy7 (clone N418, BioLegend, catalog number 117318), F4 / 80 BV605 (clone BM8, catalog number 123133), CD8a BUV805 (clone 53-6.7, BD Biosciences, catalog number 612898), Xcr1 BV421 (clone ZET, BioLegend, catalog number 148216), human Clec9a APC (clone 8F9, BioLegend, catalog number 353806, targeting the extracellular domain portion of human CLEC9a protein), CD86 PerCP-Cy-5.5 (clone GL-1, BioLegend, catalog number 105028), PDL1 BV711 (clone 10.F.9G2, catalog number 124319), mouse Clec9a PE (clone 42D2, eBioscience, catalog number L2129905, targeting the extracellular domain of mouse Clec9a), Gr-1 FITC (clone RB6-8C5, BioLegend, catalog number 108406, targeting Gr-1, a protein expressed on monocytes, granulocytes, and neutrophils), CD19 APC-Cy7 (clone 6D5, BioLegend, catalog number 115530), CD4 BV786 (clone RM4-5, BD Biosciences, catalog number 563727), and NK1.1 BV650 (clone PKI36, BD Biosciences, catalog number 564143).
[0091] Total RNA was purified according to the manufacturer's specifications using the MagMAX™-96 for Microarrays Total RNA Isolation Kit, catalog number AM1839 (Ambion by Life Technologies). Genomic DNA was removed using the MagMAX™ Turbo™ DNase Buffer and TURBO DNase of the MagMAX kit (Ambion by Life Technologies) listed above. mRNA (up to 2.5 μg) was reverse transcribed to cDNA using SuperScript® VILO® Master Mix, catalog number 11755500 (Invitrogen by Life Technologies). The cDNA was diluted to 0.5 - 5 ng / μL. Using the ABI 7900HT Sequence Detection System (Applied Biosystems), 2.5 - 25 ng of cDNA input was amplified using SensiFAST Hi-ROX MasterMix (1×100 mL), catalog number CSA-01113 (Bioline).
[0092] For the in vivo proliferation assay, CD4+ or CD8+ T cells were purified from B6.Cg-Tg(TcraTcrb)425Cbn / mouse splenocytes using the EasySep Mouse CD4+ or CD8+ T Cell Isolation Kit (StemCell Technologies, catalog number 19852 for CD4+ T cell isolation and catalog number 19853 for CD8+ T cell isolation). These purified T cells were then labeled with the CFSE cell trace dye (Invitrogen, catalog number C34554A). 2E+06 cells were retro-orbitally injected into each HumIn mouse expressing humanized Clec9a. 24 hours later, the following treatment groups were administered subcutaneously to the mice: 15 μg of anti-hCLEC9a-peptide (n = 3), 7.5 μg of anti-hCLEC9a-peptide (n = 3), 3.75 μg of anti-hCLEC9a-peptide (n = 3). The anti-hCLEC9a-peptide is an antibody-peptide fusion, where the antibody is directed against the extracellular domain of human CLEC9a and the peptide contains the OT-I (OVA amino acids 257-264) antigen and the OT-II (OVA amino acids 323-339) antigen. An isotype-peptide fusion (i.e., a fusion of an isotype antibody and a peptide containing the OT-I antigen and the OT-II antigen) was administered in a similar manner. Separate groups of mice were administered 5 mg / kg of ovalbumin endofit ((n = 3) Invitrogen, catalog number vac-pova), and 0.03366 mg / kg of peptide alone (n = 3). 72 hours later, the mice were euthanized, the spleens were harvested and purified for flow.
Claims
1. A genetically modified rodent, Rodent Clec9a nucleic acid sequence and The genome contains a humanized Clec9a gene, which includes the human CLEC9A nucleic acid sequence. The humanized Clec9a gene encodes a humanized Clec9a polypeptide that contains an extracellular domain that is at least 90% identical to the extracellular domain of the human CLEC9A protein. A genetically modified rodent in which the aforementioned rodent is a mouse or a rat.
2. (a) The human CLEC9A protein comprises the amino acid sequence described in Sequence ID No. 4, and / or (b) The humanized Clec9a protein contains a cytoplasmic-transmembrane sequence that is at least 90% identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein, and optionally the rodent Clec9a protein is the endogenous rodent Clec9a protein, and / or (c) The human CLEC9A nucleic acid sequence includes exons 3 to 6 of the human CLEC9A gene, A genetically modified rodent according to claim 1.
3. (a) The rodent Clec9a nucleic acid sequence comprises exons 1 and 2 of the rodent Clec9a gene, and / or (b) The rodent Clec9a nucleic acid sequence includes the 3' untranslated region of exon 6 of the rodent Clec9a gene, If necessary, the rodent Clec9a gene is the endogenous rodent Clec9a gene. A genetically modified rodent according to claim 1 or 2.
4. (a) The humanized Clec9a gene is operably linked to a rodent Clec9a promoter, and optionally the rodent Clec9a promoter is an endogenous rodent Clec9a promoter, and / or (b) The humanized Clec9a gene is located at the endogenous rodent Clec9a locus, and optionally the humanized Clec9a gene is formed as a result of replacing rodent Clec9a genomic DNA with human CLEC9A nucleic acid at the endogenous rodent Clec9a locus, and optionally the humanized Clec9a gene is formed as a result of replacing rodent genomic DNA, including exons 3 to the stop codon of exon 6 of the endogenous rodent Clec9a gene, with human CLEC9A nucleic acid, and the human CLEC9A nucleic acid includes exons 3 to exon 6 of the human CLEC9A gene, and / or (c) The aforementioned rodents (i) He is heterozygous for the humanized Clec9a gene, or (ii) The humanized Clec9a gene is homozygous, A genetically modified rodent according to claim 1 or 2.
5. (a) The rodent expresses the humanized Clec9a polypeptide on dendritic cells, and / or (b) The rodent is a mouse, and the humanized Clec9a polypeptide contains the amino acid sequence described in Sequence ID No.
7. A genetically modified rodent according to claim 1 or 2.
6. Isolated rodent tissue or cells having a genome containing a 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 having an extracellular domain that is at least 90% identical to the extracellular domain of the human CLEC9A protein, and the rodent tissue or cells are mouse tissue or cells, or rat tissue or cells.
7. (a) The rodent cells are (i) It is a rodent embryonic stem cell, or (ii) rodent germ cells, and / or (b) The human CLEC9A protein contains the amino acid sequence described in SEQ ID NO: 4, and / or (c) The humanized Clec9a protein contains a cytoplasmic-transmembrane sequence that is at least 90% identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein, and / or (d) The rodent tissue or cells are mouse tissue or cells, and the humanized Clec9a protein contains the amino acid sequence of SEQ ID NO: 7 The isolated rodent tissue or cells according to claim 6.
8. A rodent embryo comprising rodent embryonic stem cells as described in claim 7(a)(i).
9. A method for creating genetically modified rodents, Modifying a 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 that includes an extracellular domain that is at least 90% identical to the extracellular domain of the human Clec9A protein. The aforementioned modification is Introducing a nucleic acid molecule containing the aforementioned human CLEC9A nucleic acid sequence into the genome of rodent embryonic stem (ES) cells, Obtain rodent ES cells in which the human CLEC9A nucleic acid sequence is incorporated into the endogenous CLEC9a gene locus to replace the rodent CLEC9a genomic DNA, thereby forming the humanized CLEC9a gene. This includes generating rodents from the aforementioned obtained rodent ES cells, The aforementioned rodent is a mouse or a rat. method.
10. (a) The human CLEC9A protein comprises the amino acid sequence described in Sequence ID No. 4, and / or (b) The humanized Clec9a protein contains a cytoplasmic-transmembrane sequence that is at least 90% identical to the cytoplasmic-transmembrane sequence of the rodent Clec9a protein, and optionally the rodent Clec9a protein is the endogenous rodent Clec9a protein, and / or (c) The human CLEC9A nucleic acid sequence comprises exons 3 to 6 of the human CLEC9A gene, and / or (d) The rodent Clec9a nucleic acid sequence includes exons 1 and 2 of the rodent Clec9a gene, and / or (e) The rodent Clec9a nucleic acid sequence includes the 3' untranslated region of exon 6 of the rodent Clec9a gene, and / or (f) The humanized Clec9a gene is operably linked to the endogenous rodent Clec9a promoter at the endogenous rodent Clec9a locus, and / or (g) The rodents said above, (i) He is heterozygous for the humanized Clec9a gene, or (ii) The humanized Clec9a gene is homozygous, The method according to claim 9.
11. A human CLEC9A nucleic acid sequence incorporated into the rodent CLEC9a gene at the rodent CLEC9a locus, wherein a 5' nucleotide sequence and a 3' nucleotide sequence homologous to the nucleotide sequence at the endogenous rodent CLEC9a locus are positioned on the sides. By incorporating the human CLEC9A nucleic acid sequence into the rodent Clec9a gene, the rodent Clec9a genomic DNA is replaced by the human CLEC9A nucleic acid sequence, thereby forming a humanized Clec9a gene. The humanized Clec9a gene encodes a humanized Clec9a polypeptide that contains an extracellular domain that is at least 90% identical to the extracellular domain of the human CLEC9A protein. The aforementioned rodent is a mouse or a rat. Targeting nucleic acid constructs.
12. The targeted nucleic acid construct according to claim 11, wherein the human CLEC9A nucleic acid sequence comprises exons 3 to 6 of the human CLEC9A gene.
13. A method for evaluating the pharmacokinetic characteristics of candidate drugs, Administering the candidate drug to a rodent according to claim 1 or 2, A method comprising: performing one or more assays to determine the pharmacokinetic properties of the candidate drug in the rodent.
14. A method for screening candidate drugs that target human CLEC9A, Administering the candidate drug to a rodent according to claim 1 or 2, A method comprising performing one or more assays to determine whether the candidate drug is effective in the rodent.
15. (a) The one or more assays include an assay for measuring T cell proliferation, and optionally, (i) The assay measures the proliferation of CD4+ T cells, or (ii) The assay measures the proliferation of CD8+ T cells, and / or (b) The candidate drug is an antibody that binds to human CLEC9A. The method according to claim 14.