WO2024012578A1 - 一种tlr7和/或tlr8基因人源化修饰的非人动物 - Google Patents

一种tlr7和/或tlr8基因人源化修饰的非人动物 Download PDF

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WO2024012578A1
WO2024012578A1 PCT/CN2023/107520 CN2023107520W WO2024012578A1 WO 2024012578 A1 WO2024012578 A1 WO 2024012578A1 CN 2023107520 W CN2023107520 W CN 2023107520W WO 2024012578 A1 WO2024012578 A1 WO 2024012578A1
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tlr7
human
animal
protein
seq
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PCT/CN2023/107520
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French (fr)
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张淑金
周小飞
姚佳维
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百奥赛图(北京)医药科技股份有限公司
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Publication of WO2024012578A1 publication Critical patent/WO2024012578A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention provides a non-human animal expressing human or chimeric (eg, humanized) TLR7 and/or TLR8 proteins and methods of using the same.
  • the present application provides an animal model with human or chimeric TLR7 and/or TLR8 proteins.
  • the animal model can express human or chimeric TLR7 (eg, humanized TLR7) protein and/or human or chimeric TLR8 (eg, humanized TLR8) protein. It can be used to study the functions of TLR7 and TLR8 genes, and can also be used to screen and evaluate modulators of the TLR7/TLR8 signaling pathway (e.g., anti-human TLR7 and/or TLR8 antibodies and small molecule agonists).
  • the animal model prepared by the method described herein can be used for drug screening, pharmacodynamic research, treatment of immune-related diseases and cancer treatment of human TLR7/TLR8 target sites; the model can also be used to promote the development and design of new drugs, Save time and costs.
  • the present invention provides a powerful tool for studying the function of TLR7/TLR8 proteins and a platform for screening anti-cancer drugs.
  • the invention provides a genetically modified non-human animal whose genome comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric Toll-like receptor 7 (TLR7) protein .
  • TLR7 Toll-like receptor 7
  • the nucleotide sequence encoding a human or chimeric TLR7 protein is operably linked to an endogenous regulatory element (e.g., an endogenous 5'UTR and/or 3'UTR).
  • the human or chimeric TLR7 protein comprises portions of the signal peptide, extracellular, transmembrane and/or cytoplasmic regions of the human TLR7 protein.
  • the human or chimeric TLR7 protein comprises all or part of the extracellular domain of human TLR7 protein.
  • the amino acid sequence of the extracellular region of the human TLR7 protein is consistent with positions 27-802 of SEQ ID NO: 2 or positions 27-802 of SEQ ID NO: 2. The identity of the amino acid sequence shown at positions 27-839 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the human or chimeric TLR7 protein comprises all or part of the human TLR7 protein signal peptide.
  • the amino acid sequence of the human or chimeric TLR7 protein signal peptide has an identity of at least 70% with the amino acid sequence shown in positions 4-26 of SEQ ID NO: 2 or positions 1-26 of SEQ ID NO: 2 %, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the amino acid sequence of the human or chimeric TLR7 protein is consistent with positions 4-802 of SEQ ID NO: 2, positions 1-802 of SEQ ID NO: 2 or positions 1-839 of SEQ ID NO: 2
  • the amino acid sequence identity indicated by the position is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the amino acid sequence of the human or chimeric TLR7 protein is at least 70%, 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 9. 99% or 100%.
  • the animal is a mammal, such as a monkey, rodent, mouse, or rat. In some embodiments, the animal is a mouse. In some embodiments, the animal's endogenous TLR7 protein is not expressed or is expressed at a reduced level compared to TLR7 in a wild-type animal. In some embodiments, one or more cells of the animal express human or chimeric TLR7 protein.
  • the invention provides a genetically modified non-human animal whose genome comprises a nucleotide sequence encoding a region of endogenous TLR7 at the endogenous TLR7 locus substituted by a nucleotide sequence of the corresponding region of human TLR7 replace.
  • the nucleotide sequence encoding the corresponding region of human TLR7 is operably linked to an endogenous regulatory element (e.g., endogenous 5'UTR and/or 3'UTR) of the endogenous TLR7 locus, and One or more cells of the animal express human or chimeric TLR7 protein.
  • the animal has no expression of endogenous TLR7 protein or a reduced level of protein expression compared to TLR7 in a wild-type animal.
  • the nucleotide sequence encoding the corresponding region of human TLR7 comprises part of exon 3 of the human TLR7 gene.
  • the identity of the nucleotide sequence encoding the corresponding region of human TLR7 to the nucleotide sequence shown in SEQ ID NO: 5 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99% or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human TLR7 to the nucleotide sequence shown in SEQ ID NO: 8 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99% or 100%.
  • the nucleotide sequence encoding the endogenous TLR7 region comprises part of exon 3 of the mouse TLR7 gene.
  • the modified TLR7 gene in the animal's genome is homozygous or heterozygous for the endogenously replaced locus.
  • the invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence encoding a human or chimeric TLR7 protein, wherein the human or chimeric TLR7 protein comprises a region corresponding to a human
  • the continuous amino acid sequence is at least 50, 60, 70, 80, 90, 100, 200, 300, 500, 600, 700, 720, 740, 760, 790, 792, 795, 798, 799, 802, 839, 900, 1000, 1020, 1030, 1040, 1042, 1044, 1046, 1048 or 1049 consecutive amino acids are identical.
  • the human or chimeric TLR7 protein comprises portions of the signal peptide, extracellular, transmembrane and/or cytoplasmic regions of the human TLR7 protein. In some embodiments, the human or chimeric TLR7 protein comprises human All or part of the extracellular domain of TLR7 protein. In some embodiments, the amino acid sequence of the extracellular region of the human TLR7 protein has at least 70% identity with the amino acid sequence shown in positions 27-802 of SEQ ID NO: 2 or positions 27-839 of SEQ ID NO: 2. 75%, 80%, 85%, 90%, 95%, 99% or 100%. In some embodiments, the human or chimeric TLR7 protein comprises all or part of the human TLR7 protein signal peptide.
  • the amino acid sequence of the human or chimeric TLR7 protein signal peptide has at least an identity with the amino acid sequence shown in positions 4-26 of SEQ ID NO: 2 or positions 1-26 of SEQ ID NO: 2. 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In some embodiments, the amino acid sequence of the human or chimeric TLR7 protein is consistent with positions 4-802 of SEQ ID NO: 2, positions 1-802 of SEQ ID NO: 2 or positions 1-839 of SEQ ID NO: 2 The amino acid sequence identity indicated by the position is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the amino acid sequence of the human or chimeric TLR7 protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the amino acid sequence shown in SEQ ID NO: 9 Or 100%.
  • the nucleotide sequence encoding a human or chimeric TLR7 protein is operably linked to an endogenous regulatory element (e.g., endogenous 5'UTR and/or 3'UTR) of the endogenous TLR7 locus of at least one chromosome. 'UTR).
  • the nucleotide sequence encoding a human or chimeric TLR7 protein can be integrated into the animal's endogenous TLR7 locus.
  • the humanized TLR7 protein has at least one activity of mouse TLR7 and/or an activity of human TLR7.
  • the invention provides a method for constructing a genetically modified non-human animal, characterized in that, in at least one cell of the animal, at the animal's endogenous TLR7 locus, a nucleoside encoding the endogenous TLR7 region is The acid sequence was replaced by the nucleotide sequence encoding the corresponding region of human TLR7.
  • the nucleotide sequence encoding the corresponding region of human TLR7 includes all or part of the signal peptide and extracellular region encoding the human TLR7 protein.
  • the nucleotide sequence encoding the corresponding region of human TLR7 includes part of exon 3 of the human TLR7 gene.
  • the nucleotide sequence encoding the corresponding region of human TLR7 includes the encoded amino acid sequence and positions 4-802 of SEQ ID NO: 2, positions 1-802 of SEQ ID NO: 2 or SEQ ID NO: 2
  • the identity of the amino acid sequence shown at positions 1-839 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human TLR7 to the nucleotide sequence shown in SEQ ID NO: 5 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99% or 100%.
  • the nucleotide sequence encoding the endogenous TLR7 region comprises part of exon 3 of the mouse TLR7 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human TLR7 is operably linked to a regulatory element of endogenous TLR7, such as a promoter.
  • the animal is a mammal, such as a monkey, rodent, mouse, or rat. In some embodiments, the animal is a mouse.
  • the invention provides a method for constructing cells that express human or chimeric TLR7 protein genetically modified non-human animals, the method comprising nucleotides encoding the endogenous TLR7 region at the endogenous mouse TLR7 locus. The sequence is replaced with a nucleotide sequence encoding the corresponding region of human TLR7, resulting in a genetically modified non-human animal cell, wherein the animal cell expresses human or Chimeric TLR7 protein.
  • the human or chimeric TLR7 protein comprises all or part of the signal peptide and extracellular domain of the human TLR7 protein.
  • the nucleotide sequence encoding the corresponding region of human TLR7 includes part of exon 3 of the human TLR7 gene.
  • the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human TLR7 includes positions 4-802 of SEQ ID NO: 2, positions 1-802 of SEQ ID NO: 2 or SEQ ID NO: 2 The identity of the amino acid sequence shown at positions 1-839 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the identity of the nucleotide sequence encoding the corresponding region of human TLR7 to the nucleotide sequence shown in SEQ ID NO: 5 is at least 70%, 75%, 80%, 85%, 90%, 95 %, 99% or 100%.
  • the nucleotide sequence encoding the endogenous TLR7 region comprises part of exon 3 of the mouse TLR7 gene.
  • the nucleotide sequence encoding a human or chimeric TLR7 protein is operably linked to a regulatory element of endogenous TLR7, such as a promoter.
  • the animal is a mammal, such as a monkey, rodent, mouse, or rat.
  • the animal is a mouse.
  • the methods described above also include nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from the group consisting of TLR8, LAG-3, BTLA, PD-1, PD - at least one of L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3 or OX40.
  • the human or chimeric protein is a TLR8 protein.
  • the nucleotide sequence encoding a human or chimeric TLR8 protein is at least 70%, 75%, 80%, 85%, 90% identical to the nucleotide sequence shown in SEQ ID NO: 22 %, 95%, 99% or 100%.
  • the amino acid sequence of the human or chimeric TLR8 protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99 identical to the amino acid sequence shown in SEQ ID NO: 23 % or 100%.
  • the non-human animals described above also include nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins are selected from the group consisting of TLR8, LAG-3, BTLA, PD-1 , at least one of PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3 or OX40.
  • the human or chimeric protein is a TLR8 protein.
  • the nucleotide sequence encoding a human or chimeric TLR8 protein is at least 70%, 75%, 80%, 85%, 90% identical to the nucleotide sequence shown in SEQ ID NO: 22 %, 95%, 99% or 100%.
  • the amino acid sequence of the human or chimeric TLR8 protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99 identical to the amino acid sequence shown in SEQ ID NO: 23 % or 100%.
  • the invention provides a method for determining the effectiveness of an anti-TLR7 therapeutic agent in treating cancer, the method comprising: 1) administering an anti-TLR7 therapeutic agent to the non-human animal described herein, wherein the animal has a tumor; 2) Determine the inhibitory effect of anti-TLR7 therapeutic agents on tumors.
  • the tumor contains one or more tumor cells, wherein the tumor cells are injected into the animal.
  • determining the tumor inhibitory effect of an anti-TLR7 therapeutic agent includes measuring tumor volume in the animal.
  • the tumor is a solid tumor, bladder cancer, superficial urothelial cancer, cervical cancer, endometrial cancer, esophageal cancer, squamous cell carcinoma, renal cancer, non-small cell lung cancer, ovarian cancer, Squamous cell carcinoma, gastric cancer, uterine cancer, colorectal metastasis, liver cancer, gastrointestinal cancer.
  • the invention provides a method for determining the effectiveness of an anti-TLR7 therapeutic agent and other therapeutic agents in treating cancer, the method comprising: 1) administering an anti-TLR7 therapeutic agent to the non-human animal described in the present application, wherein said The animals have tumors; 2) Determine the inhibitory effect of anti-TLR7 therapeutic agents on tumors.
  • the animal further includes sequences encoding human or chimeric PD-1, human or chimeric PD-L1, and/or human or chimeric CTLA4.
  • the other therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and/or an anti-CTLA4 antibody.
  • the tumor contains one or more cells expressing PD-L1 protein.
  • the tumor contains one or more tumor cells, wherein the tumor cells are injected into the animal.
  • determining the tumor inhibitory effect of an anti-TLR7 therapeutic agent includes measuring tumor volume in the animal.
  • the tumor is a solid tumor, bladder cancer, superficial urothelial cancer, cervical cancer, endometrial cancer, esophageal cancer, squamous cell carcinoma, renal cancer, non-small cell lung cancer, ovarian cancer, Squamous cell carcinoma, gastric cancer, uterine cancer, colorectal metastasis, liver cancer, gastrointestinal cancer.
  • the invention provides a method for determining the effectiveness of an anti-TLR7 therapeutic agent in treating metabolic diseases, the method comprising: 1) administering an anti-TLR7 therapeutic agent to the non-human animal described in the present application, wherein the non-human animal The animals suffer from autoimmune diseases; 2) determine the effect of anti-TLR7 therapeutic agents in treating autoimmune diseases.
  • the autoimmune disease is asthma, rhinitis, systemic lupus erythematosus, or psoriasis.
  • the invention provides a method for determining the effectiveness of an anti-TLR7 therapeutic agent in treating inflammation, the method comprising: 1) administering an anti-TLR7 therapeutic agent to the non-human animal described in the present application, wherein the non-human animal suffers from There is inflammation; 2) Determine the effect of anti-TLR7 therapeutic agents in treating inflammation.
  • the inflammation is chronic obstructive pulmonary disease, sepsis, dermatitis.
  • the invention provides a method for determining the toxicity of an anti-TLR7 therapeutic agent, the method comprising: 1) administering the anti-TLR7 therapeutic agent to the non-human animal described in the present application; 2) determining the toxicity of the anti-TLR7 therapeutic agent to the animal effect.
  • determining the effect of an anti-TLR7 therapeutic on an animal involves measuring the animal's body weight, red blood cell count, hematocrit, and/or hemoglobin.
  • the present invention provides a humanized TLR7 protein, characterized in that the humanized protein includes all or part of the signal peptide and extracellular region of the human TLR7 protein.
  • the amino acid sequence of the humanized TLR7 protein is the same as SEQ ID NO: 2 Nos. 4-802, SEQ ID NO: 2 Nos. 1-802 or SEQ ID NO: 2 Nos. 1-839.
  • the amino acid sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the amino acid sequence identity of the humanized TLR7 protein and the amino acid sequence shown in SEQ ID NO: 9 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the present invention provides a humanized TLR7 gene encoding the humanized TLR7 protein described above.
  • the humanized TLR7 gene comprises part of exon 3 of the human TLR7 gene.
  • the humanized TLR7 gene contains a nucleotide sequence consistent with SEQ ID NO: 3, 4, The nucleotide sequence identity shown in 5, 6, 7, 8, 10 and 11 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
  • the present invention provides a cell, characterized in that the cell contains the humanized TLR7 protein and humanized TLR7 gene described above.
  • the present invention provides an animal model, characterized in that the animal model includes the above-mentioned humanized TLR7 protein and the above-mentioned humanized TLR7 gene.
  • Figure 1 Schematic diagram comparing the mouse TLR7 locus and the human TLR7 locus (not to scale);
  • FIG. 1 Schematic diagram of humanized transformation of mouse TLR7 locus (not to scale);
  • FIG. 3 Schematic diagram 1 of TLR7 gene targeting strategy and targeting vector V1 design (not to scale);
  • FIG. 4 Schematic diagram 2 of TLR7 gene targeting strategy and targeting vector V2 design (not to scale);
  • FIG. 5 F0 generation mouse genotype identification results, M is Marker, WT is wild-type control, H 2 O is water control, among which F0-2 is a positive mouse and has no random insertion;
  • FIG. 6 Southern blot test results, in which WT is the wild-type control, F1-4, F1-5, F1-8, F1-9, F1-10, and F1-11 are positive mice without random insertion, and other small Rats have hybrid bands;
  • Figure 7 Schematic diagram comparing the mouse TLR8 locus and the human TLR8 locus (not to scale);
  • FIG. 8 Schematic diagram of humanized transformation of mouse TLR8 locus (not to scale);
  • Figure 9 Percentages of leukocyte subtypes (A) and T cell subtypes (B) in the spleens of wild-type C57BL/6 mice and TLR7/TLR8 double-gene humanized mice;
  • Figure 10 Percentage of leukocyte subtypes (A) and percentage of T cell subtypes (B) in lymph nodes of wild-type C57BL/6 mice and TLR7/TLR8 dual-gene humanized mice;
  • Figure 11 Percentage of leukocyte subtypes (A) and T cell subtypes (B) in the blood of wild-type C57BL/6 mice and TLR7/TLR8 double-gene humanized mice;
  • FIG. 12 Wild-type C57BL/6 mice and TLR7/TLR8 dual-gene humanized mice were stimulated with TLR7/TLR8 agonists. Detection of TNF- ⁇ cytokine secretion after stimulation, where ctrl is the control group without TLR7/TLR8 agonist treatment;
  • Figure 13 Human TLR7 amino acid sequence (NP_057646.1; SEQ ID NO: 2) and mouse TLR7 amino acid sequence (NP_573474.1; SEQ ID NO: 1);
  • Figure 14 Human TLR7 amino acid sequence (NP_057646.1; SEQ ID NO: 2) and rat TLR7 amino acid sequence (NP_001091051.1; SEQ ID NO: 24).
  • TLRs Toll-like receptors
  • TLRs are a class of innate immune sensors that can recognize conserved microbial structures, and activation of TLRs can initiate innate and adaptive immune responses.
  • the ability of TLRs to promote the activation of antigen-presenting cells (such as dendritic cells or macrophages) and the induction of immunomodulatory cytokines is critical to the formation of adaptive immune responses.
  • the human TLRs family has 10 members including TLR1 to TLR10. Among them, TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are expressed on the cell surface, while TLR3, TLR7, TLR8, and TLR9 are mainly located on the endosome membrane in the cell. .
  • TLR7 and TLR8 are the two most studied Toll-like receptor targets in tumor immunity.
  • TLR7 is located on the X chromosome and is a type I transmembrane protein, including a signal peptide, extracellular region, transmembrane region and intracellular region.
  • the extracellular region is rich in leucine repeat sequences, and its main function is to recognize the corresponding ligand;
  • the intracellular region has a highly conserved protein interaction region Toll/IL-1 receptor region (TIR), which plays a role in TLR signaling. plays an important role in transmission.
  • TIR Toll/IL-1 receptor region
  • TLR7 is widely expressed in a variety of cells, such as plasmacytoid dendritic cells (pDC), macrophages, T cells and B cells, and is also expressed in eosinophils and neutrophils. Studies have shown that TLR7 is functionally expressed in cancer cells and surrounding stromal cells in patients with lung cancer, urinary tract cancer, breast cancer, and pancreatic cancer, and is also expressed in malignant B cells and myeloid cells.
  • TLR7 The ligands of TLR7 include ssRNA viruses and some synthetic small molecule compounds. Among them, Imiquomid (R837) is the most widely studied imidazoquinoline family series of compounds. It mainly acts as an agonist of TLR7/TLR8 to exert anti-tumor effects.
  • TLR7 is a MyD88-dependent signaling pathway. After ssRNA is recognized by TLR7, it causes the secretion of various inflammatory cytokines such as IFN, IL1, IL6, IL12, and TNF through a series of cascade reactions, exerting an antiviral effect mechanism. In addition to viral RNA, occasionally, TLR7 can also be activated by its own RNA, causing autoimmune diseases.
  • MyD88 myeloid differentiation factor 88
  • INF- ⁇ MyD88-independent/TRIF
  • TLR7 is increased in the active phase of patients with systemic lupus erythematosus; TLR7 is abnormally low-expressed in CIA models and may have negative regulation. It inhibits the occurrence of RA synovitis and is a potential therapeutic target for RA.
  • TLR7 and TLR8 are phylogenetically and structurally related.
  • TLR7 is mainly expressed on pDCs
  • TLR8 is mainly expressed on myeloid dendritic cells (mDCs).
  • mDCs myeloid dendritic cells
  • both can secrete type 1 interferons and express costimulatory molecules, activating killer cells to attack tumor cells.
  • dendritic cells can be derived from dead tumor cell debris Obtain tumor-specific antigens. Therefore, activating these two types of receptors at the same time can make full use of dendritic cells as a bridge between innate immunity and acquired immunity.
  • the TLR7 gene (Gene ID: 51284) contains 3 exons, namely exon 1, exon 2 and exon 3 ( Figure 1).
  • the nucleotide sequence of human TLR7 mRNA is NM_016562.4
  • the amino acid sequence of human TLR7 is NP_057646.1 (SEQ ID NO: 2).
  • SEQ ID NO: 2 the amino acid sequence of transcript NM_016562.4 and its encoded protein NP_057646.1
  • the human TLR7 gene (NCBI Gene ID: 51284) is located at positions 12867072 to 12890361 of NC_000023.11 on chromosome X (GRCh38.p13 (GCF_000001405.40).
  • the specific position of each exon based on transcript NM_016562.4 is : 5'UTR is located at NC_000023.11 at positions 12867072 to 12867123 and 12867481 to 12867578, exon 1 is located at NC_000023.11 at positions 12867072 to 12867123, intron 1 is located at NC_000023.11 at positions 12867124 to 128674 Position 80, exon 2 Located at positions 12867481 to 12867581 of NC_000023.11, intron 2 is located at positions 12867582 to 12885511 of NC_000023.11, exon 3 is located at positions 12885512 to 12890361 of NC_000023.11, and 3'UTR is located at position 128 of NC_0000
  • the TLR7 gene (Gene ID: 170743) contains 3 exons, namely exon 1, exon 2 and exon 3 ( Figure 1).
  • the nucleotide sequence of mouse TLR7 mRNA is NM_133211.4
  • the amino acid sequence of mouse TLR7 is NP_573474.1 (SEQ ID NO: 1).
  • the corresponding position of each exon is as follows:
  • the mouse TLR7 gene (NCBI Gene ID: 170743) is located at positions 166086376 to 166113570 of NC_000086.8 on chromosome X (GRCm39 (GCF_000001635.27)).
  • the specific position of each exon based on transcript NM_133211.4 is: 5'UTR is located at positions 166113554 to 166113433 and 166113095 to 166113000 of NC_000086.8, exon 1 is located at positions 166113554 to 166113433 of NC_000086.8, inclusive
  • Sub1 is located at positions 166113432 to 166113096 of NC_000086.8,
  • exon 2 is located at positions 166113095 to 166112997 of NC_000086.8, intron 2 is located at positions 166112996 to 166091482 of NC_000086.8, and exon 3 is located at NC_000086.
  • the 3’UTR is located at positions 166088331 to 166087927 of NC_000086.8. All the above relevant information about the mouse TLR7 locus can be retrieved on the NCBI website (Gene ID: 170743). The entire contents of which are incorporated herein by reference.
  • Figure 13 shows the alignment of the human TLR7 amino acid sequence (NP_057646.1; SEQ ID NO: 2) and the mouse TLR7 amino acid sequence (NP_573474.1; SEQ ID NO: 1). Therefore, the corresponding amino acid residues or regions between human and mouse TLR7 can be found in Figure 13.
  • TLR7 genes, proteins and gene loci for other species are also known in the art.
  • the Gene ID of Rattus norvegicus (rat) TLR7: 317468 the Gene ID of Macaca mulatta (rhesus monkey) TLR7: 574291
  • the Gene ID of Sus scrofa (pig) TLR7 ID: 100037296 Relevant information of these genes (eg, intron sequences, exon sequences, and amino acid sequences) can be found in NCBI, the entire content of which is incorporated herein by reference.
  • Figure 14 shows the human TLR7 amino acid sequence (NP_057646.1; SEQ ID NO: 2) and the rat TLR7 amino acid sequence (NP_001091051.1; SEQ ID NO: 24). Therefore, the corresponding amino acid residues or regions between human and rat TLR7 can be retrieved in Figure 14.
  • the invention provides a human or chimeric (eg, humanized) TLR7 nucleotide sequence or amino acid sequence.
  • all or part of the nucleotide sequence of exon 1, exon 2 and/or exon 3 of the mouse TLR7 gene is replaced with the corresponding nucleotide sequence of the human TLR7 gene.
  • "portions" of exon 1, exon 2 and/or exon 3 of the mouse TLR7 gene are replaced with the corresponding nucleotide sequence or amino acid sequence of the human TLR7 gene.
  • the "part” refers to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 800, 1400, 1800, 2200, 2300, 2340, 2380, 2390, 2391, 16 000, 20000, 24000, 2500 or 25628bp contiguous nucleotide sequence, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 , 100, 200, 300, 500, 600, 700, 800, 801, 802, 803, 820, 830, 831, 833, 836, 839, 840, 900, 1000, 1020, 1030, 1040 or 1050 consecutive amino acid sequences .
  • the "portion" is at least 50%, 55%, 60%, 65%, 70% identical to the amino acid sequence encoded by exon 1, exon 2 and/or exon 3 , 75%, 80%, 85%, 90%, 95% or at least 100%.
  • "part" or "all” of the mouse TLR7 exon 1, exon 2 and/or exon 3 (e.g., part of exon 3) sequences are excreted by the human TLR7 gene "Part” or "all” sequence replacement of exon 1, exon 2 and/or exon 3 (eg, part of exon 3).
  • portions of endogenous exon 1, exon 2, and/or exon 3 are deleted.
  • the invention provides a genetically modified non-human animal whose genome includes a human, chimeric or humanized TLR7 nucleotide sequence.
  • the protein encoded by the human, chimeric or humanized TLR7 nucleotide sequence is at least 70%, 80%, 85%, 90% identical to the amino acid sequence shown in SEQ ID NO: 9 , 95% or 100%.
  • the non-human animal genome contains a nucleotide sequence that has at least 70%, 80%, 85%, 90%, 95% or 100%.
  • the non-human animals described herein comprise a nucleotide sequence encoding a human or chimeric TLR7 protein.
  • the human or chimeric TLR7 protein comprises all or part of the signal peptide, extracellular region, transmembrane region and/or cytoplasmic region of the human TLR7 protein.
  • the human or chimeric TLR7 protein includes all or part of the signal peptide of the human TLR7 protein. Further, the part of the signal peptide of the human TLR7 protein includes at least 5 consecutive amino acids, such as at least 5, 7, 9, 10, 15, 17, 19, 20, 21, 22, 23, 24, 25, 26 consecutive amino acids, the human or chimeric TLR7 protein signal peptide includes the same as SEQ ID NO: 2 No. 4-26 position or the amino acid sequence identity shown in positions 1-26 of SEQ ID NO: 2 is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 100%.
  • the human or chimeric TLR7 protein includes all or part of the extracellular region of the human TLR7 protein.
  • the part of the extracellular region of the human TLR7 protein includes at least 200 consecutive amino acids, for example, includes at least 200, 300, 400, 500, 700, 750, 760, 770, 775, 776, 777, 778, 779, 780, 800, 813 consecutive Amino acid
  • the extracellular region of the human or chimeric TLR7 protein contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 100%.
  • the human or chimeric TLR7 protein comprises SEQ ID NO: 2, positions 4-802, SEQ ID NO: 2, positions 1-802, or SEQ ID NO: 2, positions 1-839.
  • the amino acid sequence identity is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 100%.
  • the non-human animals described herein comprise a human or chimeric TLR7 gene.
  • the chimeric TLR7 gene contains 3 human or chimeric exons.
  • the chimeric TLR7 gene comprises chimeric exon 3.
  • the chimeric TLR7 gene comprises human or endogenous 5'UTR.
  • the chimeric TLR7 gene comprises a human or endogenous 3'UTR.
  • the chimeric TLR7 gene comprises an endogenous 5'UTR.
  • the chimeric TLR7 gene comprises an endogenous 3'UTR.
  • genetically modified non-human animals can express human TLR7 and/or chimeric (e.g., humanized) TLR7 proteins, the non-human animal's endogenous TLR7 gene and/or nucleotide sequence being modified by human TLR7 gene and/or nucleotide sequence replacement.
  • the human TLR7 gene and/or nucleotide sequence encoding the amino acid sequence of human TLR7 has at least 10%, 20%, 30%, 40%, 50% identity with the amino acid sequence shown in human TLR7 SEQ ID NO: 2 , 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 100%.
  • the endogenous TLR7 gene is replaced in whole or in part with a nucleotide sequence encoding a mature TLR7 protein.
  • the genetically modified non-human animal expresses human TLR7 and/or chimeric TLR7 protein (eg, humanized TLR7 protein) under a mouse endogenous promoter and/or regulatory elements. Replacement of the endogenous mouse locus provides a non-human animal that expresses a human or chimeric TLR7 protein (eg, a humanized TLR7 protein) in the same cell type.
  • the genetically modified mice do not develop the underlying diseases known in the art to be observed in certain other transgenic mice.
  • Human TLR7 or chimeric TLR7 proteins (eg, humanized TLR7 proteins) expressed in non-human animals can maintain the function of one or more wild-type or human TLR7 proteins.
  • the genetically modified non-human animal does not express endogenous TLR7 protein. In some embodiments, the genetically modified non-human animal has reduced expression of endogenous TLR7 protein.
  • endogenous TLR7 protein refers to the TLR7 protein encoded by the nucleotide sequence of the endogenous TLR7 gene of non-human animals (eg, mice) before genetic modification.
  • the genome of the non-human animal contains the protein encoding the human TLR7 protein (NP_057646.1; SEQ ID NO: 2 positions 4-802, SEQ ID NO: 2 positions 1-802 or SEQ ID NO: 2 positions 1-839). Represents a nucleotide sequence of amino acids whose amino acid sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In some embodiments, the genome comprises at least 70%, 75%, 80%, 85%, 90%, 95%, nucleotide sequence identity to SEQ ID NO: 5 and SEQ ID NO: 8. 99% or at least 100% nucleotide sequence.
  • nucleotide sequence encoding the endogenous TLR7 region in the genome of the non-human animal is replaced with the nucleotide sequence encoding the corresponding region of human TLR7.
  • the nucleotide sequence encoding the endogenous TLR7 region is any sequence of the endogenous TLR7 locus, such as exon 1, exon 2, exon 3, 5'UTR, 3'UTR, intron 1, intron 2, or any combination thereof.
  • the nucleotide sequence encoding the endogenous TLR7 region is located within the endogenous TLR7 regulatory region.
  • nucleotide sequence encoding the endogenous TLR7 region is exon 1, exon 2 and/or exon 3, or a portion thereof.
  • One or more cells of the genetically modified non-human animal express human or chimeric TLR7 protein (eg, humanized TLR7 protein).
  • human or chimeric TLR7 protein e.g., humanized TLR7 protein
  • the genetically modified non-human animal genome includes all or part of exon 1, exon 2 and/or exon 3 of the human TLR7 gene, or the nucleotides shown in SEQ ID NO: 5 All or part of the sequence.
  • the genetically modified non-human animal genome includes a portion of exon 3 of the human TLR7 gene.
  • the portion of exon 3 of the human TLR7 gene includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 350, 450, 600, 1000, 1500, 2000, 2200, 2300, 2340, 2380, 2390, 2392, 2394, 2396, 2397, 2400, 2420, 2430, 2434, 2438, 2439, 3000, 3500, 4000, 4500, 4600, 4800, 4830, 4840 or 4850 bp contiguous nucleotide sequence.
  • the portion of exon 3 includes 2397 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 3 includes at least 100-500 bp, 1000-1500 bp, or 2000-2300 bp of nucleotide sequence. In some embodiments, the nucleotide sequence encoding the corresponding region of human TLR7 is located at nucleotide sequence 160-2556 of the human TLR7 gene transcript NM_016562.4.
  • the TLR7 gene of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.
  • the chimeric TLR7 genome lacks the 5'UTR of the human TLR7 gene.
  • the chimeric TLR7 genome comprises an endogenous (eg, mouse) 5'UTR.
  • the chimeric TLR7 genome comprises an endogenous (eg, mouse) 3'UTR.
  • chimeric TLR7 mice comprise a replacement of the endogenous mouse locus that retains endogenous mouse regulatory elements but includes the chimeric TLR7 coding sequence. Expression of TLR7 in genetically modified heterozygous mice or homozygous mice is completely normal.
  • the invention provides a genetically modified non-human animal, the non-human animal genome comprising a deletion of the endogenous TLR7 gene, wherein the deletion of the endogenous TLR7 gene includes exon 1, exon 2 and/or or exon 3, or part of the endogenous TLR7 locus.
  • the deletion of the endogenous TLR7 gene includes one or more exons or portions of exons selected from exon 3.
  • the deletions include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 400, 600, 1000, 1500, 2200, 2500, 3000, 3500, 3700, 3740, 3780, 3790, 3791, 3792, 3793, 3794, 4000, 5000, 6000, 8000, 12000, 16000, 20000 , 24000, 2500 or 25628 bp contiguous nucleotide sequence or more.
  • the deletion of the endogenous TLR7 gene comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 400, 600, 800, 1000, 1400, 1800, 2000 of exon 3 ⁇ 2100 ⁇ 2300 ⁇ 2320 ⁇ 2340 ⁇ 2360 ⁇ 2390 ⁇ 2394 ⁇ 2396 ⁇ 2397 ⁇ 2398 ⁇ 2399 ⁇ 2400 ⁇ 2420 ⁇ 2430 ⁇ 2431 ⁇ 2432 ⁇ 2433 ⁇ 2700 ⁇ 2900 ⁇ 3000 ⁇ 3100 ⁇ 3300 ⁇ 3500 ⁇ 3560bp ⁇ nucleotide sequence or more nucleotide sequence.
  • the invention provides a humanized mouse TLR7 genomic DNA sequence; a construct expressing the amino acid sequence of the humanized TLR7 protein; a cell containing the construct; and a tissue containing the cell. .
  • the invention provides a chimeric (e.g., humanized) TLR7 nucleotide sequence and/or amino acid sequence, wherein in some embodiments, the chimeric nucleotide sequence Sequence consistent with that shown in mouse endogenous TLR7 mRNA (e.g., NM_133211.4), mouse TLR7 amino acid sequence (e.g., NP_573474.1, SEQ ID NO: 1), or a portion thereof (e.g., part of exon 3) Identity of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
  • the chimeric nucleotide sequence is identical to the human TLR7 mRNA sequence (e.g., NM_016562.4), the TLR7 amino acid sequence (e.g., NP_057646.1, SEQ ID NO: 2), or a portion thereof (e.g., exosomes part of sub-3) showing a sequence identity of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25 %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the human TLR7 mRNA sequence e.g., NM_016562.4
  • the TLR7 amino acid sequence e.g., NP_057646.1, SEQ ID NO: 2
  • a portion thereof e.g
  • the chimeric nucleic acid sequence described above is operably linked to a promoter or regulatory element, for example, an endogenous mouse TLR7 promoter, an inducible promoter, an enhancer and/or a mouse or human Adjustment element.
  • a promoter or regulatory element for example, an endogenous mouse TLR7 promoter, an inducible promoter, an enhancer and/or a mouse or human Adjustment element.
  • At least a portion of the chimeric nucleic acid sequences described herein e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) that differ in whole or in part from the mouse TLR7 nucleotide sequence (e.g., Part of exon 3 of mouse TLR7 gene transcript NM_133211.4).
  • At least a portion of the chimeric nucleic acid sequence described above e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) identical to all or part of the mouse TLR7 nucleotide sequence (e.g., All of exon 1 to exon 2 and part of exon 3 of mouse TLR7 gene transcript NM_133211.4).
  • At least a portion of the chimeric nucleic acid sequence described above e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., a contiguous or non-contiguous nucleotide sequence
  • the human TLR7 nucleotide sequence in whole or in part (e.g., human TLR7 All of exon 1 to exon 2 and part of exon 3 of the gene transcript NM_016562.4).
  • At least a portion of the chimeric nucleic acid sequence described above e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., contiguous or non-contiguous nucleotide sequences) that are identical in whole or in part to the human TLR7 nucleotide sequence (e.g., human TLR7 part of exon 3 of gene transcript NM_016562.4).
  • human TLR7 nucleotide sequence e.g., human TLR7 part of exon 3 of gene transcript NM_016562.4.
  • the chimeric nucleic acid sequence encodes at least a portion of the amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) different from all or part of the mouse TLR7 protein amino acid sequence (e.g., Mouse TLR7 protein sequence NP_573474.1 amino acids 4-803, 1-803 or 1-840 (SEQ ID NO: 1)).
  • the amino acids e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • mouse TLR7 protein amino acid sequence e.g., Mouse TLR7 protein sequence NP_573474.1 amino acids 4-803, 1-803 or 1-840 (SEQ ID NO: 1)
  • the chimeric nucleic acid sequence encodes at least a portion of the amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues (e.g., contiguous or non-contiguous amino acid residues) are identical to all or part of the mouse TLR7 protein amino acid sequence (e.g., Mouse TLR7 protein sequence NP_573474.1 amino acids 1-3, 804-1050 or 841-1050 (SEQ ID NO: 1)).
  • At least a portion of the amino acid sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30 , 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) different from all or part of the human TLR7 protein amino acid sequence (e.g., human TLR7 protein sequence NP_057646.1 Amino acids 803-1049 or 840-1049 (SEQ ID NO: 2)).
  • At least a portion of the amino acid sequence e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30 , 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues
  • amino acid residues e.g., contiguous or non-contiguous amino acid residues
  • the present invention also provides a humanized TLR7 mouse amino acid sequence, wherein the amino acid sequence includes any one of the following groups:
  • the present invention also provides a humanized TLR7 amino acid sequence, wherein the amino acid sequence includes any one of the following groups:
  • SEQ ID NO: 2 bits 4-802 Same as SEQ ID NO: 2 bits 4-802, SEQ ID NO: 2 bits 1-802 or SEQ ID NO: 2 bits 1-839, including substitution, deletion and/or insertion of one or more amino acid sequence of amino acid residues.
  • the invention also provides a humanized TLR7 nucleotide (e.g., DNA or RNA) sequence, wherein the nucleotide sequence includes any one of the following groups:
  • the encoded amino acid sequence is the same as SEQ ID NO: 2 at positions 4-802, SEQ ID NO: 2 at positions 1-802 or
  • the amino acid sequence identity shown in positions 1-839 of SEQ ID NO: 2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 %;
  • the encoded amino acid sequence is the same as that shown in positions 4-802 of SEQ ID NO:2, positions 1-802 of SEQ ID NO:2 or positions 1-839 of SEQ ID NO:2, including substitutions, deletions and/or An amino acid sequence in which one or more amino acid residues are inserted.
  • the present invention further provides a TLR7 genomic DNA sequence of humanized mice.
  • the DNA sequence is obtained by reverse transcription of the transcribed mRNA and is consistent or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 5 or 8.
  • the "genetically modified non-human animal” mentioned in the present invention refers to a non-human animal in which at least one chromosome of the animal's genome has exogenous TLR7. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% in at least one or more cells, e.g., in a genetically modified non-human animal , 50% of cells contain foreign DNA.
  • Cells with exogenous DNA can be various cells, such as endogenous cells, somatic cells, immune cells, T cells, B cells, NK cells, antigen-presenting cells, macrophages, dendritic cells, germ cells, cysts, etc. embryonic or endogenous tumor cells.
  • a genetically modified non-human animal comprising an endogenous TLR7 locus and an exogenous TLR7 locus (e.g., a human sequence), e.g., replacing one with one or more human sequences or multiple non-human sequences, or insert one or more human and/or non-human sequences.
  • Animals are often able to pass genetic modifications to their offspring through germline transmission.
  • chimeric gene or “chimeric nucleic acid” used in the present invention refers to a gene or nucleic acid in which two or more parts of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is identical to that in an animal.
  • the wild-type nucleic acid is different.
  • the chimeric gene or chimeric nucleic acid has at least a portion of the sequence derived from two or more different species, for example, a sequence encoding different proteins or the same (or homologous) sequence encoding two or more different species. ) protein sequence.
  • a chimeric gene or chimeric nucleic acid refers to a humanized gene or humanized nucleic acid.
  • chimeric protein or “chimeric polypeptide” used in the present invention refers to a protein or polypeptide in which two or more parts of the polypeptide or protein are from different species, or at least one sequence of the protein or polypeptide is identical to that in an animal.
  • the wild-type amino acid sequence is different.
  • at least a portion of the sequence of the chimeric protein or chimeric polypeptide is derived from two or more different species, for example, the same (or homologous) protein from different species.
  • a chimeric protein or chimeric polypeptide refers to a humanized protein or humanized polypeptide.
  • humanized protein or humanized polypeptide refers to a protein or polypeptide, wherein at least a part of the protein or polypeptide is derived from a human protein or human polypeptide.
  • a humanized protein or humanized polypeptide refers to a human protein or polypeptide.
  • Humanized nucleic acid refers to nucleic acid, wherein at least a portion of the nucleic acid is derived from human nucleic acid. In some embodiments, the nucleic acids in the humanized nucleic acid are entirely of human origin. In some embodiments, the humanized nucleic acid refers to a humanized exon, which may be a human exon or a chimeric exon.
  • the chimeric gene or chimeric nucleic acid is a humanized TLR7 gene or humanized TLR7 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human TLR7 gene, or at least a portion of the gene or nucleic acid is derived from a non-human TLR7 gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding a TLR7 protein. In some embodiments, the encoded TLR7 protein has the activity of at least one human TLR7 protein or non-human animal TLR7 protein.
  • the chimeric protein or chimeric polypeptide is a humanized TLR7 protein or humanized TLR7 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a human TLR7 protein, or at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human TLR7 protein. .
  • the humanized TLR7 protein or humanized TLR7 polypeptide is functional, or has at least the activity of a human TLR7 protein or a non-human animal TLR7 protein.
  • Genetically modified non-human animals can be various animals, for example, mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, Primates (e.g., marmosets, rhesus monkeys).
  • ES embryonic stem
  • Such methods include, for example, modifying the genome of a non-ES cell (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transfer to transfer the modified genome to an appropriate cell, such as an oocyte, and under appropriate conditions in Modified cells (eg, modified oocytes) are gestated in non-human animals to form embryos.
  • a non-ES cell e.g., fibroblasts or induced pluripotent stem cells
  • Modified cells eg, modified oocytes
  • the animal is a mammal.
  • the genetically modified non-human animal is a rodent.
  • the rodent may be selected from mice, rats and hamsters.
  • the rodent is selected from the mouse family.
  • the genetically modified animal is selected from the group consisting of the family Hamsteridae (e.g., mouse-like hamsters), the family Hamsteridae (e.g., hamsters, New World rats and mice, voles), Muroidea (e.g., true mice and Rats, gerbils, spiny rats, crested rats), Dormoids (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Dormoids (e.g.
  • the family Hamsteridae e.g., mouse-like hamsters
  • the family Hamsteridae e.g., hamsters, New World rats and mice, voles
  • Muroidea e.g., true mice and Rats, gerbils, spiny
  • the genetically modified rodent is selected from the group consisting of true mice or rats (superfamily Muridae), gerbils, spiny rats, and crested rats.
  • the genetically modified mouse is from a member of the family Muridae.
  • the animal is a rodent.
  • the rodent is selected from the group consisting of mice and rats.
  • the non-human animal is a mouse.
  • the animal is a mouse of a C57BL strain selected from the group consisting of C57BL/a, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/min, C57BL6J, C57B1/6ByJ, C57BL/ 6NJ, C57BL/10, C57BL10SnSn, C57BL/10Cr and C57BL/Ola.
  • a C57BL strain selected from the group consisting of C57BL/a, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/min, C57BL6J, C57B1/6ByJ, C57BL/ 6NJ, C57BL/10, C57BL10SnSn, C57BL/10Cr and C57BL/Ola.
  • the mouse is selected from 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129 strains of 129S8, 129T1, and 129T2.
  • 129P1, 129P2, 129P3, 129X1, 129S1 e.g., 129S1/SV, 129S1/SvIm
  • 129S7 e.g., 129S8, 129T1, and 129T2.
  • the genetically modified mouse is a cross between the 129 strain and the C57BL/6 strain.
  • the mice are a cross of 129 strains, or a cross of BL/6 strains.
  • the mouse is a BALB strain, such as the BALB/c strain.
  • the mouse is a cross between a BALB strain and another strain.
  • the mice are from a hybrid strain (eg, 50% BALB/c-50% 12954/Sv; or 50% C57BL/6-50% 129).
  • the non-human animal is a rodent.
  • the non-human animal is a person with BALB/c, a, a/He, a/J, a/WySN, AKR, AKR/a, AKR/J, AKR/N, TA1, TA2, RF, SWR , C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/a, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL6, C57L/6J, C57BL/6ByJ, C5C57BL /6NJ mice.
  • Genetically modified non-human animals include modification of the endogenous non-human TLR7 gene locus.
  • the modifications comprise a nucleotide sequence encoding at least a portion of the mature TLR7 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistency).
  • cells e.g., ES cells, somatic cells
  • genetically modified non-human animals include modification of the endogenous TLR7 gene locus in the animal. Grooming.
  • the present invention further provides non-human mammals constructed using the above methods.
  • the non-human mammal comprises a human genome.
  • the non-human mammal is a rodent, and further preferably, the rodent is a mouse.
  • the non-human mammal expresses a protein encoded by a humanized TLR7 gene.
  • the present invention also provides a tumor-bearing non-human mammal, characterized in that the non-human mammal model is obtained by the method described herein.
  • the non-human mammal is a rodent (eg, mouse).
  • the invention also provides a cell or cell line derived from a non-human mammal or its progeny, or a tumor-bearing non-human mammal, or a primary cell culture derived from a non-human mammal or its progeny, or Tumor-bearing non-human mammals, tissues, organs or cultures derived from non-human mammals or their offspring. Tumor tissue derived from a non-human mammal or its offspring when it carries a tumor, or a non-human mammal carrying a tumor.
  • the invention provides a non-human mammal produced by any of the methods described herein.
  • non-human mammals genetically modified non-human animals whose genomes comprise DNA for human or humanized TLR7 are provided.
  • a non-human mammal includes a genetic construct described herein (eg, a genetic construct as shown in Figures 3 and 4).
  • a non-human mammal expressing a human or humanized TLR7 protein is provided.
  • tissue-specific expression of a human or humanized TLR7 protein is provided.
  • expression of the non-human animal human or humanized TLR7 protein is controllable. Such as by adding specific inducers or repressors.
  • the specific inducer is selected from Tet-Off System/Tet-On System or Tamoxifen System.
  • the non-human mammal can be any non-human animal known in the art, which can be used in the methods described herein.
  • Preferred non-human mammals are mammals (eg, rodents).
  • the non-human mammal is a mouse.
  • the present invention provides an offspring produced by mating with a non-human mammal of the same genotype or another genotype.
  • the present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny.
  • cell culture-based models can be prepared by the following method.
  • Cell cultures can be obtained by isolation from non-human mammals, or cells can be obtained from cell cultures established using the same constructs and cell transfection techniques. Integration of a genetic construct containing a DNA sequence encoding human TLR7 protein can be detected by a variety of methods.
  • RT-PCR reverse transcription-polymerase chain reaction
  • RNAdot RNA dot blot assays
  • a genetically modified animal described herein eg, a mouse homozygous for a humanized TLR7 gene
  • the present invention provides a targeting vector targeting the TLR7 gene, including: a) a DNA fragment (5' arm) homologous to the 5' end of the conversion region to be changed, which is selected from 100-100-100-100 of non-human animal TLR7 genomic DNA. 10,000 nucleotides in length; b) DNA sequence encoding the donor region; c) DNA fragment (3' arm) homologous to the 3' end of the conversion region to be changed, which is selected from non-human animal TLR7 gene genomic DNA, Length is 100-10000 nucleotides.
  • a) the DNA fragment homologous to the 5' end of the conversion region to be changed is selected from a nucleotide sequence having at least 90% homology with the NCBI accession number NC_000086.8; c) the DNA fragment to be converted to be changed The DNA fragment homologous to the 3' end of the region is selected from a nucleotide sequence that has at least 90% homology with the NCBI accession number NC_000086.8;
  • a) the homologous DNA fragment at the 5' end of the conversion region to be changed is selected from the nucleotide sequence at positions 166091476 to 166095762 with NCBI accession number NC_000086.8; c) the 3' end of the conversion region to be changed The homologous DNA fragment is selected from the nucleotide sequence from 166083092 to 166087410 with NCBI accession number NC_000086.8;
  • a) the homologous DNA fragment at the 5' end of the conversion region to be changed is selected from the nucleotide sequence at positions 166091476 to 166092724 with NCBI accession number NC_000086.8; c) the 3' end of the conversion region to be changed Homologous DNA fragments are selected from NCBI accession numbers that are at least 95% identical to the nucleotide sequence at positions 166087878 to 166089075 of NC_000086.8;
  • the selected genomic nucleotide sequence length of the targeting vector can exceed about 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 5.5kb, 6kb, 6.5kb, 7kb, 7.5kb, 8kb, 8.5kb , 9kb, 9.5kb or 10kb.
  • the conversion region to be changed is located on exons 1 to 3 of the TLR7 gene of non-human animals.
  • the switching region to be changed is located on exon 3 of the TLR7 gene of non-human animals (for example, positions 160-2556 of NM_016562.4).
  • the targeting vector further includes one or more marker genes.
  • a positive selection marker gene or a negative selection marker gene the resistance gene screened for positive clones is the neomycin phosphotransferase coding sequence Neo.
  • the gene encoding the negative selection marker is the gene encoding diphtheria toxin A subunit (DTA).
  • the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 3; the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 10; the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 11.
  • the 5' arm is a nucleotide having at least 90% homology with the NCBI accession number NC_000086.8. Further preferably, the 5' arm sequence includes SEQ ID NO: 3 or 10 The nucleotide sequence is shown. In some embodiments, the 3' arm is a nucleotide having at least 90% homology with the NCBI accession number NC_000086.8. Further preferably, the 3' arm sequence includes SEQ ID NO: 4 or 11 The nucleotide sequence is shown.
  • the targeting vector comprises human sequences (eg, positions 12885518-12887914 of NC_000023.11).
  • the targeting region in the targeting vector includes: all or part of the nucleotide sequence of the human TLR7 gene, preferably part of exon 3 of the human TLR7 gene.
  • the nucleotide sequence of the humanized TLR7 gene encodes all or part of the nucleotide sequence of the human TLR7 protein, and the NCBI protein number is NP_057646.1 (SEQ ID NO: 2).
  • the present invention also provides vectors for constructing humanized animal models or knockout models.
  • the vector contains an sgRNA sequence, wherein the sgRNA sequence targets the TLR7 gene, and the sgRNA is unique on the target sequence of the gene to be changed, and satisfies 5'-NNN(20)-NGG3' or 5'-CCN -N(20)-3' sequence arrangement rule; and in some embodiments, the targeting site of sgRNA in the mouse TLR7 gene is located in exon 1, intron 1, exon 2, intron 2. Exon 3, upstream of exon 3 of mouse TLR7 gene, or downstream of exon 3.
  • the targeting sequences are shown as SEQ ID NOs: 12 and 13.
  • the present disclosure relates to plasmid constructs comprising sgRNA sequences (eg, pT7-sgRNA) and/or cells comprising the constructs.
  • the present disclosure also relates to cells comprising a targeting vector as described above.
  • the present invention provides a non-human mammalian cell having any of the targeting vectors described above, and one or more in vitro transcripts of a construct described herein.
  • the cell contains Cas9 mRNA or an in vitro transcript thereof.
  • the genes in the cells are heterozygous. In some embodiments, the genes in the cells are homozygous.
  • the non-human mammalian cells are mouse cells.
  • the cell is a fertilized egg cell.
  • the cells are embryonic stem cells.
  • Genetically modified non-human animals can be prepared through several technologies known in the art, including gene targeting technology using embryonic stem cells, CRISPR/Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, and homing. Nested endonucleases or other molecular biology techniques. In some embodiments, it is preferred to use homologous recombination technology.
  • CRISPR/Cas9 gene editing technology can create genetically modified non-human animals.
  • CRISPR/Cas9 genome editing is used to create genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in Yin et al., “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6(2017):387-399, the entire content of which is available through Incorporated herein by reference.
  • the present invention also provides many other methods for genome editing, such as microinjecting a transgenic cell into an enucleated oocyte and fusing the enucleated oocyte with another transgenic cell.
  • the nucleoside encoding the endogenous TLR7 region in the endogenous genome of at least one cell of the non-human animal The acid sequence was replaced by the nucleotide sequence encoding the corresponding region of human TLR7.
  • the expression level of the endogenous TLR7 protein of the non-human animal is reduced or deleted compared with wild-type TLR7.
  • replacement occurs in germ cells, somatic cells, blastocysts, fibroblasts, or the like. Nuclei from somatic cells or fibroblasts can be inserted into the enucleated oocyte.
  • FIGS 3 and 4 show the humanized targeting strategy targeting the mouse TLR7 locus.
  • the targeting vector contains a 5' homology arm, a vector composed of a human or humanized TLR7 gene fragment and a 3' homology arm. This process involves the use of homologous recombination to replace the endogenous corresponding TLR7 nucleotide sequence with a human or humanized nucleotide sequence.
  • cleavage upstream and downstream of the target site e.g., by zinc finger nucleases, TALENs, or CRISPR
  • homologous recombination is used to replace human or humanized TLR7 sequences in mice.
  • Source TLR7 sequence e.g., by zinc finger nucleases, TALENs, or CRISPR
  • the nucleotide sequence encoding the corresponding region of human TLR7 includes all or part of the nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region and/or cytoplasmic region of the human TLR7 protein, preferably , comprising all or part of the nucleotide sequence encoding the signal peptide and extracellular region of the TLR7 protein.
  • the nucleotide sequence encoding the corresponding region of human TLR7 includes at least 50 to at least 1049 nucleotides encoding human TLR7 protein, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 600, 700, 720, 740, 760, 790, 792, 795, 798, 799, 800, 801, 802, 803, 804, 805, 810, 820, 830, 831, 833, 836, 839, 840, 850, 900, 1000, 1020, 1030, 1040, 1042, 1044, 1046, 1048 or 1049 consecutive amino acid nuclei
  • the nucleotide sequence further comprising a nucleotide sequence encoding the amino acid sequence shown in positions 4-802 of SEQ ID NO: 2, positions 1-802 of SEQ ID NO: 2 or positions 1-839 of SEQ ID NO: 2; Alternatively, it contains an amino acid sequence that
  • the nucleotide sequence encoding the corresponding region of human TLR7 includes all or part of exon 1, exon 2 and/or exon 3 of the human TLR7 gene, and further preferably includes the human TLR7 gene. All or part of exon 3, further preferably includes part of exon 3 of human TLR7 gene, wherein part of exon 3 of human TLR7 gene includes at least 50 bp to at least 4850 bp of exon 3 of human TLR7 gene, preferably 50, 60, 70, 80, 90, 100, 150, 200, 250, 350, 450, 600, 1000, 1500, 2000, 2200, 2300, 2340, 2380, 2390, 2392, 2394, 2396, 2397, 2400, 2420, 2430, 2434, 2438, 2439, 3000, 3500, 4000, 4500, 4600, 4800, 4830, 4840 or 4850 bp continuous nucleotide sequence, or the part of exon 3 of the human TLR7 gene including the nucleotide
  • the nucleotide sequence encoding the corresponding region of human TLR7 includes the nucleotide sequence shown in SEQ ID NO: 5; or, the nucleotide sequence including the nucleotide sequence shown in SEQ ID NO: 5 has at least 0.
  • nucleotide sequence contains a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; Alternatively, include a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5, including substitution, deletion and/or insertion of one or more nucleotides.
  • the nucleotide sequence encoding a corresponding region of human TLR7 is operably linked to an endogenous regulatory element of an endogenous TLR7 gene on at least one chromosome.
  • the endogenous TLR7 nucleotide sequence that is replaced in a non-human animal includes all or part of exon 1, exon 2 and exon 3 of the mouse TLR7 gene, preferably non-human All or part of exon 3 of the animal's TLR7 gene is replaced. More preferably, part of exon 3 of the non-human animal TLR7 gene is replaced.
  • the replaced endogenous TLR7 nucleotide sequence encodes positions 4-803 of SEQ ID NO:1, positions 1-803 of SEQ ID NO:1 or positions 1-840 of SEQ ID NO:1 The amino acid sequence shown in the position.
  • the human or humanized TLR7 gene is regulated in non-human animals through regulatory elements, including but not limited to endogenous promoters.
  • the regulatory element may be an endogenous or exogenous regulatory element.
  • the endogenous regulatory element is derived from the non-human animal TLR7 gene.
  • the exogenous regulatory element is derived from the human TLR7 gene.
  • the construction method includes replacing exon 1, exon 2 and exon 3 of the non-human animal TLR7 gene with all or part of exon 1, exon 2 and exon 3 of the human TLR7 gene. All or part of exon 2 and exon 3, preferably all or part of exon 3 of the non-human animal TLR7 gene is replaced with all or part of exon 3 of the human TLR7 gene.
  • the construction method includes replacing all or part of exon 3 of the non-human animal TLR7 gene with a part including exon 3 of the human TLR7 gene.
  • the construction method includes replacing the non-human animal genome with a nucleotide sequence encoding a human or humanized TLR7 protein, or a nucleotide sequence encoding a human or humanized TLR7 gene.
  • the construction method includes replacing the nucleotide encoding the amino acid sequence shown in SEQ ID NO: 1 in the genome of the non-human animal with a genomic DNA sequence, a cDNA sequence or a CDS sequence containing the human TLR7 gene sequence.
  • the construction method includes replacing the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1 in the non-human animal genome with a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2. nucleotide sequence.
  • the construction method includes replacing the non-human animal genome with a nucleotide sequence encoding a human or humanized TLR7 protein, or a nucleotide sequence encoding a human or humanized TLR7 gene.
  • the construction method includes replacing positions 4-803 of SEQ ID NO: 1, SEQ ID The nucleotide sequence of the amino acid sequence shown in positions 1-803 of NO:1 or positions 1-840 of SEQ ID NO:1.
  • the construction method includes encoding SEQ ID NO: 2 bits 4-802, SEQ ID NO: 2 bits 1-802 or SEQ ID NO: 2 bits 1-839
  • the nucleotide sequence of the amino acid sequence shown is replaced by the 4-803rd position of SEQ ID NO:1, the 1-803rd position of SEQ ID NO:1 or the 1-840th position of SEQ ID NO:1 in the non-human animal genome. Nucleotide sequence of amino acid sequence.
  • the construction method includes replacing positions 4-803 of the non-human animal genome encoding SEQ ID NO: 1 with the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 1 No. 1-803 or the nucleotide sequence of the amino acid sequence shown in SEQ ID NO: 1 No. 1-840.
  • the method for constructing a non-human animal includes the construction of a genetically modified non-human animal TLR7 gene coding frame, wherein the genetically modified non-human animal TLR7 gene coding frame may be constructed by knocking out the non-human animal TLR7 gene.
  • the functional region may be used to insert a sequence so that the non-human animal TLR7 protein is not expressed or the expression is reduced or the expressed protein has no function.
  • the genetically modified non-human animal TLR7 gene coding cassette construct includes knockout of all or part of the nucleotide sequence of exon 3 of the non-human animal TLR7 gene.
  • the modified non-human animal TLR7 gene coding frame construction can knock out the partial nucleotide sequence of exon 3 of the non-human animal TLR7 gene.
  • methods for constructing genetically modified non-human animals include inserting nucleotide sequences encoding human or humanized TLR7 proteins and/or auxiliary sequences after the endogenous regulatory elements of the TLR7 gene of the non-human animal.
  • the auxiliary sequence can be a stop codon, so that the humanized animal model of the TLR7 gene expresses the human TLR7 protein in vivo and does not express the non-human animal TLR7 protein.
  • the auxiliary sequence is WPRE, STOP and/or PolyA.
  • sgRNA targeting the TLR7 gene can also be used to construct non-human animals together with the above-mentioned TLR7 gene targeting vector.
  • the sgRNA targets the TLR7 gene of non-human animals, and the sequence of the sgRNA is on the target sequence of the TLR7 gene to be changed.
  • the sgRNA target site is located in the sequence of exon 1 to exon 3 of the TLR7 gene.
  • the sgRNA target site is located on exon 3 of the TLR7 gene.
  • the target sequence of the sgRNA on the TLR7 gene is shown in SEQ ID NO: 12 or SEQ ID NO: 13.
  • the construction method includes introducing the above-mentioned TLR7 gene targeting vector, TLR7 gene targeting sgRNA and Cas9 into non-human animal cells, culturing the cells (preferably fertilized eggs), and then The cultured cells are transplanted into the fallopian tubes of female non-human animals, allowed to develop, and non-human animals with TLR7 gene modification are identified and screened.
  • the construction method includes introducing the above-mentioned targeting vector into embryonic stem cells of non-human animals, briefly culturing it and then introducing it into previously isolated blastocysts, and transplanting the obtained chimeric blastocysts into In the fallopian tube of the recipient female mouse, it is allowed to develop, and non-human animals with humanized TLR7 gene are identified and screened.
  • the construction method further includes: mating non-human animals with humanized TLR7 genes with other genetically modified non-human animals, in vitro fertilization or direct gene editing, and screening to obtain Polygenetically modified nonhuman animals.
  • the other genes are at least one of TLR8, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3 or OX40. kind.
  • the non-human animal further expresses human or chimeric TLR8, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3 Or at least one of OX40.
  • some of the non-human animals are described in patents PCT/CN2019/127084, PCT/CN2017/106024, PCT/CN2018/110069, PCT/CN2017/099574, PCT/CN2017/117984, PCT/CN2018 /091846, PCT/CN2018/081628, PCT/CN2017/120388, PCT/CN2017/110494, PCT/CN2018/091845, PCT/CN2017/099576, PCT/CN2017/110494 and PCT/CN2017/099575 have been described, which All The contents are incorporated herein by reference.
  • the other gene is a TLR8 gene and the TLR8 gene is a chimeric TLR8 gene.
  • the chimeric TLR8 gene includes all or part of exons 1 to 2 of the human TLR8 gene. In some embodiments, the chimeric TLR8 gene includes part of exon 2 of the human TLR8 gene.
  • the mRNA transcribed from the nucleotide sequence of the chimeric TLR8 gene is the nucleotide sequence shown in SEQ ID NO: 22; or, the transcribed mRNA is the same as the nucleotide sequence shown in SEQ ID NO: 22.
  • Sequence identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100 % of the nucleotide sequence; alternatively, the difference between the transcribed mRNA and the nucleotide sequence shown in SEQ ID NO: 22 does not exceed 10, 9, 8, 7, 6, 5, 4, 3, 2 or does not exceed 1 nucleus
  • the nucleotide sequence of the nucleotide; alternatively, the transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 22, including the nucleotide sequence of substitution, deletion and/or insertion of one or more nucleotides.
  • the chimeric TLR8 protein comprises at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or at least 100% of the amino acid sequence; or, containing an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 23 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or an amino acid sequence of not more than 1 amino acid; alternatively, comprising the amino acid sequence shown with SEQ ID NO: 23, including substitution, deletion and/or insertion of one or more amino acids. sequence.
  • each of the plurality of genes modified in the genome of the multigene modified non-human animal is homozygous or heterozygous for the endogenous modified locus.
  • the non-human animals can be selected from rodents, pigs, rabbits, monkeys, and any other non-human animals that can undergo gene editing to produce gene humanization.
  • the non-human animal is a non-human mammal.
  • the non-human mammal is a rodent. More preferably, the rodent is a rat or mouse.
  • the non-human animal is an immunodeficient non-human mammal.
  • the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey.
  • the immunodeficient rodent is an immunodeficient mouse or rat.
  • the immunodeficient mice are NOD-Prkdc scid IL-2r ⁇ null mice, NOD-Rag 1 -/- -IL2rg -/- mice, Rag 2 -/- -IL2rg -/- mice, NOD/SCID mice or nude mice.
  • the insertion of human sequences into a non-human animal can produce a non-human animal with qualities and characteristics that may be significantly different from a typical knockout plus transgenic animal.
  • the endogenous locus is removed or disrupted and the fully human transgene is inserted into the animal's genome, possibly integrating randomly into the genome.
  • the location of the integrated transgene is unknown; human protein expression is measured by transcription of human gene and/or protein assays and/or functional assays.
  • the human sequences upstream and/or downstream provide suitable support for expression and/or regulation of the transgene.
  • transgenes with human regulatory elements are expressed in a non-physiological or otherwise unsatisfactory manner and may actually be harmful to the animal.
  • the present invention demonstrates that the replacement or insertion of human sequences at endogenous loci under the control of endogenous regulatory elements to produce humanized animals provides physiologically appropriate expression patterns and levels with respect to the physiology of the replaced genes in the humanized animals. are meaningful and appropriate in the context of physiology.
  • Genetically modified animals expressing human or humanized TLR7 proteins provide a variety of uses, including, but not limited to, the development of treatments for human diseases and conditions, and the evaluation of these human treatments in animals. Toxicity and/or efficacy in models.
  • the present invention also provides an application of a non-human animal modified by the above-mentioned TLR7 and/or TLR7/TLR8 genes or a non-human animal obtained by any of the above-mentioned construction methods.
  • the application includes:
  • TLR7 and/or TLR7/TLR8 Application as a model system related to TLR7 and/or TLR7/TLR8 for pharmacological, immunological, microbiological and medical research;
  • TLR7 and/or TLR8 genes Study the function of TLR7 and/or TLR8 genes, study the drugs and efficacy of human TLR7 and/or TLR8 target sites, and study the application of immune-related disease drugs and anti-tumor drugs related to TLR7 and/or TLR8.
  • the invention provides a non-human animal expressing human or humanized TLR7 and/or TLR8 protein, which can be used for screening of human TLR7 and/or TLR8 specific modulators.
  • the non-human animal is an animal model of human disease.
  • the disease is genetically induced (knock-in or knock-out).
  • the genetically modified non-human animal further comprises a compromised immune system, such as a genetically modified xenograft of human-derived tissue, including human solid tumors (e.g., bladder cancer) or hematological tumors (e.g., Lymphocytic neoplasms, B or T cell neoplasms).
  • genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (eg, anti-TLR7 antibodies and/or anti-TLR8 antibodies) in treating various immune diseases.
  • the immune diseases include, but are not limited to, GVHD (graft versus host disease), psoriasis, allergies, asthma, myocarditis, rhinitis, hepatitis (preferably non-alcoholic steatohepatitis), systemic erythema Lupus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, etc.
  • GVHD graft versus host disease
  • psoriasis e.g., allergies, asthma, myocarditis, rhinitis, hepatitis (preferably non-alcoholic steatohepatitis), systemic erythema Lupus, rheumatoid arthritis,
  • genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (eg, anti-TLR7 antibodies and/or anti-TLR8 antibodies) in treating various inflammatory infections.
  • the inflammation includes acute inflammation and chronic inflammation. Specifically, they include but are not limited to chronic obstructive pulmonary disease, sepsis, dermatitis, etc.
  • genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (eg, anti-TLR7 antibodies and/or anti-TLR8 antibodies) in treating cancer.
  • a therapeutic agent eg, an anti-TLR7 antibody and/or an anti-TLR8 antibody
  • the detection includes determining the size and/or proliferation rate of tumor cells.
  • the detection method includes vernier caliper measurement, flow cytometry detection and/or animal live imaging detection.
  • the detection includes assessment of the individual's body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, including changes in food consumption or water consumption.
  • the tumor cells comprise one or more cancer cells that are injected into an animal (eg, the cancer cells are derived from a human or non-human animal).
  • the therapeutic agent inhibits TLR7/TLR8-mediated signaling pathways. In some embodiments, the therapeutic agent does not inhibit TLR7/TLR8-mediated signaling pathways.
  • genetically modified non-human animals can be used to detect whether anti-TLR7 antibodies and/or anti-TLR8 antibodies are agonists or antagonists.
  • the methods described herein can be used to test the function of a therapeutic agent (e.g., an anti-TLR7 antibody and/or an anti-TLR8 antibody), e.g., whether the therapeutic agent can upregulate an immune response or downregulate an immune response, and/ or whether the therapeutic is capable of inducing complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC).
  • CMC complement-mediated cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • genetically modified non-human animals can be used to determine effective doses of therapeutic agents to treat a disease (eg, an immune disease) in a subject.
  • the inhibitory effect on tumors can also be determined by methods known in the art, for example, measuring the tumor volume in the animal, and/or determining the tumor (volume) inhibition rate (TGI TV ).
  • therapeutic agents can be used to treat various cancers.
  • Cancer refers to cells with the ability to grow autonomously, that is, an abnormal state or disease characterized by rapid cell growth and proliferation. The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage.
  • Tumor includes but is not limited to lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, Glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocyte tumors, melanoma, myelodysplastic syndromes, and sarcomas.
  • the leukemia is selected from the group consisting of acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia;
  • the lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia;
  • the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma,
  • the tumor is a solid tumor, bladder cancer, superficial urothelial cancer, cervical cancer, endometrial cancer, esophageal cancer, squamous cell carcinoma, renal cancer, non-small cell lung cancer , ovarian cancer, squamous cell carcinoma, gastric cancer, uterine cancer, colorectal metastasis, liver cancer, gastrointestinal cancer.
  • the invention also provides an assay for determining the toxicity of a therapeutic agent (e.g., anti-TLR7 antibody and/or anti-TLR8 antibody). Law.
  • the method includes administering the antibody to the non-human animal as described above, and assessing the animal's weight change, red blood cell count, hematocrit and/or hemoglobin.
  • the antibody can reduce red blood cells (RBCs), hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%.
  • the animal's body weight is at least 5%, 10%, 20%, 30%, or 40% less than a control group (eg, the average body weight of animals not treated with the antibody).
  • the present invention also provides an animal model constructed by the method described herein for developing products related to human cellular immune processes, manufacturing human antibodies, or a model system for pharmacological, immunological, microbiological and medical research.
  • an animal model generated by the methods described herein is provided for producing and utilizing human cells for animal experimental disease models, studying pathogens, or developing new diagnostic and/or therapeutic strategies for immune processes.
  • the present invention also provides for screening, validating, evaluating or studying TLR7 and/or TLR8 gene functions, human TLR7 and/or TLR8 antibodies, human TLR7 and/or TLR8 target sites, or drugs using animal models generated by the methods described herein. Effectiveness, drugs for immune-related diseases and anti-tumor drugs.
  • the invention also provides a non-human animal with two or more human or chimeric genes, the animal model comprising a human or chimeric TLR7 gene and a nucleic acid sequence encoding other human or chimeric proteins.
  • the other genes are genetically modified at least one of TLR8, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3 or OX40 Non-human animals.
  • the above-mentioned non-human animals also express human or humanized TLR8, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM- 3 or OX40 at least one.
  • the present invention also provides a method for constructing non-human animals with two or more human or chimeric genes.
  • the construction method includes:
  • step (2) Mating the non-human animal provided in step (1) with other genetically modified non-human animals, performing in vitro fertilization or direct gene editing, and conducting screening to obtain multi-gene modified non-human animals.
  • the other genetically modified non-human animals include genes TLR8, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, or OX40 One or a combination of two or more humanized non-human animals.
  • TLR7 is humanized directly with human or chimeric TLR8, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, or OX40 Genetically modified non-human animals.
  • combination therapies targeting two or more of these proteins may be a more effective treatment.
  • many relevant clinical trials are ongoing and showing good results.
  • many Genetically modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, e.g., anti-TLR7 antibodies or anti-TLR8 antibodies, and for the treatment of cancer or metabolic diseases (e.g., obesity or Add-on therapeutic agents for cardiovascular disease).
  • the methods include administering an anti-TLR7 antibody or an anti-TLR8 antibody and an additional therapeutic agent to an animal, wherein the animal has a tumor or immune disease, and determining the effect of the combination treatment on the immune tumor or immune disease.
  • the additional therapeutic agent is an antibody that specifically binds TLR8, LAG-3, BTLA, PD-1, PD-L1, CD27, CD28, CD40, CD47, CD137, TIGIT, TIM-3, or OX40 .
  • the additional therapeutic agent is an anti-CTLA4 antibody (eg, ipilimumab), an anti-PD-1 antibody (eg, nivolumab), or an anti-PD-L1 antibody.
  • the non-human animals described above also include sequences encoding human or humanized PD-1, sequences encoding human or humanized PD-L1, or sequences encoding human or humanized CTLA-4. .
  • the additional therapeutic agent is an anti-PD-1 antibody (eg, nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.
  • the tumor described above includes one or more tumor cells expressing PD-L1 and/or PD-L2.
  • the combination therapy may also be used to treat various cancers described herein, such as solid tumors, bladder cancer, superficial urothelial cancer, cervical cancer, endometrial cancer, esophageal cancer, squamous cell carcinoma , kidney cancer, non-small cell lung cancer, ovarian cancer, squamous cell carcinoma, gastric cancer, uterine cancer, colorectal metastasis, liver cancer, gastrointestinal cancer.
  • various cancers described herein such as solid tumors, bladder cancer, superficial urothelial cancer, cervical cancer, endometrial cancer, esophageal cancer, squamous cell carcinoma , kidney cancer, non-small cell lung cancer, ovarian cancer, squamous cell carcinoma, gastric cancer, uterine cancer, colorectal metastasis, liver cancer, gastrointestinal cancer.
  • the treatment methods described above can be used in combination with conventional cancer chemotherapy agents.
  • methods of treating cancer can be used alone or in combination with methods described herein, including treating the subject with chemotherapy, such as camphorine, doxorubicin, cisplatin, carboplatin, procarbazine, Methylchloride, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorambucil, endosulfan, nitrosula, actinomycin, daunorubicin, bleomycin , primycin, mitomycin, etoposide, verapir, podophyllotoxin, tamoxifen, paclitaxel, transplatinum, 5-fluoruracil, vincristine, vinblastin and/or Triamterin.
  • the method may include performing surgery on the subject to remove at least a portion of the cancer, such as removing a portion or all of a tumor from the patient
  • BsrGI and BgIII enzymes were purchased from NEB, with catalog numbers R0575S and R0144S respectively;
  • mice were purchased from the National Rodent Experimental Animal Seed Center, China Institute of Food and Drug Control;
  • PE/Cy TM 7anti-mouse/rat Foxp3 was purchased from eBioscience, the product number is 25-5773-82;
  • FITC anti-mouse F4/80Antibody was purchased from Biolegend, the product number is 123108;
  • V450Rat Anti-mouse CD11b was purchased from BD Horizon, the item number is 560455;
  • PerCP anti-mouse Ly-6G/Ly-6C(Gr-1) Antibody was purchased from Biolegend, the product number is 108426;
  • APC anti-human CD288 (TLR8) Antibody was purchased from Biolegend, product number is 395505;
  • TLR8 Monoclonal Antibody (44C143), PE was purchased from eBioscience, the product number is MA5-16194;
  • TLR7 Antibody was purchased from Biolegend, the product number is 160003;
  • PE anti-human TLR7Antibody was purchased from Biolegend, product number is 376903;
  • Red blood cell lysate was purchased from Beyotime, the product number is C3702;
  • Mouse IFN- ⁇ ELISA Kit was purchased from Biolegend, product number is 447904;
  • CL264 was purchased from MCE, the product number is HY-135905;
  • GS-9688 was purchased from MCE, the product number is HY-109137;
  • TL8-506 was purchased from Invivogen with the product number Tlrl-tl8506.
  • Mouse TLR7 gene (NCBI Gene ID: 170743, Primary source: MGI: 2176882, UniProt: P58681, located at positions 166086376 to 166113570 of the X chromosome NC_000086.8, based on the transcript NM_133211.4 and its encoded protein NP_573474.1 (SEQ ID NO: 1) and human TLR7 gene (NCBI Gene ID: 51284, Primary source: HGNC: 15631, UniProt ID: Q9NYK1, located at positions 12867072 to 12890361 of the X chromosome NC_000023.11, based on transcript NM_016562.4 and its encoding
  • the comparison diagram of protein NP_057646.1 (SEQ ID NO: 2) is shown in Figure 1.
  • the nucleotide sequence encoding the human TLR7 protein can be introduced into the endogenous TLR7 locus of the mouse, so that the mouse expresses the human or humanized TLR7 protein.
  • the partial sequence of exon 3 of the human TLR7 gene was used to replace the corresponding mouse nucleotide sequence to achieve humanized transformation of the mouse TLR7 locus.
  • the schematic diagram of the humanized TLR7 locus is shown in Figure 2.
  • FIG. 2 a schematic diagram of the targeting strategy shown in Figure 3 is further designed.
  • the targeting vector V1 contains the upstream and downstream homology arm sequences of the mouse TLR7 gene, as well as the nucleotides encoding the human TLR7 protein. sequential A1 fragment.
  • the upstream homology arm sequence (5' homology arm, SEQ ID NO: 3) is the same as the nucleotide sequence at positions 166091476 to 166095762 with NCBI registration number NC_000086.8, and the downstream homology arm sequence (3' homology Arm, SEQ ID NO: 4) is identical to the nucleotide sequence at positions 166083092 to 166087410 of the NCBI accession number NC_000086.8; the human TLR7 nucleotide sequence (SEQ ID NO: 5) contained in the A1 fragment is identical to the NCBI accession number The nucleotide sequences at positions 12885518 to 12887914 of NC_000023.11 are identical.
  • the targeting vector V1 also includes a resistance gene for positive clone selection, namely the hygromycin coding sequence HygR, and is equipped with two Frt recombination bits of the site-specific recombination system arranged in the same direction on both sides of the resistance gene. points to form HygR box 1 (HygR cassette).
  • connection between the upstream HygR box and mouse TLR7 is designed as (SEQ ID NO: 6), where the last “C” in the sequence " TCCAC " is the last nucleotide of mouse TLR7, the sequence The "G” in is the first nucleotide of the HygR box;
  • connection between the downstream HygR box and the mouse is designed as (SEQ ID NO: 7), where the last "C” in the sequence " GATCC " is the last nucleotide of the HygR box, the sequence The "C” in is the first nucleotide of mouse.
  • a coding gene (gene coding for diphtheria toxin A subunit (DTA)) with a negative selection marker was constructed downstream of the 3' homology arm of the targeting vector.
  • DTA diphtheria toxin A subunit
  • the mRNA sequence of the modified humanized mouse TLR7 is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 9.
  • the CRISPR/Cas9 system can also be used for gene editing.
  • Figure 2 a schematic diagram of the targeting strategy shown in Figure 4 is designed. The figure shows that the targeting vector V2 contains an upstream homology arm (5' homology arm) and a downstream The homology arm (3' homology arm) sequence, and the A2 fragment containing the nucleotide sequence encoding part of exon 3 of the human TLR7 gene.
  • the upstream homology arm sequence (5' homology arm, SEQ ID NO: 10) is the same as the nucleotide sequence from 166091476 to 166092724 of the NCBI accession number NC_000086.8, and the downstream homology arm sequence (3' homology Arm, SEQ ID NO: 11) is 99% identical to the nucleotide sequence 166087878 to 166089075 of the NCBI accession number NC_000086.8.
  • the difference is that the base A at positions 166089040 and 166089043 has been replaced by base G;
  • the human TLR7 nucleotide sequence contained in the A2 fragment is shown in SEQ ID NO: 5.
  • the mRNA sequence of the modified humanized mouse TLR7 is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 9.
  • the target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize the 5’ and 3’ end target sites, and select sgRNAs with better activity and higher sequence specificity for subsequent experiments. Exemplary target sequences are as follows:
  • sgRNA1 target site (SEQ ID NO: 12): 5’-ACTTCACAAGGTAGAGTTTTAGG-3’
  • sgRNA2 target site (SEQ ID NO: 13): 5'-GCCACTGATGTGACTTGTGTAGG-3'
  • pT7-sgRNA vector is synthesized from a plasmid.
  • the company synthesizes the fragment DNA (SEQ ID NO: 14) containing the T7 promoter and sgRNA scaffold and sequentially connects it to the backbone vector (from Takara, Cat. No. 3299) through enzyme digestion (EcoRI and BamHI). After sequencing verification by a professional sequencing company, the results showed that the target plasmid was obtained.
  • mice such as C57BL/6 mice
  • a microinjector to inject the in vitro transcription products of the obtained expression vector pT-TLR7-1 and pT-TLR7-2 plasmids (using the Ambion in vitro transcription kit, Transcribe according to the instructions)
  • the targeting vector and Cas9mRNA are premixed and injected into the cytoplasm or nucleus of mouse fertilized eggs.
  • mice were briefly cultured and then transplanted into the fallopian tubes of recipient female mice for development.
  • the obtained mice were crossed and selfed to expand the population and establish a stable humanized mouse strain of the TLR7 gene.
  • the genotype of somatic cells of F0 generation mice can be identified through conventional detection methods (such as PCR analysis).
  • the exemplary identification results of some F0 generation mice are shown in Figure 5.
  • the mouse numbered F0-2 in Figure 5 is a positive mouse.
  • PCR primers are shown in Table 3.
  • F1 generation mice were obtained by mating TLR7 gene humanized mice identified as positive by F0 with wild-type mice. By mating F1 generation mice with each other, mice homozygous for the humanized TLR7 gene can be obtained. Mice identified as positive by PCR were subjected to Southern blot testing to confirm whether there was random insertion. Cut the mouse tail to extract genomic DNA, use BsrGI enzyme or BgIII enzyme to digest the genome, transfer to membrane, and hybridize. The lengths of specific probes and target fragments are shown in Table 4. The Southern blot detection results are shown in Figure 6. The combined results of 3' probe and 5' probe show that there is no random insertion in F1-4, F1-5, F1-8, F1-9, F1-10 and F1-11 mice. . This shows that this method can be used to construct humanized mice with the TLR7 gene that can be stably passaged without random insertion.
  • the probe synthesis primers are as follows:
  • Mouse TLR8 gene (NCBI Gene ID: 170744, Primary source: MGI: 2176887, UniProt: P58682, located at positions 167241123 to 167264329 of the X chromosome NC_000086.7, based on the transcript NM_133212.3 and its encoded protein NP_57345.2) and Human TLR8 gene (NCBI Gene ID: 51311, Primary source: HGNC: 15632, UniProt ID: Q9NR97, located at positions 12906620 to 12923169 of the X chromosome NC_000023.11, based on transcript NM_138636.5 and its encoded protein NP_619542.1) comparison
  • Figure 7 The schematic diagram is shown in Figure 7.
  • the 2415bp nucleotide sequence of exon 3 of the mouse TLR8 gene is replaced with the corresponding 2442bp nucleotide sequence of exon 2 of the human TLR8 gene to achieve humanized transformation of the mouse TLR8 gene.
  • the schematic diagram of the modified mouse TLR8 locus is shown in Figure 8.
  • the mRNA sequence of the modified humanized mouse TLR8 is shown in SEQ ID NO: 22, and the expressed protein sequence is shown in SEQ ID NO: 23.
  • mice The expression of humanized TLR8 protein in mice was detected by flow cytometry. Select one 10-week-old wild-type C57BL/6 mouse (+/+) and one mouse homozygous for humanized TLR8 gene (H/H), intraperitoneally inject 7.5 ⁇ g/200 ⁇ L mCD3, and remove the spleen 24 hours later. Cells were analyzed by flow cytometry.
  • the staining scheme is as follows: use anti-mouse CD45 antibodies Brilliant Violet 510 TM anti-mouse CD45 (mCD45), Brilliant Violet 711 TM anti-mouse TCR ⁇ chain (mTCR ⁇ ), Brilliant Violet 421 TM anti-mouse CD4 (mCD4) to label T cells, and use anti-mouse CD45 Mouse TLR8 antibody TLR8 Monoclonal Antibody (44C143), PE (mTLR8) or anti-human TLR8 antibody APC anti-human CD288 (TLR8) Antibody (hTLR8) is recognized and stained for flow cytometry detection.
  • T cells are mCD45+mTCR ⁇ +, among which the characteristics of mouse TLR8-positive (mTLR8+) T cells are mCD45+mTCR ⁇ +mTLR8+, and the characteristics of human TLR8-positive (hTLR8+) T cells are mCD45+mTCR ⁇ +hTLR8+.
  • the results of flow cytometry analysis showed that humanized TLR8 protein can be detected in mice homozygous for the TLR8 gene, indicating that the humanized TLR8 protein can be successfully expressed in mice with humanized TLR8 genes.
  • Flow cytometry was further performed on the immune phenotyping of wild-type C57BL/6 mice and TLR8 humanized homozygous mice.
  • leukocyte subtypes including: The percentages of T cells, B cells, NK cells, granulocytes, macrophages, and monocytes
  • T cell subtypes including: CD4+T cells, CD8+T cells, and Treg cells
  • CD4+T cells, CD8+T cells, and Treg cells were smaller than those of C57BL/6 wild-type Basically the same as that of mice, indicating that the TLR8 gene is of human origin.
  • Chemical modification did not affect the overall development, differentiation and distribution of immune cells in TLR8 gene humanized mice.
  • Example 2 Because the mouse TLR7 and TLR8 genes are both located on the mouse X chromosome, in order to achieve double-gene humanization of TLR7 and TLR8, the method described in Example 1 can be followed on the basis of obtaining TLR8 gene humanized mice in Example 2. Perform gene targeting. Specifically, the embryonic stem cells (ES cells) or fertilized eggs of the TLR8 gene humanized mouse prepared in Example 2 were genetically modified according to the method shown in Figure 3 or Figure 4 to obtain TLR7/TLR8 double gene human source. ized mice.
  • ES cells embryonic stem cells
  • FIG. 4 the embryonic stem cells
  • mice The expression of humanized TLR7 and TLR8 proteins in mice was detected by flow cytometry.
  • the characteristics of DC cells are mCD45+mCD3-mCD11c+, the characteristics of mouse TLR7 and TLR8 positive macrophages are mCD45+mCD3-mCD11c+mTLR7+ and mCD45+mCD3-mCD11c+mTLR8+ respectively, and the characteristics of human TLR7 and TLR8 positive macrophages. They are mCD45+mCD3-mCD11c+hTLR7+ and mCD45+mCD3-mCD11c+hTLR8+ respectively.
  • the characteristics of neutrophils are mCD45+mGr-1+, among which the characteristics of mouse TLR7- and TLR8-positive neutrophils are mCD45+mGr-1+mTLR7+ and mCD45+mGr-1+mTLR8+ respectively, and the characteristics of human TLR7- and TLR8-positive macrophages are The characteristics of the cells are mCD45+mGr-1+hTLR7+ and mCD45+mGr-1+hTLR8+ respectively;
  • the characteristics of monocytes are: mCD45+mGr-1-mCD11b+mF4/80-, among which the characteristics of mouse TLR7- and TLR8-positive monocytes are mCD45+mGr-1-mCD11b+mF4/80-mTLR7+ and mCD45+mGr- respectively.
  • 1-mCD11b+mF4/80-mTLR8+ the characteristics of human TLR7 and TLR8 positive monocytes are mCD45+mGr-1-mCD11b+mF4/80-hTLR7+ and mCD45+mGr-1-mCD11b+mF4/80-hTLR8+ respectively;
  • the characteristics of macrophages are mCD45+mGr-1-mCD11b+mF4/80+, among which the characteristics of murine TLR7- and TLR8-positive macrophages are mCD45+mGr-1-mCD11b+mF4/80+mTLR7+ and mCD45+mGr- respectively.
  • 1-mCD11b+mF4/80+mTLR8+, human TLR7 and TLR8 positive macrophages were characterized as mCD45+mGr-1- respectively mCD11b+mF4/80+hTLR7+ and mCD45+mGr-1-mCD11b+mF4/80+hTLR8+;
  • mice with humanized TLR7/TLR8 genes prepared by this method can successfully express humanized TLR7 and TLR8 proteins.
  • flow cytometry was performed to detect the in vivo immune phenotyping of wild-type C57BL/6 mice (+/+) and TLR8/TLR7 double-gene humanized homozygous mice (H/H; H/H).
  • the spleens of each mouse were , leukocyte subtypes in lymph nodes and blood (including: T cells, B cells, NK cells, granulocytes, macrophages, monocytes, dendritic cells (DC)) and T
  • the detection results of cell subtypes including CD4+T cells, CD8+T cells and Treg cells are shown in Figure 9, Figure 10 and Figure 11 respectively.
  • the data shows that the percentages of leukocyte subtypes and T cell subtypes in the spleen, lymph nodes and blood of TLR8/TLR7 double-gene humanized mice are basically the same, indicating that the humanized transformation of TLR8 and TLR7 genes does not affect the TLR8/TLR7 double-gene humanized mice.
  • TLR7 and TLR8 signaling pathways in TLR7/TLR8 double-gene humanized mice were detected by ELISA. Specifically, four 6- to 7-week-old female wild-type C57BL/6 mice (+/+) and TLR7/TLR8 double-gene humanized homozygous mice (H/H) were taken, spleen cells were collected, and Resuspend the single cells into 10mL PBS, add red blood cell lysis solution to lyse the red blood cells, resuspend the cells with an appropriate amount of RPMI-1640 culture medium to a final concentration of 3x10 6 cells/mL; take 100uL (3x10 5 cells) of cells from each well and plate them, and then Add 100uL of culture medium containing different concentrations of TLR8 agonist (GS-9688 and TL8-506), TLR7 agonist CL264 or control, and mix well; centrifuge after 24 hours of culture, collect the cell culture supernatant, and use LEGEND MAX TM Mouse TNF- ⁇
  • TLR7/TLR8 agonists show that after stimulation with TLR8/TLR7 agonists, mTNF- ⁇ secreted by cells is dose-dependent.
  • TLR7 agonists are more sensitive to wild-type C57BL/6 mice, while TLR8 agonists are more sensitive to TLR7/TLR7 agonists.
  • TLR8 double-gene humanized mice are more sensitive.
  • TLR7/TLR 8 agonist stimulation can increase the secretion of mTNF- ⁇ cytokines in TLR7/TLR8 double-gene humanized homozygous mice, proving that The TLR7/TLR8 signaling pathway in mice homozygous for TLR7/TLR8 double-gene humanization is normal.
  • mice homozygous for humanized TLR7 and/or TLR8 genes are inoculated subcutaneously with colon cancer cells MC38. After the tumor volume grows to about 100 mm, they are divided into a control group or a treatment group according to the tumor volume. The treatment group is injected with targeted Human TLR7 and/or TLR8 drugs, and the control group was injected with an equal volume of normal saline or PBS. Regularly measure the tumor volume and weigh the mice. By comparing the changes in mouse weight and tumor volume, the safety and in vivo efficacy of antibody drugs in humanized TLR7 and/or TLR8 mice can be effectively evaluated.
  • mice prepared by this method can also be used to construct an inflammatory bowel disease model to verify the efficacy of drugs targeting TLR7 and/or TLR8.
  • mice homozygous for humanized TLR7 and/or TLR8 genes are induced to construct an inflammatory bowel disease model (IBD).
  • IBD inflammatory bowel disease model
  • the mice were divided into a control group or a treatment group.
  • the treatment group was injected with antibody drugs targeting human TLR7 and/or TLR8, and the control group was injected with an equal volume of normal saline or PBS.
  • the TLR7 and/or TLR8 gene humanized mice prepared by this method can also be used to construct experimental arthritis animal models (CIA).
  • the mice were divided into a control group or a treatment group.
  • the treatment group was injected with antibody drugs targeting human TLR7 and/or TLR8, and the control group was injected with an equal volume of normal saline or PBS.
  • the body weight, toe and arthritis index of mice in each group were monitored, the paw thickness was measured and the arthritis score of each mouse was recorded, which can effectively evaluate the use of antibody drugs in humanized TLR7 and/or TLR8 mice. safety and in vivo efficacy.
  • Multi-humanized mouse models can also be prepared using this method or the TLR7 and/or TLR8-based humanized mice prepared.
  • the embryonic stem cells used for microinjection can be derived from mice containing genetic modifications such as PD-L1, HER2, EGFR, CTLA4, VEGFR2, etc., or they can also be derived from humanized TLR7 and/or
  • dual-humanized or multi-humanized mouse models are obtained by using isolation of mouse ES embryonic stem cells and gene recombination targeting technology.
  • the homozygous or heterozygous TLR7 and/or TLR8 mice obtained by this method can also be mated with other genetically modified mice, and their offspring can be screened.
  • the laws of Mendelian inheritance there is a certain probability of obtaining humanized TLR7 and/or Polygenic mice modified with the TLR8 gene and other genes can be mated with heterozygotes to obtain homozygotes with double or multiple gene modifications.
  • the TLR7/TLR8/TLR9 three-gene humanized mouse obtained in Example 3 was mated with the humanized TLR9 gene to obtain the simultaneous expression of human or humanized TLR7 protein and TLR8. protein and multigene humanized mice of TLR9 protein. Alternatively, take embryonic stem cells or fertilized eggs with humanized TLR7 and/or TLR8 genes, and then target the TLR9 gene to obtain TLR7/TLR8/TLR9 multi-gene humanized mice.
  • any combination of various embodiments of the present invention can also be carried out. As long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

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Abstract

一种表达人或嵌合TLR7和/或TLR8蛋白的非人动物及其使用方法。

Description

一种TLR7和/或TLR8基因人源化修饰的非人动物
优先权要求
本专利申请要求于2022年7月15日提交的中国专利申请号202210831117.9的优先权，该专利申请的全部内容通过引用并入本文。
技术领域
本发明提供一种表达人或嵌合(例如，人源化)TLR7和/或TLR8蛋白的非人动物及其使用方法。
背景
传统的药物研发通常使用体外筛选方法，然而这些筛选方法无法提供机体环境(如肿瘤微环境、基质细胞、细胞外基质成分和免疫细胞相互作用等)，导致药物开发失败率较高。此外，鉴于人与动物之间的差异，使用常规实验动物进行体内药理试验获得的试验结果可能无法反映真实的疾病状态和靶向部位的相互作用，导致许多临床试验的结果与动物实验结果存在显著差异。
因此，开发适合人抗体筛选和评价的人源化动物模型将显著提高新药开发效率，降低药物研发成本。
概述
本申请提供一种具有人或嵌合TLR7和/或TLR8蛋白的动物模型。该动物模型可以表达人或嵌合TLR7(如，人源化TLR7)蛋白和/或人或嵌合TLR8(如，人源化TLR8)蛋白。它可用于TLR7和TLR8基因功能的研究，还可用于TLR7/TLR8信号通路调节剂(例如，抗人TLR7和/或TLR8抗体和小分子激动剂)的筛选和评估。此外，通过本文所述方法制备的动物模型可用于药物筛选、药效学研究、免疫相关疾病的治疗和人TLR7/TLR8靶位点的癌症治疗；该模型还可以用于促进新药开发和设计，节省时间和成本。综上所述，本发明为研究TLR7/TLR8蛋白的功能提供了强有力的工具，为筛选抗癌药物提供了平台。
在一方面，本发明提供了一种基因修饰的非人动物，所述动物的基因组包含至少一条染色体，所述染色体包含编码人或嵌合Toll样受体7(TLR7)蛋白的核苷酸序列。在一些实施例中，所述编码人或嵌合TLR7蛋白的核苷酸序列可操作地连接至至少一条染色体的内源TLR7基因座的内源调控元件(如，内源5’UTR和/或3’UTR)。在一些实施例中，所述的人或嵌合TLR7蛋白包含人TLR7蛋白的信号肽、胞外、跨膜和/或胞质区的部分。在一些实施例中，所述的人或嵌合TLR7蛋白包含人TLR7蛋白胞外区的全部或部分。在一些实施例中，所述的人TLR7蛋白胞外区的氨基酸序列与SEQ ID NO：2第27-802位或SEQ ID NO：2第 27-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述的人或嵌合TLR7蛋白包含人TLR7蛋白信号肽的全部或部分。在一些实施例中，所述的人或嵌合TLR7蛋白信号肽的氨基酸序列与SEQ ID NO：2第4-26位或SEQ ID NO：2第1-26所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述的人或嵌合TLR7蛋白的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述的人或嵌合TLR7蛋白的氨基酸序列与SEQ ID NO：9所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述动物是哺乳动物，如猴子、啮齿动物、小鼠或大鼠。在一些实施例中，所述动物是小鼠。在一些实施例中，所述动物内源TLR7蛋白不表达或与野生型动物中TLR7相比表达水平降低。在一些实施例中，所述动物的一个或多个细胞表达人或嵌合TLR7蛋白。
在一方面，本发明提供了一种基因修饰的非人动物，所述动物的基因组包含在内源TLR7基因座处编码内源TLR7区域的核苷酸序列被人TLR7相应区域的核苷酸序列替换。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列可操作地连接到内源TLR7基因座的内源调控元件(如，内源5’UTR和/或3’UTR)，并且所述动物的一个或多个细胞表达人或嵌合TLR7蛋白。在一些实施例中，所述动物的内源TLR7蛋白不表达或与野生型动物中TLR7相比蛋白表达水平降低。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列包含人TLR7基因的外显子3的部分。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列与SEQ ID NO：5所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列与SEQ ID NO：8所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码内源TLR7区域的核苷酸序列包含小鼠TLR7基因外显子3的部分。在一些实施例中，所述动物基因组中修饰的TLR7基因对于内源被替换的基因座为纯合或杂合。
在一方面，本发明提供了一种非人动物，所述动物包含至少一个编码人或嵌合TLR7蛋白的核苷酸序列的细胞，其中所述人或嵌合TLR7蛋白包含与人相应区域的连续氨基酸序列至少50、60、70、80、90、100、200、300、500、600、700、720、740、760、790、792、795、798、799、802、839、900、1000、1020、1030、1040、1042、1044、1046、1048或1049个连续氨基酸一致。在一些实施例中，所述人或嵌合TLR7蛋白包含人TLR7蛋白的信号肽、胞外、跨膜和/或胞质区的部分。在一些实施例中，所述人或嵌合TLR7蛋白包含人 TLR7蛋白的胞外区的全部或部分。在一些实施例中，所述人TLR7蛋白胞外区的氨基酸序列与SEQ ID NO：2第27-802位或SEQ ID NO：2第27-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述的人或嵌合TLR7蛋白包含人TLR7蛋白信号肽的全部或部分。在一些实施例中，所述的人或嵌合TLR7蛋白信号肽的氨基酸序列与SEQ ID NO：2第4-26位或SEQ ID NO：2第1-26位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述的人或嵌合TLR7蛋白的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述人或嵌合TLR7蛋白氨基酸序列与SEQ ID NO：9所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码人或嵌合TLR7蛋白核苷酸序列可操作地连接至至少一条染色体的内源TLR7基因座的内源调控元件(如，内源5’UTR和/或3’UTR)。在一些实施例中，所述编码人或嵌合TLR7蛋白的核苷酸序列可被整合至所述动物内源TLR7基因座。在一些实施例中，所述人源化TLR7蛋白具有至少一种小鼠TLR7的活性和/或人TLR7的活性。
在一方面，本发明提供了一种基因修饰的非人动物的构建方法，其特征在于，所述动物的至少一个细胞中，在动物内源TLR7基因座处，编码内源TLR7区域的核苷酸序列被编码人TLR7相应区域的核苷酸序列替换。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列包含编码人TLR7蛋白信号肽和胞外区的全部或部分。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列包含人TLR7基因外显子3的部分。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列包含编码的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列与SEQ ID NO：5所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码内源TLR7区域的核苷酸序列包含小鼠TLR7基因外显子3的部分。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列可操作地连接至内源TLR7的调控元件，如，启动子。在一些实施例中，所述动物为哺乳动物，如猴子、啮齿动物、小鼠或大鼠。在一些实施例中，所述动物是小鼠。
在一方面，本发明提供了一种表达人或嵌合TLR7蛋白基因修饰非人动物的细胞构建方法，所述方法包括在内源小鼠TLR7基因座处，编码内源TLR7区域的核苷酸序列被编码人TLR7相应区域的核苷酸序列替换，产生基因修饰的非人动物细胞，其中动物细胞表达人或 嵌合TLR7蛋白。在一些实施例中，所述人或嵌合TLR7蛋白包含人TLR7蛋白信号肽和胞外区的全部或部分。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列包含人TLR7基因外显子3的部分。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列与SEQ ID NO：5所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述编码内源TLR7区域的核苷酸序列包含小鼠TLR7基因外显子3的部分。在一些实施例中，所述编码人或嵌合TLR7蛋白的核苷酸序列可操作地连接至内源TLR7的调控元件，如，启动子。在一些实施例中，所述动物为哺乳动物，如猴子、啮齿动物、小鼠或大鼠。在一些实施例中，所述动物是小鼠。在一些实施例中，上述所述的方法还包括其他基因编码的人或嵌合蛋白的核苷酸序列，所述人或嵌合蛋白选自TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40的至少一种。在一些实施例中，所述人或嵌合蛋白为TLR8蛋白。在一些实施例中，所述编码人或嵌合TLR8蛋白的核苷酸序列与SEQ ID NO：22所示的核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述人或嵌合TLR8蛋白氨基酸序列与SEQ ID NO：23所示的氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，上述所述的非人动物还包括其他基因编码的人或嵌合蛋白的核苷酸序列，所述人或嵌合蛋白选自TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40的至少一种。在一些实施例中，所述人或嵌合蛋白为TLR8蛋白。在一些实施例中，所述编码人或嵌合TLR8蛋白的核苷酸序列与SEQ ID NO：22所示的核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述人或嵌合TLR8蛋白氨基酸序列与SEQ ID NO：23所示的氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
在一方面，本发明提供了一种测定抗TLR7治疗剂治疗癌症有效性的方法，所述方法包括：1)向本申请所述非人动物施用抗TLR7治疗剂，其中所述动物具有肿瘤；2)测定抗TLR7治疗剂对肿瘤的抑制作用。在一些实施例中，所述肿瘤包含一个或多个肿瘤细胞，其中肿瘤细胞被注射到动物体内。在一些实施例中，所述测定抗TLR7治疗剂对肿瘤的抑制作用包含测量动物体内的肿瘤体积。在一些实施例中，所述肿瘤为实体瘤、膀胱癌、浅表尿路上皮癌、宫颈癌、子宫内膜癌、食道癌、鳞状细胞癌、肾癌、非小细胞肺癌、卵巢癌、鳞状细胞癌、胃癌、子宫癌、结直肠转移癌、肝癌、胃肠癌。
在一方面，本发明提供了一种测定抗TLR7治疗剂和其它治疗剂治疗癌症有效性的方法，所述方法包括：1)向本申请所述非人动物施用抗TLR7治疗剂，其中所述动物具有肿瘤；2)测定抗TLR7治疗剂对肿瘤的抑制作用。在一些实施例中，所述动物还包括编码人或嵌合PD-1、人或嵌合PD-L1和/或人或嵌合CTLA4的序列。在一些实施例中，所述其它治疗剂是抗PD-1抗体、抗PD-L1抗体和/或抗CTLA4抗体。在一些实施例中，所述肿瘤包含一个或多个细胞表达PD-L1蛋白。在一些实施例中，所述肿瘤包含一个或多个肿瘤细胞，其中肿瘤细胞被注射到动物体内。在一些实施例中，所述测定抗TLR7治疗剂对肿瘤的抑制作用包含测量动物体内的肿瘤体积。在一些实施例中，所述肿瘤为实体瘤、膀胱癌、浅表尿路上皮癌、宫颈癌、子宫内膜癌、食道癌、鳞状细胞癌、肾癌、非小细胞肺癌、卵巢癌、鳞状细胞癌、胃癌、子宫癌、结直肠转移癌、肝癌、胃肠癌。
在一方面，本发明提供了一种测定抗TLR7治疗剂治疗代谢性疾病有效性的方法，所述方法包括：1)向本申请所述非人动物施用抗TLR7治疗剂，其中所述非人动物患有自身免疫性疾病；2)测定抗TLR7治疗剂对治疗自身免疫性疾病中的作用。在一些实施例中，所述自身免疫性疾病为哮喘、鼻炎、系统性红斑狼疮、银屑病。
在一方面，本发明提供了一种测定抗TLR7治疗剂治疗炎症有效性的方法，所述方法包括：1)向本申请所述非人动物施用抗TLR7治疗剂，其中所述非人动物患有炎症；2)测定抗TLR7治疗剂对治疗炎症中的作用。在一些实施例中，所述炎症为慢性阻塞性肺病、脓毒症、皮炎。
在一方面，本发明提供了一种测定抗TLR7治疗剂毒性的方法，所述方法包括：1)向本申请所述非人动物施用抗TLR7治疗剂；2)测定抗TLR7治疗剂对动物的作用。在一些实施例中，所述测定抗TLR7治疗剂对动物的作用涉及测量动物的体重、红细胞计数、血细胞比容和/或血红蛋白。
在一方面，本发明提供了一种人源化TLR7蛋白，其特征在于，所述的人源化蛋白包含人TLR7蛋白信号肽和胞外区的全部或部分。在一些实施例中，所述人源化TLR7蛋白氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。在一些实施例中，所述人源化TLR7蛋白氨基酸序列与SEQ ID NO：9所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
在一方面，本发明提供了一种人源化TLR7基因，所述人源化TLR7基因编码上述所述的人源化TLR7蛋白。在一些实施例中，所述人源化TLR7基因包含人TLR7基因外显子3的部分。在一些实施例中，所述人源化TLR7基因包含的核苷酸序列与SEQ ID NO：3、4、 5、6、7、8、10和11所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
在一方面，本发明提供了一种细胞，其特征在于，所述细胞包含权利上述所述的人源化TLR7蛋白和人源化TLR7基因。
在一方面，本发明提供了一种动物模型，其特征在于，所述的动物模型包含上述所述的人源化TLR7蛋白和上述所述人源化TLR7基因。
除非另有定义，本文使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。本文描述了用于本发明的方法和材料；可以使用本领域已知的其他合适的方法和材料。材料、方法和实施例仅是示例性的而非限制性的。本文提及的所有出版物、专利申请、专利、序列、数据库条目和其他参考文献均通过引用整体并入。在冲突的情况下,以本说明书(包括定义)为准。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方便和优势。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：小鼠TLR7基因座和人TLR7基因座对比示意图(非按比例)；
图2：小鼠TLR7基因座人源化改造示意图(非按比例)；
图3：TLR7基因打靶策略及靶向载体V1设计示意图一(非按比例)；
图4：TLR7基因打靶策略及靶向载体V2设计示意图二(非按比例)；
图5：F0代小鼠基因型鉴定结果，M为Marker，WT为野生型对照，H2O为水对照，其中F0-2为阳性小鼠且无随机插入；
图6：Southern blot检测结果，其中WT为野生型对照，其中F1-4、F1-5、F1-8、F1-9、F1-10、F1-11为阳性小鼠且无随机插入，其他小鼠有杂带；
图7：小鼠TLR8基因座和人TLR8基因座对比示意图(非按比例)；
图8：小鼠TLR8基因座人源化改造示意图(非按比例)；
图9：野生型C57BL/6小鼠和TLR7/TLR8双基因人源化小鼠脾脏中白细胞亚型百分比(A)以及T细胞亚型百分比(B)；
图10：野生型C57BL/6小鼠和TLR7/TLR8双基因人源化小鼠淋巴结中白细胞亚型百分比(A)以及T细胞亚型百分比(B)；
图11：野生型C57BL/6小鼠和TLR7/TLR8双基因人源化小鼠血液中白细胞亚型百分比(A)以及T细胞亚型百分比(B)；
图12：野生型C57BL/6小鼠和TLR7/TLR8双基因人源化小鼠经TLR7/TLR8激动剂刺 激后TNF-α细胞因子的分泌检测，其中ctrl为未经TLR7/TLR8激动剂处理的对照组；
图13：人TLR7氨基酸序列(NP_057646.1；SEQ ID NO：2)和小鼠TLR7氨基酸序列(NP_573474.1；SEQ ID NO：1)；
图14：人TLR7氨基酸序列(NP_057646.1；SEQ ID NO：2)和大鼠TLR7氨基酸序列(NP_001091051.1；SEQ ID NO：24)。
详细说明
Toll样受体(TLRs)是一类先天免疫传感器，可以识别保守的微生物结构，TLR的激活可以启动先天性和适应性免疫反应。TLRs促进抗原递呈细胞(如树突状细胞或巨噬细胞)激活和诱导免疫调节细胞因子的能力对形成适应性免疫反应至关重要。人类TLRs家族有包括TLR1至TLR10的10个成员，其中TLR1、TLR2、TLR4、TLR5、TLR6、TLR10表达于细胞表面，而TLR3、TLR7、TLR8、TLR9则主要定位于细胞内的核内体膜上。
TLR7和TLR8是肿瘤免疫中研究较多的两个Toll样受体靶点。TLR7位于X染色体上，属于I型跨膜蛋白，包括信号肽、胞外区、跨膜区和胞内区。其中，胞外区富含亮氨酸重复序列，主要功能是来识别相应配体；胞内区有一个高度保守的蛋白相互作用区Toll/IL-1受体区域(TIR)，在TLR的信号传导中发挥着重要作用。TLR7广泛表达于多种细胞中，如浆胞细胞样树突状细胞(pDC)、巨噬细胞、T细胞和B细胞，在嗜酸性粒细胞和中性粒细胞中也有表达。研究表明，TLR7在肺癌、尿道癌、乳腺癌和胰腺癌患者的癌细胞及其周围的基质细胞功能性表达，在恶性B细胞和髓样细胞中也有表达。
TLR7的配体包括ssRNA病毒以及一些合成的小分子化合物，其中Imiquomid(R837)是研究最广泛的咪唑喹啉家族系列的化合物，主要作为TLR7/TLR8的激动剂发挥抗肿瘤的效应。
TLRs家族的信号转导方式主要分为两种：一种是髓样分化因子88(MyD88)依赖型信号通路，另一种是MyD88非依赖型/TRIF(INF-β)依赖型信号转导通路。TLR7为MyD88依赖型的信号通路。ssRNA被TLR7识别后，经系列级联反应引起IFN、IL1、IL6、IL12、TNF等多种炎性细胞因子分泌，发挥抗病毒效应机制。除了病毒RNA，偶然情况下，TLR7也能被自身RNA活化，引起自身免疫性疾病，例如，系统性红斑狼疮患者活动期TLR7表达水平升高；TLR7在CIA模型中异常低表达，可能具有负调控作用抑制RA滑膜炎发生，为RA潜在治疗靶点。
TLR7和TLR8在系统发育和结构上是相关的。TLR7主要表达在pDC上，TLR8主要表达在髓样树突状细胞(mDC)上，两者在受到激活后都可以分泌1型干扰素并表达共刺激分子，使杀伤性细胞得到激活进攻肿瘤细胞，而树突状细胞可以从死亡的肿瘤细胞碎片中 获得肿瘤特异性抗原。因此同时激活这两类受体，可以充分利用树突状细胞作为先天免疫和后天免疫之间的桥梁角色。
在人的基因组中，TLR7基因(Gene ID：51284)包含3个外显子，即外显子1、外显子2和外显子3(图1)。人TLR7mRNA的核苷酸序列为NM_016562.4，人TLR7的氨基酸序列为NP_057646.1(SEQ ID NO：2)。基于转录本NM_016562.4及其编码蛋白NP_057646.1的核苷酸序列和氨基酸序列中每个外显子对应位置如下：
表1
人TLR7基因(NCBI Gene ID：51284)位于X号染色体上的NC_000023.11的第12867072至12890361位(GRCh38.p13(GCF_000001405.40)。基于转录本NM_016562.4每个外显子的具体位置为：5’UTR位于NC_000023.11第12867072至12867123和第12867481至12867578，外显子1位于NC_000023.11第12867072至12867123位，内含子1位于NC_000023.11第12867124至12867480位，外显子2位于NC_000023.11第12867481至12867581位，内含子2位于NC_000023.11第12867582至12885511位，外显子3位于NC_000023.11第12885512至12890361位，3’UTR位于NC_000023.11第12888659至12890361位。以上关于人TLR7基因座的所有相关信息都可以在NCBI网站上(Gene ID：51284)检索到。其全部内容通过引用并入本文。
在小鼠的基因组中，TLR7基因(Gene ID：170743)包含3个外显子，即外显子1、外显子2和外显子3(图1)。小鼠TLR7mRNA的核苷酸序列为NM_133211.4，小鼠TLR7的氨基酸序列为NP_573474.1(SEQ ID NO：1)。基于转录本NM_133211.4及其编码蛋白NP_573474.1的核苷酸序列和氨基酸序列中每个外显子对应位置如下：
表2

小鼠TLR7基因(NCBI Gene ID：170743)位于X号染色体上的NC_000086.8的第166086376至166113570位(GRCm39(GCF_000001635.27))。基于转录本NM_133211.4每个外显子的具体位置为：5’UTR位于NC_000086.8第166113554至166113433和第166113095至166113000位，外显子1位于NC_000086.8第166113554至166113433位，内含子1位于NC_000086.8第166113432至166113096位，外显子2位于NC_000086.8第166113095至166112997位，内含子2位于NC_000086.8第166112996至166091482位，外显子3位于NC_000086.8第166091481至166087927位。3’UTR位于NC_000086.8第166088331至166087927位。以上关于鼠TLR7基因座的所有相关信息都可以在NCBI网站上(Gene ID：170743)检索到。其全部内容通过引用并入本文。
图13显示了人TLR7氨基酸序列(NP_057646.1；SEQ ID NO：2)和小鼠TLR7氨基酸序列(NP_573474.1；SEQ ID NO：1)比对。因此，在图13中可以找到人与小鼠的TLR7之间相对应氨基酸残基或区域。
本领域中其他物种的TLR7基因、蛋白和基因位点也是已知的。例如，Rattus norvegicus(大鼠)TLR7的Gene ID：317468、Macaca mulatta(恒河猴)TLR7的Gene ID：574291、Canis lupus familiaris(狗)TLR7的Gene ID：491743，Sus scrofa(猪)TLR7的Gene ID：100037296。这些基因的相关信息(如，内含子序列、外显子序列和氨基酸序列)均可以在NCBI中查找到，其全部内容通过引用并入本文。
图14显示了人TLR7氨基酸序列(NP_057646.1；SEQ ID NO：2)和大鼠TLR7氨基酸序列(NP_001091051.1；SEQ ID NO：24)。因此，在图14中可以检索到人与大鼠的TLR7之间相对应氨基酸残基或区域。
本发明提供一种人或嵌合(如，人源化)TLR7核苷酸序列或氨基酸序列。在一些实施例中，小鼠TLR7基因外显子1、外显子2和/或外显子3的核苷酸序列的全部或部分被人TLR7基因相应核苷酸序列替换。在一些实施例中，小鼠TLR7基因外显子1、外显子2和/或外显子3的“部分”被人TLR7基因相应核苷酸序列或氨基酸序列替换。所述“部分”是指至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、 130、140、150、160、170、180、190、200、250、300、350、400、450、500、550、600、800、1400、1800、2200、2300、2340、2380、2390、2391、2392、2394、2395、2398、2399、2400、2600、3000、3300、3600、3700、3730、3750、3770、3790、3791、3792、3793、3794、4000、5000、6000、8000、12000、16000、20000、24000、2500或25628bp连续核苷酸序列，或者至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、200、300、500、600、700、800、801、802、803、820、830、831、833、836、839、840、900、1000、1020、1030、1040或1050个连续氨基酸序列。在一些实施例中，所述“部分”与外显子1、外显子2和/或外显子3编码的氨基酸序列同一性至少为50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或至少100％。在一些实施例中，小鼠TLR7外显子1、外显子2和/或外显子3(例如，外显子3的部分)的“部分”或“全部”序列被人TLR7基因外显子1、外显子2和/或外显子3(例如，外显子3的部分)的“部分”或“全部”序列替换。
在一些实施例中，内源外显子1、外显子2和/或外显子3的“部分”缺失。
在一些实施例中，本发明提供了一种基因修饰的非人动物，所述非人动物的基因组包括人、嵌合或人源化的TLR7核苷酸序列。在一些实施例中，所述人、嵌合或人源化的TLR7核苷酸序列编码的蛋白与SEQ ID NO：9所示氨基酸序列同一性至少为70％、80％、85％、90％、95％或100％。在一些实施例中，所述非人动物基因组包含的核苷酸序列与SEQ ID NO：3、4、5、8、10、11所示核苷酸序列同一性至少为70％、80％、85％、90％、95％或100％。
在一些实施例中，本文所述非人动物包含编码人或嵌合TLR7蛋白的核苷酸序列。在一些实施例中，所述人或嵌合TLR7蛋白包含人TLR7蛋白的信号肽、胞外区、跨膜区和/或胞质区的全部或部分。
在一些实施例中，所述人或嵌合TLR7蛋白包含人TLR7蛋白的信号肽的全部或部分，进一步的，所述人TLR7蛋白信号肽的部分包含至少5个连续氨基酸，例如包含至少5、7、9、10、15、17、19、20、21、22、23、24、25、26个连续氨基酸，所述人或嵌合TLR7蛋白信号肽包含与SEQ ID NO：2第4-26位或SEQ ID NO：2第1-26位所示氨基酸序列同一性至少为10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％或100％。
在一些实施例中，所述人或嵌合TLR7蛋白包含人TLR7蛋白的胞外区的全部或部分，进一步的，所述人TLR7蛋白胞外区的部分包含至少200个连续氨基酸，例如包含至少200、300、400、500、700、750、760、770、775、776、777、778、779、780、800、813个连续 氨基酸，所述人或嵌合TLR7蛋白胞外区包含与SEQ ID NO：2第27-802位或SEQ ID NO：2第27-839位所示氨基酸序列同一性至少为10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％或100％。
在一些实施例中，所述人或嵌合TLR7蛋白包含与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％或100％。
在一些实施例中，本文所述非人动物包含人或嵌合TLR7基因。在一些实施例中，所述嵌合TLR7基因包含3个人或嵌合外显子。在一些实施例中，所述嵌合TLR7基因包含嵌合外显子3。在一些实施例中，所述嵌合TLR7基因包含人或内源5’UTR。在一些实施中，所述嵌合TLR7基因包含人或内源3’UTR。在一些实施例中，所述嵌合TLR7基因包含内源5’UTR。在一些实施例中，所述嵌合TLR7基因包含内源3’UTR。
在一些实施例中，基因修饰的非人动物可以表达人TLR7和/或嵌合(如，人源化)TLR7蛋白，所述的非人动物内源TLR7基因和/或核苷酸序列被人TLR7基因和/或核苷酸序列替换。进一步的，所述人TLR7基因和/或核苷酸序列编码人TLR7的氨基酸序列与人TLR7SEQ ID NO：2所示氨基酸序列同一性至少为10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％或100％。在各种实施例中，内源TLR7基因被编码成熟的TLR7蛋白的核苷酸序列全部或部分替换。
在一些实施例中，基因修饰的非人动物在小鼠内源启动子和/或调控元件下表达人TLR7和/或嵌合TLR7蛋白(如，人源化TLR7蛋白)。小鼠内源基因座的替换提供了一种在相同细胞类型中表达人或嵌合TLR7蛋白(如，人源化TLR7蛋白)的非人动物。经基因修饰的小鼠并未出现本领域已知的在某些其它转基因小鼠中观察到的潜在疾病。在非人动物中表达的人TLR7或嵌合TLR7蛋白(如，人源化TLR7蛋白)可以维持一种或多种野生型或人TLR7蛋白的功能。进一步地，在一些实施例中，基因修饰的非人动物不表达内源TLR7蛋白。在一些实施例中，基因修饰的非人动物内源TLR7蛋白表达降低。本文所述的“内源TLR7蛋白”是指基因修饰前的非人动物(如，小鼠)内源TLR7基因核苷酸序列编码的TLR7蛋白。
非人动物的基因组包含编码与人TLR7蛋白(NP_057646.1；SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位)所示氨基酸序列的同一性至少为70％、75％、80％、85％、90％、95％、99％或100％的氨基酸的核苷酸序列。在一些实施例中，所述基因组包含与SEQ ID NO：5和SEQ ID NO：8所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或至少100％的核苷酸序列。
非人动物基因组中编码内源TLR7区域的核苷酸序列被编码人TLR7相应区域的核苷酸序列替换。在一些实施例中，所述编码内源TLR7区域的核苷酸序列是内源TLR7基因座的任一序列，如，外显子1、外显子2、外显子3、5’UTR、3’UTR、内含子1、内含子2或其任意组合。在一些实施例中，所述编码内源TLR7区域的核苷酸序列位于内源TLR7调控区内。在一些实施例中，所述编码内源TLR7区域的核苷酸序列为外显子1、外显子2和/或外显子3，或其部分。
基因修饰的非人动物一个或多个细胞表达人或嵌合TLR7蛋白(如，人源化TLR7蛋白)。在一些实施例中，人或嵌合TLR7蛋白(如，人源化TLR7蛋白)至少包含与SEQ ID NO：2所示的氨基酸序列1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、200、300、500、600、700、720、740、760、790、792、795、798、799、800、801、802、803、804、805、810、820、830、831、833、836、839、840、850、900、1000、1020、1030、1040、1042、1044、1046、1048或1049个连续的氨基酸序列。
在一些实施例中，基因修饰的非人动物基因组中包含人TLR7基因外显子1、外显子2和/或外显子3的全部或部分，或SEQ ID NO：5所示核苷酸序列的全部或部分。
在一些实施例中，基因修饰的非人动物基因组中包含人TLR7基因外显子3的部分。在一些实施例中，所述的人TLR7基因外显子3的部分包含至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、350、450、600、1000、1500、2000、2200、2300、2340、2380、2390、2392、2394、2396、2397、2400、2420、2430、2434、2438、2439、3000、3500、4000、4500、4600、4800、4830、4840或4850bp连续核苷酸序列。在一些实施例中，外显子3的部分包含2397bp的连续核苷酸序列。在一些实施例中，外显子3的部分包括至少100-500bp、1000-1500bp或2000-2300bp的核苷酸序列。在一些实施例中，所述编码人TLR7相应区域的核苷酸序列位于人TLR7基因转录本NM_016562.4的第160-2556位核苷酸序列。
在一些实施例中，基因修饰的非人动物的TLR7基因对于内源被修饰基因座是杂合的或者是纯合的。
在一些实施例中，所述嵌合TLR7基因组缺少人TLR7基因的5’UTR。在一些实施例中，所述嵌合TLR7基因组包含内源的(如，小鼠)5’UTR。在一些实施例中，所述嵌合TLR7基因组包含内源的(如，小鼠)3’UTR。在适当的情况下，基于5’侧翼序列的相似性，可以合理地推测小鼠和人TLR7基因受到相似的调控。如本发明所述，嵌合TLR7小鼠包含内源小鼠基因座的替换，该替换保留小鼠内源调控元件，但包含嵌合TLR7编码序列。基因修饰的杂合子小鼠或纯合子小鼠中TLR7的表达是完全正常的。
另一方面，本发明提供了一种基因修饰的非人动物，所述非人动物基因组包含内源TLR7基因的缺失，其中内源TLR7基因的缺失包含外显子1、外显子2和/或外显子3，或内源TLR7基因座的部分。
在一些实施例中，内源TLR7基因的缺失包含一个或多个外显子或外显子的部分，所述外显子选自外显子3。
在一些实施例中，其中所述缺失包含至少1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、400、600、1000、1500、2200、2500、3000、3500、3700、3740、3780、3790、3791、3792、3793、3794、4000、5000、6000、8000、12000、16000、20000、24000、2500或25628bp连续核苷酸序列或更多的核苷酸序列。
在一些实施例中，所述内源TLR7基因的缺失包含外显子3的至少50、60、70、80、90、100、150、200、400、600、800、1000、1400、1800、2000、2100、2300、2320、2340、2360或2390、2394、2396、2397、2398、2399、2400、2420、2430、2431、2432、2433、2700、2900、3000、3100、3300、3500或3560bp连续核苷酸序列或更多的核苷酸序列。
本发明提供了一种人源化小鼠TLR7基因组DNA序列；提供了一个表达人源化TLR7蛋白的氨基酸序列的构建体；一种包含所述构建体的细胞；一种包含所述细胞的组织。因此，在一些实施例中，本发明提供了一种嵌合的(如，人源化)TLR7核苷酸序列和/或氨基酸序列，其中在一些实施例中，所述嵌合的核苷酸序列与小鼠内源TLR7mRNA(如，NM_133211.4)、小鼠TLR7氨基酸序列(如，NP_573474.1，SEQ ID NO：1)或其部分(如，外显子3的部分)所示的序列同一性至少为1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。在一些实施例中，所述嵌合核苷酸序列与人TLR7mRNA序列(如，NM_016562.4)、TLR7氨基酸序列(如，NP_057646.1，SEQ ID NO：2)或其部分(如，外显子3的部分)所示的序列同一性至少为1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。
在一些实施例中，上述所述嵌合的核酸序列可操作地连接到启动子或调节元件上，例如，内源小鼠TLR7启动子、诱导型启动子、增强子和/或小鼠或人调节元件。
在一些实施方案中，本文所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)不同于小鼠TLR7核苷酸序列全部或部分(例如， 小鼠TLR7基因转录本NM_133211.4外显子3的部分)。
在一些实施方案中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)与小鼠TLR7核苷酸序列的全部或部分相同(例如，小鼠TLR7基因转录本NM_133211.4的外显子1至外显子2的全部和外显子3的部分)。
在一些实施方案中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)不同于人TLR7核苷酸序列全部或部分(例如，人TLR7基因转录本NM_016562.4的外显子1至外显子2的全部和外显子3的部分)。
在一些实施方案中，上述所述嵌合的核酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个核苷酸，例如，连续或非连续核苷酸序列)与人TLR7核苷酸序列全部或部分相同(例如，人TLR7基因转录本NM_016562.4的外显子3的部分)。
在一些实施方案中，所述嵌合的核酸序列编码的氨基酸至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，如，连续或非连续氨基酸残基)不同于小鼠TLR7蛋白氨基酸序列的全部或部分(例如，小鼠TLR7蛋白序列NP_573474.1第4-803位，第1-803位或1-840位氨基酸(SEQ ID NO：1))。
在一些实施方案中，所述嵌合的核酸序列编码的氨基酸至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，如，连续或非连续氨基酸残基)与小鼠TLR7蛋白氨基酸序列的全部或部分相同(例如，小鼠TLR7蛋白序列NP_573474.1第1-3，第804-1050或第841-1050位氨基酸(SEQ ID NO：1))。
在一些实施方案中，所述氨基酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，例如，连续或非连续氨基酸残基)不同于人TLR7蛋白氨基酸序列的全部或部分(例如，人TLR7蛋白序列NP_057646.1第803-1049位或第840-1049位氨基酸(SEQ ID NO：2))。
在一些实施方案中，所述氨基酸序列至少有一部分(例如，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90或100个氨基酸残基，例如，连续或非连续氨基酸残基)与人TLR7蛋白氨基酸序列的全部或部分相同(例如，人TLR7蛋白序列NP_057646.1第4-802位，第1-802位或1-839位氨基酸(SEQ ID NO： 2))。
本发明还提供一种人源化的TLR7小鼠氨基酸序列，其中所述氨基酸序列包含下列组中的任一种：
A)SEQ ID NO：1、2、9所示氨基酸序列；
B)与SEQ ID NO：1、2、9所示氨基酸序列同一性至少为90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％；
C)与SEQ ID NO：1、2、9所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；或
D)与SEQ ID NO：1、2、9所示的，包括替换、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明还提供一种人源化的TLR7氨基酸序列，其中所述氨基酸序列包含下列组中的任一种：
A)SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示的氨基酸序列；
B)与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％；
C)与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；或
D)与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示的，包括替换、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明还提供一种人源化的TLR7核苷酸(如，DNA或RNA)序列，其中所述核苷酸列包含下列组中的任一种：
A)如SEQ ID NO：3、4、5、6、7、8、10和11所示的核酸序列或编码人源化小鼠TLR7同源氨基酸序列的核酸序列；
B)能够在低严格条件或严格条件下与SEQ ID NO：3、4、5、6、7、8、10和11所示核苷酸序列杂交的核酸序列；
C)具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同源性的核酸序列，与SEQ ID NO：3、4、5、6、7、8、10和11所示核苷酸序列90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％相同；
D)其编码的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或 SEQ ID NO：2第1-839位所示的氨基酸序列同一性至少为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％；
E)编码的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸；或
F)编码的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示的，包括替换、缺失和/或插入一个或多个氨基酸残基的氨基酸序列。
本发明进一步提供了一种人源化小鼠的TLR7基因组DNA序列。该DNA序列由其转录得到的mRNA逆转录获得，与SEQ ID NO：5或8所示序列同源的DNA序列一致或互补。
基因修饰的非人动物
本发明所述“基因修饰的非人动物”是指该动物基因组中至少一条染色体具有外源TLR7的非人动物。在一些实施例中，至少一个或多个细胞中，例如，基因修饰的非人动物中至少1％、2％、3％、4％、5％、10％、20％、30％、40％、50％的细胞具有外源DNA。具有外源DNA的细胞可以是各种细胞，例如，内源细胞、体细胞、免疫细胞、T细胞、B细胞、NK细胞、抗原呈递细胞、巨噬细胞、树突状细胞、生殖细胞、囊胚或内源肿瘤细胞。在一些实施例中，提供了一种基因修饰的非人动物，所述动物包含内源TLR7基因座和外源TLR7基因座(如，人序列)，例如，用一个或多个人源序列替换一个或多个非人序列，或插入一个或多个人源和/或非人序列。动物通常能够通过种系传播将基因修饰传递给后代。
本发明所述“嵌合基因”或“嵌合核酸”是指基因或核酸，其中所述基因或核酸的两个或多个部分来自不同物种，或者该基因或核酸的至少一个序列与动物中的野生型核酸不同。在一些实施例中，嵌合基因或嵌合核酸具有至少一部分序列来源于两个或多个不同的物种，例如，编码不同蛋白的序列或编码两个或多个不同物种的相同(或同源)蛋白的序列。在一些实施例中，嵌合基因或嵌合核酸是指人源化基因或人源化核酸。
本发明所述“嵌合蛋白”或“嵌合多肽”是指蛋白或多肽，其中所述多肽或蛋白的两个或多个部分来自不同物种，或者该蛋白或多肽的至少一个序列与动物中的野生型氨基酸序列不同。在一些实施例中，嵌合蛋白或嵌合多肽的至少一部分序列具有两个或多个不同物种来源，例如，不同物种的相同(或同源)蛋白。在一些实施例中，嵌合蛋白或嵌合多肽是指人源化蛋白或人源化多肽。
本发明所述“人源化蛋白”或“人源化多肽”是指蛋白或多肽，其中所述蛋白或多肽的至少一部分来自人蛋白或人多肽。在一些实施例中，人源化蛋白或人源化多肽是指人蛋白或多肽。
本发明所述“人源化核酸”是指核酸，其中所述核酸的至少一部分来自人核酸。在一些实施例中，人源化核酸中的核酸全部来源于人。在一些实施例中，人源化核酸是指人源化外显子，所述人源化外显子可以是人的外显子或嵌合外显子。
在一些实施例中，嵌合基因或嵌合核酸是人源化TLR7基因或人源化TLR7核酸。在一些实施例中，所述基因或核酸的至少一部分来源于人TLR7基因，或者所述基因或核酸的至少一部分来源于非人TLR7基因。在一些实施例中，所述基因或核酸包含编码TLR7蛋白的序列。在一些实施例中，所述的编码的TLR7蛋白至少具有一种人TLR7蛋白或非人动物TLR7蛋白的活性。
在一些实施例中，所述嵌合蛋白或嵌合多肽是人源化TLR7蛋白或人源化TLR7多肽。在一些实施例中，所述蛋白或多肽的氨基酸序列中的至少一个或多个部分来源于人TLR7蛋白，或者所述蛋白或多肽的氨基酸序列的至少一个或多个部分来源于非人TLR7蛋白。人源化TLR7蛋白或人源化TLR7多肽是功能性的，或至少具有一种人TLR7蛋白或非人动物TLR7蛋白的活性。
基因修饰的非人动物可以是各种动物，例如，小鼠、大鼠、兔子、猪、牛(例如，牛、公牛、水牛)、鹿、绵羊、山羊、鸡、猫、狗、雪貂、灵长类动物(例如，狨猴、恒河猴)。对于不容易获得合适的可遗传修饰胚胎干细胞(ES)的非人动物，采用其他方法来构建包含遗传修饰的非人动物。这样的方法包括，例如，修饰非ES细胞基因组(例如，成纤维细胞或诱导多能干细胞)并采用核移植将修饰的基因组转移到合适的细胞，例如卵母细胞，以及在适当的条件下在非人动物中孕育修饰的细胞(例如，修饰的卵母细胞)以形成胚胎。上述所述构建方法在本领域中是已知的，并且在“A.Nagy,et al.,“Manipulating the Mouse Embryo:A Laboratory Manual(Third Edition),”Cold Spring Harbor Laboratory Press,2003”有所描述，其全部内容通过引用并入本文。
在一个方面，所述动物是哺乳动物。在一些实施例中，基因修饰的非人动物是啮齿动物。啮齿动物可以选自小鼠、大鼠和仓鼠。在一个实施例中，所述啮齿动物选自鼠家族。在一个实施例中，所述基因修饰的动物选自丽仓鼠科(例如小鼠样仓鼠)、仓鼠科(例如仓鼠、新世界大鼠和小鼠、田鼠)、鼠总科(真小鼠和大鼠、沙鼠、刺毛鼠、冠毛大鼠)、马岛鼠科(登山小鼠、岩小鼠、有尾大鼠、马达加斯加大鼠和小鼠)、刺睡鼠科(例如多刺睡鼠)和鼹形鼠科(例如摩尔大鼠、竹大鼠和鼢鼠)家族。在一个特定实施例中，所述基因修饰的啮齿动物选自真小鼠或大鼠(鼠总科)、沙鼠、刺毛鼠和冠毛大鼠。在一个实施例中，所述基因修饰的小鼠来自鼠科家族成员。在一个实施例中，所述动物是啮齿动物。在一个特定实施例中，所述啮齿动物选自小鼠和大鼠。在一个实施例中，所述非人动物是小鼠。
在一些实施例中，所述动物是C57BL品系的小鼠，所述C57BL品系选自C57BL/a、C57BL/An、C57BL/GrFa、C57BL/KaLwN、C57BL/min、C57BL6J、C57B1/6ByJ、C57BL/6NJ、C57BL/10、C57BL10SnSn、C57BL/10Cr和C57BL/Ola。在一些实施例中，小鼠是选自129P1、129P2、129P3、129X1、129S1(例如129S1/SV、129S1/SvIm)、129S2、129S4、129S5、129S9/SvEvH、129S6(129/SvEvTac)、129S7、129S8、129T1、129T2的129品系。这些小鼠描述于例如Festing et al.,Revised nomenclature for strain 129mice,Mammalian Genome 10:836(1999)；Auerbach et al.,Establishment and Chimera Analysis of129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines(2000)，上述文献相关内容通过引用整体并入本文。在一些实施例中，遗传修饰的小鼠是129品系和C57BL/6品系的杂交。在一些实施例中，小鼠是129个品系的杂交，或BL/6品系的杂交。在一些实施例中，小鼠是BALB品系，例如BALB/c品系。在一些实施例中，小鼠是BALB品系和另一品系的杂交。在一些实施例中，小鼠来自杂交系(例如，50％BALB/c-50％12954/Sv；或50％C57BL/6-50％129)。在一些实施例中，非人动物是啮齿动物。在一些实施例中，非人类动物是具有BALB/c、a、a/He、a/J、a/WySN、AKR、AKR/a、AKR/J、AKR/N、TA1、TA2、RF、SWR、C3H、C57BR、SJL、C57L、DBA/2、KM、NIH、ICR、CFW、FACA、C57BL/a、C57BL/An、C57BL/GrFa、C57BL/KaLwN、C57BL6、C57L/6J、C57BL/6ByJ、C5C57BL/6NJ的小鼠。C57BL/10、C57BL/10ScSn、C57BL(C57BL/10Cr和C57BL/Ola)、C58、CBA/Br、CBA/Ca、CBA/J、CBA/st或CBA/H品系的小鼠及NOD、NOD/SCID、NOD-Prkdcscid IL-2rgnull背景的小鼠。
基因修饰的非人动物包括内源非人TLR7基因位点的修饰。在一些实施例中，所述修饰包含编码至少一部分成熟TLR7蛋白的核苷酸序列(例如，与成熟的TLR7蛋白氨基酸序列至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或100％一致性)。虽然在本发明中提供了可包含本文所述基因修饰的细胞(例如，ES细胞、体细胞)，但在许多实施例中，基因修饰的非人动物包括对动物中内源TLR7基因位点的修饰。
本发明进一步提供了利用上述方法构建的非人哺乳动物。在一些实施例中，所述非人哺乳动物包含人基因组。在一些实施例中，所述非人哺乳动物为啮齿类动物，进一步优选的，所述啮齿类动物为小鼠。在一些实施例中，所述非人哺乳动物表达由人源化TLR7基因编码的蛋白。
此外，本发明还提供了一种携带肿瘤的非人哺乳动物，其特征在于所述非人哺乳动物模型是通过本文所述方法获得的。在一些实施例中，非人哺乳动物是啮齿类动物(如，小鼠)。
本发明还提供了一种来源于非人哺乳动物或其后代、或携带肿瘤的非人哺乳动物的细胞或细胞系，或原代细胞培养物，其来源于非人类哺乳动物或其后代、或携带肿瘤的非人类哺乳动物、来源于非人类哺乳动物或其后代的组织、器官或其培养物。当其携带肿瘤时来源于非人哺乳动物或其后代的肿瘤组织或携带肿瘤的非人哺乳动物。
本发明提供了一种通过本文描述的任一方法产生的非人哺乳动物。在一些实施例中，提供了非人哺乳动物、基因修饰的非人动物，所述基因修饰的非人动物基因组包含人或人源化TLR7的DNA。
在一些实施例中，非人哺乳动物包括本文所述遗传构建体(例如，如图3和4所示的基因构建体)。在一些实施例中，提供了一种表达人或人源化TLR7蛋白的非人哺乳动物。在一些实施例中，提供了一种人或人源化TLR7蛋白的组织特异性表达。
在一些实施例中，非人动物人或人源化TLR7蛋白的表达是可控的。如通过添加特异性诱导物或阻遏物。在一些实施例中，所述特异性诱导物选自四环素系统(Tet-Off System/Tet-On System)或他莫昔芬系统(Tamoxifen System)。
非人哺乳动物可以是本领域已知的任何非人动物，其可用于本文所述方法中。优选的非人哺乳动物是哺乳动物(例如，啮齿类动物)。在一些实施例中，非人哺乳动物是小鼠。
对上述描述的非人哺乳动物进行遗传、分子和行为分析。本发明提供了一种与相同基因型或其他基因型非人哺乳动物交配产生的后代。
本发明提供了一种来源于非人哺乳动物或其后代的细胞系或原代细胞培养物。例如可以通过以下方法制备基于细胞培养的模型。细胞培养物可以通过从非人哺乳动物中分离获得，或者可以使用相同构建体和细胞转染技术建立的细胞培养物中获得细胞。包含编码人TLR7蛋白的DNA序列的遗传结构的整合可以通过多种方法检测。
有许多分析方法可用于检测外源性DNA，包括核酸水平的方法(包含使用逆转录-聚合酶链反应(RT-PCR)或Southern Blot以及原位杂交)和蛋白水平的方法(包括组织化学分析、免疫印迹分析和体外结合研究)。此外，目的基因的表达水平可以通过本领域技术人员熟知的ELSA方法进行量化。许多标准的分析方法可用于完成定量检测。例如，可以使用RT-PCR和杂交方法检测转录水平，包括RNA酶保护分析法、Southern Blot、RNA斑点杂交分析(RNAdot)。免疫组织化学染色、流式细胞术、Western blot也可用于检测人源或人源化TLR7蛋白的存在。
在一些实施例中，本文所述的经遗传修饰的动物(例如，TLR7基因人源化纯合小鼠)可以在一个或多个B细胞中表达人或人源化TLR7。
载体
本发明提供了一种靶向TLR7基因的靶向载体，包括：a)与待改变转换区5’端同源的DNA片段(5’臂)，其选自非人动物TLR7基因组DNA的100-10000个长度的核苷酸；b)编码供体区域的DNA序列；c)与待改变转换区3’端同源的DNA片段(3’臂)，其选自非人动物TLR7基因基因组DNA，长度为100-10000个核苷酸。
在一些实施例中，a)与待改变转换区5’端同源的DNA片段选自与NCBI登录号为NC_000086.8至少具有90％同源性的核苷酸序列；c)与待改变转换区3’端同源的DNA片段选自与NCBI登录号为NC_000086.8至少具有90％同源性的核苷酸序列；
在一些实施例中，a)待改变转换区5’端同源的DNA片段选自于NCBI登录号为NC_000086.8的第166091476至166095762位核苷酸序列；c)待改变转换区3’端同源的DNA片段选自于NCBI登录号为NC_000086.8的第166083092至166087410位核苷酸序列；
在一些实施例中，a)待改变转换区5’端同源的DNA片段选自于NCBI登录号为NC_000086.8的第166091476至166092724位核苷酸序列；c)待改变转换区3’端同源的DNA片段选自于NCBI登录号与NC_000086.8的第166087878至166089075位核苷酸序列至少95％一致性；
在一些实施例中，靶向载体所选的基因组核苷酸序列长度可以超过约3kb、3.5kb、4kb、4.5kb、5kb、5.5kb、6kb、6.5kb、7kb、7.5kb、8kb、8.5kb、9kb、9.5kb或10kb。
在一些实施例中，所述待改变转换区位于非人动物TLR7基因的1号至3号外显子上。
在一些实施例中，所述待改变转换区位于位于非人动物TLR7基因的3号外显子上(例如NM_016562.4第160-2556位)。
在一些实施例中，所述靶向载体还包含一个或多个标记基因。例如，阳性筛选标记基因或阴性筛选标记基因。在一些实施例中，阳性克隆筛选的抗性基因为新霉素磷酸转移酶编码序列Neo。在一些实施例中，负筛选标记的编码基因为白喉毒素A亚基的编码基因(DTA)。
在一些实施例中，所述5’臂序列如SEQ ID NO：3所示核苷酸序列；所述3’臂序列如SEQ ID NO：4所示核苷酸序列。在一些实施例中，所述5’臂序列如SEQ ID NO：10所示核苷酸序列；所述3’臂序列如SEQ ID NO：11所示核苷酸序列。
在一些实施例中，所述5’臂为与NCBI登录号为NC_000086.8至少具有90％同源性的核苷酸，进一步优选的，所述5’臂序列包含SEQ ID NO：3或10所示核苷酸序列。在一些实施例中，所述3’臂为与NCBI登录号为NC_000086.8至少具有90％同源性的核苷酸，进一步优选的，所述3’臂序列包含SEQ ID NO：4或11所示核苷酸序列。
在一些实施例中，所述靶向载体包含人序列(例如，NC_000023.11的第12885518-12887914位)。例如，靶向载体中的靶向区域包括：人TLR7基因的全部或部分核苷酸序列，优选人TLR7基因的外显子3的部分。在一些实施例中，人源化TLR7基因的核苷酸序列编码人TLR7蛋白的全部或部分核苷酸序列，NCBI的蛋白号为NP_057646.1(SEQ ID NO：2)。
本发明还提供了用于构建人源化动物模型或敲除模型的载体。在一些实施例中，载体包含sgRNA序列，其中sgRNA序列靶向TLR7基因，并且sgRNA在待改变基因的靶序列上是唯一的，并且满足5'-NNN(20)-NGG3'或5'-CCN-N(20)-3'的序列排列规则；并且在一些实施例中，小鼠TLR7基因中sgRNA的靶向位点位于外显子1、内含子1、外显子2、内含子2、外显子3，小鼠TLR7基因外显子3的上游，或外显子3的下游。
在一些实施例中，靶向序列显示为SEQ ID NO：12和13。在一些实施例中，本公开涉及包括sgRNA序列的质粒构建体(例如pT7-sgRNA)和/或包括该构建体的细胞。
本公开还涉及包含如上所述的靶向载体的细胞。
此外，本发明还提供了一种非人哺乳动物细胞，其具有上述靶向载体中的任何一种，以及本文所述构建体的一种或多种体外转录物。在一些实施例中，细胞包含Cas9mRNA或其体外转录物。
在一些实施例中，所述细胞中基因是杂合的。在一些实施例中，所述细胞中的基因是纯合的。
在一些实施例中，所述非人哺乳动物细胞是小鼠细胞。在一些实施例中，所述细胞是受精卵细胞。在一些实施例中，所述细胞是胚胎干细胞。
基因修饰的非人动物的构建方法
基因修饰的非人动物可以通过本领域已知的几种技术制备获得，包括利用胚胎干细胞的基因打靶技术、CRISPR/Cas9技术、锌指核酸酶技术、转录激活子样效应因子核酸酶技术、归巢核酸内切酶或其他分子生物学技术。在一些实施例中，优选使用同源重组技术。在一些实施例中，CRISPR/Cas9基因编辑技术可以构建基因修饰的非人动物。在一些实施例中，CRISPR/Cas9基因组编辑用于产生基因修饰的非人动物。这些基因组编辑技术中的许多技术是本领域已知的，并且在Yin等人的“Delivery technologies for genome editing,”Nature Reviews Drug Discovery 16.6(2017):387-399中进行了描述，其全部内容通过引用并入本文。
本发明还提供了许多其他方法用于基因组编辑，例如，将转基因细胞显微注射到去核卵母细胞中，并将去核卵母细胞与另一个转基因细胞融合。
在一些实施例中，非人动物的至少一个细胞的内源基因组中编码内源TLR7区域的核苷 酸序列被编码人TLR7相应区域的核苷酸序列替换。在一些实施例中，所述非人动物内源TLR7蛋白与野生型TLR7相比表达量降低或缺失。在一些实施例中，替换发生在生殖细胞、体细胞、囊胚或成纤维细胞等中。体细胞或成纤维细胞的细胞核可以插入去核卵母细胞中。
图3和图4显示了靶向小鼠TLR7位点的人源化打靶策略。靶向载体包含5’同源臂、人或人源化TLR7基因片段和3’同源臂组成的载体。该过程涉及利用同源重组将人或人源化核苷酸序列替换内源相应TLR7核苷酸序列。在一些实施例中，靶位点上游和下游的的切割(例如，通过锌指核酸酶、TALEN或CRISPR)可导致DNA双链断裂，利用同源重组将人或人源化TLR7序列替换鼠内源TLR7序列。
在一些实施例中，所述编码人TLR7相应区域的核苷酸序列包含编码人TLR7蛋白的信号肽、胞外区、跨膜区和/或胞质区的全部或部分核苷酸序列，优选的，包含编码TLR7蛋白信号肽和胞外区全部或部分的核苷酸序列。
在一些实施中，所述编码人TLR7相应区域的核苷酸序列包含编码人TLR7蛋白至少50个到至少1049个，优选为1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、200、300、500、600、700、720、740、760、790、792、795、798、799、800、801、802、803、804、805、810、820、830、831、833、836、839、840、850、900、1000、1020、1030、1040、1042、1044、1046、1048或1049个连续氨基酸的核苷酸序列；更进一步包含编码SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列的核苷酸序列；或者，包含编码与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或至少100％的核苷酸序列；或者，包含编码与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸的核苷酸序列；或者，包含编码与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列所示的，包括替换、缺失和/或插入一个或多个氨基酸的核苷酸序列。
在一些实施例中，所述编码人TLR7相应区域的核苷酸序列包含人TLR7基因的外显子1、外显子2和/或外显子3的全部或部分，进一步优选包含人TLR7基因外显子3的全部或部分，更进一步优选包含人TLR7基因外显子3的部分，其中，人TLR7基因外显子3的部分包含人TLR7基因外显子3至少50bp到至少4850bp，优选为50、60、70、80、90、100、150、200、250、350、450、600、1000、1500、2000、2200、2300、2340、2380、2390、2392、2394、2396、2397、2400、2420、2430、2434、2438、2439、3000、3500、4000、 4500、4600、4800、4830、4840或4850bp连续核苷酸序列，或者，人TLR7基因外显子3的部分包含编码区的核苷酸序列。
在一些实施例中，所述编码人TLR7相应区域的核苷酸序列包含SEQ ID NO：5所示核苷酸序列；或者，包含与SEQ ID NO：5所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或至少100％的核苷酸序列；或者，包含与SEQ ID NO：5所示核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸的核苷酸序列；或者，包含具有SEQ ID NO：5所示核苷酸序列所示的，包括替换、缺失和/或插入一个或多个核苷酸的核苷酸序列。
在一些实施例中，所述编码人TLR7相应区域的核苷酸序列可操作连接到至少一条染色体上内源TLR7基因的内源调控元件。
在一些实施例中，非人动物中被替换的内源TLR7核苷酸序列包括小鼠TLR7基因的外显子1、外显子2和外显子3的全部或部分被替换，优选非人动物TLR7基因外显子3的全部或部分被替换。更优选的，非人动物TLR7基因的外显子3的部分被替换。在一些实施例中，所述被替换的内源TLR7核苷酸序列编码SEQ ID NO：1第4-803位，SEQ ID NO：1第1-803位或SEQ ID NO：1第1-840位所示氨基酸序列。
在一些实施例中，所述人或人源化TLR7基因在非人动物体内通过调控元件进行调控，所述调控元件包括但不限于内源启动子。例如，所述调控元件可以是内源或者外源的调控元件。在本发明的一个具体实施例中，所述内源调控元件来源于非人动物TLR7基因。所述外源性调控元件来源于人TLR7基因。
在本发明的一个具体实施例中，所述构建方法包括用包含人TLR7基因外显子1、外显子2和外显子3的全部或部分替换非人动物TLR7基因的外显子1、外显子2和外显子3的全部或部分，优选用包含人TLR7基因的外显子3的全部或部分替换非人动物TLR7基因的外显子3的全部或部分。
在本发明的一个具体实施例中，所述构建方法包括用包含人TLR7基因的外显子3的部分替换非人动物TLR7基因的外显子3的全部或部分。
在本发明的一个具体实施例中，所述构建方法包括用包含编码人或人源化TLR7蛋白的核苷酸序列，或，人或人源化TLR7基因的核苷酸序列替换非人动物基因组中编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含人TLR7基因的基因组DNA序列、cDNA序列或CDS序列替换非人动物基因组中编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含编码SEQ ID NO：2所示氨基酸序列的核苷酸序列替换非人动物基因组中编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含编码人或人源化TLR7蛋白的核苷酸序列，或，人或人源化TLR7基因的核苷酸序列替换非人动物基因组中编码SEQ ID NO：1第4-803位，SEQ ID NO：1第1-803位或SEQ ID NO：1第1-840位所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含人TLR7基因的基因组DNA序列、cDNA序列或CDS序列替换非人动物基因组中编码SEQ ID NO：1第4-803位，SEQ ID NO：1第1-803位或SEQ ID NO：1第1-840位所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含编码SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列的核苷酸序列替换非人动物基因组中编码SEQ ID NO：1第4-803位，SEQ ID NO：1第1-803位或SEQ ID NO：1第1-840位所示氨基酸序列的核苷酸序列。
在本发明的一个具体实施例中，所述构建方法包括用包含SEQ ID NO：5所示核苷酸序列替换非人动物基因组中编码SEQ ID NO：1第4-803位，SEQ ID NO：1第1-803位或SEQ ID NO：1第1-840位所示氨基酸序列的核苷酸序列。
在一些实施例中，非人动物的构建方法包含基因修饰的非人动物TLR7基因编码框的构建，其中所述的基因修饰的非人动物TLR7基因编码框构建可以采用敲除非人动物TLR7基因的功能区或者采用插入一段序列，使得非人动物TLR7蛋白不表达或表达降低或表达的蛋白无功能。在一些实施例中，所述基因修饰的非人动物TLR7基因编码框构建包含敲除非人动物TLR7基因的外显子3的全部或部分核苷酸序列。在一些实施例中，所述修饰非人动物TLR7基因编码框构建可以敲除非人动物TLR7基因外显子3的部分核苷酸序列。
在一些实施例中，基因修饰的非人动物的构建方法包括在非人动物TLR7基因的内源调控元件之后插入编码人或人源化TLR7蛋白的核苷酸序列和/或辅助序列。在一些实施例中，辅助序列可以是终止密码子，使得TLR7基因人源化动物模型体内表达人TLR7蛋白，不表达非人动物TLR7蛋白，进一步的，所述辅助序列为WPRE、STOP和/或PolyA。
在一些实施例中，为提高重组效率，还可以使用靶向TLR7基因的sgRNA与上述TLR7基因的靶向载体一起进行非人动物的构建。其中，所述的sgRNA靶向非人动物TLR7基因，同时所述sgRNA的序列在待改变的TLR7基因上的靶序列上。在一些实施 例中，所述的sgRNA靶位点位于TLR7基因的1号外显子至3号外显子序列上。在一些实施例中，所述的sgRNA靶位点位于TLR7基因位于3号外显子序列上。在一些实施例中，所述的sgRNA在TLR7基因上的靶序列如SEQ ID NO：12或SEQ ID NO：13所示。
在本发明的一个具体实施例中，所述构建方法包括将上述TLR7基因的靶向载体、靶向TLR7基因的sgRNA及Cas9导入非人动物细胞中，培养该细胞(优选为受精卵)，然后将培养后的细胞移植至雌性非人动物输卵管内，允许其发育，鉴定筛选获得TLR7基因修饰的非人动物。
在本发明的另一个具体实施例中，所述构建方法包括将上述靶向载体导入非人动物的胚胎干细胞中，短暂培养后导入事先分离好的囊胚中，得到的嵌合囊胚移植至受体母鼠的输卵管中，允许其发育，鉴定筛选获得TLR7基因人源化的非人动物。
在本发明的一些实施例，所述的该构建方法进一步包括：将TLR7基因人源化的非人动物与其他基因修饰的非人动物交配、体外受精或直接进行基因编辑，并进行筛选，得到多基因修饰的非人动物。在本发明的一些实施例中，所述的其他基因为TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40至少一种。在一些实施例中，所述的非人动物还表达人或嵌合的TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40的至少一种。
在一些实施例中，所述非人动物中的其中一些在专利PCT/CN2019/127084、PCT/CN2017/106024、PCT/CN2018/110069、PCT/CN2017/099574、PCT/CN2017/117984、PCT/CN2018/091846、PCT/CN2018/081628、PCT/CN2017/120388、PCT/CN2017/110494、PCT/CN2018/091845、PCT/CN2017/099576、PCT/CN2017/110494和PCT/CN2017/099575已被描述，其全部内容通过引用并入本文。
在一些实施例中，所述其他基因为TLR8基因，所述的TLR8基因为嵌合TLR8基因。在一些实施例中，所述的嵌合TLR8基因包含人TLR8基因的1号至2号外显子的全部或部分。在一些实施例中，所述的嵌合TLR8基因包括人TLR8基因的2号外显子的部分。
在一些实施例中，所述的嵌合TLR8基因的核苷酸序列转录的mRNA为SEQ ID NO：22所示核苷酸序列；或者，转录的mRNA与SEQ ID NO：22所示核苷酸序列的同一性至少为70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或至少100％的核苷酸序列；或者，转录的mRNA与SEQ ID NO：22所示核苷酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个核苷酸的核苷酸序列；或者，转录的mRNA具有SEQ ID NO：22所示核苷酸序列的，包括取代、缺失和/或插入一个或多个核苷酸的核苷酸序列。
在一些实施例中，所述的嵌合TLR8蛋白包含与SEQ ID NO：23所示的氨基酸序列同一性至少为70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、99％或至少100％的氨基酸序列；或者，包含与SEQ ID NO：23所示的氨基酸序列差异不超过10、9、8、7、6、5、4、3、2或不超过1个氨基酸的氨基酸序列；或者，包含具有SEQ ID NO：23的所示的氨基酸序列，包括取代、缺失和/或插入一个或多个氨基酸的氨基酸序列。
在一些实施例中，所述的多基因修饰的非人动物的基因组中修饰的多个基因中的每一个基因均对于内源被修饰基因座为纯合或杂合的。
在一些实施例中，所述非人动物可以选自啮齿类动物、猪、兔子、猴子等任何可以进行基因编辑制备基因人源化的非人动物。
优选的，所述非人动物为非人哺乳动物。进一步优选的，所述非人哺乳动物为啮齿类动物。更进一步优选的，所述啮齿类动物为大鼠或小鼠。
优选的，所述非人动物是免疫缺陷的非人哺乳动物。进一步优选的，所述免疫缺陷的非人哺乳动物为免疫缺陷的啮齿类动物、免疫缺陷的猪、免疫缺陷的兔子或免疫缺陷的猴子。更进一步优选的，所述免疫缺陷的啮齿类动物为免疫缺陷的小鼠或大鼠。再进一步优选的，所述免疫缺陷鼠是NOD-Prkdcscid IL-2rγnull小鼠、NOD-Rag 1-/--IL2rg-/-小鼠、Rag 2-/--IL2rg-/-小鼠、NOD/SCID小鼠或者裸鼠。
基因修饰的非人动物的应用
在内源非人动物基因座并在内源启动子和/或调控元件的控制下，用同源或直系同源人基因或人序列替换非人动物基因或将同源或直系同源人基因或人序列插入非人动物中，可以产生具有可能与典型的敲除加转基因动物显著不同的品质和特征的非人动物。在典型的敲除加转基因动物中，内源基因座被移除或破坏，全人转基因被插入动物的基因组中，并可能随机整合到基因组中。通常，整合转基因的位置是未知的；通过人基因和/或蛋白质测定和/或功能测定的转录来测量人类蛋白质的表达。在人转基因中，人序列的上游和/或下游为转基因的表达和/或调节提供合适的支持。
在某些情况下，具有人调控元件的转基因以非生理学或其他方面不令人满意的方式表达，并且实际上可能对动物有害。本发明证明在内源调控元件控制下，在内源基因座用人序列替换或插入产生人源化动物，提供了生理上合适的表达模式和水平，其关于被替换基因的生理学在人源化动物的生理学的背景下是有意义的和合适的。
表达人或人源化TLR7蛋白的基因修饰动物，例如，以生理学上合适的方式，提供了多种用途，包括但不限于开发人类疾病和病症的治疗方法，以及评估这些人类治疗方法在动物 模型中的毒性和/或功效。
本发明还提供了一种上述TLR7和/或TLR7/TLR8基因修饰的非人动物或上述任一构建方法获得的非人动物的应用。
在一些实施例中，所述应用包含：
A)涉及人类细胞的与TLR7和/或TLR8相关的免疫过程的产品开发中的应用；
B)作为药理学、免疫学、微生物学和医学研究的与TLR7和/或TLR7/TLR8相关的模型系统中的应用；
C)涉及生产和利用动物实验疾病模型用于与TLR7和/或TLR8相关的病原学研究和/或用于开发诊断策略和/或用于开发治疗策略中的应用；
D)在体内研究人TLR7/TLR8信号通路调节剂的筛选、药效检测、评估疗效、验证或评价中的应用；或者，
E)研究TLR7和/或TLR8基因功能，研究针对人TLR7和/或TLR8靶位点的药物、药效，研究与TLR7和/或TLR8相关的免疫相关疾病药物和抗肿瘤药物方面的应用。
本发明提供了一种表达人或人源化TLR7和/或TLR8蛋白非人动物，该动物可用于人TLR7和/或TLR8特异性调节剂的筛选。在一些实施例中，所述非人动物是人疾病动物模型。如，疾病是遗传诱导的(敲入或敲除)。在不同的实施例中，基因修饰的非人动物还包含受损的免疫系统，如，经过基因修饰的人源性组织异种移植，包括人实体瘤(例如，膀胱癌)或血细胞肿瘤(例如，淋巴细胞肿瘤、B或T细胞肿瘤)。
在一些实施例中，基因修饰的非人动物可用于确定治疗剂(如，抗TLR7抗体和/或抗TLR8抗体)在治疗各种免疫疾病方面的有效性。在一些实施例中，所述免疫疾病包括但不限于GVHD(移植物抗宿主病)、银屑病、过敏、哮喘、心肌炎、鼻炎、肝炎(优选为非酒精性脂肪性肝炎)、系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺功能亢进、原发性血小板减少性紫癜、自身免疫性溶血性贫血、溃疡性结肠炎等。
在一些实施例中，基因修饰的非人动物可用于确定治疗剂(如，抗TLR7抗体和/或抗TLR8抗体)在治疗各种炎症感染方面的有效性。在一些实施例中，所述炎症包括急性炎症，也包括慢性炎症。具体的，包括但不限于慢性阻塞性肺病、脓毒症和皮炎等。
在一些实施例中，基因修饰的非人动物可用于确定治疗剂(如，抗TLR7抗体和/或抗TLR8抗体)对治疗癌症的有效性。在一些实施例中，向非人动物施用治疗剂(如，抗TLR7抗体和/或抗TLR8抗体)，其中所述非人动物具有癌症或肿瘤，检测治疗剂对癌症或肿瘤的抑制作用。在一些实施例中，所述检测包括测定肿瘤细胞的大小和/或增殖速率。在一些实施例中，所述检测方法包括游标卡尺测量、流式细胞检测和/或动物活体成像检测。 在一些实施例中，所述检测包括评估个体体重、脂肪量、活化途径、神经保护活性或代谢变化，所述代谢变化包括食物消耗或水消耗的变化。
在一些实施例中，所述肿瘤细胞包括一个或多个被注射到动物体内的癌细胞(如，癌细胞来源于人或非人动物)。在一些实施例中，治疗剂抑制TLR7/TLR8介导的信号通路。在一些实施例中，治疗剂不抑制TLR7/TLR8介导的信号通路。
在一些实施例中，基因修饰的非人动物可用于检测抗TLR7抗体和/或抗TLR8抗体是激动剂还是拮抗剂。在一些实施例中，本文描述的方法可以用来检测治疗剂(如，抗TLR7抗体和/或抗TLR8抗体)的功能，例如，所述治疗剂是否可以上调免疫应答或下调免疫应答，和/或该治疗剂是否能够诱导补体介导的细胞毒性(CMC)或抗体依赖性细胞毒性(ADCC)。在一些实施例中，基因修饰的非人动物可用于确定治疗受试者疾病(例如免疫疾病)的治疗剂的有效剂量。对肿瘤的抑制作用也可以通过本领域已知的方法来确定，例如，测量动物中的肿瘤体积，和/或确定肿瘤(体积)抑制率(TGITV)。肿瘤生长抑制率可以使用公式TGITV(％)＝(1–TVt/TVc)x100计算，其中TVt和TVc是治疗组和对照组的平均肿瘤体积(或重量)。
在一些实施例中，治疗剂(如，抗TLR7抗体和/或抗TLR8抗体)可以被用于治疗各种癌症。本发明所述“癌症”是指具有自主生长能力的细胞，即以细胞生长迅速增殖为特征的异常状态或病症。该术语旨在包括所有类型的癌性生长或致癌过程、转移性组织或恶性转化的细胞、组织或器官，无论组织病理学类型或侵袭性阶段如何。本发明所述“肿瘤”包括但不限于淋巴瘤、非小细胞肺癌、宫颈癌、白血病、卵巢癌、鼻咽癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、胃癌、膀胱癌、脑胶质瘤、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、肝和胆管癌、食管癌、肾癌、甲状腺癌、头颈部癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、以及肉瘤。其中，所述白血病选自急性淋巴细胞性(成淋巴细胞性)白血病、急性骨髓性白血病、髓性白血病、慢性淋巴细胞性白血病、多发性骨髓瘤、浆细胞白血病、以及慢性骨髓性白血病；所述淋巴瘤选自霍奇金淋巴瘤和非霍奇金淋巴瘤，包括B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、边缘区B细胞淋巴瘤、T细胞淋巴瘤、和瓦尔登斯特伦巨球蛋白血症；所述肉瘤选自骨肉瘤、尤文肉瘤、平滑肌肉瘤、滑膜肉瘤、软组织肉瘤、血管肉瘤、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤、以及软骨肉瘤。在本发明的一个具体实施方式中，所述肿瘤为实体瘤、膀胱癌、浅表尿路上皮癌、宫颈癌、子宫内膜癌、食道癌、鳞状细胞癌、肾癌、非小细胞肺癌、卵巢癌、鳞状细胞癌、胃癌、子宫癌、结直肠转移癌、肝癌、胃肠癌。
本发明还提供了一种确定治疗剂(如，抗TLR7抗体和/或抗TLR8抗体)毒性的检测方 法。所述方法包括向上述所述非人动物施用抗体，评估动物的体重变化、红细胞计数、血细胞比容和/或血红蛋白。在一些实施例中，抗体可使红细胞(RBC)、血细胞比容或血红蛋白降低20％、30％、40％或50％以上。在一些实施例中，动物的体重与对照组(如，未用抗体处理的动物的平均体重)相比至少小5％、10％、20％、30％或40％。
本发明还提供了一种通过本文所述方法构建的动物模型在开发与人类细胞免疫过程相关的产品、制造人抗体、或用于药理学、免疫学、微生物学和医学研究的模型系统。
在一些实施例中，提供了一种通过本文描述的方法生成的动物模型在生产和利用人体细胞的免疫过程的动物实验疾病模型、研究病原体、或制定新的诊断策略和/或治疗策略。
本发明还提供了通过本文所述方法生成的动物模型来筛选、验证、评估或研究TLR7和/或TLR8基因功能、人TLR7和/或TLR8抗体、人TLR7和/或TLR8靶位点的药物或有效性、免疫相关疾病的药物和抗肿瘤药物。
两个或多个人或嵌合基因的非人动物模型
本发明还提供了一种具两个或多个人或嵌合基因的非人动物，所述动物模型包含人或嵌合TLR7基因以及编码其他人或嵌合蛋白的核酸序列。在一些实施例中，所述其他基因为TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40至少一种基因修饰的非人动物。在一些实施例中，上述所述非人动物还表达人或人源化的TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40至少一种。
本发明还提供了一种两个或多个人或嵌合基因的非人动物的构建方法，所述构建方法包括：
(一)提供上述的构建方法获得非人动物；
(二)将步骤(一)提供的非人动物与其他基因修饰的非人动物交配、体外受精或直接进行基因编辑，并进行筛选，得到多基因修饰的非人动物。
在一些实施例中，所述其他基因修饰的非人动物包括基因TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40中的一种或两种以上的组合人源化的非人动物。
在一些实施例中，TLR7人源化直接在具有人或嵌合TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40基因修饰的非人动物上进行。
由于这些蛋白可能涉及不同的机制，因此靶向其中两种或多种蛋白的联合疗法可能是一种更有效的治疗方法。事实上，许多相关的临床试验正在进行中，并显示出良好的效果。多 基因修饰的非人动物模型可用于确定靶向两种或多种蛋白的联合疗法的有效性，例如，抗TLR7抗体或抗TLR8抗体，以及用于治疗癌症或代谢性疾病(例如，肥胖症或写心血管疾病)的附加治疗剂。所述方法包括向动物施用抗TLR7抗体或抗TLR8抗体和附加治疗剂，其中动物具有肿瘤或免疫疾病，并确定联合治疗对免疫肿瘤或免疫疾病的影响。在一些实施例中，所述附加治疗剂是特异性结合TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40的抗体。在一些实施例中，所述附加治疗剂是抗CTLA4抗体(例如，ipilimumab)、抗PD-1抗体(例如，nivolumab)或抗PD-L1抗体。在一些实施例中，上述所述非人动物还包括编码人或人源化PD-1的序列、编码人或人源化PD-L1的序列、或编码人或人源化CTLA-4的序列。在一些实施例中，附加治疗剂是抗PD-1抗体(例如，纳武利尤单抗、帕博利珠单抗)、抗PD-L1抗体或抗CTLA-4抗体。在一些实施例中，上述所述肿瘤包括一个或多个表达PD-L1和/或PD-L2的肿瘤细胞。
在一些实施例中，所述联合疗法还可用于治疗本文所述各种癌症，例如实体瘤、膀胱癌、浅表尿路上皮癌、宫颈癌、子宫内膜癌、食道癌、鳞状细胞癌、肾癌、非小细胞肺癌、卵巢癌、鳞状细胞癌、胃癌、子宫癌、结直肠转移癌、肝癌、胃肠癌。
在一些实施例中，上述描述的治疗方法可与常规癌症化疗药联合使用。在一些实施例中，治疗癌症的方法可以单独使用或与本文描述的方法组合使用，包括，用化疗治疗受试者，如樟树碱、多柔比星、顺铂、卡铂、丙卡巴肼、甲氯乙胺、环磷酰胺、阿霉素、异环磷酰胺、美法仑、苯丁酸氮芥、硫丹、硝基苏拉、放线菌素、柔红霉素、博来霉素、普利霉素、丝裂霉素、依托泊苷、维拉皮尔、鬼臼毒素、他莫昔芬、紫杉醇、反铂、5-氟拉嘧啶、长春新碱、长春爆蛋白和/或甲氨蝶呤。所述方法可以包括对受试者进行手术去除至少一部分癌症，如从患者身上切除肿瘤的一部分或全部。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
在下述每一实施例中，设备和材料是从以下所指出的几家公司获得：
BsrGI和BgIII酶购自NEB，货号分别为R0575S和R0144S；
C57BL/6小鼠购自中国食品药品检定研究院国家啮齿类实验动物种子中心；
Brilliant Violet 711TManti-mouse TCRβchain Antibody购自Biolegend，货号为109243；
Brilliant Violet 510TManti-mouse CD45Antibody购自Biolegend，货号为103138；
PE/CyTM7anti-mouse/rat Foxp3购自eBioscience，货号为25-5773-82；
Brilliant Violet 421TManti-mouse CD4购自Biolegend，货号为100438；
FITC anti-mouse F4/80Antibody购自Biolegend，货号为123108；
V450Rat Anti-mouse CD11b购自BD Horizon，货号为560455；
PerCP anti-mouse Ly-6G/Ly-6C(Gr-1)Antibody购自Biolegend，货号为108426；
FITC Rat Anti-Mouse CD3Molecular Complex购自BD Pharmingen，货号为561798；
Brilliant Violet 711TManti-mouse NK-1.1Antibody购自Biolegend，货号为108745；
APC anti-human CD288(TLR8)Antibody购自Biolegend，货号为395505；
TLR8Monoclonal Antibody(44C143),PE购自eBioscience，货号为MA5-16194；
Brilliant Violet 605TManti-mouse CD11c Antibody购自Biolegend，货号为117334；
PE anti-mouse CD287(TLR7)Antibody购自Biolegend，货号为160003；
PE anti-human TLR7Antibody购自Biolegend，货号为376903；
红细胞裂解液购自碧云天，货号为C3702；
LEGEND MAXTMMouse TNF-αELISA Kit购自Biolegend，货号为430907；
Mouse IFN-αELISA Kit购自Biolegend，货号为447904；
CL264购自MCE，货号为HY-135905；
GS-9688购自MCE，货号为HY-109137；
TL8-506购自Invivogen，货号为Tlrl-tl8506。
实施例1 TLR7基因人源化小鼠的构建方法
小鼠TLR7基因(NCBI Gene ID：170743，Primary source：MGI：2176882，UniProt：P58681，位于X染色体NC_000086.8的第166086376至166113570位，基于转录本NM_133211.4及其编码蛋白NP_573474.1(SEQ ID NO：1)和人TLR7基因(NCBI Gene ID:51284，Primary source：HGNC：15631，UniProt ID：Q9NYK1，位于X染色体NC_000023.11的第12867072至12890361位，基于转录本NM_016562.4及其编码蛋白NP_057646.1(SEQ ID NO：2)对比示意图如图1所示。
为了达到本发明的目的，可在小鼠内源TLR7基因座引入编码人TLR7蛋白的核苷酸序列，使得该小鼠表达人或人源化TLR7蛋白。具体来说，用人TLR7基因3号外显子部分序列替换小鼠相应核苷酸序列，实现对小鼠TLR7基因座的人源化改造，得到人源化TLR7基因座示意图如图2所示。
根据图2进一步设计了如图3所示的打靶策略示意图，图中显示了靶向载体V1上含有小鼠TLR7基因的上游和下游的同源臂序列，以及包含编码人TLR7蛋白的核苷酸序列的 A1片段。其中，上游同源臂序列(5’同源臂，SEQ ID NO：3)与NCBI登录号为NC_000086.8的第166091476至166095762位核苷酸序列相同，下游同源臂序列(3’同源臂，SEQ ID NO：4)与NCBI登录号为NC_000086.8的第166083092至166087410位核苷酸序列相同；A1片段中包含的人TLR7核苷酸序列(SEQ ID NO：5)与NCBI登录号为NC_000023.11的第12885518至12887914位核苷酸序列相同。
靶向载体V1上还包括用于阳性克隆筛选的抗性基因，即潮霉素编码序列HygR，并在抗性基因的两侧装上两个同向排列的位点特异性重组系统Frt重组位点，组成HygR盒1(HygR cassette)。其中，HygR盒上游与鼠TLR7的连接设计为 (SEQ ID NO：6)，其中序列“TCCAC”中最后一个的“C”是鼠TLR7的最后一个核苷酸，序列中的“G”是HygR盒的第一个核苷酸；HygR盒下游与鼠的连接设计为 (SEQ ID NO：7)，其中序列“GATCC”中最后一个“C”是HygR盒的最后一个核苷酸，序列中的“C”是鼠的第一个核苷酸。此外，还在靶向载体3’同源臂下游构建了具有负筛选标记的编码基因(白喉毒素A亚基的编码基因(DTA))。改造后的人源化小鼠TLR7的mRNA序列如SEQ ID NO：8所示，表达的蛋白序列如SEQ ID NO：9所示。
此外，还可采用CRISPR/Cas9系统进行基因编辑，根据图2设计如图4所示的打靶策略示意图，图中显示了靶向载体V2上含有上游同源臂(5’同源臂)和下游同源臂(3’同源臂)序列，以及包含编码人TLR7基因3号外显子部分核苷酸序列A2片段。其中，上游同源臂序列(5’同源臂，SEQ ID NO：10)与NCBI登录号为NC_000086.8的第166091476至166092724位核苷酸序列相同，下游同源臂序列(3’同源臂，SEQ ID NO：11)与NCBI登录号为NC_000086.8的第166087878至166089075位核苷酸序列一致性99％，区别在于166089040位和166089043位的碱基A均被替换为碱基G；A2片段中包含的人TLR7核苷酸序列如SEQ ID NO：5所示。改造后的人源化小鼠TLR7的mRNA序列如SEQ ID NO：8所示，表达的蛋白序列如SEQ ID NO：9所示。
靶序列决定了sgRNA的靶向特异性和诱导Cas9切割目的基因的效率。因此，高效特异的靶序列选择和设计是构建sgRNA表达载体的前提。设计并合成识别5’端和3’端靶位点的sgRNA序列，筛选出活性较好、序列特异性较高的sgRNA进行后续实验。示例性靶序列如下所示：
sgRNA1靶位点(SEQ ID NO：12)：5’-ACTTCACAAGGTAGAGTTTTAGG-3’
sgRNA2靶位点(SEQ ID NO：13)：5’-GCCACTGATGTGACTTGTGTAGG-3’
在sgRNA的5’端及互补链上分别加上酶切位点得到正向寡核苷酸和反向寡核苷酸序列，退火后将退火产物连接至pT7-sgRNA质粒(质粒先用BbsI线性化)，获得表达载体pT-TLR7-1和pT-TLR7-2。pT-sgRNA载体由质粒合成，公司合成含有T7启动子及sgRNA scaffold的片段DNA(SEQ ID NO：14)并依次通过酶切(EcoRI及BamHI)连接至骨架载体(来源Takara，货号3299)上，经专业测序公司测序验证，结果表明获得了目的质粒。
取小鼠的原核期受精卵，例如C57BL/6小鼠，利用显微注射仪将获得的表达载体pT-TLR7-1和pT-TLR7-2质粒的体外转录产物(使用Ambion体外转录试剂盒，按照说明书方法进行转录)、靶向载体与Cas9mRNA预混好后注射至小鼠受精卵细胞质或细胞核中。按照《小鼠胚胎操作实验手册(第三版)》(安德拉斯·纳吉，化学工业出版社，2006)中的方法进行受精卵的显微注射，注射后的受精卵转移至培养液中短暂培养，然后移植至受体母鼠的输卵管中发育，将获得的小鼠(F0代)通过杂交和自交，扩大种群数量，建立稳定的TLR7基因人源化小鼠品系。
可通过常规检测方法(如PCR分析)鉴定F0代小鼠体细胞的基因型，部分F0代小鼠的示例性鉴定结果见图5。结合PCR引物检测结果，并经测序进一步验证图5中编号为F0-2小鼠为阳性小鼠。PCR引物如表3所示。
表3 F0代基因型PCR检测引物序列及重组片段大小
将F0鉴定为阳性的TLR7基因人源化小鼠与野生型小鼠交配得到F1代小鼠。将F1代小鼠相互交配，可获得TLR7基因人源化纯合小鼠。对PCR鉴定为阳性的小鼠进行Southern blot检测，确认是否存在随机插入。剪取鼠尾提取基因组DNA，选用BsrGI酶或BgIII酶消化基因组，转膜，杂交。具体探针及目的片段的长度见表4。Southern blot检测结果见图6，综合3’探针和5’探针的结果表明，F1-4、F1-5、F1-8、F1-9、F1-10和F1-11鼠均无随机插入。这表明使用本方法能构建出可稳定传代，且无随机插入的TLR7基因人源化小鼠。
表4具体探针及目的片段长度
探针合成引物如下：
5’Probe:
5’Probe-F：5’-ctgcagtgacttccaatagcaatcc-3’(SEQ ID NO：18)；
5’Probe-R：5’-gttgctgcgaagagtgctgtaac-3’(SEQ ID NO：19)；
3’Probe:
3’Probe-F：5’-ctctccataaagttccatagaagagg-3’(SEQ ID NO：20)；
3’Probe-R：5’-catcatggtgtctttgggaatgattc-3’(SEQ ID NO：21)；
实施例2 TLR8基因人源化小鼠的构建方法
小鼠TLR8基因(NCBI Gene ID：170744，Primary source：MGI：2176887，UniProt：P58682，位于X染色体NC_000086.7的第167241123至167264329位，基于转录本NM_133212.3及其编码蛋白NP_57345.2)和人TLR8基因(NCBI Gene ID:51311，Primary source：HGNC：15632，UniProt ID：Q9NR97，位于X染色体NC_000023.11的第12906620至12923169位，基于转录本NM_138636.5及其编码蛋白NP_619542.1)对比示意图如图7所示。
通过基因编辑技术将小鼠TLR8基因的3号外显子2415bp核苷酸序列替换为对应的人TLR8基因的2号外显子2442bp核苷酸序列替换，实现对小鼠TLR8基因的人源化改造，小鼠TLR8基因座改造后的示意图如图8所示。改造后的人源化小鼠TLR8的mRNA序列如SEQ ID NO：22所示，表达的蛋白序列如SEQ ID NO：23所示。
通过流式细胞术检测小鼠体内人源化TLR8蛋白的表达情况。选取10周龄的野生型C57BL/6小鼠(+/+)和TLR8基因人源化纯合小鼠(H/H)各1只，腹腔注射7.5μg/200μL的mCD3，24小时后取脾脏细胞进行流式检测。染色方案如下：用抗鼠CD45抗体Brilliant Violet 510TManti-mouse CD45(mCD45)、Brilliant Violet 711TManti-mouse TCRβchain(mTCRβ)、Brilliant Violet 421TManti-mouse CD4(mCD4)标记T细胞，用抗鼠TLR8抗体TLR8Monoclonal Antibody(44C143),PE(mTLR8)或抗人TLR8抗体APC anti-human CD288(TLR8)Antibody(hTLR8)识别染色后进行流式检测。
T细胞的特征为mCD45+mTCRβ+，其中鼠TLR8阳性(mTLR8+)T细胞特征为mCD45+mTCRβ+mTLR8+，人TLR8阳性(hTLR8+)T细胞特征为mCD45+mTCRβ+hTLR8+。流式分析结果显示在TLR8基因纯合小鼠体内可以检测到人源化TLR8蛋白，表明TLR8基因人源化小鼠体内可成功表达人源化TLR8蛋白。
进一步对野生型C57BL/6小鼠、TLR8人源化纯合小鼠体内免疫分型情况进行流式检测，数据显示，TLR8基因人源化小鼠脾脏、淋巴结和血液中白细胞亚型(包括：T细胞、B细胞、NK细胞、粒细胞、巨噬细胞、单核细胞)百分比和T细胞亚型(包括：CD4+T细胞、CD8+T细胞和Treg细胞)百分比与C57BL/6野生型小鼠基本一致，表明TLR8基因的人源 化改造未影响TLR8基因人源化小鼠体内免疫细胞的整体发育、分化和分布。
实施例3 TLR7/TLR8双人源化鼠的制备
因为小鼠TLR7和TLR8基因均处于小鼠X染色体上，为实现TLR7和TLR8双基因人源化改造，可按照实施例1所述方法，在实施例2获得TLR8基因人源化小鼠基础上进行基因打靶。具体来说，取实施例2制得的TLR8基因人源化小鼠的胚胎干细胞(ES细胞)或受精卵，按照图3或图4所示方法进行基因改造，获得TLR7/TLR8双基因人源化小鼠。
通过流式细胞术检测小鼠体内人源化TLR7和TLR8蛋白的表达情况。选取10周龄的野生型C57BL/6小鼠(+/+)和TLR7/TLR8基因人源化纯合小鼠(H/H；H/H)各1只，脱颈安乐死后，取脾脏细胞，用抗鼠CD45抗体Brilliant Violet 510TManti-mouse CD45(mCD45)、抗鼠CD3抗体FITC Rat Anti-Mouse CD3(mCD3)、抗鼠CD19抗体FITC anti-Mouse CD19(mCD19)、鼠DC细胞标记抗体Brilliant Violet 605TManti-mouse CD11c antibody(mCD11c)、鼠中性粒细胞标记抗体PerCP anti-mouse Ly-6G/Ly-6C(Gr-1)Antibody(mGr-1)；鼠单核细胞抗体V450Rat Anti-mouse CD11b(mCD11b)、鼠巨噬细胞标记抗体FITC anti-mouse F4/80(mF4/80)、人鼠TLR8交叉识别抗体TLR8Monoclonal Antibody(44C143),PE(mTLR8)、抗人TLR8抗体APC anti-human CD288(TLR8)Antibody(hTLR8)、抗鼠TLR7抗体PE anti-mouse CD287(TLR7)Antibody(mTLR7)或抗人TLR7抗体PE anti-human TLR7Antibody(hTLR7)分别识别染色后进行流式检测。
DC细胞的特征为mCD45+mCD3-mCD11c+，其中鼠TLR7和TLR8阳性巨噬细胞的特征分别为mCD45+mCD3-mCD11c+mTLR7+和mCD45+mCD3-mCD11c+mTLR8+，人TLR7和TLR8阳性巨噬细胞的特征分别为mCD45+mCD3-mCD11c+hTLR7+和mCD45+mCD3-mCD11c+hTLR8+。
中性粒细胞特征为mCD45+mGr-1+,其中鼠TLR7和TLR8阳性中性粒细胞的特征分别为mCD45+mGr-1+mTLR7+和mCD45+mGr-1+mTLR8+，人TLR7和TLR8阳性巨噬细胞的特征分别为mCD45+mGr-1+hTLR7+和mCD45+mGr-1+hTLR8+；
单核细胞特征为：mCD45+mGr-1-mCD11b+mF4/80-，其中鼠TLR7和TLR8阳性单核细胞的特征分别为mCD45+mGr-1-mCD11b+mF4/80-mTLR7+和mCD45+mGr-1-mCD11b+mF4/80-mTLR8+，人TLR7和TLR8阳性单核细胞的特征分别为mCD45+mGr-1-mCD11b+mF4/80-hTLR7+和mCD45+mGr-1-mCD11b+mF4/80-hTLR8+；
巨噬细胞的特征为mCD45+mGr-1-mCD11b+mF4/80+，其中鼠TLR7和TLR8阳性巨噬细胞的特征分别为mCD45+mGr-1-mCD11b+mF4/80+mTLR7+和mCD45+mGr-1-mCD11b+mF4/80+mTLR8+，人TLR7和TLR8阳性巨噬细胞的特征分别为mCD45+mGr-1- mCD11b+mF4/80+hTLR7+和mCD45+mGr-1-mCD11b+mF4/80+hTLR8+；
结果如表5所示，数据表明，本方法制备的TLR7/TLR8基因人源化小鼠脾脏DC、单核细胞、巨噬细胞可成功表达人源化TLR7和TLR8蛋白。
表5 TLR7/TLR8双基因人源化小鼠体内TLR7和TLR8流式检测结果
进一步对野生型C57BL/6小鼠(+/+)、TLR8/TLR7双基因人源化纯合小鼠(H/H；H/H)体内免疫分型情况进行流式检测，各小鼠脾脏、淋巴结和血液中白细胞亚型(包括：T细胞、B细胞、NK细胞、粒细胞(Granulocyte)、巨噬细胞(Macrophages)、单核细胞(Monocytes)、树突状细胞(DC))和T细胞亚型(包括：CD4+T细胞、CD8+T细胞和Treg细胞)检测结果分别如图9、图10和图11所示。数据显示，TLR8/TLR7双基因人源化小鼠脾脏、淋巴结和血液中白细胞亚型百分比和T细胞亚型百分比基本一致，表明TLR8和TLR7基因的人源化改造未影响TLR8/TLR7双基因人源化小鼠体内免疫细胞的整体发育、分化和分布。
通过ELISA对TLR7/TLR8双基因人源化小鼠体内TLR7和TLR8信号通路情况进行检测。具体来说，取6-7周龄雌性野生型C57BL/6小鼠(+/+)和TLR7/TLR8双基因人源化纯合小鼠(H/H)各四只，取脾脏细胞，制成单细胞悬液于10mL PBS中，加入红细胞裂解液裂解红细胞，用适量RPMI-1640培养液重悬细胞终浓度为3x106个/mL；每孔取100uL(3x105个)细胞数铺板，再加入含有不同浓度的TLR8激动剂(GS-9688和TL8-506)、TLR7激动剂CL264或对照的培养基100uL，混匀；培养24h后离心，收集细胞培养上清，使用LEGEND MAXTMMouse TNF-αELISA Kit和Mouse IFN-αELISA Kit检测小鼠TNF-α(mTNF-a)的浓度。检测结果见图12，结果表明经TLR8/TLR7激动剂刺激后，细胞分泌的mTNF-α均具有剂量依赖性，TLR7激动剂对野生型C57BL/6小鼠更加敏感，而TLR8激动剂对TLR7/TLR8双基因人源化纯合小鼠更敏感。综上所述，TLR7/TLR 8激动剂刺激后可以引起TLR7/TLR8双基因人源化纯合小鼠体内mTNF-α细胞因子的分泌增加，证明 TLR7/TLR8双基因人源化纯合小鼠体内TLR7/TLR8信号通路正常。
实施例4 药效实验
利用本方法制备的TLR7和/或TLR8基因人源化鼠构建肿瘤模型，可用于靶向TLR7和/或TLR8的药物的筛选及药效验证。例如，取TLR7和/或TLR8基因人源化纯合子小鼠，皮下接种结肠癌细胞MC38，待肿瘤体积生长到约100mm3后，根据肿瘤体积分为对照组或治疗组，治疗组注射靶向人TLR7和/或TLR8的药物，对照组注射等体积的生理盐水或PBS。定期测量肿瘤体积并称量小鼠的体重，通过比较小鼠体重变化和肿瘤体积，可有效评估抗体药物在人源化TLR7和/或TLR8小鼠体内安全性和体内药效。
还可以利用本方法制备的TLR7和/或TLR8基因人源化鼠构建炎性肠病模型，验证靶向TLR7和/或TLR8的药物的药效。例如，取TLR7和/或TLR8基因人源化纯合子小鼠诱导构建炎性肠病模型(IBD)。将小鼠分为对照组或治疗组，治疗组注射靶向人TLR7和/或TLR8的抗体药物，对照组注射等体积的生理盐水或PBS。然后对各组小鼠的体重、结肠重量和长度指数情况进行检测，测量每只小鼠的结肠病理评分，可有效评估抗体药物在人源化TLR7和/或TLR8小鼠体内的安全性和体内药效。
此外，利用本方法制备的TLR7和/或TLR8基因人源化鼠还可构建实验性关节炎动物模型(CIA)。将小鼠分为对照组或治疗组，治疗组注射靶向人TLR7和/或TLR8的抗体药物，对照组注射等体积的生理盐水或PBS。然后对各组小鼠的体重、足趾和关节炎指数情况进行监测，测量爪厚并记录每只小鼠的关节炎评分，可有效评估抗体药物在人源化TLR7和/或TLR8小鼠体内的安全性和体内药效。
实施例5 多基因人源化鼠的制备
利用本方法或制得的TLR7和/或TLR8基人源化小鼠还可以制备多人源化小鼠模型。例如，前述实施例1中，显微注射使用的胚胎干细胞可选择来源于含有PD-L1、HER2、EGFR、CTLA4、VEGFR2等基因修饰的小鼠，或者，也可在人源化TLR7和/或TLR8小鼠的基础上，利用分离小鼠ES胚胎干细胞和基因重组打靶技术，获得双人源化或多人源化小鼠模型。也可将本方法得到TLR7和/或TLR8小鼠纯合子或杂合子与其它基因修饰小鼠交配，对其后代进行筛选，根据孟德尔遗传规律，可有一定机率得到人源化TLR7和/或TLR8基因与其他基因修饰的多基因小鼠，再将杂合子相互交配可以得到双基因或多基因修饰的纯合子。
以TLR7/TLR8/TLR9三基因人源化鼠为例，将实施例3获得TLR7/TLR8双基因人源化鼠与TLR9基因人源化进行交配，获得同时表达人或人源化TLR7蛋白、TLR8蛋白和TLR9蛋白的多基因人源化鼠。或者，取TLR7和/或TLR8基因人源化的胚胎干细胞或受精卵，在其基础上对TLR9基因进行打靶，获得TLR7/TLR8/TLR9多基因人源化鼠。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (83)

  1. 一种基因修饰的非人动物，其特征在于，所述动物的基因组包含至少一条染色体，所述染色体包含编码人或嵌合Toll样受体7(TLR7)蛋白的核苷酸序列。
  2. 根据权利要求1所述的动物，其特征在于，所述编码人或嵌合TLR7蛋白的核苷酸序列可操作地连接至至少一条染色体的内源TLR7基因座的内源调控元件(如，内源5’UTR和/或3’UTR)。
  3. 根据权利要求1或2所述的动物，其特征在于，所述的人或嵌合TLR7蛋白包含人TLR7蛋白的信号肽、胞外、跨膜和/或胞质区的部分。
  4. 根据权利要求1-3任一所述的动物，其特征在于，所述的人或嵌合TLR7蛋白包含人TLR7蛋白胞外区的全部或部分。
  5. 根据权利要求1-4任一所述的动物，其特征在于，所述的人TLR7蛋白胞外区的氨基酸序列与SEQ ID NO：2第27-802位或SEQ ID NO：2第27-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  6. 根据权利要求1-5任一所述的动物，其特征在于，所述的人或嵌合TLR7蛋白包含人TLR7蛋白信号肽的全部或部分。
  7. 根据权利要求1-6任一所述的动物，其特征在于，所述的人或嵌合TLR7蛋白信号肽的氨基酸序列与SEQ ID NO：2第4-26位或SEQ ID NO：2第1-26所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  8. 根据权利要求1-7任一所述的动物，其特征在于，所述的人或嵌合TLR7蛋白的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  9. 根据权利要求1-8任一所述的动物，其特征在于，所述的人或嵌合TLR7蛋白的氨基酸序列与SEQ ID NO：9所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  10. 根据权利要求1-9任一所述的动物，其特征在于，所述动物是哺乳动物，如猴子、啮齿动物、小鼠或大鼠。
  11. 根据权利要求1-10任一所述的动物，其特征在于，所述动物是小鼠。
  12. 根据权利要求1-11任一所述的动物，其特征在于，所述动物内源TLR7蛋白不表达或与野生型动物中TLR7相比表达水平降低。
  13. 根据权利要求1-12任一所述的动物，其特征在于，所述动物的一个或多个细胞表达人或嵌合TLR7蛋白。
  14. 一种基因修饰的非人动物，其特征在于，所述动物的基因组包含在内源TLR7基因 座处编码内源TLR7区域的核苷酸序列被人TLR7相应区域的核苷酸序列替换。
  15. 根据权利要求14所述的动物，其特征在于，所述编码人TLR7相应区域的核苷酸序列可操作地连接到内源TLR7基因座的内源调控元件(如，内源5’UTR和/或3’UTR)，并且所述动物的一个或多个细胞表达人或嵌合TLR7蛋白。
  16. 根据权利要求14或15所述的动物，其特征在于，所述动物的内源TLR7蛋白不表达或与野生型动物中TLR7相比蛋白表达水平降低。
  17. 根据权利要求14-16任一所述的动物，其特征在于，所述编码人TLR7相应区域的核苷酸序列包含人TLR7基因的外显子3的部分。
  18. 根据权利要求14-17任一所述的动物，其特征在于，所述编码人TLR7相应区域的核苷酸序列与SEQ ID NO：5所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  19. 根据权利要求14-18任一所述的动物，其特征在于，所述编码人TLR7相应区域的核苷酸序列与SEQ ID NO：8所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  20. 根据权利要求14-19任一所述的动物，其特征在于，所述编码内源TLR7区域的核苷酸序列包含小鼠TLR7基因外显子3的部分。
  21. 根据权利要求14-20任一所述的动物，其特征在于，所述动物基因组中修饰的TLR7基因对于内源被替换的基因座为纯合或杂合。
  22. 一种非人动物，其特征在于，所述动物包含至少一个编码人或嵌合TLR7蛋白的核苷酸序列的细胞，其中所述人或嵌合TLR7蛋白包含与人相应区域的连续氨基酸序列至少50、60、70、80、90、100、200、300、500、600、700、720、740、760、790、792、795、798、799、802、839、900、1000、1020、1030、1040、1042、1044、1046、1048或1049个连续氨基酸一致。
  23. 根据权利要求22所述的动物，其特征在于，所述人或嵌合TLR7蛋白包含人TLR7蛋白的信号肽、胞外、跨膜和/或胞质区的部分。
  24. 根据权利要求22或23所述的动物，其特征在于，所述人或嵌合TLR7蛋白包含人TLR7蛋白的胞外区的全部或部分。
  25. 根据权利要求22-24任一所述的动物，其特征在于，所述人TLR7蛋白胞外区的氨基酸序列与SEQ ID NO：2第27-802位或SEQ ID NO：2第27-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  26. 根据权利要求22-25任一所述的动物，其特征在于，所述的人或嵌合TLR7蛋白包 含人TLR7蛋白信号肽的全部或部分。
  27. 根据权利要求22-26任一所述的动物，其特征在于，所述的人或嵌合TLR7蛋白信号肽的氨基酸序列与SEQ ID NO：2第4-26位或SEQ ID NO：2第1-26位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  28. 根据权利要求22-27任一所述的动物，其特征在于，所述的人或嵌合TLR7蛋白的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  29. 根据权利要求22-28任一所述的动物，其特征在于，所述人或嵌合TLR7蛋白氨基酸序列与SEQ ID NO：9所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  30. 根据权利要求22-29任一所述的动物，其特征在于，所述编码人或嵌合TLR7蛋白核苷酸序列可操作地连接至至少一条染色体的内源TLR7基因座的内源调控元件(如，内源5’UTR和/或3’UTR)。
  31. 根据权利要求22-30任一所述的动物，其特征在于，所述编码人或嵌合TLR7蛋白的核苷酸序列可被整合至所述动物内源TLR7基因座。
  32. 根据权利要求22-31任一所述的动物，其特征在于，所述人源化TLR7蛋白具有至少一种小鼠TLR7的活性和/或人TLR7的活性。
  33. 一种基因修饰的非人动物的构建方法，其特征在于，所述动物的至少一个细胞中，在动物内源TLR7基因座处，编码内源TLR7区域的核苷酸序列被编码人TLR7相应区域的核苷酸序列替换。
  34. 根据权利要求33所述的方法，其特征在于，所述编码人TLR7相应区域的核苷酸序列包含编码人TLR7蛋白信号肽和胞外区的全部或部分。
  35. 根据权利要求33或34所述的方法，其特征在于，所述编码人TLR7相应区域的核苷酸序列包含人TLR7基因外显子3的部分。
  36. 根据权利要求33-35任一所述的方法，其特征在于，所述编码人TLR7相应区域的核苷酸序列包含编码的氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  37. 根据权利要求33-36任一所述的方法，其特征在于，所述编码人TLR7相应区域的核苷酸序列与SEQ ID NO：5所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  38. 根据权利要求33-37任一所述的方法，其特征在于，所述编码内源TLR7区域的核苷酸序列包含小鼠TLR7基因外显子3的部分。
  39. 根据权利要求33-38任一所述的方法，其特征在于，所述编码人TLR7相应区域的核苷酸序列可操作地连接至内源TLR7的调控元件，如，启动子。
  40. 根据权利要求33-39任一所述的方法，其特征在于，所述动物为哺乳动物，如猴子、啮齿动物、小鼠或大鼠。
  41. 根据权利要求33-40任一所述的方法，其特征在于，所述动物是小鼠。
  42. 一种表达人或嵌合TLR7蛋白基因修饰非人动物的细胞构建方法，所述方法包括在内源小鼠TLR7基因座处，编码内源TLR7区域的核苷酸序列被编码人TLR7相应区域的核苷酸序列替换，产生基因修饰的非人动物细胞，其中动物细胞表达人或嵌合TLR7蛋白。
  43. 根据权利要求42所述的方法，其特征在于，所述人或嵌合TLR7蛋白包含人TLR7蛋白信号肽和胞外区的全部或部分。
  44. 根据权利要求42或43所述的方法，其特征在于，所述编码人TLR7相应区域的核苷酸序列包含人TLR7基因外显子3的部分。
  45. 根据权利要求42-44任一所述的方法，其特征在于，所述编码人TLR7相应区域的核苷酸序列编码的氨基酸序列包含与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  46. 根据权利要求42-45任一所述的方法，其特征在于，所述编码人TLR7相应区域的核苷酸序列与SEQ ID NO：5所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  47. 根据权利要求42-46任一所述的方法，其特征在于，所述编码内源TLR7区域的核苷酸序列包含小鼠TLR7基因外显子3的部分。
  48. 根据权利要求42-47任一所述的方法，其特征在于，所述编码人或嵌合TLR7蛋白的核苷酸序列可操作地连接至内源TLR7的调控元件，如，启动子。
  49. 根据权利要求42-48任一所述的方法，其特征在于，所述动物为哺乳动物，如猴子、啮齿动物、小鼠或大鼠。
  50. 根据权利要求42-49任一所述的方法，其特征在于，所述动物是小鼠。
  51. 根据权利要求33-50任一所述的方法，其特征在于，所述非人动物包括其他基因编码的人或嵌合蛋白的核苷酸序列，所述人或嵌合蛋白选自TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40的至少一种。
  52. 根据权利要求51任一所述的方法，其特征在于，所述人或嵌合蛋白为TLR8蛋白。
  53. 根据权利要求51或52所述的方法，其特征在于，所述编码人或嵌合TLR8蛋白的核苷酸序列与SEQ ID NO：22所示的核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  54. 根据权利要求51-53任一所述的方法，其特征在于，所述人或嵌合TLR8蛋白氨基酸序列与SEQ ID NO：23所示的氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  55. 根据权利要求1-32任一所述的非人动物，其特征在于，非人动物包括其他基因编码的人或嵌合蛋白的核苷酸序列，所述人或嵌合蛋白选自TLR8、LAG-3、BTLA、PD-1、PD-L1、CD27、CD28、CD40、CD47、CD137、TIGIT、TIM-3或OX40的至少一种。
  56. 根据权利要求55任一所述的非人动物，其特征在于，所述人或嵌合蛋白为TLR8蛋白。
  57. 根据权利要求55或56所述的动物，其特征在于，所述编码人或嵌合TLR8蛋白的核苷酸序列与SEQ ID NO：22所示的核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  58. 根据权利要求55-57任一所述的动物，其特征在于，所述人或嵌合TLR8蛋白氨基酸序列与SEQ ID NO：23所示的氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  59. 一种测定抗TLR7治疗剂治疗癌症有效性的方法，其特征在于，所述方法包括：
    1)向权利要求1-32和55-58任一所述的动物施用抗TLR7治疗剂，其中所述动物具有肿瘤；
    2)测定抗TLR7治疗剂对肿瘤的抑制作用。
  60. 根据权利要求59所述的方法，其特征在于，所述肿瘤包含一个或多个肿瘤细胞，其中肿瘤细胞被注射到动物体内。
  61. 根据权利要求59或60所述的方法，其特征在于，所述测定抗TLR7治疗剂对肿瘤的抑制作用包含测量动物体内的肿瘤体积。
  62. 根据权利要求59-61任一所述的方法，其特征在于，所述肿瘤为实体瘤、膀胱癌、浅表尿路上皮癌、宫颈癌、子宫内膜癌、食道癌、鳞状细胞癌、肾癌、非小细胞肺癌、卵巢癌、鳞状细胞癌、胃癌、子宫癌、结直肠转移癌、肝癌、胃肠癌。
  63. 一种测定抗TLR7治疗剂和其它治疗剂治疗癌症有效性的方法，其特征在于，所述方法包括：
    1)向权利要求1-32和55-58任一所述的动物施用抗TLR7治疗剂，其中所述动物具有肿瘤；
    2)测定抗TLR7治疗剂对肿瘤的抑制作用。
  64. 根据权利要求63所述的方法，其特征在于，所述动物还包括编码人或嵌合PD-1、人或嵌合PD-L1和/或人或嵌合CTLA4的序列。
  65. 根据权利要求63或64所述的方法，其特征在于，所述其它治疗剂是抗PD-1抗体、抗PD-L1抗体和/或抗CTLA4抗体。
  66. 根据权利要求3-65任一所述的方法，其特征在于，所述肿瘤包含一个或多个细胞表达PD-L1蛋白。
  67. 根据权利要求63-66任一所述的方法，其特征在于，所述肿瘤包含一个或多个肿瘤细胞，其中肿瘤细胞被注射到动物体内。
  68. 根据权利要求63-67任一所述的方法，其特征在于，所述测定抗TLR7治疗剂对肿瘤的抑制作用包含测量动物体内的肿瘤体积。
  69. 根据权利要求63-68任一所述的方法，其特征在于，所述肿瘤为实体瘤、膀胱癌、浅表尿路上皮癌、宫颈癌、子宫内膜癌、食道癌、鳞状细胞癌、肾癌、非小细胞肺癌、卵巢癌、鳞状细胞癌、胃癌、子宫癌、结直肠转移癌、肝癌、胃肠癌。
  70. 一种测定抗TLR7治疗剂治疗代谢性疾病有效性的方法，其特征在于，所述方法包括：
    1)向权利要求1-32和55-58任一所述非人动物施用抗TLR7治疗剂，其中所述非人动物患有自身免疫性疾病；
    2)测定抗TLR7治疗剂对治疗自身免疫性疾病中的作用。
  71. 根据权利要求70所述的方法，其特征在于，所述自身免疫性疾病为哮喘、鼻炎、系统性红斑狼疮、银屑病。
  72. 一种测定抗TLR7治疗剂治疗炎症有效性的方法，其特征在于，所述方法包括：
    1)向权利要求1-32和55-58任一所述非人动物施用抗TLR7治疗剂，其中所述非人动物患有炎症；
    2)测定抗TLR7治疗剂对治疗炎症中的作用。
  73. 根据权利要求72所述的方法，其特征在于，所述炎症为慢性阻塞性肺病、脓毒症、皮炎。
  74. 一种测定抗TLR7治疗剂毒性的方法，其特征在于，所述方法包括：
    1)向权利要求1-32和55-58任一所述的动物施用抗TLR7治疗剂；
    2)测定抗TLR7治疗剂对动物的作用。
  75. 根据权利要求74所述的方法，其特征在于，所述测定抗TLR7治疗剂对动物的作用涉及测量动物的体重、红细胞计数、血细胞比容和/或血红蛋白。
  76. 一种人源化TLR7蛋白，其特征在于，所述的人源化蛋白包含人TLR7蛋白信号肽和胞外区的全部或部分。
  77. 根据权利要求76所述的人源化蛋白，其特征在于，所述人源化TLR7蛋白氨基酸序列与SEQ ID NO：2第4-802位，SEQ ID NO：2第1-802位或SEQ ID NO：2第1-839位所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  78. 根据权利要求76或77所述的人源化蛋白，其特征在于，所述人源化TLR7蛋白氨基酸序列与SEQ ID NO：9所示氨基酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  79. 一种人源化TLR7基因，其特征在于，所述人源化基因编码权利要求76-78任一所述的人源化蛋白。
  80. 根据权利要求79所述的人源化TLR7基因，其特征在于，所述人源化TLR7基因包含人TLR7基因外显子3的部分。
  81. 根据权利要求79或80所述的人源化TLR7基因，其特征在于，所述人源化TLR7基因包含的核苷酸序列与SEQ ID NO：3、4、5、6、7、8、10和11任一所示核苷酸序列同一性至少为70％、75％、80％、85％、90％、95％、99％或100％。
  82. 一种细胞，其特征在于，所述细胞包含权利要求76-78任一所述的人源化TLR7蛋白和权利要求79-81任一所述人源化TLR7基因。
  83. 一种动物模型，其特征在于，所述的动物模型包含权利要求76-78任一所述的人源化TLR7蛋白和权利要求79-81任一所述人源化TLR7基因。
PCT/CN2023/107520 2022-07-15 2023-07-14 一种tlr7和/或tlr8基因人源化修饰的非人动物 WO2024012578A1 (zh)

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