OSM, OSMR, IL31RA and / or IL31 gene modified non-human animal

Abstract

A non-human animal expressing human or chimeric (e.g., humanized) OSMR, OSM, IL31RA, and / or IL31 proteins, and methods of use thereof.

Description

A non-human animal modified with OSM, OSMR, IL31RA and / or IL31 genes

[0001] Priority claim

[0002] This patent application claims priority to Chinese patent application No. 202210752534.4 filed on June 29, 2022, and priority to Chinese patent application No. 202310261885.X filed on March 17, 2023. The entire contents of the above applications are incorporated herein by reference. Technical Field

[0003] The present invention provides a non-human animal expressing human or chimeric (eg, humanized) OSM, OSMR, IL31RA and / or IL31 protein and methods of use thereof.

[0004] background

[0005] Traditional drug development typically uses in vitro screening methods. However, these screening methods cannot provide the body's environment (such as the tumor microenvironment, stromal cells, extracellular matrix components, and immune cell interactions), resulting in a high rate of drug development failure. In addition, given the differences between humans and animals, the results of in vivo pharmacology trials using conventional experimental animals may not reflect the actual disease state and the interaction of target sites, resulting in significant differences between the results of many clinical trials and those of animal experiments.

[0006] Therefore, developing humanized animal models suitable for screening and evaluation of human antibodies will significantly improve the efficiency of new drug development and reduce drug development costs.

[0007] Overview

[0008] The present application provides an animal model with human or chimeric OSMR, OSM, IL31RA, and / or IL31 proteins. The animal model can express human or chimeric OSMR (e.g., humanized OSMR) protein, human or chimeric OSM (e.g., humanized OSM) protein, human or chimeric IL31RA (e.g., humanized IL31RA) protein, and / or human or chimeric IL31 (e.g., humanized IL31) protein. It can be used to study the functions of OSMR, OSM, IL31RA, and IL31 genes, and can also be used to screen and evaluate OSM / IL31 signaling pathway regulators (e.g., anti-human OSMR antibodies, anti-human OSM antibodies, anti-human IL31RA antibodies, or anti-human IL31 antibodies). In addition, the animal model prepared by the method described herein can be used for drug screening, pharmacodynamic studies, treatment of immune-related diseases, and treatment of diseases targeting human OSMR / OSM / IL31RA / IL31 target sites; the model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the functions of OSMR / OSM / IL31RA / IL31 proteins and a platform for screening related drugs.

[0009] In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric Oncostatin M Receptor (OSMR) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric OSMR protein is operably linked to endogenous regulatory elements (e.g., endogenous 5'UTR and / or 3'UTR) of an endogenous OSMR locus on at least one chromosome. In some embodiments, the chimeric OSMR protein comprises a human signal peptide, a human or humanized extracellular region, and endogenous intracellular and transmembrane regions. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human OSMR (NP_001310434.1, SEQ ID NO: 10). In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 1-737 of SEQ ID NO: 10. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 16. In some embodiments, 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 does not express endogenous OSMR protein or its expression level is reduced compared to OSMR in wild-type animals. In some embodiments, one or more cells of the animal express the human or chimeric OSMR protein.

[0010] In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises a nucleotide sequence encoding a human or chimeric OSMR corresponding region inserted into an endogenous OSMR locus. In some embodiments, the nucleotide sequence encoding the human or chimeric OSMR corresponding region is operably linked to an endogenous regulatory element of the endogenous OSMR locus, and one or more cells of the animal express a human or humanized OSMR protein. In some embodiments, the endogenous OSMR protein of the animal is not expressed or is expressed at a reduced level compared to OSMR in a wild-type animal. In some embodiments, the nucleotide sequence encoding the human or chimeric OSMR corresponding region is inserted into exon 2 of the endogenous OSMR locus. In some embodiments, the nucleotide sequence encoding the human or chimeric OSMR corresponding region is inserted before nucleotide position 129 of NM_011019.3. In some embodiments, the nucleotide sequence encoding the human or chimeric OSMR corresponding region is inserted between nucleotide positions 125 and 129 of NM_011019.3. In some embodiments, at least 3 bp of exon 2 of the endogenous OSMR locus (e.g., nucleotides 126 to 128 of NM_011019.3) are deleted. In some embodiments, the inserted sequence comprises, optionally, from 5' to 3': A) a first sequence encoding all or part of the human OSMR signal peptide and extracellular region; and B) a second sequence encoding all or part of the endogenous OSMR transmembrane region and intracellular region, optionally further comprising a portion of the endogenous OSMR extracellular region. In some embodiments, the amino acid sequence encoded by the first sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-737 of SEQ ID NO: 10, and the amino acid sequence encoded by the second sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 735-971 of SEQ ID NO: 9. In some embodiments, the inserted sequence further comprises one or more auxiliary sequences. In some embodiments, the auxiliary sequence is a polyA sequence. In some embodiments, the genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 13, 14, 15 or 51. In some embodiments, the inserted sequence in the genome of the animal is homozygous and / or heterozygous for the endogenous OSMR locus.

[0011] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a chimeric OSMR protein, wherein the chimeric OSMR protein comprises at least 50, 100, 200, 300, 400, 500, 600, 700, 710, 720, 730, 737, 740, 760, 800, 900, or 979 consecutive amino acids that are identical to the corresponding human amino acid sequence, and the animal expresses the chimeric OSMR protein. In some embodiments, the chimeric OSMR protein comprises at least 50, 100, 200, 300, 400, 500, 600, 700, 710, 720, 730, 735, 737, 738, 739, or 740 consecutive amino acids that are identical to the corresponding human signal peptide and extracellular region. In some embodiments, the chimeric OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 1-737 of SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the chimeric OSMR protein is operably linked to an endogenous OSMR regulatory element. In some embodiments, the chimeric OSMR protein comprises the transmembrane and intracellular regions of an endogenous OSMR, and optionally also comprises a portion of the extracellular region of an endogenous OSMR. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSMR is integrated into the animal's endogenous OSMR locus. In some embodiments, the chimeric OSMR protein exhibits at least one of a mouse OSMR activity and / or a human OSMR activity.

[0012] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein a nucleotide sequence encoding a chimeric OSMR polypeptide is introduced into at least one cell of the animal at the animal's endogenous OSMR locus. In some embodiments, the chimeric OSMR polypeptide comprises: A) all or part of the human OSMR signal peptide and extracellular region; and B) all or part of the endogenous OSMR transmembrane region and intracellular region, optionally further comprising part of the endogenous OSMR extracellular region. In some embodiments, the nucleotide sequence encoding the chimeric OSMR polypeptide is inserted into exon 2 of the animal's endogenous OSMR locus. In some embodiments, at least 3 bp of exon 2 of the endogenous OSMR locus (e.g., nucleotides 126 to 128 of NM_011019.3) is deleted. In some embodiments, the nucleotide sequence encoding the corresponding region of the chimeric OSMR is operably linked to an endogenous OSMR regulatory element, such as a promoter. In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat.

[0013] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing a chimeric OSMR, the method comprising inserting the following nucleotide sequences at the endogenous mouse OSMR locus (e.g., exon 2 of the endogenous OSMR gene), in order from 5' to 3': A) a nucleotide sequence encoding all or part of the human OSMR signal peptide and extracellular region; B) a nucleotide sequence encoding all or part of the endogenous OSMR transmembrane region and intracellular region, optionally also comprising part of the endogenous OSMR extracellular region; C) optionally one or more auxiliary sequences (e.g., a polyA sequence), thereby producing a genetically modified non-human animal cell comprising a nucleotide sequence encoding a chimeric OSMR protein, wherein the animal cell expresses the chimeric OSMR protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSMR comprises a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, and / or a portion of exon 15 of the human OSMR gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSMR is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 13 or 14. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous OSMR comprises a portion of exon 15, exon 16, exon 17, and / or a portion of exon 18 of the endogenous OSMR gene. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous OSMR is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 13 or 15. In some embodiments, the nucleotide sequence encoding the chimeric OSMR protein is operably linked to a regulatory element of the endogenous OSMR, such as a promoter. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding a human or chimeric protein selected from at least one of OSM, IL31RA, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT and CD73. In some embodiments, the human or chimeric protein is selected from OSM, IL31RA and / or IL31 protein.In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by another gene, wherein the human or chimeric protein is selected from at least one of OSM, IL31RA, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT, and CD73. In some embodiments, the human or chimeric protein is selected from OSM, IL31RA, and / or IL31 protein.

[0014] In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric Oncostatin M (OSM) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric OSM protein is operably linked to endogenous regulatory elements (e.g., endogenous 5'UTR and / or 3'UTR) of an endogenous OSM locus on at least one chromosome. In some embodiments, the nucleotide sequence encoding the human or chimeric OSM protein encodes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human OSM (NP_065391.1, SEQ ID NO: 2). In some embodiments, 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 does not express endogenous OSM protein or its expression is reduced compared to OSM in wild-type animals. In some embodiments, one or more cells of the animal express the human or chimeric OSM protein.

[0015] In one aspect, the present invention provides a genetically modified non-human animal, wherein the genome of the animal comprises a nucleotide sequence encoding an endogenous OSM region at an endogenous OSM locus replaced with a nucleotide sequence encoding a corresponding region of human OSM. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSM is operably linked to endogenous regulatory elements of the endogenous OSM locus, and one or more cells of the animal express a human or humanized OSM protein. In some embodiments, the endogenous OSM protein in the animal is not expressed or is expressed at a reduced level compared to OSM in a wild-type animal. In some embodiments, a contiguous genomic segment from the ATG start codon of exon 1 to exon 3 of the animal's endogenous OSM gene is replaced with a contiguous genomic sequence comprising the ATG start codon of exon 1 to exon 3 of the human OSM gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSM is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence encoding the human or chimeric OSM protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, a portion of exon 1 to exon 3 of the endogenous OSM gene of the animal is replaced. In some embodiments, the modified OSM gene in the genome of the animal is homozygous and / or heterozygous for the endogenous replaced locus.

[0016] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence encoding a human or humanized OSM protein, wherein the humanized OSM protein comprises at least 50, 100, 150, 200, 250, 251, or 252 consecutive amino acids identical to a corresponding region of human OSM, and the animal expresses the human or humanized OSM protein. In some embodiments, the human or humanized OSM protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the human or humanized OSM protein is operably linked to an endogenous OSM regulatory element. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSM is integrated into the animal's endogenous OSM locus. In some embodiments, the human or humanized OSM protein exhibits at least one of a mouse OSM activity and / or a human OSM activity.

[0017] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein in at least one cell of the animal, a nucleotide sequence encoding an endogenous OSM region is replaced at the animal's endogenous OSM gene locus with a nucleotide sequence encoding a corresponding region of human OSM. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSM comprises a portion of exon 1 to a portion of exon 3 of the human OSM gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human OSM is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSM is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the endogenous OSM region comprises a portion of exon 1, exon 2, and / or a portion of exon 3 of the mouse OSM gene. In some embodiments, the nucleotide sequence encoding the human OSM corresponding region is operably linked to an endogenous OSM regulatory element, such as a promoter. In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat.

[0018] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell that expresses human or chimeric OSM. The method comprises replacing a nucleotide sequence encoding an endogenous mouse OSM region with a nucleotide sequence encoding a corresponding region of human OSM at the endogenous mouse OSM gene locus, thereby producing a genetically modified non-human animal cell that expresses a human or chimeric OSM protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSM comprises a portion of exon 1, exon 2, and / or a portion of exon 3 of the human OSM gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human OSM is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSM is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the nucleotide sequence of endogenous OSM comprises a portion of exon 1, exon 2, and / or exon 3 of the mouse OSM gene. In some embodiments, the nucleotide sequence encoding the human or chimeric OSM protein is operably linked to a regulatory element of the endogenous OSM, such as a promoter. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises nucleotide sequences encoding human or chimeric proteins from other genes, the human or chimeric proteins being selected from at least one of OSMR, IL31RA, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT, and CD73. In some embodiments, the human or chimeric protein is selected from OSMR, IL31RA, and / or IL31 proteins. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, wherein the human or chimeric protein is selected from at least one of OSMR, IL31RA, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT, and CD73. In some embodiments, the human or chimeric protein is selected from OSMR, IL31RA, and / or IL31 protein.

[0019] In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric interleukin-31 receptor A (IL31RA) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31RA protein is operably linked to endogenous regulatory elements (e.g., endogenous 5'UTR and / or 3'UTR) of the endogenous IL31RA locus on at least one chromosome. In some embodiments, the chimeric IL31RA protein comprises a human signal peptide, a human or humanized extracellular region, and endogenous intracellular and transmembrane regions. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric IL31RA protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human IL31RA (NP_001229567.1, SEQ ID NO: 61). In some embodiments, the nucleotide sequence encoding the human or chimeric IL31RA protein encodes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 1-520 of SEQ ID NO: 61. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31RA protein encodes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 68. In some embodiments, 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 does not express endogenous IL31RA protein or expresses it at a reduced level compared to IL31RA in wild-type animals. In some embodiments, one or more cells of the animal express the human or chimeric IL31RA protein.

[0020] In one aspect, the present invention provides a genetically modified non-human animal having a genome comprising a nucleotide sequence encoding a human or chimeric IL31RA corresponding region inserted into an endogenous IL31RA locus. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31RA corresponding region is operably linked to an endogenous regulatory element of the endogenous IL31RA locus, and one or more cells of the animal express a human or humanized IL31RA protein. In some embodiments, the endogenous IL31RA protein of the animal is not expressed or is expressed at a reduced level compared to IL31RA in wild-type animals. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31RA corresponding region is inserted into exon 4 of the endogenous IL31RA locus. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31RA corresponding region is inserted before nucleotide position 425 of NM_139299.2. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31RA corresponding region is inserted between nucleotide positions 421 and 425 of NM_139299.2. In some embodiments, at least 3 bp of exon 4 of the endogenous IL31RA locus (e.g., nucleotides 422 to 424 of NM_139299.2) are deleted. In some embodiments, the insert sequence comprises, optionally, from 5' to 3': A) a first sequence encoding all or part of the human IL31RA signal peptide and extracellular region; and B) a second sequence encoding all or part of the endogenous IL31RA transmembrane and intracellular regions, optionally further comprising a portion of the endogenous IL31RA extracellular region. In some embodiments, the first sequence encodes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-520 of SEQ ID NO: 61, and the second sequence encodes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 495-716 of SEQ ID NO: 60. In some embodiments, the insert sequence further comprises one or more auxiliary sequences. In some embodiments, the helper sequence is a WPRE sequence and / or a poly A sequence. In some embodiments, the genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 64, 65, 66, or 67. In some embodiments, the inserted sequence in the genome of the animal is homozygous and / or heterozygous for the endogenous IL31RA locus.

[0021] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a chimeric IL31RA protein, wherein the chimeric IL31RA protein comprises at least 50, 100, 200, 300, 400, 450, 500, 520, 550, 600, 660, 661, or 662 consecutive amino acids identical to a human corresponding region, and the animal expresses the chimeric IL31RA protein. In some embodiments, the chimeric IL31RA protein comprises at least 50, 100, 200, 300, 400, 450, 500, 520, 525, 530, 531, or 532 consecutive amino acids identical to a human signal peptide and extracellular region. In some embodiments, the chimeric IL31RA protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 1-520 of SEQ ID NO: 61. In some embodiments, the nucleotide sequence encoding the chimeric IL31RA protein is operably linked to an endogenous IL31RA regulatory element. In some embodiments, the chimeric IL31RA protein comprises the transmembrane region and intracellular region of endogenous IL31RA, and optionally further comprises a portion of the extracellular region of endogenous IL31RA. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31RA is integrated into the animal's endogenous IL31RA locus. In some embodiments, the chimeric IL31RA protein has at least one mouse IL31RA activity and / or human IL31RA activity.

[0022] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein a nucleotide sequence encoding a chimeric IL31RA polypeptide is introduced into at least one cell of the animal at the animal's endogenous IL31RA locus. In some embodiments, the nucleotide sequence encoding the chimeric IL31RA polypeptide is inserted into exon 4 of the animal's endogenous IL31RA locus. In some embodiments, the chimeric IL31RA polypeptide comprises: A) all or part of the human IL31RA signal peptide and extracellular region; and B) all or part of the endogenous IL31RA transmembrane region and intracellular region, optionally further comprising a portion of the endogenous IL31RA extracellular region. In some embodiments, at least 3 bp of exon 4 of the endogenous IL31RA locus (e.g., nucleotides 422 to 424 of NM_139299.2) are deleted. In some embodiments, the nucleotide sequence encoding the corresponding region of the chimeric IL31RA is operably linked to an endogenous IL31RA regulatory element, such as a promoter. In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat.

[0023] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell that expresses a chimeric IL31RA, the method comprising inserting the following nucleotide sequences, from 5' to 3', into the endogenous mouse IL31RA locus (e.g., exon 4 of the endogenous IL31RA gene): A) a nucleotide sequence encoding all or part of the human IL31RA signal peptide and extracellular region; B) a nucleotide sequence encoding all or part of the endogenous IL31RA transmembrane region and intracellular region, optionally further comprising a portion of the endogenous IL31RA extracellular region; C) optionally one or more auxiliary sequences (e.g., a polyA sequence and / or a WPRE sequence), thereby generating a genetically modified non-human animal cell comprising a nucleotide sequence encoding a chimeric IL31RA protein, wherein the animal cell expresses the chimeric IL31RA protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31RA comprises a portion of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and / or a portion of exon 14 of the human IL31RA gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31RA is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 64 or 66. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous IL31RA comprises a portion of exon 14, exon 15, exon 16, and / or a portion of exon 17 of the endogenous IL31RA gene. In some embodiments, the nucleotide sequence encoding the corresponding region of endogenous IL31RA is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 65 or 66. In some embodiments, the nucleotide sequence encoding the chimeric IL31RA protein is operably linked to a regulatory element of endogenous IL31RA, such as a promoter. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding a human or chimeric protein selected from at least one of OSM, OSMR, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT and CD73. In some embodiments, the human or chimeric protein is selected from OSM, OSMR and / or IL31 protein.In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by another gene, wherein the human or chimeric protein is selected from at least one of OSM, OSMR, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT, and CD73. In some embodiments, the human or chimeric protein is selected from OSM, OSMR, and / or IL31 protein.

[0024] In one aspect, the present invention provides a genetically modified non-human animal, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric interleukin 31 (IL31) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31 protein is operably linked to an endogenous regulatory element (e.g., endogenous 5'UTR and / or 3'UTR) of an endogenous IL31 locus of at least one chromosome. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric IL31 protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human IL31 (NP_001014358.1, SEQ ID NO: 53). In some embodiments, 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 IL31 protein is not expressed or is expressed at a reduced level compared to IL31 in wild-type animals. In some embodiments, one or more cells of the animal express human or chimeric IL31 protein.

[0025] In one aspect, the present invention provides a genetically modified non-human animal, wherein the genome of the animal comprises a nucleotide sequence encoding an endogenous IL31 region at an endogenous IL31 locus replaced with a nucleotide sequence encoding a corresponding region of human IL31. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 is operably linked to an endogenous regulatory element of the endogenous IL31 locus, and one or more cells of the animal express a human or humanized IL31 protein. In some embodiments, the endogenous IL31 protein of the animal is not expressed or is expressed at a reduced level compared to IL31 in wild-type animals. In some embodiments, a contiguous genomic segment from the ATG start codon of exon 1 to exon 3 of the animal's endogenous IL31 gene is replaced with a contiguous genomic sequence comprising the ATG start codon of exon 1 to exon 3 of the human IL31 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 56. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31 protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 57. In some embodiments, a portion of exon 1 to exon 3 of the animal's endogenous IL31 gene is replaced. In some embodiments, the modified gene in the animal's genome is homozygous and / or heterozygous for the endogenous replaced locus.

[0026] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence encoding a human or humanized IL31 protein, wherein the humanized IL31 protein comprises at least 50, 100, 150, 160, 161, 162, 163, or 164 consecutive amino acids identical to the corresponding region of human IL31, and the animal expresses the human or humanized IL31 protein. In some embodiments, the human or humanized IL31 protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the nucleotide sequence encoding the human or humanized IL31 protein is operably linked to an endogenous IL31 regulatory element. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 is integrated into the animal's endogenous IL31 locus. In some embodiments, the human or humanized IL31 protein exhibits at least one of a mouse IL31 activity and / or a human IL31 activity.

[0027] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein in at least one cell of the animal, a nucleotide sequence encoding an endogenous IL31 region is replaced at the animal's endogenous IL31 locus with a nucleotide sequence encoding a corresponding region of human IL31. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 comprises a portion of exon 1 to a portion of exon 3 of the human IL31 gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human IL31 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 56. In some embodiments, the nucleotide sequence encoding the endogenous IL31 region comprises a portion of exon 1, exon 2, and / or a portion of exon 3 of the mouse IL31 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 is operably linked to an endogenous IL31 regulatory element, such as a promoter. In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat.

[0028] In one aspect, the present invention provides a method for constructing genetically modified non-human animal cells that express human or chimeric IL31. The method comprises replacing a nucleotide sequence encoding an endogenous IL31 region at the endogenous mouse IL31 locus with a nucleotide sequence encoding a corresponding region of human IL31, thereby producing genetically modified non-human animal cells that express human or chimeric IL31 protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 comprises a portion of exon 1, exon 2, and / or a portion of exon 3 of the human IL31 gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human IL31 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 56. In some embodiments, the nucleotide sequence of the endogenous IL31 comprises a portion of exon 1, exon 2, and / or a portion of exon 3 of the mouse IL31 gene. In some embodiments, the nucleotide sequence encoding the human or chimeric IL31 protein is operably linked to a regulatory element of endogenous IL31, such as a promoter. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, the human or chimeric protein being selected from at least one of OSMR, IL31RA, OSM, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT, and CD73. In some embodiments, the human or chimeric protein is selected from OSMR, IL31RA, and / or OSM protein. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, wherein the human or chimeric protein is selected from at least one of OSMR, IL31RA, OSM, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT, and CD73. In some embodiments, the human or chimeric protein is selected from OSMR, IL31RA, and / or OSM protein.

[0029] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer, the method comprising: 1) administering a therapeutic agent to an animal described herein, wherein the animal suffers from cancer; and 2) determining the inhibitory effect of the therapeutic agent on the cancer. In some embodiments, the therapeutic agent is an anti-OSM antibody, an anti-OSMR antibody, an anti-IL31 antibody, and / or an anti-IL31RA antibody. In some embodiments, the cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume in the animal. In some embodiments, the cancer comprises the injection of one or more cancer cells into the animal. In some embodiments, the cancer is ovarian cancer, female reproductive system cancer, head and neck cancer, nasopharyngeal carcinoma, skin cancer, melanoma, and the like.

[0030] In one aspect, the present invention provides a method for determining the effectiveness of an anti-OSM antibody, an anti-OSMR antibody, an anti-IL31 antibody, and / or an anti-IL31RA antibody and an additional therapeutic agent in treating cancer, the method comprising: 1) administering an anti-OSM antibody, an anti-OSMR antibody, an anti-IL31 antibody, and / or an anti-IL31RA antibody and an additional therapeutic agent to an animal described herein, wherein the animal has a tumor; and 2) determining the inhibitory effect of the OSMR, OSM, IL31RA, and / or IL31 therapeutic agent on the tumor. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the tumor comprises one or more cancer cells injected into the animal. In some embodiments, the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume in the animal. In some embodiments, the cancer is ovarian cancer, female reproductive system cancer, head and neck cancer, nasopharyngeal carcinoma, skin cancer, melanoma, etc.

[0031] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating an immune disease, the method comprising: 1) administering the therapeutic agent to an animal described herein, wherein the animal suffers from an immune disease; and 2) determining the effectiveness of the therapeutic agent in treating the immune disease. In some embodiments, the immune disease is rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, atherosclerosis, interstitial lung disease, psoriasis, allergy, Crohn's disease, pulmonary fibrosis, and the like.

[0032] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation, the method comprising: 1) administering a therapeutic agent to an animal described herein, wherein the animal suffers from inflammation; and 2) determining the effectiveness of the therapeutic agent in treating the inflammation. In some embodiments, the inflammation is atopic dermatitis, pruritus, prurigo, dermatitis, or the like.

[0033] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, the method comprising: 1) administering the therapeutic agent to an animal as described herein; and 2) determining the effect of the therapeutic agent on the animal. In some embodiments, determining the effect of the therapeutic agent on the animal comprises measuring the animal's weight, red blood cell count, hematocrit, and / or hemoglobin.

[0034] In one aspect, the present invention provides a humanized OSM gene comprising any one of the following nucleotide sequences: A) a nucleotide sequence encoding SEQ ID NO: 2; B) a sequence of SEQ ID NO: 3, 4, 5, 6, 7 or 8; C) a sequence having at least 90% identity to SEQ ID NO: 3, 4, 5, 6, 7 or 8; and D) a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3, 4, 5, 6, 7 or 8.

[0035] In one aspect, the present invention provides a humanized OSMR protein comprising all or a portion of a human OSMR protein. In some embodiments, the humanized OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0036] In one aspect, the present invention provides a humanized OSMR gene, wherein the humanized OSMR gene encodes the humanized protein of any one of claims 202 to 203. In some embodiments, the humanized OSMR gene comprises a portion of exon 2, all of exon 3 to exon 14, and / or a portion of exon 15 of a human OSMR gene, and the humanized OSMR gene further comprises a portion of exon 15, all of exon 16 to exon 17, and / or a portion of exon 18 of a non-human animal OSMR gene. In some embodiments, the nucleotide sequence comprised by the humanized OSMR gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 13.

[0037] In one aspect, the present invention provides a humanized IL31 gene comprising any one of the following nucleotide sequences: A) a nucleotide sequence encoding SEQ ID NO: 53; B) a sequence of SEQ ID NO: 54, 55, 56, 57, 58 or 59; C) a sequence having at least 90% identity to SEQ ID NO: 54, 55, 56, 57, 58 or 59; and D) a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 54, 55, 56, 57, 58 or 59.

[0038] In one aspect, the present invention provides a humanized IL31RA protein comprising all or a portion of a human IL31RA protein. In some embodiments, the humanized IL31RA protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 68.

[0039] In one aspect, the present invention provides a humanized IL31RA gene, which encodes the humanized protein of any one of claims 208 to 209. In some embodiments, the humanized IL31RA gene comprises a portion of exon 3, all of exon 4 to exon 13, and / or a portion of exon 14 of a human IL31RA gene, and further comprises a portion of exon 14, all of exon 15 to exon 16, and / or a portion of exon 17 of a non-human animal IL31RA gene. In some embodiments, the nucleotide sequence comprised by the humanized IL31RA gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 66.

[0040] In one aspect, the present invention provides a cell, comprising the humanized gene described herein, and / or expressing the humanized protein described herein.

[0041] In one aspect, the present invention provides an animal model, comprising the humanized gene described herein, and / or expressing the humanized protein described herein.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of conflict, the present specification, including definitions, will control.

[0043] Those skilled in the art can easily discern other conveniences and advantages of the present application from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0045] Figure 1: Schematic comparison of the mouse OSM gene and the human OSM locus (not to scale);

[0046] Figure 2: Schematic diagram of the humanized mouse OSM gene (not to scale);

[0047] Figure 3: Schematic diagram of OSM gene targeting strategy and targeting vector design (not to scale);

[0048] Figure 4: Schematic diagram of the FRT recombination process for OSM gene humanized mice (not to scale);

[0049] Figure 5: Schematic diagram of OSM gene targeting strategy and targeting vector design (not to scale);

[0050] Figure 6: PCR identification results of the tail of F1 generation of OSM humanized mice, where WT is the wild-type control, H2O is the water control, and M is the marker;

[0051] Figure 7: Schematic diagram of Southern Blot detection results, where WT is the wild-type control;

[0052] Figure 8: Schematic comparison of the mouse OSMR gene and the human OSMR locus (not to scale);

[0053] Figure 9: Schematic diagram of the humanized mouse OSMR gene (not to scale);

[0054] Figure 10: Schematic diagram of OSMR gene targeting strategy and targeting vector design (not to scale);

[0055] Figure 11: Schematic diagram of the FRT recombination process of OSMR gene humanized mice (not to scale);

[0056] Figure 12: PCR identification results of OSMR gene humanized F1 mouse tails, where WT is the wild-type control, PC1 and PC2 are positive controls, H2O is the water control, and M is a marker;

[0057] Figure 13: Western blot test results, where + / + represents wild-type C57BL / 6 mice, H / H represents OSMR gene homozygous mice, 293T represents human embryonic kidney epithelial cell line (positive control), (A) represents the Goat Anti-OSMR antibody test result, (B) represents the OSMR Polyclonal Antibody test result, and β-actin is the internal control of β-actin;

[0058] Figure 14: RT-PCR test results of C57BL / 6 wild-type mice (+ / +) and OSMR gene homozygous mice (H / H), where H2O is the water control and GAPDH is the internal control for glyceraldehyde-3-phosphate dehydrogenase;

[0059] Figure 15: Flow cytometry analysis of the proportions of leukocyte subsets in the spleens of C57BL / 6 wild-type mice (+ / +) and OSMR gene homozygous mice (H / H);

[0060] Figure 16: Flow cytometry analysis of the proportions of T cell subsets in the spleens of C57BL / 6 wild-type mice (+ / +) and OSMR gene homozygous mice (H / H);

[0061] Figure 17: Flow cytometry analysis of the proportions of leukocyte subsets in the peripheral blood of C57BL / 6 wild-type mice (+ / +) and OSMR gene homozygous mice (H / H);

[0062] Figure 18: Flow cytometry analysis of the proportions of T cell subsets in the peripheral blood of C57BL / 6 wild-type mice (+ / +) and OSMR gene homozygous mice (H / H);

[0063] Figure 19: Flow cytometry analysis of the proportions of leukocyte subsets in lymph nodes of C57BL / 6 wild-type mice (+ / +) and OSMR gene homozygous mice (H / H);

[0064] Figure 20: Flow cytometry analysis of the proportion of T cell subsets in the lymph nodes of C57BL / 6 wild-type mice (+ / +) and OSMR gene homozygous mice (H / H);

[0065] Figure 21: Blood routine test results, where + / + indicates C57BL / 6 wild-type mice, and H / H indicates OSMR gene humanized homozygous mice;

[0066] Figure 22: Schematic comparison of the mouse IL31 gene and the human IL31 locus (not to scale);

[0067] Figure 23: Schematic diagram of the humanized mouse IL31 gene (not to scale);

[0068] Figure 24: Schematic diagram of IL31 gene targeting strategy and targeting vector design (not to scale);

[0069] Figure 25: Schematic diagram of Southern Blot detection results, where WT is the wild-type control;

[0070] Figure 26: Schematic diagram of the FRT recombination process of IL31 gene humanized mice (not to scale);

[0071] Figure 27: PCR identification results of the tail of F1 generation of humanized mice carrying the IL31 gene, where WT is the wild-type control, H2O is the water control, M is the marker, and PC1 and PC2 are positive controls;

[0072] Figure 28: RT-PCR test results of C57BL / 6 wild-type mice (+ / +) and IL31 gene homozygous mice (H / H), where H2O is the water control and GAPDH is the internal control for glyceraldehyde-3-phosphate dehydrogenase;

[0073] Figure 29: Schematic comparison of the mouse IL31RA gene and the human IL31RA locus (not to scale);

[0074] Figure 30: Schematic diagram of the humanized mouse IL31RA gene (not to scale);

[0075] Figure 31: Schematic diagram of IL31RA gene targeting strategy and targeting vector design (not to scale);

[0076] Figure 32: Schematic diagram of Southern Blot detection results, where WT is the wild-type control;

[0077] Figure 33: Schematic diagram of the FRT recombination process of the IL31RA gene humanized mouse (not to scale);

[0078] Figure 34: PCR identification results of the tail of F1 generation mice carrying the humanized IL31RA gene, where WT is the wild-type control, PC is the positive control, H2O is the water control, and M is a marker;

[0079] Figure 35: RT-PCR test results of C57BL / 6 wild-type mice (+ / +) and IL31RA gene homozygous mice (H / H), where H2O is the water control and GAPDH is the internal control for glyceraldehyde-3-phosphate dehydrogenase;

[0080] Figure 36: RT-PCR test results of C57BL / 6 wild-type mice (+ / +) and OSM / OSMR double humanized homozygous mice (H / H), where H2O is the water control and GAPDH is the internal control for glyceraldehyde-3-phosphate dehydrogenase;

[0081] Figure 37: RT-qPCR test results of C57BL / 6 wild-type mice (+ / +) and OSM / OSMR double humanized homozygous mice (H / H);

[0082] Figure 38: Western blot results, where + / + represents wild-type C57BL / 6 mice, H / H represents OSM / OSMR double humanized homozygous mice, PC represents the positive control, and β-actin represents the internal control of β-actin;

[0083] Figure 39: ELISA test results, where + / + represents wild-type C57BL / 6 mice, H / H represents OSM / OSMR double humanized homozygous mice, (A) represents the mouse OSM test result, and (B) represents the human OSM test result;

[0084] Figure 40: RT-PCR test results of C57BL / 6 wild-type mice (+ / +) and IL31 / IL31RA humanized heterozygous mice (H / +), where H2O is the water control, GAPDH is the internal reference for glyceraldehyde-3-phosphate dehydrogenase, and Hprt is the internal reference for hypoxanthine phosphoribosyltransferase;

[0085] Figure 41: ELISA detection of the expression of mouse IL-6 in embryonic fibroblasts (MEFs) of C57BL / 6 wild-type mice (+ / +) after stimulation with mouse or human OSM;

[0086] Figure 42: ELISA detection of mouse IL-6 expression in embryonic fibroblasts (MEFs) from OSM / OSMR double humanized homozygous mice (H / H) after stimulation with human OSM or human OSM and anti-human OSMR (anti-hOSMR);

[0087] Figure 43: Alignment of the human OSMR amino acid sequence (NP_001310434.1; SEQ ID NO: 10) and the mouse OSMR amino acid sequence (NP_035149.2; SEQ ID NO: 9);

[0088] Figure 45: Alignment of the human OSMR amino acid sequence (NP_001310434.1; SEQ ID NO: 10) and the rat OSMR amino acid sequence (NP_001005384.2; SEQ ID NO: 115);

[0089] Figure 45: Alignment of the human OSM amino acid sequence (NP_065391.1; SEQ ID NO: 2) and the mouse OSM amino acid sequence (NP_001013383.1; SEQ ID NO: 1);

[0090] Figure 46: Alignment of the human OSM amino acid sequence (NP_065391.1; SEQ ID NO: 2) and the rat OSM amino acid sequence (NP_001006962.1; SEQ ID NO: 116);

[0091] Figure 47: Alignment of the human IL31RA amino acid sequence (NP_001229567.1; SEQ ID NO: 61) and the mouse IL31RA amino acid sequence (NP_647460.2; SEQ ID NO: 60);

[0092] FIG48 : Alignment of the amino acid sequence of human IL31RA (NP_001229567.1; SEQ ID NO: 61) and rat IL31RA (NP_001244207.1; SEQ ID NO: 117);

[0093] Figure 49: Alignment of the human IL31 amino acid sequence (NP_001014358.1; SEQ ID NO: 53) and the mouse IL31RA amino acid sequence (NP_083870.1; SEQ ID NO: 52);

[0094] Figure 50: Alignment between the human IL31 amino acid sequence (NP_001014358.1; SEQ ID NO: 53) and the rat IL31RA amino acid sequence (NP_001006962.1; SEQ ID NO: 118).

[0095] Detailed description

[0096] OSMR

[0097] In the human genome, the OSMR gene (Gene ID: 9180) contains 18 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and exon 18 ( Figure 8 ). The human OSMR protein comprises a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region from the N-terminus to the C-terminus. The nucleotide sequence of human OSMR mRNA is NM_001323505.2, and the amino acid sequence of human OSMR is NP_001310434.1 (SEQ ID NO: 10). Based on the nucleotide and amino acid sequences of transcript NM_001323505.2 and its encoded protein NP_001310434.1, the corresponding positions of each exon and each region are as follows:

[0098] Table 1

[0099] The human OSMR gene (NCBI Gene ID: 9180) is located on chromosome 5 at positions 38846012 to 38945579 of NC_000005.10, based on transcript NM_001323505.2. Among them, the 5'UTR is located at positions 38846012 to 38846387 and 38869032 to 38869044, exon 1 is located at positions 38846012 to 38846387, intron 1 is located at positions 38846388 to 38869031, exon 2 is located at positions 38869032 to 38869117, intron 2 is located at positions 38869118 to 38876200, exon 3 is located at positions 38876201 to 38876373, intron 3 is located at positions 38876374 to 38881592, and exon 4 is located at positions 38881593 to 38881594. 881764, intron 4 is located at positions 38881765 to 38883826, exon 5 is located at positions 38883827 to 38884111, intron 5 is located at positions 38884112 to 38885348, exon 6 is located at positions 38885349 to 38885484, intron 6 is located at positions 38885485 to 38886038, exon 7 is located at positions 38886039 to 38886190, intron 7 is located at positions 38886191 to 38903881, exon 8 is located at positions 38903882 to 38904024, intron 8 is located at positions 38903883 to 38904024. 8904025 to 38904352, exon 9 is located at positions 38904353 to 38904503, intron 9 is located at positions 38904504 to 38917545, exon 10 is located at positions 38917546 to 38917622, ​​intron 10 is located at positions 38917623 to 38918839, exon 11 is located at positions 38918840 to 38919062, intron 11 is located at positions 38919063 to 38921614, exon 12 is located at positions 38921615 to 38921794, and intron 12 is located at positions 38921795 to 389218860. 923149, exon 13 is located at position 38923150 to 38923254, intron 13 is located at position 38923255 to 38924421, exon 14 is located at position 38924422 to 38924595, intron 14 is located at position 38924596 to 38925203, exon 15 is located at position 38925204 to 38925371, intron 15 is located at position 38925372 to 38931882, exon 16 is located at position 38931883 to 38931964, intron 16 is located at position 38931965 to 38932462,Exon 17 is located at positions 38932463 to 38932535, intron 17 is located at positions 38932536 to 38932871, exon 18 is located at positions 38932872 to 38935641, and 3'-UTR is located at positions 38933445 to 38935641. All relevant information about the human OSMR locus can be retrieved on the NCBI website (GeneID: 9180). The entire content is incorporated herein by reference.

[0100] In the mouse genome, the OSMR gene (Gene ID: 18414) contains 18 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and exon 18 ( Figure 8 ). The mouse OSMR protein comprises a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region from the N-terminus to the C-terminus. The nucleotide sequence of mouse OSMR mRNA is NM_011019.3, and the amino acid sequence of mouse OSMR is NP_035149.2 (SEQ ID NO: 9). Based on the nucleotide and amino acid sequences of transcript NM_011019.3 and its encoded protein NP_035149.2, the corresponding positions of each exon and each region are as follows:

[0101] Table 2

[0102] The mouse OSMR gene (NCBI Gene ID: 18414) is located on chromosome 15 at positions 6843049 to 6904434 of NC_000081.7, based on transcript NM_001323505.2.Among them, the 5'UTR is located at positions 6904450 to 6903682 and 6889858 to 6889847, exon 1 is located at positions 6904450 to 6903682, intron 1 is located at positions 6903681 to 6889859, exon 2 is located at positions 6889858 to 6889774, intron 2 is located at positions 6889773 to 6882070, exon 3 is located at positions 6882069 to 6881900, intron 3 is located at positions 6881899 to 6876680, exon 4 is located at positions 6876679 to 6876508, and intron 5 is located at positions 6876507 to 6876508. 3981, exon 5 is located at position 6873980 to 6873702, intron 5 is located at position 6873701 to 6872399, exon 6 is located at position 6872398 to 6872263, intron 6 is located at position 6872262 to 6871586, exon 7 is located at position 6871585 to 6871434, intron 7 is located at position 6871433 to 6867313, exon 8 is located at position 6867312 to 6867173, intron 8 is located at position 6867172 to 6866551, exon 9 is located at position 6866550 to 6866397, intron 9 is located at position 6866658 6396 to 6858191, exon 10 is located at positions 6858190 to 6858114, intron 10 is located at positions 6858113 to 6856793, exon 11 is located at positions 6856792 to 6856570, intron 11 is located at positions 6856569 to 6854134, exon 12 is located at positions 6854133 to 6853954, intron 12 is located at positions 6853953 to 6853138, exon 13 is located at positions 6853137 to 6853033, intron 13 is located at positions 6853032 to 6851669, and exon 14 is located at positions 6851668 to 6851670. The amino acid sequence of the 5′ endonuclease protein 51495 is located at position 6851494 to 6850570, exon 15 is located at position 6850569 to 6850402, intron 15 is located at position 6850401 to 6846470, exon 16 is located at position 6846469 to 6846388, intron 16 is located at position 6846387 to 6846136, exon 17 is located at position 6846135 to 6846063, intron 17 is located at position 6846062 to 6845410, exon 18 is located at position 6845409 to 6843058, and 3'UTR is located at position 6844851 to 6843058. All relevant information on the mouse OSMR locus can be retrieved from the NCBI website (GeneID: 18414), the entire contents of which are incorporated herein by reference.

[0103] Figure 43 shows an alignment of the human OSMR amino acid sequence (NP_001310434.1; SEQ ID NO: 10) and the mouse OSMR amino acid sequence (NP_035149.2; SEQ ID NO: 9). Therefore, the corresponding amino acid residues or regions between human and mouse OSMR can be found in Figure 43.

[0104] OMSR genes, proteins, and gene loci for other species are also known in the art. For example, Rattus norvegicus (rat) OSMR has Gene ID: 310132, Macaca mulatta (rhesus monkey) OSMR has Gene ID: 693569, Canis lupus familiaris (dog) OSMR has Gene ID: 489223, and Sus scrofa (pig) OSMR has Gene ID: 100519398. Relevant information (e.g., intron sequences, exon sequences, and amino acid sequences) for these genes can be found in NCBI, the entire contents of which are incorporated herein by reference.

[0105] Figure 44 shows an alignment of the human OSMR amino acid sequence (NP_001310434.1; SEQ ID NO: 10) and the rat OSMR amino acid sequence (NP_001005384.2; SEQ ID NO: 115). Thus, corresponding amino acid residues or regions between human and rat OSMR can be found in Figure 44.

[0106] The present invention provides a human or chimeric (e.g., humanized) OSMR nucleotide sequence or amino acid sequence. In some embodiments, the entire nucleotide sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of the mouse OSMR gene is replaced by the corresponding nucleotide sequence of the human OSMR gene. In some embodiments, "part" of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of the mouse OSMR gene is replaced by the corresponding sequence of the human OSMR gene. By "portion" is meant 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, 650, 700, 750, 800, 850, 900, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 930, 940, 950, 460, 465, 466, 467, 468, 469, 470, 500, 600, 700, 800, 900, or 971 amino acids. In some embodiments, the "portion" is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100% identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, a signal peptide, an extracellular region, a transmembrane region, or a cytoplasmic region.In some embodiments, a "partial" or "complete" sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and / or exon 18 of a mouse OSMR gene (e.g., a portion of exon 2, all of exons 3-13, and a portion of exon 14) is included. The present invention relates to a method for replacing a portion of an exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18 of the human OSMR gene (e.g., part of exon 2, all of exons 3-13 and part of exon 14).

[0107] In some embodiments, “partial” deletion of the signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, intron 17, and / or exon 18 of endogenous OSMR.

[0108] In some embodiments, the present invention provides a genetically modified non-human animal, wherein the genome of the non-human animal comprises a chimeric OSMR nucleotide sequence. In some embodiments, the chimeric OSMR nucleotide sequence encodes a protein comprising a human or humanized signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the encoded protein is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NOs: 9, 10, or 16. In some embodiments, the genome of the non-human animal comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 11, 12, 13, 14, 15, 38, 39, 40, 41, or 51.

[0109] In some embodiments, the non-human animal described herein comprises a nucleotide sequence encoding a human or humanized OSMR protein. In some embodiments, the human or humanized OSMR protein comprises four homology domains of a human OSMR protein, wherein the homology domains comprise DI, DII, DIII, and DIV.

[0110] In some embodiments, the non-human animals described herein comprise a sequence encoding a human or humanized OSMR protein. In some embodiments, the OSMR protein comprises, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized OSMR protein comprises a human or humanized signal peptide. For example, the human or humanized OSMR signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 1-27 of SEQ ID NO: 10. In some embodiments, the humanized OSMR protein comprises an endogenous signal peptide. For example, the endogenous OSMR signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 1-23 of SEQ ID NO: 9. In some embodiments, the humanized OSMR protein comprises a human or humanized extracellular region. For example, the humanized extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 28-737 of SEQ ID NO: 10. In some embodiments, the humanized OSMR protein comprises at least 1, 2, 3, 4, or 5 amino acids C-terminal to the endogenous extracellular region, such as amino acids 735-736 of SEQ ID NO: 9. In some embodiments, the humanized OSMR protein comprises an endogenous extracellular region. In some embodiments, the humanized OSMR protein comprises a human or humanized transmembrane region. In some embodiments, the humanized OSMR protein comprises an endogenous transmembrane region. For example, the endogenous OSMR transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 737-758 of SEQ ID NO: 9. In some embodiments, the humanized OSMR protein comprises a human or humanized cytoplasmic region. In some embodiments, the humanized OSMR protein comprises an endogenous cytoplasmic region. For example, the endogenous OSMR cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 759-971 of SEQ ID NO: 9. In some embodiments, the humanized OSMR protein comprises an endogenous OSMR sequence of amino acids 735-971 of SEQ ID NO: 9. In some embodiments, the humanized OSMR protein comprises a human OSMR sequence of amino acids 1-737 of SEQ ID NO: 10.

[0111] In some embodiments, the non-human animals described herein comprise a human or humanized OSMR gene. In some embodiments, the humanized OSMR gene comprises, from 5' to 3': endogenous exon 1, a portion of endogenous exon 2 (e.g., nucleotide sequence 114-125 of NM_011019.3), a portion of human exon 2 (e.g., nucleotide sequence 129-201 of NM_001323505.2), human exons 3-14, a portion of human exon 15 (e.g., nucleotide sequence 2173-2339 of NM_001323505.2), a portion of endogenous exon 15 (e.g., nucleotide sequence 2328 of NM_011019.3), endogenous exons 16-17, and a portion of endogenous exon 18 (e.g., nucleotide sequence 2484-3041 of NM_011019.3). In some embodiments, the humanized OSMR comprises 18 exons. In some embodiments, the humanized OSMR comprises endogenous exon 1, humanized exon 2, human exon 3, human exon 4, human exon 5, human exon 6, human exon 7, human exon 8, human exon 9, human exon 10, human exon 11, human exon 12, human exon 13, human exon 14, humanized exon 15, endogenous exon 16, endogenous exon 17, and / or endogenous exon 18. In some embodiments, the humanized OSMR further comprises endogenous exon 2, endogenous exon 3, endogenous exon 4, endogenous exon 5, endogenous exon 6, endogenous exon 7, endogenous exon 8, endogenous exon 9, endogenous exon 10, endogenous exon 11, endogenous exon 12, endogenous exon 13, endogenous exon 14 and / or endogenous exon 15. In some embodiments, the humanized OSMR does not comprise introns. In some embodiments, the humanized OSMR gene further comprises one or more auxiliary sequences (e.g., a polyA sequence). In some embodiments, the humanized OSMR gene further comprises a human or humanized 5'UTR. In some embodiments, the humanized OSMR gene further comprises a human or humanized 3'UTR. In some embodiments, the humanized OSMR gene further comprises an endogenous 5'UTR. In some embodiments, the humanized OSMR gene further comprises an endogenous 3'UTR.

[0112] In some embodiments, the present invention also provides chimeric (e.g., humanized) OSMR nucleotide sequences and / or amino acid sequences, in which, in some embodiments, 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% sequence identity to a mouse OSMR mRNA sequence (e.g., NM_011019.3), a mouse OSMR amino acid sequence (e.g., SEQ ID NO: 9), or a portion thereof (e.g., exon 1, and a portion of exon 2, and / or a portion of exon 15, exons 16-17, and a portion of exon 18). In some embodiments, 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% of the sequence is identical to the human OSMR mRNA sequence (e.g., NM_001323505.2), the amino acid sequence of human OSMR (e.g., SEQ ID NO: 10), or a portion thereof (e.g., a portion of exon 2, exons 3-14, and a portion of exon 15).

[0113] In some embodiments, the amino acid sequence encoding mouse OSMR positions 1-734 (SEQ ID NO: 9) is replaced. In some embodiments, the sequence is replaced with a sequence encoding the corresponding region of human OSMR (e.g., the amino acid sequence encoding human OSMR positions 1-737 (SEQ ID NO: 10).

[0114] In some embodiments, the nucleic acids described herein are operably linked to a promoter or regulatory element, such as an endogenous mouse OSMR promoter, an inducible promoter, an enhancer, and / or mouse or human regulatory elements.

[0115] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein differs from all or a portion of a mouse OSMR nucleotide sequence (e.g., a portion of exon 2, exons 3-14, and a portion of exon 15 of mouse OSMR gene transcript NM_011019.3).

[0116] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein is identical to all or a portion of a mouse OSMR nucleotide sequence (e.g., exon 1, a portion of exon 2, a portion of exon 15, exons 16-17, and a portion of exon 18 of mouse OSMR gene transcript NM_011019.3).

[0117] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein differs from all or a portion of a human OSMR nucleotide sequence (e.g., exon 1, a portion of exon 2, a portion of exon 15, and exons 16-18 of the human OSMR gene transcript NM_001323505.2).

[0118] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein is identical to all or a portion of a human OSMR nucleotide sequence (e.g., a portion of exon 2, exons 3-14, and a portion of exon 15 of the human OSMR gene transcript NM_001323505.2).

[0119] In some embodiments, the chimeric nucleic acid sequence described herein 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, such as contiguous or non-contiguous amino acid residues) that differs from all or a portion of the amino acid sequence of the mouse OSMR protein (e.g., amino acids 1-734 of the mouse OSMR protein sequence NP_035149.2 (SEQ ID NO: 9)).

[0120] In some embodiments, at least a portion (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) of an amino acid sequence described herein is identical to all or part of an amino acid sequence of a mouse OSMR protein (e.g., amino acids 735-971 of mouse OSMR protein sequence NP_035149.2 (SEQ ID NO: 9)).

[0121] In some embodiments, at least a portion (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) of an amino acid sequence described herein differs from all or a portion of an amino acid sequence of a human OSMR protein (e.g., amino acids 738-979 of the human OSMR protein sequence NP_001310434.1 (SEQ ID NO: 10)).

[0122] In some embodiments, at least a portion (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) of an amino acid sequence described herein is identical to all or part of a human OSMR protein amino acid sequence (e.g., amino acids 1-737 of human OSMR protein sequence NP_001310434.1 (SEQ ID NO: 10)).

[0123] The present invention also provides a humanized OSMR mouse amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0124] A) the amino acid sequence shown in SEQ ID NO: 9, 10 or 16;

[0125] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 9, 10 or 16;

[0126] C) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is capable of hybridizing under low stringency conditions or high stringency conditions to a nucleotide sequence encoding an amino acid as set forth in SEQ ID NO: 9, 10, or 16;

[0127] D) differs from the amino acid sequence of SEQ ID NO: 9, 10 or 16 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0128] E) an amino acid sequence as shown in SEQ ID NO: 9, 10 or 16, comprising substitution, deletion and / or insertion of one or more amino acid residues.

[0129] The present invention also provides a humanized OSMR amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0130] A) the amino acid sequence shown in SEQ ID NO: 10, positions 1-737;

[0131] B) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:10, positions 1-737;

[0132] C) differs from the amino acid sequence of SEQ ID NO: 10 at positions 1-737 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0133] D) an amino acid sequence comprising substitutions, deletions and / or insertions of one or more amino acid residues as shown in positions 1 to 737 of SEQ ID NO: 10.

[0134] The present invention also provides a humanized OSMR amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0135] A) the amino acid sequence shown in SEQ ID NO: 9, positions 735-971;

[0136] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:9 as shown in positions 735-971;

[0137] C) differs from the amino acid sequence as set forth in positions 735-971 of SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0138] D) an amino acid sequence comprising substitutions, deletions and / or insertions of one or more amino acid residues as shown in positions 735-971 of SEQ ID NO: 9.

[0139] The present invention also provides a humanized OSMR nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

[0140] A) a nucleic acid sequence as shown in SEQ ID NO: 11, 12, 13, 14, 15, 17, 18 or 51, or a nucleic acid sequence encoding a humanized mouse OSMR homologous amino acid sequence;

[0141] B) a nucleic acid sequence capable of hybridizing to the nucleotide sequence shown in SEQ ID NO: 11, 12, 13, 14, 15, 17, 18 or 51 under low stringency conditions or stringent conditions;

[0142] C) a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence of SEQ ID NO: 11, 12, 13, 14, 15, 17, 18 or 51;

[0143] D) the amino acid sequence it encodes is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9, 10 or 16;

[0144] E) encodes an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 9, 10, or 16 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0145] F) The amino acid sequence encoded by the amino acid sequence is the same as that shown in SEQ ID NO: 9, 10 or 16, including substitution, deletion and / or insertion of one or more amino acid residues.

[0146] The present invention further provides a humanized mouse OSMR genomic DNA sequence, which is obtained by reverse transcription of the mRNA transcribed from the OSMR and is identical to or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 13, 14, 15, or 51.

[0147] OSM

[0148] In the human genome, the OSM gene (Gene ID: 5008) contains three exons: exon 1, exon 2, and exon 3 (Figure 1). The nucleotide sequence of human OSM mRNA is NM_020530.6, and the amino acid sequence of human OSM is NP_065391.1 (SEQ ID NO: 2). Based on the nucleotide and amino acid sequences of transcript NM_020530.6 and its encoded protein NP_065391.1, the corresponding positions of each exon are as follows:

[0149] Table 3

[0150] The human OSM gene (NCBI Gene ID: 5008) is located at positions 30262829 to 30266851 of NC_000022.11 on chromosome 22, based on transcript NM_020530.6. The 5' UTR is located at positions 30266851 to 30266800, exon 1 is located at positions 30266851 to 30266766, intron 1 is located at positions 30266765 to 30265145, exon 2 is located at positions 30265144 to 30265002, intron 2 is located at positions 30265001 to 30264465, exon 3 is located at positions 30264464 to 30262829, and the 3' UTR is located at positions 30263882 to 30262829. All relevant information on the human OSM locus can be retrieved from the NCBI website (Gene ID: 5008), the entire contents of which are incorporated herein by reference.

[0151] In the mouse genome, the OSM gene (Gene ID: 18413) contains three exons: exon 1, exon 2, and exon 3 (Figure 1). The nucleotide sequence of mouse OSM mRNA is NM_001013365.2, and the amino acid sequence of mouse OSM is NP_001013383.1 (SEQ ID NO: 1). Based on the nucleotide and amino acid sequences of transcript NM_001013365.2 and its encoded protein NP_001013383.1, the corresponding positions of each exon and region are as follows:

[0152] Table 4

[0153] The mouse OSM gene (NCBI Gene ID: 18413) is located at positions 4186785 to 4191026 of NC_000077.7 on chromosome 11, based on transcript NM_001013365.2. The 5' UTR is located at positions 4186420 to 4186882, exon 1 is located at positions 4186420 to 4186913, intron 1 is located at positions 4186914 to 4188405, exon 2 is located at positions 4188406 to 4188542, intron 2 is located at positions 4188543 to 4189385, exon 3 is located at positions 4189386 to 4191026, and the 3' UTR is located at positions 4190010 to 4191026. All relevant information on the mouse OSM locus can be retrieved from the NCBI website (Gene ID: 18413), the entire contents of which are incorporated herein by reference.

[0154] Figure 45 shows an alignment of the human OSM amino acid sequence (NP_065391.1; SEQ ID NO: 2) and the mouse OSM amino acid sequence (NP_001013383.1; SEQ ID NO: 1). Therefore, corresponding amino acid residues or regions between human and mouse OSM can be found in Figure 45.

[0155] OSM genes, proteins, and gene loci from other species are also known in the art. For example, Rattus norvegicus (rat) OSM has Gene ID: 289747, Macaca mulatta (rhesus monkey) OSM has Gene ID: 717994, and Canis lupus familiaris (dog) OSM has Gene ID: 611921. Relevant information (e.g., intron sequences, exon sequences, and amino acid sequences) for these genes can be found at NCBI, the entire contents of which are incorporated herein by reference.

[0156] Figure 46 shows the amino acid sequence of human OSM (NP_065391.1; SEQ ID NO: 2) and rat OSM (NP_001006962.1; SEQ ID NO: 116). Therefore, the corresponding amino acid residues or regions between human and rat OSM can be retrieved in Figure 46.

[0157] The present invention provides a human or chimeric (e.g., humanized) OSM nucleotide sequence and / or amino acid sequence. In some embodiments, the entire nucleotide sequence of exon 1, exon 2, exon 3, and / or signal peptide of the mouse OSM gene is replaced with the corresponding nucleotide sequence of the human OSM gene. In some embodiments, "partial" exon 1, exon 2, exon 3, and / or signal peptide of the mouse OSM gene is replaced with the corresponding nucleotide sequence or amino acid sequence of the human OSM gene. The term "portion" 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, 650, 700, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010 770, 780, 790, 791, 792, 793, 800, 1000 or 1500 bp of continuous nucleotide sequence, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 220, 240, 250, 260, 261, 262 or 263 continuous amino acid sequence. In some embodiments, the "portion" is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100% identical to exon 1, exon 2, exon 3, or the signal peptide. In some embodiments, a "partial" or "complete" sequence of exon 1, exon 2, and / or exon 3 of the mouse OSM gene (e.g., a portion of exon 1, all of exon 2, and a portion of exon 3) is replaced with a "partial" or "complete" sequence of the corresponding exon 1, exon 2, and / or exon 3 of the human OSM gene (e.g., a portion of exon 1, all of exon 2, and a portion of exon 3).

[0158] In some embodiments, "portions" of the signal peptide, exon 1, exon 2, and / or exon 3 are deleted.

[0159] In some embodiments, the present invention provides a genetically modified non-human animal, wherein the genome of the non-human animal comprises a human, chimeric, or humanized OSM nucleotide sequence. In some embodiments, the protein encoded by the human, chimeric, or humanized OSM nucleotide sequence is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the genome of the non-human animal comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 17, or 18.

[0160] In some embodiments, the non-human animals described herein comprise a human or humanized OSM protein. In some embodiments, the OSM protein comprises an N-terminal signal peptide. In some embodiments, the humanized OSM protein comprises a human or humanized signal peptide. In some embodiments, the humanized OSM protein comprises an endogenous signal peptide.

[0161] In some embodiments, the non-human animals described herein comprise a human or humanized OSM gene. In some embodiments, the humanized OSM gene comprises three exons. In some embodiments, the humanized OSM gene comprises humanized exon 1, human exon 2, and / or human exon 3. In some implementations, the humanized OSM gene comprises two introns. In some implementations, the humanized OSM gene comprises human intron 1 and human intron 2. In some embodiments, the humanized OSM gene comprises a human or humanized 5'UTR. In some implementations, the humanized OSM gene comprises a human or humanized 3'UTR. In some embodiments, the humanized OSM gene comprises an endogenous 5'UTR. In some embodiments, the humanized OSM gene comprises an endogenous 3'UTR.

[0162] In some embodiments, the genetically modified non-human animal can express human OSM and / or chimeric (e.g., humanized) OSM protein, wherein the endogenous OSM gene sequence is replaced by a human OSM gene and / or nucleotide sequence. Furthermore, the human OSM gene and / or nucleotide sequence encodes an amino acid sequence of human OSM that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of human OSM set forth in SEQ ID NO: 2. In some embodiments, the nucleotide sequence of the endogenous non-human animal's OSM gene is replaced in whole or in part by a nucleotide sequence encoding a mature human OSM protein.

[0163] In some embodiments, the genetically modified non-human animal expresses human OSM and / or chimeric OSM proteins (e.g., humanized OSM) under endogenous promoters and / or regulatory elements. Replacement of the endogenous locus provides a non-human animal that expresses a human or chimeric OSM protein (e.g., humanized OSM) in the same cell type. The genetically modified mice do not exhibit potential diseases observed in certain other transgenic mice known in the art. The human OSM or chimeric OSM protein expressed in the non-human animal can maintain the function of one or more wild-type or human OSM proteins, for example, the expressed OSM protein can bind to human or non-human OSM proteins. Furthermore, in some embodiments, the genetically modified non-human animal does not express endogenous OSM proteins. In some embodiments, the genetically modified non-human animal has reduced expression of endogenous OSM proteins. As used herein, "endogenous OSM protein" refers to an OSM protein encoded by an endogenous OSM nucleotide sequence of the non-human animal (e.g., mouse) prior to genetic modification.

[0164] The genome of the non-human animal comprises a nucleotide sequence encoding an amino acid that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of human OSM protein (NP_065391.1; SEQ ID NO: 2). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or at least 100% identical to the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8.

[0165] The nucleotide sequence encoding the endogenous OSM region in the non-human animal genome is replaced with a nucleotide sequence encoding the corresponding human OSM region. In some embodiments, the nucleotide sequence encoding the endogenous OSM region is any sequence within the endogenous OSM locus, such as exon 1, exon 2, exon 3, 5' UTR, 3' UTR, intron 1, intron 2, or any combination thereof. In some embodiments, the nucleotide sequence encoding the endogenous OSM region is located within an endogenous OSM regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous OSM region is exon 1, exon 2, and / or exon 3, or portions thereof.

[0166] One or more cells of the genetically modified non-human animal express a human or chimeric OSM protein (e.g., a humanized OSM protein). In some embodiments, the human or chimeric OSM protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 220, 240, 250, 251, or 252 consecutive amino acids of the amino acid sequence of SEQ ID NO: 2.

[0167] In some embodiments, the genome of the genetically modified non-human animal comprises all or part of exon 1, exon 2, and / or exon 3 of the human OSM gene, or all or part of the nucleotide sequence shown in SEQ ID NO: 7.

[0168] In some embodiments, the genetically modified non-human animal genome comprises a portion of exon 1, all of exon 2, and a portion of exon 3 of the human OSM gene. In some embodiments, the portion of exon 1 comprises at least 5, 10, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 85, or 86 bp of contiguous nucleotide sequence of exon 1 of the human OSM gene. In some embodiments, the portion of exon 1 comprises 34 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 1 comprises at least 15 bp of nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 550, 560, 570, 580, 581, 582, 583, 585, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1635, or 1636 bp of contiguous nucleotide sequence of human OSM gene exon 3. In some implementations, the portion of exon 3 comprises 582 bp of contiguous nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 200 bp of nucleotides. A portion of exon 1, all of exon 2, and a portion of exon 3 of the human OSM gene include at least 100-500 bp, 500-700 bp, or 700-800 bp of contiguous nucleotide sequence. In some embodiments, the nucleotide sequence encoding the corresponding region of human OSM is located at nucleotides 53-811 of the human OSM gene transcript NM_020530.6.

[0169] In some embodiments, the OSM gene of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.

[0170] In some embodiments, the humanized OSM genome comprises a 5'UTR of the human OSM gene. In some embodiments, the humanized OSM genome comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanized OSM genome comprises a 3'UTR of the human OSM gene. In some embodiments, the humanized OSM genome comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, based on the similarity of the 5' flanking sequences, it is reasonable to infer that the mouse and human OSM genes are similarly regulated. As described herein, humanized OSM mice comprise a replacement of an endogenous mouse locus that retains the mouse endogenous regulatory elements but contains a humanized OSM coding sequence. OSM expression in genetically modified heterozygous or homozygous mice is completely normal.

[0171] In another aspect, the present invention provides a genetically modified non-human animal, wherein the genome of the non-human animal comprises a deletion of an endogenous OSM gene, wherein the deletion of the endogenous OSM gene comprises exon 1, exon 2, and / or exon 3, or a portion of the endogenous OSM locus.

[0172] In some embodiments, the deletion of the endogenous OSM gene comprises one or more exons or portions of exons selected from exon 1, exon 2, and / or exon 3.

[0173] In some embodiments, the endogenous OSM gene deletion further comprises one or more introns or portions of introns, wherein the introns are selected from intron 1 and / or intron 2 of the OSM gene.

[0174] In some embodiments, the deletion comprises 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, 650, 700, 750, 760, 770, 780, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825 2, 793, 795, 800, 850, 900, 950, 1000, 1300, 1350, 1400, 1450, 1500, 1600, 1800, 2000, 2300, 2400, 2500, 2600, 2700, 2750, 2766, 2767, 3000, 3100, 3125, 3126, 3127, 4000, 4100, 4200 or 4242 bp of consecutive nucleotide sequence or more.

[0175] In some embodiments, the deletion of the endogenous OSM gene comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, or 800 consecutive nucleotide sequences or more of exon 1, exon 2, and / or exon 3 (e.g., a deletion of at least 15 bp of consecutive nucleotide sequences of exon 1, all of exon 2, and at least 200 nucleotide sequences of exon 3).

[0176] The present invention provides a humanized mouse OSM DNA sequence, a construct expressing the amino acid sequence of humanized OSM protein, a cell comprising the construct, and a tissue comprising the cell.

[0177] Therefore, in some embodiments, the present invention provides a chimeric (e.g., humanized) OSM nucleotide sequence and / or amino acid sequence, wherein in some embodiments, the chimeric nucleotide sequence is fused to mouse endogenous OSM mRNA (e.g., NM_001013365.2), mouse OSM amino acid sequence (e.g., NP_001013383.1, SEQ ID NO: 1), or a portion thereof (e.g., 5' UTR, a portion of exon 1, a portion of exon 3, and the 3' UTR) having 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%. In some embodiments, the chimeric nucleotide sequence has 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% sequence identity to a human OSM mRNA sequence (e.g., NM_020530.6), a human OSM amino acid sequence (e.g., NP_065391.1, SEQ ID NO: 2), or a portion thereof (e.g., a portion of exon 1, a portion of exon 2, and a portion of exon 3).

[0178] In some embodiments, the nucleotide sequence encoding amino acids 1-263 of mouse OSM (SEQ ID NO: 1) is replaced with the corresponding nucleotide sequence encoding amino acids 1-252 of human OSM (SEQ ID NO: 2).

[0179] In some embodiments, the nucleotide sequence is operably linked to a promoter or regulatory element, such as an endogenous mouse OSM promoter, an inducible promoter, an enhancer, and / or a mouse or human regulatory element.

[0180] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein differs from all or a portion of a mouse OSM nucleotide sequence (e.g., a portion of exon 1, exon 2, and a portion of exon 3 of mouse OSM gene transcript NM_001013365.2).

[0181] In some embodiments, at least a portion (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) of the chimeric nucleic acid sequence is identical to all or a portion of a mouse OSM nucleotide sequence (e.g., a portion of exon 1 and a portion of exon 3 of mouse OSM gene transcript NM_001013365.2).

[0182] In some embodiments, at least a portion (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) of the chimeric nucleic acid sequence differs from all or a portion of a human OSM nucleotide sequence (e.g., a portion of exon 1 and a portion of exon 3 of the human OSM gene transcript NM_020530.6).

[0183] In some embodiments, at least a portion (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) of the chimeric nucleic acid sequence is identical to all or part of a human OSM nucleotide sequence (e.g., a portion of exon 1, exon 2, and a portion of exon 3 of the human OSM gene transcript NM_020530.6).

[0184] In some embodiments, at least a portion (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) of the amino acid sequence encoded by the chimeric nucleic acid sequence differs from all or a portion of the mouse OSM protein amino acid sequence (e.g., amino acids 1-263 of mouse OSM protein sequence NP_001013383.1 (SEQ ID NO: 1)).

[0185] In some embodiments, 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) is identical to all or part of the amino acid sequence of mouse OSM protein (e.g., amino acids 1-263 of mouse OSM protein sequence NP_001013383.1 (SEQ ID NO: 1)).

[0186] In some embodiments, at least a portion (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) of the amino acid sequence differs from all or a portion of the amino acid sequence of human OSM protein (e.g., amino acids 1-252 of human OSM protein sequence NP_065391.1 (SEQ ID NO: 2)).

[0187] In some embodiments, 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) is identical to all or part of the amino acid sequence of human OSM protein (e.g., amino acids 1-252 of human OSM protein sequence NP_065391.1 (SEQ ID NO: 2)).

[0188] The present invention also provides a humanized OSM amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0189] A) the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0190] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 1 or 2;

[0191] C) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is capable of hybridizing under low stringency conditions or high stringency conditions to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 1 or 2;

[0192] D) differs from the amino acid sequence of SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0193] E) an amino acid sequence as shown in SEQ ID NO: 1 or 2, comprising substitution, deletion and / or insertion of one or more amino acid residues.

[0194] The present invention also provides a humanized OSM amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0195] A) the amino acid sequence shown in positions 1-252 or 26-252 of SEQ ID NO: 2;

[0196] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence as set forth in positions 1-252 or 26-252 of SEQ ID NO: 2;

[0197] C) differs from the amino acid sequence of SEQ ID NO: 2 at positions 1-252 or 26-252 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0198] D) an amino acid sequence comprising substitutions, deletions and / or insertions of one or more amino acid residues as shown in positions 1-252 or 26-252 of SEQ ID NO: 2.

[0199] The present invention also provides a humanized OSM nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

[0200] A) the nucleic acid sequence shown in SEQ ID NO: 7 and 8 or a nucleic acid sequence encoding a humanized mouse OSM homologous amino acid sequence;

[0201] B) a nucleic acid sequence capable of hybridizing to the nucleotide sequences shown in SEQ ID NOs: 7 and 8 under low stringency conditions or stringent conditions;

[0202] C) a nucleic acid sequence that is identical to, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 90% homology to, the nucleotide sequences set forth in SEQ ID NOs: 7 and 8;

[0203] D) the amino acid sequence it encodes is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 2;

[0204] E) the encoded amino acid sequence differs from the amino acid sequence of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0205] F) The encoded amino acid sequence is the same as that shown in SEQ ID NO: 2, including the amino acid sequence in which one or more amino acid residues are substituted, deleted and / or inserted.

[0206] The present invention further provides a humanized mouse OSM genomic DNA sequence, which is obtained by reverse transcription of mRNA transcribed from the OSM genomic DNA sequence and is identical to or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 7 or 8.

[0207] IL31RA

[0208] In the human genome, the IL31RA gene (Gene ID: 133396) contains 18 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and exon 18 ( FIG29 ). The human IL31RA protein comprises a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region from the N-terminus to the C-terminus. The nucleotide sequence of human IL31RA mRNA is NM_001242638.1, and the amino acid sequence of human IL31RA is NP_001229567.1 (SEQ ID NO: 61). Based on the nucleotide and amino acid sequences of transcript NM_001242638.1 and its encoded protein NP_001229567.1, the corresponding positions of each exon and each region are as follows:

[0209] Table 5

[0210] The human IL31RA gene (NCBI Gene ID: 133396) is located on chromosome 5 at positions 55840334 to 55922854 of NC_000005.10, based on transcript NM_001242638.1. Among them, the 5'UTR is located at position 55853314 to 55853561, exon 1 is located at position 55853314 to 55853567, intron 1 is located at position 55853568 to 55859508, exon 2 is located at position 55859509 to 55859599, intron 2 is located at position 55859600 to 55868790, exon 3 is located at position 55868791 to 55868908, intron 3 is located at position 55868909 to 55872269, exon 4 is located at position 55872270 to 55872451, and intron 4 is located at position 55872271 to 55872452. 55872452 to 55883043, exon 5 is located at position 55883044 to 55883195, intron 5 is located at position 55883196 to 55889969, exon 6 is located at position 55889970 to 55890135, intron 6 is located at position 55890136 to 55896349, exon 7 is located at position 55896350 to 55896429, intron 7 is located at position 55896430 to 55899915, exon 8 is located at position 55899916 to 55900132, intron 8 is located at position 55900133 to 55900134. 906105, exon 9 is located at positions 55906106 to 55906288, intron 9 is located at positions 55906289 to 55907358, exon 10 is located at positions 55907359 to 55907460, intron 10 is located at positions 55907461 to 55908264, exon 11 is located at positions 55908265 to 55908411, intron 11 is located at positions 55908412 to 55910531, exon 12 is located at positions 55910532 to 55910672, intron 12 is located at positions 55910673 to 5591347 6, exon 13 is located at position 55913477 to 55913570, intron 13 is located at position 55913571 to 55914846, exon 14 is located at position 55914847 to 55914928, intron 14 is located at position 55914929 to 55916643, exon 15 is located at position 55916644 to 55916834, intron 15 is located at position 55916835 to 55922395, exon 16 is located at position 55922396 to 55922853, and 3'UTR is located at position 55922433 to 55922853.All relevant information on the human IL31RA locus can be retrieved from the NCBI website (GeneID: 133396), the entire contents of which are incorporated herein by reference.

[0211] In the mouse genome, the IL31RA gene (Gene ID: 218624) contains 17 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and exon 17 ( FIG29 ). The mouse IL31RA protein comprises a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region from the N-terminus to the C-terminus. The nucleotide sequence of mouse OSMR mRNA is NM_139299.2, and the amino acid sequence of mouse IL31RA is NP_647460.2 (SEQ ID NO: 60). Based on the nucleotide and amino acid sequences of transcript NM_139299.2 and its encoded protein NP_647460.2, the corresponding positions of each exon and each region are as follows:

[0212] Table 6

[0213] The mouse IL31RA gene (NCBI Gene ID: 218624) is located on chromosome 13 at positions 112649439 to 112717266 of NC_000079.7, based on transcript NM_139299.2. Among them, the 5'UTR is located at positions 112717196 to 112717172, 112702435 to 112702272, 112698591 to 112698384, and 112695760 to 112695737, exon 1 is located at positions 112717196 to 112717172, intron 1 is located at positions 112717171 to 112702436, exon 2 is located at positions 112702435 to 112702272, intron 2 is located at positions 112698591 to 112698384, and exon 3 is located at positions 112695760 to 112695737. 91 to 112698384, intron 3 is located at positions 112698383 to 112695761, exon 4 is located at positions 112695760 to 112695682, intron 4 is located at positions 112695681 to 112688521, exon 5 is located at positions 112688520 to 112688403, intron 5 is located at positions 112688402 to 112686115, exon 6 is located at positions 112686114 to 112685951, intron 6 is located at positions 112685950 to 112684034, and exon 7 is located at positions 112684 033 to 112683891, intron 7 is located at positions 112683890 to 112682868, exon 8 is located at positions 112682867 to 112682714, intron 8 is located at positions 112682713 to 112680501, exon 9 is located at positions 112680500 to 112680421, intron 9 is located at positions 112680420 to 112677821, exon 10 is located at positions 112677820 to 112677604, intron 10 is located at positions 112677603 to 112670456, exon 11 is located at positions 1 12670455 to 112670270, intron 11 is located at position 112670269 to 112669547, exon 12 is located at position 112669546 to 112669445, intron 12 is located at position 112669444 to 112668349, exon 13 is located at position 112668348 to 112668202, intron 13 is located at position 112668201 to 112666954, exon 14 is located at position 112666953 to 112666813, intron 14 is located at position 112666812 to 112664088,Exon 15 is located at positions 112664087 to 112663994, intron 15 is located at positions 112663993 to 112662450, exon 16 is located at positions 112662449 to 112662368, intron 16 is located at positions 112662367 to 112660905, exon 17 is located at positions 112660904 to 112659329, and the 3'UTR is located at positions 112660436 to 112659329. All relevant information about the mouse IL31RA locus can be retrieved on the NCBI website (Gene ID: 218624). The entire content is incorporated herein by reference.

[0214] Figure 47 shows an alignment of the human IL31RA amino acid sequence (NP_001229567.1; SEQ ID NO: 61) and the mouse IL31RA amino acid sequence (NP_647460.2; SEQ ID NO: 60). Therefore, the corresponding amino acid residues or regions between human and mouse IL31RA can be found in Figure 47.

[0215] IL31RA genes, proteins, and gene loci from other species are also known in the art. For example, Rattus norvegicus (rat) IL31RA has Gene ID: 688622, Macaca mulatta (rhesus monkey) IL31RA has Gene ID: 709719, Canis lupus familiaris (dog) IL31RA has Gene ID: 487212, and Sus scrofa (pig) IL31RA has Gene ID: 100522044. Relevant information (e.g., intron sequences, exon sequences, and amino acid sequences) for these genes can be found at NCBI, the entire contents of which are incorporated herein by reference.

[0216] Figure 48 shows an alignment of the human IL31RA amino acid sequence (NP_001229567.1; SEQ ID NO: 61) and the rat IL31RA amino acid sequence (NP_001244207.1; SEQ ID NO: 117). Therefore, corresponding amino acid residues or regions between human and rat IL31RA can be found in Figure 48.

[0217] The present invention provides a human or chimeric (e.g., humanized) IL31RA nucleotide sequence or amino acid sequence. In some embodiments, the entire nucleotide sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of the mouse IL31RA gene is replaced by the corresponding nucleotide sequence of the human IL31RA gene. In some embodiments, "portions" of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of the mouse IL31RA gene are replaced by corresponding sequences of the human IL31RA gene. The term "portion" 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, 650, 700, 750, 800, 850, 900, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 930, 940, 950, 1000, 1100, 1200, 1 470, 480, 490, 491, 492, 493, 494, 495, 500, 600, 700, 710, 715 or 716 amino acids. In some embodiments, the "portion" is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100% identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, a signal peptide, an extracellular region, a transmembrane region, or a cytoplasmic region.In some embodiments, "partial" or "full" sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and / or exon 17 of the mouse IL31RA gene (e.g., part of exon 4, all of exons 5-13, and part of exon 14) is expressed. "Partial" or "full" sequence replacement of the corresponding exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18 of the human IL31RA gene (e.g., part of exon 3, all of exons 3-13 and part of exon 14).

[0218] In some embodiments, a “portion” of the signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, and / or exon 17 of endogenous IL31RA is deleted.

[0219] In some embodiments, the present invention provides a genetically modified non-human animal, wherein the genome of the non-human animal comprises a chimeric IL31RA nucleotide sequence. In some embodiments, the chimeric IL31RA nucleotide sequence encodes a protein comprising a human or humanized signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the encoded protein is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 60, 61, or 68. In some embodiments, the genome of the non-human animal comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of SEQ ID NO: 62, 63, 64, 65, 66, 67, 69, or 70.

[0220] In some embodiments, the non-human animals described herein comprise a nucleotide sequence encoding a human or humanized IL31RA protein. In some embodiments, the human or humanized IL31RA protein comprises five homology domains of the human IL31RA protein, wherein the homology domains comprise DI, DII, DIII, DIV, and DV.

[0221] In some embodiments, the non-human animals described herein comprise a sequence encoding a human or humanized IL31RA protein. In some embodiments, the IL31RA protein comprises, from the N-terminus to the C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the humanized IL31RA protein comprises a human or humanized signal peptide. For example, the human or humanized IL31RA signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 1-32 of SEQ ID NO: 61. In some embodiments, the humanized IL31RA protein comprises an endogenous signal peptide. For example, the endogenous IL31RA signal peptide comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 1-18 of SEQ ID NO: 60. In some embodiments, the humanized IL31RA protein comprises a human or humanized extracellular region. For example, the humanized extracellular region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 33-520 of SEQ ID NO: 61, and further comprises at least 1, 2, 3, 4, or 5 amino acids C-terminal to the endogenous extracellular region, such as amino acids 495-499 of SEQ ID NO: 60. In some embodiments, the humanized IL31RA protein comprises an endogenous extracellular region. In some embodiments, the humanized IL31RA protein comprises a human or humanized transmembrane region. In some embodiments, the humanized IL31RA protein comprises an endogenous transmembrane region. For example, the endogenous IL31RA transmembrane region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 500-520 of SEQ ID NO: 60. In some embodiments, the humanized IL31RA protein comprises a human or humanized cytoplasmic region. In some embodiments, the humanized IL31RA protein comprises an endogenous cytoplasmic region. For example, the endogenous IL31RA cytoplasmic region comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to amino acids 521-716 of SEQ ID NO: 60. In some embodiments, the humanized IL31RA protein comprises an endogenous IL31RA sequence of amino acids 495-716 of SEQ ID NO: 60. In some embodiments, the humanized IL31RA protein comprises a human IL31RA sequence of amino acids 1-520 of SEQ ID NO: 61.

[0222] In some embodiments, the non-human animals described herein comprise a human or humanized IL31RA gene. In some embodiments, the humanized IL31RA gene comprises, from 5' to 3', endogenous exon 1, endogenous exon 2, endogenous exon 3, a portion of endogenous exon 4 (e.g., nucleotide sequence 398-421 of NM_139299.2), a portion of human exon 3 (e.g., nucleotide sequence 614-619 of NM_001242638.1), human exons 4-13, a portion of human exon 14 (e.g., nucleotide sequence 2058-2173 of NM_001242638.1), a portion of endogenous exon 14 (e.g., nucleotide sequence 1904-1928 of NM_139299.2), endogenous exons 15-16, and a portion of endogenous exon 17 (e.g., nucleotide sequence 2105-2572 of NM_139299.2). In some embodiments, humanized IL31RA comprises 17 exons. In some embodiments, humanized IL31RA comprises endogenous exon 1, endogenous exon 2, endogenous exon 3, humanized exon 4, human exon 5, human exon 6, human exon 7, human exon 8, human exon 9, human exon 10, human exon 11, human exon 12, human exon 13, humanized exon 14, endogenous exon 15, endogenous exon 16, and / or endogenous exon 17. In some embodiments, humanized IL31RA further comprises endogenous exon 4, endogenous exon 5, endogenous exon 6, endogenous exon 7, endogenous exon 8, endogenous exon 9, endogenous exon 10, endogenous exon 11, endogenous exon 12, endogenous exon 13, and / or endogenous exon 14. In some embodiments, the humanized IL31RA gene does not contain introns. In some embodiments, the humanized IL31RA gene further comprises one or more auxiliary sequences (e.g., a WPRE sequence and / or a polyA sequence). In some embodiments, the humanized IL31RA gene further comprises a human or humanized 5'UTR. In some embodiments, the humanized IL31RA gene comprises a human or humanized 3'UTR. In some embodiments, the humanized IL31RA gene comprises an endogenous 5'UTR. In some embodiments, the humanized IL31RA gene comprises an endogenous 3'UTR.

[0223] In some embodiments, the present invention also provides chimeric (e.g., humanized) IL31RA nucleotide sequences and / or amino acid sequences, in which, in some embodiments, 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% sequence identity to a mouse IL31RA mRNA sequence (e.g., NM_139299.2), a mouse IL31RA amino acid sequence (e.g., SEQ ID NO: 60), or a portion thereof (e.g., exons 1-3, a portion of exon 4, and / or a portion of exon 14, exons 15-16, and a portion of exon 17). In some embodiments, 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% of the sequence is identical to the human IL31RA mRNA sequence (e.g., NM_001242638.1), the amino acid sequence of human IL31RA (e.g., SEQ ID NO: 61), or a portion thereof (e.g., a portion of exon 3, exons 4-13, and a portion of exon 14).

[0224] In some embodiments, the amino acid sequence encoding mouse IL31RA positions 1-494 (SEQ ID NO: 60) is replaced. In some embodiments, the sequence is replaced with a sequence encoding the corresponding region of human IL31RA (e.g., amino acid sequence encoding human IL31RA positions 1-520 (SEQ ID NO: 61).

[0225] In some embodiments, the nucleic acids described herein are operably linked to a promoter or regulatory element, such as an endogenous mouse IL31RA promoter, an inducible promoter, an enhancer, and / or mouse or human regulatory elements.

[0226] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein differs from all or a portion of a mouse IL31RA nucleotide sequence (e.g., a portion of exon 4, exons 5-13, and a portion of exon 14 of the mouse IL31RA gene transcript NM_139299.2).

[0227] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein is identical to all or a portion of a mouse IL31RA nucleotide sequence (e.g., exons 1-3, a portion of exon 4, a portion of exon 14, exons 15-16, and a portion of exon 17 of mouse IL31RA gene transcript NM_139299.2).

[0228] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein differs from all or a portion of a human IL31RA nucleotide sequence (e.g., exons 1-2, a portion of exon 3, a portion of exon 14, and exons 15-18) of the human IL31RA gene transcript NM_001242638.1.

[0229] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein is identical to all or a portion of a human IL31RA nucleotide sequence (e.g., a portion of exon 3, exons 4-13, and a portion of exon 14 of the human IL31RA gene transcript NM_001242638.1).

[0230] In some embodiments, the chimeric nucleic acid sequences described herein encode at least a portion (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) of amino acids that differ from all or a portion of the amino acid sequence of a mouse IL31RA protein (e.g., amino acids 1-494 of mouse IL31RA protein sequence NP_647460.2 (SEQ ID NO: 60)).

[0231] In some embodiments, at least a portion (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) of an amino acid sequence described herein is identical to all or a portion of a mouse IL31RA protein amino acid sequence (e.g., amino acids 495-716 of mouse IL31RA protein sequence NP_647460.2 (SEQ ID NO: 60)).

[0232] In some embodiments, at least a portion (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) of an amino acid sequence described herein differs from all or a portion of an amino acid sequence of a human IL31RA protein (e.g., amino acids 521-662 of the human IL31RA protein sequence NP_001229567.1 (SEQ ID NO: 61)).

[0233] In some embodiments, at least a portion (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) of an amino acid sequence described herein is identical to all or a portion of a human IL31RA protein amino acid sequence (e.g., amino acids 1-520 of human IL31RA protein sequence NP_001229567.1 (SEQ ID NO: 61)).

[0234] The present invention also provides a humanized IL31RA mouse amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0235] A) the amino acid sequence set forth in SEQ ID NO: 60, 61, or 68;

[0236] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 60, 61 or 68;

[0237] C) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is capable of hybridizing under low stringency conditions or high stringency conditions to a nucleotide sequence encoding an amino acid as set forth in SEQ ID NO: 60, 61 or 68;

[0238] D) differs from the amino acid sequence of SEQ ID NO: 60, 61 or 68 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0239] E) an amino acid sequence as shown in SEQ ID NO: 60, 61 or 68, comprising substitution, deletion and / or insertion of one or more amino acid residues.

[0240] The present invention also provides a humanized IL31RA amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0241] A) the amino acid sequence shown in positions 1-520 of SEQ ID NO: 61;

[0242] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:61 as shown in positions 1-520;

[0243] C) differs from the amino acid sequence of SEQ ID NO: 61 at positions 1-520 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0244] D) an amino acid sequence comprising substitutions, deletions and / or insertions of one or more amino acid residues as shown in positions 1 to 520 of SEQ ID NO: 61.

[0245] The present invention also provides a humanized IL31RA amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0246] A) the amino acid sequence shown in positions 495-716 of SEQ ID NO: 60;

[0247] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 60, positions 495-716;

[0248] C) differs from the amino acid sequence of SEQ ID NO: 60 at positions 495-716 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0249] D) an amino acid sequence comprising substitutions, deletions and / or insertions of one or more amino acid residues as shown in positions 495-716 of SEQ ID NO: 60.

[0250] The present invention also provides a humanized IL31RA nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

[0251] A) a nucleic acid sequence as shown in SEQ ID NO: 62, 63, 64, 65, 66, 67, 69 or 70, or a nucleic acid sequence encoding a humanized mouse IL31RA homologous amino acid sequence;

[0252] B) a nucleic acid sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO: 62, 63, 64, 65, 66, 67, 69 or 70 under low stringency conditions or stringent conditions;

[0253] C) a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence of SEQ ID NO: 62, 63, 64, 65, 66, 67, 69 or 70;

[0254] D) the amino acid sequence it encodes is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 60, 61 or 68;

[0255] E) encodes an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 60, 61, or 68 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid; or

[0256] F) The amino acid sequence encoded by the amino acid sequence is the same as that shown in SEQ ID NO: 60, 61 or 68, including substitution, deletion and / or insertion of one or more amino acid residues.

[0257] The present invention further provides a humanized mouse IL31RA genomic DNA sequence, which is obtained by reverse transcription of the mRNA transcribed therefrom and is identical to or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 64, 65, 66, or 67.

[0258] IL31

[0259] In the human genome, the IL31 gene (Gene ID: 386653) contains three exons: exon 1, exon 2, and exon 3 ( Figure 22 ). The nucleotide sequence of human IL31 mRNA is NM_001014336.1, and the amino acid sequence of human IL31 is NP_001014358.1 (SEQ ID NO: 53). Based on the nucleotide and amino acid sequences of transcript NM_001014336.1 and its encoded protein NP_001014358.1, the corresponding positions of each exon are as follows:

[0260] Table 7

[0261] The human IL31 gene (NCBI Gene ID: 386653) is located on chromosome 12 at positions 122172029 to 122174221 of NC_000012.12, based on transcript NM_001014336.1. Among them, the 5'UTR is located at positions 122174221 to 122174173, exon 1 is located at positions 122174221 to 122174157, intron 1 is located at positions 122174156 to 122173993, exon 2 is located at positions 122173992 to 122173844, intron 2 is located at positions 122173843 to 122172742, exon 3 is located at positions 122172741 to 122172029, and the 3'UTR is located at positions 122172411 to 122172029. All relevant information about the human IL31 locus can be retrieved on the NCBI website (Gene ID: 386653). The entire content of which is incorporated herein by reference.

[0262] In the mouse genome, the IL31 gene (Gene ID: 76399) contains three exons: exon 1, exon 2, and exon 3 ( Figure 22 ). The nucleotide sequence of mouse IL31 mRNA is NM_029594.1, and the amino acid sequence of mouse IL31 is NP_083870.1 (SEQ ID NO: 52). Based on the nucleotide and amino acid sequences of the transcript NM_029594.1 and its encoded protein NP_083870.1, the corresponding positions of each exon and region are as follows:

[0263] Table 8

[0264] The mouse IL31 gene (NCBI Gene ID: 76399) is located on chromosome 5 at positions 123618215 to 123620686 of NC_000071.7, based on transcript NM_029594.1. The 5' UTR is located at positions 123620164 to 123620113, exon 1 is located at positions 123620164 to 123620097, intron 1 is located at positions 123620096 to 123619917, exon 2 is located at positions 123619916 to 123619765, intron 2 is located at positions 123619764 to 123618806, exon 3 is located at positions 123618805 to 123618220, and the 3' UTR is located at positions 123618481 to 123618220. All relevant information regarding the murine IL31 locus can be retrieved on the NCBI website (Gene ID: 76399), the entire contents of which are incorporated herein by reference.

[0265] Figure 49 shows an alignment of the human IL31 amino acid sequence (NP_001014358.1; SEQ ID NO: 53) and the mouse IL31 amino acid sequence (NP_083870.1; SEQ ID NO: 52). Thus, corresponding amino acid residues or regions between human and mouse IL31 can be found in Figure 49.

[0266] IL31 genes, proteins, and gene loci from other species are also known in the art. For example, Rattus norvegicus (rat) IL31 has Gene ID: 690028, Macaca mulatta (rhesus monkey) IL31 has Gene ID: 701655, Canis lupus familiaris (dog) IL31 has Gene ID: 100302725, and Sus scrofa (pig) IL31 has Gene ID: 102167534. Relevant information (e.g., intron sequences, exon sequences, and amino acid sequences) for these genes can be found at NCBI, the entire contents of which are incorporated herein by reference.

[0267] Figure 50 shows the amino acid sequence of human IL31 (NP_001014358.1; SEQ ID NO: 53) and rat IL31 (XP_017454003.1; SEQ ID NO: 118). Therefore, the corresponding amino acid residues or regions between human and rat IL31 can be retrieved in Figure 50 .

[0268] The present invention provides a human or chimeric (e.g., humanized) IL31 nucleotide sequence and / or amino acid sequence. In some embodiments, the entire nucleotide sequence of exon 1, exon 2, exon 3, and / or signal peptide of the mouse IL31 gene is replaced with the corresponding nucleotide sequence of the human IL31 gene. In some embodiments, "partial" exon 1, exon 2, exon 3, and / or signal peptide of the mouse IL31 gene is replaced with the corresponding nucleotide sequence or amino acid sequence of the human IL31 gene. The term "portion" 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, 491, 492, 493, 500, 550, 600, 650, 700, 750, 760, 770, 780, 790, 800 or 810 bp of continuous nucleotide sequence, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 163 continuous amino acid sequence. In some embodiments, the "portion" is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100% identical to exon 1, exon 2, exon 3, or the signal peptide. In some embodiments, a "portion" or "entire" sequence of exon 1, exon 2, and / or exon 3 of the mouse IL31 gene (e.g., a portion of exon 1, all of exon 2, and a portion of exon 3) is replaced with a "portion" or "entire" sequence of the corresponding exon 1, exon 2, and / or exon 3 of the human IL31 gene (e.g., a portion of exon 1, all of exon 2, and a portion of exon 3).

[0269] In some embodiments, "portions" of the signal peptide, exon 1, exon 2, and / or exon 3 are deleted.

[0270] In some embodiments, the present invention provides a genetically modified non-human animal, wherein the genome of the non-human animal comprises a human, chimeric, or humanized IL31 nucleotide sequence. In some embodiments, the protein encoded by the human, chimeric, or humanized IL31 nucleotide sequence is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the genome of the non-human animal comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of SEQ ID NO: 54, 55, 56, 57, 58, or 59.

[0271] In some embodiments, the non-human animal described herein comprises a human or humanized IL31 protein. In some embodiments, the IL31 protein comprises an N-terminal signal peptide. In some embodiments, the humanized IL31 protein comprises a human or humanized signal peptide. In some embodiments, the humanized IL31 protein comprises an endogenous signal peptide.

[0272] In some embodiments, the non-human animal described herein comprises a human or humanized IL31 gene. In some embodiments, the humanized IL31 gene comprises 3 exons. In some embodiments, the humanized IL31 gene comprises humanized exon 1, human exon 2, and / or human exon 3. In some implementations, the humanized IL31 gene comprises 2 introns. In some implementations, the humanized IL31 gene comprises human intron 1 and human intron 2. In some embodiments, the humanized IL31 gene comprises a human or humanized 5'UTR. In some implementations, the humanized IL31 gene comprises a human or humanized 3'UTR. In some embodiments, the humanized IL31 gene comprises an endogenous 5'UTR. In some embodiments, the humanized IL31 gene comprises an endogenous 3'UTR.

[0273] In some embodiments, the genetically modified non-human animal can express human IL31 and / or chimeric (e.g., humanized) IL31 protein, wherein the endogenous IL31 gene sequence is replaced with a human IL31 gene and / or nucleotide sequence. Furthermore, the human IL31 gene and / or nucleotide sequence encodes an amino acid sequence of human IL31 that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of human IL31 set forth in SEQ ID NO: 53. In some embodiments, the nucleotide sequence of the endogenous non-human animal's IL31 gene is replaced in whole or in part with a nucleotide sequence encoding a mature human IL31 protein.

[0274] In some embodiments, genetically modified non-human animals express human IL31 and / or chimeric IL31 proteins (e.g., humanized IL31) under endogenous promoters and / or regulatory elements. Replacement of the endogenous locus provides a non-human animal that expresses human or chimeric IL31 proteins (e.g., humanized IL31) in the same type of cells. Genetically modified mice do not show potential diseases observed in certain other transgenic mice known in the art. Human IL31 or chimeric IL31 proteins expressed in non-human animals can maintain the functions of one or more wild-type or human IL31 proteins, for example, the expressed IL31 proteins can be combined with human or non-human IL31 proteins. Further, in some embodiments, genetically modified non-human animals do not express endogenous IL31 proteins. In some embodiments, genetically modified non-human animals have reduced endogenous IL31 protein expression. "Endogenous IL31 protein" described herein refers to the IL31 protein encoded by the endogenous IL31 nucleotide sequence of a non-human animal (e.g., mouse) before genetic modification.

[0275] The genome of the non-human animal comprises a nucleotide sequence encoding an amino acid that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of human IL31 protein (NP_001014358.1; SEQ ID NO: 53). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or at least 100% identical to the nucleotide sequences of SEQ ID NO: 56 and SEQ ID NO: 57.

[0276] The nucleotide sequence encoding the endogenous IL31 region in the non-human animal genome is replaced by the nucleotide sequence encoding the corresponding region of human IL31. In some embodiments, the nucleotide sequence encoding the endogenous IL31 region is any sequence of the endogenous IL31 locus, such as exon 1, exon 2, exon 3, 5'UTR, 3'UTR, intron 1, intron 2, or any combination thereof. In some embodiments, the nucleotide sequence encoding the endogenous IL31 region is located within the endogenous IL31 regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous IL31 region is exon 1, exon 2, and / or exon 3, or a portion thereof.

[0277] One or more cells of the genetically modified non-human animal express a human or chimeric IL31 protein (e.g., a humanized IL31 protein). In some embodiments, the human or chimeric IL31 protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 160, 163, or 164 consecutive amino acids of the amino acid sequence of SEQ ID NO: 53.

[0278] In some embodiments, the genome of the genetically modified non-human animal comprises all or part of exon 1, exon 2, and / or exon 3 of the human IL31 gene, or all or part of the nucleotide sequence shown in SEQ ID NO: 56.

[0279] In some embodiments, the genetically modified non-human animal genome comprises a portion of exon 1, all of exon 2, and a portion of exon 3 of the human IL31 gene. In some embodiments, the portion of exon 1 comprises at least 5, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, 35, 40, or 43 bp of contiguous nucleotide sequence of exon 1 of the human IL31 gene. In some embodiments, the portion of exon 1 comprises a 16 bp contiguous nucleotide sequence. In some embodiments, the portion of exon 1 comprises at least 5 bp of nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 310, 320, 330, 350, 400, 500, 550, 600, 700, 710, or 712 bp of contiguous nucleotide sequence of exon 3 of the human IL31 gene. In some embodiments, the portion of exon 3 comprises a 330 bp continuous nucleotide sequence. In some embodiments, the portion of exon 3 comprises at least 150 bp of nucleotides. The portion of exon 1, all of exon 2, and the portion of exon 3 of the human IL31 gene comprise at least 100-300 bp, 300-400 bp, or 400-500 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence encoding the corresponding region of human IL31 is located at nucleotides 28-522 of human IL31 gene transcript NM_001014336.1.

[0280] In some embodiments, the IL31 gene of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.

[0281] In some embodiments, the humanized IL31 genome comprises the 5'UTR of the human IL31 gene. In some embodiments, the humanized IL31 genome comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanized IL31 genome comprises the 3'UTR of the human IL31 gene. In some embodiments, the humanized IL31 genome comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, based on the similarity of the 5' flanking sequences, it is reasonable to infer that the mouse and human IL31 genes are subject to similar regulation. As described herein, the humanized IL31 mouse comprises a replacement of the endogenous mouse locus, which retains the mouse endogenous regulatory elements but comprises the humanized IL31 coding sequence. The expression of IL31 in the genetically modified heterozygous or homozygous mice is completely normal.

[0282] In another aspect, the present invention provides a genetically modified non-human animal, wherein the genome of the non-human animal comprises a deletion of an endogenous IL31 gene, wherein the deletion of the endogenous IL31 gene comprises exon 1, exon 2, and / or exon 3, or a portion of the endogenous IL31 locus.

[0283] In some embodiments, the deletion of the endogenous IL31 gene comprises one or more exons or portions of exons selected from exon 1, exon 2, and / or exon 3.

[0284] In some embodiments, the endogenous IL31 gene deletion further comprises one or more introns or portions of introns, wherein the introns are selected from intron 1 and / or intron 2 of the IL31 gene.

[0285] In some embodiments, the deletion comprises 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, 1600, 1631, 1700, 1800, 2000, 2300, 2400, 2470 or 2472 bp of consecutive nucleotide sequence or more.

[0286] In some embodiments, the deletion of the endogenous IL31 gene comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400 or 500 bp of contiguous nucleotide sequence or more of exon 1, exon 2 and / or exon 3 (e.g., deletion of at least 5 bp of contiguous nucleotide sequence of exon 1, all of exon 2, and at least 150 nucleotides of exon 3).

[0287] The present invention provides a humanized mouse IL31 DNA sequence, a construct expressing the amino acid sequence of humanized IL31 protein, a cell comprising the construct, and a tissue comprising the cell.

[0288] Therefore, in some embodiments, the present invention provides a chimeric (e.g., humanized) IL31 nucleotide sequence and / or amino acid sequence, wherein in some embodiments, the chimeric nucleotide sequence is fused to mouse endogenous IL31 mRNA (e.g., NM_029594.1), mouse IL31 amino acid sequence (e.g., NP_083870.1, SEQ ID NO: 52), or a portion thereof (e.g., a 5' UTR, a portion of exon 1, a portion of exon 3, and a 3' UTR) having 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%. In some embodiments, the chimeric nucleotide sequence has 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% sequence identity to a human IL31 mRNA sequence (e.g., NM_001014336.1), a human IL31 amino acid sequence (e.g., NP_001014358.1, SEQ ID NO: 53), or a portion thereof (e.g., a portion of exon 1, a portion of exon 2 and a portion of exon 3).

[0289] In some embodiments, the nucleotide sequence encoding amino acids 1-163 of mouse IL31 (SEQ ID NO: 52) is replaced with the corresponding nucleotide sequence encoding amino acids 1-164 of human IL31 (SEQ ID NO: 53).

[0290] In some embodiments, the nucleotide sequence is operably linked to a promoter or regulatory element, such as an endogenous mouse IL31 promoter, an inducible promoter, an enhancer, and / or a mouse or human regulatory element.

[0291] In some embodiments, at least a portion (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) of a chimeric nucleic acid sequence described herein differs from all or a portion of a mouse IL31 nucleotide sequence (e.g., a portion of exon 1, exon 2, and a portion of exon 3 of the mouse IL31 gene transcript NM_029594.1).

[0292] In some embodiments, at least a portion (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) of the chimeric nucleic acid sequence is identical to all or a portion of a mouse IL31 nucleotide sequence (e.g., a portion of exon 1 and a portion of exon 3 of the mouse IL31 gene transcript NM_029594.1).

[0293] In some embodiments, at least a portion (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) of the chimeric nucleic acid sequence differs from all or a portion of the human IL31 nucleotide sequence (e.g., a portion of exon 1 and a portion of exon 3 of the human IL31 gene transcript NM_001014336.1).

[0294] In some embodiments, at least a portion (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) of the chimeric nucleic acid sequence is identical to all or part of a human IL31 nucleotide sequence (e.g., a portion of exon 1, exon 2, and a portion of exon 3 of the human IL31 gene transcript NM_001014336.1).

[0295] In some embodiments, at least a portion of the amino acids encoded by the chimeric nucleic 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, such as contiguous or non-contiguous amino acid residues) differs from all or a portion of the amino acid sequence of the mouse IL31 protein (e.g., amino acids 1-163 of the mouse IL31 protein sequence NP_083870.1 (SEQ ID NO: 52)).

[0296] In some embodiments, 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) is identical to all or part of the amino acid sequence of a mouse IL31 protein (e.g., amino acids 1-163 of mouse IL31 protein sequence NP_083870.1 (SEQ ID NO: 52)).

[0297] In some embodiments, 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) differs from all or a portion of the amino acid sequence of a human IL31 protein (e.g., amino acids 1-164 of the human IL31 protein sequence NP_001014358.1 (SEQ ID NO: 53)).

[0298] In some embodiments, 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) is identical to all or part of the amino acid sequence of a human IL31 protein (e.g., amino acids 1-164 of human IL31 protein sequence NP_001014358.1 (SEQ ID NO: 53)).

[0299] The present invention also provides a humanized IL31 amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0300] A) the amino acid sequence set forth in SEQ ID NO: 52 or 53;

[0301] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 52 or 53;

[0302] C) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is capable of hybridizing under low stringency conditions or high stringency conditions to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 52 or 53;

[0303] D) differs from the amino acid sequence of SEQ ID NO: 52 or 53 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0304] E) an amino acid sequence as shown in SEQ ID NO: 52 or 53, comprising substitution, deletion and / or insertion of one or more amino acid residues.

[0305] The present invention also provides a humanized IL31 amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0306] A) the amino acid sequence shown in positions 1-164 or 24-164 of SEQ ID NO: 53;

[0307] B) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence as set forth in positions 1-164 or 24-164 of SEQ ID NO:53;

[0308] C) differs from the amino acid sequence of SEQ ID NO: 53 at positions 1-164 or 24-164 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0309] D) an amino acid sequence comprising substitutions, deletions and / or insertions of one or more amino acid residues as shown in positions 1-164 or 24-164 of SEQ ID NO: 53.

[0310] The present invention also provides a humanized IL31 nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

[0311] A) the nucleic acid sequence shown in SEQ ID NOs: 56 and 57 or a nucleic acid sequence encoding a humanized mouse IL31 homologous amino acid sequence;

[0312] B) a nucleic acid sequence capable of hybridizing to the nucleotide sequences shown in SEQ ID NOs: 56 and 57 under low stringency conditions or stringent conditions;

[0313] C) a nucleic acid sequence that is identical to, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 90% homologous to, the nucleotide sequences set forth in SEQ ID NOs: 56 and 57;

[0314] D) the amino acid sequence it encodes is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 53;

[0315] E) encodes an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 53 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid sequence; or

[0316] F) The amino acid sequence encoded by the amino acid sequence is the same as that shown in SEQ ID NO: 53, including substitutions, deletions and / or insertions of one or more amino acid residues.

[0317] The present invention further provides a humanized mouse IL31 genomic DNA sequence, which is obtained by reverse transcription of the mRNA transcribed from the genomic DNA sequence and is identical to or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 56 or 57.

[0318] The present invention also provides an amino acid sequence that has at least 90% homology or at least 90% identity to the sequence shown in SEQ ID NO: 1, 2, 9, 10, 16, 52, 53, 60, 61 or 68 and has protein activity. In some embodiments, the homology to the sequence shown in SEQ ID NO: 1, 2, 9, 10, 16, 52, 53, 60, 61 or 68 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%. In some embodiments, the homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80% or 85%.

[0319] The present invention also provides a nucleotide sequence having at least 90% homology or at least 90% identity to the sequence shown in SEQ ID NO: 7, 8, 13, 14, 15, 51, 56, 57, 64, 65, 66, or 67, and encoding a polypeptide having protein activity. In some embodiments, the homology to the sequence shown in SEQ ID NO: 7, 8, 13, 14, 15, 51, 56, 57, 64, 65, 66, or 67 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

[0320] In some embodiments, the polypeptide is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 17, 18, 38, 39, 40, 41, 51, 54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 66, 67, 69, or 70. In some embodiments, the aforementioned percentage of identity is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

[0321] The present invention also provides nucleic acids that are 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 any of the nucleotide sequences described herein. Sequences, and amino acid sequences that are 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% identical to any amino acid sequence described herein. In some embodiments, the present invention relates to nucleotide sequences encoding any polypeptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 5000, 10000, 11000, or 12000 nucleotides. In some embodiments, the amino acid sequence is less than 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, 210, 220, 230, 240, 245, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 amino acid residues.

[0322] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0323] In order to determine the percent identity of two amino acid sequences or two nucleotide sequences, for the purpose of best comparison, sequence is compared (for example, for best comparison, can introduce gap in one or both of the first and second amino acid or nucleotide sequences, and can ignore non-homologous sequences for the purpose of comparison).Then the amino acid residue or the nucleotide on the corresponding amino acid position or nucleotide position are compared.When a position in the first sequence is occupied by the amino acid residue or the nucleotide identical with the corresponding position in the second sequence, the molecule is identical at this position.The percent identity between the two sequences is a function of the quantity of the same positions shared by the sequences, and considering the number of gaps and the length of each gap, this needs to be introduced to realize the best comparison of the two sequences.For example, the determination of the percent identity between the comparison of the sequence and the two sequences can be completed using the Blossum 62 scoring matrix of gap penalty 12, gap extension penalty 4 and frameshift gap penalty 5.

[0324] The percentage ratio (homology percentage) of conserved residues with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine and isoleucine) and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage ratio is higher than the identity percentage ratio.

[0325] The present invention also provides cells, tissues, and animals (e.g., mice) comprising the nucleotide sequences described herein, as well as animals (e.g., mice) that express human or chimeric (e.g., humanized) OSMR from an endogenous non-human OSMR locus, human or chimeric (e.g., humanized) OSM from an endogenous non-human OSM locus, human or chimeric (e.g., humanized) IL31RA from an endogenous non-human IL31RA locus, and human or chimeric (e.g., humanized) IL31 from an endogenous non-human IL31 locus.

[0326] Genetically modified non-human animals

[0327] The "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome of the animal has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells with exogenous DNA can be various cells, for example, endogenous cells, somatic cells, immune cells, T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells, germ cells, blastocysts or endogenous tumor cells. In some embodiments, a genetically modified non-human animal is provided, which comprises a modified endogenous OSMR, OSM, IL31RA and / or IL31 locus, comprising an exogenous sequence (e.g., a human sequence), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. Animals are generally able to pass genetic modifications to offspring through germline transmission.

[0328] The "chimeric gene" or "chimeric nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least one sequence of the gene or nucleic acid is different from the nucleic acid in the wild-type animal. In some embodiments, the chimeric gene or chimeric nucleic acid has at least a portion of the sequence having two or more different species origins, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, the chimeric gene or chimeric nucleic acid refers to a humanized gene or humanized nucleic acid.

[0329] As used herein, a "chimeric protein" or "chimeric polypeptide" refers to a protein or polypeptide in which two or more portions of the polypeptide or protein are derived from different species, or at least one sequence of the protein or polypeptide differs from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of a chimeric protein or chimeric polypeptide is derived from two or more different species, for example, the same (or homologous) proteins from different species. In some embodiments, a chimeric protein or chimeric polypeptide is a humanized protein or polypeptide.

[0330] The "humanized protein" or "humanized polypeptide" of the present invention refers to a protein or polypeptide, wherein at least a portion of the protein or polypeptide is derived from a human protein or polypeptide. In some embodiments, the humanized protein or polypeptide refers to a human protein or polypeptide.

[0331] As used herein, a "humanized nucleic acid" refers to a nucleic acid wherein at least a portion of the nucleic acid is human. In some embodiments, the nucleic acid in the humanized nucleic acid is entirely human. In some embodiments, the humanized nucleic acid refers to humanized exons, which can be human exons or chimeric exons.

[0332] Animals with humanized OSMR loci

[0333] In some embodiments, the chimeric gene or nucleic acid is a humanized OSMR gene or nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human OSMR gene, and at least a portion of the gene or nucleic acid is derived from a non-human OSMR gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding an OSMR protein. The encoded OSMR protein has at least one activity of a human OSMR protein or a non-human animal OSMR protein.

[0334] In some embodiments, the chimeric protein or polypeptide is a humanized OSMR protein or 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 OSMR protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human animal OSMR protein. The humanized OSMR protein or polypeptide is functional or has at least one activity of a human OSMR protein or a non-human animal OSMR protein.

[0335] In some embodiments, the humanized OSMR protein comprises a polypeptide sequence of 5-979 amino acids (continuous or non-continuous) identical to the human OSMR protein. In some embodiments, the polypeptide sequence is 5-979, 10-737, or 10-979 amino acids in length. In some embodiments, the humanized OSMR gene comprises a nucleotide sequence of 20-99568 bp (continuous or non-continuous) identical to the human OSMR gene. In some embodiments, the nucleotide sequence is 20-5265 bp or 20-2211 bp.

[0336] In some embodiments, the OSMR extracellular region is human or humanized. In some embodiments, the OSMR signal peptide is human or humanized. In some embodiments, the OSMR cytoplasmic region is human or humanized. In some embodiments, the OSMR transmembrane region is human or humanized. In some embodiments, both the OSMR extracellular region and the signal peptide are human or humanized. In some embodiments, both the OSMR transmembrane region and the cytoplasmic region are endogenous.

[0337] Genetically modified non-human animals include modifications to an endogenous non-human animal OSMR gene site. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature OSMR protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a mature OSMR protein amino acid sequence). Although the present invention provides cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein, in many embodiments, the genetically modified non-human animal includes modifications to an endogenous OSMR gene site in the animal.

[0338] The genetically modified animal can express a human OSMR and / or a chimeric (e.g., humanized) OSMR at an endogenous mouse locus, wherein the endogenous mouse OSMR gene has been replaced or inserted with a human OSMR gene and / or a nucleotide sequence encoding a region of a human OSMR sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% identical to a human OSMR sequence. In various embodiments, the endogenous non-human animal OSMR locus is modified to include a human full or partial nucleic acid sequence encoding a mature OSMR protein.

[0339] In some embodiments, the genetically modified mouse can express a human OSMR and / or a chimeric OSMR (e.g., a humanized OSMR) under the control of a mouse promoter and / or mouse regulatory elements. Insertion or substitution at the mouse endogenous locus provides a non-human animal that expresses a human OSMR or a chimeric OSMR (e.g., a humanized OSMR) in suitable cells in a manner that does not cause the potential pathology observed in some other transgenic mice known in the art. The human OSMR or chimeric OSMR (e.g., a humanized OSMR) expressed in the animal can maintain one or more functions of a wild-type mouse or human OSMR in the animal. In addition, in some embodiments, the animal does not express an endogenous OSMR. In some embodiments, the animal's endogenous OSMR expression level is reduced compared to the OSMR expression level in a wild-type animal. As used herein, the term "endogenous OSMR" refers to an OSMR protein expressed by an endogenous OSMR nucleotide sequence of a non-human animal (e.g., a mouse) prior to any genetic modification.

[0340] The genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of human OSMR (NP_001310434.1; SEQ ID NO: 10). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 13, 14, 15, or 51. In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to positions 129-2339 of NM_001323505.2.

[0341] The genome of the genetically modified animal can include replacement of the sequence encoding the endogenous OSMR region at the endogenous OSMR locus with the sequence encoding the corresponding region of human OSMR. In some embodiments, the replaced sequence is any sequence of the endogenous OSMR locus, such as exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, 5' UTR, 3' UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, or any combination thereof. In some embodiments, the replaced sequence is within the regulatory region of the endogenous OSMR gene. In some embodiments, the replaced sequence is a portion of exon 2, exons 3-14, and a portion of exon 15 of the endogenous mouse OSMR locus.

[0342] The genetically modified animal can have one or more cells expressing a human or chimeric OSMR (e.g., a humanized OSMR) having, from N-terminus to C-terminus, a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the signal peptide of human OSMR. In some embodiments, the signal peptide of humanized OSMR has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids (e.g., continuous or non-contiguous) that is identical to the signal peptide of human OSMR. In some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the extracellular region of human OSMR. In some embodiments, the extracellular region of a humanized OSMR has a sequence of at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 710, 711, 712, or 713 amino acids (contiguous or non-contiguous) identical to the extracellular region of a human OSMR. The amino acids are identical to at least 1 or 2 amino acids at the C-terminus of the extracellular region of an endogenous OSMR (e.g., a mouse OSMR). In some embodiments, the extracellular region described herein includes a signal peptide. In some embodiments, the extracellular region described herein does not include a signal peptide. Because in many cases, the sequences of human OSMR and non-human OSMR (e.g., mouse OSMR) are different, antibodies that bind to human OSMR do not necessarily have the same affinity or activity against non-human OSMR. Therefore, transgenic animals with human or humanized extracellular regions can be used to better evaluate the effects of anti-human OSMR antibodies in animal models.

[0343] In some embodiments, the transmembrane region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the transmembrane region of human OSMR (e.g., amino acids 741-761 of SEQ ID NO: 10). In some embodiments, the transmembrane region of a humanized OSMR has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids (contiguous or non-contiguous) that are identical to the transmembrane region of an endogenous OSMR. In some embodiments, the cytoplasmic region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the cytoplasmic region of human OSMR (e.g., amino acids 762-979 of SEQ ID NO: 10). In some embodiments, the cytoplasmic region of a humanized OSMR has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 210, 211, 212, or 213 amino acids (contiguous or non-contiguous) identical to the cytoplasmic region of an endogenous OSMR (e.g., mouse OSMR).

[0344] In some embodiments, the entire transmembrane region and the entire cytoplasmic region of a humanized OSMR described herein are derived from endogenous sequences.

[0345] In some embodiments, the genome of the animal comprises: a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, and / or a portion of exon 15 of the human OSMR gene; or a nucleotide sequence encoding amino acids 1-737, 28-737, 1-979, or 28-979 of SEQ ID NO: 10.

[0346] In some embodiments, the genome of the genetically modified animal comprises a portion of exon 2, all of exons 3-14, and a portion of exon 15 of the human OSMR gene. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 72, 73, 75, 80, 85, or 86 nucleotides. In some embodiments, the portion of exon 2 comprises 73 nucleotides. In some embodiments, the portion of exon 2 comprises at least 30 nucleotides. In some embodiments, the portion of exon 15 comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 165, 167, or 168 nucleotides. In some embodiments, the portion of exon 15 comprises 167 nucleotides. In some embodiments, the portion of exon 15 comprises at least 50 bp of nucleotides.

[0347] In some embodiments, the genome of the genetically modified animal comprises a portion of exon 15, all of exons 16-17, and a portion of exon 18 of an endogenous OSMR gene (e.g., mouse OSMR). In some embodiments, the portion of exon 15 comprises at least 1, 2, 3, 4, 5, 6, 10, 20, 50, 100, 110, 120, 130, 140, 150, 160, 165, 166, 167, or 168 nucleotides. In some embodiments, the portion of exon 15 comprises 1 nucleotide. In some embodiments, the portion of exon 15 comprises at least 1 bp of nucleotides. In some embodiments, the portion of exon 18 includes at least 10, 20, 30, 40, 45, 50, 51, 52, 53, 54, 55, 60, 70, 80, 500, 550, 555, 558, 600, 800, 1000, 1500, 2000, 2300, or 2355 nucleotides. In some embodiments, the portion of exon 18 includes 561 nucleotides. In some embodiments, the portion of exon 18 includes at least 200 bp of nucleotides.

[0348] In some embodiments, a non-human animal harbors a nucleotide sequence encoding a chimeric human / non-human OSMR polypeptide at an endogenous OSMR locus, and the animal expresses functional OSMR on the surface of its cells. The human portion of the chimeric human / non-human OSMR polypeptide can comprise an amino acid sequence encoded by a portion of exon 2, exons 3-14, and / or a portion of exon 15 of a human OSMR gene. In some embodiments, the human portion of the chimeric human / non-human OSMR polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 10.

[0349] In some embodiments, the non-human portion of the chimeric human / non-human OSMR polypeptide includes the entire transmembrane region and / or the entire cytoplasmic region of an endogenous non-human OSMR polypeptide.

[0350] In addition, the modified gene in the genome of the modified animal is homozygous or heterozygous for the endogenous replaced locus. In a specific embodiment, the modified OSMR gene in the genome is homozygous or heterozygous for the endogenous replaced locus.

[0351] In some embodiments, the humanized OSMR locus comprises a human 5'UTR. In some embodiments, the humanized OSMR locus comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, it is reasonable to assume that based on the similarity of the 5' flanking sequences of the mouse and human OSMR genes, they appear to be similarly regulated. As shown in the application, humanized OSMR mice comprising insertions or substitutions in the endogenous mouse OSMR locus, which retain mouse regulatory elements but comprise humanization of the OSMR coding sequence, do not exhibit pathology. Both genetically modified mice, heterozygous or homozygous for humanized OSMR, are normal.

[0352] In another aspect, the present invention also provides a genetically modified non-human animal whose genome comprises a disruption of the animal's endogenous OSMR gene, wherein the disruption of the endogenous OSMR gene comprises a deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and / or exon 18, or a partial deletion thereof.

[0353] In some embodiments, disruption of the endogenous OSMR gene further comprises deletion of one or more introns selected from intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, and intron 17.

[0354] In some embodiments, the deletion may comprise the deletion of 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, 300, 400, 500, 600, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 61000, 61386 or more nucleotides.

[0355] In some embodiments, disruption of the endogenous OSMR gene includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and / or 18 of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and / or exon 18. , 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 500, 1000, 1500, 1794, 1800, 2000, 2300, 2327, or 4835 nucleotides.

[0356] Animals with humanized OSM loci

[0357] In some embodiments, the chimeric gene or nucleic acid is a humanized OSM gene or nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human OSM gene, and at least a portion of the gene or nucleic acid is derived from a non-human OSM gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding an OSM protein. The encoded OSM protein exhibits at least one activity of a human OSM protein or a non-human animal OSM protein.

[0358] In some embodiments, the chimeric protein or polypeptide is a humanized OSM protein or 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 OSM protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human animal OSM protein. The humanized OSM protein or polypeptide is functional or possesses at least one activity of a human OSM protein or a non-human animal OSM protein.

[0359] In some embodiments, the humanized OSM protein comprises a polypeptide sequence of 5-252 amino acids (continuous or non-continuous) identical to a human OSM protein. In some embodiments, the polypeptide sequence is 10-252 amino acids in length. In some embodiments, the humanized OSM gene comprises a nucleotide sequence of 20-4023 bp (continuous or non-continuous) identical to a human OSM gene. In some embodiments, the nucleotide sequence is 20-2917 bp, 20-1907 bp, or 20-792 bp.

[0360] Genetically modified non-human animals include modifications to an endogenous non-human animal OSM gene locus. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature OSM protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a mature OSM protein amino acid sequence). While cells (e.g., ES cells, somatic cells) that may include the genetic modifications described herein are provided herein, in many embodiments, the genetically modified non-human animal includes modifications to an endogenous OSM gene locus in the animal.

[0361] The genetically modified animal can express human OSM and / or chimeric (e.g., humanized) OSM at an endogenous mouse locus, wherein the endogenous mouse OSM gene has been replaced or inserted with a human OSM gene and / or a nucleotide sequence encoding a region of a human OSM sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% identical to a human OSM sequence. In various embodiments, the endogenous non-human animal OSM locus is modified to include all or part of a human nucleic acid sequence encoding a mature OSM protein.

[0362] In some embodiments, the genetically modified mouse can express human OSM and / or chimeric OSM (e.g., humanized OSM) under the control of a mouse promoter and / or mouse regulatory elements. Insertion or substitution at the mouse endogenous locus provides a non-human animal that expresses human OSM or chimeric OSM (e.g., humanized OSM) in suitable cells in a manner that does not cause the potential pathology observed in some other transgenic mice known in the art. The human OSM or chimeric OSM (e.g., humanized OSM) expressed in the animal can maintain one or more functions of wild-type mouse or human OSM in the animal. In addition, in some embodiments, the animal does not express endogenous OSM. In some embodiments, the animal's endogenous OSM expression level is reduced compared to the OSM expression level in a wild-type animal. As used herein, the term "endogenous OSM" refers to an OSM protein expressed by an endogenous OSM nucleotide sequence of a non-human animal (e.g., mouse) prior to any genetic modification.

[0363] The genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of human OSM (NP_001013383.1; SEQ ID NO: 2). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7 or 8. In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to positions 99-890 of NM_001013365.2.

[0364] The genome of the genetically modified animal can include replacement of a sequence encoding a region of the endogenous OSM gene locus with a sequence encoding the corresponding region of human OSM. In some embodiments, the replaced sequence is any sequence of the endogenous OSM gene locus, such as exon 1, exon 2, exon 3, 5' UTR, 3' UTR, intron 1, intron 2, or any combination thereof. In some embodiments, the replaced sequence is within the regulatory region of the endogenous OSM gene. In some embodiments, the replaced sequence is a portion of exon 1, exon 2, and a portion of exon 3 of the endogenous mouse OSM gene locus. In some embodiments, a contiguous genomic segment from the ATG start codon of exon 1 to exon 3 of the animal's endogenous OSM gene is replaced with a contiguous genomic sequence comprising the human OSM gene from the ATG start codon of exon 1 to exon 3.

[0365] The genetically modified animal can have one or more cells expressing human or chimeric OSM (e.g., humanized OSM) having an N-terminal signal peptide. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the signal peptide of human OSM. In some embodiments, the signal peptide of humanized OSM has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids (e.g., continuous or non-contiguous) that are identical to the signal peptide of human OSM. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the signal peptide of endogenous OSM. In some embodiments, the signal peptide of humanized OSM has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids (e.g., continuous or non-contiguous) that is identical to the endogenous OSM signal peptide.

[0366] In some embodiments, the genome of the animal comprises: all or part of exon 1, exon 2, and / or exon 3 of the human OSM gene; or the entire sequence of human OSM; or a nucleotide sequence encoding amino acids 1-252 or 26-252 of SEQ ID NO: 2.

[0367] In some embodiments, the genome of the genetically modified animal comprises a portion of exon 1, all of exon 2, and a portion of exon 3 of the human OSM gene. In some embodiments, the portion of exon 1 comprises at least 5, 10, 20, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 75, 80, 85, or 86 nucleotides. In some embodiments, the portion of exon 1 comprises 34 nucleotides. In some embodiments, the portion of exon 1 comprises at least 15 bp of nucleotides. In some embodiments, the portion of exon 3 includes at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 550, 580, 581, 582, 600, 800, 1000, 1200, 1500, 1600, or 1636 nucleotides. In some embodiments, the portion of exon 3 includes 582 nucleotides. In some embodiments, the portion of exon 3 includes at least 200 bp of nucleotides.

[0368] In some embodiments, a non-human animal harbors a nucleotide sequence encoding a chimeric human / non-human OSM polypeptide at an endogenous OSM locus, and the animal expresses functional OSM on the surface of its cells. The human portion of the chimeric human / non-human OSM polypeptide can comprise an amino acid sequence encoded by a portion of exon 1, exon 2, and / or a portion of exon 3 of a human OSM gene. In some embodiments, the human portion of the chimeric human / non-human OSM polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2.

[0369] In addition, the modified gene in the genome of the modified animal is homozygous or heterozygous for the endogenous replaced gene locus. In a specific embodiment, the modified OSM gene in the genome is homozygous or heterozygous for the endogenous replaced gene locus.

[0370] In some embodiments, the humanized OSM locus comprises a human 5'UTR. In some embodiments, the humanized OSM locus comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, it is reasonable to assume that, based on the similarity in the 5' flanking sequences of the mouse and human OSM genes, they appear to be similarly regulated. As shown in the application, humanized OSM mice comprising insertions or substitutions in the endogenous mouse OSM locus, which retain mouse regulatory elements but include humanization of the OSM coding sequence, do not exhibit pathology. Both genetically modified mice, heterozygous or homozygous for humanized OSM, are normal.

[0371] In another aspect, the present invention also provides a genetically modified non-human animal, whose genome comprises a disruption of an endogenous OSM gene of the animal, wherein the disruption of the endogenous OSM gene comprises a deletion of exon 1, exon 2, and / or exon 3, or a partial deletion thereof.

[0372] In some embodiments, the disruption of the endogenous OSM gene further comprises deletion of one or more introns selected from intron 1 and intron 2.

[0373] In some embodiments, the deletion may comprise the deletion of 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, 300, 400, 500, 600, 800, 900, 1000, 2000, 3000, 3100, 3127, 3130, 3200, 4000, 4100, 4200, 4242, or more nucleotides.

[0374] In some embodiments, the disruption of the endogenous OSM gene comprises a deletion of 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, 300, 400, 500, 600, 700, 710, 730, 750, 790, or 792 nucleotides of exon 1, exon 2, and / or exon 3.

[0375] Animals with a humanized IL31RA locus

[0376] In some embodiments, the chimeric gene or nucleic acid is a humanized IL31RA gene or nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human IL31RA gene, and at least a portion of the gene or nucleic acid is derived from a non-human IL31RA gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding an IL31RA protein. The encoded IL31RA protein has at least one activity of a human IL31RA protein or a non-human animal IL31RA protein.

[0377] In some embodiments, the chimeric protein or polypeptide is a humanized IL31RA protein or 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 IL31RA protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human IL31RA protein. The humanized IL31RA protein or polypeptide is functional or has at least one activity of a human IL31RA protein or a non-human IL31RA protein.

[0378] In some embodiments, the humanized IL31RA protein comprises a polypeptide sequence of 5-662 amino acids (continuous or non-continuous) identical to the human IL31RA protein. In some embodiments, the polypeptide sequence is 5-662, 10-520, or 10-662 amino acids in length. In some embodiments, the humanized IL31RA gene comprises a nucleotide sequence of 20-82521 bp (continuous or non-continuous) identical to the human IL31RA gene. In some embodiments, the nucleotide sequence is 20-3036 bp or 20-1560 bp.

[0379] In some embodiments, the IL31RA extracellular region is human or humanized. In some embodiments, the IL31RA signal peptide is human or humanized. In some embodiments, the IL31RA cytoplasmic region is human or humanized. In some embodiments, the IL31RA transmembrane region is human or humanized. In some embodiments, both the IL31RA extracellular region and the signal peptide are human or humanized. In some embodiments, both the IL31RA transmembrane region and the cytoplasmic region are endogenous.

[0380] Genetically modified non-human animals include modifications to an endogenous non-human animal IL31RA gene locus. In some embodiments, the modifications comprise a nucleotide sequence encoding at least a portion of a mature IL31RA protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of a mature IL31RA protein). While cells (e.g., ES cells, somatic cells) that may include the genetic modifications described herein are provided herein, in many embodiments, the genetically modified non-human animal includes modifications to an endogenous IL31RA gene locus in the animal.

[0381] The genetically modified animal can express human IL31RA and / or chimeric (e.g., humanized) IL31RA at the endogenous mouse locus, wherein the endogenous mouse IL31RA gene has been replaced or inserted with a human IL31RA gene and / or a nucleotide sequence encoding a region of the human IL31RA sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% identical to the human IL31RA sequence. In various embodiments, the endogenous non-human animal IL31RA locus is modified to include all or part of a human nucleic acid sequence encoding a mature IL31RA protein.

[0382] In some embodiments, the genetically modified mouse can express human IL31RA and / or chimeric IL31RA (e.g., humanized IL31RA) under the control of a mouse promoter and / or mouse regulatory elements. Insertion or substitution at the mouse endogenous locus provides a non-human animal that expresses human IL31RA or chimeric IL31RA (e.g., humanized IL31RA) in suitable cells in a manner that does not cause the potential pathology observed in some other transgenic mice known in the art. The human IL31RA or chimeric IL31RA (e.g., humanized IL31RA) expressed in the animal can maintain one or more functions of wild-type mouse or human IL31RA in the animal. In addition, in some embodiments, the animal does not express endogenous IL31RA. In some embodiments, the animal's endogenous IL31RA expression level is reduced compared to the IL31RA expression level in the wild-type animal. As used herein, the term "endogenous IL31RA" refers to the IL31RA protein expressed by the endogenous IL31RA nucleotide sequence of a non-human animal (e.g., mouse) before any genetic modification.

[0383] The genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of human IL31RA (NP_001229567.1; SEQ ID NO: 61). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 64, 65, 66 or 67. In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to positions 614-2173 of NM_001242638.1.

[0384] The genome of the genetically modified animal can include replacement of a sequence encoding a region of the endogenous IL31RA locus with a sequence encoding a corresponding region of human IL31RA. In some embodiments, the replaced sequence is any sequence of the endogenous IL31RA locus, such as exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, 5'UTR, 3'UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, or any combination thereof. In some embodiments, the replaced sequence is within the regulatory region of the endogenous IL31RA gene. In some embodiments, the replaced sequence is a portion of exon 4, exons 5-13, and a portion of exon 14 of the endogenous mouse IL31RA locus.

[0385] The genetically modified animal can have one or more cells expressing human or chimeric IL31RA (e.g., humanized IL31RA) having a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region from the N-terminus to the C-terminus. In some embodiments, the signal peptide comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the signal peptide of human IL31RA. In some embodiments, the signal peptide of humanized IL31RA has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids (e.g., continuous or non-continuous) that is identical to the human IL31RA signal peptide. In some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the extracellular region of human IL31RA. In some embodiments, the extracellular region of humanized IL31RA has a sequence of at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 450, 480, 485, 488, or 500 amino acids (contiguous or non-contiguous) identical to the extracellular region of human IL31RA. The amino acids are identical to at least 1-5 amino acids at the C-terminus of the extracellular region of endogenous IL31RA (e.g., mouse IL31RA). In some embodiments, the extracellular region described herein includes a signal peptide. In some embodiments, the extracellular region described herein does not include a signal peptide. Because human IL31RA and non-human IL31RA (e.g., mouse IL31RA) sequences are often different, antibodies that bind to human IL31RA do not necessarily have the same affinity or effect on non-human IL31RA. Therefore, transgenic animals with human or humanized extracellular regions can be used to better evaluate the effects of anti-human IL31RA antibodies in animal models.

[0386] In some embodiments, the transmembrane region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the transmembrane region of human IL31RA (e.g., amino acids 533-553 of SEQ ID NO: 61). In some embodiments, the transmembrane region of humanized IL31RA has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids (contiguous or non-contiguous) that are identical to the transmembrane region of endogenous IL31RA. In some embodiments, the cytoplasmic region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the cytoplasmic region of human IL31RA (e.g., amino acids 554-662 of SEQ ID NO: 62). In some embodiments, the cytoplasmic region of humanized IL31RA has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 160, 170, 180, 190, or 196 amino acids (contiguous or non-contiguous) identical to the cytoplasmic region of endogenous IL31RA (e.g., mouse IL31RA).

[0387] In some embodiments, the entire transmembrane region and the entire cytoplasmic region of the humanized IL31RA described herein are derived from endogenous sequences.

[0388] In some embodiments, the genome of the animal comprises: a portion of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and / or a portion of exon 14 of the human IL31RA gene; or a nucleotide sequence encoding amino acids 1-520, 33-520, 1-662, or 33-662 of SEQ ID NO: 61.

[0389] In some embodiments, the genome of the genetically modified animal comprises a portion of exon 3, all of exons 4-13, and a portion of exon 14 of the human IL31RA gene. In some embodiments, the portion of exon 3 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 90, 100, 150, 200, 240, or 246 nucleotides. In some embodiments, the portion of exon 3 comprises 6 nucleotides. In some embodiments, the portion of exon 3 comprises at least 3 bp of nucleotides. In some embodiments, the portion of exon 14 comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 115, 116, 120, 130, 140, or 141 nucleotides. In some embodiments, the portion of exon 14 comprises 116 nucleotides. In some embodiments, the portion of exon 14 comprises at least 50 bp of nucleotides.

[0390] In some embodiments, the genome of the genetically modified animal comprises a portion of exon 14, all of exons 15-16, and a portion of exon 17 of an endogenous IL31RA gene (e.g., mouse IL31RA). In some embodiments, the portion of exon 14 comprises at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 50, 100, 110, 120, 130, 140, or 141 nucleotides. In some embodiments, the portion of exon 14 comprises 25 nucleotides. In some embodiments, the portion of exon 14 comprises at least 10 bp of nucleotides. In some embodiments, the portion of exon 17 includes at least 10, 20, 30, 40, 45, 50, 60, 70, 80, 100, 200, 300, 400, 450, 460, 465, 468, 500, 560, 600, 800, 1000, 1200, 1300, 1500, or 1576 nucleotides. In some embodiments, the portion of exon 17 includes 561 nucleotides. In some embodiments, the portion of exon 17 includes at least 200 bp of nucleotides.

[0391] In some embodiments, a non-human animal harbors a nucleotide sequence encoding a chimeric human / non-human IL31RA polypeptide at an endogenous IL31RA locus, and the animal expresses functional IL31RA on the surface of its cells. The human portion of the chimeric human / non-human IL31RA polypeptide can comprise an amino acid sequence encoded by a portion of exon 3, exons 4-13, and / or a portion of exon 14 of a human IL31RA gene. In some embodiments, the human portion of the chimeric human / non-human IL31RA polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 61.

[0392] In some embodiments, the non-human portion of the chimeric human / non-human IL31RA polypeptide includes the entire transmembrane region and / or the entire cytoplasmic region of an endogenous non-human IL31RA polypeptide.

[0393] In addition, the modified gene in the genome of the modified animal is homozygous or heterozygous for the endogenous replaced gene locus. In a specific embodiment, the modified IL31RA gene in the genome is homozygous or heterozygous for the endogenous replaced gene locus.

[0394] In some embodiments, the humanized IL31RA locus comprises a human 5'UTR. In some embodiments, the humanized IL31RA locus comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, it is reasonable to assume that based on the similarity of the 5' flanking sequences of the mouse and human IL31RA genes, they appear to be similarly regulated. As shown in the application, humanized IL31RA mice comprising insertions or substitutions in the endogenous mouse IL31RA locus, which retain mouse regulatory elements but comprise humanization of the IL31RA coding sequence, do not exhibit pathology. Both genetically modified mice, heterozygous or homozygous for humanized IL31RA, are normal.

[0395] In another aspect, the present invention also provides a genetically modified non-human animal whose genome comprises a disruption of the animal's endogenous IL31RA gene, wherein the disruption of the endogenous IL31RA gene comprises a deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and / or exon 17, or a partial deletion thereof.

[0396] In some embodiments, disruption of the endogenous IL31RA gene further comprises deletion of one or more introns selected from intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, and intron 16.

[0397] In some embodiments, the deletion may comprise the deletion of 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, 300, 400, 500, 600, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 61000, 62000, 65000, 67000, 67828 or more nucleotides.

[0398] In some embodiments, disruption of the endogenous IL31RA gene comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and / or 17 of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, and / or exon 17. or a deletion of 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 1000, 1108, 1500, 1800, 1903, or 3680 nucleotides.

[0399] Animals with a humanized IL31 locus

[0400] In some embodiments, the chimeric gene or chimeric nucleic acid is a humanized IL31 gene or humanized IL31 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human IL31 gene, and at least a portion of the gene or nucleic acid is derived from a non-human IL31 gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding an IL31 protein. The encoded IL31 protein has at least one activity of a human IL31 protein or a non-human animal IL31 protein.

[0401] In some embodiments, the chimeric protein or polypeptide is a humanized IL31 protein or 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 IL31 protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human animal IL31 protein. The humanized IL31 protein or polypeptide is functional or possesses at least one activity of a human IL31 protein or a non-human animal IL31 protein.

[0402] In some embodiments, the humanized IL31 protein comprises a polypeptide sequence of 5-164 amino acids (continuous or non-continuous) identical to the human IL31 protein. In some embodiments, the polypeptide sequence is 10-164 amino acids in length. In some embodiments, the humanized IL31 gene comprises a nucleotide sequence of 20-2193 bp (continuous or non-continuous) identical to the human IL31 gene. In some embodiments, the nucleotide sequence is 20-1761 bp, 20-904 bp, or 20-495 bp.

[0403] Genetically modified non-human animals include modifications of endogenous non-human animal IL31 gene loci. In some embodiments, the modifications comprise a nucleotide sequence encoding at least a portion of a mature IL31 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a mature IL31 protein amino acid sequence). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein are provided herein, in many embodiments, the genetically modified non-human animals include modifications of an endogenous IL31 gene locus in the animal.

[0404] The genetically modified animal can express human IL31 and / or chimeric (e.g., humanized) IL31 at the endogenous mouse locus, wherein the endogenous mouse IL31 gene has been replaced or inserted with a human IL31 gene and / or a nucleotide sequence encoding a region of the human IL31 sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 100% identical to the human IL31 sequence. In various embodiments, the endogenous non-human animal IL31 locus is modified with a human nucleic acid sequence that encodes all or part of the mature IL31 protein.

[0405] In certain embodiments, genetically modified mice can express human IL31 and / or chimeric IL31 (e.g., humanized IL31) under the control of mouse promoter and / or mouse regulatory elements. Insertion or replacement at mouse endogenous locus provides a non-human animal expressing human IL31 or chimeric IL31 (e.g., humanized IL31) in suitable cells and in a manner that does not result in the potential pathological changes observed in some other transgenic mice known in the art. Human IL31 or chimeric IL31 (e.g., humanized IL31) expressed in animals can maintain one or more functions of wild-type mice or human IL31 in animals. In addition, in certain embodiments, animals do not express endogenous IL31. In certain embodiments, compared with the IL31 expression level in wild-type animals, animal endogenous IL31 expression level is reduced. As used herein, the term "endogenous IL31" refers to the IL31 protein expressed by the endogenous IL31 nucleotide sequence of a non-human animal (e.g., mouse) before any genetic modification.

[0406] The genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of human IL31 (NP_001014358.1; SEQ ID NO: 53). In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 56 or 57. In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to positions 28-522 of NM_001014336.1.

[0407] The genome of the genetically modified animal can include replacing the sequence encoding the endogenous IL31 region at the endogenous IL31 locus with the sequence encoding the corresponding region of human IL31. In some embodiments, the replaced sequence is any sequence of the endogenous IL31 locus, such as exon 1, exon 2, exon 3, 5'UTR, 3'UTR, intron 1, intron 2, or any combination thereof. In some embodiments, the replaced sequence is within the regulatory region of the endogenous IL31 gene. In some embodiments, the replaced sequence is a portion of exon 1, exon 2, and exon 3 of the endogenous mouse IL31 locus. In some embodiments, the continuous genomic segment of the animal's endogenous IL31 gene from the ATG start codon of exon 1 to exon 3 is replaced by a continuous genomic sequence comprising the human IL31 gene from the ATG start codon of exon 1 to exon 3.

[0408] Genetically modified animals may have one or more cells expressing human or chimeric IL31 (e.g., humanized IL31) having an N-terminal signal peptide. In some embodiments, the signal peptide comprises a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the signal peptide of human IL31. In some embodiments, the signal peptide of humanized IL31 has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids (e.g., continuous or non-continuous) that is consistent with the human IL31 signal peptide. In some embodiments, the signal peptide comprises a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the signal peptide of endogenous IL31. In some embodiments, the signal peptide of humanized IL31 has a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids (e.g., continuous or non-contiguous) that is identical to the endogenous IL31 signal peptide.

[0409] In some embodiments, the genome of the animal comprises: all or part of exon 1, exon 2 and / or exon 3 of the human IL31 gene; or the entire sequence of human IL31; or a nucleotide sequence encoding amino acids 1-164 or 24-164 of SEQ ID NO: 53.

[0410] In some embodiments, the genome of the genetically modified animal comprises a portion of exon 1, all of exon 2, and a portion of exon 3 of the human IL31 gene. In some embodiments, the portion of exon 1 comprises at least 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or 43 nucleotides. In some embodiments, the portion of exon 1 comprises 16 nucleotides. In some embodiments, the portion of exon 1 comprises at least 5 bp of nucleotides. In some embodiments, the portion of exon 3 comprises at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 330, 350, 400, 500, 550, 600, 650, 700, 710, or 712 nucleotides. In some embodiments, the portion of exon 3 comprises 330 nucleotides. In some embodiments, the portion of exon 3 comprises at least 150 bp of nucleotides.

[0411] In some embodiments, a non-human animal harbors a nucleotide sequence encoding a chimeric human / non-human IL31 polypeptide at an endogenous IL31 locus, and the animal expresses functional IL31 on the surface of its cells. The human portion of the chimeric human / non-human IL31 polypeptide can comprise an amino acid sequence encoded by a portion of exon 1, exon 2, and / or a portion of exon 3 of a human IL31 gene. In some embodiments, the human portion of the chimeric human / non-human IL31 polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 53.

[0412] In addition, the modified gene in the genome of the modified animal is homozygous or heterozygous for the endogenous replaced gene locus. In a specific embodiment, the modified IL31 gene in the genome is homozygous or heterozygous for the endogenous replaced gene locus.

[0413] In some embodiments, the humanized IL31 locus comprises a human 5'UTR. In some embodiments, the humanized IL31 locus comprises an endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'UTR. Where appropriate, it is reasonable to assume that, based on the similarity of the 5' flanking sequences of the mouse and human IL31 genes, they appear to be similarly regulated. As shown in the application, humanized IL31 mice comprising insertions or replacements in the endogenous mouse IL31 locus retain mouse regulatory elements but comprise humanization of the IL31 coding sequence and do not exhibit pathological phenomena. Both genetically modified mice, either heterozygous or homozygous for humanized IL31, are normal.

[0414] In another aspect, the present invention also provides a genetically modified non-human animal, whose genome comprises a disruption of the animal's endogenous IL31 gene, wherein the disruption of the endogenous IL31 gene comprises a deletion of exon 1, exon 2, and / or exon 3, or a partial deletion thereof.

[0415] In some embodiments, the disruption of the endogenous IL31 gene further comprises deletion of one or more introns selected from intron 1 and intron 2.

[0416] In some embodiments, the deletion may comprise the deletion of 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, 300, 400, 500, 600, 800, 900, 1000, 1500, 1600, 1630, 1631, 1635, 2000, 2400, 2472, or more nucleotides.

[0417] In some embodiments, the disruption of the endogenous IL31 gene comprises a deletion of 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, 300, 400, 450, 490, or 492 nucleotides of exon 1, exon 2, and / or exon 3.

[0418] The non-human animal of genetic modification can be various animals, for example, mouse, rat, rabbit, pig, cattle (for example, cattle, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (for example, marmoset, rhesus monkey).For the non-human animal of suitable genetically modified embryonic stem cell (ES) that is not easy to obtain, adopt alternative methods to construct the non-human animal comprising genetic modification.Such method includes, for example, modifying non-ES cell genome (for example, fibroblast or induced pluripotent stem cell) and adopting nuclear transplantation to transfer the modified genome to suitable cell, for example oocyte, and incubating the modified cell (for example, modified oocyte) to form embryo in non-human animal under appropriate conditions.Above-mentioned construction method is known in the art, and is described in " A.Nagy, et al., " Manipulating the Mouse Embryo:A Laboratory Manual (Third Edition), " Cold Spring Harbor Laboratory Press, 2006 ", its entire contents are incorporated herein by reference.

[0419] In one aspect, the animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified animal is from a family selected from the family Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Malinomys (climbing mice, rock mice, tailed rats, Madagascar rats, and mice), Spiny Mouse (e.g., spiny dormouse), and Muridae (e.g., mole rats, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (Muroidea), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0420] In some embodiments, the animal is a mouse of the C57BL strain selected from the group consisting of C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / min, C57BL6J, C57Bl / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL10SnSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of 129 strains and C57BL / 6 strains. In some embodiments, the mouse is a hybrid of 129 strains, or a hybrid of BL / 6 strains. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid system (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a BALB / c, 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, or C5C57BL / 6NJ.Mice of the C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, or CBA / H strains.

[0421] In some embodiments, the animal is a rat. The rat can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a hybrid of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0422] The animal may have one or more other genetic modifications and / or other modifications that are suitable for the specific purpose of preparing a humanized animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) may have one or more modifications that damage, inactivate, or destroy all or part of the immune system of the non-human animal. Damage, inactivation, or destruction of the immune system of the non-human animal can include, for example, by chemical means (e.g., administration of a toxin), physical means (e.g., irradiating the animal), and / or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc null Mice (Ito, M. et al., NOD / SCID / γc null mouse:an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can optionally be irradiated or otherwise treated to destroy one or more immune cell types. Therefore, in various embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of the endogenous non-human OSMR, OSM, IL31RA and / or IL31 loci, and also includes modifications that damage, inactivate or partially destroy the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the mouse modification type is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null Mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scidIL-2Rγ null Mouse, NOD Rag 1 - / - IL2rg - / - (NRG) mice, Rag2 - / - IL2rg - / - Modification of (RG) mice and combinations thereof. These transgenic animals are described, for example, in US Pat. No. 10,820,580 B2, which is incorporated herein by reference in its entirety. In some embodiments, the mice can include replacement of all or part of the mouse endogenous mature OSMR, OSM, IL31RA, and / or IL31 coding sequence with all or part of the human mature OSMR, OSM, IL31RA, and / or IL31 coding sequence, respectively.

[0423] The present invention further relates to a non-human mammal produced by the above method. In some embodiments, its genome comprises human genes.

[0424] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0425] In some embodiments, the non-human mammal expresses proteins encoded by humanized OSMR, OSM, IL31RA, IL31 genes.

[0426] In addition, the present invention also provides a non-human mammal model carrying a tumor, characterized in that the non-human mammal model is obtained by the method described herein. In some embodiments, the non-human mammal is a rodent (eg, mouse).

[0427] The present invention also provides a cell or cell line, or primary cell culture derived from a non-human mammal or its progeny, or a non-human mammal carrying a tumor, wherein the cell or cell line is derived from a non-human mammal or its progeny, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its progeny. In the case of a tumor, the cell or cell line is derived from a tumor tissue of a non-human mammal or its progeny, or a non-human mammal carrying a tumor.

[0428] The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal comprises DNA of human or humanized OSMR, OSM, IL31RA, or IL31.

[0429] In some embodiments, a non-human mammal comprises a gene construct described herein (e.g., a gene construct as shown in Figures 2, 3, 4, 5, 9, 10, 11, 23, 24, 26, 30, 31, and 33). In some embodiments, a non-human mammal expressing human or humanized OSMR, OSM, IL31RA, or IL31 protein is provided. In some embodiments, a tissue specifically expressing human or humanized OSMR, OSM, IL31RA, or IL31 protein is provided.

[0430] In some embodiments, the expression of human or humanized OSMR, OSM, IL31RA, or IL31 protein in non-human animals is controllable, such as by adding specific inducers or repressors. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0431] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferred non-human mammals are mammals (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0432] The non-human mammal described above is subjected to genetic, molecular and behavioral analysis. The present invention provides offspring produced by mating with a non-human mammal of the same genotype or another genotype.

[0433] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its descendants. For example, a cell culture-based model can be prepared by the following methods. The cell culture can be obtained by isolating the cells from the non-human mammal, or by using the same construct and establishing a cell culture using standard cell transfection techniques. Integration of a genetic construct comprising DNA sequences encoding human OSMR, OSM, IL31RA, or IL31 proteins can be detected by various methods.

[0434] There are many analytical methods that can be used to detect exogenous DNA, including nucleic acid level methods (including reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, immunoblotting analysis and in vitro binding studies). In addition, the expression level of the target gene can be quantified by the ELSA method well known to those skilled in the art. Many standard analytical methods can be used to perform quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcription levels, including RNase protection assays, Southern Blots, and RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blots can also be used to detect the presence of human or humanized OSMR, OSM, IL31RA, and IL31 proteins.

[0435] carrier

[0436] The present invention provides a targeting vector comprising: a) a DNA fragment homologous to the 5' end of the region to be changed (5' arm), which is selected from the genomic DNA of the OSMR gene and has a length of 100 to 10,000 nucleotides; b) a desired donor DNA sequence encoding the donor region; and c) a second DNA fragment homologous to the 3' end of the region to be changed (3' arm), which is selected from the genomic DNA of the OSMR gene and has a length of 100 to 10,000 nucleotides.

[0437] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000081.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000081.7.

[0438] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 6889847 to 6894350 of NCBI Accession No. NC_000081.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 6885855 to 6889843 of NCBI Accession No. NC_000081.7.

[0439] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 6.8 kb, or 7 kb.

[0440] In some embodiments, the switch region to be altered is located in exon 2 of the OSMR gene of a non-human animal.

[0441] In some embodiments, the region to be altered is exon 2 and / or intron 2 of the endogenous OSMR gene (eg, positions 126-128 of NM_010513.2).

[0442] In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 11; and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 12.

[0443] In some embodiments, the targeting vector comprises a human sequence (e.g., positions 129-2339 of NM_001323505.2). For example, the targeting region in the targeting vector includes: part or all of the nucleotide sequence of the human OSMR gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and / or exon 18 of the human OSMR gene. In some embodiments, the nucleotide sequence of the humanized OSMR gene encodes all or part of the human OSMR protein, and the NCBI protein number is NP_001310434.1 (SEQ ID NO: 10). In some embodiments, the protein encoded by the nucleotide sequence of the humanized OSMR gene is SEQ ID NO: 16.

[0444] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the OSMR gene, and the sgRNA is unique on the target sequence of the gene to be changed and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the targeting site of the sgRNA in the mouse OSMR gene is located at exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 1 Exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, intron 17, exon 18, upstream of exon 1, or downstream of exon 18 of the mouse OSMR gene.

[0445] The present invention provides a targeting vector comprising: a) a DNA fragment homologous to the 5' end of the region to be altered (5' arm), which is selected from the genomic DNA of the OSM gene and has a length of 100 to 10,000 nucleotides; b) a desired donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3' end of the region to be altered (3' arm), which is selected from the genomic DNA of the OSM gene and has a length of 100 to 10,000 nucleotides.

[0446] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000077.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000077.7.

[0447] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 4182438 to 4186882 of NCBI Accession No. NC_000077.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 4191473 to 4196114 of NCBI Accession No. NC_000077.7.

[0448] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 4185375 to 4186882 of NCBI Accession No. NC_000077.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 4190010 to 4191085 of NCBI Accession No. NC_000077.7.

[0449] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, or 3 kb.

[0450] In some embodiments, the switch region to be altered is located on exons 1 to 3 of the OSM gene of a non-human animal.

[0451] In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 3, and 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: 5, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 6.

[0452] In some embodiments, the targeting vector comprises a human sequence (e.g., positions 30263883 to 30266799 of NC_000022.11). For example, the targeting region in the targeting vector includes: a partial or complete nucleotide sequence of the human OSM gene, preferably exon 1, exon 2, and / or exon 3 of the human OSM gene. In some embodiments, the nucleotide sequence of the humanized OSM gene encodes all or part of the human OSM protein, and the NCBI protein number is NP_065391.1 (SEQ ID NO: 2).

[0453] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the OSM gene, and the sgRNA is unique on the target sequence of the gene to be altered and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the targeting site of the sgRNA in the mouse OSM gene is located in exon 1, intron 1, exon 2, intron 2, exon 3, upstream of exon 1 of the mouse OSM gene, or downstream of exon 3.

[0454] In some embodiments, the targeting sequence is shown as SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32. Thus, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 25 and 27. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 26 and 28. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 29 and 31. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 30 and 32.

[0455] The present invention provides a targeting vector comprising: a) a DNA fragment homologous to the 5' end of the region to be altered (5' arm), which is selected from the genomic DNA of the IL31RA gene and has a length of 100 to 10,000 nucleotides; b) a desired donor DNA sequence encoding the donor region; and c) a second DNA fragment homologous to the 3' end of the region to be altered (3' arm), which is selected from the genomic DNA of the IL31RA gene and has a length of 100 to 10,000 nucleotides.

[0456] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000079.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000079.7.

[0457] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 112695737 to 112699971 of NCBI Accession No. NC_000079.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 112691844 to 112695733 of NCBI Accession No. NC_000079.7.

[0458] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 4.9 kb, or 5 kb.

[0459] In some embodiments, the switch region to be altered is located in exon 4 of the IL31RA gene of a non-human animal.

[0460] In some embodiments, the region to be altered is exon 4 and / or intron 4 of the endogenous IL31RA gene (eg, positions 422-424 of NM_139299.2).

[0461] In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 62; and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 63.

[0462] In some embodiments, the targeting vector comprises a human sequence (e.g., positions 614-2173 of NM_001242638.1). For example, the targeting region in the targeting vector includes: a portion or all of the nucleotide sequence of the human IL31RA gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and / or exon 18 of the human IL31RA gene. In some embodiments, the nucleotide sequence of the humanized IL31RA gene encodes all or part of the human IL31RA protein, and the NCBI protein number is NP_001229567.1 (SEQ ID NO: 61). In some embodiments, the protein encoded by the nucleotide sequence of the humanized IL31RA gene is SEQ ID NO: 68.

[0463] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the IL31RA gene, and the sgRNA is unique in the target sequence of the gene to be changed and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the targeting site of the sgRNA in the mouse IL31RA gene is located at exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4 , intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, upstream of exon 1, or downstream of exon 17 of the mouse IL31RA gene.

[0464] The present invention provides a targeting vector comprising: a) a DNA fragment homologous to the 5' end of the region to be changed (5' arm), which is selected from the genomic DNA of the IL31 gene and has a length of 100 to 10,000 nucleotides; b) a desired donor DNA sequence encoding the donor region; and c) a second DNA fragment homologous to the 3' end of the region to be changed (3' arm), which is selected from the genomic DNA of the IL31 gene and has a length of 100 to 10,000 nucleotides.

[0465] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000071.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000071.7.

[0466] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence at positions 123620113 to 123625584 of NCBI Accession No. NC_000071.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence at positions 123614373 to 123617890 of NCBI Accession No. NC_000071.7.

[0467] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 0.5 kb, 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, or 1.8 kb.

[0468] In some embodiments, the switch region to be altered is located on exons 1 to 3 of the IL31 gene of a non-human animal.

[0469] In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 54; and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 55.

[0470] In some embodiments, the targeting vector comprises a human sequence (e.g., positions 122172412 to 122174172 of NC_000012.12). For example, the targeting region in the targeting vector includes: a partial or complete nucleotide sequence of the human IL31 gene, preferably exon 1, exon 2, and / or exon 3 of the human IL31 gene. In some embodiments, the nucleotide sequence of the humanized IL31 gene encodes all or part of the human IL31 protein, and the NCBI protein number is NP_001014358.1 (SEQ ID NO: 53).

[0471] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the IL31 gene, and the sgRNA is unique on the target sequence of the gene to be changed and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the targeting site of the sgRNA in the mouse IL31 gene is located in exon 1, intron 1, exon 2, intron 2, exon 3, upstream of exon 1 of the mouse IL31 gene, or downstream of exon 3.

[0472] In some embodiments, the targeting vector further comprises one or more marker genes. For example, a positive screening marker gene or a negative screening marker gene. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo. In some embodiments, the coding gene for the negative screening marker is a gene encoding the diphtheria toxin A subunit (DTA).

[0473] In some embodiments, the present disclosure relates to a plasmid construct (e.g., pT7-sgRNA) comprising an sgRNA sequence and / or a cell comprising the construct.

[0474] The present invention also relates to a cell comprising a targeting vector as described above.

[0475] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned targeting vectors and one or more in vitro transcripts of the constructs described herein. In some embodiments, the cell comprises Cas9 mRNA or its in vitro transcript.

[0476] In some embodiments, the cell is heterozygous for the gene. In some embodiments, the cell is homozygous for the gene.

[0477] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

[0478] Method for constructing genetically modified non-human animals

[0479] Genetically modified non-human animals can be prepared by several techniques 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, homing endonuclease or other molecular biology techniques. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR-Cas9 genome editing is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in Yin et al. “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0480] In some embodiments, the nucleotide sequence encoding the endogenous OSMR region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding the corresponding region of human OSMR. In some embodiments, the non-human animal has reduced or absent expression of the endogenous OSMR protein compared to wild-type. In some embodiments, the replacement occurs in a cell, such as a germ cell, somatic cell, blastocyst, or fibroblast. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0481] Figure 10 shows a humanized targeting strategy for the mouse OSMR locus. The targeting vector comprises a 5' homology arm, a human or humanized OSMR gene (DNA or cDNA) fragment, and a 3' homology arm. The process involves replacing the endogenous OSMR sequence with a human / non-human sequence, or inserting a human / non-human OSMR sequence by homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPR) can result in a double-strand break in the DNA, and homologous recombination is used to replace the endogenous OSMR sequence with a human / non-human sequence, or to insert a human / non-human OSMR sequence.

[0482] In some embodiments, the non-human animal is constructed by introducing any of the following nucleotide sequences into the non-human animal OSMR locus:

[0483] A) A portion of the human OSMR gene, preferably comprising all or part of exons 1 to 18 of the human OSMR gene, further preferably comprising all or part of one, two or more, or a combination of two or more consecutive exons of exons 1 to 18 of the human OSMR gene, more preferably comprising all or part of exon 2 to all or part of exon 15 of the human OSMR gene, further preferably comprising the nucleotide sequence of the coding region of part of exon 2, all of exons 3 to 14, and part of exon 15 of the human OSMR gene, wherein the portion of exon 2 of the human OSMR gene comprises at least 50 bp to at least 86 bp of exon 2 of the human OSMR gene, e.g., 50, 55 , 60, 61, 62, 63, 64, 65, 70, 72, 73, 75, 80, 85 or 86 bp continuous nucleotide sequence, preferably the portion of exon 2 of the human OSMR gene includes the nucleotide sequence of the coding region; the portion of exon 15 of the human OSMR gene includes at least 100 bp to at least 168 bp of exon 15 of the human OSMR gene, for example, 100, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 165, 167 or 168 bp continuous nucleotide sequence, preferably the portion of exon 15 of the human OSMR gene includes the nucleotide sequence of the coding region; more preferably, the portion of the human OSMR gene includes SEQ or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence of SEQ ID NO: 14; or a nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence of SEQ ID NO: 14; or a nucleotide sequence having SEQ ID NO: 14; NO: 14, comprising a nucleotide sequence of substitution, deletion and / or insertion of one or more nucleotides; or, the portion of the human OSMR gene comprises the nucleotide sequence of positions 129-2339 of NM_001323505.2; or, comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of positions 129-2339 of NM_001323505.2; or, comprises a nucleotide sequence that differs from the nucleotide sequence of positions 129-2339 of NM_001323505.2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises NM_001323505.2.2 shown in the nucleotide sequence shown at positions 129-2339, including substitution, deletion and / or insertion of one or more nucleotides; or,.

[0484] B) all or part of a nucleotide sequence encoding a human OSMR protein, preferably comprising all or part of a nucleotide sequence encoding a signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of a human OSMR protein, further preferably comprising all or part of a nucleotide sequence encoding the extracellular region of a human OSMR protein, more preferably comprising a nucleotide sequence encoding at least 100 to at least 713, such as 100, 200, 300, 400, 500, 600, 700, 710, 711, 712 or 713 consecutive amino acids of the extracellular region of a human OSMR protein, further preferably comprising a nucleotide sequence encoding at least 1 to at least 27, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 consecutive amino acids of a human OSMR protein signal peptide, further preferably comprising a nucleotide sequence encoding SEQ ID NO: 10; or, a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-737 or 28-737 of SEQ ID NO: 10; or, a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-737 or 28-737 of SEQ ID NO: 10 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference; or, a nucleotide sequence that comprises a substitution, deletion and / or insertion of one or more nucleotides from the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-737 or 28-737 of SEQ ID NO: 10, and further preferably, comprises a nucleotide sequence encoding the amino acid sequence as set forth at positions 1-737 or 28-737 of SEQ ID NO: 10, NO:10; or, comprising a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-737 or 28-737 of SEQ ID NO:10; or, comprising a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-737 or 28-737 of SEQ ID NO:10 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide. Or, comprising a nucleotide sequence having a nucleotide sequence encoding the amino acid sequence as set forth at positions 1-737 or 28-737 of SEQ ID NO:10, including substitutions, deletions and / or insertions of one or more nucleotides.

[0485] Further preferably, the nucleotide sequence introduced into the non-human animal OSMR locus further comprises:

[0486] C) A portion of the OSMR gene of a non-human animal, preferably comprising all or part of exons 1 to 18 of the OSMR gene of a non-human animal, further preferably comprising all or part of one, two or more, or a combination of two or more consecutive exons of exons 1 to 18 of the OSMR gene of a non-human animal, more preferably comprising all or part of exon 15 to all or part of exon 18 of the OSMR gene of a non-human animal, further preferably comprising the nucleotide sequence of the coding region of part of exon 15, all of exons 16 to 17 and part of exon 18 of the OSMR gene of a non-human animal, wherein the portion of exon 15 of the OSMR gene of a non-human animal comprises at least 100 bp to at least 168 bp of exon 15 of the OSMR gene of a non-human animal, for example, 100, 110, 120, 130, 140, 150, 160, 165, 166, 167 or 168 bp of consecutive nucleotides. Acid sequence, or, the portion of exon 15 of the non-human animal OSMR gene comprises at most 20bp of exon 15 of the non-human animal OSMR gene, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20bp continuous nucleotide sequence, preferably, the portion of exon 15 of the non-human animal OSMR gene comprises the nucleotide sequence of the coding region; the portion of exon 18 of the non-human animal OSMR gene comprises at least 100bp to at least 2355bp of exon 18 of the non-human animal OSMR gene, such as 100, 500, 550, 555, 558, 600, 800, 1000, 1500, 2000, 2300 or 2355bp continuous nucleotide sequence, preferably, the portion of exon 18 of the non-human animal OSMR gene comprises the nucleotide sequence of the coding region; more preferably, the portion of the non-human animal OSMR gene comprises SEQ SEQ ID NO: 15; or, comprising a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 15; or, comprising a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 15 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprising a nucleotide sequence having a substitution, deletion and / or insertion of one or more nucleotides as set forth in the nucleotide sequence of SEQ ID NO: 15; or,

[0487] D) All or part of the nucleotide sequence encoding the non-human animal OSMR protein, preferably comprising all or part of the nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the non-human animal OSMR protein, further preferably comprising all or part of the nucleotide sequence encoding the extracellular region, transmembrane region and / or cytoplasmic region of the non-human animal OSMR protein, more preferably comprising a nucleotide sequence encoding at least one or at least two, for example, one or two consecutive amino acids at the C-terminus of the extracellular region of the non-human animal OSMR protein; more preferably comprising a nucleotide sequence encoding at least one amino acid sequence at the transmembrane region of the non-human animal OSMR protein. 1 to at least 22, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive amino acids of the nucleotide sequence; more preferably comprising a nucleotide sequence encoding at least 20 to at least 213, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 210, 211, 212 or 213 consecutive amino acids of the cytoplasmic region of the non-human animal OSMR protein; further preferably comprising a nucleotide sequence encoding SEQ or a nucleotide sequence encoding the amino acid sequence as set forth in positions 735-971 or 737-971 of SEQ ID NO:9; or a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence encoding the amino acid sequence as set forth in positions 735-971 or 737-971 of SEQ ID NO:9; or a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth in positions 735-971 or 737-971 of SEQ ID NO:9 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide. Or a nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides from the nucleotide sequence encoding the amino acid sequence as set forth in positions 735-971 or 737-971 of SEQ ID NO:9.

[0488] More preferably, the non-human animal is constructed by introducing any one of the following nucleotide sequences into the non-human animal OSMR locus:

[0489] E) a nucleotide sequence encoding the above-mentioned humanized OSMR protein; or

[0490] F) The humanized OSMR gene described above.

[0491] Preferably, the non-human animal is constructed by introducing the nucleotide sequence shown in SEQ ID NO: 13 into the OSMR locus of a non-human animal; or, introducing a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 13 into the OSMR locus of a non-human animal; or, introducing a nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 13 into the OSMR locus of a non-human animal; or, introducing a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 13, including substitution, deletion and / or insertion of one or more nucleotides, into the OSMR locus of a non-human animal.

[0492] Preferably, the non-human animal is constructed using the above-mentioned targeting vector.

[0493] Preferably, the non-human animal further comprises other gene modifications, more preferably, the other genes are selected from at least one of OSM, IL31RA, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT and CD73.

[0494] Preferably, the human or humanized OSMR gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) locus.

[0495] Preferably, the human or humanized OSMR gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

[0496] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to produce humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0497] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0498] Therefore, the present invention provides a method for constructing a non-human animal with a humanized OSMR gene, wherein the non-human animal expresses human or humanized OSMR protein in vivo, and / or the genome of the non-human animal contains a portion of a human OSMR gene or a humanized OSMR gene.

[0499] In some embodiments, the method for preparing a genetically modified humanized animal comprises replacing a nucleic acid sequence encoding an endogenous OSMR region with a nucleotide sequence encoding a corresponding region of human OSMR at an endogenous OSMR locus (or site). In some embodiments, the method may comprise inserting a nucleic acid sequence encoding a human OSMR region and a sequence encoding an endogenous OSMR region at an endogenous OSMR locus (or site). The inserted sequence may comprise a region (e.g., part or all) of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, and / or exon 15 of a human OSMR gene. In some embodiments, the sequence comprises a portion of exon 2, exons 3-14, and a portion of exon 15 of a human OSMR gene (e.g., the nucleotide sequence of positions 129-2339 of NM_001323505.2). In some embodiments, the sequence includes a portion of exon 15, exons 16-17, and a portion of exon 18 of an endogenous OSMR gene (e.g., nucleotide sequence 2328-3041 of NM_011019.3), a portion of the extracellular region of human OSMR (e.g., amino acids 28-737 of SEQ ID NO: 10), an endogenous OSMR transmembrane region (e.g., amino acids 737-758 of SEQ ID NO: 9), and an endogenous OSMR cytoplasmic region (e.g., amino acids 759-971 of SEQ ID NO: 9). In some embodiments, the endogenous OSMR locus (or site) is exon 2 and / or intron 2 of mouse OSMR. In some embodiments, the sequence is inserted after the 5'UTR of the mouse OSMR gene and before nucleotide 129 of NM_011019.3, and 1-3 bp of nucleotides are deleted (eg, inserted between nucleotides 125 to 129 of NM_011019.3, and nucleotides 126-128 are deleted).

[0500] In some embodiments, a method of modifying the OSMR locus of a mouse to express a chimeric human / mouse OSMR polypeptide can include replacing a nucleotide sequence encoding a mouse OSMR at an endogenous mouse OSMR locus with a nucleotide sequence encoding a human OSMR, thereby generating a chimeric human / mouse OSMR sequence. In some embodiments, the method can include inserting a nucleotide sequence encoding a chimeric human / mouse OSMR at an endogenous mouse OSMR locus, thereby generating a chimeric human / mouse OSMR sequence.

[0501] In some embodiments, the nucleotide sequence encoding the chimeric human / mouse OSMR may include a first nucleotide sequence encoding all or part of the extracellular region of human OSMR; and a second nucleotide sequence encoding all or part of the extracellular region, cytoplasmic region, and transmembrane region of mouse OSMR.

[0502] The present invention also provides a method for establishing an OSMR gene humanized animal model, comprising the following steps:

[0503] (a) providing a cell (e.g., a fertilized egg cell) according to the method described herein;

[0504] (b) culturing the cells in a liquid culture medium;

[0505] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0506] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0507] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0508] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pseudo-pregnancy).

[0509] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0510] The fertilized egg can be from any non-human animal, such as any of the non-human animals described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The genetic construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which can then give birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0511] In some embodiments, the method for preparing a genetically modified animal comprises modifying the coding frame of the OSMR gene of a non-human animal, for example, by replacing the nucleic acid sequence encoding the endogenous OSMR region (e.g., a DNA or cDNA sequence) with a nucleotide sequence encoding the corresponding region of human OSMR under the control of the endogenous regulatory elements of the OSMR gene of the non-human animal. For example, one or more functional region sequences of the OSMR gene of the non-human animal can be knocked out or inserted into a sequence so that the endogenous OSMR protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the modified non-human animal OSMR gene can be all or part of the nucleotide sequence of exon 1 to exon 18 of the OSMR gene of the non-human animal.

[0512] In some embodiments, a method for preparing a genetically modified animal comprises inserting a nucleotide sequence encoding a human or humanized OSMR protein and / or an auxiliary sequence after the endogenous regulatory elements of the OSMR gene of a non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, such that the OSMR gene humanized animal model can express the human or humanized OSMR protein in vivo, but does not express the OSMR protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0513] In some embodiments, the method for preparing a transgenic animal comprises:

[0514] (1) providing a plasmid comprising a human OSMR gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous OSMR;

[0515] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous OSMR gene;

[0516] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0517] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express humanized OSMR protein;

[0518] (5) mating the offspring mice obtained in step (4) to obtain homozygous mice,

[0519] In some embodiments, fertilized eggs are modified using CRISPR technology with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal target site.

[0520] In some embodiments, the sequence encoding the humanized OSMR protein is operably linked to endogenous regulatory elements at the endogenous OSMR locus.

[0521] In some embodiments, the genetically modified animal does not express endogenous OSMR protein.

[0522] In some embodiments, the method for preparing a transgenic animal comprises:

[0523] (1) providing a plasmid comprising a human or chimeric OSMR gene fragment, said plasmid being flanked by 5' homology arms and 3' homology arms, wherein said 5' and 3' homology arms target endogenous OSMR;

[0524] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous OSMR gene;

[0525] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric OSMR gene fragment into the genome.

[0526] In some embodiments, a nucleotide sequence encoding an endogenous OSM region in the endogenous genome of at least one cell of a non-human animal is replaced with a nucleotide sequence encoding a corresponding region of human OSM. In some embodiments, the expression of endogenous OSM protein in the non-human animal is reduced or absent compared to wild-type. In some embodiments, the replacement occurs in a cell, such as a germ cell, somatic cell, blastocyst, or fibroblast. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0527] Figure 3 shows a humanized targeting strategy for the mouse OSM locus. The targeting vector comprises a 5' homology arm, a human or humanized OSM gene fragment, and a 3' homology arm. This process involves replacing the endogenous OSM sequence with the human or humanized OSM sequence via homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPR) results in DNA double-strand breaks, allowing homologous recombination to replace the endogenous mouse OSM sequence with the human or humanized OSM sequence.

[0528] Therefore, in some embodiments, the method for preparing a genetically modified humanized animal comprises replacing a nucleic acid sequence encoding an endogenous OSM region at the endogenous OSM gene locus (or site) with a nucleotide sequence encoding a corresponding region of human OSM. The replaced sequence may include regions (e.g., part or all) of exon 1, exon 2, and / or exon 3 of the human OSM gene. In some embodiments, the sequence includes a portion of exon 1, exon 2, and exon 3 of the human OSM gene (e.g., the nucleotide sequence of positions 53-811 of NM_020530.6). In some embodiments, the sequence includes a portion of exon 1 and a portion of exon 3 of the endogenous OSM gene (e.g., the nucleotide sequence of positions 1-98 and 891-1907 of NM_001013365.2).

[0529] The present invention also provides a method for establishing an OSM gene humanized animal model, comprising the following steps:

[0530] (a) providing a cell (e.g., a fertilized egg cell) according to the method described herein;

[0531] (b) culturing the cells in a liquid culture medium;

[0532] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0533] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0534] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0535] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pseudo-pregnancy).

[0536] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0537] The fertilized egg can be from any non-human animal, such as any of the non-human animals described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The genetic construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which can then give birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0538] In some embodiments, methods for preparing genetically modified animals include modifying the coding frame of a non-human animal's OSM gene, for example, by replacing a nucleic acid sequence (e.g., a DNA or cDNA sequence) encoding an endogenous OSM region with a nucleotide sequence encoding a corresponding region of human OSM under the control of endogenous regulatory elements of the non-human animal's OSM gene. For example, one or more functional region sequences of the non-human animal's OSM gene can be deleted or inserted, such that the non-human animal's endogenous OSM protein is not expressed or its expression level is reduced. In some embodiments, the modified coding frame of the non-human animal's OSM gene can be all or part of the nucleotide sequence of exon 1 to exon 3 of the non-human animal's OSM gene.

[0539] In some embodiments, a method for preparing a genetically modified animal comprises inserting a nucleotide sequence encoding a human or humanized OSM protein and / or an auxiliary sequence after the endogenous regulatory elements of the OSM gene of a non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, such that the humanized OSM gene animal model can express the human or humanized OSM protein in vivo without expressing the OSM protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0540] In some embodiments, the method for preparing a transgenic animal comprises:

[0541] (1) providing a plasmid comprising a human OSM gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous OSM;

[0542] (2) providing one or more guide RNAs (sgRNAs) targeting endogenous OSM genes;

[0543] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0544] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express humanized OSM protein;

[0545] (5) mating the offspring mice obtained in step (4) to obtain homozygous mice,

[0546] In some embodiments, fertilized eggs are modified using CRISPR technology with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal target site.

[0547] In some embodiments, the sequence encoding the humanized OSM protein is operably linked to endogenous regulatory elements at the endogenous OSM locus.

[0548] In some embodiments, the genetically modified animal does not express endogenous OSM protein.

[0549] In some embodiments, the method for preparing a transgenic animal comprises:

[0550] (1) providing a plasmid comprising a human or chimeric OSM gene fragment, said plasmid being flanked by 5' homology arms and 3' homology arms, wherein said 5' and 3' homology arms target endogenous OSM;

[0551] (2) providing one or more guide RNAs (sgRNAs) targeting endogenous OSM genes;

[0552] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric OSM gene fragment into the genome.

[0553] In some embodiments, the nucleotide sequence encoding the endogenous IL31RA region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding the corresponding region of human IL31RA. In some embodiments, the expression level of the endogenous IL31RA protein in the non-human animal is reduced or absent compared to wild-type. In some embodiments, the replacement occurs in a cell such as a germ cell, somatic cell, blastocyst, or fibroblast. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0554] Figures 30, 31, and 33 show strategies for humanizing the mouse IL31RA locus. The targeting vector comprises a 5' homology arm, a human or humanized IL31RA gene (DNA or cDNA) fragment, and a 3' homology arm. This process involves replacing the endogenous IL31RA sequence with a human / non-human sequence or inserting a human / non-human IL31RA sequence via homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPR) results in DNA double-strand breaks, which allow homologous recombination to replace the endogenous IL31RA sequence with a human / non-human sequence or insert a human / non-human IL31RA sequence.

[0555] In some embodiments, the non-human animal is constructed by introducing any of the following nucleotide sequences into the IL31RA locus of the non-human animal:

[0556] A) a portion of the human IL31RA gene, preferably comprising all or part of exons 1 to 18 of the human IL31RA gene, further preferably comprising all or part of one, two, or three or more, or a combination of two or more consecutive exons of exons 1 to 18 of the human IL31RA gene, more preferably comprising all or part of exon 3 to all or part of exon 14 of the human IL31RA gene, further preferably comprising a nucleotide sequence comprising a portion of exon 3, all of exons 4 to 13, and a portion of the coding region of exon 14 of the human IL31RA gene, wherein the portion of exon 3 of the human IL31RA gene comprises at least 1 bp to at least 246 bp of exon 3 of the human IL31RA gene, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 90, 100, 150, 200, The present invention relates to a human IL31RA gene comprising a 240 or 246 bp continuous nucleotide sequence, preferably a portion of exon 3 of the human IL31RA gene comprising the nucleotide sequence of the coding region; a portion of exon 14 of the human IL31RA gene comprises at least 50 bp to at least 141 bp, for example, 50, 60, 70, 80, 90, 100, 115, 116, 120, 130, 140 or 141 bp continuous nucleotide sequence of exon 14 of the human IL31RA gene, preferably a portion of exon 14 of the human IL31RA gene comprising the nucleotide sequence of the coding region; further preferably, the portion of the human IL31RA gene comprises the nucleotide sequence of SEQ ID NO: 64; or comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 64; or comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: A nucleotide sequence having a nucleotide sequence with a difference of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide from the nucleotide sequence of NO: 64; or a nucleotide sequence having a nucleotide sequence with ... NO: 64, comprising a nucleotide sequence of substitution, deletion and / or insertion of one or more nucleotides; or, the portion of the human IL31RA gene comprises the nucleotide sequence of positions 614-2173 of NM_001242638.1; or, comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of positions 614-2173 of NM_001242638.1; or, comprises a nucleotide sequence that differs from the nucleotide sequence of positions 614-2173 of NM_001242638.1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide. or, comprises a nucleotide sequence having NM_001242638.1.1 shown in the nucleotide sequence shown at positions 614-2173, including substitution, deletion and / or insertion of one or more nucleotides; or,.

[0557] B) all or part of the nucleotide sequence encoding the human IL31RA protein, preferably comprising all or part of the nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human IL31RA protein, further preferably comprising all or part of the nucleotide sequence encoding the extracellular region of the human IL31RA protein, more preferably comprising at least 50 to at least 500, e.g., 50, 100, 200, 300, 400, 450, 480, 490, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2500, 2600, 3000, 3500, 3600, 3700, 3800, 4000, 4500, 4800, 5000, 5000, 6000, 7000, 8000, 9000, 18000, 2500 85, 488 or 500 consecutive amino acids, further preferably comprising a nucleotide sequence encoding at least 1 to at least 32, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 consecutive amino acids of the human IL31RA protein signal peptide, further preferably comprising a nucleotide sequence encoding SEQ 520 or 33-520 of SEQ ID NO:61; or, comprising a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-520 or 33-520 of SEQ ID NO:61; or, comprising a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-520 or 33-520 of SEQ ID NO:61 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference; or, comprising a nucleotide sequence having a substitution, deletion and / or insertion of one or more nucleotides from the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-520 or 33-520 of SEQ ID NO:61, further preferably, comprising a nucleotide sequence encoding the amino acid sequence as set forth at positions 1-520 or 33-520 of SEQ ID NO:61, NO:61; or, comprising a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-520 or 33-520 of SEQ ID NO:61; or, comprising a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-520 or 33-520 of SEQ ID NO:61 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide(s); or, comprising a nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides as set forth in the nucleotide sequence encoding the amino acid sequence as set forth at positions 1-520 or 33-520 of SEQ ID NO:61.

[0558] More preferably, the nucleotide sequence introduced into the non-human animal IL31RA locus further comprises:

[0559] C) a portion of the IL31RA gene of a non-human animal, preferably comprising all or part of exons 1 to 17 of the IL31RA gene of a non-human animal, more preferably comprising all or part of one, two, three or more, or a combination of two or more consecutive exons of the IL31RA gene of a non-human animal, more preferably comprising all or part of exons 14 to 17 of the IL31RA gene of a non-human animal, and even more preferably comprising all or part of exons 14 to 17 of the IL31RA gene of a non-human animal. The nucleotide sequence of the coding region of part of exon 14, all of exons 15 to 16, and part of exon 17 of the non-human animal IL31RA gene, wherein the part of exon 14 of the non-human animal IL31RA gene comprises at least 10 bp to at least 141 bp, such as 10, 15, 20, 25, 50, 100, 110, 120, 130, 140 or 141 bp of exon 14 of the non-human animal IL31RA gene, or the non-human animal IL The portion of exon 14 of the non-human IL31RA gene comprises up to 50 bp of exon 14 of the non-human IL31RA gene, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40 or 50 bp of a continuous nucleotide sequence, preferably, the portion of exon 14 of the non-human IL31RA gene comprises the nucleotide sequence of the coding region; the portion of exon 17 of the non-human IL31RA gene comprises the nucleotide sequence of the non-human IL31RA gene. A portion of exon 17 of the IL31RA gene of a non-human animal comprises at least 100 bp to at least 1576 bp, for example, a continuous nucleotide sequence of 100, 200, 300, 400, 450, 460, 465, 468, 500, 560, 600, 800, 1000, 1200, 1300, 1500 or 1576 bp, preferably a portion of exon 17 of the IL31RA gene of a non-human animal comprises a nucleotide sequence of the coding region; more preferably, the portion of the IL31RA gene of a non-human animal comprises SEQ SEQ ID NO: 65; or, comprising a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 64; or, comprising a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 65 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprising a nucleotide sequence having a substitution, deletion and / or insertion of one or more nucleotides as set forth in the nucleotide sequence of SEQ ID NO: 65; or,

[0560] D) all or part of a nucleotide sequence encoding a non-human animal IL31RA protein, preferably comprising all or part of a nucleotide sequence encoding a signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of a non-human animal IL31RA protein, further preferably comprising all or part of a nucleotide sequence encoding an extracellular region, transmembrane region and / or cytoplasmic region of a non-human animal IL31RA protein, more preferably comprising a nucleotide sequence encoding at least 1-5, for example, 1, 2, 3, 4 or 5 consecutive amino acids at the C-terminus of the extracellular region of a non-human animal IL31RA protein; more preferably comprising a nucleotide sequence encoding a non-human animal IL31RA protein. A nucleotide sequence encoding at least 1 to at least 21, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive amino acids of a protein transmembrane region; more preferably, a nucleotide sequence encoding at least 20 to at least 196, for example, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 160, 170, 180, 190, or 196 consecutive amino acids of a non-human animal IL31RA protein cytoplasmic region; further preferably, a nucleotide sequence encoding SEQ or a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the nucleotide sequence encoding the amino acid sequence as set forth in positions 495-716 or 500-716 of SEQ ID NO:60; or a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth in positions 495-716 or 500-716 of SEQ ID NO:60 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide. Alternatively, the nucleotide sequence comprises a nucleotide sequence having substitutions, deletions, and / or insertions of one or more nucleotides from the nucleotide sequence encoding the amino acid sequence as set forth in positions 495-716 or 500-716 of SEQ ID NO:60.

[0561] More preferably, the non-human animal is further constructed by introducing any one of the following nucleotide sequences into the IL31RA locus of the non-human animal:

[0562] E) a nucleotide sequence encoding the above-mentioned humanized IL31RA protein; or

[0563] F) The humanized IL31RA gene described above.

[0564] Preferably, the non-human animal is constructed by introducing the nucleotide sequence shown in SEQ ID NO: 66 into the IL31RA locus of a non-human animal; or, by introducing a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 66 into the IL31RA locus of a non-human animal; or, by introducing a nucleotide sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 66 into the IL31RA locus of a non-human animal; or, by introducing a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 66, including substitution, deletion and / or insertion of one or more nucleotides, into the IL31RA locus of a non-human animal.

[0565] Preferably, the non-human animal is constructed using the above-mentioned targeting vector.

[0566] Preferably, the non-human animal further comprises other gene modifications, more preferably, the other genes are selected from at least one of OSMR, OSM, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT and CD73.

[0567] Preferably, the human or humanized IL31RA gene and / or other genes are homozygous for the endogenous modified (preferably replaced or inserted) locus.

[0568] Preferably, the human or humanized IL31RA gene and / or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

[0569] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to produce humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0570] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0571] Therefore, the present invention provides a method for constructing a non-human animal with a humanized IL31RA gene, wherein the non-human animal expresses a human or humanized IL31RA protein, and / or the genome of the non-human animal comprises a portion of a human IL31RA gene or a humanized IL31RA gene.

[0572] In some embodiments, a method for preparing a genetically modified humanized animal comprises replacing a nucleic acid sequence encoding a region of endogenous IL31RA with a nucleotide sequence encoding a corresponding region of human IL31RA at an endogenous IL31RA locus (or site). In some embodiments, the method can comprise inserting a nucleic acid sequence encoding a region of human IL31RA and a sequence encoding a region of endogenous IL31RA at an endogenous IL31RA locus (or site). The inserted sequence can comprise regions (e.g., partial or complete) of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and / or exon 14 of the human IL31RA gene. In some embodiments, the sequence comprises a portion of exon 3, exons 4-13, and a portion of exon 14 of the human IL31RA gene (e.g., nucleotide sequence 614-2173 of NM_001242638.1). In some embodiments, the sequence includes a portion of exon 14, exons 15-16, and a portion of exon 17 of the endogenous IL31RA gene (e.g., nucleotide sequence 1904-2572 of NM_139299.2), a portion of the extracellular region of human IL31RA (e.g., amino acids 33-520 of SEQ ID NO: 61), an endogenous IL31RA transmembrane region (e.g., amino acids 500-520 of SEQ ID NO: 60), and an endogenous IL31RA cytoplasmic region (e.g., amino acids 521-716 of SEQ ID NO: 60). In some embodiments, the endogenous IL31RA locus (or site) is exon 4 and / or intron 4 of mouse IL31RA. In some embodiments, the sequence is inserted after the 5'UTR of the mouse IL31RA gene and before nucleotide 425 of NM_139299.2, and 1-3 bp of nucleotides are deleted (eg, inserted between nucleotides 421 to 425 of NM_139299.2, and nucleotides 422-424 are deleted).

[0573] In some embodiments, a method of modifying the IL31RA locus of a mouse to express a chimeric human / mouse IL31RA polypeptide can include replacing a nucleotide sequence encoding mouse IL31RA at an endogenous mouse IL31RA locus with a nucleotide sequence encoding human IL31RA, thereby generating a chimeric human / mouse IL31RA sequence. In some embodiments, the method can include inserting a nucleotide sequence encoding a chimeric human / mouse IL31RA at an endogenous mouse IL31RA locus, thereby generating a chimeric human / mouse IL31RA sequence.

[0574] In some embodiments, the nucleotide sequence encoding the chimeric human / mouse IL31RA can include a first nucleotide sequence encoding all or part of the extracellular region of human IL31RA; and a second nucleotide sequence encoding all or part of the extracellular, cytoplasmic, and transmembrane regions of mouse IL31RA.

[0575] The present invention also provides a method for establishing an IL31RA gene humanized animal model, comprising the following steps:

[0576] (a) providing a cell (e.g., a fertilized egg cell) according to the method described herein;

[0577] (b) culturing the cells in a liquid culture medium;

[0578] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0579] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0580] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0581] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pseudo-pregnancy).

[0582] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0583] The fertilized egg can be from any non-human animal, such as any of the non-human animals described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The genetic construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which can then give birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0584] In some embodiments, a method for preparing a genetically modified animal comprises modifying the coding frame of the IL31RA gene of a non-human animal, for example, by replacing a nucleic acid sequence (e.g., a DNA or cDNA sequence) encoding a region of endogenous IL31RA with a nucleotide sequence encoding a corresponding region of human IL31RA under the control of an endogenous regulatory element of the IL31RA gene of the non-human animal. For example, one or more functional region sequences of the IL31RA gene of the non-human animal can be deleted or inserted into a sequence such that the endogenous IL31RA protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the modified IL31RA gene of the non-human animal can be all or part of the nucleotide sequence of exon 1 to exon 17 of the IL31RA gene of the non-human animal.

[0585] In some embodiments, the method of preparing a genetically modified animal comprises inserting a nucleotide sequence encoding a human or humanized IL31RA protein and / or an auxiliary sequence after the endogenous regulatory elements of the IL31RA gene of a non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, such that the IL31RA gene humanized animal model can express the human or humanized IL31RA protein in vivo, but does not express the IL31RA protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0586] In some embodiments, the method for preparing a transgenic animal comprises:

[0587] (1) providing a plasmid comprising a human IL31RA gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous IL31RA;

[0588] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous IL31RA gene;

[0589] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0590] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express the humanized IL31RA protein;

[0591] (5) mating the offspring mice obtained in step (4) to obtain homozygous mice,

[0592] In some embodiments, fertilized eggs are modified using CRISPR technology with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal target site.

[0593] In some embodiments, the sequence encoding the humanized IL31RA protein is operably linked to endogenous regulatory elements at the endogenous IL31RA locus.

[0594] In some embodiments, the genetically modified animal does not express endogenous IL31RA protein.

[0595] In some embodiments, the method for preparing a transgenic animal comprises:

[0596] (1) providing a plasmid comprising a human or chimeric IL31RA gene fragment, said plasmid being flanked by 5' homology arms and 3' homology arms, wherein said 5' and 3' homology arms target endogenous IL31RA;

[0597] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous IL31RA gene;

[0598] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric IL31RA gene fragment into the genome.

[0599] In some embodiments, the nucleotide sequence encoding the endogenous IL31 region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding the corresponding region of human IL31. In some embodiments, the non-human animal's endogenous IL31 protein expression level is reduced or absent compared to wild-type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts, or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0600] Figure 24 shows a humanized targeting strategy for the mouse IL31 locus. The targeting vector comprises a 5' homology arm, a human or humanized IL31 gene fragment, and a 3' homology arm. The process involves using homologous recombination to replace the endogenous corresponding IL31 sequence with the human or humanized IL31 sequence. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace the mouse endogenous IL31 sequence with the human or humanized IL31 sequence.

[0601] Therefore, in some embodiments, the method for preparing a genetically modified humanized animal includes replacing the nucleic acid sequence encoding the endogenous IL31 region with a nucleotide sequence encoding the corresponding region of human IL31 at the endogenous IL31 locus (or site). The replaced sequence can include regions (e.g., part or all of) of exon 1, exon 2, and / or exon 3 of the human IL31 gene. In some embodiments, the sequence includes a portion of exon 1, exon 2, and exon 3 of the human IL31 gene (e.g., a nucleotide sequence of positions 28-522 of NM_001014336.1). In some embodiments, the sequence includes a portion of exon 1 and a portion of exon 3 of the endogenous IL31 gene (e.g., a nucleotide sequence of positions 1-52 and 545-810 of NM_029594.1).

[0602] The present invention also provides a method for establishing an IL31 gene humanized animal model, comprising the following steps:

[0603] (a) providing a cell (e.g., a fertilized egg cell) according to the method described herein;

[0604] (b) culturing the cells in a liquid culture medium;

[0605] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0606] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0607] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0608] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pseudo-pregnancy).

[0609] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0610] The fertilized egg can be from any non-human animal, such as any of the non-human animals described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The genetic construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which can then give birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0611] In some embodiments, the method for preparing a genetically modified animal includes modifying the coding frame of the IL31 gene of a non-human animal, for example, by replacing the nucleic acid sequence encoding the endogenous IL31 region with a nucleotide sequence encoding the corresponding region of human IL31 under the control of the endogenous regulatory elements of the IL31 gene of the non-human animal (e.g., DNA or cDNA sequence). For example, one or more functional region sequences of the IL31 gene of the non-human animal can be knocked out or inserted into a sequence so that the endogenous IL31 protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the IL31 gene of the modified non-human animal can be all or part of the nucleotide sequence of exon 1 to exon 3 of the IL31 gene of the non-human animal.

[0612] In some embodiments, the method for preparing a genetically modified animal comprises inserting a nucleotide sequence encoding a human or humanized IL31 protein and / or an auxiliary sequence after the endogenous regulatory elements of the IL31 gene of a non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the IL31 gene humanized animal model can express the human or humanized IL31 protein in vivo without expressing the IL31 protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0613] In some embodiments, the method for preparing a transgenic animal comprises:

[0614] (1) providing a plasmid comprising a human IL31 gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' and 3' homology arms target endogenous IL31;

[0615] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous IL31 gene;

[0616] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2), and Cas9;

[0617] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express the humanized IL31 protein;

[0618] (5) mating the offspring mice obtained in step (4) to obtain homozygous mice,

[0619] In some embodiments, fertilized eggs are modified using CRISPR technology with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal target site.

[0620] In some embodiments, the sequence encoding the humanized IL31 protein is operably linked to endogenous regulatory elements at the endogenous IL31 locus.

[0621] In some embodiments, the genetically modified animal does not express endogenous IL31 protein.

[0622] In some embodiments, the method for preparing a transgenic animal comprises:

[0623] (1) providing a plasmid comprising a human or chimeric IL31 gene segment, said plasmid being flanked by 5' homology arms and 3' homology arms, wherein said 5' and 3' homology arms target endogenous IL31;

[0624] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous IL31 gene;

[0625] (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric IL31 gene fragment into the genome.

[0626] In some embodiments, the nucleotide sequences described herein do not overlap with each other (eg, the first nucleotide sequence, the second nucleotide sequence, and / or the third nucleotide sequence do not overlap). In some embodiments, the amino acid sequences described herein do not overlap with each other.

[0627] Use of genetically modified non-human animals

[0628] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at an endogenous non-human locus and under the control of an endogenous promoter and / or regulatory elements can produce a non-human animal with qualities and characteristics that may be significantly different from those of a typical knockout plus transgenic animal. In a typical knockout plus transgenic animal, the endogenous locus is removed or destroyed and a fully human transgene is inserted into the genome of the animal and may integrate randomly into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and / or protein assays and / or functional assays. In a human transgene, the human sequences upstream and / or downstream provide suitable support for expression and / or regulation of the transgene.

[0629] In some cases, transgenes with human regulatory elements are expressed in a non-physiological or otherwise unsatisfactory manner and may actually be harmful to the animal. The present invention demonstrates that the replacement or insertion of human sequences at endogenous gene loci under the control of endogenous regulatory elements to generate humanized animals provides physiologically appropriate expression patterns and levels that are meaningful and appropriate in the context of the physiology of the humanized animal with respect to the physiology of the replaced gene.

[0630] Genetically modified animals that express human or humanized OSMR, OSM, IL31RA and / or IL31 proteins, e.g., in a physiologically appropriate manner, provide a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0631] The present invention also provides a use of the above-mentioned OSMR, OSM, IL31RA and / or IL31 gene-modified non-human animal, or a non-human animal obtained by any of the above-mentioned construction methods.

[0632] In some embodiments, the application comprises:

[0633] A) Applications in the development of products involving immune processes associated with OSMR, OSM, IL31RA and / or IL31 in human cells;

[0634] B) Use as a model system for pharmacological, immunological, microbiological and medical research related to OSMR, OSM, IL31RA and / or IL31;

[0635] C) applications involving the production and use of animal experimental disease models for the study of the etiology associated with OSMR, OSM, IL31RA and / or IL31 and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0636] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human OSMR, OSM, IL31RA and / or IL31 signaling pathway modulators; or

[0637] E) Study the functions of OSMR, OSM, IL31RA and / or IL31 genes, study the drugs and drug efficacy targeting human OSMR, OSM, IL31RA and / or IL31 target sites, and study the applications of OSMR, OSM, IL31RA and / or IL31 in inflammatory and immune-related disease drugs and anti-tumor drugs.

[0638] The present invention provides a non-human animal expressing human or humanized OSMR, OSM, IL31RA, and / or IL31 proteins, which can be used to screen for therapeutic agents specific for human OSMR, OSM, IL31RA, and / or IL31. The therapeutic agent can reduce or block the interaction between OSM and the OSM receptor complex (or the interaction between IL31 and the IL31 receptor complex), and / or IL31 can bind to its target antigen, to test whether the therapeutic agent can increase or decrease the immune response, and / or to determine whether the therapeutic agent is an agonist or antagonist of OSMR, OSM, IL31RA, and / or IL31. In some embodiments, the non-human animal is an animal model for human disease. For example, the disease is genetically induced (knock-in or knock-out). In various embodiments, the genetically modified non-human animal further comprises a compromised immune system, such as a genetically modified human tissue xenograft, including a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocytic tumor, a B or T cell tumor).

[0639] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-OSMR, OSM, IL31RA and / or IL31 antibodies; or drugs targeting the OSM / IL31 signaling pathway) in treating tumors. In some embodiments, the method involves administering a therapeutic agent to an animal as described herein, wherein the animal has cancer or a tumor; and determining the inhibitory effect of the therapeutic agent on the cancer or tumor. Inhibitory effects that can be determined include, for example, a reduction in tumor size or tumor volume, a reduction in tumor growth, a reduction in the rate of increase of tumor volume in a subject (e.g., compared to the rate of increase of tumor volume in the same subject before treatment or in another subject that has not received such treatment), a reduction in the risk of metastasis or the risk of one or more additional metastases, an increase in survival rate, and an increase in life expectancy. The tumor volume of a subject can be determined by various methods, such as by direct measurement, MRI, or CT. In some embodiments, antibodies can directly target the expression of OSMR, OSM, IL31RA, and / or IL31.

[0640] In some embodiments, the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) injected into the animal. In some embodiments, the antibody activates the OSM and / or IL31 signaling pathway. In some embodiments, the antibody does not activate the OSM and / or IL31 signaling pathway.

[0641] In some embodiments, genetically modified animals can be used to determine whether an antibody is an agonist or antagonist of OSMR, OSM, IL31RA, and / or IL31. In some embodiments, the methods described herein are also designed to determine the effect of a therapeutic agent (e.g., an antibody targeting OSMR, OSM, IL31RA, and / or IL31; or a drug targeting the OSM / IL31 signaling pathway) on OSMR, OSM, IL31RA, and / or IL31, for example, whether the therapeutic agent can upregulate or downregulate an immune response, and / or whether the agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, transgenic animals can be used to determine the effective dose of a therapeutic agent to treat a disease, such as cancer, in a subject.

[0642] The inhibitory effect on tumors can also be determined by methods known in the art, for example, by measuring the tumor volume in animals, and / or determining the tumor (volume) inhibition rate (TGI). TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%)=(1-TV t / TVc)×100, where TV t and TVc are the mean tumor volumes (or weights) of the treatment and control groups.

[0643] In some embodiments, therapeutic agents (e.g., antibodies targeting OSMR, OSM, IL31RA and / or IL31; or drugs targeting the OSM / IL31 signaling pathway) are designed to treat various cancers. As used herein, the term "cancer" refers to cells with autonomous growth capacity, i.e., an abnormal state or state characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. The term "tumor...

Claims

1. A genetically modified non-human animal, characterized in that The genome of the animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric Oncostatin M Receptor (OSMR) protein.

2. The animal according to claim 1, characterized in that The nucleotide sequence encoding the human or chimeric OSMR protein is operably linked to endogenous regulatory elements (eg, endogenous 5'UTR and / or 3'UTR) of the endogenous OSMR locus on at least one chromosome.

3. The animal according to claim 1 or 2, characterized in that The chimeric OSMR protein comprises a human signal peptide, a human or humanized extracellular region, and endogenous intracellular and transmembrane regions.

4. The animal according to claim 1 or 2, characterized in that The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human OSMR (NP_001310434.1, SEQ ID NO: 10).

5. The animal according to claim 1 or 2, characterized in that The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-737 of SEQ ID NO:

10.

6. The animal according to claim 1 or 2, characterized in that The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:

16.

7. The animal according to any one of claims 1 to 6, characterized in that The animal is a mammal, such as a monkey, rodent, mouse or rat.

8. The animal according to any one of claims 7, characterized in that The animal is a mouse.

9. The animal according to any one of claims 1 to 8, characterized in that The animal does not express endogenous OSMR protein or the expression level is reduced compared to OSMR in wild-type animals.

10. The animal according to any one of claims 1 to 9, characterized in that One or more cells of the animal express human or chimeric OSMR protein.

11. A genetically modified non-human animal, characterized in that The genome of the animal comprises a nucleotide sequence encoding the corresponding region of human or chimeric OSMR inserted at the endogenous OSMR locus.

12. The animal according to claim 11, characterized in that The nucleotide sequence encoding the corresponding region of human or chimeric OSMR is operably linked to endogenous regulatory elements of the endogenous OSMR locus, and one or more cells of the animal express the human or humanized OSMR protein.

13. The animal according to claim 11 or 12, characterized in that The animal's endogenous OSMR protein is not expressed or is expressed at a reduced level compared to OSMR in wild-type animals.

14. The animal according to any one of claims 11 to 13, characterized in that The nucleotide sequence encoding the corresponding region of human or chimeric OSMR is inserted into exon 2 of the endogenous OSMR locus.

15. The animal according to any one of claims 11 to 14, characterized in that The nucleotide sequence encoding the corresponding region of human or chimeric OSMR is inserted before the 129th nucleotide of NM_011019.

3.

16. The animal according to claim 15, characterized in that The nucleotide sequence encoding the corresponding region of human or chimeric OSMR is inserted between nucleotides 125 and 129 of NM_011019.

3.

17. The animal according to any one of claims 11 to 16, characterized in that At least 3 bp of exon 2 of the endogenous OSMR locus (such as nucleotides 126 to 128 of NM_011019.3) are deleted.

18. The animal according to any one of claims 11 to 17, characterized in that The insert sequence comprises, optionally, 5'-3': A) the first sequence encodes the entire or part of the human OSMR signal peptide and extracellular region; and B) The second sequence encodes all or part of the endogenous OSMR transmembrane region and intracellular region, and optionally also includes part of the endogenous OSMR extracellular region.

19. The animal according to claim 18, characterized in that The amino acid sequence encoded by the first sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-737 of SEQ ID NO: 10, and the amino acid sequence encoded by the second sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 735-971 of SEQ ID NO:

9.

20. The animal according to claim 18 or 19, characterized in that The insert sequence further comprises one or more auxiliary sequences.

21. The animal according to claim 20, characterized in that The auxiliary sequence is a polyA sequence.

22. The animal according to any one of claims 11 to 21, characterized in that The genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 13, 14, 15 or 51.

23. The animal according to any one of claims 11 to 22, characterized in that The inserted sequence in the genome of the animal is homozygous and / or heterozygous for the endogenous OSMR locus.

24. A non-human animal, characterized in that The animal comprises at least one cell that encodes a nucleotide sequence of a chimeric OSMR protein, wherein the chimeric OSMR protein comprises at least 50, 100, 200, 300, 400, 500, 600, 700, 710, 720, 730, 737, 740, 760, 800, 900, or 979 consecutive amino acids that are identical to the consecutive amino acid sequence of the corresponding region in humans, and the animal expresses the chimeric OSMR protein.

25. The animal according to claim 24, characterized in that The chimeric OSMR protein comprises at least 50, 100, 200, 300, 400, 500, 600, 700, 710, 720, 730, 735, 737, 738, 739 or 740 consecutive amino acids that are identical to the corresponding consecutive amino acid sequences of the human signal peptide and extracellular region.

26. The animal according to claim 24 or 25, characterized in that The chimeric OSMR protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-737 of SEQ ID NO:

10.

27. The animal according to any one of claims 24 to 26, characterized in that The nucleotide sequence encoding the chimeric OSMR protein is operably linked to an endogenous OSMR regulatory element.

28. The animal according to any one of claims 24 to 27, characterized in that The chimeric OSMR protein comprises the transmembrane and intracellular regions of endogenous OSMR, and optionally also comprises a portion of the extracellular region of endogenous OSMR.

29. The animal according to any one of claims 24 to 28, characterized in that The nucleotide sequence encoding the corresponding region of human OSMR is integrated into the animal's endogenous OSMR locus.

30. The animal according to any one of claims 24 to 29, characterized in that The chimeric OSMR protein has at least one of a mouse OSMR activity and / or a human OSMR activity.

31. A method for constructing a genetically modified non-human animal, characterized in that: A nucleotide sequence encoding a chimeric OSMR polypeptide is introduced into at least one cell of the animal at the animal's endogenous OSMR locus.

32. The method according to claim 31, wherein: The chimeric OSMR polypeptide comprises: A) all or part of the human OSMR signal peptide and extracellular region; and B) All or part of the endogenous OSMR transmembrane region and intracellular region, and optionally also part of the endogenous OSMR extracellular region.

33. The method according to claim 31 or 32, characterized in that: The nucleotide sequence encoding the chimeric OSMR polypeptide is inserted into exon 2 of the animal's endogenous OSMR gene locus.

34. The method according to any one of claims 31 to 33, characterized in that: At least 3 bp of exon 2 of the endogenous OSMR locus (such as nucleotides 126 to 128 of NM_011019.3) are deleted.

35. The method according to any one of claims 31 to 34, characterized in that: The nucleotide sequence encoding the chimeric OSMR corresponding region is operably linked to an endogenous OSMR regulatory element, such as a promoter.

36. The method according to any one of claims 31 to 35, characterized in that: The animal is a mammal, such as a monkey, rodent, mouse or rat.

37. A method for constructing a genetically modified non-human animal cell expressing a chimeric OSMR, the method comprising inserting the following nucleotide sequence at the endogenous mouse OSMR locus (e.g., exon 2 of the endogenous OSMR gene), from 5' to 3': A) Nucleotide sequence encoding all or part of the human OSMR signal peptide and extracellular region; B) the nucleotide sequence of all or part of the endogenous OSMR transmembrane region and intracellular region, and optionally also comprising part of the endogenous OSMR extracellular region; C) optionally one or more auxiliary sequences (e.g., polyA sequences), Thereby, a genetically modified non-human animal cell comprising a nucleotide sequence encoding a chimeric OSMR protein is produced, wherein the animal cell expresses the chimeric OSMR protein.

38. The method according to claim 37, wherein The nucleotide sequence encoding the corresponding region of human OSMR includes part of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, and / or part of exon 15 of the human OSMR gene.

39. The method according to claim 37 or 38, characterized in that The nucleotide sequence encoding the corresponding region of human OSMR is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 13 or 14.

40. The method according to any one of claims 37 to 39, characterized in that: The nucleotide sequence encoding the corresponding region of endogenous OSMR includes a portion of exon 15, exon 16, exon 17, and / or a portion of exon 18 of the endogenous OSMR gene.

41. The method according to any one of claims 37 to 40, characterized in that: The nucleotide sequence encoding the corresponding region of endogenous OSMR is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 13 or 15.

42. The method according to any one of claims 37 to 41, characterized in that: The nucleotide sequence encoding the chimeric OSMR protein is operably linked to a regulatory element of endogenous OSMR, such as a promoter.

43. The method according to any one of claims 37 to 42, characterized in that: The non-human animal is a mouse.

44. The animal according to any one of claims 1 to 30, characterized in that The non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins are selected from at least one of OSM, IL31RA, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT and CD73.

45. The animal according to claim 44, characterized in that The human or chimeric protein is selected from OSM, IL31RA and / or IL31 protein.

46. ​​The method according to any one of claims 31 to 43, characterized in that The non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins are selected from at least one of OSM, IL31RA, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT and CD73.

47. The method according to claim 46, wherein The human or chimeric protein is selected from OSM, IL31RA and / or IL31 protein.

48. A genetically modified non-human animal, characterized in that The genome of the animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric interleukin 31 receptor A (IL31RA) protein.

49. The animal according to claim 48, characterized in that The nucleotide sequence encoding the human or chimeric IL31RA protein is operably linked to endogenous regulatory elements (eg, endogenous 5'UTR and / or 3'UTR) of the endogenous IL31RA locus on at least one chromosome.

50. The animal according to claim 48 or 49, characterized in that The chimeric IL31RA protein comprises a human signal peptide, a human or humanized extracellular region, and endogenous intracellular and transmembrane regions.

51. The animal according to claim 48 or 49, characterized in that The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric IL31RA protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to human IL31RA (NP_001229567.1, SEQ ID NO: 61).

52. The animal according to claim 48 or 49, characterized in that The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric IL31RA protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-520 of SEQ ID NO:

61.

53. The animal according to claim 48 or 49, characterized in that The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric IL31RA protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:

68.

54. The animal according to any one of claims 48 to 53, characterized in that The animal is a mammal, such as a monkey, rodent, mouse or rat.

55. The animal according to any one of claims 54, characterized in that The animal is a mouse.

56. The animal according to any one of claims 48 to 55, characterized in that The animal does not express endogenous IL31RA protein or the expression level is reduced compared to IL31RA in wild-type animals.

57. The animal according to any one of claims 48 to 56, characterized in that One or more cells of the animal express human or chimeric IL31RA protein.

58. A genetically modified non-human animal, characterized in that The genome of the animal comprises a nucleotide sequence encoding the corresponding region of human or chimeric IL31RA inserted at the endogenous IL31RA locus.

59. The animal according to claim 58, characterized in that The nucleotide sequence encoding the corresponding region of human or chimeric IL31RA is operably linked to endogenous regulatory elements of the endogenous IL31RA locus, and one or more cells of the animal express the human or humanized IL31RA protein.

60. The animal according to claim 58 or 59, characterized in that The animals do not express endogenous IL31RA protein or express it at a reduced level compared to IL31RA in wild-type animals.

61. The animal according to any one of claims 58 to 61, characterized in that The nucleotide sequence encoding the corresponding region of human or chimeric IL31RA is inserted into exon 4 of the endogenous IL31RA locus.

62. The animal according to any one of claims 58 to 62, characterized in that The nucleotide sequence encoding the corresponding region of human or chimeric IL31RA is inserted before the 425th nucleotide of NM_139299.

2.

63. The animal according to claim 62, characterized in that The nucleotide sequence encoding the corresponding region of human or chimeric IL31RA is inserted between nucleotides 421 to 425 of NM_139299.

2.

64. The animal according to any one of claims 58 to 63, characterized in that At least 3 bp of exon 4 of the endogenous IL31RA locus (such as nucleotides 422 to 424 of NM_139299.2) are deleted.

65. The animal according to any one of claims 58 to 64, characterized in that The insert sequence comprises, optionally, 5'-3': A) the first sequence encodes the human IL31RA signal peptide and all or part of the extracellular region; and B) The second sequence encodes all or part of the endogenous IL31RA transmembrane region and intracellular region, and optionally also includes part of the endogenous IL31RA extracellular region.

66. The animal according to claim 65, characterized in that The amino acid sequence encoded by the first sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-520 of SEQ ID NO: 61, and the amino acid sequence encoded by the second sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 495-716 of SEQ ID NO:

60.

67. The animal according to claim 65 or 66, characterized in that The insert sequence further comprises one or more auxiliary sequences.

68. The animal according to claim 67, characterized in that The auxiliary sequence is a WPRE sequence and / or a polyA sequence.

69. The animal according to any one of claims 65 to 68, characterized in that The genome of the animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 64, 65, 66 or 67.

70. The animal according to any one of claims 65 to 69, characterized in that The inserted sequence in the genome of the animal is homozygous and / or heterozygous for the endogenous IL31RA locus.

71. A non-human animal, characterized in that The animal comprises at least one cell that contains a nucleotide sequence encoding a chimeric IL31RA protein, wherein the chimeric IL31RA protein comprises at least 50, 100, 200, 300, 400, 450, 500, 520, 550, 600, 660, 661, or 662 consecutive amino acids that are identical to the corresponding region in a human, and the animal expresses the chimeric IL31RA protein.

72. The animal according to claim 71, characterized in that The chimeric IL31RA protein comprises at least 50, 100, 200, 300, 400, 450, 500, 520, 525, 530, 531 or 532 consecutive amino acids that are identical to the corresponding consecutive amino acid sequences of the human signal peptide and extracellular region.

73. The animal according to claim 71 or 72, characterized in that The chimeric IL31RA protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to amino acids 1-520 of SEQ ID NO:

61.

74. The animal according to any one of claims 71 to 73, characterized in that The nucleotide sequence encoding the chimeric IL31RA protein is operably linked to endogenous IL31RA regulatory elements.

75. The animal according to any one of claims 71 to 74, characterized in that The chimeric IL31RA protein comprises the transmembrane and intracellular regions of endogenous IL31RA, and optionally also comprises a portion of the extracellular region of endogenous IL31RA.

76. The animal according to any one of claims 71 to 75, characterized in that The nucleotide sequence encoding the corresponding region of human IL31RA is integrated into the endogenous IL31RA gene locus of the animal.

77. The animal according to any one of claims 71 to 76, characterized in that The chimeric IL31RA protein has at least one of a mouse IL31RA activity and / or a human IL31RA activity.

78. A method for constructing a genetically modified non-human animal, characterized in that: A nucleotide sequence encoding a chimeric IL31RA polypeptide is introduced into at least one cell of the animal at the animal's endogenous IL31RA locus.

79. The method according to claim 78, characterized in that The nucleotide sequence encoding the chimeric IL31RA polypeptide is inserted into exon 4 of the animal's endogenous IL31RA gene locus.

80. The method according to claim 78 or 79, characterized in that: The chimeric IL31RA polypeptide comprises: A) all or part of the human IL31RA signal peptide and extracellular region; and B) All or part of the endogenous IL31RA transmembrane region and intracellular region, and optionally also part of the endogenous IL31RA extracellular region.

81. The method according to any one of claims 78 to 80, characterized in that At least 3 bp of exon 4 of the endogenous IL31RA locus (such as nucleotides 422 to 424 of NM_139299.2) are deleted.

82. The method according to any one of claims 78 to 81, characterized in that The nucleotide sequence encoding the chimeric IL31RA corresponding region is operably linked to an endogenous IL31RA regulatory element, such as a promoter.

83. The method according to any one of claims 78 to 82, characterized in that The animal is a mammal, such as a monkey, rodent, mouse or rat.

84. A method for constructing a genetically modified non-human animal cell expressing a chimeric IL31RA, the method comprising inserting the following nucleotide sequence, from 5' to 3', at the endogenous mouse IL31RA locus (e.g., exon 4 of the endogenous IL31RA gene): A) Nucleotide sequence encoding all or part of the human IL31RA signal peptide and extracellular region; B) the nucleotide sequence of all or part of the endogenous IL31RA transmembrane region and intracellular region, and optionally also comprising part of the endogenous IL31RA extracellular region; C) optionally one or more auxiliary sequences (e.g., a polyA sequence and / or a WPRE sequence), Thereby, a genetically modified non-human animal cell comprising a nucleotide sequence encoding a chimeric IL31RA protein is generated, wherein the animal cell expresses the chimeric IL31RA protein.

85. The method according to claim 84, characterized in that The nucleotide sequence encoding the corresponding region of human IL31RA includes part of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and / or part of exon 14 of the human IL31RA gene.

86. The method according to claim 84 or 85, characterized in that The nucleotide sequence encoding the corresponding region of human IL31RA is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 64 or 66.

87. The method according to any one of claims 84-86, characterized in that The nucleotide sequence encoding the corresponding region of endogenous IL31RA includes a portion of exon 14, exon 15, exon 16, and / or a portion of exon 17 of the endogenous IL31RA gene.

88. The method according to any one of claims 84-87, characterized in that The nucleotide sequence encoding the corresponding region of endogenous IL31RA is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 65 or 66.

89. The method according to any one of claims 84-88, characterized in that The nucleotide sequence encoding the chimeric IL31RA protein is operably linked to a regulatory element of endogenous IL31RA, such as a promoter.

90. The method according to any one of claims 84-89, characterized in that The non-human animal is a mouse.

91. The animal according to any one of claims 48 to 77, characterized in that The non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric protein is selected from at least one of OSM, OSMR, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT or CD73.

92. The animal according to claim 91, characterized in that The human or chimeric protein is selected from OSM, OSMR and / or IL31 protein.

93. The method according to any one of claims 78 to 90, characterized in that The non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the human or chimeric proteins are selected from at least one of OSM, OSMR, IL31, PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, IL6ST, IL6, IL4, IL13, 4-1BB, CD47, TIGIT and CD73.

94. The animal according to claim 93, characterized in that The human or chimeric protein is selected from OSM, OSMR and / or IL31 protein.

95. A method for determining the effectiveness of a therapeutic agent in treating cancer, characterized in that The method comprises: 1) administering a therapeutic agent to an animal according to any one of claims 1-30, 44-45, 48-77 and 91-92, wherein the animal has cancer; 2) Determine the inhibitory effect of therapeutic agents on cancer.

96. The method according to claim 95, characterized in that The therapeutic agent is an anti-OSM antibody, an anti-OSMR antibody, an anti-IL31 antibody and / or an anti-IL31RA antibody.

97. The method according to claim 95 or 96, characterized in that The cancer is a tumor, and the inhibitory effect of the therapeutic agent on the tumor is determined by measuring the tumor volume of the animal.

98. The method according to any one of claims 95-97, characterized in that The cancer comprises injecting one or more cancer cells into an animal.

99. The method according to any one of claims 95-98, characterized in that The cancers include ovarian cancer, female reproductive system cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, etc.

100. A method for determining the effectiveness of an anti-OSM antibody, an anti-OSMR antibody, an anti-IL31 antibody and / or an anti-IL31RA antibody and an additional therapeutic agent in treating cancer, characterized in that The method comprises: 1) administering an anti-OSM antibody, an anti-OSMR antibody, an anti-IL31 antibody and / or an anti-IL31RA antibody and an additional therapeutic agent to the animal of any one of claims 1-30, 44-45, 48-77 and 91-92, wherein the animal has a tumor; 2) Determine the inhibitory effect of OSMR, OSM, IL31RA and / or IL31 therapeutic agents on tumors.

101. The method according to claim 100, characterized in that The additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.

102. The method according to claim 100 or 101, characterized in that The tumor comprises injecting one or more cancer cells into the animal.

103. The method according to any one of claims 100-102, characterized in that: The tumor volume of the animals was measured to determine the inhibitory effect of the therapeutic agent on the tumor.

104. The method according to any one of claims 100-103, characterized in that: The cancers include ovarian cancer, female reproductive system cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, etc.

105. A method for determining the effectiveness of a therapeutic agent in treating an immune disease, characterized in that: The method comprises: 1) administering a therapeutic agent to an animal according to any one of claims 1-30, 44-45, 48-77 and 91-92, wherein the animal suffers from an immune disease; 2) Determine the therapeutic effect of therapeutic agents on immune diseases.

106. The method according to claim 105, characterized in that The immune diseases include rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, atherosclerosis, interstitial lung disease, psoriasis, allergy, Crohn's disease, pulmonary fibrosis, etc.

107. A method for determining the effectiveness of a therapeutic agent in treating inflammation, characterized in that The method comprises: 1) administering a therapeutic agent to an animal according to any one of claims 1-30, 44-45, 48-77 and 91-92, wherein the animal suffers from inflammation; 2) Determine the therapeutic effect of the therapeutic agent on inflammation.

108. The method according to claim 107, characterized in that The inflammation is atopic dermatitis, pruritus, prurigo, dermatitis and the like.

109. A method for determining the toxicity of a therapeutic agent, characterized in that The method comprises: 1) administering a therapeutic agent to an animal according to any one of claims 1-30, 44-45, 48-77 and 91-92; 2) Determine the effects of the therapeutic agent on the animals.

110. The method according to claim 109, characterized in that Determining the effect of the therapeutic agent on the animal comprises measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

111. A humanized OSMR protein, characterized in that The humanized OSMR protein comprises all or part of the human OSMR protein.

112. The humanized OSMR protein according to claim 111, characterized in that The humanized OSMR protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:

13.

113. A humanized OSMR gene, characterized in that The humanized OSMR gene encodes the humanized protein according to any one of claims 202-203.

114. The humanized OSMR gene according to claim 113, characterized in that The humanized OSMR gene includes part of exon 2, all of exon 3 to exon 14, and / or part of exon 15 of the human OSMR gene. The humanized OSMR gene also includes part of exon 15, all of exon 16 to exon 17, and / or part of exon 18 of the non-human animal OSMR gene.

115. The humanized OSMR gene according to claim 113 or 114, characterized in that The nucleotide sequence comprised by the humanized OSMR gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO:

13.

116. A humanized IL31RA protein, characterized in that The humanized IL31RA protein comprises all or part of the human IL31RA protein.

117. The humanized IL31RA protein according to claim 116, characterized in that The humanized IL31RA protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:

68.

118. A humanized IL31RA gene, characterized in that The humanized IL31RA gene encodes the humanized protein according to any one of claims 208-209.

119. The humanized IL31RA gene according to claim 118, wherein The humanized IL31RA gene comprises a portion of exon 3, all of exon 4 to exon 13, and / or a portion of exon 14 of the human IL31RA gene. The humanized IL31RA gene further comprises a portion of exon 14, all of exon 15 to exon 16, and / or a portion of exon 17 of the non-human animal IL31RA gene.

120. The humanized IL31RA gene according to claim 118 or 119, wherein The nucleotide sequence comprised by the humanized IL31RA gene is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

66.

121. A cell, characterized in that The cell comprises the humanized gene of any one of claims 113-115, 118-120, and / or the cell expresses the humanized protein of any one of claims 111-112, 116-117.

122. An animal model, characterized in that The animal model comprises the humanized gene of any one of claims 113-115, 118-120, and / or the animal model expresses the humanized protein of any one of claims 111-112, 116-117.

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