Genetically modified non-human animal with human or chimeric genes

Abstract

The present disclosure relates to genetically modified non-human animals that express a human or chimeric (e.g., humanized) IL4R and/or IL4, and methods of use thereof.

What is claimed is: 
     
         1 . A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric interleukin-4 receptor (IL4R). 
     
     
         2 . The animal of  claim 1 , wherein the sequence encoding the human or chimeric IL4R is operably linked to an endogenous regulatory element at the endogenous IL4R gene locus in the at least one chromosome. 
     
     
         3 . The animal of  claim 1 , wherein the sequence encoding a human or chimeric IL4R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human IL4R (NP_000409.1; SEQ ID NO: 42). 
     
     
         4 . The animal of  claim 1 , wherein the sequence encoding a human or chimeric IL4R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 44. 
     
     
         5 . The animal of  claim 1 , wherein the sequence encoding a human or chimeric IL4R comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 30-216 of SEQ ID NO: 42. 
     
     
         6 . The animal of any one of  claims 1 - 5 , wherein the animal is a mammal, e.g., a monkey, a rodent or a mouse. 
     
     
         7 . The animal of any one of  claims 1 - 5 , wherein the animal is a mouse. 
     
     
         8 . The animal of any one of  claims 1 - 7 , wherein the animal does not express endogenous IL4R. 
     
     
         9 . The animal of  claim 1 , wherein the animal has one or more cells expressing human or chimeric IL4R. 
     
     
         10 . The animal of  claim 1 , wherein the animal has one or more cells expressing human or chimeric IL4R, and the expressed human or chimeric IL4R can bind to endogenous IL4. 
     
     
         11 . The animal of  claim 1 , wherein the animal has one or more cells expressing human or chimeric IL4R, and the expressed human or chimeric IL4R can bind to human IL4. 
     
     
         12 . A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL4R with a sequence encoding a corresponding region of human IL4R at an endogenous IL4R gene locus. 
     
     
         13 . The animal of  claim 12 , wherein the sequence encoding the corresponding region of human IL4R is operably linked to an endogenous regulatory element at the endogenous IL4R locus, and one or more cells of the animal express a chimeric IL4R. 
     
     
         14 . The animal of  claim 12 , wherein the animal does not express endogenous IL4R. 
     
     
         15 . The animal of  claim 12 , wherein the replaced locus is the extracellular region of IL4R. 
     
     
         16 . The animal of  claim 12 , wherein the animal has one or more cells expressing a chimeric IL4R having an extracellular region, wherein 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 IL4R. 
     
     
         17 . The animal of  claim 16 , wherein the extracellular region of the chimeric IL4R has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 150 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human IL4R. 
     
     
         18 . The animal of  claim 12 , wherein the animal is a mouse, and the replaced endogenous IL4R region is exon 4, exon 5, exon 6, and/or exon 7 of the endogenous mouse IL4R gene. 
     
     
         19 . The animal of  claim 12 , wherein the animal is heterozygous with respect to the replacement at the endogenous IL4R gene locus. 
     
     
         20 . The animal of  claim 12 , wherein the animal is homozygous with respect to the replacement at the endogenous IL4R gene locus. 
     
     
         21 . A method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous IL4R gene locus, a sequence encoding a region of an endogenous IL4R with a sequence encoding a corresponding region of human IL4R. 
     
     
         22 . The method of  claim 21 , wherein the sequence encoding the corresponding region of human IL4R comprises exon 4, exon 5, exon 6, and/or exon 7 of a human IL4R gene. 
     
     
         23 . The method of  claim 21 , wherein the sequence encoding the corresponding region of IL4R comprises at least 100, 200, or 300 nucleotides of exon 4, exon 5, exon 6 and/or exon 7 of a human IL4R gene. 
     
     
         24 . The method of  claim 21 , wherein the sequence encoding the corresponding region of human IL4R encodes a sequence that is at least 90% identical to amino acids 30-216 of SEQ ID NO: 42. 
     
     
         25 . The method of  claim 21 , wherein the locus is located within the extracellular region of IL4R. 
     
     
         26 . The method of  claim 21 , wherein the animal is a mouse, and the locus is exons 4, exon 5, exon 6 and/or exon 7 of the mouse IL4R gene. 
     
     
         27 . A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL4R polypeptide, wherein the chimeric IL4R polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4R, wherein the animal expresses the chimeric IL4R. 
     
     
         28 . The animal of  claim 27 , wherein the chimeric IL4R polypeptide has at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4R extracellular region. 
     
     
         29 . The animal of  claim 27 , wherein the chimeric IL4R polypeptide comprises a sequence that is at least 90%, 95%, or 99% identical to amino acids 30-216 of SEQ ID NO: 42. 
     
     
         30 . The animal of  claim 27 , wherein the nucleotide sequence is operably linked to an endogenous IL4R regulatory element of the animal. 
     
     
         31 . The animal of  claim 27 , wherein the chimeric IL4R polypeptide comprises an endogenous IL4R transmembrane region and/or an endogenous cytoplasmic region. 
     
     
         32 . The animal of  claim 27 , wherein the nucleotide sequence is integrated to an endogenous IL4R gene locus of the animal. 
     
     
         33 . The animal of  claim 27 , wherein the chimeric IL4R has at least one mouse IL4R activity and/or at least one human IL4R activity. 
     
     
         34 . A method of making a genetically-modified mouse cell that expresses a chimeric IL4R, the method comprising:
 replacing, at an endogenous mouse IL4R gene locus, a nucleotide sequence encoding a region of mouse IL4R with a nucleotide sequence encoding a corresponding region of human IL4R, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the chimeric IL4R, wherein the mouse cell expresses the chimeric IL4R.   
     
     
         35 . The method of  claim 34 , wherein the chimeric IL4R comprises:
 an extracellular region of human IL4R;   a transmembrane region of mouse IL4R; and/or   a cytoplasmic region of mouse IL4R.   
     
     
         36 . The method of  claim 35 , wherein the nucleotide sequence encoding the chimeric IL4R is operably linked to an endogenous IL4R regulatory region, e.g., promoter. 
     
     
         37 . The animal of any one of  claims 1 - 20  and  27 - 33 , wherein the animal further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4, IL33, IL13, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), CD137, TNF Receptor Superfamily Member 4 (OX40), CD47, or Signal regulatory protein α (SIRPa)). 
     
     
         38 . The animal of  claim 37 , wherein the additional human or chimeric protein is IL4. 
     
     
         39 . The method of any one of  claims 21 - 26  and  34 - 36 , wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4, IL33, PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, TIGIT, TIM-3, GITR, CD137, OX40, CD47 or SIRPa). 
     
     
         40 . The method of  claim 39 , wherein the additional human or chimeric protein is IL4. 
     
     
         41 . A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric IL4. 
     
     
         42 . The animal of  claim 41 , wherein the sequence encoding the human or chimeric IL4 is operably linked to an endogenous regulatory element at the endogenous IL4 gene locus in the at least one chromosome. 
     
     
         43 . The animal of  claim 41 , wherein the sequence encoding the human or chimeric IL4 is operably linked to a human regulatory element at the endogenous IL4 gene locus in the at least one chromosome. 
     
     
         44 . The animal of  claim 41 , wherein the sequence encoding a human or chimeric IL4 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human IL4 (NP_000580.1; SEQ ID NO: 4). 
     
     
         45 . The animal of  claim 41 , wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8 or SEQ ID NO: 9. 
     
     
         46 . The animal of  claim 41 , wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10 or SEQ ID NO: 11. 
     
     
         47 . The animal of  claim 41 , wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 23. 
     
     
         48 . The animal of  claim 41 , wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 24. 
     
     
         49 . The animal of  claim 41 , wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 25. 
     
     
         50 . The animal of any one of  claims 41 - 49 , wherein the animal is a mammal, e.g., a monkey, a rodent or a mouse. 
     
     
         51 . The animal of any one of  claims 41 - 49 , wherein the animal is a mouse. 
     
     
         52 . The animal of any one of  claims 41 - 51 , wherein the animal does not express endogenous IL4. 
     
     
         53 . The animal of  claim 41 , wherein the animal has one or more cells expressing human IL4. 
     
     
         54 . The animal of  claim 41 , wherein the animal has one or more cells expressing human or chimeric IL4, and the expressed human or chimeric IL4 can bind to endogenous IL4R. 
     
     
         55 . The animal of  claim 41 , wherein the animal has one or more cells expressing human or chimeric IL4, and the expressed human or chimeric IL4 can bind to human IL4R. 
     
     
         56 . A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL4 with a sequence encoding a corresponding region of human IL4 at an endogenous IL4 gene locus. 
     
     
         57 . The animal of  claim 56 , wherein the sequence encoding the corresponding region of human IL4 is operably linked to an endogenous regulatory element at the endogenous IL4 locus, and one or more cells of the animal expresses a human IL4. 
     
     
         58 . The animal of  claim 56 , wherein the sequence encoding the corresponding region of human IL4 is operably linked to a human regulatory element at the endogenous IL4 locus, and one or more cells of the animal expresses a human IL4. 
     
     
         59 . The animal of  claim 56 , wherein the animal does not express endogenous IL4. 
     
     
         60 . The animal of  claim 56 , wherein the replaced locus comprises a sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. 
     
     
         61 . The animal of  claim 56 , wherein the animal is a mouse, and the replaced endogenous IL4 region is exon 1, exon 2, exon 3 and/or exon 4 of the endogenous mouse IL4 gene. 
     
     
         62 . The animal of  claim 56 , wherein the animal is heterozygous with respect to the replacement at the endogenous IL4 gene locus. 
     
     
         63 . The animal of  claim 56 , wherein the animal is homozygous with respect to the replacement at the endogenous IL4 gene locus. 
     
     
         64 . A method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous IL4 gene locus, a sequence encoding a region of an endogenous IL4 with a sequence encoding a corresponding region of human IL4. 
     
     
         65 . The method of  claim 64 , wherein the sequence encoding the corresponding region of human IL4 comprises exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene. 
     
     
         66 . The method of  claim 64 , wherein the sequence encoding the corresponding region of IL4 comprises at least 30, 50, 75, 100, or 150 nucleotides of exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene. 
     
     
         67 . The method of  claim 64 , wherein the sequence encoding the corresponding region of human IL4 encodes a sequence that is at least 90% identical to SEQ ID NO: 4. 
     
     
         68 . The method of  claim 64 , wherein replaced locus comprises a sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. 
     
     
         69 . The method of  claim 64 , wherein the animal is a mouse, and the locus is exon 1, exon 2, exon 3 and exon 4 of the mouse IL4 gene. 
     
     
         70 . A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a human or chimeric IL4 polypeptide, wherein the chimeric IL4 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4, wherein the animal expresses the chimeric IL4. 
     
     
         71 . The animal of  claim 70 , wherein the chimeric IL4 polypeptide has at least 100 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4. 
     
     
         72 . The animal of  claim 70 , wherein the nucleotide sequence is operably linked to an endogenous IL4 regulatory element of the animal. 
     
     
         73 . The animal of  claim 70 , wherein the nucleotide sequence is operably linked to a human IL4 regulatory element of the animal. 
     
     
         74 . The animal of  claim 70 , wherein the nucleotide sequence is integrated to an endogenous IL4 gene locus of the animal. 
     
     
         75 . The animal of  claim 70 , wherein the chimeric IL4 has at least one mouse IL4 activity and/or at least one human IL4 activity. 
     
     
         76 . A method of making a genetically-modified mouse cell that expresses a human or chimeric IL4, the method comprising:
 replacing, at an endogenous mouse IL4 gene locus, a nucleotide sequence encoding a region of mouse IL4 with a nucleotide sequence encoding a corresponding region of human IL4, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the human or chimeric IL4, wherein the mouse cell expresses the human or chimeric IL4.   
     
     
         77 . The method of  claim 76 , wherein the nucleotide sequence encoding the human or chimeric IL4 is operably linked to an endogenous IL4 regulatory region, e.g., promoter. 
     
     
         78 . The method of  claim 76 , wherein the nucleotide sequence encoding the human or chimeric IL4 is operably linked to a human IL4 regulatory region, e.g., promoter. 
     
     
         79 . The animal of any one of  claims 41 - 63  and  70 - 75 , wherein the animal further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4R, IL33, IL13, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), CD137, TNF Receptor Superfamily Member 4 (OX40), CD47, or SIRPa). 
     
     
         80 . The animal of  claim 79 , wherein the additional human or chimeric protein is IL4R. 
     
     
         81 . The method of any one of  claims 64 - 69  and  76 - 78 , wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4R, IL33, IL13, PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, TIGIT, TIM-3, GITR, CD137, OX40, CD47, or SIRPa). 
     
     
         82 . The method of  claim 81 , wherein the additional human or chimeric protein is IL4R. 
     
     
         83 . A method of determining effectiveness of an IL4-IL4R pathway inhibitor for treating an allergic disorder, comprising:
 administering the IL4-IL4R pathway inhibitor to the animal of any one of  claims 1 - 20 ,  27 - 33 ,  37 - 38 ,  41 - 63 ,  70 - 75 , and  79 - 80 ; and   determining the inhibitory effects of the IL4-IL4R pathway inhibitor.   
     
     
         84 . The method of  claim 83 , wherein the allergic disorder is asthma. 
     
     
         85 . The method of  claim 83 , wherein the allergic disorder is atopic dermatitis. 
     
     
         86 . The method of  claim 83 , wherein the allergic disorder is chromic sinusitis. 
     
     
         87 . The method of  claim 83 , wherein the IL4-IL4R pathway inhibitor is an anti-human IL4 antibody. 
     
     
         88 . The method of  claim 83 , wherein the IL4-IL4R pathway inhibitor is an anti-human IL4R antibody. 
     
     
         89 . The method of  claim 83 , wherein the inhibitory effects are evaluated by serum IgE levels; pathological lung histology features; number of leukocytes (CD45+ cells), eosinophils (Eos) or neutrophils in bronchoalveolar lavage fluid (BALF); or ratio of eosinophils or neutrophils cells in CD45+ cells in bronchoalveolar lavage fluid (BALF). 
     
     
         90 . A method of determining effectiveness of an IL4-IL4R pathway inhibitor for reducing inflammation, comprising:
 administering the IL4-IL4R pathway inhibitor to the animal of any one of  claims 1 - 20 ,  27 - 33 ,  37 - 38 ,  41 - 63 ,  70 - 75 , and  79 - 80 ; and   determining the inhibitory effects of the IL4-IL4R pathway inhibitor.   
     
     
         91 . A method of determining effectiveness of an IL4-IL4R pathway inhibitor for treating autoimmune disorder, comprising:
 administering the IL4-IL4R pathway inhibitor to the animal of any one of  claims 1 - 20 ,  27 - 33 ,  37 - 38 ,  41 - 63 ,  70 - 75 , and  79 - 80 ; and   determining the inhibitory effects of the IL4-IL4R pathway inhibitor.   
     
     
         92 . A method of determining effectiveness of an IL4-IL4R pathway inhibitor for treating cancer, comprising:
 administering the IL4-IL4R pathway inhibitor to the animal of any one of  claims 1 - 20 ,  27 - 33 ,  37 - 38 ,  41 - 63 ,  70 - 75 , and  79 - 80 ; and   determining the inhibitory effects of the IL4-IL4R pathway inhibitor.   
     
     
         93 . The method of  claim 92 , wherein determining the inhibitory effects of the treatment involves measuring the tumor volume in the animal. 
     
     
         94 . A method of determining toxicity of an anti-IL4R antibody or an anti-IL4 antibody, the method comprising
 administering the anti-IL4R antibody or the anti-IL4 antibody to the animal of any one of 1-20, 27-33, 37-38, 41-63, 70-75, and 79-80; and   determining weight change of the animal.   
     
     
         95 . The method of  claim 94 , the method further comprising performing a blood test (e.g., determining red blood cell count). 
     
     
         96 . A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:
 (a) an amino acid sequence set forth in SEQ ID NO: 44;   (b) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44;   (c) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and   (d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 44.   
     
     
         97 . A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:
 (a) a sequence that encodes the protein of  claim 96 ;   (b) SEQ ID NO: 8, 9, 10, 11, 23, 24, 25, 43, 48, 49, 50, or 51; or   (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, 9, 10, 11, 23, 24, 25, 43, 48, 49, 50, or 51.   
     
     
         98 . A cell comprising the protein of  claim 96  and/or the nucleic acid of  claim 97 . 
     
     
         99 . An animal comprising the protein of  claim 96  and/or the nucleic acid of  claim 97 .