CN118956959A

CN118956959A - IFNAR1 and/or IFNAR2 gene modified non-human animal

Info

Publication number: CN118956959A
Application number: CN202411289702.6A
Authority: CN
Inventors: 王琳琳; 张淑金; 王淑峰
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2023-09-15
Filing date: 2024-09-14
Publication date: 2024-11-15

Abstract

The present invention provides a non-human animal expressing human or chimeric (e.g., humanized) IFNAR1 and/or IFNAR2 proteins, methods of construction and uses thereof.

Description

IFNAR1 and/or IFNAR2 gene modified non-human animal

Technical Field

The invention provides a non-human animal expressing human or chimeric (e.g., humanized) IFNAR1 and/or IFNAR2 proteins, methods of construction thereof and application in the biomedical field.

Background

In vitro screening methods are generally used in traditional drug development, however, these screening methods cannot provide body environment (such as tumor microenvironment, stromal cells, extracellular matrix components, immune cell interactions, etc.), resulting in higher failure rate in drug development. Furthermore, given the differences between humans and animals, the experimental results obtained from in vivo pharmacological tests using conventional laboratory animals may not reflect the actual disease state and interaction at the targeted site, resulting in significant differences between the results of many clinical trials and animal experimental results.

Therefore, developing humanized animal models suitable for human antibody screening and evaluation will significantly improve the development efficiency of new drugs and reduce the cost of drug development.

However, it is difficult to construct a humanized animal model, for example, non-patent document ：Generation and utility of genetically humanized mouse models(Scheer N,Snaith M,Wolf CR,Seibler J.,Drug Discovery Today;18(23-24):1200-11,2013) discloses that humanization of any gene carries a certain risk, and even if a carefully designed strategy is adopted, expression and function of human genes in an animal body cannot be ensured.

Disclosure of Invention

The present application provides an animal model having human or chimeric IFNAR1 and/or IFNAR2 proteins. The animal model may express human or chimeric IFNAR1 and/or IFNAR2 (e.g., humanized IFNAR1 and/or IFNAR 2) proteins. It is useful for the study of IFNAR1 and/or IFNAR2 gene function, and for the screening and evaluation of IFNAR1 and/or IFNAR2 signaling pathway modulators (e.g., anti-human IFNAR1 and/or IFNAR2 antibodies, nucleic acid agents (e.g., oligonucleotide agents) and/or polypeptide agents). Furthermore, the non-human animals or animal models prepared by the method can be used for drug screening, pharmacodynamic studies, and treatment of immune diseases, inflammation or cancer and treatment of diseases at the target sites of human IFNAR1 and/or IFNAR 2; the model can also be used for promoting new medicine development and design, and saving time and cost. In conclusion, the application provides a powerful tool for researching the functions of the IFNAR1 and/or IFNAR2 proteins and provides a platform for screening relevant disease treatment medicines.

In one aspect, the invention provides a genetically modified non-human animal, or a method of constructing the same, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric type i interferon receptor 1 (IFNAR 1) protein. In some embodiments, the chimeric IFNAR1 protein is a humanized IFNAR1 protein. In some embodiments, the nucleotide sequence encoding a human or chimeric IFNAR1 protein is operably linked to endogenous regulatory elements (e.g., endogenous 5'utr and/or 3' utr) of an endogenous IFNAR1 locus of at least one chromosome. in some embodiments, the chimeric IFNAR1 protein comprises a human IFNAR1 extracellular domain, or comprises at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% identity to a human IFNAR1 extracellular domain. In some embodiments, the chimeric IFNAR1 protein comprises at least 10 to 409, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 406, or 409 consecutive amino acids of the extracellular domain of human IFNAR 1. In some embodiments, the chimeric IFNAR1 protein comprises a human IFNAR1 extracellular domain with 1-10 (e.g., 1,2,3,4, 5,6, 7, 8, 9, or 10) amino acids removed from the C-terminus and/or the N-terminus. In some embodiments, the chimeric IFNAR1 protein comprises 1-10 (e.g., 1,2, 3,4, 5, 6, 7, 8, 9, or 10) amino acids of the C-terminal and/or N-piece of the extracellular region of the non-human animal IFNAR 1. In some embodiments, the chimeric IFNAR1 protein comprises a humanized extracellular region comprising a portion of a human IFNAR1 extracellular region and a portion of a non-human animal extracellular region. In some embodiments, the chimeric IFNAR1 protein comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the chimeric IFNAR1 protein further comprises a human or humanized signal peptide. In some embodiments, the chimeric IFNAR1 protein further comprises an endogenous signal peptide. In some embodiments, the amino acid sequence of the human or chimeric IFNAR1 protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to human IFNAR1 (np_ 000620.2,SEQ ID NO:2). In some embodiments, the amino acid sequence of the human or chimeric IFNAR1 protein comprises SEQ ID NO:2 at positions 28-430 or 28-436 or comprising a nucleotide sequence identical to SEQ ID NO: the amino acid identity of positions 28-430 or 28-436 of 2 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the amino acid sequence of the human or chimeric IFNAR1 protein comprises SEQ ID NO:7, or comprises a sequence identical to SEQ ID NO:7 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the non-human animal is a mammal, such as a monkey, rodent (e.g., mouse or rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous IFNAR1 protein of the non-human animal is not expressed or the level of expression is reduced compared to IFNAR1 in a wild-type animal. in some embodiments, the non-human animal one or more cells express a human or chimeric IFNAR1 protein. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by another gene selected from at least one of IFNAR2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4.

In one aspect, the invention provides a genetically modified non-human animal, or a method of constructing the same, the genome of which comprises a nucleotide sequence encoding a human or chimeric IFNAR1 region at an endogenous IFNAR1 locus in place of a nucleotide sequence encoding a corresponding region of endogenous IFNAR 1. In some embodiments, the nucleotide sequence encoding a human or chimeric IFNAR1 corresponding region is operably linked to an endogenous regulatory element of an endogenous IFNAR1 locus, and the one or more cells of the non-human animal express a human or humanized IFNAR1 protein. In some embodiments, the endogenous IFNAR1 protein of the non-human animal is not expressed or the level of expression is reduced compared to IFNAR1 in a wild-type animal. In some embodiments, the region encoding the endogenous IFNAR1 corresponding is an endogenous IFNAR1 extracellular region. In some embodiments, the one or more cells of the non-human animal express a chimeric IFNAR1, the chimeric IFNAR1 comprising an extracellular region, a transmembrane region, and a cytoplasmic region, the extracellular region being a human IFNAR1 extracellular region, or having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99.5% identity to a human IFNAR1 extracellular region. In some embodiments, the extracellular region is identical to at least 10 to 409, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 406, or 409 consecutive amino acids of the extracellular region of human IFNAR 1. In some embodiments, the nucleotide sequence encoding the corresponding region of human or chimeric IFNAR1 comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of the human IFNAR1 gene, preferably further comprising intron 2 and/or intron 8. In some embodiments, the nucleotide sequence encoding a corresponding region of human IFNAR1 comprises SEQ ID NO:5, or comprises a sequence identical to SEQ ID NO:5 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding the endogenous IFNAR1 region comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of the mouse IFNAR1 gene, preferably further comprising intron 2 and/or intron 8. In some embodiments, the nucleotide sequence encoding the extracellular region of human IFNAR1 replaces the corresponding region of non-human animal endogenous IFNAR1 at the non-human animal endogenous IFNAR1 locus. In some embodiments, at the non-human animal endogenous IFNAR1 locus, a nucleotide sequence encoding an extracellular region of IFNAR1 with 1-10 amino acids removed from the C-and/or N-terminus of the human is substituted for a corresponding region of non-human animal endogenous IFNAR 1. In some embodiments, at the non-human animal endogenous IFNAR1 locus, the sequence encoding SEQ ID NO:2 nucleotide sequence at positions 28-430 or 28-436 replacing the corresponding region of endogenous IFNAR1 of the non-human animal. In some embodiments, the modified IFNAR1 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

In one aspect, the invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or chimeric IFNAR1 protein, wherein the human or chimeric IFNAR1 protein comprises at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 500, 550 or 557 consecutive amino acid sequences identical to the consecutive amino acid sequences of the corresponding region of the human, and the non-human animal expresses the human or chimeric IFNAR1 protein. In some embodiments, the chimeric IFNAR1 protein comprises at least 50 consecutive amino acids of the extracellular region of human IFNAR 1. In some embodiments, the human or chimeric IFNAR1 protein comprises the amino acid sequence of SEQ ID NO:2 at positions 28-430 or 28-436 or comprising a nucleotide sequence identical to SEQ ID NO: the amino acid identity of positions 28-430 or 28-436 of 2 is at least 70%,75%,80%,85%,90%,95%,99% or 99.5%. In some embodiments, the nucleotide sequence encoding a human or chimeric IFNAR1 protein is operably linked to an endogenous IFNAR1 regulatory element. In some embodiments, the nucleotide sequence encoding a human or chimeric IFNAR1 corresponding region may be integrated into the non-human animal endogenous IFNAR1 locus. In some embodiments, the human or chimeric IFNAR1 protein has at least one mouse IFNAR1 activity and/or human IFNAR1 activity.

In one aspect, the invention provides a method of constructing a genetically modified non-human animal by replacing, at an endogenous IFNAR1 locus of the non-human animal, a corresponding region of the endogenous IFNAR1 gene with a nucleotide sequence comprising human IFNAR 1. In some embodiments, the endogenous IFNAR1 protein of the non-human animal is not expressed or the level of expression is reduced compared to IFNAR1 in a wild-type animal. In some embodiments, the nucleotide sequence of human IFNAR1 comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the nucleotide sequence of human IFNAR1 comprises a portion of exon 2 to a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the human IFNAR1 gene exon 2 portion comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 119, 120, or 124bp contiguous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a contiguous nucleotide sequence of 119 bp. In some embodiments, the human IFNAR1 gene exon 9 portion comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 145, 146, 147, 148, 149, 150 or 151bp contiguous nucleotide sequence. In some embodiments, the portion of exon 9 comprises a 147bp contiguous nucleotide sequence. In some embodiments, the corresponding region of the endogenous IFNAR1 gene comprises a nucleotide sequence endogenous to the non-human animal encoding an extracellular region of IFNAR 1. In some embodiments, the corresponding region of the endogenous IFNAR1 gene comprises a portion of exon 2 to a portion of exon 9 of the non-human animal IFNAR1 gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence of human IFNAR1 comprises SEQ ID NO:2 or 7, or comprises a sequence identical to SEQ ID NO:2 or 7 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence of human IFNAR1 comprises SEQ ID NO:5, or comprises a sequence identical to SEQ ID NO:5 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the endogenous IFNAR1 gene comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of the non-human animal IFNAR1 gene. In some embodiments, the corresponding region of the endogenous IFNAR1 gene comprises a portion of exon 2 to a portion of exon 9 of the non-human animal IFNAR1 gene. In some embodiments, the mRNA transcribed from the modified IFNAR1 gene in the genome of the non-human animal comprises the amino acid sequence of SEQ ID NO:6, or comprises a sequence identical to SEQ ID NO:6 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the modified IFNAR1 gene in the non-human animal's gene is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the nucleotide sequence of human IFNAR1 is operably linked to an endogenous IFNAR1 regulatory element, such as a promoter. in some embodiments, the non-human animal is a mammal, such as a monkey, rodent (e.g., mouse or rat).

In one aspect, the invention provides a method of constructing a genetically modified non-human animal cell expressing a human or chimeric IFNAR1, the method comprising replacing at an endogenous mouse IFNAR1 locus a nucleotide sequence encoding an endogenous IFNAR1 region with a nucleotide sequence encoding a corresponding region of human IFNAR1, resulting in a genetically modified non-human animal cell expressing a human or chimeric IFNAR1 protein. In some embodiments, the chimeric IFNAR1 protein comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the chimeric IFNAR1 protein further comprises an endogenous IFNAR1 signal peptide. In some embodiments, the nucleotide sequence encoding a corresponding region of human IFNAR1 comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the nucleotide sequence encoding a corresponding region of human IFNAR1 comprises a portion of exon 2 to a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human IFNAR1 comprises SEQ ID NO:2, or comprises a sequence identical to SEQ ID NO:2 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human IFNAR1 comprises SEQ ID NO:2 at positions 28-430 or 28-436 or comprising a nucleotide sequence identical to SEQ ID NO:2 from 28 to 430 or from 28 to 436, at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the region encoding endogenous IFNAR1 is an endogenous IFNAR1 extracellular region. In some embodiments, the nucleotide sequence encoding an endogenous IFNAR1 region comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of a non-human animal (e.g., mouse) IFNAR1 gene. In some embodiments, the nucleotide sequence encoding the endogenous IFNAR1 region comprises a portion of exon 2 to a portion of exon 9 of a non-human animal (e.g., mouse) IFNAR1 gene. In some embodiments, the nucleotide sequence encoding a human or chimeric IFNAR1 protein is operably linked to regulatory elements, such as a promoter, of endogenous IFNAR 1. 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 another gene selected from at least one of IFNAR2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4. In some embodiments, the human or chimeric protein is an IFNAR2 protein.

In one aspect, the invention provides a targeting vector comprising a 5 'arm, a donor region, and a 3' arm. In some embodiments, the 5 'arm is homologous to the 5' end of the region to be altered. In some embodiments, the 3 'arm is homologous to the 3' end of the region to be altered. In some embodiments, the donor region includes a nucleotide sequence encoding a human or chimeric IFNAR1 protein. In some embodiments, the donor region comprises a nucleotide sequence encoding a human IFNAR1 extracellular region, or comprises a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% identical to a human IFNAR1 extracellular region. In some embodiments, the donor region comprises a nucleotide sequence encoding at least 10 to 409, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 406, or 409 consecutive amino acids of the extracellular region of human IFNAR 1. In some embodiments, the donor region comprises a nucleotide sequence encoding a human IFNAR1 extracellular region with 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids removed from the C-terminus and/or the N-terminus. In some embodiments, the donor region includes all or part of the human IFNAR1 gene, such as any exon or any intron or combination thereof. In some embodiments, the donor region comprises a portion of exon 2 to a portion of exon 9 of human IFNAR 1. In some embodiments, the human IFNAR1 gene exon 2 portion comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 119, 120, or 124bp contiguous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a contiguous nucleotide sequence of 119 bp. In some embodiments, the human IFNAR1 gene exon 9 portion comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 145, 146, 147, 148, 149, 150 or 151bp contiguous nucleotide sequence. In some embodiments, the portion of exon 9 comprises a 147bp contiguous nucleotide sequence. In some embodiments, the region to be altered is located from exon 2 to exon 9 of the non-human animal IFNAR1 gene. The 5' arm is shown as SEQ ID NO: 3. The 3' arm is shown as SEQ ID NO: 4.

In one aspect, the invention provides a genetically modified non-human animal, or a method of constructing the same, the genome of which comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric type i interferon receptor 2 (IFNAR 2) protein. In some embodiments, the chimeric IFNAR2 protein is a humanized IFNAR2 protein. In some embodiments, the nucleotide sequence encoding a human or chimeric IFNAR2 protein is operably linked to endogenous regulatory elements (e.g., endogenous 5'utr and/or 3' utr) of an endogenous IFNAR2 locus of at least one chromosome. In some embodiments, the chimeric IFNAR2 protein comprises a human IFNAR2 extracellular domain, or comprises at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% identity to a human IFNAR2 extracellular domain. In some embodiments, the chimeric IFNAR2 protein comprises at least 10 to 217, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 210, 215, 216, or 217 consecutive amino acids of the extracellular domain of human IFNAR 2. In some embodiments, the chimeric IFNAR1 protein comprises a human IFNAR2 extracellular domain with 1-10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids removed from the C-terminus and/or the N-terminus. In some embodiments, the chimeric IFNAR2 protein comprises a human IFNAR2 signal peptide. In some embodiments, the chimeric IFNAR2 protein comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the chimeric IFNAR2 protein further comprises a human or humanized signal peptide. In some embodiments, the amino acid sequence of the human or chimeric IFNAR2 protein comprises human IFNAR2 (np_ 997468.1,SEQ ID NO:22), or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to human IFNAR2 (np_ 997468.1,SEQ ID NO:22). In some embodiments, the amino acid sequence of the human or chimeric IFNAR2 protein comprises SEQ ID NO:22 at positions 1-243 or 1-242, or comprises a sequence identical to SEQ ID NO:22 at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 1-243 or 1-242. In some embodiments, the amino acid sequence of the chimeric IFNAR2 protein comprises SEQ ID NO:29 or 58, or comprises a sequence identical to SEQ ID NO:29 or 58 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the non-human animal is a mammal, such as a monkey, rodent (e.g., mouse or rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal endogenous IFNAR2 protein is not expressed or the expression level is reduced compared to IFNAR2 in a wild-type animal. In some embodiments, the non-human animal one or more cells express a human or chimeric IFNAR2 protein.

In one aspect, the invention provides a method of constructing a genetically modified non-human animal by introducing a nucleotide sequence comprising human IFNAR2 at an endogenous IFNAR2 locus in the non-human animal. In some embodiments, the nucleotide sequence of human IFNAR2 comprises a portion of exon 2 to a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 91, or 92bp contiguous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a contiguous nucleotide sequence of 55 bp. In some embodiments, the portion of exon 8 comprises at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, or 131bp contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises a 20bp contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises a 17bp contiguous nucleotide sequence. In some embodiments, the nucleotide sequence of human IFNAR2 comprises SEQ ID NO:26 or 57, or comprises a sequence identical to SEQ ID NO:26 or 57 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the introduced sequence further includes a nucleotide sequence of non-human animal IFNAR 2. In some embodiments, the introduced sequence includes a portion of exon 8 to exon 9 of non-human animal IFNAR 2. In some embodiments, the introduced sequence comprises SEQ ID NO:27, or comprises a sequence identical to SEQ ID NO:27 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the introducing is inserting or replacing. In some embodiments, the nucleotide sequences encoding the signal peptide and extracellular region of endogenous IFNAR2 are replaced. In some embodiments, part of exon 2 to exon 3 is replaced. In some embodiments, the portion of exon 2 to the portion of exon 8 is replaced. In some embodiments, the nucleotide sequence of human IFNAR2 is operably linked to an endogenous IFNAR2 regulatory element, such as a promoter. In some embodiments, the endogenous IFNAR2 protein of the non-human animal is not expressed or the level of expression is reduced compared to IFNAR2 in a wild-type animal. In some embodiments, the non-human animal is a mammal, e.g., monkey, rodent. In some embodiments, the rodent comprises a mouse or a rat. In some embodiments, the mRNA transcribed from the modified IFNAR2 gene in the genome of the non-human animal comprises the amino acid sequence of SEQ ID NO:28, or comprises a sequence identical to SEQ ID NO:28 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the modified IFNAR2 gene in the non-human animal's gene is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by another gene selected from at least one of IFNAR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4.

In one aspect, the invention provides a genetically modified non-human animal, or a method of constructing the same, the genome of which comprises a nucleotide sequence encoding a human or chimeric IFNAR2 at an endogenous IFNAR2 locus in place of the nucleotide sequence encoding the endogenous IFNAR2, or a nucleotide sequence encoding a human or chimeric IFNAR2 inserted into an endogenous IFNAR2 locus. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 is operably linked to an endogenous regulatory element of an endogenous IFNAR2 locus, and the one or more cells of the non-human animal express a human or humanized IFNAR2 protein. In some embodiments, the endogenous IFNAR2 protein of the non-human animal is not expressed or the level of expression is reduced compared to IFNAR2 in a wild-type animal. In some embodiments, the replacement encoding endogenous IFNAR2 nucleotide sequence is an endogenous IFNAR2 signal peptide and/or extracellular region. In some embodiments, the one or more cells of the non-human animal express chimeric IFNAR2, the chimeric IFNAR2 comprising a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region, wherein the signal peptide and/or extracellular region is identical to the human IFNAR2 signal peptide and/or extracellular region, or is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.5% identical. In some embodiments, the signal peptide and/or extracellular region is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 consecutive amino acids identical to the human IFNAR2 signal peptide and/or extracellular region. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 comprises a portion of exon 2 to a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 comprises a portion of exon 2, all of exons 3-7, and a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 comprises SEQ ID NO:26 or 57, or comprises a sequence identical to SEQ ID NO:26 or 57 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding endogenous IFNAR2 comprises a portion of exon 2 to a portion of exon 8 of a non-human animal (e.g., mouse) IFNAR2 gene. In some embodiments, the nucleotide sequence encoding endogenous IFNAR2 comprises a portion of exon 2, all of exons 3-7, and a portion of exon 8 of a non-human animal (e.g., mouse) IFNAR2 gene. In some embodiments, the nucleotide sequence encoding endogenous IFNAR2 comprises a portion of exon 2 of a non-human animal (e.g., mouse) IFNAR2 gene to exon 3. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 is inserted into exon 2-exon 3 of the endogenous IFNAR2 locus. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 is inserted after the 5' utr of nm_ 010509.2. In some embodiments, the endogenous IFNAR2 gene locus exon 2-exon 3 is deleted by at least 20bp (e.g., NM_010509.2 th to 427 bp). In some embodiments, the endogenous IFNAR2 locus nm_010509.2 nucleotide sequence 331-427 and 333bp downstream of exon 3 are deleted. In some embodiments, the insertion sequence comprises: a) A first nucleotide sequence encoding all or part of the human signal peptide and extracellular region, B) a second nucleotide sequence encoding all or part of the mouse transmembrane region and cytoplasmic region. In some embodiments, the amino acid sequence encoded by the first nucleotide sequence comprises SEQ ID NO:22 at positions 1-243 or 1-242, or comprises a sequence identical to SEQ ID NO:22 at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to amino acids 1-243 or 1-242. The amino acid sequence encoded by the second nucleotide sequence comprises SEQ ID NO:21 from positions 243 to 513 or comprises a sequence corresponding to SEQ ID NO: amino acids 243-513 of 21 are at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical. In some embodiments, the insertion sequence further comprises one or more helper sequences. In some embodiments, the helper sequence is a STOP sequence. In some embodiments, the non-human animal comprises in its genome the sequence of SEQ ID NO: 25. 26, 27, 28 or 57, or a nucleotide sequence comprising SEQ ID NO: 25. 26, 27, 28 or 57 is at least 70%,75%,80%,85%,90%,95%,99% or 99.5%. In some embodiments, the modified IFNAR2 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

In one aspect, the invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or chimeric IFNAR2 protein, wherein the human or chimeric IFNAR2 protein comprises at least 50, 60, 70, 80, 90, 100, 200, 240, 241, 242, 243, 244, 245, 300, 400, 500, 510, 511, 512, 513, 514 or 515 consecutive amino acid sequences identical to the consecutive amino acid sequences of the corresponding region of the human, or a method of constructing the same, the non-human animal expressing the human or chimeric IFNAR2 protein. In some embodiments, the chimeric IFNAR2 protein comprises a human IFNAR2 signal peptide and/or an extracellular amino acid of at least 20 consecutive amino acids. In some embodiments, the human or chimeric IFNAR2 protein comprises SEQ ID NO:22 at positions 1-243 or 1-242, or comprises a sequence identical to SEQ ID NO:22 at least 70%,75%,80%,85%,90%,95%,99% or 99.5% identical to amino acids 1-243 or 1-242. In some embodiments, the nucleotide sequence encoding a human or chimeric IFNAR2 protein is operably linked to an endogenous IFNAR2 regulatory element. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 may be integrated into the non-human animal endogenous IFNAR2 locus. In some embodiments, the human or chimeric IFNAR2 protein has at least one mouse IFNAR2 activity and/or human IFNAR2 activity.

In one aspect, the invention provides a method of constructing a genetically modified non-human animal in which, in at least one cell of the non-human animal, at the non-human animal endogenous IFNAR2 locus, the nucleotide sequence encoding the endogenous IFNAR2 region is replaced with a nucleotide sequence encoding a human or chimeric IFNAR2, or a sequence encoding a human or chimeric IFNAR2 protein is inserted into the non-human animal endogenous IFNAR2 locus. In some embodiments, the endogenous IFNAR2 protein of the non-human animal is not expressed or the level of expression is reduced compared to IFNAR2 in a wild-type animal. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 comprises a portion of exon 2, all of exons 3-7, and a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 comprises a portion of exon 2 to a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the amino acid sequence of the human or chimeric IFNAR2 protein comprises SEQ ID NO:22 at positions 1-242 or 1-243, or comprising a nucleotide sequence identical to SEQ ID NO:22 at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 1-242 or 1-243. In some embodiments, the region encoding endogenous IFNAR2 is an endogenous IFNAR2 signal peptide and an extracellular region. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 encodes an amino acid sequence comprising SEQ ID NO: 22. 29 or 58, or comprises a sequence identical to SEQ ID NO: 22. 29 or 58 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 comprises SEQ ID NO:26 or 57, or comprises a sequence identical to SEQ ID NO:26 or 57 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%. In some embodiments, the nucleotide sequence encoding the endogenous IFNAR2 region comprises a portion of exon 2 to a portion of exon 8 of a non-human animal (e.g., mouse) IFNAR2 gene. In some embodiments, the nucleotide sequence encoding an endogenous IFNAR2 region comprises a portion of exon 2, all of exons 3-7, and a portion of exon 8 of a non-human animal (e.g., mouse) IFNAR2 gene. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 is inserted into exon 2-exon 3 of the endogenous IFNAR2 locus. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 is inserted after the 5' utr of nm_ 010509.2. In some embodiments, the endogenous IFNAR2 gene locus exon 2-exon 3 is deleted by at least 20bp (e.g., NM_010509.2 th to 427 bp). In some embodiments, the endogenous IFNAR2 locus nm_010509.2 nucleotide sequence 331-427 and 333bp downstream of exon 3 are deleted. In some embodiments, the chimeric IFNAR2 polypeptide comprises: a) All or part of the signal peptide and extracellular region of human IFNAR 2; b) All or part of the transmembrane and cytoplasmic regions of endogenous IFNAR 2. In some embodiments, the nucleotide sequence encoding human or chimeric IFNAR2 is operably linked to an endogenous IFNAR2 regulatory element, such as a promoter. in some embodiments, the non-human animal is a mammal, such as a monkey, rodent (e.g., mouse or rat).

In one aspect, the invention provides a method of constructing a genetically modified non-human animal cell expressing human or chimeric IFNAR2, the method comprising replacing at an endogenous mouse IFNAR2 locus a nucleotide sequence encoding an endogenous IFNAR2 region with a nucleotide sequence encoding human IFNAR2, or inserting a sequence encoding a human or chimeric IFNAR2 protein into the endogenous IFNAR2 locus, to produce a genetically modified non-human animal cell. In some embodiments, the non-human animal cell expresses a human or chimeric IFNAR2 protein. In some embodiments, the chimeric IFNAR2 protein comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region. In some embodiments, the chimeric IFNAR2 protein further comprises a human or humanized IFNAR2 signal peptide. In some embodiments, the nucleotide sequence encoding human IFNAR2 comprises a portion of exon 2, all of exons 3-7, and a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the nucleotide sequence encoding human IFNAR2 comprises a portion of exon 2 to a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding human IFNAR2 comprises SEQ ID NO:22 at positions 1-243 or 1-242, or comprises a sequence identical to SEQ ID NO:22 at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% amino acid sequence identity between positions 1-243 or 1-242. In some embodiments, the region encoding endogenous IFNAR2 is an endogenous IFNAR2 signal peptide and an extracellular region. In some embodiments, the nucleotide sequence encoding an endogenous IFNAR2 region comprises a portion of exon 2, all of exons 3-7, and a portion of exon 8 of a non-human animal (e.g., mouse) IFNAR2 gene. In some embodiments, the nucleotide sequence encoding the endogenous IFNAR2 region comprises a portion of exon 2 to a portion of exon 8 of a non-human animal (e.g., mouse) IFNAR2 gene. In some embodiments, the insertion sequence comprises: a) A nucleotide sequence encoding all or part of the human IFNAR2 signal peptide and extracellular region; B) A nucleotide sequence encoding all or part of the endogenous IFNAR2 transmembrane and cytoplasmic regions; optionally C) one or more auxiliary sequences (e.g., STOP sequences and/or 3' UTRs). In some embodiments, the nucleotide sequence encoding a human or chimeric IFNAR2 protein is operably linked to regulatory elements of endogenous IFNAR2, 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 another gene selected from at least one of IFNAR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4. In some embodiments, the human or chimeric protein is an IFNAR1 protein.

In one aspect, the invention provides a targeting vector comprising a5 'arm, a donor region, and a 3' arm. In some embodiments, the 5 'arm is homologous to the 5' end of the region to be altered. In some embodiments, the 3 'arm is homologous to the 3' end of the region to be altered. In some embodiments, the donor region includes a nucleotide sequence encoding a human or chimeric IFNAR2 protein. In some embodiments, the donor region comprises a nucleotide sequence encoding a human IFNAR2 extracellular region, or comprises a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, 95% or 99.5% identical to a human IFNAR2 extracellular region. In some embodiments, the donor region comprises a nucleotide sequence encoding at least 10 to 217, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 210, 215, 216, or 217 consecutive amino acids of the extracellular region of human IFNAR 2. In some embodiments, the donor region comprises a nucleotide sequence encoding a human IFNAR2 extracellular region with 1-10 (e.g., 1,2, 3, 4, 5,6, 7, 8, 9, or 10) amino acids removed from the C-terminus and/or the N-terminus. In some embodiments, the donor region comprises a nucleotide sequence encoding a human IFNAR2 signal peptide. In some embodiments, the donor region comprises a nucleotide sequence encoding a human IFNAR2 signal peptide and an extracellular region. In some embodiments, the donor region comprises a nucleotide sequence encoding a human IFNAR2 signal peptide and an extracellular region, and a nucleotide sequence encoding an IFNAR2 transmembrane region and a cytoplasmic region in a non-human animal. In some embodiments, the donor region comprises a portion of exon 2 to a portion of exon 8 of human IFNAR 2. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 91, or 92bp contiguous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a contiguous nucleotide sequence of 55 bp. In some embodiments, the portion of exon 8 comprises at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, or 131bp contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises a 20bp contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises a 17bp contiguous nucleotide sequence. In some embodiments, the donor region further comprises a portion of exon 8 to exon 9 of non-human animal IFNAR 2. In some embodiments, the region to be altered is located from exon 2 to exon 8 or from exon 2 to exon 3 of the non-human animal IFNAR2 gene. The 5' arm is shown as SEQ ID NO: 23. The 3' arm is shown as SEQ ID NO: shown at 24. The 5' arm is shown as SEQ ID NO: 52. The 3' arm is shown as SEQ ID NO: 53. The 5' arm is shown as SEQ ID NO: shown at 55. The 3' arm is shown as SEQ ID NO: shown at 56.

In one aspect, the invention provides a non-human animal genome comprising at least one chromosome comprising a nucleotide sequence encoding a human or chimeric IFNAR1 and/or IFNAR2 protein. In some embodiments, the chromosome comprises a human or chimeric IFNAR1 and/or IFNAR2 gene.

In one aspect, the invention provides a non-human animal genome comprising: at the non-human animal endogenous IFNAR1 and/or IFNAR2 locus, a nucleotide sequence comprising human IFNAR1 and/or IFNAR2 is introduced. In some embodiments, the nucleotide sequence comprising a human or chimeric IFNAR1 and/or IFNAR2 gene or protein replaces a non-human animal endogenous IFNAR1 and/or IFNAR2 gene.

In one aspect, the invention provides a cell comprising the non-human animal genome described above.

In one aspect, the invention provides a non-human animal comprising a genome of the non-human animal or a cell as described above.

In one aspect, the invention provides a method of determining the effectiveness of a therapeutic agent in treating a disease, the method comprising: 1) Administering a therapeutic agent to a non-human animal obtained by the construction method, wherein the non-human animal has a disease; 2) The inhibition of the disease by the therapeutic agent is measured. In some embodiments, the therapeutic agent is an anti-IFNAR 1 antibody and/or an anti-IFNAR 2 antibody; preferably, additional therapeutic agents (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA 4 antibodies) are also included. In some embodiments, the disease is a disease associated with IFNAR1 and/or IFNAR2 expression or abnormal expression (e.g., over-expression). In some embodiments, the disease is a disease that targets IFNAR1 and/or IFNAR2 or down regulates expression of IFNAR1 and/or IFNAR2, which is beneficial for treatment. In some embodiments, the disease is cancer, an immune disease, or inflammation. In some embodiments, the cancer is a digestive tract cancer (e.g., colon cancer, gastrointestinal cancer, rectal cancer), leukemia, liver cancer, kidney cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, or lymphoma. In some embodiments, the immune disorder is systemic lupus erythematosus, psoriasis, transplant rejection, diabetes, immune neuromuscular disease, multiple sclerosis, rheumatoid arthritis. In some embodiments, the inflammation is inflammatory bowel disease, hepatitis, arthritis.

In one aspect, the invention provides a method of determining the effectiveness of a therapeutic agent in treating cancer, the method comprising: 1) Administering a therapeutic agent to said non-human animal, wherein said non-human animal has cancer; 2) The inhibition of cancer by a therapeutic agent is measured. In some embodiments, the therapeutic agent is an anti-IFNAR 1 antibody and/or an anti-IFNAR 2 antibody. In some embodiments, the cancer is a tumor and the inhibition of the tumor by the therapeutic agent is determined by measuring the tumor volume of the non-human animal. In some embodiments, the cancer comprises injecting one or more cancer cells into the non-human animal. In some embodiments, the cancer is a digestive tract cancer (e.g., colon cancer, gastrointestinal cancer, rectal cancer), leukemia, liver cancer, kidney cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, or lymphoma.

In one aspect, the invention provides a method of determining the effectiveness of an anti-IFNAR 1 antibody and/or an anti-IFNAR 2 antibody and an additional therapeutic agent in treating cancer, the method comprising: 1) Administering an anti-IFNAR 1 antibody and an additional therapeutic agent to the non-human animal, wherein the non-human animal has cancer; 2) The inhibition of tumors by anti-IFNAR 1 antibodies and/or anti-IFNAR 2 antibodies and additional therapeutic agents is determined. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA 4 antibody. In some embodiments, the tumor comprises injection of one or more cancer cells into the non-human animal. In some embodiments, the inhibition of the tumor by a therapeutic agent is determined by measuring the tumor volume of a non-human animal. In some embodiments, the cancer is a digestive tract cancer (e.g., colon cancer, gastrointestinal cancer, rectal cancer), leukemia, liver cancer, kidney cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, or lymphoma.

In one aspect, the invention provides a method of determining the effectiveness of a therapeutic agent in treating an immune disorder, the method comprising: 1) Administering a therapeutic agent to the non-human animal, wherein the non-human animal has an immune disease; 2) The therapeutic effect of the therapeutic agent on the immune disease is measured. In some embodiments, the immune disorder is systemic lupus erythematosus, psoriasis, transplant rejection, diabetes, immune neuromuscular disease, multiple sclerosis, rheumatoid arthritis.

In one aspect, the invention provides a method of determining the effectiveness of an IFNAR1 therapeutic agent in treating inflammation, the method comprising: 1) Administering a therapeutic agent to said non-human animal, wherein said non-human animal has inflammation; 2) The effectiveness of a therapeutic agent for treating inflammation is determined. In some embodiments, the inflammation is inflammatory bowel disease, hepatitis, arthritis.

In one aspect, the invention provides a method of determining toxicity of a therapeutic agent, the method comprising: 1) Administering a therapeutic agent to said non-human animal or to a non-human animal obtained by a method of construction; 2) The effect of the therapeutic agent on the non-human animal is determined. In some embodiments, the determining the effect of the therapeutic agent on the non-human animal involves measuring the weight or blood test of the non-human animal. In some embodiments, the blood test includes one or more of red blood cell count, hematocrit, or hemoglobin content.

In one aspect, the invention provides a humanized IFNAR1 protein, the humanized IFNAR1 protein comprising all or part of a human IFNAR1 protein. In some embodiments, the humanized IFNAR1 protein comprises SEQ ID NO:7, or comprises a sequence identical to SEQ ID NO:7 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

In one aspect, the invention provides a humanized IFNAR1 gene, the humanized IFNAR1 gene encoding the above-described humanized IFNAR1 protein. In some embodiments, the humanized IFNAR1 gene comprises a portion of exon 2, all of exons 3-8, and/or a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the humanized IFNAR1 gene comprises a portion of exon 2 to a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the humanized IFNAR1 gene further comprises a non-human animal IFNAR1 gene exon 1, a portion of exon 2, a portion of exon 9, and/or all of exons 10-11. In some embodiments, the humanized IFNAR1 gene comprises a nucleotide sequence comprising SEQ ID NO: 3. 4, 5, 6, 8 or 9, or a sequence comprising a nucleotide sequence identical to SEQ ID NO: 3. 4, 5, 6, 8 or 9 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

In one aspect, the invention provides a humanized IFNAR2 protein, the humanized IFNAR2 protein comprising all or part of a human IFNAR2 protein. In some embodiments, the humanized IFNAR2 protein comprises SEQ ID NO:29 or 58, or comprises a sequence identical to SEQ ID NO:29 or 58 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

In one aspect, the invention provides a humanized IFNAR2 gene, the humanized IFNAR2 gene encoding the above-described humanized IFNAR2 protein. In some embodiments, the humanized IFNAR2 gene comprises a portion of exon 2, all of exons 3-7, and/or a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the humanized IFNAR2 gene comprises a portion of exon 2 to a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the humanized IFNAR2 gene further comprises non-human animal IFNAR2 gene exon 1, a portion of exon 2, and all of exons 4-9. In some embodiments, the humanized IFNAR2 gene further comprises all of exon 1, part of exon 2, part of exon 8, and exon 9 of the non-human animal IFNAR2 gene. In some embodiments, the humanized IFNAR2 gene comprises SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 52, 53, 55, 56, or 57, or a polypeptide comprising a sequence identical to SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 52, 53, 55, 56, or 57 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5%.

In one aspect, the invention provides a cell, tissue or organ comprising a humanized IFNAR1 protein, a humanized IFNAR1 gene, a humanized IFNAR2 protein or a humanized IFNAR2 gene as described above.

In one aspect, the invention provides an animal model comprising a humanized IFNAR1 protein, a humanized IFNAR1 gene, a humanized IFNAR2 protein, a humanized IFNAR2 gene, or a cell, tissue or organ as described above.

In one aspect, the invention provides a non-human animal, the non-human animal or animal model, the targeting vector, the humanized IFNAR1 protein, the humanized IFNAR1 gene, the humanized IFNAR2 protein, the humanized IFNAR2 gene, the cell, tissue or organ obtained by the method of construction, the use comprising:

a) Use in product development involving immune processes related to IFNAR1 and/or IFNAR2 of human cells;

B) Use in model systems related to IFNAR1 and/or IFNAR2 as pharmacological, immunological, microbiological and medical studies;

C) To the use of animal experimental disease models for the production and use in etiology studies associated with IFNAR1 and/or IFNAR2 and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

D) Application in screening, efficacy detection, evaluation of therapeutic efficacy, validation or evaluation of human IFNAR1 and/or IFNAR2 signaling pathway modulators in vivo; or alternatively

E) The functions of IFNAR1 and/or IFNAR2 genes are researched, medicines and medicine effects aiming at target sites of human IFNAR1 and/or IFNAR2 are researched, and the application of medicines for inflammatory and immune diseases related to IFNAR1 and/or IFNAR2 is researched.

The term "locus" according to the present invention refers broadly to the position occupied by a gene on a chromosome, and in a narrow sense to a DNA fragment on a gene, either a gene or a part of a gene. For example, the "IFNAR1 gene locus" means the IFNAR1 gene exon 1-11 in the optional DNA fragment. In some embodiments, the non-human animal endogenous IFNAR1 locus to be replaced may be a DNA fragment of an optional stretch of exons 1-11 of the non-human animal endogenous IFNAR1 gene. As another example, the "IFNAR2 gene locus" means a DNA fragment of an optional stretch of exons 1 to 9 of the IFNAR2 gene. In some embodiments, the non-human animal endogenous IFNAR2 locus to be replaced may be a DNA fragment of an optional stretch of exons 1-9 of the non-human animal endogenous IFNAR2 gene.

The term "exon XX to exon XXX" or "exons XX-XXX" or "all of exons XX to exons XXX" in the present invention is meant to include exons and introns during which, for example, exons 1 to 11 include exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, and all nucleotide sequences of exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10 and exon 11. Also for example, "part of exon 2 to part of exon 8" or "part of exon 2-part of exon 8" includes part of exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7 and part of the nucleotide sequence of exon 8. Also for example, "part of exon 2 to (-) exon 3" includes part of exon 2, intron 2 and exon 3.

The term "intron xx" in the present application refers to an intron between two exons, for example, intron 1 is an intron between exon 1 and exon 2.

The term "comprising" or "comprises" is an open-ended writing that includes the specified components or steps described, as well as other specified components or steps that are not materially affected. When used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

The term "and/or" in this disclosure encompasses all combinations of items to which the term is attached, and should be taken as individual combinations having been listed individually in this disclosure. For example, "a and/or B" includes "a", "a and B", and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".

Unless defined otherwise, 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 application belongs. The present application describes methods and materials for use in the present application; other suitable methods and materials known in the art may be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description.

IFNAR1

In the human genome, the IFNAR1 Gene (Gene ID: 3454) contains 11 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and exon 11 (FIG. 1). The nucleotide sequence of human IFNAR1 mRNA is NM_000629.3, and the amino acid sequence of human IFNAR1 is NP-000620.2 (SEQ ID NO: 2). The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM-000629.3 and its encoded protein NP-000620.2 are shown in Table 1.

TABLE 1

In the genome of the mice, the IFNAR1 Gene (Gene ID: 15975) comprises 11 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and exon 11 (FIG. 1). The nucleotide sequence of mouse IFNAR1 mRNA is NM_010508.2, and the amino acid sequence of mouse IFNAR1 is NP-034638.2 (SEQ ID NO: 1). The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM-010508.2 and its encoded protein NP-034638.2 are shown in Table 2.

TABLE 2

Other species of IFNAR1 genes, proteins and gene loci are also known in the art. For example, rattus norvegicus (rat) IFNAR1 Gene ID:288264, macaca mulatta (rhesus) IFNAR1 Gene ID:699604, canis lupus familiaris (dog) IFNAR1 Gene ID:609830,Sus scrofa (pig) IFNAR1 Gene ID:396658 relevant information about these genes (e.g., intron sequences, exon sequences, and amino acid sequences) can be found in NCBI, the entire contents of which are incorporated herein by reference.

The present invention provides a human or chimeric (e.g., humanized) IFNAR1 gene or protein. 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 and/or exon 11 of the mouse IFNAR1 gene is replaced with the corresponding nucleotide sequence of the human IFNAR1 gene. In some embodiments, the signal peptide, extracellular region, transmembrane region and/or cytoplasmic region of the mouse IFNAR1 protein are all replaced with the corresponding amino acid sequence of the human IFNAR1 protein. In some embodiments, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or "part" of exon 11 of the mouse IFNAR1 gene are replaced with the corresponding sequences of the human IFNAR1 gene. In some embodiments, the signal peptide, extracellular region, transmembrane region, and/or "portion" of the cytoplasmic region of the mouse IFNAR1 protein is replaced with a corresponding sequence of human IFNAR 1. The "portion" refers to a contiguous nucleotide sequence of at least 1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、200、300、400、500、600、700、800、1000、1100、1191、1200、1398、2000、3000、4000、5000、6000、7000、7014 or 7025bp, or a contiguous amino acid sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 350, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 423, 500, 550, or 590. In some embodiments of the present invention, in some embodiments, the "part" is associated with exon 1, exon 2, exon 3, intron 3, exon 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 and/or exon 11, or signal peptide, extracellular region, transmembrane region and/or cytoplasmic region identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99.5%. In some embodiments, the mouse IFNAR1 gene of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and exon 11 "part" or "all" sequences (e.g., part of exon 2, all of exon 3-8 and part of exon 9, and also e.g., part of exon 2 to part of exon 9) are replaced by a corresponding human IFNAR1 gene of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and exon 11 "part" or "all" sequences (e.g., Part of exon 2, all of exons 3-8 and part of exon 9, again for example part of exon 2 to part of exon 9). in some embodiments, the extracellular region comprises a signal peptide. In some embodiments, the extracellular region does not comprise a signal peptide.

In some embodiments, the "portion" of endogenous signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or exon 11 is deleted.

In some embodiments, the invention provides a genetically modified non-human animal whose genome comprises a chimeric IFNAR1 protein or gene, or a method of constructing the same. In some embodiments, the chimeric IFNAR1 protein comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, an endogenous cytoplasmic region. In some embodiments, the chimeric IFNAR1 protein comprises SEQ ID NO: 1.2 or 7, or comprises a sequence identical to SEQ ID NO: 1.2 or 7 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the non-human animal genome comprises a nucleotide sequence comprising SEQ ID NO: 3. 4,5,6, 8, 9, 10 and 11, or comprises a sequence identical to SEQ ID NO: 3. 4,5,6, 8, 9, 10 and 11 is at least 70%, 80%, 85%, 90%, 95% or 99.5%.

In some embodiments, the non-human animal comprises a sequence encoding a human or humanized IFNAR1 protein. In some embodiments, the IFNAR1 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 IFNAR1 protein comprises a human or humanized signal peptide, e.g., the human or humanized IFNAR1 signal peptide comprises the amino acid sequence of SEQ ID NO:2, or from positions 1 to 27, or from SEQ ID NO: the amino acid identity at positions 1-27 of 2 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR1 protein comprises an endogenous signal peptide, e.g., the endogenous IFNAR1 signal peptide comprises the amino acid sequence of SEQ ID NO:1 from position 1 to position 26, or comprises a sequence identical to SEQ ID NO: amino acid identity of at least 70%, 80%, 85%, 90%, 95% or 99.5% between positions 1 and 26. In some embodiments, the humanized IFNAR1 protein comprises a human or humanized extracellular region, e.g., the humanized extracellular region comprises the amino acid sequence of SEQ ID NO:2 at positions 28-430 or 28-436 or comprising a nucleotide sequence identical to SEQ ID NO: the amino acid identity of positions 28-430 or 28-436 of 2 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR1 protein comprises an endogenous extracellular region, e.g., the endogenous IFNAR1 extracellular region comprises the amino acid sequence of SEQ ID NO:1 from position 424 to 429 or comprises a sequence identical to SEQ ID NO: the amino acid identity of 1 at positions 424-429 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR1 protein comprises a human or humanized transmembrane region, e.g., the humanized transmembrane region comprises the amino acid sequence of SEQ ID NO: positions 437-457 of 2, or comprising a sequence identical to SEQ ID NO: the amino acid identity at positions 437-457 of 2 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR1 protein comprises an endogenous transmembrane region, e.g., the endogenous IFNAR1 transmembrane region comprises SEQ ID NO:1, or from positions 430-449, or from SEQ ID NO: the amino acid identity at positions 430-449 of 1 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR1 protein comprises a human or humanized cytoplasmic region, e.g., the humanized cytoplasmic region comprises the amino acid sequence of SEQ ID NO: positions 458-557 of 2, or comprising a nucleotide sequence identical to SEQ ID NO: the identity of amino acids 458-557 of 2 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR1 protein comprises an endogenous cytoplasmic region. For example, the endogenous IFNAR1 cytoplasmic region comprises SEQ ID NO:1 from positions 450 to 590 or comprises a sequence corresponding to SEQ ID NO: the amino acid identity of positions 450-590 of 1 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR1 protein comprises an endogenous IFNAR1 sequence of SEQ ID NO:1 from 1 to 26 and 424 to 590, or from 1 to 26 and 430 to 590. In some embodiments, the humanized IFNAR1 protein comprises a human IFNAR1 sequence of SEQ ID NO:2 amino acids 28-430 or 28-436.

In some embodiments, the nucleotide sequence encoding all or part of the extracellular region of mouse IFNAR1 (SEQ ID NO: 1) is replaced or inactivated. In some embodiments, the sequence is replaced by a nucleotide sequence encoding all or part of the extracellular region of human IFNAR1 (SEQ ID NO: 2). In some embodiments, the nucleic acid encoding SEQ ID NO:1 from amino acid 27 to 429. In some embodiments, the nucleic acid encoding SEQ ID NO:1 from amino acid 27 to 423.

In some embodiments, the non-human animal comprises a human or humanized IFNAR1 gene. In some embodiments, the humanized IFNAR1 gene comprises, in order from 5' -3: endogenous exon 1, a portion of endogenous exon 2 (e.g., nucleotide sequences NM_010508.2, 203-207), a portion of human exon 2 (e.g., nucleotide sequences NM_000629.3, 168-286), human exons 3-8, a portion of human exon 9 (e.g., nucleotide sequences NM_000629.3, 1230-1376), a portion of endogenous exon 9 (e.g., nucleotide sequences NM_010508.2, 1399-1402), endogenous exons 10-11.

In some embodiments, the humanized IFNAR1 comprises 11 exons. In some embodiments, humanized IFNAR1 comprises murine exon 1, humanized exon 2, human exon 3, human exon 4, human exon 5, human exon 6, human exon 7, human exon 8, humanized exon 9, murine exon 10, and/or murine exon 11. In some embodiments, the humanized IFNAR1 gene further comprises a human or humanized 5' utr. In some embodiments, the humanized IFNAR1 gene further comprises a human or humanized 3' utr. In some embodiments, the humanized IFNAR1 gene further comprises an endogenous 5' utr. In some embodiments, the humanized IFNAR1 gene further comprises an endogenous 3' utr.

In some embodiments, the genetically modified non-human animal may express human IFNAR1 and/or chimeric (e.g., humanized) IFNAR1 proteins, with endogenous IFNAR1 gene sequences replaced with human IFNAR1 genes and/or nucleotide sequences. Further, the amino acid sequence encoded by the human IFNAR1 gene and/or nucleotide sequence comprises SEQ ID NO:2 or 7, or comprises a sequence identical to SEQ ID NO:2 or 7 is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99.5%. In some embodiments, the nucleotide sequence of the endogenous non-human animal IFNAR1 gene is replaced with all or part of the nucleotide sequence encoding the mature human IFNAR1 protein.

In some embodiments, the genetically modified non-human animal expresses human IFNAR1 and/or chimeric IFNAR1 proteins (e.g., humanized IFNAR 1) under endogenous regulatory elements (e.g., promoters). Replacement of the endogenous locus provides a non-human animal expressing a human or chimeric IFNAR1 protein (e.g., humanized IFNAR 1) in the same type of cell. Genetically modified mice do not present the underlying disease observed in certain other transgenic mice known in the art. The human IFNAR1 or chimeric IFNAR1 protein expressed in the non-human animal may maintain the function of one or more wild-type or human IFNAR1 proteins, e.g., the expressed IFNAR1 protein may bind to a human or non-human IFNAR1 protein. Further, in some embodiments, the genetically modified non-human animal does not express an endogenous IFNAR1 protein. In some embodiments, the genetically modified non-human animal has reduced expression of endogenous IFNAR1 protein. As used herein, the term "endogenous IFNAR1 protein" refers to an IFNAR1 protein encoded by an endogenous IFNAR1 nucleotide sequence of a non-human animal (e.g., a mouse) prior to genetic modification.

The genome of the non-human animal comprises a nucleotide sequence encoding an amino acid that is identical to, or at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to, the amino acid sequence shown for the human IFNAR1 protein (np_ 000620.2;SEQ ID NO:2). In some embodiments, the genome comprises SEQ ID NO:5 or 6, or comprises a sequence identical to SEQ ID NO:5 or 6 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or at least 99.5%.

The nucleotide sequence encoding the endogenous IFNAR1 region in the genome of the non-human animal is replaced by a nucleotide sequence encoding a corresponding region of human IFNAR 1. In some embodiments, the nucleotide sequence encoding the endogenous IFNAR1 region is any sequence of the endogenous IFNAR1 locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, 5'utr, 3' utr, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, or any combination thereof. In some embodiments, the nucleotide sequence encoding an endogenous IFNAR1 region is located within an endogenous IFNAR1 regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous IFNAR1 region is all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or exon 11.

One or more cells of the genetically modified non-human animal express a human or chimeric IFNAR1 protein (e.g., a humanized IFNAR1 protein). In some embodiments, the human or chimeric IFNAR1 protein comprises at least one amino acid sequence that hybridizes to SEQ ID NO:2, 50, 60, 70, 80, 90, 100, 200, 300, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 500, 550, or 557 consecutive amino acid sequences.

In some embodiments, the genetically modified non-human animal genome comprises all or part of human IFNAR1 gene exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or exon 11, or encodes SEQ ID NO:2 or a part of the nucleotide sequence of the amino acid sequence shown in 2.

In some embodiments, the genetically modified non-human animal genome comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the genetically modified non-human animal genome comprises a portion of human IFNAR1 gene exon 2 to a portion of exon 9. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 119, 120, or 124bp contiguous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a contiguous nucleotide sequence of 119 bp. In some embodiments, the portion of exon 9 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 145, 146, 147, 148, 149, 150, or 151bp contiguous nucleotide sequence. In some embodiments, the portion of exon 9 comprises a 147bp contiguous nucleotide sequence. The human IFNAR1 gene exon 2 part, exon 3-8 all and exon 9 part including at least 500-1000, 1000-5000bp or 5000-6000bp consecutive nucleotide sequences. In some embodiments, the nucleotide sequence encoding the corresponding region of human IFNAR1 is located at nucleotide sequences 168-1376 of human IFNAR1 gene transcript nm_ 000629.3.

In some embodiments, the IFNAR1 gene of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.

In some embodiments, the humanized IFNAR1 genome comprises a 5' utr of a human IFNAR1 gene. In some embodiments, the humanized IFNAR1 genome comprises an endogenous (e.g., mouse) 5' utr. In some embodiments, the humanized IFNAR1 genome comprises a 3' utr of a human IFNAR1 gene. In some embodiments, the humanized IFNAR1 genome comprises an endogenous (e.g., mouse) 3' utr. Based on the similarity of the 5' flanking sequences, it is reasonable to speculate that the mouse and human IFNAR1 genes are similarly regulated, where appropriate. The humanized IFNAR1 mice according to the invention comprise a substitution of an endogenous mouse locus that retains the mouse endogenous regulatory elements but comprises the humanized IFNAR1 coding sequence. The expression of IFNAR1 in the genetically modified heterozygous mice or homozygous mice was completely normal.

In another aspect, the invention provides a genetically modified non-human animal comprising a deletion of an endogenous IFNAR1 gene, wherein the deletion of the endogenous IFNAR1 gene comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11, or a portion of an endogenous IFNAR1 locus.

In some embodiments, the deletion of the endogenous IFNAR1 gene comprises one or more exons or portions of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or exon 11.

In some embodiments, the endogenous IFNAR1 gene deletion further comprises one or more introns or portions of introns selected from the group consisting of IFNAR1 gene intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, and/or intron 10. In some embodiments, the intron is intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, and/or intron 8.

In some embodiments, the deletion of the endogenous IFNAR1 gene comprises a nucleotide sequence of at least 50、60、70、80、90、100、200、300、400、500、600、700、800、900、1000、1100、1191、1200、1500、1800、1900、2000、2100、2200、2500、3000、3500、4000、5000、6000、7000 or 7025bp consecutive nucleotide sequences or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11.

The invention provides a humanized mouse IFNAR1 genome DNA sequence, and provides a construct for expressing the amino acid sequence of a humanized IFNAR1 protein; a cell comprising the construct described above; a tissue comprising the above-described cells.

Thus, in some embodiments, the invention provides a chimeric (e.g., humanized) IFNAR1 nucleotide sequence and/or amino acid sequence, wherein in some embodiments, the chimeric nucleotide sequence is identical to a mouse endogenous IFNAR1 mRNA (e.g., nm_ 010508.2), a mouse IFNAR1 amino acid sequence (e.g., np_034638.2,SEQ ID NO:1), or a portion thereof (e.g., 5'utr, exon 1, a portion of exon 2, a portion of exon 9, and all of exons 10-11 and 3' utr) sequence, or is 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 99.5% identical. In some embodiments, the chimeric nucleotide sequence is identical to a human IFNAR1 mRNA sequence (e.g., nm_ 000629.3), a human IFNAR1 amino acid sequence (e.g., np_000620.2,SEQ ID NO:2), or a portion thereof (e.g., a portion of exon 2, all of exons 3-8, and a portion of exon 9) sequence, or is 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 99.5% identical.

In some embodiments, the nucleotide sequence encoding amino acids 27-423 or 27-429 of mouse IFNAR1 (SEQ ID NO: 1) is replaced by a corresponding nucleotide sequence encoding amino acids 28-430 or 28-436 of human IFNAR1 (SEQ ID NO: 2).

In some embodiments, the above-described nucleotide is operably linked to regulatory elements, such as an endogenous mouse IFNAR1 promoter, an inducible promoter, an enhancer, and/or a mouse or human regulatory element.

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., contiguous or non-contiguous nucleotide sequences) of the chimeric nucleic acid sequence differs from all or a portion of the mouse IFNAR1 nucleotide sequence (e.g., a portion of exon 2, all of exons 3-8, and a portion of exon 9 of mouse IFNAR1 gene transcript nm_010508.2, or a portion of exon 2 to a portion of exon 9).

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., contiguous or non-contiguous nucleotide sequences) of the chimeric nucleic acid sequence is identical to all or a portion of a mouse IFNAR1 nucleotide sequence (e.g., exon 1, part of exon 2, part of exon 9, and all of exons 10-11 of mouse IFNAR1 gene transcript nm_ 010508.2.)

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., contiguous or non-contiguous nucleotide sequences) of the chimeric nucleic acid sequence differs from all or a portion of the human IFNAR1 nucleotide sequence (e.g., exon 1, part of exon 2, part of exon 9, and all of exons 10-11 of human IFNAR1 gene transcript nm_ 000629.3).

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 human IFNAR1 nucleotide sequence (e.g., a portion of exon 2, all of exons 3-8, and a portion of exon 9 of human IFNAR1 gene transcript nm_000629.3, or a portion of exon 2 to a portion of exon 9).

In some embodiments, the chimeric nucleic acid sequence encodes an amino acid having 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) that is different from all or a portion of the amino acid sequence of a mouse IFNAR1 protein (e.g., amino acids 27-423 or 27-429 of the mouse IFNAR1 protein sequence np_034638.2 (SEQ ID NO: 1)).

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 is identical to all or a portion of the amino acid sequence of a mouse IFNAR1 protein (e.g., amino acids 1-26 and 424-590 or 430-590 of the mouse IFNAR1 protein sequence np_034638.2 (SEQ ID NO: 1)).

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 a human IFNAR1 protein (e.g., amino acids 1-27 and 431-557 or 437-557 of the human IFNAR1 protein sequence np_000620.2 (SEQ ID NO: 2)).

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 is identical to all or a portion of the amino acid sequence of a human IFNAR1 protein (e.g., amino acids 28-430 or 28-436 of human IFNAR1 protein sequence np_000620.2 (SEQ ID NO: 2)).

The present invention also provides a humanized IFNAR1 protein having an amino acid sequence comprising any one of the following groups:

a) SEQ ID NO: 1.2 or 7;

B) And SEQ ID NO: 1.2 or 7 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

C) An amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is capable of hybridizing to a nucleic acid sequence encoding SEQ ID NO: 1.2 or 7, and hybridizing the nucleotide sequence of the amino acid shown in 2 or 7;

D) And SEQ ID NO: 1.2 or 7, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or (b)

E) And SEQ ID NO: 1.2 or 7, comprising substitution, deletion and/or insertion of one or more amino acid residues.

A) SEQ ID NO:2 from positions 28 to 430 or from positions 28 to 436;

B) And SEQ ID NO:2 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 28-430 or 28-436;

C) And SEQ ID NO: the amino acid sequences shown at positions 28-430 or 28-436 of 2 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or (b)

D) And SEQ ID NO:2 from positions 28-430 or 28-436, comprising substitutions, deletions and/or insertions of one or more amino acid residues.

A) SEQ ID NO:1 from positions 1 to 26 and 424 to 590 or 430 to 590;

B) And SEQ ID NO:1 from 1 to 26 and 424 to 590 or 430 to 590 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) And SEQ ID NO: the amino acid sequences shown at positions 1-26 and 424-590 or 430-590 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or (b)

D) And SEQ ID NO:1 from positions 1-26 and 424-590 or 430-590, including substitution, deletion and/or insertion of one or more amino acid residues.

The invention also provides a humanized IFNAR1 gene (e.g., DNA or RNA) sequence whose nucleotide sequence comprises any one of the following groups:

a) As set forth in SEQ ID NO: 3.4, 5, 6, 8, 9, 10 and 11 or a nucleic acid sequence encoding a humanized mouse IFNAR1 homologous amino acid sequence;

b) Capable of hybridizing to SEQ ID NO: 3. 4, 5, 6, 8, 9, 10 and 11;

C) And SEQ ID NO: 3. 4, 5, 6, 8, 9, 10 and 11 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 90% homology;

D) The coded amino acid sequence is identical with SEQ ID NO: 1.2 or 7 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

E) The encoded amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 1.2 or 7, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or (b)

F) The encoded amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 1.2 or 7, comprising substitution, deletion and/or insertion of one or more amino acid residues.

The invention further provides an IFNAR1 genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by reverse transcription of mRNA transcribed from the DNA sequence and is identical to SEQ ID NO:5 or 6, and the DNA sequences homologous to the sequences shown in 5 or 6 are identical or complementary.

IFNAR2

In the human genome, the IFNAR2 Gene (Gene ID: 3455) contains 9 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and exon 9 (FIG. 5). The nucleotide sequence of human IFNAR2 mRNA is NM_207585.2, and the amino acid sequence of human IFNAR2 is NP-997468.1 (SEQ ID NO: 22). The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM-207585.2 and its encoded protein NP-997468.1 are shown in Table 3.

TABLE 3 Table 3

In the genome of the mice, the IFNAR2 Gene (Gene ID: 15976) contained 9 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and exon 9 (FIG. 5). The nucleotide sequence of mouse IFNAR2 mRNA is NM_010509.2, and the amino acid sequence of mouse IFNAR2 is NP-034639.2 (SEQ ID NO: 21). The corresponding positions of each exon in the nucleotide sequence and amino acid sequence based on transcript NM-010509.2 and its encoded protein NP-034639.2 are shown in Table 4.

TABLE 4 Table 4

Other species IFNAR2 genes, proteins and gene loci are also known in the art. For example, rattus norvegicus (rat) IFNAR2 Gene ID:686326, macaca mulatta (rhesus) IFNAR2 Gene ID:699726, canis lupus familiaris (dog) IFNAR2 Gene ID:100856468,Sus scrofa (pig) IFNAR2 Gene ID:100533555 relevant information about these genes (e.g., intron sequences, exon sequences, and amino acid sequences) can be found in NCBI, the entire contents of which are incorporated herein by reference.

The present invention provides a human or chimeric (e.g., humanized) IFNAR2 gene or protein. 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, a signal peptide, an extracellular region, a transmembrane region, and/or a cytoplasmic region of the mouse IFNAR2 gene is replaced with the nucleotide sequence of human IFNAR 2. In some embodiments, the mouse IFNAR2 gene exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, a signal peptide, an extracellular region, a transmembrane region, and/or a "portion" of a cytoplasmic region is replaced with a nucleotide sequence of human IFNAR 2. The term "portion" refers to a contiguous nucleotide sequence of at least 1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、200、300、400、500、600、700、720、725、726、727、728、729、730、800、1000、1100、1191、1200、2000、3000 or 3045bp, or at least 5, 10, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 510, or 513 contiguous amino acid sequences. In some embodiments, the "part" or "all" sequences of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and exon 9 of the mouse IFNAR2 gene (e.g., part of exon 2 and all of exon 3, or part of exon 2, all of exons 3-7, and part of exon 8, or part of exon 2 to part of exon 3) are replaced with the "part" or "all" sequences of the human IFNAR2 gene (e.g., part of exon 2, all of exons 3-7, and part of exon 8, or part of exon 2 to part of exon 8) by the "part" or "all" sequences of the human IFNAR2 gene, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and exon 9. In some embodiments, the extracellular region comprises a signal peptide. In some embodiments, the extracellular region does not comprise a signal peptide.

In some embodiments, the "portion" of the endogenous signal peptide, extracellular region, transmembrane region, cytoplasmic region, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9 is deleted.

In some embodiments, the invention provides a genetically modified non-human animal whose genome comprises a chimeric IFNAR2 gene. In some embodiments, the chimeric IFNAR2 gene encodes a protein comprising a human or humanized signal peptide, a human or humanized extracellular region, an endogenous transmembrane region, an endogenous cytoplasmic region. In some embodiments, the encoded protein comprises SEQ ID NO: 21. 22, 29 or 58, or comprises a sequence identical to SEQ ID NO: 21. 22, 29 or 58 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the non-human animal genome comprises a nucleotide sequence comprising SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 32, 33, 52, 53, 55, 56 and 57, or comprises a sequence identical to SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 32, 33, 52, 53, 55, 56, and 57 is at least 70%, 80%, 85%, 90%, 95%, or 99.5%.

In some embodiments, the non-human animal comprises a sequence encoding a human or humanized IFNAR2 protein. In some embodiments, the IFNAR2 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 IFNAR2 protein comprises a human or humanized signal peptide, e.g., the human or humanized IFNAR2 signal peptide comprises the amino acid sequence of SEQ ID NO:22, or comprises a sequence corresponding to positions 1-26 of SEQ ID NO:22 at least 70%, 80%, 85%, 90%, 95% or 99.5% amino acid identity between positions 1 and 26. In some embodiments, the humanized IFNAR2 protein comprises an endogenous signal peptide, e.g., the endogenous IFNAR2 signal peptide comprises the amino acid sequence of SEQ ID NO:21, or comprises a sequence corresponding to positions 1-21 of SEQ ID NO:21 from amino acid position 1 to 21 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR2 protein comprises a human or humanized extracellular region, e.g., the humanized extracellular region comprises the amino acid sequence of SEQ ID NO:22 at positions 27-242 or 27-243, or comprises a sequence identical to SEQ ID NO:22 at least 70%, 80%, 85%, 90%, 95% or 99.5% identical to amino acids 27-242 or 27-243. In some embodiments, the humanized IFNAR2 protein comprises an endogenous extracellular region, e.g., the endogenous IFNAR2 extracellular region comprises the amino acid sequence of SEQ ID NO:21, or comprising amino acid sequences corresponding to positions 33-242 of SEQ ID NO: the amino acid identity at positions 33-242 of 21 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR2 protein comprises a human or humanized transmembrane region, e.g., the humanized transmembrane region comprises the amino acid sequence of SEQ ID NO:22, or from positions 244 to 264, or from SEQ ID NO:22 at least 70%, 80%, 85%, 90%, 95% or 99.5% amino acid identity between positions 244 and 264. In some embodiments, the humanized IFNAR2 protein comprises an endogenous transmembrane region, e.g., the endogenous IFNAR2 transmembrane region comprises SEQ ID NO:21 at positions 243-263 or comprising a sequence identical to SEQ ID NO: the amino acid identity of positions 243-263 of 21 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR2 protein comprises a human or humanized cytoplasmic region, e.g., the humanized cytoplasmic region comprises the amino acid sequence of SEQ ID NO:22 from positions 265 to 515 or comprises a sequence corresponding to SEQ ID NO: the amino acid identity of positions 265-515 of 22 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. in some embodiments, the humanized IFNAR2 protein comprises an endogenous cytoplasmic region. For example, the endogenous IFNAR2 cytoplasmic region comprises SEQ ID NO:21, or comprising a sequence identical to SEQ ID NO: the amino acid identity of positions 264-513 of 21 is at least 70%, 80%, 85%, 90%, 95% or 99.5%. In some embodiments, the humanized IFNAR2 protein comprises an endogenous IFNAR2 sequence of SEQ ID NO:21 amino acids 243-513 or 33-513. In some embodiments, the humanized IFNAR2 protein comprises a human IFNAR2 sequence of SEQ ID NO:22 amino acids 1-242 or 1-243.

In some embodiments, the nucleotide sequence encoding all or part of the extracellular region of mouse IFNAR2 (SEQ ID NO: 21) is replaced or inactivated. In some embodiments, the sequence is replaced by a nucleotide sequence encoding all or part of the extracellular region of human IFNAR2 (SEQ ID NO: 22). In some embodiments, the nucleic acid encoding SEQ ID NO:21 from amino acid 1 to 242. In some embodiments, the nucleic acid encoding SEQ ID NO: the nucleotide sequence of amino acids 1-32 of 21 is replaced.

In some embodiments, the non-human animal comprises a human or humanized IFNAR2 gene. In some embodiments, the humanized IFNAR2 gene comprises, in order from 5' -3: endogenous exon 1, a portion of endogenous exon 2 (e.g., nucleotide sequences No. 293-330 of NM-010509.2), a portion of human exon 2 (e.g., nucleotide sequences No. 317-371 of NM-207585.2), human exons 3-7, a portion of human exon 8 (e.g., nucleotide sequences No. 1026-1045 of NM-207585.2), a portion of endogenous exon 8 (e.g., nucleotide sequences No. 1057-1167 of NM-010509.2), endogenous exon 9.

In some embodiments, the non-human animal comprises a human or humanized IFNAR2 gene. In some embodiments, the humanized IFNAR2 gene comprises, in order from 5' -3: endogenous exon 1, a portion of endogenous exon 2 (e.g., nucleotide sequences No. 293-330 of NM-010509.2), a portion of human exon 2 (e.g., nucleotide sequences No. 317-371 of NM-207585.2), human exons 3-7, a portion of human exon 8 (e.g., nucleotide sequences No. 1026-1042 of NM-207585.2), a portion of endogenous exon 8 (e.g., nucleotide sequences No. 1057-1167 of NM-010509.2), endogenous exon 9.

In some embodiments, the humanized IFNAR2 comprises 9 exons. In some embodiments, humanized IFNAR2 comprises murine exon 1, humanized exon 2, human exon 3, human exon 4, human exon 5, human exon 6, human exon 7, humanized exon 8, and/or murine exon 9. In some embodiments, the humanized IFNAR2 gene does not comprise an intron. In some embodiments, the humanized IFNAR2 gene further comprises one or more auxiliary sequences (e.g., STOP sequences). In some embodiments, the humanized IFNAR2 gene further comprises a human or humanized 5' utr. In some embodiments, the humanized IFNAR2 gene further comprises a human or humanized 3' utr. In some embodiments, the humanized IFNAR2 gene further comprises an endogenous 5' utr. In some embodiments, the humanized IFNAR2 gene further comprises an endogenous 3' utr.

In some embodiments, the genetically modified non-human animal may express human IFNAR2 and/or chimeric (e.g., humanized) IFNAR2 proteins, with endogenous IFNAR2 gene sequences replaced with human IFNAR2 genes and/or nucleotide sequences. Further, the amino acid sequence encoded by the human IFNAR2 gene and/or nucleotide sequence comprises SEQ ID NO: 22. 29 or 58, or comprises a sequence identical to SEQ ID NO: 22. 29 or 58 is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99.5%. In some embodiments, the nucleotide sequence of the endogenous non-human animal IFNAR2 gene is replaced with all or part of the nucleotide sequence encoding the mature human IFNAR2 protein.

In some embodiments, the genetically modified non-human animal expresses human IFNAR2 and/or chimeric IFNAR2 proteins (e.g., humanized IFNAR2 proteins) under endogenous regulatory elements (e.g., promoters). Replacement of the endogenous locus provides a non-human animal expressing a human or chimeric IFNAR2 protein (e.g., a humanized IFNAR2 protein) in the same type of cell. Genetically modified mice do not present the underlying disease observed in certain other transgenic mice known in the art. The human IFNAR2 or chimeric IFNAR2 protein expressed in the non-human animal may maintain the function of one or more wild-type or human IFNAR2 proteins, e.g., the expressed IFNAR2 protein may bind to a human or non-human IFNAR2 protein. Further, in some embodiments, the genetically modified non-human animal does not express an endogenous IFNAR2 protein. In some embodiments, the genetically modified non-human animal has reduced expression of endogenous IFNAR2 protein. As used herein, the term "endogenous IFNAR2 protein" refers to an IFNAR2 protein encoded by an endogenous IFNAR2 nucleotide sequence of a non-human animal (e.g., a mouse) prior to genetic modification.

The genome of the non-human animal comprises an amino acid sequence encoding the human IFNAR2 protein (np_ 997468.1;SEQ ID NO:22), or comprises a nucleotide sequence encoding an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence encoding the human IFNAR2 protein (np_ 997468.1;SEQ ID NO:22). In some embodiments, the genome comprises SEQ ID NO: 26. 57 or 28, or comprises a sequence identical to SEQ ID NO: 26. 57 or 28 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or at least 99.5%.

The nucleotide sequence encoding the endogenous IFNAR2 region in the genome of the non-human animal is replaced by the nucleotide sequence encoding human IFNAR 2. In some embodiments, the nucleotide sequence encoding the endogenous IFNAR2 region is any sequence of the endogenous IFNAR2 locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, 5'utr, 3' utr, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, or any combination thereof. In some embodiments, the nucleotide sequence encoding an endogenous IFNAR2 region is located within an endogenous IFNAR2 regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous IFNAR2 region is all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9.

One or more cells of the genetically modified non-human animal express a human or chimeric IFNAR2 protein (e.g., a humanized IFNAR2 protein). In some embodiments, the human or chimeric IFNAR2 protein comprises at least one amino acid sequence that hybridizes to SEQ ID NO:22, 50, 60, 70, 80, 90, 100, 200, 240, 241, 242, 243, 244, 245, 300, 400, 500, 510, 511, 512, 513, 514, or 515 consecutive amino acid sequences.

In some embodiments, the genetically modified non-human animal genome comprises all or part of human IFNAR2 gene exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9, or encodes SEQ ID NO:22 or a portion of the nucleotide sequence of the amino acid sequence shown in seq id no.

In some embodiments, the genetically modified non-human animal genome comprises a portion of exon 2, all of exons 3-7, and a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the genetically modified non-human animal genome comprises a portion of human IFNAR2 gene exon 2 to a portion of exon 8. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 91, or 92bp contiguous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a contiguous nucleotide sequence of 55 bp. In some embodiments, the portion of exon 8 comprises at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, or 131bp contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises a 20bp contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises a 17bp contiguous nucleotide sequence. The human IFNAR2 gene exon 2 part, exon 3-7 all and exon 8 part including at least 500-1000bp, 1000-5000bp or 5000-6000bp consecutive nucleotide sequences. In some embodiments, the nucleotide sequence encoding the corresponding region of human IFNAR2 is located at nucleotide sequence 317-1045 or 317-1042 of human IFNAR2 gene transcript NM_ 207585.2.

In some embodiments, the IFNAR2 gene of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.

In some embodiments, the humanized IFNAR2 genome comprises a 5' utr of a human IFNAR2 gene. In some embodiments, the humanized IFNAR2 genome comprises an endogenous (e.g., mouse) 5' utr. In some embodiments, the humanized IFNAR2 genome comprises a 3' utr of a human IFNAR2 gene. In some embodiments, the humanized IFNAR2 genome comprises an endogenous (e.g., mouse) 3' utr. Based on the similarity of the 5' flanking sequences, it is reasonable to speculate that the mouse and human IFNAR2 genes are similarly regulated, where appropriate. The humanized IFNAR2 mice according to the invention comprise a substitution of an endogenous mouse locus that retains the mouse endogenous regulatory elements but comprises the humanized IFNAR2 coding sequence. The expression of IFNAR2 in the genetically modified heterozygous mice or homozygous mice was completely normal.

In another aspect, the invention provides a genetically modified non-human animal comprising a deletion of an endogenous IFNAR2 gene, wherein the deletion of the endogenous IFNAR2 gene comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and/or exon 9, or a portion of an endogenous IFNAR2 locus.

In some embodiments, the deletion of the endogenous IFNAR2 gene comprises one or more exons or portions of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9.

In some embodiments, the endogenous IFNAR2 gene deletion further comprises one or more introns, or portions of introns, selected from the group consisting of IFNAR2 gene intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, and/or intron 8. In some embodiments, the intron is intron 2 and/or intron 3. In some embodiments, the intron is intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7.

In some embodiments, the deletion of the endogenous IFNAR2 gene comprises a nucleotide sequence of at least 50、60、70、80、90、95、96、97、98、99、100、200、300、400、500、600、700、726、800、900、1000、1100、1200、1500、1800、1900、2000、2100、2200、2500、3000 or 3045bp contiguous nucleotide sequence or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9.

The invention provides a humanized mouse IFNAR2 genome DNA sequence, and provides a construct for expressing the amino acid sequence of a humanized IFNAR2 protein; a cell comprising the construct described above; a tissue comprising the above-described cells.

Thus, in some embodiments, the invention provides a chimeric (e.g., humanized) IFNAR2 nucleotide sequence and/or amino acid sequence. In some embodiments, the chimeric nucleotide sequence is identical to a mouse endogenous IFNAR2 mRNA (e.g., nm_ 010509.2), a mouse IFNAR2 amino acid sequence (e.g., np_034639.2,SEQ ID NO:21), or a portion thereof (e.g., 5'utr, portion of exon 1, exon 2, portion of exon 8, and all of exon 9 and 3' utr) sequence, or has an 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 99.5%. In some embodiments, the chimeric nucleotide sequence is identical to a human IFNAR2 mRNA sequence (e.g., nm_ 207585.2), a human IFNAR2 amino acid sequence (e.g., np_997468.1,SEQ ID NO:22), or a portion thereof (e.g., a portion of exon 2, all of exons 3-7, and a portion of exon 8, or a portion of exon 2 to a portion of exon 8), or 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 99.5% identity.

In some embodiments, the nucleotide sequence encoding amino acids 1-32 or 1-242 of mouse IFNAR2 (SEQ ID NO: 21) is replaced with the nucleotide sequence encoding amino acids 1-242 or 1-243 of human IFNAR2 (SEQ ID NO: 22).

In some embodiments, the above-described nucleotide is operably linked to regulatory elements, such as an endogenous mouse IFNAR2 promoter, an inducible promoter, an enhancer, and/or a mouse or human regulatory element.

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., contiguous or non-contiguous nucleotide sequences) of the chimeric nucleic acid sequence is different from all or a portion of the mouse IFNAR2 nucleotide sequence (e.g., a portion of exon 2 and all of exon 3 of mouse IFNAR2 gene transcript nm_010509.2, or a portion of exon 2, all of exons 3-7, and a portion of exon 8, or a portion of exon 2 to a portion of exon 8).

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., contiguous or non-contiguous nucleotide sequences) of the chimeric nucleic acid sequence is identical to all or a portion of a mouse IFNAR2 nucleotide sequence (e.g., exon 1, a portion of exon 2, all of exons 4-9 or all of exon 1, a portion of exon 2, a portion of exon 8, and all of exon 9 of mouse IFNAR2 gene transcript nm_ 010509.2).

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., contiguous or non-contiguous nucleotide sequences) of the chimeric nucleic acid sequence differs from all or a portion of the human IFNAR2 nucleotide sequence (e.g., exon 1, part of exon 2, part of exon 8, and all of exon 9 of human IFNAR2 gene transcript nm_ 207585.2).

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 human IFNAR2 nucleotide sequence (e.g., a portion of exon 2, all of exons 3-7, and a portion of exon 8 of human IFNAR2 gene transcript nm_207585.2, or a portion of exon 2 to a portion of exon 8).

In some embodiments, the chimeric nucleic acid sequence encodes an amino acid having 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) that is different from all or a portion of the amino acid sequence of a mouse IFNAR2 protein (e.g., amino acids 1-32 or 1-242 of the mouse IFNAR2 protein sequence np_034639.2 (SEQ ID NO: 21)).

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 is identical to all or a portion of a mouse IFNAR2 protein amino acid sequence (e.g., amino acids 32-513 or 243-513 (SEQ ID NO: 21) of mouse IFNAR2 protein sequence np_ 034639.2).

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 a human IFNAR2 protein (e.g., amino acids 243-515 or 244-515 of human IFNAR2 protein sequence np_000620.2 (SEQ ID NO: 22)).

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 is identical to all or a portion of the amino acid sequence of a human IFNAR2 protein (e.g., amino acids 1-242 or 1-243 (SEQ ID NO: 22) of the human IFNAR2 protein sequence np_ 000620.2).

The present invention also provides a humanized IFNAR2 protein having an amino acid sequence comprising any one of the following groups:

a) SEQ ID NO: 21. 22, 29 or 58;

B) And SEQ ID NO: 21. 22, 29 or 58 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) An amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is capable of hybridizing to a nucleic acid sequence encoding SEQ ID NO: 21. 22 29 or 58, and a nucleotide sequence of an amino acid shown in seq id no;

D) And SEQ ID NO: 21. 22, 29 or 58, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or (b)

E) And SEQ ID NO: 21. 22, 29 or 58, comprising substitution, deletion and/or insertion of one or more amino acid residues.

a) SEQ ID NO:22 amino acid sequence shown in positions 1-242 or 1-243;

B) And SEQ ID NO:22 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 1-242 or 1-243;

C) And SEQ ID NO:22 at positions 1-242 or 1-243 by no more than 10, 9, 8, 7, 6, 5, 4, 3,2 or no more than 1 amino acid; or (b)

D) And SEQ ID NO:22 from positions 1-242 or 1-243, including substitutions, deletions and/or insertions of one or more amino acid residues.

a) SEQ ID NO:21 from position 33 to 513 or 243 to 513;

B) And SEQ ID NO:21 from 33 to 513 or 243 to 513, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions;

C) And SEQ ID NO: the amino acid sequences shown at positions 33-513 or 243-513 of 21 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or (b)

D) And SEQ ID NO:21 from positions 33-513 or 243-513, including substitutions, deletions and/or insertions of one or more amino acid residues.

The invention also provides a humanized IFNAR2 gene (e.g., DNA or RNA) sequence whose nucleotide sequence comprises any one of the following groups:

a) As set forth in SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 32, 33, 52, 53, 55, 56 and 57 or a nucleic acid sequence encoding a humanized mouse IFNAR2 homologous amino acid sequence;

b) Capable of hybridizing to SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 32, 33, 52, 53, 55, 56, and 57;

C) And SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 32, 33, 52, 53, 55, 56, and 57, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 90% homology;

D) The coded amino acid sequence is identical with SEQ ID NO: 21. 22, 29 or 58 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

e) The encoded amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 21. 22, 29 or 58, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or (b)

F) The encoded amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 21. 22, 29 or 58, comprising substitution, deletion and/or insertion of one or more amino acid residues.

The invention further provides an IFNAR2 genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by reverse transcription of mRNA transcribed from the DNA sequence and is identical to SEQ ID NO: 26. 27 is arranged 28 or 57, and the DNA sequences homologous to the sequences shown in figures 28 or 57 are identical or complementary.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be omitted for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to achieve optimal alignment of the two sequences, taking into account the number of gaps and the length of each gap. For example, the comparison of sequences and the determination of percent identity between two sequences can be accomplished using Blossum 62 scoring matrices with gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5.

Percentages of conserved residues (percent homology) with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. The art has defined families of amino acid residues with similar physicochemical properties. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophane, histidine). In many cases, the percent homology is higher than the percent identity.

The application also provides cells, tissues and animals (e.g., mice) comprising the nucleotide sequences of the application, as well as cells, tissues and animals (e.g., mice) expressing human or chimeric (e.g., humanized) IFNAR1 and/or IFNAR2 at endogenous non-human IFNAR1 and/or IFNAR2 loci.

Genetically modified non-human animals

The term "genetically modified non-human animal" or "genetically modified non-human animal" as used herein refers to a non-human animal having exogenous DNA (e.g., of human origin) on at least one chromosome in the genome of the non-human animal. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells having exogenous DNA may be various cells, for example, somatic cells, immune cells (e.g., T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blasts, or tumor cells. In some embodiments, a genetically modified non-human animal is provided that comprises a modified endogenous IFNAR1 and/or IFNAR2 locus, comprises an exogenous sequence (e.g., a human sequence), e.g., replaces one or more non-human sequences with one or more human sequences, or inserts one or more human and/or non-human sequences. Non-human animals are typically capable of transmitting genetic modifications to offspring through germ line transmission.

The term "chimeric gene" or "chimeric nucleic acid" or "chimeric nucleotide sequence" as used herein refers to a gene or nucleic acid in which two or more portions of the gene or nucleic acid are from different species or at least one sequence of the gene or nucleic acid is different from that of a nucleic acid in a wild-type animal. In some embodiments, the chimeric gene or chimeric nucleic acid or chimeric nucleotide sequence has at least a portion of the sequence from two or more different species sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, a chimeric gene or chimeric nucleic acid or chimeric nucleotide sequence refers to a humanized gene or humanized nucleic acid.

The term "chimeric protein" or "chimeric polypeptide" or "chimeric amino acid sequence" as used herein refers to a protein or polypeptide in which two or more portions of the polypeptide or protein are from different species or at least one sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric protein or chimeric polypeptide or chimeric amino acid sequence has two or more sources of different species, e.g., the same (or homologous) protein of different species. In some embodiments, a chimeric protein or chimeric polypeptide or chimeric amino acid sequence refers to a humanized protein or humanized polypeptide.

The term "humanized protein" or "humanized polypeptide" as used herein 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, a humanized protein or humanized polypeptide refers to a human protein or polypeptide.

The term "humanized nucleic acid" as used herein refers to nucleic acids, wherein at least a portion of the nucleic acids are derived from a human. In some embodiments, the nucleic acids in the humanized nucleic acids are all derived from humans. In some embodiments, a humanized nucleic acid refers to a humanized exon, which may be a human exon or a chimeric exon.

Non-human animals with humanized IFNAR1 loci

In some embodiments, the chimeric gene or chimeric nucleic acid or chimeric nucleotide sequence is a humanized IFNAR1 gene or a humanized IFNAR1 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human IFNAR1 gene and at least a portion of the gene or nucleic acid is derived from a non-human IFNAR1 gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding an IFNAR1 protein. The encoded IFNAR1 protein has at least one activity of human IFNAR1 protein or non-human animal IFNAR1 protein.

In some embodiments, the chimeric protein or chimeric polypeptide or chimeric amino acid sequence is a humanized IFNAR1 protein or a humanized IFNAR1 polypeptide. In some embodiments, the protein or polypeptide amino acid sequence of at least one or more portions from human IFNAR1 protein, and the protein or polypeptide amino acid sequence of at least one or more portions from non-human animal IFNAR1 protein. The humanized IFNAR1 protein or the humanized IFNAR1 polypeptide is functional or at least has the activity of a human IFNAR1 protein or a non-human animal IFNAR1 protein.

In some embodiments, the humanized IFNAR1 protein includes and human IFNAR1 protein identical 5-557 amino acid (continuous or discontinuous) polypeptide sequence. In some embodiments, the polypeptide sequence is 5-557, 10-403, 10-409 or 10-557 amino acids in length, for example, 5, 10, 20, 50, 100, 200, 300, 400, 403, 409, 500, 550 or 557 amino acid sequences identical to a human IFNAR1 protein. In some embodiments, the humanized IFNAR1 gene includes 20-35470bp (continuous or discontinuous) nucleotide sequence, which is identical to human IFNAR1 gene. In some embodiments, the contiguous 20-17376bp nucleotide sequence is identical to a human IFNAR1 gene, e.g., 20、50、100、200、500、1000、1209、2000、3000、4000、5000、6000、6075、7000、7043、8000、9000、10000、20000、30000、35000 or 35470bp nucleotide sequence is identical to a human IFNAR1 gene.

In some embodiments, the IFNAR1 extracellular region is human or humanized. In some embodiments, the IFNAR1 signal peptide is human or humanized. In some embodiments, the IFNAR1 cytoplasmic region is human or humanized. In some embodiments, the IFNAR1 transmembrane region is human or humanized. In some embodiments, the IFNAR1 signal peptide, the transmembrane region and the cytoplasmic region are endogenous.

Genetically modified non-human animals include modification of the endogenous non-human animal IFNAR1 gene locus (or locus). In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature IFNAR1 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the mature IFNAR1 protein). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein are provided in the present application, in many embodiments, the genetically modified non-human animal includes modifications to the endogenous IFNAR1 gene locus (or loci) in the non-human animal.

The genetically modified non-human animal may express human IFNAR1 and/or chimeric (e.g., humanized) IFNAR1 at an endogenous mouse locus, wherein the endogenous mouse IFNAR1 gene has been substituted or inserted with a gene of human IFNAR1 and/or a nucleotide sequence encoding a region of human IFNAR1 sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99.5% identical to the human IFNAR1 sequence. In various embodiments, the endogenous non-human animal IFNAR1 locus is modified by inclusion of a human full or partial nucleic acid sequence encoding a mature IFNAR1 protein.

In some embodiments, the genetically modified mice can express human IFNAR1 and/or chimeric IFNAR1 (e.g., humanized IFNAR 1) under the control of mouse regulatory elements (e.g., promoters). Insertion or substitution at a mouse endogenous locus provides a non-human animal that expresses human IFNAR1 or chimeric IFNAR1 (e.g., humanized IFNAR 1) in a suitable cell and in a manner that does not result in the underlying pathology observed in some other transgenic mice known in the art. Human IFNAR1 or chimeric IFNAR1 (e.g., humanized IFNAR 1) expressed in a non-human animal may maintain one or more functions of wild-type mouse or human IFNAR1 in the non-human animal. Furthermore, in some embodiments, the non-human animal does not express endogenous IFNAR1. In some embodiments, the non-human animal has a reduced level of endogenous IFNAR1 expression compared to the level of IFNAR1 expression in the wild-type animal. The term "endogenous IFNAR1" as used herein refers to an IFNAR1 protein expressed by the endogenous IFNAR1 nucleotide sequence of a non-human animal (e.g., mouse) prior to any genetic modification.

The genome of the non-human animal comprises an amino acid sequence encoding human IFNAR1 (np_ 000620.2;SEQ ID NO:2) or comprises a nucleotide sequence encoding at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence of human IFNAR1 (np_ 000620.2;SEQ ID NO:2). In some embodiments, the genome comprises SEQ ID NO:5 or 6, or comprises a sequence identical to SEQ ID NO:5 or 6, at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical. In some embodiments, the genome comprises positions 168-1376 of nm_000629.3, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to positions 168-1376 of nm_ 000629.3.

The genome of the genetically modified non-human animal may comprise a replacement of the endogenous IFNAR1 locus with a sequence encoding a corresponding region of human IFNAR 1. In some embodiments, the sequence that is replaced is any sequence of the endogenous IFNAR1 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, 5'utr, 3' utr, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, or any combination thereof. In some embodiments, the substituted sequence is within the regulatory region of the endogenous IFNAR1 gene. In some embodiments, the sequence replaced is part of exon 2, all of exons 3-8, and part of exon 9 of the endogenous mouse IFNAR1 locus. In some embodiments, the sequence that is replaced is a portion of exon 2 to a portion of exon 9 of the endogenous mouse IFNAR1 locus.

The genetically modified non-human animal may have one or more cells expressing human or chimeric IFNAR1 (e.g., humanized IFNAR 1) 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 signal peptide of human IFNAR1 (e.g., amino acids 1-27 of SEQ ID NO: 2), or comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to a signal peptide of human IFNAR1 (e.g., amino acids 1-27 of SEQ ID NO: 2). In some embodiments, the signal peptide of the humanized IFNAR1 has a sequence of at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 26 amino acids (e.g., contiguous or non-contiguous) that is identical to the endogenous IFNAR1 signal peptide. In some embodiments, the extracellular region comprises the extracellular region of human IFNAR1, or comprises a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the extracellular region of human IFNAR 1. In some embodiments, the extracellular region of the humanized IFNAR1 has a sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 401, 402, 403, 404, 405, 406, 407, 408, or 409 amino acids (contiguous or non-contiguous) that is identical to the extracellular region of human IFNAR 1. In some embodiments, the extracellular region of the humanized IFNAR1 has a sequence of at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 401, 402, or 403 amino acids (contiguous or non-contiguous) that is identical to the extracellular region of the endogenous IFNAR 1. In some embodiments, the extracellular region of the application comprises a signal peptide. In some embodiments, the extracellular regions described herein do not include a signal peptide. Because in many cases the human IFNAR1 and non-human IFNAR1 (e.g., mouse IFNAR 1) sequences are different, antibodies that bind to human IFNAR1 do not necessarily have the same affinity as non-human IFNAR1 or have the same effect on non-human IFNAR 1. Thus, transgenic animals with human or humanized extracellular regions can be used to better assess the role of anti-human IFNAR1 antibodies in non-human animals or animal models.

In some embodiments, the transmembrane region comprises the transmembrane region of human IFNAR1 (e.g., amino acids 437-457 of SEQ ID NO: 2), or comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the transmembrane region of human IFNAR1 (e.g., amino acids 437-457 of SEQ ID NO: 2). In some embodiments, the transmembrane region of the humanized IFNAR1 has at least 1,2,3,4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids (contiguous or non-contiguous) that are identical to the transmembrane region of the endogenous IFNAR 1. In some embodiments, the cytoplasmic region comprises a cytoplasmic region of human IFNAR1 (e.g., amino acids 458-557 of SEQ ID NO: 2), or comprises a sequence which is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to a cytoplasmic region of human IFNAR1 (e.g., amino acids 458-557 of SEQ ID NO: 2). In some embodiments, the cytoplasmic region of the humanized IFNAR1 has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 141 amino acids (contiguous or non-contiguous) that is identical to the cytoplasmic region of an endogenous IFNAR1 (e.g., mouse IFNAR 1).

In some embodiments, the entire signal peptide, the entire transmembrane region and the entire cytoplasmic region of the humanized IFNAR1 are derived from an endogenous IFNAR1 sequence.

In some embodiments, the genome of the non-human animal comprises: part of exon 2, all of exons 3-8 and/or part of exon 9 of the human IFNAR1 gene; or encodes SEQ ID NO:2 from amino acid position 28 to 430 or 28 to 436.

In some embodiments, the genome of the genetically modified non-human animal comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 119, 120, or 124bp contiguous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a contiguous nucleotide sequence of 119 bp. In some embodiments, the portion of exon 9 comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 145, 146, 147, 148, 149, 150, or 151bp contiguous nucleotide sequence. In some embodiments, the portion of exon 9 comprises a 147bp contiguous nucleotide sequence.

In some embodiments, the genome of the genetically modified non-human animal includes exon 1, part of exon 2, part of exon 9, and all of exons 10-11 of an endogenous IFNAR1 gene (e.g., mouse IFNAR 1). In some embodiments, all of exon 1, part of exon 2, part of exon 9, and exons 10-11 comprise at least 1,2,3,4, 5, 6, 10, 20, 30, 35, 38, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides.

In some embodiments, the non-human animal has a nucleotide sequence encoding a chimeric human/non-human IFNAR1 polypeptide at an endogenous IFNAR1 locus, which expresses functional IFNAR1 on the cell surface of the non-human animal. In some embodiments, the human portion of the chimeric human/non-human IFNAR1 polypeptide may comprise an amino acid sequence encoded by a portion of exon 2, all of exons 3-8, and/or a portion of exon 9 of the human IFNAR1 gene. In some embodiments, the human portion of the chimeric human/non-human IFNAR1 polypeptide comprises SEQ ID NO:2, or comprises a sequence identical to SEQ ID NO:2, at least 80%, 85%, 90%, 95% or 99% identical.

Furthermore, the modified gene in the genome of the modified non-human animal is homozygous or heterozygous for the endogenous replaced locus. In a specific embodiment, the modified IFNAR1 gene in the genome is homozygous or heterozygous for the endogenous replaced locus.

In some embodiments, the humanized IFNAR1 locus comprises a human 5' utr. In some embodiments, the humanized IFNAR1 locus comprises an endogenous (e.g., mouse) 5' utr. In some embodiments, humanization comprises an endogenous (e.g., mouse) 3' utr. Where appropriate, it may reasonably be assumed that, based on the similarity of the 5' flanking sequences of the mouse and human IFNAR1 genes, they appear to be similarly regulated. As shown herein, humanized IFNAR1 mice comprising insertions or substitutions at the endogenous mouse IFNAR1 locus retain mouse regulatory elements but comprise humanization of the IFNAR1 coding sequence, and do not exhibit pathological phenomena. Both genetically modified mice, heterozygous or homozygous for the humanized IFNAR1, were normal.

In another aspect, the invention also provides a genetically modified non-human animal whose genome comprises a disruption of a non-human animal endogenous IFNAR1 gene, wherein the disruption of the endogenous IFNAR1 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, and/or exon 11, or a partial deletion thereof.

In some embodiments, the disruption of the endogenous IFNAR1 gene further comprises a deletion of one or more introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10.

In some embodiments, the deletion may include deleting at least 1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、200、300、400、500、600、700、800、1000、1100、1191、1200、2000、3000、4000、5000、6000、7000、7014 or 7025bp consecutive nucleotide sequences or more.

Non-human animals with humanized IFNAR2 loci

In some embodiments, the chimeric gene or chimeric nucleic acid or chimeric nucleotide sequence is a humanized IFNAR2 gene or a humanized IFNAR2 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human IFNAR2 gene and at least a portion of the gene or nucleic acid is derived from a non-human IFNAR2 gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding an IFNAR2 protein. The encoded IFNAR2 protein has at least one activity of human IFNAR2 protein or non-human animal IFNAR2 protein.

In some embodiments, the chimeric protein or chimeric polypeptide or chimeric amino acid sequence is a humanized IFNAR2 protein or a humanized IFNAR2 polypeptide. In some embodiments, the protein or polypeptide amino acid sequence of at least one or more portions from human IFNAR2 protein, and the protein or polypeptide amino acid sequence of at least one or more portions from non-human animal IFNAR2 protein. The humanized IFNAR2 protein or the humanized IFNAR2 polypeptide is functional or at least has the activity of a human IFNAR2 protein or a non-human animal IFNAR2 protein.

In some embodiments, the humanized IFNAR2 protein includes and human IFNAR2 protein identical 5-515 amino acid (continuous or discontinuous) polypeptide sequence. In some embodiments, the polypeptide sequence is 5-515, 10-242, 10-243, or 10-515 amino acids in length, e.g., 10, 20, 30, 40, 50, 100, 150, 200, 242, 243, 250, 300, 350, 400, 450, 500, or 515. In some embodiments, the humanized IFNAR2 gene comprises a nucleotide sequence of 20-35727bp (contiguous or non-contiguous, e.g., 20, 50, 100, 200, 300, 400, 500, 600, 700, 726, 729, 800, 900, 1000, 2000, 5000, 10000, 15000, 18691, 20000, 30000, or 35727 bp) that is identical to a human IFNAR2 gene. In some embodiments, 20-729bp, 20-726bp consecutive with the human IFNAR2 gene is the same. In some embodiments, 20-18691bp consecutive with the human IFNAR2 gene is the same.

In some embodiments, the IFNAR2 extracellular region is human or humanized. In some embodiments, the IFNAR2 signal peptide is human or humanized. In some embodiments, the IFNAR2 cytoplasmic region is human or humanized. In some embodiments, the IFNAR2 transmembrane region is human or humanized. In some embodiments, both the IFNAR2 transmembrane region and cytoplasmic region are endogenous.

Genetically modified non-human animals include modification of the endogenous non-human animal IFNAR2 gene locus (or locus). In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature IFNAR2 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the mature IFNAR2 protein). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modifications described herein are provided in the present application, in many embodiments, the genetically modified non-human animal includes modifications to the endogenous IFNAR2 gene locus (or loci) in the non-human animal.

The genetically modified non-human animal may express human IFNAR2 and/or chimeric (e.g., humanized) IFNAR2 at an endogenous mouse locus, wherein the endogenous mouse IFNAR2 gene has been substituted or inserted with a gene for human IFNAR2 and/or a nucleotide sequence encoding a region of human IFNAR2 sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% identical to the human IFNAR2 sequence. In various embodiments, the endogenous non-human animal IFNAR2 locus is modified by inclusion of a human full or partial nucleic acid sequence encoding a mature IFNAR2 protein.

In some embodiments, the genetically modified mice can express human IFNAR2 and/or chimeric IFNAR2 (e.g., humanized IFNAR 2) under the control of mouse regulatory elements (e.g., promoters). Insertion or substitution at a mouse endogenous locus provides a non-human animal that expresses human IFNAR2 or chimeric IFNAR2 (e.g., humanized IFNAR 2) in a suitable cell and in a manner that does not result in the underlying pathology observed in some other transgenic mice known in the art. Human IFNAR2 or chimeric IFNAR2 (e.g., humanized IFNAR 2) expressed in a non-human animal may maintain one or more functions of wild-type mouse or human IFNAR2 in the non-human animal. Furthermore, in some embodiments, the non-human animal does not express endogenous IFNAR2. In some embodiments, the non-human animal has a reduced level of endogenous IFNAR2 expression compared to the level of IFNAR2 expression in the wild-type animal. The term "endogenous IFNAR2" as used herein refers to an IFNAR2 protein expressed by the endogenous IFNAR2 nucleotide sequence of a non-human animal (e.g., mouse) prior to any genetic modification.

The genome of the non-human animal comprises an amino acid sequence encoding human IFNAR2 (np_ 997468.1;SEQ ID NO:22) or comprises a nucleotide sequence encoding at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence of human IFNAR2 (np_ 997468.1;SEQ ID NO:22). In some embodiments, the genome comprises SEQ ID NO: 26. 28 or 57, or comprising a sequence identical to SEQ ID NO: 26. 28 or 57, at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical. In some embodiments, the genome comprises positions 317 to 1045 or 317 to 1042 of nm_207585.2, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to positions 317 to 1045 or 317 to 1042 of nm_ 207585.2.

The genome of the genetically modified non-human animal may include a replacement of the endogenous IFNAR2 locus with a sequence encoding a human IFNAR2 region. In some embodiments, the sequence that is replaced is any sequence of the endogenous IFNAR2 locus, such as exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, 5'utr, 3' utr, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, or any combination thereof. In some embodiments, the substituted sequence is within the regulatory region of the endogenous IFNAR2 gene. In some embodiments, the sequence replaced is part of exon 2 and all of exon 3 of the endogenous mouse IFNAR2 locus. In some embodiments, the sequence replaced is part of exon 2, all of exons 3-7, and part of exon 8 of the endogenous mouse IFNAR2 locus.

The genetically modified non-human animal may have one or more cells expressing human or chimeric IFNAR2 (e.g., humanized IFNAR 2) 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 signal peptide of endogenous IFNAR2 (e.g., amino acids 1-21 of SEQ ID NO: 21), or comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to a signal peptide of endogenous IFNAR2 (e.g., amino acids 1-21 of SEQ ID NO: 21). In some embodiments, the signal peptide of the humanized IFNAR2 has a sequence of at least 1,2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 26 amino acids (e.g., contiguous or non-contiguous) that is identical to the human IFNAR2 signal peptide. In some embodiments, the extracellular region comprises the extracellular region of human IFNAR2, or comprises a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the extracellular region of human IFNAR 2. In some embodiments, the extracellular region of the humanized IFNAR2 has a sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 210, 215, 216, or 217 amino acids (contiguous or non-contiguous) that is identical to the extracellular region of human IFNAR 2. In some embodiments, the extracellular region of the humanized IFNAR2 has a sequence of at least 1,2, 3,4, 5, 6, 7, 8, 9, 10, 15, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 210, 220, or 221 amino acids (contiguous or non-contiguous) that is identical to the endogenous IFNAR2 extracellular region. In some embodiments, the extracellular region comprises a signal peptide. In some embodiments, the extracellular region does not include a signal peptide. Because in many cases the human IFNAR2 and non-human IFNAR2 (e.g., mouse IFNAR 2) sequences are different, antibodies that bind to human IFNAR2 do not necessarily have the same affinity as non-human IFNAR2 or have the same effect on non-human IFNAR 2. Thus, transgenic animals with human or humanized extracellular regions can be used to better assess the role of anti-human IFNAR2 antibodies in non-human animals or animal models.

In some embodiments, the transmembrane region comprises the transmembrane region of human IFNAR2 (e.g., amino acids 244-264 of SEQ ID NO: 22), or comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the transmembrane region of human IFNAR2 (e.g., amino acids 244-264 of SEQ ID NO: 22). In some embodiments, the transmembrane region of the humanized IFNAR2 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 the endogenous IFNAR 2. In some embodiments, the cytoplasmic region comprises a cytoplasmic region of human IFNAR2 (e.g., amino acids 265-515 of SEQ ID NO: 22), or comprises a sequence which is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to a cytoplasmic region of human IFNAR2 (e.g., amino acids 265-515 of SEQ ID NO: 22). In some embodiments, the cytoplasmic region of the humanized IFNAR2 has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 200, or 250 amino acids (contiguous or non-contiguous) that is identical to the cytoplasmic region of an endogenous IFNAR2 (e.g., mouse IFNAR 2).

In some embodiments, the entire transmembrane region and the entire cytoplasmic region of the humanized IFNAR2 are derived from an endogenous IFNAR2 sequence.

In some embodiments, the humanized IFNAR2 whole signal peptide and whole extracellular region derived from human IFNAR2 sequence.

In some embodiments, the genome of the non-human animal comprises: part of exon 2, all of exons 3-7 and part of exon 8 of the human IFNAR2 gene; or from the part of exon 2 to the part of exon 8 of the human IFNAR2 gene; or encodes SEQ ID NO:22 amino acid 1-242 or 1-243.

In some embodiments, the genome of the genetically modified non-human animal comprises a portion of exon 2, all of exons 3-7, and a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the portion of exon 2 comprises at least 5, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 91, or 92bp contiguous nucleotide sequence. In some embodiments, the portion of exon 2 comprises a contiguous nucleotide sequence of 55 bp. In some embodiments, the portion of exon 8 comprises at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, or 131bp contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises a 20bp contiguous nucleotide sequence. In some embodiments, the portion of exon 8 comprises a 17bp contiguous nucleotide sequence.

In some embodiments, the genome of the genetically modified non-human animal includes exon 1, part of exon 2, all of exons 4-9 of an endogenous IFNAR2 gene (e.g., mouse IFNAR 2). In some embodiments, all of exon 1, part of exon 2, exons 4-9 comprise at least 1,2, 3, 4, 5, 6, 10, 20, 30, 35, 38, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides.

In some embodiments, the genome of the genetically modified non-human animal includes exon 1, part of exon 2, part of exon 8, and all of exon 9 of the endogenous IFNAR2 gene (e.g., mouse IFNAR 2). In some embodiments, all of exon 1, part of exon 2, part of exon 8, and exon 9 comprise at least 1,2,3, 4, 5, 6, 10, 20, 30, 35, 38, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides.

In some embodiments, the non-human animal has a nucleotide sequence encoding a chimeric human/non-human IFNAR2 polypeptide at an endogenous IFNAR2 locus, which expresses functional IFNAR2 on the cell surface of the non-human animal. In some embodiments, the human portion of the chimeric human/non-human IFNAR2 polypeptide may comprise an amino acid sequence encoded by a portion of exon 2, all of exons 3-7, and/or a portion of exon 8 of the human IFNAR2 gene. In some embodiments, the human portion of the chimeric human/non-human IFNAR2 polypeptide comprises SEQ ID NO:22 or comprises a sequence identical to SEQ ID NO:22 at least 80%, 85%, 90%, 95% or 99% identical.

Furthermore, the modified gene in the genome of the modified non-human animal is homozygous or heterozygous for the endogenous replaced locus. In a specific embodiment, the modified IFNAR2 gene in the genome is homozygous or heterozygous for the endogenous replaced locus.

In some embodiments, the humanized IFNAR2 locus comprises a human 5' utr. In some embodiments, the humanized IFNAR2 locus comprises an endogenous (e.g., mouse) 5' utr. In some embodiments, humanization comprises an endogenous (e.g., mouse) 3' utr. Where appropriate, it may reasonably be assumed that, based on the similarity of the 5' flanking sequences of the mouse and human IFNAR2 genes, they appear to be similarly regulated. As shown herein, humanized IFNAR2 mice comprising insertions or substitutions at the endogenous mouse IFNAR2 locus retain mouse regulatory elements but comprise humanization of the IFNAR2 coding sequence, and do not exhibit pathological phenomena. Both genetically modified mice, heterozygous or homozygous for the humanized IFNAR2, were normal.

In another aspect, the invention also provides a genetically modified non-human animal whose genome comprises a disruption of a non-human animal endogenous IFNAR2 gene, wherein the disruption of the endogenous IFNAR2 gene comprises a deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9, or a partial deletion thereof.

In some embodiments, the disruption of the endogenous IFNAR2 gene further comprises a deletion of one or more introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, and intron 8.

In some embodiments, the deletion may include deletion of at least 50、60、70、80、90、97、100、200、300、400、500、600、700、720、726、800、900、1000、1100、1200、1500、1800、1900、2000、2100、2200、2500、3000、3050 or 3045bp contiguous nucleotide sequences or more.

The genetically modified non-human animal can be a variety of animals, e.g., mice, rats, zebra fish, rabbits, pigs, cattle (e.g., cattle, bull, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmoset, rhesus). For non-human animals for which suitable genetically modifiable embryonic stem cells (ES) are not readily available, other methods are employed to construct non-human animals comprising genetic modifications. Such methods include, for example, modifying a non-ES cell genome (e.g., a fibroblast or induced pluripotent stem cell) and transferring the modified genome to a suitable cell, such as an oocyte, using a nuclear transfer, and seeding the modified cell (e.g., modified oocyte) under appropriate conditions in a non-human animal to form an embryo. The above-described construction methods are known in the art and described at "A.Nagy,et al.,"Manipulating the Mouse Embryo:A Laboratory Manual(Third Edition),"Cold Spring Harbor Laboratory Press,2006", the entire contents of which are incorporated herein by reference.

In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent may be selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from a murine family. In one embodiment, the genetically modified non-human animal is from a family selected from the group consisting of the hamstocidae (e.g., hamster-like), hamster family (e.g., hamster, new world rats and mice, field mice), murine superfamily (true mice and rats, gerbil, spiny mice, coronary rats), equine island murine family (mountain climbing mice, rock mice, tailed rats, motor gas rats and mice), spiny murine family (e.g., spiny sleeping mice) and mole murine family (e.g., mole rats, bamboo rats and zokor). In a particular embodiment, the genetically modified rodent is selected from the group consisting of a true mouse or rat (murine superfamily), a gerbil, a spiny mouse, and a coronary rat. In one embodiment, the genetically modified mouse is from a member of the murine family. In one embodiment, the non-human animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

In some embodiments, the non-human animal may be an immunodeficient non-human mammal. Such as immunodeficient rodents, immunodeficient rabbits, immunodeficient pigs, immunodeficient monkeys, etc. In some embodiments, the animal is a mouse of the C57BL strain, the C57BL strain is selected from the group consisting of C57BL/a, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL6, C57BL/10, C57BL10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of 129/J, 129/ReJ, 129/OlaHsd, 129/Sv, 129/SvJ, 129/Re, 129/RrJ, 129/Sv-ter/+. These mice are described, for example, in Festing et al.,Revised nomenclature for strain 129mice,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 literature references cited above, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a cross of the 129 strain and the C57BL/6 strain. In some embodiments, the mice are a 129 strain of crosses, or a BL/6 strain of crosses. In some embodiments, the mice are BALB strains, e.g., BALB/c strains. In some embodiments, the mouse is a cross of a BALB strain and another strain. In some embodiments, the mice are from a hybrid line (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 strain of BALB/C, BALB/cHean, BALB/cJ, BALB/cRl, BALB/cWt, C57BL/10ScSn, C57BL (C57 BL/10Cr and C57 BL/Ola), C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, or CBA/H, and in some embodiments, the non-human animal is a rat. Rats may 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 species of two or more strains selected from Wistar, LEA, sprague-Dawley, fischer, F344, F6, and Dark Agouti.

The non-human animal may have one or more other genetic modifications and/or other modifications that are suitable for the particular purpose of preparing the 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 would damage, inactivate, or destroy all or part of the immune system of a non-human animal. Damage, inactivation, or disruption 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., irradiation of the animal), and/or genetic modification (e.g., knockout of 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/yc ^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 may optionally be irradiated, or otherwise treated to destroy one or more immune cell types. Thus, in various embodiments, a genetically modified mouse is provided that may include humanization of at least a portion of endogenous non-human IFNAR1 and/or IFNAR2 loci, and further include modification that damages, inactivates, or partially disrupts 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 the group consisting of modifications of NOD mice, SCID mice, NOD/SCID mice, IL-2rγ knockout mice, NOD/SCID/yc ^null mice, nude mice, rag1 and/or Rag2 knockout mice, NOD Prkdc ^scid IL-2Rγ^null mice, NOD Rag1 ^-/-IL2rg^-/- (NRG) mice, rag2 ^-/-IL2rg^-/- (RG) mice, and combinations thereof. These transgenic animals are described, for example, in US10820580B2, which is incorporated by reference in its entirety. In some embodiments, the mouse may include replacement of all or part of the mouse endogenous mature IFNAR1 and/or IFNAR2 coding sequence with all or part of the human mature IFNAR1 and/or IFNAR2 coding sequence, respectively.

The invention further relates to a non-human mammal produced by the above method. In some embodiments, the genome comprises a human gene.

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

In some embodiments, the non-human mammal expresses a protein encoded by a humanized IFNAR1 and/or IFNAR2 gene.

Furthermore, the application provides a non-human mammal model carrying a tumor, said non-human mammal model being obtained by the method according to the application. In some embodiments, the non-human mammal is a rodent (e.g., a mouse).

The invention also provides a cell or cell line derived from a non-human mammal or a progeny thereof, or a non-human mammal bearing a tumor, or a primary cell culture derived from a non-human mammal or a progeny thereof, or a non-human mammal bearing a tumor, a tissue, organ or culture thereof derived from a non-human mammal or a progeny thereof. Tumor tissue derived from or carrying a tumor when it carries a tumor.

The application provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal, a genetically modified non-human animal is provided, the genetically modified non-human animal genome comprising DNA of a human or humanized IFNAR1 and/or IFNAR 2.

In some embodiments, the non-human mammal comprises a gene construct according to the application (e.g., a gene construct as shown in figures 2 and 6-8). In some embodiments, a non-human mammal expressing a human or humanized IFNAR1 and/or IFNAR2 protein is provided. In some embodiments, a tissue is provided that specifically expresses a human or humanized IFNAR1 and/or IFNAR2 protein.

In some embodiments, expression of human or humanized IFNAR1 and/or IFNAR2 proteins in a non-human animal is controllable. Such as by the addition of specific inducers or repressors. In some embodiments, the specific inducer is selected from the group consisting of a tetracycline System (Tet-Off System/Tet-On System) or a Tamoxifen System (Tamoxifen System).

The non-human mammal may be any non-human animal known in the art which may be used in the method of the application. Preferred non-human mammals are mammals (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

Genetic, molecular and behavioral analysis was performed on the non-human mammal described above. The invention provides a offspring produced by mating with a non-human mammal of the same genotype or other genotypes.

The present invention provides a cell line or primary cell culture derived from a non-human mammal or a progeny thereof. A cell culture-based model can be prepared, for example, by the following method. The cell culture may be obtained by isolation from a non-human mammal, or the cells may be obtained from a cell culture established using the same construct and using standard cell transfection techniques. Integration of the genetic structure comprising a DNA sequence encoding a human IFNAR1 and/or IFNAR2 protein may be detected by a variety of methods.

There are a number of analytical methods available for detecting exogenous DNA, including methods at the nucleic acid level, including methods using reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization, and methods at the protein level, including histochemical analysis, immunoblot analysis and in vitro binding studies. In addition, the expression level of the gene of interest can be quantified by ELSA methods well known to those skilled in the art. A number of standard analytical methods are available for accomplishing quantitative detection. For example, transcription levels can be detected using RT-PCR and hybridization methods, including RNase protection assays, southern Blot, RNA spot hybridization assays (RNAdot). Immunohistochemical staining, flow cytometry, western blot may also be used to detect the presence of human or humanized IFNAR1 and/or IFNAR2 proteins.

Carrier body

The invention provides a targeting vector, comprising: a) A DNA fragment (5 'arm) homologous to the 5' end of the region to be altered, selected from the group consisting of genomic DNA of the IFNAR1 gene, from 100 to 10000 nucleotides in length; b) A donor area; and c) a DNA fragment (3 'arm) homologous to the 3' end of the region to be altered, selected from the group consisting of genomic DNA of the IFNAR1 gene, of 100 to 10000 nucleotides in length.

In some embodiments, a) the DNA fragment homologous to the 5' end of the region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI accession nc_ 000082.7; c) The DNA fragment homologous to the 3' end of the region to be altered is selected from the group consisting of nucleotide sequences having at least 90% homology to NCBI accession No. NC-000082.7.

In some embodiments, a) the 5' homologous DNA fragment of the region to be altered is selected from the nucleotide sequences 91282641 to 91286588 of NCBI accession nc_ 000082.7; c) The 3' -homologous DNA fragment of the region to be altered is selected from the nucleotide sequences 91300904 to 91305823 of NCBI accession No. NC_ 000082.7.

In some embodiments, the genomic nucleotide sequence selected for the targeting vector may be more than about 0.8kb、1kb、1.5kb、2kb、2.5kb、3kb、3.5kb、4kb、4.5kb、6.5kb、7kb、7.5kb、8kb、8.5kb、9kb、9.5kb、10kb、15kb、16kb、18kb、19kb or 20kb in length.

In some embodiments, the region to be altered is located on exons 2 to 9 of the non-human animal IFNAR1 gene.

In some embodiments, the 5 'arm is a nucleotide having at least 90% homology to NCBI accession nc_000082.7, further preferably, the 5' arm sequence comprises SEQ ID NO:3, and a nucleotide sequence shown in 3. In some embodiments, the 3 'arm is a nucleotide having at least 90% homology to NCBI accession nc_000082.7, further preferably, the 3' arm sequence comprises SEQ ID NO:4, and a nucleotide sequence shown in the specification.

In some embodiments, the targeting vector comprises a human sequence (e.g., positions 33335529 to 33352904 of nc_ 000021.9). For example, the targeting region in the targeting vector includes: some or all of the nucleotide sequence of the human IFNAR1 gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11 of the human IFNAR1 gene. In some embodiments, the nucleotide sequence of the humanized IFNAR1 gene encodes all or a portion of a human IFNAR1 protein, NCBI protein number NP-000620.2 (SEQ ID NO: 2). In some embodiments, the nucleotide sequence of the humanized IFNAR1 gene encodes a protein of SEQ ID NO:7.

The invention also provides vectors for constructing humanized animal models or knockout models. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the IFNAR1 gene and the sgRNA is unique in the target sequence of the gene to be altered and satisfies the sequence alignment rules of 5'-NNN (20) -NGG3' or 5'-CCN-N (20) -3'; and in some embodiments, the targeting site for sgRNA in the mouse IFNAR1 gene is located on or upstream of exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, exon 6, intron 6, exon 7, intron 7, exon 8, exon 9, intron 9, exon 10, intron 10 and/or exon 11, on or downstream of exon 2 of the mouse IFNAR1 gene.

The invention provides a targeting vector, comprising: a) A DNA fragment homologous to the 5 'end of the region to be altered (5' arm) selected from the group consisting of genomic DNA of the IFNAR2 gene, 100 to 10000 nucleotides in length; b) A donor area; and c) a second DNA fragment homologous to the 3 'end of the region to be altered (3' arm) selected from the group consisting of genomic DNA of the IFNAR2 gene, of length 100 to 10000 nucleotides.

In some embodiments, a) the DNA fragment homologous to the 5' end of the region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI accession nc_ 000082.7; c) The DNA fragment homologous to the 3' end of the region to be altered is selected from the group consisting of nucleotide sequences having at least 90% homology to NCBI accession No. NC_ 000082.7.

In some embodiments, a) the 5' homologous DNA fragment of the region to be altered is selected from the nucleotide sequences 91177130 to 91180786 of NCBI accession nc_ 000082.7; c) The 3' -homologous DNA fragment of the region to be altered is selected from the nucleotide sequences 91182246 to 91186421 of NCBI accession No. NC_ 000082.7.

In some embodiments, a) the 5' homologous DNA fragment of the region to be altered is selected from the nucleotide sequences 91179365 to 91180786 of NCBI accession nc_ 000082.7; c) The 3' -homologous DNA fragment of the region to be altered is selected from the nucleotide sequences 91182246 to 91183528 of NCBI accession No. NC_ 000082.7.

In some embodiments, a) the 5' homologous DNA fragment of the region to be altered is selected from the nucleotide sequences 91179313 to 91180786 of NCBI accession nc_ 000082.7; c) The 3' -homologous DNA fragment of the region to be altered is selected from the nucleotide sequences 91196123 to 91197502 of NCBI accession No. NC_ 000082.7.

In some embodiments, the region to be altered is located on exons 2 to 8 of the non-human animal IFNAR2 gene.

In some embodiments, the region to be altered is located on exon 2 through exon 3 of the non-human animal IFNAR2 gene.

In some embodiments, the 5 'arm is a nucleotide having at least 90% homology to NCBI accession nc_000082.7, further preferably, the 5' arm sequence comprises SEQ ID NO: 23. 52 or 55. In some embodiments, the 3 'arm is a nucleotide having at least 90% homology to NCBI accession nc_000082.7, further preferably, the 3' arm sequence comprises SEQ ID NO: 24. 53 or 56.

In some embodiments, the targeting vector comprises a human sequence (e.g., positions 317 to 1045 of nm_207585.2 or positions 33241923 to 33260613 of nc_ 000021.9). For example, the targeting region in the targeting vector includes: some or all of the nucleotide sequence of the human IFNAR2 gene, preferably exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and/or exon 9 of the human IFNAR2 gene. In some embodiments, the nucleotide sequence of the humanized IFNAR2 gene encodes all or a portion of a human IFNAR2 protein, NCBI protein number NP-997468.1 (SEQ ID NO: 22). In some embodiments, the nucleotide sequence of the humanized IFNAR2 gene encodes a protein of SEQ ID NO:29.

The invention also provides vectors for constructing humanized animal models or knockout models. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the IFNAR2 gene and the sgRNA is unique in the target sequence of the gene to be altered and satisfies the sequence alignment rules of 5'-NNN (20) -NGG3' or 5'-CCN-N (20) -3'; and in some embodiments, the targeting site for sgRNA in the mouse IFNAR2 gene is located on or upstream of 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 and/or exon 9, on or downstream of exon 2 of the mouse IFNAR2 gene.

In some embodiments, the targeting sequence is shown as SEQ ID NO: 42. 43, 44, 45, 46, 47, 48, 49, 50 and 51. Thus, the invention provides sgRNA sequences for use in constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequence is set forth in SEQ ID NO:44 and 46. In some embodiments, the oligonucleotide sgRNA sequence is set forth in SEQ ID NO: listed in 45 and 47. In some embodiments, the oligonucleotide sgRNA sequence is set forth in SEQ ID NO:48 and 50. In some embodiments, the oligonucleotide sgRNA sequence is set forth in SEQ ID NO:49 and 51.

In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a positive selectable marker gene or a negative selectable marker gene. In some embodiments, the resistance gene screened for positive clones is neomycin phosphotransferase coding sequence Neo. Preferably, the targeting vector further comprises two Frt recombination sites which are arranged in the same direction and are arranged at two sides of the marker gene. In some embodiments, the gene encoding the negative selection marker is the diphtheria toxin a subunit encoding gene (DTA).

In some embodiments, the disclosure relates to plasmid constructs (e.g., pT 7-sgRNA) comprising a sgRNA sequence and/or cells comprising the constructs.

The invention also relates to cells comprising a targeting vector or sgRNA vector as described above.

In addition, the application provides a non-human mammalian cell having any of the targeting vectors described above, and one or more in vitro transcripts of the constructs of the application. In some embodiments, the cell comprises Cas9 mRNA or an in vitro transcript thereof.

In some embodiments, the gene in the cell is heterozygous. In some embodiments, the gene in the cell is homozygous.

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.

Construction method of genetically modified non-human animal

Genetically modified non-human animals can be prepared by several gene editing techniques known in the art, including homologous recombination techniques using embryonic stem cells, gene targeting techniques, CRISPR/Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases or other molecular biology techniques. In some embodiments, it is preferred to use homologous recombination techniques. In some embodiments, CRISPR/Cas9 gene editing techniques can construct genetically modified non-human animals. In some embodiments, CRISPR-Cas9 genome editing is used to produce a genetically modified non-human animal. Many of these genome editing techniques are known in the art and are described in Yin et al, "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6.6 (2017): 387-399, the disclosure of which is incorporated herein by reference. The application also provides many other methods for genome editing, for example, microinjection of a transgenic cell into an enucleated oocyte, and fusion of the enucleated oocyte with another transgenic cell.

In some embodiments, the nucleotide sequence encoding the endogenous IFNAR1 region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding a corresponding region of human IFNAR 1. In some embodiments, the non-human animal endogenous IFNAR1 protein expression is reduced or absent as 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 a fibroblast may be inserted into the enucleated oocyte.

FIG. 2 shows a humanized targeting strategy for the mouse IFNAR1 locus. The targeting vector comprises a vector consisting of a 5 'homology arm, a human or humanized IFNAR1 gene fragment and a 3' homology arm. The process involves replacing the endogenous corresponding IFNAR1 sequence with a human or humanized IFNAR1 sequence using homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs or CRISPRs) can result in DNA double strand breaks, replacing the murine endogenous IFNAR1 sequence with a human or humanized IFNAR1 sequence using homologous recombination to form a chimeric IFNAR1 gene.

In some embodiments, the non-human animal is obtained by introducing into the non-human animal IFNAR1 locus construct any one of the following nucleotide sequences:

A) A portion of a human IFNAR1 gene, preferably comprising all or part of exons 1 to 11 of a human IFNAR1 gene, further preferably comprising one, two or more of exons 1 to 11 of a human IFNAR1 gene, further preferably comprising part of exon 2 of a human IFNAR1 gene, all of exons 3-8 and part of exon 9, wherein part of exon 2 comprises a nucleotide sequence of at least 5 to 124bp (e.g. 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 119, 120 or 124 bp), and part of exon 9 comprises a nucleotide sequence of at least 5 to 151bp (e.g. 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 145, 146, 147, 149, 150 or 151 bp), still further preferably comprising SEQ ID NO:5, a nucleotide sequence shown in seq id no; or comprises a sequence identical to SEQ ID NO:5 is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%; or comprises a sequence identical to SEQ ID NO:5, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or comprises a polypeptide having the sequence of SEQ ID NO:5, a nucleotide sequence comprising one or more substitutions, deletions and/or insertions of nucleotides;

B) All or part of the nucleotide sequence encoding the human IFNAR1 protein, preferably all or part of the nucleotide sequence encoding a signal peptide, extracellular region, cytoplasmic region and/or transmembrane region of the human IFNAR1 protein, further preferably all or part of the nucleotide sequence encoding the extracellular region of the human IFNAR1 protein, further preferably a nucleotide sequence encoding at least 100 consecutive amino acids of the extracellular region of the human IFNAR1 protein, still further preferably the nucleotide sequence encoding SEQ ID NO:2 from 28 to 430 or from 28 to 436; or comprises a sequence encoding a sequence corresponding to SEQ ID NO:2 at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% amino acid sequence identity at positions 28-430 or 28-436; or comprises a sequence encoding a sequence corresponding to SEQ ID NO:2, the amino acid sequences shown at positions 28-430 or 28-436 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or comprises a sequence encoding a sequence corresponding to SEQ ID NO:2 from positions 28-430 or 28-436, including substitutions, deletions and/or insertions of one or more amino acid residues;

c) A nucleotide sequence encoding a human or humanized IFNAR1 protein; or alternatively, the first and second heat exchangers may be,

D) Nucleotide sequence of human or humanized IFNAR1 gene.

Preferably, the non-human animal is constructed using the targeting vector described above.

Preferably, the non-human animal further comprises additional genetic modifications, more preferably, the additional genes are selected from at least one of IFNAR2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4.

Preferably, the human or humanized IFNAR1 gene and/or other genes are homozygous for the endogenous modified (preferably replacement or insertion) locus.

Preferably, the human or humanized IFNAR1 gene and/or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

Preferably, the non-human animal is selected from any non-human animal that can be genetically edited to produce a humanized gene, such as rodent, pig, rabbit, monkey, etc.

Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or mouse.

Accordingly, the present invention provides a method of constructing a non-human animal humanized with an IFNAR1 gene, wherein the non-human animal expresses a human or humanized IFNAR1 protein in vivo, and/or wherein the genome of the non-human animal comprises a portion of the human IFNAR1 gene or the humanized IFNAR1 gene.

Thus, in some embodiments, the method of making a genetically modified humanized animal comprises replacing at an endogenous IFNAR1 locus (or locus) a nucleic acid sequence encoding a region of endogenous IFNAR1 with a nucleotide sequence encoding a corresponding region of human IFNAR 1. The sequence of substitution may include regions (e.g., partial or full regions) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11 of the human IFNAR1 gene. In some embodiments, the sequence comprises a portion of exon 2, all of exons 3-8, and a portion of exon 9 of the human IFNAR1 gene (e.g., nucleotide sequences 168-1376 of nm_ 000629.3), or comprises a portion of exon 2 to a portion of exon 8 of the human IFNAR1 gene.

In some embodiments, the method of modifying a mouse IFNAR1 locus to express a chimeric human/mouse IFNAR1 polypeptide may comprise replacing a nucleotide sequence encoding mouse IFNAR1 at an endogenous mouse IFNAR1 locus with a nucleotide sequence encoding human IFNAR1, thereby producing a chimeric human/mouse IFNAR1 sequence. In some embodiments, the methods may include inserting a nucleotide sequence encoding a chimeric human/mouse IFNAR1 at an endogenous mouse IFNAR1 locus, thereby producing a chimeric human/mouse IFNAR1 encoding sequence.

The invention also provides a method for establishing the IFNAR1 gene humanized animal model, which comprises the following steps:

(a) Providing a cell (e.g., a fertilized egg cell) based on the methods described herein;

(b) Culturing the cells, preferably in a liquid medium;

(c) Transplanting the cultured cells to the oviduct or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

(d) Identifying germ line transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

In some embodiments, the non-human mammal in the above methods is a mouse (e.g., a C57BL/6 mouse).

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

In some embodiments, the fertilized egg used in the above method is a C57BL/6 fertilized egg. Other fertilized eggs that may also be used in the methods of the present application include, but are not limited to, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs, and DBA/2 fertilized eggs.

Fertilized eggs may be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct may be used to introduce DNA into fertilized eggs by microinjection. For example, by culturing fertilized eggs after microinjection, the cultured fertilized eggs may be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal grows a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

In some embodiments, methods of making a genetically modified animal include modifying the coding framework of an IFNAR1 gene of a non-human animal, e.g., by replacing a nucleic acid sequence (e.g., genomic DNA, CDS, or cDNA sequence) encoding an endogenous IFNAR1 region with a nucleotide sequence encoding a corresponding region of a human IFNAR1 under the control of an endogenous regulatory element of the IFNAR1 gene of the non-human animal. For example, one or more of the functional region sequences of the IFNAR1 gene of the non-human animal may be knocked out or inserted such that the endogenous IFNAR1 protein of the non-human animal is not expressed or the expression level is reduced. In some embodiments, the coding box of the modified non-human animal IFNAR1 gene may be all or part of the nucleotide sequence of exons 1 to 11 of the non-human animal IFNAR1 gene.

In some embodiments, methods of making genetically modified animals include inserting nucleotide sequences and/or helper sequences encoding human or humanized IFNAR1 proteins following endogenous regulatory elements of the IFNAR1 genes of non-human animals. In some embodiments, the helper sequence may be a stop codon such that the IFNAR1 gene humanized animal model may express human or humanized IFNAR1 protein in vivo, but not non-human animal IFNAR1 protein. In some embodiments, the helper sequences include WPRE (WHP post transcriptional response element), loxP, STOP, and/or polyA.

In some embodiments, a method for preparing a transgenic animal comprises:

(1) Providing a plasmid comprising a fragment of a human IFNAR1 gene flanked by a 5 'homology arm and a 3' homology arm, wherein the 5 'and 3' homology arms target endogenous IFNAR1;

(2) Providing one or more guide RNAs (sgrnas) targeting an endogenous IFNAR1 gene;

(3) Modifying the genome of the fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2) and Cas 9;

(4) Transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudopregnant female mouse, or transplanting the embryonic stem cell obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudopregnant female mouse to produce a offspring mouse functionally expressing the humanized IFNAR1 protein;

(5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

In some embodiments, the fertilized egg is modified by CRISPR with sgrnas targeting the 5 '-end targeting site and the 3' -end targeting site.

In some embodiments, the sequence encoding the humanized IFNAR1 protein is operably linked to an endogenous regulatory element at an endogenous IFNAR1 locus.

In some embodiments, the genetically modified non-human animal does not express an endogenous IFNAR1 protein.

In some embodiments, a method for preparing a transgenic animal comprises:

(1) Providing a plasmid comprising a fragment of a human or chimeric IFNAR1 gene flanked by a 5 'homology arm and a 3' homology arm, wherein the 5 'and 3' homology arms target endogenous IFNAR1;

(3) The genome of a fertilized egg or embryonic stem cell is modified by inserting the human or chimeric IFNAR1 gene fragment into the genome.

In some embodiments, the nucleotide sequence encoding the endogenous IFNAR2 region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding human IFNAR 2. In some embodiments, the non-human animal endogenous IFNAR2 protein expression is reduced or absent as 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 a fibroblast may be inserted into the enucleated oocyte.

Fig. 6, 7, 8 show the humanized targeting strategy of the mouse IFNAR2 locus. The targeting vector comprises a vector consisting of a 5 'homology arm, a human or humanized IFNAR2 gene fragment and a 3' homology arm. The process involves replacing the endogenous corresponding IFNAR2 sequence with a human or humanized IFNAR2 sequence using homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs or CRISPRs) can result in DNA double strand breaks, replacing the murine endogenous IFNAR2 sequence with a human or humanized IFNAR2 sequence using homologous recombination to form a chimeric IFNAR2 gene.

In some embodiments, the non-human animal is obtained by introducing into the non-human animal IFNAR2 locus construct any one of the following nucleotide sequences:

A) A portion of a human IFNAR2 gene, preferably comprising all or part of exons 1 to 9 of a human IFNAR2 gene, further preferably comprising one, two or more than three of exons 1 to 9 of a human IFNAR2 gene, further preferably comprising part of exon 2, all of exons 3-7 and part of exon 8 of a human IFNAR2 gene, or comprising part of exon 2 to part of exon 8 of a human IFNAR2 gene, wherein part of exon 2 comprises a nucleotide sequence of at least 20 to 92bp (e.g. at least 5, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 91 or 92 bp), and part of exon 8 comprises a nucleotide sequence of at least 5 to 131bp (e.g. at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130 or 131 bp), further preferably comprising the nucleotide sequence of SEQ ID NO:26 or 57, and a nucleotide sequence shown in seq id no; or comprises a sequence identical to SEQ ID NO:26 or 57 is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%; or comprises a sequence identical to SEQ ID NO:26 or 57, no more than 10, 9, 8,7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or comprises a polypeptide having the sequence of SEQ ID NO:26 or 57, including substitutions, deletions and/or insertions of one or more nucleotides;

B) All or part of the nucleotide sequence encoding the human IFNAR2 protein, preferably all or part of the nucleotide sequence encoding a signal peptide, an extracellular region, a cytoplasmic region and/or a transmembrane region of the human IFNAR2 protein, further preferably all or part of the nucleotide sequence encoding a signal peptide and an extracellular region of the human IFNAR2 protein, further preferably a nucleotide sequence encoding at least 100 consecutive amino acids of the extracellular region of the human IFNAR2 protein, still further preferably a nucleotide sequence encoding SEQ ID NO:22 amino acid sequence shown in positions 1-242 or 1-243; or comprises a sequence encoding a sequence corresponding to SEQ ID NO:22 at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% amino acid sequence identity between the amino acids shown at positions 1-242 or 1-243; or comprises a sequence encoding a sequence corresponding to SEQ ID NO:22, the nucleotide sequences of amino acids 1-242 or 1-243 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or comprises a sequence encoding a sequence corresponding to SEQ ID NO:22 from positions 1 to 242 or from positions 1 to 243, including substitutions, deletions and/or insertions of one or more amino acid residues;

c) A nucleotide sequence encoding a human or humanized IFNAR2 protein; or alternatively, the first and second heat exchangers may be,

D) Nucleotide sequence of human or humanized IFNAR2 gene.

Preferably, the non-human animal further comprises additional genetic modifications, more preferably, the additional genes are selected from at least one of IFNAR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4.

Preferably, the human or humanized IFNAR2 gene and/or other genes are homozygous for the endogenous modified (preferably replacement or insertion) locus.

Preferably, the human or humanized IFNAR2 gene and/or other genes are heterozygous for the endogenous modified (preferably replaced or inserted) locus.

Accordingly, the present invention provides a method of constructing a non-human animal humanized with an IFNAR2 gene, wherein the non-human animal expresses a human or humanized IFNAR2 protein in vivo, and/or wherein the genome of the non-human animal comprises a portion of the human IFNAR2 gene or the humanized IFNAR2 gene.

Thus, in some embodiments, the method of making a genetically modified humanized animal comprises replacing, at an endogenous IFNAR2 locus (or site), a nucleic acid sequence encoding a region of endogenous IFNAR2 with a nucleotide sequence encoding human IFNAR 2. The replacement sequence may include exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and/or regions of exon 9 (e.g., partial or full regions) of the human IFNAR2 gene. In some embodiments, the sequence includes the human IFNAR2 gene exon 2 part, exon 3-7 all and exon 8 part, or exon 2 part to exon 8 part (for example, NM_207585.2 in 317-1045 or 317-1042 nucleotide sequence).

In some embodiments, the methods may include inserting a nucleic acid sequence encoding a human IFNAR2 region at an endogenous IFNAR2 locus (or site). The inserted sequence may include regions (e.g., partial or full regions) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, and/or exon 9 of the human IFNAR2 gene. In some embodiments, the sequence includes a portion of exon 2, all of exons 3-7, and a portion of exon 8 of the human IFNAR2 gene, or a portion of exon 2 to exon 8 of the human IFNAR2 gene (e.g., nucleotide sequence 317-1045 or 317-1042 of nm_ 207585.2). In some embodiments, the sequence includes a portion of exon 8 and all of exon 9 (e.g., nucleotide sequence at positions 1057-3045 of NM-010509.2) of the endogenous IFNAR2 gene and 309bp downstream of exon 9. In some embodiments, the sequence does not comprise an intron. In some embodiments, the sequence comprises an intron. In some embodiments, the sequence includes the human IFNAR2 signal peptide, extracellular region (such as SEQ ID NO:22 amino acids 1-242 or 1-243), endogenous transmembrane region (such as SEQ ID NO:21 amino acids 243-263) and endogenous cytoplasmic region (such as SEQ ID NO:21 amino acids 264-513). In some embodiments, the endogenous IFNAR2 locus (or site) is exon 2, intron 2, exon 3, and/or intron 3 of mouse IFNAR 2. In some embodiments, the sequence is inserted after the 5'UTR of the mouse IFNAR2 gene and 1-1459bp of nucleotide is deleted (e.g., after insertion of the 5' UTR of NM_010509.2 and deletion of nucleotide from exon 2 to nucleotide 331-427 and nucleotide 333bp downstream of exon 3).

In some embodiments, the method of modifying a mouse IFNAR2 locus to express a chimeric human/mouse IFNAR2 polypeptide may comprise replacing a nucleotide sequence encoding mouse IFNAR2 at an endogenous mouse IFNAR2 locus with a nucleotide sequence encoding human IFNAR2, thereby producing a chimeric human/mouse IFNAR2 sequence. In some embodiments, the method may comprise inserting a nucleotide sequence encoding a chimeric human/mouse IFNAR2 at an endogenous mouse IFNAR2 locus, thereby producing a chimeric human/mouse IFNAR2 encoding sequence.

The invention also provides a method for establishing the IFNAR2 gene humanized animal model, which comprises the following steps:

(b) Culturing the cells, preferably in a liquid medium;

In some embodiments, methods of making a genetically modified animal include modifying the coding framework of an IFNAR2 gene of a non-human animal, e.g., by replacing a nucleic acid sequence (e.g., genomic DNA, CDS, or cDNA sequence) encoding the endogenous IFNAR2 region with a nucleotide sequence encoding a corresponding region of a human IFNAR2 under the control of an endogenous regulatory element of the IFNAR2 gene of the non-human animal. For example, one or more of the functional region sequences of the IFNAR2 gene of the non-human animal may be knocked out or inserted such that the endogenous IFNAR2 protein of the non-human animal is not expressed or the expression level is reduced. In some embodiments, the coding box of the modified non-human animal IFNAR2 gene may be all or part of the nucleotide sequence of exons 1 to 9 of the non-human animal IFNAR2 gene.

In some embodiments, methods of making genetically modified animals include inserting nucleotide sequences and/or helper sequences encoding human or humanized IFNAR2 proteins following endogenous regulatory elements of the IFNAR2 genes of non-human animals. In some embodiments, the helper sequence may be a stop codon such that the IFNAR2 gene humanized animal model may express human or humanized IFNAR2 protein in vivo, but not non-human animal IFNAR2 protein. In some embodiments, the helper sequences include WPRE (WHP post transcriptional response element), loxP, STOP, and/or polyA.

In some embodiments, a method for preparing a transgenic animal comprises:

(1) Providing a plasmid comprising a fragment of a human IFNAR2 gene flanked by a 5 'homology arm and a 3' homology arm, wherein the 5 'and 3' homology arms target endogenous IFNAR2;

(2) Providing one or more guide RNAs (sgrnas) targeting an endogenous IFNAR2 gene;

(4) Transplanting the fertilized egg obtained in the step (3) into the oviduct of a pseudopregnant female mouse, or transplanting the embryonic stem cell obtained in the step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudopregnant female mouse to produce a offspring mouse functionally expressing the humanized IFNAR2 protein;

(5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

In some embodiments, the fertilized egg is modified by CRISPR with sgrnas targeting the 5 '-end targeting site and the 3' -end target site.

In some embodiments, the sequence encoding the humanized IFNAR2 protein is operably linked to an endogenous regulatory element at an endogenous IFNAR2 locus.

In some embodiments, the genetically modified non-human animal does not express an endogenous IFNAR2 protein.

In some embodiments, a method for preparing a transgenic non-human animal comprises:

(1) Providing a plasmid comprising a fragment of a human or chimeric IFNAR2 gene flanked by a 5 'homology arm and a 3' homology arm, wherein the 5 'and 3' homology arms target endogenous IFNAR2;

(3) The genome of a fertilized egg or embryonic stem cell is modified by inserting the human or chimeric IFNAR2 gene fragment into the genome.

Use of genetically modified non-human animals

Replacement of a non-human animal gene with or insertion of a homologous or orthologous human gene or human sequence into a non-human animal under the control of endogenous non-human animal loci and endogenous regulatory elements (e.g., promoters) can result in a non-human animal having qualities and characteristics that can vary significantly from typical knockout plus transgenic animals. In a typical knockout plus transgenic animal, the endogenous locus is removed or disrupted, and the fully human transgene is inserted into the animal's genome and may randomly integrate into the genome. Typically, the location of the integration transgene is unknown; the expression of human proteins is measured by transcription of human genes and/or protein assays and/or functional assays. In human transgenes, the upstream and/or downstream of the human sequence provides suitable support for expression and/or regulation of the transgene.

In some cases, transgenes with human regulatory elements are expressed in a non-physiological or otherwise unsatisfactory manner and may in fact be harmful to animals. The present invention demonstrates that substitution or insertion of human sequences at endogenous loci under the control of endogenous regulatory elements produces a humanized animal, providing 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 gene being substituted.

Genetically modified animals expressing human or humanized IFNAR1 and/or IFNAR2 proteins, for example, in a physiologically suitable manner, provide for a variety of uses including, but not limited to, developing methods of treatment of human diseases and disorders, and assessing toxicity and/or efficacy of these methods of treatment in animal models.

The invention also provides a non-human animal modified by the IFNAR1 and/or IFNAR2 genes or a progeny thereof, and application of the non-human animal or the progeny thereof obtained by any construction method.

In some embodiments, the application comprises:

The present invention provides a non-human animal expressing human or humanized IFNAR1 and/or IFNAR2 proteins, which can be used for screening of therapeutic agents specific for human IFNAR1 and/or IFNAR 2. Testing whether the therapeutic agent can increase or decrease the immune response, and/or determining whether the therapeutic agent is an IFNAR1 and/or IFNAR2 agonist or antagonist. In some embodiments, the non-human animal is a disease animal model. For example, the disease is genetically induced (knockin or knockout). In various embodiments, the genetically modified non-human animal further comprises an impaired immune system, e.g., genetically modified human tissue xenografts, including human solid tumors (e.g., breast cancer) or blood cell tumors (e.g., lymphocytic tumors, B or T cell tumors).

In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of a therapeutic agent (e.g., an anti-IFNAR 1 and/or IFNAR2 antibody; or a drug targeting JAK-STAT signaling pathway) in treating a tumor. In some embodiments, the methods involve administering a therapeutic agent to a non-human animal as described herein, wherein the non-human animal has a cancer or tumor; and determining the inhibition of the cancer or tumor by the therapeutic agent. Inhibition that can be determined includes, for example, a decrease in tumor size or tumor volume, a decrease in tumor growth, a decrease in the rate of increase in tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or another subject not undergoing such treatment), a decrease 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, etc. Tumor volume of a subject can be determined by various methods, such as by direct measurement, MRI, or CT. In some embodiments, the antibody may be directed to express IFNAR1 and/or IFNAR2.

In some embodiments, the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) injected into a non-human animal. In some embodiments, the antibody activates the JAK-STAT signaling pathway. In some embodiments, the antibody does not activate the JAK-STAT signaling pathway.

In some embodiments, the genetically modified non-human animal may be used to determine whether the antibody is an IFNAR1 and/or IFNAR2 agonist or antagonist. In some embodiments, the methods of the application are further designed to determine the effect of a therapeutic agent (e.g., an antibody targeting IFNAR1 and/or IFNAR 2; or a drug targeting the JAK-STAT signaling pathway) on IFNAR1 and/or IFNAR2, e.g., whether the therapeutic agent can up-regulate or down-regulate an immune response, and/or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cytotoxicity (ADCC). In some embodiments, the transgenic animals can be used to determine an effective dose of a therapeutic agent to treat a disease, such as cancer, in a subject.

Inhibition of tumors can also be determined by methods known in the art, for example, measuring tumor volume in a non-human animal, and/or determining tumor (volume) inhibition ratio (TGI _TV). Tumor growth inhibition can be calculated using the formula TGI _TV(％)＝(1–TV_t/TVc) ×100, where TV _t and TVc are the average tumor volumes (or weights) of the treatment group and the control group.

In some embodiments, therapeutic agents (e.g., antibodies targeting IFNAR1 and/or IFNAR 2; or drugs targeting the JAK-STAT signaling pathway) are designed to treat a variety of cancers. As used herein, the term "cancer" refers to a cell that has the ability to grow autonomously, i.e., an abnormal state or condition characterized by the growth of rapidly proliferating cells. The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the type of histopathology or invasive stage. The term "tumor" as used herein refers to cancer cells, such as a mass of cancer cells. Cancers that may be treated or diagnosed using the methods of the application include malignant tumors of various organ systems, such as those affecting the lung, breast, thyroid, lymph, gastrointestinal and genitourinary tracts, as well as adenocarcinomas, including malignant tumors such as most colon, renal cell carcinoma, prostate cancer and/or testicular tumor, non-small cell lung cancer, cancer and cancer. In some embodiments, the therapeutic agents of the application are designed to treat or diagnose cancer in a subject. The term "cancer" is well recognized and refers to malignant tumors of epithelial or endocrine tissues, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testis, breast, prostate, endocrine system and melanoma. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include cancers formed by cervical, lung, prostate, breast, head-neck, colon, and ovarian tissue. The term also includes carcinomatous tumors, for example, including malignant tumors composed of carcinomatous and sarcomatous tissues. "adenocarcinoma" refers to a cancer derived from glandular tissue or tumor cells that forms recognizable glandular structures. The term "sarcoma" is well-known to refer to malignant tumors of mesenchymal origin.

In some embodiments, the cancer described herein is a digestive tract cancer, gastrointestinal cancer, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, glioma, cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. In some embodiments, the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from hodgkin's lymphoma and non-hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and T-cell lymphoma, and Waldenstrom's macroglobulinemia. In some embodiments, the sarcoma is selected from the group consisting of osteosarcoma, ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In specific embodiments, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer, or cancer.

In some embodiments, the therapeutic agent (e.g., an antibody targeting IFNAR1 and/or IFNAR 2; or a drug targeting JAK-STAT signaling pathway) is designed to treat a variety of immune disorders, including systemic lupus erythematosus, psoriasis, transplant rejection, diabetes, immune neuromuscular disorders, multiple sclerosis, rheumatoid arthritis. Thus, the methods described herein can be used to determine the effectiveness of a therapeutic agent (e.g., an antibody targeting IFNAR1 and/or IFNAR 2; or a drug targeting JAK-STAT signaling pathway) in suppressing an immune response. In some embodiments, the immune disease described herein is Graft Versus Host Disease (GVHD), psoriasis, allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or a neurological disease, and the like. In some embodiments, therapeutic agents (e.g., antibodies targeting IFNAR1 and/or IFNAR 2; or drugs targeting JAK-STAT signaling pathways) are designed to treat various inflammations, such as inflammatory bowel disease, hepatitis, arthritis. In some embodiments, the inflammation described herein includes acute inflammation and chronic inflammation. In particular, inflammation includes, but is not limited to, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrin inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (e.g., tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, the inflammation described herein includes infection, and infection refers to local tissue and systemic inflammatory responses caused by bacteria, viruses, fungi, parasites and/or other pathogens that invade the human body.

The invention also provides a method of determining toxicity of a therapeutic agent (e.g., an anti-IFNAR 1 and/or IFNAR2 antibody). The method comprises administering a therapeutic agent to the non-human animal described above, assessing the non-human animal for weight change, red blood cell count, hematocrit, and/or hemoglobin. In some embodiments, the antibody may reduce Red Blood Cells (RBCs), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than the weight of a control group (e.g., the average weight of non-human animals not treated with the antibody).

The application also provides a model system for developing products related to the human cellular immune process, manufacturing human antibodies, or for pharmacological, immunological, microbiological and medical research in animal models constructed by the method of the application.

In some embodiments, an animal model generated by the methods described herein is provided for animal experimental disease models, studying pathogens, or developing new diagnostic and/or therapeutic strategies in the production and use of immune processes of human cells.

The application also provides an animal model generated by the method for screening, verifying, evaluating or researching the functions of the IFNAR1 and/or IFNAR2 genes, the human IFNAR1 and/or IFNAR2 antibodies, medicaments or effectiveness of the target sites of the human IFNAR1 and/or IFNAR2, medicaments for immune diseases and antitumor medicaments.

In some embodiments, the present disclosure provides a method of verifying the in vivo efficacy of TCR-T, CAR-T and/or other immunotherapies (e.g., T cell adoptive transfer therapies). For example, the method comprises transplanting human tumor cells into a non-human animal as described herein, and applying the human CAR-T to the non-human animal having human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the animal is selected from the group consisting of an IFNAR1 and/or IFNAR2 gene humanized non-human animal prepared by the methods described herein, an IFNAR1 and/or IFNAR2 gene humanized non-human animal described herein, a double or multiple humanized non-human animal (or progeny thereof) produced by the methods described herein, a non-human animal expressing a human or humanized IFNAR1 protein, or a tumor-bearing or inflammatory animal model described herein. In some embodiments, TCR-T, CAR-T and/or other immunotherapies may treat IFNAR1 and/or IFNAR2 related diseases described herein. In some embodiments, TCA-T, CAR-T and/or other immunotherapies provide an assessment method for treating IFNAR1 and/or IFNAR2 related diseases described herein.

Non-human animal model of two or more human or chimeric genes

The invention also provides a method for generating a transgenic animal model having two or more human or chimeric genes. The non-human animal may comprise a human or chimeric IFNAR1 and/or IFNAR2 gene and a sequence encoding an additional human or chimeric protein. In some embodiments, the non-human animal comprises a human or humanized IFNAR1 and/or IFNAR2 gene.

In some embodiments, the additional gene is a non-human animal genetically modified with at least one of LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4. In some embodiments, the non-human animal described above further expresses at least one of human or humanized LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 proteins.

In some embodiments, each of the individual genes modified in the genome of the non-human animal of the two or more human or chimeric genes is homozygous or heterozygous for the endogenous modified (preferably replacement) locus.

The invention also provides a method of constructing a non-human animal of two or more human or chimeric genes, the method comprising:

(one) providing a non-human animal obtained by the above construction method;

and (II) mating the non-human animal provided in the step (I) with other non-human animals modified by genes, performing in vitro fertilization or directly performing gene editing, and screening to obtain the non-human animal modified by multiple genes.

In some embodiments, the other genetically modified non-human animal comprises a non-human animal humanized with one or a combination of two or more of the genes LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4.

In some embodiments, humanization is performed directly on non-human animals with human or chimeric LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 genetic modifications.

Because these proteins may be involved in different mechanisms, combination therapies targeting two or more of these proteins may be a more effective treatment. In fact, many relevant clinical trials are underway and show good results. The polygenic modified non-human animal model can be used to determine the effectiveness of combination therapies targeting two or more proteins, e.g., anti-IFNAR 1 and/or IFNAR2 antibodies, as well as additional therapeutic agents for the treatment of cancer or immune diseases (e.g., asthma or atopic dermatitis). The method comprises administering an anti-IFNAR 1 and/or IFNAR2 antibody and an additional therapeutic agent to a non-human animal, wherein the non-human animal has a tumor or immune disease, and determining the effect of the combination therapy on the immune tumor or immune disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4. In some embodiments, the additional therapeutic agent is an anti-CTLA 4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., nivolumab), or an anti-PD-L1 antibody. In some embodiments, the non-human animals described above further include a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nal Wu Liyou mab, palbociclib mab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor described above comprises one or more tumor cells expressing PD-L1 and/or PD-L2.

In some embodiments, the combination therapy is used to treat various cancers described herein, e.g., breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer, or cancer. In some embodiments, the combination therapy is designed to treat an immune disorder described herein, such as psoriasis.

In some embodiments, the methods described herein can be used to evaluate combination therapies with some other methods. Methods of treating cancer that may be used alone or in combination with the methods described herein include, for example, treating a subject with chemotherapy, e.g., camphorine, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine Lei Ta, cyclophosphamide, doxorubicin, ifosfamide, malcyamide, marflange, neopyrimidine, bithiofen, nitrosourea, dyclonine, daunorubicin, bleomycin, plicin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouremic acid, vincristine, vinblastine, and/or methotrexate. Alternatively or in addition, the method may include performing surgery on the subject to remove at least a portion of the cancer, e.g., to remove a portion or all of the tumor from the patient.

Drawings

Fig. 1: schematic representation of the comparison of the mouse IFNAR1 locus and the human IFNAR1 locus (not to scale);

fig. 2: IFNAR1 gene targeting strategy and targeting vector V1 design schematic (not to scale);

Fig. 3: the F1 generation PCR identification result of the IFNAR1 gene humanized mouse, wherein WT is wild type contrast, M is Marker, and H ₂ O is water contrast;

Fig. 4: RT-PCR detection results, wherein +/+ is a wild C57BL/6 mouse, H/+ is an IFNAR1 gene humanized heterozygote mouse, and H ₂ O is a water control;

Fig. 5: schematic representation of the comparison of the mouse IFNAR2 locus and the human IFNAR2 locus (not to scale);

fig. 6: IFNAR2 gene targeting strategy and targeting vector V2 design schematic (not to scale);

fig. 7: IFNAR2 gene targeting strategy and targeting vector V3 design schematic (not to scale);

Fig. 8: IFNAR2 gene targeting strategy and targeting vector V4 design schematic (not to scale);

Fig. 9: the F1 generation southern identification result of the IFNAR2 gene humanized mouse, wherein WT is a wild type control;

fig. 10: RT-PCR detection results, wherein +/+ is a wild C57BL/6 mouse, H/+ is an IFNAR2 gene humanized heterozygote mouse, and H ₂ O is a water control;

fig. 11: degree of phosphorylation of Stat1 in T cells, B cells and NK cells after stimulation of C57BL/6 mice (+/+) and IFNAR1/IFNAR2 double gene humanized homozygous mouse (H/H) spleen cells with human IFNA2 or murine IFNA2 protein.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Materials and methods

In each of the following examples, the devices and materials were obtained from several companies as indicated below:

C57BL/6 mice were purchased from national rodent laboratory animal seed center of China food and drug verification institute;

BsrGI and NcoI enzymes were purchased from NEB under the accession number: R3575S and R3193S;

interferon alpha 2/IFNA2 Protein, human, recombinant available from Yiqiao Shenzhou under the trade designation: 13833-HNAY;

Interferon alpha 2/IFNA2 Protein, human, recombinant available from Yiqiao Shenzhou under the trade designation: 50225-MNAY;

Human TruStrain FcX available from Biolegend under the designation: 422302;

Purified anti-mouse CD16/32 anti-body was purchased from bioleged under the trade designation: 101302;

eBioscience ^TMFixable Viability Dye eFluor^TM 780 was purchased from eBioscience under the trade designation: 65-0865-14;

Alexa 700anti-mouse CD45 anti-body was purchased from bioleged under the trade designation: 103128;

Brilliant Violet 510 ^TM anti-mouse CD3 ε anti-body available from bioleged under the trade designation: 100353;

FITC anti-mouse CD19 anti-body was purchased from bioleged under the designation: 115506;

Brilliant Violet 421 ^TM anti-mouse NK1.1 anti-body is available from bioleged under the trade designation: 108732;

APC anti-mouse/human CD11b anti-body was purchased from Biolegend under the designation: 101212;

Brilliant Violet 605 ^TM anti-mouse F4/80 anti-body available from bioleged under the trade designation: 123133;

Brilliant Violet 711 ^TM anti-mouse CD11c Antibody available from bioleged under the designation: 117349;

PE/Cy ^TM anti-mouse-6G anti-body was purchased from Biolegend under the trade designation: 127618;

R-Phycoerythrin AffiniPure F (ab') 2Fragment Donkey Anti-Rabbit IgG (H+L) was purchased from Jackson Immuno Research under the designation: 711-116-152;

The Phospho-Stat1 (Tyr 701) (58D 6) Rabbit mAb was purchased from CELL SIGNALING Technology under the trade designation: 9167S;

Anti-Hu CD45, eBioscience eFluor, 506 was purchased from eBioscience under the trade designation: 69-0459-42;

Anti-Hu CD3, eBioscience Super Bright 702,702 was purchased from eBioscience under the trade designation: 67-0037-42;

Alexa 700anti-human CD14 anti-body was purchased from bioleged under the trade designation: 301822;

APC anti-human HLA-DR antibodies were purchased from Biolegend under the designation: 307610;

APC/Cy7 anti-human CD19 anti-body was purchased from Biolegend under the trade designation: 302218;

brilliant Violet 605 ^TM anti-human CD11c Antibody was purchased from bioleged under the trade designation: 301635;

Brilliant Violet 421 ^TM anti-human CD56 (NCAM) antibodies were purchased from bioleged under the trade designation: 318328;

BD PHARMINGEN PERM Buffer III was purchased from BD under the trade designation: 558050;

BD PHARMINGEN LYSE/Fix Buffer 5X was purchased from BD under the trade designation: 558049.

EXAMPLE 1IFNAR1 Gene humanized mice

Mouse IFNAR1 Gene (NCBI Gene ID:15975,Primary source:MGI:107658,UniProt ID:P33896, located at positions 91281016 to 91307411 of chromosome 16 NC_000082.7, based on transcript NM_010508.2 and its encoded protein NP_034638.2 (SEQ ID NO: 1)) and the human IFNAR1 Gene (NCBI Gene ID:3454,Primary source:HGNC:5432,UniProt ID:P17181-1, located at positions 33324395 to 33359864 of chromosome 21 NC_000021.9, based on transcript NM_000629.3 and its encoded protein NP_000620.2 (SEQ ID NO: 2)), are shown in FIG. 1.

For the purposes of the present invention, a nucleotide sequence encoding a human IFNAR1 protein may be introduced at the endogenous IFNAR1 locus of a mouse, such that the mouse expresses the human or humanized IFNAR1 protein. Specifically, under the control of regulatory elements of the mouse IFNAR1 gene, the humanized IFNAR1 locus is obtained by replacing the partial sequence of mouse exon 2 to the partial sequence of exon 9 by about 17kb containing the partial sequence of exon 2 to the partial sequence of exon 9 of the human IFNAR1 gene with about 12kb containing the partial sequence of exon 9 by using the gene editing technique.

Targeting strategies were designed as shown in FIG. 2, which shows targeting vector V1 containing homologous arm sequences upstream and downstream of the mouse IFNAR1 gene, and fragment A comprising the human IFNAR1 gene fragment. Wherein the upstream 5 'homology arm sequence (SEQ ID NO: 3) is identical to nucleotide sequence 91282641 to 91286588 of NCBI accession NC_000082.7 and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to nucleotide sequence 91300904 to 91305823 of NCBI accession NC_ 000082.7. The nucleotide sequence of the human IFNAR1 gene fragment (SEQ ID NO: 5) is identical to nucleotide sequence 33335529 to 33352904 of NCBI accession NC_ 000021.9; the ligation of the human IFNAR1 fragment sequence upstream to the mouse was designed as: 5' -

Wherein "A" in the sequence "GTGGA" is the last nucleotide of the mouse, the sequenceThe first "A" of (3) is the first nucleotide of the human sequence. The linkage of the human IFNAR1 fragment sequence downstream to the mouse was designed as: 5' -

Wherein the last "A" in sequence "CAAAA" is the last nucleotide of the human sequence, the sequenceThe first "C" of (3) is the first nucleotide of the mouse sequence.

The targeting vector also comprises a resistance gene for positive clone screening, namely neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo box (neocassette). Wherein the ligation of the 5' end of Neo cassette to the murine IFNAR1 gene is designed to: 5' -

Wherein the last "A" of the sequence "ATACA" is the last nucleotide of the murine IFNAR1 gene, the sequenceThe first "C" of (3) is the first nucleotide of the Neo cassette; the ligation of the Neo cassette 3' end to the murine IFNAR1 gene was designed as: 5' -

Wherein the last "C" in the sequence "GATCC" is the last nucleotide of the Neo cassette, the sequenceThe "A" in (2) is the first nucleotide of the murine IFNAR1 gene. The mRNA sequence of the modified humanized mouse IFNAR1 is shown as SEQ ID NO:6, the expressed protein sequence is shown as SEQ ID NO: shown at 7.

Targeting vector construction can be performed by conventional methods, such as enzyme digestion ligation, and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification. And (3) electroporation transfection of the targeting vector with correct sequencing verification into embryonic stem cells of a C57BL/6 mouse, and screening the obtained cells by using a positive clone screening marker gene to obtain correct positive clone cells. The correctly positive cloned cells (black mice) are introduced into the isolated blasts (white mice) according to the known technique in the art, and the obtained chimeric blasts are transferred to a culture solution for short culture and then transplanted into oviducts of recipient mice (white mice), so that F0 generation chimeric mice (black-white interphase) can be produced. And backcrossing the F0 generation chimeric mice and the wild mice to obtain F1 generation mice, and then mating the F1 generation heterozygous mice to obtain F2 generation homozygous mice. The positive mice and the Flp tool mice can also be mated to remove the positive clone screening marker genes, and then the IFNAR1 gene humanized homozygote mice can be obtained through the mutual mating.

The mice prepared by the above method can be identified as successful by conventional methods. For example, the genotype of the somatic cells of F1 mice can be identified by PCR (primers shown in Table 5), and the identification results of exemplary F1 mice are shown in FIG. 3, wherein the mice numbered F1-01 and F1-02 are positive heterozygote mice. This shows that the IFNAR1 gene humanized mice which can be stably passaged and have no random insertion can be constructed by using the method.

Table 5: f1 generation genotype PCR detection primer sequence and recombinant fragment size

The expression of mRNA in IFNAR1 gene humanized mice was examined by RT-PCR, specifically, C57BL/6 mice (+/+) and 1 IFNAR1 gene humanized heterozygotes (H/+) prepared in this example were selected, respectively, spleens of the mice were taken after neck-free euthanasia, and RT-PCR was performed using the primer sequences shown in Table 6, and the results of the examination are shown in FIG. 4. As can be seen from the figure, only mouse IFNAR1 mRNA was detected in wild type C57BL/6 mice, and no human IFNAR1 mRNA was detected; human IFNAR1 mRNA was detected only in IFNAR1 gene humanized heterozygous mice.

TABLE 6RT-PCR primer sequences and fragment sizes of interest

EXAMPLE 2IFNAR2 Gene humanized mice

Mouse IFNAR2 Gene (NCBI Gene ID:15976,Primary source:MGI:1098243,UniProt ID:O35664-1, located at positions 91166517 to 91202477 of chromosome 16 nc_000082.7, based on transcript nm_010509.2 and its encoded protein np_034639.2 (SEQ ID NO: 21)) and the human IFNAR2 Gene (NCBI Gene ID:3455,Primary source:HGNC:5433,UniProt ID:P48551-1, located at positions 33229938 to 33265664 of chromosome 21 NC_000021.9, based on transcript NM_207585.2 and its encoded protein NP_997468.1 (SEQ ID NO: 22)) are shown in FIG. 5.

For the purposes of the present invention, a nucleotide sequence encoding a human IFNAR2 protein may be introduced at the endogenous IFNAR2 locus of a mouse, such that the mouse expresses the human or humanized IFNAR2 protein. Specifically, the humanized IFNAR2 locus was obtained by substituting a chimeric CDS sequence comprising about 0.7kb of the coding region sequence of the portion from exon 2 to exon 8 of the human IFNAR2 gene and about 2.3kb of the entire sequence from portion to exon 9 of the mouse exon 8 for about 1.5kb of the sequence from portion to exon 3 downstream of the mouse exon 2 gene under the control of the regulatory element of the mouse IFNAR2 gene by using the gene editing technique.

Targeting strategies were designed as shown in FIG. 6, which shows targeting vector V2 containing homologous arm sequences upstream and downstream of the mouse IFNAR2 gene, and the A3 fragment comprising the human IFNAR2 gene fragment. Wherein the upstream 5 'homology arm sequence (SEQ ID NO: 23) is identical to nucleotide sequence 91177130 to 91180786 of NCBI accession NC_000082.7 and the downstream 3' homology arm sequence (SEQ ID NO: 24) is identical to nucleotide sequence 91182246 to 91186421 of NCBI accession NC_ 000082.7. The nucleotide sequence of the human IFNAR2 gene fragment (SEQ ID NO: 26) is identical to nucleotide sequences 317 to 1045 of NCBI accession No. NM-207585.2; the nucleotide sequence of the mouse IFNAR2 fragment in the A3 fragment is shown as SEQ ID NO: shown at 27. The ligation of the human IFNAR2 fragment sequence upstream to the mouse was designed as: 5' - Wherein the last "G" in the sequence "GCAGG" is the last nucleotide of the mouse, the sequence"A" in (2) is the first nucleotide of the human sequence. The linkage of the human IFNAR2 fragment sequence downstream to the mouse was designed as: 5' - Wherein the last "A" in sequence "CCAAA" is the last nucleotide of the human sequence, the sequenceThe first "A" of (2) is the first nucleotide of the mouse sequence.

The targeting vector also comprises a resistance gene for positive clone screening, namely neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo box (neocassette). Wherein the ligation of the 5' end of the Neo cassette to the STOP sequence is designed to be: 5' - Wherein the last "T" in sequence "TTAAT" is the last nucleotide of the STOP sequence, the sequenceThe first "G" of (a) is the first nucleotide of the Neo cassette; the ligation of the Neo cassette 3' end to the murine IFNAR2 gene was designed as: 5' -

Wherein the last "C" in the sequence "ACTTC" is the last nucleotide of the Neo cassette, the sequenceThe "A" in (2) is the first nucleotide of the murine IFNAR2 gene. The mRNA sequence of the modified humanized mouse IFNAR2 is shown as SEQ ID NO:28, the expressed protein sequence is shown as SEQ ID NO: 29.

Targeting vector construction can be performed by conventional methods, such as enzyme digestion ligation, and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification. And (3) electroporation transfection of the targeting vector with correct sequencing verification into embryonic stem cells of a C57BL/6 mouse, and screening the obtained cells by using a positive clone screening marker gene to obtain correct positive clone cells. The correctly positive cloned cells (black mice) are introduced into the isolated blasts (white mice) according to the known technique in the art, and the obtained chimeric blasts are transferred to a culture solution for short culture and then transplanted into oviducts of recipient mice (white mice), so that F0 generation chimeric mice (black-white interphase) can be produced. And backcrossing the F0 generation chimeric mice and the wild mice to obtain F1 generation mice, and then mating the F1 generation heterozygous mice to obtain F2 generation homozygous mice. The positive mice and the Flp tool mice can also be mated to remove the positive clone screening marker genes, and then the IFNAR2 gene humanized homozygote mice can be obtained through the mutual mating.

In addition, gene editing can be performed by using CRISPR/Cas9 system, and a targeting strategy shown in FIG. 7 is further designed, wherein the targeting vector V3 contains homologous arm sequences upstream and downstream of the mouse IFNAR2 gene, and A2 fragment (SEQ ID NO: 25) containing the human IFNAR2 gene. Wherein the upstream 5 'homology arm sequence (SEQ ID NO: 52) is identical to nucleotide sequence 91179365 to 91180786 of NCBI accession NC_000082.7 and the downstream 3' homology arm sequence (SEQ ID NO: 53) is identical to nucleotide sequence 91182246 to 91183528 of NCBI accession NC_ 000082.7. The nucleotide sequence of the human IFNAR2 fragment (SEQ ID NO: 26) is identical to the 317 to 1045 nucleotide sequence of NCBI accession No. NM-207585.2, and the nucleotide sequence of the mouse IFNAR2 fragment in the A2 fragment is shown as SEQ ID NO: shown at 27. The mRNA sequence of the modified humanized IFNAR2 mouse is shown as SEQ ID NO:28, the expressed protein sequence is shown as SEQ ID NO: 29.

The targeting vector construction can be carried out by conventional methods, such as enzyme digestion, ligation, direct synthesis and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification was used for subsequent experiments.

The target sequence determines the targeting specificity of the sgrnas and the efficiency of inducing Cas9 cleavage of the gene of interest. Therefore, efficient and specific target sequence selection and design are a prerequisite for construction of sgRNA expression vectors. The sgrnas sequence that recognizes the target site were designed and synthesized, and the target sequence of an exemplary sgRNA on the IFNAR2 gene was as follows:

SgRNA1 target site (SEQ ID NO: 42): 5'-CGGTGCACCGTCTCTGCCGTCGG-3';

SgRNA2 target site (SEQ ID NO: 43): 5'-GCCACCTTCTAAACGCCACCGGG-3';

The UCA kit is used for detecting the activity of sgRNA, after determining that the efficiency of high-efficiency cleavage can be mediated, enzyme cleavage sites are respectively added on the 5' end and the complementary strand of the sgRNA to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in table 7, annealing products are connected to pT7-sgRNA plasmid (the plasmid is linearized by BbsI first), and expression vectors pT7-IFNAR2-1 and pT7-IFNAR2-2 are obtained.

TABLE 7sgRNA1 and sgRNA2 sequence listing

PT7-sgRNA vector A fragment DNA (SEQ ID NO: 54) containing the T7 promoter and sgRNA scaffold was synthesized by plasmid synthesis company and ligated to a backbone vector (source Takara, cat. No. 3299) by cleavage (EcoRI and BamHI) in sequence, and the results were verified by sequencing by a professional sequencing company, indicating that the plasmid of interest was obtained. The prokaryotic fertilized eggs of the mice, such as C57BL/6 mice, are taken, and the in vitro transcription products of pT7-IFNAR2-1 and pT7-IFNAR2-2 plasmids (transcribed according to the instruction method using an Ambion in vitro transcription kit) and targeting vectors are premixed with Cas9 mRNA by a microinjection instrument and injected into the cytoplasmic region or nucleus of the fertilized eggs of the mice. Microinjection of fertilized eggs was performed according to the method of the "mouse embryo handling laboratory Manual (third edition)" (andela, nagel, chemical industry Press, 2006), the fertilized eggs after injection were transferred into a culture medium for short-term culture, then transplanted into oviducts of recipient mice for development, and the obtained mice (F0 generation) were subjected to hybridization and selfing to expand population numbers and establish stable IFNAR2 gene humanized mouse strains.

Gene editing can also be performed using the CRISPR/Cas9 system, and targeting strategies as shown in fig. 8, which show that targeting vector V4 contains homologous arm sequences upstream and downstream of the mouse IFNAR2 gene, as well as human IFNAR2 gene fragments, are designed. Wherein the upstream 5 'homology arm sequence (SEQ ID NO: 55) is identical to nucleotide sequence 91179313 to 91180786 of NCBI accession NC-000082.7 and the downstream 3' homology arm sequence (SEQ ID NO: 56) is identical to nucleotide sequence 91196123 to 91197502 of NCBI accession NC-000082.7. The nucleotide sequence of the human IFNAR2 fragment (SEQ ID NO: 57) is identical to the nucleotide sequence at positions 33241923 to 33260613 of NCBI accession NC-000021.9. The protein sequence expressed by the modified humanized IFNAR2 mouse is shown as SEQ ID NO: shown at 58.

The somatic cell genotype of F1-generation mice can be identified by PCR, and the positive mice can be identified by PCR, and then Southern blot detection (digestion of cellular DNA with BsrGI or NcoI and hybridization using 2 probes, the probe and the target fragment length are shown in Table 8), to confirm the presence or absence of random insertion. Exemplary results are shown in FIG. 9, where 4 mice numbered F1-01, F1-02, F1-03 and F1-04 were randomly inserted in combination with PCR and sequencing results. This shows that the IFNAR2 gene humanized mice which can be stably passaged and have no random insertion can be constructed by using the method.

TABLE 8 lengths of specific probes and fragments of interest

Restriction enzyme	Probe with a probe tip	Wild fragment size	Recombinant sequence fragment size
				BsrGI	3’Probe	8.6kb	11.0kb
NcoI	5’Probe	6.8kb	4.6kb

3’Probe-F：5’-GTGTTGGCATAACATTCTTAATCTAC-3’(SEQ ID NO：34)；

3’Probe-R：5’-GCTCACAACCTTATCCGTGTCTATG-3’(SEQ ID NO：35)；

5’Probe-F：5’-GAATCCGTGAACTCAAAACAGTTC-3’(SEQ ID NO：36)；

5’Probe-R：5’-GGCCAGACTTGTTCTCTAATTCC-3’(SEQ ID NO：37)；

The expression of mRNA in IFNAR2 gene humanized mice was examined by RT-PCR, specifically, 8-week-old female C57BL/6 mice (+/+) and 1 female IFNAR2 gene humanized heterozygotes (H/+) of 6-week-old females prepared in this example were selected, respectively, spleens of mice were taken after neck-removing euthanasia, and RT-PCR was performed using the primer sequences shown in Table 9, and the results of the examination are shown in FIG. 10. As can be seen from the figure, only mouse IFNAR2 mRNA was detected in wild type C57BL/6 mice, and no human IFNAR2 mRNA was detected; human IFNAR2 mRNA was detected only in IFNAR2 gene humanized heterozygous mice.

TABLE 9RT-PCR primer sequences and fragment sizes of interest

EXAMPLE 3 preparation of double-or Multi-Gene humanized mice

The method or the prepared IFNAR1 or IFNAR2 gene humanized mice can also be used for preparing a polygenic humanized mouse model. For example, in example 1, the embryonic stem cells used for microinjection may be selected from mice containing the genetic modifications such as IFNAR2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4, or may be obtained by isolating mouse ES embryonic stem cells and gene recombination targeting techniques based on humanized IFNAR1 or IFNAR2 mice. The method can also be used for obtaining homozygote or heterozygote of the IFNAR1 or IFNAR2 mice and other genetically modified mice, screening offspring thereof, obtaining humanized IFNAR1 or IFNAR2 genes and other genetically modified polygenic mice with a certain probability according to Mendelian genetic law, and then mutually mating heterozygotes to obtain the polygenic or polygenic modified homozygote. Using the foregoing methods, it is possible to obtain IFNAR2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4 humanized mice, e.g., IFNAR1/IFNAR2 humanized mice.

And (3) taking the IFNAR1/IFNAR2 double-gene humanized mice prepared by the method, and detecting the signal paths in the IFNAR1/IFNAR2 double-gene humanized homozygous mice by flow cytometry. Specifically, 1 each of 6-8-week-old male C57BL/6 mice (+/+) and 6-8-week-old male IFNAR1/IFNAR2 double-gene humanized homozygous mice (H/H) prepared in this example were taken, spleen cells of the mice were taken, and then stimulated in vitro with Interferon alpha/IFNA 2 Protein, human, recombinant (hIFNA 2) (1-10000 ng/mL) or Interferon alpha/IFNA 2 Protein, mouse, recombinant (mIFNA 2) (1-10000 ng/mL) at different concentrations for 30min, using Purified anti-mouse CD16/32Antibody、PE/Cy^TM7anti-mouse ly-6G Antibody、Phospho-Stat1(Tyr701)(58D6)Rabbit mAb、Alexa The results of the flow-through detection after staining with antibodies such as 700anti-human CD14 Antibody, APC/Cy7 anti-human CD19 are shown in FIG. 11. The results in FIG. 11 show that C57BL/6 mice (+/+) and IFNAR1/IFNAR2 double-gene humanized homozygous mice had elevated levels of Stat1 phosphorylation in spleen cells after stimulation with different concentrations mIFNA2 and exhibited dose-dependence, but the mIFNA 2-stimulated group had overall lower phosphorylation levels than the hIFNA-stimulated group. However, only IFNAR1/IFNAR2 double-gene humanized homozygous mice were detected to have an increased level of Stat1 phosphorylation in spleen cells after stimulation with hIFNA different concentrations and exhibited dose dependence. In conclusion, the IFNAR1/IFNAR2 signal pathway in the modified IFNAR1/IFNAR2 double-gene humanized mouse body is normal.

Example 4 model and efficacy

The humanized mice disclosed by the invention can be used for preparing various human disease models including autoimmune disease models such as multiple systemic lupus erythematosus, psoriasis, transplant rejection and the like by induction, and can be used for testing the in vivo efficacy of human specific antibodies. For example, IFNAR1 and/or IFNAR2 gene humanized mice can be used to assess the efficacy of antagonists of human-specific IFNAR1 and/or IFNAR2 signaling pathways, pharmacokinetics, and efficacy of in vivo therapies in various disease models known in the art.

Taking the preparation of a systemic lupus erythematosus (systemic lupus erythematosus, SLE) model as an example, IFNAR1 humanized homozygous mice, IFNAR2 humanized homozygous mice and/or IFNAR1/IFNAR2 double-gene humanized homozygous mice prepared by the invention can be used to induce SLE symptoms through reagents. The mice are weighed and continuously observed daily after the first induction, and the mice are grouped after the onset of the disease and are administrated through various routes such as intragastric injection, intraperitoneal injection or rat tail intravenous injection. The in vivo efficacy of different human drugs can be assessed by multiple detection indexes such as behavioral scoring, erythema degree, HE pathology examination, cytokine detection and the like.

The IFNAR1 humanized mice, IFNAR2 humanized mice and/or IFNAR1/IFNAR2 double-gene humanized mice prepared by the method can be used for evaluating the drug effect of a regulator targeting human IFNAR1 and/or IFNAR 2. For example, IFNAR1 humanized homozygous mice, IFNAR2 humanized homozygous mice and/or IFNAR1/IFNAR2 double-gene humanized homozygous mice were inoculated subcutaneously with colon cancer cells MC38, and after the tumor volume had grown to about 100mm ³, the treatment group was injected with an antibody drug targeting human IFNAR1 and/or IFNAR2 according to the tumor volume fraction, and the control group was injected with an equal volume of physiological saline. Tumor volumes were measured periodically and the body weight of the mice was weighed, and the in vivo safety and in vivo efficacy of the antibody drug in humanized IFNAR1 and/or IFNAR2 mice could be effectively assessed by comparing the change in body weight of the mice with the tumor volumes.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims

1. A method of constructing a genetically modified non-human animal, wherein the genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric type i interferon receptor 1 (IFNAR 1) protein.

2. The method of claim 1, wherein the nucleotide sequence encoding a human or chimeric IFNAR1 protein is operably linked to endogenous regulatory elements (e.g., endogenous 5'utr and/or 3' utr) of an endogenous IFNAR1 locus of at least one chromosome.

3. A method of construction according to claim 1 or 2, wherein the chimeric IFNAR1 protein comprises a human or humanised extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region; preferably also endogenous signal peptides.

4. A method of construction according to any one of claims 1 to 3, wherein the amino acid sequence of the human or chimeric IFNAR1 protein comprises SEQ ID NO:2 at positions 28-430 or 28-436 or comprising a nucleotide sequence identical to SEQ ID NO: the amino acid identity of positions 28-430 or 28-436 of 2 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

5. The method of construction according to any one of claims 1 to 4, wherein the amino acid sequence of the chimeric IFNAR1 protein comprises the amino acid sequence of SEQ ID NO:7, or comprises a sequence identical to SEQ ID NO:7 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

6. A method of constructing a genetically modified non-human animal, wherein at the non-human animal endogenous IFNAR1 locus, the corresponding region of the endogenous IFNAR1 gene is replaced with a nucleotide sequence comprising human IFNAR 1.

7. The method of construction according to claim 6, wherein the nucleotide sequence of human IFNAR1 comprises a portion of exon 2 to a portion of exon 9 of the human IFNAR1 gene; preferably, the portion of exon 2 comprises at least a 5bp contiguous nucleotide sequence; the portion of exon 9 comprises at least a 5bp contiguous nucleotide sequence;

further preferred, the nucleotide sequence of human IFNAR1 comprises SEQ ID NO:5, or comprises a sequence identical to SEQ ID NO:5 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

8. The method of construction according to claim 6 or 7, wherein the corresponding region of the endogenous IFNAR1 gene comprises a portion of exon 2 to a portion of exon 9 of the non-human animal IFNAR1 gene; or the corresponding region of the endogenous IFNAR1 gene comprises a nucleotide sequence encoding an extracellular region of IFNAR1 in a non-human animal.

9. A method of construction according to any one of claims 6 to 8, wherein the nucleotide sequence of human IFNAR1 is operably linked to an endogenous IFNAR1 regulatory element, such as a promoter;

preferably, the endogenous IFNAR1 protein of the non-human animal is not expressed or the expression level is reduced compared to IFNAR1 in a wild-type animal;

preferably, the modified IFNAR1 gene in the non-human animal gene is homozygous or heterozygous for the endogenous replaced locus.

10. The method of any one of claims 1-9, wherein the non-human animal is a mammal, e.g., a monkey, rodent; preferably, the rodent is a mouse or a rat.

11. The method of any one of claims 1-10, wherein the modified IFNAR1 gene transcribed mRNA in the genome of the non-human animal comprises the amino acid sequence of SEQ ID NO:6, or comprises a sequence identical to SEQ ID NO:6 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

12. The method of construction according to any one of claims 1 to 11, wherein the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by another gene, the human or chimeric protein being selected from at least one of IFNAR2, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA 4.

13. A method of constructing a genetically modified non-human animal, wherein the genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric type i interferon receptor 2 (IFNAR 2) protein.

14. A method of construction according to claim 13, wherein the nucleotide sequence encoding a human or chimeric IFNAR2 protein is operably linked to endogenous regulatory elements (e.g. endogenous 5'utr and/or 3' utr) of the endogenous IFNAR2 locus of at least one chromosome.

15. A method of construction according to claim 13 or 14, wherein the chimeric IFNAR2 protein comprises a human or humanised extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region; preferably also human or humanized signal peptides.

16. A method of construction according to any one of claims 13 to 15, wherein the amino acid sequence of the human or chimeric IFNAR2 protein comprises the amino acid sequence of SEQ ID NO:22 at positions 1-243 or 1-242, or comprises a sequence identical to SEQ ID NO:22 at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to amino acids 1-243 or 1-242.

17. The method of any one of claims 13-16, wherein the amino acid sequence of the chimeric IFNAR2 protein comprises the amino acid sequence of SEQ ID NO:29 or 58, or comprises a sequence identical to SEQ ID NO:29 or 58 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

18. A method of constructing a genetically modified non-human animal, comprising introducing a nucleotide sequence comprising human IFNAR2 at a non-human animal endogenous IFNAR2 locus.

19. The method of construction according to claim 18, wherein the nucleotide sequence of human IFNAR2 comprises a portion of exon 2 to a portion of exon 8 of the human IFNAR2 gene; preferably, the portion of exon 2 comprises at least a 5bp contiguous nucleotide sequence; preferably, the portion of exon 8 comprises at least a 5bp contiguous nucleotide sequence;

Further preferred, the nucleotide sequence of human IFNAR2 comprises SEQ ID NO:26 or 57, or comprises a sequence identical to SEQ ID NO:26 or 57 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

20. The method of construction according to claim 18 or 19, wherein the introduced sequence further comprises a nucleotide sequence of non-human animal IFNAR 2; preferably, the introduced sequence comprises part of exon 8 to exon 9 of non-human animal IFNAR 2; further preferred, the introduced sequence comprises SEQ ID NO:27, or comprises a sequence identical to SEQ ID NO:27 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

21. The method of any one of claims 18-20, wherein the introducing is insertion or substitution;

preferably, the nucleotide sequences encoding the signal peptide and extracellular region of endogenous IFNAR2 are replaced;

preferably, part of exon 2 to exon 3 is replaced;

preferably, the portion from exon 2 to exon 8 is replaced.

22. A method of construction according to any one of claims 18 to 21, wherein the nucleotide sequence of human IFNAR2 is operably linked to an endogenous IFNAR2 regulatory element, such as a promoter;

Preferably, the endogenous IFNAR2 protein of the non-human animal is not expressed or the level of expression is reduced compared to IFNAR2 in a wild-type animal;

Preferably, the modified IFNAR2 gene in the non-human animal gene is homozygous or heterozygous for the endogenous replaced locus.

23. The method of any one of claims 18-22, wherein the non-human animal is a mammal, e.g., a monkey, rodent; preferably, the rodent comprises a mouse or a rat.

24. The method of any one of claims 18-23, wherein the mRNA transcribed from the modified IFNAR2 gene in the genome of the non-human animal comprises the sequence set forth in SEQ ID NO:28, or comprises a sequence identical to SEQ ID NO:28 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

25. The method of construction according to any one of claims 18 to 24, wherein the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by another gene, the human or chimeric protein being selected from at least one of IFNAR1, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA 4.

26. A method of determining the effectiveness of a therapeutic agent in treating a disease, the method comprising:

1) Administering a therapeutic agent to a non-human animal obtained by the construction method of any one of claims 1-25, wherein the non-human animal has a disease;

2) The inhibition of the disease by the therapeutic agent is measured.

27. The method of claim 26, wherein the therapeutic agent is an anti-IFNAR 1 antibody and/or an anti-IFNAR 2 antibody; preferably, additional therapeutic agents (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA 4 antibodies) are also included.

28. The method of claim 26 or 27, wherein the disease is cancer, an immune disease or inflammation;

preferably, the cancer is a digestive tract cancer (e.g., colon cancer, gastrointestinal cancer, rectal cancer), leukemia, liver cancer, kidney cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, or lymphoma;

Preferably, the immune disease is systemic lupus erythematosus, psoriasis, transplant rejection, diabetes, immune neuromuscular disease, multiple sclerosis, rheumatoid arthritis;

preferably, the inflammation is inflammatory bowel disease, hepatitis, arthritis.

29. A method of determining toxicity of a therapeutic agent, the method comprising:

1) Administering a therapeutic agent to a non-human animal obtained by the construction method of any one of claims 1-25;

2) The effect of the therapeutic agent on the non-human animal is determined.

30. The method of claim 29, wherein determining the effect of the therapeutic agent on the non-human animal involves measuring the weight of the non-human animal or a blood test; preferably, the blood test includes one or more of red blood cell count, hematocrit, or hemoglobin content.

31. A humanized IFNAR1 protein, wherein the humanized IFNAR1 protein comprises the amino acid sequence of SEQ ID NO:7, or comprises a sequence identical to SEQ ID NO:7 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

32. A humanized IFNAR1 gene, wherein the humanized IFNAR1 gene encodes the humanized IFNAR1 protein of claim 31.

33. The humanized IFNAR1 gene of claim 32, wherein the humanized IFNAR1 gene comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 3. 4, 5, 6, 8 or 9, or a sequence comprising a nucleotide sequence identical to SEQ ID NO: 3. 4, 5, 6, 8 or 9 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

34. A humanized IFNAR2 protein, wherein the humanized IFNAR2 protein comprises the amino acid sequence of SEQ ID NO:29 or 58, or comprises a sequence identical to SEQ ID NO:29 or 58 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5%.

35. A humanized IFNAR2 gene, wherein the humanized IFNAR2 gene encodes the humanized IFNAR2 protein of claim 34.

36. The humanized IFNAR2 gene of claim 35, wherein the humanized IFNAR2 gene comprises the amino acid sequence of SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 52, 53, 55, 56, or 57, or a polypeptide comprising a sequence identical to SEQ ID NO: 23. 24, 25, 26, 27, 28, 30, 31, 52, 53, 55, 56, or 57 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5%.

37. A cell, tissue or organ comprising the humanized IFNAR1 protein of claim 31, the humanized IFNAR1 gene of any one of claims 32-33, the humanized IFNAR2 protein of claim 34 or the humanized IFNAR2 gene of any one of claims 35-36.

38. Use of a non-human animal obtained by the construction method according to any one of claims 1-25, the humanized IFNAR1 protein according to claim 31, the humanized IFNAR1 gene according to any one of claims 32-33, the humanized IFNAR2 protein according to claim 34, the humanized IFNAR2 gene according to any one of claims 35-36, the cell, tissue or organ according to claim 37, characterized in that said use comprises: