CFB genetically modified non-human animals

Abstract

The present invention provides non-human animals expressing human CFB protein or chimeric (e.g., humanized) CFB protein and methods for using the same. The present invention further provides a non-human animal genome, a humanized CFB gene, and cells, tissues, organs, or non-human animals containing the non-human animal genome and the humanized CFB gene. The non-human animals obtained in this application successfully express human CFB protein or humanized CFB protein, which can be cleaved by CFD, bind to C3b, and exert similar effects in the human body. Furthermore, the non-human animals obtained in this application have complete renal function, normal blood biochemical indicators, and, more remarkably, do not exhibit the potential diseases observed in other transgenic mice known in this art.

Description

Cross - reference to related applications

[0001] This application claims the priority of Chinese Patent Application for Invention No. 202310778275.7 filed on June 28, 2023, Chinese Patent Application for Invention No. 202410169335.X filed on February 6, 2024, Chinese Patent Application for Invention No. 202410462699.7 filed on April 17, 2024, and Chinese Patent Application for Invention No. 202410555788.6 filed on May 7, 2024, and all the contents of these four applications are incorporated herein by reference.

Technical Field

[0002] The present invention provides non - human animals that express human CFB protein or chimeric (e.g., humanized) CFB protein and methods of using the same.

Background Art

[0003] In conventional drug development, in vitro screening methods are usually used. However, since these screening methods cannot provide an in - vivo environment (such as the interaction between the tumor micro - environment, stromal cells, extracellular matrix components, and immune cells), the failure rate of drug development is relatively high. Also, in view of the differences between humans and animals, the test results obtained from in - vivo pharmacological tests using ordinary experimental animals may not reflect the actual disease state and the interaction of the target site, so the results of many clinical trials are significantly different from those of animal experiments.

[0004] Therefore, the development of a humanized animal model suitable for screening and evaluating human antibodies will significantly improve the efficiency of new drug development and reduce drug development costs.

[0005] However, constructing humanized animal models is extremely difficult. For example, the 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 there are certain risks associated with the humanization of any gene, and that even with carefully designed strategies, it is not possible to ensure the expression and function of human genes in animal bodies. [Overview of the project]

[0006] Based on the lack of prior art, this application provides a genetically modified non-human animal. This non-human animal normally expresses human CFB protein in its body, and this protein can be cleaved by CFD, bind to C3b, and exert a similar effect in the human body. Furthermore, the non-human animal obtained in this application has complete renal function, normal blood biochemical indicators, and, more remarkably, does not exhibit the potential diseases observed in certain other transgenic mice known in this field.

[0007] This application provides an animal model having human CFB protein or chimeric CFB protein. The animal model can express human CFB protein or chimeric CFB (e.g., humanized CFB) protein. It can be used to study CFB gene function and further to screen and evaluate CFB signaling pathway modulators (e.g., anti-human CFB antibodies, oligonucleotide drugs, and / or polypeptide drugs). Furthermore, non-human animals or animal models produced by the method described herein can be used for drug screening, pharmacodynamic studies, treatment of immune-related diseases, and treatment of diseases of human CFB target sites. These non-human animals or animal models can also be used to accelerate the development and design of new drugs, saving time and cost. As described above, the present invention provides a powerful tool for studying the function of CFB protein and a platform for screening related drugs.

[0008] In one embodiment, the present invention provides a genetically modified non-human animal whose genome comprises at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human complement factor B (CFB) protein or a chimeric complement factor B (CFB) protein. In some examples, the expression of the nucleotide sequence encoding the human CFB protein or chimeric CFB protein is regulated by a regulatory element (e.g., an endogenous regulatory element or a human-derived regulatory element, preferably the regulatory element being the 5'UTR and / or 3'UTR). In some examples, the chimeric CFB protein is a humanized CFB protein, which comprises a portion of the human CFB protein. In some examples, the portion of the human CFB protein comprises a Ba region and / or a Bb region, preferably the Bb region comprising a VWA domain, and more preferably further comprising von Willebrand factor and / or peptidase S1. In some examples, the humanized CFB protein includes those whose amino acid sequence matches at least 50 to 764 amino acids to a portion of the continuous amino acid sequence of a human CFB protein. In some examples, the non-human animal is a mammal, for example, a monkey or a rodent (for example, a mouse or a rat). In some examples, the human CFB protein or chimeric CFB protein includes SEQ ID NO: 2 or positions 26-764 thereof, or includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2 or positions 26-764, or includes an amino acid sequence that differs from SEQ ID NO: 2 or positions 26-764 by no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids, or no more than 1 amino acid, or includes a sequence that has one or more amino acid substitutions, deletions, and / or insertions relative to SEQ ID NO: 2 or positions 26-764 thereof.In some examples, the non-human endogenous CFB protein is not expressed or its expression level is reduced compared to CFB in wild-type animals. In some examples, the nucleotide sequence encoding the endogenous CFB protein in the non-human animal is silenced or disrupted (e.g., deleted). In some examples, the nucleotide sequence encoding the endogenous CFB protein in the non-human animal is deleted, for example, the nucleotide sequence encoding SEQ ID NO: 1 or its positions 18-712 is deleted. In some examples, the modified CFB gene in the genome of the non-human animal is homozygous or heterozygous for the endogenously substituted locus. In some examples, the human CFB protein or chimeric CFB protein expressed in the non-human animal has at least one CFB activity, for example, non-human animal CFB activity and / or human CFB activity. In some examples, the non-human animal has a renal function state that is essentially consistent with that of wild-type non-human animals. In some examples, the levels of urea, serum creatinine, and / or total protein in the non-human animals were not significantly different compared to wild-type non-human animals. In some examples, the human CFB protein or chimeric CFB protein expressed in the non-human animals could be cleaved by human CFD or non-human animal CFD. In some examples, the human CFB protein or chimeric CFB protein expressed in the non-human animals could bind to human C3b or non-human animal C3b. In some examples, the non-human animals further comprised nucleotide sequences of human proteins or chimeric proteins encoded by other genes, and the human proteins or chimeric proteins included, but not limited to, at least one of CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4.

[0009] In one embodiment, the present invention provides a genetically modified non-human animal in which the endogenous CFB gene is replaced with a nucleotide sequence containing human CFB at the endogenous CFB locus of the non-human animal. In some examples, the expression of the human CFB nucleotide sequence is regulated by a regulatory element (e.g., an endogenous regulatory element or a human-derived regulatory element). In some examples, one or more cells of the non-human animal express human CFB protein or chimeric CFB protein. In some examples, the endogenous CFB protein of the non-human animal is not expressed or its expression level is reduced compared to CFB in wild-type animals. In some examples, the endogenous CFB gene is silenced or disrupted (e.g., deleted). In some embodiments, the nucleotide sequence of human CFB includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, for example, a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or a portion of exon 18, preferably including the nucleotide sequence of the coding region of the human CFB gene, and more preferably including the nucleotide sequence from the start codon to the stop codon of the human CFB gene. In some embodiments, the nucleotide sequence of the human CFB further includes the 5'UTR and / or 3'UTR of the human CFB, and further includes a sequence of nucleotides from at least 50 bp to at least 10,000 bp upstream of the 5'UTR of the human CFB and / or a sequence of nucleotides from at least 50 bp to at least 5,000 bp downstream of the 3'UTR of the human CFB.In several embodiments, the nucleotide sequence of the human CFB is a sequence of nucleotides from at least 50 bp to at least 10,000 bp (e.g., 50 bp, 100 bp, 500 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 5500 bp, 5600 bp, 5700 bp, 5800 bp, 5900 bp, 6000 bp, 6100 bp, 6200 bp, 7000 bp, 8000 bp, 9000 bp, or 10000 bp, preferably 3000 bp to 8000 bp, more preferably 5000 bp to 7000 bp) of the 5'UTR of the human CFB, and further upstream therefrom. The nucleotide sequence from the start codon to the stop codon of the gene, as well as the 3'UTR of the human CFB and the subsequent nucleotides from at least 50 bp to at least 5000 bp downstream thereof (e.g., 50 bp, 100 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 910 bp, 920 bp, 930 bp, 940 bp, 950 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 2000 bp, 3000 bp, 4000 bp, or 5000 bp, preferably 500 bp to 3000 bp, and more preferably 500 bp to 1500 bp). In some embodiments, the nucleotide sequence of the human CFB includes the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 39, or includes a nucleotide sequence whose homology to SEQ ID NO: 7 or SEQ ID NO: 39 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or includes a nucleotide sequence whose difference from SEQ ID NO: 7 or SEQ ID NO: 39 does not exceed 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 6 bp, 3 bp, or 1 bp, or includes a nucleotide sequence having one or more nucleotide substitutions, deletions, and / or insertions compared to that shown in SEQ ID NO: 7 or SEQ ID NO: 39.In some examples, exons 2 to exons 17 of the endogenous CFB gene are substituted, preferably the substituted endogenous CFB gene further includes part of exon 1, all of intron 1 and / or part of intron 17, and more preferably the substituted endogenous CFB gene further includes part of exon 18. In some examples, the nucleotide sequence from the start codon to the stop codon of the endogenous CFB gene is substituted, or part of exon 1 to part of intron 17 of the endogenous CFB gene is substituted, of which part of exon 1 includes 70 or fewer nucleotides, 60 or fewer nucleotides, 50 or fewer nucleotides, 40 or fewer nucleotides, 30 or fewer nucleotides, and 20 or fewer nucleotides at the 3' end of exon 1. In some examples, the non-human animal is a mammal, for example, a monkey or a rodent (for example, a mouse or a rat). In some examples, the modified CFB gene in the genome of the non-human animal is homozygous or heterozygous to the endogenously substituted locus. In some examples, the nucleotide sequence of human CFB in the genome of the non-human animal is such that the upstream linkage sequence with mouse is sequence number 9, and the downstream linkage sequence with mouse is sequence number 10. In some examples, the nucleotide sequence of human CFB in the genome of the non-human animal is such that the upstream linkage sequence with mouse is sequence number 41, and the downstream linkage sequence with mouse is sequence number 42.In some embodiments, the mRNA transcribed by the endogenous CFB-modified nucleotide sequence in the genome of the non-human animal described above includes SEQ ID NO: 8 or SEQ ID NO: 40, or includes a nucleotide sequence whose homology to SEQ ID NO: 8 or SEQ ID NO: 40 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or includes a nucleotide sequence whose difference from SEQ ID NO: 8 or SEQ ID NO: 40 does not exceed 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 6 bp, 3 bp, or 1 bp, or includes a nucleotide sequence having one or more nucleotide substitutions, deletions, and / or insertions relative to what is shown in SEQ ID NO: 8 or SEQ ID NO: 40. In some examples, the human CFB protein or chimeric CFB protein expressed in the non-human animal has at least one CFB activity, for example, non-human animal CFB activity and / or human CFB activity. In some examples, the non-human animal has a renal function state that is essentially consistent with that of a wild-type non-human animal. In some examples, there is no significant difference in the levels of urea, serum creatinine and / or total protein contained in the non-human animal compared to a wild-type non-human animal. In some examples, the human CFB protein or chimeric CFB protein expressed in the non-human animal can be cleaved by human CFD or non-human animal CFD. In some examples, the human CFB protein or chimeric CFB protein expressed in the non-human animal can bind to human C3b or non-human animal C3b. In some examples, the non-human animal further comprises a nucleotide sequence of a human protein or chimeric protein encoded by another gene, the human protein or chimeric protein comprising, but not limited to, at least one of CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4.

[0010] In one embodiment, the present invention provides a non-human animal comprising a cell having at least one nucleotide sequence encoding a human CFB protein or a humanized CFB protein, wherein the humanized CFB protein comprises at least 50, 100, 200, 300, 400, 500, 600, 700, 710, 720, 730, 740, 750, 760, 763, or 764 consecutive amino acid sequences matching the human CFB protein, and the non-human animal expresses the human CFB protein or the humanized CFB protein. In some embodiments, the nucleotide sequence encoding the human CFB protein or the humanized CFB protein is operably linked to an endogenous CFB regulator or a human-derived CFB regulator. In some embodiments, the nucleotide sequence encoding the human CFB protein or the humanized CFB protein can be incorporated into the endogenous CFB locus of the non-human animal. In some examples, the human CFB protein or humanized CFB protein has at least one CFB activity, for example, mouse CFB activity and / or human CFB activity.

[0011] In one embodiment, the present invention provides a non-human animal genome comprising at least one chromosome, the chromosome comprising a nucleotide sequence encoding human complement factor B (CFB) protein or chimeric complement factor B (CFB) protein. In some embodiments, the endogenous CFB gene is silenced or disrupted (e.g., deleted). In some embodiments, the endogenous CFB gene is deleted, and the deleted region is replaced with a nucleotide sequence encoding human CFB protein or chimeric CFB protein. In some embodiments, the endogenous CFB gene is deleted, and the deleted region is replaced with a nucleotide sequence of human CFB. In some embodiments, the chimeric CFB protein is a humanized CFB protein, and the humanized CFB protein comprises a portion of human CFB protein and a portion of non-human animal CFB protein. In some examples, a portion of the human CFB protein includes a Ba region and / or a Bb region, preferably the Bb region includes a VWA domain, and more preferably further includes von Willebrand factor and / or peptidase S1. In some examples, the humanized CFB protein includes those in which at least 50 to 764 amino acids match the continuous amino acid sequence of a portion of the human CFB protein. In some examples, the human CFB protein or chimeric CFB protein includes SEQ ID NO: 2 or positions 26-764 thereof, or includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2 or positions 26-764, or includes an amino acid sequence that differs from SEQ ID NO: 2 or positions 26-764 by no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids, or no more than 1 amino acid, or includes a sequence that has one or more amino acid substitutions, deletions, and / or insertions relative to SEQ ID NO: 2 or positions 26-764 thereof.In a non-human animal endogenous CFB gene locus, the endogenous CFB gene is replaced with a nucleotide sequence encompassing human CFB. In some examples, the human CFB nucleotide sequence includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, of which the portion of exon 1 of the human CFB gene includes at least 5 bp of a continuous nucleotide sequence of exon 1 of the human CFB gene, and the portion of exon 18 of the human CFB gene includes at least 20 bp of a continuous nucleotide sequence of exon 18 of the human CFB gene. Preferably, it includes the nucleotide sequence of the coding region of the human CFB gene, and more preferably, it includes the nucleotide sequence from the start codon to the stop codon of the human CFB gene. In some examples, the human CFB nucleotide sequence further includes the 5'UTR and / or 3'UTR of human CFB, and further includes at least 50 bp of a continuous nucleotide sequence upstream outside the 5'UTR of human CFB and / or at least 50 bp of a continuous nucleotide sequence downstream outside the 3'UTR of human CFB. In some embodiments, the nucleotide sequence of human CFB includes the 5'UTR of human CFB and at least 50 bp of consecutive nucleotides upstream thereout, the nucleotide sequence from the start codon to the stop codon of the human CFB gene, and at least 50 bp of consecutive nucleotides downstream thereout of the 3'UTR of human CFB.

[0012] In some embodiments, the nucleotide sequence of the human CFB includes the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 39, or includes a nucleotide sequence whose homology to SEQ ID NO: 7 or SEQ ID NO: 39 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or includes a nucleotide sequence whose difference from SEQ ID NO: 7 or SEQ ID NO: 39 does not exceed 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 6 bp, 3 bp, or 1 bp, or includes a nucleotide sequence having one or more nucleotide substitutions, deletions, and / or insertions compared to what is shown in SEQ ID NO: 7 or SEQ ID NO: 39. In some embodiments, the deleted endogenous CFB gene includes a nucleotide sequence encoding the endogenous CFB protein, preferably including SEQ ID NO: 1 or the nucleotide sequence encoding its positions 18 to 712. In some examples, exons 2 to 17 of the endogenous CFB gene are substituted, preferably the substituted endogenous CFB gene further includes a portion of exon 1, all of intron 1 and / or a portion of intron 17, and more preferably the substituted endogenous CFB gene further includes a portion of exon 18. In some examples, the nucleotide sequence from the start codon to the stop codon of the endogenous CFB gene is substituted, or a portion of exon 1 to a portion of intron 17 of the endogenous CFB gene is substituted, of which the portion of exon 1 includes 70 or fewer nucleotides at the 3' end of exon 1. In some examples, the nucleotide sequence encoding the human CFB protein or chimeric CFB protein, or the nucleotide sequence of human CFB, is operably linked to a regulatory element (e.g., an endogenous regulatory element or a human-derived regulatory element) of the CFB locus on at least one chromosome. In some examples, the non-human animal is a mammal, e.g., a monkey or a rodent (e.g., a mouse or a rat). In some examples, the non-human animal is a mouse.In some examples, the nucleotide sequence of human CFB in the above non-human animal genome is such that the upstream ligation sequence with mouse is sequence number 9, and the downstream ligation sequence with mouse is sequence number 10. In some examples, the nucleotide sequence of human CFB in the above non-human animal genome is such that the upstream ligation sequence with mouse is sequence number 41, and the downstream ligation sequence with mouse is sequence number 42. In some embodiments, the mRNA transcribed by the nucleotide sequence modified by the endogenous CFB in the non-human animal genome includes SEQ ID NO: 8 or SEQ ID NO: 40, or includes a nucleotide sequence whose homology to SEQ ID NO: 8 or SEQ ID NO: 40 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or includes a nucleotide sequence whose difference from SEQ ID NO: 8 or SEQ ID NO: 40 does not exceed 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 6 bp, 3 bp, or 1 bp, or includes a nucleotide sequence having one or more nucleotide substitutions, deletions, and / or insertions relative to what is shown in SEQ ID NO: 8 or SEQ ID NO: 40.

[0013] In one embodiment, the present invention provides cells containing the above-mentioned non-human animal genome.

[0014] In one embodiment, the present invention provides a non-human animal comprising the above-mentioned non-human animal genome or cells.

[0015] In one embodiment, the present invention provides a method for constructing a genetically modified non-human animal that expresses human CFB protein or chimeric CFB protein. In some examples, in at least one cell of the non-human animal, the endogenous CFB gene is replaced at the non-human animal endogenous CFB locus with a nucleotide sequence encompassing human CFB. In some examples, the non-human animal endogenous CFB protein is either not expressed or its expression level is reduced compared to CFB in wild-type animals. In some examples, the human CFB nucleotide sequence encompasses a portion of exon 1 to a portion of exon 18 of the human CFB gene, preferably the nucleotide sequence of the coding region of the human CFB gene, and more preferably the nucleotide sequence from the start codon to the stop codon of the human CFB gene. In some embodiments, the nucleotide sequence of the human CFB further includes the 5'UTR and / or 3'UTR of the human CFB, and further includes at least 50 bp of consecutive nucleotides outside the 5'UTR of the human CFB and / or at least 50 bp of consecutive nucleotides outside the 3'UTR of the human CFB. In some embodiments, the nucleotide sequence of the human CFB includes the 5'UTR of the human CFB and at least 50 bp of consecutive nucleotides outside its upstream, the nucleotide sequence from the start codon to the stop codon of the human CFB gene, and the 3'UTR of the human CFB and at least 50 bp of consecutive nucleotides outside its downstream.In some embodiments, the nucleotide sequence of the human CFB includes the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 39, or includes a nucleotide sequence whose homology to SEQ ID NO: 7 or SEQ ID NO: 39 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or includes a nucleotide sequence whose difference from SEQ ID NO: 7 or SEQ ID NO: 39 does not exceed 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 6 bp, 3 bp, or 1 bp, or includes a nucleotide sequence having one or more nucleotide substitutions, deletions, and / or insertions compared to that shown in SEQ ID NO: 7 or SEQ ID NO: 39. In some examples, the nucleotide sequence of the endogenously substituted CFB region includes the nucleotide sequence encoding the endogenous CFB protein, preferably including SEQ ID NO: 1 or the nucleotide sequence encoding positions 18 to 712 thereof. In some examples, the nucleotide sequence of the endogenously substituted CFB region includes parts of exon 1 to exon 18 of the endogenous CFB gene, for example, parts of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18, for example, including the nucleotide sequence of the coding region. In some examples, the nucleotide sequence of the endogenously substituted CFB region includes parts of exon 1 to intron 17 of the endogenous CFB gene. In some embodiments, the nucleotide sequence of the human CFB is operably linked to a CFB regulatory element at the CFB locus of at least one chromosome, such as an endogenous or exogenous regulatory element, where the exogenous regulatory element is, for example, a human-derived regulatory element, and the regulatory element may be a promoter. In some embodiments, the non-human animal is a mammal, such as a monkey or rodent (e.g., mouse or rat).In some examples, the non-human animal further comprises a nucleotide sequence of a human protein or chimeric protein encoded by another gene, the human protein or chimeric protein comprising, but not limited to, at least one of CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4.

[0016] In one embodiment, the present invention provides a method for constructing genetically modified non-human animal cells expressing human CFB protein or chimeric CFB protein, the method comprising substituting the endogenous CFB gene with the nucleotide sequence of human CFB at the endogenous CFB locus of a non-human animal. In some examples, the nucleotide sequence of human CFB includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, for example, a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or a portion of exon 18, preferably including the nucleotide sequence of the coding region of the human CFB gene, and more preferably including the nucleotide sequence from the start codon to the stop codon of the human CFB gene. In some examples, the nucleotide sequence of human CFB further includes the 5'UTR and / or 3'UTR of human CFB, and further includes at least 50 bp of consecutive nucleotides outside the 5'UTR of human CFB and / or at least 50 bp of consecutive nucleotides outside the 3'UTR of human CFB. In some examples, the nucleotide sequence of human CFB includes the 5'UTR of human CFB and at least 50 bp of consecutive nucleotides outside its upstream, the nucleotide sequence from the start codon to the stop codon of the human CFB gene, and at least 50 bp of consecutive nucleotides outside the 3'UTR of human CFB and outside its downstream. In some examples, the expression of the nucleotide sequence of human CFB is regulated by a regulatory element, such as an endogenous regulatory element or a human-derived regulatory element, which may be a promoter. In some examples, the non-human animal is a mammal, such as a monkey or rodent (e.g., mouse or rat). In some examples, the non-human animal is a mouse.In some examples, the non-human animal cells further comprise a nucleotide sequence of a human protein or chimeric protein encoded by another gene, the human protein or chimeric protein comprising, but not limited to, at least one of CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4.

[0017] In one embodiment, the present invention provides a humanized CFB gene that includes a non-human endogenous CFB gene locus in which the endogenous CFB gene is replaced with a human CFB nucleotide sequence. In some examples, the humanized CFB gene includes any one of the following nucleotide sequences.

[0018] A) The nucleotide sequence encoding Sequence ID No. 2 or its positions 26 to 764, B) Sequence IDs 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46, C) A nucleotide sequence having at least 90% homology with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46, and D) A nucleotide sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46.

[0019] In one embodiment, the present invention provides a polypeptide encoded by the humanized CFB gene.

[0020] In one embodiment, the present invention provides a target vector comprising a 5' arm, a donor region, and a 3' arm, wherein the 5' arm is homologous to the 5' end of the region to be altered, the 3' arm is homologous to the 3' end of the region to be altered, and the donor region comprises a nucleotide sequence encoding a human CFB protein or a chimeric CFB protein. In some embodiments, the region to be altered is located at a non-human animal endogenous CFB locus, preferably on at least one exon or at least one intron of the non-human animal endogenous CFB gene, for example, from a portion of exon 1 to a portion of intron 17, or from a portion of exon 1 to a portion of exon 18 of the endogenous CFB gene. In some embodiments, the 5' end of the region to be altered is located at exon 1 of the non-human animal endogenous CFB gene, and / or the 3' end of the region to be altered is located at exon 18 of the non-human animal endogenous CFB gene. In some examples, the 5' end of the region to be altered is located in exon 1 of the non-human endogenous CFB gene, and / or the 3' end of the region to be altered is located in intron 17 of the non-human endogenous CFB gene. In some examples, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 3, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 4. In some examples, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 5, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 6. In some examples, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 37, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 38. In some examples, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 45, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 46. In some examples, the chimeric CFB protein is a humanized CFB protein containing a portion of human CFB protein and a portion of non-human CFB protein.In some examples, a portion of the human CFB protein includes a Ba region and / or a Bb region, preferably the Bb region includes a VWA domain, and more preferably further includes von Willebrand factor and / or peptidase S1. In some examples, the humanized CFB protein includes those in which at least 50 to 764 amino acids match the continuous amino acid sequence of a portion of the human CFB protein. In some examples, the human CFB protein or chimeric CFB protein includes SEQ ID NO: 2 or positions 26-764 thereof, or includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2 or positions 26-764, or includes an amino acid sequence that differs from SEQ ID NO: 2 or positions 26-764 by no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids, or no more than 1 amino acid, or includes a sequence that has one or more amino acid substitutions, deletions, and / or insertions relative to SEQ ID NO: 2 or positions 26-764 thereof. In some examples, the donor region includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, preferably encompassing the nucleotide sequence of the coding region of the human CFB gene, and more preferably encompassing the nucleotide sequence from the start codon to the stop codon of the human CFB gene. In some examples, the donor region further includes the 5'UTR and / or 3'UTR of human CFB, and further includes at least 50 bp of consecutive nucleotides outside the 5'UTR of human CFB and / or at least 50 bp of consecutive nucleotides outside the 3'UTR of human CFB. In some examples, the donor region includes the 5'UTR of human CFB and at least 50 bp of consecutive nucleotides outside its upstream, the nucleotide sequence from the start codon to the stop codon of the human CFB gene, and the 3'UTR of human CFB and at least 50 bp of consecutive nucleotides outside its downstream.In some embodiments, the nucleotide sequence of the donor region includes the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 39, or includes a nucleotide sequence whose homology to SEQ ID NO: 7 or SEQ ID NO: 39 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or includes a nucleotide sequence whose difference from SEQ ID NO: 7 or SEQ ID NO: 39 does not exceed 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 6 bp, 3 bp, or 1 bp, or includes a nucleotide sequence having one or more nucleotide substitutions, deletions, and / or insertions compared to what is shown in SEQ ID NO: 7 or SEQ ID NO: 39.

[0021] In one embodiment, the present invention provides cells, tissues, or organs that contain the humanized CFB gene or express the polypeptide.

[0022] In one embodiment, the present invention provides an animal model comprising the humanized CFB gene or the polypeptide or the cells, tissues or organs.

[0023] In one embodiment, the present invention provides the use of the above-mentioned non-human animals, non-human animals obtained by the above-mentioned construction method, the above-mentioned non-human animal genome, the above-mentioned cells obtained by the above-mentioned construction method, the above-mentioned humanized CFB gene, the above-mentioned polypeptide, the above-mentioned cells, tissues or organs, the above-mentioned animal model, and the above-mentioned target vector, the above-mentioned use includes: A) use in product development relating to CFB-related immune processes in human cells; B) use as a CFB-related model system for pharmacological, immunological, microbiological, and medical studies; C) use relating to the production and utilization of animal experimental disease models for CFB-related etiological studies and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) use in in vivo studies for screening, detection of drug efficacy, evaluation, verification or assessment of therapeutic effects of human CFB signaling pathway modulators; or E) use in studies of CFB gene function, studies of drugs and drug efficacy against human CFB target sites, and studies of drugs for CFB-related inflammation and immune-related diseases.

[0024] In one embodiment, the present invention provides a method for measuring the effectiveness of a CFB therapeutic agent for treating a disease, the method comprising: 1) administering the CFB therapeutic agent to a non-human animal or animal model described herein, wherein the non-human animal is suffering from a disease; and 2) measuring the therapeutic effect of the CFB therapeutic agent on the disease. In some embodiments, the disease is an immune disease, inflammation, or tumor. In some embodiments, the immune disease is one or more of the following: age-related macular degeneration (AMD), rheumatoid arthritis, colitis (including ulcerative colitis or Crohn's disease, e.g., perianal Crohn's disease), rheumatism, multiple sclerosis, Parkinson's disease, asthma, myasthenia gravis, or complement disease. In some embodiments, the inflammation described above includes one or more of the following: IgA nephropathy, C3 glomerulopathy, glomerulonephritis, degenerative inflammation, exudative inflammation (e.g., serous inflammation, cellulosic inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, or catarrhal inflammation), proliferative inflammation, or specific inflammation (e.g., tuberculosis, syphilis, leprosy, or lymphogranuloma). In some embodiments, the tumor described above includes solid tumors (e.g., breast cancer) or hematopoietic tumors (e.g., lymphocyte tumors, B-cell tumors, or T-cell tumors).

[0025] In one aspect, the present invention provides a method for measuring the toxicity of a CFB therapeutic agent, the method comprising: 1) administering a CFB therapeutic agent to a non-human animal or animal model described herein; and 2) measuring the effect of the CFB therapeutic agent on the non-human animal. In some embodiments, measuring the effect of the CFB therapeutic agent on the non-human animal relates to measuring the body weight of the non-human animal or a blood test, and preferably, the blood test includes one or more of the number of red blood cells, hematocrit, or hemoglobin content.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials for the present invention are described herein, other suitable methods and materials known in the art can be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) will prevail.

[0027] The term "all or part" of the present invention means that "all" is the whole, and "part" is a local part within the whole or a part of the individuals constituting the whole.

[0028] The term "humanized CFB protein" of the present invention includes a part derived from human CFB protein, preferably further includes a part of non-human animal CFB protein. For example, the above "human CFB protein" is the same as the whole human CFB protein, that is, the amino acid sequence is identical to the full-length amino acid sequence of human CFB protein. The above "a part of human CFB protein" is 5 to 764, preferably 10 to 764 or 50 to 764 consecutive or non-consecutive amino acid sequences, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 730, 735, 739, 750, 760 or 764 amino acid sequences are identical to the human CFB protein sequence.

[0029] The term "humanized CFB gene" of the present invention includes a part derived from human CFB gene and a part of non-human animal CFB gene. For example, the above "human CFB gene" is the same as the whole human CFB gene, that is, its nucleotide sequence is identical to the full-length nucleotide sequence of human CFB gene. The above "a part of human CFB gene" is 20bp to 599o bp, or 20bp to 2476bp, or 20bp to 2295bp consecutive or non-consecutive nucleotide sequences that are identical to the nucleotide sequence of human CFB gene. For example, 20bp, 50bp, 100bp, 200bp, 500bp, 1000bp, 2000bp, 2200bp, 2200bp, 2250bp, 2290bp, 2295bp, 2soobp, 2400bp, 2470bp, soobp, 2476bp, 2500bp, 3000bp, 3500bp, 4000bp, 4500bp, 5000bp, 5500bp, 5900bp, 5990bp nucleotide sequences are identical to the nucleotide sequence of human CFB gene.

[0030] In this invention, the term "locus" broadly refers to the position of a gene on a chromosome, and narrowly refers to a single DNA fragment in a given gene; that is, it may be a single gene or a part of a single gene. For example, the "CFB locus" above refers to an optional single DNA fragment in exons 1 to 18 of the CFB gene. In some embodiments, the substituted non-human endogenous CFB locus may be an optional single DNA fragment in exons 1 to 18 of the non-human endogenous CFB gene.

[0031] In this invention, the term "part of an exon" refers to a sequence of several, tens, or hundreds of nucleotides, whether continuous or discontinuous, that matches the entire exon nucleotide sequence. For example, a portion of exon 1 of the human CFB gene is a sequence of 5 bp to 191 bp, preferably 50 bp to 100 bp, such as 5 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, or 55 bp. This includes those in which the nucleotide sequence of 60bp, 61bp, 62bp, 63bp, 64bp, 65bp, 66bp, 67bp, 68bp, 69bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, or 191bp matches the nucleotide sequence of exon 1 of the human CFB gene.

[0032] In this invention, the terms "exon XX to exon XXX" or "all of exon XX to exon XXX" refer to including exons and introns between them. For example, exons 1 to 18 include all nucleotide sequences 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, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, intron 17, and exon 18. For example, "from part of exon XX to part of exon XXX" or "part of exon XX to part of exon XXX" includes nucleotide sequences of part 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, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, intron 17, and part of exon 18.

[0033] In this application, the term "intron xx" refers to an intron between two exons. For example, intron 1 is the intron between exon 1 and exon 2, and intron 17 is the intron between exon 17 and exon 18.

[0034] The terms “encompassing” or “including” in this invention are open notation and include the designated components or steps described and other designated components or steps that do not substantially affect them. When used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence described, 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.

[0035] The term "and / or" in this invention encompasses all combinations of items to which the term is linked, and each combination should be considered to have already been described separately in this application. For example, "A and / or B" encompasses "A," "A and B," and "B." Also, for example, "A, B and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A, B and C."

[0036] Those skilled in the art will readily understand other aspects and advantages of the present invention from the following detailed description.

[0037] CFB Complement factor B (CFB) is a single-chain protein. When the alternative complement pathway is activated, CFB is cleaved by CFD into a Ba region (~33kD) and a Bb region (~60kD). The Ba region (residues 1-259) contains three complement regulatory protein (CCP) domains, which are important for the initial binding of CFB to C3b. The Bb region (residues 260-764) contains one von Willebrand factor, a type A domain (VWA), and a peptidase S1 (PA family) domain. The VWA domain consists of approximately 200 residues and forms a single α / β opening sheet. The MIDAS site in the VWA domain is also important for the binding of the protein to C3b.

[0038] CFD is a key regulator of the bypass complement pathway, acting on the C3b / CFB complex to cleave CFB to Ba and release it, while Bb remains attached to C3b. Such a C3bBb complex forms a C3 convertase. While the direct biological effects of Ba and Bb have already been reported, the physiological importance of their effects remains unclear. Ba may have chemotactic activity against neutrophils and macrophages and may restrict B cell proliferation. Bb has already been reported to affect the function of macrophages and monocytes and may promote B cell growth.

[0039] In the human genome, the CFB gene (Gene ID: 629) contains 18 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and exon 18 (Figure 1). The nucleotide sequence of human CFB mRNA is NM_001710.6, and the amino acid sequence of human CFB is NP_001701.2 (SEQ ID NO: 2). The corresponding positions of each exon in the nucleotide and amino acid sequences based on the transcript NM_001710.6 and its coding protein NP_001701.2 are shown in Table 1.

[0040] [Table 1]

[0041] The human CFB gene (NCBI Gene ID: 629) is located at positions 31946095 to 31952084 of NC_000006.12 on chromosome 6 (based on transcript NM_001710.6). Of these, 5'UTR is located at 31,946,095~31,946,221, Exon 1 is located at 31,946,095~31,946,285, Intron 1 is located at 31,946,286~31,946,372, Exon 2 is located at 31,946,373~31,946,606, Intron 2 is located at 31,946,607~31,947,006, Exon 3 is located at 31,947,007~31,947,192, Intron 3 is located at 31,947,193~31,94 It is ranked 7,347th, Exxon-4 is ranked 31,947,348-31,947,521st, Intron-4 is ranked 31,947,522-31,947,741st, Exxon-5 is ranked 31,947,742-31,947,843rd, Intron-5 is ranked 31,947,844-31,947,944th, Exxon-6 is ranked 31,947,945-31,948,081st, Intron-6 is ranked 31,948,082-31,948,373rd, Exxon-7 is ranked 31,948, Intron 7 is located at 374th to 31,948,512th place, Intron 7 is located at 31,948,513th to 31,948,829th place, Exxon 8 is located at 31,948,830th to 31,948,961st place, Intron 8 is located at 31,948,962nd to 31,949,242nd place, Exxon 9 is located at 31,949,243rd to 31,949,344th place, Intron 9 is located at 31,949,345th to 31,949,419th place, Exxon 10 is located at 31,949,420th to 31,949,557th place, Intron 10 is It is located at 31,949,558~31,950,049th, Exxon-11 is located at 31,950,050~31,950,147th, Intron-11 is located at 31,950,148~31,950,285th, Exxon-12 is located at 31,950,286~31,950,403rd, Intron-12 is located at 31,950,404~31,950,618th, Exxon-13 is located at 31,950,619~31,950,772nd, Intron-13 is located at 31,950,773~31,950,It is ranked 867th, Exxon-14 is ranked 31,950,868-31,950,944th, Intron-14 is ranked 31,950,945-31,951,143rd, Exxon-15 is ranked 31,951,144-31,951,244th, Intron-15 is ranked 31,951,245-31,951,340th, and Exxon-16 is ranked 31,951,341-31,951,47th Located at position 3, intron 16 is located at positions 31,951,474-31,951,554, exon 17 at positions 31,951,555-31,951,604, intron 17 at positions 31,951,605-31,951,874, exon 18 at positions 31,951,875-31,952,084, and the 3'UTR is located at positions 31952031-31952084. All relevant information regarding the human CFB locus can be found on the NCBI website (Gene ID: 629). All of its contents are incorporated herein by reference.

[0042] In the mouse genome, the CFB gene (Gene ID: 14962) contains 18 exons, namely exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and exon 18 (Figure 1). The nucleotide sequence of mouse CFB mRNA is NM_008198.2, and the amino acid sequence of mouse CFB is NP_032224.2 (SEQ ID NO: 1). The corresponding positions of each exon in the nucleotide and amino acid sequences based on the transcript NM_008198.2 and its coding protein NP_032224.2 are shown in Table 2.

[0043] [Table 2]

[0044] The mouse CFB gene (NCBI Gene ID: 14962) is located at positions 35075350 to 35081492 of NC_000083.7 on chromosome 17 (based on transcript NM_008198.2). Of these, 5'UTR is located at rank 35,081,490~35,081,089, Exon 1 is located at rank 35,081,490~35,081,028, Intron 1 is located at rank 35,081,027~35,080,941, Exon 2 is located at rank 35,080,940~35,080,707, Intron 2 is located at rank 35,080,706~35,080,190, Exon 3 is located at rank 35,080,189~35,080,004, Intron 3 is located at rank 35,080,003~35,079 Exxon-4 is ranked 35,079,912-35,079,739, Intron-4 is ranked 35,079,738-35,079,602, Exxon-5 is ranked 35,079,601-35,079,500, Intron-5 is ranked 35,079,499-35,079,399, Exxon-6 is ranked 35,079,398-35,079,262, Intron-6 is ranked 35,079,261-35,079,051, Exxon-7 is ranked 35,079,0 Intron 7 is located at 35,078,911 to 35,078,550, Exxon 8 is located at 35,078,549 to 35,078,418, Intron 8 is located at 35,078,417 to 35,078,308, Exxon 9 is located at 35,078,307 to 35,078,206, Intron 9 is located at 35,078,205 to 35,078,123, Exxon 10 is located at 35,078,122 to 35,077,985, Intro Exxon-10 is located at 35,077,984 to 35,077,572nd place, Exxon-11 is located at 35,077,571 to 35,077,474th place, Intron-11 is located at 35,077,473 to 35,077,131st place, Exxon-12 is located at 35,077,130 to 35,077,013th place, Intron-12 is located at 35,077,012 to 35,076,798th place, Exxon-13 is located at 35,076,797 to 35,076,644th place, Intron-13 is located at 35,076,643 to 35,It is located at 076,545th, Exxon-14 is located at 35,076,544~35,076,468th, Intron-14 is located at 35,076,467~35,076,296th, Exxon-15 is located at 35,076,295~35,076,195th, Intron-15 is located at 35,076,194~35,076,113th, Exxon-16 is located at 35,076,112~35,075th, It is located at position 980, intron 16 is located at positions 35,075,979-35,075,906, exon 17 is located at positions 35,075,905-35,075,856, intron 17 is located at positions 35,075,855-35,075,559, exon 18 is located at positions 35,075,558-35,075,360, and the 3'UTR is located at positions 35,075,402-35,075,360. All relevant information regarding the mouse CFB gene locus can be found on the NCBI (Gene ID: 14962) website. All of its contents are incorporated herein by reference.

[0045] Figure 10 and Table 3 show the alignment of the human CFB amino acid sequence (NP_001701.2, SEQ ID NO: 2) and the mouse CFB amino acid sequence (NP_032224.2, SEQ ID NO: 1). Therefore, in Figure 10, corresponding amino acid residues or regions between the human CFB and the mouse CFB can be found.

[0046] [Table 3]

[0047] Other species of CFB genes, proteins, and gene regions in this field are also known. For example, Gene ID: 294257 for Rattus norvegicus (rat) CFB, Gene ID: 716809 for Macaca mulatta (rhesus monkey) CFB, Gene ID: 100688056 for Canis lupus familiaris (dog) CFB, and Gene ID: 100124383 for Sus scrofa (pig) CFB. Relevant information for these genes (e.g., intron sequences, exon sequences, and amino acid sequences) can all be searched in NCBI, and all of its contents are incorporated into this application by reference.

[0048] Figure 11 and Table 4 show the amino acid sequences of human CFB (NP_001701.2, SEQ ID NO: 2) and rat CFB (NP_997631.2, SEQ ID NO: 57). Therefore, in Figure 11, it is possible to search for corresponding amino acid residues or regions between human CFB and rat CFB.

[0049] [Table 4]

[0050] The present invention provides a human CFB protein or a chimeric (e.g., humanized) CFB protein, or a human CFB gene or a chimeric (e.g., humanized) CFB gene.

[0051] In some examples, the humanized CFB protein includes a portion of the human CFB protein. Preferably, it further includes a portion of the non-human animal CFB protein. The portion of the human CFB protein includes a Ba region and / or a Bb region. Preferably, the Bb region includes a VWA domain, and more preferably, it further includes a von Willebrand factor and / or peptidase S1. In some examples, the humanized CFB protein includes the Ba region of the human CFB protein and the Bb region of the non-human animal CFB protein. In some examples, the humanized CFB protein includes the Bb region of the human CFB protein and the Ba region of the non-human animal CFB protein. In some examples, the humanized CFB protein includes the VWA domain of the human CFB protein and the von Willebrand factor, peptidase S1, and Ba region of the non-human animal CFB protein. In some examples, the humanized CFB protein includes a form in which the VWA domain of the non-human animal CFB protein is replaced with the VWA domain region of the human CFB. In some examples, the humanized CFB protein includes a non-human animal CFB protein in which the Bb region is replaced with the Bb region of human CFB. In some examples, the humanized CFB protein includes a non-human animal CFB protein in which the Ba region is replaced with the Ba region of human CFB. In some examples, the humanized CFB protein includes those in which at least 50 to 764 amino acids (for example, 50, 60, 70, 80, 90, 100, 150, 200, 220, 240, 260, 280, 300, 310, 350, 400, 450, 500, 550, 600, 650, 700, 710, 720, 730, 739, 740, 750, 760, 761, 762, 763, or 764) of the amino acid sequence matches a portion of the continuous amino acid sequence of a human CFB protein.In some examples, the humanized CFB protein includes SEQ ID NO: 2 or positions 26-764 thereof, or includes an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to SEQ ID NO: 2 or positions 26-764, or includes an amino acid sequence that differs from SEQ ID NO: 2 or positions 26-764 by no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids, or no more than 1 amino acid, or includes a sequence that has one or more amino acid substitutions, deletions, and / or insertions relative to SEQ ID NO: 2 or positions 26-764 thereof.

[0052] In some examples, the humanized CFB gene encodes the humanized CFB protein. Preferably, it includes a portion of the non-human endogenous CFB gene and a portion of the human CFB gene. In some examples, the portion of the human CFB gene includes a nucleotide sequence encoding the Ba region and / or Bb region of the human CFB protein. In some examples, the portion of the human CFB gene includes a nucleotide sequence encoding the VWA domain of the human CFB protein. In some examples, the humanized CFB gene includes a substitution of the endogenous CFB gene with a nucleotide sequence containing human CFB at the non-human endogenous CFB locus. In some examples, the non-human animal may be a mouse. In some examples, the nucleotide sequence of human CFB includes a nucleotide sequence encoding the Ba region and / or Bb region. In some examples, the nucleotide sequence of human CFB includes a nucleotide sequence encoding the VWA domain. In some examples, the nucleotide sequence of human CFB may be a genome sequence, CDS, or cDNA sequence. In some examples, the nucleotide sequence of the human CFB includes at least one exon and / or at least one intron of the human CFB gene. In some examples, the nucleotide sequence of the human CFB includes one or more of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the human CFB gene. In some examples, the nucleotide sequence of the human CFB includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, the portion of exon 1 includes the nucleotide sequence of the coding region in exon 1, and the portion of exon 18 includes the nucleotide sequence of the coding region in exon 18. Preferably, the nucleotide sequence of the human CFB includes the nucleotide sequence of the coding region of the human CFB gene, and more preferably, includes the nucleotide sequence from the start codon to the stop codon of the human CFB gene.In some embodiments, the nucleotide sequence of human CFB comprises the nucleotide sequence from the start codon to the stop codon of the human CFB gene, preferably further comprising the 5'UTR and / or 3'UTR of human CFB, and further comprising at least 50 bp of consecutive nucleotides outside the 5'UTR of human CFB and / or at least 50 bp of consecutive nucleotides outside the 3'UTR of human CFB. In some embodiments, the nucleotide sequence of human CFB comprises the 5'UTR of human CFB and at least 50 bp of consecutive nucleotides outside its upstream, the nucleotide sequence from the start codon to the stop codon of the human CFB gene, and the 3'UTR of human CFB and at least 50 bp of consecutive nucleotides outside its downstream. In some embodiments, the amino acid sequence of the human CFB includes SEQ ID NO: 2 or positions 26-764 thereof, or includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to SEQ ID NO: 2 or positions 26-764, or includes an amino acid sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids or no more than 1 amino acid difference from SEQ ID NO: 2 or positions 26-764 thereof, or includes a sequence having one or more amino acid substitutions, deletions, and / or insertions relative to SEQ ID NO: 2 or positions 26-764 thereof.In some embodiments, the nucleotide sequence of the human CFB includes the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 39, or includes a nucleotide sequence whose homology to SEQ ID NO: 7 or SEQ ID NO: 39 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or includes a nucleotide sequence whose difference from SEQ ID NO: 7 or SEQ ID NO: 39 is not more than 50, 40, 30, 20, 10, 9, 6, 3, or 1 nucleotide, or includes a nucleotide sequence having one or more nucleotide substitutions, deletions, and / or insertions compared to what is shown in SEQ ID NO: 7 or SEQ ID NO: 39. In some embodiments, the nucleotide sequence encoding the Ba region and / or Bb region of the endogenous CFB is substituted. In some examples, the nucleotide sequence encoding the von Willebrand factor and / or peptidase S1 region of endogenous CFB is substituted. In some examples, the nucleotide sequence encoding SEQ ID NO: 1 or its positions 18-712 or SEQ ID NO: 57 is substituted. In some examples, at least one exon and / or at least one intron of the endogenous CFB gene is substituted. In some examples, one or more of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the endogenous CFB are substituted. In some examples, exons 2 to 17 of the endogenous CFB gene are substituted, preferably the substituted endogenous CFB gene further includes a portion of exon 1, all of intron 1 and / or a portion of intron 17, and more preferably the substituted endogenous CFB gene further includes a portion of exon 18.In some examples, a portion of exon 1 to exon 18 of the endogenous CFB gene, for example, a nucleotide sequence encompassing the coding region or encompassing the start codon to the stop codon, is substituted. In some examples, a portion of exon 1 to intron 17 of the endogenous CFB gene is substituted. In some examples, the non-human animal is a mammal, for example, a monkey or a rodent (for example, a mouse or a rat). In some examples, the entire nucleotide sequence of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18 of the non-human animal (e.g., mouse) CFB gene is substituted with the nucleotide sequence of human CFB. In some embodiments, "parts" of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and / or 18 of the non-human animal (e.g., mouse) CFB gene are replaced with nucleotide or amino acid sequences of human CFB.The term "part" above refers to at least 1 bp to 2767 bp, or at least 1 bp to at least 2449 bp, of the CFB gene in non-human animals (e.g., mice), for example, at least 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp. p, 170bp, 180bp, 190bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 550bp, 600bp, 650bp, 700bp, 750bp, 800bp, 850bp , 900bp, 910bp, 911bp, 912bp, 913bp, 914bp, 915bp, 916bp, 917bp, 918bp, 919bp, 920bp, 930bp, 940bp, 950bp, 1000bp, 1100bp , a continuous nucleotide sequence of 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1750bp, 1800bp, 1900bp, 2000bp, 2080bp, 2085bp, 2100bp, 2200bp, 2292bp, 2295bp, 2449bp, 2700bp or 2767bp, or at least one to 763 amino acids of a non-human animal (e.g., mouse) CFB protein, for example, at least one, two, three, This refers to a sequence of 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 220, 240, 260, 280, 300, 310, 350, 400, 450, 500, 550, 600, 650, 690, 695, 700, 710, 720, 730, 740, 750, 760, 761, 762, or 763 consecutive amino acid sequences.In some embodiments, the “part” described above is one in which homology is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% with exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, and / or exon 18 of the CFB gene. In several examples, the sequences of "part" or "all" of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and / or 18 of the CFB gene of a non-human animal (e.g., mouse) (e.g., part of exon 1, all of exons 2-17 and part of exon 18) are used to represent the sequences of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or of human CFB. The sequence is replaced with "part" or "all" of xon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18 (for example, from part of exon 1 to part of exon 18 of the human CFB gene, for example, from the start codon to the stop codon, or using at least 50 bp of consecutive nucleotides from the 5'UTR of human CFB and its outer upstream side, the nucleotide sequence from the start codon to the stop codon of the human CFB gene, and at least 50 bp of consecutive nucleotides from the 3'UTR of human CFB and its outer downstream side, more preferably including SEQ ID NO: 7 or SEQ ID NO: 39). In some examples, all or part of the nucleotide sequence encoding the endogenous CFB protein is replaced with nucleotides encoding the human CFB protein.

[0053] In some examples, the humanized CFB gene includes, in order from the 5' end to the 3' end, a portion of exon 1 of the mouse CFB gene, a portion of exon 1 to a portion of exon 18 of the human CFB gene, and a portion of exon 18 of the mouse CFB gene. In some examples, the humanized CFB gene includes, in order from the 5' end to the 3' end, a portion of exon 1 of the mouse CFB gene, the 5'UTR of human CFB and at least 50 bp of consecutive nucleotides upstream of it, the nucleotide sequence of the human CFB gene from the start codon to the stop codon, the 3'UTR of human CFB and at least 50 bp of consecutive nucleotides downstream of it, a portion of intron 17 of the mouse CFB gene, and exon 18 of the mouse CFB gene. In some examples, the nucleotide sequence of human CFB in the humanized CFB gene has sequence number 9 as the upstream linking sequence with mouse, and sequence number 10 as the downstream linking sequence with mouse. In some examples, the nucleotide sequence of human CFB in the humanized CFB gene is such that the upstream linkage sequence with mouse is SEQ ID NO: 41, and the downstream linkage sequence with mouse is SEQ ID NO: 42. In some examples, the humanized CFB gene includes, in order from the 5' end to the 3' end, A) SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 4, B) SEQ ID NO: 3, SEQ ID NO: 39, and SEQ ID NO: 4, C) SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 6, D) SEQ ID NO: 5, SEQ ID NO: 39, and SEQ ID NO: 6, E) SEQ ID NO: 37, SEQ ID NO: 7, and SEQ ID NO: 38, F) SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 38, G) SEQ ID NO: 45, SEQ ID NO: 7, and SEQ ID NO: 46, and H) SEQ ID NO: 45, SEQ ID NO: 39, and SEQ ID NO: 46. In some examples, the humanized CFB gene includes one or more of SEQ ID NOs: 11, SEQ ID NO: 12, SEQ ID NO: 43, or SEQ ID NO: 44.In some examples, the mRNA transcribed by the humanized CFB gene includes SEQ ID NO: 8 or SEQ ID NO: 40, or includes a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with SEQ ID NO: 8 or SEQ ID NO: 40, or includes a nucleotide sequence whose difference from SEQ ID NO: 8 or SEQ ID NO: 40 does not exceed 50, 40, 30, 20, 10, 9, 6, 3, or 1 nucleotide, or includes a nucleotide sequence having one or more nucleotide substitutions, deletions, and / or insertions relative to what is shown in SEQ ID NO: 8 or SEQ ID NO: 40. In some examples, the humanized CFB gene further includes a resistance gene, preferably the humanized CFB gene further includes two Frt recombination sites arranged in the same direction on both sides of the resistance gene. In some examples, the resistance gene is the neomycin phosphotransferase coding gene Neo. Preferably, the humanized CFB gene further comprises a specific inducer or repressor, and more preferably, the specific inducer or repressor may be a conventional induceable or repressible substance. In one specific embodiment of the present invention, the specific inducer comprises, but is not limited to, a tetracycline system (Tet-Off System / Tet-On System) or a tamoxifen system. Preferably, the humanized CFB gene is regulated in a non-human animal by a regulatory element, and more preferably, the regulatory element is an endogenous or exogenous regulatory element (e.g., a human regulatory element). Preferably, the regulatory element is a promoter.

[0054] In some embodiments, a portion of endogenous exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, intron 17 and / or a portion of exon 18 is deleted.

[0055] In some embodiments, a portion of endogenous exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, and a portion of intron 17 are deleted.

[0056] In some embodiments, the present invention provides a genetically modified non-human animal whose genome comprises a human CFB gene, a chimeric CFB gene, or a humanized CFB gene. In some embodiments, the protein encoded by the nucleotide sequence of the human CFB gene, chimeric CFB gene, or humanized CFB gene has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology with SEQ ID NO: 2 or the amino acid sequence shown at positions 26 to 764 thereof. In some embodiments, the nucleotide sequences included in the non-human animal genome have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology with the nucleotide sequences shown in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46.

[0057] In some embodiments, the non-human animal described herein includes a human CFB gene or a humanized CFB gene. In some embodiments, the humanized CFB gene includes 18 exons. In some embodiments, the humanized CFB gene includes a human exon or humanized exon 1, human exon 2, human exon 3, human exon 4, human exon 5, human exon 6, human exon 7, human exon 8, human exon 9, human exon 10, human exon 11, human exon 12, human exon 13, human exon 14, human exon 15, human exon 16, human exon 17 and / or a human exon or humanized exon 18. In some examples, the humanized CFB gene includes human intron 1, human intron 2, human intron 3, human intron 4, human intron 5, human intron 6, human intron 7, human intron 8, human intron 9, human intron 10, human intron 11, human intron 12, human intron 13, human intron 14, human intron 15, human intron 16 and / or human intron or humanized intron 17. In some examples, the humanized CFB gene includes human 5'UTR or endogenous 5'UTR or humanized 5'UTR. In some examples, the humanized CFB gene includes human 3'UTR or endogenous 3'UTR or humanized 3'UTR. In some examples, the humanized CFB gene includes endogenous 5'UTR or human 5'UTR. In some examples, the humanized CFB gene includes endogenous 3'UTR or human 3'UTR.

[0058] In some examples, genetically modified non-human animals can express human CFB protein and / or chimeric (e.g., humanized) CFB protein, and the endogenous CFB gene sequence is replaced with the human CFB gene and / or nucleotide sequence. Furthermore, the amino acid sequence of human CFB encoded by the above human CFB gene and / or nucleotide sequence has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% homology to SEQ ID NO: 2 or positions 26-764 of the amino acid sequence represented by human CFB. In some examples, the nucleotide sequence of the endogenous non-human animal's CFB gene is replaced with all or part of the nucleotide sequence encoding a mature human CFB protein.

[0059] In several examples, genetically modified non-human animals express human CFB and / or chimeric CFB protein (e.g., humanized CFB protein) under endogenous or human-derived regulatory elements (e.g., promoters). Substitution of endogenous loci provides non-human animals that express human CFB protein or chimeric CFB protein (e.g., humanized CFB protein) in the same type of cells. The genetically modified mice do not exhibit the potential diseases observed in certain other transgenic mice known in this field. The human CFB protein or chimeric CFB protein expressed in non-human animals can maintain the function of one or more wild-type or human CFB proteins; for example, the expressed CFB protein can bind to human CFB protein or non-human CFB protein. Furthermore, in several examples, the genetically modified non-human animals do not express endogenous CFB protein. In several examples, the expression of endogenous CFB protein is reduced in the genetically modified non-human animals. The term "endogenous CFB protein" as used in this application refers to a CFB protein encoded by the endogenous CFB nucleotide sequence of a non-human animal (e.g., mouse) before genetic modification.

[0060] The genome of a non-human animal includes nucleotide sequences encoding amino acids that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homology with the amino acid sequence represented by the human CFB protein (NP_001701.2, SEQ ID NO: 2). In some examples, the genome includes nucleotide sequences that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homology with the nucleotide sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 39, or SEQ ID NO: 40.

[0061] The nucleotide sequence encoding the endogenous CFB region in a non-human animal genome is replaced with the nucleotide sequence encoding human CFB. In some examples, the nucleotide sequence encoding the endogenous CFB region is any one sequence of the endogenous CFB locus, for example, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, 5'UTR, 3'UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, or any combination thereof. In some embodiments, the nucleotide sequence encoding the endogenous CFB region is located within the endogenous CFB regulatory region. In some embodiments, the nucleotide sequence encoding the endogenous CFB region is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18, or a portion thereof.

[0062] One or more cells from a genetically modified non-human animal express human CFB protein or chimeric CFB protein (e.g., humanized CFB protein). In some examples, the human CFB protein or chimeric CFB protein contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 220, 240, 260, 280, 300, 310, 350, 400, 450, 500, 550, 600, 650, 700, 710, 720, 730, 739, 740, 750, 760, 761, 762, 763, or 764 consecutive amino acid sequences relative to the amino acid sequence indicated by SEQ ID NO: 2 or its positions 26-764.

[0063] In some embodiments, the genetically modified non-human animal genome comprises all or part of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and / or 18 of the human CFB gene, or all or part of the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 39, or SEQ ID NO: 40.

[0064] In some examples, the genetically modified non-human animal genome comprises a portion of exon 1, all of exons 2 to 17, and a portion of exon 18 of the human CFB gene, preferably further comprising intron 1 and / or intron 17. In some examples, the portion of exon 1 comprises a sequence of nucleotides of at least 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 70 bp, 75 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, or 191 bp of exon 1 of the human CFB gene. In some examples, the portion of exon 1 comprises a sequence of 64 bp of nucleotides. In some examples, a portion of exon 1 includes a nucleotide sequence of at least 30 bp, preferably encompassing the nucleotide sequence of the coding region in exon 1. In some examples, a portion of exon 18 includes a continuous nucleotide sequence of at least 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 150 bp, 151 bp, 152 bp, 153 bp, 154 bp, 155 bp, 156 bp, 157 bp, 158 bp, 159 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, or 210 bp in exon 18 of the human CFB gene. In some examples, a portion of exon 18 includes a continuous nucleotide sequence of 156 bp. In some examples, a portion of exon 18 includes a nucleotide sequence of at least 50 bp, preferably encompassing the nucleotide sequence of the coding region in exon 18. Part of exon 1, all of exons 2 to 17, and part of exon 18 of the human CFB gene contain a sequence of nucleotides of at least 100 bp to 500 bp, 500 bp to 1000 bp, 1000 bp to 2000 bp, or 2000 bp to 2500 bp.In several examples, the genetically modified non-human animal genome encompasses all of exons 1 to 18 of the human CFB gene, and also encompasses the 5'UTR and / or 3'UTR of the human CFB, and further encompasses at least 50 bp to at least 10000 bp outside the 5'UTR of the human CFB (e.g., 50 bp, 100 bp, 500 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 5500 bp, 5600 bp, 5700 bp, 5800 bp, 5900 bp, 6000 bp, 6100 bp, 6200 bp, 7000 bp, 8000 bp, 9000 bp or 10000 bp, preferably 3000 bp). The compound includes a sequence of nucleotides (bp to 8000bp, more preferably 5000bp to 7000bp) and / or a sequence of nucleotides (e.g., 50bp, 100bp, 500bp, 600bp, 700bp, 800bp, 900bp, 910bp, 920bp, 930bp, 940bp, 950bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 2000bp, 3000bp, 4000bp, or 5000bp, preferably 500bp to 3000bp, more preferably 500bp to 1500bp) outside the 3'UTR of human CFB. In some examples, the nucleotide sequence of the human CFB is the nucleotide sequence from position 1 to 2476 or from position 128 to 2422 of the human CFB gene transcript NM_001710.6.

[0065] In several examples, the CFB gene in genetically modified non-human animals is heterozygous or homozygous for the endogenously modified locus.

[0066] In some embodiments, the humanized CFB genome includes the 5'UTR of the human CFB gene. In some embodiments, the humanized CFB genome includes the endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanized CFB genome includes the 3'UTR of the human CFB gene. In some embodiments, the humanized CFB genome includes the endogenous (e.g., mouse) 3'UTR. Where appropriate, it can be reasonably inferred, based on sequence similarity, that the mouse CFB gene and the human CFB gene are subject to similar regulation. As described in the present invention, the humanized CFB mouse includes a substitution at the endogenous mouse locus, which includes the humanized CFB coding sequence, whether it retains or does not retain the mouse endogenous regulatory element. CFB expression in genetically modified heterozygous or homozygous mice is completely normal.

[0067] In another embodiment, the present invention provides a genetically modified non-human animal genome comprising a deletion of the endogenous CFB gene.

[0068] In some embodiments, the deletion of the endogenous CFB gene encompasses one or more exons or a portion of an exon, the exon being selected from exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18.

[0069] In some embodiments, the deletion of the endogenous CFB gene further includes one or more introns or a portion of an intron, the intron being selected from introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and / or 17 of the CFB gene.

[0070] In some examples, the above deletion encompasses deletions of at least 1 bp to 6143 bp in the endogenous CFB gene. In some examples, the above deletion encompasses deletions of at least 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 950 bp, and 1000 bp. The sequence of nucleotides is a sequence of p, 1300bp, 1350bp, 1400bp, 1450bp, 1500bp, 1600bp, 1800bp, 2000bp, 2292bp, 2293bp, 2300bp, 2400bp, 2500bp, 2600bp, 2700bp, 2750bp, 2766bp, 2767bp, 3000bp, 4000bp, 5000bp, 5500bp, 5686bp, 5687bp, 5688bp, 5689bp, 5690bp, 5700bp, 5800bp, 5900bp, 6000bp, 6100bp, or 6143bp, or longer.

[0071] In some examples, the above deletions include deletions of at least 1 bp to 7011 bp in the endogenous CFB gene. In some examples, the above deletions include at least 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 42 bp.

[0072] In several examples, the deletion of the endogenous CFB gene is at least 50 bp to at least 2767 bp, or at least 50 bp to 2449 bp, in exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and / or 18, for example, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1500 bp, 2000 bp. The sequence includes a continuous nucleotide sequence of p, 2080bp, 2085bp, 2100bp, 2200bp, 2290bp, 2292bp, 2295bp, 2449bp, 2500bp, 2600bp, 2700bp, 2750bp, 2760bp, 2765bp, or 2767bp or more (for example, a deletion of at least 30bp of a continuous nucleotide sequence in exon 1, all of exons 2 to 17, or at least 50bp of a continuous nucleotide sequence in exon 18, preferably further a deletion of intron 1 and / or intron 17, or, for example, a deletion of at least 10bp of exon 1, all of exons 2 to 17, and part of intron 17).

[0073] The present invention provides a humanized mouse CFB genomic DNA sequence, a construct of an amino acid sequence expressing a humanized CFB protein, cells containing the construct, and tissue containing the cells.

[0074] Accordingly, in some embodiments, the present invention provides a chimeric (e.g., humanized) CFB nucleotide sequence and / or amino acid sequence, in some embodiments, the above-mentioned chimeric CFB nucleotide sequence and / or amino acid sequence is derived from mouse endogenous CFB The homology between the mRNA (e.g., NM_008198.2), the mouse CFB amino acid sequence (e.g., NP_032224.2, SEQ ID NO: 1), or a portion thereof (e.g., a portion of exon 1 (including the coding region and possibly the 5'UTR), a portion of exon 18 (including the coding region and possibly the 3'UTR)) 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 100%. In some examples, the above chimeric nucleotide sequence is human CFB mRNA sequence (e.g., NM_001710.6), human CFB amino acid sequence (e.g., NP_001701.2, SEQ ID NO: 2), or a part thereof (e.g., a part of exon 1, exons 2 to 17, or, for example, the 5'UTR and at least 50 bp of consecutive nucleotides upstream of it, the nucleotide sequence from the start codon to the stop codon of the human CFB gene, and the 3'UTR and of human CFB) Its homology to the outer downstream sequence of at least 50 bp of consecutive nucleotides 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 100%.

[0075] In some examples, the mouse CFB (SEQ ID NO: 1) or the nucleotide sequence encoding the amino acids at positions 25 to 763 therein is replaced with the human CFB (SEQ ID NO: 2) or the nucleotide sequence encoding the amino acids at positions 26 to 764 therein.

[0076] In some examples, the nucleotide sequence encoding mouse CFB (SEQ ID NO: 1) or the amino acids therefrom from position 18 to 712 is replaced with the nucleotide sequence encoding the amino acids of human CFB (SEQ ID NO: 2).

[0077] In some embodiments, the nucleotides are operably linked to regulatory elements (preferably promoters), such as human CFB promoters or endogenous mouse CFB promoters, inducible promoters, enhancers, and / or mouse or human regulatory elements.

[0078] In some embodiments, the chimeric nucleotide sequences described herein differ in at least a portion (for example, 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, for example, a continuous nucleotide sequence or a discontinuous nucleotide sequence) from all or part of the mouse CFB nucleotide sequence (for example, a portion of exon 1, exons 2 to 17 and exon 18 of the mouse CFB gene transcript NM_008198.2, or for example, exons 1 to 18 of the mouse CFB gene transcript NM_008198.2).

[0079] In some examples, the above chimeric nucleotide sequence is identical in all or part to the mouse CFB nucleotide sequence (for example, part of exon 1 and all or part of exon 18 of the mouse CFB gene transcript NM_008198.2) in at least a portion of it (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 continuous nucleotide sequence or a discontinuous nucleotide sequence).

[0080] In some embodiments, the above chimeric nucleotide sequence differs in at least a portion (for example, 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, for example, a continuous nucleotide sequence or a discontinuous nucleotide sequence) from all or part of the human CFB nucleotide sequence (for example, part of exon 1 and all or part of exon 18 of the human CFB gene transcript NM_001710.6).

[0081] In some embodiments, the above chimeric nucleotide sequence is identical in part to all or part of the human CFB nucleotide sequence (for example, part of exon 1, exons 2 to 17 and part of exon 18 of the human CFB gene transcript NM_001710.6, or for example, exons 1 to 18 of the human CFB gene transcript NM_001710.6) in terms of at least a portion of it (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 continuous nucleotide sequence or a discontinuous nucleotide sequence).

[0082] In some examples, the amino acid sequence encoded by the above chimeric nucleotide sequence differs in part from the amino acid sequence of the mouse CFB protein (for example, the mouse CFB protein sequence NP_032224.2 (SEQ ID NO: 1) or the amino acids therefrom) in at least one part (e.g., at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, or one hundred amino acid residues, e.g., consecutive or discontinuous amino acid residues) in all or part of the amino acid sequence of the mouse CFB protein (e.g., the mouse CFB protein sequence NP_032224.2 (SEQ ID NO: 1) or the amino acids therefrom from positions 18 to 712).

[0083] In some examples, the above amino acid sequence is identical in all or part to the amino acid sequence of the mouse CFB protein in at least a portion of it (for example, 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, for example, consecutive amino acid residues or discontinuous amino acid residues).

[0084] In some embodiments, the above amino acid sequence differs in all or part from the amino acid sequence of the human CFB protein in at least a portion of it (for example, 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, for example, consecutive amino acid residues or discontinuous amino acid residues).

[0085] In some examples, the above amino acid sequence is identical in all or part to the amino acid sequence of the human CFB protein (for example, the human CFB protein sequence NP_001701.2 (SEQ ID NO: 2) or the amino acids therefrom) in at least a portion of it (for example, 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, for example, consecutive or discontinuous amino acid residues).

[0086] The present invention further provides a humanized CFB protein whose amino acid sequence includes one of the following groups.

[0087] A) The amino acid sequence shown in SEQ ID NO: 2 or from position 26 to 764 therein, B) The homology to the amino acid sequence shown in SEQ ID NO: 2 or from position 26 to 764 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. C) Amino acid sequences encoded by nucleic acid sequences that can hybridize with nucleotide sequences encoding amino acids shown in SEQ ID NO: 2 or at positions 26-764 therein under low stringency or stringency conditions. D) The difference between the amino acid sequence shown in SEQ ID NO: 2 or at positions 26-764 and the actual amino acid sequence does not exceed 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids, or does not exceed 1 amino acid, E) An amino acid sequence, represented by SEQ ID NO: 2 or positions 26-764 thereof, comprising one or more amino acid residue substitutions, deletions, and / or insertions.

[0088] The present invention further provides a humanized CFB gene (e.g., DNA or RNA) whose nucleotide sequence includes one of the following groups.

[0089] A) Nucleic acid sequences encoding the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 39, or SEQ ID NO: 40, or the homologous amino acid sequence of humanized mouse CFB, B) Nucleic acid sequences that can hybridize with the nucleotide sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 39, or SEQ ID NO: 40 under low stringency conditions or stringency conditions, C) A nucleic acid sequence that is the same as the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 39, or SEQ ID NO: 40, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology. D) The homology between the amino acid sequence encoded thereby and the amino acid sequence shown at position 26-764 of Sequence ID No. 2 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. E) The difference between the encoded amino acid sequence and the amino acid sequence shown in Sequence ID No. 2 or at positions 26 to 764 thereof does not exceed 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids, or does not exceed 1 amino acid, or F) An amino acid sequence containing one or more amino acid residue substitutions, deletions, and / or insertions, the encoded amino acid sequence shown in SEQ ID NO: 2 or its positions 26-764.

[0090] The present invention further provides DNA sequences of the CFB genome of a humanized mouse. These DNA sequences are obtained by reverse transcription of mRNA obtained by transcription and are homologous to or complementary to DNA sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 39, or SEQ ID NO: 40.

[0091] To determine the percentage of identity between two amino acid sequences or two nucleotide sequences, the sequences are aligned for the purpose of optimal comparison (for example, gaps can be introduced between the first and second amino acid sequences or between the first and second nucleic acid sequences, or between the first and second nucleic acid sequences, or both, for optimal alignment, and non-homologous sequences can be ignored for the purpose of comparison). Next, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If one position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are the same at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, and considering the number of gaps and the length of each gap, these need to be introduced to achieve optimal alignment of the two sequences. For example, the comparison of sequences and the determination of the percentage of identity between two sequences can be completed using a Blossum 62 score matrix with a gap penalty 12, a gap extension penalty 4, and a frameshift gap penalty 5.

[0092] The percentage of conserved residues with similar physicochemical properties (homology percentage), such as leucine and isoleucine, may be used to measure sequence similarity. In this art, families of amino acid residues with similar physicochemical properties are defined. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Often, the homology percentage is higher than the identity percentage.

[0093] In some examples, the term "homology" above means that, in the use of an amino acid sequence or nucleotide sequence, a person skilled in the art can adjust the sequence as needed for the actual work, while ensuring that the structure or function is similar to a known sequence, and that the sequence used is similar to a sequence obtained by the prior art by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, and 37%. %, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% This refers to having (including, but not limited to) the identity or consistency of 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9%.

[0094] The present invention further provides cells, tissues, and animals (e.g., mice) that contain the nucleotide sequence described in this application, as well as cells, tissues, and animals (e.g., mice) that express human CFB or chimeric (e.g., humanized) CFB at the endogenous non-human CFB locus.

[0095] Genetically modified non-human animals The term "genetically modified non-human animal" or "genetically modified non-human animal" as used in this invention refers to a non-human animal having exogenous DNA on at least one chromosome in its genome. In some examples, at least one or more cells, for example, 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 types of cells, such as somatic cells, immune cells (e.g., T cells, B cells, NK cells, antigen-presenting cells, macrophages, dendritic cells), germ cells, blastocyst cells, or tumor cells. In several embodiments, genetically modified non-human animals are provided, which include an endogenous CFB locus to be modified and an exogenous sequence (e.g., a human-derived sequence), for example, by substituting one or more non-human animal sequences with one or more human-derived sequences, or by inserting one or more human-derived and / or non-human sequences. Non-human animals can typically transmit genetic modification to their offspring through germline transmission.

[0096] The “chimeric(x) gene” or “chimeric(x) nucleic acid” described in the present invention refers to a gene or nucleic acid whose two or more parts originate from different species, or whose at least one sequence differs from the nucleic acid in wild-type animals. In some examples, the chimeric(x) gene or chimeric(x) nucleic acid has at least a portion of the sequence that originates from two or more different species, for example, a sequence that codes for a different protein or a sequence that codes for the same (or homologous) protein from two or more different species. In some examples, the chimeric(x) gene or chimeric(x) nucleic acid refers to a humanized gene or humanized nucleic acid.

[0097] The “chimeric(x) protein” or “chimeric(x) polypeptide” described in the present invention refers to a protein or polypeptide whose two or more portions originate from different species, or whose at least one sequence differs from the amino acid sequence in a wild-type animal. In some examples, at least a portion of the sequence of the chimeric(x) protein or chimeric(x) polypeptide originates from two or more different species, for example, having the same (or homologous) protein of different species. In some examples, the chimeric(x) protein or chimeric(x) polypeptide refers to a humanized(x) protein or humanized(x) polypeptide.

[0098] The "humanized (x) protein" or "humanized (x) polypeptide" described in the present invention refers to a protein or polypeptide that is at least partly derived from a human protein or human polypeptide. In some examples, the humanized (x) protein or humanized (x) polypeptide refers to a human protein or polypeptide.

[0099] The "humanized (x) nucleic acid" or "humanized (x) gene" described in the present invention refers to a nucleic acid that is at least partially derived from humans. In some examples, all nucleic acids in the humanized (x) nucleic acid or humanized (x) gene are derived from humans. In some examples, the humanized (x) nucleic acid or humanized (x) gene refers to a humanized exon, and the humanized exon may be a human exon or a chimeric exon. In some examples, the humanized (x) nucleic acid or humanized (x) gene refers to a humanized exon and a humanized intron, and the humanized intron may be a human intron or a chimeric intron.

[0100] In some examples, the chimeric (x) gene or chimeric (x) nucleic acid is a humanized CFB gene or humanized CFB nucleic acid. In some examples, at least a portion of the gene or nucleic acid is derived from a human CFB gene, and at least a portion of the gene or nucleic acid is derived from a non-human CFB gene. In some examples, the gene or nucleic acid includes a sequence encoding a CFB protein. The encoded CFB protein has the activity of at least one human CFB protein or a non-human animal CFB protein.

[0101] In some examples, the chimeric(x) protein or chimeric(x) polypeptide is a humanized CFB protein or humanized CFB polypeptide. In some examples, at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a human CFB protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human animal CFB protein. The humanized CFB protein or humanized CFB polypeptide is functional or has the activity of at least one human CFB protein or non-human animal CFB protein.

[0102] Genetically modified non-human animals may be any animal species, such as mice, rats, zebrafish, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, or primates (e.g., marmosets, rhesus monkeys). Non-human animals for which suitable genetically modifiable embryonic stem cells (ES) are difficult to obtain may be constructed by other methods that incorporate genetic modification. Such methods may include, for example, modifying a non-ES cell genome (e.g., fibroblasts or induced pluripotent stem cells), transferring the modified genome to a suitable cell, such as an oocyte, using nuclear transfer, and culturing the modified cells (e.g., modified oocytes) in a non-human animal under appropriate conditions to form an embryo. The above construction method is known in this field and is described in "A. Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)," Cold Spring Harbor Laboratory Press, 2006," and all of its contents are incorporated herein by reference.

[0103] In one embodiment, the non-human animal is a non-human mammal. In some examples, the genetically modified non-human animal is a rodent. The rodent may be selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the mouse family. In one embodiment, the genetically modified non-human animal is derived from families selected from the family Stercidae (e.g., mouse-like hamster), Stercidae (e.g., hamster, New World rat and New World mouse, field vole), Muroidea (true mouse and true rat, gerbil, spiny rat, crested rat), Muridae (tree mouse, rock mouse, withtailed rat, Madagascar rat and Madagascar mouse), Glididae (e.g., spiny dormouse), and Muridae (e.g., mor rat, taklerat, and zokol). In one particular embodiment, the genetically modified rodent is selected from true mouse or true rat (superfamily Muroidea), gerbil, spiny rat, and crested rat. In one embodiment, the genetically modified mouse is derived from a member of the Muridae family. In one embodiment, the non-human animal is a rodent. In one particular embodiment, the rodent is selected from mouse and rat. In one embodiment, the non-human animal is a mouse.

[0104] In some embodiments, the non-human animal may be an immunodeficient non-human mammal. For example, an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, or an immunodeficient monkey.

[0105] In some examples, the non-human animal is a mouse of the C57BL strain, which is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / min, C57BL6J, C57B1 / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL10SnSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mice are 129 strains selected from 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); and Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), and the relevant content of the above literature is incorporated into this application by reference. In some examples, the genetically modified mice are hybridizations of the 129 strain and the C57BL / 6 strain. In some examples, the mice are hybridizations of the 129 strain or the BL / 6 strain. In some examples, the mice are from the BALB strain, e.g., the BALB / c strain. In some examples, the mice are hybridizations of the BALB strain and another strain. In some examples, the mice were derived from hybridization strains (e.g., 50%BALB / c-50%12954 / Sv, or 50%C57BL / 6-50%129). In some examples, the non-human animals were rodents.In some examples, the non-human animals are mice having BALB / c, a, a / He, a / J, a / WySN, AKR, AKR / a, AKR / J, AKR / N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA / 2, KM, NIH, ICR, CFW, FACA, C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57L / 6J, C57BL / 6ByJ, and C5C57BL / 6NJ. Mice of the C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, or CBA / H strains, as well as NOD, NOD / SCID, and NOD-Prkdc. scid IL-2rg null It's a mouse in the background.

[0106] The genetically modified non-human animals include modifications to the endogenous non-human animal CFB gene site (or locus). In some examples, the modifications include nucleotide sequences encoding at least some of the mature CFB protein (for example, having 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 CFB protein). The present invention provides cells (e.g., ES cells, somatic cells) that can include the genetic modifications described herein, but in many examples, the genetically modified non-human animals include modifications to the endogenous CFB gene site in the non-human animal.

[0107] Genetically modified non-human animals can express human CFB and / or chimeric (e.g., humanized) CFB at non-human animal (e.g., mouse) endogenous loci, wherein the non-human animal (e.g., mouse) endogenous CFB gene is replaced or inserted with the human CFB gene and / or a nucleotide sequence encoding human CFB, or a nucleotide sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% homology to the human CFB sequence. In various examples, the non-human animal endogenous CFB locus is modified by incorporating all or part of a nucleic acid sequence encoding a mature human CFB protein.

[0108] In several examples, genetically modified non-human animals (e.g., mice) can express human CFB and / or chimeric CFB (e.g., humanized CFB) under the control of non-human endogenous regulators or human-derived regulators (including 5'UTR, 3'UTR, enhancers, or promoters). By making insertions or substitutions at non-human animal (e.g., mouse) endogenous loci, non-human animals are provided that express human CFB or chimeric CFB (e.g., humanized CFB) in appropriate cells and do not cause the potential pathologies observed in some other transgenic mice known in this art. The human CFB or chimeric CFB (e.g., humanized CFB) expressed in non-human animals can maintain the function of one or more species of wild-type mouse CFB or human CFB in the non-human animal. In some examples, the non-human animals do not express endogenous CFB. In some examples, the expression level of non-human endogenous CFB is reduced compared to the CFB expression level in wild-type animals. For example, the term "endogenous CFB" as used in this invention refers to the CFB protein expressed by the endogenous CFB nucleotide sequence of a non-human animal (e.g., mouse) before any genetic recombination.

[0109] The genome of a genetically modified non-human animal contains nucleotide sequences encoding at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homology to the human CFB (NP_001701.2, SEQ ID NO: 2) amino acid sequence. In some examples, the genome includes nucleotide sequences that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homology to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 39, or SEQ ID NO: 40. In some examples, the genome includes nucleotide sequences that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homology to positions 1-2476 or 128-2422 of NM_001710.6.

[0110] In some embodiments, the genome of a non-human animal includes a portion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18 of the human CFB gene, or all of exons 1 to 18 of the human CFB gene, or at least 50 bp of consecutive nucleotides in the 5'UTR and upstream outside of it of the human CFB gene, the nucleotide sequence from the start codon to the stop codon of the human CFB gene and at least 50 bp of consecutive nucleotides in the 3'UTR and downstream outside of it of the human CFB gene, or a nucleotide sequence encoding all or part of the amino acid sequence of the human CFB gene, or a nucleotide sequence encoding SEQ ID NO: 2 or positions 26 to 764 thereof.

[0111] In some examples, the genome of a genetically modified non-human animal includes a portion of exon 1 of the human CFB gene, all of exons 2 to 17, and a portion of exon 18, preferably further including intron 1 and / or intron 17. In some examples, a portion of exon 1 includes a continuous nucleotide sequence of at least 5 bp to 191 bp, for example, nucleotides of 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 70 bp, 75 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, or 191 bp. In some examples, a portion of exon 1 includes a nucleotide of 64 bp. In some examples, a portion of exon 1 contains at least 30 bp of nucleotides. In some examples, a portion of exon 18 contains a continuous nucleotide sequence of at least 20 bp to 210 bp, for example, nucleotides of 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 150 bp, 151 bp, 152 bp, 153 bp, 154 bp, 155 bp, 156 bp, 157 bp, 158 bp, 159 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, or 210 bp. In some examples, a portion of exon 18 contains 156 bp of nucleotides. In some examples, a portion of exon 18 contains at least 50 bp of nucleotides.

[0112] In some examples, the genome of a genetically modified non-human animal includes a portion of exon 1 and exon 18 of the endogenous CFB gene (e.g., mouse CFB). In some examples, the portion of exon 1 contains at least 1, 2, 3, 4, 5, 6, 10, 20, 50, 100, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 170, 180, 190, 200, 300, 400, 401, 402, 403, 404, 405, 410, 450, 460, or 463 nucleotides. In some examples, the portion of exon 1 contains 452 bp of nucleotides. In some examples, a portion of exon 1 contains nucleotides of at least 100 bp, 150 bp, or 200 bp.

[0113] In some examples, the genome of a genetically modified non-human animal includes a portion of exon 1 and a portion of exon 18 of the endogenous CFB gene (e.g., mouse CFB). In some examples, the portion of exon 1 includes at least 1, 2, 3, 4, 5, 6, 10, 20, 50, 100, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 170, 180, 190, 200, 300, 400, 401, 402, 403, 404, 405, 410, 450, 460, or 463 nucleotides. In some examples, the portion of exon 1 includes 402 bp or 155 bp of nucleotides. In some examples, a portion of exon 1 contains at least 100 bp, 150 bp, or 200 bp of nucleotides. In some examples, a portion of exon 18 contains at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 45, 50, 51, 52, 53, 54, 55, 60, 70, 80, 90, 100, 150, 200, 205, 208, or 209 nucleotides. In some examples, a portion of exon 18 contains 209 bp or 53 bp of nucleotides. In some examples, a portion of exon 18 contains at least 20 bp or 150 bp of nucleotides.

[0114] In some examples, non-human animals have a nucleotide sequence encoding a chimeric human CFB polypeptide / non-human CFB polypeptide at the endogenous CFB gene locus, and functional CFB is expressed on the cell surface of the non-human animals. The human portion of the chimeric human polypeptide / non-human CFB polypeptide may include an amino acid sequence encoded by part of exon 1, exons 2 to 17 and / or part of exon 18 of the human CFB gene, or an amino acid sequence encoded by all of exons 1 to 18 of the human CFB gene. In some examples, the human portion of the chimeric human polypeptide / non-human CFB polypeptide includes a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 2 or positions 26 to 764 thereof.

[0115] Furthermore, the modified gene in the modified non-human animal genome is homozygous or heterozygous with respect to the endogenously substituted locus. In one specific example, the modified CFB gene in the genome is homozygous or heterozygous with respect to the endogenously substituted locus.

[0116] In some embodiments, the humanized CFB locus encompasses the human 5'UTR. In some embodiments, the humanized CFB locus encompasses the non-human endogenous (e.g., mouse) 5'UTR. In some embodiments, the humanized CFB locus encompasses the human 3'UTR. In some embodiments, the humanized CFB locus encompasses the non-human endogenous (e.g., mouse) 3'UTR. Where appropriate, based on the similarity between the mouse CFB gene sequence and the human CFB gene sequence, it can be reasonably assumed that they appear to be similarly regulated. As shown in the application, humanized CFB mice that incorporate insertions or substitutions into the non-human endogenous CFB locus retain the mouse regulatory elements, but humanization that incorporates the CFB coding sequence does not exhibit pathological phenomena. Both heterozygous and homozygous humanized CFB recombinant mice are normal.

[0117] In another aspect, the present invention further provides a genetically modified non-human animal whose genome includes a disruption of the non-human animal endogenous CFB gene, of which the disruption of the endogenous CFB gene includes deletions of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18, or deletions of a portion thereof.

[0118] In some embodiments, disruption of the non-human endogenous CFB gene further includes the deletion of one or more introns selected from intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, and intron 17.

[0119] In some embodiments, the above-mentioned deletions are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, and 30. This may include the deletion of 0, 400, 450, 460, 465, 466, 467, 468, 469, 470, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 5500, 5600, 5686 or more nucleotides.

[0120] In several examples, disruption of the endogenous CFB gene resulted in the disruption of at least one, two, three, four, or five exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and / or 18. Includes deletions of 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides.

[0121] The present invention further relates to a CFB genomic DNA sequence of a humanized mouse in which the DNA sequence obtained by reverse transcription of mRNA matches or is complementary to the said DNA sequence, a construct that expresses the amino acid sequence thereof, cells that contain the construct, and tissues containing the cells.

[0122] The present invention further relates to non-human mammals produced by the above method. In some embodiments, the genome contains human genes.

[0123] In some embodiments, the non-human mammal is a rodent, preferably a mouse.

[0124] In several examples, non-human mammals express the protein encoded by the humanized CFB gene.

[0125] Furthermore, the present invention provides a non-human mammalian model having tumors, which can be obtained by the method described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse).

[0126] The present invention further provides cells or cell lines derived from non-human mammals or their offspring, or non-human mammals with tumors, or primary cell cultures derived from non-human mammals or their offspring, or non-human mammals with tumors, or tissues, organs or cultures thereof derived from non-human mammals or their offspring. If it has a tumor, it is derived from tumor tissue of a non-human mammal or its offspring or from a non-human mammal with a tumor.

[0127] The present invention provides a non-human mammal produced by any one of the methods described herein. In some examples, a non-human mammal, a genetically modified non-human animal, is provided, wherein the genome of the genetically modified non-human animal comprises human CFB or humanized CFB DNA.

[0128] In some embodiments, the non-human mammal includes the gene construct described in this application (for example, the gene constructs shown in Figures 2 to 5 and Figures 17 to 19). In some embodiments, a non-human mammal expressing human CFB protein or humanized CFB protein is provided. In some embodiments, a tissue specifically expressing human CFB protein or humanized CFB protein is provided.

[0129] In some examples, the expression of human CFB protein or humanized CFB protein in non-human animals is controllable. This can be done, for example, by adding specific inducers or inhibitors. In some examples, the specific inducers include, but are not limited to, tetracycline systems (Tet-Off System / Tet-On System) or tamoxifen systems.

[0130] The non-human mammal may be any non-human animal known in this art and can be used in the method of this application. A preferred non-human mammal is a mammal (e.g., a rodent). In some embodiments, the non-human mammal is a mouse.

[0131] The above-mentioned non-human mammals will undergo genetic, molecular, and behavioral analysis. The present invention provides offspring produced by mating with non-human mammals of the same genotype or different genotypes.

[0132] This invention provides cell lines or primary cell cultures derived from non-human mammals or their offspring. Cell culture-based models can be produced, for example, by the following methods. Cell cultures can be obtained by isolation from non-human mammals, or cells can be obtained from cell cultures established using the same construct and standard cell transfection techniques. The incorporation of gene structures containing DNA sequences encoding human CFB proteins can be detected by several methods.

[0133] Many analytical methods are available to detect exogenous DNA, including nucleic acid-level methods (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein-level methods (including histochemical analysis, Western blot analysis, and in vitro binding studies). Furthermore, the expression level of target genes can be quantified by the ELSA method, which is well known to those skilled in the art. Many standard analytical methods can be used to complete quantitative detection. For example, transcription levels can be detected using RT-PCR and hybridization methods, including RNA enzyme protection analysis, Southern Blot, and RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blot can also be used to detect the presence of human CFB protein or humanized CFB protein.

[0134] In some embodiments, the genetically modified non-human animals described in this application (e.g., homozygous mice with the humanized CFB gene) can express human CFB or humanized CFB in one or more liver tissue cells.

[0135] vector The present invention provides a target vector comprising: a) a DNA fragment selected from the genomic DNA of the CFB gene, homologous to the 5' end of the region to be changed (5' arm), which has a length of 100 to 10,000 nucleotides; b) a donor DNA sequence necessary to encode the donor region; and c) a second DNA fragment selected from the genomic DNA of the CFB gene, homologous to the 3' end of the region to be changed (3' arm), which has a length of 100 to 10,000 nucleotides.

[0136] In several examples, 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 registry number NC_000083.7, and c) the DNA fragment homologous to the 3' end of the region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI registry number NC_000083.7.

[0137] In several examples, a) a DNA fragment homologous to the 5' end of the region to be altered is selected from the nucleotide sequence from positions 35081089 to 35084696 of NCBI registry number NC_000083.7, and c) a DNA fragment homologous to the 3' end of the region to be altered is selected from the nucleotide sequence from positions 35070668 to 35074536 of NCBI registry number NC_000083.7.

[0138] In several examples, a) a DNA fragment homologous to the 5' end of the region to be altered is selected from the nucleotide sequence from positions 35081089 to 35082539 of NCBI registry number NC_000083.7, and c) a DNA fragment homologous to the 3' end of the region to be altered is selected from the nucleotide sequence from positions 35073826 to 35075402 of NCBI registry number NC_000083.7.

[0139] In several examples, a) a DNA fragment homologous to the 5' end of the conversion region to be changed is selected from the nucleotide sequence from positions 35081039 to 35084904 of NCBI registry number NC_000083.7, and c) a DNA fragment homologous to the 3' end of the conversion region to be changed is selected from the nucleotide sequence from positions 35071591 to 35075667 of NCBI registry number NC_000083.7.

[0140] In several examples, a) the DNA fragment homologous to the 5' end of the transformation region to be changed is selected from the nucleotide sequence from positions 35081039 to 35082302 of NCBI registry number NC_000083.7, and c) the DNA fragment homologous to the 3' end of the transformation region to be changed is selected from the nucleotide sequence from positions 35074350 to 35075667 of NCBI registry number NC_000083.7.

[0141] In some examples, the length of the genomic nucleotide sequence selected for the target vector may exceed approximately 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 5.5kb, 5.8kb, or 6kb.

[0142] In some embodiments, the region to be modified is located at the non-human endogenous CFB gene locus. For example, it is located at exons 1 to 18 of the non-human CFB gene. Alternatively, for example, it is located at exons 1 to 17 of the non-human CFB gene.

[0143] In some examples, the target vector further includes one or more marker genes (or resistance genes), for example, a positive screening marker gene or a negative screening marker gene. In some examples, the resistance gene for positive clonal screening is the neomycin phosphotransferase coding sequence Neo. Preferably, the target vector further includes two Frt recombination sites arranged in the same direction on both sides of the marker gene. In some examples, the coding gene for the negative screening marker is the coding gene for diphtheria toxin A subunit (DTA).

[0144] In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 3, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 5, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 6.

[0145] In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 37, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 38. In some embodiments, the 5' arm sequence is the nucleotide sequence shown in SEQ ID NO: 45, and the 3' arm sequence is the nucleotide sequence shown in SEQ ID NO: 46.

[0146] In some embodiments, the 5' arm is a nucleotide having at least 90% homology to NCBI registration number NC_000083.7, and more preferably, the 5' arm sequence includes the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5. More preferably, the 5' arm sequence includes the nucleotide sequence shown in SEQ ID NO: 37 or SEQ ID NO: 45. In some embodiments, the 3' arm is a nucleotide having at least 90% homology to NCBI registration number NC_000083.7, and more preferably, the 3' arm sequence includes the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6. More preferably, the 3' arm sequence includes the nucleotide sequence shown in SEQ ID NO: 38 or SEQ ID NO: 46.

[0147] In some examples, the target vector contains a human sequence (e.g., positions 31946222 to 31952030 of NC_000006.12). For example, the donor region in the target vector contains the nucleotide sequence of part or all of the human CFB gene, preferably exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and / or 18 of the human CFB gene. In some examples, the nucleotide sequence of the humanized CFB gene codes for all or part of the human CFB protein, and the NCBI protein number is NP_001701.2 (SEQ ID NO: 2).

[0148] In some examples, the target vector includes, in order from the 5' end to the 3' end, a portion of exon 1 of the mouse CFB gene, a portion of exon 1 to a portion of exon 18 of the human CFB gene, and a portion of exon 18 of the mouse CFB gene.

[0149] In some examples, the target vector contains, in order from the 5' end to the 3' end, a portion of exon 1 of the mouse CFB gene, exons 1 to 18 of the human CFB gene, and exon 18 of the mouse CFB gene.

[0150] In some examples, the target vector includes, in order from the 5' end to the 3' end, a portion of exon 1 of the mouse CFB gene, the 5'UTR of the human CFB gene and at least 50 bp of consecutive nucleotides upstream of it, the nucleotide sequence from the start codon to the stop codon of the human CFB gene, the 3'UTR of the human CFB gene and at least 50 bp of consecutive nucleotides downstream of it, and exon 18 of the mouse CFB gene.

[0151] In some embodiments, the donor region sequence in the target vector is such that the linkage sequence between the 5' end and the 5' homologous arm is sequence number 9, and the linkage sequence between the 3' end and the 3' homologous arm is sequence number 10.

[0152] In some embodiments, the donor region sequence in the target vector is such that the linkage sequence between the 5' end and the 5' homologous arm is sequence number 41, and the linkage sequence between the 3' end and the 3' homologous arm is sequence number 42.

[0153] In some examples, the target vector contains the following in order from the 5' end to the 3' end.

[0154] A) Sequence ID 3, Sequence ID 7 and Sequence ID 4, B) Sequence ID 3, Sequence ID 39 and Sequence ID 4, C) Sequence ID 5, Sequence ID 7 and Sequence ID 6, D) Sequence ID 5, Sequence ID 39 and Sequence ID 6, E) Sequence ID 37, Sequence ID 7 and Sequence ID 38, F) Sequence IDs 37, 39 and 38, G) Sequence ID 45, Sequence ID 7 and Sequence ID 46, and H) Sequence IDs 45, 39, and 46.

[0155] In some embodiments, the target vector includes a Neo cassette. The Neo cassette can be inserted into a 5' homologous arm, a 3' homologous arm, or a donor region sequence.

[0156] In some embodiments, the target vector further comprises one or more of SEQ ID NOs: 11, 12, 43, or 44.

[0157] The present invention further provides sgRNA vectors for constructing humanized animal models or knockout models. In some examples, the vector contains an sgRNA sequence, of which the sgRNA sequence targets the CFB gene, and the sgRNA is unique in the target sequence of the gene to be altered, and satisfies the arrangement rule of the sequence 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3', and in some examples, the target site of the sgRNA in the mouse CFB gene is exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5 , located in intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, intron 17, exon 18, for example, in exon 1, intron 17 and / or exon 18 of the mouse CFB gene.

[0158] In some examples, the target sequence is represented by one or more of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22. In some examples, the target sequence is represented by one or more of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, 55, and 56. Accordingly, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some examples, oligonucleotide sgRNA sequences are described in SEQ ID NOs: 15 and 17. In some examples, oligonucleotide sgRNA sequences are described in SEQ ID NOs: 16 and 18. In some examples, oligonucleotide sgRNA sequences are described in SEQ ID NOs: 19 and 21. In some examples, oligonucleotide sgRNA sequences are described in SEQ ID NOs: 20 and 22. In some examples, oligonucleotide sgRNA sequences are described in SEQ ID NOs: 49 and 51. In some examples, the oligonucleotide sgRNA sequences are described in SEQ ID NOs. 50 and 52. In some examples, the oligonucleotide sgRNA sequences are described in SEQ ID NOs. 53 and 55. In some examples, the oligonucleotide sgRNA sequences are described in SEQ ID NOs. 54 and 56.

[0159] In some embodiments, the present invention relates to plasmid constructs containing sgRNA sequences (e.g., pT7-sgRNA) and / or cells containing said constructs.

[0160] The present invention further relates to cells containing the above-mentioned target vector or sgRNA vector.

[0161] Furthermore, the present invention provides non-human mammalian cells having any one of the target vectors described above and one or more in vitro transcripts of the constructs described in this application. In some examples, the cells contain Cas9 mRNA or its in vitro transcript.

[0162] In some examples, the genes in the cells are heterozygous. In some examples, the genes in the cells are homozygous.

[0163] In some examples, the non-human mammalian cells are mouse cells. In some examples, the cells are fertilized egg cells. In some examples, the cells are embryonic stem cells.

[0164] The present invention further relates to the use of the above-mentioned target vector or sgRNA or sgRNA vector in CFB gene recombination.

[0165] Method for constructing genetically modified non-human animals Genetically modified non-human animals can be produced by several gene editing technologies known in the art, including homologous recombination technologies using embryonic stem cells (including gene targeting technologies and CRISPR / Cas9 gene editing technologies), zinc finger nuclease technologies, transcription activator-like effector nuclease technologies, homing endonucleases, or other molecular biological technologies. In some examples, homologous recombination technologies are preferably used. In some examples, CRISPR / Cas9 gene editing technologies can be used to construct genetically modified non-human animals. In some examples, CRISPR-Cas9 gene editing technologies are used to produce genetically modified non-human animals. Many of these gene editing technologies are known in the art and are described in Yin et al., “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6(2017):387-399, which are incorporated herein by reference. The present invention further provides many other methods for gene editing, for example, by microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with other transgenic cells.

[0166] In some embodiments, the nucleotide sequence encoding the endogenous CFB region in the endogenous genome of at least one cell of a non-human animal is replaced with the nucleotide sequence encoding the human CFB region. In some embodiments, the expression level of the non-human animal endogenous CFB protein is reduced or absent compared to the wild type. In some embodiments, the substitution occurs in cells such as germ cells, somatic cells, blastocyst cells, or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0167] Figures 3, 5, 18, and 19 illustrate humanization targeting strategies for the mouse CFB locus. Target vectors include vectors consisting of a 5' homologous arm, a human CFB gene fragment or a humanized CFB gene fragment, and a 3' homologous arm. The process involves replacing the endogenous CFB sequence with a human or humanized CFB sequence using homologous recombination. In some examples, upstream and downstream cleavage of the target site (e.g., by zinc finger nuclease, TALEN, or CRISPR) can induce DNA double-strand breaks, and a chimeric CFB gene (e.g., a humanized CFB gene) is formed by replacing the mouse endogenous CFB sequence with a human or humanized CFB sequence using homologous recombination.

[0168] Accordingly, in some examples, the method for producing a genetically modified humanized animal includes substituting the nucleic acid sequence encoding the endogenous CFB region at the endogenous CFB locus (or site) with the nucleotide sequence encoding human CFB. The substituted sequence may include the regions (e.g., part or all of the regions) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17 and / or exon 18 of the human CFB gene. In some examples, the sequence includes a portion of exon 1, exons 2 to 17 and exon 18 of the human CFB gene (e.g., the nucleotide sequence from position 1 to 2476 or position 128 to 2422 of NM_001710.6). In some examples, the sequence includes exons 1 to 18 of the human CFB gene (e.g., positions 1 to 2476 of NM_001710.6). In some examples, the sequence includes part of exon 1 and all or part of exon 18 of the endogenous CFB gene (e.g., nucleotide sequences from positions 1 to 402 and 2695 to 2747 of NM_008198.2 or nucleotide sequences from positions 1 to 452 and 2539 to 2747 of NM_008198.2 or nucleotide sequences from positions 1 to 155 and 2241 to 2449 of NM_008198.3).

[0169] The present invention further provides a method for establishing a humanized animal model of the CFB gene, comprising the following steps.

[0170] (a) To provide cells (e.g., fertilized egg cells) based on the method described herein, (b) Culturing the above cells (preferably in a liquid medium), (c) transplanting cultured cells into the fallopian tube or uterus of a female non-human mammal that serves as a receptor, and growing the cells in the uterus of the female non-human mammal, and (d) Identify germline transmission in the offspring of a pregnant female genetically modified humanized non-human mammal in step (c).

[0171] In some embodiments, the non-human mammal in the above method is a mouse (e.g., C57BL / 6 mouse).

[0172] In some embodiments, the non-human mammal in step (c) is a female having a pseudopregnancy (or pregnancy).

[0173] In some embodiments, the embryo used in the above method is a C57BL / 6 embryo. Other embryos that can be further used in the method of the present invention include, but are not limited to, FVB / N embryos, BALB / c embryos, DBA / 1 embryos, and DBA / 2 embryos.

[0174] The fertilized egg may be derived from any non-human animal, for example, any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection. For example, the non-human mammal mentioned in the above method can be produced by culturing the fertilized egg after microinjection, transferring the cultured fertilized egg to a pseudopregnant non-human animal, and then producing a non-human mammal from the pseudopregnant non-human animal.

[0175] In some embodiments, a method for producing a genetically modified non-human animal involves modifying the coding framework of the non-human animal's CFB gene, for example, by substituting a nucleic acid sequence (e.g., genomic DNA, CDS, or cDNA sequence) encoding the endogenous CFB protein with a nucleotide sequence encoding human CFB under the control of an endogenous regulatory element of the non-human animal's CFB gene. For example, one or more functional region sequences of the non-human animal's CFB gene may be knocked out or inserted into the sequence so that the non-human animal's endogenous CFB protein is not expressed or its expression level is reduced. In some embodiments, the coding framework of the modified non-human animal's CFB gene may be all or part of the nucleotide sequence of exons 1 to exons 18 of the non-human animal's CFB gene.

[0176] In some embodiments, a method for producing a genetically modified non-human animal comprises inserting a nucleotide sequence encoding a human CFB protein or a humanized CFB protein after an endogenous regulatory element of the non-human animal's CFB gene, and may further include an auxiliary sequence. In some embodiments, the auxiliary sequence may be a stop codon so that the CFB gene humanized animal model can express a human CFB protein or a humanized CFB protein in vivo, but not the non-human animal's CFB protein. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcription reaction element), loxP, STOP, and / or polyA.

[0177] In some examples, a method for producing genetically modified non-human animals includes using the above-mentioned vectors, for example, target vectors and / or sgRNA vectors.

[0178] In some examples, the method for producing transgenic non-human animals includes the following:

[0179] (1) To provide a plasmid containing a human CFB gene fragment, wherein the lateral wings of the plasmid are a 5' homologous arm and a 3' homologous arm, and of these, the 5' homologous arm and the 3' homologous arm target endogenous CFB. (2) To provide one or more guide RNAs (sgRNAs) that target the endogenous CFB gene. (3) Modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid from step (1), the sgRNA from step (2), and Cas9, (4) To produce offspring non-human animals (e.g., mice) that functionally express humanized CFB protein by transplanting the fertilized eggs obtained in step (3) into the fallopian tubes of a pseudopregnant female non-human animal (e.g., mouse), or by transplanting the embryonic stem cells obtained in step (3) into blastocysts and then transplanting the blastocysts into the fallopian tubes of a pseudopregnant female non-human animal (e.g., mouse).

[0180] Preferably, the process further includes step (5) of mating the offspring non-human animals (e.g., mice) obtained in step (4) to obtain homozygous non-human animals (e.g., mice).

[0181] In several examples, fertilized eggs are modified by CRISPR with sgRNAs targeting the 5'-terminal and 3'-terminal target sites.

[0182] In some embodiments, the sequence encoding the humanized CFB protein is operably linked to an endogenous regulatory element or a human-derived regulatory element at the endogenous CFB locus.

[0183] In some examples, genetically modified non-human animals do not express the endogenous CFB protein.

[0184] In some examples, the method for producing transgenic non-human animals includes the following:

[0185] (1) To provide a plasmid containing a human CFB gene fragment or a chimeric CFB gene fragment, wherein the lateral wings of the plasmid are a 5' homologous arm and a 3' homologous arm, and of these, the 5' homologous arm and the 3' homologous arm target endogenous CFB. (2) To provide one or more guide RNAs (sgRNAs) that target the endogenous CFB gene, and (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the above-mentioned human CFB gene fragment or chimeric CFB gene fragment into the genome.

[0186] Use of genetically modified non-human animals By substituting non-human animal genes with homologous human genes or sequences, or direct-derived human genes or sequences, at endogenous non-human animal loci and under the control of endogenous or human-derived regulators (e.g., promoters), or by inserting homologous human genes or sequences, or direct-derived human genes or sequences, into non-human animals, it is possible to produce non-human animals with qualities and characteristics that can differ significantly from typical knockout transgenic animals. In typical knockout transgenic animals, the endogenous locus is removed or disrupted, and a fully human transgenic is inserted into the animal's genome and may be randomly integrated into the genome. Typically, the site of transgenic integration is unknown, and human protein expression is measured by transcription of human genes and / or protein assays and / or functional assays. In human transgenic animals, the upstream and / or downstream of the human sequence provide appropriate support for transgenic expression and / or regulation.

[0187] Genetically modified non-human animals expressing human CFB protein or humanized CFB protein offer multiple uses, for example, in physiologically appropriate ways, including, but not limited to, the development of treatments for human diseases and conditions, and the evaluation of the toxicity and / or efficacy of these human treatments in animal models.

[0188] The present invention further provides the use of CFB genetically modified non-human animals or their offspring, or non-human animals or their offspring obtained by any one of the above-described construction methods.

[0189] In some embodiments, the above use includes the following:

[0190] A) Use in product development related to CFB-associated immune processes in human cells, B) Use as a CFB-related model system for pharmacological, immunological, microbiological, and medical research. C) Use for the production and utilization of animal disease models for etiological research related to CFB, and / or for the development of diagnostic strategies, and / or for the development of therapeutic strategies. D) Use in in vivo studies for screening, detection of drug efficacy, evaluation, verification, or evaluation of human CFB signaling pathway modulators, or E) Use in research on CFB gene function, research on drugs and their efficacy against human CFB target sites, and research on drugs for CFB-related inflammation and immune-related diseases.

[0191] The present invention provides non-human animals expressing human CFB protein or humanized CFB protein, which can be used for screening therapeutic agents (e.g., anti-human CFB modulators, oligonucleotide drugs and / or polypeptide drugs). In some embodiments, the non-human animals are human disease animal models. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animals further encompass damaged immune systems and are genetically modified human-derived tissue xenografts, including, for example, human solid tumors (e.g., breast cancer) or hematopoietic tumors (e.g., lymphocyte tumors, B-cell tumors or T-cell tumors).

[0192] In some examples, genetically modified non-human animals can be used to determine the efficacy of therapeutic agents in the treatment of various diseases. In some examples, the diseases are related to CFB expression or abnormal expression (e.g., overexpression). In some examples, the diseases are those in which targeting CFB or downregulating CFB expression is beneficial for treatment.

[0193] In some examples, genetically modified non-human animals can be used to determine the efficacy of therapeutic agents (e.g., anti-human CFB regulators, oligonucleotide drugs and / or polypeptide drugs) in the treatment of various immune disorders. In some examples, the immune disorders include, but are not limited to, age-related macular degeneration (AMD), rheumatoid arthritis, colitis (including ulcerative colitis or Crohn's disease, e.g., perianal Crohn's disease), rheumatism, multiple sclerosis, Parkinson's disease, asthma, myasthenia gravis, or complement disorders.

[0194] In some examples, genetically modified non-human animals can be used to determine the efficacy of therapeutic agents (e.g., anti-human CFB regulators, oligonucleotide drugs and / or polypeptide drugs) in the treatment of various inflammatory infections. In some examples, the inflammation includes both acute and chronic inflammation. Specifically, this includes, but is not limited to, IgA nephropathy, C3 glomerulopathy, glomerulonephritis, degenerative inflammation, exudative inflammation (e.g., serous inflammation, cellulosic inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation or catarrhal inflammation), proliferative inflammation or specific inflammation (such as tuberculosis, syphilis, leprosy or lymphogranuloma).

[0195] In some examples, genetically modified non-human animals can be used to detect whether an anti-CFB antibody or anti-CFB nucleic acid drug is an agonist or an antagonist. In some examples, the method of the present invention can be used to detect the function of a therapeutic agent (e.g., an anti-human CFB modulator, an oligonucleotide drug, and / or a polypeptide drug), for example, whether the therapeutic agent can upregulate or downregulate an immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cytotoxicity (ADCC). In some examples, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent to treat a subject's disease (e.g., an immune disorder).

[0196] The present invention further provides a detection method for determining the toxicity of therapeutic agents (e.g., anti-human CFB regulators, oligonucleotide drugs, and / or polypeptide drugs). The method comprises administering the therapeutic agent to a non-human animal and evaluating the change in body weight, red blood cell count, hematocrit, and / or hemoglobin of the non-human animal. In some examples, the antibody can reduce red blood cells (RBCs), hematocrit, or hemoglobin by 20%, 30%, 40%, or 50% or more. In some examples, the body weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% lower than that of a control group (e.g., the average body weight of a non-human animal not treated with the antibody).

[0197] The present invention further provides for the use of animal models constructed by the method described herein in the development of products related to human cellular immune processes, the production of human antibodies, or in model systems for pharmacological, immunological, microbiological, and medical research.

[0198] In several embodiments, the animal models produced by the method of this application provide for the production and utilization of animal disease models of human cellular immune processes, for pathogen research, or for the development of novel diagnostic and / or therapeutic strategies.

[0199] The present invention also further provides for screening, validating, evaluating, or studying CFB gene function, human CFB antibodies, drugs or efficacy of human CFB target sites, drugs for immune-related diseases, and antitumor drugs using animal models produced by the method described herein.

[0200] Non-human animal models of two or more human (x) genes or chimeric (x) genes The present invention further provides a method for producing a transgenic animal model having two or more human(x) genes or chimeric(x) genes. The animal model may include a human CFB gene or a chimeric CFB gene and a sequence encoding an additional human(x) protein or chimeric(x) protein.

[0201] In some examples, the additional human(x) protein or chimeric(x) protein includes at least one of CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4. In some examples, the animal model further expresses at least one of human or humanized CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4 proteins.

[0202] The present invention further provides a method for constructing two or more human (x) genes or chimeric (x) genes in non-human animals, the method of construction of which includes the following:

[0203] (i) Provide the above-mentioned non-human animals, or obtain non-human animals or animal models by the construction method.

[0204] (ii) The non-human animals provided in step (i) are crossed with other genetically modified non-human animals, fertilized in vitro, or directly subjected to gene editing and screening to obtain multiple genetically modified non-human animals.

[0205] In some examples, the other genetically modified non-human animals include humanized non-human animals that combine one or more of the genes CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, or CTLA4.

[0206] In some examples, CFB humanization is carried out directly in at least one non-human animal having human or chimeric CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4 gene recombinants.

[0207] Each of the modified genes in the genomes of non-human animals or animal models of the two or more human genes or chimeric genes described above is homozygous or heterozygous to the endogenously modified (preferably substituted) locus.

[0208] Because these proteins may be involved in different mechanisms, combination therapies targeting two or more of these proteins may be a more effective treatment method. In fact, many relevant clinical trials are underway and showing good efficacy. Multiple recombinant non-human animal models can be used to determine the efficacy of combination therapies targeting two or more proteins, for example, anti-CFB antibodies and additional therapeutic agents for treating cancer or immune diseases (e.g., asthma or specific dermatitis). The above method involves administering anti-CFB antibodies and additional therapeutic agents to non-human animals with tumors or immune diseases and determining the effect of the combination therapy on the immunotumor or immune disease. In some examples, the additional therapeutic agents are antibodies that specifically bind to CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4. In some examples, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., nivolumab), or an anti-PD-L1 antibody. In some examples, the non-human animal further comprises a sequence encoding human PD-1 or humanized PD-1, a sequence encoding human PD-L1 or humanized PD-L1, or a sequence encoding human CTLA-4 or humanized CTLA-4. In some examples, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some examples, the tumor comprises one or more tumor cells expressing PD-L1 and / or PD-L2. [Brief explanation of the drawing]

[0209] The embodiments of the present invention will be described in detail below, along with the drawings, and among them, [Figure 1] This is a schematic diagram comparing the mouse CFB gene locus and the human CFB gene locus (not to scale). [Figure 2] This is a schematic diagram (not to scale) of the humanization modification of the mouse CFB gene locus. [Figure 3]This is a schematic diagram (not to scale) of the targeting strategy for the CFB gene and the design of the target vector V1. [Figure 4] This is a schematic diagram (not to scale) of the FRT recombination process in CFB gene-humanized mice. [Figure 5] This is a schematic diagram (not to scale) of the targeting strategy for the CFB gene and the design of the target vector V2. [Figure 6] These are the PCR identification results for the F1 generation of humanized CFB genes. In A, the primers used are WT-F / WT-R, and in B, the primers used are WT-F / Mut-R. M is the marker, WT is the wild-type control, H2O is the water control, and PC is the positive control. [Figure 7] This is the Southern blot identification result for the F1 generation of humanized CFB mice, with WT being the wild-type control. [Figure 8] These are RT-PCR detection results, where + / + indicates wild-type C57BL / 6 mice, H / H indicates CFB gene humanized homozygous mice, and H2O is the water control. [Figure 9] This is the result of detecting human CFB protein expression in CFB humanized mice using ELISA. Of these, + / + represents wild-type C57BL / 6 mice, and H / H represents CFB gene humanized homozygous mice. [Figure 10] This shows the alignment results between the human CFB amino acid sequence (NP_001701.2, SEQ ID NO: 2) and the mouse CFB amino acid sequence (NP_032224.2, SEQ ID NO: 1). [Figure 11] This shows the alignment results between the human CFB amino acid sequence (NP_001701.2, SEQ ID NO: 2) and the rat CFB amino acid sequence (NP_997631.2, SEQ ID NO: 57). [Figure 12] This is the result of detecting human Ba ​​protein expression in CFB humanized mice using ELISA, and among them, H / H indicates a CFB gene humanized homozygous mouse. [Figure 13]This is HE staining analysis of kidney tissue from CFB gene-humanized mice. [Figure 14A] This study measured the complement II pathway activity of human C3b protein (71.4 μg / mL) and human C3b protein (71.4 μg / mL) + C5 protein (107 μg / mL) using hemolysis experiments, with Nc serving as the solvent control. [Figure 14B] This study measured the complement II pathway activity of human C3b protein (71.4 μg / mL) + C5 protein (107 μg / mL) and human C3 protein (143 μg / mL) + human C5 protein (107 μg / mL) groups using hemolysis experiments, with Nc serving as the solvent control. [Figure 15] This involves measuring complement pathway 2 activity using a pathway 2 reagent kit. [Figure 16] This represents the inhibition rate of small molecule nucleic acid drugs against CFB protein (A) and mRNA (B) expression levels. [Figure 17] This is a schematic diagram (not to scale) of the humanized modification of the mouse CFB gene locus. [Figure 18] This is a schematic diagram (not to scale) of the targeting strategy for the CFB gene and the design of the target vector V3. [Figure 19] This is a schematic diagram (not to scale) of the targeting strategy for the CFB gene and the design of the target vector V4. [Modes for carrying out the invention]

[0210] The present invention will be further described below with specific examples, and the advantages and features of the present invention will become clearer with the description. However, these examples are merely illustrative and do not limit the scope of the present invention. Details and forms of the technical proposals of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, and those skilled in the art will understand that any such modifications and replacements will fall within the scope of the claims of the present invention.

[0211] In each of the following embodiments, the equipment and materials are obtained from several companies listed below.

[0212] C57BL / 6 mice, Flp transgenic mice, were purchased from the National Center for Experimental Animals and Seeds of the Chinese Academy of Food and Drug Certification. The human Factor B ELISA reagent kit was purchased from Abcam, catalog number ab137973.

[0213] Unless otherwise specified, the implementation of this invention involves the use of prior arts in cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are described in detail in the following literature. For example, Molecular Cloning A Laboratory Manual,2ndEd.,ed.By Sambrook,FritschandManiatis(Cold Spring Harbor Laboratory Press:1989);DNA Cloning,Volumes I and II(DNGlovered.,1985);Oligonucleotide Synthesis(MJGaited.,1984);Mullisetal.USPat.No.4,683,195;Nucleic Acid Hybridization(BDHames&S.J.Higginseds.1984);Transcription And Translation(BDHames&S.J.Higginseds.1984);Culture Of Animal Cells(RIFreshney,AlanR.Liss,Inc.,1987);Immobilized Cells And Enzymes(IRL Press,1986);B.Perbal,A Practical Guide To Molecular Cloning(1984);the series,Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wuetal. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and MP Caloseds.,1987,Cold Spring Harbor Laboratory);Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker,eds.,Academic Press,London,1987);Handbook Of Experimental Immunology,Volumes V(DMWeir and CCBlackwell,eds.,1986);and Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY, 1986). .

[0214] Example 1: Method for constructing a humanized mouse with the CFB gene. A schematic comparison between the mouse CFB gene (NCBI Gene ID: 14962, Primary source: MGI: 105975, UniProt ID: B8JJM5, located at positions 35075350 to 5081492 on chromosome 17, NC_000083.7, based on transcript NM_008198.2 and its coding protein NP_032224.2 (SEQ ID NO: 1)) and the human CFB gene (NCBI Gene ID: 629, Primary source: HGNC: 1037, UniProt ID: P00751-1, located at positions 31946095 to 31952084 on chromosome 6, NC_000006.12, based on transcript NM_001710.6 and its coding protein NP_001701.2 (SEQ ID NO: 2)) is shown in Figure 1.

[0215] To achieve the objectives of the present invention, a mouse can express human CFB protein or humanized CFB protein by introducing a nucleotide sequence encoding human CFB protein into the mouse endogenous CFB gene locus. Specifically, using gene editing technology, under the control of mouse CFB gene regulatory elements, a coding region of approximately 5.8 kb encompassing a portion of the sequence from a portion of exon 1 to a portion of exon 18 of the human CFB gene (e.g., from the start codon ATG to the stop codon TAA) is replaced with a portion of the sequence from a portion of exon 1 to a portion of exon 18 of the mouse (e.g., from the start codon ATG to the stop codon TAA) to obtain a humanized CFB gene locus, as shown in the schematic diagram in Figure 2, thereby realizing humanized modification of the mouse CFB gene.

[0216] Based on Figure 2, a targeting strategy was further designed as shown in Figure 3, where the V1 target vector contains the upstream and downstream homologous arm sequences of the mouse CFB gene and a fragment A containing the human CFB fragment. Of these, the upstream 5' homologous arm sequence (SEQ ID NO: 3) is the same as the nucleotide sequence from positions 35081089 to 35084696 of NCBI registration number NC_000083.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 4) is the same as the nucleotide sequence from positions 35070668 to 35074536 of NCBI registration number NC_000083.7. The nucleotide sequence of the human CFB fragment (SEQ ID NO: 7) is the same as the nucleotide sequence from positions 31946222 to 31952030 of NCBI registration number NC_000006.12, and the ligation design of the upstream of the human CFB fragment sequence to the mouse is 5'-AGGGCTTTGGGCCACTGGACTCTCTGGTGC TTTCCATGGG GAGCAATCTCAGCCCCCAACTCTGCCTGATGCCCTTTATCTTGGGC-3' (sequence number 9), and among them, the sequence " TTTCC The last "C" in the sequence is the last nucleotide of the mouse, and the sequence " ATGGGThe "A" in the above is the first nucleotide of the human sequence. The downstream linkage design of the human CFB fragment sequence to the mouse is 5'-TGCTGCCCTGGCTGAAGGAGAAACTCCAAGATGAGGATTTGGGTTTTC TATAAAGAGC TTCCTGCAGGGAGAGTGTGAGGACAGATTAAAGCAGTTACA-3' (Sequence ID 10), and among them, the sequence " TATAA The last "A" in the sequence is the last nucleotide in the human sequence, and the sequence " AGAGC The first "A" in the sequence is the first nucleotide of the mouse sequence.

[0217] The target vector further contains a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites are located on both sides of the resistance gene, arranged in the same direction, to form the Neo cassette. Of these, the ligation design between the 5' end of the Neo cassette and the mouse CFB gene is 5'-GGGGTTGTTCTTAGCTTGGACTCTTCTTCATGGCTCCTTTGCTG CAGACGTCG ACGGTATCGATAAGCTTGATATCGAATTCCGAAGTTC-3' (sequence number 11), and among them, the sequence " CAGAC The last "C" in the sequence is the last nucleotide of the mouse CFB gene, and the sequence is GTCG The first "G" in the sequence is the first nucleotide of the Neocassette, and the ligation design between the 3' end of the Neocassette and the mouse CFB gene is 5'-TATAGGAACTTCATCAGTCAGGTACATAATGGTG GATCCTTAAC TTGGGCCTAGCAAGCCAGGACTGCTGGAGGT-3' (Sequence ID 12), and among them, the sequence " GATCC The last "C" in the sequence is the last nucleotide of the Neo cassette, and the sequence is TTAAC The first "T" in the sequence is the first nucleotide of the mouse CFB gene. The mRNA sequence of the modified humanized mouse CFB is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0218] The target vector can be constructed using standard methods such as enzymatic cleavage and ligation. After preliminary validation of the constructed target vector by enzymatic cleavage, it is sent to a sequencing company for sequencing validation. The target vector, validated by sequencing, is transfected into embryonic stem cells of C57BL / 6 mice by electroporation, and the resulting cells are screened using a positive clone screening marker gene to screen for correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into isolated blastocysts (white mice) according to techniques known in this field, and the resulting chimeric blastocysts are transferred to a culture medium and cultured for a while. After transplantation into the fallopian tubes of receptor parent mice (white mice), F0 generation chimeric mice (black and white) can be produced. The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and further crosses of the F1 generation heterozygous mice with each other can yield F2 generation homozygous mice. Furthermore, by crossing positive mice with Flp tool mice to remove the positive clonal screening marker gene (a schematic diagram of this process is shown in Figure 4), and then crossing them, CFB gene humanized homozygous mice can be obtained.

[0219] Furthermore, gene editing may be performed using CRISPR / Cas9 technology, and a targeting strategy as shown in Figure 5 may be further designed. In the figure, target vector V2 is shown to contain the upstream and downstream homologous arm sequences of the mouse CFB gene and a human CFB fragment. A schematic diagram of the humanized CFB locus after construction is shown in Figure 2. Of these, the upstream 5' homologous arm sequence (SEQ ID NO: 5) is the same as the nucleotide sequence at positions 35081089 to 35082539 of NCBI registration number NC_000083.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 6) is the same as the nucleotide sequence at positions 35073826 to 35075402 of NCBI registration number NC_000083.7. The nucleotide sequence of the human CFB fragment (SEQ ID NO: 7) is the same as the nucleotide sequence at positions 31946222 to 31952030 of NCBI registration number NC_000006.12. The mRNA sequence of the modified humanized CFB mouse is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0220] Target vectors can be constructed using standard methods such as enzymatic cleavage and ligation, or direct synthesis. After preliminary validation of the constructed target vectors by enzymatic cleavage, they are sent to a sequencing company for sequencing validation. Target vectors validated by sequencing are then used in subsequent experiments.

[0221] The target sequence determines the target specificity of the sgRNA and the efficiency of inducing Cas9 cleavage target genes. Therefore, the selection and design of highly efficient and specific target sequences is a prerequisite for constructing sgRNA expression vectors. The target sequences of the CFB gene for example sgRNAs designed and synthesized to recognize target sites are as follows.

[0222] sgRNA1 target site (SEQ ID NO: 13): 5'-AGCCTAAGACCAAGAGGACGAGG-3', sgRNA2 target site (SEQ ID NO: 14): 5'-GTGGAAGTCCCGGGCATAAGAGG-3', After detecting the activity of sgRNA using a UCA reagent kit and confirming that it can mediate highly efficient cleavage, the enzyme cleavage sites were added to its 5' end and complementary strand, respectively, to obtain forward oligonucleotide sequences and reverse oligonucleotide sequences as shown in Table 5. After annealing, the annealed products were ligated to pT7-sgRNA plasmids (the plasmids were first linearized with BbsI) to obtain expression vectors pT7-CFB-1 and pT7-CFB-2.

[0223] [Table 5]

[0224] The pT7-sgRNA vector was synthesized by a plasmid synthesis company using fragment DNA (SEQ ID NO: 23) containing the T7 promoter and sgRNA scaffold. This fragment was then sequentially ligated to a backbone vector (Takara, catalog number 3299) by enzymatic cleavage (EcoRI and BamHI), and then sequenced and validated by a specialized sequencing company, revealing that the target plasmid was obtained. Pronuclear stage fertilized eggs of mice such as C57BL / 6 mice were collected, and the in vitro transcripts of the pT7-CFB-1 plasmid and pT7-CFB-2 plasmid (transcribed using the Ambion in vitro transcription reagent kit according to the instructions), the target vector, and Cas9 mRNA were pre-mixed using a microinjector and then injected into the cytoplasm or nucleus of the mouse fertilized egg. Following the method described in "Manual for Experimental Manipulation of Mouse Embryos (3rd Edition)" (Andras Nagy, Chemical Industry Press, 2006), microinjection of fertilized eggs was performed. After injection, the fertilized eggs were transferred to a culture medium and cultured for a period of time. They were then transplanted into the oviducts of receptor parent mice for development. The resulting mice (F0 generation) were then hybridized and self-pollinated to expand the population and establish a stable CFB gene humanized mouse line.

[0225] The somatic genotype of F1 generation mice can be identified by PCR (primers are shown in Table 6). Figure 6 shows the identification results for example F1 generation mice, and all 14 mice numbered F1-01 to F1-14 are positive.

[0226] [Table 6]

[0227] Southern blot detection was performed on mice identified as PCR-positive in the F1 generation to confirm the presence or absence of random insertions. The mouse tails were excised to extract genomic DNA, which was then digested using either BglII or ScaI enzymes, transcribed into membranes, and hybridized. The 5' probe (5'Probe) and 3' probe (A Probe(3')) were located in the 5' homologous arm and human CFB genome fragment, respectively, and the lengths of the specific probes and target fragments are shown in Table 7. Exemplary results are shown in Figure 7. Combining the PCR and sequencing results, eight mice numbered F1-03, F1-08 to F1-14 were identified as positive. This demonstrates that this method can be used to construct CFB gene humanized mice that can be stably passaged and do not contain random insertions.

[0228] [Table 7]

[0229] 5' probe-F (sequence number 27): 5'-GCTCACTGCTTCCATGACATTCAG-3', 5' probe-R (Sequence ID 28): 5'-GGGTTCAGGCACCTGGCATGGGTGG-3', A probe (3')-F (Sequence No. 29): 5'-CTGGGGAGATGCCAAGTGGTCAGC-3', A probe(3')-R(SEQ ID NO: 30):5'-AGCTGATTACACCAACCTGCAGA-3', RT-PCR can be used to detect the expression status of mRNA in CFB gene humanized mice. Specifically, one 8-week-old male C57BL / 6 mouse (+ / +) and one 8-week-old male CFB gene humanized homozygous mouse (H / H) produced in this example were selected and euthanized by cervical dislocation. Liver tissue was collected, and RT-PCR detection was performed using the primer sequences shown in Table 8. The detection results are shown in Figure 8. As can be seen from the figure, only mouse CFB mRNA was detected in the body of wild-type C57BL / 6 mice, and no human CFB mRNA was detected. In contrast, only human CFB mRNA was detected in the body of CFB gene humanized homozygous mice.

[0230] [Table 8]

[0231] Furthermore, the expression status of human CFB protein in CFB-humanized mice can be detected using conventional methods such as ELISA. Specifically, Five 8-week-old male C57BL / 6 mice (+ / +) and five 8-week-old male CFB gene humanized homozygous mice (H / H) produced in this example were selected, serum was collected, and the detection results using the human Factor B ELISA reagent kit (Abcam, catalog number ab137973, detection range: 2.188 ng / mL to 140 ng / mL) are shown in Figure 9. Together with the above RT-PCR results, this demonstrates that human CFB protein can be successfully expressed in CFB gene humanized homozygous mice.

[0232] Example 2: Functional detection in CFB humanized mice Complement factor B, encoded by the CFB gene, is one of the components of the alternative complement activation pathway. Complement factor B circulates in the blood as a single-chain polypeptide. When the alternative pathway is activated, it can be cleaved by complement factor D to produce the non-catalytic chain Ba (also called Fba) and the catalytic subunit Bb. Furthermore, the expression status of human Ba ​​protein in the CFB humanized mice produced in Example 1 was detected by ELISA. Specifically, CFB gene humanized homozygotes (H / H) were selected, serum was collected, and detection was performed using a human mouse cross-ELISA reagent kit (Ba ELISA reagent kit, QuidelOrtho, catalog number A033). The detection results are shown in Figure 12.

[0233] As can be seen in Figure 12, when detected using the Ba ELISA reagent kit, Ba protein is detected in all CFB humanized homozygous mice. This indicates that human CFB protein in CFB gene humanized homozygous mice can be cleaved by CFD to produce Ba fragments, and that the CFB signaling pathway in these mice is normal.

[0234] In another experiment, six 7-week-old male wild-type C57BL / 6 mice (+ / +) and six CFB humanized homozygous mice (H / H) were selected, and peripheral blood was collected for blood biochemical detection. The indicators for blood biochemical detection included urea (UREA), serum creatinine (CREA), and total protein (TP), and the detection results are shown in Table 9. As a result, it was revealed that humanization modification of the CFB gene did not affect UREA, CREA, and TP levels in the mice, and that the modified mice had the same renal function as wild-type mice.

[0235] [Table 9]

[0236] Furthermore, three 7-week-old male wild-type C57BL / 6 mice (+ / +) and three CFB humanized homozygous mice (H / H) were selected and euthanized by cervical vertebral dislocation. Kidney tissue was then collected, stained with HE, and analyzed. As shown in Figure 13, no kidney damage was observed in either the G1 (C57BL / 6 mouse) group or the G2 (CFB humanized homozygous mouse) group of mice, clearly demonstrating that humanization of the CFB gene does not affect the kidney health of mice.

[0237] Furthermore, the hemolytic activity of the complement activation pathway II (OD415 at the time of hemolysis) in CFB humanized mice can also be measured. Specifically, three male CFB humanized homozygous mice (H / H) are selected, euthanized by cervical vertebral dislocation, and their serum is collected. Rabbit erythrocyte separatory is added and mixed together, and the mice are divided into four groups. Of these, the control group (Nc) is added with an equal volume of H2O or PBS, and the experimental groups are added with either human C3b protein (71.4 μg / mL) or human C3b protein (71.4 μg / mL) + C5 protein (107 μg / mL). The OD415 value is read using a spectrophotometer to measure the mouse serum complement pathway II activity. In another similar experiment, CFB-humanized homozygous mouse serum was collected and divided into three groups. The control group (Nc) was added with an equal volume of H2O or PBS, while the experimental groups were added with either human C3b protein (71.4 μg / mL) + C5 protein (107 μg / mL) or human C3 protein (143 μg / mL) + human C5 protein (107 μg / mL). The OD415 values ​​were read using a spectrophotometer to measure the mouse serum complement pathway II activity.

[0238] As a result, as shown in Figures 14A and 14B, the second pathway can be rescued by adding human C3b+C5 and C3+C5 recombinant proteins compared to the control group.

[0239] The complement activation pathway II activity in CFB humanized mice (homozygous H / H) was detected using the Second Pathway ELISA reagent kit (HyCult Biotech, catalog number HIT422). Specifically, 5-6 week old humanized CFB male mice were taken, euthanized by cervical dislocation, and their serum was collected and divided into 7 groups. C3b, C3, C5, C3b+C5, or C3+C5 recombinant protein were added to the serum, and the complement pathway II activity was detected using the Second Pathway ELISA reagent kit for each different component. The results in Figure 15 show that the addition of both C3b+C5 and C3+C5 can rescue complement pathway II activity.

[0240] Humanized CFB mice (8 weeks old, male, homozygous H / H) were randomly divided into a control group or a treatment group (n=3 / group). On the day of group division (day 0), the treatment group was randomly injected with 1mpk(G2) or 3mpk(G3) siRNA drug (sense is represented by SEQ ID NO: 58 with N-[tris(GalNAc-alkyl)-amidedecanoyl]-4-hydroxyprolinol ligated to the 3' end, and antisense is represented by SEQ ID NO: 59), while the control group (G1) was subcutaneously injected with an equal volume of PBS solvent. Liver tissue and plasma were collected from the mice on day 7 after group division, and the mRNA and protein levels of human CFB were detected, respectively. The detection results showed that the protein concentration of human CFB in the plasma of the treatment groups (G2, G3) was significantly reduced compared to the control group (G1) (Figure 16A), and the mRNA level of human CFB in the liver tissue of the treatment groups (G2, G3) was also significantly reduced (Figure 16B), demonstrating a dose-correlation. This indicates that humanized CFB mice can be used for preclinical drug screening of small molecule nucleic acid drugs.

[0241] Sequence ID 58: Sense (5'-3') Gm-s-Am-s-Am-Um-Um-Cm-Cm-Um-Gf-Af-Af-Um-Um-Um-Um-Am-Um-Gm-Am-Cm-Um Sequence ID 59: Antisense (5'-3') Am-s-dG-s-Um-Cm-dA-Um-dA-Am-Am-Am-Um-dT-Cm-Af-Gm-Gm-Am-Am-Um-Um-Cm-s-Cm-s-Um Of these, m is 2-O-methyl, s is a phosphorothioate bond, f is a 2'-fluoro modification, dG is 2'-deoxyguanosine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, and dT is 2'-deoxythymidine-3'-phosphate.

[0242] Example 3: Method for constructing a CFB gene-humanized mouse (Part 2) The genes used are the mouse CFB gene (NCBI gene ID: 14962, primary source: MGI: 105975, UniProt ID: B8JJM5, located at positions 35075350 to 5081492 on chromosome 17, NC_000083.7, based on transcript NM_008198.3 and its coding protein NP_032224.2 (SEQ ID NO: 1)) and the human CFB gene (NCBI gene ID: 629, primary source: HGNC: 1037, UniProt ID: P00751-1, located at positions 31946095 to 31952084 on chromosome 6, NC_000006.12, based on transcript NM_001710.6 and its coding protein NP_001701.2 (SEQ ID NO: 2)).

[0243] Furthermore, in order to achieve the objectives of the present invention, it is also possible to further replace approximately 5.4 kb from a portion of mouse exon 1 to exon 17 with approximately 13.0 kb from the upstream nucleotide sequence of the 5'UTR to the downstream nucleotide sequence of the 3'UTR that encompasses the human CFB gene, as shown in Figure 17, thereby obtaining a schematic diagram of the humanized CFB gene locus and realizing humanization modification of the mouse CFB gene.

[0244] Based on Figure 17, a targeting strategy as shown in Figure 18 was further designed, and the target vector V3 (see Figure 18) contains the upstream and downstream homologous arm sequences of the mouse CFB gene and the A3 fragment containing the human CFB fragment. Of these, the upstream 5' homologous arm sequence (SEQ ID NO: 37) is the same as the nucleotide sequence at positions 35081039 to 35084904 of NCBI registry number NC_000083.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 38) is the same as the nucleotide sequence at positions 35071591 to 35075667 of NCBI registry number NC_000083.7. The nucleotide sequence of the human CFB gene fragment (SEQ ID NO: 39) is the same as the nucleotide sequence from positions 31940095 to 31953084 of NCBI registration number NC_000006.12. The ligation design between the upstream of the human CFB fragment sequence and the mouse is 5'-TGACAATGGAGAGCCCCCAGCTCTGCCTCGTCCTCTTGGTCTTA GGCTTAATTA TTATTTTTAATCAACAGCTTTAGACAGAGAACCTTGGTTT-3' (Sequence ID 41), and among them, the sequence " GGCTT The last "T" in the sequence is the last nucleotide of the mouse, and the sequence " AATTA The first "A" in the sequence is the first nucleotide of the human sequence. The downstream linkage design of the human CFB fragment sequence to the mouse is 5'-GATCTCTGTGCCTCAACACTGCTGGCTACTCCCTCT TTCTCGAAGT TCCTATTCTCTAGAAAGTATAGGAACTTCAGGTCTGAAGA-3' (Sequence ID 42), and among them, the sequence " TTCTC The last "C" in the sequence is the last nucleotide in the human sequence, and the sequence " GAAGT The first "G" in the sequence is the first nucleotide of the mouse sequence.

[0245] The target vector further contains a resistance gene for positive clone screening, namely, the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites arranged in the same direction are located on both sides of the resistance gene to constitute a Neo cassette. Among them, the ligation design between the 5'-end of the Neo cassette and the human CFB gene is 5'-CCATCCTGTGATCTCTGTGCCTCAACACTGCTGGCTACTCCCTC TTTCTCGAAGT TCCTATTCTCTAGAAAGTATAGGAACTTCA-3'(SEQ ID NO: 43), among which the last "C" in the sequence " TTTCTC " is the last nucleotide of the human CFB gene, and the first "G" in the sequence " GAAGT " is the first nucleotide of the Neo cassette. The ligation design between the 3'-end of the Neo cassette and the mouse CFB gene is 5'-GAAGTTCCTATTCTCTAGAAAGTATAGGA ACTTCCAGAA GACAGGGGGAGCTAGGCTCCCAGTGCACTTCTTTAG-3'(SEQ ID NO: 44), among which the last "C" in the sequence " ACTTC " is the last nucleotide of the Neo cassette, and the "C" in the sequence " CAGAA " is the first nucleotide of the mouse CFB gene. The mRNA sequence of the humanized mouse CFB after modification is shown by SEQ ID NO: 40, and the expressed protein sequence is shown by SEQ ID NO: 2.

[0246] The target vector can be constructed using standard methods such as enzymatic cleavage and ligation. After preliminary validation of the constructed target vector by enzymatic cleavage, it is sent to a sequencing company for sequencing validation. The target vector, validated by sequencing, is transfected into embryonic stem cells of C57BL / 6 mice by electroporation, and the resulting cells are screened using a positive clone screening marker gene to screen for correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into isolated blastocysts (white mice) according to techniques known in this field, and the resulting chimeric blastocysts are transferred to a culture medium and cultured for a while. After transplantation into the fallopian tubes of receptor parent mice (white mice), F0 generation chimeric mice (black and white) can be produced. The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and further crosses of the F1 generation heterozygous mice with each other can yield F2 generation homozygous mice. Furthermore, by crossing positive mice with Flp tool mice to remove the positive clonal screening marker gene, and then crossing them with each other, CFB gene humanized homozygous mice can be obtained.

[0247] In addition, gene editing may be performed by adopting the CRISPR / Cas9 technology, and the target vector V4 may be further designed (see Figure 19). The vector contains homologous arm sequences upstream and downstream of the mouse CFB gene and a human CFB fragment. The schematic diagram of the humanized CFB locus after construction is as shown in Figure 17. Among them, the upstream 5' homologous arm sequence (SEQ ID NO: 45) is the same as the nucleotide sequence at positions 35081039 to 35082302 of NCBI accession number NC_000083.7, and the downstream 3' homologous arm sequence (SEQ ID NO: 46) is the same as the nucleotide sequence at positions 35074350 to 35075667 of NCBI accession number NC_000083.7. The nucleotide sequence of the human CFB fragment (SEQ ID NO: 39) is the same as the nucleotide sequence at positions 31940095 to 31953084 of NCBI accession number NC_000006.12. The mRNA sequence of the humanized CFB mouse after modification is represented by SEQ ID NO: 40, and the expressed protein sequence is represented by SEQ ID NO: 2.

[0248] The construction of the target vector can be carried out by ordinary methods such as enzymatic cleavage ligation and direct synthesis. After the constructed target vector is preliminarily verified by enzymatic cleavage, it is sent to a sequencing company for sequencing verification. The target vector verified to be correct by sequencing is used in subsequent experiments.

[0249] The target sequence determines the target specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, the selection and design of a highly efficient and specific target sequence are the premise for constructing the sgRNA expression vector. Design and synthesize the sgRNA sequence that recognizes the target site. The target sequences of exemplary sgRNAs in the CFB gene are as follows.

[0250] sgRNA3 target site (SEQ ID NO: 47): 5'-TGCAGGTTCGAGTCTGCACGGGG-3', sgRNA4 target site (SEQ ID NO: 48): 5'-TCTTCTACAGGAGATTCCGGGGG-3', After detecting the activity of sgRNA using a UCA reagent kit and confirming that it can mediate highly efficient cleavage, the enzyme cleavage sites were added to its 5' end and complementary strand, respectively, to obtain forward oligonucleotide sequences and reverse oligonucleotide sequences as shown in Table 10. After annealing, the annealed products were ligated to pT7-sgRNA plasmids (the plasmids were first linearized with BbsI) to obtain expression vectors pT7-CFB-1 and pT7-CFB-2.

[0251] [Table 10]

[0252] The pT7-sgRNA vector was synthesized by a plasmid synthesis company using fragment DNA (SEQ ID NO: 23) containing the T7 promoter and sgRNA scaffold. This fragment was then sequentially ligated to a skeletal vector (Takara, catalog number 3299) by enzymatic cleavage (EcoRI and BamHI), and then sequenced and validated by a specialized sequencing company, revealing that the target plasmid was obtained. Pronuclear stage fertilized eggs of mice such as C57BL / 6 mice were collected, and the in vitro transcripts of the pT7-CFB-3 plasmid and pT7-CFB-4 plasmid (transcribed using the Ambion in vitro transcription reagent kit according to the instructions), the target vector, and Cas9 mRNA were pre-mixed using a microinjector and then injected into the cytoplasm or nucleus of the mouse fertilized egg. Following the method described in "Manual for Experimental Manipulation of Mouse Embryos (3rd Edition)" (Andras Nagy, Chemical Industry Press, 2006), microinjection of fertilized eggs was performed. After injection, the fertilized eggs were transferred to a culture medium and cultured for a period of time. They were then transplanted into the oviducts of receptor parent mice for development. The resulting mice (F0 generation) were then hybridized and self-pollinated to expand the population and establish a stable CFB gene humanized mouse line.

[0253] Example 4: Pharmacological Efficacy Model CFB-humanized mice produced by this method can be used to evaluate the efficacy of regulators targeting human CFB. For example, CFB-humanized homozygous mice are taken and randomly divided into a control group and a treatment group. On day 0 after group division, the treatment group is injected with a drug targeting human CFB, and the control group is injected with an equal volume of PBS. Peripheral blood is collected on day 14 after administration, and the amount of human CFB protein expression in the mouse serum is detected. The mice are then euthanized on day 14, liver tissue is collected, and the mRNA expression level in the mouse liver is detected. The body weight of the mice is weighed periodically during the experiment, and the body weight data and the amount of human CFB protein expression in the mice can be used to effectively evaluate the in vivo safety and efficacy of the drug in humanized CFB mice.

[0254] Example 5 Production of a dual-gene humanized mouse or a multiple-gene humanized mouse Multiple humanized mouse models can also be produced using this method or the CFB gene humanized mice produced. For example, in Example 1 above, the embryonic stem cells used for microinjection can be selected from mice containing recombinant genes such as CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4. Alternatively, a dual humanized mouse model or multiple humanized mouse model can be obtained by using humanized CFB mice as a base and utilizing the isolation of mouse ES embryonic stem cells and recombinant targeting technology. Furthermore, CFB mouse homozygotes or CFB mouse heterozygotes obtained by this method can be crossed with other recombinant mice, and their offspring can be screened. Based on Mendelian inheritance laws, multiple genetic mice with humanized CFB gene recombinant and other recombinants can be obtained with a certain probability. By further crossing heterozygotes with each other, dual genetically modified or multiple genetically modified homozygotes can be obtained. Although preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the specific details of the above embodiments, and within the scope of the technical concept of the present invention, several simple modifications can be made to the technical proposal of the present invention, and all of these simple modifications fall within the scope of the claims of the present invention.

[0255] Furthermore, it should be noted that each specific technical feature described in the above-described specific embodiments can be combined in any suitable manner, as long as they do not contradict each other, and in order to avoid unnecessary redundancy, the present invention does not separately describe the various possible combinations. Also, the various different embodiments of the present invention can be combined in any way, and as long as they do not deviate from the spirit of the present invention, they should be considered as being disclosed in the present invention as well.

Claims

1. A genetically modified non-human animal, wherein the genome of the non-human animal comprises at least one chromosome, and the chromosome comprises a nucleotide sequence encoding human complement factor B (CFB) protein or chimeric complement factor B (CFB) protein.

2. The non-human animal according to claim 1, characterized in that the nucleotide sequence encoding the human CFB protein or chimeric CFB protein is regulated by a regulatory element, the regulatory element being an endogenous regulatory element or a human regulatory element.

3. The chimeric CFB protein is a humanized CFB protein, and the humanized CFB protein comprises a portion of a human CFB protein, preferably the portion of the human CFB protein comprises a Ba region and / or a Bb region. The non-human animal according to claim 1, more preferably the Bb region comprises a VWA domain, and even more preferably the Bb region further comprises von Willebrand factor and / or peptidase S1.

4. The non-human animal according to claim 3, characterized in that the humanized CFB protein includes at least 50 to 764 consecutive amino acid sequences that are identical to a portion of the human CFB protein.

5. The non-human animal according to any one of claims 1 to 4, characterized in that the non-human animal is a mammal, for example, a monkey or a rodent (for example, a mouse or a rat).

6. The non-human animal according to any one of claims 1 to 5, characterized in that the human CFB protein or chimeric CFB protein includes SEQ ID NO: 2 or positions 26 to 764 thereof, or includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 or positions 26 to 764 thereof, or includes an amino acid sequence in which the difference from SEQ ID NO: 2 or positions 26 to 764 thereof does not exceed 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids or does not exceed 1 amino acid, or includes a sequence that includes substitutions, deletions, and / or insertions of one or more amino acids relative to what is indicated by SEQ ID NO: 2 or positions 26 to 764 thereof.

7. The non-human animal is characterized in that it does not express endogenous CFB protein, or expresses endogenous CFB at a reduced expression level compared to CFB in wild-type animals, according to any one of claims 1 to 6.

8. A genetically modified non-human animal characterized in that, at the endogenous CFB gene locus, the endogenous CFB gene nucleotide sequence is replaced with a nucleotide sequence that includes the human CFB gene.

9. The expression of the nucleotide sequence of the human CFB is regulated by a regulatory element, the regulatory element being an endogenous regulatory element or a human-derived regulatory element. Preferably, the non-human animal has one or more cells that express human CFB protein or chimeric CFB protein, as described in claim 8.

10. The animal according to claim 8, characterized in that the non-human animal does not express endogenous CFB protein, or expresses endogenous CFB at an expression level reduced compared to the expression level of CFB in wild-type animals.

11. The non-human animal according to any one of claims 8 to 10, characterized in that the nucleotide sequence of the human CFB gene includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, preferably the nucleotide sequence of the human CFB gene includes the nucleotide sequence of the coding region of the human CFB gene, and more preferably the nucleotide sequence of the human CFB gene includes the nucleotide sequence from the start codon to the stop codon of the human CFB gene.

12. The non-human animal according to claim 11, characterized in that the nucleotide sequence of the human CFB gene further includes the 5'UTR and / or 3'UTR of the human CFB gene, and further includes at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the human CFB gene and / or at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human CFB.

13. The non-human animal according to any one of claims 8 to 12, characterized in that the nucleotide sequence of the human CFB gene includes the 5' UTR of human CFB and at least 50 bp of consecutive nucleotides upstream thereof, the nucleotide sequence of the human CFB gene from the start codon to the stop codon, and the 3' UTR of human CFB and at least 50 bp of consecutive nucleotides downstream thereof.

14. The non-human animal according to any one of claims 8 to 13, characterized in that the nucleotide sequence of the human CFB includes the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 39, or includes a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 7 or SEQ ID NO: 39, or includes a nucleotide sequence in which the difference from SEQ ID NO: 7 or SEQ ID NO: 39 does not exceed 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 6 bp, 3 bp, or 1 bp of nucleotides, or includes a nucleotide sequence that includes one or more nucleotide substitutions, deletions, and / or insertions relative to what is shown in SEQ ID NO: 7 or SEQ ID NO:

39.

15. A non-human animal according to any one of claims 8 to 14, characterized in that exons 2 to 17 of the endogenous CFB gene are substituted, preferably the endogenous CFB gene further comprises a portion of the substituted exon 1, all of intron 1 and / or a portion of intron 17, and more preferably the endogenous CFB gene further comprises a portion of the substituted exon 18.

16. The non-human animal according to any one of claims 8 to 15, characterized in that the endogenous CFB gene has a nucleotide sequence from its start codon to its stop codon that is substituted, or the endogenous CFB gene has a nucleotide sequence from a portion of exon 1 to a portion of intron 17 that is substituted, and of which the portion of exon 1 contains 70 or fewer nucleotides at the 3' end of exon 1.

17. The non-human animal according to any one of claims 8 to 16, characterized in that the non-human animal is a mammal, for example, a monkey or a rodent (for example, a mouse or a rat).

18. The animal according to any one of claims 8 to 17, characterized in that the mRNA transcribed by the nucleotide sequence after endogenous CFB modification in the genome of the non-human animal includes SEQ ID NO: 8 or SEQ ID NO: 40, or includes a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8 or SEQ ID NO: 40, or includes a nucleotide sequence whose difference from SEQ ID NO: 8 or SEQ ID NO: 40 does not exceed 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, 9 bp, 6 bp, 3 bp, or 1 bp of nucleotides, or includes a nucleotide sequence that includes one or more nucleotide substitutions, deletions, and / or insertions relative to what is shown by SEQ ID NO: 8 or SEQ ID NO:

40.

19. The non-human animal according to any one of claims 1 to 18, characterized in that the animal is homozygous or heterozygous for the substitution at the endogenous CFB gene locus.

20. The non-human animal is characterized by expressing the human CFB protein or chimeric CFB protein having at least one mouse CFB activity and / or at least one human CFB activity, according to any one of claims 1 to 19.

21. A non-human animal according to any one of claims 1 to 20, further comprising a sequence encoding an additional human protein or chimeric protein encoded by another gene, wherein the additional human protein or chimeric protein is at least one selected from the group consisting of CFD, C3, C5, C5AR1, ANGPTL3, IL36R, IGF1R, TSLP, TLR8, OX40, PD-1, PD-L1, and CTLA4.

22. A non-human animal genome characterized by containing at least one chromosome containing a nucleotide sequence encoding human complement factor B (CFB) protein or chimeric complement factor B (CFB) protein.

23. The non-human animal genome according to claim 22, characterized in that, at the endogenous CFB gene locus, the endogenous CFB gene nucleotide sequence is substituted with a nucleotide sequence that includes the human CFB gene.

24. The non-human animal genome according to claim 23, characterized in that the nucleotide sequence of the human CFB gene includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, preferably the nucleotide sequence of the human CFB gene includes the nucleotide sequence of the coding region of the human CFB gene, and more preferably the nucleotide sequence of the human CFB gene includes the nucleotide sequence from the start codon to the stop codon of the human CFB gene.

25. The animal genome according to claim 24, characterized in that the nucleotide sequence of the human CFB gene further includes the 5'UTR and / or 3'UTR of the human CFB gene, and further includes at least 50 bp of continuous nucleotide sequences upstream of the 5'UTR of the human CFB gene and / or at least 50 bp of continuous nucleotide sequences downstream of the 3'UTR of the human CFB gene.

26. The animal genome according to claim 23, characterized in that the nucleotide sequence of the human CFB gene includes the 5' UTR of the human CFB and at least 50 bp of continuous nucleotide sequences upstream thereof, the nucleotide sequence of the human CFB gene from the start codon to the stop codon, and the 3' UTR of the human CFB and at least 50 bp of continuous nucleotide sequences downstream thereof.

27. The non-human animal genome according to any one of claims 23 to 26, characterized in that exons 2 to 17 of the endogenous CFB gene are substituted, preferably the endogenous CFB gene further comprises a portion of the substituted exon 1, all of intron 1 and / or a portion of intron 17, and more preferably the endogenous CFB gene further comprises a portion of the substituted exon 18.

28. The non-human animal genome according to claim 27, characterized in that the nucleotide sequence from the start codon to the stop codon of the endogenous CFB gene is substituted, or the portion from exon 1 to intron 17 of the endogenous CFB gene is substituted, and of which the portion of exon 1 includes 70 or fewer nucleotides at the 3' end of exon 1.

29. A method for producing a genetically modified non-human animal, characterized in that the nucleotide sequence of the endogenous CFB gene in at least one cell of the non-human animal is replaced with a nucleotide sequence that includes the nucleotide sequence of the human CFB gene.

30. The method according to claim 29, characterized in that the nucleotide sequence of the human CFB gene includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, preferably the nucleotide sequence of the human CFB gene includes the nucleotide sequence of the coding region of the human CFB gene, and more preferably the nucleotide sequence of the human CFB gene includes the nucleotide sequence from the start codon to the stop codon of the human CFB gene.

31. The method according to claim 30, characterized in that the nucleotide sequence of the human CFB gene further includes the 5'UTR and / or 3'UTR of the human CFB gene, and further includes at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the human CFB gene and / or at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human CFB gene.

32. The method according to any one of claims 29 to 31, characterized in that the nucleotide sequence of the human CFB gene includes the 5' UTR of the human CFB and at least 50 bp of consecutive nucleotides upstream thereof, the nucleotide sequence of the human CFB gene from the start codon to the stop codon, and the 3' UTR of the human CFB and at least 50 bp of consecutive nucleotides downstream thereof.

33. A method for producing genetically modified non-human animal cells that express human CFB protein or chimeric CFB protein, the method comprising substituting the nucleotide sequence of the endogenous CFB gene with a nucleotide sequence containing the nucleotide sequence of the human CFB gene at the endogenous CFB gene locus of a non-human animal.

34. The method according to claim 33, characterized in that the nucleotide sequence of the human CFB gene includes a portion of exon 1 to a portion of exon 18 of the human CFB gene, preferably the nucleotide sequence of the human CFB gene includes the nucleotide sequence of the coding region of the human CFB gene, and more preferably the nucleotide sequence of the human CFB gene includes the nucleotide sequence from the start codon to the stop codon of the human CFB gene.

35. The nucleotide sequence of the human CFB gene further includes the 5'UTR and / or the 3'UTR of the human CFB gene, and further includes at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the human CFB gene and / or at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human CFB gene. Preferably, the method according to claim 34, characterized in that the nucleotide sequence of the human CFB includes the 5' UTR of the human CFB and at least 50 bp of consecutive nucleotides upstream thereof, the nucleotide sequence of the human CFB gene from the start codon to the stop codon, and the 3' UTR of the human CFB and at least 50 bp of consecutive nucleotides downstream thereof.

36. The method according to any one of claims 33 to 35, characterized in that the expression of the nucleotide sequence of the human CFB gene is regulated by a regulatory element, the regulatory element being an endogenous regulatory element or a human-derived regulatory element.

37. The nucleotide sequence includes, A) The nucleotide sequence encoding Sequence ID No. 2 or its positions 26 to 764, B) Sequence IDs 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46, C) Nucleotide sequences having at least 90% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46, and D) Nucleotide sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46. A humanized CFB gene characterized by being one of the following.

38. A cell, tissue, or organ characterized by containing the humanized CFB gene described in claim 37.

39. An animal model characterized by comprising the humanized CFB gene described in claim 37 or the cell, tissue, or organ described in claim 38.

40. A target vector comprising a 5' arm, a donor region, and a 3' arm, wherein the 5' arm is homologous to the 5' end of the region to be altered, the 3' arm is homologous to the 3' end of the region to be altered, and the donor region comprises a nucleotide sequence encoding a human CFB protein or a chimeric CFB protein.

41. The target vector according to claim 40, wherein the region to be altered is located at the endogenous CFB gene locus of a non-human animal, and preferably the region to be altered includes at least one exon or at least one intron of the endogenous CFB gene of a non-human animal, for example, from a portion of exon 1 to a portion of intron 17, or from a portion of exon 1 to a portion of exon 18 of the endogenous CFB gene.

42. The target vector according to claim 40, wherein the donor region comprises a portion of exon 1 to a portion of exon 18 of the human CFB gene, preferably the donor region encompasses the nucleotide sequence of the coding region of the human CFB gene, and more preferably the donor region encompasses the nucleotide sequence from the start codon to the stop codon of the human CFB gene.

43. The donor region includes the 5'UTR and / or 3'UTR of the human CFB, and includes at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the human CFB gene and / or at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human CFB. Preferably, the donor region comprises the 5' UTR of the human CFB and at least 50 bp of consecutive nucleotides upstream thereof, the nucleotide sequence of the human CFB gene from the start codon to the stop codon, and the 3' UTR of the human CFB and at least 50 bp of consecutive nucleotides downstream thereof, as described in claim 42.

44. The use of a non-human animal according to any one of claims 1 to 21, a non-human animal genome according to any one of claims 22 to 28, a non-human animal obtained by the method described in any one of claims 29 to 32, a cell obtained by the method described in any one of claims 33 to 36, a humanized CFB gene according to claim 37, a cell, tissue or organ according to claim 38, an animal model according to claim 39, or a target vector according to any one of claims 40 to 43, wherein the use is A) Use in product development related to CFB-associated immune processes in human cells, B) Use as a CFB-related model system for pharmacological, immunological, microbiological, and medical research. C) Use in the production and utilization of animal disease models for etiological research related to CFB, and / or for the development of diagnostic strategies, and / or for the development of therapeutic strategies. D) Use in in vivo studies for screening, detection of drug efficacy, evaluation, verification, or evaluation of human CFB signaling pathway modulators, or E) Use in research on CFB gene function, research on drugs and their efficacy against human CFB target sites, and research on drugs for CFB-related inflammation and immune-related diseases. Includes, use.

45. A method for measuring the effectiveness of a CFB treatment agent for treating a disease, wherein the method is: 1) Administering a CFB therapeutic agent to an animal according to any one of claims 1 to 21, an animal obtained by the production method according to any one of claims 29 to 32, or an animal model according to claim 39, in which the non-human animal is suffering from a disease, and 2) Measuring the effect of the CFB therapeutic agent in the treatment of the disease, Preferably, the method is characterized in that the disease is an immune disease, inflammation, or tumor.

46. The method according to claim 45, characterized in that the immune disease is one or more selected from the group consisting of age-related macular degeneration (AMD), rheumatoid arthritis, colitis (including ulcerative colitis or Crohn's disease, such as perianal Crohn's disease), rheumatism, multiple sclerosis, Parkinson's disease, asthma, myasthenia gravis, and complement disorders.

47. The method according to claim 45, characterized in that the inflammation is one or more selected from the group consisting of IgA nephropathy, C3 glomerulopathy, glomerulonephritis, degenerative inflammation, exudative inflammation (e.g., serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation or catarrhal inflammation), proliferative inflammation and specific inflammation (e.g., tuberculosis, syphilis, leprosy or lymphogranuloma).

48. The method according to claim 45, characterized in that the tumor is a solid tumor (e.g., breast cancer) or a hematopoietic tumor (e.g., a lymphocyte tumor, a B-cell tumor, or a T-cell tumor).

49. A method for measuring the toxicity of CFB therapeutic agents, 1) Administering a CFB therapeutic agent to a non-human animal according to any one of claims 1 to 21, or a non-human animal obtained by the manufacturing method according to any one of claims 29 to 32, or an animal model according to claim 39, and 2) To measure the effect of the CFB therapeutic agent on the aforementioned animals. A method characterized by including

50. The method according to claim 49, wherein measuring the effect of the CFB therapeutic agent on the non-human animal relates to measuring the weight of the non-human animal or performing a blood test, and preferably, the blood test is characterized in that it includes one or more selected from the group consisting of red blood cell count, hematocrit and hemoglobin content.

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