Genetically modified non-human animal

Abstract

Provided are: a genetically modified non-human animal that lacks endogenous FVIII / FIX / FX gene expression and expresses a human FIX / FX gene at a physiologically reasonable level; a method for producing the non-human animal; and a method for evaluating a test substance using the non-human animal.

Description

Genetically modified non-human animals

[0001] The present disclosure relates to genetically modified non-human animals that lack expression of endogenous blood clotting factor VIII (FVIII), blood clotting factor IX (FIX), and blood clotting factor X (FX), and that functionally express human FIX and FX. The disclosure also relates to methods of making and using the genetically modified non-human animals.

[0002] Hemophilia A is a bleeding disorder caused by a congenital deficiency or dysfunction of blood coagulation factor VIII (FVIII). Patients with hemophilia A typically receive FVIII preparations for bleeding (on-demand administration). Recently, FVIII preparations have also been administered prophylactically to prevent bleeding events (Non-Patent Documents 1 and 2) (prophylactic administration). The blood half-life of FVIII preparations is approximately 12 to 16 hours. Therefore, for continuous prevention, FVIII preparations are administered to patients three times a week (Non-Patent Documents 3 and 4). In on-demand administration, additional FVIII preparations are administered at regular intervals as needed to prevent rebleeding. Furthermore, FVIII preparations are administered intravenously. Therefore, there is a strong need for a drug that is less invasive than FVIII preparations.

[0003] Occasionally, antibodies (inhibitors) against FVIII develop in hemophilia patients. Inhibitors counteract the effectiveness of FVIII drugs. Patients with inhibitors (inhibitor patients) are given bypassing agents to treat bleeding. Their mechanism of action is independent of FVIII's function, namely, its ability to catalyze the activation of blood coagulation factor X (FX) by activated blood coagulation factor IX (FIXa). Therefore, bypassing agents may not adequately stop bleeding in some cases. Therefore, there is a strong need for a drug that can replace the function of FVIII and is not affected by the presence of inhibitors.

[0004] As a means to solve these problems, bispecific antibodies that substitute for the function of FVIII and their use have been reported (Patent Documents 1, 2, 3, and 4). Bispecific antibodies against FIXa and FX can substitute for the function of FVIII by positioning both factors in close proximity, thereby exerting FVIII cofactor substituting activity (Non-Patent Document 5). The FVIII cofactor substituting activity of bispecific antibodies against FIXa and FX refers to the activity calculated, for example, from a colorimetric FIXa-FX activation reaction test and a thrombin generation test using hemophilia A plasma. It has been reported that the FVIII cofactor substituting activity of such antibodies can be improved by optimizing their affinity for FIXa and FX (Non-Patent Document 6). It is also known that the FVIII cofactor substituting activity of such antibodies is affected by the IgG isotype, disulfide bond pattern, amino acid sequence of the hinge region, and the presence or absence of glycans in the Fc region (Non-Patent Document 7).

[0005] Recently, one such antibody, emicizumab (trade names Hemlibra, ACE910, RO5534262), has been approved in many countries, including the United States, Europe, and Japan, as a recombinant drug for suppressing bleeding tendency in patients with congenital hemophilia A (congenital blood coagulation factor VIII deficiency), and in Japan, it has also been approved for the additional indication of acquired hemophilia A. Emicizumab has been reported to exert hemostatic effects in a monkey hemophilia model (Non-Patent Documents 8 and 9), and has also been confirmed to have excellent pharmacokinetics (long half-life) and tolerability in clinical trials involving healthy subjects (Non-Patent Document 10), as well as a significant reduction in the number of bleeding episodes in patients with hemophilia A, regardless of the presence or absence of FVIII inhibitors (Non-Patent Document 11).

[0006] Thus, emicizumab has been shown to have an effect of suppressing the number of bleeding episodes in clinical trials. However, the improvement effect of emicizumab on the maximum thrombin generation amount (Peak height) in a thrombin generation test using FVIII-deficient plasma is lower than the normal level of FVIII activity of 100 U / dL (Non-Patent Document 8). Therefore, in hopes of further enhancing the efficacy and further reducing the dose by improving the specific activity, research is being conducted on FVIII functional substitute antibodies that have a higher maximum activity than emicizumab (maximum activity of FVIII cofactor functional substitute activity) and can exert FVIII cofactor functional substitute activity at a lower concentration than emicizumab (Patent Documents 5 and 6).

[0007] To evaluate the activity of such FVIII substitutes, it is desirable to measure coagulation parameters using blood samples and confirm blood coagulation in vivo. However, because emicizumab, for example, does not cross-link to mouse FIX / FX, preclinical evaluation requires testing in monkeys, which is very costly. Therefore, there is a need to establish a simpler and more accurate in vivo experimental system.

[0008] To develop therapeutics with high specificity for molecular targets, such as antibody drugs, it is desirable to establish humanized non-human animals, such as humanized mice, capable of expressing foreign genes at physiologically relevant levels for more appropriate preclinical evaluation. Gene targeting, in which mouse genes are replaced with human genes, is one method for creating humanized mice. Previously, methods for replacing human genomic genes with mouse homologous genes have been reported, but the expression level of the replaced human gene is lower than that of the mouse homologous gene, making expression difficult to control (Non-Patent Document 12). When the coding sequence of a full-length human gene is inserted into a target mouse gene, the resulting transcribed mRNA contains a premature termination codon (PTC) far upstream of the mouse gene's termination codon, and an exon-exon junction derived from the mouse gene downstream of this termination codon. This structure is recognized by nonsense-mediated mRNA decay (NMD), resulting in mRNA degradation, often resulting in failure to achieve the desired gene expression levels. One approach to address this issue has been reported: inserting a sequence called hp7 into the 3' end of DNA encoding any foreign gene, thereby preventing mRNA degradation by the NMD mechanism and enabling stable expression of the foreign gene in mice (Patent Document 7). In addition to hp7, another method for avoiding NMD has been to add a poly(A) addition signal directly downstream of the cDNA sequence of any foreign gene and then insert the gene into mice. This results in a structure in the transcribed mRNA that does not contain exon-exon junctions derived from the target gene downstream of the PTC, preventing NMD. Other factors contributing to mRNA stability include the 3' untranslated region (3'UTR) and splicing mechanisms. It has been reported that the poly(A) addition signal in the 3'UTR contributes to mRNA stability (Non-Patent Document 13), and that the presence of adenine / uridine-rich elements (Non-Patent Document 14) and GU-rich elements (Non-Patent Document 15) contributes to protein translation regulation.It has also been reported that the expression level of genes without introns, i.e., mRNA that has not undergone splicing out, decreases (Non-Patent Document 16). This report indicates that if the length of a genomic region having an exon-intron structure exceeds several hundred kilobases, it is difficult to replace the genomic region with the desired genomic region, but it is possible to replace a short genomic region of several tens of kilobases (Non-Patent Document 16).

[0009] Under these technical circumstances, non-human animals have been generated in which human blood coagulation-related factors such as FIX, FX, and FVII have been knocked in (Patent Document 8), and mice in which the endogenous FVIII gene has been knocked out and the human FIX / FX gene has been knocked in (Non-Patent Document 17). However, the expression levels of human FIX / FX in these humanized non-human animals are low compared to both the endogenous FIX / FX expression levels in non-human animals and in humans, and do not represent the actual state in vivo. This makes them unsuitable for use in evaluating the efficacy of FVIII functional substitutes. Thus, there is no versatile method for generating non-human animals that can suppress endogenous gene expression and express foreign genes at physiologically appropriate levels. Furthermore, there are no non-human animals that lack the expression of the endogenous FVIII / FIX / FX genes in non-human animals and can express human FIX / FX genes at physiologically appropriate levels, and the method for generating such animals has also been unknown.

[0010] WO2005 / 035754WO2005 / 035756WO2006 / 109592WO2012 / 067176WO2017 / 110980WO2018 / 021450WO2014 / 042251WO2009 / 088876

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[0012] The present invention has been made in light of the above-mentioned circumstances, and aims to provide, in a non-limiting aspect, genetically modified non-human animals that lack expression of endogenous FVIII, FIX, and FX genes and express human FIX and FX genes, methods for producing such genetically modified non-human animals, and methods for evaluating / screening test substances using such genetically modified non-human animals.

[0013] As a result of intensive research conducted in accordance with the above objectives, the inventors surprisingly succeeded in producing a non-human animal that expresses the human FIX / FX gene at a level equivalent to or greater than that of a healthy human by inserting, in that order, an artificial exon-intron sequence, a human FIX / FX cDNA sequence, and a poly(A) addition signal sequence into the endogenous FIX / FX gene locus of the non-human animal.

[0014] Furthermore, the present inventors have found that when emicizumab is administered to genetically modified non-human animals that lack the expression of the endogenous FVIII, FIX, and FX genes of the non-human animal and are capable of functionally expressing human FIX and FX genes, high blood coagulation activity is detected in the blood of the non-human animals. In other words, by using these non-human animals as models, it is possible to accurately and simply evaluate the in vivo blood coagulation activity of test substances that have human FVIII cofactor functional substituting activity. Furthermore, by utilizing such an evaluation system, it is possible to efficiently develop antibodies and other substances that have desired properties in terms of efficacy, safety, pharmacokinetics, biodistribution, and the like.

[0015] More specifically, for example, the following inventions are provided: [A1] A genetically modified non-human animal that lacks expression of endogenous FVIII, FIX, and FX (F8, F9, and F10) and expresses human FIX and FX (F9 and F10). [A2] A genetically modified non-human animal that is functionally deficient in endogenous FVIII, FIX, and FX (F8, F9, and F10) genes on its genome and functionally expresses human FIX and FX (F9 and F10) genes. [A3] A genetically modified non-human animal that lacks expression of endogenous FVIII, FIX, and FX (F8, F9, and F10) polypeptides and expresses human FIX and FX (F9 and F10) polypeptides. [A4] The genetically modified non-human animal according to any one of [A1] to [A3], wherein the first exon of the endogenous FIX locus is deleted from the start codon ATG onwards (e.g., from the start codon ATG to a part of the intron region), and a human FIX gene has been inserted in its place. [A5] The genetically modified non-human animal according to any one of [A1] to [A4], wherein the second exon of the endogenous FX locus is deleted from the start codon ATG onwards (e.g., from the start codon ATG to a part of the intron region), and a human FX gene has been inserted in the same reading frame as the start codon ATG. [A6] The genetically modified non-human animal according to any one of [A1] to [A5], wherein the human FIX and FX gene regions have been inserted with the exon and intron sequences of the beta-actin (ACTB) gene of the non-human animal, the exon and intron sequences of the beta-globin (Hbb-bs) gene of the non-human animal, and / or the 3' untranslated region of SV40, in addition to the regions encoding human FIX and FX proteins. [A7] The genetically modified non-human animal according to any one of [A1] to [A5], wherein an artificial exon-intron structural sequence has been inserted into the 5' side of the region encoding the human FIX and FX proteins. [A8] The genetically modified non-human animal according to any one of [A1] to [A5], wherein an exon-intron structural sequence derived from the gene of the non-human animal has been inserted into the 5' side of the region encoding the human FIX and FX proteins.[A9] The genetically modified non-human animal according to any one of [A1] to [A5], wherein two sets of exon-intron structural sequences derived from the genes of the non-human animal have been inserted into the 5' side of the region encoding the human FIX and FX proteins. [A10] The genetically modified non-human animal according to any one of [A1] to [A5], wherein exon-intron structural sequences derived from the beta-actin (ACTB) gene and / or the beta-globin (Hbb-bs) gene of the non-human animal have been inserted into the 5' side of the region encoding the human FIX and FX proteins. [A11] The genetically modified non-human animal according to any one of [A1] to [A5], wherein a sequence comprising the first exon sequence, the intron sequence, and the second exon sequence of the beta-actin (ACTB) gene of the non-human animal, or the second exon sequence, the intron sequence, and the third exon sequence of the beta-globin (Hbb-bs) gene has been inserted into the 5' side of the region encoding the human FIX and FX proteins. [A12] The genetically modified non-human animal according to any one of [A1] to [A5], wherein an artificial 3' untranslated region sequence, for example, a poly(A) addition signal sequence, has been inserted into the 3' side of the region encoding the human FIX and FX proteins. [A13] The genetically modified non-human animal according to any one of [A1] to [A5], wherein a 3' untranslated region sequence of a non-human gene, for example, a poly(A) addition signal sequence, has been inserted into the 3' side of the region encoding the human FIX and FX proteins. [A14] The genetically modified non-human animal according to any one of [A1] to [A5], wherein a 3' untranslated region sequence of SV40, for example, a poly(A) addition signal sequence, has been inserted into the 3' side of the region encoding the human FIX and FX proteins. [A15] The genetically modified non-human animal according to any one of [A1] to [A14], wherein a human FX cDNA has been inserted into the region encoding the FX protein. [A16] The genetically modified non-human animal according to any one of [A1] to [A14], wherein a human FIX cDNA has been inserted into the region encoding the FIX protein.[A17] The genetically modified non-human animal according to any one of [A1] to [A3], comprising DNA encoding human FIX and FX genes, inserted into the same reading frame of the non-human animal's endogenous FIX and FX genes, to which a nucleotide sequence comprising an exon-intron structure has been added to the 5' side and the 3' untranslated region of the non-human gene has been added to the 3' side. [A18] The genetically modified non-human animal according to [A17], wherein the DNA encoding the human FIX and FX genes is cDNA. [A19] The genetically modified non-human animal according to [A17] or [A18], wherein the nucleotide sequence comprising an exon-intron structure comprises the second exon sequence, the intron sequence, and the third exon sequence of beta globin. [A20] The genetically modified non-human animal according to [A19], wherein the beta globin is beta globin of the non-human animal. [A21] The genetically modified non-human animal according to any one of [A17] to [A20], wherein the base sequence comprising the exon-intron structure comprises the first exon sequence, the intron sequence, and the second exon sequence of beta-actin. [A22] The genetically modified non-human animal according to [A21], wherein the beta-actin is beta-actin from the non-human animal. [A23] The genetically modified non-human animal according to any one of [A17] to [A22], wherein the 3' untranslated region of the non-human gene comprises a poly(A) addition signal sequence.

[0016] The following inventions are also provided, for example: [B1] A genetically modified non-human animal according to any one of [A1] to [A23], which expresses human FIX and FX polypeptides at physiologically appropriate levels; [B2] A genetically modified non-human animal according to [B1], which expresses human FIX and FX polypeptides at blood concentrations equivalent to those of human FIX and FX in healthy humans; [B3] A genetically modified non-human animal according to [B1] or [B2], wherein the blood concentration of human FIX polypeptide in the non-human animal is 2 μg / mL or more, 3 μg / mL or more, or 4 μg / mL or more, and / or the blood concentration of human FX polypeptide in the non-human animal is 3 μg / mL or more, 4 μg / mL or more, 5 μg / mL or more, 6 μg / mL or more, 7 μg / mL or more, or 8 μg / mL or more. [B4] The genetically modified non-human animal according to any one of [B1] to [B3], wherein the expression levels of human FIX and FX polypeptides in the blood of the non-human animal are 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or 80% or more, respectively, relative to the expression levels of human FIX and FX polypeptides in human blood, which are taken as 100%. [B5] The genetically modified non-human animal according to any one of [B1] to [B4], wherein the expression levels of human FIX and FX polypeptides in the blood of the non-human animal are 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or 80% or more, respectively, relative to the expression levels of FIX and FX polypeptides in the blood of a wild-type non-human animal, which are taken as 100%. [B6] A genetically modified non-human animal described in [B1], wherein the expression levels of human FIX and FX in the liver of the non-human animal are 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or 80% or more, respectively, when the expression levels of human FIX and FX in the human liver are taken as 100%.[B7] The genetically modified non-human animal according to [B1], wherein the expression levels of human FIX and FX in the liver of the non-human animal are 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or 80% or more, respectively, when the expression levels of FIX and FX in the liver of the wild-type non-human animal are taken as 100%. [B8] The genetically modified non-human animal according to [B6] or [B7], wherein the expression levels of human FIX and FX are the expression levels of human FIX and FX polypeptides, the expression levels of mRNA encoding human FIX and FX polypeptides, or the copy numbers of human FIX and FX mRNA in total RNA in the liver. [B9] The genetically modified non-human animal according to any of [B1] to [B8], wherein the non-human animal is a non-human mammal. [B10] The genetically modified non-human animal according to any of [B1] to [B9], wherein the non-human animal is a rodent. [B11] The genetically modified non-human animal according to any one of [B1] to [B10], wherein the non-human animal is a mouse. [B12] The genetically modified non-human animal according to any one of [B1] to [B11], wherein, when emicizumab is administered to the non-human animal, one or more selected from the group consisting of human FX activation, increased thrombin generation, shortened thrombin generation time, shortened blood clotting time, and suppression of bleeding episodes in the non-human animal are detected. [B13] The genetically modified non-human animal according to [B12], wherein the thrombin generation and blood clotting time are measured using a biological sample, such as a blood, serum, or plasma sample, collected from the non-human animal. [B14] A blood, serum, or plasma sample isolated from the genetically modified non-human animal according to any one of [B1] to [B13]. [B15] The genetically modified non-human animal according to any one of [B1] to [B13] or the sample according to [B14], for use in measuring the blood clotting ability of a test substance. [B16] The genetically modified non-human animal according to any one of [B1] to [B13] or the sample according to [B14], for use in predicting the blood coagulation ability of a test substance in humans. [B17] The genetically modified non-human animal according to any one of [B1] to [B13] or the sample according to [B14], for use in screening for a candidate substance exhibiting blood coagulation ability in humans.[B18] A genetically modified non-human animal according to any one of [B1] to [B13] or a sample according to [B14] for use in screening for therapeutic or preventive agents for bleeding, diseases accompanied by bleeding, or diseases caused by bleeding.

[0017] The following inventions are also provided, for example: [C1] A method for measuring the blood coagulation ability of a test substance, comprising: (1) administering the test substance to a genetically modified non-human animal described in any of [A1] to [A23] or [B1] to [B13]; and (2) collecting a biological sample from the genetically modified non-human animal after the administration of the test substance in step (1), and measuring the blood coagulation ability using the biological sample. [C2] The method described in [C1], wherein the biological sample is a blood, serum, or plasma sample. [C3] A method for predicting the blood coagulation ability of a test substance in humans, comprising: (1) measuring the blood coagulation ability of the test substance in a genetically modified non-human animal using the method described in [C1] or [C2] for the test substance; and (2) predicting the blood coagulation ability of the test substance when administered to humans, based on the blood coagulation ability of the test substance in the genetically modified non-human animal measured in (1) above. [C4] The method according to [C3], in which a test substance that exhibits high blood coagulation ability in a genetically modified non-human animal in step (1) is predicted to also exhibit high blood coagulation ability when administered to a human in step (2). [C5] A method for screening a candidate substance that exhibits blood coagulation ability in humans, comprising: (1) measuring the blood coagulation ability of the test substance in a genetically modified non-human animal using the method described in [C1] or [C2], and (2) selecting the test substance that exhibits blood coagulation ability in the genetically modified non-human animal as a candidate substance that exhibits blood coagulation ability in humans. [C6] A method for screening for a therapeutic or preventive agent for bleeding, a disease accompanied by bleeding, or a disease caused by bleeding, comprising: (1) a step of administering a test substance to a genetically modified non-human animal described in any of [A1] to [A23] or [B1] to [B13], (2) a step of measuring at least one evaluation index selected from the group consisting of blood coagulation ability, safety, pharmacokinetics, and biodistribution characteristics in the genetically modified non-human animal administered the test substance, and (3) a step of selecting, as a candidate for the therapeutic or preventive agent, a test substance for which the evaluation index measured in step (2) is superior to that of a control. [C7] The method described in [C6], wherein the disease is hemophilia A.[C8] The method according to [C6] or [C7], wherein the evaluation index of the control is the evaluation index measured in step (2) in the genetically modified non-human animal that has not been administered the test substance. [C9] A method for evaluating the safety of a test substance, comprising the steps of: (1) administering the test substance to a genetically modified non-human animal according to any of [A1] to [A23] or [B1] to [B13], (2) measuring cytokine release in the genetically modified non-human animal administered the test substance, and (3) comparing the cytokine levels measured in step (2) with those of a control, wherein a change in the cytokine levels indicates a safety risk. [C10] A method for evaluating the pharmacokinetic properties of a test substance, comprising: (1) a step of administering the test substance to a genetically modified non-human animal described in any of [A1] to [A23] or [B1] to [B13], and (2) a step of measuring changes in the blood concentration of the test substance over time in the genetically modified non-human animal to which the test substance has been administered. [C11] A method for evaluating the biodistribution properties of a test substance, comprising: (1) a step of administering the test substance to a genetically modified non-human animal described in any of [A1] to [A23] or [B1] to [B13], and (2) a step of measuring the biodistribution of the test substance in the genetically modified non-human animal to which the test substance has been administered, wherein the biodistribution properties of the test substance are indicated by the localization of the test substance. [C12] The method of any of [C1] to [C11], wherein the test substance is a bispecific antigen-binding molecule comprising a human FIX-binding domain and a human FX-binding domain. [C13] The method of [C12], wherein the antigen-binding molecule is an antibody. [C14] The method of any of [C1] to [C13], wherein the blood coagulation ability is measured by measuring the number of bleeding episodes of the genetically modified non-human animal, and one or more blood coagulation parameters, such as activated partial thromboplastin time (APTT) and thrombin generation (TG), in a biological sample, such as a blood, serum, or plasma sample, obtained from the genetically modified non-human animal.

[0018] The following inventions are also provided, for example: [D1] A DNA construct comprising DNA encoding a human FIX or FX protein, to which a nucleotide sequence comprising an exon-intron structure has been added to the 5' side and a 3' untranslated region of a non-human gene has been added to the 3' side. [D2] The DNA construct according to [D1], wherein the DNA encoding the human FIX or FX protein is cDNA. [D3] The DNA construct according to [D1] or [D2], wherein the nucleotide sequence comprising an exon-intron structure comprises the second exon sequence, the intron sequence, and the third exon sequence of a beta globin gene. [D4] The DNA construct according to [D3], wherein the beta globin is mouse beta globin. [D5] The DNA construct according to any of [D1] to [D4], wherein the nucleotide sequence comprising an exon-intron structure comprises the first exon sequence, the intron sequence, and the second exon sequence of a beta-actin gene. [D6] The DNA construct according to [D5], wherein the beta-actin is beta-actin from the non-human animal. [D7] The DNA construct according to any one of [D1] to [D6], wherein the 3' untranslated region of the non-human gene comprises a poly(A) addition signal sequence. [D8] A knock-in vector carrying the DNA construct according to any one of [D1] to [D7]. [D9] The knock-in vector according to [D8], characterized in that the 5' side of the DNA construct comprises a nucleotide sequence homologous to the 5' upstream region of the target region of the FIX or FX gene of a non-human animal, and the 3' side of the DNA construct comprises a nucleotide sequence homologous to the 3' downstream region of the target region of the FIX or FX gene of a non-human animal. [D10] A non-human animal cell into which the knock-in vector according to [D8] or [D9] has been introduced. [D11] The non-human animal cell according to [D10], wherein the cell is an embryonic stem cell (ES cell), an induced pluripotent stem cell (iPS cell), a germline stem cell, or a fertilized egg. [D12] A method for producing a non-human animal that expresses human FIX and / or FX polypeptide, comprising the step of introducing the knock-in vector described in [D8] or [D9] into a host cell.

[0019] Furthermore, for example, the following inventions are provided: [E1] A method for producing a genetically modified non-human animal that is functionally deficient in endogenous FVIII, FIX, and FX (F8, F9, and F10) genes on its genome and that functionally expresses human FIX and FX (F9 and F10) genes, the method comprising: (1) a step of producing a genetically modified non-human animal that is functionally deficient in endogenous FVIII gene on its genome and that does not functionally express the human FVIII gene, (2) a step of producing a genetically modified non-human animal that is functionally deficient in endogenous FIX gene on its genome and that functionally expresses the human FIX gene, (3) a step of producing a genetically modified non-human animal that is functionally deficient in endogenous FX gene on its genome and that functionally expresses the human FX gene, and (4) a step of mating the genetically modified non-human animals produced by steps (1) to (3).

[0020] This photograph shows a representative example of electrophoresis of PCR products in the selection of FVIII-deficient mice. Electrophoresis was performed on a 1% agarose gel. Lane 1: 1 Kb Plus DNA Ladder (10787018, Invitrogen), Lanes 2-5: sample, Lanes 6 and 7: C57BL / 6J Jcl-derived genomic DNA (wild-type with no endogenous FVIII deficiency), Lane 8: non-template control. This photograph shows a representative example of electrophoresis of PCR products to detect 5' homologous recombination in the selection of human FIX-introduced mice. Electrophoresis was performed on a 1% agarose gel. Lane 1: 1 Kb Plus DNA Ladder (10787018, Invitrogen), Lane 2: sample, Lane 3: C57BL / 6J Jcl-derived genomic DNA (wild-type), Lane 4: positive control, Lane 5: non-template control. This photograph shows a representative example of electrophoresis of PCR products used to detect 3' homologous recombination during selection of human FIX transgenic mice. Electrophoresis was performed on a 1% agarose gel. Lane 1: 1 Kb Plus DNA Ladder (10787018, Invitrogen), Lane 2: sample, Lane 3: C57BL / 6J Jcl-derived genomic DNA (wild-type), Lane 4: positive control, Lane 5: non-template control. This photograph shows a representative example of electrophoresis of PCR products used to detect 5' homologous recombination during selection of human FX transgenic mice. Electrophoresis was performed on a 1% agarose gel. Lane 1: 1 Kb Plus DNA Ladder (10787018, Invitrogen), Lane 2-13: sample, Lane 14: C57BL / 6J Jcl-derived genomic DNA (wild-type), Lane 15: positive control, Lane 16: non-template control. This is a photograph showing a representative example of electrophoresis of PCR products used to detect 3' homologous recombination during selection of human FX-introduced mice. Electrophoresis was performed on a 1% agarose gel.Lane 1: 1 Kb Plus DNA Ladder (10787018, Invitrogen), Lanes 2-13: sample, Lane 14: C57BL / 6J Jcl-derived genomic DNA (wild-type), Lane 15: positive control, Lane 16: non-template control. Representative electrophoretic images of genotyping of FVIII-deficient mice after hybridization. Capillary electrophoresis was performed using a LabChip GX Touch HT. Lanes 1-7: sample, Lane 8: C57BL / 6J Jcl-derived genomic DNA (wild-type with no endogenous FVIII deficiency), Lane 9: Hetero KO, Lane 10: Homo KO, Lane 11: non-template control. Representative electrophoretic images of genotyping of human FIX-introduced mice after hybridization. Capillary electrophoresis was performed using a LabChip GX Touch HT. Lanes 1-7: sample; lane 8: C57BL / 6J Jcl-derived genomic DNA (wild-type); lane 9: hetero KI; lane 10: homo KI; lane 11: non-template control. This photograph shows a representative example of electrophoresis for genotyping of human FX-introduced mice after hybridization. Capillary electrophoresis was performed using a LabChip GX Touch HT. Lanes 1-3: sample; lane 4: C57BL / 6J Jcl-derived genomic DNA (wild-type); lane 5: hetero KI; lane 6: homo KI; lane 7: non-template control. This graph shows the results of a thrombin generation test using mice with the genotypes of FVIII deficiency, human FIX introduction, and human FX introduction. The plasma samples were divided into three groups: a control group (cont), a rhFVIII group containing 0.1, 0.3, or 1 IU / mL recombinant human FVIII (rhFVIII), and an Emi group containing 50 μg / mL emicizumab (Emi).In the graph, "a" indicates that the p-value between "cont" and "Emi" by t-test is p<0.05, and "b" indicates that the p-value between "cont" and "rhFVIII" by Dunnett's multiple comparison test is p<0.05.

[0021] 1. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety.

[0022] In this specification, unless a term is explained by describing a limitation indicating a quantity such as "one" or "multiple," the terms described in this specification are not construed as being particularly limited in quantity, but are understood to be terms having the meaning of "one or multiple."

[0023] "Conservative substitutions" are those that occur within a family of amino acids related by their side chains and chemical properties. For example, amino acids can be grouped by shared side chain properties: (1) hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) acidic: aspartic acid (Asp), glutamic acid (Glu); (4) basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for another class. In one non-limiting embodiment, the present invention includes genetically modified non-human animals that express human FIX / FX polypeptides containing conservative amino acid substitutions in the amino acid sequences described herein.

[0024] The term "target gene" includes an endogenous gene of a non-human animal into which an exogenous gene is to be inserted. The term "target region" includes a specific region of an endogenous gene of a non-human animal into which an exogenous gene is to be inserted. In a non-limiting embodiment, the term "target region" refers to a region including the endogenous gene flanking the 5' and 3' sides of the inserted exogenous gene. In another embodiment, the term "target region" refers to a region of an endogenous gene whose expression is impaired by the insertion of an exogenous gene.

[0025] "Endogenous" includes substances that are naturally occurring within an organism, tissue, or cell, for example. For example, an endogenous nucleic acid or peptide refers to a nucleic acid or peptide that is present in a cell and has not been introduced into the cell using recombinant engineering techniques. As used herein, the term "endogenous" may be used interchangeably with the term "indwelling."

[0026] The term "exogenous" includes substances derived from organisms, tissues, cells, etc. other than the organism, tissue, or cell in question. For example, "exogenous genes" include genes introduced into the non-human animals of the present invention. The exogenous genes of the present invention can be used regardless of the species of origin, but are preferably human genes. Furthermore, reporter genes such as green fluorescent protein (GFP) and β-galactosidase, and selectable marker genes such as drug (e.g., neomycin) resistance genes can also be used as exogenous genes. Combinations of two or more genes can also be used as exogenous genes. Furthermore, an enhancer or the like that regulates the expression of the exogenous gene may be added. The form of the exogenous gene is not particularly limited and may be, for example, cDNA or genomic DNA. In this specification, the term "exogenous" may be used interchangeably with the terms "exogenous" and "exogenous."

[0027] "Isolated" means separated from a component of its original environment.

[0028] "Operably linked" includes a state in which each gene is linked in a state in which it can perform its intended function. For example, a nucleic acid sequence encoding a protein can be operably linked to a control sequence (e.g., a promoter, an enhancer, a silencer sequence, etc.) to maintain appropriate transcriptional regulation, but the promoter does not need to be contiguous with the sequence as long as it functions in this manner. In this specification, the term "operably linked" may be used interchangeably with the term "operably linked."

[0029] Examples of "vectors" include, but are not limited to, genetically engineered plasmids or viruses derived from bacteriophage, adenovirus, retrovirus, poxvirus, herpesvirus, or artificial chromosomes.

[0030] The term "non-human animal" is not particularly limited as long as it is an animal other than a human, and examples thereof include mice, rats, guinea pigs, hamsters, rabbits, goats, cows, horses, pigs, dogs, cats, and monkeys. Non-human animals are preferably non-human mammals, and particularly preferably rodents, such as mice. Preferred examples of mice include, but are not limited to, C57 / BL / 6, ICR, and BALB / c.

[0031] "Wild-type" includes having the normal structure and / or activity found in nature. Wild-type nucleic acids or peptides include multiple different forms and polymorphisms, such as allelic variants. A "wild-type" non-human animal also optionally includes an animal that is wild-type with respect to the FVIII / FIX / FX gene, i.e., an animal that has not been genetically engineered with respect to FVIII / FIX / FX.

[0032] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0033] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0034] Emicizumab (Hemlibra, ACE910, RO5534262) is a bispecific antigen-binding molecule that recognizes (a) blood coagulation factor IX (FIX) and / or activated blood coagulation factor IX (FIXa) and (b) blood coagulation factor X (FX) and / or activated blood coagulation factor X (FXa), and has activity that substitutes for the function of blood coagulation factor VIII (FVIII). As used herein, "FVIII cofactor functional substitution activity," "FVIII substitution activity," and "activity that substitutes for the function of FVIII" are used interchangeably and refer to the activity of recognizing FIX and / or FIXa and FX and promoting FX activation (promoting FXa production).

[0035] In the present invention, the phrase "substitutes for the function of FVIII" means that (a) FIX and / or FIXa and (b) FX and / or FXa are recognized and the activation of FX by FIXa is promoted (FXa production by FIXa is promoted). FXa production-promoting activity can be evaluated, for example, using an assay system containing FIXa, FX, the synthetic substrate S-2222 (a synthetic substrate for FXa), and phospholipids. Such an assay system shows a correlation between the severity of disease and clinical symptoms in hemophilia A cases (Rosen S, Andersson M, Blomback M et al. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985; 54: 811-23). ​​In the present invention, the phrases "substitutes for the function of FVIII" and "substitutes for the function of FVIIIa" are used interchangeably.

[0036] In addition to emicizumab, such antigen-binding molecules (e.g., antibodies) that recognize (a) FIX and / or FIXa and (b) FX and / or FXa are described in, for example, WO2005 / 035754, WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, WO2017 / 110980, and WO2018 / 021450, and can be obtained according to the methods described in those documents. Specifically, for example, such antibodies can be produced using genetic recombination techniques known to those skilled in the art based on the sequences of antibodies against FIX and / or FIXa and antibodies against FX and / or FXa. Based on the sequences of antibodies against FIX and / or FIXa and antibodies against FX and / or FXa, polynucleotides encoding the antibodies can be constructed, inserted into expression vectors, and then expressed in appropriate host cells (e.g., Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; and Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

[0037] Emicizumab is a bispecific antibody in which a first polypeptide is associated with a third polypeptide and a second polypeptide is associated with a fourth polypeptide, and has the following structure: (a) a bispecific antibody comprising a first polypeptide that is a heavy chain containing a heavy chain variable region containing the amino acid sequences of CDRs 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively; a second polypeptide that is a heavy chain containing a heavy chain variable region containing the amino acid sequences of CDRs 1, 2, and 3 of SEQ ID NOs: 6, 7, and 8, respectively; and third and fourth polypeptides that are shared light chains containing a light chain variable region containing the amino acid sequences of CDRs 1, 2, and 3 of SEQ ID NOs: 11, 12, and 13, respectively; (b) a first polypeptide that is a heavy chain containing the heavy chain variable region amino acid sequence of SEQ ID NO: 4; a second polypeptide which is a heavy chain comprising the amino acid sequence of SEQ ID NO:10; and third and fourth polypeptides which are a shared light chain comprising the amino acid sequence of SEQ ID NO:15; or (c) a bispecific antibody (Q499-z121 / J327-z119 / L404-k) comprising a first polypeptide which is a heavy chain comprising the amino acid sequence of SEQ ID NO:5; a second polypeptide which is a heavy chain comprising the amino acid sequence of SEQ ID NO:10; and third and fourth polypeptides which are a shared light chain comprising the amino acid sequence of SEQ ID NO:15.

[0038] 2. FVIII, FIX, and FX Blood coagulation factors VIII, IX, and X are proteins that make up the blood coagulation cascade and are also called FVIII, FIX, and FX, or FVIII, FIX, and FX, or F8, F9, and F10, respectively. The activated forms of blood coagulation factors VIII, IX, and X are called blood coagulation factors VIIIa, IXa, and Xa, or FVIIIa, FIXa, and FXa, or FVIIIa, FIXa, and FXa, or F8a, F9a, and F10a, respectively. Hemophilia is a bleeding disorder in which the inability to clot blood is caused by a deficiency or impairment of the function of these components. When FVIII is the cause, it is called hemophilia A. FVIII is converted to FVIIIa upon activation by thrombin, and FVIIIa functions as a cofactor for FIXa, promoting the activation of FX. FVIII, FIX, and FX are primarily produced in the liver and secreted into the blood. In humans and mice, the FVIII and FIX loci are located on chromosome X, and the FX locus is located on chromosome 13.

[0039] Examples of FIX polypeptide sequences include SEQ ID NO: 1 (human, GenBank: CCA61111.1) and SEQ ID NO: 2 (mouse, GenBank: AAA37629.1), and examples of mRNA sequences include SEQ ID NO: 3 (human, GenBank: FR846239.1) and SEQ ID NO: 4 (mouse, GenBank: M23109.1). Examples of FX polypeptide sequences include SEQ ID NO: 5 (human, GenBank: AAH46125.1) and SEQ ID NO: 6 (mouse, GenBank: CAA10933.1), and examples of mRNA sequences include SEQ ID NO: 7 (human, GenBank: BC046125.1) and SEQ ID NO: 8 (mouse, GenBank: AJ222677.1).

[0040] Herein, when FIX and FX are collectively described, they may be referred to as FIX / FX. For example, the phrase "incorporating a gene encoding human FIX / FX into an endogenous FIX / FX allele" is intended to collectively describe "incorporating a gene encoding human FIX into an endogenous FIX allele" and "incorporating a gene encoding human FX into an endogenous FX allele," but does not intend to mean "incorporating a gene encoding human FX into an endogenous FIX allele" or "incorporating a gene encoding human FIX into an endogenous FX allele." Similarly, when FVIII, FIX, and FX are collectively described, they may be referred to as FVIII / FIX / FX. Herein, the terms FVIII / FIX / FX polypeptide and FVIII / FIX / FX protein are used interchangeably.

[0041] In a non-limiting embodiment, the "FVIII gene," "FIX gene," and "FX gene" are not particularly limited as long as they are genes encoding FVIII, FIX, and FX polypeptides, respectively, and may be genomic DNA or cDNA. Furthermore, the FVIII, FIX, and FX genes include polymorphisms or mutants thereof.

[0042] 3. Non-human animals lacking expression of endogenous FVIII, FIX, and FX and expressing human FIX and FX In one aspect, the present invention relates to genetically modified non-human animals lacking expression of endogenous FVIII, FIX, and FX and expressing human FIX and FX. In a non-limiting embodiment, the genetically modified non-human animals of the present invention are functionally deficient in endogenous FVIII, FIX, and FX genes on their genomes and functionally express human FIX and FX. In a non-limiting embodiment, the genetically modified non-human animals of the present invention are deficient in expression of endogenous FVIII, FIX, and FX polypeptides and express human FIX and FX polypeptides.

[0043] In a non-limiting embodiment, a non-human animal "lacking expression of endogenous FVIII / FIX / FX" or "having a functional deficiency in the endogenous FVIII / FIX / FX gene in the genome" means that the non-human animal does not express mRNA encoding endogenous FVIII / FIX / FX polypeptides and, as a result, does not express functional endogenous FVIII / FIX / FX polypeptides, and the embodiment is not particularly limited thereto. The non-human animal may lack part or all of the gene region encoding endogenous FVIII / FIX / FX polypeptides in its genome, or may lack mRNA encoding endogenous FVIII / FIX / FX polypeptides due to the introduction of a foreign gene into the endogenous FVIII / FIX / FX gene locus in the genome. Whether or not an animal lacks expression of endogenous FVIII / FIX / FX polypeptides can be determined by methods known to those skilled in the art, such as detection methods using antibodies that specifically recognize endogenous FVIII / FIX / FX polypeptides, as well as methods measuring the biological activity of endogenous FVIII / FIX / FX polypeptides. In a non-limiting embodiment, since functional endogenous FVIII polypeptides are not expressed in the genetically modified non-human animals of the present invention, activation of FX by FIXa and / or blood coagulation are inhibited when exogenous FVIII is not expressed or when a substance having FVIII polypeptide or FVIII cofactor functional substituting activity is not administered. FX activation and blood coagulation can be measured by methods known to those skilled in the art, as described elsewhere herein.

[0044] In a non-limiting embodiment, "expressing human FIX and FX" and "functionally expressing human FIX and FX genes" in a non-human animal refer to expressing mRNA encoding (preferably full-length) human FIX and FX polypeptides in the non-human animal, preferably referring to expressing (preferably full-length) human FIX and FX polypeptides in the non-human animal. In a non-limiting embodiment, "expressing human FIX and FX polypeptides" in a non-human animal refers to expressing functional human FIX and FX polypeptides that can exhibit biological activity in the non-human animal, for example, a state in which human FX can be activated by positioning the human FIX and FX polypeptides in close proximity with human FVIII or a substance with human FVIII cofactor functional replacement activity (e.g., emicizumab). In one embodiment, "functionally expressing human FIX and FX genes / polypeptides" in the non-human animal of the present invention is achieved by expressing human FIX and FX polypeptides at physiologically relevant levels in the non-human animal (e.g., levels equivalent to those of healthy humans). In one embodiment, the non-human animal of the present invention functionally expressing human FIX and FX polypeptides has a blood coagulation system that functions normally in the presence of human FVIII or a substance with human FVIII cofactor functional substituting activity (e.g., emicizumab). Functional expression of human FIX and FX in the non-human animal of the present invention can be assessed by measuring the activation of human FX in the non-human animal administered human FVIII or a substance with human FVIII cofactor functional substituting activity (e.g., emicizumab), as well as by measuring blood coagulation or hemostatic activity, such as thrombin generation amount, thrombin generation time, shortening of blood clotting time (APTT), blood clotting time, and number of bleeding episodes. In one embodiment, the genetically modified non-human animal of the present invention is capable of activating human FX, exhibiting blood coagulation or hemostatic activity, upon administration of human FVIII or a substance with human FVIII cofactor functional substituting activity (e.g., emicizumab).The activation, blood coagulation, and hemostatic activity of human FX can be measured by various methods known to those skilled in the art. In a non-limiting embodiment, the activation, blood coagulation, or hemostatic activity of human FX in the genetically modified non-human animals of the present invention is comparable to that of wild-type non-human animals. In a non-limiting embodiment, the activation, blood coagulation, or hemostatic activity of human FX in the genetically modified non-human animals of the present invention is comparable to that of healthy humans (i.e., humans not deficient in FVIII, FIX, and FX). In a non-limiting embodiment, the activation of human FX in plasma obtained from the genetically modified non-human animals of the present invention is comparable to that of healthy human plasma. In one non-limiting embodiment, "similar levels of FX activation, blood coagulation activity, or hemostatic activity" means that, when one activation / activity is taken as 100%, the other activation / activity is 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or 80% or more, and 500% or less, 450% or less, 400% or less, 350% or less, 300% or less, 250% or less, 200% or less, 150% or less, 130% or less, or 120% or less, for example, 40-400%, 50-400%, 60-400%, 70-400%, or 80-400%.

[0045] In a non-limiting embodiment of the present invention, the genetically modified non-human animals of the present invention have enhanced blood coagulation ability compared to control animals that do not express human FIX and FX genes (animals lacking endogenous FVIII, FIX, and FX genes in the same combination as the animal) and non-human animals that express low levels of human FIX and FX genes. Whether blood coagulation ability is elevated can be determined by comparing the blood coagulation or hemostatic activity of these non-human animals when administered human FVIII or a substance with human FVIII cofactor functional replacement activity (e.g., emicizumab). The genetically modified non-human animals of the present invention have blood coagulation ability that is, for example, 1.3-fold or more, preferably 1.5-fold or more, more preferably 1.6-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 50-fold or more, or 100-fold or more, compared to the control animals. The animals to be compared for blood coagulation ability are preferably of similar ages, but may be of any age as long as the animals have comparable blood coagulation ability in wild-type non-human animals.

[0046] In a non-limiting embodiment, "expressing human FIX / FX polypeptide at a physiologically relevant level" includes, for example, the expression level of human FIX / FX polypeptide or human FIX / FX mRNA in a non-human animal being equivalent to any one of the following expression levels (i) to (iii): (i) the expression level of mouse FIX / FX polypeptide or mouse FIX / FX mRNA in a wild-type mouse, (ii) the expression level of monkey FIX / FX polypeptide or monkey FIX / FX mRNA in a wild-type monkey, and (iii) the expression level of human FIX / FX polypeptide or human FIX / FX mRNA in a healthy human. It is also known that FIX / FX is primarily biosynthesized in the liver, released into the blood, and secreted into the blood. Therefore, "expressing human FIX / FX polypeptide at a physiologically relevant level" includes, for example, the expression level of human FIX / FX polypeptide or human FIX / FX mRNA in the liver and / or blood of a non-human animal being equivalent to any one of the following expression levels (i) to (iii): (i) the expression level of mouse FIX / FX polypeptide or mouse FIX / FX mRNA in the liver and / or blood of a wild-type mouse, (ii) the expression level of monkey FIX / FX polypeptide or monkey FIX / FX mRNA in the liver and / or blood of a wild-type monkey, and (iii) the expression level of human FIX / FX polypeptide or human FIX / FX mRNA in the liver and / or blood of a healthy human. Preferably, the expression level of human FIX / FX polypeptide or human FIX / FX mRNA in the liver and / or blood of the non-human animal of the present invention is equivalent to or greater than the expression level of human FIX / FX polypeptide or human FIX / FX mRNA in the liver and / or blood of a human. In a non-limiting embodiment, when the expression level of one gene is taken as 100%, the expression level of the other gene can be considered to be equivalent if it is 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or 80% or more.In a non-limiting embodiment, when the expression level of one protein is taken as 100%, the expression level of the other protein can be considered equivalent if it is 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or 80% or more, and 500% or less, 450% or less, 400% or less, 350% or less, 300% or less, 250% or less, 200% or less, 150% or less, 130% or less, or 120% or less. In a specific embodiment, when the expression level of one protein is taken as 100%, the expression level of the other protein can be considered equivalent if it is 40-400%, 50-400%, 60-400%, 70-400%, or 80-400%. It is also desirable to compare the expression levels of polypeptides or mRNAs between common tissues or cells.

[0047] In a non-limiting embodiment, the expression level of FIX / FX polypeptide can be expressed as the concentration of FIX / FX polypeptide in blood, serum, or plasma. The blood concentration of human FIX in healthy humans is known to be approximately 5 μg / mL. In a non-limiting embodiment, the blood concentration of human FIX in the genetically modified non-human animals of the present invention is a physiologically relevant level, for example, approximately 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, or 8 μg / mL, preferably approximately 4 to 8 μg / mL. The blood concentration of human FX in healthy humans is known to be approximately 8 μg / mL. In one non-limiting embodiment, the human FX blood concentration in the genetically modified non-human animal of the present invention is a physiologically relevant level, for example, about 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, or 30 μg / mL, preferably about 8 to 30 μg / mL.

[0048] In another embodiment, the expression level of FIX / FX polypeptide can be expressed as the weight of FIX / FX polypeptide per total protein weight in an organ such as the liver, and the expression level of FIX / FX mRNA can be expressed as the copy number of the FIX / FX mRNA in total RNA. Methods for quantitatively assessing RNA copy number are known. Specifically, for example, real-time PCR can be used to perform an amplification reaction using a standard sample with a known amount of RNA as a sample, and a calibration curve (standard curve) can be plotted based on the number of reaction cycles required to reach a certain signal intensity. A similar amplification reaction can also be performed on the RNA sample to be quantified, and the number of reaction cycles required to reach the same signal intensity can be measured. The RNA amount can then be determined by applying the results to a previously prepared calibration curve. Meanwhile, methods for determining the amount of total RNA are known, such as electrophoresis or absorbance measurement. In the present invention, total RNA may also refer to total mRNA.

[0049] In another aspect, an antibody that binds to human FIX / FX can be administered to a genetically modified non-human animal that expresses human FIX / FX polypeptide, and the pharmacokinetic properties of the antibody, such as its half-life in the blood, can be determined to be similar to those of the antibody administered to a human, to determine that the animal "expresses human FIX / FX polypeptide at a physiologically appropriate level."

[0050] In one non-limiting embodiment, a genetically modified non-human animal expressing a human FIX / FX polypeptide exhibits immune tolerance to the human FIX / FX polypeptide. Specifically, because living organisms activate the adaptive immune response system against antigens that are foreign to the organism (self), when a human FIX / FX polypeptide is administered to a non-human animal or cells expressing the human FIX / FX polypeptide are transplanted into the non-human animal, the non-human animal recognizes the human FIX / FX polypeptide or cells expressing the human FIX / FX polypeptide as foreign and eliminates it. However, because the genetically modified non-human animal of the present invention expresses a human FIX / FX polypeptide, the non-human animal regards the human FIX / FX polypeptide as a self-biological component and no longer exhibits an immune response to it. Whether the genetically modified non-human animal of the present invention exhibits immune tolerance to human FIX / FX can be confirmed by various methods known to those skilled in the art, such as administering a human FIX / FX polypeptide or a fragment thereof to the non-human animal and measuring the anti-human FIX / FX antibody titer in the non-human animal's body.

[0051] In a non-limiting embodiment, whether a non-human animal exhibits immune tolerance to a human FIX / FX polypeptide can be confirmed by administering a human FIX / FX polypeptide or a fragment thereof to the non-human animal and then measuring the titer of anti-human FIX / FX antibodies in the plasma of the human animal. An adjuvant may also be administered together with the human FIX / FX polypeptide or its fragment. The antibody titer can be measured, for example, on days 0, 14, 21, 28, 35, 42, and / or 49 after the administration of the human FIX / FX polypeptide or its fragment. For example, if the titer of anti-human FIX / FX antibodies in the plasma of a genetically modified non-human animal expressing a human FIX / FX polypeptide is significantly lower than the titer of anti-human FIX / FX antibodies in a wild-type non-human animal, the genetically modified non-human animal can be evaluated as exhibiting immune tolerance to a human FIX / FX polypeptide.

[0052] In a non-limiting embodiment, a genetically modified non-human animal expressing a human FIX / FX polypeptide exhibits human FX activation, blood coagulation, or hemostatic activity in the presence of a human FVIII polypeptide or a substance with human FVIII cofactor functional replacement activity (e.g., emicizumab). This means that the human FIX / FX polypeptide functions in the blood coagulation reaction of the genetically modified non-human animal of the present invention, i.e., the genetically modified non-human animal of the present invention functionally expresses the human FIX / FX polypeptide. Whether the genetically modified non-human animal of the present invention functionally expresses the human FIX / FX polypeptide can be determined by measuring the activation or blood coagulation activity of human FX using various methods known to those skilled in the art. For example, this can be confirmed by administering a human FVIII polypeptide or a substance with human FVIII cofactor functional replacement activity (e.g., emicizumab) and measuring the number of bleeding episodes or blood coagulation parameters in the non-human animal. In one non-limiting embodiment, the genetically modified non-human animal of the present invention can be determined to functionally express human FIX / FX polypeptide if the number of bleeding episodes or blood coagulation parameters in the genetically modified non-human animal of the present invention are improved by administration of a human FVIII polypeptide or a substance having activity of functionally substituting for a human FVIII cofactor (e.g., emicizumab). In another embodiment, the genetically modified non-human animal of the present invention can be determined to functionally express human FIX / FX polypeptide if the number of bleeding episodes or blood coagulation parameters in the genetically modified non-human animal of the present invention after administration of a human FVIII polypeptide or a substance having activity of functionally substituting for a human FVIII cofactor (e.g., emicizumab) are improved compared to non-human animals that lack endogenous FVIII expression and do not functionally express human FIX and / or FX.In yet another embodiment, it can be determined that the genetically modified non-human animal of the present invention functionally expresses human FIX / FX polypeptide when blood coagulation parameters measured using plasma obtained from the genetically modified non-human animal of the present invention after administration of a human FVIII polypeptide or a substance having human FVIII cofactor functional substituting activity (e.g., emicizumab) are comparable to or greater than blood coagulation parameters measured using plasma from a human not suffering from hemophilia A. Methods for measuring blood coagulation parameters are well known to those skilled in the art, and include, for example, activated partial thromboplastin time (APTT) tests and thrombin generation assays (TGA) using plasma obtained from the non-human animal.

[0053] In a non-limiting embodiment, the genetically modified non-human animal of the present invention is a non-human animal in which the endogenous FVIII locus has been disrupted, the endogenous FIX gene has been replaced with a human FIX gene at the endogenous FIX locus, and the endogenous FX gene has been replaced with a human FX gene at the endogenous FX locus. In a non-limiting embodiment of the present invention, a disruption of the endogenous FVIII locus refers to a state in which the endogenous FVIII gene is functionally deleted in the genome. This is not necessarily limited to a state in which the entire endogenous FVIII locus is deleted, but also refers to a state in which, for example, a frameshift occurs in the generated mRNA due to deletion of some exons, resulting in the failure to express functional endogenous FVIII polypeptides. In a non-limiting embodiment of the present invention, the genetically modified non-human animal of the present invention is a genetically modified non-human animal in which the full-length nucleotide sequences of the human FIX and FX genes have been inserted into the genome. In a non-limiting embodiment, the full-length nucleotide sequences of the human FIX and FX genes inserted into the genome comprise cDNAs encoding full-length human FIX and FX polypeptides. In one non-limiting embodiment, the full-length nucleotide sequence inserted into the genome can utilize the endogenous sequence of the non-human animal for the portion that is completely identical between the human FIX / FX gene and the non-human animal FIX / FX gene (e.g., the start codon ATG). For example, the nucleotide sequence from the start codon ATG of the human FIX / FX gene (3' side from the start codon ATG) onward is inserted into the non-human animal in the same reading frame (in frame) as the start codon ATG of the non-human animal FIX / FX gene. In one non-limiting embodiment of the present invention, the "full-length nucleotide sequence" of the human FIX / FX gene refers to the nucleotide sequence from the start codon ATG of human FIX / FX (3' ​​side from the start codon ATG) onward, and may further include the 3' untranslated region (UTR).

[0054] In one non-limiting embodiment, the genetically modified non-human animal of the present invention, which is deficient in the expression of endogenous FVIII, FIX, and FX and expresses human FIX and FX, can be obtained by crossing a knockout non-human animal in which the endogenous FVIII locus has been disrupted with a knockin non-human animal in which the endogenous FIX gene has been replaced with a human FIX gene at the endogenous FIX locus, and a knockin non-human animal in which the endogenous FX gene has been replaced with a human FIX gene at the endogenous FX locus. In another embodiment, the genetically modified non-human animal of the present invention, which is deficient in the expression of endogenous FVIII, FIX, and FX and expresses human FIX and FX, can be obtained by crossing a knockout non-human animal in which the endogenous FVIII locus has been disrupted with a knockin non-human animal in which the endogenous FIX gene has been replaced with a human FIX gene at the endogenous FIX locus and a knockin non-human animal in which the endogenous FX gene has been replaced with a human FIX gene at the endogenous FX locus. In this case, whether the desired non-human animal has been obtained can be determined by analyzing the genotype of the next generation individuals obtained by mating these non-human animals and confirming that the non-human FVIII gene region deletion allele, the human FIX knock-in allele, and the human FX knock-in allele have been transmitted.

[0055] In one non-limiting embodiment, the present invention provides a non-human animal comprising DNA encoding the human FIX / FX gene and expressing a human FIX / FX polypeptide. In another embodiment, the non-human animal of the present invention includes a non-human animal characterized in that DNA encoding the human FIX / FX gene has been inserted in the same reading frame as an endogenous FIX / FX gene present in the genome of the non-human animal. The DNA encoding the human FIX / FX gene may be genomic DNA or cDNA, but is preferably cDNA, and in a specific embodiment, it is cDNA comprising an amino acid sequence coding region (CDS). The amino acid sequence coding region also comprises a signal sequence.

[0056] In a non-limiting embodiment, "the same reading frame" refers to the unit of every three bases of the base sequence that is read when mRNA is translated into protein. "Insertion in the same reading frame" or "insertion in frame" includes inserting the human FIX / FX gene so that the ATG start codon of the endogenous FIX / FX gene of the nonhuman animal coincides with the ATG start codon of the human FIX / FX gene. "Insertion in the same reading frame" or "insertion in frame" also includes inserting the human FIX / FX gene so that an exon-intron structure sequence is added to the 5' side of the ATG start codon of the human FIX / FX gene and the 5' end of the exon-intron structure coincides with the ATG start codon of the endogenous FIX / FX gene of the nonhuman animal.

[0057] This operably links the promoter of the endogenous FIX / FX gene in the non-human animal to the inserted human FIX / FX gene, allowing the human FIX / FX gene to be expressed in response to activation of the promoter. When this configuration is adopted, the exogenous human FIX / FX is expressed under the control of the endogenous FIX / FX expression regulatory system, allowing for the expression of human FIX / FX to be controlled at the same timing and location (tissue) as endogenous FIX / FX. FIX / FX is a gene involved in the blood coagulation cascade. Therefore, the adoption of this configuration is expected to reduce the effects of genetic modification on blood coagulation in non-human animals.

[0058] In another embodiment, when inserting the human FIX / FX gene, in order to prevent the disrupted endogenous FIX / FX gene from being translated again in the original reading frame, it is preferable to delete a sequence of bases from the ATG of the endogenous FIX / FX gene that is not a multiple of 3. Furthermore, in the present invention, it is preferable that the human FIX / FX gene is inserted only into the exon containing the original translation initiation site of the endogenous FIX / FX gene (i.e., homologous recombination occurs only with the target exon of the endogenous FIX / FX gene).

[0059] In a non-limiting embodiment, the human FIX gene possessed by the non-human animal of the present invention comprises a human FIX gene having at least 50%, 60%, or 70%, preferably 75% or more, 80% or more, 85% or more, and more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO: 9. In another embodiment, the human FIX gene includes a polymorphism or mutant thereof. In another embodiment, the human FIX gene may be a gene encoding a human FIX polypeptide having an amino acid insertion, deletion, or conservative amino acid substitution. In one non-limiting embodiment, the human FX gene possessed by the non-human animal of the present invention comprises a human FX gene having at least 50%, 60%, or 70%, preferably 75% or more, 80% or more, 85% or more, and more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO: 10. In another embodiment, the human FX gene comprises a polymorphism or mutant thereof. In another embodiment, the human FX gene may be a gene encoding a human FX polypeptide having an amino acid insertion, deletion, or conservative amino acid substitution.

[0060] In one non-limiting embodiment, the human FIX polypeptide expressed by the non-human animal of the present invention comprises a human FIX polypeptide having at least 50%, 60%, or 70%, preferably 75% or more, 80% or more, 85% or more, and more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO: 1. In another embodiment, the human FIX polypeptide includes polymorphisms or variants thereof. In another embodiment, the human FIX polypeptide may contain amino acid insertions, deletions, or conservative amino acid substitutions. In one non-limiting embodiment, the human FX polypeptide expressed by the non-human animal of the present invention comprises a human FX polypeptide having at least 50%, 60%, or 70%, preferably 75% or more, 80% or more, or 85% or more, and more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to SEQ ID NO: 5. In another embodiment, the human FX polypeptide includes a polymorphism or variant thereof. In another embodiment, the human FX polypeptide may contain amino acid insertions, deletions, or conservative amino acid substitutions. In the present invention, the homology of nucleotide sequences or amino acid sequences can be determined using known algorithms. Algorithms for determining the homology between multiple sequences are known. Sequence homology can be determined by taking synonymous codons into account when comparing nucleotide sequences or by considering the commonality of different amino acid residues when comparing amino acid sequences, or by comparing sequence information purely without considering these factors. The result of the latter comparison can preferably be expressed as identity.

[0061] In a non-limiting embodiment, the non-human animal of the present invention lacks the expression of endogenous FVIII, FIX, and FX polypeptides of the non-human animal and is capable of expressing human FIX and FX polypeptides at physiologically relevant levels. The human FIX / FX gene (including mRNA) inserted into the non-human animal of the present invention or the human FIX / FX polypeptide expressed from the gene can be detected by various methods well known to those skilled in the art, such as PCR, Southern blot, RFLP (Restriction Fragment Length Polymorphism) method, Western blot, IHC, ELISA, etc.

[0062] 4. DNA Construct and Knock-in Vector In one non-limiting aspect, the present invention provides a DNA construct for producing a non-human animal that expresses a human FIX / FX polypeptide, a knock-in vector harboring the DNA construct, and a transformed cell or a progeny cell thereof into which the knock-in vector has been introduced.

[0063] In one non-limiting embodiment, the DNA construct of the present invention includes a DNA construct comprising DNA encoding a human FIX / FX gene, to which an exon-intron structural sequence has been added on the 5' side and a 3' untranslated region of a non-human gene has been added on the 3' side. In another embodiment, the DNA construct of the present invention may further comprise a recombinase substrate sequence (e.g., a loxP sequence, which is a substrate sequence for Cre), a drug selection marker (e.g., the neo gene), and / or other sequences.

[0064] In one non-limiting embodiment, the DNA construct of the present invention comprises a DNA construct containing DNA encoding the human FIX / FX gene, to which a portion of the exon-intron structural sequence of the beta-actin gene and / or beta-globin gene has been added on the 5' side and the 3' untranslated region of a non-human gene has been added on the 3' side. In another embodiment, the DNA construct of the present invention may further comprise a recombinase substrate sequence (e.g., a loxP sequence, which is a substrate sequence for Cre), a drug selection marker (e.g., the neo gene), and / or other sequences.

[0065] In a non-limiting embodiment, the term "exon-intron structure sequence" refers to a sequence containing both exons that are not removed by splicing and introns that are removed by splicing. The exon-intron structure sequence is not particularly limited as long as it contains both exons and introns and the introns are removed by splicing. Examples of the exon-intron structure sequence of the present invention include, but are not limited to, a sequence containing the first exon sequence, the intron sequence, and the second exon sequence of beta-actin, or a sequence containing the second exon sequence, the intron sequence, and the third exon sequence of beta-globin.

[0066] In one non-limiting embodiment, the DNA construct of the present invention includes a DNA construct comprising DNA encoding the human FIX or FX gene, to which a sequence including the first exon sequence, the intron sequence, and the second exon sequence of beta-actin and / or a sequence including the second exon sequence, the intron sequence, and the third exon sequence of beta-globin has been added on the 5' side, and to which a 3' untranslated region of SV40 (e.g., a polyA addition signal sequence) has been added on the 3' side. In another embodiment, the DNA construct of the present invention includes a DNA construct comprising DNA encoding the human FIX / FX gene, to which a sequence including the second exon sequence, the intron sequence, and the third exon sequence of beta-actin and / or a sequence including the second exon sequence, the intron sequence, and the third exon sequence of beta-globin has been added on the 5' side, and to which a 3' untranslated region of SV40 (e.g., a polyA addition signal sequence), a drug selection marker, and a recombinase substrate sequence have been added on the 3' side. The beta-actin and beta-globin are not particularly limited, but are preferably beta-actin and beta-globin derived from the non-human animal. For example, when the genetically modified non-human animal is a mouse, mouse beta-actin and beta-globin are preferred, but other beta-actin and beta-globin (e.g., rabbit beta-globin) may also be used. The SV40 3' untranslated region added to the 3' end preferably contains a poly(A) addition signal. On the other hand, in the knock-in vector of the present invention, the DNA encoding the human FIX / FX gene is typically a cDNA. The human FIX / FX cDNA preferably consists of its coding sequence. For example, the nucleotide sequence shown in SEQ ID NO: 9, which includes the initiation codon (atg) through the termination codon (taa) in the human FIX cDNA, encodes the full-length amino acid sequence of human FIX (461 amino acids including the signal sequence; SEQ ID NO: 1). For example, the base sequence shown in SEQ ID NO: 10 contains the start codon (atg) to the stop codon (tga) in the human FX cDNA and encodes the full-length amino acid sequence of human FX (488 amino acids including the signal sequence; SEQ ID NO: 5).Without being limited by theory, it is believed that in the genetically modified non-human animals of the present invention, splicing occurs due to the introduction of artificial exon / intron sequences, and the addition of a poly(A) addition signal sequence stabilizes mRNA containing the cDNA sequence of human FIX / FX, resulting in the expression of mRNA encoding human FIX / FX at physiologically appropriate levels.

[0067] Examples of the structure of the above DNA constructs include those containing the following nucleotide sequences: 5'-mouse beta globin exon 2 (SEQ ID NO: 11)-intron (SEQ ID NO: 12), exon 3 (SEQ ID NO: 13)-hFIX / FX gene coding sequence (SEQ ID NO: 9 / 10)-SV40 polyA addition signal sequence (SEQ ID NO: 14)-3'; or 5'-mouse beta actin exon 1 (SEQ ID NO: 15)-intron (SEQ ID NO: 16), exon 2 (ttcgcca)-mouse beta globin exon 2 (SEQ ID NO: 11)-intron (SEQ ID NO: 12), exon 3 (SEQ ID NO: 13)-hFIX / FX gene coding sequence (SEQ ID NO: 9 / 10)-SV40 polyA addition signal sequence (SEQ ID NO: 14)-3'. Furthermore, in the above constructs, the 5' upstream sequence of the mouse FIX / FX gene can also be located 5' upstream. The 5' upstream sequence of the mouse FIX / FX gene can be, for example, 1500 bp upstream of the translation initiation site. Similarly, the 3' downstream sequence of the mouse FIX / FX gene can also be located downstream of the above structure. The 3' downstream sequence of the mouse FIX / FX gene can be, for example, 1500 bp downstream of the termination codon.

[0068] In one non-limiting embodiment, the "knock-in vector carrying a DNA construct" of the present invention is a vector capable of inserting a DNA construct for generating a non-human animal into a target gene region in a host by homologous recombination, and the DNA construct for generating a non-human animal has a 5' arm (a nucleotide sequence homologous to the nucleotide sequence 5' upstream of the target region) located on the 5' side and a 3' arm (a nucleotide sequence homologous to the nucleotide sequence 3' downstream of the target region) located on the 3' side. In the present invention, the knock-in vector is constructed so that the DNA construct for generating a non-human animal is inserted into the same reading frame as the target gene in the host. In one embodiment, in the knock-in vector, it is preferable that a foreign gene is inserted into an exon containing the translation start point of the target gene so that its translation start point coincides with the translation start point of the target gene. In this case, it is preferable that a nucleotide sequence upstream of the translation start point of the target gene is located 5' to the translation start point of the foreign gene in the knock-in vector. In another embodiment, when an exon-intron structure sequence is added to the 5' end of any foreign gene in the knock-in vector, the 5' end of the exon-intron structure is preferably inserted into the exon containing the translation start point of the target gene so that the 5' end of the exon-intron structure coincides with the translation start point of the target gene. In this case, it is preferable that the knock-in vector has a nucleotide sequence upstream of the translation start point of the target gene located 5' upstream of the 5' end of the exon-intron structure.

[0069] In addition, the knock-in vector of the present invention preferably has the ability to replicate in host cells. Such a vector can be constructed, for example, by inserting the DNA for producing the non-human animal into a known vector. The knock-in vector is not particularly limited as long as it is a vector used in genetic engineering. Known vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacterial artificial chromosome (BAC) vectors, yeast artificial chromosome (YAC) vectors, retroviral vectors, lentiviral vectors and other viral vectors.

[0070] In one non-limiting embodiment, the "transformed cell into which a knock-in vector has been introduced" of the present invention is a cell into which a knock-in vector carrying DNA for producing the non-human animal has been introduced. In another embodiment, the transformed cell of the present invention is a cell in which DNA encoding a human FIX / FX gene, to which an exon-intron structural sequence has been added on the 5' side and the 3' untranslated region of the non-human gene has been added on the 3' side, has been inserted in the same reading frame as the endogenous FIX / FX gene present in the genome of the non-human animal.

[0071] In yet another embodiment, the transformed cells of the present invention are cells in which DNA encoding a human FIX / FX gene, to which an exon-intron structural sequence has been added at the 5' end and an SV40 poly(A) addition signal sequence has been added at the 3' end, is inserted in the same reading frame as an endogenous FIX / FX gene present in the genome of a non-human animal. The host cells into which the knock-in vector is introduced are cells of the non-human animal or cells (including populations of cells) capable of differentiating into cells of the non-human animal. Various types of cells can be used as host cells depending on the purpose, including pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), germline stem cells capable of differentiating into germ cells such as spermatogonial stem cells, and fertilized eggs. The knock-in vector can be introduced into the host cells by known methods such as electroporation. Alternatively, the present invention provides cells transformed with the knock-in vectors of the present invention, genetically modified non-human animals developed from the cells, and model animals in which cancer cells are transplanted or carcinogenesis is induced into the genetically modified non-human animals. The present invention also relates to methods for evaluating the properties, such as blood coagulation activity, safety, and pharmacokinetics, of antigen-binding substances and test substances for evaluating the therapeutic effect on hemophilia A using these genetically modified non-human animals and model animals.

[0072] 5. Method for Producing a Non-Human Animal Deficient in Expression of Endogenous FVIII, FIX, and FX and Expressing Human FIX and FX In one aspect, the present invention relates to a method for producing a genetically modified non-human animal that is functionally deficient in endogenous FVIII, FIX, and FX genes on its genome and functionally expresses human FIX and FX genes. Such a genetically modified non-human animal can be produced, for example, by mating a genetically modified non-human animal that is functionally deficient in the endogenous FVIII gene on its genome with a genetically modified non-human animal that is functionally deficient in the endogenous FIX gene on its genome and functionally expresses the human FIX gene, and another genetically modified non-human animal that is functionally deficient in the endogenous FX gene on its genome and functionally expresses the human FX gene. In one limited embodiment, the method for producing a genetically modified non-human animal of the present invention comprises the following steps (1) to (4): (1) producing a genetically modified non-human animal in which the endogenous FVIII gene is functionally deleted on the genome and which does not functionally express the human FVIII gene; (2) producing a genetically modified non-human animal in which the endogenous FIX gene is functionally deleted on the genome and which functionally expresses the human FIX gene; (3) producing a genetically modified non-human animal in which the endogenous FX gene is functionally deleted on the genome and which functionally expresses the human FX gene; and (4) mating the genetically modified non-human animals produced in steps (1) to (3).

[0073] In a non-limiting embodiment, a method for producing a non-human animal lacking expression of endogenous FVIII / FIX / FX comprises deleting (disabling) or disrupting the endogenous FVIII / FIX / FX gene. Specifically, the endogenous FVIII / FIX / FX gene may be deleted (disabled) in the genome of the non-human animal using knockout techniques based on genome editing techniques such as homologous recombination, the Cre / loxP system, CRISPR / Cas9, zinc finger nucleases, or TALEN, or a technique may be used to completely suppress expression of the endogenous FVIII / FIX / FX gene using siRNA or the like. For example, the mouse FVIII locus can be disrupted by deleting exons 17 and 18 of mouse FVIII using the Cre-loxP system, resulting in a frameshift in mRNA produced from the disrupted locus, resulting in the loss of expression of functional mouse FVIII polypeptides (Fahs SA et al., Blood, 2014, 123(24), 3706-13.). Alternatively, expression of mRNA encoding (e.g., full-length) endogenous FVIII / FIX / FX polypeptides can be disrupted by knocking in an exogenous polynucleotide sequence into the endogenous FVIII / FIX / FX locus.

[0074] In the production methods of the present invention, a foreign gene may be inserted into the FIX / FX gene position in the genome of a non-human animal, for example, using knock-in technology, as long as endogenous FIX / FX polypeptides are not expressed. In a non-limiting embodiment, the method for producing a genetically modified non-human animal of the present invention comprises deleting the first exon of mouse FIX (e.g., from the initiation codon ATG to a portion of the intron region) and inserting a human FIX gene in that position. In a specific embodiment, the inserted human FIX gene is a human FIX cDNA. In a specific embodiment, the inserted human FIX gene has an exon-intron structure sequence of a non-human gene (e.g., an exon-intron structure or sequence derived from the mouse beta-actin gene and an exon-intron structure sequence derived from the mouse beta-globin gene) added to the 5' side, and a 3'UTR sequence of a non-human gene (e.g., an SV40 polyA addition signal sequence) added to the 3' side of the human FIX gene. In one non-limiting embodiment, the method of the present invention for producing a genetically modified non-human animal comprises deleting the second exon of mouse FX from the initiation codon ATG onwards (e.g., from the initiation codon ATG to a portion of the intron region) and inserting the human FX gene in its place. In a specific embodiment, the inserted human FX gene is a human FX cDNA. In a specific embodiment, the inserted human FX gene has an exon-intron structural sequence of a non-human gene (e.g., an exon-intron structure or sequence derived from the mouse beta-actin gene and an exon-intron structural sequence derived from the mouse beta-globin gene) added to its 5' side, and a 3' UTR sequence of a non-human gene (e.g., an SV40 poly(A) addition signal sequence) added to its 3' side.

[0075] In one non-limiting embodiment, a method for producing a non-human animal that expresses human FIX / FX polypeptide comprises introducing a knock-in vector carrying DNA encoding the human FIX / FX gene into a host cell. The method for introducing the knock-in vector carrying DNA encoding the human FIX / FX gene is not particularly limited, and any known method can be used, such as microinjection of the knock-in vector into the pronucleus of a fertilized egg, or introduction into pluripotent stem cells such as ES cells and iPS cells, or germline stem cells such as spermatogonial stem cells, by electroporation, lipofection, viral infection, or transformation.

[0076] In one non-limiting embodiment, the knock-in vector is injected into a host cell together with an artificial nuclease such as a Zinc Finger Nuclease (ZFN) or a Transcription Activator-like Effector Nuclease (TALEN), which binds to and cleaves a target sequence specific to a target region in the genome, or Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) / Cas9.

[0077] In one non-limiting embodiment, chimeric animals can be obtained by injecting pluripotent stem cells into early embryos using known methods such as microinjection, and then transplanting the resulting embryos into foster parents for development. Furthermore, by breeding these chimeric animals, individuals homozygous for the knock-in allele can be obtained from their progeny. In another embodiment, when germline stem cells are used, knock-in animals can be produced by introducing a knock-in vector into cells undergoing targeted recombination using known methods, transplanting the cells into the gonads of an animal, and allowing them to differentiate into germ cells. The resulting animals can then be mated, or germ cells collected from the animal can be used to produce knock-in animals (Kanatsu-Shinohara, M. et al. (2008) Biol. Reprod. 79, 1121-1128).

[0078] In another embodiment, when using fertilized eggs, knock-in animals can be produced by implanting fertilized eggs injected with a knock-in vector together with artificial nucleases into a foster mother and allowing the fertilized eggs to develop. Methods using ZFN and TALEN are described in the literature (Cui, X. et al. (2011) Nat. Biotechnol. 29, 64-67) and (Li, T. et al. (2011) Nucleic Acids Res. 39, 6315-6325), respectively. Methods using CRISPR / Cas9 are described in the literature (Yang, H. et al. (2013) Cell. in press.). Furthermore, in another embodiment, it is possible to inject a knock-in vector into the testis or ovary of an animal, directly modify the germ cells by techniques such as electroporation, and then obtain individuals carrying the knock-in allele by mating (Niu, Y. et al. (2008) J. Genet. Genomics. 35, 701-714).

[0079] In the present invention, genetically modified non-human animals can be homozygous (having both knock-in alleles) or hemizygous (having one copy of the knock-in allele). Homozygous animals are preferred for stable preservation of modified traits during breeding. Because FIX is located on the X chromosome, females (XX) can be homozygous, but males (XY) will be hemizygous.

[0080] Alternatively, the present invention relates to a method for producing a non-human animal that expresses human FIX / FX polypeptide, comprising the following steps: (A) introducing the knock-in vector of the present invention into stem cells of a non-human animal to integrate a gene encoding human FIX / FX into the endogenous FIX / FX allele in the stem cell genome of the non-human animal; (B) transplanting the non-human animal stem cells of step (A) into an early embryo of the same non-human animal; (C) transplanting the early embryo of step (B) into the uterus of a surrogate non-human animal to allow it to develop, thereby obtaining a chimeric non-human animal having a genome in which the knock-in vector has been introduced into somatic cells; and (D) breeding the chimeric animal of step (C) to obtain from its progeny individuals in which the knock-in allele is homozygous.

[0081] Alternatively, the present invention relates to a method for producing a non-human animal that expresses human FIX / FX polypeptide, comprising the following steps: (A) introducing the knock-in vector of the present invention into a fertilized egg of a non-human animal to integrate a gene encoding human FIX / FX into the endogenous FIX / FX allele in the genome of the fertilized egg of the non-human animal; (B) implanting the fertilized egg of the non-human animal obtained in step (A) into the uterus of a foster non-human animal to allow development, thereby obtaining a non-human animal having a genome in which the knock-in vector has been introduced into somatic cells; and (C) breeding the non-human animal obtained in step (B) to obtain from its progeny individuals in which the knock-in allele is homozygous. The knock-in vector of the present invention typically contains an expression cassette that carries human FIX / FX cDNA in an expressible state and contains the human FIX / FX cDNA and its expression control region, and is sandwiched between recombination regions for integration into the endogenous FIX / FX allele in the genome of the non-human animal.

[0082] 6. Method for Evaluating Test Substances Using Non-Human Animals Expressing Human FIX / FX Polypeptides In one non-limiting embodiment, the non-human animals of the present invention can be used for various evaluations of test substances, such as their safety, therapeutic efficacy for diseases, pharmacokinetics, and biodistribution. Accordingly, the present invention also provides a method for evaluating test substances using such non-human animals of the present invention. In another embodiment, the present invention includes non-human animals used for various evaluations and / or screenings of test substances, such as their safety, therapeutic efficacy for diseases, pharmacokinetics, and biodistribution. In yet another embodiment, the present invention also includes use of the non-human animals of the present invention for various evaluations and / or screenings of test substances, such as their safety, therapeutic efficacy for diseases, pharmacokinetics, and biodistribution.

[0083] The "test substance" used in the evaluation method of the present invention is not particularly limited and may include, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, etc. Preferably, it is a bispecific antibody against human FIX / FX, such as known antibodies described in WO2005 / 035754, WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, WO2017 / 110980, WO2018 / 021450, etc. Test substances can be administered to non-human animals via, for example, tail vein administration, subcutaneous administration, intraperitoneal administration, oral administration, nasal administration, transdermal administration, pulmonary administration, etc., but are not limited to these.

[0084] Examples of evaluation of test substances include the following. For example, the safety of a test substance in a living body can be evaluated by administering the test substance to a non-human animal of the present invention and measuring the plasma concentration of cytokines (e.g., including, but not limited to, IFN-gamma, IL-10, IL-17, IL-2, IL-4, IL-6, and TNF). More specifically, a difference between the cytokine level and the level of the same cytokine in a control indicates a safety risk of the test compound. Here, "safety risk" refers to the prediction of a biological response that may be detrimental to the living body due to a change in cytokine level. The in vivo roles of cytokines such as IFN-gamma, IL-10, IL-17, IL-2, IL-4, IL-6, and TNF, as exemplified above, are well known, and a wealth of information has been accumulated regarding the relationship between changes in each cytokine level and biological responses in disease treatment. Therefore, those skilled in the art can predict safety risks suggested by changes in cytokine levels. In this case, if the expression level of the human FIX / FX gene in the liver or blood of the non-human animal is equivalent to that of mice, monkeys, or humans, it will be possible to extrapolate the results of safety tests using the non-human animal to mice, monkeys, or humans and predict the effects in mice, monkeys, or humans.

[0085] Furthermore, the therapeutic effect of a test substance can be evaluated by administering the substance to a non-human animal of the present invention expressing a human FIX / FX polypeptide and measuring its FVIII cofactor substituting activity or blood coagulation activity. Here, the therapeutic effect refers to the therapeutic effect on bleeding, bleeding-associated diseases, or bleeding-related diseases, and may also be referred to as the therapeutic effect on hemophilia A, blood coagulation effect, or hemostatic effect. The FVIII cofactor substituting activity in a non-human animal can be evaluated, for example, by a colorimetric FIXa-based FX activation reaction test, an activated partial thromboplastin time (APTT) test, or a thrombin generation test using plasma obtained from the non-human animal. The non-human animal of the present invention lacks expression of functional FVIII polypeptide and expresses human FIX / FX polypeptide at physiologically relevant levels. Therefore, the use of the non-human animal of the present invention as a model enables more accurate and convenient evaluation of the human FVIII cofactor substituting activity of a test substance ex vivo or in vivo.

[0086] Therefore, the present invention provides an animal model for evaluating the therapeutic, blood coagulation, or hemostatic effects of a test compound on hemophilia A. The animal model for evaluating the therapeutic, blood coagulation, or hemostatic effects of a test compound on hemophilia A based on the present invention can be produced using the genetically modified non-human animal of the present invention. Specifically, the present invention relates to a method for producing a model animal for evaluating the therapeutic effect, blood coagulation effect, or hemostatic effect of a test compound on hemophilia A, comprising the following steps: (A) introducing the knock-in vector of the present invention into stem cells of a non-human animal to integrate a gene encoding human FIX / FX into the endogenous FIX / FX allele in the stem cell genome of the non-human animal; (B) transplanting the non-human animal stem cells of step (A) into an early embryo of the same non-human animal; (C) transplanting the early embryo of step (B) into the uterus of a surrogate non-human animal to allow it to develop, thereby obtaining a chimeric non-human animal having a genome in which the knock-in vector has been introduced into somatic cells; (D) breeding the chimeric animal of step (C) to obtain from its offspring individuals in which the knock-in allele is homozygous; and (E) mating the homozygous individual of step (D) with a genetically modified non-human animal that is deficient in the expression of endogenous FVIII.

[0087] The present invention also provides another model animal for evaluating the therapeutic, blood coagulation, or hemostatic effects of a test compound on hemophilia A. The model animal for evaluating the therapeutic, blood coagulation, or hemostatic effects of a test compound on hemophilia A based on the present invention can be produced using the genetically modified non-human animal of the present invention. Specifically, the present invention relates to a method for producing a model animal for evaluating the therapeutic effect, blood coagulation effect, or hemostatic effect of a test compound for hemophilia A, comprising the following steps: (A) introducing the knock-in vector of the present invention into a fertilized egg of a non-human animal to integrate a gene encoding human FIX / FX into the endogenous FIX / FX allele in the genome of the fertilized egg of the non-human animal; (B) implanting the fertilized egg of the non-human animal of step (A) into the uterus of a surrogate non-human animal to allow it to develop, thereby obtaining a non-human animal having a genome in which the knock-in vector has been introduced into somatic cells; and (C) breeding the non-human animal of step (B) to obtain from its offspring individuals in which the knock-in allele is homozygous; and (D) mating the homozygous individual of step (C) with a genetically modified non-human animal that is deficient in the expression of endogenous FVIII.

[0088] Furthermore, the effective blood concentration or pharmacokinetic properties of a test substance can be evaluated by measuring the blood concentration of the test substance in a non-human animal of the present invention administered with the test substance. Here, "pharmacokinetic properties" refers to characteristics of the test substance in the animal's body, such as the blood half-life and elimination rate. For example, in a non-limiting embodiment, a substance with a lower effective blood concentration, a longer blood half-life, or a slower elimination rate is considered to have better pharmacokinetic properties. The method for measuring the blood concentration of a test substance is not particularly limited. For example, when the test substance is a protein (including an antibody), ELISA is an example, and when the test substance is a low-molecular-weight compound, liquid chromatography-mass spectrometry (LC-MS) is an example. For methodologies for evaluating pharmacokinetic properties from blood concentrations, see Igawa et al. (2010) Nat. Biotechnol. 28:1203-1207. By utilizing the above-described method for evaluating test substances of the present invention, it is possible to efficiently select human FIX / FX-targeted therapeutic agents having desired activities. Therefore, the present invention also provides a method for selecting such antibodies.

[0089] Furthermore, by observing the biodistribution of a test substance in a non-human animal of the present invention administered with the test substance, the degree to which the test substance reaches the target site and its biodistribution characteristics can be evaluated. For example, a human FIX / FX targeted therapeutic drug conjugated with an imaging agent can be administered to a non-human animal of the present invention as a test substance, and the biodistribution of the test substance can be observed by detecting the imaging agent. Radionuclides used for imaging include, but are not limited to, I-131, I-123, In-111, and Tc-99m for SPECT imaging, and F-18, I-124, Cu-64, and Y-86 for PET imaging. Furthermore, the non-human animal of the present invention can also be used for live imaging of the blood coagulation process.

[0090] In a non-limiting aspect, the present invention further provides a method for screening for a therapeutic agent for hemophilia A, comprising the following steps (1) and (2): (1) administering a test substance to the non-human animal of the present invention; and (2) selecting a candidate test substance using either or both of the efficacy and toxicity of the test substance in the non-human animal as an index.

[0091] In one non-limiting embodiment, the efficacy (therapeutic effect) and / or safety (toxicity) of a test substance can be measured in a non-human animal of the present invention, and a test substance with recognized or high efficacy can be selected, or a test substance with low or no toxicity can be selected. Alternatively, a similar measurement can be performed using a substance with pharmacological efficacy as a control, and a test substance with higher efficacy or lower toxicity than the control can be selected. Examples of pharmacological efficacy include, but are not limited to, blood coagulation effects and hemostatic effects.

[0092] For example, the present invention provides a method for screening for a therapeutic drug for hemophilia A, comprising the following steps (1) to (3): (1) administering a bispecific antibody comprising a human FIX / FX-binding domain as a test substance to a first individual non-human animal of the present invention, (2) measuring one or more of the blood coagulation effect, safety, and pharmacokinetic properties of the test substance in the non-human animal, and (3) comparing one or more of the blood coagulation effect, safety, and pharmacokinetic properties of the test substance with one or more of the blood coagulation effect, safety, and pharmacokinetic properties of a control antibody administered to a second individual non-human animal of the present invention that is different from the first individual. The screening step of the present invention can additionally comprise (4) selecting a test substance that exhibits superior one or more of the blood coagulation effect, safety, and pharmacokinetic properties as a result of step (3). In the present invention, the blood coagulation effect, safety, and pharmacokinetic properties of the control antibody do not necessarily have to satisfy all of the properties simultaneously. Rather, by repeating the process of screening candidates using substances that have received a certain level of evaluation in one of the properties, it is possible to efficiently narrow down the test substances to those that have superior effects in multiple properties.

[0093] By utilizing the above-described method for evaluating a test substance of the present invention, it is possible to efficiently select anti-human FIX / FX bispecific antibodies having desired activities. Therefore, the present invention also provides a method for selecting such antibodies.

[0094] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0095] Example 1: Generation of FVIII-deficient, human FIX-introduced, and human FX-introduced mice (1) Design of various gRNAs The CRISPR / Cas9 system was used to generate FVIII-deficient, human FIX-introduced, and human FX-introduced mice. The gRNAs were designed as follows: i. FVIII region: To delete exon 17 and exon 18 of the mouse FVIII gene, mFVIII_ex16-17_1 (SEQ ID NO: 21) and mFVIII_ex18-19_1 (SEQ ID NO: 22) were designed for intron 16 and intron 18 of the mouse FVIII gene, respectively. ii. FIX region: To introduce a sequence containing the human FIX cDNA sequence into the mouse FIX gene region, mF95_4 (SEQ ID NO: 23) was designed for exon 1 of the mouse FIX gene, and mF95_5 (SEQ ID NO: 24) was designed for the region spanning exon 1 to intron 1. iii. FX Region In order to introduce a sequence containing the human FX cDNA sequence into the mouse FX gene region, mF105_2 (SEQ ID NO: 25) was designed in the region spanning from exon 3 to intron 3 of the mouse FX gene.

[0096] (2) Vector Construction: Targeting vectors were constructed to generate human FIX- and human FX-transfected mice. i. Construction of a vector for generating human FIX-transfected mice: The following nucleic acid sequences were cloned into pBluescript SK(-) as a base: mouse FIX gene sequence (SEQ ID NO: 28 and SEQ ID NO: 29), mouse beta-actin exon 1 (SEQ ID NO: 15)-intron (SEQ ID NO: 16), exon 2 (SEQ ID NO: 17), mouse beta-globin exon 2 (SEQ ID NO: 11)-intron (SEQ ID NO: 12), exon 3 (SEQ ID NO: 13), hFIX gene coding sequence (SEQ ID NO: 9), and SV40 polyA addition signal sequence (SEQ ID NO: 14). ii. Construction of a vector for generating human FX-transfected mice: The following nucleic acid sequences were cloned into pBluescript SK(-) as a base: Mouse FX gene sequence (SEQ ID NO: 30 and SEQ ID NO: 31), mouse beta-actin first exon (SEQ ID NO: 15)-intron (SEQ ID NO: 16), second exon (SEQ ID NO: 17), mouse beta-globin second exon (SEQ ID NO: 11)-intron (SEQ ID NO: 12), third exon (SEQ ID NO: 13), hFX gene coding sequence (SEQ ID NO: 10), and SV40 polyA addition signal sequence (SEQ ID NO: 14).

[0097] (3) Generation of Various Genetically Modified Mice Using the designed gRNA and constructed vectors described above, FVIII-deficient, human FIX-transfected, and human FX-transfected mice were generated. i. FVIII-Deficient Mice Because FVIII is encoded on the same X chromosome as FIX, it is difficult to obtain FVIII-deficient, human FIX-transfected mice by crossbreeding. Therefore, experiments were performed using pronuclear fertilized eggs of previously generated human FIX-transfected mice. The mFVIII_ex16-17_1 (FASMAC, SEQ ID NO: 21) and mFVIII_ex18-19_1 (FASMAC, SEQ ID NO: 22) designed in (1) above, tracrRNA (FASMAC, SEQ ID NO: 26), Cas9 protein (NIPPON GENE, Code No. 316-08651), and FVIII oligo (Eurofins, SEQ ID NO: 27) were dissolved in Opti-MEM I Reduced Serum medium (Thermo Fisher Scientific, 31985062) to prepare a nucleic acid mixture solution. The final concentrations of mFVIII_ex16-17_1, mFVIII_ex18-19_1, tracrRNA, Cas9 protein, and FVIII oligo were adjusted to 45 ng / μL, 150 ng / μL, 50 ng / μL, and 100 ng / μL, respectively. Human FIX-transfected mouse pronuclear zygotes were cultured in this nucleic acid mixture, and the RNA / Cas9 complex (RNP) was introduced into the pronuclear zygotes from human FIX-transfected mice using electroporation (NEPAGENE, NEPA21). After RNP transfection, the zygotes were cultured overnight, and the resulting 2-cell embryos were transferred into the oviducts of 0.5-day-old pseudopregnant ICR recipient females. Founder pups were obtained 19 days later.

[0098] ii. Generation of human FIX-transfected mice. The targeting vectors prepared in (2) were purified by phenol-chloroform extraction and used in the experiments. The mF95_4 (FASMAC, SEQ ID NO: 23) and mF95_5 (FASMAC, SEQ ID NO: 24) designed in (1), tracrRNA (FASMAC, SEQ ID NO: 26), and Cas9 protein (NIPPON GENE, Code No. 316-08651) were dissolved in buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0)) to prepare a nucleic acid mixture solution. The buffer was prepared using 1 M Tris-HCl (pH 8.0) and 500 mM EDTA (pH 8.0). The final concentrations of mF95_4, mF95_5, tracrRNA, Cas9 protein, and targeting vector were adjusted to 0.3 pmol / μL, 0.61 pmol / μL, 30 ng / μL, and 10 ng / μL, respectively. This nucleic acid mixture was microinjected into pronuclear fertilized eggs derived from C57BL / 6J Jcl. The microinjected fertilized eggs were cultured overnight, and the resulting two-cell embryos were transferred into the oviducts of 0.5-day-old pseudopregnant ICR recipient females. Founder offspring were obtained 19 days later.

[0099] iii. Generation of human FX-transfected mice. The targeting vector prepared in (2) was purified by phenol-chloroform extraction and used in the experiment. The mf105_2 (FASMAC, SEQ ID NO: 25) and tracrRNA (FASMAC, SEQ ID NO: 26) designed in (1) and Cas9 protein (NIPPON GENE, Code No. 316-08651) were dissolved in buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0)) to prepare a nucleic acid mixture solution. The buffer was prepared using 1 M Tris-HCl (pH 8.0) and 500 mM EDTA (pH 8.0). The final concentrations of mf105_2, tracrRNA, Cas9 protein, and targeting vector were adjusted to 0.61 pmol / μL, 0.61 pmol / μL, 30 ng / μL, and 5 ng / μL, respectively. The nucleic acid mixture was microinjected into pronuclear-stage fertilized eggs derived from C57BL / 6J Jcl. The microinjected fertilized eggs were cultured overnight, and the resulting 2-cell embryos were transferred into the oviducts of 0.5-day-old pseudopregnant ICR recipient females. Founder offspring were obtained 19 days later.

[0100] (4) Selection of founder mice Ear samples were collected from the obtained founder offspring, DNA was extracted, and PCR was performed to select offspring having the desired genotype.

[0101] i. Selection of FVIII-deficient mice. A DNA solution was prepared using Cell Lysis Solution (QIAGEN, 158906) and Proteinase K, recombinant, PCR grade (Roche, 03115828001). Pups with the desired genotype were selected by PCR using the prepared DNA solution. The PCR reaction mixture consisted of 1 μL of DNA sample prepared from founder pups, 12.5 μL of 2x GC buffer I, 2.5 μL of dNTP mix, 0.2 μL each of two primers (50 μM each), 0.25 μL of LA Taq (TAKARA), and 8.35 μL of distilled water (total volume: 25 μL). PCR conditions included 35 cycles of 96°C for 10 seconds, 55°C for 15 seconds, and 72°C for 3 minutes. The primers used were mFVIII 121710- (SEQ ID NO: 32) and mFVIII -124827 (SEQ ID NO: 33). As shown in Figure 1, when the FVIII gene was intact, a 3117-bp band was amplified, and when the FVIII gene was deleted, a band corresponding to the deleted region was amplified.

[0102] ii. Selection of Human FIX-Transgenic Mice. A DNA solution was prepared using Maxwell-16 (Promega). PCR was performed using the prepared DNA solution to select offspring with the desired genotype. The PCR reaction mixture consisted of 1 μL of DNA sample prepared from founder offspring, 5 μL of 5x buffer, 2 μL of dNTP mix, 0.2 μL each of two primers (50 μM each), 0.5 μL of PrimeSTAR GXL (TAKARA), and 16.1 μL of distilled water (total volume: 25 μL). The PCR conditions were 98°C for 10 seconds, 55°C for 15 seconds, and 68°C for 7 minutes (35 cycles). PCR was performed separately on the 5' and 3' ends of the gene to detect homologous recombination. The primers used were as follows: mFIX-1705F (SEQ ID NO: 34) was engineered 5' from the homologous region cloned into the targeting vector, and mFIX 1947R (SEQ ID NO: 35) was engineered 3' from the homologous region cloned into the targeting vector. hFIX 336F (SEQ ID NO: 36) and hFIX cDNA R1 (SEQ ID NO: 37) were engineered within the human FIX cDNA sequence. PCR was performed to detect homologous recombination at the 5' end using mFIX-1705F (SEQ ID NO: 34) and hFIX cDNA R1 (SEQ ID NO: 37). As shown in Figure 2, a 4837-bp band was amplified in the presence of the desired genotype. PCR to detect homologous recombination at the 3' end was performed using hFIX 336F (SEQ ID NO: 36) and mFIX 1947R (SEQ ID NO: 35). As shown in Figure 3, a 3145-bp band was amplified in the presence of the desired genotype.

[0103] iii. Selection of Human FX-Transgenic Mice. A DNA solution was prepared using Maxwell-16 (Promega). PCR was performed using the prepared DNA solution to select offspring with the desired genotype. The PCR reaction mixture consisted of 1 μL of DNA sample prepared from founder offspring, 5 μL of 5x buffer, 2 μL of dNTP mix, 0.2 μL each of two primers (50 μM each), 0.5 μL of PrimeSTAR GXL (TAKARA), and 16.1 μL of distilled water (total volume: 25 μL). PCR conditions consisted of 35 cycles of 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 5 minutes. The primers used to detect each genotype are listed in the table below. PCR was performed separately on the 5' and 3' ends of the gene to detect homologous recombination. The primers used were as follows: mFX-2055 F (SEQ ID NO: 38) was engineered 5' from the homologous region cloned into the targeting vector, and mFX 1676R (SEQ ID NO: 39) was engineered 3' from the homologous region cloned into the targeting vector. hFX 61F (SEQ ID NO: 40) and hFX 1227R (SEQ ID NO: 41) were engineered within the human FIX cDNA sequence. PCR was performed to detect homologous recombination at the 5' end using mFX-2055 F (SEQ ID NO: 38) and hFX 1227R (SEQ ID NO: 41). As shown in Figure 4, a 5046 bp band was amplified in the presence of the desired genotype. PCR was performed to detect homologous recombination at the 3' end using hFX 61F (SEQ ID NO: 40) and mFX 1676R (SEQ ID NO: 39). As shown in Figure 5, a 3244 bp band was amplified in the presence of the desired genotype.

[0104] (5) Selection of Mouse Lines Expression analysis and protein function analysis were performed on the selected founder mice to select mouse lines expressing the target protein.

[0105] i. Selection of FVIII-deficient mice After sexual maturity, the founder mice were mated with human FIX-transduced mice to confirm the transmission of mouse FVIII deficiency to the next generation of mice. The PCR method used for confirmation was the same as that used for the selection of founder mice described above.

[0106] ii. Selection of human FIX-transfected mice. Blood was collected from the tail vein of founder mice and the concentration and activity of human FIX were measured. Human FIX concentrations in human FIX-transfected mice were measured by electrochemiluminescent (ECL) bridging immunoassay. Mouse plasma samples diluted 1:1000 were incubated with a solution containing 0.5 μg / ml biotin-labeled anti-FIX antibody and 1.5 μg / ml ruthenium-labeled anti-FIX antibody. The solution was added to a streptavidin-immobilized plate, and ECL was detected using a MESO SECTOR S 600 Reader (Meso Scale Diagnostics, LLC.). Based on the detection results, the human FIX concentrations in the plasma of human FIX-transfected mice were calculated. Plasma FIX activity (FIX:C) was measured using a synthetic substrate method using Rebohem FIX synthetic substrate (Sysmex), standard human plasma for blood coagulation tests (Sysmex), and a CS-2400 (Sysmex) measuring instrument, and FIX activity was confirmed. Based on this, it is believed that the human FIX-transfected mice express active human FIX. After sexual maturity, the mice were crossed with C57BL / 6J Jcl mice, and transmission of the human FIX gene to the next generation of mice was confirmed. The PCR method used for confirmation was the same as that used for selecting the founder mice described above.

[0107] iii. Selection of human FX transgenic mice. Blood was collected from the tail vein of founder mice and human FX concentration and activity were measured. Human FX concentrations in human FX transgenic mice were measured by ECL bridging immunoassay. Plasma diluted 1:1000 was incubated with a solution containing 0.5 μg / ml biotin-labeled anti-FX antibody and 1.5 μg / ml ruthenium-labeled anti-FX antibody. The solution was added to a streptavidin-immobilized plate, and ECL was detected using a MESO SECTOR S 600. Based on the detection results, human FX concentrations in the plasma of human FX transgenic mice were calculated. Plasma FX activity (FX:C) was measured by a one-stage coagulation assay using a Thrombocheck PT (Sysmex) and standard human plasma for blood coagulation testing on a CS-2400 analyzer. FX activity was confirmed. These results suggest that human FX transgenic mice express active human FX. After sexual maturity, the offspring were crossed with C57BL / 6J Jcl mice, and the transmission of the human FX gene to the offspring was confirmed using the same PCR method as in the selection of the founder mice described above.

[0108] (6) Hybridization: The resulting offspring were crossbred to obtain hybridized offspring. Ear samples were collected from the offspring, and DNA solutions were prepared using Maxwell-16 (Promega) and 50 mM sodium hydroxide solution. PCR was performed using the prepared DNA solutions to select offspring with the genotypes of FVIII deficiency, human FIX transduction, and human FX transduction. The PCR reaction mixture consisted of 1 μL of DNA sample prepared from the offspring, 5 μL of KOD One PCR Master Mix, 0.06 μL each of three primers (50 μM each), and 3.82 μL of distilled water (total 10 μL). PCR conditions consisted of 30 cycles of 98°C for 10 seconds, 60°C for 5 seconds, and 68°C for 1 second. The primers used and the lengths of the amplified bands are as follows: PCR to determine FVIII was performed using mFVIII Fw1 (SEQ ID NO: 42), mFVIII WT Rv1 (SEQ ID NO: 43), and mFVIII KO Rv1 (SEQ ID NO: 44). As shown in Figure 6, an 855-bp band was amplified for the wild-type allele, and a 520-bp band was amplified for the KO allele. PCR to determine FIX was performed using hFIX KI -1945F (SEQ ID NO: 45), hFIX KI -1334R (SEQ ID NO: 46), and hFIX KI WTR1_1 (SEQ ID NO: 47). As shown in Figure 7, a 273-bp band was amplified for the wild-type allele, and a 612-bp band was amplified for the KI allele. PCR to determine FX was performed using hFX KI v5 -105F (SEQ ID NO: 48), hFX KI v5 88R (SEQ ID NO: 49), and hFX KI v5 429R (SEQ ID NO: 50). As shown in FIG. 8, a 193-bp band was amplified for the wild-type allele, and a 534-bp band was amplified for the KI allele.

[0109] (7) Expression Analysis. Using the obtained mice with the genotypes of FVIII-deficient, human FIX-transfected, and human FX-transfected, human FVIII, FIX, and FX were used to measure the concentrations of human FIX and human FX, as well as the activities of mouse FVIII, FIX, and FX. Five male and five female mice with the genotypes of FVIII-deficient, human FIX-transfected, and human FX-transfected were used. Human FIX and FX concentrations in each mouse model were measured by ECL bridging immunoassay. A 1000-fold diluted plasma sample was incubated with a solution containing 0.5 μg / ml of biotin-labeled anti-FIX or biotin-labeled anti-FX antibody and 1.5 μg / ml of ruthenium-labeled anti-FIX or ruthenium-labeled anti-FX antibody. The solution was applied to a streptavidin-immobilized plate, and ECL was detected using a MESO SECTOR S 600. The plasma human FIX concentrations in mice with FVIII deficiency, human FIX transfection, and human FX transfection were 4598.7 ± 2789.7 (ng / mL) in males and 6388.7 ± 1076.3 (ng / mL) in females, and human FX concentrations were 32591.5 ± 4391.4 (ng / mL) in males and 35357.2 ± 2355.4 (ng / mL) in females. Plasma FVIII activity (FVIII:C, aPTT basis), FIX activity (FIX:C, aPTT basis), and FX activity (FX:C, PT basis) were measured by a one-stage coagulation assay using a CS-2400 (Sysmex) using FVIII-deficient plasma (SIEMENS), FIX-deficient plasma (SIEMENS), and FX-deficient plasma (SIEMENS), respectively. In the above measurements, ThrombocheckAPTT-SLA was used for aPTT-based analysis, and ThrombocheckPT was used for PT-based analysis. Plasma FIX activity in FVIII-deficient, human FIX-transfected, and human FX-transfected mice was 319.1 ± 188.8% (males) and 435.2 ± 46.8% (females), respectively. FX activity was 279.9 ± 101.9% (males) and 446.7 ± 27.4% (females). FVIII activity was not measured.

[0110] A thrombin generation assay (TGA) was performed using four male mice with FVIII deficiency, human FIX transfection, and human FX transfection. Plasma samples were collected from the control group (no addition), the rhFVIII group (0.1, 0.3, or 1 IU / mL recombinant human FVIII), and the rhFVIII group (50 μg / mL emicizumab (Emi)). TGs in mouse plasma were measured according to the method reported by Tchaikovski et al. (J Thromb Haemost. 2007;5(10):2079-86.) with minor modifications. The assay was performed in a 96-well plate. Each well contained 60 μl of the final reaction mixture. First, 20 μL of trigger reagent (10.8 μmol / L ellagic acid (Elg), 24 μg / mL phospholipid (PL, 10% phosphatidylserine, 60% phosphatidylcholine, 30% phosphatidylethanolamine) was added to each well and thoroughly mixed with 20 μL of mouse plasma. The plate was placed in a Fluoroskan Ascent microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) and heated at 33°C for 10 minutes. At the start of the measurement, 20 μL of substrate solution was dispensed into each well. The development of fluorescent signal was monitored at 10-second intervals for 60 minutes. A calibration curve was created using a thrombin calibrator (Diagnostica Stago). Peak thrombin (Th-peak) was calculated using Thrombinoscope software. All values ​​are expressed as mean + standard deviation (SD) (Fig. 9). Statistical analysis was performed using JMP 15.0.0 (SAS The Institute t-test and Dunnett's test were used, and a P value of <0.05 was considered statistically significant. The 50 μg / mL emicizumab group showed a significant increase compared to the control group, and the rhFVIII group showed a significant increase compared to the control group.

[0111] The present invention provides genetically modified non-human animals that lack expression of the endogenous FVIII / FIX / FX genes and express the human FIX / FX genes at physiologically appropriate levels, methods for producing such non-human animals, and methods for evaluating test substances using such non-human animals. The genetically modified non-human animals of the present invention express the human FIX / FX genes in their blood at levels equivalent to those of healthy humans. Therefore, the genetically modified non-human animals of the present invention enable more accurate evaluation than conventional methods, particularly of the efficacy of compounds that substitute for the function of human blood coagulation factor VIII.

Claims

A genetically modified non-human animal in which endogenous FVIII, FIX, and FX genes are functionally deleted on the genome and which functionally expresses human FIX and FX genes, A genetically modified non-human animal, in which, in addition to regions encoding human FIX and FX proteins, a gene region including the exon and intron sequences of the beta-actin (ACTB) gene of the non-human animal, the exon and intron sequences of the beta-globin (Hbb-bs) gene of the non-human animal, or the 3' untranslated region of SV40 has been inserted as the human FIX and FX gene region.

2. The genetically modified non-human animal of claim 1, wherein human FX cDNA is inserted as the region encoding the human FX protein. 　 The genetically modified non-human animal of claim 2, wherein the non-human animal is a non-human mammal. 　 A method for measuring the blood coagulation ability of a test substance, comprising the following steps (1) and (2): (1) administering a test substance to the genetically modified non-human animal according to any one of claims 1 to 3; and (2) A step of collecting a biological sample from the genetically modified non-human animal after administration of the test substance in step (1), and measuring blood coagulation ability using the biological sample. A method for predicting the blood coagulation ability of a test substance in humans, comprising the following steps (1) and (2): (1) measuring the blood coagulation ability of the test substance in a genetically modified non-human animal by the method of claim 4; and (2) A step of predicting the blood coagulation ability of the test substance when administered to a human, based on the blood coagulation ability of the test substance in the genetically modified non-human animal measured in (1) above. 　 A screening method for a candidate substance that exhibits blood clotting ability in humans, comprising the steps of measuring the blood clotting ability of the test substance in a genetically modified non-human animal using the method described in claim 4, and selecting the test substance that exhibits blood clotting ability in the genetically modified non-human animal as a candidate substance that exhibits blood clotting ability in humans.

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