Recombinant coagulation factor VIII and its uses

Abstract

A recombinant coagulation factor VIII and its application uses are provided. The recombinant coagulation factor VIII contains more than 80% of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. Through genetic modification of the B domain, the recombinant coagulation factor VIII is rich in glycosylation sites, has good coagulation function, is easily secreted outside the cell, and easily interacts with cofactors such as thrombin (EIIa). In addition, the recombinant coagulation factor VIII has a weak ability to induce antibody response, which can effectively realize the therapeutic effect, reduce the risk of immune rejection, and reduce the treatment cost.

Description

[Technical field]

[0001] This application claims priority to Chinese Patent Application No. CN202110455812.5, filed on April 26, 2021, the disclosure of which is incorporated by reference herein in its entirety.

[0002] The present application is in the field of biotechnology and relates to recombinant coagulation factor VIII and uses thereof. [Background technology]

[0003] Hemophilia A (HA), also known as hereditary antihemophilic globulin deficiency or FVIII deficiency, is a clotting disorder caused by a genetic deficiency in the coagulation factor VIII gene (FVIII gene or F8 gene). Currently, HA is mainly treated with protein replacement therapy (RPT) based on plasma-derived clotting factors or exogenously cultivated recombinant proteins. However, PRT has limitations such as a short half-life, high cost, and long treatment duration.

[0004] Gene therapy refers to the introduction of normal foreign genes into target cells to correct or compensate for defective or abnormal genes, with the aim of treating diseases caused by defective or abnormal genes. Currently, gene therapy is considered the most promising treatment for HA.

[0005] Analysis of the human F8 gene indicates that the expressed protein of the human F8 gene has distinct domains expressed as A1-A2-B-A3-C1-C2. The B domain is encoded by a very large exon and contains a highly conserved region consisting of asparagine (N) linked to oligosaccharides. Miao et al. pointed out that a partial deletion of the B domain, retaining the N-terminal 226 amino acids containing six intact asparagine-linked glycosylation sites, could increase the in vitro secretion of F8 by 10-fold (see Miao, HZ, Sirachainan, N., Palmer, L, Kucab, P., Cunningham, MA et al. Bioengineering of coagulation factor VIII for improved secretion. Blood, 2004, 103(9), pp. 3412-3419). However, gene therapy using the B-domain deleted F8 gene (F8-BDD) has problems such as reduced protein secretion and function, reduced transduction efficiency of the F8 viral vector, and responses associated with antibody and inhibitor formation (immune rejection).

[0006] In summary, a recombinant coagulation factor VIII is provided which has good coagulation function and weak induction of inhibitory antibody responses in vivo, and which is of great significance in the field of HA gene therapy. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Miao, HZ, Sirachainan, N., Palmer, L, Kucab, P., Cunningham, MA et al. Bioengineering of coagulation factor VIII for improved secretion. Blood, 2004, 103(9), pp. 3412-3419 Summary of the Invention [Problem to be solved by the invention]

[0008] (Summary) The present application provides a recombinant coagulation factor VIII and its use, which is rich in glycosylation sites, has good coagulation function and weak response to antibodies, is easily secreted outside cells, and can effectively correct hemophilia A. [Means for solving the problem]

[0009] In a first aspect, the present application provides a recombinant coagulation factor VIII comprising more than 80% of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0010] The recombinant coagulation factor VIII of the present application is rich in the sequence of glycosylation sites, has good coagulation function, is easily secreted outside the cells, and easily interacts with cofactors such as thrombin (FIIa). In addition, the recombinant coagulation factor VIII has a weak antibody-inducing response, which can effectively achieve the therapeutic effect, reduce the risk of immune rejection, and reduce the treatment cost.

[0011] SEQ ID NO:1: (The underlined portion is the synthetic B domain [N8] in this application, and the A2 and A3 domains precede and follow the B domain, respectively.)

[0012] [ka]

[0013] SEQ ID NO:2: (The underlined portion is the synthetic B domain

[0299] in this application, and the A2 and A3 domains precede and follow the B domain, respectively.)

[0014] [ka]

[0015] In some embodiments of the present application, the recombinant coagulation factor VIII contains a B domain comprising the amino acid sequence set forth in SEQ ID NO:8.

[0016] SEQ ID NO:8: (B domain [N8])

[0017] [ka]

[0018] In some embodiments of the present application, the recombinant coagulation factor VIII contains a B domain comprising the amino acid sequence set forth in SEQ ID NO:9.

[0019] SEQ ID NO: 9: (B domain

[0299] )

[0020] [ka]

[0021] In a second aspect, the present application provides gene sequences as shown in SEQ ID NO:3 and SEQ ID NO:4, which encode the B domain of the recombinant coagulation factor VIII of the present application.

[0022] SEQ ID NO:3:

[0023] [ka]

[0024] SEQ ID NO:4:

[0025] [ka]

[0026] In certain embodiments, the gene encoding the recombinant coagulation factor VIII comprises the nucleic acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.

[0027] SEQ ID NO: 5: (Nucleic acid sequence encoding the full-length F8-BDD-N8 protein)

[0028] [ka] TIFF0007655586000008.tif242169TIFF0007655586000009.tif63169

[0029] SEQ ID NO:6: (Nucleic acid sequence encoding full-length F8-BDD-299 protein)

[0030] [ka] TIFF0007655586000011.tif220169

[0031] The gene encoding the recombinant coagulation factor VIII of the present application is rich in sequences of glycosylation sites, and the expressed coagulation factor VIII (F8 protein) has good coagulation function, is easily secreted outside the cells, and easily interacts with cofactors such as thrombin (FIIa). In addition, the expressed coagulation factor VIII has a weak ability to induce antibody response, which can effectively realize the therapeutic effect, reduce the risk of immune rejection, and reduce the treatment cost.

[0032] In a third aspect, the present application provides a recombinant expression vector comprising a gene encoding the recombinant coagulation factor VIII according to the second aspect.

[0033] Preferably, the recombinant expression vector comprises a viral or plasmid vector containing a gene encoding the recombinant coagulation factor VIII according to the second aspect.

[0034] Preferably, the viral vector comprises the lentiviral vector pEGWI.

[0035] Preferably, the 5' splice donor site GT of the lentiviral vector pEGWI is mutated to CA.

[0036] Preferably, the enhancer in the U3 region of the lentiviral vector pEGWI is deleted.

[0037] Preferably, the U3 region of the lentiviral vector pEGWI contains an insulator.

[0038] Preferably, the recombinant expression vector comprises the EF1α promoter.

[0039] In this application, the lentiviral vector pEGWI is modified: the wild-type 5' splice donor site GT is mutated to CA, the enhancer in the U3 region is deleted, and an insulator (cHS4) is added to the U3 region, which can effectively improve the transduction and expression efficiency of pEGWI to reduce the vector production cost, and further improve the safety.

[0040] In a fourth aspect, the present application provides a recombinant lentivirus comprising a gene encoding the recombinant coagulation factor VIII according to the second aspect.

[0041] Preferably, the method for preparing a recombinant lentivirus comprises: Packaging the lentiviral vector according to the third aspect using a packaging plasmid to obtain a recombinant lentivirus. Includes.

[0042] Preferably, the packaging plasmids include pNHP and pHEF-VSV-G.

[0043] Preferably, the method for preparing a recombinant lentivirus comprises the following steps: (1) co-transfecting the lentiviral vector according to the third aspect and the packaging plasmids pNHP and pHEF-VSV-G into mammalian cells HEK293T, and culturing the mammalian cells HEK293T for 24 to 72 hours; and (2) Purifying and concentrating to obtain the recombinant lentivirus.

[0044] In a fifth aspect, the present application provides a recombinant cell, the recombinant cell comprising a gene encoding the recombinant coagulation factor VIII according to the second aspect.

[0045] Preferably, the genome of the recombinant cell is fused to a gene encoding the recombinant coagulation factor VIII according to the second aspect.

[0046] Preferably the recombinant cell contains a recombinant expression vector according to the third aspect.

[0047] In a sixth aspect, the present application provides a method for preparing a recombinant cell according to the fifth aspect, the method comprising: A gene encoding the recombinant coagulation factor VIII according to the second aspect, a recombinant expression vector according to the third aspect, or a recombinant lentivirus according to the fourth aspect is introduced into a host cell to obtain a recombinant cell. Includes.

[0048] Preferably, the introduction is performed by a method comprising any one of the following: electrotransduction, a viral vector system, a non-viral vector system, or direct gene injection.

[0049] Preferably, the host cells comprise hematopoietic stem cells.

[0050] In a seventh aspect, the present application provides a pharmaceutical composition comprising any one or combination of recombinant coagulation factor VIII according to the first aspect, a gene encoding recombinant coagulation factor VIII according to the second aspect, a recombinant expression vector according to the third aspect, a recombinant lentivirus according to the fourth aspect or a recombinant cell according to the fifth aspect.

[0051] Preferably, the pharmaceutical composition further comprises any one of a pharma- ceutically acceptable carrier, excipient or diluent, or a combination of at least two of these.

[0052] In an eighth aspect, the present application provides the use of recombinant coagulation factor VIII according to the first aspect, a gene encoding recombinant coagulation factor VIII according to the second aspect, a recombinant expression vector according to the third aspect, a recombinant lentivirus according to the fourth aspect, a recombinant cell according to the fifth aspect or a pharmaceutical composition according to the seventh aspect for the preparation of a medicament for treating hemophilia.

[0053] Compared with existing technologies, the present application has the following beneficial effects:

[0054] (1) Through genetic modification, the recombinant coagulation factor VIII of the present application is rich in glycosylation sites, has good coagulation function, is easily secreted outside the cells, and easily interacts with cofactors such as thrombin (FIIa). In addition, the recombinant coagulation factor VIII has a weak response to antibodies, which can effectively achieve therapeutic effects, reduce the risk of immune rejection, and reduce treatment costs.

[0055] (2) In this application, the lentiviral vector pEGWI is modified, which can effectively improve the transduction efficiency and expression efficiency of pEGWI so as to reduce vector costs, and further improve safety.

[0056] (3) In this application, the expression vector constructed by using the gene encoding recombinant coagulation factor VIII and the lentivirus vector can be successfully expressed in vivo in HA mice and can correct the bleeding phenotype of HA mice to a certain extent. The expression vector has a weak response to antibodies, which is very significant in realizing the effectiveness of gene therapy, which is the basis for alleviating HA symptoms more quickly and achieving a more extensive and long-lasting gene therapy effect. [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 is a diagram showing the structure of the lentiviral vector pEGWI. [Figure 2A] FIG. 1 is a diagram showing the structure of the normal F8-BDD gene. [Figure 2B] FIG. 1 is a diagram showing the structure of the F8-BDD-N8 gene. [Figure 2C] FIG. 1 is a diagram showing the structure of the F8-BDD-299 gene. [Figure 2D] FIG. 1 is a structural diagram of a recombinant lentiviral vector. [Diagram 3] 1 is a graph showing viral copy numbers of recombinant lentiviruses. [Figure 4] 1 is a flow chart for analysis of protein expression of cells transfected with recombinant lentiviruses. [Diagram 5] 1 is a graph showing the results of protein expression of cells transduced with recombinant lentiviruses. [Figure 6] 1 is a graph showing the results of in vitro plasma detection by APTT assay. [Figure 7] 1 is a graph showing the results of in vitro plasma detection by substrate luminescence assay. [Figure 8] FIG. 1 is a graph showing the results of Western blotting of factor VIII treated with a glycosylation inhibitor. [Figure 9] 1 is a graph showing the in vitro activity of Factor VIII. [Figure 10] 1 is a flow chart for the treatment of HA mice. [Figure 11] 1 is a graph showing the in vivo activity of Factor VIII in mice. [Figure 12] 1 is a graph showing the results of detection in mouse plasma by APTT assay. [Figure 13] 1 is a graph showing the results of detection in mouse plasma by enzyme-linked immunosorbent assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] In order to further elaborate on the technical means adopted and the effects achieved in the present application, the present application is further described below in conjunction with examples and figures. It should be understood that the specific embodiments described herein are intended to illustrate, but not to limit, the present application.

[0059] In the examples, experiments in which no specific techniques or conditions are mentioned are carried out according to the techniques or conditions described in the literature or product specifications in the art. Reagents or equipment used herein whose manufacturers are not specified are conventional products commercially available from the appropriate channels. EXAMPLES

[0060] [Example 1] A lentiviral vector was constructed. The method specifically includes the following steps:

[0061] (1) The structure of the lentivirus vector pEGWI is shown in Figure 1. The wild-type 5' splice donor site GT was mutated to CA, the enhancer in U3 was deleted, and an insulator (cHS4) was added to U3. For specific modification methods, see "Contributions of Viral Splice Sites and cis-Regulatory Elements to Lentivirus Vector Function, Cui et al. Journal of Virology, July 1999, pp. 6171-6176."

[0062] (2) The promoter and F8-BDD / F8-BDD-N8 / F8-BDD-299 genes were inserted.

[0063] The normal unmodified F8-BDD gene sequence (shown in SEQ ID NO: 7), F8-BDD-N8 gene sequence (shown in SEQ ID NO: 5) and F8-BDD-299 gene sequence (shown in SEQ ID NO: 6) were chemically synthesized with the sequences of the selected possible glycosylation sites and added with the human EF1α (hEF1α) promoter sequence. The gene structure of normal F8-BDD is shown in Figure 2A. Synthetic glycosylation site-related sequences were inserted into F8-BDD-N8 and F8-BDD-299 individually, and their gene structures are shown in Figures 2B and 2C. Normal F8-BDD, F8-BDD-N8 and F8-BDD-299 were inserted into the lentiviral vector pEGWI via the restriction enzyme digestion sites. The resulting products were identified by sequencing and double digestion (for the best reaction conditions, refer to the original recommendation of New England Biolab [NEB]), and the BamHI cloning site (ggatcc-acc)-AUG was used at the 5' end and the SpeI cloning site (actagt) was used at the 3' end to obtain the correctly ligated lentiviral vectors carrying the normal F8-BDD, F8-BDD-N8 or F8-BDD-299 genes controlled by hEF1α. The specific ligation positions and composition of the lentiviral vectors are shown in Figure 2D.

[0064] SEQ ID NO:7:

[0065] [ka] TIFF0007655586000013.tif241169TIFF0007655586000014.tif21169

[0066] [Example 2] In this example, the lentiviral vector constructed in Example 1 was further packaged, purified, and concentrated to obtain recombinant lentivirus. For experimental methods, see [1] Chang LJ, Urlacher V, Iwakuma T, et al. Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system [J]. Gene Therapy, 1999, 6(5): 715-728. [2] Chang LJ, Zaiss A K. Chang, LJ, and Zaiss, AK. Lentiviral vectors. Preparation and use. Methods Mol Med 69: 303-318 [J]. Methods in Molecular Medicine, 2002, 69: 303-318.

[0067] For specific steps, please refer to the references listed above. The experimental method is briefly described as follows.

[0068] (1) The lentiviral vector constructed in Example 1 and the packaging plasmids pNHP and pHEF-VSV-G were co-transfected into mammalian cells HEK293T, and then the mammalian cells HEK293T were cultured for 48 hours, and the viral vector supernatant was collected.

[0069] (2) The lentivirus collected from the culture was purified and concentrated to obtain recombinant lentivirus.

[0070] (3) The viral copy number (VCN) of each lentivirus was detected. The detection results are shown in Figure 3. At the same multiplicity of infection, LV-F8-BDD, LV-F8-BDD-N8 and LV-F8-BDD-299 lentiviruses have essentially similar copy numbers.

[0071] [Example 3] In this example, the recombinant lentiviruses prepared in Example 2 were tested in vitro.

[0072] The three lentiviruses (LV-F8-BDD, LV-F8-BDD-N8, and LV-F8-BDD-299) carrying normal F8-BDD, F8-BDD-N8, or F8-BDD-299 genes prepared in Example 2 were transduced into EA-hy926 endothelial cells individually. The lentiviruses were transduced by the following method.

[0073] DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution was added to a 6-well plate (Corning, USA). 4 × 10 cells were added per well. 4 EA-hy926 endothelial cell line were seeded and incubated at 37°C for 18 h under 5% CO2 and transfected with lentivirus at MOI 50. Polybrene (8 μg / mL, Sigma-Aldrich) was added to a final medium volume of 600 μL. Transduction was performed for 24 h, and then the medium was replaced with fresh medium daily until cell confluence reached 90.0%. Cells were transferred to T75 flasks (Corning, USA).

[0074] Protein expression was detected to determine the expression of F8-BDD, F8-BDD-N8 and F8-BDD-299 genes in cells. The specific steps are shown in Figure 4. The supernatant secreted by transduced EA-hy926 cells was collected and concentrated, and the intracellular extract was also collected. Protein expression was detected by Western blotting. Cells transduced without lentivirus were used as negative control (NC). Using GAPDH as the internal standard, the results are shown in Figure 5, where L indicates the detection result of cell extract and S indicates the detection result of cell culture supernatant. As shown in FIG. 5, a small amount of factor VIII (F8 protein) was expressed in cells transduced without lentivirus (low activity, 43 kDa), while a relatively large amount of F8-BDD, F8-BDD-N8 or F8-BDD-299 was obviously expressed in cells transduced with lentivirus carrying normal F8-BDD, F8-BDD-N8 or F8-BDD-299 genes. Compared with F8-BDD, more complete F8 protein (110-200 kDa) is obtained in the supernatant of cells transduced with F8-BDD-N8 or F8-BDD-299. This indicates that factor VIII expressed by genetically modified F8-BDD genes: F8-BDD-N8 and F8-BDD-299 in the present application, is more likely to be secreted outside the cells to achieve effective coagulation.

[0075] Coagulation function is mainly evaluated by two methods: Activated Partial Thromboplastin Time (APTT) assay and substrate luminescence assay. APTT (Siemens Healthcare Diagnostics Products GmbH, Germany) assay was performed as follows: 50 μL of cell supernatant was added to 50 μL of plasma to be tested at 37°C, then 100 μL of actin reagent (factor XII activator and cerebral phospholipids) was added in APTT, mixed thoroughly, incubated at 7°C for 3 minutes, and finally 100 μL of calcium ions (CaCl2) was added to observe the time required for plasma clotting, i.e., the activated partial thromboplastin time.

[0076] The substrate luminescence assay is a method for determining activity with the F8 chromogenic assay kit (Hyphen BioMed, France). The substrate luminescence assay was performed as follows: the plasma to be tested and the blank control group were diluted 40 times with Tris-BSA buffer (R4+), 50 μL were added to a microplate, 50 μL of factor X (R1), 50 μL of activated factor IX mixture (R2) and 50 μL of SXa-11 substrate (R3) were added individually, incubated at 37° C. for 5 minutes, and the reaction was stopped by adding 50 μL of 20% acetic acid. The absorbance was read at 405 nm.

[0077] The previously collected supernatants of virus-transduced EA-hy926 cells were removed from -80°C and thawed on ice. Each supernatant was mixed with plasma from an F8-deficient patient. The plasma from an F8-deficient patient was used as a negative control (NC), and plasma from a healthy volunteer was used as a positive control (PC). Detection was performed by APTT assay and substrate luminescence assay.

[0078] The detection results of the APTT assay are shown in Figure 6. Compared with the negative control, the supernatants of cells transduced with F8-BDD, cells transduced with F8-BDD-N8 and cells transduced with F8-BDD-299 have good coagulation activity and significant coagulation effect in that the plasma coagulation time is significantly reduced, respectively. The supernatants of cells transduced with F8-BDD-299 have the best effect in that the coagulation time is 94.3 seconds, which has a certain statistical difference (p<0.05) compared with the effect of the positive control (57.5s). The coagulation time of the supernatants of cells transduced with F8-BDD-N8 is 113.7 seconds, which has a certain statistical difference (p<0.05) compared with the coagulation time of F8-BDD-299 and the positive control. The clotting time of the supernatant of cells transfected with F8-BDD was 156.7 seconds, which has a relatively large statistical difference (p-average)<0.001) compared with the clotting times of F8-BDD-N8, F8-BDD-299 and the positive control, indicating that the factor VIII expressed by the modified F8-BDD gene: F8-BDD-N8 and F8-BDD-299 in the present application, has a good clotting effect.

[0079] Figure 7 shows the detection results of substrate luminescence assay. Activity was obtained using F8 chromogenic assay kit, and then factor VIII was quantified by ELISA to obtain unit activity. The unit activity of factor VIII expressed by F8-BDD-299 is 100 times that of factor VIII expressed by F8-BDD, and 2.8 times that of factor VIII expressed by F8-BDD-N8.

[0080] In summary, in this application, lentiviral vectors are constructed using modified F8-BDD genes: F8-BDD-N8 and F8-BDD-299, respectively, which are successfully expressed in cells, and the factor VIII expressed by F8-BDD-N8 and F8-BDD-299 can be more easily secreted outside the cells and have good coagulation effect.

[0081] [Example 4] In this example, N-glycosylation of factor VIII was detected.

[0082] The supernatants of endothelial cells transduced with F8-BDD, F8-BDD-N8 and F8-BDD-299 were treated with glycosylation inhibitors: neuraminidase (N) and peptide-N-glycosidase F (G) individually. Glycosylation inhibitors can induce deglycosylation and reduce the molecular weight. The results of Western blotting are shown in FIG. 8, where "+" indicates treatment with the corresponding enzyme and "-" indicates no treatment with the corresponding enzyme. The protein bands with reduced protein molecular weight after deglycosylation after treatment with glycosylation inhibitors are indicated by arrows. The molecular weight of factor VIII expressed by F8-BDD-N8 and F8-BDD-299 was significantly reduced (++ lane compared to - lane), while the molecular weight of factor VIII expressed by F8-BDD was not reduced, indicating that the modification of the F8-BDD gene in this application can effectively introduce glycosylation sites.

[0083] Thrombin (FIIa) is an important cofactor for factor VIII. FIIa is used to treat the supernatant of transduced endothelial cells to examine whether the modification of the B domain also affects the interaction with the procoagulant cofactor. The addition of thrombin enhances the activation of F8, and more protein related to the A2 region of factor VIII (with a molecular weight of 43 kDa) is generated. After the addition of thrombin, activity detection was performed every 2 minutes (by the substrate luminescence assay described above). The results are shown in FIG. 9. From the second minute onwards, the activity of factor VIII expressed by F8-BDD-299 increases significantly, and the activity of factor VIII expressed by F8-BDD and F8-BDD-N8 increases slightly.

[0084] [Example 5] Three lentiviruses carrying F8-BDD, F8-BDD-N8 or F8-BDD-299 (LV-F8-BDD, LV-F8-BDD-N8 and LV-F8-BDD-299) were prepared as shown in Example 2 and transduced into mouse hematopoietic stem cells. HA mice were treated as shown in Figure 10. C57BL / 6 mice (6 weeks old, purchased from Beijing Biosubstrate Technologies) with knockout of F8 gene were used as HA mice. All mice were placed in a pathogen-free environment and irradiated (9.5 Gy / mouse) using an X-ray irradiation cabinet (Faxitron, Tucson, AZ, USA). Bone marrow cells were collected from the tibia and femur of HA mice. Hematopoietic stem cells were isolated from bone marrow cells and transduced with lentiviruses LV-F8-BDD, LV-F8-BDD-N8 and LV-F8-BDD-299, respectively, to obtain stem cells carrying F8-BDD, F8-BDD-N8 or F8-BDD-299 genes. The transduced stem cells were injected back into HA mice by intravenous injection for disease treatment.

[0085] On days 15, 30, 45 and 60 after allogeneic bone marrow transplantation, blood was collected from the mice and plasma was isolated from the blood. The activity of factor VIII in plasma was determined by a two-step substrate luminescence assay, using untreated hemophilic mice (sham) and wild-type mice (WT) as controls. The results are shown in Figure 11. The activity of factor VIII expressed by F8-BDD-299 increases continuously from 5% on day 15 to 8% on day 60. The activity of factor VIII expressed by F8-BDD and F8-BDD-N8 remains at about 3.0%, which has a certain statistical difference (P<0.05) with that of F8-BDD-299. The coagulation function of plasma was determined by APTT assay. The results are shown in Figure 12. The clotting time of factor VIII expressed by F8-BDD-299 is the shortest and is closest to that of wild-type mice (p<0.05).

[0086] Further, for antibody response, the orbital peripheral blood of the previously treated mice was collected and centrifuged at 3000 rpm for 15 minutes to obtain plasma. The plasma was diluted 1:200 with Tris-BSA buffer, placed in a PVC microplate, and peroxidase-conjugated goat anti-mouse total IgG was added. Then, the luminescent substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added for enzyme-linked immunosorbent assay (ELISA) to evaluate the antibody response to factor VIII. HA mice injected with monoclonal antibody to factor VIII were used as positive control (Ctrl+). The results are shown in Figure 13. Factor VIII expressed by F8-BDD-N8 and F8-BDD-299 had a weaker response to IgG antibody than factor VIII expressed by F8-BDD, and factor VIII expressed by F8-BDD-299 had the weakest response to IgG antibody.

[0087] In summary, in this application, F8-BDD gene is genetically modified and added with a glycosylation site sequence, and an expression vector is constructed by using a modified lentivirus vector. The expression vector has high transduction and expression efficiency, and the expressed factor VIII can be easily secreted outside the cell, has efficient coagulation function, can correct the bleeding phenotype of HA mice to a certain extent, and has a weak ability to induce antibody response, which is of great significance in realizing the efficacy of gene therapy, and is the basis for alleviating HA symptoms more quickly and achieving more extensive and long-lasting gene therapy.

[0088] The applicant specifies that the detailed method of the present application is described by the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, which means that the implementation of the present application does not necessarily depend on the above-mentioned detailed method. Any improvements made to the present application, equivalent substitution of various raw materials of the product, addition of auxiliary components and selection of specific modes, etc. in the present application should be obvious to those skilled in the art and fall within the protection scope and disclosure scope of the present application.

Claims

1. Recombinant coagulation factor VIII comprising a B domain having the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO:

9.

2. The recombinant coagulation factor VIII described in claim 1, containing the amino acid sequence shown in sequence number 1 or sequence number 2.

3. A gene encoding the recombinant coagulation factor VIII of claim 1 or 2, comprising the nucleic acid sequence shown in SEQ ID NO:5 or SEQ ID NO:

6.

4. A recombinant expression vector comprising a gene encoding the recombinant coagulation factor VIII of claim 1 or 2.

5. The recombinant expression vector described in claim 4, comprising a viral vector or a plasmid vector containing a gene encoding recombinant coagulation factor VIII.

6. The recombinant expression vector described in claim 5, wherein the viral vector comprises a lentiviral vector.

7. A recombinant lentivirus comprising a gene encoding the recombinant coagulation factor VIII of claim 1 or 2.

8. A recombinant cell comprising a gene encoding the recombinant coagulation factor VIII of claim 1 or 2.

9. The recombinant cell of claim 8, wherein the genome of the recombinant cell is fused to a gene encoding recombinant coagulation factor VIII.

10. A pharmaceutical composition comprising any one of the recombinant coagulation factor VIII described in claim 1 or 2, a gene encoding the recombinant coagulation factor VIII described in claim 3, a recombinant expression vector described in claim 4, 5 or 6, a recombinant lentivirus described in claim 7, or a recombinant cell described in claim 8 or 9, or a combination thereof.

11. The pharmaceutical composition of claim 10, further comprising any one of a pharma- ceutically acceptable carrier, excipient or diluent, or a combination of at least two of these.

12. Use of the recombinant coagulation factor VIII described in claim 1 or 2, the gene encoding the recombinant coagulation factor VIII described in claim 3, the recombinant expression vector described in claim 4, 5 or 6, the recombinant lentivirus described in claim 7, the recombinant cell described in claim 8 or 9, or the pharmaceutical composition described in claim 10 or 11 for the preparation of a medicament for treating hemophilia.

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