Coagulation factor fviii protein variant, expression vector and use

Abstract

Provided are a coagulation factor FVIII protein variant, an expression vector and a use. The coagulation factor FVIII protein variant has a shorter coding nucleic acid sequence than a wild-type coagulation factor FVIII protein, can easily achieve normal packaging of virus vectors, represents enhanced protein expression activity, and has high expression quantity of the coagulation factor FVIII protein when transferred into cells.

Description

Coagulation factor FVIII protein variants, expression vectors and uses Technical Field

[0001] The present invention relates to the technical field of recombinant coagulation factor FVIII, and in particular to an improved coagulation factor FVIII protein variant and its encoding nucleic acid, corresponding expression vector and use. Background Art

[0002] Hemophilia A (HA) is an X-linked recessive genetic disease caused by abnormal coagulation function due to mutations in the FVIII gene on the X chromosome. It affects approximately 1 in 5,000 males and is characterized by a tendency to bleed, with widespread bleeding, often recurring, and potentially forming hematomas and joint deformities. Clinically, the severity of hemophilia A depends on the level of FVIII protein in the patient's blood. Patients with severe hemophilia usually have FVIII levels below 1% of normal, experience bleeding after injury, and often experience spontaneous bleeding in the joints. Moderate patients have FVIII levels of 1-5% of normal, while mild patients have FVIII levels of 6% or more of normal and only bleed after severe injury, trauma, or surgery.

[0003] Patients with hemophilia A typically require intravenous infusions of human plasma or recombinant FVIII protease replacement therapy, with the frequency of treatment based on the severity of the hemophilia. Mild patients are usually treated as needed after an injury, while patients with severe hemophilia require lifelong infusions of human plasma or recombinant FVIII protein to prevent bleeding. The half-life of FVIII protein is very short (8-18 hours), and patients require frequent infusions. However, due to the high cost, less than 30% of hemophilia A patients worldwide receive this treatment. In addition, among patients receiving FVIII protease replacement therapy, approximately 25% will develop neutralizing antibodies against FVIII, rendering the treatment ineffective. In contrast, gene therapy offers the potential for a cure, with a single infusion of an adeno-associated virus (AAV) expressing the FVIII protein, which can continuously express the FVIII protein in the liver and avoid the production of FVIII neutralizing antibodies.

[0004] While gene therapy offers the potential for a cure compared to enzyme replacement therapy, gene therapy for hemophilia A also faces several challenges: First, the FVIII protein coding sequence is 7.0 kb in size, far exceeding the normal packaging capacity of AAV vectors. Second, compared with other proteins of similar size, FVIII protein expression is inefficient, which typically requires a higher dosage to achieve the desired FVIII protein expression level. By bioengineering the FVIII protein, using a 14-amino acid short linker peptide (SQ) to replace the B domain that does not affect FVIII protein activity, the FVIII protein coding sequence can be significantly shortened to 4.4 kb, enabling normal AAV vector packaging. Currently, several companies have launched or are applying for clinical trials for hemophilia A gene therapy products (BioMarin-BMN270 - already on the market; Sangamo-SB-525 - Phase 3 clinical trial; Spark-SPK-8011 - Phase 3 clinical trial; Bayer-BAY2599023 - Phase 1 / 2 clinical trial). All of these companies use a FVIII-SQ linker peptide to replace the FVIII protein B domain, achieving effective expression of the FVIII protein and improving clinical symptoms. However, due to the low expression efficiency of the FVIII protein, many products require very high dosages (BMN270 - 6e13 vg / kg; SB-525 - 3e13 vg / kg; BAY2599023 - 2e13 vg / kg), which increases the risk of liver damage and the pressure of viral production.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to optimize the coagulation factor FVIII protein by providing an improved coagulation factor FVIII protein variant and its encoding nucleic acid, corresponding expression vector and use, thereby increasing its expression level and effectively reducing the dosage.

[0007] In one aspect, the present invention provides a coagulation factor FVIII protein variant, the amino acid sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] In another aspect, the present invention provides a nucleic acid encoding a coagulation factor FVIII protein variant, the nucleotide sequence of which is shown in any one of SEQ ID NOs: 3 to 15 and SEQ ID NOs: 60 to 68.

[0009] In another aspect, the present invention provides an expression vector comprising the nucleic acid provided by the present invention.

[0010] In another aspect, the present invention provides a host cell comprising the nucleic acid or the expression vector provided by the present invention.

[0011] In another aspect, the present invention provides a pharmaceutical composition comprising the coagulation factor FVIII protein variant provided by the present invention, the nucleic acid and / or the expression vector, and pharmaceutically acceptable excipients.

[0012] In another aspect, the present invention provides use of the coagulation factor FVIII protein variant, the nucleic acid and / or the expression vector in expressing the coagulation factor FVIII protein.

[0013] In another aspect, the present invention provides use of the coagulation factor FVIII protein variant, the nucleic acid and / or the expression vector in preparing a medicament for treating a disease requiring the coagulation factor FVIII protein.

[0014] In another aspect, the present invention provides a method for treating a disease, comprising administering the FVIII protein variant, the nucleic acid, and / or the expression vector provided herein to a subject in need thereof. The disease is a disease requiring a FVIII protein. Preferably, the disease is hemophilia; more preferably, the disease is hemophilia A.

[0015] In another aspect, the present invention provides the blood coagulation factor FVIII protein variant, the nucleic acid and / or the expression vector of the present invention for use as a medicament.

[0016] In another aspect, the present invention provides the blood coagulation factor FVIII protein variant, the nucleic acid and / or the expression vector of the present invention for use in therapy.

[0017] In another aspect, the present invention provides a method for producing a coagulation factor FVIII protein, comprising:

[0018] Expressing the nucleic acid and / or the expression vector provided by the present invention in a cell;

[0019] The coagulation factor FVIII protein produced by the cells is recovered.

[0020] The nucleic acid sequence encoding the FVIII protein variant provided by the present invention is shorter than that of the wild-type FVIII protein, making it easy to package the protein in a viral vector and exhibiting enhanced protein expression activity. When transferred into cells, the protein is expressed at a higher level. The present invention further codon-optimizes the encoding nucleic acid to further increase the expression level and activity of the FVIII protein, thereby significantly reducing the dosage of the viral vector and improving safety and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is a schematic structural diagram of an hFVIII gene expression cassette provided in one embodiment of the present invention;

[0022] FIG2 is a comparison of the amount of hFVIII protein expressed by different hFVIII structures in HepG2-AAVR cells in Example 1 of the present invention;

[0023] FIG3 is a comparison of the hFVIII protein activity expressed in HepG2-AAVR cells with different hFVIII structures in Example 1 of the present invention;

[0024] Figures 4-5 are comparative results of comparing the amount of hFVIII protein expressed by different hFVIII optimized genes in wild-type C57BL / 6 mice in Example 2 of the present invention; wherein, Figure 4 shows the hFVIII protein expression in mouse plasma 4 weeks after tail vein administration of AAV8-OTE-hFVIII-L1-WT\1\2\3\9\10-pA and AAV8-OTE-hFVIII-L1-pA viruses to wild-type C57BL / 6 mice; Figure 5 shows the hFVIII protein expression in mouse plasma 4 weeks after tail vein administration of AAV8-OTE-hFVIII-L1-WT\4\5\6\8-pA and AAV8-OTE-hFVIII-L1-pA viruses to wild-type C57BL / 6 mice;

[0025] FIG6 is a comparison of the results of hFVIII expression in wild-type C57BL / 6 mice using different combinations of elements in Example 3 of the present invention;

[0026] FIG7 shows the hFVIII protein activity in mouse plasma at 1 to 6 weeks after administration of different hFVIII structure viruses to FVIII KO mice in Example 4 of the present invention;

[0027] FIG8 shows the bleeding time after tail docking of FVIII KO mice at the end of the 8-week experiment after administration of different hFVIII structure viruses in Example 4 of the present invention;

[0028] Figure 9 shows the hFVIII protein expression in mouse plasma at 2, 4, and 6 weeks after AAV8-OTE-hFVIII-L1-pA virus was administered to FVIII KO mice at doses of 4e12 and 8e11 vg / kg in Example 5 of the present invention;

[0029] Figure 10 shows the hFVIII protein activity in mouse plasma at 2, 4, and 6 weeks after AAV8-OTE-hFVIII-L1-pA virus was administered to FVIII KO mice at doses of 4e12 and 8e11 vg / kg in Example 5 of the present invention;

[0030] FIG11 shows the bleeding time after tail amputation of FVIII KO mice at the end of the 6-week experiment after AAV8-0TE-hFVIII-L1-pA virus was administered at doses of 4e12 and 8e11 vg / kg in Example 5 of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be further described below through specific examples. The examples of the present invention are only for illustration of the present invention and do not limit the scope of the present invention.

[0032] According to the wild-type human FVIII gene (GeneID: 2157), its nucleic acid sequence and amino acid sequence can be known. The wild-type human FVIII protein structure includes a signal peptide, an A1-a1 domain, an A2-a2 domain, a B domain, an a3-A3 domain, and a C1-C2 domain. hFVIII-SQ (whose amino acid sequence is shown in SEQ ID NO: 59) is an SQ connecting peptide instead of the B domain to connect the A2-a2 domain and the a3-A3 domain. Its structure is a signal peptide, an A1-a1 domain, an A2-a2 domain, an SQ connecting peptide, an a3-A3 domain, and a C1-C2 domain. hFVIII-SQ is widely used in gene therapy for hemophilia A. However, although hFVIII-SQ achieves effective expression of FVIII protein by shortening the coding sequence, there is still a problem of low protein expression efficiency, which increases the risk of liver damage and the pressure of virus production. Based on this, the inventors of the present application obtained the nucleic acid of the coagulation factor FVIII protein and the coagulation factor FVIII protein variant encoded by it through research, so as to further improve the expression efficiency of the FVIII protein.

[0033] hFVIII-SQ (SEQ ID NO: 59):

[0034] The present invention provides nucleic acids encoding human coagulation factor FVIII protein and encoded FVIII protein variants thereof, wherein the nucleic acids are different from the nucleic acids encoding wild-type human coagulation factor FVIII protein. The encoding nucleic acids provided by the present invention are expressed at high levels in cells and / or animals, thereby providing high FVIII protein expression levels and expression efficiency.

[0035] Specifically, an embodiment of the present invention provides a coagulation factor FVIII protein variant, specifically named hFVIII-L1 and hFVIII-L2, whose amino acid sequences are shown in SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

[0036] hFVIII-L1 (SEQ ID NO: 1):

[0037] hFVIII-L2 (SEQ ID NO: 2):

[0038] Accordingly, the present invention also provides a nucleic acid encoding the above-mentioned coagulation factor FVIII protein variant, the nucleotide sequence of which is shown in any one of SEQ ID NOs: 3 to 15 and SEQ ID NOs: 60 to 68.

[0039] The encoding nucleic acid provided by the embodiments of the present invention significantly shortens the length of the hFVIII sequence without affecting the function of the FVIII protein encoded thereby.

[0040] Nucleic acid hFVIII-L1-WT (SEQ ID NO: 3)

[0041] Nucleic acid hFVIII-L1-1 (SEQ ID NO: 4)

[0042] Nucleic acid hFVIII-L1-2 (SEQ ID NO: 5)

[0043] Nucleic acid hFVIII-L1-3 (SEQ ID NO: 6)

[0044] Nucleic acid hFVIII-L1-4 (SEQ ID NO: 7)

[0045] Nucleic acid hFVIII-L1-5 (SEQ ID NO: 8)

[0046] Nucleic acid hFVIII-L1-6 (SEQ ID NO: 9)

[0047] Nucleic acid hFVIII-L1-7 (SEQ ID NO: 10)

[0048] Nucleic acid hFVIII-L1-8 (SEQ ID NO: 11)

[0049] Nucleic acid hFVIII-L1-9 (SEQ ID NO:12)

[0050] Nucleic acid hFVIII-L1-10 (SEQ ID NO:13)

[0051] Nucleic acid hFVIII-L2-WT (SEQ ID NO:14)

[0052] Nucleic acid hFVIII-L2-7 (SEQ ID NO:15)

[0053] Nucleic acid hFVIII-L2-1 (SEQ ID NO:60)

[0054] Nucleic acid hFVIII-L2-2 (SEQ ID NO:61)

[0055] Nucleic acid hFVIII-L2-3 (SEQ ID NO:62)

[0056] Nucleic acid hFVIII-L2-4 (SEQ ID NO:63)

[0057] Nucleic acid hFVIII-L2-5 (SEQ ID NO:64)

[0058] Nucleic acid hFVIII-L2-6 (SEQ ID NO:65)

[0059] Nucleic acid hFVIII-L2-8 (SEQ ID NO:66)

[0060] Nucleic acid hFVIII-L2-9 (SEQ ID NO:67)

[0061] Nucleic acid hFVIII-L2-10 (SEQ ID NO:68)

[0062] Among the above nucleic acids, hFVIII-L1-WT and hFVIII-L1-1 to hFVIII-L1-10 are all nucleic acids encoding hFVIII-L1, wherein hFVIII-L1-1 to hFVIII-L1-10 are codon-optimized based on hFVIII-L1-WT; hFVIII-L2-WT and hFVIII-L2-1 to hFVIII-L2-10 are all nucleic acids encoding hFVIII-L2, wherein hFVIII-L2-1 to hFVIII-L2-10 are codon-optimized based on hFVIII-L2-WT.

[0063] Preferably, the nucleic acid encoding the coagulation factor FVIII protein variant is hFVIII-L1-5, hFVIII-L1-9, hFVIII-L1-10, hFVIII-L2-5, hFVIII-L2-9 or hFVIII-L2-10. Further preferably, the nucleic acid encoding the coagulation factor FVIII protein variant is hFVIII-L1-7 and hFVIII-L2-7.

[0064] Accordingly, an embodiment of the present invention further provides an expression vector comprising the nucleic acid described in the embodiment of the present invention.

[0065] Viral vectors such as lentiviral and parvoviral vectors, including AAV serotypes and variants thereof, provide a means of delivering nucleic acids into cells in vitro, in vivo, and in vitro, so that the cells express the encoded protein. AAV is a virus that can be used for gene therapy vectors because it can penetrate cells and introduce nucleic acids / genetic materials so that the nucleic acids / genetic materials can be stably maintained in cells. Viral vectors that can be used in the present invention include, but are not limited to, adeno-associated virus (AAV) vectors of various serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 serotypes. In a specific embodiment, the expression vector is AAV8.

[0066] In some embodiments, the expression vector further comprises a promoter, an enhancer, an intron, one or more terminal inverted repeats (e.g., 5' ITR, 3' ITR), a Kozak sequence, and / or a polyA tail operably linked to the nucleic acid. In some embodiments, the enhancer and the promoter are sequentially operably linked to the sequence of the nucleic acid, or the intron is located within or on both sides of the sequence of the nucleic acid, or the terminal inverted repeat is located at the 5' or 3' end of the sequence of the nucleic acid, or the Kozak sequence is located between the 5' end of the sequence of the nucleic acid and the intron, or the polyA tail is located between the 3' end of the sequence of the nucleic acid and the terminal inverted repeat. Figure 1 shows the structure of an hFVIII gene expression cassette involved in a specific embodiment of the present invention, which comprises, from 5' to 3' end, a 5' ITR, an enhancer, a promoter, an intron, a Kozak sequence, an hFVIII target gene (i.e., the nucleic acid sequence), polyA, and a 3' ITR. However, the structure of the hFVIII gene expression cassette provided by the present invention is not limited thereto.

[0067] As used herein, the term "operably linked" means that regulatory sequences necessary for expression of a coding sequence are placed at appropriate positions relative to the coding sequence, thereby resulting in expression of the coding sequence. The term also applies to the arrangement of a coding sequence and transcriptional control elements (e.g., promoters, enhancers, etc.) in an expression vector.

[0068] In some embodiments, the promoter is TTRmut promoter (mutated mini human transthyretin promoter), TTRm promoter (mini human transthyretin promoter) or HLP promoter (human α1 antitrypsin core promoter), and the nucleotide sequence of the promoter is shown in any one of SEQ ID NOs: 16 to 18.

[0069] TTRmut promoter (SEQ ID NO: 16)

[0070] TTRm promoter (SEQ ID NO: 17)

[0071] HLP promoter (SEQ ID NO: 18)

[0072] In some embodiments, the enhancer is a TTR enhancer (human transthyretin enhancer), a SerpinA1 enhancer (human α1 antitrypsin enhancer), or a CRMSB2 enhancer (modified human α1 antitrypsin enhancer), and the nucleotide sequence of the enhancer is shown in any one of SEQ ID NOs: 19 to 21.

[0073] TTR enhancer (SEQ ID NO: 19)

[0074] SerpinA1 enhancer (SEQ ID NO: 20)

[0075] CRMSB2 enhancer (SEQ ID NO: 21)

[0076] In some embodiments, the intron is EF1a intron (human elongation factor 1a intron), MVM intron (minute virus of mice intron) or SBR intron 3 (modified minute virus of mice intron), and the nucleotide sequence of the intron is shown in any one of SEQ ID NOs: 22 to 24.

[0077] EF1a intron (SEQ ID NO: 22)

[0078] MVM intron (SEQ ID NO:23)

[0079] SBR intron 3 (SEQ ID NO: 24)

[0080] In some embodiments, the terminal inverted repeat sequence is a 5'ITR located at the 5' end and a 3'ITR located at the 3' end, and the nucleotide sequence of the terminal inverted repeat sequence is shown in SEQ ID NO: 25 or SEQ ID NO: 26.

[0081] 5' ITR (SEQ ID NO: 25)

[0082] 3' ITR (SEQ ID NO: 26)

[0083] In some embodiments, the Kozak sequence is: gccacc

[0084] The polyA tail is a polyadenylation signal. In some embodiments, the nucleotide sequence of the polyA tail is as shown in SEQ ID NO: 27.

[0085] polyA tail (SEQ ID NO: 27)

[0086] The embodiment of the present invention further optimizes the combination of enhancer, promoter, and intron elements in the hFVIII gene expression cassette to obtain an element combination with better expression effect, as shown in Table 1.

[0087] Table 1

[0088] Element combination OTE (SEQ ID NO: 44)

[0089] Element combination TTM (SEQ ID NO: 45)

[0090] Element combination HLP (SEQ ID NO: 46)

[0091] Element combination STM (SEQ ID NO: 47)

[0092] Element combination CTS (SEQ ID NO: 48)

[0093] In some embodiments, the expression vector comprises any one of the hFVIII protein expression cassettes shown in SEQ ID NOs: 28-43.

[0094] Protein expression cassette AAV8-OTE-hFVIII-L1-WT-pA (SEQ ID NO: 28)

[0095] Protein expression cassette AAV8-OTE-hFVIII-L1-1-pA (SEQ ID NO: 29)

[0096] Protein expression cassette AAV8-OTE-hFVIII-L1-2-pA (SEQ ID NO: 30)

[0097] Protein expression cassette AAV8-OTE-hFVIII-L1-3-pA (SEQ ID NO: 31)

[0098] Protein expression cassette AAV8-OTE-hFVIII-L1-4-pA (SEQ ID NO: 32)

[0099] Protein expression cassette AAV8-OTE-hFVIII-L1-5-pA (SEQ ID NO: 33)

[0100] Protein expression cassette AAV8-OTE-hFVIII-L1-6-pA (SEQ ID NO: 34)

[0101] Protein expression cassette AAV8-OTE-hFVIII-L1-pA (SEQ ID NO: 35)

[0102] Protein expression cassette AAV8-OTE-hFVIII-L1-8-pA (SEQ ID NO: 36)

[0103] Protein expression cassette AAV8-OTE-hFVIII-L1-9-pA (SEQ ID NO: 37)

[0104] Protein expression cassette AAV8-OTE-hFVIII-L1-10-pA (SEQ ID NO: 38)

[0105] Protein expression cassette AAV8-OTE-hFVIII-L2-pA (SEQ ID NO: 39)

[0106] Protein expression cassette AAV8-TTM-hFVIII-L1-pA (SEQ ID NO: 40)

[0107] Protein expression cassette AAV8-HLP-hFVIII-L1-pA (SEQ ID NO: 41)

[0108] Protein expression cassette AAV8-STM-hFVIII-L1-pA (SEQ ID NO: 42)

[0109] Protein expression cassette AAV8-CTS-hFVIII-L1-pA (SEQ ID NO: 43)

[0110] The present invention also provides a host cell comprising the expression vector provided herein. In some embodiments, the host cell produces the FVIII protein encoded by the nucleic acid and recovers the produced FVIII protein. The FVIII protein produced by the cell is optionally isolated and / or purified for use in preparing a corresponding medicament or for administration to a subject in need thereof. In a specific embodiment, the host cell is a HepG2 cell.

[0111] The present invention also provides a pharmaceutical composition comprising the coagulation factor FVIII protein variant provided by the present invention, the nucleic acid and / or the expression vector, and a pharmaceutically acceptable excipient. The pharmaceutical composition exhibits one or more of the following properties: 1) enhanced expression in cells or animals; 2) enhanced cellular secretion; and 3) enhanced activity, such as increased coagulation.

[0112] The nucleic acid and expression vector of the present invention can be incorporated into the pharmaceutical composition for in vivo or in vitro administration, or delivered to a subject in need.

[0113] The FVIII protein variants, nucleic acids, and / or expression vectors provided in the embodiments of the present invention can be used to express the FVIII protein. The FVIII protein variants, nucleic acids, and / or expression vectors provided in the present invention can also be used to prepare medicaments for treating diseases requiring the FVIII protein. In some embodiments, the diseases requiring the FVIII protein include hemophilia A, von Willebrand disease, and related traumatic bleeding, injuries, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC), and the like.

[0114] The uses provided by the embodiments of the present invention include uses that reduce the need or use of other compounds, agents, drugs, treatment regimens, treatment plans, processes or remedies. For example, for coagulation disorders, the uses provided by the embodiments of the present invention are beneficial in that the coagulation factor FVIII protein is administered less frequently or at a lower dose or is stopped in a subject in need thereof to supplement the deficiency or defect of the subject's endogenous coagulation factor.

[0115] In some embodiments, the subject in need includes animals, such as mammals, for example, humans, non-human primates, domestic animals (such as dogs, cats, etc.), livestock (such as chickens, ducks, horses, cows, goats, sheep, pigs, etc.), experimental animals (such as mice, rats, rabbits, guinea pigs), etc.

[0116] In another aspect, the present invention provides a method for producing a coagulation factor FVIII protein, comprising:

[0117] S1. Expressing the nucleic acid and / or the expression vector provided by the present invention in a cell;

[0118] S2. Recovering the coagulation factor FVIII protein produced by the cells.

[0119] Specifically, in S1, the cell may be the host cell described above. The method for causing the cell to express the nucleic acid and / or the expression vector may be a method known in the art. In S2, the recovery method may also be a method known in the art and will not be described in detail here.

[0120] The present invention will be further described below in conjunction with specific examples. However, the following specific examples are only used to illustrate but not to limit the scope of the present invention in any way.

[0121] Experimental Materials and Methods

[0122] hFVIII expression cassette and AAV vector

[0123] The wild-type and codon-optimized FVIII gene DNA sequences used in the examples of this invention were synthesized by GenScript or GeneWeiZ. The AAV-mediated hFVIII expression cassette is shown in Figure 1. The expression cassette sequence was cloned into a shuttle plasmid vector to generate a shuttle plasmid containing the AAV-mediated expression of the hFVIII target gene. The hFVIII shuttle plasmids used in the examples of this invention are shown in Table 2.

[0124] Table 2

[0125] Among them, the codon-optimized sequence of hFVIII-SQ (hFVIII-SQ-7) is the sequence shown in SEQ ID NO: 49, and the sequence of AAV8-OTE-hFVIII-SQ-pA is shown in SEQ ID NO: 50.

[0126] hFVIII-SQ-7 (SEQ ID NO: 49):

[0127] AAV8-0TE-hFVIII-SQ-pA (SEQ ID NO:50):

[0128] AAV8 vector production and purification methods

[0129] The production of AAV8 vector adopts a three-plasmid system, that is, a shuttle plasmid containing the hFVIII target gene, a pAAV-R2C8-Kan plasmid (SEQ ID NO: 51) containing the AAV vector rep2 and cap8 genes, and a helper plasmid pHelper-Kan (SEQ ID NO: 52), using PEI as a transfection reagent, to co-transfect HEK293 cells and recombinantly package the AAV8 viral vector. Harvest 48-72 hours after transfection, and the harvested fluid is purified to obtain a recombinant AAV8 viral vector of a certain purity. The purification method is as follows:

[0130] First, the harvested liquid is pretreated: HEK293 cells are fully lysed to release the AAV8 viral vector in the cells, and nuclease is added to digest the free nucleic acid. After digestion, deep filtration is used to remove large molecular impurities and cell fragments. After deep filtration, the filtrate is filtered twice to obtain a clarified liquid for affinity loading.

[0131] Affinity chromatography utilizes the specific adsorption of ligands and proteins to capture the AAV8 viral vector in the harvested fluid and remove most process-related impurities, achieving both concentration and impurity removal. The collected eluate is mixed, neutralized with neutralization buffer, and stored in a sterile reservoir bottle as the sample for anion chromatography.

[0132] Anion chromatography uses the difference in isoelectric points of different components to separate solid and empty AAV8 viruses, while continuing to remove residual impurities. The eluate is collected in a new sterile storage bottle, and then the buffer is replaced with a preparation-stable buffer by ultrafiltration and concentration. Finally, it is sterilized, filtered, and packaged for later use.

[0133] pAAV-R2C8-Kan plasmid (SEQ ID NO: 51):

[0134] Helper plasmid pHelper-Kan (SEQ ID NO: 52):

[0135] Quantification of AAV8 vector titer

[0136] After AAV8 virus purification is complete, the viral content needs to be measured. Genomic titer is the most classic test item to characterize the physical titer of AAV. The most common method for genomic titer detection is to design primer probes based on the rAAV genome sequence and then perform Q-PCR detection.

[0137] Considering that the codon optimization of the hFVIIIORF coding frame was carried out in the present invention, which involves the screening of multiple different vector structures, two sets of primers and probes were designed in the present invention for titer detection of AAV8 virus. The first set of primers and probes were designed for the OTE element combination (SEQ ID NO: 44) region and used for titer detection of AAV8-OTE-hFVIII-L1-WT-pA (SEQ ID NO: 28), AAV8-OTE-hFVIII-L1-1~6-pA (SEQ ID NO: 29~34), AAV8-OTE-hFVIII-L1-8~10-pA (SEQ ID NO: 36~38), and AAV8-OTE-hFVIII-L1-pA (SEQ ID NO: 35). The F1 primer sequence is: 5'-gccgtcacacagatccacaag-3' (SEQ ID NO: 53), and the R1 primer sequence is: 5'-agtggatgtcagtgtcagtaattca-3' (SEQ ID NO: NO:54), and the P1 probe sequence was: 5'-Fam-ttcccgcgggcctggcctcttt-BHQ1-3' (SEQ ID NO:55). The second set of primers and probes were designed for the hFVIII-L1-7 (SEQ ID NO: 10) region and were used for titer detection of AAV8-OTE-hFVIII-L1\L2\SQ-pA (SEQ ID NOs: 35, 39, 50), and AAV8-TTM, HLP, STM, CTS-hFVIII-L1-pA (SEQ ID NOs: 40-43) viruses, wherein the F2 primer sequence is: 5'-gtctcatgtggacctggtga-3' (SEQ ID NO: 56), the R2 primer sequence is: 5'-ggtcttttccttagccaggc-3' (SEQ ID NO: 57), and the P2 probe sequence is: 5'-Fam-ctccctacacaccagcagagccccaatc-BHQ1-3' (SEQ ID NO: 58).

[0138] In the process of genome titer detection, the standard curve should be established first. The positive standard should be diluted to 2×10 7 , 2×10 6 , 2×105 , 2×10 4 , 2×10 3 , 2×10 2 Copies / μl is used as a standard curve template. The standard curve needs to control its linearity and amplification efficiency, generally requiring R2>0.99 and an amplification efficiency between 90% and 110%. The pretreated rAAV sample is then diluted and tested by QPCR to ensure that the sample detection Ct value is within the standard curve range. The sample Ct value is substituted into the standard curve to calculate the rAAV sample genome titer and identify the product content.

[0139] Virus infection of HepG2-AAVR cells

[0140] HepG2-AAVR cells were digested and seeded into 24-well cell culture dishes at 1.5e5 cells / well. After 24 hours, cells from one well were digested and counted. The amount of virus to be added to each well was calculated based on the MOI of viral infection, and the viral stock solution was diluted to an appropriate concentration with DMEM + 10% FBS medium. The medium in the 24-well cell culture dish was removed, and 500 μl of the viral solution was added to each well for infection. The cells were cultured in a CO2 constant temperature incubator for 5 days. After 5 days, the cell culture supernatant was collected and quantified to 500 μl by adding an appropriate volume of DMEM + 10% FBS medium.

[0141] Inject virus samples into the mouse tail vein

[0142] Place the mouse in a suitable container under an infrared lamp, turn on the switch, and irradiate for several minutes. Remove the mouse and wipe the tail with an alcohol cotton ball to fully dilate the tail vein. Place the mouse in a mouse holder, exposing the tail. Use a suitable syringe and needle to extract the sample to be injected. For injection, use a 1mL syringe needle and a matching 1mL syringe barrel. Draw the corresponding volume of sample to be injected and inject it through the tail vein. The injection should be completed in more than 10 seconds. The injection should be slow and uniform. After the injection is completed, press the injection site with a dry cotton ball to stop bleeding.

[0143] Mouse plasma collection

[0144] Mice were anesthetized with isoflurane, and 180 μl of whole blood was collected from the mouse orbit using a capillary tube. 20 μl of 3.8% sodium citrate anticoagulant was added, and the blood was mixed by inversion and centrifuged at 5000 rpm for 10 min. The supernatant was collected as mouse plasma.

[0145] ELISA method for measuring hFVIII protein expression in mouse plasma

[0146] The expression of hFVIII protein in mouse plasma was detected using a Matched-Pair Antibody Set for ELISA of human Factor VIII antigen (FVIII:C) kit (F8C-E1A, Affinity Biologicals). The experimental procedure was as follows: the capture antibody was diluted 1:100 in 50 mM carbonate buffer, 100 μl / well was added to a 96-well ELISA plate, and incubated at room temperature for 2 hours; the wells were removed and washed five times with 300 μl of PBST solution, and 100 μl of standard (0.59 ng / ml to 37.5 ng / ml) or test sample (the standard or test sample was diluted with the sample diluent in the kit) was added and incubated at room temperature for 2 hours; the wells were removed and washed five times with 300 μl of PBST solution, and 100 μl of detecting antibody was added and incubated at room temperature for 1 hour; the wells were removed and washed five times with 300 μl of PBST solution, and 100 μl of The TMB substrate was incubated at room temperature for 15 min, 100 μl of stop solution was added, and OD450 nm was measured using a microplate reader. The hFVIII protein expression level in the mouse plasma sample was calculated based on the standard curve.

[0147] CSA method for measuring hFVIII protein activity in mouse plasma

[0148] Using Chromogenix The hFVIII protein activity in mouse plasma was detected using the SP4 Factor VIII kit (K824094, Diapharma). The experimental procedure was as follows: 1 volume of sample diluent storage solution (10×) and 9 volumes of sterile water were mixed to prepare sample diluent; 2.5% to 80% standard curve diluent was prepared according to the method in the kit (the concentration of the standard stock solution was 1 IU / ml); 5 μl of the sample to be tested was added to 400 μl of diluent to dilute the sample; 1 volume of phospholipid reagent and 5 volumes of coagulation factor reagent were mixed to prepare RP reagent; 40 μl of RP solution and 20 μl of standard or sample to be tested were added to a 96-well plate, mixed and incubated at 37°C for 5 min; 20 μl of CaCl2 solution (preheated at 37°C) was added, mixed and incubated at 37°C for 10 min; 40 μl S-2765 + I-2581 solution (preheated at 37°C) was mixed and incubated at 37°C for 10 minutes. 20 μl of 20% acetic acid solution was added and mixed, and the absorbance at A405 nm was measured using a microplate reader. The hFVIII protein activity in the mouse plasma sample was calculated based on the standard curve.

[0149] ELISA method for measuring hFVIII binding antibodies in mouse plasma

[0150] hFVIII standard (Xyntha, Pfizer) was diluted to 1 μg / mL with PBS and added to a 96-well Elisa plate at 100 μl / well and incubated at 4°C overnight; the wells were removed, washed 5 times with 300 μl PBST solution, blocked with 300 μl blocking solution (5% skim milk powder), and incubated at room temperature for 2 hours; the wells were removed, washed 5 times with 300 μl PBST solution, and 100 μl Murine Anti-Factor VIII standard (GMA-8024, Green Mountain Antibodies) (1.56 ng / mL to 100 ng / ml) or the test sample (the standard or the test sample was diluted with casein buffer) was added and incubated at room temperature for 2 hours at 300 rpm; the wells were removed, washed 5 times with 300 μl PBST solution, and 100 μl of 1:100,000 diluted goat anti-mouse IgG (H+L) secondary antibody (Goat anti-Mouse IgG) was added. IgG (H+L) Secondary Antibody), HRP antibody, incubate at room temperature at 300 rpm for 2 hours; remove the liquid in the wells, add 300 μl PBST solution to wash 5 times, add 100 μl TMB substrate and incubate at room temperature for 15 minutes, add 100 μl stop solution, and measure OD450nm with a microplate reader.

[0151] Mouse tail docking experiment

[0152] Mice were anesthetized with isoflurane and maintained in an anesthesia state with a gas anesthesia machine throughout the experiment. The mice were placed on a 37°C warming pad to maintain body temperature, and their tails were placed in a 37°C constant-temperature water bath for 5 minutes. The tails were removed and a 6mm transverse cut was made from the tip using a disposable scalpel blade. After resection, the tails were immediately placed in 15 mL of normal saline preheated to 37°C, and the bleeding time of the mice was recorded within 30 minutes.

[0153] Quantitative PCR method to measure hFVIII DNA copy number in mouse liver tissue

[0154] hFVIII DNA copy number analysis was performed using a TaqMan probe-based qPCR method. Genomic DNA was extracted from mouse liver tissue using the DNeasy Blood & Tissue Kit (69506, Qiagen). qPCR reactions were set up with the following system: 10 μl 2x TaqMan Gene Expression Master Mix (4369016, ABI), 6 μl ddH2O, 0.5 μl 10 μM F2 primer, 0.5 μl 10 μM R2 primer, 0.5 μl 10 μM P2 probe, 2.5 μl genomic DNA (500 ng) or 2.5 μl positive standard (2e1-2e7 copies / μl), with a total reaction volume of 20 μl. Two replicate wells were set up for each standard or sample to be tested. qPCR reactions were performed using a Quant studio 5 (ABI) qPCR instrument under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. Amplicons corresponding to the hFVIII vector genome were detected using fluorescence at 483-533 nm. The hFVIII DNA copy number per diploid genome was calculated using the linear regression equation of the standard curve and the sample Ct values.

[0155] Quantitative PCR method to measure the copy number of hFVIII mRNA in mouse liver tissue

[0156] Total RNA was extracted from mouse liver tissue using the RNeasy Mini Kit (74106, Qiagen). Genomic DNA removal and reverse transcription were performed according to the instructions for the PrimeScript RT reagent kit with gDNA Eraser (RR047A, TaKaRa). qPCR reactions were set up with the following system: 5 μl of 2x TB Green Premix EX Taq II (RR820A, TaKaRa), 0.4 μl of 10 μM F2 primer or mTbp-F internal reference primer (5'-ccgtgaatcttggctgtaaact-3', SEQ ID NO: 69), 0.4 μl of 10 μM R2 primer or mTbp-R internal reference primer (5'-tgtccgtggctctcttattc-3', SEQ ID NO: 70), 0.2 μl of Rox II, and 4 μl of reverse transcription product (7x dilution). The total reaction volume was 10 μl. Two replicate wells were set up for each standard or test sample. qPCR reactions were performed using a Quant studio 5 (ABI) qPCR instrument. The reaction conditions were as follows: 95°C for 30 seconds, 95°C for 10 seconds, and 60°C for 30 seconds for 40 cycles. hFVIII and mTbp internal reference signals were detected using fluorescence at 483-533 nm. The fold expression of hFVIII mRNA relative to mTbp mRNA was calculated based on the difference between the hFVII ICt value of the sample and the Ct value of the mTbp internal reference.

[0157] Example 1: Comparison of the expression effects of different hFVIII structures in HepG2-AAVR cells

[0158] This example evaluated the expression effects of hFVIII-L1, hFVIII-L2, and hFVIII-SQ in HepG2-AAVR cells.

[0159] HepG2-AAVR cells were seeded into 24-well cell culture dishes one day in advance, with 1.5e5 cells per well, and the culture medium was DMEM + 10% fetal bovine serum. 24 hours later, AAV8-OTE-hFVIII-L1-pA, AAV8-OTE-hFVIII-L2-pA, and AAV8-OTE-hFVIII-SQ-pA viruses were infected into HepG2-AAVR cells at an MOI of 2e4, 1e5, and 4e5, with three replicates per group. Five days after virus infection, the culture supernatants were collected and hFVIII protein expression was measured using the ELISA method and hFVIII protein activity was measured using the CSA method.

[0160] The results of hFVIII protein expression and activity assays after infection of HepG2-AAVR cells with viruses containing different hFVIII structures are shown in Figures 2 and 3. The results showed that both hFVIII protein expression and activity increased in a dose-dependent manner with the MOI of viral infection; at the same MOI, the hFVIII protein expression and activity of the AAV8-OTE-hFVIII-L1-pA and AAV8-OTE-hFVIII-L2-pA viruses were significantly higher than those of the AAV8-OTE-hFVIII-SQ-pA virus. The AAV8-0TE-hFVIII-L1-pA virus had the highest hFVIII protein expression level and activity. The hFVIII protein expression levels were 8 times (MOI: 2e4), 16 times (MOI: 1e5), and 34 times (MOI: 4e5) of the AAV8-0TE-hFVIII-SQ-pA virus, and the hFVIII protein activity was 5 times (MOI: 2e4), 17 times (MOI: 1e5), and 24 times (MOI: 4e5) of the AAV8-0TE-hFVIII-SQ-pA virus.

[0161] Example 2: Comparison of the effects of different hFVIII optimized gene expression in wild-type C57BL / 6 mice

[0162] This example evaluated the expression effects of different hFVIII optimized genes in wild-type C57BL / 6 mice.

[0163] Male wild-type C57BL / 6 mice aged 8 to 10 weeks (n = 4 / group) were selected and AAV8-0TE-hFVIII-L1-1~6-pA, AAV8-0TE-hFVIII-L1-8~10-pA, AAV8-0TE-hFVIII-L1-pA and AAV8-0TE-hFVIII-L1-WT-pA viruses were administered via the tail vein at a dose of 4e12 vg / kg. At 4 weeks after administration, plasma was collected from the mouse orbits, and the hFVIII protein expression level was measured using the ELISA method.

[0164] The first batch of mice were administered AAV8-0TE-hFVIII-L1-WT\1\2\3\9\10-pA and AAV8-0TE-hFVIII-L1-pA viruses. The results of hFVIII protein expression detection are shown in Figure 4. The results showed that after 4 weeks of administration, the expression levels of AAV8-0TE-hFVIII-L1-pA, AAV8-0TE-hFVIII-L1-9-pA and AAV8-0TE-hFVIII-L1-10-pA were significantly higher than those of AAV8-0TE-hFVIII-L1-WT-pA, which were 431±186 ng / ml, 324±78 ng / ml and 317±79 ng / ml, respectively, which were 17 times, 13 times and 13 times that of AAV8-0TE-hFVIII-L1-WT-pA. The second batch of mice were administered AAV8-0TE-hFVIII-L1-WT-pA\4\5\6\8-pA and AAV8-0TE-hFVIII-L1-pA viruses. The results of hFVIII protein expression detection are shown in Figure 5. The results showed that after 4 weeks of administration, the expression levels of AAV8-0TE-hFVIII-L1-pA and AAV8-0TE-hFVIII-L1-5-pA were significantly higher than those of AAV8-0TE-hFVIII-L1-WT-pA, which were 674±463 ng / ml and 379±172 ng / ml, respectively, which were 19 times and 11 times that of AAV8-0TE-hFVIII-L1-WT-pA.

[0165] Example 3: Comparison of hFVIII expression effects of different element combinations in wild-type C57BL / 6 mice

[0166] This example evaluated the effect of a combination of OTE, TTM, HLP, STM, and CTS elements on hFVIII expression in wild-type C57BL / 6 mice.

[0167] Male wild-type C57BL / 6 mice aged 8 to 10 weeks (n = 4 / group) were selected and AAV8-OTE-hFVIII-L1-pA, AAV8-TTM-hFVIII-L1-pA, AAV8-HLP-hFVIII-L1-pA, AAV8-STM-hFVIII-L1-pA, and AAV8-CTS-hFVIII-L1-pA viruses were administered via the tail vein at a dose of 4e12 vg / kg. After 4 weeks of administration, plasma was collected from the mouse orbits, and the hFVIII protein expression level was measured using the ELISA method.

[0168] After 4 weeks of administration to mice, the results of hFVIII protein expression detection are shown in Figure 6. The results show that AAV8-0TE-hFVIII-L1-pA, AAV8-TTM-hFVIII-L1-pA, and AAV8-CTS-hFVIII-L1-pA viruses have similar hFVIII expression levels, which are slightly better than AAV8-HLP-hFVIII-L1-pA and AAV8-STM-hFVIII-L1-pA viruses.

[0169] Example 4: Evaluation of the efficacy of different hFVIII structural viruses in FVIII KO mice

[0170] This example evaluated the expression activity of hFVIII-L1, hFVIII-L2, and hFVIII-SQ structural viruses in FVIII KO mice and the improvement of the abnormal coagulation phenotype of the mice.

[0171] Male FVIII KO mice aged 8 to 10 weeks (n = 5 / group) were selected and AAV8-OTE-hFVIII-L1-pA, AAV8-OTE-hFVIII-L2-pA, and AAV8-OTE-hFVIII-SQ-pA viruses were administered via the tail vein at a dose of 4e12 vg / kg. Plasma was collected from the mouse orbits at 1, 2, 3, 4, and 6 weeks after administration, and hFVIII protein activity was measured using the CSA method. At the experimental endpoint of 8 weeks after administration, a tail-cutting experiment was performed on mice, and the bleeding time of the mice was measured to evaluate the improvement of the abnormal coagulation phenotype of the mice.

[0172] The results of hFVIII protein activity detection at 1, 2, 3, 4, and 6 weeks after administration to mice are shown in Figure 7. The results show that the hFVIII protein activity of mice administered with AAV8-OTE-hFVIII-L1-pA and AAV8-OTE-hFVIII-L2-pA viruses was higher than that of mice administered with AAV8-OTE-hFVIII-SQ-pA virus. At the 6-week time point when hFVIII was stably expressed, the hFVIII protein activity was 7.1 times (hFVIII-L1) and 5.9 times (hFVIII-L2) that of the AAV8-OTE-hFVIII-SQ-pA virus, respectively.

[0173] At 8 weeks after administration, the bleeding time of the mice after tail amputation was measured. The test results are shown in Figure 8, which show that the mice administered with the AAV8-0TE-hFVIII-L1-pA virus had the most significant improvement in the abnormal coagulation phenotype of the mice.

[0174] Example 5: Evaluation of the efficacy of different doses of hFVIII virus in FVIII KO mice

[0175] This example evaluates the expression activity of AAV8-OTE-hFVIII-L1-pA virus at different doses in FVIII KO mice and the improvement of the abnormal coagulation phenotype of mice.

[0176] Male FVIII KO mice aged 8 to 10 weeks (n = 6 / group) were selected and administered AAV8-OTE-hFVIII-L1-pA virus via the tail vein at a dose of 4e12 and 8e11 vg / kg. A FVIII KO mouse vehicle group and a wild-type C57BL / 6 mouse vehicle group control were also set up. At 2, 4, and 6 weeks after administration, plasma was collected from the mouse orbits, and hFVIII protein expression was measured using the Elisa method and hFVIII protein activity was measured using the CSA method. At the experimental endpoint of 6 weeks after administration, a tail-breaking experiment was performed on mice, and the bleeding time of the mice was measured to evaluate the improvement of the abnormal coagulation phenotype of the mice; at the same time, liver tissue was collected from the mice, and the hFVIII DNA copy number was measured by qPCR.

[0177] The results of hFVIII protein expression and hFVIII protein activity testing in mice at 2, 4, and 6 weeks after administration are shown in Figures 9 and 10. The results show that effective hFVIII protein expression was detected at doses of 4e12 vg / kg and 8e11 vg / kg. At 6 weeks after administration, hFVIII protein expression levels were 513±222 ng / ml and 53±34 ng / ml, respectively, and hFVIII protein activities were 279%±144% and 39%±21%, respectively. Table 3 lists the in vivo expression data of similar products in FVIII KO mice, demonstrating that the nucleic acid encoding the human coagulation factor FVIII protein provided by the present invention can achieve higher hFVIII protein expression at a lower dose.

[0178] At the end of the experiment, six weeks after dosing, the mice underwent tail docking to measure bleeding time. The test results are shown in Figure 11. The results showed that at doses of 4e12 vg / kg and 8e11 vg / kg, the bleeding time after tail docking was 3.7±5.1 and 7.2±7.3 minutes, respectively. These were 85.5% and 71.8% shorter than those in the vehicle-treated FVIII KO mice, respectively, demonstrating a significant improvement in the abnormal coagulation phenotype of the mice.

[0179] Table 3

[0180] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A coagulation factor FVIII protein variant, whose amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A nucleic acid encoding a coagulation factor FVIII protein variant, wherein the nucleotide sequence is as shown in any one of SEQ ID NOs: 3 to 15 and SEQ ID NOs: 60 to 68. An expression vector comprising the nucleic acid according to claim 2.

4. The expression vector according to claim 3, It is characterized in that The expression vector is selected from any one of the group consisting of an adeno-associated virus vector, a retrovirus vector, an adenovirus vector, a plasmid or a lentivirus vector; Preferably, the adeno-associated viral vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 serotype.

5. The expression vector according to claim 3 or 4, It is characterized in that The expression vector further comprises a promoter, an enhancer, an intron, one or more terminal inverted repeat sequences, a Kozak sequence and / or a polyA tail operably linked to the nucleic acid; Optionally, the enhancer and the promoter are operably linked to the sequence of the nucleic acid in sequence, or the intron is located inside or on both sides of the sequence of the nucleic acid, or the terminal inverted repeat sequence is located at the 5' or 3' end of the sequence of the nucleic acid, or the Kozak sequence is located between the 5' end of the sequence of the nucleic acid and the intron, or the polyA tail is located between the 3' end of the sequence of the nucleic acid and the terminal inverted repeat sequence.

6. The expression vector according to claim 5, It is characterized in that The nucleotide sequence of the promoter is shown in any one of SEQ ID NOs: 16 to 18; the nucleotide sequence of the enhancer is shown in any one of SEQ ID NOs: 19 to 21; the nucleotide sequence of the intron is shown in any one of SEQ ID NOs: 22 to 24; the nucleotide sequence of the terminal inverted repeat sequence is shown in SEQ ID NO: 25 or SEQ ID NO: 26; the Kozak sequence is gccacc; the nucleotide sequence of the polyA tail is shown in SEQ ID NO: 27; Preferably, the expression vector comprises any one of the sequences shown in SEQ ID NOs: 28 to 43.

7. A host cell comprising the nucleic acid of claim 2 or the expression vector of any one of claims 3 to 6.

8. A pharmaceutical composition comprising the coagulation factor FVIII protein variant according to claim 1, the nucleic acid according to claim 2 and / or the expression vector according to any one of claims 3 to 6, and a pharmaceutically acceptable excipient.

9. Use of the blood coagulation factor FVIII protein variant according to claim 1, the nucleic acid according to claim 2 and / or the expression vector according to any one of claims 3 to 6 in expressing the blood coagulation factor FVIII protein, or in preparing a medicament for treating a disease requiring the blood coagulation factor FVIII protein.

10. A method for producing a coagulation factor FVIII protein, It is characterized in that include: Expressing the nucleic acid of claim 2 and / or the expression vector of any one of claims 3 to 6 in a cell; The blood coagulation factor FVIII protein produced by the cells is recovered.

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