High specific activity / high secretion expression-type factor viii variant, nucleic acid encoding high expression-type factor viii, and use thereof

Abstract

The present invention provides: a human factor VIII variant characterized in that at least one amino acid residue selected from the group consisting of K213, S367, and F2196 of a wild-type human factor VIII is substituted with another amino acid, and characterized by having a higher specific activity and / or secretion expression efficiency as compared to the wild type; a nucleic acid encoding the human factor VIII variant; and a therapeutic agent for hemophilia A using the same.

Description

High specific activity / high secretory expression type of modified factor VIII, highly expressed type of nucleic acid encoding factor VIII, and uses thereof

[0001] The present invention relates to a human factor VIII variant with high specific activity and high secretory expression, a nucleic acid encoding the same, a nucleic acid encoding a highly expressed form of human factor VIII, and uses thereof. More specifically, the present invention relates to a human factor VIII variant with enhanced secretory expression efficiency and / or specific activity compared to the wild-type, a nucleic acid encoding the same, a nucleic acid encoding human factor VIII (including variants) whose expression efficiency has been enhanced by modifying the base sequence, and the use thereof for the treatment of hemophilia A, etc.

[0002] Hemophilia is a disease caused by a genetic deficiency in blood coagulation factors involved in hemostasis. According to the World Federation of Hemophilia (WFH), there are approximately 400,000 hemophilia patients worldwide, with approximately 6,000 patients in Japan. There are two types of hemophilia: hemophilia A, which is caused by a deficiency in factor VIII, and hemophilia B, which is caused by a deficiency in factor IX. Hemophilia A patients predominate, with a ratio of 5:1.

[0003] Traditionally, hemophilia A has been treated with factor VIII replacement therapy. However, the half-life of this coagulation factor is short, and patients must receive intravenous administration two to three times a week from childhood to prevent bleeding. Furthermore, when factor VIII is continuously administered to hemophilia A patients, it is known that they may develop antibodies against factor VIII, known as inhibitors (25-30%), which reduces the effectiveness of replacement therapy.

[0004] In recent years, the antibody drug emicizumab, which acts as a substitute for factor VIII, has been launched as a treatment for hemophilia A. Emicizumab can treat hemophilia A with subcutaneous injections less frequently (once a month) than factor VIII preparations. However, it is no different from conventional coagulation factor preparations in that it requires continuous administration, and because it is produced using recombinant animal cells, the manufacturing costs are high, placing a significant burden on the medical economy.

[0005] Therefore, gene therapy using viral vectors, which are expected to maintain coagulation factor activity for a long period of time with a single administration, has attracted attention as a new treatment for hemophilia. Hemophilia is a hereditary disease caused by a single gene deficiency. Because therapeutic effects can be easily confirmed by measuring blood coagulation factor levels and because even a relatively small increase in blood levels can be expected to be effective, it has long been considered a good target disease for gene therapy. Furthermore, preclinical studies have shown that continuous expression of coagulation factors through gene therapy can induce immune tolerance via regulatory T cells and eliminate inhibitors. Recently, partial successes in human gene therapy using adeno-associated virus (AAV) vectors have been reported one after another (see, for example, Non-Patent Documents 1 and 2). Coagulation factors are produced in the liver, and AAV vectors can deliver genes to the liver with high efficiency via intravenous administration.

[0006] In BioMarin's clinical trial, high factor VIII activity was maintained in the high-dose administration group even after one year (Non-Patent Document 1), but a tendency for blood levels to decrease was observed after three years. One possible cause of this is that factor VIII expression from high-dose AAV vectors in liver cells may induce endoplasmic reticulum stress (Non-Patent Document 3). On the other hand, in Spark's clinical trial, a serotype with high hepatotropism was used and a lower dose of AAV vector than BioMarin's was administered, but 2 × 10 １２ Hepatotoxicity was observed in the vg / kg group (Non-Patent Document 2). Attempts have been made to reduce the dose and improve safety by achieving high expression through the use of liver-specific promoters or codon optimization of factor VIII, but these approaches are not sufficient when high protein expression itself impairs the long-term stability of the therapeutic effect.

[0007] As a means to avoid attenuation of therapeutic efficacy due to high-dose viral vector administration, it is desirable to develop a variant of factor VIII with high specific activity that can exert coagulation activity equivalent to or greater than that of wild-type factor VIII expressed at an appropriate protein level, or a variant of factor VIII with improved extracellular secretion efficiency to eliminate adverse effects on cells such as endoplasmic reticulum stress. Xiao et al. reported that substituting 10 amino acid residues in the A1 domain with porcine amino acid residues significantly increases secretion efficiency, with five amino acid substitutions being particularly important (Patent Document 1 and Non-Patent Document 4). Furthermore, the same group reported that substituting 12 amino acid residues in the human factor VIII light chain with canine amino acid residues can increase specific activity while maintaining wild-type secretion efficiency (Non-Patent Document 5). Sabatino et al. also disclosed that substituting one or more amino acids in the furin recognition site and the a3 domain in the human factor VIII light chain with canine amino acid residues or deleting them can increase specific activity (Patent Document 2).

[0008] WO 2014 / 209942US 2019 / 0144524

[0009] Rangarajan, S. et al., N. Eng. J. Med., 377: 2519-2530 (2017)Perrin, GQ et al., Blood, 133: 407-414 (2019)Zolotukhin, I. et al., Mol. Ther. Methods Clin. Dev., 3: 16063 (2016)Cao, W. et al. al., Mol. Ther. Methods Clin. Dev., 19: 486-495 (2020)Firrman, J. et al., Mol. Ther. Methods Clin. Dev., 17: 328-336 (2020)

[0010] The first object of the present invention is to provide novel human factor VIII variants and nucleic acids encoding them that can stably complement factor VIII over a long period of time in gene therapy for hemophilia A, thereby establishing a more sustained-effect gene therapy for hemophilia A. The second object of the present invention is to provide recombinant factor VIII preparations containing the variants and mRNA pharmaceuticals encoding the variants.

[0011] As a result of extensive research aimed at achieving the above object, the present inventors have found that various variants of wild-type human factor VIII in which at least one amino acid residue selected from the group consisting of lysine at position 213 (K213), serine at position 367 (S367), and phenylalanine at position 2196 (F2196) is substituted with another amino acid exhibit higher specific activity and / or higher secretory expression than the wild-type (herein, these variants may be abbreviated as "high specific activity / high secretory expression type"). (Herein, the positions of amino acid residues in human factor VIII are represented by the amino acid numbering in the mature full-length single-chain polypeptide (2332 amino acids), and the nth amino acid residue, X (X is the one-letter code for the amino acid) is abbreviated as "Xn." When n is a negative integer, this indicates the position of an amino acid residue in the signal peptide. When the nth amino acid residue, X, is substituted with another amino acid, Y (Y is the one-letter code for the other amino acid), it is abbreviated as "XnY.")

[0012] Furthermore, in order to improve the expression efficiency of the variants containing the above amino acid substitutions in humans, the inventors optimized the nucleotide sequence of the nucleic acid encoding the variants for human use using a codon optimization algorithm. However, the resulting nucleotide sequence contained many CpG sequences. The inventors predicted that CpG sequences may induce an immune response in the host and inhibit continuous transgene expression, so they performed base substitutions to remove these CpG sequences without changing the amino acid sequence of the variants, and then performed codon optimization again. The resulting variant-encoding nucleic acid was ligated downstream of a promoter sequence from which CpG sequences had also been removed, and then incorporated into an AAV vector. When the resulting nucleic acid encoding the variants was administered to a human liver cell line and a hemophilia model mouse, factor VIII expression was significantly increased compared to the variant-encoding nucleic acid before CpG sequence removal. To demonstrate that the effect of CpG sequence removal on enhancing expression is not limited to the variant but is common to any human factor VIII, including wild-type, the expression of factor VIII was similarly compared between a codon-optimized nucleic acid encoding wild-type human factor VIII and a nucleic acid that had been further codon-optimized after CpG sequence removal. Again, significant enhancement of expression was observed by CpG sequence removal. Based on these findings, the present inventors conducted further studies, which led to the completion of the present invention.

[0013] That is, the present invention provides the following: [Item 1] A variant of human factor VIII, in which at least one amino acid residue selected from the group consisting of K213, S367, and F2196 of wild-type human factor VIII is substituted with another amino acid, and which exhibits higher specific activity and / or higher secretory expression efficiency compared to the wild-type. [Item 2] The variant of Item 1, in which the amino acid substitution at K213 is K213N or K213H, and / or the amino acid substitution at S367 is S367P, S367N, or S367Q, and / or the amino acid substitution at F2196 is F2196L or F2196M. [Item 3] The variant of Item 2, which comprises the amino acid substitutions K213N, S367P, and F2196L. [Item 4] The variant of any one of Items 1 to 3, wherein at least one amino acid residue selected from the group consisting of R-5, P25, A28, L152, M217, W228, Q410, Y487, R489, F501, M539, I566, L603, I642, S727, A736, S1657, Q1659, E1661, I1668, D1681, R1776, H1859, A1993, H2007, N2019, K2085, K2207, F2275, S2296, V2314, Q2316, and M2321 is further substituted with another amino acid. [Item 5] R-5P, P25H, A28T, L152A, L152D, L152E, L152H, L152I, L152M, L152N, L152P, L152Q or L152V, M217T, W228Q, Q410L, Y487H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657 [Item 6] The variant according to Item 4, comprising at least one amino acid substitution selected from the group consisting of L152A, L152D, L152E, L152H, L152I, L152M, L152N, L152P, L152Q, or L152V, and / or I1668F and D1681G.[Item 7] The variant of Item 1, further comprising the amino acid substitutions: K2207Q, Q2316H, and M2321L, and optionally at least one amino acid substitution in the C2 domain selected from the group consisting of F2275L, S2296A, and V2314A. [Item 8] The variant of Item 7, further comprising the amino acid substitutions: I1668F and D1681G. [Item 9] The variant of Item 3, further comprising amino acid substitutions L152P, Y487H, and L603P, and optionally further comprising at least one amino acid substitution selected from the group consisting of S727P, Q1659E, H1859R, A1993V, H2007Q, N2019K, and K2085M. [Item 10] The variant of Item 3, further comprising amino acid substitutions M539L, I566M, L603P, I642V, K2207Q, F2275L, S2296A, V2314A, Q2316H, and M2321L. [Item 11] The variant of Item 10, further comprising amino acid substitutions I1668F and D1681G. [Item 12] A human factor VIII variant having the following amino acid substitutions (a) to (n) in wild-type human factor VIII: (a) K213N, S367P, and F2196L; (b) K213N, S367P, F2196L, I1668F, and D1681G; (c) K213N, M539L, I566M, I642V, K2207Q, F2275L, Q2316H, and M2321L; or (d) K213N, M539L, I566M, L603P, I642V, F2196L, K2207Q, F2275L, Q2316H, and M2321L. (e) K213N, M539L, I566M, I642V, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L (f) K213N, M539L, I566M, I642V, I1668F, D1681G, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L(g) L152P, K213N, S367P and F2196L (h) L152P, K213N, S367P, Y487H, L603P and F2196L (i) L152P, K213N, S367P, Y487H, L603P, S727P, Q1659E and F2196L (j) L152P, K213N, S367P, Y487H, L603P, S727P, Q1659E, H1859R, A1993V, H2007Q, K2085M and F2196L (k) K213N, S367P, M539L, I566M, L603P, I642V, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L (l) P25H, A28T, K213N, M217T, S367P, Q410L, Y487H, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P , Q1659E, E1661K, I1668F, D1681G, R1776K, N2019K, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L (m) R-5P, P25H, A28T, K213N, M217T, W228Q, S367P, Q410L, Y487H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736 V, S1657P, Q1659E, E1661K, I1668F, D1681G, R1776K, N2019K, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L (n) R-5P, P25H, A28T, L152P, K213N, M217T, W228Q, S367P, Q410L, Y487H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P, Q1659E, E1661K, I1668F, D1681G, R1776K, H1859R, A1993V, H2007Q, N2019K, K2085M, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L [Item 13] [Item 14] The variant according to any one of Items 1 to 12, which lacks the B domain.The variant according to Item 13, wherein the B domain deletion is a deletion of the amino acid sequence from positions 763 to 1656 in the amino acid sequence represented by SEQ ID NO: 1. [Item 15] A nucleic acid encoding the variant according to any one of Items 1 to 14. [Item 16] The nucleic acid according to Item 15, which has been codon-optimized for human use. [Item 17] The nucleic acid according to Item 16, or a nucleic acid encoding wild-type human factor VIII codon-optimized for human use, which has been further modified so as to contain no CpG sequences without changing the amino acid sequence it encodes. [Item 18] An expression vector comprising the nucleic acid according to any one of Items 15 to 17. [Item 19] The expression vector according to Item 18, wherein the nucleic acid is under the control of a liver-specific promoter. [Item 20] The expression vector according to Item 18 or 19, wherein the promoter has been modified so as to contain no CpG sequences. [Item 21] The expression vector according to any one of Items 18 to 20, wherein the vector is a viral vector. [Item 22] The expression vector of Item 21, wherein the virus is an adeno-associated virus having liver tropism. [Item 23] A host cell into which the expression vector of any one of Items 18 to 22 has been introduced. [Item 24] A method for producing human factor VIII or a variant thereof, comprising culturing the host cell of Item 23 and recovering human factor VIII or a variant thereof from the resulting culture. [Item 25] A pharmaceutical comprising the variant of any one of Items 1 to 14, the nucleic acid of any one of Items 15 to 17, the expression vector of any one of Items 18 to 22, or the host cell of Item 23. [Item 26] The pharmaceutical of Item 25, which is used for treating hemophilia A. [Item 26a] A method for treating hemophilia A, comprising administering to a patient with hemophilia A an effective amount of the variant of any one of Items 1 to 14, the nucleic acid of any one of Items 15 to 17, the expression vector of any one of Items 18 to 22, or the host cell of Item 23. [Item 26b] The variant according to any one of Items 1 to 14, the nucleic acid according to any one of Items 15 to 17, the expression vector according to any one of Items 18 to 22, or the host cell according to Item 23, for use in treating hemophilia A. [Item 26c]Use of the variant according to any one of Items 1 to 14, the nucleic acid according to any one of Items 15 to 17, the expression vector according to any one of Items 18 to 22, or the host cell according to Item 23 for the manufacture of a therapeutic agent for hemophilia A.

[0014] The present invention provides human factor VIII variants with high specific activity and / or high secretory expression efficiency, and nucleic acids encoding the same, which can be used to stably supplement factor VIII for a long period of time to treat hemophilia A. The present invention also provides human factor VIII-encoding nucleic acids with enhanced protein expression efficiency compared to nucleic acids encoding the same amino acid sequence.

[0015] The amino acid substitutions of the variants of the present invention (Ver. 1 to Ver. 12) are shown. The in vitro (left) and in vivo coagulation factor activity of Ver. 1 to Ver. 10 are shown. The in vitro coagulation factor activity of variants such as S3, S5, S7, S4, S6, S8, and S12 is shown (left: one-stage coagulation assay; right: synthetic substrate assay). The effect of each amino acid substitution contained in the S8 variant on the coagulation factor activity alone is shown. The coagulation factor activity of XNPL variants when L152 is substituted with various other amino acids is shown. The coagulation factor activity of PXPL variants when K213 is substituted with various other amino acids is shown. The coagulation factor activity of PNXL variants when S367 is substituted with various other amino acids is shown. The coagulation factor activity of PNPX variants when F2196 is substituted with various other amino acids is shown. The in vivo coagulation factor activity and blood FVIII antigen levels of various variants are shown.

[0033] Figure 1 shows the intracellular and extracellular localization of wild-type and various variants of FVIII. Figure 2 shows the intracellular localization of wild-type and FG-mutated FVIII. Figure 3 shows the in vitro coagulation factor activity of variant Ver. 12. Figure 4 shows the amino acid substitutions of variants of the present invention (F8-Tochigi-3, 5, 8, 10, 11, 13, 15). Figure 5 shows the in vitro coagulation factor activity and antigen amount of variants of the present invention (F8-Tochigi-3, 5, 8, 10, 11, 13, 15, 28). Figure 6 shows the in vitro and in vivo expression enhancement effect of the Ver. 4 variant by CpG sequence removal. Figure 7 shows the in vitro and in vivo expression enhancement effect of wild-type hFVIII by CpG sequence removal. Figure 8 shows that an AAV8 vector carrying the CpG sequence-removed Ver. 4 variant can significantly increase factor VIII activity in a cynomolgus monkey model at the dose used in clinical trials. This shows that an AAV5 vector carrying the Ver. 4 variant with CpG sequences removed can significantly increase factor VIII activity in a cynomolgus monkey model at a dose of 1 / 10 (top) and 1 / 30 (bottom) of the commercially available drug. The figure also shows the effect of shortening the polyA addition signal, the sequence between the promoter and the human factor VIII coding sequence, on enhancing factor VIII activity.

[0016] The single-chain full-length human factor VIII protein, after its 19-amino acid signal peptide has been cleaved in the endoplasmic reticulum, consists of three A domains (A1, A2, A3), two C domains (C1 and C2), a B domain, and three peptide regions rich in acidic amino acids (a1, a2, a3). These regions are arranged in the following order from the amino terminus: A1 (positions 1-336), a1 (positions 337-372), A2 (positions 373-710), a2 (positions 711-740), B (positions 741-1648), a3 (positions 1649-1689), A3 (positions 1690-2019), C1 (positions 2020-2172), and C2 (positions 2173-2332). The B domain is cleaved at R1313 and R1648 in the Golgi apparatus, resulting in a two-chain heavy and light chain. Since the coagulation activity is hardly impaired even when the B domain is deleted, B domain deleted (BDD) factor VIII is often used and is also preferably used in the present invention.

[0017] The present invention provides human factor VIII variants (hereinafter also referred to as "variants of the present invention") that have enhanced specific activity and / or secretory expression efficiency compared to wild-type human factor VIII. The variants of the present invention are characterized in that at least one amino acid residue selected from the group consisting of K213, S367, and F2196 of wild-type human factor VIII is substituted with another amino acid.

[0018] As used herein, the term "wild-type human factor VIII" refers to a protein comprising the amino acid sequence of naturally occurring normal human factor VIII, and specifically includes: (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 (registered in UniProtKB under accession number P00451); or (b) a protein consisting of the amino acid sequence of an allelic variant (minor allele frequency (MAF) of less than 1%) or genetic polymorphism (MAF of 1% or more; e.g., SNP) of the protein of (a) (for example, a variant listed as a non-disease-associated variant in the "Variant" column of UniProtKB under accession number P00451; excluding the amino acid mutations at positions K213, S367, and F2196), which protein exhibits a specific activity and secretory expression efficiency equivalent to those of the protein of (a). Here, "equivalent" in specific activity or secretory expression efficiency means that the specific activity or secretory expression efficiency is 0.8 to 1.2 times, preferably 0.9 to 1.1 times, and more preferably 0.95 to 1.05 times that of the protein (a). In a preferred embodiment, the wild-type human factor VIII is the protein (a).

[0019] As used herein, the term "variant human factor VIII" refers to a protein having an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence (reference sequence) of wild-type human factor VIII (a) or (b) above, and which has a substantially altered specific activity and / or secretory expression efficiency compared to human factor VIII consisting of the reference sequence. Here, "substantially altered" preferably means a statistically significant change, but also encompasses cases in which there is an increase or decrease even if there is no significant difference.

[0020] The variants of the present invention are human factor VIII variants having higher specific activity and / or secretory expression efficiency than the wild-type. The specific activity of human factor VIII can be evaluated by measuring the coagulation factor activity using a known one-stage coagulation assay (OSA) or chromogenic assay (CSA), measuring the protein amount using a known protein quantification method (e.g., immunoassay such as ELISA), and calculating the coagulation factor activity per unit protein amount. The secretory expression efficiency of human factor VIII can be evaluated by measuring the human factor VIII concentration in the culture supernatant of cells transfected with a nucleic acid encoding it or in the plasma of animals administered the nucleic acid using a known method (e.g., immunoassay such as ELISA).

[0021] In the variants of the present invention, at least one, preferably two (e.g., K213 and S367, K213 and F2196, or S367 and F2196), more preferably three (K213, S367 and F2196) amino acid residues selected from the group consisting of K213, S367, and F2196 of wild-type human factor VIII are substituted with other amino acids. When the wild-type human factor VIII is the protein (b) above and the positions of the amino acid residues therein differ from those in SEQ ID NO: 1 due to deletion or insertion / addition of one or more amino acids, the amino acid residues corresponding to K213, S367, and F2196 can be identified as the amino acid residues corresponding to K213, S367, and F2196 in SEQ ID NO: 1, respectively, when the amino acid sequence (b) and the amino acid sequence of SEQ ID NO: 1 are appropriately aligned using any homology search algorithm.

[0022] The other amino acids are not particularly limited as long as the resulting human factor VIII variant has enhanced specific activity and / or secretory expression efficiency compared to the wild-type, but an amino acid substitution at K213 is preferably K213N or K213H, with K213N being more preferred. An amino acid substitution at S367 is preferably S367P, S367N, or S367Q, with S367P being more preferred. An amino acid substitution at F2196 is preferably F2196L or F2196M, with F2196L being more preferred.

[0023] In a particularly preferred embodiment, the variant of the present invention contains the amino acid substitutions K213N, S367P, and F2196L in wild-type human factor VIII (hereinafter, sometimes referred to as "NPL mutation" after the substituted amino acid residues). A variant having the NPL mutation is characterized by a higher specific activity than the wild-type, provided that there are no other mutations (combinations of mutations) that would negate the effect of the NPL mutation. As described above, a high-specific-activity variant can achieve coagulation activity equivalent to or greater than that achieved by overexpressing wild-type factor VIII at an appropriate protein expression level that does not impair the long-term stability of the therapeutic effect, and is therefore useful for gene therapy for hemophilia A.

[0024] As long as the variant of the present invention maintains the properties of higher specific activity and / or higher secretory expression efficiency compared to the wild-type, in addition to at least one amino acid substitution selected from the group consisting of K213, S367, and F2196, amino acid residues at positions other than K213, S367, and F2196 may be substituted with other amino acids. Examples of such substitution sites include R-5, P25, A28, L152, M217, W228, Q410, Y487, R489, F501, M539, I566, L603, I642, S727, A736, S1657, Q1659, E1661, I1668, D1681, R1776, H1859, A1993, H2007, N2019, K2085, K2207, F2275, S2296, V2314, Q2316, and M2321. Thus, in one embodiment, the variant of the present invention is one in which at least one of the amino acid residues is further substituted with another amino acid.

[0025] The other amino acids are not particularly limited as long as the human factor VIII variant obtained as a result of the amino acid substitution still has enhanced specific activity and / or secretory expression efficiency compared to the wild-type, but are preferably R-5P, P25H, A28T, L152A, L152D, L152E, L152H, L152I, L152M, L152N, L152P, L152Q or L152V, M217T, W228Q, Q410L, Y487H, and at least one amino acid substitution selected from the group consisting of R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P, Q1659E, E1661K, I1668F, D1681G, R1776K, H1859R, A1993V, H2007Q, N2019K, K2085M, K2207Q, F2275L, S2296A, V2314A, Q2316H, and M2321L. More preferably, the other amino acid substitutions include: (i) L152A, L152D, L152E, L152H, L152I, L152M, L152N, L152P, L152Q or L152V, and / or (ii) I1668F and D1681G.

[0026] A particularly preferred embodiment of the present invention is a variant containing the amino acid substitution (i) above in addition to the NPL mutation (hereinafter, this may be referred to as an "XNPL mutation," and when L152 is substituted with proline (P), this may be referred to as a "PNPL mutation"). Variants containing XNPL mutations, including PNPL mutations, may not only have higher specific activity than the wild-type, but also exhibit higher secretory expression than the wild-type. Therefore, in addition to the advantages conferred by the NPL mutation, these variants may have the further advantage of suppressing the induction of endoplasmic reticulum stress by improving secretion efficiency, thereby realizing long-term stability of the therapeutic effects of human factor VIII.

[0027] Another preferred embodiment of the present invention includes a variant containing the amino acid substitution (ii) above (hereinafter sometimes referred to as "FG mutation") in addition to the NPL or XNPL mutation. The variant containing the FG mutation exhibits significantly reduced intracellular retention of the light chain compared to the corresponding human factor VIII lacking either the I1668F or D1681G mutation, and therefore may exhibit further improvements in secretion efficiency and / or heavy and light chain reconstitution.

[0028] In a preferred embodiment, the variant of the present invention comprises: the amino acid substitution at K213 is K213N; it further comprises M539L and, optionally, at least one amino acid substitution in the A2 domain selected from the group consisting of I566M, L603P, and I642V; it further comprises at least one amino acid substitution in the C2 domain selected from the group consisting of K2207Q, Q2316H, and M2321L and, optionally, F2275L, S2296A, and V2314A. By including at least the amino acid substitutions K213N, M539L, K2207Q, Q2316H, and M2321L, the variant can be conferred the property of high secretory expression compared to wild-type human factor VIII, even without the NPL mutation. The secretory expression efficiency of the variant can be further enhanced by further including the FG mutation.

[0029] In another preferred embodiment, the variant of the present invention further comprises, in addition to the NPL mutation, amino acid substitutions L152P, Y487H, and L603P, and optionally further comprises at least one amino acid substitution selected from the group consisting of S727P, Q1659E, H1859R, A1993V, H2007Q, N2019K, and K2085M. Because the variant comprises the PNPL mutation, it has a higher specific activity and is expressed at a higher level of secretion compared to the wild-type.

[0030] In another preferred embodiment, the variant of the present invention further comprises the following amino acid substitutions in addition to the NPL mutation: M539L, I566M, L603P, I642V, K2207Q, F2275L, S2296A, V2314A, Q2316H, and M2321L. While the high specific activity characteristic of the NPL mutation may be attenuated by the inclusion of additional amino acid mutations, including the 10 amino acid substitutions described above, the variant exhibits enhanced secretory expression efficiency regardless of the presence or absence of an amino acid substitution at L152 (e.g., L152P) (i.e., even without the XNPL mutation). The secretory expression efficiency of the variant can be further enhanced by further including the FG mutation.

[0031] Preferred embodiments of the present invention include human factor VIII variants having the following amino acid substitutions (a) to (n) in wild-type human factor VIII: (a) K213N, S367P, and F2196L; (b) K213N, S367P, F2196L, I1668F, and D1681G; (c) K213N, M539L, I566M, I642V, K2207Q, F2275L, Q2316H, and M2321L; and (d) K213N, M539L, I566M, L603P, I642V, F2196L, K2207Q, F2275L, Q2316H, and M2321L. (e) K213N, M539L, I566M, I642V, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L; (f) K213N, M539L, I566M, I642V, I1668F, D1681G, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L; (g) L152P, K213N, S367P and F2196L; (h) L152P, K213N, S367P, Y487H, L603P and F2196L. (i) L152P, K213N, S367P, Y487H, L603P, S727P, Q1659E and F2196L; (j) L152P, K213N, S367P, Y487H, L603P, S727P, Q1659E, H1859R, A1993V, H2007Q, K2085M and F2196L; (k) K213N, S367P, M539L, I566M, L603P, I642V, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L. (l) P25H, A28T, K213N, M217T, S367P, Q410L, Y487H, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P , Q1659E, E1661K, I1668F, D1681G, R1776K, N2019K, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L(m) R-5P, P25H, A28T, K213N, M217T, W228Q, S367P, Q410L, Y487H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736 V, S1657P, Q1659E, E1661K, I1668F, D1681G, R1776K, N2019K, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L (n) R-5P, P25H, A28T, L152P, K213N, M217T, W228Q, S367P, Q410L, Y487 H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P, Q1 659E, E1661K, I1668F, D1681G, R1776K, H1859R, A1993V, H2007Q, N201 9K, K2085M, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L

[0032] The B domain of human factor VIII does not contain regions that interact with various proteins and does not contribute to cofactor activity in circulating blood. Therefore, even if the B domain is deleted, coagulation factor activity is maintained. Therefore, the variants of the present invention may lack the B domain. "B domain deleted" refers not only to a complete deletion of the amino acid sequence of the B domain (positions 741 to 1648) but also to a variant in which an amino acid sequence of approximately 1 to 15 amino acid residues at the N-terminus and / or C-terminus of the B domain is retained between the a2 and a3 regions. B domain-deleted (BDD) human factor VIII variants are advantageous when using AAV vectors in gene therapy for hemophilia A. Although the nucleic acid size that can be carried by AAV vectors is small (4.7 kbp), full-length human factor VIII consists of 2351 amino acids, so the coding region alone exceeds 7 kbp and cannot be carried by AAV vectors. On the other hand, the coding sequence of BDD-human factor VIII is less than 4.4 kbp, and the entire expression cassette including the promoter sequence and the like fits within the packaging size of an AAV vector.

[0033] In a preferred embodiment, the variant of the present invention lacks positions 744 to 1637 of the B domain (corresponding to positions 763 to 1656 in the amino acid sequence represented by SEQ ID NO: 1) (i.e., S743 is linked to Q1638; this may be referred to as "FVIII SQ" or "SQ"). BDD-human factor VIII that leads to efficient human factor VIII processing, such as deletion of the B domain leaving portions of both termini of the B domain or substitution of the B domain with 3 to 4 arginine (R) residues, other than FVIII SQ, are known (e.g., Lind et al., Eur. J. Biochem. 232, 19-27 (1995)), and these can also be used in the present invention.

[0034] On the other hand, since the B domain is known to be involved in the intracellular transport of factor VIII, the variant of the present invention may contain all or a part of the B domain in order to improve secretion efficiency. For example, when a lentivirus (LV) vector or adenovirus (AdV) vector with a larger packaging size is used in gene therapy, when mRNA encoding the variant of the present invention is used as an mRNA drug, or when the variant of the present invention is recombinantly produced and used as a protein preparation, it is also preferable to use a variant containing the B domain.

[0035] The variants of the present invention may be expressed in an animal to be treated by administering a nucleic acid encoding them in vivo or ex vivo to the animal, or may be recombinant proteins obtained by introducing the nucleic acid into an appropriate host cell, allowing it to be expressed, and culturing the host cell. In either case, the variants of the present invention are produced based on nucleic acids encoding them, and therefore the present invention also provides nucleic acids encoding any of the above variants of the present invention.

[0036] The "nucleic acid encoding a human factor VIII variant" used in the present invention may be DNA or RNA, or may be a DNA / RNA chimera. The nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. If single-stranded, it may be a sense strand (i.e., coding strand) or an antisense strand (i.e., non-coding strand). The type of nucleic acid can be appropriately selected depending on its application (such as the type of vector used), but is preferably DNA, more preferably double-stranded DNA. The nucleic acid may also be a physiologically acceptable salt with an acid or base, for example, a physiologically acceptable acid addition salt. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid). DNA encoding human factor VIII variants includes genomic DNA, cDNA (cRNA) derived from cells or tissues of humans or other mammals, and synthetic DNA (RNA).

[0037] More specifically, examples of "nucleic acids encoding variants of the present invention" include any nucleotide sequence encoding a variant amino acid sequence having any of the above-mentioned amino acid substitutions in the amino acid sequence of wild-type human factor VIII represented by SEQ ID NO: 1, or the amino acid sequence of a natural allelic variant or genetic polymorphism (e.g., SNP, etc.) of a protein consisting of said amino acid sequence. Preferably, the nucleotide sequence is a codon-optimized sequence for expression in the host cell to be used, preferably a human cell. During gene expression, converting the nucleotide sequence to codons frequently used in the host organism can be expected to increase the amount of protein expression. Data on codon usage in the host to be used can be obtained, for example, from the genetic code usage database published on the website of the Kazusa DNA Research Institute (http: / / www.kazusa.or.jp / codon / index.html), or references listing codon usage in each host can be referenced. Alternatively, as used in the Examples described below, codon optimization can be performed using a known codon optimization algorithm (e.g., GeneArt Codon Optimizer). In such algorithms, in addition to the frequency of codon usage in the host, multiple parameters can be taken into consideration, such as GC content, removal of destabilizing RNA elements, removal of hidden splice sites, removal of intragenic polyA sites, removal of repetitive sequences, avoidance of RNA secondary structures, and removal of IRES. DNA encoding the desired variant of the present invention can be constructed by chemically synthesizing a DNA strand of the codon-optimized sequence obtained as described above, or by connecting chemically synthesized, partially overlapping short oligo-DNA strands using PCR or Gibson Assembly.

[0038] In a preferred embodiment, the nucleic acid encoding the variant of the present invention is a nucleic acid encoding a wild-type human factor VIII consisting of the amino acid sequence represented by SEQ ID NO: 1, wherein the nucleic acid encoding the variant of the present invention is a nucleic acid encoding the variant of the wild-type human factor VIII consisting of the amino acid sequence represented by SEQ ID NO: 1, wherein the nucleic acid encoding the variant of the wild-type human factor VIII consists of the amino acid sequence represented by SEQ ID NO: 1, and ... An example of such a nucleic acid is a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 2, which is codon-optimized for human use and encodes a variant having the amino acid substitutions Q1659E, E1661K, I1668F, D1681G, R1776K, H1859R, A1993V, H2007Q, N2019K, K2085M, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H, and M2321L.

[0039] Nucleotide sequences codon-optimized for a target host (humans in the case of gene therapy for hemophilia A or mRNA pharmaceuticals) may contain one or more CpG sequences. However, it is predicted that CpG sequences may induce an immune response in the host and inhibit continuous transgene expression. Therefore, base substitutions are made to remove these CpG sequences without changing the amino acid sequence of the variant, followed by re-codon optimization, thereby significantly increasing the expression efficiency in host cells. For example, a nucleic acid encoding a variant codon-optimized for humans, consisting of the nucleotide sequence represented by SEQ ID NO: 2, can be modified to eliminate CpG sequences without changing the encoded amino acid sequence. An example of such a CpG-removed and codon-optimized nucleotide sequence is the nucleotide sequence represented by SEQ ID NO: 3.

[0040] The improvement in expression efficiency achieved by removing CpG sequences is effective not only for the specific human factor VIII variants described above, but also for any variants, and even for nucleic acids encoding wild-type human factor VIII. Therefore, the present invention also provides a human factor VIII-encoding nucleic acid comprising a nucleotide sequence obtained by modifying a nucleic acid encoding human factor VIII codon-optimized for a target host, preferably a human, to eliminate CpG sequences without changing the encoded amino acid sequence, and then further codon-optimizing the nucleotide sequence. For example, an example of a nucleotide sequence encoding wild-type human factor VIII consisting of the amino acid sequence represented by SEQ ID NO: 1 and codon-optimized for humans is the nucleotide sequence represented by SEQ ID NO: 4. An example of a nucleotide sequence obtained by modifying the nucleotide sequence represented by SEQ ID NO: 4 to eliminate CpG sequences without changing the encoded amino acid sequence, and then further codon-optimizing the nucleotide sequence represented by SEQ ID NO: 5, is an example of a nucleotide sequence obtained by modifying the nucleotide sequence represented by SEQ ID NO: 4 to eliminate CpG sequences without changing the encoded amino acid sequence, and then further codon-optimizing the nucleotide sequence represented by SEQ ID NO: 5.

[0041] A nucleic acid encoding a variant of the present invention, or a nucleic acid encoding human factor VIII whose expression efficiency in host cells has been enhanced by codon optimization and removal of CpG sequences (hereinafter, these may be collectively referred to as the "nucleic acid of the present invention") can be linked downstream of a promoter functional in liver cells and inserted into a vector to construct an expression vector that can be expressed in liver cells.

[0042] The liver is composed of hepatic parenchymal cells (hepatocytes), which are responsible for the liver's main functions, such as bile production and metabolism, as well as non-parenchymal hepatic cells, such as hepatic sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, pit cells, bile duct epithelial cells, and mesothelial cells. As used herein, "liver cells" refers to one or more of the above cell groups that make up the liver (including cancer cells and cell lines derived from them).

[0043] As used herein, a "promoter functional in liver cells" refers to a promoter capable of inducing transcription of a gene (a nucleic acid encoding a protein) linked downstream in liver cells. Considering that the preferred application of the present invention is in vivo gene therapy, it is desirable for the promoter to function specifically in liver cells (inducing transcription of a downstream gene). Here, the terms "specifically in liver cells" and "liver-specific" are used to encompass not only cases in which expression is limited to liver cells, but also cases in which expression in liver cells is significantly higher than that in cells of other organs or tissues. Even when inducing gene expression in cells other than liver cells, substantial liver-specific gene expression (e.g., to the extent that undesirable side effects are not induced) can be achieved by combining the promoter with, for example, a vector with high organ / tissue / cell tropism (e.g., various serotypes of viral vectors). As long as the promoter contains a minimal nucleotide sequence that exerts basal transcriptional activity, it may further contain other regulatory sequences (e.g., endogenous proximal or distal enhancer sequences, enhancer sequences derived from other genes, etc.).

[0044] The promoter functional in liver cells is not particularly limited as long as it can induce transcription of a downstream gene in liver cells. Preferably, it is a promoter of a gene highly expressed in the liver, and more preferably, it is a liver-specific promoter. Here, "liver-specific" has the same meaning as above. Examples of promoters functional in liver cells that can be used in the expression vector of the present invention include, but are not limited to, the transthyretin (TTR) promoter, the α1-antitrypsin (AAT) promoter, the albumin promoter, the α-fetoprotein promoter, and the thyroxine-binding globulin promoter. In a preferred embodiment, for example, when using an AAV vector for gene therapy of hemophilia A, the TTR promoter or the AAT promoter can be used as a promoter functional in liver cells. In another preferred embodiment, a chimeric promoter (HCRhAAT) of the human AAT promoter and the liver-type regulatory region (HCR) of the Apo E / C1 gene can also be used as a promoter functional in liver cells.

[0045] Promoters functional in liver cells can be obtained by known methods based on the sequence information of the gene (genomic DNA) from which each promoter is derived. Such sequence information is registered in publicly available databases such as NCBI, EMBL, FASTA, and DDBJ, and is also described in literature. Those skilled in the art can easily access such sequence information.

[0046] When the nucleotide sequence of a promoter functional in liver cells contains one or more CpG sequences, it is desirable to modify the promoter to remove the CpG sequences, as in the case of the nucleic acid of the present invention, in order to enhance the expression efficiency in host cells of the nucleic acid of the present invention linked downstream. For example, while the wild-type mouse TTR promoter consists of the nucleotide sequence set forth in SEQ ID NO:6, a modified promoter consisting of the nucleotide sequence set forth in SEQ ID NO:7, in which the CpG sequences in the wild-type mouse TTR promoter have been removed, can be used.

[0047] The promoter functional in liver cells can be linked to the nucleic acid of the present invention by methods known in the art. For example, when the promoter and nucleic acid fragments each have blunt ends, the two fragments can be ligated using DNA ligase. Alternatively, any adapter sequence can be added to the ends of both fragments, and the two fragments can be ligated after treating them with an appropriate restriction enzyme to generate sticky ends.

[0048] In a preferred embodiment, the nucleic acid of the present invention linked downstream of a promoter functional in liver cells is preferably inserted into a vector in the form of an expression cassette further linked downstream of a transcription termination signal functional in liver cells, i.e., a polyA addition signal (e.g., an SV40 polyA addition signal).

[0049] For example, (i) downstream of a mouse TTR promoter (SEQ ID NO: 7) that has been modified so as not to contain a CpG sequence, (ii) in wild-type human factor VIII, (n) R-5P, P25H, A28T, L152P, K213N, M217T, W228Q, S367P, Q410L, Y487H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P, Q1659E, E1661K Preferably, an expression cassette is used that encodes a variant having the amino acid substitutions of I1668F, D1681G, R1776K, H1859R, A1993V, H2007Q, N2019K, K2085M, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H, and M2321L, that has been codon-optimized for human use, and that has been further CpG-deleted and re-codon-optimized (SEQ ID NO: 3), and that is linked to an SV40 polyA additional signal sequence. An example of such an expression cassette is a nucleic acid consisting of the nucleotide sequence (4814 bp) set forth in SEQ ID NO: 8. This nucleic acid contains a 17-nucleotide 5'-upstream sequence (positions 283-299) between the promoter sequence (positions 1-282) of SEQ ID NO: 7 and the coding sequence (positions 300-4673) of SEQ ID NO: 3. By deleting 11 nucleotides from positions 283-293 of this 5'-upstream sequence to shorten the sequence, the efficiency of secretory expression of human factor VIII can be improved. SEQ ID NO: 9 shows the nucleotide sequence (4803 bp) in which these 11 nucleotides have been deleted from the nucleotide sequence of SEQ ID NO: 8. Furthermore, SEQ ID NO: 10 shows the nucleotide sequence (4790 bp) in which 13 nucleotides from the 3'-end of the SV40 polyA addition signal sequence (positions 4663-4803) of SEQ ID NO: 9 have been deleted, and SEQ ID NO: 11 shows the nucleotide sequence (4768 bp) in which 35 nucleotides from the 3'-end have been deleted. By modifying (shortening) these polyA addition signals, the efficiency of secretory expression of human factor VIII can be further improved.

[0050] The vector into which the nucleic acid of the present invention is inserted is not particularly limited as long as it is one generally used in gene therapy. For example, viral vectors such as AAV vectors, LV vectors, retroviral vectors, AdV vectors, Sindbis virus vectors, rabies virus vectors, Sendai virus vectors, and herpes simplex virus vectors, as well as non-viral vectors such as plasmids for animal cells, can be used. AAV and LV vectors are preferred from the viewpoints of high gene transfer and expression efficiency, ability to be transferred to non-dividing cells, and long-term expression of the transgene. AAV vectors are more preferred from the viewpoints of low frequency of chromosomal integration, no risk of insertional mutagenesis, low immunogenicity, and high safety. AAV vectors are diluted in dividing cells as they grow, but adult hepatocytes rarely divide, so transgene expression can be maintained for a long period of time. However, because the gene size that can be carried is small (4.7 kbp), depending on the size of the nucleic acid to be introduced, the use of LV or AdV vectors, which can carry a larger insert, may also be preferable.

[0051] For example, when an AAV vector is used as the vector, it is desirable to use a vector derived from a serotype with high liver tropism, such as AAV1, AAV2, AAV3, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, and AAV-DJ / 8. Below, the procedure for vector construction will be described using an AAV vector as an example. However, those skilled in the art will be able to construct other viral vectors and non-viral vectors (e.g., plasmid vectors) using known methods [e.g., Current Protocols in Molecular Biology, F. Ausubel et al. eds. (1994) John Wiley & Sons, Inc.; Molecular Cloning (A Laboratory Manual), 3rd ed. Volumes 1-3, Joseph Sambrook & David W. Russell eds., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001); Culture of Animal Cells; A Manual of Basic Technique, R. Freshney eds., 2nd ed. (1987), Wiley-Liss; Frank L. Graham, Manipulation of adenovirus vector, Chapter 11, pp. 109-128; E. J. Murray eds., Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols (1991); [Chen, SH. et al., Combination gene therapy for liver metastases of colon carcinoma in vivo., Proc. Natl. Acad. Sci. USA (1995) 92, 2477-2581, etc.], a desired expression vector can be easily constructed.

[0052] In preparing an AAV expression vector, the synthetic promoter of the present invention is first inserted between the 5'- and 3'-ITRs of AAV into a plasmid that can be amplified in a suitable host cell (e.g., Escherichia coli, Bacillus subtilis, yeast, etc.). Examples of such plasmids include E. coli-derived plasmids (e.g., pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, pC194), and yeast-derived plasmids (e.g., pSH19, pSH15). Although ITRs derived from the desired serotype may be used as the AAV ITRs, AAV2-derived ITRs are commonly used. Although the synthetic promoter of the present invention alone can be inserted between the two ITR sequences, it is desirable to insert a polyA addition signal (e.g., an SV40 polyA addition signal) downstream thereof, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) can also be inserted between the synthetic promoter and the polyA addition signal. A shortened WPRE (0.25 kb; Mol. Brain, 7:17, 2014) can also be used. Inserting one or more restriction enzyme recognition sites, preferably a multicloning site (MCS), between the promoter of the present invention and the polyA addition signal (or between the promoter and WPRE if the WPRE is included) can facilitate insertion of the nucleic acid of the present invention (construction of an expression cassette for human factor VIII).

[0053] An expression cassette for human factor VIII is constructed in the obtained AAV vector expression plasmid by inserting the nucleic acid of the present invention to be expressed in liver cells between the promoter and the polyA addition signal (between the promoter and WPRE if WPRE is contained).

[0054] If desired, the expression vector may further contain a 5'-UTR that is functional in the host downstream of the promoter, and a 3'-UTR that is functional in the host downstream of the DNA encoding human factor VIII. It may also contain an enhancer, a splicing signal, etc. These components may be those that are known per se.

[0055] An AAV vector can be produced from an AAV vector expression plasmid containing the nucleic acid of the present invention downstream of a promoter functional in liver cells by methods known per se, such as the plasmid transfection method, the recombinant baculovirus method, the recombinant herpes virus vector method, the yeast method, etc. For example, in the plasmid transfection method, the AAV vector expression plasmid, a plasmid containing the AAV Rep and Cap genes, and a pHelper plasmid containing the adenovirus-derived E2A, E4orf6, and VARNA genes are transfected into HEK293 cells or the like to produce AAV virus particles. The Rep gene does not need to be derived from the target serotype; the Rep gene derived from AAV2 is generally used. On the other hand, the Cap gene must be derived from the target serotype, and the cell tropism of the AAV serotype is determined by the capsid protein encoded by the Cap gene. That is, in the expression vector of the present invention that targets liver cells, Cap genes that have liver tropism and are derived from AAV1, AAV2, AAV3, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV-DJ, AAV-DJ / 8, etc. can be used as the Cap gene.

[0056] On the other hand, when a non-viral vector is used as the expression vector of the present invention, the expression vector can be introduced using a polymer carrier such as a poly-L-lysine-nucleic acid complex or by encapsulation in a liposome. A liposome is a capsule made of phospholipids with a particle size of several tens to several hundreds of nanometers, and a plasmid vector can be encapsulated inside it.

[0057] An expression vector containing a nucleic acid of the present invention downstream of a promoter functional in liver cells can be administered to a subject suffering from a disease for which human factor VIII exerts a therapeutic effect, particularly hemophilia A, to treat the disease. Therefore, the present invention also provides a gene therapy agent for hemophilia A, comprising the expression vector of the present invention.

[0058] The gene therapy agent of the present invention may be the expression vector of the present invention in its original form, or it may be mixed with a pharmacologically acceptable carrier as needed to form various formulations such as injections and then used as a medicine.

[0059] Here, various organic or inorganic carrier substances commonly used as pharmaceutical ingredients are used as pharmacologically acceptable carriers, and are incorporated into liquid preparations as solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. Furthermore, pharmaceutical additives such as preservatives, antioxidants, and coloring agents can also be used as needed.

[0060] Preferable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, and the like.

[0061] Suitable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0062] Suitable examples of suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates; and polyoxyethylene hydrogenated castor oil.

[0063] Suitable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, and the like.

[0064] Suitable examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.

[0065] Suitable examples of soothing agents include benzyl alcohol.

[0066] Suitable examples of the preservative include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0067] Suitable examples of antioxidants include sulfites and ascorbic acid salts.

[0068] Suitable examples of the coloring agent include water-soluble food tar dyes (e.g., food dyes such as Food Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and Food Blue Nos. 1 and 2), water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble food tar dyes), and natural dyes (e.g., β-carotene, chlorophyll, red iron oxide, etc.).

[0069] The dosage form of the pharmaceutical composition may be, for example, parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.) and drip infusions.

[0070] The gene therapy agent of the present invention can be produced by a method commonly used in the field of pharmaceutical technology, such as the method described in the Japanese Pharmacopoeia. The content of the viral vector, which is the active ingredient in the formulation, varies depending on the dosage form, the dose of the active ingredient, etc., but is, for example, about 0.1 to 100% by weight. The viral titer is, for example, 1 x 10 １０ ~10 １３ The concentration can be appropriately adjusted to about vp / mL, but is not limited to this range.

[0071] Preparations suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. The most suitable dosage form in the present invention is an injection solution.

[0072] The dosage of the preparation varies depending on the type of vector, promoter activity, administration route, severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc. of the recipient. For example, when an AAV vector (e.g., AAV8) with the HCRhAAT promoter as a basal promoter is used as a human factor VIII expression vector for hemophilia A, when the AAV vector is administered systemically, particularly via a peripheral vein, a single dose of, for example, about 5 × 10 １２ ~Approx. 5×10 １３ In recent clinical trials of AAV vectors, high doses (3 x 10 １４ Because deaths due to severe liver damage were reported in the 100 mg / kg body weight (Audentes Therapeutics. Letter to the MTM disease community. https: / / myotubulartrust.org / audentes-therapeutics-letter-23-june-2020 / ; Hum Gene Ther 2020; 31: 695-696), １４ For example, in a preferred embodiment, the gene therapy agent of the present invention is administered at a dose of about 1 x 10 vp / kg body weight or less as an AAV vector. １０ ~Approx. 5×10 １３ vp / kg body weight, preferably about 1 x 10 １１ ~Approx. 1×10 １３ vp / kg body weight, more preferably about 5 x 10 １１ ~Approx. 5×10 １２ vp / kg body weight or approximately 1 x 10 １２ ~Approx. 1×10 １３ It can be administered systemically in a dose of vp / kg body weight.

[0073] The present invention also provides a method for expressing human factor VIII in liver cells, which comprises introducing into liver cells in vitro a gene therapy agent containing the expression vector or nucleic acid of the present invention. Transplanting liver cells transfected with the gene therapy agent of the present invention into a subject with hemophilia A can provide an ex vivo treatment for the disease. Therefore, the present invention also provides a pharmaceutical (cell preparation), particularly a therapeutic agent for hemophilia, containing liver cells transfected with the gene therapy agent of the present invention as an active ingredient.

[0074] When the nucleic acid of the present invention is a single-stranded RNA, the nucleic acid can be formulated in the form of an mRNA pharmaceutical. For example, the expression vector is introduced into a suitable host (e.g., mammalian cells) and cultured, and the mRNA is recovered using a method known per se (e.g., the LiCl method), and the mRNA encoding human factor VIII is purified to obtain the mRNA.

[0075] Alternatively, the mRNA can be obtained by excising the human factor VIII coding sequence (including the 5'- and 3'-UTRs in addition to the CDS) from the expression vector and using it as a template to convert it into mRNA encoding the human factor VIII using a known in vitro transcription system. More specifically, the mRNA coding region is excised using an appropriate restriction enzyme and a phage (T7, T3, SP6, etc.) promoter is ligated to the 5' end, or a fragment of the mRNA coding region linked to the phage promoter is obtained by PCR using the expression vector as a template and a primer containing the phage promoter sequence. The resulting DNA fragment can be used as a template to react with phage (T7, T3, SP6, etc.) RNA polymerase to synthesize mRNA encoding human factor VIII in vitro. In this case, by adding pseudouridine (Ψ) or N1-methylpseudouridine (N1mΨ) triphosphate instead of UTP as an RNA monomer (NTP) to the reaction solution, the resulting mRNA will contain Ψ or N1mΨ instead of U. This makes it possible to avoid attacks by natural immunity against the mRNA, improving mRNA stability and translation efficiency. Similarly, modified NTPs with base substitutions that have been reported to avoid natural immunity (e.g., inosine triphosphate instead of ATP, 5-methylcytidine triphosphate instead of CTP, etc.) can also be used for other NTPs.

[0076] It is also desirable to add a 5'-cap structure and a polyA tail, which are necessary for stabilizing mRNA and improving translation efficiency. The 5'-cap structure can be added by adding a Cap 0 structure using a capping enzyme after mRNA synthesis, and then converting it to a Cap 1 structure using an mRNA 2'-O-methyltransferase. Alternatively, an RNA cap analog (e.g., 3'-O-Me-m) can be used. ７ G(5')ppp(5')G,m ７ G(5')ppp(5')G,3'-O-Me-m ７ G(5') ppp(5') A, m ７By adding a poly(5')ppp(5')A or other polynucleotides to the transcription reaction solution, transcription and 5'-capping can be performed simultaneously. Poly(A) tailing can also be added to the 3' end using poly(A) polymerase after mRNA synthesis, or can be performed simultaneously with the transcription reaction by adding a poly(A) sequence to the transcription template in advance. The mRNA obtained as described above can be purified by removing the template DNA using DNase I, for example, by the LiCl method.

[0077] To promote the introduction of the nucleic acid of the present invention into target cells, a nucleic acid introduction reagent may be used, such as atelocollagen, liposomes, nanoparticles, lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or a cationic lipid such as poly(ethyleneimine) (PEI).

[0078] In one embodiment, the nucleic acid of the present invention may be a pharmaceutical composition encapsulated in a liposome. Liposomes are minute closed vesicles having an internal phase surrounded by one or more lipid bilayers, and can typically hold a water-soluble substance in the internal phase and a lipid-soluble substance within the lipid bilayer. Here, the term "encapsulated" refers to the nucleic acid of the present invention, which may be held in the internal phase of the liposome or within the lipid bilayer. The liposomes used in the present invention may be monolayer or multilayer membranes, and the particle size can be appropriately selected, for example, within the range of 10 to 1,000 nm, preferably 50 to 300 nm. Considering delivery to target tissues, the particle size may be, for example, 200 nm or less, preferably 100 nm or less.

[0079] Methods for encapsulating water-soluble compounds such as polynucleotides into liposomes include, but are not limited to, the lipid film method (vortex method), reverse phase evaporation, surfactant removal method, freeze-thaw method, and remote loading method, and any known method can be appropriately selected.

[0080] In a preferred embodiment, the nucleic acid of the present invention is encapsulated in a lipid nanoparticle as a carrier. As used herein, "lipid nanoparticle" (sometimes abbreviated as "LNP") refers to a particle having a membrane structure in which the hydrophilic groups of an amphipathic lipid are aligned toward the aqueous phase at the interface, and having a particle diameter of less than 1 μm. "Amphipathic lipid" refers to a lipid having both a hydrophilic group and a hydrophobic group.

[0081] The particle size of the lipid nanoparticles used in the present invention is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. Measurement of particle size can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles can be appropriately adjusted depending on the method for producing the lipid nanoparticles. In this specification, "particle size" refers to the average particle size (zeta mean) measured by dynamic light scattering.

[0082] Examples of amphipathic lipids include cationic lipids, ionic lipids, phospholipids, PEG lipids, etc. As used herein, "cationic lipid" refers to a lipid having a constitutively positively charged hydrophilic group. As used herein, "ionizable lipid" refers to a lipid that is neutral at physiological pH but becomes positively charged by protonation at low pH. As used herein, "PEG" refers to polyethylene glycol, and "PEG lipid" refers to a lipid modified with PEG, i.e., a lipid to which PEG is bound.

[0083] In a preferred embodiment, the lipid nanoparticles encapsulating the nucleic acid of the present invention include lipid nanoparticles containing: (A) a cationic lipid or an ionic lipid, (B) a phospholipid, (C) a steroid, and (D) a PEG lipid. Each of the component lipids (A) to (D) includes known lipids that are commonly used in lipid nanoparticles, and a person skilled in the art can easily select an appropriate type of lipid and its composition.

[0084] The present invention also provides host cells into which an expression vector containing the nucleic acid of the present invention has been introduced; a method for producing human factor VIII or a variant thereof, which comprises culturing the host cells and recovering human factor VIII or a variant thereof from the resulting culture; and a pharmaceutical (recombinant protein preparation), particularly a therapeutic agent for hemophilia A, containing the human factor VIII variant obtained by the method.

[0085] An expression vector for producing recombinant human factor VIII can be produced by ligating the nucleic acid of the present invention (i.e., the nucleic acid encoding the variant of the present invention or the nucleic acid encoding a highly expressed form of human factor VIII) downstream of a promoter in an appropriate expression vector. Examples of expression vectors that can be used include plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194); yeast-derived plasmids (e.g., pSH19, pSH15); insect cell expression plasmids (e.g., pFast-Bac); animal cell expression plasmids (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as λ phage; insect virus vectors such as baculovirus (e.g., BmNPV, AcNPV); and animal virus vectors such as retrovirus, lentivirus, vaccinia virus, adenovirus, adeno-associated virus, and herpes virus.

[0086] The promoter may be any promoter appropriate for the host used to express the gene. For example, when the host is an animal cell, the SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. may be used. For other hosts, a publicly known promoter may be appropriately selected.

[0087] In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 origin of replication (hereinafter sometimes abbreviated as SV40 ori), etc. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, and a neomycin resistance gene.

[0088] Human factor VIII or a modified form thereof can be produced by transforming a host with an expression vector containing the nucleic acid of the present invention and culturing the resulting transformant. Examples of hosts that can be used include Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, and animal cells. Examples of mammalian cells include monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), and dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO(dhfr) － ) cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, HeLa cells, HepG2 cells, HEK293 cells, etc. For other hosts, known cells can be appropriately selected.

[0089] Transformation can be carried out according to known methods depending on the type of host. Animal cells can be transformed, for example, according to the methods described in Cell Engineering, Special Issue 8, New Cell Engineering Experimental Protocols, pp. 263-267 (1995) (published by Shujunsha) and Virology, Vol. 52, p. 456 (1973).

[0090] The transformant can be cultured according to known methods depending on the type of host. For example, when the host is an animal cell, a medium such as minimum essential medium (MEM) containing about 5 to about 20% fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, 199 medium, or Ham's F-12 medium can be used. The pH of the medium is preferably about 6 to about 8. The culture is usually carried out at about 30°C to about 40°C for about 15 to about 60 hours. Aeration or stirring may be performed as necessary. In this manner, human factor VIII can be produced intracellularly or extracellularly by the transformant.

[0091] Human factor VIII can be separated and purified from the culture obtained by culturing the transformant by a method known per se. Examples of such methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that primarily utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing. These methods can also be combined as appropriate.

[0092] When the human factor VIII thus obtained is in the free form, the free form can be converted into a salt by a method known per se or a method similar thereto. When the human factor VIII is obtained as a salt, the salt can be converted into the free form or another salt by a method known per se or a method similar thereto.

[0093] Human factor VIII has low toxicity and can be administered to humans or other mammals parenterally (e.g., intravascular administration (intravenous administration, intraarterial administration, etc.), subcutaneous administration, intradermal administration, intraperitoneal administration, intramuscular injection, topical administration, etc.) as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form.

[0094] Compositions for parenteral administration include, for example, injections, suppositories, etc., and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip infusion injections. Such injections can be prepared according to known methods. For example, injections can be prepared by dissolving, suspending, or emulsifying one or more active ingredients in a sterile aqueous or oily liquid typically used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and the like, which may be used in combination with an appropriate solubilizing agent, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), or nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and solubilizing agents such as benzyl benzoate, benzyl alcohol, etc. may be used in combination. The prepared injection solution is preferably filled into an appropriate ampule. Suppositories for rectal administration may be prepared by mixing the active ingredient with a conventional suppository base.

[0095] The above-mentioned parenteral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories, and each dosage unit usually contains 100 to 5,000 units, preferably 250 to 3,000 units.

[0096] The dosage of human factor VIII varies depending on the subject, symptoms, administration route, etc., but when used to treat hemophilia A, for example, it is convenient to administer a single dose of typically about 10 to 100 units / kg body weight, preferably about 20 to 50 units / kg body weight, preferably about two to three times a week, by intravenous or intraperitoneal administration. Similar amounts can also be administered in other parenteral administrations. When symptoms are particularly severe, the dosage may be increased according to the symptoms.

[0097] The present invention will be described in more detail below by showing examples, but these are merely illustrative and do not limit the scope of the present invention in any way.

[0098] In the following examples, all animal experimental protocols were approved by the Jichi Medical University Animal Care and Related Institutional Committee, and animal care was conducted in accordance with the committee's guidelines. Mice were purchased from Japan SLC. Factor VIII-deficient mice were obtained from Jackson Laboratory (USA).

[0099] Example 1: Preparation of nucleic acids encoding B domain-deleted human factor VIII (hFVIIISQ) and its variants, and coagulation factor activity of each variant hFVIIISQ The DNA sequence of human coagulation factor VIII (hFVIIISQ) with almost all of the B domain deleted was designed based on the NCBI reference sequence (NM_000132.4), and codon-optimized and gene synthesized using GeneArt (Thermo Scientific) (SEQ ID NO: 4). Based on the sequences of Ver. 1-Ver. 12 hFVIIISQ, canine FVIIISQ, porcine FVIIISQ, ovine FVIIISQ, and bovine FVIIISQ, gene synthesis and mutagenesis PCR were used to generate variant Ver. 1, in which 72 amino acid sites were substituted with canine-type amino acids (however, codons were optimized for human use). Furthermore, variants Ver. 2 and Ver. 3 were prepared by reducing the number of substitutions to 58 and 50, respectively. Ver. 4, which has 36 substitutions and only contains the amino acid substitutions common to Ver. 2 and Ver. 3, was also prepared. Ver. 5, which has 56 substitutions, was prepared by removing overlaps with the X5 and X10 mutations previously reported in Ver. 2 (WO 2014 / 209942); Ver. 6, which has 35 substitutions, was prepared by removing overlaps with the X5 mutation from Ver. 4; Ver. 7, which has 51 substitutions, was prepared by removing overlaps with the JF12 mutation previously reported in Ver. 2 (Mol Ther Methods Clin Dev. 2020 Jan 15; 17:328-336); and Ver. 8, which has 32 substitutions, was prepared by removing overlaps with the JF12 mutation from Ver. 4. Ver. 9 had 49 substitutions, obtained by removing overlaps with the X5, X10, and JF12 mutations from Ver. 2; Ver. 10 had 31 substitutions, obtained by removing overlaps with the X5 and JF12 mutations from Ver. 4; Ver. 11 had 28 substitutions, obtained by removing three substitutions from Ver. 10; and Ver. 12 had 13 substitutions, obtained by removing 15 substitutions from Ver. 11 (amino acid substitutions in Ver. 1 to Ver. 12 are summarized in Figure 1). For comparison, a variant containing the previously reported X5 mutation (codon-optimized for human use) was also prepared.

[0100] After cloning, each hFVIIISQ was introduced into a plasmid (p1.1HCRhAAT) carrying the HCRhAAT promoter, which combines HCR, a liver-specific enhancer of the ApoE gene, with the human α1-antitrypsin promoter.

[0101] To evaluate FVIII expression after transfection of each FVIII gene (Ver. 1 to Ver. 10), the amount of FVIII transiently secreted into the culture supernatant was measured after plasmid transfection into Huh7 cells (human hepatoma cell line). The Huh7 cell culture medium was RPMI-1640 (Sigma) supplemented with 10% FBS, GlutaMAX (Thermo Scientific), and penicillin-streptomycin. 1.5 × 10 ５ Huh7 cells were seeded in a 24-well plate at 10 cells / well and transfected the following day using Lipoefectamin 3000 (Thermo Scientific). The medium was replaced the day after transfection, and the medium was collected the following day after 24 hours of culture. The collected medium was centrifuged, and the supernatant was used as a sample for measuring factor VIII activity and antigen.

[0102] The activity of factor VIII was measured by the synthetic substrate method using a fully automated blood coagulation analyzer CS-1600 (Sysmex).

[0103] AAV8 vectors carrying each FVIII gene (Ver. 1, 2, 4, 10) were injected into factor VIII-deficient mice at a dose of 1 × 10 per gram of mouse body weight. ８ The factor VIII activity in mouse plasma was measured by the synthetic substrate method.

[0104] The results are shown in Figure 2. All variants had increased coagulation factor activity compared to wild-type hFVIIISQ. In particular, Ver. 4 showed high activity in both in vitro and in vivo experiments.

[0105] Example 2: Identification of Mutation Sites Important for Increased Activity of Ver. 4 The number of substitutions was reduced from Ver. 4, and a variant S12 with 12 substitutions was identified using the one-stage clotting assay. Further reductions in the number of substitutions led to the identification of PNPL (L152P, K213N, S367P, F2196L) using the synthetic substrate assay. S7, with 7 substitutions, was created by removing five substitutions from S12 that overlap with the X5 and JF12 mutations. S5 and S3, with 5 and 3 substitutions, respectively, were further reduced, and S8, S6, and S4, with the addition of the L152P substitution to S7, S5, and S3, respectively, were created. These were transfected with plasmids as in Example 1, and the coagulation factor activity in the culture supernatant was measured using the one-stage clotting assay and the synthetic substrate assay. These results are shown in Figure 3 . S12, S8, and S6, which contain the PNPL mutation, exhibited high activity. Furthermore, Ver. 10, with the number of substitutions reduced to 28, was also developed. Ver. 11 also showed coagulation factor activity equivalent to that of Ver. 4.

[0106] Next, the effect of each amino acid substitution in S8 was evaluated using the single-stage clotting assay and the synthetic substrate assay. The results are shown in Figure 4. L152P, K213N, S367P, S727P, and F2196L increased the coagulation factor activity compared to the wild-type.

[0107] Example 3: Substitution of PNPL variants with other amino acids A series of variants (XNPL, PXPL, PNXL, PNPX) were prepared by substituting any of the substitution sites in PNPL with 19 other amino acids. These variants were transfected with plasmids in the same manner as in Example 1, and the coagulation factor activity in the culture supernatant was measured by the one-stage coagulation assay and the synthetic substrate assay. The results are shown in Figures 5-1 to 5-4. It was revealed that substitutions of L152 with Q, V, M, etc., K213 with N, H, etc., S367 with N, Q, etc., and F2196 with M, etc., also showed high activity similar to that of PNPL.

[0108] Example 4: Administration Experiment to FVIII KO Mice The Ver. 4, 10, and 11, S12, S8, and PNPL genes, which showed particularly high activity in Examples 1 and 2, were loaded onto AAV8 vectors and administered to FVIII knockout hemophilia A model mice. Four weeks later, blood samples were collected and coagulation factor activity was measured using a one-stage clotting assay and a synthetic chromogenic assay. Antigen levels were also measured using ELISA. The results are shown in Figure 6. S12 and PNPL, which contain the PNPL mutation, almost matched the activity of Ver. 4, but the antigen levels were significantly lower. This suggests that these variants have increased specific activity.

[0109] Example 5: Localization of FVIII in and outside FVIII-expressing cells. Huh7 cells were transfected with Ver. 4, S12, and PNPL, and the amount of FVIII present in the culture supernatant and cell extract was analyzed by Western blot. The results are shown in Figure 7-1. Intracellular retention of the light chain was observed with wild-type hFVIIISQ, whereas retention was abolished with Ver. 4. S12 and PNPL did not improve intracellular retention of the light chain. The inventors predicted that the improvement in intracellular retention of the light chain in Ver. 4 was due to the two mutations, I1668F and D1681G, and prepared FVIIISQ variants containing only these two substitutions, and similarly examined the amount of FVIII present in cell extracts. As shown in Figure 7-2, the FG mutation demonstrated improved intracellular retention of the light chain.

[0110] Example 6 Identification of Mutation Sites Important for High Secretion and Activity of Ver. 4 (2) Based on Ver. 11, Ver. 12 (Figure 1) was identified by reducing the number of substitutions to 13 using the synthetic substrate method. The results are shown in Figure 8. The number of substitutions was further reduced based on Ver. 12 to create highly secreted variants with 8 to 15 substitutions (F8-Tochigi-3 (NPL), 5, 8, 10, 11, 13, and 15) (the amino acid substitutions of these variants are shown in Figure 9). Plasmid transfection was performed as in Example 1, and the coagulation factor activity in the culture supernatant was measured using the one-stage clotting method and the synthetic substrate method. The antigen amount in the culture supernatant was also measured using ELISA. The results are shown in Figure 10. In the figure, F8-Tochigi-28 represents the same variant as Ver. 11.

[0111] Example 7 Construction of a High-Expression hFVIII Gene The nucleotide sequences of the codon-optimized wild-type hFVIII DNA (SEQ ID NO: 4) and the Ver. 4 variant DNA (SEQ ID NO: 2) contain numerous CpG sequences. The inventors predicted that these CpG sequences may induce an immune response in the host, impairing FVIII expression. Therefore, they removed all of the CpG sequences without changing the amino acid sequence of the FVIII they encode. Then, they re-optimized the codons, taking into account other parameters such as GC content, and designed the nucleotide sequences using GeneArt Codon Optimizer (Thermo Scientific). The nucleotide sequences of the CpG-removed and re-optimized wild-type hFVIII DNA and the Ver. 4 variant DNA are shown in SEQ ID NOs: 5 and 3, respectively. These DNAs were ligated downstream of a mouse transthyretin (mTTR) promoter sequence (SEQ ID NO: 7) from which the CpG sequence had also been removed, and then loaded into an AAV6 vector. The vector was then plasmid transfected and introduced into Huh7 cells in the same manner as in Example 1. The expression cassette was also loaded into an AAV8 vector and administered to mice. The coagulation factor activity in the culture supernatant and blood was measured by the one-stage clotting assay and the synthetic substrate assay. The results are shown in Figures 11-1 and 11-2. For both Ver. 4 and the wild-type, removal of the CpG sequence significantly increased the expression efficiency.

[0112] Example 8 Administration to a Cynomolgus Monkey Model The DNA prepared in Example 7, in which the CpG-deleted and re-optimized Ver. 4 variant coding sequence was ligated downstream of the CpG-deleted mTTR promoter sequence, was inserted into an AAV8 vector and administered at the vector dose (2 x 10) used in a clinical trial (NCT0300183) conducted by the University College of London. １２The vector was administered at a dose of 1 / 10 (6 × 10) of that of Rectavian (registered trademark) (a therapeutic drug for hemophilia A marketed by BioMarin) to a cynomolgus monkey hemophilia model treated with immunosuppressants (prednisolone 1 mg / kg / day and tacrolimus 0.05 mg / kg / day), and factor VIII activity and antigen amount were measured every one or two weeks after administration. The results are shown in Figure 12-1. Although the time course differed between individuals, factor VIII activity increased significantly in all individuals after vector administration. In addition, the above DNA was inserted into an AAV5 vector and administered at a dose of 1 / 10 (6 × 10) of that of Rectavian (registered trademark) (a therapeutic drug for hemophilia A marketed by BioMarin). １２ vg / kg) and 1 / 30 (2 × 10 １２ The vector was administered at a dose of 100 mg / kg (100 mg / kg) to a cynomolgus monkey model treated in the same manner, and factor VIII activity and antigen levels were measured every one or two weeks after administration. The results are shown in Figure 12-2. Factor VIII activity was significantly increased after vector administration at all doses.

[0113] Example 9 Optimization of Expression Cassette Sequence Huh7 cells were transfected with the AAV6 vector (original) prepared in Example 7, in which DNA was inserted into the CpG-deleted and re-optimized Ver. 4 variant coding sequence ligated downstream of the CpG-deleted mTTR promoter sequence (which further contained an SV40 polyA addition signal downstream of the coding sequence and contained the nucleotide sequence shown in SEQ ID NO: 8 as an expression cassette), the AAV6 vector (v1mTTRp) containing, as an expression cassette, a nucleotide sequence (SEQ ID NO: 9) in which the sequence between the promoter and the coding sequence was shortened, and the AAV6 vector (v1-v1, v1-v2, and v1-v3, respectively) containing, as expression cassettes, three nucleotide sequences (SEQ ID NOs: 10, 11, and 12) in which the 3'-end of the polyA addition signal was shortened by different lengths. The medium was replaced after 24 hours, and the culture supernatant and cells were collected after another 24 hours. The Factor VIII activity and antigen content in the supernatant, as well as the AAV genome content per cell, were measured. The results are shown in Figure 13. Activity increased when the sequence between the promoter and coding sequence was shortened, and activity was further improved by shortening the polyA addition signal in combination. v1-v2 showed the highest activity. However, when the 3' end of the polyA addition signal was deleted up to v1-v3, Factor VIII activity was significantly reduced.

[0114] The variants of the present invention, nucleic acids encoding them, expression vectors containing the nucleic acids, and liver cells transfected with the expression vectors are useful in in vivo or ex vivo gene therapy, mRNA medicines, and the like for hemophilia A. Furthermore, recombinant human factor VIII variants obtained by culturing host cells transfected with the expression vectors are useful as biological preparations for the treatment of hemophilia A. Furthermore, nucleic acids encoding highly expressed forms of human factor VIII are useful in gene therapy and mRNA medicines for hemophilia A, and are also useful for producing hosts that highly produce recombinant human factor VIII.

Claims

1. A human factor VIII variant in which at least one amino acid residue selected from the group consisting of K213, S367, and F2196 of wild-type human factor VIII is substituted with another amino acid, and which is characterized by having higher specific activity and / or secretory expression efficiency compared to the wild-type.

2. The variant according to claim 1, wherein the amino acid substitution at K213 is K213N or K213H, and / or the amino acid substitution at S367 is S367P, S367N or S367Q, and / or the amino acid substitution at F2196 is F2196L or F2196M.

3. The variant of claim 2, comprising the amino acid substitutions K213N, S367P and F2196L.

4. The variant according to any one of claims 1 to 3, wherein at least one amino acid residue selected from the group consisting of R-5, P25, A28, L152, M217, W228, Q410, Y487, R489, F501, M539, I566, L603, I642, S727, A736, S1657, Q1659, E1661, I1668, D1681, R1776, H1859, A1993, H2007, N2019, K2085, K2207, F2275, S2296, V2314, Q2316, and M2321 is further substituted with another amino acid.

5. R-5P, P25H, A28T, L152A, L152D, L152E, L152H, L152I, L152M, L152N, L152P, L152Q or L152V, M217T, W228Q, Q410L, Y487H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P 5. The variant of claim 4, comprising at least one amino acid substitution selected from the group consisting of Q1659E, E1661K, I1668F, D1681G, R1776K, H1859R, A1993V, H2007Q, N2019K, K2085M, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L.

6. The variant of claim 3, further comprising the amino acid substitutions L152A, L152D, L152E, L152H, L152I, L152M, L152N, L152P, L152Q or L152V, and / or I1668F and D1681G.

7. The variant of claim 1, wherein the amino acid substitution at K213 is K213N, and further comprising M539L, and optionally at least one amino acid substitution in the A2 domain selected from the group consisting of I566M, L603P, and I642V, and further comprising K2207Q, Q2316H, and M2321L, and optionally at least one amino acid substitution in the C2 domain selected from the group consisting of F2275L, S2296A, and V2314A.

8. The variant of claim 7, further comprising the amino acid substitutions I1668F and D1681G.

9. The variant of claim 3, further comprising amino acid substitutions L152P, Y487H and L603P, and optionally at least one amino acid substitution selected from the group consisting of S727P, Q1659E, H1859R, A1993V, H2007Q, N2019K and K2085M.

10. The variant of claim 3 further comprising the amino acid substitutions M539L, I566M, L603P, I642V, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L.

11. The variant of claim 10, further comprising the amino acid substitutions I1668F and D1681G.

12. A human factor VIII variant having the following amino acid substitutions (a) to (n) in wild-type human factor VIII: (a) K213N, S367P, and F2196L; (b) K213N, S367P, F2196L, I1668F, and D1681G; (c) K213N, M539L, I566M, I642V, K2207Q, F2275L, Q2316H, and M2321L; and (d) K213N, M539L, I566M, L603P, I642V, F2196L, K2207Q, F2275L, Q2316H, and M2321L. (e) K213N, M539L, I566M, I642V, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L; (f) K213N, M539L, I566M, I642V, I1668F, D1681G, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L; (g) L152P, K213N, S367P and F2196L; (h) L152P, K213N, S367P, Y487H, L603P and F2196L. (i) L152P, K213N, S367P, Y487H, L603P, S727P, Q1659E and F2196L; (j) L152P, K213N, S367P, Y487H, L603P, S727P, Q1659E, H1859R, A1993V, H2007Q, K2085M and F2196L; (k) K213N, S367P, M539L, I566M, L603P, I642V, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L. (l) P25H, A28T, K213N, M217T, S367P, Q410L, Y487H, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P , Q1659E, E1661K, I1668F, D1681G, R1776K, N2019K, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L(m) R-5P, P25H, A28T, K213N, M217T, W228Q, S367P, Q410L, Y487H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P, Q1659E, E1661K, I1668F, D1681G, R1776K, N2019K, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L (n) R-5P, P25H, A28T, L152P, K213N, M217T, W228Q, S367P, Q410L, Y487H, R489G, F501M, M539L, I566M, L603P, I642V, S727P, A736V, S1657P, Q1659E, E1661K, I1668F, D1681G, R1776K, H1859R, A1993V, H2007Q, N2019K, K2085M, F2196L, K2207Q, F2275L, S2296A, V2314A, Q2316H and M2321L 13. A variant according to any one of claims 1 to 12, which lacks the B domain.

14. The variant according to claim 13, wherein the deletion of the B domain is a deletion of the amino acid sequence from positions 763 to 1656 in the amino acid sequence represented by SEQ ID NO:

1.

15. A nucleic acid encoding a variant according to any one of claims 1 to 14.

16. The nucleic acid of claim 15, which is codon-optimized for human use.

17. The nucleic acid of claim 16, or a nucleic acid encoding human codon-optimized wild-type human Factor VIII, which has been further modified to be free of CpG sequences without changing the amino acid sequence it encodes.

18. An expression vector comprising the nucleic acid of any one of claims 15 to 17.

19. The expression vector of claim 18, wherein the nucleic acid is under the control of a liver-specific promoter.

20. The expression vector according to claim 18 or 19, wherein the promoter has been modified so as not to contain any CpG sequences.

21. The expression vector of any one of claims 18 to 20, wherein the vector is a viral vector.

22. The expression vector of claim 21, wherein the virus is an adeno-associated virus having liver tropism.

23. A host cell into which the expression vector according to any one of claims 18 to 22 has been introduced.

24. A method for producing human factor VIII or a variant thereof, comprising culturing the host cell according to claim 23 and recovering human factor VIII or a variant thereof from the resulting culture.

25. A pharmaceutical comprising the variant described in any one of claims 1 to 14, the nucleic acid described in any one of claims 15 to 17, the expression vector described in any one of claims 18 to 22, or the host cell described in claim 23.

26. The pharmaceutical composition according to claim 25, for use in the treatment of hemophilia A.

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