Compositions and Methods for Modulating Factor VIII Function

Abstract

Factor VIII variants and methods of use thereof are disclosed.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 749,182, filed Oct. 23, 2018, under 35 U.S.C. § 119(e). The above application is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under Grant No. R01 HL-137335-01A1, awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0003] The present invention relates to the fields of medicine and hematology. More specifically, the present invention provides novel Factor VIII variants and methods of using the same to modulate the coagulation cascade in a patient in need thereof. Background Art

[0004] Throughout the specification, several publications and patent documents are cited to describe the prior art relevant to the present invention. Each of these citations is hereby incorporated by reference in its entirety.

[0005] Mutations in factor VIII (FVIII) can result in severe bleeding disorders and are associated with hemophilia A. FVIII deficiency or lack of FVIII activity leads to an inability to effectively form clots. To date, due to high costs, only 20% of hemophilia A patients worldwide receive routine treatment with FVIII replacement therapy. Typically, the FVIII is plasma-derived or recombinantly produced. Gene therapy for hemophilia A based on AAV vectors is promising, but there are safety limitations due to abnormal immune responses to the vectors. Such abnormal immune responses have been found to be vector dose-dependent. Moreover, the immunogenicity of the delivered or expressed FVIII can be problematic, regardless of whether protein or gene therapy is used. In fact, 20% to 30% of hemophilia A patients develop inhibitors (e.g., anti-FVIII neutralizing antibodies) to the treatment (Peyvandi, et al., N. Engl. J. Med. (2016) 374:2054-2064; Walsh, et al., Am. J. Hematol. (2015) 90:400-405; Eckhardt, et al., J. Thromb. Haemost. (2015) 13:1217-1225; Darby, et al., J. Thromb. Haemost. (2004) 2:1047-1054; Donfield, et al., Blood (2007) 110:3656-3661; Witmer, et al., Br. J. Haematol. (2011) 152:211-216; Hoots, W.K., Semin. Hematol. (2008) 45(2 Suppl 1):S42-S49; Guh, et al., Haemophilia (2012) 18:268-275; Lindvall, et al., Pediatr. Blood Cancer (2014) 61:706-711). Thus, generating enhanced FVIII molecules would benefit the treatment of hemophilia by reducing the production cost of FVIII, increasing the safety of AAV gene therapy, and / or reducing immunogenicity. Accordingly, there is clearly a need for FVIII molecules with improved biological properties. SUMMARY OF THE INVENTION

[0006] According to the present invention, compositions and methods for modulating blood coagulation in patients in need thereof are provided. More specifically, factor VIII (FVIII) variants that modulate (e.g., increase) blood coagulation are provided. In one particular embodiment, the B domain of the FVIII variant is replaced with an amino acid sequence having at least 90% identity to SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In one particular embodiment, the B domain of the FVIII variant is replaced with the amino acid sequence comprising SEQ ID NO:18. In one particular embodiment, the factor VIII variant comprises at least one mutation at positions 560, 561, 712, 713, and / or 659, optionally with a B domain replacement. In one particular embodiment, the FVIII variant comprises a substitution of Lys at position 659 with another amino acid. In a specific embodiment, Lys at position 659 is replaced with Trp, Arg, Ala, His, Tyr, Asp, Thr, Ser, Val, Phe, Gln, or Cys, particularly with Ser, Gln, or Cys. The FVIII variant may comprise a B domain replacement and a substitution at position 659. Compositions comprising at least one FVIII variant of the present invention and at least one pharmaceutically acceptable carrier are also provided. Nucleic acid molecules encoding the FVIII variants of the present invention and methods of using the same are also disclosed. Another aspect of the present invention includes host cells expressing the FVIII variants described herein. Methods for isolating and purifying the FVIII variants are also disclosed.

[0007] Pharmaceutical compositions are also provided, which comprise the FVIII variants of the present invention and / or nucleic acid molecules encoding the FVIII variants in a carrier. The present invention also includes methods for treating coagulation-related disorders in patients in need thereof, the methods comprising administering a therapeutically effective amount of the FVIII variant or the nucleic acid molecule encoding the FVIII variant, particularly in a pharmaceutical composition. These methods are efficacious in the treatment of disorders that require a procoagulant, the diseases including but not limited to hemophilia, particularly hemophilia A. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A A schematic representation of the factor VIII protein is provided. The length of the full-length protein is 2332 amino acids, and the B domain is 908 amino acids. Various cleavage sites are also indicated in the schematic representation. Figure 1BA schematic diagram of the processing of factor VIII protein is provided. FVIII is translated as a single polypeptide chain (single chain) with a domain structure of A1-α1-A2-α2-B-α3-A3-C1-C2. The protease furin cleaves the FVIII protein at R-1313 and / or R-1648 (triangles), resulting in heterodimer formation. The FVIII heavy chain (A1-α1-A2-α2-B) and light chain (α3-A3-C1-C2) are held associated by a non-covalent metal ion-dependent interaction (dashed line) that exists between the A1 and A3 domains. After secretion, the B domain undergoes additional non-specific proteolysis in plasma. During blood coagulation, the FVIII single chain or heterodimer is activated to its heterotrimeric cofactor form by cleavage with thrombin at R-372, R-740, and R-1689 (triangles). A2 remains associated with A1-α1 by a non-covalent interaction (dashed line). Inactivation of FVIIIa occurs by spontaneous dissociation and / or proteolytic cleavage of A2 at R-336 and R-562 (triangles), mainly by activated protein C.

[0009] Figure 2 Graphs showing the expression of various human FVIII B domain variants in hemophilia A mice are provided.

[0010] Figure 3 Graphs showing the specific activity of FVIII variants having an amino acid substitution at position 659 are provided.

[0011] Figure 4 The amino acid sequence of FVIII (SEQ ID NO:1) is provided. The amino acids at positions 560, 561, 659, 712, and / or 713 are shown in bold and underlined. The B domain is also shown in italics and bold. The provided amino acid sequence lacks a 19-amino acid signal peptide (MQIELSTCFFLCLLRFCFS (SEQ ID NO:2)) at the N-terminus.

[0012] Figure 5 Graphs showing the specific activity of FVIII variants having amino acid substitutions at positions 560, 561, 712, and 713 are provided. Detailed Description

[0013] Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders caused by hereditary deficiencies of coagulation factor VIII (FVIII) or coagulation factor IX (FIX), respectively (Peyvandi, et al., Lancet (2016) 388:187-197; Konkle, et al., Hemophilia A. In GeneReviews, Adam et al. eds, University of Washington (1993)). The bleeding phenotype is usually related to residual factor activity: individuals with severe disease (factor activity < 1% of normal values) often bleed spontaneously; those with moderate disease (factor activity 1%-5% of normal values) rarely bleed spontaneously but bleed upon minor trauma; and those with mild disease (factor activity 5%-40% of normal values) bleed during invasive surgery or trauma. Given this well-defined relationship between factor activity and bleeding phenotype, HA and HB are attractive targets for gene therapy because a small increase in factor levels is expected to have a meaningful clinical impact. Although multiple strategies have been investigated for decades, the field has focused on using adeno-associated virus (AAV) vectors to deliver transgenes encoding engineered FVIII or FIX variants that have therapeutic advantage properties not present in the wild-type (WT) protein (Hough, et al., J. Thromb. Haemost. (2005) 3:1195-1205; Lheriteau, et al., Blood Rev. (2015) 29:321-328; Rogers, et al., Front. Biosci. (2015) 20:556-603; Arruda, et al., Expert Opin. Orphan Drugs (2015) 3:997-1010; High, K. A., Hematology Am. Soc. Hematol. Educ. Program (2012) 2012:375-381; Zinn, et al., Curr. Opin. Virol. (2014) 8:90-97; Mingozzi, et al., Nat. Rev. Genet. (2011) 12:341-355; Colella, et al., Mol. Ther. Methods Clin. Dev. (2017) 8:87-104). Notably, the full-length FVIII cDNA (7 kb) exceeds the packaging capacity of AAV vectors (approximately 4.7 kb). Removal of the B domain of FVIII can reduce the cDNA to approximately 4.4 kb. AAV-based clinical trials for HA have reported positive results using this approach (Rangarajan, et al., N. Engl. J. Med. (2017) 377:2519-2530).

[0014] As explained above, factor VIII is crucial for clotting activity, and mutations in the FVIII gene result in hemophilia A, the most common form of hemophilia. In this article, specific alterations in the amino acid sequence of FVIII are shown to be associated with enhanced protein production and activity. Accordingly, the present invention provides rationally designed amino acid residue modifications that provide gain-of-function variants.

[0015] Full-length FVIII is a large 280 kDa protein expressed primarily in liver sinusoidal endothelial cells (LSEC) and extrahepatic endothelial cells (Fahs, et al., Blood (2014) 123:3706-3713; Everett, et al., Blood (2014) 123:3697-3705). FVIII circulates mainly as a heterodimer of a heavy chain and a light chain bound by non-covalent metal-dependent interactions (Lenting, et al., Blood (1998) 92:3983-3996). Factor VIII contains several domains. Generally, the domains are referred to as A1-A2-B-A3-C1-C2, as shown in FIG. 1. The heavy chain of FVIII contains A1-A2-B, while the light chain contains A3-C1-C2. Initially, FVIII is in an inactive form bound to von Willebrand factor (vWF). FVIII is activated and the B domain is released by cleavage with thrombin (factor IIa). The activated form of FVIII (FVIIIa) dissociates from vWF and interacts with coagulation factor IXa - resulting in the formation of a blood clot through the coagulation cascade.

[0016] Said B domain contains 40% (908 amino acids) of the protein and is not essential for the procoagulant activity of the protein (Brinkhous, et al., Proc. Natl. Acad. Sci. (1985) 82:8752-8756). The most common B domain deleted (BDD) FVIII contains 14 original amino acid residues as a linker (Lind, et al. (1995) Eur. J. Biochem., 232(1):19-27). This BDD FVIII is commonly referred to as BDD-SQ or hFVIII-SQ (see Table 1). This form of BDD FVIII is commonly used for the production of recombinant BDD-FVIII (about 4.4 Kb) as well as for gene therapy (Berntorp, E., Semin. Hematol. (2001) 38(2 Suppl 4):1-3; Gouw, et al., N. Engl. J. Med. (2013) 368:231-239; Xi, et al., J. Thromb. Haemost. (2013) 11:1655-1662; Recht, et al., Haemophilia (2009) 15:869-880; Sabatino, et al., Mol. Ther. (2011) 19:442-449; Scallan, et al., Blood (2003) 102:2031-2037). As described above, due to the limited packaging capacity of AAV (4.7 Kb) and other vector systems, gene therapy using AAV vectors can only use shortened FVIII molecules, such as BDD-FVIII (Lind, et al. (1995) Eur. J. Biochem., 232(1):19-27). U.S. Patent 8,816,054 also provides BDD FVIII molecules with linkers of different lengths and sequences (see, e.g., Table 1). However, the linkers in Table 1 contain more than one new epitope, which can lead to the production of FVIII inhibitors).

[0017]

[0018] Table 1: Replacement of the B domain in FVIII variants with short peptide linkers (Lind, et al. (1995) Eur. J. Biochem., 232(1):19-27; Pittman, et al., Blood (1993) 81:2925-2935; Toole, et al., Proc. Natl. Acad. Sci. (1986) 83:5939-5942). The furin recognition motif is underlined. *Also known as hFVIII-BDD. Abbreviations: aa, amino acid; c, canine; cl, cell line; F, factor; h, human; NA, not applicable; p, porcine. The amino acid sequences provided are SEQ ID NOs: 3-12 from top to bottom.

[0019] The present invention provides novel Factor VIII variants. The invention includes FVIII variants, which include FVIIIa variants and FVIII propeptide variants. For simplicity, throughout the application the variants are mainly described in the context of FVIII. However, the invention contemplates and encompasses Factor FVIIIa and FVIII propeptide molecules having the same amino acid substitutions and / or linkers as described in FVIII. In a particular embodiment, the FVIII variants of the present invention are expressed as single-chain molecules or at least almost exclusively as single-chain molecules.

[0020] The FVIII variants of the present invention can be from any mammalian species. In a specific embodiment, the FVIII variant is from a human. Gene ID: 2157 and GenBank accession numbers NM_000132.3 and NP_000123.1 provide examples of the amino acid and nucleotide sequences of wild-type human FVIII (particularly the propeptide containing the signal peptide). Figure 4 SEQ ID NO:1 is provided, which is an example of the amino acid sequence of human Factor FVIII. SEQ ID NO:1 lacks the 19-amino acid signal peptide (MQIELSTCFFLCLLRFCFS (SEQ ID NO:2)) at its N-terminus. The nucleic acid molecules encoding the FVIII variants can be readily determined from the provided amino acid sequences and the provided GenBank accession numbers.

[0021] According to one aspect of the present invention, the FVIII variant is a B domain deleted (BDD) FVIII molecule comprising a linker. In a specific embodiment, the linker comprises the sequences listed in Table 2. As shown herein, replacing the B domain of factor VIII with the sequences listed in Table 2 yields FVIII variants with enhanced factor VIII activity. The expression levels of these FVIII variants are also higher than those of other BDD FVIII. Moreover, each of the linkers provided in Table 2 has only one new epitope, while each of the linkers in Table 1 has more than one new epitope. By reducing or minimizing the number of new epitopes in the linker region, the adverse immunogenicity of the FVIII variant is reduced with the linkers in Table 2.

[0022]

[0023] Table 2: Novel B domain linkers with the fewest new epitopes. The amino acid sequences provided are SEQ ID NO: 13 - 18 from top to bottom.

[0024] In a particular embodiment, the invention encompasses FVIII variants in which the B domain (e.g., amino acids 741-1648 of SEQ ID NO:1) is replaced with an amino acid sequence comprising SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18 or consisting of the same. In a particular embodiment, the B domain is replaced with an amino acid sequence that is at most about 50, at most about 45, at most about 40, at most about 35, at most about 30, at most about 25, at most about 20, at most about 15, at most about 10, or at most about 5 amino acids in length. In a particular embodiment, the B domain (e.g., amino acids 741-1648 of SEQ ID NO:1) is replaced with an amino acid sequence comprising SEQ ID NO:17 or SEQ ID NO:18 or consisting of the same. In a particular embodiment, the B domain (e.g., amino acids 741-1648 of SEQ ID NO:1) is replaced with an amino acid sequence comprising SEQ ID NO:18 or consisting of the same. In a particular embodiment, the B domain (e.g., amino acids 741-1648 of SEQ ID NO:1) is replaced with an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity), particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity), with SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In a particular embodiment, the B domain (e.g., amino acids 741-1648 of SEQ ID NO:1) is replaced with an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity), particularly at least 90%, 95%, 97%, 99%, or 100% homology (identity), with SEQ ID NO:18.

[0025] According to another aspect of the present invention, the Factor VIII variant comprises at least one mutation at positions 560, 561, 712, 713, and / or 659. As described herein, these FVIII variants have a higher specific activity than wild-type FVIII. In certain embodiments, the Factor VIII variant comprises a mutation at position 659. In one specific embodiment, Lys (K) at position 659 is not replaced by Pro (P), Gly (G), Met (M), or Leu (L). In one specific embodiment, Lys at position 659 is replaced by Trp (W), Arg (R), Ala (A), His (H), Tyr (Y), Asp (D), Thr (T), Ser (S), Val (V), Phe (F), Gln (Q), or Cys (C). In one specific embodiment, Lys at position 659 is replaced by Asp (D), Thr (T), Ser (S), Val (V), Phe (F), Gln (Q), or Cys (C). In one specific embodiment, Lys at position 659 is replaced by Ser (S), Val (V), Phe (F), Gln (Q), or Cys (C). In one specific embodiment, Lys at position 659 is replaced by Ser (S), Gln (Q), or Cys (C). In one specific embodiment, Lys at position 659 is replaced by Gln (Q), or Cys (C).

[0026] In certain embodiments, the Factor VIII variant comprises a mutation at position 560. In one specific embodiment, Asp (D) at position 560 is replaced by Ala (A), Val (V), Ile (I), Leu (L), His (H), Arg (R), or Lys (K). In one specific embodiment, Asp (D) at position 560 is replaced by Ala (A), Val (V), Ile (I), or Leu (L). In one specific embodiment, Asp (D) at position 560 is replaced by His (H), Arg (R), or Lys (K). In one specific embodiment, Asp (D) at position 560 is replaced by Ile (I) or His (H).

[0027] In certain embodiments, the Factor VIII variant comprises a mutation at position 561. In one specific embodiment, Gln (Q) at position 561 is not replaced by Leu (L), Arg (R), or Asn (N). In one specific embodiment, Gln (Q) at position 561 is replaced by Asp (D) or Glu (E). In one specific embodiment, Gln (Q) at position 561 is replaced by Asp (D).

[0028] In certain embodiments, the Factor VIII variant comprises a mutation at position 712. In one specific embodiment, the Asp (D) at position 712 is replaced with an amino acid other than Glu (E). In one specific embodiment, the Asp (D) at position 712 is replaced with Ala (A), Val (V), Ile (I), or Leu (L). In one specific embodiment, the Asp (D) at position 712 is replaced with Ile (I) or Leu (L). In one specific embodiment, the Asp (D) at position 712 is replaced with Leu (L).

[0029] In certain embodiments, the Factor VIII variant comprises a mutation at position 713. In one specific embodiment, the Lys (K) at position 713 is replaced with Ala (A), Arg (R), Met (M), Tyr (Y), Asp (D), Glu (E), Cys (C), or Gly (G). In one specific embodiment, the Lys (K) at position 713 is replaced with Arg (R), Met (M), Tyr (Y), Asp (D), Cys (C), or Gly (G). In one specific embodiment, the Lys (K) at position 713 is replaced with Asp (D) or Glu (E). In one specific embodiment, the Lys (K) at position 713 is replaced with Cys (C). In one specific embodiment, the Lys (K) at position 713 is replaced with Ala (A) or Gly (G). In one specific embodiment, the Lys (K) at position 713 is replaced with Gly (G).

[0030] The FVIII variants of the present invention as described herein may comprise at least one mutation at positions 560, 561, 712, 713, and / or 659 and / or may comprise a linker replacing the B domain as described herein. In other words, the present invention encompasses FVIII variants having only mutations at positions 560, 561, 712, 713, and / or 659 (e.g., FVIII comprises the full B domain), FVIII variants having only a linker replacing the B domain (e.g., the amino acids at positions 560, 561, 712, 713, and 659 are wild type), and FVIII variants comprising mutations at positions 560, 561, 712, 713, and / or 659 and a linker replacing the B domain.

[0031] In a specific embodiment, the Factor VIII variant comprises mutations at positions 560, 561, 712, 713, and / or 659 and a linker replacing the B domain. In a particular embodiment, the Factor VIII variant comprises mutations at positions 560, 561, 712, 713, and / or 659 and a linker replacing the B domain, wherein the B domain has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, particularly an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology. In a particular embodiment, the Factor VIII variant comprises mutations at positions 560, 561, 712, 713, and / or 659 and a linker replacing the B domain, wherein the B domain has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with SEQ ID NO:18, particularly an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology (identity). In certain embodiments, the Factor VIII variant comprises a mutation at position 659. In a specific embodiment, Lys (K) at position 659 is not replaced by Pro (P), Gly (G), Met (M), or Leu (L). In a specific embodiment, Lys at position 659 is replaced by Trp (W), Arg (R), Ala (A), His (H), Tyr (Y), Asp (D), Thr (T), Ser (S), Val (V), Phe (F), Gln (Q), or Cys (C). In a specific embodiment, Lys at position 659 is replaced by Asp (D), Thr (T), Ser (S), Val (V), Phe (F), Gln (Q), or Cys (C). In a specific embodiment, Lys at position 659 is replaced by Ser (S), Val (V), Phe (F), Gln (Q), or Cys (C). In a specific embodiment, Lys at position 659 is replaced by Ser (S), Gln (Q), or Cys (C). In a specific embodiment, Lys at position 659 is replaced by Gln (Q) or Cys (C). In certain embodiments, the Factor VIII variant comprises a mutation at position 560. In a specific embodiment, Asp (D) at position 560 is replaced by Ala (A), Val (V), Ile (I), Leu (L), His (H), Arg (R), or Lys (K).In a specific embodiment, Asp (D) at position 560 is replaced by Ala (A), Val (V), Ile (I), or Leu (L). In a specific embodiment, Asp (D) at position 560 is replaced by His (H), Arg (R), or Lys (K). In a specific embodiment, Asp (D) at position 560 is replaced by Ile (I) or His (H). In certain embodiments, the Factor VIII variant contains a mutation at position 561. In a specific embodiment, Gln (Q) at position 561 is not replaced by Leu (L), Arg (R), or Asn (N). In a specific embodiment, Gln (Q) at position 561 is replaced by Asp (D) or Glu (E). In a specific embodiment, Gln (Q) at position 561 is replaced by Asp (D). In certain embodiments, the Factor VIII variant contains a mutation at position 712. In a specific embodiment, Asp (D) at position 712 is replaced by an amino acid other than Glu (E). In a specific embodiment, Asp (D) at position 712 is replaced by Ala (A), Val (V), Ile (I), or Leu (L). In a specific embodiment, Asp (D) at position 712 is replaced by Ile (I) or Leu (L). In a specific embodiment, Asp (D) at position 712 is replaced by Leu (L). In certain embodiments, the Factor VIII variant contains a mutation at position 713. In a specific embodiment, Lys (K) at position 713 is replaced by Ala (A), Arg (R), Met (M), Tyr (Y), Asp (D), Glu (E), Cys (C), or Gly (G). In a specific embodiment, Lys (K) at position 713 is replaced by Arg (R), Met (M), Tyr (Y), Asp (D), Cys (C), or Gly (G). In a specific embodiment, Lys (K) at position 713 is replaced by Asp (D) or Glu (E). In a specific embodiment, Lys (K) at position 713 is replaced by Cys (C). In a specific embodiment, Lys (K) at position 713 is replaced by Ala (A) or Gly (G). In a specific embodiment, Lys (K) at position 713 is replaced by Gly (G).

[0032] As described above, the FVIII variants of the present invention can be human. In a specific embodiment, the FVIII variant of the present invention has at least 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% homology (identity) with SEQ ID NO: 1 (or its activated FVIII fragment), particularly at least 90%, 95%, 97% or 99% homology (identity). In a specific embodiment, the FVIII variant comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% homology (identity) with amino acids 1-740 of SEQ ID NO: 1 (or its activated FVIII fragment), particularly at least 90%, 95%, 97% or 99% homology (identity), and an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% homology (identity) with amino acids 1649-2332 of SEQ ID NO: 1 (or its activated FVIII fragment), particularly at least 90%, 95%, 97% or 99% homology (identity). The percentage of homology (identity) does not include substitutions at positions 560, 561, 712, 713 and / or 659.

[0033] The FVIII variants of the present invention can also be post-translationally modified. The FVIII variant can be post-translationally modified in cells (particularly human cells) or in vitro.

[0034] In a particular embodiment, the FVIII variant of the present invention has increased expression compared to wild-type FVIII or hFVIII-SQ. In a particular embodiment, the FVIII variant of the present invention has increased FVIII activity or increased specific activity compared to wild-type FVIII.

[0035] Nucleic acid molecules encoding the above FVIII variants are also included in the present invention. The nucleic acid molecule encoding the variant can be prepared by any method known in the art. The nucleic acid molecule can be maintained in any convenient vector, specifically, an expression vector.

[0036] Compositions comprising at least one FVIII variant and at least one vector are also included in the present invention. In a specific embodiment, the FVIII is isolated and / or substantially pure in the composition. Compositions comprising at least one nucleic acid molecule of an FVIII variant and at least one vector are also included in the present invention. Its use in pharmaceutical compositions is contemplated unless any conventional vector is incompatible with the variant to be administered. In a specific embodiment, the vector is a pharmaceutically acceptable vector for intravenous administration.

[0037] Definition

[0038] A variety of terms related to the biomolecules of the present invention are used above, throughout the specification, and in the claims.

[0039] The phrase "coagulation-related disorder" refers to hemorrhagic disorders, such as but not limited to hemophilia A, hemophilia B, patients with hemophilia A and B, those with inhibitory antibodies, defective in at least one coagulation factor (e.g., factor VII, VIII, IX, X, XI, V, XII, II, and / or von Willebrand factor; specifically, factor VIII), combined FV / FVIII deficiency, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency hemophilia, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocoagulability), disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, or small molecule antithrombotic drugs (e.g., FXa inhibitors); and platelet disorders such as Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency. In a particular embodiment, the term "coagulation-related disorder" refers to a hemorrhagic disorder characterized by excessive and / or uncontrolled bleeding (e.g., a disorder that can be treated with a procoagulant). In a specific embodiment, the coagulation-related disorder is hemophilia. In a specific embodiment, the coagulation-related disorder is hemophilia A.

[0040] Regarding the nucleic acids of the present invention, the term "isolated nucleic acid" is sometimes used. When applied to DNA, this term refers to a DNA molecule that has been separated from the sequences that are immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originated. For example, an "isolated nucleic acid" can include a DNA or cDNA molecule inserted into a vector (e.g., a plasmid or viral vector), or integrated into the DNA of a prokaryotic or eukaryotic organism. Regarding the RNA molecules of the present invention, the term "isolated nucleic acid" mainly refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been sufficiently separated from the RNA molecules with which it is associated in its natural state (i.e., in a cell or tissue) such that it exists in a "substantially pure" form.

[0041] Regarding proteins, the term "isolated protein" is sometimes used herein. This term can refer to a protein produced by expressing an isolated nucleic acid molecule of the present invention. Alternatively, the term can refer to a protein that is sufficiently separated from other proteins with which it is naturally associated (e.g., such that it exists in a "substantially pure" form). "Isolated" does not mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the exclusion of the presence of impurities that do not interfere with the essential activity (and impurities may be present, e.g., due to incomplete purification), or the exclusion of added stabilizers.

[0042] The term "vector" refers to a vector nucleic acid molecule (e.g., RNA or DNA) into which a nucleic acid sequence for introduction into a host cell can be inserted, and the nucleic acid sequence will be replicated in the host cell. An "expression vector" is a specialized vector containing a gene or nucleic acid sequence having the necessary regulatory regions (e.g., a promoter) required for expression in a host cell.

[0043] The term "operably linked" means placing regulatory sequences necessary for the expression of a coding sequence in a DNA molecule in a proper position relative to the coding sequence to effect the expression of the coding sequence. Sometimes this same definition is applied to the arrangement of a coding sequence and transcriptional control elements (e.g., a promoter, enhancer, and termination element) in an expression vector. This definition is sometimes also applicable to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule, where a hybrid nucleic acid molecule is produced.

[0044] The term "substantially pure" means that a preparation contains at least 50 - 60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.), specifically, at least 75% by weight, or at least 90 - 99% by weight or more of the compound of interest. Purity can be measured by methods suitable for the compound of interest (e.g., chromatography, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0045] "Pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias and can be used in animals, particularly humans.

[0046] "Carrier" means, for example, a diluent, adjuvant, preservative (e.g., thimerosal, benzyl alcohol), antioxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), antibacterial agent, filling substance (e.g., lactose, mannitol), excipient, adjuvant or vehicle for administering the active agent of the present invention. A pharmaceutically acceptable carrier can be a sterile liquid such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or saline solutions, as well as aqueous dextrose and glycerol solutions, are preferably used as carriers, especially for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A.R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); edited by Liberman et al., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and edited by Kibbe et al., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0047] Preparation of Nucleic Acid Molecules and Polypeptides with Coding Variants

[0048] The nucleic acid molecules encoding the variants of the present invention can be prepared by using methods of recombinant DNA technology. The availability of nucleotide sequence information enables the preparation of the isolated nucleic acid molecules of the present invention by a variety of means. For example, the nucleic acid sequences encoding the variants can be isolated from suitable biological sources using standard protocols well known in the art.

[0049] The nucleic acids of the present invention can be maintained as RNA or DNA in any convenient cloning vector. In one specific embodiment, the clone is maintained in a plasmid cloning / expression vector (e.g., pBluescript (Stratagene, La Jolla, CA)), which is propagated in a suitable Escherichia coli host cell. Alternatively, the nucleic acid can be maintained in a vector suitable for expression in mammalian cells. In cases where post-translational modifications affect the function of the variant, it is preferred to express the molecule in mammalian cells, particularly human cells.

[0050] The nucleic acid molecules encoding FVIII variants of the present invention include cDNA, genomic DNA, RNA, and fragments thereof, which may be single- or double-stranded. Thus, the present invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing to at least one sequence of the nucleic acid molecules of the present invention. Such oligonucleotides can be used as probes for detecting variant expression.

[0051] According to known methods, the FVIII variants of the present invention can be prepared in a variety of ways. The protein can be purified from a suitable source, such as transformed bacteria or animal (e.g., mammalian or human) cultured cells or tissues that express the FVIII variant, for example, by immunoaffinity purification. The availability of the nucleic acid molecules encoding the variant enables the production of the variant using in vitro expression methods known in the art. For example, cDNA or a gene can be cloned into a suitable in vitro transcription vector, such as pSP64 or pSP65 for in vitro transcription, and then subjected to cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysate. In vitro transcription and translation systems are commercially available, for example, from Promega or Life Technologies.

[0052] Alternatively, larger amounts of the variant can be produced by expression in a suitable prokaryotic or eukaryotic expression system. For example, part or all of the DNA molecule encoding the FVIII variant can be inserted into a plasmid vector suitable for expression in bacterial cells, such as Escherichia coli, or in mammalian cells (especially human cells), such as CHO or HeLa cells. Alternatively, a tagged fusion protein containing the variant can be produced. Such a variant-tagged fusion protein is encoded by part or all of the DNA molecule, which is ligated in the correct codon reading frame to a nucleotide sequence encoding part or all of the desired polypeptide tag, and the polypeptide tag is inserted into a plasmid vector suitable for expression in bacterial cells or eukaryotic cells, such as Escherichia coli for bacterial cells, and yeast and mammalian cells, especially human cells for eukaryotic cells, but not limited thereto. The vectors described above contain regulatory elements necessary for expressing the DNA in a host cell, and the regulatory elements are positioned in a manner that allows the DNA to be expressed in the host cell. Such regulatory elements required for expression include, but are not limited to, promoter sequences, transcription initiation sequences, and enhancer sequences.

[0053] FVIII variant proteins produced by gene expression in recombinant prokaryotic or eukaryotic systems, particularly human, can be purified according to methods known in the art. In a specific embodiment, commercially available expression / secretion systems can be used, whereby the recombinant protein is expressed and then secreted from the host cell for easy purification from the surrounding culture medium. If an expression / secretion vector is not used, another method involves purifying the recombinant protein by affinity separation, such as by immunological interaction using an antibody that specifically binds to the recombinant protein or by nickel column to separate recombinant proteins bearing a 6-8 histidine residue tag at their N-terminus or C-terminus. Other tags can include, but are not limited to, FLAG epitope, GST or hemagglutinin epitope. These methods are generally used by skilled practitioners.

[0054] FVIII variant proteins prepared by the above methods can be analyzed according to standard procedures. For example, according to known methods, amino acid sequence analysis can be performed on these proteins.

[0055] As described above, a convenient way to produce the polypeptide of the present invention is to express the nucleic acid encoding it by using nucleic acid in an expression system. Various expression systems for use in the methods of the present invention are well known to those skilled in the art.

[0056] Accordingly, the present invention also includes methods for preparing (as disclosed) polypeptides, which methods comprise expressing from a nucleic acid (usually a nucleic acid) encoding the polypeptide. This can be conveniently achieved by culturing host cells containing such a vector under conditions that cause or permit production of the polypeptide. The polypeptide can also be produced in an in vitro system, such as in a reticulocyte lysate.

[0057] Use of FVIII Variant Proteins and Nucleic Acids with Coding Variants

[0058] The FVIII variant proteins and nucleic acids of the present invention can be used, for example, as therapeutic and / or prophylactic agents for modulating the coagulation cascade system. It is demonstrated herein that the FVIII variants have excellent properties and can provide effective coagulation.

[0059] In a specific embodiment of the present invention, the FVIII variant can be administered to a patient by injection in a biocompatible carrier, such as by intravenous injection. The FVIII variant of the present invention can optionally be encapsulated in liposomes or mixed with other phospholipids or micelles to increase the stability of the molecule. The FVIII variant can be administered alone or in combination with other agents known to modulate coagulation, such as vFW, factor IX, factor IXa, etc. Suitable compositions for delivering the FVIII variant can be determined by a physician considering various physiological variables, including but not limited to the patient's condition and hemodynamic status. Various compositions suitable for different applications and administration routes are well known in the art and are described hereinafter.

[0060] Preparations containing FVIII variants may contain a physiologically acceptable matrix and are formulated as pharmaceutical preparations. The preparations can be formulated using substantially known prior art methods and can be mixed with a buffer containing salts (such as NaCl, CaCl2) and amino acids (such as glycine and / or lysine) and having a pH range of 6 to 8. The purified preparations containing FVIII variants can be stored for later use in the form of a finished solution or in a lyophilized or deeply frozen form. In a specific embodiment, the preparation is stored in a lyophilized form and dissolved in a visually clear solution using an appropriate reconstitution solution. Alternatively, the preparations of the present invention can also be obtained as a liquid preparation or as a deeply frozen liquid. The preparations according to the present invention can be particularly stable, i.e., they can be left in a dissolved form for a long time before application.

[0061] The preparations of the present invention can be obtained as a pharmaceutical preparation with FVIII variants in the form of a single-component preparation or in combination with other factors in the form of a multi-component preparation.

[0062] Before processing the purified protein into a pharmaceutical preparation, the purified protein can be subjected to routine quality control and made into a therapeutic form. Specifically, during recombinant preparation, it can be tested whether there is cellular nucleic acid and nucleic acid derived from the expression vector in the purified preparation.

[0063] Another feature of the present invention relates to allowing the preparation of a preparation containing an FVIII variant with high stability and structural integrity, and, specifically, free of inactive FVIII intermediates and / or proteolytic degradation products, and by formulating it into a suitable preparation.

[0064] As an example, the pharmaceutical preparation may contain a dose of about 1 - 1000 μg / kg, about 10 - 500 μg / kg, about 10 - 250 μg / kg or about 10 - 100 μg / kg. In a specific embodiment, the pharmaceutical protein preparation may contain a dose of 30 - 100 IU / kg (e.g., a single injection per day or up to 3 or more times per day). The patient can receive treatment immediately when visiting the clinic due to bleeding or before bleeding is caused by a cut / wound. Alternatively, the patient can receive a bolus injection every one to three, eight or twelve hours, or, if sufficient improvement is observed, an injection of the FVIII variant described herein once a day.

[0065] According to the present invention, nucleic acids encoding FVIII variants can be used for a variety of purposes. In a specific embodiment of the present invention, there is provided a nucleic acid delivery vehicle (e.g., an expression vector, such as a viral vector) for modulating blood coagulation, wherein the expression vector comprises a nucleic acid sequence encoding an FVIII variant as described herein. Administration of an expression vector encoding an FVIII variant to a patient results in the expression of the FVIII variant for altering the blood coagulation cascade. According to the present invention, the nucleic acid sequence encoding an FVIII variant can encode a variant polypeptide as described herein, and the expression of the variant polypeptide increases blood coagulation. In a specific embodiment, the nucleic acid sequence encodes a human FVIII variant.

[0066] An expression vector comprising an FVIII variant nucleic acid sequence can be administered alone or in combination with other molecules for modulating blood coagulation. According to the present invention, the expression vector or combination of therapeutic agents can be administered to a patient alone or in a pharmaceutically acceptable or biologically compatible composition.

[0067] In a specific embodiment of the present invention, the expression vector comprising a nucleic acid sequence encoding an FVIII variant is a viral vector. Viral vectors that can be used in the present invention include, but are not limited to: adenovirus vectors (with or without tissue-specific promoters / enhancers), adeno-associated virus (AAV) vectors of various serotypes (e.g., AAV-1 to AAV-12, especially AAV-2, AAV-5, AAV-7, and AAV-8) and hybrid AAV vectors, lentiviral vectors and pseudotyped lentiviral vectors (e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)), herpes simplex virus vectors, vaccinia virus vectors, and retroviral vectors. In a particular embodiment, the vector is an adeno-associated virus (AAV) vector. In a particular embodiment, the vector is a lentiviral vector.

[0068] In a specific embodiment of the present invention, there is provided a method for administering a viral vector comprising a nucleic acid sequence encoding an FVIII variant. Adenovirus vectors that can be used in the method of the present invention preferably comprise at least the essential part of the adenovirus vector DNA. As described herein, the expression of the FVIII variant after administration of such an adenovirus vector is used for modulating blood coagulation, particularly enhancing the procoagulant activity of proteases.

[0069] Recombinant adenovirus vectors have been found to be widely useful for a variety of gene therapy applications. Their use in such applications is largely due to the high efficiency of in vivo gene transfer achieved in various organ environments.

[0070] Adenovirus particles can advantageously be used as vehicles for appropriate gene delivery. Such virions have many desirable characteristics for such applications, including: structural and biological characteristics associated with being a double-stranded DNA non-enveloped virus, such as tropism for the human respiratory and gastrointestinal tracts. In addition, adenoviruses are known to infect a variety of cell types in vivo and in vitro via receptor-mediated endocytosis. With regard to the demonstration of the overall safety of adenovirus vectors, adenovirus infections result in a mild disease state in humans, including mild flu-like symptoms.

[0071] Due to the large size of the adenovirus genome (about 36 kilobases), they are well-suited as gene therapy vehicles because they can accommodate the insertion of foreign DNA after removal of the adenovirus genes and non-essential regions necessary for replication. Such substitutions render the viral vectors impaired in terms of replication function and infectivity. Notably, adenoviruses have been used as vectors for gene therapy and the expression of heterologous genes.

[0072] There is a desire to introduce a vector that can provide, for example, multiple copies of a desired gene and thus provide a greater amount of the product of that gene. Improved adenovirus vectors and methods for preparing these vectors have been described in detail in many references, patents, and patent applications, including: Wright (Hum Gen Ther. (2009) 20:698 - 706); Mitani and Kubo (Curr Gene Ther. (2002) 2(2):135 - 44); Olmsted-Davis et al. (Hum Gene Ther. (2002) 13(11):1337 - 47); Reynolds et al. (Nat Biotechnol. (2001) 19(9):838 - 42); U.S. Patent No. 5,998,205 (which provides a tumor-specific replication vector containing multiple DNA copies); 6,228,646 (which describes a helper-free, fully defective adenovirus vector); 6,093,699 (which provides vectors and methods for gene therapy); 6,100,242 (in which a replication-deficient adenovirus vector into which a transgene is inserted is effective for in vivo gene therapy of peripheral vascular disease and heart disease); and International Patent Application Nos. WO 94 / 17810 and WO 94 / 23744.

[0073] For certain applications, the expression construct may further comprise regulatory elements for driving expression in specific cell or tissue types. Such regulatory elements are known to those skilled in the art and are discussed in depth in Sambrook et al. (1989) and Ausubel et al. (1992). Incorporation of tissue-specific regulatory elements into the expression constructs of the present invention provides at least partial tissue tropism for the expression of the variant or its functional fragments. For example, an E1-deleted adenovirus type 5 vector containing a nucleic acid sequence encoding the variant under the control of the cytomegalovirus (CMV) promoter can be used in the methods of the present invention. Hematopoietic or liver-specific promoters can also be used.

[0074] AAV has been generated for recombinant gene expression in the human embryonic kidney cell line 293 (Wright, Hum Gene Ther (2009) 20:698 - 706; Graham et al. (1977) J. Gen. Virol. 36:59 - 72). Briefly, AAV vectors are generally engineered from wild-type AAV, a non-pathogenic single-stranded DNA virus. The parental virus is non-pathogenic, the vectors have a broad host range, and can infect both dividing and non-dividing cells. The viral vectors are typically engineered by deleting the rep and cap genes and replacing them with the transgene of interest under the control of a specific promoter. For recombinant AAV preparations, the upper size limit of the sequence that can be inserted between the two ITRs is approximately 4.7 kb. A plasmid expressing the FVIII variant under the control of the CMV promoter / enhancer, a second plasmid providing adenovirus helper functions, and a third plasmid containing the AAV-2 rep and cap genes can be used to prepare AAV-2 vectors, while plasmids containing AAV-1, AAV-6, or AAV-8 cap genes, the AAV-2 rep gene, and the ITRs can be used to prepare the respective alternative serotype vectors (e.g., Gao et al. (2002) Proc. Natl. Acad. Sci. USA 99:11854 - 11859; Xiao et al., (1999) J. Virol. 73:3994 - 4003; Arruda et al., (2004) Blood 103:85 - 92). The AAV vectors can be purified by repeated CsCl density gradient centrifugation, and the titer of the purified vectors can be determined by quantitative dot blot hybridization. In a specific embodiment, the vectors can be prepared by the Vector Core at The Children's Hospital of Philadelphia.

[0075] The present invention also includes methods of modulating blood coagulation, the methods comprising providing a nucleic acid delivery vehicle encoding an FVIII variant to cells of an individual and allowing the cells to grow under conditions that express the FVIII variant.

[0076] As can be seen from the foregoing discussion, FVIII variants and nucleic acid carriers expressing FVIII variants can be used for the treatment of disorders associated with abnormal blood clotting.

[0077] The expression vectors of the present invention can be incorporated into pharmaceutical compositions that can be delivered to a subject, thereby allowing the production of a bioactive protein (such as an FVIII variant) or the induction of the expression of an FVIII variant in vivo by gene- and / or cell-based therapies or by ex vivo modification / transduction of patient or donor cells. In a specific embodiment of the present invention, a pharmaceutical composition containing sufficient genetic material to enable a receptor to produce a therapeutically effective amount of an FVIII variant can affect blood clotting in a subject. Alternatively, as described above, an effective amount of an FVIII variant can be directly injected into a patient in need. The composition can be administered alone or in combination with at least one other agent (such as a stabilizing compound), and the other agent can be administered in any sterile, biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, dextrose, and water. The composition can be administered to a patient alone or in combination with other agents that affect blood clotting (such as cofactors).

[0078] In a specific embodiment, the pharmaceutical composition further contains a pharmaceutically acceptable excipient / carrier. These excipients include any agent that does not itself induce an immune response harmful to the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include but are not limited to liquids such as water, saline, glycerol, sugars, and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, etc. can be present in such carriers. A detailed discussion of pharmaceutically acceptable excipients is provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., 18th ed., Easton, Pa.

[1990] ).

[0079] Pharmaceutical preparations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compound can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (such as sesame oil), or synthetic fatty acid esters (such as ethyl oleate or triglycerides), or liposomes. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound to prepare highly concentrated solutions.

[0080] The pharmaceutical composition can be provided in the form of a salt, and the salt can be formed with many acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. Compared with the corresponding free base form, the salt tends to be more soluble in water or other protic solvents. In other cases, the preparation can be a lyophilized powder, which can contain any or all of the following: 1 - 50 mM histidine, 0.1% - 2% sucrose, and 2 - 7% mannitol, with a pH range of 4.5 - 5.5, and is used in combination with a buffer before use.

[0081] After the pharmaceutical compositions are prepared, they can be placed in suitable containers and labeled for treatment. For the administration of the FVIII variant or the vector encoding the FVIII variant, such labeling should include the dosage, frequency of administration, and method of administration.

[0082] Pharmaceutical compositions suitable for the present invention include those containing an effective amount of the active ingredient to achieve the intended therapeutic purpose. Using the techniques and guidance provided by the present invention, determining the therapeutically effective dose is well within the ability of a skilled physician. The therapeutic dose will depend on the age and general condition of the subject, the severity of the abnormal coagulation phenotype, the strength of the control sequence regulating the expression level of the variant polypeptide, and so on. Thus, the therapeutically effective amount in humans will fall within a relatively wide range, which can be determined by the physician based on the response of the individual patient to the vector-based variant treatment.

[0083] The FVIII variant, alone or in combination with other agents, can be directly injected into a patient in a suitable biological vector as described above. The expression vectors of the present invention containing the nucleic acid sequence encoding the variant or its functional fragment can be administered to a patient in a variety of ways (see below) to achieve and maintain a prophylactic and / or therapeutically effective level of the variant polypeptide. Those skilled in the art can readily determine the specific protocol for using the expression vectors encoding the variant of the present invention for the therapeutic treatment of a particular patient. Protocols for generating adenoviral vectors and administering them to patients are described in: U.S. Patent Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; and International Patent Application Nos. WO 94 / 17810 and WO 94 / 23744, which are hereby incorporated by reference in their entirety.

[0084] The adenoviral vectors encoding FVIII variants of the present invention can be administered to a patient by any known method. Direct delivery of the pharmaceutical composition in vivo can generally be accomplished by injection using a conventional syringe, but other delivery methods are conceivable, such as convection enhanced delivery (see, e.g., U.S. Patent No. 5,720,720). In this regard, the composition can be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intraarterially, orally, intrahepatically or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories and transdermal applications. Clinicians specializing in the treatment of patients with coagulation disorders can determine the optimal route of administration of the adenoviral vector containing the variant nucleic acid sequence based on a number of criteria, including but not limited to: the condition of the patient and the purpose of the treatment (e.g., enhancing or reducing coagulation).

[0085] The present invention also includes AAV vectors containing nucleic acid sequences encoding FVIII variants. Lentiviral or pseudotyped lentiviral vectors containing nucleic acid sequences encoding FVIII variants are also provided. Also included are naked plasmids or expression vectors containing nucleic acid sequences encoding FVIII variants.

[0086] The following examples are provided to illustrate various embodiments of the present invention. The examples are illustrative and are not intended to limit the present invention in any way.

[0087] Example 1

[0088] Non-viral vectors (naked DNA, 5 μg / mouse) expressing various human FVIII B domain variants and FVIII-SQ under the control of a liver-specific promoter were injected via the tail vein route under hydrodynamic conditions (5 mice / variant). The variant B domains tested were: B1: SFSQNSRHPS (SEQ ID NO:13); B2: SFSQNSRHPSTRQKQ (SEQ ID NO:14); B3: SFSQNSRHPSTRQKQFNATT (SEQ ID NO:15); B4: SFSQN (SEQ ID NO:16); B5: SFSQNSRH (SEQ ID NO:17); and B6: SFSQNSRHPSTRQKQFNATTIPENDIEKTD (SEQ ID NO:18). After 24 hours, blood was collected and FVIII antigen levels were measured by ELISA using the Affinity Biologicals Matched Pair Antibody Set Product#F8C-EIA. As Figure 2 shown, all human FVIII B domain variants of the present invention expressed higher levels in hemophilia A mice than FVIII-SQ.

[0089] Example 2

[0090] Wild-type FVIII (659K) and its amino acid substitution variants were transiently expressed in BHK cells. The specific activity of the expressed FVIII was determined by a one-stage aPTT assay performed in conditioned expression medium. As Figure 3 shown, most of the FVIII variants showed increased specific activity compared to wild-type FVIII.

[0091] Example 3

[0092] In addition to FVIII variants having an amino acid substitution at position 659, FVIII variants having substitutions at 560, 561, 712, and 713 were also tested by transient expression in BHK cells. The specific activity of the expressed FVIII was determined by a one-stage aPTT assay performed in conditioned expression medium. As Figure 5 shown, the variants having an amino acid substitution at each position exhibited enhanced specific activity relative to wild-type FVIII. Combinations of these substitutions, as well as those provided in Example 2, can give rise to FVIII variants having even higher specific activity.

[0093] Although certain preferred embodiments of the invention have been described and specifically exemplified above, this does not mean that the invention is limited to these embodiments. As set forth in the appended claims, various modifications can be made thereto without departing from the scope and spirit of the invention. SEQUENCE LISTING <110> Wald R. Arruda Ben Samelson-Jones <120> Compositions and Methods for Modulating Factor VIII Function <130> 3460-P06618WO00 <150> 62 / 749,182 <151> 2018-10-23 <160> 18 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 2332 <212> PRT <213> Homo sapiens <400> 1 Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310 315 320 Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335 Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430 Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445 Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740 745 750 Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765 Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770 775 780 Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro 785 790 795 800 His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815 Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830 Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845 Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860 Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser 865 870 875 880 Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895 Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910 Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925 Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940 Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp 945 950 955 960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn 1010 1015 1020 Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys 1025 1030 1035 1040 Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala 1045 1050 1055 Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys 1060 1065 1070 Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp 1075 1080 1085 Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu 1090 1095 1100 Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser 1105 1110 1115 1120 Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys 1125 1130 1135 Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val 1140 1145 1150 Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe 1155 1160 1165 Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu 1170 1175 1180 Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys 1185 1190 1195 1200 Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr 1205 1210 1215 Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr 1220 1225 1230 Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245 Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His 1250 1255 1260 Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu 1265 1270 1275 1280 Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg 1285 1290 1295 Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys 1300 1305 1310 Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu 1315 1320 1325 Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met 1330 1335 1340 Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys 1345 1350 1355 1360 Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg 1365 1370 1375 Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys 1380 1385 1390 Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu 1395 1400 1405 Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1410 1415 1420 Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys 1425 1430 1435 1440 Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln 1445 1450 1455 Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr 1460 1465 1470 Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485 Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp 1490 1495 1500 Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu 1505 1510 1515 1520 Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn 1525 1530 1535 Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu 1540 1545 1550 Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp 1555 1560 1565 Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu 1570 1575 1580 Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser 1585 1590 1595 1600 Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly 1605 1610 1615 Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr 1620 1625 1630 Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg 1635 1640 1645 Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1650 1655 1660 Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr 1665 1670 1675 1680 Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg 1685 1690 1695 His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser 1700 1705 1710 Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720 1725 Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr 1730 1735 1740 Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly 1745 1750 1755 1760 Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg 1765 1770 1775 Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr 1780 1785 1790 Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 1795 1800 1805 Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala 1810 1815 1820 Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp 1825 1830 1835 1840 Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu 1845 1850 1855 Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr 1860 1865 1870 Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser 1875 1880 1885 Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1890 1895 1900 Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala 1905 1910 1915 1920 Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln 1925 1930 1935 Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn 1940 1945 1950 Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965 Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu 1970 1975 1980 Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys 1985 1990 1995 2000 Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val 2005 2010 2015 Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile 2020 2025 2030 Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 2035 2040 2045 Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr 2050 2055 2060 Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile 2065 2070 2075 2080 Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu 2085 2090 2095 Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp 2100 2105 2110 Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly 2115 2120 2125 Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 2130 2135 2140 Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser 2145 2150 2155 2160 Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met 2165 2170 2175 Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala 2180 2185 2190 Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205 Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn 2210 2215 2220 Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val 2225 2230 2235 2240 Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr 2245 2250 2255 Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr 2260 2265 2270 Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 2275 2280 2285 Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg 2290 2295 2300 Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg 2305 2310 2315 2320 Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 2325 2330 <210> 2 <211> 19 <212> PRT <213> Homo sapiens <400> 2 Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15 Cys Phe Ser <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 3 Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg 1 5 10 <210> 4 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 4 Ser Phe Ser Gln Asn Ser Arg His Pro Ser Gln Asn Pro Pro Val Leu 1 5 10 15 Lys Arg His Gln Arg 20 <210> 5 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 5 Ser Phe Ala Gln Asn Ser Arg Pro Pro Ser Ala Ser Ala Pro Lys Pro 1 5 10 15 Pro Val Leu Arg Arg His Gln Arg 20 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 6 Ser Phe Ser Gln Asn Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly 1 5 10 15 <210> 7 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 7 Ser Phe Ser Gln Asn Ala Thr Asn Val Ser Asn Asn Ser Asn Thr Ser 1 5 10 15 Asn Asp Ser Asn Val Ser Pro Pro Val Leu Lys Arg His Gln Arg 20 25 30 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 8 Ser Phe Ser Gln Asn Pro Pro Val Leu Lys His His Gly Arg 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 9 Ser Phe Ser Gln Asn Pro Pro Val Leu Lys 1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 10 Ser Phe Ser Gln Asn Pro Pro Val Ser Lys His His Gln Arg 1 5 10 <210> 11 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 11 Ser Phe Ser Gln Asn Pro Pro Val Ser Lys Arg His Gln Arg 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 12 Ser Phe Ser Gln Asn Pro Pro Val Ser Lys 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 13 Ser Phe Ser Gln Asn Ser Arg His Pro Ser 1 5 10 <210> 14 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 14 Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg Gln Lys Gln 1 5 10 15 <210> 15 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 15 Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg Gln Lys Gln Phe 1 5 10 15 Asn Ala Thr Thr 20 <210> 16 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 16 Ser Phe Ser Gln Asn 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 17 Ser Phe Ser Gln Asn Ser Arg His 1 5 <210> 18 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 18 Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg Gln Lys Gln Phe 1 5 10 15 Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys Thr Asp 20 25 30

Claims

1. A Factor VIII (FVIII) variant consisting of the sequence obtained by replacing amino acids 741 - 1648 of SEQ ID NO: 1 with SEQ ID NO: 13, wherein amino acids 1 - 740 and 1649 - 2332 of SEQ ID NO: 1 are joined by SEQ ID NO:

13.

2. A composition comprising the FVIII variant according to claim 1 and at least one pharmaceutically acceptable carrier.

3. Use of the FVIII variant according to claim 1 in the preparation of a medicament for the treatment of hemophilia A.

4. An isolated nucleic acid molecule encoding the FVIII variant according to claim 1.

5. An expression vector comprising the nucleic acid molecule according to claim 4 operably linked to a regulatory sequence.

6. The vector according to claim 5, which is selected from the group consisting of an adenovirus vector, an adeno - associated vector, a retrovirus vector, and a plasmid.

7. The vector according to claim 5, which is a lentiviral vector.

8. A host cell comprising the vector according to any one of claims 5 - 7.

9. The host cell according to claim 8, wherein the host cell is a human cell.

10. Use of the vector according to any one of claims 5 - 7 in the preparation of a medicament for the treatment of hemophilia A.

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