Compositions and methods for modulating factor VIII function

Abstract

The present invention relates to the field of medicine and hematology, and provides compositions and methods for modulating Factor VIII function. In particular, the present invention provides novel factor VIII variants, compositions comprising the variants, nucleic acid molecules encoding the variants, expression vectors comprising the nucleic acid molecules, and host cells comprising the vectors. The present invention also provides methods of using the variants and / or compositions to modulate the coagulation cascade in a patient in need thereof for the treatment of diseases including, but not limited to, hemophilia, particularly hemophilia A.

Description

[0001] This application is a divisional application of the invention patent application filed on October 23, 2019, with application number 201980070273.4 and entitled "Composition and Method for Regulating the Function of Factor VIII".

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 749,182, filed October 23, 2018, pursuant to 35 U.S.SC §119(e). The above application is incorporated herein by reference.

[0003] This invention was completed with government support under license number R01 HL-137335-01A1 granted by the National Institutes of Health in the United States. The government holds certain rights in this invention. Technical Field

[0004] This invention relates to the fields of medicine and hematology. More specifically, this invention provides novel factor VIII variants and methods for using them to modulate coagulation cascades in patients in need. Background Technology

[0005] Several publications and patent documents are cited throughout the specification to describe prior art relating to the present invention. Each of these citations is incorporated herein by reference in its entirety.

[0006] Mutations in factor VIII (FVIII) can cause severe bleeding disorders and are associated with hemophilia A. FVIII deficiency or lack of FVIII activity results in the inability to form clots effectively. To date, only 20% of hemophilia A patients worldwide receive routine FVIII replacement therapy due to its high cost. Typically, the FVIII is plasma-derived or recombinant. Gene therapy for hemophilia A based on AAV vectors is promising, but safety limitations exist due to aberrant immune responses to these vectors. This aberrant immune response has been found to be vector dose-dependent. Furthermore, 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 will develop inhibitors to the aforementioned treatment (e.g., anti-FVIII neutralizing antibodies) (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) 13:1). 217-1225; 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, WK, Semin. Hematol. (2008) 45(2Suppl 1):S42-S49; Guh et al., Haemophilia (2012) 18:268-275; Lindvall et al., Pediatr. Blood Cancer (2014) 61:706-711). Therefore, producing enhanced FVIII molecules will benefit the treatment of hemophilia by reducing the cost of FVIII production, increasing the safety of AAV gene therapy, and / or reducing immunogenicity. Therefore, it is clear that FVIII molecules with improved biological properties are needed. Summary of the Invention

[0007] According to the present invention, compositions and methods for regulating coagulation in patients with such need are provided. More specifically, a factor VIII (FVIII) variant for regulating (e.g., increasing) coagulation is provided. In one particular embodiment, the B domain of the FVIII variant is replaced with an amino acid sequence having at least 90% 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 one particular embodiment, the B domain of the FVIII variant is replaced with an amino acid sequence comprising SEQ ID NO:18. In one particular embodiment, the factor VIII variant contains at least one mutation at positions 560, 561, 712, 713, and / or 659, optionally with a B domain substitution. In one particular embodiment, the FVIII variant comprises replacing Lys at position 659 with another amino acid. In one specific embodiment, the Lys at position 659 is substituted with Trp, Arg, Ala, His, Tyr, Asp, Thr, Ser, Val, Phe, Gln, or Cys, particularly with Ser, Gln, or Cys. The FVIII variant may contain B-domain substitution and substitution at position 659. Compositions comprising at least one FVIII variant of the present invention and at least one pharmaceutically acceptable vector are also provided. Nucleic acid molecules encoding the FVIII variants of the present invention and methods of using them are also disclosed. Another aspect of the invention includes host cells expressing the FVIII variants described herein. Methods for isolating and purifying the FVIII variants are also disclosed.

[0008] Pharmaceutical compositions are also provided that contain, within a carrier, the FVIII variant of the invention and / or a nucleic acid molecule encoding the FVIII variant. The invention also includes methods for treating coagulation-related conditions in patients with such need, the methods comprising administering a therapeutically effective amount of the FVIII variant or a nucleic acid molecule encoding the FVIII variant, particularly in a pharmaceutical composition. These methods are effective in treating conditions requiring procoagulants, including but not limited to hemophilia, particularly hemophilia A. Attached Figure Description

[0009] Figure 1A A schematic diagram of the factor VIII protein is provided. The complete protein is 2332 amino acids long, and the B domain is 908 amino acids long. Various cleavage sites are also indicated in the schematic diagram. Figure 1BA schematic diagram of the processing factor VIII protein is provided. FVIII is translated into a single polypeptide chain (single chain) with a structure of A1-α1-A2-α2-B-α3-A3-C1-C2 domains. On the reverse side, Golgi furin cleaves the FVIII protein at R-1313 and / or R-1648 (triangles), leading to heterodimer formation. The FVIII heavy chain (A1-α1-A2-α2-B) and light chain (α3-A3-C1-C2) are linked by non-covalent metal-ion-dependent interactions (dashed lines) between the A1 and A3 domains. After secretion, the B domain undergoes further non-specific proteolytic cleavage in the plasma. During coagulation, the FVIII single chain or heterodimer is activated to its heterotrimeric cofactor form by thrombin cleavage at R-372, R-740, and R-1689 (triangles). A2 is associated with A1-α1 through non-covalent interactions (dashed lines). Inactivation of FVIIIa occurs through spontaneous A2 dissociation and / or proteolytic cleavage at R-336 and R-562 (triangles), primarily via activated protein C.

[0010] Figure 2 Expression maps of various human FVIII B domain variants in hemophilia A mice are provided.

[0011] Figure 3 A graph of the specific activity of FVIII for variants with amino acid substitution at position 659 is provided.

[0012] Figure 4 The amino acid sequence of FVIII is provided (SEQ ID NO:1). Amino acids at positions 560, 561, 659, 712 and / or 713 are shown in bold and underlined. The B domain is also indicated in italics and bold. The provided amino acid sequence is a signal peptide missing 19 amino acids at the N-terminus (MQIELSTCFFLCLLRFCFS(SEQ ID NO:2)).

[0013] Figure 5 A graph of FVIII specific activity for variants with amino acid substitutions at positions 560, 561, 712, and 713 is provided. Detailed Implementation

[0014] Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders caused by inherited defects in coagulation factor VIII (FVIII) or coagulation factor IX (FIX), respectively (Peyvandi et al., Lancet (2016) 388:187-197; Konkle et al., Hemophilia A. In Gene Reviews, Adam et al., University of Washington (1993)). Bleeding phenotypes are generally associated with residual factor activity: individuals with severe disease (factor activity <1% of normal) frequently bleed spontaneously; those with moderate disease (factor activity 1%–5% of normal) rarely bleed spontaneously but bleed from minor trauma; and those with mild disease (factor activity 5%–40% of normal) 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, as small increases in factor levels are expected to have meaningful clinical effects. Although various strategies have been studied over the decades, the field has focused on delivering engineered FVIII or FIX variants of transgenic proteins using adeno-associated virus (AAV) vectors, which possess therapeutic advantages not found in wild-type (WT) proteins (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, KA, 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 (7kb) exceeded the packaging capacity of the AAV vector (approximately 4.7kb). Removing the B domain of FVIII reduced the cDNA to approximately 4.4kb. Positive results have been reported in clinical trials of AAV-based HA using this method (Rangarajan, et al., N. Engl. J. Med. (2017) 377: 2519-2530).

[0015] As explained above, factor VIII is crucial for coagulation activity, and mutations in the FVIII gene lead to hemophilia A, the most common form of hemophilia. This paper demonstrates that specific alterations to the amino acid sequence of FVIII are associated with enhanced protein production and activity. Therefore, this invention provides rationally designed amino acid residue modifications that offer gain-of-function variants.

[0016] Full-length FVIII is a large 280 kDa protein primarily expressed in hepatic sinusoidal endothelial cells (LSECs) and extrahepatic endothelial cells (Fahs et al., Blood (2014) 123:3706-3713; Everett et al., Blood (2014) 123:3697-3705). FVIII primarily functions as a heterodimer cycle of heavy and light chains bound via non-covalent metal-dependent interactions (Lenting et al., Blood (1998) 92:3983-3996). Factor VIII contains several domains. These domains are typically referred to as A1-A2-B-A3-C1-C2, as shown in Figure 1. The heavy chain of FVIII contains A1-A2-B, while the light chain contains A3-C1-C2. Initially, FVIII exists in an inactive form bound to the von Willebrand factor (vWF). FVIII is activated by thrombin (factor IIa) and releases its B domain. The activated form of FVIII (FVIIIa) separates from vWF and interacts with coagulation factor IXa—leading to the formation of a blood clot through the coagulation cascade.

[0017] The B domain comprises 40% (908 amino acids) of the protein and is not essential for its procoagulant activity (Brinkhous et al., Proc. Natl. Acad. Sci. (1985) 82: 8752-8756). The most common B domain-deficient (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 BDD FVIII form is commonly used to produce recombinant BDD-FVIII (approximately 4.4 kb) and 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 mentioned above, due to the limited packaging capacity of AAV (4.7Kb) and other vector systems, gene therapy using AAV vectors can only utilize shortened FVIII molecules, such as BDD-FVIII (Lind, et al. (1995) Eur. J. Biochem., 232(1):19-27). US Patent 8,816,054 also provides BDD FVIII molecules with adapters of different lengths and sequences (see, for example, Table 1). However, the adapters in Table 1 contain more than one novel epitope that can lead to the production of FVIII inhibitors.

[0018]

[0019] Table 1: Short peptide linkers replacing the B domain in FVIII variants (Lind, et al. (1995) Eur. J. Biochem., 232(1): 19-27; Pittman, et al., Blood (1993) 81: 2925-2935; Tote, et al., Proc. Natl. Acad. Sci. (1986) 83: 5939-5942). Furin recognition motifs are underlined. *Also known as hFVIII-BDD. Abbreviations: aa, amino acid; c, canine; cl, cell line; F, factor; h,

[0020] Human; NA, not applicable; p, pig. The provided amino acid sequence from top to bottom is SEQ ID NO: 3-12.

[0021] This document provides novel factor VIII variants. The invention includes FVIII variants, which include FVIIIa variants and FVIII propeptide variants. For simplicity, the variants described throughout this application are primarily described in the context of FVIII. However, the invention contemplates and covers 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 invention are expressed as single-chain molecules or at least almost exclusively as single-chain molecules.

[0022] The FVIII variants of this invention can be derived from any mammalian species. In one specific embodiment, the FVIII variant is derived from humans. Examples of amino acid and nucleotide sequences of wild-type human FVIII (particularly the precursor containing the signal peptide) are provided in Gene ID: 2157 and GenBank accessions NM_000132.3 and NP_000123.1. 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 a 19-amino acid signal peptide (MQIELSTCFFLCLLRFCFS (SEQ ID NO:2)) at its N-terminus. The nucleic acid molecule encoding the factor FVIII variant can be readily identified from the provided amino acid sequence and the provided GenBank accession number.

[0023] According to one aspect of the invention, the FVIII variant is an FVIII molecule containing a B-domain deletion (BDD) of the linker. In a particular 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 produces factor VIII variants with enhanced factor VIII activity. The expression levels of these FVIII variants are also higher than those of other BDD FVIII variants. Moreover, the linkers provided in Table 2 each have only one novel epitope, while each of the linkers in Table 1 has more than one novel epitope. By reducing or minimizing the number of novel epitopes in the linker region, the adverse immunogenicity of the FVIII variants is reduced with the linkers in Table 2.

[0024]

[0025] Table 2: Novel B-domain linkers with minimal new epitopes. The provided amino acid sequences, from top to bottom, are SEQ ID NO: 13-18.

[0026] In one particular embodiment, the invention covers the FVIII variant, wherein the B domain (e.g., amino acids 741-1648 of SEQ ID NO:1) is replaced with an amino acid sequence comprising or consisting of 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 is replaced by an amino acid sequence of up to about 50, up to about 45, up to about 40, up to about 35, up to about 30, up to about 25, up to about 20, up to about 15, up to about 10, or up to about 5 amino acids in length. In one particular embodiment, the B domain (e.g., amino acids 741-1648 of SEQ ID NO:1) is replaced with an amino acid sequence comprising or consisting of SEQ ID NO:17 or SEQ ID NO:18. In one particular embodiment, the B domain (e.g., amino acids 741-1648 of SEQ ID NO:1) is replaced with an amino acid sequence comprising or consisting of SEQ ID NO:18. In another 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) 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 having less than 90%, 95%, 97%, 99%, or 100% homology (identity). In one 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) with SEQ ID NO:18, particularly having less than 90%, 95%, 97%, 99%, or 100% homology (identity).

[0027] According to another aspect of the invention, the factor VIII variant contains 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 some embodiments, the factor VIII variant contains a mutation at position 659. In one specific embodiment, Lys(K) at position 659 is not substituted by Pro(P), Gly(G), Met(M), or Leu(L). In one specific embodiment, Lys at position 659 is substituted 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 substituted by Asp(D), Thr(T), Ser(S), Val(V), Phe(F), Gln(Q), or Cys(C). In one specific implementation, Lys at position 659 is replaced by Ser(S), Val(V), Phe(F), Gln(Q), or Cys(C). In one specific implementation, Lys at position 659 is replaced by Ser(S), Gln(Q), or Cys(C). In one specific implementation, Lys at position 659 is replaced by Gln(Q) or Cys(C).

[0028] In some embodiments, the factor VIII variant contains 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).

[0029] In some embodiments, the factor VIII variant contains a mutation at position 561. In one specific embodiment, Gln(Q) at position 561 is not substituted with Leu(L), Arg(R), or Asn(N). In one specific embodiment, Gln(Q) at position 561 is substituted with Asp(D) or Glu(E). In one specific embodiment, the glutamic acid in Gln(Q) at position 561 is substituted with Asp(D).

[0030] In some embodiments, the factor VIII variant contains a mutation at position 712. In one specific embodiment, Asp(D) at position 712 is substituted with an amino acid other than Glu(E). In one specific embodiment, Asp(D) at position 712 is substituted with Ala(A), Val(V), Ile(I), or Leu(L). In one specific embodiment, Asp(D) at position 712 is substituted with Ile(I) or Leu(L). In one specific embodiment, Asp(D) at position 712 is substituted with Leu(L).

[0031] In some embodiments, the factor VIII variant contains a mutation at position 713. In one 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 one specific embodiment, Lys(K) at position 713 is replaced by Arg(R), Met(M), Tyr(Y), Asp(D), Cys(C), or Gly(G). In one specific embodiment, Lys(K) at position 713 is replaced by Asp(D) or Glu(E). In one specific embodiment, Lys(K) at position 713 is replaced by Cys(C). In one specific embodiment, Lys(K) at position 713 is replaced by Ala(A) or Gly(G). In one specific embodiment, Lys(K) at position 713 is replaced by Gly(G).

[0032] The FVIII variants of the present invention, as described herein, may contain at least one mutation at positions 560, 561, 712, 713, and / or 659 and / or may contain a linker that substitutes for the B domain, as described herein. In other words, the present invention covers FVIII variants having only mutations at positions 560, 561, 712, 713, and / or 659 (e.g., FVIII containing the complete B domain), FVIII variants having only linkers that substitute for the B domain (e.g., amino acids at positions 560, 561, 712, 713, and 659 are wild-type), and FVIII variants containing mutations at positions 560, 561, 712, 713, and / or 659 and linkers that substitute for the B domain.

[0033] In one specific embodiment, the VIII variant comprises mutations at positions 560, 561, 712, 713, and / or 659, and a linker replacing the B domain. In another specific embodiment, the VIII variant comprises mutations at positions 560, 561, 712, 713, and / or 659, and a linker replacing the B domain, the B domain having amino acid sequences having 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 having less than 90%, 95%, 97%, 99%, or 100% homology. In one particular embodiment, the VIII variant includes mutations at positions 560, 561, 712, 713, and / or 659, and a linker replacing the B domain, the B domain having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with SEQ ID NO:18, particularly having less than 90%, 95%, 97%, 99%, or 100% homology (identity). In some embodiments, the factor VIII variant includes a mutation at position 659. In one specific embodiment, Lys(K) at position 659 is not substituted with Pro(P), Gly(G), Met(M), or Leu(L). In one specific implementation, 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 implementation, 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 implementation, Lys at position 659 is replaced by Ser(S), Val(V), Phe(F), Gln(Q), or Cys(C). In one specific implementation, Lys at position 659 is replaced by Ser(S), Gln(Q), or Cys(C). In one specific implementation, Lys at position 659 is replaced by Gln(Q) or Cys(C). In some implementations, the factor VIII variant contains a mutation at position 560. In one specific implementation, 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 substituted with Ala(A), Val(V), Ile(I), or Leu(L). In one specific embodiment, Asp(D) at position 560 is substituted with His(H), Arg(R), or Lys(K). In one specific embodiment, Asp(D) at position 560 is substituted with Ile(I) or His(H). In some embodiments, the factor VIII variant contains a mutation at position 561. In one specific embodiment, Gln(Q) at position 561 is not substituted with Leu(L), Arg(R), or Asn(N). In one specific embodiment, Gln(Q) at position 561 is substituted with Asp(D) or Glu(E). In one specific embodiment, the glutamic acid in Gln(Q) at position 561 is substituted with Asp(D). In some embodiments, the factor VIII variant contains a mutation at position 712. In one specific embodiment, Asp(D) at position 712 is substituted with an amino acid other than Glu(E). In one specific embodiment, Asp(D) at position 712 is replaced by Ala(A), Val(V), Ile(I), or Leu(L). In one specific embodiment, Asp(D) at position 712 is replaced by Ile(I) or Leu(L). In one specific embodiment, Asp(D) at position 712 is replaced by Leu(L). In some embodiments, the factor VIII variant contains a mutation at position 713. In one 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 one specific embodiment, Lys(K) at position 713 is replaced by Arg(R), Met(M), Tyr(Y), Asp(D), Cys(C), or Gly(G). In one specific embodiment, Lys(K) at position 713 is replaced by Asp(D) or Glu(E). In one specific implementation, Lys(K) at position 713 is replaced by Cys(C). In another specific implementation, Lys(K) at position 713 is replaced by Ala(A) or Gly(G). In yet another specific implementation, Lys(K) at position 713 is replaced by Gly(G).

[0034] As described above, the FVIII variant of the present invention can be human. In one 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%. In one 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 comprising 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.

[0035] The FVIII variants of the present invention can also be post-translational modified. These FVIII variants can be post-translational modified in cells (particularly human cells) or in vitro.

[0036] In one particular embodiment, the FVIII variant of the present invention has increased expression compared to wild-type FVIII or hFVIII-SQ. In one particular embodiment, the FVIII variant of the present invention has increased FVIII activity or increased specific activity compared to wild-type FVIII.

[0037] Nucleic acid molecules encoding the aforementioned FVIII variants are also included in this invention. Nucleic acid molecules encoding these variants can be prepared by any method known in the art. These nucleic acid molecules can be stored in any convenient vector, specifically, an expression vector.

[0038] Compositions comprising at least one FVIII variant and at least one carrier are also included in this invention. In one specific embodiment, the FVIII is isolated and / or substantially pure in the composition. Compositions comprising at least one FVIII variant nucleic acid molecule and at least one carrier are also included in this invention. The use of any conventional carrier in pharmaceutical compositions is considered unless it is incompatible with the variant to be administered. In one specific embodiment, the carrier is a pharmaceutically acceptable carrier for intravenous administration.

[0039] definition

[0040] Various terms related to the biomolecules of the present invention have been used in the foregoing, throughout the specification, and in the claims.

[0041] The phrase “coagulation-related disorders” refers to bleeding disorders such as, but not limited to, hemophilia A, hemophilia B, hemophilia A and B patients, hemophilia with inhibitory antibodies, deficiency of 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, γ-carboxylase deficiency, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocoagulability), disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharides, 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 one particular implementation, the term "coagulation-related disorder" refers to a bleeding disorder characterized by excessive and / or uncontrolled bleeding (e.g., a disorder that can be treated with a procoagulant). In one particular implementation, the coagulation-related disorder is hemophilia. In one particular implementation, the coagulation-related disorder is hemophilia A.

[0042] 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 isolated from a sequence immediately following (in the 5' and 3' directions) of the naturally occurring genome of the organism of its origin. For example, "isolated nucleic acid" can comprise DNA or cDNA molecules inserted into a vector (e.g., a plasmid or viral vector) or integrated into prokaryotic or eukaryotic DNA. Regarding the RNA molecules of the present invention, the term "isolated nucleic acid" primarily 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 adequately isolated from RNA molecules associated with it in its natural state (i.e., in cells or tissues), so that it exists in a "substantially pure" form.

[0043] Regarding proteins, the term "isolated protein" is sometimes used herein. This term can refer to a protein produced by expressing the isolated nucleic acid molecules of the present invention. Alternatively, the term can refer to a protein that is sufficiently isolated from other proteins naturally associated with it (e.g., thus existing in a "substantially pure" form). "Isolated" does not imply 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 the addition of stabilizers.

[0044] The term "vector" refers to a carrier nucleic acid molecule (e.g., RNA or DNA) into which a nucleic acid sequence for introduction into a host cell can be inserted, where the nucleic acid sequence will be replicated. An "expression vector" is a specialized vector containing a gene or nucleic acid sequence having the necessary regulatory regions (e.g., promoters) required for expression in a host cell.

[0045] The term "operably ligated" refers to placing a regulatory sequence necessary for the expression of a coding sequence in the appropriate position within a DNA molecule relative to the coding sequence to achieve expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. This definition also sometimes applies to the arrangement of nucleic acid sequences in first and second nucleic acid molecules, resulting in hybrid nucleic acid molecules.

[0046] The term "substantially pure" means that the formulation contains at least 50-60% by weight of the target compound (e.g., nucleic acid, oligonucleotide, protein, etc.), specifically at least 75% by weight, or at least 90-99% by weight or more of the target compound. Purity can be measured by methods suitable for the target compound (e.g., chromatography, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0047] "Pharmaceutical acceptable" means that it has been approved by federal or state regulatory agencies or is listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, especially humans.

[0048] "Carrier" refers to, for example, diluents, adjuvants, preservatives (e.g., thimerosal, benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), solubilizers (e.g., polysorbate 80), emulsifiers, buffers (e.g., Tris HCl, acetates, phosphates), antimicrobial agents, fillers (e.g., lactose, mannitol), excipients, adjuvants, or mediators for administering the active agent of the present invention. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin. Water or saline solutions, as well as dextran and glycerol solutions, are preferred as carriers, particularly for injectable solutions. Suitable drug delivery systems are described in EW Martin's "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, PA); Gennaro, AR, Remington: The Science and Practice of Pharmacy (Lippincott, Williams and Wilkins); Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0049] Preparation of nucleic acid molecules and peptides encoding variants

[0050] Nucleic acid molecules encoding variants of the present invention can be prepared using recombinant DNA techniques. The availability of nucleotide sequence information allows for the preparation of isolated nucleic acid molecules of the present invention by a variety of methods. For example, standard protocols well known in the art can be used to isolate nucleic acid sequences encoding variants from suitable biological sources.

[0051] 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 *E. coli* host cell. Alternatively, the nucleic acids can be maintained in a vector suitable for expression in mammalian cells. In cases where post-translational modifications affect the function of the variant, expression of the molecule in mammalian cells, particularly human cells, is preferred.

[0052] 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. Therefore, the present invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of the nucleic acid molecules of the present invention. Such oligonucleotides can be used as probes for detecting variant expression.

[0053] The FVIII variants of the present invention can be prepared in a variety of ways according to known methods. The protein can be purified from a suitable source (e.g., transformed bacteria expressing the FVIII variant or cells or tissues cultured from animals (e.g., mammals or humans), for example, by immunoaffinity purification. The availability of nucleic acid molecules encoding the variants makes it possible to generate variants 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 translated cell-free in a suitable cell-free translation system (e.g., wheat germ or rabbit reticulocyte lysate). In vitro transcription and translation systems are commercially available, for example from Promega or Life Technologies.

[0054] Alternatively, a larger number of variants can be generated 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 *E. coli* or in mammalian cells (particularly human cells) such as CHO or HeLa cells. Alternatively, a labeled fusion protein containing the variant can be generated. This variant-labeled fusion protein is encoded by part or all of a DNA molecule linked in the correct codon reading frame to a nucleotide sequence encoding part or all of the desired polypeptide tag, which is inserted into a plasmid vector suitable for expression in bacterial cells such as *E. coli*, eukaryotic cells such as, but not limited to, yeast, and mammalian cells, particularly human cells. Vectors as described above contain regulatory elements necessary for DNA expression in the host cell, which are positioned in a manner that allows DNA expression in the host cell. Regulatory elements required for such expression include, but are not limited to, promoter sequences, transcription initiation sequences, and enhancer sequences.

[0055] FVIII variant proteins produced by gene expression in recombinant prokaryotic or eukaryotic systems (particularly humans) can be purified according to methods known in the art. In one specific embodiment, a commercially available expression / secretion system can be used to express the recombinant protein, which is then secreted from a 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 through an immune interaction using an antibody that specifically binds to the recombinant protein, or by separating recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C-terminus using a nickel column. Other tags may include, but are not limited to, FLAG epitopes, GST, or hemagglutinin epitopes. These methods are typically used by skilled practitioners.

[0056] The FVIII variant proteins prepared by the above method can be analyzed according to standard procedures. For example, these proteins can be analyzed by amino acid sequence analysis according to known methods.

[0057] As described above, a convenient way to generate the polypeptides of the present invention is by expressing the nucleic acid encoding it using a nucleic acid in an expression system. Various expression systems used in the methods of the present invention are well known to those skilled in the art.

[0058] Therefore, the present invention also includes a method for preparing (as disclosed) polypeptides, the method comprising expression from a nucleic acid (typically a nucleic acid) encoding the polypeptide. This can be conveniently achieved by culturing host cells containing such a vector under suitable conditions that induce or allow the production of the polypeptide. The polypeptide can also be produced in an in vitro system, for example in reticulocyte lysate.

[0059] Uses of FVIII variant protein and the nucleic acid encoding the variant

[0060] The FVIII variant protein and nucleic acid of this invention can be used, for example, as therapeutic and / or preventative agents for regulating the coagulation cascade system. This paper demonstrates that the FVIII variant possesses superior properties and can provide effective coagulation.

[0061] In one specific embodiment of the invention, the FVIII variant can be administered to a patient by infusion in a biocompatible carrier, for example, via intravenous injection. The FVIII variant of the invention may optionally be encapsulated in liposomes or mixed with other phospholipids or micelles to increase molecular stability. The FVIII variant can be administered alone or in combination with other known coagulation modulators (e.g., vFW, factor IX, factor IXa, etc.). Suitable compositions for delivering the FVIII variant can be determined by a physician taking into account various physiological variables, including but not limited to the patient's condition and hemodynamic status. Various compositions suitable for different applications and routes of administration are well known in the art and are described below.

[0062] Formulations containing FVIII variants may contain a physiologically acceptable matrix and are formulated into pharmaceutical preparations. These preparations can be formulated using substantially known prior art methods, and can be mixed with a buffer containing salts (e.g., NaCl, CaCl2) and amino acids (e.g., glycine and / or lysine) and a pH range of 6 to 8. Purified formulations containing FVIII variants can be stored for later use as a finished solution or in lyophilized or deep-frozen form. In one specific embodiment, the formulation is stored in lyophilized form and dissolved in a visually clear solution using a suitable reconstitution solution. Alternatively, the formulations of the present invention can also be obtained as liquid formulations or as deep-frozen liquids. Formulations according to the present invention can be particularly stable, i.e., can be allowed to stand in dissolved form for a long period of time prior to application.

[0063] The formulations of the present invention can be obtained as a single-component formulation of a pharmaceutical formulation having an FVIII variant or as a multi-component formulation in combination with other factors.

[0064] Before processing purified proteins into pharmaceutical formulations, the purified proteins can undergo routine quality control and be formulated into therapeutic forms. Specifically, during recombinant preparation, the presence of cellular nucleic acids and nucleic acids derived from expression vectors in the purified formulation can be tested.

[0065] Another feature of the invention relates to allowing the preparation of a formulation containing an FVIII variant with high stability and structural integrity, and, specifically, free of inactive FVIII intermediates and / or protein hydrolysis degradation products, and by formulating it into a suitable formulation.

[0066] As an example, the pharmaceutical formulation 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 one specific embodiment, the pharmaceutical protein formulation may contain a dose of 30-100 IU / kg (e.g., once daily injection or up to three or more times daily). Patients may receive treatment immediately upon arrival at an outpatient clinic for bleeding or before bleeding occurs from a cut / wound. Alternatively, patients may receive a bolus injection every one to three, eight, or twelve hours, or, if sufficient improvement is observed, a once-daily injection of the FVIII variant described herein.

[0067] According to the present invention, nucleic acids encoding FVIII variants can be used for a variety of purposes. In one specific embodiment of the invention, a nucleic acid delivery medium (e.g., an expression vector, such as a viral vector) for regulating coagulation is provided, wherein the expression vector comprises a nucleic acid sequence encoding an FVIII variant as described herein. Administration of the expression vector encoding an FVIII variant to a patient results in the expression of an FVIII variant for altering the coagulation cascade. According to the present invention, the nucleic acid sequence encoding an FVIII variant may encode a variant polypeptide as described herein, the expression of which increases coagulation. In one specific embodiment, the nucleic acid sequence encodes a human FVIII variant.

[0068] Expression vectors containing FVIII variant nucleic acid sequences can be administered alone or in combination with other molecules for regulating coagulation. According to the invention, the expression vectors or therapeutic combinations can be administered to patients alone or in pharmaceutically acceptable or biologically compatible compositions.

[0069] In one specific embodiment of the invention, the expression vector comprising the nucleic acid sequence encoding the FVIII variant is a viral vector. Viral vectors that can be used in this 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, particularly 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 one particular embodiment, the vector is an adeno-associated virus (AAV) vector. In another particular embodiment, the vector is a lentiviral vector.

[0070] In one specific embodiment of the invention, a method for administering a viral vector comprising a nucleic acid sequence encoding an FVIII variant is provided. The adenoviral vector available in the method of the invention preferably comprises at least a necessary portion of adenoviral vector DNA. As described herein, expression of the FVIII variant following administration of such an adenoviral vector is used to regulate coagulation, particularly enhancing the procoagulant activity of proteases.

[0071] Recombinant adenovirus vectors have been found to have wide applications in various gene therapy applications. Their usefulness in these applications is largely due to the high efficiency of in vivo gene transfer achieved in various organ environments.

[0072] Adenovirus particles can be advantageously used as a medium for appropriate gene delivery. These virions possess many desirable characteristics for such applications, including structural features associated with being double-stranded DNA non-enveloped viruses, as well as biological characteristics such as tropism for the human respiratory and gastrointestinal systems. Furthermore, adenoviruses are known to infect a variety of cell types in vivo and in vitro via receptor-mediated endocytosis. Regarding the overall safety of adenovirus vectors, adenovirus infection has been demonstrated to cause mild illness states in humans, including mild flu-like symptoms.

[0073] Because of their large genome size (approximately 36 kilobases), adenoviruses are well-suited for use as gene therapy vectors, as they can accommodate the insertion of foreign DNA after the removal of adenoviral genes essential for replication and non-essential regions. Such substitution impairs the viral vector's replication function and infectivity. Notably, adenoviruses have been used as vectors for gene therapy and for expressing heterologous genes.

[0074] It is desirable to introduce a vector that can provide, for example, multiple copies of a desired gene, and thus provide a larger quantity of the gene product. Improved adenovirus vectors and methods for preparing these vectors have been described in detail in numerous 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); US Patent No. 5,998,205 (which provides a tumor-specific replication vector containing multiple copies of DNA); 6,228,646 (which describes an unhelper-free, fully defective adenovirus vector); 6,093,699 (which provides a vector and method for gene therapy); 6,100,242 (which provides a replication-defective adenovirus vector with inserted transgenes that is effective for in vivo gene therapy for peripheral vascular disease and heart disease); and International Patent Application Nos. WO 94 / 17810 and WO 94 / 23744.

[0075] For certain applications, the expression construct may further include regulatory elements for driving expression in specific cell or tissue types. Such regulatory elements are known to those skilled in the art and have been discussed in detail in Sambrook et al. (1989) and Ausubel et al. (1992). Incorporation of tissue-specific regulatory elements into the expression construct of the present invention provides at least partial tissue specificity for the expression of said variants or functional fragments thereof. For example, an E1-deleted adenovirus type 5 vector containing a nucleic acid sequence encoding a variant, under the control of a cytomegalovirus (CMV) promoter, can be used in the methods of the present invention. Hematopoietic or liver-specific promoters may also be used.

[0076] AAV vectors for recombinant gene expression have been generated in human embryonic kidney cell line 293 (Wright, HumGene Ther (2009) 20:698-706; Graham et al. (1977) J. Gen. Virol. 36:59-72). In short, AAV vectors are typically derived from wild-type AAV (a non-pathogenic single-stranded DNA virus). The parent virus is non-pathogenic, and the vector has a broad host range and can infect both dividing and non-dividing cells. Vectors are usually modified from viruses by deleting the rep and cap genes and replacing them with the desired transgene under the control of a specific promoter. For recombinant AAV formulations, the upper limit of the sequence size that can be inserted between two ITRs is approximately 4.7 kb. AAV-2 vectors can be prepared using plasmids expressing the FVIII variant under the control of CMV promoters / enhancers, a second plasmid providing adenovirus helper functions, and a third plasmid containing the AAV-2rep and cap genes. Plasmids containing the AAV-1, AAV-6, or AAV-8 cap genes, the AAV-2rep gene, and the ITR can be used to prepare their 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). AAV vectors can be purified by repeated CsCl density gradient centrifugation, and the titer of the purified vector can be determined by quantitative dot blot hybridization. In one specific embodiment, the vectors can be prepared by Vector Core at Children's Hospital of Philadelphia.

[0077] The present invention also includes a method for regulating coagulation, the method comprising providing an individual’s cells with a nucleic acid delivery medium encoding an FVIII variant and allowing the cells to grow under conditions expressing the FVIII variant.

[0078] As can be seen from the preceding discussion, FVIII variants and nucleic acid vectors expressing FVIII variants can be used to treat conditions related to abnormal coagulation.

[0079] The expression vector of the present invention can be incorporated into a pharmaceutical composition deliverable to a subject, thereby allowing the production of a bioactive protein (e.g., an FVIII variant) or in vivo induction of FVIII variant expression via gene- and / or cell-based therapies or via in vitro modification / transduction of patient or donor cells. In one specific embodiment of the invention, a pharmaceutical composition containing sufficient genetic material to induce a therapeutically effective amount of the FVIII variant in the recipient can affect the subject's coagulation. Alternatively, as described above, an effective amount of the 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 (e.g., a stabilizing compound), which can be administered in any sterile, biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, dextran, and water. The composition can be administered to a patient alone or in combination with other agents affecting coagulation (e.g., cofactors).

[0080] In specific embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients / carriers. These excipients include any pharmaceutical agent that does not 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, sugar, and ethanol. Pharmaceutically acceptable salts may also be included therein, such as 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 buffers, etc., may be present in such carriers. A detailed discussion of pharmaceutically acceptable excipients is provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., 18th edition, Easton, Pa.

[1990] ).

[0081] Pharmaceutical formulations suitable for parenteral administration can be prepared in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively, suspensions of active compounds can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or mediators include fatty oils (e.g., sesame oil), or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to prepare high-concentration solutions.

[0082] The pharmaceutical composition may be provided in the form of a salt, and the salt may be formed with a variety of acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. Salts tend to be more soluble in water or other protic solvents compared to their corresponding free base forms. In other cases, the formulation may be a lyophilized powder containing 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 used in combination with a buffer solution prior to use.

[0083] After the pharmaceutical compositions are prepared, they can be placed in suitable containers and labeled for treatment. For administration of FVIII variants or carriers encoding FVIII variants, such labeling should include the dosage, frequency of administration, and method of administration.

[0084] Pharmaceutical compositions applicable to this invention include compositions containing an effective amount of the active ingredient to achieve the intended therapeutic purpose. Using the techniques and guidance provided by this invention, determining the therapeutically effective dose is entirely within the capabilities of a skilled physician. The therapeutic dose will depend on the subject's age and general condition, the severity of the abnormal coagulation phenotype, and the strength of the control sequence regulating the expression level of the variant peptide, etc. Therefore, the therapeutically effective dose in humans will fall into a relatively wide range, which can be determined by a physician based on the individual patient's response to the vector-based variant therapy.

[0085] The FVIII variant, alone or in combination with other agents, can be directly injected into patients in suitable biological vectors as described above. Expression vectors of the present invention, containing nucleic acid sequences encoding variants or functional fragments thereof, can be administered to patients in a variety of ways (see below) to achieve and maintain effective levels of the variant peptide for prevention and / or treatment. Those skilled in the art can readily determine specific protocols for the therapeutic treatment of particular patients using expression vectors encoding variants of the present invention. Protocols for generating adenovirus 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, all of which are incorporated herein by reference in their entirety.

[0086] The adenovirus vector encoding the FVIII variant 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 contemplated, such as convection-enhanced delivery (see, for example, U.S. Patent No. 5,720,720). In this regard, the composition can be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosa, intraperitoneally, intravenously, intraarterially, intraorally, intrahepatically, or intramuscularly. Other routes of administration include oral and pulmonary administration, suppositories, and percutaneous application. Clinicians specializing in the treatment of patients with coagulation disorders can determine the optimal route of administration of the adenovirus vector containing the variant nucleic acid sequence based on a number of criteria, including but not limited to: the patient's condition and the purpose of treatment (e.g., enhancing or reducing coagulation).

[0087] The present invention also includes an AAV vector containing a nucleic acid sequence encoding an FVIII variant. Lentiviral or pseudolentiviral vectors containing nucleic acid sequences encoding an FVIII variant are also provided. Naked plasmids or expression vectors containing nucleic acid sequences encoding an FVIII variant are also included.

[0088] The following examples are provided to illustrate various embodiments of the present invention. These examples are illustrative and are not intended to limit the invention in any way.

[0089] Example 1

[0090] Various human FVIII B domain variants and non-viral vectors (naked DNA, 5 μg / mouse) expressing FVIII-SQ under liver-specific promoter control were injected via tail vein under hydrodynamic conditions (5 mice / variant). The variants tested included: 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). Twenty-four hours later, blood samples were collected, and FVIII antigen levels were measured by ELISA using Affinity Biologicals Matched Pair Antibody Set Product#F8C-EIA. Figure 2 As shown, all human FVIII B domain variants of the present invention were expressed at higher levels than FVIII-SQ in hemophilia A mice.

[0091] Example 2

[0092] Wild-type FVIII (659K) and its amino acid substitution variants were transiently expressed in BHK cells. The specific activity of expressed FVIII was determined by a one-stage aPTT assay performed in conditioned expression medium. Figure 3 As shown, most FVIII variants exhibit increased specific activity compared to wild-type FVIII.

[0093] Example 3

[0094] In addition to the FVIII variant with an amino acid substitution at position 659, FVIII variants with substitutions at positions 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. Figure 5 As shown, variants with amino acid substitutions at each position exhibit enhanced specific activity compared to wild-type FVIII. Combinations of these substitutions, as well as those provided in Example 2, can produce FVIII variants with even higher specific activity.

[0095] Although certain preferred embodiments of the invention have been described and specifically illustrated above, this does not mean that the invention is limited to these embodiments. As stated in the appended claims, various modifications can be made thereto without departing from the scope and spirit of the invention.

Claims

1. A factor VIII (FVIII) variant containing a Lys substitution mutation at position 659.

2. The FVIII variant of claim 1, wherein Lys at position 659 is replaced by Trp, Arg, Ala, His, Tyr, Asp, Thr, Ser, Val, Phe, Gln, or Cys.

3. The FVIII variant of claim 1, wherein Lys at position 659 is replaced by Asp, Thr, Ser, Val, Phe, Gln, or Cys.

4. The FVIII variant of claim 1, wherein Lys at position 659 is replaced by Gln or Cys.

5. The FVIII variant of claim 1, wherein Lys at position 659 is replaced by Val.

6. The FVIII variant according to claim 1, wherein the FVIII comprises amino acids 1-740 and 1649-2332 of SEQ ID NO:

1.

7. A composition comprising at least one FVIII variant according to any one of claims 1-6 and at least one pharmaceutically acceptable carrier.

8. Use of the FVIII variant according to any one of claims 1-6 in the preparation of a medicament for treating hemophilia A.

9. An isolated nucleic acid molecule encoding the FVIII variant of claim 1.

10. An expression vector comprising a nucleic acid molecule according to claim 9 operably linked to a regulatory sequence.

11. The vector according to claim 10, wherein the vector is selected from the group consisting of adenovirus vectors, adenovirus-associated vectors, retroviral vectors, and plasmids.

12. The vector according to claim 10, wherein it is a lentiviral vector.

13. A host cell comprising the vector according to any one of claims 10-12.

14. The host cell of claim 13, wherein the host cell is a human cell.

15. Use of the carrier according to any one of claims 10-12 in the preparation of a medicament for treating hemophilia A.

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