Compositions and methods for modulating factor VIII function

Abstract

Factor VIII variants and methods of use thereof are disclosed.

Description

[Technical field]

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 749,182, filed October 23, 2018, which is incorporated herein by reference.

[0002] This invention was made with Government support under Grant No. R01HL-137335-01A1 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] FIELD OF THEINVENTION 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 same to regulate the coagulation cascade in patients in need thereof. [Background technology]

[0004] Several publications and patent documents are cited herein to describe the state of the art to which the invention pertains, and these citations are incorporated by reference as if set forth in full.

[0005] Mutations in factor VIII (FVIII) can lead to severe bleeding disorders and are associated with hemophilia A. FVIII deficiency or insufficient FVIII activity results in the inability to form blood clots effectively. Currently, only 20% of patients with hemophilia A worldwide receive regular FVIII replacement therapy, due in part to the high cost. Typically, FVIII is plasma-derived or recombinantly produced. AAV vector-based gene therapy for hemophilia A shows promise but has safety limitations due to an aberrant immune response to the vector. This aberrant immune response has been found to be vector-dosage dependent. Furthermore, immunogenicity of the delivered or expressed FVIII can be problematic, regardless of the administered protein or use of gene therapy. Indeed, 20-30% of patients with hemophilia A develop inhibitors to 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) 13:1217-1225; 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, WK, 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).

[0006] Therefore, generating an enhanced FVIII molecule would benefit the treatment of hemophilia by lowering the cost of FVIII production, increasing the safety and / or reducing the immunogenicity of AAV gene therapy. Therefore, there is a clear need for FVIII molecules with improved biological properties. Summary of the Invention

[0007] According to the present invention, compositions and methods are provided for regulating hemostasis in a patient in need thereof. More specifically, factor VIII (FVIII) variants are provided that regulate (e.g., increase) hemostasis. In certain embodiments, 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 certain embodiments, the B domain of the FVIII variant is replaced with an amino acid sequence comprising SEQ ID NO: 18. In certain embodiments, the factor VIII variant comprises at least one mutation at positions 560, 561, 712, 713, and / or 659, optionally with a substitution of the B domain. In certain embodiments, the FVIII variant comprises a substitution of Lys at position 659 with another amino acid. In certain embodiments, Lys at position 659 is substituted with Trp, Arg, Ala, His, Tyr, Asp, Thr, Ser, Val, Phe, Gln, or Cys, particularly Ser, Gln, or Cys. The FVIII variant may include a substitution in the B domain and a substitution at position 659. Also provided are compositions comprising at least one FVIII variant of the invention and at least one pharma- ceutically acceptable carrier. Also disclosed are nucleic acid molecules encoding the FVIII variants of the invention, as well as methods of use thereof. Another aspect of the invention includes host cells expressing the FVIII variants described herein. Methods of isolating and purifying the FVIII variants are also disclosed.

[0008] Also provided is a pharmaceutical composition comprising the FVIII variants of the invention and / or a nucleic acid molecule encoding a FVIII variant in a carrier.The invention also includes a method for treating a hemostasis-related disorder in a patient in need thereof, comprising administering a therapeutically effective amount of a FVIII variant and / or a nucleic acid molecule encoding a FVIII variant, particularly in a pharmaceutical composition.Such a method is useful for treating diseases in which a procoagulant is required, including but not limited to hemophilia, particularly hemophilia A. [Brief description of the drawings]

[0009] [Figure 1] Figure 1A provides a schematic diagram of the factor VIII protein. The complete protein is 2332 amino acids long, and the B domain is 908 amino acids long. The various cleavage sites are also shown in the schematic diagram. Figure 1B shows an overview of the processing of the factor VIII protein. FVIII is translated as a single peptide chain with the domain structure A1-α1-A2-α2-B-α3-A3-C1-C2. Heterodimers are formed upon proteolytic cleavage of R-1313 and / or R-1648 of FVIII by the trans-Golgi protease Furin (triangle). The FVIII heavy chain (A1-α1-A2-α2-B) and light chain (α3-A3-C1-C2) are kept linked by non-covalent metal ion-dependent interactions between the A1 and A3 domains (dashes). The B domain undergoes further non-specific proteolysis in plasma after secretion. During coagulation, FVIII single chains or heterodimers are activated to their heterotrimeric coenzyme form by cleavage by thrombin at R-372, R-740, and R-1689 (triangles). A2 remains noncovalently bound to A1-α1 (dashes). Inactivation of FVIIIa occurs by spontaneous A2 dissociation and / or proteolytic cleavage primarily by activated protein C at R-336 and R-562 (triangles).

[0010] [Diagram 2]FIG. 2 provides a graph of the expression of various human FVIII B domain variants in hemophilia A mice.

[0011] [Diagram 3] FIG. 3 provides a graph of the FVIII specific activity of variants with an amino acid substitution at position 659.

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

[0013] [Diagram 5] FIG. 5 provides a graph of the FVIII specific activity of variants with amino acid substitutions at positions 560, 561, 712, and 713. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Invention Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders caused by inherited deficiencies of either coagulation factor VIII (FVIII) or 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)).

[0015] The bleeding phenotype is generally associated with residual factor activity, with severely affected individuals (factor activity <1% of normal) having frequent spontaneous bleeding, moderately affected individuals (factor activity 1%-5% of normal) rarely bleeding spontaneously but bleeding with minor trauma, and mildly affected individuals (factor activity 5%-40% of normal) bleeding with invasive procedures or trauma. Given this clear 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 important clinical benefits. Although various strategies have been explored for decades, the field has centered on the use of adeno-associated virus (AAV) vectors to deliver transgenes for modified FVIII or FIX variants that have therapeutically advantageous 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, 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).

[0016] Of note, full-length FVIII cDNA (7 kb) exceeds the packing capacity of AAV vectors (~4.7 kb). Deletion of the B domain of FVIII reduces the cDNA to ~4.4 kb. Good results have been reported using this method in clinical trials of HA (Rangarajan, et al., N. Engl. J. Med. (2017) 377:2519-2530).

[0017] As explained above, factor VIII is central to coagulation activity, and mutations in the FVIII gene lead to hemophilia A, the most common form of hemophilia. It is shown herein that specific changes in the amino acid sequence of FVIII are associated with enhanced protein production and activity. Thus, the present invention provides rationally designed modifications of amino acid residues that provide gain-of-function variants.

[0018] Full-length FVIII is a large 280 kDa protein that is expressed primarily 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 circulates primarily as a heterodimer of heavy and light chains, which are held together by non-covalent metal-dependent interactions (Lenting, et al., Blood (1998) 92:3983-3996). Factor VIII is composed of several domains. In general, the domains are referred to as A1-A2-B-A3-C1-C2, as shown in Figure 1. The heavy chain of FVIII contains A1-A2-B, and the light chain contains A3-C1-C2. Initially, FVIII is in an inactive form bound to von Willebrand factor (vWF). FVIII is activated when it is cleaved by thrombin (factor IIa) and the B domain is released. The activated form of FVIII (FVIIIa) separates from vWF and interacts with the coagulation factor factor IXa, which then progresses through the coagulation cascade to form a clot.

[0019] The B domain comprises 40% of the protein (908 amino acids) and is not required 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 BDD FVIII form is commonly used to produce recombinant BDD-FVIII (~4.4Kb) 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).

[0020] As mentioned above, in gene therapy using AAV vectors, only short FVIII molecules such as BDD-FVIII can be used due to the limited packaging capacity of vector systems such as AAV (4.7Kb) (Lind, et al. (1995) Eur. J. Biochem., 232(1):19-27). U.S. Patent No. 8,816,054 also provides BDD FVIII molecules with linkers of different lengths and sequences (see, for example, Table 1). However, the linkers in Table 1 contain one or more neoepitopes, which may lead to the generation of FVIII inhibitors.

[0021] [Table 1] Table 1: Short peptide linkers substituted into the B domain of FVIII variants (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 called 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.

[0022] Provided herein is a novel factor VIII variant. The present invention encompasses FVIII variants, including FVIIIa variants and FVIII prepeptide variants. For simplicity, throughout this application, variants are generally described in the context of FVIII. However, the present invention contemplates and encompasses factor FVIIIa and FVIII prepeptide molecules with the same amino acid substitutions and / or linkers as described for FVIII. In certain embodiments, the FVIII variants of the present invention are expressed as single-chain molecules, or at least mostly as single-chain molecules.

[0023] The FVIII variants of the present invention can be derived from any mammalian species. In a particular embodiment, the FVIII variant is human. Gene ID: 2157 and GenBank Accession Nos. NM_000132.3 and NP_000123.1 provide examples of amino acid and nucleotide sequences of wild-type human FVIII (particularly the pre-peptide including the signal peptide). Figure 4 provides SEQ ID NO: 1, an example of an amino acid sequence of human FVIII. SEQ ID NO: 1 lacks the 19 amino acid signal peptide at its N-terminus (MQIELSTCFFLCLLRFCFS (SEQ ID NO: 2)). Nucleic acid molecules encoding variants of factor FVIII can be easily determined from the provided amino acid sequences and provided GenBank Accession numbers.

[0024] According to one aspect of the present invention, the FVIII variant is a B-domain deleted (BDD) FVIII molecule comprising a linker. In a particular embodiment, the linker comprises a sequence as set forth in Table 2. As shown herein, by replacing the B-domain of factor VIII with the sequence as set forth in Table 2, factor VIII variants exhibiting enhanced factor VIII activity were obtained. These FVIII variants were also expressed at higher levels than other BDD FVIII. Furthermore, the linkers provided in Table 2 each have only one neoepitope, whereas each of the linkers in Table 1 has one or more neoepitopes. By reducing or minimizing the number of neoepitopes in the linker region, the linkers in Table 2 reduce the adverse immunogenicity of the FVIII variant.

[0025] [Table 2] Table 2: New B domain linkers with minimal neoepitopes. The amino acid sequences provided are SEQ ID NOs: 13 to 18, from top to bottom.

[0026] In certain embodiments, the invention encompasses FVIII variants in which the B-domain (e.g., amino acids 741-1648 of SEQ ID NO:1) has been 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 certain embodiments, the B-domain has been replaced with 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 certain embodiments, the B-domain (e.g., amino acids 741-1648 of SEQ ID NO:1) has been replaced with an amino acid sequence comprising or consisting of SEQ ID NO:17 or SEQ ID NO:18. In certain embodiments, the B-domain (e.g., amino acids 741-1648 of SEQ ID NO:1) has been replaced with an amino acid sequence comprising or consisting of SEQ ID NO:18. In certain embodiments, 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, in particular at least 90%, 95%, 97%, 99%, or 100% homology (identity). In certain embodiments, 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, in particular at least 90%, 95%, 97%, 99%, or 100% homology (identity).

[0027] According to another aspect of the invention, the factor VIII variant comprises at least one mutation at positions 560, 561, 712, 713, and / or 659. As seen herein, these FVIII variants have higher specific activity than wild-type FVIII. In certain embodiments, the factor VIII variant comprises a mutation at position 659. In certain embodiments, Lys (K) at position 659 is not substituted with Pro (P), Gly (G), Met (M), or Leu (L). In certain embodiments, Lys at position 659 is substituted with 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 certain embodiments, the Lys at position 659 is substituted with Asp(D), Thr(T), Ser(S), Val(V), Phe(F), Gln(Q), or Cys(C). In certain embodiments, the Lys at position 659 is substituted with Ser(S), Val(V), Phe(F), Gln(Q), or Cys(C). In certain embodiments, the Lys at position 659 is substituted with Ser(S), Gln(Q), or Cys(C). In certain embodiments, the Lys at position 659 is substituted with Gln(Q) or Cys(C).

[0028] In certain embodiments, the factor VIII variant comprises a mutation at position 560. In certain embodiments, the Asp(D) at position 560 is replaced with Ala(A), Val(V), Ile(I), Leu(L), His(H), Arg(R), or Lys(K). In certain embodiments, the Asp(D) at position 560 is replaced with Ala(A), Val(V), Ile(I), or Leu(L). In certain embodiments, the Asp(D) at position 560 is replaced with His(H), Arg(R), or Lys(K). In certain embodiments, the Asp(D) at position 560 is replaced with Ile(I) or His(H).

[0029] In certain embodiments, the factor VIII variant comprises a mutation at position 561. In certain embodiments, the Gln(Q) at position 561 is not substituted with Leu(L), Arg(R), or Asn(N). In certain embodiments, the Gln(Q) at position 561 is substituted with Asp(D) or Glu(E). In certain embodiments, the Gln(Q) at position 561 is substituted with Asp(D).

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

[0031] In certain embodiments, the factor VIII variant comprises a mutation at position 713. In certain embodiments, Lys(K) at position 713 is substituted with Ala(A), Arg(R), Met(M), Tyr(Y), Asp(D), Glu(E), Cys(C), or Gly(G). In certain embodiments, Lys(K) at position 713 is substituted with Arg(R), Met(M), Tyr(Y), Asp(D), Cys(C), or Gly(G). In certain embodiments, Lys(K) at position 713 is substituted with Asp(D) or Glu(E). In certain embodiments, Lys(K) at position 713 is substituted with Cys(C). In certain embodiments, Lys(K) at position 713 is substituted with Ala(A) or Gly(G). In a particular embodiment, Lys (K) at position 713 is substituted with Gly (G).

[0032] FVIII variants of the invention may comprise at least one mutation at positions 560, 561, 712, 713, and / or 659 as described herein, and / or comprise a linker in place of the B domain as described herein. That is, the invention encompasses FVIII variants that only have mutations at positions 560, 561, 712, 713, and / or 659 (e.g., the FVIII comprises an intact B domain), FVIII variants that only have a linker in place of the B domain (e.g., the amino acids at positions 560, 561, 712, 713, and 659 are wild type), and FVIII variants that comprise mutations at positions 560, 561, 712, 713, and / or 659 and a linker in place of the B domain.

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

[0034] As mentioned above, the FVIII variant of the present invention may be human. In a particular embodiment, the FVIII variant of the present invention has at least 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% homology (identity), in particular at least 90%, 95%, 97%, 99% or 100% homology (identity) with SEQ ID NO: 1 (or an activated FVIII fragment thereof). In certain embodiments, 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 to 740 of SEQ ID NO: 1 (or an activated FVIII fragment thereof), in particular at least 90%, 95%, 97%, 99% or 100% 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 to 2332 of SEQ ID NO: 1 (or an activated FVIII fragment thereof), in particular at least 90%, 95%, 97%, 99% or 100% homology (identity). The above percentages of homology (identity) exclude substitutions at positions 560, 561, 712, 713, and / or 659.

[0035] Furthermore, the FVIII variants of the invention may be post-translationally modified. The FVIII variants may be post-translationally modified in cells, particularly human cells, or in vitro.

[0036] In certain embodiments, the FVIII variants of the invention have increased expression compared to wild-type FVIII or hFVIII-SQ, hi certain embodiments, the FVIII variants of the invention have increased FVIII activity or increased specific activity compared to wild-type FVIII.

[0037] The present invention also includes nucleic acid molecules encoding the above FVIII variants. The nucleic acid molecules encoding the variants can be prepared by any method known in the art. The nucleic acid molecules can be maintained in any convenient vector, in particular an expression vector.

[0038] The present invention also includes compositions comprising at least one FVIII variant and at least one carrier. In certain embodiments, the FVIII is isolated and / or substantially pure in the composition. The present invention also includes compositions comprising at least one FVIII variant nucleic acid molecule and at least one carrier. Use in pharmaceutical compositions is contemplated, except where conventional carriers are incompatible with the variant to be administered. In certain embodiments, the carrier is a pharma- ceutically acceptable carrier for intravenous administration.

[0039] (definition) Various terms relating to the biological molecules of the present invention are used above and throughout the specification and claims.

[0040] The term "hemostasis-related disorder" includes, but is not limited to, hemophilia A, hemophilia B, hemophilia A and B patients, hemophilia due to inhibitory antibodies, deficiency of at least one coagulation factor (e.g., factor VII, factor VIII, factor IX, factor X, factor XI, factor V, factor XII, factor II, and / or von Willebrand factor (particularly factor VIII), combined FV / FVIII deficiency, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency, bleeding following trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocalgic), disseminated Examples of hemostasis-related disorders include disseminated intravascular coagulation (DIC), excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharides, warfarin, small molecule antithrombotic agents (e.g., FXa inhibitors), and platelet disorders such as Bernard-Soulier syndrome, Glanzmann thrombosis, and storage pool deficiency. In certain embodiments, the term "hemostasis-related disorder" refers to bleeding disorders characterized by excessive and / or uncontrolled bleeding (e.g., disorders treatable with procoagulants). In certain embodiments, the hemostasis-related disorder is hemophilia. In certain embodiments, the hemostasis-related disorder is hemophilia A.

[0041] With respect to 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 is separated from sequences that are immediately adjacent (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates. For example, "isolated nucleic acid" can include a DNA or cDNA molecule inserted into a vector, such as a plasmid or viral vector, or integrated into the DNA of a prokaryotic or eukaryotic organism. With respect to the RNA molecules of the present invention, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, it can refer to an RNA molecule that has been sufficiently separated from the RNA molecules with which it would be associated in its natural state (i.e., in cells and tissues) and exists in a "substantially pure" form.

[0042] With respect to proteins, the term "isolated protein" may be used herein. This term may refer to a protein produced by expression of an isolated nucleic acid molecule of the invention. This term may also refer to a protein that has been sufficiently separated from other proteins with which it is naturally associated (e.g., so as to be present in "substantially pure" form). "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the essential activity, but may be present, for example, by incomplete purification or addition of stabilizers.

[0043] The term "vector" refers to a carrier nucleic acid molecule (e.g., RNA or DNA) into which a nucleic acid sequence can be inserted for introduction into a host cell where it will be replicated. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with regulatory regions (e.g., promoter) required for expression in a host cell.

[0044] The term "operably linked" means that regulatory sequences required for expression of a coding sequence are positioned on the DNA molecule in the appropriate position relative to the coding sequence to effect expression of the coding sequence. This same definition may also be applied to the arrangement of a coding sequence and transcription control elements (promoters, enhancers, termination elements, etc.) in an expression vector. This definition may also be applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule such that a hybrid nucleic acid molecule is produced.

[0045] The term "substantially pure" refers to a preparation that contains at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.), particularly at least 75% by weight, or at least 90-99% by weight of the compound of interest. Purity can be measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0046] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia, and approved for use in animals, and especially humans.

[0047] The term "carrier" refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), antioxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris-HCl, acetate, phosphate), antibacterial agent, bulking agent (e.g., lactose, mannitol), excipient, auxiliary, or vehicle with which the active agent of the present invention is administered. Pharmaceutically acceptable carriers are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Preferred carriers include water, saline, and aqueous solutions of dextrose or glycerol, particularly for injections. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin (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.

[0048] Preparation of nucleic acid molecules and polypeptides encoding variants The nucleic acid molecule encoding the variant of the present invention can be prepared by recombinant DNA technology.By obtaining nucleotide sequence information, the isolated nucleic acid molecule of the present invention can be prepared by various means.For example, the nucleic acid sequence encoding the variant can be isolated from a suitable biological source by using standard protocols well known in the art.

[0049] The nucleic acids of the invention can be maintained as RNA or DNA in any convenient cloning vector. In certain embodiments, they are maintained in a plasmid cloning / expression vector clone (e.g., pBluescript (Stratagene, La Jolla, Calif.)) which is propagated in a suitable E. coli host cell. Alternatively, the nucleic acid may 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 molecule encoding the FVIII variant of the present invention may be single-stranded or double-stranded, and includes cDNA, genomic DNA, RNA, and fragments thereof.Thus, the present invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having a sequence that can hybridize with at least one sequence of the nucleic acid molecule of the present invention.Such oligonucleotides are useful as probes for detecting the expression of the variant.

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

[0052] Larger quantities of the variants can also be produced by expression in suitable prokaryotic or eukaryotic expression systems. For example, part or all of a DNA molecule encoding the FVIII variant can be inserted into a plasmid vector adapted for expression in bacterial cells such as E. coli or mammalian cells, particularly human cells, such as CHO cells or HeLa cells. Alternatively, tagged fusion proteins containing the variants can be generated. Such variant-tagged fusion proteins are encoded by part or all of a DNA molecule, ligated in the correct codon reading frame to a nucleotide sequence encoding part or all of a desired polypeptide tag inserted into a plasmid vector adapted for expression in bacterial cells such as E. coli or eukaryotic cells, such as yeast or mammalian cells, particularly human cells. Such vectors contain regulatory elements necessary for expression of the DNA in a host cell, arranged to allow expression of the DNA in the host cell. Such regulatory elements necessary 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 (especially human) can be purified according to methods known in the art. In certain embodiments, commercially available expression / secretion systems can be used, whereby recombinant proteins are expressed and then secreted from the host cells and easily purified from the surrounding medium. If no expression / secretion vector is used, alternative approaches include purifying recombinant proteins by affinity separation, such as immunological interactions with antibodies that specifically bind to the recombinant protein or nickel columns to isolate recombinant proteins tagged with 6-8 histidine residues at the N-terminus or C-terminus. Alternative tags include, but are not limited to, FLAG epitope, GST, hemagglutinin epitope, etc. Such methods are well-known to skilled artisans.

[0054] The FVIII variant protein products prepared by the above-described methods can be analyzed according to standard procedures, for example, such proteins can be subjected to amino acid sequence analysis according to known methods.

[0055] As noted above, a convenient way of producing a polypeptide according to the invention is to express it by using a nucleic acid encoding it in an expression system. A variety of expression systems useful in the methods of the invention are well known to those skilled in the art.

[0056] Thus, the present invention also encompasses a method for producing a (disclosed) polypeptide, which comprises expression from a nucleic acid (typically a nucleic acid) encoding the polypeptide. This can be conveniently achieved by culturing a host cell containing such a vector under appropriate conditions that cause or allow production of the polypeptide. Polypeptides can also be produced in in vitro systems such as reticulocyte lysates.

[0057] Uses of FVIII variant proteins and nucleic acids encoding the variants The FVIII variant proteins and nucleic acids of the present invention can be used, for example, as therapeutic and / or prophylactic agents to regulate the blood coagulation cascade. It is shown herein that FVIII variants have excellent properties and can provide effective hemostasis.

[0058] In a particular embodiment of the invention, the FVIII variant may be administered to a patient by infusion, for example, by intravenous injection, in a biologically compatible carrier. The FVIII variant of the invention may be optionally encapsulated in liposomes or mixed with other phospholipids or micelles to enhance the stability of the molecule. The FVIII variant may be administered alone or in combination with other agents known to regulate hemostasis (e.g., vFW, factor IX, factor IXa, etc.). The appropriate composition for administering the FVIII variant can be determined by a medical practitioner, taking into account various physiological variables, including, but not limited to, the condition and hemodynamic status of the patient. Various compositions suitable for various applications and routes of administration are well known in the art and are described below.

[0059] The preparation comprising the FVIII variant may comprise a physiologically acceptable matrix and may be formulated as a pharmaceutical preparation. The preparation may be formulated using substantially known prior art methods, which may be mixed with a buffer containing salts such as NaCl, CaCl2, and amino acids such as glycine and / or lysine, with a pH ranging from 6 to 8. The purified preparation comprising the FVIII variant may be stored in the form of a finished solution or in lyophilized or deep-frozen form until required. In a particular embodiment, the preparation is stored in a lyophilized state and dissolved into a visually clear solution using a suitable reconstitution solution. Alternatively, the preparation according to the invention may be made available as a liquid preparation or as a deep-frozen liquid. The preparation according to the invention may be particularly stable, i.e., it may be left in dissolved form for a long period of time before application.

[0060] The preparations according to the invention can be used as pharmaceutical preparations with FVIII variants in the form of monocomponent preparations or in combination with other agents in the form of multicomponent preparations.

[0061] Prior to processing the purified protein into a pharmaceutical product, the purified protein can be subjected to conventional quality control and finalized to a therapeutic presentation. In particular, during recombinant production, purified preparations can be tested for the absence of cellular nucleic acid and nucleic acid derived from the expression vector.

[0062] Another feature of the present invention is that formulations are made available which contain FVIII variants with high stability and structural integrity, and in particular free of inactive FVIII intermediates and / or proteolytic products, by incorporating them into suitable formulations.

[0063] The pharmaceutical formulation may, by way of example, comprise 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 certain embodiments, the pharmaceutical protein formulation may comprise a dose of 30-100 IU / kg (e.g., as a single injection per day, or three or more injections per day). If a patient presents bleeding or with a cut or wound causing bleeding, they can be treated immediately. Alternatively, the patient may receive a bolus injection every 1-3, 8, or 12 hours, or may receive a single daily injection of the FVIII variant described herein once sufficient improvement is observed.

[0064] Nucleic acids encoding FVIII variants can be used in various applications in accordance with the present invention. In certain embodiments of the present invention, a nucleic acid delivery vehicle (e.g., an expression vector, such as a viral vector) for regulating blood clotting is provided, where the expression vector comprises a nucleic acid sequence encoding a FVIII variant as described herein. When the expression vector encoding the FVIII variant is administered to a patient, the FVIII variant is expressed and plays a role in altering the coagulation cascade. According to the present invention, the FVIII variant encoding nucleic acid sequence may encode a variant polypeptide as described herein, the expression of which increases hemostasis. In certain embodiments, the nucleic acid sequence encodes a human FVIII variant.

[0065] Expression vectors containing the nucleic acid of the FVIII variant can be administered alone or in combination with other molecules useful for regulating hemostasis. According to the invention, the expression vector or therapeutic combination may be administered to a patient alone or in a pharma- ceutically acceptable or biologically compatible composition.

[0066] In certain embodiments of the present invention, the expression vector comprising the nucleic acid sequence encoding the FVIII variant is a viral vector. The viral vectors that can be used in the present invention include, but are not limited to, adenoviral vectors (with or without tissue-specific promoters / enhancers), adeno-associated viral (AAV) vectors of multiple 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), feline immunodeficiency virus (FIV)), herpes simplex viral vectors, vaccinia viral vectors, and retroviral vectors. In certain embodiments, the vector is an adeno-associated viral (AAV) vector. In certain embodiments, the vector is a lentiviral vector.

[0067] In a particular embodiment of the present invention, a method is provided for administering a viral vector comprising a nucleic acid sequence encoding a FVIII variant.The adenoviral vector useful in the method of the present invention preferably comprises at least the essential part of adenoviral vector DNA.As described herein, the expression of FVIII variant after administration of such adenoviral vector serves to regulate hemostasis, in particular enhancing the procoagulant activity of protease.

[0068] Recombinant adenoviral vectors have found broad utility in a variety of gene therapy applications, largely due to the high efficiency of in vivo gene transfer achieved in multiple organ contexts.

[0069] Adenovirus particles can be advantageously used as vehicles for efficient gene transfer. Such virions have many desirable features for such applications, including structural features related to being double-stranded DNA non-enveloped viruses, and biological features such as tropism for the human respiratory and gastrointestinal systems. Furthermore, adenoviruses are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis. To prove the overall safety of adenovirus vectors, infection with adenoviruses produces minimal pathology in humans, including mild flu-like symptoms.

[0070] The large size of the adenovirus genome (~36 kilobases) makes it suitable for use as a gene therapy vehicle, since foreign DNA can be inserted after removal of adenoviral genes essential for replication and non-essential regions. Such replacements render the viral vector impaired in terms of replication function and infectivity. Notably, adenoviruses have been used as vectors for gene therapy and expression of heterologous genes.

[0071] For example, it may be desirable to introduce a vector that carries multiple copies of a desired gene and thus provides greater amounts of the product of that gene. Improved adenoviral vectors and methods for producing these vectors are described in detail in many publications, patents, and patent applications, such as: 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. Pat. No. 5,998,205 (providing tumor-specific replicating vectors containing multiple DNA copies); 6,228,646 (describes helper-free fully defective adenoviral vectors); 6,093,699 (providing vectors and methods for gene therapy); 6,100,242 (replication-deficient adenoviral vectors containing inserted transgenes have been successfully used for in vivo gene therapy of peripheral vascular and cardiac diseases); and International Patent Applications WO94 / 17810 and WO94 / 23744.

[0072] In some applications, the expression construct may further comprise regulatory elements that serve to drive expression in specific cell or tissue types. Such regulatory elements are known to those skilled in the art and are described in detail in Sambrook et al. (1989) and Ausubel et al. (1992). The incorporation of tissue-specific regulatory elements into the expression construct of the invention provides at least partial tissue tropism for the expression of the variant or functional fragment thereof. For example, an E1-deleted type 5 adenoviral vector that contains a nucleic acid sequence encoding the variant under the control of a cytomegalovirus (CMV) promoter can be advantageously used in the methods of the invention. Hematopoietic or liver specific promoters can also be used.

[0073] AAV for recombinant gene expression has been produced 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 usually engineered from wild-type AAV, a non-pathogenic, single-stranded DNA virus. The parent virus is non-pathogenic, and the vectors have a broad host range and can infect dividing and non-dividing cells. The vectors are usually engineered by deleting the rep and cap genes from the virus and replacing them with the transgene of interest under the control of a specific promoter. For recombinant AAV preparations, the upper limit of the size of the sequence that can be inserted between the two ITRs is approximately 4.7 kb. AAV-2 vectors can be produced using a plasmid expressing a FVIII variant under the control of the CMV promoter / enhancer and a second plasmid supplying adenovirus helper functions, as well as a third plasmid containing the AAV-2 rep and cap genes, and plasmids containing either the AAV-1, AAV-6, or AAV-8 cap genes and the AAV-2 rep gene and ITR's can be used to produce 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). AAV vectors can be purified by repeated CsCl density gradient centrifugation, and the titer of purified vector can be measured by quantitative dot blot hybridization. In a specific embodiment, vectors can be prepared at the Vector Core at The Children's Hospital of Philadelphia.

[0074] The invention also includes a method of modulating hemostasis comprising providing cells of an individual with a nucleic acid delivery vehicle encoding a FVIII variant and growing the cells under conditions in which the FVIII variant is expressed.

[0075] From the above discussion it can be seen that FVIII variants and nucleic acid vectors expressing FVIII variants can be used to treat diseases associated with abnormal blood clotting.

[0076] The expression vectors of the invention may be incorporated into pharmaceutical compositions delivered to a subject to allow production of biologically active proteins (e.g., FVIII variants), or expression of FVIII variants may be induced in vivo by gene and / or cell-based therapy, or patient or donor cells may be modified / introduced ex vivo. In certain embodiments of the invention, pharmaceutical compositions containing sufficient genetic material to allow the recipient to produce a therapeutically effective amount of FVIII variants may affect hemostasis in a subject. Alternatively, as described above, an effective amount of FVIII variants may be directly injected into a patient in need thereof. The composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound, and may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, water, and the like. The composition may be administered alone to a patient, or in combination with other agents (e.g., coenzymes) that affect hemostasis.

[0077] In certain embodiments, the pharmaceutical composition also includes a pharma- ceutically acceptable excipient / carrier. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition and that can be administered without undue toxicity. Pharmaceutically acceptable excipients include liquids such as water, saline, glycerol, sugars, ethanol, and the like. Examples include mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. In addition, such vehicles may include auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. Pharmaceutically acceptable excipients are described in detail in Remington's Pharmaceutical Sciences (Mack Pub.Co., 18th Edition, Easton, Pa.

[1990] ).

[0078] Pharmaceutical preparations suitable for parenteral administration are formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, physiologically buffered saline, etc. Aqueous injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, etc. 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 may contain suitable stabilizers or agents that increase the solubility of the compound, allowing the preparation of highly concentrated solutions.

[0079] Pharmaceutical compositions may be provided as salts, which can be formed with many acids, including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, and succinic acid. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free bases. In other cases, the formulation may be a lyophilized powder that can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, with a pH in the range of 4.5-5.5, combined with a buffer prior to use.

[0080] After the pharmaceutical composition has been prepared, it can be placed in an appropriate container and labeled for treatment. If a FVIII variant or a vector encoding a FVIII variant is administered, such labeling would include amount, frequency, and method of administration.

[0081] Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredient is contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capabilities of a skilled medical practitioner using the techniques and guidance provided in the present invention. The therapeutic dose will depend on the age and general condition of the subject, the severity of the abnormal blood clotting phenotype, the strength of the sequence controlling the expression level of the variant polypeptide, etc. Thus, the therapeutically effective amount in humans will fall within a relatively broad range that can be determined by a medical practitioner based on the response of an individual patient to variant treatment with a vector.

[0082] The FVIII variants, alone or in combination with other agents, may be directly injected into a patient in a suitable biological carrier as described herein. The expression vectors of the present invention, including nucleic acid sequences encoding the variants or functional fragments thereof, may be administered to a patient by various means (see below) to achieve and maintain prophylactically and / or therapeutically effective levels of the variant polypeptide. Those skilled in the art can easily determine the specific protocol for using the variant encoding expression vectors of the present invention for therapeutic treatment of a particular patient. Protocols for the manufacture of adenoviral vectors and administration to patients are described in U.S. Patent Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242, and International Patent Applications WO 94 / 17810 and WO 94 / 23744, the entireties of which are incorporated herein by reference.

[0083] The FVIII variant encoding adenoviral vector of the present invention can be administered to a patient by any known means. Direct in vivo delivery of the pharmaceutical composition can generally be achieved by injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery are also envisioned (see, for example, U.S. Pat. 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 administration methods include oral administration, pulmonary administration, suppository, transdermal administration, etc. Clinicians who specialize in treating patients with blood clotting 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 patient's condition and the goal of treatment (e.g., enhancing or reducing blood clotting).

[0084] The invention also encompasses AAV vectors comprising a nucleic acid sequence encoding a FVIII variant. Also provided are lentiviral or pseudotyped lentiviral vectors comprising a nucleic acid sequence encoding a FVIII variant. Also encompassed are naked plasmids and expression vectors comprising the nucleic acid sequence and the FVIII variant.

[0085] The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any way. EXAMPLES

[0086] Example 1 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). Blood was collected 24 hours later and FVIII antigen levels were measured by ELISA using Affinity Biologicals Matched Pair Antibody Set Product #F8C-EIA. As can be seen in FIG. 2, all of the human FVIII B domain variants of the present invention were expressed at higher levels in hemophilia A mice than FVIII-SQ.

[0087] Example 2 Wild-type FVIII (659K) and amino acid substitution variant products were transiently expressed in BHK cells. The expressed FVIII specific activity was determined by a one-stage aPTT assay using conditioned expression medium. As seen in Figure 3, most of the FVIII variants showed increased specific activity compared to wild-type FVIII.

[0088] Example 3 In addition to the FVIII variants with amino acid substitutions at position 659, FVIII variants with amino acid 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 using conditioned expression medium. As seen in FIG. 5, the variants with amino acid substitutions at each position showed enhanced specific activity compared to wild-type FVIII. Combining these substitutions with those provided in Example 2 can produce FVIII variants with even higher specific activity.

[0089] While some preferred embodiments of the present invention have been described and specifically exemplified, it is not intended that the invention be limited to such embodiments, and various modifications may be made thereto without departing from the scope and spirit of the invention, as set forth in the following claims.

Claims

1. 1. A Factor VIII (FVIII) variant comprising amino acids 1-740 and 1649-2332 of SEQ ID NO:1, further comprising a substitution mutation of Lys at position 659, wherein Lys at position 659 is substituted with Asp, Thr, Ser, Val, Phe, or Cys.

2. 2. The FVIII variant of claim 1, wherein the variant consists of amino acids 1-740 and 1649-2332 of SEQ ID NO:1 and has a substitution mutation of Lys at position 659, where Lys at position 659 is substituted with Asp, Thr, Ser, Val, Phe, or Cys.

3. A factor VIII (FVIII) variant comprising amino acids 1 to 740 and 1649 to 2332 of SEQ ID NO:1, and further comprising a substitution mutation of Lys at position 659, wherein Lys at position 659 is replaced with Met.

4. 3. The FVIII variant of claim 1, wherein Lys at position 659 is substituted with Ser or Cys.

5. 3. The FVIII variant of claim 1, wherein Lys at position 659 is substituted with Cys.

6. 3. The FVIII variant of claim 1 or 2, wherein Lys at position 659 is substituted with Ser, Val, Phe, or Cys.

7. 3. The FVIII variant of claim 1, wherein Lys at position 659 is substituted with Val.

8. The FVIII variant according to any one of claims 1 to 7, wherein the FVIII variant has a higher specific activity than wild-type FVIII.

9. 9. The FVIII variant according to any one of claims 1 to 8, wherein amino acids 1 to 740 and 1649 to 2332 of SEQ ID NO:1 are linked by 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.

10. 9. The FVIII variant according to any one of claims 1 to 8, wherein amino acids 1 to 740 and 1649 to 2332 of SEQ ID NO:1 are linked by an amino acid sequence selected from the group 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.

11. The FVIII variant of claim 10, wherein amino acids 1 to 740 and 1649 to 2332 of SEQ ID NO:1 are linked by the amino acid sequence of SEQ ID NO:

13.

12. A composition comprising at least one FVIII variant according to any one of claims 1 to 11 and at least one pharma- ceutically acceptable carrier.

13. The composition according to claim 12 for the treatment of hemostasis-related disorders.

14. The composition of claim 13, wherein the hemostasis-related disorder is hemophilia A.

15. An isolated nucleic acid molecule encoding a FVIII variant according to any one of claims 1 to 11.

16. The nucleic acid molecule of claim 15 , wherein the FVIII variant comprises a signal peptide.

17. 17. An expression vector comprising the nucleic acid molecule of claim 15 or 16 operably linked to a regulatory sequence.

18. 18. The vector of claim 17, which is selected from the group consisting of an adenoviral vector, an adenovirus-associated vector, a retroviral vector, a plasmid, and a lentiviral vector.

19. A host cell comprising the vector of claim 18.

20. The host cell of claim 19 , wherein the host cell is a human cell.

21. The vector according to claim 17 for the treatment of a hemostasis-related disorder.

22. Activated form of the FVIII variant according to any one of claims 1 to 11.

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