Compositions and methods for modulating factor VIII function

Abstract

Factor VIII variants and methods of use thereof are disclosed. In accordance with the present invention, compositions and methods are provided for modulating hemostasis in patients in need thereof. More specifically, factor VIII (FVIII) variants that modulate (e.g., increase) hemostasis are provided. In certain embodiments, the FVIII variants contain mutations at least at positions 336 and / or 562.

Description

[Technical Field]

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 944,718, filed on 6 December 2019. The aforementioned application is incorporated herein by reference.

[0002] This invention was made with government support under grant number NHLBI K08HL146991-01, awarded by the National Institutes of Health. The U.S. Government reserves certain rights in this invention. [Technical field]

[0003] This invention relates to the fields of medicine and hematology. More specifically, the invention provides a novel factor VIII variant and a method for using it to modulate the coagulation cascade in patients who require it. [Background technology]

[0004] To illustrate the state of the art to which this invention relates, several publications and patent documents are referenced throughout this specification. All of these referenced documents are incorporated herein by reference.

[0005] Coagulation factor VIII (FVIII) circulates in the bloodstream by strongly binding to its carrier protein, von Willebrand factor (vWF) (Eaton, et al. (1986) Biochemistry 25(2):505-512; Vehar, et al. (1984) Nature 312(5992):337-342; Lollar, et al. (1988) J. Biol. Chem., 263(21):10451-10455). Through proteolytic treatment with thrombin, FVIII is released from vWF and generates the active coenzyme species (FVIIIa). FVIIIa is a heterotrimer consisting of a metal ion-stabilized A1 / A3-C1-C2 heterodimer and a weakly bound A2 domain (Vehar, et al. (1984) Nature (1984) 312(5992):337-342; Fay, et al. (1992) J. Biol.Chem., 267(19):13246-13250). Factor VIIIa associates with activated FIX (FIXa) on the surface of anionic phospholipids, forming an endogenous Xase enzyme complex, one of the two enzymes that activate FX (Eaton, et al. (1986) Biochemistry 25(2):505-512; Hill-Eubanks, et al. (1990) J. Biol.Chem., 265(29):17854-17858; Lenting, et al. (1994) J. Biol.Chem., 269(10):7150-7155; Venkateswarlu, D. (2014) Biochem.Biophys.Res.Comm., 452(3):408-414; Kolkman, et al. (1999) Biochem J., 339(Pt 2):217-221; Fay, et al. (1998) J. Biol. Chem., 273(30):19049-19054; Kolkman, et al. (1999) 274(41):29087-29093; Kolkman, et al. (2000) Biochemistry 39(25):7398-7405).The importance of the function of FVIIIa coenzyme has been highlighted, as a deficiency or dysfunction of FVIII leads to hemophilia A (HA). Downregulation of endogenous Xase function is achieved through inhibition of FIXa by antithrombin and possibly protein S (PS), and inactivation of FVIIIa by spontaneous dissociation of the A2 domain or proteolytic cleavage at Arg336 and Arg562 by activated protein C (APC) (Lollar, et al. (1991) J. Biol.Chem., 266(19):12481-12486; Hultin, et al. (1981) Blood 57(3):476-482; Lollar, et al. (1984) Blood 63(6):1303-1308; Lollar, et al. (1990) J. Biol.Chem., 265(3):1688-1692; Walker, et al. (1987) Arch.Biochem.Biophys., 252(1):322-328; Plautz, et al. (2018) Arterioscler.Thromb.Vasc.Biol., 38(4):816-828; Fay, et al. (1991) J. Biol.Chem., 266(30):20139-20145). FVIIIa has a very large effect in increasing FIXa function (10. 3 ~10 6 Since it has a multiplier, its inactivation is important for regulating endogenous Xase function (van Dieijen, et al. (1981) J. Biol.Chem., 256(7):3433-3442; Mertens, et al. (1984) Biochem.J., 223(3):599-605).

[0006] Following activation by thrombin, FVIIIa loses its activity within minutes due to spontaneous dissociation of the A2 domain (Lollar, et al.(1991) J. Biol.Chem., 266(19):12481-12486; Hultin, et al.(1981) Blood 57(3):476-482; Lollar, et al.(1984) Blood 63(6):1303-1308; Lollar, et al.(1990) J. Biol.Chem., 265(3):1688-1692; Lu, et al.(1996) Blood 87(11):4708-4717; Fay, et al.(1991) J. Biol.Chem., 266(14):8957-8962). The physiological relevance of this mechanism is exemplified by numerous mild HA mutations that reduce A2 affinity in FVIIIa heterotrimers (McGinniss, et al.(1993) Genomics 15(2):392-398; Duncan, et al.(1994) Br.J. Haematol., 87(4):846-848; Rudzki, et al.(1996) Br.J. Haematol., 94(2):400-406; Hakeos, et al.(2002) Thromb.Haemost., 88(5):781-787; Pipe, et al.(2001) Blood 97(3):685-691; Pipe, et al.(1999) Blood 93(1):176-183). The presumed importance of A2 domain dissociation in regulating FVIIIa function has been demonstrated by the successful bioengineering of variants with enhanced interdomain interactions that confer improved hemostatic function (Leong, et al. (2015) Blood 125(2):392-398; Wakabayashi, et al. (2008) Blood 112(7):2761-2769; Gale, et al. (2003) J. Thromb.Haemostasis 1(9):1966-1971; Gale, et al. (2008) J. Biol.Chem., 283(24):16355-16362).Integrating biochemical, clinical, and in vivo data supports the idea that A2 domain dissociation is a crucial mechanism for regulating FVIIIa function. On the other hand, previous biochemical studies have shown that APC-mediated inactivation of FVIIIa takes several hours (Fay, et al. (1991) J. Biol.Chem., 266(30):20139-20145; Lu, et al. (1996) Blood 87(11):4708-4717). Since A2 dissociation occurs faster than APC cleavage, the former is considered to be the main mechanism of FVIIIa inactivation (Lollar, et al.(1991) J. Biol.Chem., 266(19):12481-12486; Hultin, et al.(1981) Blood 57(3):476-482; Lollar, et al.(1984) Blood 63(6):1303-1308; Lollar, et al.(1990) J. Biol.Chem., 265(3):1688-1692; Lu, et al.(1996) Blood 87(11):4708-4717; Fay, et al.(1991) J. Biol.Chem., 266(14):8957-8962). Consistent with this understanding, no clinical phenotypes associated with changes in APC cleavage in FVIII / FVIIIa have been described (Bezemer, et al. (2008) JAMA 299(11):1306-1314; EAHAD F8 Gene Variant Database).This is in contrast to FV, which is similar to FVIII, and APC resistance (FV-Leiden, Arg506Gln) confers a 50-100 times and 5-10 times risk of venous thrombosis in homozygous and heterozygous states, respectively, and is the most common hereditary thrombosis (Bertina, et al. (1994) Nature 369(6475):64-67; Zoller, et al. (1994) Lancet 343(8912):1536-1538; Zoller, et al. (1994) J. Clin.Invest., 94(6):2521-2524; Juul, et al. (2002) Blood 100(1):3-10; Suzuki, et al. (1983) J. Biol.Chem., 258:1914-1920).

[0007] As explained above, mutations in factor VIII (FVIII) cause severe bleeding disorders and are associated with hemophilia A. If FVIII is deficient or its activity is insufficient, effective thrombus formation is impossible. Currently, FVIII replacement therapy is expensive, and only 20% of hemophilia A patients worldwide receive regular treatment. Generally, FVIII is produced from plasma or recombinantly. Gene therapy for hemophilia A using AAV vectors is promising, but there are safety limitations due to abnormal immune responses to the vectors. Therefore, generating enhanced FVIII molecules is considered beneficial for treating hemophilia. Thus, there is a clear need for FVIII molecules with improved biological properties. [Overview of the project]

[0008] The present invention provides compositions and methods for modulating hemostasis in patients requiring it. More specifically, factor VIII (FVIII) variants that modulate (e.g., increase) hemostasis are provided. In certain embodiments, the factor VIII variant comprises at least one mutation at positions 336 and / or 562. In certain embodiments, Arg at positions 336 and / or 562 is replaced with Gln. Compositions comprising at least one FVIII variant of the present invention and at least one pharmaceutically acceptable carrier are also provided. Nucleic acid molecules encoding the FVIII variant of the present invention are also disclosed, as well as methods for their use. Another aspect of the present invention comprises host cells expressing the FVIII variant described herein. Methods for isolating and purifying the FVIII variant are also disclosed.

[0009] The present invention also provides pharmaceutical compositions containing the FVIII variant and / or a nucleic acid molecule encoding the FVIII variant on a carrier. The present invention also includes a method for treating hemostatic disorders in patients in need, comprising administration of a therapeutically effective amount of the FVIII variant and / or a nucleic acid molecule encoding the FVIII variant, particularly by a pharmaceutical composition. Such methods are effective in treating diseases requiring coagulation accelerators, including, but not limited to, hemophilia, particularly hemophilia A. [Brief explanation of the drawing]

[0010] [Figure 1A] Figure 1A shows the amino acid sequence of FVIII (SEQ ID NO: 1). Amino acids at positions 336 and 562 are shown in bold and underlined. The B domain is also shown in italics and bold. Arginine at positions 372, 740, and 1689, which are thrombin cleavage sites, are shown in italics and underlined. The provided amino acid sequence lacks the N-terminal 19-amino acid signal peptide (MQIELSTCFFLCLLRFCFS (SEQ ID NO: 2)).

[0011] [Figure 1B]Figure 1B is a schematic diagram of the FVIII domain structure, showing the thrombin and APC cleavage sites.

[0012] [Figure 2A] Figure 2A provides SDS-PAGE analysis of 1.5 μM FVIII-WT and FVIII-QQ before and after 20-minute incubation with 10 nM thrombin. The gel was stained with Coomassie Blue. SC is single chain, HC is heavy chain, and LC is light chain.

[0013] [Figure 2B] Figure 2B shows representative traces of thrombin generation in HA human plasma reconstituted with various concentrations of FVIII-WT (solid line) or FVIII-QQ (dashed line), initiated with 1 pM FXIa in the presence of 4 μM PCPS and 7.5 mM CaCl2.

[0014] [Figure 2C] Figure 2C shows the decrease in FVIIIa activity due to dissociation of the A2 domain measured in the intrinsic Xase assay. 5 nM FVIIIa-WT (squares) or FVIIIa-QQ (triangles) were incubated with 100 nM thrombin for 30 seconds, and the residual activity of FVIIIa was evaluated in a 15-minute incubation. The data shown are representative of three independent experiments.

[0015] [Figure 3A] Figure 3A provides Western blot analysis of 10 nM FVIIIWT and FVIII-QQ after incubation with 6 nM APC, 20 μM PCPS, and 6 nM hirudin for 30 minutes. The FVIII fragments were visualized with an anti-A2 antibody (GMA-012).

[0016] [Figure 3B]Figure 3B provides Western blot analysis of 10 nM FVIII-WT, FVIII-QQ, and FVIII-R372Q after 30-minute incubation with 6 nM APC, 20 μM PCPS, and 6 nM hirudin. 30 ng of purified protein was loaded onto the gel and the FVIII fragment was visualized with GMA-012.

[0017] [Figure 3C] Figure 3C shows a graph of the inactivation of 10 nM FVIII-WT (filled squares) and FVIII-QQ (filled triangles) by 6 nM APC in the presence of 20 μM PCPS and 6 nM hirudin in a purified intrinsic Xase assay, compared to the inactivation of 10 nM FVIII-WT (open squares) and FVIII-QQ (open triangles) by 6 nM APC containing 100 nM PS in the presence of 20 μM PCPS and 6 nM hirudin. The initial rate of FXa generation during incubation was compared to the 0-minute time point and the residual FVIII activity was measured. Representative plots of duplicate experiments are plotted. The data was fit to an exponential decay or linear regression (FVIII-QQ with only APC).

[0018] [Figure 3D] Figure 3D provides the results of Western blot analysis of 10 nM FVIII-WT, FVIII-QQ, and FVIII-R372Q, 20 μM PCPS, and 6 nM hirudin after 2- and 10-minute incubation with either 100 nM PS or 6 nM APC or 6 nM APC and 100 nM PS. 20 ng of purified protein was loaded onto the gel and the FVIII fragment was visualized with GMA-012. FVIII-R372Q is resistant to cleavage at Arg372.

[0019] [Figure 3E] Figure 3E provides a Western blot of the time course of cleavage of WT FVIII and FVIII-QQ by APC.

[0020] [Figure 4A-4D]Figures 4A-4D show the effect of APCs (Amphibole-Protein Compounds) of FVIII-WT / FVIIIa-WT versus FVIII-QQ / FVIIIa-QQ on thrombin generation in reconstituted HA human and mouse plasma. Thrombin generation was evaluated in HA plasma reconstituted with FVIII in 4 μM PCPS and 7.5 mM CaCl2, while increasing the APC concentration. Figure 4A: HA human plasma was reconstituted with 1 nM FVIII-WT (square) or FVIII-QQ (triangle), and thrombin generation was initiated at 1 pM FXIa. Figure 4B: 1.5 nM FVIII was activated with 30 nM thrombin for 30 seconds and quenched with 60 nM hirudin. HA human plasma was reconstituted with 0.2 nM FVIIIa-WT or FVIIIa-QQ. Thrombin generation was initiated at 10 pM FXIa. Figure 4C: HA mouse plasma was reconstituted with 1 nM FVIII-WT (square) or FVIII-QQ (triangle), and thrombin generation was initiated with 30 pM FXIa. Figure 4D: 1.5 nM FVIII was activated with 30 nM thrombin for 30 seconds and quenched with 60 nM hirudin. HA mouse plasma was reconstituted with 0.2 nM FVIIIa-WT or FVIIIa-QQ. Thrombin generation was initiated with 400 pM FXIa. In both panels, residual peak thrombin shows the peak thrombin relative to the 0 nM APC condition. Mean ± SEM of four independent experiments are plotted. Figure 4E shows the effect of sTM of FVIII-WT vs. FVIII-QQ on thrombin generation in HA human plasma. Thrombin generation was assessed in HA human plasma reconstituted with 1 nM FVIII-WT (square) or FVIII-QQ (triangle) by increasing the sTM concentration in the presence of 4 μM PCPS, 0.1 pM FXIa, and 7.5 mM CaCl2. Residual peak thrombin represents the peak thrombin compared to the 0 nM sTM condition. The mean ± SEM of four independent experiments is plotted. Peak thrombin values ​​(nM) under the 0 nM sTM condition: FVIII-WT: 533.65 ± 4.69, FVIII-QQ: 561.85 ± 6.10; lag time (mins): FIII-WT: 14.5 ± 1.5, FVIII-QQ: 14.0 ± 1.0.

[0021] [Figures 5A-5D] Figures 5A–5D show that FVIII-QQ exhibits superior in vivo hemostatic or thrombus-forming ability compared to FVIII-WT in HA mice. HA mice were injected with PBS (diamond shape), or with or without 10 mg / kg mAb1609 as shown, or with or without 10 mg / kg mAb1609, before being injured by a tail clip (Figure 5A) or 7.5% FeCl3 injury (Figure 5C). WT mice injected with PBS (black circles) are hemostatically normal controls. Each point represents one mouse, and the median and interquartile range are shown. Kruskal-Wallis tests were used for comparison with WT PBS controls, with a p-value ≤0.1 being statistically significant (p ≤0.1 = *, p ≤0.05 = **, p ≤0.01 = ***). Dose-dependent vascular occlusion of FVIII-WT and FVIII-QQ was determined by empirically fitting data from tail clipping (Figure 5B) and 7.5% FeCl3 injury (Figure 5D) to a logistic function (solid line). The dots represent the median, and the error bars represent the IQR. EC50 and EC80 values ​​were obtained from logistic fitting. The dotted line shows the median of the normal control. ns are not statistically significant.

[0022] [Figure 5E] Figure 5E shows the half-life studies of FVIII-WT and FVIII-QQ in HA mice. FVIII activity was measured at specified time points after administering 125 IU / kg of FVIII-WT or FVIII-QQ to HA mice. Each point represents three individual mice, and the mean and its standard error are plotted. Half-life values ​​were calculated by fitting the data to an exponential decay curve.

[0023] [Figure 6A-6B]Figures 6A-6B show the effect of APCs (Aquatic Protein Concentrates) of FVIII-WT / FVIIIa-WT versus FVIII-QQ / FVIIIa-QQ on thrombin generation in reconstituted HA / FVL mouse plasma. Thrombin generation was evaluated by adding 4 μM PCPS and 7.5 mM CaCl2 to HA / FVL mouse plasma reconstituted with FVIII, increasing the APC concentration. Figure 6A: HA / FVL plasma was reconstituted with 1 nM FVIII-WT (square) or FVIII-QQ (triangle), and thrombin generation was initiated with 30 nM FXIa. Figure 6B: 10 nM FVIII was activated with thrombin (30 nM) for 30 seconds and quenched with 60 nM hirudin. HA / FVL mouse plasma was reconstituted with 0.2 nM FVIIIa-WT or FVIIIa-QQ. Thrombin generation was initiated with 400 pM FXIa. In both panels, residual peak thrombin represents the peak thrombin relative to the 0 nM APC condition. The mean ± SEM of four independent experiments is plotted.

[0024] [Figure 7] Figure 7 shows that the enhancement of the hemostatic effect of FVIII-QQ compared to FVIII-WT is APC-dependent. HA / FVL mice were injected with PBS (diamond shape), FVIII-WT (square shape), and FVIII-QQ (triangle shape) at 2 μg / kg, with or without 10 mg / kg of mAPC anticoagulant antibody (mAb1609), as described, and tail clip injuries were performed. Each point represents one mouse, and the median and IQR are shown. The Kruskal-Wallis test was used to determine relative significance with respect to the FVL PBS control, with a p-value ≤0.1 being considered significant (p ≤0.1 = *, p ≤0.05 = **, p ≤0.01 = ***).

[0025] [Figure 8A] Figure 8A provides graphs of thrombin production in HA / FVL plasma as the concentrations of APC and WT FVIII, FVIII-QQ, FVIII-R336Q, and FVIII-R562Q are increased.

[0026] [Figure 8B] Figure 8B provides a graph of blood loss in HA / FVL mice after a tail clip assay. Control FVL mice are also shown. Mice were treated with PBS or WT FVIII, FVIII-QQ, FVIII-R336Q, and FVIII-R562Q. Detailed description of the invention

[0027] Hemophilia A (HA) and hemophilia B (HB) are X-linked hemorrhagic disorders caused by a genetic deficiency in 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)). The bleeding phenotype is generally related to residual factor activity; those with severe hemophilia (factor activity less than 1%) frequently experience spontaneous bleeding, those with moderate hemophilia (factor activity between 1% and 5%) rarely experience spontaneous bleeding but bleed with minor trauma, and those with mild hemophilia (factor activity between 5% and 40%) bleed with invasive procedures or trauma. Given the clear relationship between factor activity and bleeding phenotype, HA and HB, where even slight increases in factor levels are expected to have clinically significant effects, are attractive targets for protein infusion and gene therapy.

[0028] As described above, factor VIII is central to coagulation activity, and mutations in the FVIII gene cause hemophilia A, the most common type of hemophilia. This specification has shown that specific changes in the amino acid sequence of FVIII are associated with increased resistance of the protein to inactivation by proteolysis. Therefore, the present invention provides rationally designed amino acid residue modifications that offer superior variants.

[0029] 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 circulates mainly as a heavy-light chain heterodimer linked by non-covalent metal-dependent interactions (Lenting, et al., Blood (1998) 92:3983-3996). Factor VIII is composed of multiple domains and has a full-length total of 2332 amino acids (mature form without signal peptides). Generally, the domains are named A1-A2-B-A3-C1-C2, etc. FVIII is translated as a single peptide chain (monochain) with the domain structure A1-α1-A2-α2-B-α3-A3-C1-C2. FVIII is proteolytically cleaved by the trans-Golgi protease furin at R-1313 and / or R-1648, forming a heterodimer. The FVIII heavy chain (A1-α1-A2-α2-B) and light chain (α3-A3-C1-C2) remain bound by a non-covalent metal-ion-dependent interaction between the A1 and A3 domains. FVIII is initially inactive, bound to vWF (von Willebrand factor). FVIII is activated by thrombin (factor IIa) cleavage and release of the B domain. Activated FVIII (FVIIIa) separates from vWF and interacts with coagulation factor IXa, forming a thrombus via the coagulation cascade. During the coagulation process, FVIII single chains or heterodimers are activated into heterotrimeric coenzyme forms by thrombin-mediated cleavage at R-372, R-740, and R-1689. A2 remains noncovalently bound to A1-α1. Inactivation of FVIIIa occurs through spontaneous A2 dissociation and / or proteolysis at R-336 and R-562, primarily by activated protein C.

[0030] The B domain accounts for 40% (908 amino acids) of the protein and is not necessary for the protein's coagulation-promoting activity (Brinkhous, et al., Proc.Natl.Acad.Sci.(1985) 82:8752-8756). The most common B-domain deletion (BDD) FVIII consists of 14 original amino acid residues (SFSQNPPVLKRHQR (SEQ ID NO: 3)) as a linker (Lind, et al.(1995) Eur.J. Biochem., 232(1):19-27). This BDD FVIII is usually called BDD-SQ or hFVIII-SQ. Short peptide linkers substituted in the B domain (e.g., 25 or fewer amino acids, 20 or fewer amino acids, 15 or fewer amino acids, or 10 or fewer amino acids) can be used in the FVIII variant (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). In certain embodiments, the peptide linker contains basic amino acids (e.g., Arg, His, or Lys) at positions -1 and -4 relative to Glu1649. This BDD FVIII form is commonly used not only for gene therapy but also for producing recombinant BDD-FVIII (~4.4Kb) (Berntorp, E., Semin.Hematol.(2001) 38(2 Suppl 4):1-3; Gouw, et al., N. Engl.J. Med.(2013) 368:231-239; Xi, et al., J. Thromb.Haemost.(2013) 11:1655-1662; Recht, et al., Haemophilia (2009) 15:869-880; Sabatino, et al., Mol.Ther.(2011) 19:442-449; Scallan, et al., Blood (2003) 102:2031-2037).As described above, gene therapy using AAV vectors is limited to truncated FVIII molecules such as BDD-FVIII due to the AAV packaging capacity (4.7 Kb) and other vector system limitations (Lind, et al. (1995) Eur.J. Biochem., 232(1):19-27). U.S. Patent 8,816,054, incorporated herein by reference, also provides BDD FVIII molecules having linkers of different lengths and sequences.

[0031] FVIIIa functions as a cofactor of FXIa in the endogenous Xase complex, generating FXa and advancing the coagulation cascade. Inactivation of FVIIIa is thought to be primarily involved in the downregulation of endogenous Xase. FVIIIa inactivation is due to 1) spontaneous A2 dissociation and 2) proteolytic cleavage of activated protein C (APC) (e.g., cleavage of A2 into A2N and A2C). Biochemical and clinical data support the importance of A2 dissociation. In fact, 90% of FVIIIa activity is lost after 5 minutes in the purification system (Lollar, et al. (1991) J. Biol. Chem., 266:12481-12486). Furthermore, clinical data have shown that one-third of patients with mild hemophilia have mutations that enhance A2 dissociation. Regarding cleavage, APC cleavage results in the loss of 90% of FVIII activity after 4 hours in the purification system (Lu et al. (1996) Blood 87(11):4708-17). However, unlike changes in A2 dissociation, there are no known clinical phenotypes associated with changes in APC cleavage.

[0032] While there is no data to identify the crucial role of APC in the functional regulation of FVIIIa, the lack of clinical phenotypes does not negate the importance of APC-mediated cleavage in FVIIIa inactivation. Furthermore, attempting to derive physiological significance from FVIII A2 domain dissociation or APC inactivation solely from in vitro inactivation rates should be approached with caution. To study these mechanisms, numerous experimental conditions (many of which are non-physiological) have been used, complicating interpretation and recognition of their significance. Surprisingly, despite decades of FVIII research, the role of APC in the regulation of FVIIIa in vivo has not been investigated.

[0033] To investigate the contribution of APC cleavage to FVIIIa inactivation, Gln missense mutations were introduced into the known FVIII APC cleavage sites, Arg336 and Arg562, to create an APC-resistant FVIII variant (FVIII-R336Q / R562Q [FVIII-QQ]). Similar to the presence of APC, which plays a crucial role in FVIIIa regulation in vivo, FVIII-QQ showed superior hemostatic efficacy compared to wild-type FVIII in an APC-dependent manner.

[0034] Novel factor VIII variants are provided according to the present invention. The present invention encompasses FVIII variants, including FVIIIa variants and variants of the FVIII prepeptide. For simplicity, throughout this application, variants are generally described in the context of FVIII. However, the present invention intends to encompass factor FVIIIa and FVIII prepeptide molecules, as well as factor VIII domains (e.g., A1 and / or A2 domains) having the same amino acid substitutions and / or linkers as described in FVIII. In certain embodiments, the FVIII variants of the present invention are expressed as single-chain molecules, or at least nearly single-chain molecules. In certain embodiments, the FVIII variant is B-domain deletion (BDD) FVIII (optionally including a linker instead of a B-domain). In certain embodiments, the FVIII variant includes A1-α1-A2-α2-B-α3-A3-C1-C2. In certain embodiments, the FVIII variant includes A1-α1-A2-α2-α3-A3-C1-C2. In certain embodiments, the FVIII variant includes A1-α1-A2-α2-A3-C1-C2. In certain embodiments, the FVIII variant includes light and heavy chains (e.g., as single-chain molecules).

[0035] As shown herein, the FVIII variant of the present invention exhibits greater resistance to APC cleavage than WT FVIII. Furthermore, it is demonstrated herein that the FVIII variant of the present invention has unexpectedly superior hemostatic efficacy compared to WT FVIII. Conventionally, APC-resistant FVIII was thought to have in vivo function nearly equivalent to WT FVIII, which is because, as mentioned above, the main mechanism of FVIIIa inactivation is thought to be A2 dissociation. As shown herein, the FVIII variant of the present invention has the same activity properties as WT FVIII, but unexpectedly demonstrated ~5 times superior in vivo hemostatic function compared to the wild-type protein.

[0036] That is, the FVIII variant of the present invention can originate from any mammalian species. In certain embodiments, the FVIII variant is human. Gene ID 2157, 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 prepeptide constituting the signal peptide). Figure 1 shows SEQ ID NO: 1, an example of the amino acid sequence of human FVIII. SEQ ID NO: 1 is missing a 19-amino acid signal peptide (MQIELSTCFFLCLLRFCFS (SEQ ID NO: 2)) at its N-terminus. Nucleic acid molecules encoding variants of the FVIII factor can be readily determined from the provided amino acid sequences and provided GenBank Accession Nos.

[0037] According to another aspect of the present invention, the factor VIII variant includes at least one mutation at positions 336 and / or 562. Thus, these FVIII variants have greater resistance to cleavage by APC than wild-type FVIII. In certain embodiments, the factor VIII variant includes a mutation at position 336. In certain embodiments, Arg(R) at position 336 is not substituted with Lys(K). In certain embodiments, Arg at position 336 is substituted with Asp(D), Glu(E), Asn(N), or Gln(Q). In certain embodiments, Arg at position 336 is substituted with Asn(N) or Gln(Q). In certain embodiments, Arg at position 336 is substituted with Gln(Q).

[0038] In certain embodiments, the factor VIII variant includes a mutation at position 562. In certain embodiments, Arg(R) at position 562 is not substituted with Lys(K). In certain embodiments, Arg at position 562 is substituted with Asp(D), Glu(E), Asn(N), or Gln(Q). In certain embodiments, Arg at position 562 is substituted with Asn(N) or Gln(Q). In certain embodiments, Arg at position 562 is substituted with Gln(Q).

[0039] As described herein, the FVIII variant of the present invention may be human. In certain embodiments, 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 fragment or domain or its activated FVIII fragment), and in particular, at least 90%, 95%, 97%, 99%, or 100% homology (identity). In certain embodiments, the FVIII variant includes 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 fragment or domain, or its activated FVIII fragment), particularly 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 1649-2332 or 1690-2332 of SEQ ID NO: 1 (or its fragment or domain, or its activated FVIII fragment). The above percentages of homology (identity) exclude substitutions at positions 336 and / or 562.

[0040] The FVIII variant of the present invention may also be post-translationally modified. The FVIII variant may be post-translationally modified in cells (particularly human cells) or in vitro.

[0041] In certain embodiments, the FVIII variant of the present invention exhibits increased resistance to cleavage and / or inactivation (e.g., by APC) compared to wild-type FVIII.

[0042] Nucleic acid molecules encoding the above-mentioned FVIII variant (or its fragment or domain, or activation fragment) are also included in the present invention. Nucleic acid molecules encoding the variant can be prepared by any method known in the art. The nucleic acid molecules may be held in any convenient vector, in particular an expression vector.

[0043] Compositions comprising at least one FVIII variant and at least one carrier (e.g., a pharmaceutically acceptable carrier) are also encompassed by the present invention. In certain embodiments, the FVIII is isolated and / or substantially pure within the composition. Compositions comprising at least one FVIII variant nucleic acid molecule and at least one carrier are also encompassed by the present invention. Use in a pharmaceutical composition is intended unless any conventional carrier is incompatible with the variant to be administered. In certain embodiments, the carrier is a pharmaceutically acceptable carrier for intravenous administration.

[0044] definition Various terms relating to the biomolecules of the present invention are used throughout the above and the specification and claims of this invention.

[0045] "Hemostasis-related disorders" include, but are not limited to, hemophilia A, hemophilia B, patients with hemophilia A and B, hemophilia due to inhibitory antibodies, deficiencies of at least one coagulation factor (e.g., factors VII, VIII, IX, X, XI, V, XII, II, and / or von Willebrand factor, especially factor VIII), combined FV / FVIII deficiency, vitamin K epoxide reductase C1 deficiency, gamma carboxylase deficiency, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulation disorders (hypocortic), disseminated intravascular coagulation (DIC), hyperanticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin or low molecular weight antithrombotic agents (e.g., FXa inhibitors); and hemorrhagic disorders such as Bernard-Soulier syndrome, Granzman thrombosis, and platelet disorders such as storage pool deficiency. In certain embodiments, the term “hemostatic disorder” refers to a hemorrhagic disorder characterized by excessive and / or uncontrolled bleeding (e.g., a disorder treatable with coagulation accelerators). In certain embodiments, the hemostatic disorder is hemophilia. In certain embodiments, the hemostatic disorder is hemophilia A.

[0046] 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 has been separated from its directly adjacent sequences (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates. For example, “isolated nucleic acid” may include DNA or cDNA molecules inserted into vectors such as plasmids or viral vectors, or those incorporated into prokaryotic or eukaryotic DNA. With respect to RNA molecules of the present invention, the term “isolated nucleic acid” primarily refers to RNA molecules encoded by the isolated DNA molecules defined above. Alternatively, it may refer to RNA molecules that are sufficiently separated from the RNA molecules bound in their natural state (i.e., within cells or tissues) and exist in a “substantially pure” form.

[0047] With respect to proteins, the term “isolated protein” may be used herein. This may refer to a protein produced by the expression of an isolated nucleic acid molecule of the present invention. Alternatively, this term may refer to a protein that is sufficiently isolated from other naturally binding proteins (e.g., to exist in a “substantially pure” form). “Isolated” does not mean an artificial or synthetic mixture with other compounds or materials, or the exclusion of impurities that may be present, for example, by incomplete purification or the addition of stabilizers, without interfering with the basic activity.

[0048] 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 into which it can be expressed and / or replicated. An "expression vector" is a special type of vector containing a gene or nucleic acid sequence that has a regulatory region (such as a promoter) necessary for expression in a host cell.

[0049] The term "operably linked" means that regulatory sequences essential for the expression of the coding sequence are positioned within the DNA molecule at the appropriate location relative to the coding sequence, thereby resulting in the expression of the coding sequence. This same definition may apply to the arrangement of coding sequences and transcriptional regulatory elements (e.g., promoters, enhancers, and termination elements) in expression vectors. Furthermore, this definition may also apply to the arrangement of nucleic acid sequences in a first and second nucleic acid molecule when generating a hybrid nucleic acid molecule.

[0050] The term "substantially pure" means a preparation containing at least 50–60% by weight of the compound of interest (e.g., nucleic acids, oligonucleotides, proteins, etc.), and especially at least 75% by weight, or at least 90–99% by weight or more of the compound of interest. Purity can be measured by a method suitable for the compound of interest (e.g., chromatography, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0051] "Pharmacologically acceptable" means approval by a federal or state regulatory authority for use in animals, or more specifically, in humans, or approval listed in the United States Pharmacopeia or other generally accepted pharmacopoeias.

[0052] A "carrier" is, for example, a diluent, adjuvant, preservative (e.g., thimerosal, benzyl alcohol), antioxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., tris hydrochloride, acetate, phosphate), antimicrobial agent, bulking agent (e.g., lactose, mannitol), excipient, adjuvant, or vehicle on which the active agent of the present invention is administered. Pharmaceutically acceptable carriers may be sterile solutions such as water and oil, including those of petroleum, animal, plant, or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are particularly preferred as carriers for injectable solutions. Suitable drug carriers are described in E.W. Martin, "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.

[0053] Preparation of nucleic acid molecules and polypeptides encoding variants The nucleic acid molecules encoding the variant of the present invention can be prepared using recombinant DNA technology. By utilizing nucleotide sequence information, the isolated nucleic acid molecules 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 using standard protocols well known in the art.

[0054] The nucleic acids of the present invention can be retained as RNA or DNA in any convenient cloning vector. In certain embodiments, the clones are maintained in a plasmid cloning / expression vector (e.g., pBluescript (Stratagene, La Jolla, CA)), which is grown in a suitable E. coli host cell. Alternatively, the nucleic acids may be retained in a vector suitable for expression in mammalian cells. If post-translational modifications affect variant function, it is preferable to express the molecule in mammalian cells, particularly human cells.

[0055] The nucleic acid molecules encoding the FVIII variant of the present invention include cDNA, genomic DNA, RNA, and fragments thereof, which may be single-stranded or double-stranded. Accordingly, the present invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences that can hybridize with at least one sequence of the nucleic acid molecules of the present invention. Such oligonucleotides are useful as probes for detecting variant expression.

[0056] The FVIII variant of the present invention can be prepared in various ways according to known methods. The protein can be purified from a suitable source (e.g., transformed bacteria or animal (e.g., mammalian or human) cultured cells or tissues expressing the FVIII variant) by, for example, immunoaffinity purification. By utilizing the nucleic acid molecule encoding the variant, it becomes possible to produce the variant using in vitro expression methods known in the art. For example, cDNA or the gene can be cloned into a suitable in vitro transcription vector such as pSP64 or pSP65, and then cell-free translation can be performed in a suitable cell-free translation system such as wheat germ or rabbit reticulocyte lysate. In vitro transcription and translation systems are commercially available, for example, from Promega and Life Technologies.

[0057] Alternatively, larger quantities of the variant can be produced by expression in appropriate prokaryotic or eukaryotic expression systems. For example, part or all of the 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 (especially human cells) such as CHO cells or HeLa cells. Alternatively, a tagged fusion protein containing the variant can be created. Such a variant-tagged fusion protein is 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 the desired polypeptide tag, which is inserted into a plasmid vector adapted for expression in bacterial cells such as E. coli, or eukaryotic cells such as yeast, mammalian cells, and especially human cells. Such a vector is configured such that the regulatory elements necessary for DNA expression in the host cell are arranged to enable DNA expression in the host cell. Regulatory elements necessary for expression include, but are not limited to, promoter sequences, transcription start sequences, and enhancer sequences.

[0058] FVIII variant proteins produced by gene expression in recombinant prokaryotes or eukaryotes (particularly humans) can be purified according to methods known in the art. In certain embodiments, commercially available expression / secretion systems can be used to express the recombinant protein, which is then secreted from host cells and readily purified from the surrounding culture medium. If expression / secretion vectors are not used, alternative methods for purifying the recombinant protein include immunological interaction with antibodies that specifically bind to the recombinant protein, or affinity separation, such as a nickel column, to isolate recombinant proteins tagged with 6-8 histidine residues at the N-terminus or C-terminus. Alternative tags may consist of, but are not limited to, FLAG epitopes, GST, or hemagglutinin epitopes. Such methods are commonly used among those skilled in the art.

[0059] The FVIII variant protein prepared by the method described above can be analyzed using standard procedures. For example, such a protein can be subjected to amino acid sequence analysis according to known methods.

[0060] As described above, a convenient method for producing the polypeptide according to the present invention is to express the nucleic acid encoding it using an expression system. Various expression systems useful for the method of the present invention are well known to those skilled in the art.

[0061] Accordingly, the present invention also encompasses methods for producing polypeptides (as disclosed), the methods comprising expression from nucleic acids (generally nucleic acids) encoding polypeptides. This can be conveniently achieved by culturing host cells containing such vectors under appropriate conditions that induce or enable polypeptide production. Polypeptides can also be produced in in vitro systems such as reticulocyte lysates.

[0062] Applications of FVIII variant proteins and nucleic acids that encode variants The FVIII variant proteins and nucleic acids of the present invention can be used, for example, as therapeutic and / or prophylactic agents that modulate the blood coagulation cascade. That is, the FVIII variant proteins and nucleic acids of the present invention can be administered in therapeutically effective doses to modulate (e.g., increase) hemostasis and / or to form thrombi and / or to stop or suppress bleeding or abnormal bleeding. This specification demonstrates that the FVIII variants possess excellent properties and can provide effective hemostasis.

[0063] In certain embodiments of the present invention, the FVIII variant may be administered to a patient via injection in a biocompatible carrier, for example, by intravenous injection. The FVIII variant of the present invention may optionally be encapsulated in liposomes or mixed with other phospholipids or micelles to enhance molecular stability. The FVIII variant may be administered alone or in combination with other agents known to modulate hemostasis (e.g., vFW, factor IX, factor IXa, etc.). A suitable composition for administering the FVIII variant can be determined by a healthcare professional, taking into account a variety of physiological variables, including but not limited to the patient's condition and hemodynamics. Various compositions well suited to different uses and routes of administration are well known in the art and are described below.

[0064] Preparations containing the FVIII variant may contain a physiologically acceptable matrix and are formulated as pharmaceutical formulations. The formulations can be prepared using substantially known methods, by mixing with a buffer containing salts such as NaCl, CaCl2, and amino acids such as glycine and / or lysine, with a pH in the range of 6 to 8. The purified preparations containing the FVIII variant can be stored in the form of a finished solution, or in the form of lyophilization or deep freezing, until needed. In certain embodiments, the preparation is stored in lyophilized form and dissolved in a visually clear solution using a suitable reconstitution solution. Alternatively, the preparations according to the present invention may be available as liquid formulations or as deep-frozen liquids. Preparations according to the present invention may be particularly stable, i.e., they can be left standing for extended periods in a dissolved form before application.

[0065] The formulation according to the present invention can be used as a pharmaceutical formulation in the form of a single-component formulation of the FVIII variant or in the form of a multi-component formulation in combination with other factors. Before processing the purified protein into a pharmaceutical formulation, the purified protein may undergo conventional quality control and be processed into a therapeutic presentation form. In particular, during recombinant manufacturing, the purified preparation may be tested for the presence of cellular nucleic acids as well as nucleic acids derived from the expression vector.

[0066] Another feature of the present invention relates to comprising an FVIII variant having high stability and structural integrity, particularly free from inactive FVIII intermediates and / or protein degradation products, and making it available by incorporating it into a suitable formulation.

[0067] The pharmaceutical preparation may contain, for example, doses of approximately 1–1000 μg / kg, approximately 10–500 μg / kg, approximately 10–250 μg / kg, or approximately 10–100 μg / kg. In certain embodiments, the pharmaceutical protein preparation may contain a dose of 30–100 IU / kg (e.g., as one injection per day, or up to three or more times per day). Treatment can be administered immediately upon presentation of a bleeding patient or before delivery of a cut or wound that caused the bleeding. Alternatively, the patient may receive bolus infusions every 1–3 hours, 8 hours, or 12 hours, or, if sufficient improvement is observed, the FVIII variant described herein may be administered once daily.

[0068] Nucleic acids encoding the FVIII variant can be used for a variety of purposes according to the present invention. In a particular embodiment of the present invention, a nucleic acid delivery vehicle (e.g., an expression vector such as a viral vector) for regulating blood coagulation is provided, the expression vector comprising a nucleic acid sequence encoding the FVIII variant described herein. When an expression vector encoding the FVIII variant is administered to a patient, the FVIII variant is expressed, and the coagulation cascade can be altered. According to the present invention, the FVIII variant-encoding nucleic acid sequence may encode a variant polypeptide described herein whose expression increases hemostasis. In a particular embodiment, the nucleic acid sequence encodes a human FVIII variant.

[0069] Expression vectors containing the FVIII variant nucleic acid sequence may be administered alone or in combination with other molecules useful for modulating hemostasis. According to the present invention, expression vectors or combinations of therapeutic agents may be administered to a patient alone or in pharmaceutically acceptable or biocompatible compositions.

[0070] In certain embodiments of the present invention, the expression vector comprising a nucleic acid sequence encoding an FVIII variant is a viral vector. Viral vectors that may be used in the present invention include adenovirus vectors (with or without a tissue-specific promoter / enhancer), adeno-associated virus (AAV) vectors of any serotype (e.g., AAV-1 to AAV-12, particularly AAV-2, AAV-5, AAV-7, and AAV-8) and hybrid AAV vectors, lentiviral vectors and pseudo-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 certain embodiments, the vector is an adeno-associated virus (AAV) vector. In certain embodiments, the vector is a lentiviral vector.

[0071] In certain embodiments of the present invention, a method is provided for administering a viral vector comprising a nucleic acid sequence encoding an FVIII variant. An adenovirus vector useful in the method of the present invention preferably comprises at least an essential portion of adenovirus vector DNA. As described herein, the expression of the FVIII variant following administration of such an adenovirus vector is useful in regulating hemostasis, particularly in enhancing the protease-coagulation activity.

[0072] Recombinant adenovirus vectors are widely used in various gene therapy applications. Their usefulness in these applications is largely due to their high efficiency in in vivo gene transfer, which can be achieved in a variety of organs.

[0073] Adenovirus particles can be advantageously used as vehicles for sufficient gene transfer. Such viruses possess many desirable characteristics for their application, including structural features such as being non-enveloped viruses with double-stranded DNA, and biological features such as tropism towards the human respiratory and gastrointestinal systems. Furthermore, adenoviruses are known to infect various types of cells in vivo and in vitro through receptor-mediated endocytosis. Supporting the safety of adenovirus vectors, adenoviruses cause mild influenza-like symptoms in humans, and the disease state is generally mild.

[0074] Adenovirus genomes are large (approximately 36 kilobases), making them ideal for use as vehicles in gene therapy because foreign DNA can be inserted after removing essential adenovirus genes and non-essential regions. Such substitutions impair the replication function and infectivity of the viral vector. Notably, adenoviruses have been used as vectors for gene therapy and the expression of heterologous genes.

[0075] For example, it is desirable to introduce a vector that can produce multiple copies of the desired gene, thereby supplying a larger quantity of the gene's product. Improved adenovirus vectors and methods for producing these vectors are described in detail in numerous publications, patents, and patent applications, including: Wright (Hum Gen Ther. (2009) 20:698-706), Mitani and Kubo (Curr Gene Ther. (2002) 2(2):135-44); Olmsted-Davis et al. (Hum Gene Ther. (2002) 13(11):1337-47); Reynolds et al. (Nat Biotechnol. (2001) 19(9):838-42); U.S. Patent Nos. 5,998,205, 6,228,646, 6,093,699, and 6,100,242; WO 94 / 17810; and WO 94 / 23744.

[0076] In some applications, the expression construct may further include regulatory elements that play a role in promoting expression in specific cell or tissue types. Such regulatory elements are known to those skilled in the art. The incorporation of tissue-specific regulatory elements in the expression construct of the present invention provides at least partial tissue tropism for the expression of the variant or its functional fragment. For example, an E1 deletion type 5 adenovirus vector containing a nucleic acid sequence encoding the variant under the control of a cytomegalovirus (CMV) promoter can be advantageously used in the methods of the present invention. Alternatively, promoters specific to the hematopoietic system or liver can also be used.

[0077] AAV for recombinant gene expression has been constructed using 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 typically designed 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, capable of infecting both dividing and non-dividing cells. Vectors are usually genetically engineered by deleting the rep and cap genes from the virus and replacing them with the desired transgene under the control of a specific promoter. For recombinant AAV construction, the upper limit of the sequence size that can be inserted between two ITRs is approximately 4.7 kb. AAV-2 vectors may be prepared using a plasmid expressing the FVIII variant under the control of a CMV promoter / enhancer, and a second plasmid supplying adenovirus helper function, together with a third plasmid containing the AAV-2 rep and cap genes. Alternatively, each surrogate serotype vector may be prepared using plasmids containing either the AAV-1, AAV-6, or AAV-8 cap gene and the AAV-2 rep gene, as well as the ITR (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 also be purified by repeated CsCl density gradient centrifugation, and the titer of the purified vector can be determined by quantitative dot blot hybridization. In certain embodiments, the vector may be prepared by a vector core at Children's Hospital of Philadelphia.

[0078] Furthermore, the present invention also includes a method for regulating hemostasis, which involves administering a nucleic acid delivery vehicle encoding an FVIII variant to cells of an organism and growing the cells under conditions in which the FVIII variant is expressed.

[0079] From the above discussion, it can be seen that FVIII variants and FVIII variant-expressing nucleic acid vectors can be used to treat disorders related to abnormal blood coagulation.

[0080] The expression vector of the present invention may be incorporated into a pharmaceutical composition that can be delivered to a subject by enabling the production of a biologically active protein (e.g., an FVIII variant), by gene and / or cell-based therapy, or by in vivo induction of FVIII variant expression by ex vivo modification / transformation of patient or donor cells. In certain embodiments of the present invention, a pharmaceutical composition containing sufficient genetic material to enable a recipient to produce a therapeutically effective amount of FVIII variant can affect hemostasis in the subject. Alternatively, as described above, an effective amount of FVIII variant may be injected directly into a patient in need. The composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound, and may be administered on any sterile, biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, glucose, and water. The composition may be administered to a patient alone or in combination with other agents that affect hemostasis (e.g., cofactors).

[0081] In certain embodiments, the compositions of the present invention (e.g., pharmaceutical compositions) also include pharmaceutically acceptable carriers. Such carriers include any pharmaceutical agent that does not induce an adverse immune response in the individual receiving the composition and can be administered without excessive toxicity. Examples of pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol, sugars, and ethanol. Pharmaceutically acceptable salts may also be included, for example, mineral salts such as hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as acetate, propionate, malonate, and benzoate. Furthermore, auxiliary substances such as wetting agents or emulsifiers and pH buffers may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub.Co., 18th Edition, Easton, Pa.

[1990] ).

[0082] Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological buffered saline. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Furthermore, suspensions of active compounds can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to enable the preparation of high-concentration solutions.

[0083] The pharmaceutical composition may also be provided as a salt and can be formed with many acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. The salt tends to be more soluble in aqueous solvents and other protic solvents than the corresponding free base form. In other embodiments, the preparation may be a lyophilized powder containing any or all of the following: 1-50 mM histidine, 0.1-2% sucrose, 2-7% mannitol, pH in the range of 4.5-5.5, bound to buffer before use.

[0084] After the pharmaceutical composition is prepared, it can be placed in a suitable container and labeled for therapeutic use. For the administration of an FVIII variant or an FVIII variant encoding vector, such labeling may include the dosage, frequency, and method of administration. Pharmaceutical compositions suitable for use in this invention include compositions in which the active ingredient is contained in an effective amount to achieve the intended therapeutic objective.

[0085] Determining a therapeutically effective dosage is within the capabilities of a skilled healthcare professional using the techniques and guidance provided in this invention.

[0086] The therapeutic dose depends particularly on the patient's age and general health condition, the severity of the abnormal blood coagulation phenotype, and the strength of the regulatory elements that modulate the expression level of variant polypeptides.

[0087] Therefore, the therapeutically effective dose in humans will likely fall within a relatively wide range that can be determined by healthcare professionals based on the individual patient's response to vector-based variant therapy.

[0088] The FVIII variant may be injected directly into a patient on an appropriate biological / pharmaceutical carrier, either alone or in combination with other agents, as described herein. The expression vectors of the present invention, comprising nucleic acid sequences encoding the variant or a functional fragment thereof, may be administered to a patient by various means (see below) to achieve and maintain prophylactic and / or therapeutic levels of efficacy of the variant polypeptide. Those skilled in the art will readily be able to determine specific protocols for using the variant-encoding expression vectors of the present invention for therapeutic treatment of a particular patient. Protocols for the generation and administration of adenovirus vectors to patients are described in U.S. Patent Nos. 5,998,205; 6,228,646; 6,093,699; and 6,100,242; WO 94 / 17810 and WO 94 / 23744, which are incorporated herein by reference in their entirety.

[0089] The FVIII variant and / or nucleic acids (nuceleci acids) encoding the FVIII variant (e.g., adenovirus vectors) of the present invention can be administered to patients by any known means. Direct delivery of the pharmaceutical composition in vivo can generally be achieved by injection using a conventional syringe, but other delivery methods such as convection-enhanced delivery are also envisioned (see, for example, U.S. Patent No. 5,720,720). In this regard, the composition may be delivered subcutaneously, intraepidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intraarterially, orally, intrahepatically, or intramuscularly. Other methods of administration include oral administration, pulmonary administration, suppositories, and transdermal formulations. Clinicians specializing in the treatment of patients with blood coagulation disorders can determine the optimal route for administration of adenovirus vectors containing the variant nucleic acid sequence based on a number of criteria, including but not limited to the patient's condition and the objective of treatment (e.g., enhancement or reduction of blood coagulation).

[0090] The present invention also includes AAV vectors containing nucleic acid sequences encoding the FVIII variant. Furthermore, lentiviral or pseudo-lentiviral vectors containing nucleic acid sequences encoding the FVIII variant are also provided. Additionally, naked plasmids or expression vectors containing nucleic acid sequences encoding the FVIII variant are also included.

[0091] The following examples are provided to illustrate various embodiments of the present invention. These examples are for illustrative purposes only and are not intended to limit the present invention in any way. [Examples]

[0092] material and method reagent The inhibitors, benzamidine and 4-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF), were obtained from Sigma Aldrich (St. Louis, MO). Cell culture reagents were purchased from Invitrogen (Waltham, Massachusetts), with the exception of insulin-transferrin-sodium selenite purchased from Roche (Basel, Switzerland). Synthetic phospholipid vesicles (PCPS) were prepared from 75% chicken egg L-α-phosphatidylcholine (PC) and 25% porcine brain L-α-phosphatidylserine (PS) (Avanti Polar Lipids; Alabaster, AL) and quantified as described (Pittman, et al. (1993) Blood 81(11):2925-2935). Triniclot reagent (Tcoag) was used for automated activated partial thromboplastin time (aPTT) measurement. The peptidyl substrate Spectrozyme® Xa (Sekisui Diagnostics; Burlington, MA) was prepared with water, and E 342 = 8279 M -1 cm -1The concentration was confirmed using (Lottenberg, et al. (1983) Biochim. Biophys. Acta., 742(3):558-564). The fluorescent substrate 0.5 mM Z-Gly-Gly-Arg-AMC was purchased from Bachem Bioscience Inc. (Bubendorf, Switzerland), prepared with 15 mM CaCl2, and E 326 = 17,200 M -1 cm -1 (Bunce, et al. (2011) Blood 117(1):290-298). Normal human plasma and FVIII-deficient plasma with low pooled platelets were purchased from George King Biomedical (Overland Park, KS). Unless otherwise specified, all assays were performed at 25 °C in assay buffer (20 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 150 mM NaCl, 5 mM CaCl2, 0.1% polyethylene glycol-8000, pH 7.4), and all reagent or protein concentrations described are the final concentrations under the experimental conditions.

[0093] Protein Plasma-derived FX, FXa, and thrombin were purified and prepared as described (Baugh, et al. (1996) J. Biol. Chem., 271:***16126-16134***; Buddai, et al. (2002) J. Biol. Chem., 277(29):26689-26698). Factor IXa and APC were purchased from Haemtech (Essex Junction, VT). Hirudin was purchased from Calbiochem (San Diego, CA). Protein concentrations were determined based on the following molecular weight (M r ) and extinction coefficient (E) 0.1%The following values ​​were determined immediately before each experiment using the following methods: thrombin (37,500 and 1.94), FIXa (45,000 and 1.40), FX (59,000 and 1.16), FXa (46,000 and 1.16), APC (45,000 and 1.45), and PS (69,000 and 0.95), respectively. (Lundblad, et al. (1976) Thrombin 1976:156-176; Di Scipio, et al. (1977) Biochemistry 16(4):698-706; Fujikawa, et al. (1974) Biochemistry 13(22):4508-4516).

[0094] Recombinant FVIII protein production We developed and purified a baby hamster kidney (BHK) cell line that stably expresses wild-type B-domain deletion FVIII (FVIII-WT) (Pittman, et al. (1993) Blood 81(11):2925-2935; Sabatino, et al. (2009) Blood 114(20):4562-4565). Using site-directed mutagenesis (Genescript; Piscataway, NJ) of FVIII-WT cDNA, we introduced mutations from Arg to Gln at Arg336 and Arg562, which are FVIII APC cleavage sites (Figure 1B). Using ion-exchange chromatography, we purified factor VIII protein from 24 liters of prepared medium (approximately 3 mg each) (Sabatino, et al. (2009) Blood 114(20):4562-4565). Recombinant FVIII concentration is 1.60 E 0.1% Based on a molecular weight (Mr) of 165,000, the absorbance at 280 nm was determined (Curtis, et al. (1994) J. Bio.Chem., 269(8):6246-6251). Recombinant FVIII-R372Q was prepared similarly as a control.

[0095] Plasma assay FVIII-specific activity was measured using an aPTT-based one-step coagulation assay (Siner, et al. (2016) JCI Insight., 1(16):e89371). Thrombin generation in platelet-poor plasma was measured using the modified method described (Bunce, et al. (2011) Blood 117(1):290-298). Factor VIII-deficient plasma was reconstituted with 1 nM FVIII or 0.2 nM FVIIIa and 4 μM PCPS. To generate FVIIIa, FVIII (1.5 nM) was incubated with thrombin (30 nM) for 30 seconds and quenched with hirudin (60 nM). In FVIII-reconstituted plasma, thrombin generation was initiated using 1 pM or 30 pM FXIa in human and mouse plasma, respectively. In FVIIIa-reconstituted plasma, thrombin generation was initiated with 10 pM FXIa in humans and 400 pM FXIa in mice. The concentrations of FVIIIa and FXIa in these assays were selected to produce similar peak thrombin and lag time compared to experiments using FVIII in similar HA plasma (Table 1). The reaction was initiated with 0.5 mM Z-Gly-Gly-Arg-AMC (Bachem Bioscience Inc.) in 0.5 mM CaCl2. Fluorescence was measured for human and mouse plasma using Spectromax® M2 (Molecular Devices; San Jose, CA) at 37°C or 33°C for 90 minutes, with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The raw fluorescence values ​​were compared to a thrombin calibration curve using a thrombin calibrator (Technothrombin® thrombin generation assay calibrator set) to convert the data to nM thrombin and thrombin generation curves (nM / time), and peak thrombin generation and lag time were analyzed. Human soluble thrombomodulin (sTm) was used because it does not cross-react with mouse APC.

[0096] [Table 1] Peak thrombin and lag time values ​​for plasma reconstituted with FVIII and FVIIIa. Mean and standard error of the mean are displayed. Peak thrombin: maximum concentration of thrombin produced; Lag time: time to peak thrombin production; HA: hemophilia A; HA / FVL: homozygous hemophilia A, factor V Leiden.

[0097] Western blot analysis of cleavage of FVIII protein by proteolysis Factor VIII (1.5 μM) was incubated with thrombin (10 nM) for 20 minutes to generate FVIIIa, which was then quenched with hirudin (20 nM). To evaluate APC cleavage, FVIII (10 nM) was incubated with APC (6 nM), hirudin (6 nM), and PCPS (20 μM) for 30 minutes. Hirudin was added to the purification assay to quench for trace amounts of thrombin present in commercially available APC. Samples were analyzed by Western blotting. FVIII and FVIII cleavage products were analyzed using a primary antibody that recognizes the FVIII A2 domain (Fay, et al. (1991) J. Biol. Chem., 266(14):8957-8962) (GMA-012, Green Mountain Antibodies; Burlington, VT) and Dylight. TM 800 was detected by secondary detection antibody (Rockland; Pottstown, PA).

[0098] Dynamics study of factor VIII enzyme and measurement of A2 stability Kinetic analysis of FXa generation was performed by an endogenous Xase assay, modified from the description (Lollar, et al. (1989) Biochemistry 28(2):666-674). Activated FVIII (FVIIIa) was generated by incubating 25 nM FVIII with 100 nM thrombin for 30 seconds, followed by quenching with hirudin (150 nM). Factor VIIIa (0.25 nM) was immediately conjugated with FIXa (20 nM) and variable FX concentrations (0-500 nM) in the presence of 20 μM PCPS. Aliquots of the reaction mixture were quenched at various time intervals (0.25-2 min) with 20 mM HEPES, 150 mM NaCl, 25 mM EDTA, 0.1% polyethylene glycol-8000, pH 7.4. Using Spectrozyme® Xa, absorbance at 405 nm was measured with a SpectraMax® 190 Microplate reader (Molecular Devices) and compared to a prepared FXa standard curve to evaluate the amount of FXa in each quenched sample. Residual FVIII activity in the presence of APC or APC and PS was performed as described, except that the FVIII protein was incubated with 6nM APC (hemtec) or 6nM APC and 100nM PS in the presence of 20 μM PCPS and 6nM hirudin for 0–60 minutes prior to thrombin activation. FVIIIa-A2 dissociation was evaluated as described, except that variable concentrations of FVIII were activated with 100nM thrombin (5–100nM), aliquots were taken at the indicated time, and residual FVIIIa function was immediately assayed in an endogenous Xase assay (Lollar, et al. (1989) Biochemistry 28(2):666-674).

[0099] animal HA-C57BL / 6 mice were used in in vivo studies (Bi, et al. (1995) Nat. Genet., 10(1):119-121). Homozygous HA-C57BL / 6 mice were crossed with homozygous FV-Leiden (FVL)-C57BL / 6 mice to produce homozygous HA / FVL-C57BL / 6 mice (Schlachterman, et al. (2005) J. Thromb. Haemost., 3(12):2730-2737; Cui, et al. (2000) Blood 96(13):4222-4226). Factor V Leiden (FVL) 506Q The (FVL) is cleaved 10 times slower than the FV and is therefore APC resistant. Wild-type C57BL / 6 mice were purchased from Jackson Labs. Males and females aged 8-12 weeks were used for the experiments. Animal experiments were approved by the Animal Welfare and Use Committee of Children's Hospital Philadelphia.

[0100] Tail clip assay Mice were anesthetized with isoflurane, and their tails were preheated to 37°C. Factor VIII protein and / or mAb 1609 (200 μL at a dose of 10 μg / mL) was injected into a 3 mm diameter retroorbital injection 3 minutes before tail amputation (Xu, et al. (2009) J. Thromb. Haemost., 7(5):851-856). The tails were placed in conical tubes, blood was collected for 2 minutes, and then placed in normal saline for a further 10 minutes. The 10-minute samples were hemolyzed, and the absorbance was measured at 575 nm to determine the total hemoglobin present (Sambrano, et al. (2001) Nature 413(6851):74-78). Total blood volume (μL) was determined by converting the sample hemoglobin amount using an established standard curve for known amounts of hemolyzed mouse whole blood (Ivanciu, et al. (2011) Nat. Biotechnol., 29(11):1028-1033).

[0101] FeCl3 injury model Ferric chloride (FeCl3) injury was performed in HA-C57BL / 6 mice as described (Schlachterman, et al. (2005) J. Thromb. Haemost., 3(12):2730-2737). Briefly, the carotid artery was exposed and the flow rate was measured using a Doppler probe (Model 0.5VB; Transonic Systems; Ithaca, NY) placed subarterially. Approximately 3 minutes after injecting jugular vein FVIII protein, 2 mm of tissue immersed in 7.5% FeCl3 was used. 2 Carotid artery vascular injury was induced by placing filter paper on the arterial outer surface for 2 minutes. Afterward, the filter paper was removed, the affected area was washed with normal saline solution, and blood flow was continuously monitored using Doppler flow for up to 30 minutes. The time until carotid artery occlusion was defined as the absence of measurable blood flow, and this is reported in the experimental results.

[0102] Thrombin generation assay by soluble thrombomodulin titration Human FVIII-deficient plasma was reconstituted with 1 nM FVIII-WT or FVIII-QQ, 4 μM PCPS, and increasing amounts of soluble thrombomodulin (sTM). Human sTM was recombinantly produced and purified as described (Bradford, et al. (2012) J. Biol. Chem., 287(36):30414-30425; Parkinson, et al. (1990) J. Biol. Chem., 265(21):12602-12610). Thrombin generation was induced with 0.1 pM FXIa. The reaction was initiated with 0.5 mM Z-Gly-Gly-Arg-AMC (Bachem Bioscience Inc.) and 7.5 mM CaCl2 (final concentration). bFluorescence was measured using a Spectromax® M2 (Molecular Devices) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm at 37°C for 90 minutes. The raw fluorescence values ​​were compared with thrombin calibration curves using a thrombin calibrator (Technothrombin® thrombin generation assay calibration set), and the data was converted to nM thrombin and thrombin generation curves (nM / time). The thrombin generation peak and lag time were then analyzed (Figure 4E).

[0103] FVIII Half-Life Test FVIII half-life was measured after tail vein injection of FVIII-WT or FVIII-QQ at 125 IU / kg into HA-C57BL / 6 mice. Plasma samples were collected in 3.8% sodium citrate at 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours, and 48 hours after protein injection and snap-frozen for later analysis. Residual factor VIII activity was measured using the Chromogenix Coamatic® FVIII kit (Diapharma, Louisville, KY) with a protocol shortened to 4 minutes incubation time (Rosen, et al.) (1985) Thromb. Haemost., 54(4):818-823). The half-life was determined by fitting the residual FVIII activity to an exponential decay curve using Prism software (Dumont, et al. (2012) Blood 119(13):3024-3030) (Figure 5E).

[0104] Data Analysis The analysis was performed using Graphpad Prism 8 software. The specific statistical analysis method is outlined in the figure caption. The steady-state dynamics parameter of FX activation by endogenous FXase, K m and V max The results were calculated by unweighted nonlinear least-squares fitting to the Michaelis-Menten equation. The results are expressed as mean ± standard error. Mouse injury studies were analyzed by one-way ANOVA for rank (Kruskal-Wallis, nonparametric fit) with Dunn's multiple comparison test.

[0105] result Characterization of the coagulation-promoting activity of FVIII-WT and FVIII-QQ To confirm that the introduction of the two mutations did not alter the procoagulation function of FVIII, FVIII-QQ was compared with FVIII-WT using a different assay system. FVIII-WT and FVIII-QQ were purified from conditional medium in single-chain (Mr=165,000) and heterodimer (heavy chain, Mr=90,000 and light chain, Mr=80,000) forms. Thrombin cleaved both proteins, yielding fragments representing cleavage of R1689 (A3-C1-C2; Mr = 70,000) and R740 / R372 (A1, Mr = 50,000 and A2, Mr = 43,000), corresponding to FVIIIa (Figure 2A).

[0106] The specific activity of FVIII-WT (9000±700 IU / mg) and FVIII-QQ (11000±900 IU / mg) was consistent with that of commercially available B-domainless FVIII products (Table 2) (www.fda.gov / media / 70399 / download2014). Furthermore, both proteins showed similar peak thrombin generation, endogenous thrombin potential, and lag time at various concentrations evaluated in thrombin generation assays (Figure 2B). In the purification system, FVIIIa-WT and FVIIIa-QQ showed similar Km and Vmax values ​​for FX activation (Table 2), which were consistent with published values ​​(Lollar, et al. (1994) J. Clin.Invest., 93(6):2497-2504). Importantly, the introduction of the two mutations did not affect the stability of the A2 domain. Both proteins spontaneously lost almost all FVIIIa activity within 15 minutes, due to dissociation of the A2 domain (Figure 2C).

[0107] [Table 2] Biochemical characterization of FVIII-QQ. Data are expressed as mean ± SEM from at least two independent experiments. Kinetic values ​​of FX activation were determined by an endogenous Xase assay using 0.25 nM FVIIIa, 20 nM FIXa, and 0–500 nM FX in the presence of 20 μM phospholipid.

[0108] FVIII / FVIIIa-QQ is resistant to APC cutting. To confirm the resistance of FVIII-QQ to APC cleavage, FVIII-QQ and FVIII-WT were incubated with APC for 30 minutes, and the reaction products were evaluated by Western blotting. As expected, APC cleavage of FVIII-WT resulted in R336(A1 336 -A2) and R562(A2 562 Fragments consistent with cleavage were obtained in both WT and FVIII-QQ, but no similar fragments were detected for FVIII-QQ cleavage (Figure 3A). Under the conditions adopted, both FVIII-WT and FVIII-QQ were cleaved at the A2 domain by APC, yielding fragments consistent with cleavage at R372 (Figure 3A). This was confirmed by incubating the FVIII-R372Q mutant with APC, but no A2 fragment was produced (Figure 3B), which is consistent with reports of APC cleavage at the FVIII thrombin cleavage site (Fay, et al. (1991) J. Biol.Chem., 266(30):20139-20145). Furthermore, APC-specific cleavage was achieved by adding hirudin to this reaction to suppress potential trace thrombin. Similar to FVIII-QQ's APC resistance, this protein maintained over 90% activity after 1 hour of APC incubation (Figure 3C). On the other hand, FVIII-WT lost approximately 75% of its activity after 1 hour of APC incubation (Figure 3C). The loss of FVIII-WT activity due to APC cleavage was confirmed by Western blot analysis. Figure 3E shows the incubation time course for APC. WT and FVIII-QQ (450 nM) were reacted with 20 μM PCPS and 90 nM APC over a 60-minute time course. Western blots were visualized using an A2-specific antibody (GMA-8028). These data indicate that introducing Arg336Gln and Arg562Gln mutations into FVIII inhibits cleavage at these sites, conferring functional APC resistance without significantly affecting other aspects of the procoagulation function of FVIII / FVIIIa.

[0109] Consistent with published data (Lu et al. (1996) Blood 87:4708-4717), FVIII-WT lost almost all function within 15 minutes of combined incubation with APC and PS (Figure 3C). Surprisingly, combined culture with PS and APC also accelerated the loss of FVIII-QQ.

[0110] This indicates the role of APC / PS in inactivation at sites other than R336 and R562. Western blotting revealed that both FVIII-WT and FVIII-QQ showed enhanced APC cleavage at R372 in the presence of PS (Figure 3D). Thrombin-mediated cleavage of R372 converts the FVIII heterodimer to an FVIIIa heterotrimer. The coenzyme function of PS is thought to promote APC cleavage at R336, R562, and R372, leading to heterotrimer formation. The loss of FVIII function measured after incubation of APC and PS in this in vitro system may reflect APC cleavage at R336 and R562, as well as spontaneous A2 domain dissociation after APC cleavage at R372. Since R372 is already cleaved by thrombin-mediated FVIIIa heterotrimer formation, the physiological significance of APC cleavage at R372 remains unclear.

[0111] To investigate the effect of APC on the inactivation of FVIIIa in plasma, human HA plasma was reconstituted with physiological amounts of FVIII (1 nM) and APC. Factor VIII procoagulation activity was evaluated by a thrombin generation assay. FVIII-QQ showed increased thrombin generation (peak thrombin value) compared to FVIII-WT with increasing APC concentration. Factor VIII-QQ reconstituted HA plasma lost approximately 30% of its activity, while FVIII-WT reconstituted HA plasma lost 80% of its activity in the presence of 3 nM APC (Figure 4A). Similar results were obtained when the concentration of sTM was increased instead of APC (Figure 4E). In this measurement system, FVIIIa and FV / FVa are inactivated by APC, which explains the decrease in thrombin generation when using FVIII-QQ. However, reconstituted plasma containing FVIII-QQ is more resistant to APC than FVIII-WT. A similar thrombin generation assay was performed using FVIIIa. Here, FVIII-QQ and FVIII-WT were rapidly activated with thrombin and added to human HA plasma. Similar to procofactors, FVIIIa-QQ showed greater thrombinogenesis compared to FVIIIa-WT within the range of APC concentrations tested (Figure 4B). Since FVIIIa is added before thrombinogenesis, A2 dissociation is thought to play a major role in regulating FVIIIa in this experimental system. However, even under conditions of enhanced A2 dissociation, a difference in APC sensitivity was observed between FVIIIa-WT and FVIIIa-QQ. Similar results were observed using HA mouse plasma reconstituted with FVIII (Figure 4C) or FVIIIa (Figure 4D). Thrombinogenesis of FVIII / FVIIIa-WT in the presence of APC was more significantly reduced compared to FVIII / FVIIIa-QQ, supporting the role of APC in FVIIIa inactivation in this HA plasma-based system.

[0112] APC-resistant FVIII improves hemostatic effects in HA mouse injury models. To evaluate the relative in vivo effects of FVIII-WT and FVIII-QQ, tail clipping and FeCl3 assays were performed using HA mice. The tail clipping assay showed a dose-dependent reduction in bleeding for both FVIII-QQ and FVIII-WT (Figure 5A). The FVIII-QQ dose (2.5 μg / kg) was lower than the FVIII-WT dose (10 μg / kg), suggesting the in vivo contribution of APC to FVIIIa regulation. To confirm the APC-specificity of the observations, the tail clipping assay was repeated in the presence of mAb1609 (Figure 5A), an antibody that inhibits the anticoagulant function of mouse APC (Xu, et al. (2009) J. Thromb. Haemostasis 7(5):851-856). Injection of mAb1609 into HA mice did not produce a hemostatic effect, and bleeding was similar to that of the PBS control. However, when HA mice were administered mAb1609 and 2.5 μg / kg FVIII-WT (a dose of FVIII-QQ that normalizes bleeding), bleeding was reduced in a manner similar to that of normal hemostatic controls. Unlike FVIII-WT, the bleeding volume with FVIII-QQ was the same regardless of the presence or absence of mAb1609. These results suggest that the superior hemostatic effect of FVIII-QQ in vivo is specific to resistance to APC cleavage.

[0113] Based on recovery studies (Figure 5E), the dose of FVIII-WT required to normalize bleeding approximated 67% of normal plasma FVIII activity, consistent with publications (Nguyen, et al. (2017) J. Thromb. Haemost., 15(1):110-121; Siner, et al. (2013) Blood 121(21):4396-4403). Quantitatively, the EC of FVIII-QQ was... 50 It was 6 to 7 times lower than FVIII-WT (1.1 μg / kg and 7.4 μg / kg, respectively), and EC 80The EC50 levels were 8-9 times lower than those of FVIII-WT (2.2 μg / kg and 18.6 μg / kg, respectively) (see Figure 5B and Table 3). Similar to the tail clip assay, the FVIII-QQ dose (2 μg / kg) that normalized the time to vascular occlusion in the FeCl3 assay was lower than the FVIII-WT dose (10 μg / kg). In the FeCl3 assay, the EC50 levels of FVIII-QQ were lower. 50 It is three times lower than FVIII-WT (1.2 μg / kg and 3.4 μg / kg, respectively), and the EC of FVIII-QQ is lower. 80 The levels were eight times lower than those of FVIII-WT (1.5 μg / kg and 12.1 μg / kg, respectively) (Figure 5D, Table 3). The half-lives and recovery rates of FVIII-WT and FVIII-QQ were similar in HA mice (Figure 5E). These data suggest that in a large vessel injury model, APC plays a crucial role in regulating FVIIIa in vivo, and resistance to APC cleavage contributes to hemostasis.

[0114] [Table 3] In vivo hemostatic function of FVIII-QQ against FVIII-WT. EC 50 / 80 FVIII dosage required for 50% or 80% of the normal bleeding volume or time to vascular occlusion, FeCl3, 7.5% ferric chloride injury model.

[0115] The effect of APC on FVIII / FVIIIa function in HA / FVL mouse plasma and injury models. To further isolate the contribution of APC cleavage to FVIIIa inactivation, homozygous HA / FVL mice were generated. Studies with HA / FVL mice showed that FVL provided a slight improvement in microvascular hemorrhage, but no observable effect in large vessel injury models (Schlachterman, et al. (2005) J. Thromb.Haemost., 3(12):2730-2737). First, mouse HA / FVL plasma reconstituted with FVIII-WT or FVIII-QQ was subjected to repeated thrombin generation assays in the presence of various APC concentrations. As expected, inactivation of FVIII-WT and FVIII-QQ in HA plasma differed significantly from that in HA / FVL plasma (comparing Figure 4C and Figure 6A). However, in HA / FVL plasma, FVIII-QQ resulted in higher residual thrombin peak values ​​than FVIII-WT at all APC concentrations (Figure 6A). Similar results were obtained when plasma was reconstituted with FVIIIa-WT and FVIIIa-QQ (Figure 6B). Next, the tail clip assay was repeated in HA / FVL mice to compare the hemostatic effect of FVIII-QQ with that of FVIII-WT. Administration of FVIII-WT (2 μg / kg) to HA / FVL mice had only a slight effect on bleeding volume, while FVIII-QQ (2 μg / kg) normalized bleeding volume to a hemostatically normal control (Figure 7). These data were obtained using a system in which FV inactivation is significantly inhibited, suggesting that APC-mediated inactivation of FVIIIa must play an important role in controlling thrombus formation in vivo.

[0116] To further demonstrate this, a tail clip assay using HA / FVL mice was repeated in the presence of mAb1609, an antibody that inhibits mouse APC anticoagulation function (Xu, et al. (2009) J. Thromb. Haemost., 7(5):851-856). As expected, injection of mAb1609 into HA / FVL mice did not produce hemostatic effects on its own, and the amount of bleeding was similar to that of the PBS control (Figure 7). These data demonstrate that FV is resistant to APC inactivation, and that APC-mediated inactivation of FVIIIa must play an important role in the control of thrombus formation in vivo. Similar to the observations in HA mice, injection of PBS and mAb1609 into HA / FVL mice resulted in bleeding similar to that of the HA / FVL PBS control (Figure 6). Similar to observations in HA mice, administration of mAb1609 along with FVIII-QQ (2.5 μg / kg) to HA / FVL mice did not significantly alter bleeding volume. However, administration of FVIII-WT (2.5 μg / kg) reduced bleeding volume to levels seen in normal hemostatic controls. Therefore, similar hemostatic effects were effectively obtained even when the anticoagulant function of APC was eliminated (mAb1609) or when APC's coagulation-promoting substrates (FV-Leiden or FVIII-QQ) were removed. These results suggest that the superior hemostatic effect of FVIII-QQ in vivo is specific to resistance to APC cleavage.

[0117] Finally, single mutants of FVIII were evaluated using an HA / FVL plasma thrombinogenesis assay while increasing the APC concentration. As can be seen in Figure 8A, FVIII-R336Q and FVIII-R562Q retained superior activity compared to the wild-type (WT). The single mutants were also tested in a hemostatic injury model of HA / FVL mice. As can be seen in Figure 8B, FVIII-R336Q and FVIII-R562Q were superior to the wild-type FVIII.

[0118] Our research using FVIII-QQ, published here, revealed that APC plays an unexpectedly significant role in the regulation of FVIIIa in vivo. Compared to FVIII-WT, FVIII-QQ exhibited APC resistance without altering its procoagulant function or the stability of its A2 domain. Simultaneously, FVIII-QQ's resistance to APC cleavage improved hemostatic function in a large vessel injury model in HA mice compared to FVIII-WT. The superiority of FVIII-QQ over FVIII-WT was abolished by an APC inhibitory antibody, confirming that the enhanced hemostatic effect of FVIII-QQ is APC-specific. These data demonstrate the in vivo importance of APC cleavage in the inactivation of FVIIIa.

[0119] Two mechanisms are known for the inactivation of FVIIIa. Biochemical studies suggest that spontaneous dissociation of the A2 domain is the main factor involved in the inactivation of FVIIIa, and that the contribution of APC is relatively small in relation to the inactivation rate (Lollar, et al. (1991) J. Biol.Chem., 266(19):12481-12486; Fay, et al. (1991) J. Biol.Chem., 266(14):8957-8962; Lollar, et al. (1992) J. Biol.Chem., 267(33):23652-23657). Indeed, the data show rapid, spontaneous dissociation of the A2 domain and relatively slow cleavage via APC, resulting in FVIIIa inactivation occurring over several minutes or hours, respectively (Lollar, et al. (1991) J. Biol.Chem., 266(19):12481-12486; Lollar, et al. (1992) J. Biol.Chem., 267(33):23652-23657; Fay, et al. (1991) J. Biol.Chem., 266(30):20139-20145). Thus, the sensitivity of FVIII / FVIIIa-WT to increased plasma APC concentrations and the enhanced hemostatic effect of FVIII-QQ in a mouse injury model are surprising given the biochemical data on FVIIIa regulation. While this data does not negate the idea that A2 domain dissociation is an important mechanism of FVIIIa regulation, it suggests that A2 dissociation is not the only relevant mechanism of FVIIIa regulation in vivo, in stark contrast to in vitro rate constant predictions.

[0120] In particular, interactions within the endogenous Xase complex that alter the dissociation rate of the A2 domain are difficult to model simultaneously and can lead to discrepancies between the inactivation rate of FVIIIa determined in vitro and the hemostatic effect observed in vivo. This highlights the importance of combining in vitro analyses with in vivo investigations to confirm the influence of specific regulatory mechanisms. For example, the binding affinity of the A2 domain in the FVIIIa heterotrimer is nearly 300 times higher than that of plasma FVIII concentration, suggesting that rapid dissociation of the A2 domain occurs in vivo when FVIIIa is free (Lollar, et al. (1992) J. Biol.Chem., 267(33):23652-23657; Parker, et al. (2006) J. Biol.Chem., 281(20):13922-13930). However, the concentration of FVIIIa at the injury site is unknown, and it is unclear to what extent it binds to various ligands and is actually free. Importantly, FIXa is well known to stabilize the A2 domain in the FVIIIa heterotrimer within the endogenous Xase complex (Fay, et al. (1996) J. Biol.Chem., 271(11):6027-6032; Lollar, et al. (1984) Blood 63(6):1303-1308). Furthermore, APC cleavage alters the orientation of the A2 domain and reduces the affinity of FVIIIa to both FIXa and FX (Regan, et al. (1996) J. Biol.Chem., 271(8):3982-3987; Rosenblum, et al. (2002) J. Biol.Chem., 277(14):11664-11669). Therefore, it is unclear whether the A2 domain is assembled within the endogenous Xase enzyme complex at the injury site and reaches equilibrium within the FVIIIa heterotrimer.Furthermore, both protein S (PS) and FV (neither of which are present in direct measurements of FXa production) have been reported to be synergistic cofactors of APC-mediated cleavage of FVIIIa (Dahlback, et al. (1994) Proc.Natl.Acad.Sci., 91:1396-1400; Shen, et al. (1994) J. Biol.Chem., 269:18735-18738; Fay, et al. (1991) J. Biol.Chem., 266(30):20139-20145; Lu, et al. (1996) Blood 87(11):4708-4717). This study, using reconstituted HA or HA / FVL plasma containing FVIIIa and in an in vivo injury model, allowed for the simultaneous analysis of FVIIIa regulatory mechanisms (APC-mediated proteolysis and A2 domain dissociation) in the presence of PS and FV. This comprehensive evaluation demonstrated the importance of APC regulation of FVIIIa in vivo.

[0121] In addition to APC, FIXa and FXa exhibit the ability to cleave FVIIIa residues 336 and 562, respectively (Eaton, et al. (1986) Biochemistry 25(2):505-512; Lamphear, et al. (1992) Blood 80(12):3120-3126; Nogami, et al. (2003) J. Biol.Chem., 278(3):1634-1641). By disrupting these cleavage sites in the FVIII-QQ mutant, the possible role of FIXa and FXa-mediated FVIIIa cleavage in the regulation of the endogenous Xase complex was also ruled out (Nogami, et al. (2003) J. Biol.Chem., 278(3):1634-1641; Regan, et al. (1996) J. Biol.Chem., 271(8):3982-3987).

[0122] Using a gain-of-function FVIII transgene for HA gene transfer overcomes the dose-dependent safety and efficacy limitations of vectors, reduces the need for vector production, and improves efficacy (George, LA(2017) Hematology 2017(1):587-594). This method has been well adapted to gene therapy efforts for hemophilia B, and all currently enrolled clinical trials are using the highly specific FIX variant FIX-Padua (George, et al.(2017) New Eng.J. Med., 377(23):2215-2227; Chowdary, et al.(2020) Res.Pract.Thromb.Haemost., 4(Suppl 1); Majowicz, et al.(2020) Haemophilia 26(S4):20; Simioni, et al.(2009) New Eng.J. Med., 361(17):1671-1675). Furthermore, the first successful trial of HA gene therapy observed an unexpected decrease in FVIII expression (Rangarajan, et al. (2017) New Eng.J.Med., 377(26):2519-2530; Pasi, et al. (2020) New Eng.J.Med., 382(1):29-40). One proposed etiology is that FVIII expression induces unfolded protein reactions and endoplasmic reticulum stress, as demonstrated in mammalian cell cultures and mouse hepatic gene transduction, leading to the loss of FVIII expression (Malhotra, et al.(2008) Proc.Natl.Acad.Sci., 105(47):18525-18530; Lange, et al.(2016) Mol.Ther.Meth.Clin.Dev., 3:16064; Poothong, et al.(2020) Blood 135(21):1899-1911; Becker, et al.(2004) Thromb.Haemost., 92(1):23-35; Brown, et al.(2011) J. Biol.Chem., 286(27):24451-24457).This supports the idea that using a gain-of-function variant of FVIII allows for sustained effects even at lower gene expression levels. In injury models, the dose of FVIII-QQ required to normalize bleeding and thrombus formation was consistently lower, approximately five times that of FVIII-WT. The enhancement of hemostatic function by FVIII-QQ compared to FVIII-WT was higher than that of previously described gain-of-function FVIII variants (Pipe, et al. (1997) Proc.Natl.Acad.Sci., 94:11851-11856; Zakas, et al. (2017) Nat.Biotech., 35(1):35-37; Leong, et al. (2015) Blood 125(2):392-398; Wakabayashi, et al. (2008) Blood 112(7):2761-2769). Based on the above, it has been shown that APC plays an important role in the regulation of FVIIIa in vivo, and it is hoped that this will be utilized to develop new hemophilia treatments.

[0123] While preferred embodiments of the present invention have been described and illustrated above, the present invention is not limited to these embodiments. Various modifications can be made without departing from the scope and spirit of the invention, as described in the following claims.

Claims

1. A pharmaceutical composition for the treatment of hemophilia, comprising at least one Factor VIII (FVIII) variant or a nucleic acid encoding said FVIII variant in a pharmaceutically acceptable carrier, wherein said FVIII variant comprises amino acids 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, or comprises amino acids 1 to 740 and 1690 to 2332 of SEQ ID NO: 1, provided that Arg at position 336 of said FVIII variant is substituted with Gln, and Arg at position 562 of said FVIII variant is substituted with Gln.

2. The pharmaceutical composition according to claim 1, wherein said hemophilia is hemophilia A.

3. The pharmaceutical composition according to claim 1 or 2, wherein said FVIII variant lacks a B domain or the B domain is substituted with a peptide linker.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein said FVIII variant comprises a signal peptide.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the nucleic acid encoding said FVIII variant is contained within an expression vector and is operably linked to a regulatory sequence.

6. The pharmaceutical composition according to claim 5, wherein said expression vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a plasmid, and a lentivirus vector.

7. The pharmaceutical composition according to claim 5, wherein said vector is an AAV vector.

8. A pharmaceutical composition for reducing bleeding in a patient, comprising at least one Factor VIII (FVIII) variant or a nucleic acid encoding said FVIII variant in a pharmaceutically acceptable carrier, wherein said FVIII variant comprises amino acids 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, or comprises amino acids 1 to 740 and 1690 to 2332 of SEQ ID NO: 1, provided that Arg at position 336 of said FVIII variant is replaced by Gln and Arg at position 562 of said FVIII variant is replaced by Gln. **Claim 9**: The pharmaceutical composition according to claim 8, wherein said FVIII variant lacks a B domain or the B domain is replaced by a peptide linker. **Claim 10**: The pharmaceutical composition according to claim 8 or 9, wherein said FVIII variant comprises a signal peptide. **Claim 11**: The pharmaceutical composition according to any one of claims 8 to 10, wherein the nucleic acid encoding said FVIII variant is contained within an expression vector and is operably linked to regulatory sequences. **Claim 12**: The pharmaceutical composition according to claim 11, wherein said expression vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a plasmid, and a lentivirus vector. **Claim 13**: The pharmaceutical composition according to claim 11, wherein said vector is an AAV vector.

Images