Compositions and methods for expressing factor viii

Abstract

Factor VIII encoding nucleic acids and methods of use thereof are disclosed.

Description

[0001] COMPOSITIONS AND METHODS FOR EXPRESSING FACTOR VIII By Lindsey A. George Xavier Anguela Martinez

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 715,813, filed November 4, 2024. The foregoing application is incorporated by reference herein.

[0003] This invention was made with government support under grant number HL146991 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the fields of medicine and hematology. More specifically, the invention provides novel Factor VIII expression vectors and methods of using the same to modulate the coagulation cascade in patients in need thereof.

[0006] BACKGROUND OF THE INVENTION

[0007] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0008] Coagulation factor VIII (FVIII) circulates in blood tightly bound 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). Proteolytic processing by thrombin liberates FVIII from vWF and produces the active cofactor species (FVIIIa), which is a heterotrimer comprised of an A2-domain weakly associated with the metal ion-stabilized A1 / A3-C1-C2 heterodimer (Vehar, et al. (1984) Nature (1984) 312(5992):337-342; Fay, et al. (1992) J. Biol. Chem., 267(19): 13246-13250). Factor Villa associates with activated FIX (FIXa) on anionic phospholipid surfaces forming the intrinsic Xase enzyme complex, one of two enzymes that activates 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). Deficiency or dysfunction of FVIII results in hemophilia A (HA), highlighting the importance of F Villa cofactor function. Indeed, defective FVIII or a lack of FVIII activity results in an inability to effectively form clots. To date, only 20% of patients with hemophilia A worldwide receive regular treatment with FVIII replacement therapy due its high cost. Gene therapy for hemophilia A based on AAV vectors is promising, but improved vectors for optimized expression of FVIII are needed.

[0009] SUMMARY OF THE INVENTION

[0010] In accordance with the present invention, compositions and methods for the modulation of hemostasis in patients in need thereof are provided. More specifically, Factor VIII (FVIII) encoding nucleic acids which modulate (e.g., increase) hemostasis are provided. In certain embodiments, the FVIII encoding nucleic acids are codon-optimized. In certain embodiments, the FVIII encoding nucleic acids are contained in a vector, particularly a viral vector such as an AAV vector. Compositions comprising at least one FVIII encoding nucleic acid of the instant invention and at least one pharmaceutically acceptable carrier are also provided.

[0011] The invention also includes gene therapy methods. The invention also includes methods for the treatment of a hemostasis related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the FVIII encoding nucleic acids, particularly in AAV vectors, particularly within a pharmaceutical composition. Such methods have efficacy in the treatment of disorders where a procoagulant is needed and include, without limitation, hemophilia, particularly hemophilia A.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 provides a graph of FVIII activity for FVIII expressed in vitro from CHOPF8 novel expression cassettes. FVIII expression and secretion was evaluated following transient transfection of CHOPF8 plasmids in Huh-7 cells after 48 hours in culture. Mock-transfected cells (-) and positive control transfected cells were included for comparison. Brown -Forsythe and Welch ANOVA tests were used to determine differences between CHOPF8 constructs and the positive control FVIIFC levels. Significant differences are indicated: * p<0.05, ** p<0.01. Figures 2A and 2B show the in vivo FVIII expression from AAV vectors carrying CHOPF8 cassettes. HA / CD4K0 mice treated with AAV8 vectors carrying CHOPF8 expression cassettes that expressed 50% or more FVIII than the positive control expression cassette in Huh-7 cells. FVIII Ag was measured in plasma 8 weeks after vector infusion. Fig. 2A: CHOPF8 expression cassettes were initially screened in mice at a dose of 2 x 1012vg / kg. Significant differences in FVIII antigen between the CHOPF8 expression cassettes vs. the positive control were determined by Brown-Forsythe and Welch ANOVA tests. Fig. 2B: Based on expression levels achieved in Fig. 2A, CHOPF8-1, 8, 9, and 10 were chosen for further dose titration. Additional cohorts of mice were treated with 4 x 1011or 2 xlO11vg / kg and steady-state plasma FVIII expression level at 8 weeks was measured. Significant differences in FVIII Ag level between CHOPF8 expression cassettes at each dose level were determined by two-way ANOVA with Sidak’s multiple comparisons tests. * p<0.05, ** <0.01, *** <0.001. Figure 2C provides a dose response summary for CHOPF8-1, 8, 9, and 10. Shaded area indicates normal range.

[0014] Figures 3 A and 3B show liver FVIII mRNA levels and vector copy number analysis, respectively. HA / CD4KO mice treated with AAV8-CHOPF8-1 (dark bar) and AAV8-CHOP8-10 (light bar) were sacrificed 8 weeks post-gene transfer and livers were harvested. Liver FVIII mRNA and vector copy number per diploid genome (VCN) were quantified. Two way ANOVA with Sidak’s multiple comparisons test was used to determine statistical differences within in each dose level group between CHOPF8-1 and CHOPF8-10. Each point represents an individual animal and mean ± SEM is plotted.

[0015] Figure 4 provides an amino acid sequence of FVIII (SEQ ID NO: 1). The B domain is indicated with italics and bolding. The provided amino acid sequence contains the 19 amino acid signal peptide at the N-terminus (MQIELSTCFFLCLLRFCFS (SEQ ID NO: 2)).

[0016] Figures 5A and 5B provide the nucleotide sequence of CHOPF8-1 (SEQ ID NO:

[0017] 3). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 20) with the B-domain linker coding sequence underlined (SEQ ID NO: 34). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0018] Figures 6A and 6B provide the nucleotide sequence of CHOPF8-2 (SEQ ID NO:

[0019] 4). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 21) with the B-domain linker coding sequence underlined (SEQ ID NO: 35). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0020] Figures 7A and 7B provide the nucleotide sequence of CHOPF8-3 (SEQ ID NO:

[0021] 5). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 22) with the B-domain linker coding sequence underlined (SEQ ID NO: 36). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0022] Figures 8A and 8B provide the nucleotide sequence of CHOPF8-4 (SEQ ID NO:

[0023] 6). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 23) with the B-domain linker coding sequence underlined (SEQ ID NO: 37). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0024] Figures 9A and 9B provide the nucleotide sequence of CHOPF8-5 (SEQ ID NO:

[0025] 7). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 24) with the B-domain linker coding sequence underlined (SEQ ID NO: 38). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0026] Figures 10A and 10B provide the nucleotide sequence of CHOPF8-6 (SEQ ID NO: 8). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 25) with the B-domain linker coding sequence underlined (SEQ ID NO: 39). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0027] Figures 11 A and 1 IB provide the nucleotide sequence of CHOPF8-7 (SEQ ID NO: 9). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 26) with the B-domain linker coding sequence underlined (SEQ ID NO: 40). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33). Figures 12A and 12B provide the nucleotide sequence of CHOPF8-8 (SEQ ID NO: 10). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 27) with the B-domain linker coding sequence underlined (SEQ ID NO: 41). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0028] Figures 13 A and 13B provide the nucleotide sequence of CHOPF8-9 (SEQ ID NO: 11). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 28) with the B-domain linker coding sequence underlined (SEQ ID NO: 42). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0029] Figures 14A and 14B provide the nucleotide sequence of CHOPF8-10 (SEQ ID NO: 12). The 5’ ITR (SEQ ID NO: 30) and 3’ ITR (SEQ ID NO: 31) are shaded. The transthyretin (TTR) promoter (SEQ ID NO: 32) is indicated with italics and underlining. The FVIII coding sequence is bolded (SEQ ID NO: 29) with the B-domain linker coding sequence underlined (SEQ ID NO: 43). The polyadenylation (pA) signal is underlined (SEQ ID NO: 33).

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders due to inheritable deficiencies 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 the residual factor activity: people with severe disease (factor activity <1% normal) have frequent spontaneous bleeds; people with moderate disease (factor activity l%-5% normal) rarely have spontaneous bleeds, but bleed with minor trauma; and people with mild disease (factor activity 5%-40% normal) bleed during invasive procedures or trauma. Given this well-defined relationship between factor activity and bleeding phenotype, HA and HB are attractive targets for protein infusion or gene therapy as increases in factor levels have a meaningful clinical impact. As explained above, Factor VIII is central for coagulation activity and mutations in the FVIII gene result in hemophilia A, the most common form of hemophilia. Full-length FVIII is a large, 280-kDa protein primarily expressed in liver sinusoidal endothelial cells (LSECs), as well as extra-hepatic endothelial cells (Fahs, et al., Blood (2014) 123:3706-3713; Everett, et al., Blood (2014) 123:3697-3705). FVIII predominantly circulates as a heterodimer of a heavy chain and a light chain bound through noncovalent metal-dependent interactions (Lenting, et al., Blood (1998) 92:3983- 3996). F Villa is a cofactor for FIXa within the intrinsic Xase complex which functions to generate FXa, leading to the propagation of the coagulation cascade.

[0032] Factor VIII comprises several domains and is 2332 amino acids in length (mature without the 19 amino acid signal peptide at the N-terminus). Generally, the domains are referred to as A1-A2-B-A3-C1-C2. FVIII is translated as a single-peptide chain (single chain) with the domain structure of Al-al-A2-a2-B-a3-A3-Cl-C2. Proteolytic cleavage of FVIII at R-1313 and / or R-1648 by the trans-Golgi protease furin results in heterodimer formation. The FVIII heavy chain (Al-al-A2-a2-B) and light chain (a3-A3-Cl-C2) remain associated through non-covalent metal-ion-dependent interactions occurring between the Al and A3 domains. Initially, FVIII is in an inactive form bound to von Willebrand factor (vWF). FVIII is activated by cleavage by thrombin (Factor Ila) and release of the B domain. The activated form of FVIII (FVIIIa) separates from vWF and interacts with coagulation factor Factor IXa - leading to the formation of a blood clot via a coagulation cascade. During coagulation, FVIII single chain or heterodimer is activated to its heterotrimeric cofactor form by cleavage by thrombin at R-372, R-740, and R-1689. A2 remains associated with Al -al via non-covalent interactions. Inactivation of FVIIIa occurs via spontaneous A2 dissociation and / or proteolytic cleavage, primarily by activated protein C, at R-336 and R-562. In a particular embodiment, the FVIII comprises Al-al-A2-a2-B-a3-A3-Cl-C2. In a particular embodiment, the FVIII comprises Al-al-A2-a2-a3-A3-Cl-C2. In a particular embodiment, the FVIII comprises Al-al-A2-a2-A3-Cl-C2. In a particular embodiment, the FVIII comprises a light chain and a heavy chain.

[0033] The B domain comprises 40% of the protein (908 amino acids) and is not required for the protein procoagulant activity (Brinkhous, et al., Proc. Natl. Acad. Sci. (1985) 82:8752-8756). The most common B-domain deleted (BDD) FVIII comprises 14 original amino acid residues (SFSQNPPVLKRHQR; SEQ ID NO 13) as a linker (Lind, et al. (1995) Eur. J. Biochem., 232(1): 19-27). This BDD FVIII is typically referred to as BDD- SQ or hFVIII-SQ. Short peptide linkers (e.g., 25 or fewer amino acids, 20 or fewer amino acids, 15 or fewer amino acids, or 10 or fewer amino acids) substituted for the B- domain can be used in FVIII (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 a particular embodiment, the peptide linker comprises a basic amino acid (e.g., Arg, His, or Lys) at position -1 and -4 to Glul649. This BDD FVIII form is commonly used to produce recombinant BDD-FVIII as well for gene therapy (Berntorp, E., Semin. Hematol. (2001) 38(2 Suppl 4): 1-3; Gouw, et al., N. Engl. J. Med. (2013) 368:231-239; Xi, et al., J. Thromb. Haemost. (2013) 11 : 1655-1662; Recht, et al., Haemophilia (2009) 15:869-880; Sabatino, et al., Mol. Ther. (2011) 19:442-449; Scallan, et al., Blood (2003) 102:2031-2037). As noted above, gene therapy using AAV vectors can only use shortened FVIII molecules such as a BDD-FVIII due to the limited packaging capacity of the AAV (~4.7 Kb) and other vector systems (Lind, et al. (1995) Eur. J. Biochem., 232(1): 19-27). WO 2020 / 086686 and U.S. Patent 8,816,054, each incorporated by reference herein, also provide BDD FVIII molecules with linkers of different lengths and sequences.

[0034] Hemophilia A gene therapy has demonstrated proof-of-concept success leading to now one FDA approved hemophilia A adeno-associated viral vector. Despite these early successes, there are multiple limitations of first-generation approaches that may be overcome by, among other possibilities, incorporating of a gain-of-function FVIII transgene. Here, codon-optimized cassettes that are approximately 4.65 kb are provided that can be efficiently packaged within an AAV vector. The vectors express FVIII at low levels of systemic AAV vector administration. The optimized AAV8-FVIII vector permits the use of lower vector doses clinically, thereby limiting anti -vector immune responses.

[0035] A novel vector cassette (CHOPF8) is presented herein. The vector cassette contains a promoter (particularly a liver specific promoter (e.g., TTR promoter)), a FVIII codon optimized transgene, and a poly A tail. Ten codon-optimized FVIII transgene sequences were introduced into the novel cassette that is approximately 4.65 kb between the inverted terminal repeats (ITRs). Cassettes were initially screened by measuring secreted FVIII activity following transient transfection of CHOPF8 plasmids into Huh7 cells. Subsequent screening in vivo was performed following packaging into AAV8. Male HA / CD4KO 8-12 week old mice were intravenously infused with AAV8-FVIII vectors manufactured using the CHOPF8 expression cassettes. After an initial screen of plasma FVIII antigen levels, the liver FVIII mRNA levels, and liver vector copy numbers were measured 8 weeks after vector administration for lead candidates. The novel CHOPF8 expression cassettes expressed FVIII both in vitro and in vivo. At 2 xlO12vg / kg, AAV8-FVIII expression was 2-fold higher than a FVIII codon optimized AAV8-FVIII control vector. CHOPF8 cassettes were manufactured into AAV8 vectors and FVIII expression was evaluated at multiple dose cohorts for which expression from the CHOPF8-1 cassette was the highest. The AAV8 vectors manufactured from the lead candidate cassettes expressed the highest levels of FVIII and demonstrate the same vector genomes per diploid genome in the liver, i.e. the target tissue.

[0036] In accordance with the instant invention, nucleic acids encoding Factor VIII are provided. In certain embodiments, the nucleic acids are adeno-associated viruses (AAV) vectors or FVIII expression cassettes. In certain embodiments, the nucleic acid has a length less than about 5 kb, less than about 4.9 kb, or less than about 4.8 kb. In certain embodiments, the nucleic acid has a length between ITRs of less than about 4.70 kb, less than about 4.69 kb, less than about 4.675 kb, less than about 4.67 kb, less than about 4.66 kb, or less than about 4.65 kb.

[0037] In certain embodiments, the nucleic acid of the instant invention comprises a sequence encoding Factor VIII. In certain embodiments, the nucleic acid comprises a sequence encoding Factor VIII and a promoter (e.g., operably linked). In certain embodiments, the nucleic acid comprises a sequence encoding Factor VIII and a polyadenylation signal (e.g., operably linked). In certain embodiments, the nucleic acid comprises a sequence encoding Factor VIII, a promoter (e.g., operably linked), and a polyadenylation signal (e.g., operably linked). In certain embodiments, the nucleic acid comprises inverted terminal repeats (ITRs (e.g., AAV ITRs)) (e.g., a 5’ and 3’ ITR). In certain embodiments, the nucleic acid comprises a sequence encoding Factor VIII, a promoter (e.g., operably linked), and inverted terminal repeats (ITRs (e.g., AAV ITRs)) (e.g., a 5’ and 3’ ITR). In certain embodiments, the nucleic acid comprises a sequence encoding Factor VIII, a polyadenylation signal (e.g., operably linked), and inverted terminal repeats (ITRs (e.g., AAV ITRs)) (e.g., a 5’ and 3’ ITR). In certain embodiments, the nucleic acid comprises a sequence encoding Factor VIII, a promoter (e.g., operably linked), a polyadenylation signal (e.g., operably linked), and inverted terminal repeats (ITRs (e.g., AAV ITRs)) (e g., a 5’ and 3’ ITR).

[0038] In certain embodiments, the FVIII of the instant invention is human. Gene ID: 2157 and GenBank Accession Nos. NM_000132.3 and NP_000123.1 provide examples of the amino acid and nucleotide sequences of wild-type human FVIII (particularly the prepeptide comprising the signal peptide). Figure 4 provides an example of the amino acid sequence of wild-type human FVIII (SEQ ID NO: 1) including the 19 amino acid signal peptide at its N-terminus (MQIELSTCFFLCLLRFCFS; SEQ ID NO: 2).

[0039] In certain embodiments, the sequence encoding FVIII of the instant invention is not the wild-type or native sequence encoding human FVIII. In certain embodiments, the sequence encoding FVIII of the instant invention is a codon-optimized sequence, particularly for expression in human cells, particularly human liver cells or hepatocytes. In certain embodiments, the sequence encoding FVIII of the instant invention encodes wild-type FVIII (e.g., Figure 4; SEQ ID NO: 1). In certain embodiments, the sequence encoding FVIII of the instant invention encodes a FVIII variant (e.g., substitution mutations), wherein the sequence encoding FVIII is the same as that set forth herein except that the codon(s) encoding the mutation(s) of the variant are changed to the required codon. FVIII variants are known in the art (see, e.g., WO 2024 / 124227; WO 2023 / 212539; WO 2021 / 113800; and WO 2020 / 086686, each incorporated herein by reference).

[0040] In certain embodiments, the sequence encoding FVIII does not encode for a full length B domain. In certain embodiments, the sequence encoding FVIII encodes for a linker in place of the B domain (see, e.g., Figure 4). In certain embodiments, the peptide linker is up to about 25, up to about 20, up to about 15, up to about 10, or up to about 5 amino acids in length. In certain embodiments, the peptide linker comprises amino acid sequences (e.g., two sequences (e.g., one from the N terminus and one from the C- terminus)) from the B domain or is a fragment of the B domain. Examples of peptide linkers are provided, for example, in WO 2020 / 086686 and U.S. Patent 8,816,054. In certain embodiments, the peptide linker comprises a basic amino acid (e.g., Arg, His, or Lys) at positions -1 and -4. In certain embodiments, the peptide linker comprises the sequence RHQR (SEQ ID NO: 14) or HHQR (SEQ ID NO: 15) at the C-terminus. In certain embodiments, the peptide linker comprises SFSQNPPVLKRHQR (SEQ ID NO: 13). In certain embodiments, the linker has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SFSQNPPVLKRHQR (SEQ ID NO: 13), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity with SFSQNPPVLKRHQR (SEQ ID NO: 13).

[0041] In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12; SEQ ID NOs: 20-29). In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in any one of CH0PF8-1 (SEQ ID NO: 3; SEQ ID NO: 20), CHOPF8-3 (SEQ ID NO: 5; SEQ ID NO: 22), CHOPF8-4 (SEQ ID NO: 6; SEQ ID NO: 23), CHOPF8-5 (SEQ ID NO: 7; SEQ ID NO: 24), CHOPF8-6 (SEQ ID NO: 8; SEQ ID NO: 25), CHOPF8-8 (SEQ ID NO: 10; SEQ ID NO: 27), CHOPF8-9 (SEQ ID NO: 11; SEQ ID NO: 28), and CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14). In certain embodiments, the sequence encoding FVIII comprises SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SE ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29. In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20), CHOPF8-8 (SEQ ID NO: 10; SEQ ID NO: 27), or CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figures 5, 12, and 14). In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in CHOPF8-1 (see, Figure 5; SEQ ID NO: 3; SEQ ID NO: 20). In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in CHOPF8-10 (see, Figure 14; SEQ ID NO: 12; SEQ ID NO: 29).

[0042] In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12; SEQ ID NOs: 20-29), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20), CHOPF8-3 (SEQ ID NO: 5; SEQ ID NO: 22), CHOPF8-4 (SEQ ID NO: 6; SEQ ID NO: 23), CHOPF8-5 (SEQ ID NO: 7; SEQ ID NO: 24), CHOPF8-6 (SEQ ID NO: 8; SEQ ID NO: 25), CHOPF8-8 (SEQ ID NO: 10; SEQ ID NO: 27), CHOPF8-9 (SEQ ID NO: 11; SEQ ID NO: 28), and CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29, particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20), CHOPF8-8 (SEQ ID NO: 10; SEQ ID NO: 27), or CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figures 5, 12, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8-1 (see, Figure 5, SEQ ID NO: 3; SEQ ID NO: 20), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8- 10 (see, Figure 14, SEQ ID NO: 12; SEQ ID NO: 29), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity.

[0043] The linker in place of the B domain encoded by any one of CHOPF8-1 to CHOPF8-10 can be replaced with a different peptide linker as set forth hereinabove. For example, the sequence encoding FVIII of the instant invention may comprise the sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12; SEQ ID NOs: 20-29), with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker. In certain embodiments, the sequence encoding FVIII of the instant invention comprises any one of SEQ ID NOs: 20-29, with the exception that the sequence encoding the peptide linker - SEQ ID NOs: 34-43, respectively - is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20), CHOPF8-3 (SEQ ID NO: 5; SEQ ID NO: 22), CHOPF8-4 (SEQ ID NO: 6; SEQ ID NO: 23), CHOPF8-5 (SEQ ID NO: 7; SEQ ID NO: 24), CHOPF8-6 (SEQ ID NO: 8; SEQ ID NO: 25), CHOPF8-8 (SEQ ID NO: 10; SEQ ID NO: 27), CHOPF8-9 (SEQ ID NO: 11; SEQ ID NO: 28), and CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14), with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20), CHOPF8-8 (SEQ ID NO: 10; SEQ ID NO: 27), or CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figures 5, 12, and 14), with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20) (see, Figure 5), with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the sequence encoding FVIII comprises the sequence set forth in CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figure 14), with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43).

[0044] In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12; SEQ ID NOs: 20-29), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity, with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20), CHOPF8-3 (SEQ ID NO: 5; SEQ ID NO: 22), CHOPF8-4 (SEQ ID NO: 6; SEQ ID NO: 23), CHOPF8-5 (SEQ ID NO: 7; SEQ ID NO: 24), CHOPF8-6 (SEQ ID NO: 8; SEQ ID NO: 25), CHOPF8-8 (SEQ ID NO: 10; SEQ ID NO: 27), CHOPF8-9 (SEQ ID NO: 11; SEQ ID NO: 28), and CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity, with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20), CHOPF8-8 (SEQ ID NO: 10; SEQ ID NO: 27), or CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figures 5, 12, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity, with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8-1 (SEQ ID NO: 3; SEQ ID NO: 20) (see, Figure 5), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity, with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the sequence encoding FVIII has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8-10 (SEQ ID NO: 12; SEQ ID NO: 29) (see, Figure 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity, with the exception that the sequence encoding the peptide linker (e.g., SFSQNPPVLKRHQR; SEQ ID NO: 13) is replaced with a sequence encoding a different peptide linker or a different sequence encoding the same peptide linker (e.g., one of SEQ ID NOs: 34-43). In certain embodiments, the peptide linker replacing the B domain is not included in the calculation for percent identity.

[0045] In certain embodiments, the promoter is a liver specific promoter. In certain embodiments, the promoter is human. Examples of liver specific promoters include, without limitation: thyroxine binding globulin (TBG) promoter, transthyretin (TTR) promoter, phosphoglycerate kinase (PGK), hybrid liver-specific promoter (HLP), alpha- 1 -antitrypsin (AAT), albumin promoter, and hepatitis B virus core protein promoter. In certain embodiments, the promoter is the transthyretin (TTR) promoter. In certain embodiments, the promoter comprises the promoter sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12). In certain embodiments, the promoter has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the promoter sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the promoter comprises or has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 32. In certain embodiments, the polyadenylation signal or sequence causes the addition of a poly A tail, which stabilizes the mRNA within the cell. In certain embodiments, the length of the polyadenylation sequence is about 60 or fewer nucleotides, about 55 or fewer nucleotides, about 50 or fewer nucleotides, about 45 or fewer nucleotides, or about 40 or fewer nucleotides. In certain embodiments, the polyadenylation sequence comprises an AATAAA sequence, a GT rich sequence (e.g., GTGTGTTGG), and a T rich sequence (e g., TTTTTT or TTTTTTGTGTG (SEQ ID NO: 16)). In certain embodiments, the polyadenylation sequence comprises the polyadenylation sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12). In certain embodiments, the polyadenylation sequence has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the polyadenylation sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the polyadenylation sequence comprises or has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 33.

[0046] In certain embodiments, the inverted terminal repeats (ITRs) are AAV ITRs. The AAV ITRs can be from any serotype AAV. In certain embodiments, the ITRs are from any one of AAV1 to AAV8, particularly AAV1, AAV2, AAV3, AAV4, AAV6, or AAV7. In certain embodiments, the ITRs are from AAV2 or AAV3. In certain embodiments, the ITRs are hybrids (e.g., AAV2-5 ITR wherein an AAV2 ITR and an AAV5 ITR were used in a single vector). In certain embodiments, the ITRs are ITR2 (e.g., from AAV2). In certain embodiments, the ITR sequences (5’ and 3’ ITR) comprise the ITR sequences (5’ and 3’ ITR) set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12). In certain embodiments, the ITR sequences (5’ and 3’ ITR) have at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the ITR sequences (5’ and 3’ ITR) set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the 5’ ITR sequence comprises or has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 30. In certain embodiments, the 3’ ITR sequence comprises or has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with SEQ ID NO: 31. In certain embodiments, the nucleic acid comprises the sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12). In certain embodiments, the nucleic acid comprises the sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3), CHOPF8-3 (SEQ ID NO: 5), CHOPF8-4 (SEQ ID NO: 6), CHOPF8-5 (SEQ ID NO: 7), CHOPF8-6 (SEQ ID NO: 8), CHOPF8-8 (SEQ ID NO: 10), CHOPF8-9 (SEQ ID NO: 11), and CHOPF8-10 (SEQ ID NO: 12) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14). In certain embodiments, the nucleic acid comprises the sequence set forth in CHOPF8-1 (SEQ ID NO: 3), CHOPF8-8 (SEQ ID NO: 10), or CHOPF8-10 (SEQ ID NO: 12) (see, Figures 5, 12, and 14). In certain embodiments, the nucleic acid comprises the sequence set forth in CHOPF8-1 (SEQ ID NO: 3) (see, Figure 5). In certain embodiments, the nucleic acid comprises the sequence set forth in CHOPF8-10 (SEQ ID NO: 12) (see, Figure 14). In certain embodiments, the nucleic acid has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3- 12), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3), CHOPF8-3 (SEQ ID NO: 5), CHOPF8-4 (SEQ ID NO: 6), CHOPF8-5 (SEQ ID NO: 7), CHOPF8-6 (SEQ ID NO: 8), CHOPF8-8 (SEQ ID NO: 10), CHOPF8-9 (SEQ ID NO: 11), and CHOPF8-10 (SEQ ID NO: 12) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8-1 (SEQ ID NO: 3), CHOPF8-8 (SEQ ID NO: 10), or CHOPF8-10 (SEQ ID NO: 12) (see, Figures 5, 12, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8-1 (see, Figure 5; SEQ ID NO: 3), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the sequence set forth in CHOPF8-10 (see, Figure 14; SEQ ID NO: 12), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity.

[0047] The instant invention also encompasses nucleic acids encoding FVIII wherein the sequence encoding the heavy chain (with or without the B domain or linker) is selected from CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12; SEQ ID NOs: 20-29) or is wild-type and the sequence encoding the light chain is selected from CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12; SEQ ID NOs: 20-29) or is wild-type, but is from a different construct than the sequence encoding heavy chain (and both the heavy chain and light chain are not wild-type). In certain embodiments, the B domain or linker is as described hereinabove.

[0048] In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) comprises the heavy chain sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3 or 20), CHOPF8-3 (SEQ ID NO: 5 or 22), CHOPF8-4 (SEQ ID NO: 6 or 23), CHOPF8-5 (SEQ ID NO: 7 or 24), CHOPF8-6 (SEQ ID NO: 8 or 25), CHOPF8-8 (SEQ ID NO: 10 or 27), CHOPF8-9 (SEQ ID NO: 11 or 28), and CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14). In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) comprises the heavy chain sequence set forth in CHOPF8-1 (SEQ ID NO: 3 or 20), CHOPF8-8 (SEQ ID NO: 10 or 27), or CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figures 5, 12, and 14). In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) comprises the heavy chain sequence set forth in CHOPF8-1 (SEQ ID NO: 3 or 20) (see, Figure 5). In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) comprises the heavy chain sequence set forth in CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figure 14). In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the heavy chain sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12; SEQ ID NOs: 20-29), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the heavy chain sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3 or 20), CHOPF8-3 (SEQ ID NO: 5 or 22), CHOPF8-4 (SEQ ID NO: 6 or 23), CHOPF8-5 (SEQ ID NO: 7 or 24), CHOPF8-6 (SEQ ID NO: 8 or 25), CHOPF8-8 (SEQ ID NO: 10 or 27), CHOPF8-9 (SEQ ID NO: 11 or 28), and CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the heavy chain sequence set forth in CHOPF8- 1 (SEQ ID NO: 3 or 20), CHOPF8-8 (SEQ ID NO: 10 or 27), or CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figures 5, 12, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the heavy chain sequence set forth in CHOPF8-1 (see, Figure 5; SEQ ID NO: 3 or 20), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid encoding the heavy chain (with or without the B domain or linker) has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the heavy chain sequence set forth in CHOPF8-10 (see, Figure 14; SEQ ID NO: 12 or 29), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity.

[0049] In certain embodiments, the nucleic acid encoding the light chain comprises the light chain sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3 or 20), CHOPF8- 3 (SEQ ID NO: 5 or 22), CHOPF8-4 (SEQ ID NO: 6 or 23), CHOPF8-5 (SEQ ID NO: 7 or 24), CHOPF8-6 (SEQ ID NO: 8 or 25), CHOPF8-8 (SEQ ID NO: 10 or 27), CHOPF8- 9 (SEQ ID NO: 11 or 28), and CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14). In certain embodiments, the nucleic acid encoding the light chain comprises the light chain sequence set forth in CHOPF8-1 (SEQ ID NO: 3 or 20), CHOPF8-8 (SEQ ID NO: 10 or 27), or CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figures 5, 12, and 14). In certain embodiments, the nucleic acid encoding the light chain comprises the light chain sequence set forth in CHOPF8-1 (SEQ ID NO: 3 or 20) (see, Figure 5). In certain embodiments, the nucleic acid encoding the light chain comprises the light chain sequence set forth in CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figure 14). In certain embodiments, the nucleic acid encoding the light chain has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the light chain sequence set forth in any one of CHOPF8-1 to CHOPF8-10 (see, Figures 5-14; SEQ ID NOs: 3-12; SEQ ID NOs: 20-29), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid encoding the light chain has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the light chain sequence set forth in any one of CHOPF8-1 (SEQ ID NO: 3 or 20), CHOPF8-3 (SEQ ID NO: 5 or 22), CHOPF8-4 (SEQ ID NO: 6 or 23), CHOPF8-5 (SEQ ID NO: 7 or 24), CHOPF8-6 (SEQ ID NO: 8 or 25), CHOPF8-8 (SEQ ID NO: 10 or 27), CHOPF8-9 (SEQ ID NO: 11 or 28), and CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figures 5, 7, 8, 9, 10, 12, 13, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid encoding the light chain has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the light chain sequence set forth in CHOPF8-1 (SEQ ID NO: 3 or 20), CHOPF8-8 (SEQ ID NO: 10 or 27), or CHOPF8-10 (SEQ ID NO: 12 or 29) (see, Figures 5, 12, and 14), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid encoding the light chain has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the light chain sequence set forth in CHOPF8-1 (see, Figure 5; SEQ ID NO: 3 or 20), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity. In certain embodiments, the nucleic acid encoding the light chain has at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology or identity with the light chain sequence set forth in CHOPF8-10 (see, Figure 14; SEQ ID NO: 12 or 29), particularly at least 90%, 95%, 97%, 99%, or 100% homology or identity.

[0050] Nucleic acids of the instant invention may be prepared by any method known in the art. Nucleic acids of the invention may be prepared by using recombinant DNA technology methods. The nucleic acids may be maintained in any convenient vector, particularly an expression vector (e.g., for expression in mammalian cells, particularly human cells). The nucleic acids may be maintained in a cloning vector. The nucleic acids may be DNA or RNA and may be single- or double-stranded.

[0051] Compositions comprising at least one nucleic acid of the instant invention and at least one carrier (e.g., pharmaceutically acceptable carrier) are also encompassed by the instant invention. Compositions comprising at least one viral vector (e.g., viral particle) of the instant invention and at least one carrier (e.g., pharmaceutically acceptable carrier) are also encompassed by the instant invention. Except insofar as any conventional carrier is incompatible with the nucleic acid to be administered, its use in the pharmaceutical composition is contemplated. In a particular embodiment, the carrier is a pharmaceutically acceptable carrier for intravenous administration.

[0052] The present invention also encompasses method of making a polypeptide (FVIII). In certain embodiments, the method includes expression from nucleic acids encoding the polypeptide. This may conveniently be achieved by culturing a host cell, containing such a vector, under appropriate conditions which cause or allow production of the polypeptide. Polypeptides may also be produced in in vitro.

[0053] Nucleic acids of the instant invention may be used, for example, as therapeutic and / or prophylactic agents which modulate the blood coagulation cascade. The nucleic acids of the instant invention may be administered in a therapeutically effective amount to modulate (e.g., increase) hemostasis and / or form a clot and / or stop or inhibit bleeding or aberrant bleeding. It is demonstrated herein that the nucleic acids possess superior properties and can provide effective hemostasis.

[0054] In certain embodiments, nucleic acids may be administered to a patient via infusion in a biologically compatible carrier, e.g., via injection or intravenous injection. The nucleic acids of the invention may optionally be encapsulated into liposomes or mixed with other phospholipids or micelles to increase stability. Nucleic acids may be administered alone or in combination with other agents known to modulate hemostasis (e.g., vFW, Factor IX, Factor IXa, etc.). An appropriate composition in which to deliver the nucleic acids may be determined by a medical practitioner upon consideration of a variety of physiological variables, including, but not limited to, the patient’s condition and hemodynamic state. A variety of compositions well suited for different applications and routes of administration are well known in the art and are described hereinbelow.

[0055] The preparation containing the nucleic acids may contain a physiologically acceptable matrix and is formulated as a pharmaceutical preparation. The preparation can be formulated using substantially known methods, it can be mixed with a buffer containing salts, such as NaCl, CaCh, and amino acids, such as glycine and / or lysine, and in a pH range from 6 to 8. Until needed, the purified preparation containing the nucleic acids can be stored in the form of a finished solution or in lyophilized or deep-frozen form. In a particular embodiment, the preparation is stored in lyophilized form and is dissolved into a visually clear solution using an appropriate reconstitution solution. Alternatively, the preparation according to the present invention can also be made available as a liquid preparation or as a liquid that is deep-frozen. The preparation according to the present invention may be especially stable, i.e., it can be allowed to stand in dissolved form for a prolonged time prior to application.

[0056] The preparation according to the present invention can be made available as a pharmaceutical preparation with the nucleic acid in the form of a one-component preparation or in combination with other factors in the form of a multi-component preparation.

[0057] Nucleic acids of the instant invention may be used for a variety of purposes in accordance with the present invention such as gene therapy and / or gene editing. In a particular embodiment of the invention, a nucleic acid delivery vehicle (e.g., an expression vector such as a viral vector) for modulating blood coagulation is provided wherein the expression vector comprises a nucleic acid sequence coding for FVIII as described herein. Administration of the FVIII encoding expression vectors to a patient results in the expression of the FVIII which serves to alter the coagulation cascade. In accordance with the present invention, a FVIII encoding nucleic acid sequence may encode a polypeptide as described herein whose expression increases hemostasis. In a particular embodiment, the nucleic acid sequence encodes a human FVIII.

[0058] Expression vectors comprising nucleic acid sequences may be administered alone, or in combination with other molecules useful for modulating hemostasis. According to the present invention, the expression vectors or combination of therapeutic agents may be administered to the patient alone or in a pharmaceutically acceptable or biologically compatible composition.

[0059] In a particular embodiment of the invention, the expression vector comprising nucleic acid sequences encoding the FVIII is a viral vector or non-viral vector. Viral vectors which may be used in the present invention include, but are not limited to, adenoviral vectors (with or without tissue specific promoters / enhancers), 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, lentivirus vectors and pseudo-typed lentivirus vectors (e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)), herpes simplex virus vectors, vaccinia virus vectors, and retroviral vectors. Examples of non-viral means include, without limitation, gene delivery by lipid nanoparticles. In a particular embodiment, the vector is an adeno- associated virus (AAV) vector. In a particular embodiment, the vector is a lentiviral vector.

[0060] The instant invention also encompasses gene therapy methods (e.g., for modulating hemostasis) with the nucleic acids of the instant invention. In a particular embodiment of the present invention, methods are provided for the administration of a viral vector comprising nucleic acids. Viral (e.g., AAV) vectors of utility in the methods of the present invention preferably include at least the essential parts of viral (e.g., AAV) vector DNA. As described herein, expression of a FVIII following administration of such a viral (e.g., AAV) vector (particles) serves to modulate hemostasis, particularly to enhance the procoagulation activity of the protease.

[0061] Recombinant viral (e.g., AAV) vectors have found broad utility for a variety of gene therapy applications. Their utility for such applications is due largely to the high efficiency of in vivo gene transfer achieved in a variety of organ contexts. AAV particles may be used to advantage as vehicles for adequate gene delivery. Such virions possess a number of desirable features for such applications, including: structural features related to being a double stranded DNA nonenveloped virus and biological features such as a tropism for the human respiratory system and gastrointestinal tract. Moreover, AAV are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis. Attesting to the overall safety of AAV vectors, infection with AAV leads to a minimal disease state in humans comprising mild flu-like symptoms.

[0062] Viral (e.g., AAV) genomes are well suited for use as gene therapy vehicles because they can accommodate the insertion of foreign DNA following the removal of viral genes essential for replication and / or nonessential regions. Such substitutions render the viral vector impaired with regard to replicative functions and infectivity. Many viruses (e.g., AAV) have been used as vectors for gene therapy and for expression of heterologous genes.

[0063] It is desirable to introduce a vector that can provide, for example, multiple copies of a desired gene and hence greater amounts of the product of that gene. Improved viral (e.g., AAV) vectors and methods for producing these vectors have been described (e.g., Penn Vector Core; addgene; etc.).

[0064] For some applications, an expression construct may further comprise regulatory elements which serve to drive expression in a particular cell or tissue type and / or constitutively. Such regulatory elements are known to those of skill in the art. The incorporation of tissue specific regulatory elements in the expression constructs of the present invention provides for at least partial tissue tropism for the expression of the FVIII. In certain embodiments, a constitutive promoter (e.g., cytomegalovirus (CMV) promoter) may be used. Hematopoietic or liver specific promoters may also be used.

[0065] AAV for recombinant gene expression have been produced in human cells (e.g., the human embryonic kidney cell line 293). Briefly, AAV vectors may be engineered from wild-type AAV, a single-stranded DNA virus that is non-pathogenic. The parent virus is non-pathogenic, the vectors have a broad host range, and they can infect both dividing and non-dividing cells. The vector is typically engineered from the virus by deleting the rep and cap genes and replacing these with the transgene of interest under the control of a specific promoter. For recombinant AAV preparation, the upper size limit of the sequence that can be inserted between the two ITRs is about 4.7 kb. Plasmids expressing FVIII under the control of a promoter (e.g., the CMV promoter / enhancer) and a second plasmid supplying adenovirus helper functions along with a third plasmid containing the rep and cap genes (e.g., AAV-2 rep and cap genes) may be used to produce AAV vectors (e.g., AAV-2 vectors). Other AAV serotype cap genes (e.g., AAV-1, AAV- 6, or AAV-8 cap genes) may be expressed with other serotype rep genes and ITRs (e.g., AAV-2 rep gene and ITRs) to produce different vectors (e.g., Gao et al. (2002) Proc.

[0066] 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 may be purified by repeated CsCl density gradient centrifugation and the titer of purified vectors determined by quantitative dotblot hybridization. In a particular embodiment, vectors may be prepared by the Vector Core at The Children's Hospital of Philadelphia. In certain embodiments, the capsid protein is AAV8.

[0067] Also included in the present invention is a method for modulating hemostasis comprising providing cells of an individual with a nucleic acid delivery vehicle encoding a FVIII and allowing the cells to grow under conditions wherein the FVIII is expressed.

[0068] From the foregoing discussion, it can be seen that FVIII encoding nucleic acid molecules (e.g., FVIII expressing nucleic acid vectors) may be used in the treatment of disorders associated with aberrant blood coagulation.

[0069] FVIII encoding nucleic acid molecules (e.g., expression vectors) of the present invention may be incorporated into pharmaceutical compositions that may be delivered to a subject, so as to allow production of a biologically active protein (e.g., a FVIII) or by inducing expression of the FVIII in vivo by gene- and or cell-based therapies or by ex vivo modification / transduction of the patient's or donor's cells. The FVIII encoding nucleic acid molecules may be used for gene addition or gene editing to express the FVIII. In a particular embodiment of the present invention, pharmaceutical compositions comprising sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a FVIII can influence hemostasis in the subject. Alternatively, as discussed above, an effective amount of the FVIII may be directly infused into a patient in need thereof. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents (e.g., co-factors) which influence hemostasis.

[0070] In particular embodiments, the compositions (e.g., pharmaceutical compositions) of the instant invention also contain a pharmaceutically acceptable carrier. Such carriers include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences.

[0071] Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0072] The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding, free base forms. In other cases, the preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0073] After pharmaceutical compositions have been prepared, they may be placed in an appropriate container and labeled for treatment. For administration of FVIII encoding nucleic acids (e.g., vectors), such labeling could include amount, frequency, and method of administration.

[0074] Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using the techniques and guidance provided in the present invention. Therapeutic doses will depend on, among other factors, the age and general condition of the subject, the severity of the aberrant blood coagulation phenotype, and the strength of the control sequences regulating the expression levels of the polypeptide. Thus, a therapeutically effective amount in humans will fall in a relatively broad range that may be determined by a medical practitioner based on the response of an individual patient to vector-based treatment.

[0075] The FVIII, alone or in combination with other agents, may be directly infused into a patient in an appropriate biological / pharmaceutical carrier as described hereinabove. Expression vectors of the present invention comprising nucleic acid sequences encoding FVIII, may be administered to a patient by a variety of means (see below) to achieve and maintain a prophylactically and / or therapeutically effective level of the polypeptide. One of skill in the art could readily determine specific protocols for using the encoding expression vectors of the present invention for the therapeutic treatment of a particular patient.

[0076] FVIII encoding nucleic acids (e.g., AAV vectors) of the present invention may be administered to a patient by any means known. Direct delivery of the pharmaceutical compositions in vivo may generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery are envisioned. In this regard, the compositions may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intraarterially, orally, intrahepatically or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. In certain embodiments, the FVIII is administered by injection (e.g., to the bloodstream). In certain embodiments, the FVIII encoding nucleic acids (e.g., AAV vectors) is administered by injection (e.g., to the bloodstream or liver). A clinician specializing in the treatment of patients with blood coagulation disorders may determine the optimal route for administration of the vectors (e.g., AAV vectors) comprising nucleic acid sequences based on a number of criteria, including, but not limited to: the condition of the patient and the purpose of the treatment (e.g., reduced blood coagulation).

[0077] The present invention also encompasses vectors (e.g., viral vectors or AAV vectors) comprising a nucleic acid sequence encoding a FVIII. Also provided are lentiviruses or pseudo-typed lentivirus vectors comprising a nucleic acid sequence encoding a FVIII. Also encompassed are naked plasmid or expression vectors comprising a nucleic acid sequence encoding a FVIII.

[0078] Definitions

[0079] The following definitions are provided to facilitate an understanding of the present invention.

[0080] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0081] The phrase “hemostasis related disorder” refers to bleeding disorders such as, without limitation, hemophilia A, hemophilia B, hemophilia A and B patients, hemophilia with inhibitory antibodies, deficiencies in at least one coagulation factor (e.g., Factors VII, VIII, IX, X, XI, V, XII, II, and / or von Willebrand factor, particularly Factor VIII), combined FV / FVIII deficiency, vitamin K epoxide reductase Cl deficiency, gammacarboxylase deficiency, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocoagulability), disseminated intravascular coagulation (DIC), over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, or small molecule antithrombotics (e.g., FXa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency. In a particular embodiment, the term “hemostasis related disorder” refers to bleeding disorders characterized by excessive and / or uncontrolled bleeding (e.g., a disorder which can be treated with a procoagulant). In a particular embodiment, the hemostasis related disorder is hemophilia. In a particular embodiment, the hemostasis related disorder is hemophilia A.

[0082] With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates. For example, the “isolated nucleic acid” may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote. With respect to RNA molecules of the invention, the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form. With respect to protein, the term “isolated protein” is sometimes used herein. This term may refer to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated (e.g., so as to exist in “substantially pure” form). “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.

[0083] The term “vector” refers to a carrier nucleic acid molecule (e.g., RNA or DNA) into which a nucleic acid sequence can be inserted for introduction into a host cell where it will be replicated. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions (e.g., promoter) needed for expression in a host cell.

[0084] The term “operably linked” means that the regulatory sequences necessary for expression of a coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.

[0085] The term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.), particularly at least 75% by weight, or at least 90-99% or more by weight of the compound of interest. Purity may be measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0086] “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0087] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0088] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0089] The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

[0090] As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition (e.g., aberrant bleeding) resulting in a decrease in the probability that the subject will develop the condition.

[0091] A “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, treat, and / or lessen the symptoms of a particular disorder or disease.

[0092] The following example is provided to illustrate various embodiments of the present invention. The example is illustrative and is not intended to limit the invention in any way.

[0093] EXAMPLE

[0094] Materials and Methods

[0095] Vectors

[0096] Table 1 describes the recombinant AAV (rAAV) vectors compared herein. Figures 5-14 provide the nucleotide sequences of the CHOPF8 vectors. All CHOPF8 vectors contain a single-stranded expression cassette with a hepatocyte-specific to drive expression of a human FVIII transgene. The transgene expression cassettes are flanked by AAV2 inverted terminal repeats and packaged into an AAV8 capsid. All vectors contain a FVIII cDNA encoding an SQ variant of human B-domain-deleted FVIII. able 1: Vectors.

[0097] A FVIII codon optimization algorithm to enhance FVIII expression was applied to the FVIII transgene in the Control 1 expression cassette (McIntosh, et al. (2013) Blood 121 :3335-3344). rAAV packaging and quality control was performed at the Research Vector Core at CHOP using the triple transfection method with HEK293 cells. Vector genomes were titered using ddPCR. Vectors were stored at -80°C before use. Doses used herein were 5 x 109, l x IO10, 5 x IO10vg / mouse. For a 25 g mouse, these doses translate to 2 x 1011, 4 x 1011, and 2 x 1012vg / kg.

[0098] Animals

[0099] An animal model was used for experiments described herein: male C57BL / 6J hemophilia A, CD4-deficient mice (HA / CD4KO). Immunocompromised male HA / CD4KO animals are used standardly in HA hFVIII gene therapy studies in murine models as mice develop an immunological response to hFVIII expression. The CD4KO permits the ability to reliably monitor hFVIII expression in the absence of anti-FVIII antibody formation. HA / CD4KO mice were generated by crossing C57BL / 6J HA (F8 exon 16 knockout) mice with C57BL / 6J CD4-deficient mice that lack circulating CD4+ T cells.

[0100] HA / CD4KO mice were bred and maintained in the vivarium at CHOP. Vivarium staff monitored animals daily for morbidity, mortality, injury, and the availability of food and water. All care and procedures were approved by the Institutional Animal Care and Use Committee at CHOP.

[0101] 8- to 12-week-old male HA / CD4K0 mice were enrolled herein. Prior to infusion, vectors were thawed and prepared to a final volume of 200 pl in PBS and IX Pluronics®. rAAV vectors were infused into mice via lateral tail vein injection. Doses and number of animals are shown in Table 2. Total volume injected into each animal was 200 pl. Peripheral blood was collected at 8 weeks following vector administration into 3.8% sodium citrate (9: 1 vol / vol). Plasma was isolated by centrifuging citrated blood for 10 minutes at 10,000 x g at 4°C and stored at -80°C until use. At endpoint collection, the left lobe of the liver was harvested and immediately processed for further analysis.

[0102] Table 2: Experimental groups for rAAV administration to male HA / CD4KO mice.

[0103] Tissue culture and transient transfection

[0104] Liver hepatoma-derived Huh-7 cells were seeded at a density of 2 x 105cells per 6 well plate and grown overnight in DMEM / F-12 supplemented with 10% fetal bovine serum, IX L-glutamine and 2 mM CaCh. On the next day, cells were transfected in triplicate with 2.5 pg pCHOPF8 plasmids or Control 1 plasmid following Lipofectamine™ 3000 Transfection Reagent manufacture’s protocol. After overnight plasmid DNA transfection cell media was changed to expression media (DMEM / F-12 50 / 50 supplemented with 0.75 mg / ml AlbuMAX™, IX L-glutamine, 2 mM CaCh and 5 pg / ml insulin-transferrin-sodium selenite). FVIII expression was measured in culture media after 48 hours.

[0105] FVIII aPTT assay

[0106] FVIII activity was determined by an activated partial thromboplastin time-based 1 -stage (aPTT) clotting assay. A standard curve of recombinant hFVIII was prepared in expression media. 48-hour culture media from transfected cells and standards were diluted 5-fold in assay buffer (HBS, 0.1% BSA) and kept on ice until assayed. Samples were then incubated with equal volumes of aPTT reagent and human FVIII-deficient (HA) plasma for 3 minutes at 37°C. Coagulation was initiated with the addition of an equal volume of 25 mM CaCL. Clotting times were recorded and FVIIFC was determined by extrapolation using standard curve values. Each sample was run in duplicate.

[0107] ELISA for human factor VIII antigen

[0108] Circulating human FVIII (hFVIII) antigen levels in mouse plasma, intracellular hFVIII, and hFVIII secreted into culture media were determined by ELISA using matched-pair antibodies against hFVIII (Affinity Biologicals, #F8C-EIA) according to manufacturer’s protocol. Two modifications were made to the protocol. First, plates were coated with primary capture antibody overnight at 4°C. Additionally, sample volume for all steps was reduced to 50 pl per well. Standard curves of recombinant hFVIII ranging from 2 nM to 0.01 nM were prepared in C57BL / 6J HA mouse plasma or Milli-Q® water. Recombinant FVIII were produced and purified using described methods (Wilhelm, et al. (2021) Blood 137(18):2532-2543). Prior to the assay, samples and standards were diluted 1 : 10 in sample diluent provided in the kit. Sample FVIII concentration was determined by converting absorbance at 490 nm using the sample’s respective standard curve.

[0109] Isolation of DNA and total RNA from mouse liver

[0110] RNA and DNA was isolated from frozen livers using the AllPrep DNA / RNA / Protein Mini Kit (QIAGEN) following the manufacturer’s protocol. Briefly, liver samples (20-25 mg) were added to Buffer RLT Plus with B-ME and homogenized using stainless steel beads (QIAGEN) in a TissueLyser LT (QIAGEN). Eluted RNA and genomic DNA (gDNA) were quantified and stored at -80°C until further use.

[0111] Quantitative real time PCR

[0112] F8 mRNA levels from mouse liver were measured using real-time reverse transcription quantitative PCR (RT-qPCR). cDNA was synthesized from 2 pg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. Synthesized cDNA was used to quantify relative expression of FVIII mRNA. A murine GAPDH-specific TaqMan™ Gene Expression Assay was purchased from Thermo Fisher (murine GAPDH: Mm99999915_gl). Gene expression assays were designed for human FVIII (Table 3). RT-qPCR was performed using TaqMan™ Universal Master Mix II, no UNG (Applied Biosystems #4440040) according to manufacturer’s instructions on a CFX384 Touch Real-Time PCR System (Bio-Rad). Each PCR reaction was performed in triplicate. Target mRNA expression levels were calculated using the relative comparison Ct (2- AACt) method (Schmittgen, et al. (2008) Nat. Protoc., 3(6): 1101-8) after normalization to GAPDH as an endogenous control.

[0113] Table 3: Gene expression assays designed for RT-qPCR and ddPCR of FVIII.

[0114] Vector copy number quantification

[0115] Droplet digital PCR (ddPCR) was used to quantify copy number variation (CNV) in livers harvested from AAV-treated mice. Isolated gDNA (200 ng) was digested with restriction enzymes that cut at the 5’ (SacII) and 3’ (Notl) ends to excise the vector expression cassette. Digested gDNA (~20 ng) was added in duplicate to 96-well PCR plates containing a master mix of ddPCR Supermix for Probes (No dUTP) (Bio-Rad), a FAM-conjugated Gene Expression Assay for human FVIII (Table 3), and a HEX- conjugated ddPCR Copy Number Assay for mouse diploid reference gene AP3B1 (dMmuCNS801070401, Bio-Rad). Nanoliter-sized droplets were generated with a QX200™ Droplet Generator (Bio-Rad), transferred to a new 96-well PCR plate, and run in a T100 thermal cycler (Bio-Rad) following the manufacturer’s standard protocol. Droplets from each well were read and individually analyzed on a QX200™ Droplet Reader (Bio-Rad) with PCR-positive and PCR-negative droplets identified using QX Manager Software (Bio-Rad). Vector copy number (VCN) was then calculated as vector copies per diploid genome.

[0116] Results

[0117] Studies to evaluate performance of optimized FVIII expression cassettes in vitro

[0118] Clinical experience with AAV vectors demonstrates that vector dose increases the risk for immune mediated vector toxicities for which vector dose-dependent limiting toxicities are supported by been observed in non-human primates and clinical trial studies. FVIII expression cassettes were optimized to permit use of the lowest possible vector dose.

[0119] To achieve this, the TTRm-FVIII expression cassette was modified by removing the synthetic intron and modifying the polyA tail. Ten codon-optimized FVIII transgene sequences were subsequently introduced into the modified cassette (CHOPF8). Transient transfection of plasmids carrying the CHOPF8 cassettes was performed to initially screen the cassettes for FVIII expression. CHOPF8 plasmids were transfected into a liver hepatoma cell line commonly used in hemophilia gene therapy studies (Huh7 cells). A plasmid carrying the expression cassette of a previous codon optimized FVIII AAV vector was included as a positive control (Control 1). 48 hours after transfection, FVIII expression was quantified from media and FVIII activity was measured in a one- stage aPTT assay (Figure 1). Similar results were obtained in HepG2 cells. All but 2 CHOPF8 cassettes (CHOPF8-2 and CHOPF8-7) expressed FVIII at levels that were at least 50% of the positive control. These 8 plasmids were used to generate AAV8 vectors. CHOPF8-2 and CHOPF8-7 were not further pursed for clinical development.

[0120] FVIII expression in mice following AAV-mediated gene transfer of novel CHOPF8 expression cassettes

[0121] Expression of FVIII in vivo from systemic AAV8 vector infusion of AAV8 vectors containing CHOPF8 expression cassettes was initially evaluate in select cassettes. 8 to 12 week old male HA / CD4KO mice were treated with AAV8-CHOPF8 vectors at a dose of 2 x 1012vg / kg. Steady-state plasma FVIII Ag levels were measured 8 weeks after treatment (Figure 2A). Normal plasma FVIII values are generally considered to be approximately 1 nM and targeted expression of FVIII is below normal FVIII concentrations at 0.05 to < 0.4 nM. At a dose of 2 x 1012vg / kg delivered by intravenous infusion, all constructs showed robust expression of FVIII, ranging from 0.7 nM to 1.8 nM. In comparison to historical control mice treated with codon-optimized AAV8-FVIII, which had a mean FVIII Ag of 0.8 nM at 8 weeks after dosing, two constructs had statistically significant increased expression levels. AAV8-CHOPF8-1 and AAV8- CHOPF8-8 treated mice had mean FVIII: Ag levels of 1.8 nM and 1.5 nM, respectively.

[0122] Based on expression levels achieved, AAV8-CHOPF8-1, -8, -9, and -10 were selected for further dose titration. Additional cohorts of mice were infused with vectors at a dose of 4 x 1011vg / kg or 2 xlO11vg / kg. Eight weeks after dosing, dose-dependent FVIII expression was observed (Figure 2B). At the mid vector dose, AAV8-CHOPF8-9 expressed 4-5-fold less than the other vectors, which ranged from 0.82 nM - 1.16 nM. At the low dose, all vectors achieved expression within the mild HA range (0.05 to < 0.4 nM), which is the clinically targeted expression level. CHOPF8-1 and CHOPF8-10 had the highest FVIII antigen levels at 0.37 nM and 0.24 nM, respectively. Figure 2C provides a dose response summary of the tested constructs.

[0123] FVIII mRNA levels (Figure 3 A) and vector copy number (Figure 3B) were measured in livers of animals 8 weeks after treatment with AAV8-CHOPF8-1 or AAV8- CHOPF8-10. Vector doses were 2 x 1012(n = 4), 4 x 1011(n = 3), and 2 x 1011(n = 3). Similar FVIII mRNA levels and vector copy number were observed between vectors at all doses. A control vector at a dose of 2 xlO12vg / kg showed a vector copy number per diploid genome of about 0.8 and a relative F8 mRNA expression of about 1.5. Accordingly, AAV8-CHOPF8-1 and AAV8-CHOPF8-10 showed unexpectedly increased copy number and F8 mRNA expression.

[0124] The data provides numerous cassettes, particularly CHOPF8-1, which demonstrated robust transgene expression from an AAV8 vector in mice in vivo.

[0125] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

WHAT IS CLAIMED IS1. A nucleic acid comprising a Factor VIII (FVIII) encoding sequence, wherein said FVIII encoding sequence is codon-optimized.

2. The nucleic acid of claim 1, wherein the FVIII encoding sequence comprises a sequence selected from SEQ ID NOs: 20-29 or a sequence having at least 99% identity with a sequence selected from SEQ ID NOs: 20-29.

3. The nucleic acid of claim 1, wherein the FVIII encoding sequence comprises a sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 28; and SEQ ID NO: 29.

4. The nucleic acid of claim 1, wherein the FVIII encoding sequence comprises a sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO: 27; and SEQ ID NO: 29.

5. The nucleic acid of claim 1, wherein the B domain of the FVIII has been replaced by a peptide linker.

6. The nucleic acid of any one of claims 1-5, further comprising a liver specific promoter.

7. The nucleic acid of claim 6, wherein said liver specific promoter is the transthyretin promoter.

8. The nucleic acid of any one of claims 1-7, further comprising a polyadenylation signal.

9. The nucleic acid of any one of claims 1-8, further comprising inverted terminal repeats.

10. The nucleic acid of claim 1, wherein the FVIII encoding sequence comprises a sequence selected from SEQ ID NOs: 3-12 or a sequence having at least 99% identity with a sequence selected from SEQ ID NOs: 3-12.

11. A composition comprising at least one nucleic acid of any one of claims 1-10 and at least one pharmaceutically acceptable carrier.

12. An expression vector comprising the nucleic acid of any one of claims 1-10.

13. The expression vector of claim 12, wherein the vector is an adenovirus-associated viral vector.

14. An adenovirus-associated viral particle comprising the nucleic acid molecule of any one of claims 1-10.

15. A composition comprising an adenovirus-associated viral particle of claim 14 and at least one pharmaceutically acceptable carrier.

16. A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the nucleic acid of any one of claims 1-10.

17. The method of claim 16, wherein said hemostasis related disorder is hemophilia A.

18. The method of claim 16, wherein said nucleic acid is in an adenovirus-associated viral vector.

19. A method for reducing blood loss in a patient in need thereof comprising administration of a therapeutically effective amount of the nucleic acid of any one of claims 1-10.

20. The method of claim 19, wherein said nucleic acid is in an adenovirus-associated viral vector.

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