Use of lentiviral vectors expressing factor viii

Abstract

The present disclosure provides lentiviral vectors comprising codon optimized Factor VIII sequences, and methods of using such lentiviral vectors. The liver-targeted lentiviral vectors disclosed herein can be used for gene therapy, wherein the lentiviral gene delivery enables stable integration of the transgene expression cassette into the genome of targeted cells (e.g., hepatocytes) of pediatric (e.g., neonatal) or adult subjects, achieving an improvement in FVIII expression (for example, a 100-fold improvement) at low lentiviral vector doses (e.g., 5x10e10or lower, such as 1.5x10e9or lower, or 1x10e8 TU / kg or lower). The present disclosure also provides methods of treating bleeding disorders such as hemophilia (e.g., hemophilia A) comprising administering to a subject in need thereof a liver-targeted lentiviral vector comprising a codon optimized Factor VIII nucleic acid sequence at low dosages (1x10e8TU / kg or lower to 1.5x10e10TU / kg).

Description

USE OF LENTIVIRAL VECTORS EXPRESSING FACTOR VIIIRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial Nos.62 / 625,145, filed February 1 , 2018, 62 / 671 ,915, filed May 15, 2018, and 62 / 793,158, filed January 16, 2019, the entire disclosures of which are hereby incorporated herein by reference.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing in ASCII text file (Name:609628_SA9_460PC_Sequence_Listing.txt; Size: 204,203 bytes; and Date of Creation: January 31 , 2019) is incorporated herein by reference in its entirety.BACKGROUND OF THE DISCLOSURE

[0003] The blood coagulation pathway, in part, involves the formation of an enzymatic complex of Factor Villa (FVIIIa) and Factor IXa (FIXa) (Xase complex) on the surface of platelets. FIXa is a serine protease with relatively weak catalytic activity without its cofactor FVIIIa. The Xase complex cleaves Factor X (FX) into Factor Xa (FXa), which in turn interacts with Factor Va (FVa) to cleave prothrombin and generate thrombin. Hemophilia A is a bleeding disorder caused by mutations and / or deletions in the FVIII (FVIII) gene resulting in a deficiency of FVIII activity (Peyvandi et al. 2006). In some cases, patients have reduced levels of FVIII due to the presence of FVIII inhibitors, such as anti-FVI II antibodies.

[0004] The disease can be treated by replacement therapy targeting restoration of FVIII activity to prevent spontaneous bleeding. There are plasma-derived and recombinant FVIII products available to treat bleeding episodes on-demand or to prevent bleeding episodes from occurring by treating prophylactically. Based on the half-life of these products (10-12 hr) (White G.C., et al., Thromb. Haemost. 77:660-7 (1997); Morfini, M., Haemophilia 9 (suppl 1):94-99; discussion 100 (2003)), treatment regimens require frequent intravenous administration, commonly two to three times weekly for prophylaxis and one to three times daily for on-demand treatment (Manco-Johnson, M.J., et al., N. Engl. J. Med. 357:535-544 (2007)). Such frequent administration is inconvenient and costly.

[0005] A major impediment in providing a low-cost recombinant FVIII protein to patients is the high cost of commercial production. FVIII protein expresses poorly in heterologous expression systems, two to three orders of magnitude lower than similarly sized proteins. (Lynch et al., Hum. Gene. Ther. 4:259-72 (1993). Advances in our understanding of the biology of FVIII expression has led to the development of more potent FVIII variants. For instance, biochemical studiesdemonstrated that the FVIII B-domain was dispensable for FVIII cofactor activity. Deletion of the B-domain resulted in a 17-fold increase in mRNA levels over full-length wild-type FVIII and a 30% increase in secreted protein. (Toole et al., Proc Natl Acad Sci USA 83:5939-42 (1986)). Nonetheless, there still exists a need in the art for FVIII sequences that express efficiently in heterologous systems.SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides methods of treating a bleeding disorder in a subject in need thereof comprising administering to the subject at least one dose of 5X1010TU / kg transducing units / kg (TU / kg) or less(e.g., 5x109or less or 10®TU / kg or less) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has (i) at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1 ; (ii) at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2; (iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70; (iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71 ; (v) at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleotides 58- 2277 and 2320-4374 of SEQ ID NO: 3; (vi) at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4; (vii) at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5; (viii) at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6; or (ix) or any combination of (i) to (viii).

[0007] The present disclosure also provides methods of treating a bleeding disorder in a subject in need thereof comprising administering to the subject at least one dose of 5X1010TU / kg or less (e.g., 5x109or less or 10®TU / kg or less) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a Factor VIII (FVIII) polypeptide and a second nucleic acidsequence encoding a C-terminal portion of a FVIII polypeptide; (a) wherein the first nucleic acid sequence has: (i) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 3; (ii) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 4; (iii) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 5; or (iv) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 6; (b) wherein the second nucleotide sequence has: (i) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792- 2277 and 2320-4374 of SEQ ID NO: 3; (ii) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 4; (iii) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5; or (iv) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6; or (c) any combination of (a) and (b); and wherein the N-terminal portion and the C-terminal portion together have a FVI II polypeptide activity.

[0008] In some embodiments of the methods disclosed above, the dose is about 9.5x10®TU / kg, about 9x10®TU / kg, about 8.5x10®TU / kg, about 8x10®TU / kg, about 7.5x10®TU / kg, about 7x10®TU / kg, about 6.5x10®TU / kg, about 6x10®TU / kg, about 5.5x10®TU / kg, about 5x10®TU / kg, about 4.5x10®TU / kg, about 4x10®TU / kg, about 3.5x10®TU / kg, about 3x10®TU / kg, about 2.5x10®TU / kg, about 2x10®TU / kg, about 1.5x10®TU / kg, or about 1x10®TU / kg, about 5x1010TU / kg, about 4.5x1010TU / kg, about 4x1010TU / kg, about 3.5x1010TU / kg, about 3x1010TU / kg, about 2.5x1010TU / kg, about 2x1010TU / kg, about 1 .5x1010TU / kg, about 1x1010TU / kg, about 9.5x109TU / kg, about 9x109TU / kg, about 8.5x109TU / kg, about 8x109TU / kg, about 7.5x109TU / kg, about 7x109TU / kg, about 6.5x109TU / kg, about 6x109TU / kg, about 5.5x109TU / kg, about 5x109TU / kg, about 4.5x109TU / kg, about 4x109TU / kg, about 3.5x109TU / kg, about 3x109TU / kg, about 2.5x109TU / kg, about 2x109TU / kg, about 1 .5x109TU / kg, or about 1x109TU / kg.

[0009] In some embodiments, the dose is less than about 9.5x10®TU / kg, less than about9x10®TU / kg, less than about 8.5x10®TU / kg, less than about 8x10®TU / kg, less than about 7.5x10®TU / kg, less than about 7x10®TU / kg, less than about 6.5x10®TU / kg, less than about 6x10®TU / kg, less than about 5.5x10®TU / kg, less than about 5x10®TU / kg, less than about 4.5x10®TU / kg, less than about 4x10®TU / kg, less than about 3.5x10®TU / kg, less than about 3x10®TU / kg, less thanabout 2.5x10®TU / kg, less than about 2x10®TU / kg, less than about 1.5x10®TU / kg, or less than about 1x10®TU / kg, less than about 5x1010TU / kg, less than about 4.5x1010TU / kg, less than about 4x1010TU / kg, less than about 3.5x1010TU / kg, less than about 3x1010TU / kg, less than about 2.5x1010TU / kg, less than about 2x1010TU / kg, less than about 1 .5x1010TU / kg, less than about 1x1010TU / kg, less than about 9.5x109TU / kg, less than about 9x109TU / kg, less than about 8.5x109TU / kg, less than about 8x109TU / kg, less than about 7.5x109TU / kg, less than about 7x109TU / kg, less than about 6.5x109TU / kg, less than about 6x109TU / kg, less than about 5.5x109TU / kg, less than about 5x109TU / kg, less than about 4.5x109TU / kg, less than about 4x109TU / kg, less than about 3.5x109TU / kg, less than about 3x109TU / kg, less than about 2.5x109TU / kg, less than about 2x109TU / kg, less than about 1 .5x109TU / kg, or less than about 1x109TU / kg.

[0010] In some embodiments, the dose is between 1x10®and 5x1010TU / kg, between 1x10®and 5x109TU / kg, between 1x10®and 1x109TU / kg, between 1x10®and 1x1010TU / kg, between 1x109and 5x1010TU / kg, between 2x109and 5x1010TU / kg, between 3x109and 5x1010TU / kg, between 4x109and 5x1010TU / kg, between 5x109and 5x1010TU / kg, between 6x109and 5x1010TU / kg, between 7x109and 5x1010TU / kg, 8x109and 5x1010TU / kg, between 9x109and 5x1010TU / kg, between 1010and 5x1010TU / kg, between 1.5x1010and 5x1010TU / kg, between 2x1010and 5x1010TU / kg, between 2.5x1010and 5x1010TU / kg, between 3x1010and 5x1010TU / kg, between 3.5x1010and 5x1010TU / kg, between 4x1010and 5x1010TU / kg, or between 4.5x1010and 5x1010TU / kg. In some embodiments, the dose is between 1x109and 5x1010TU / kg, between 1x109and 4.5x1010TU / kg, between 1x109and 4x1010TU / kg, between 1x109and 3.5x1010TU / kg, between 1x109and 3x1010TU / kg, between 1x109and 2.5x1010TU / kg, between 1 x109and 2x1010TU / kg, between 1x109and 1 .5x1010TU / kg, between 1x109and 1010TU / kg, between 1 x109and 9x109TU / kg, between 1x109and 8x109TU / kg, between 1x109and 7x109TU / kg, between 1x109and 6x109TU / kg, between 1x109and 5x109TU / kg, between 1x109and 4x109TU / kg, between 1x109and 3x109TU / kg, and between 1x109and 2x109. In some embodiments, the dose is between 1x1010and 2x1010TU / kg, between 1 .1x1010and 1.9x1010TU / kg, between 1 .2x1010and 1 .8x1010TU / kg, between 1 .3x1010and 1 .7x1010TU / kg, or between 1 .4x1010and 1 .6x1010TU / kg. In some embodiments, the dose is about 1 .5x1010TU / kg. In some embodiments, the dose is 1 .5x109TU / kg. In some embodiments, the dose is between 2.5x109TU / kg and 3.5x109TU / kg, between 2.6 x109TU / kg and 3.4x109TU / kg, between 2.7x109TU / kg and 3.3x109TU / kg, between 2.8x109TU / kg and 3.2x109TU / kg, or between 2.9x109TU / kg and 3.1x109TU / kg. In some embodiments, the dose is about 3.0x109TU / kg. In some embodiments, the dose is between 5.5x109TU / kg and 6.5x109TU / kg, between 5.6 x109TU / kg and 6.4x109TU / kg, between 5.7x109TU / kg and 6.3x109TU / kg, between 5.8x109TU / kg and 6.2x109TU / kg, or between 5.9x109TU / kg and 6.1x109TU / kg. In some embodiments, the dose is about 6.0x109TU / kg.

[0011] In some embodiments of the methods disclosed above, plasma FVIII activity at 24 hours to 48 hours post administration of the lentiviral vector is increased relative to a subject administered a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16. In some embodiments, the plasma FVIII activity is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 1 1 -fold, at least about 12- fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 1 10-fold, at least about 120-fold, at least about 130- fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170- fold, at least about 180-fold, at least about 190-fold, or at least about 200-fold.

[0012] In some embodiments of the methods disclosed above, the lentiviral vector is administered as a single dose or multiple doses. In some embodiments, the lentiviral vector is administered via intravenous injection. In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is an adult subject.

[0013] In some embodiments, the lentiviral vector comprises a tissue specific promoter. In some embodiments, the tissue specific promoter selectively enhances expression of the polypeptide with FVIII activity in a target liver cell. In some embodiments, the tissue specific promoter that selectively enhances expression of the polypeptide with FVIII activity in a target liver cell comprises an mTTR promoter. In some embodiments, the target liver cell is a hepatocyte. In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of the hepatocyte. In some embodiments, the bleeding disorder is hemophilia A.

[0014] In some embodiments of the methods disclosed above, the isolated nucleic acid molecule comprises LV-coFVIII-6 (SEQ ID NO:71). In some embodiments, the isolated nucleic acid molecule comprises LV-coFVIII-6-XTEN (SEQ ID NO:72).

[0015] In some embodiments, the dose of lentivirus vector is administered at once or divided into two sub-doses, three sub-doses, four sub-doses, five sub-doses, or six sub-doses. In some embodiments, the dose of lentivirus vector is repeated at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity further comprises a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding a signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to: (i) nucleotides 1 to 57 of SEQ ID NO: 1 ; (ii) nucleotides 1 to 57 of SEQID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ ID NO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) nucleotides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71 ; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.

[0016] In some embodiments, the nucleic acid molecule (or the nucleotide sequence encoding a polypeptide with FVIII activity) comprises one or more property selected from the group consisting of: (a) the human codon adaptation index the nucleic acid molecule or a portion thereof is increased relative to SEQ ID NO: 16; (b) the frequency of optimal codons of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16; (d) the relative synonymous codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (e) the effective number of codons of the nucleotide sequence or a portion thereof is reduced relative SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21 and 22) relative to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) relative to SEQ ID NO: 16; and (h) any combination thereof.

[0017] In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity further comprises a heterologous nucleotide sequence encoding a heterologous amino acid sequence (e.g., a half-life extender). In some embodiments, the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence. In some embodiments, the heterologous amino acid sequence is linked to the N-terminus or the C-terminus of the amino acid sequence encoded by the nucleotide sequence or inserted between two amino acids in the amino acid sequence encoded by the nucleotide sequence at one or more insertion site selected from TABLE 3. In some embodiments, the FVIII polypeptide is a full length FVIII or a B domain deleted FVIII.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGs. 1A-1J provide the codon optimized nucleotide sequences encoding B domain-deleted Factor VIII. FIG. 1A shows the nucleotide sequence of coFVIII-3 (SEQ ID NO:1). FIG. 1 B shows the nucleotide sequence of coFVIII-4 (SEQ ID NO: 2). FIG. 1 C shows the nucleotide sequence of coFVIII-5 (SEQ ID NO: 70). FIG. 1 D shows the nucleotide sequence of coFVIII-6 (SEQ ID NO: 71). FIG. 1 E shows the nucleotide sequence of coFVIII-52 (SEQ ID NO: 3). FIG. 1 F shows the nucleotide sequence of coFVIII-62 (SEQ ID NO: 4). FIG. 1 G shows the nucleotide sequence of coFVIII-25 (SEQ ID NO: 5). FIG. 1 H shows the nucleotide sequence of coFVIII-26 (SEQ ID NO: 6). FIGs. 11 and 1J show the non-codon optimized nucleotide and amino acid sequences, respectively, of B domain-deleted (BDD-FVIII) (SEQ ID NOs: 16 and 17, respectively).

[0019] FIGs. 2A-2J show codon usage bias adjustments in the codon optimized nucleotide sequences encoding BDD-FVIII. FIG. 2A shows the relative frequency of codons in the wild-type nucleotide sequence (before codon optimization) encoding BDD-FVIII, e.g., non-optimized BDD- FVIII. The human codon adaptation index (CAI) of the non-optimized BDD-FVIII sequence is 74%. FIG. 2B shows the relative frequency of codons in the coFVIII-1 variant sequence, which has a human CAI of 88%. FIG. 2C shows the relative frequency of codons in the coFVIII-3 variant sequence, which has a human CAI of 91 %. FIG. 2D shows the relative frequency of codons in the coFVIII-4 variant sequence, which has a human CAI of 97%. FIG. 2E shows the relative frequency of codons in the coFVIII-5 variant sequence, which has a human CAI of 83%. FIG. 2F shows the relative frequency of codons in the coFVIII-6 variant sequence, which has a human CAI of 83%. FIG. 2G shows the relative frequency of codons in the coFVIII-52 variant sequence, which has a human CAI of 91 %. FIG. 2H shows the relative frequency of codons in the coFVIII-62 variant sequence, which has a human CAI of 91 %. FIG. 2I shows the relative frequency of codons in the coFVIII-25 variant sequence, which has a human CAI of 88%. FIG. 2J shows the relative frequency of codons in the coFVIII-26 variant sequence, which has a human CAI of 88%.

[0020] FIG. 3 provides a plasmid map of FVIII-303, which comprises coFVII 1-1 in a pcDNA3 backbone under the control of the ET-enhanced transthyretin promoter, which is positioned upstream of the coFVIII-1 translation start site and which comprises a synthetic enhancer, an mTIR enhancer, and an mTIR promoter.

[0021] FIG. 4 shows a graphical representation of FVIII plasma activity in HemA mice following hydrodynamic injection of 5 pg FVIII-303 (coFVIII-1 ; circles) or 5 pg FVIII-31 1 (BDD-FVIII; squares). FVIII plasma activity was determined by a FVIII specific chromogenic assay at 24, 48, and 72 hours post-injection. The relative activity levels at 72 hours, normalized to the expression level of FVIII-31 1 , are shown.

[0022] FIG. 5 shows a plasmid map of pLV-coFVIII-52, which comprises coFVIII-52 in a lentiviral plasmid underthe control of an ET promoter, which is positioned upstream of the coFVIII- 52 translation start site and which comprises a synthetic enhancer, an mTTR enhancer, and an mTTR promoter.

[0023] FIGs. 6A-6C show graphical representations of FVIII plasma activity in HemA mice following hydrodynamic injection of various FVIII encoding nucleotides. FVIII plasma activity was determined by a FVIII specific chromogenic assay at 24, 48, and 72 hours post-injection. FIG. 6A shows FVIII plasma activity in HemA mice following hydrodynamic injection of 5 pg LV-coFVIII-1 (filled circles), 5 pg LV-coFVIII-3 (triangles), 5 pg LV-coFVIII-4 (inverted triangles), 5 pg LV-coFVIII- 5 (diamonds), or 5 pg LV-coFVIII-6 (open circles). FIG. 6B shows FVIII plasma activity in HemA mice following hydrodynamic injection of 5 pg LV-coFVIII-1 (circles), 5 pg LV-coFVIII-25 (triangles),or 5 pg LV-coFVIII-26 (inverted triangles). FIG. 6C shows FVIII plasma activity in HemA mice following hydrodynamic injection of 20 pg LV-21 16 (non-codon optimized (WT) BDD-FVIII nucleotide sequence; open circles), 20 pg LV-coFVIII-1 (triangles), 20 pg LV-coFVIII-52 (squares), or 20 pg LV-coFVIII-62 (filled circles). The relative activity levels at 72 hours are shown for each plasmid, normalized to the expression levels of LV-coFVIII-1 (FIGs. 6A, 6B, and 6C) and / or LV- 21 16 (FIG. 6C), as indicated.

[0024] FIG. 7 shows plasma FVIII activity in HemA mice 24 days after injection with 1 E8TU / mouse lentiviral vector comprising coFVII 1-1 , coFVIII-5, coFVIII-52, coFVIII-6, or coFVIII-62 as compared with the LV-21 16 (BDD-FVIII) control, and as measured by a FVII l-specific chromogenic assay. Error bars indicate standard deviations.

[0025] FIGs. 8A-8C provide the various codon optimized nucleotide sequences encodingBDD-FVIII fused to an XTEN. FIG. 8A shows the nucleotide sequence of coFVIII-52-XTEN (SEQ ID NO: 19), wherein a nucleotide sequence encoding an XTEN having 144 amino acids ("CTENMT'; SEQ ID NO: 18; underlined) is inserted within the coFVIII-52 nucleotide sequence. FIG. 8B shows the nucleotide sequence of coFVIII-1-XTEN (SEQ ID NO: 20), wherein a nucleotide sequence encoding an XTEN having 144 amino acid ("XTENW; SEQ ID NO: 18; underlined) is inserted within the coFVIII-1 nucleotide sequence. FIG. 8C shows the nucleotide sequence of coFVIII-6- XTEN (SEQ ID NO: 72), wherein a nucleotide sequence encoding an XTEN having 144 amino acid ("XTEN144"; SEQ ID NO: 18; underlined) is inserted within the coFVIII-6 nucleotide sequence (e.g., amino acid residue 745 corresponding to mature FVIII sequence).

[0026] FIG. 9 provides a plasmid map of pLV-coFVIII-52-XTEN, which comprises coFVIII-52-XTEN in a lentiviral vector under the control of the ET promoter. Lentiviral vectors comprising each of the remaining codon optimized nucleic acid molecules encoding a polypeptide with FVIII activity, as described herein, were constructed in the same manner as pLV-coFVIII-52-XTEN, in which the same XTEN sequence was inserted to replace the B-domain of FVIII.

[0027] FIGs. 10A and 10B show FVIII activity in HemA mice following injection with plasmidDNA (FIG. 10A) or lentiviral vector (FIG. 10B) comprising the various codon optimized nucleotide sequences encoding BDD-FVIII. FIG. 10A shows a graphical representation of FVIII plasma activity in HemA mice following hydrodynamic injection with 5pg FVIII-31 1 (non-codon optimized, BDD-FVIII encoding nucleotide sequence; squares), 5pg FVIII-303 (coFVIII-1 ; small circles), or FVIII-306 (C0FVIII-I-XTEN144; large circles). The relative activity at 72 hours, normalized to FVIII- 31 1 , is shown for each plasmid. FIG. 10B shows plasma FVIII activity in HemA mice 21 days after injection with 1 E8 TU / mouse of lentiviral vector comprising coFVIII-52 or coFVIII-52-XTEN as compared with the LV-21 16 (BDD-FVIII) control, and as measured by a FVII l-specific chromogenic assay. Error bars indicate standard deviations.

[0028] FIG. 1 1 A shows the amino acid sequence of full-length mature human factor VIII.FIG. 1 1 B shows the amino acid sequence of full length human von Willebrand Factor (SEQ ID NO: 44). FIGs. 1 1 C and 1 1 D show the amino acid and nucleotide sequences, respectively, of an XTEN polypeptide having 42 amino acids (XTEN AE42-4; SEQ ID NOs: 46 and 47, respectively). The amino acid sequences of various XTEN polypeptides having 144 amino acids are shown in FIGs.I I E, 1 1 G, 111, 1 1 K, 1 1 M, 1 10, 1 1 Q, 1 1 S, 1 1 U, and 1 1W (SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60,62, 64, and 66, respectively), and the corresponding nucleotide sequences are shown in FIGs.I I F, 1 1 H, 11 J, 1 1 L, 1 1 N, 1 1 P, 1 1 R, 1 1T, 1 1V, and 1 1X (SEQ ID NOs: 49, 51 , 53, 55, 57, 59, 61 ,63, 65, and 67, respectively). FIG. 1 1 Y shows the nucleotide sequence of an ET promoter (SEQ ID NO: 69). FIG. 11 Z shows the nucleotide sequence for coFVIII-1 (SEQ ID NO: 68) (see International Publication No. WO 2014 / 127215, SEQ ID NO: 1).

[0029] FIG. 12A is a graphic representation of FVIII plasma activity (lll / mL) in 14-day-oldHemA mice following IV administration of about 1.5x1010TU / kg LV-wtBDD-FVIII (circles), LV- coFVIII-6 (squares), or LV-coFVIII-6XTEN (triangles). FIG. 12B is a graphic representation of vector copy number (VCN) 150 days after treatment of 14-day-old HemA mice administered by IV about 1 .5x1010TU / kg of lentiviral vectors expressing wtBDD-FVI II, coFVII 1-1 , coFVIII-3, coFVIII-4, coFVIII-5, coFVIII-6, coFVIII-52, coFVIII-62, coFVIII-25, or coFVIII-26. FIG. 12C is a graphic representation of FVIII plasma activity (lU / mL) 21 days after treatment of 14-day-old HemA mice administered by IV about 1.5x1010TU / kg of lentiviral vectors expressing wtBDD-FVIII, coFVIII-1 , coFVIII-3, coFVIII-4, coFVIII-5, coFVIII-6, coFVIII-52, coFVIII-62, coFVIII-25, or coFVIII-26.

[0030] FIGs. 13A and 13B are graphic representations that illustrate the FVIII plasma activity levels (FIG. 13A) and anti-FVIII antibody levels (FIG. 13B) in five HemA mice treated with a lentivirus expressing the coFVIII-5 variant. Fourteen-day-old HemA littermates were administered approximately 1.5x1010TU / kg of a lentivirus expressing the coFVIII-5 variant by intravenous injection. Each mouse is designated by a number ( .e., 1 , 2, 3, 4, and 5; FIGs. 13A and 13B).

[0031] FIG. 14 is a graphic representation of the correlation between LV-FVIII expression level, as evidenced by FVIII plasma activity at 21 days post lentiviral treatment, and the presence of anti-FVIII antibodies. Each data point corresponds to a single HemA mouse. Each mouse received a 1.5x1010TU / kg dose by intravenous injection of a lentivirus expressing one of the coFVIII variants disclosed herein. Horizontal lines indicate the average FVIII plasma activity.

[0032] FIG. 15 is a graphic representation of the correlation between vector copy number(VCN) per cell at 150 days post lentiviral treatment and the presence of anti-FVIII antibodies. Each data point corresponds to a single HemA mouse. Each mouse received a 1.5x1010TU / kg dose byintravenous injection of a lentivirus expressing one of the coFVIII variants disclosed herein. Horizontal lines indicate the average VCN.

[0033] FIGs. 16A and 16B are graphic representations that illustrate the FVIII plasma activity levels (FIG. 16A) and anti-FVIII antibody levels (FIG. 16B) in two HemA mice (coFVIII-52- A and coFVIII-52-B) treated with a lentivirus expressing the coFVIII-52 variant. Fourteen-day-old HemA littermates were administered approximately 1.5x1010TU / kg of a lentivirus expressing the coFVIII-52 variant by intravenous injection. FIGs. 16C and 16D are images showing RNA in situ hybridization staining for FVIII expression (dark staining) in liver tissue collected from the coFVIII- 52-A (FIG. 16C) and coFVIII-52-B (FIG. 16D) mice of FIGs. 16A and 16B.

[0034] FIG. 17 is a graphic representation that shows long-term FVIII expression in HemA neonate mice treated with a lentivirus expressing a wild-type B domain deleted FVIII (wtBDD-FVIII; triangles), coFVIII-52-XTEN (circles), or coFVIII-6-XTEN (inverted triangle) variant. Neonatal HemA mice were administered by intravenous injection approximately 1.5x1010TU / kg of a lentivirus expressing wtBDD-FVIII, coFVIII-52-XTEN, or coFVIII-6-XTEN. FVIII plasma activity was measured over approximately 16 weeks.

[0035] FIGs. 18A-18B show a graphical representation of dose-response results corresponding to treatment of HemA mice with lentivirus expressing coFVIII-6 (FIG. 18A) or coFVIII-6-XTEN (FIG. 18B).

[0036] FIG. 19 provides a schematic of a lentiviral vector for liver-targeted gene therapy.SD: splice donor site; SA: splice acceptor site; GA: truncated gag sequence; RRE: Rev responsive element; ET: Enhance transthyretin; FVIII: Factor VIII; 142T: Target sequence for miR-142; Wpre: mutated Woodchuck hepatitis virus Post-transcriptional Regulatory Element; Y (packaging signal).

[0037] FIGs. 20A-20B are graphical representations of the peak circulating FVIII levels in male pigtail macaques administered 3 x 109TU / kg lentivirus expressing coFVIII-6-XTEN produced from CD47h'9h / MHC-lfree293T cells, as measured by FVIII plasma activity (FIG. 20A) and FVIII plasma antigen levels (FIG. 20B).

[0038] FIGs. 21 A-21 B are graphical representations of peak plasma levels of human FVIII activity (FIG. 21 A) and human FVIII antigen levels (FIG. 21 B) in male pigtail macaques administered 3 x 109TU / kg or 6 x 109TU / kg lentivirus expressing coFVIII-6.

[0039] FIGs. 22A-22B show a graphical presentation of peak plasma levels of human FVIII activity (FIG. 22A) and average human FVIII antigen levels (FIG. 22B) in male pigtail macaques administered 1 x 109or 3 x 109TU / kg lentivirus expressing coFVIII-6-XTEN.DETAILED DESCRIPTION OF THE DISCLOSURE

[0040] The present disclosure describes liver-targeted lentiviral gene therapy using codon- optimized genes encoding polypeptides with Factor VIII (FVIII) activity. See, e.g., International Publ. WO2017136358, which is herein incorporated by reference in its entirety.

[0041] Accordingly, in some aspects, the present disclosure is directed to gene therapy comprising the administration of lentiviral vectors comprising codon optimized nucleic acid molecules comprising nucleic acid sequences encoding polypeptides with Factor VIII activity. In particular aspects, the present disclosure is directed to methods of treating bleeding disorders such as hemophilia (e.g., hemophilia A) comprising administering to the subject a lentiviral vector comprising a codon optimized Factor VIII nucleic acid sequence targeted to the liver (e.g., to hepatocytes). The present disclosure meets an important need in the art through a gene therapy approach that results in the stable integration of a transgene expression cassette comprising a codon optimized Factor VIII nucleic acid sequence into the genome of the targeted cells.

[0042] This system demonstrates increased long-term expression of Factor VIII in the targeted cells (e.g., hepatocytes), when the lentiviral vector is administering to the subject at least one dose of 5x1010transducing units / kg (TU / kg) or lower, e.g., about 1.5x1010TU / kg or less, or about 1.5x109TU / kg or less, or about 108TU / kg or less.

[0043] In specific embodiments, the lentiviral vectors disclosed herein comprise a codon optimized nucleic acid sequence comprising, consisting, or consisting essentially of SEQ ID NO: 71 (LV-coFVIII-6).

[0044] In some other specific embodiments, the lentiviral vectors disclosed herein comprise a codon optimized nucleic acid sequence comprising, consisting, or consisting essentially of SEQ ID NO: 72 (LV-coFVIII-6-XTEN).

[0045] The liver-targeted lentiviral vectors disclosed herein enable stable integration of the transgene expression cassette comprising a codon optimized nucleic acid encoding FVIII into the genome of targeted cells (e.g., hepatocytes) of pediatric (e.g., neonatal) or adult subjects, achieving an improvement in FVIII expression (for example, a 100-fold improvement) at low lentiviral vector doses (e.g., 5x1010or lower, such as 109TU / kg or lower, or 10®TU / kg or lower). Since the disclosed lentiviral vectors can achieve therapeutic levels of circulating FVIII at very low doses (e.g., 109TU / kg or lower, or 10®TU / kg or lower), these vectors may significantly reduce potential acute toxicity associated with lentivirus vector treatment. Furthermore, the use of lentiviral vectors, and in particular third-generation vectors, can lead to potentially life long integration in the genome of the subject. The high capacity of lentiviral vectors (1 Okb) with respect to other gene delivery systems (e.g., AAV) allows the inclusions of more regulatory elements in the transgene, e.g., promoters that would control the expression of the FVIII transgene in different tissues (e.g.,hepatocytes and liver endothelial cells). The lentiviral vectors disclosed herein can be used in vivo, in vitro, or ex vivo treatments.

[0046] The Exemplary constructs of the disclosure are illustrated in the accompanyingFigures and sequence listing.

[0047] In order to provide a clear understanding of the specification and claims, the following definitions are provided below.I. Definitions

[0048] It is to be noted that the term "a" or "an" entity refers to one or more of that entity: for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0049] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0050] The term "isolated" for the purposes of the present disclosure designates a biological material (cell, polypeptide, polynucleotide, or a fragment, variant, or derivative thereof) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered "isolated." No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0051] "Nucleic acids," "nucleic acid molecules," "oligonucleotide," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5’ to 3’ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein.

[0052] As used herein, a "coding region" or "coding sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5’ terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3’ terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then, that a single vector can contain just a single coding region, or comprise two or more coding regions.

[0053] Certain proteins secreted by mammalian cells are associated with a secretory signal peptide which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that signal peptides are generally fused to the N-terminus of the polypeptide, and are cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, e.g., a human tissue plasminogen activator (TPA) or mouse b-glucuronidase signal peptide, or a functional derivative thereof, can be used.

[0054] The term "downstream" refers to a nucleotide sequence that is located 3’ to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0055] The term "upstream" refers to a nucleotide sequence that is located 5’ to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate tosequences that are located on the 5’ side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0056] As used herein, the term "gene regulatory region" or "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' noncoding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3’ to the coding sequence.

[0057] A polynucleotide which encodes a gene product, e.g., a polypeptide, can include a promoter and / or other expression (e.g., transcription or translation) control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other expression control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[0058] "Transcriptional control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit b-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0059] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0060] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage. The term "yield," as used herein, refers to the amount of a polypeptide produced by the expression of a gene.

[0061] A "vector" refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0062] Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), b-galactosidase (LacZ), b-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.

[0063] The term "selectable marker" refers to an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene’s effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and / or to identify a cell or organism that has inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.

[0064] The term "reporter gene" refers to a nucleic acid encoding an identifying factor that is able to be identified based upon the reporter gene’s effect, wherein the effect is used to track the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and / or to measure gene expression induction or transcription. Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), b-galactosidase (LacZ), b-glucuronidase (Gus), and the like. Selectable marker genes can also be considered reporter genes.

[0065] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue- specific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

[0066] The promoter sequence is typically bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0067] The terms "restriction endonuclease" and "restriction enzyme" are used interchangeably and refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.

[0068] The term "plasmid" refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double- stranded DNA molecules. Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0069] A "cloning vector" refers to a "replicon," which is a unit length of a nucleic acid that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g. , bacteria and expression in another, e.g. , eukaryotic cells. Cloning vectors typically comprise one or more sequences that can be used for selection of cells comprising the vector and / or one or more multiple cloning sites for insertion of nucleic acid sequences of interest.

[0070] The term "expression vector" refers to a vehicle designed to enable the expression of an inserted nucleic acid sequence following insertion into a host cell. The inserted nucleic acid sequence is placed in operable association with regulatory regions as described above.

[0071] Vectors are introduced into host cells by methods well known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter.

[0072] "Culture," "to culture" and "culturing," as used herein, means to incubate cells under in vitro conditions that allow for cell growth or division or to maintain cells in a living state. "Cultured cells," as used herein, means cells that are propagated in vitro.

[0073] As used herein, the term "polypeptide" is intended to encompass a singular"polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0074] The term "amino acid" includes alanine (Ala or A); arginine (Arg or R); asparagine(Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (lie or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V). Non-traditional amino acids are also within the scope of the disclosure and include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA. Introduction of the non-traditional amino acid can also be achieved using peptide chemistries known in the art. As used herein, the term "polar amino acid" includes amino acids that have net zero charge, but have non-zero partial charges in different portions of their side chains (e.g. , M, F, W, S, Y, N, Q, C). These amino acids can participate in hydrophobic interactions and electrostatic interactions. As used herein, the term "charged amino acid" includes amino acids that can have non-zero net charge on their side chains (e.g., R, K, H, E, D). These amino acids can participate in hydrophobic interactions and electrostatic interactions.

[0075] Also included in the present disclosure are fragments or variants of polypeptides, and any combination thereof. The term "fragment" or "variant" when referring to polypeptide binding domains or binding molecules of the present disclosure include any polypeptides whichretain at least some of the properties ( e.g ., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for an FVIII variant, or FVIII binding activity for the VWF fragment) of the reference polypeptide. Fragments of polypeptides include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein, but do not include the naturally occurring full-length polypeptide (or mature polypeptide). Variants of polypeptide binding domains or binding molecules of the present disclosure include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally or non-naturally occurring. Non- naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[0076] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g. , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. , threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and / or composition of side chain family members.

[0077] The term "percent identity" as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case can be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Sequence alignments andpercent identity calculations can be performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl), the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wl), BLASTP, BLASTN, BLASTX (Altschul e / a / ., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wl 53715 USA). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters which originally load with the software when first initialized. For the purposes of determining percent identity between an optimized BDD FVIII sequence of the disclosure and a reference sequence, only nucleotides in the reference sequence corresponding to nucleotides in the optimized BDD FVIII sequence of the disclosure are used to calculate percent identity. For example, when comparing a full length FVIII nucleotide sequence containing the B domain to an optimized B domain deleted (BDD) FVIII nucleotide sequence of the disclosure, the portion of the alignment including the A1 , A2, A3, C1 , and C2 domain will be used to calculate percent identity. The nucleotides in the portion of the full length FVIII sequence encoding the B domain (which will result in a large "gap" in the alignment) will not be counted as a mismatch. In addition, in determining percent identity between an optimized BDD FVIII sequence of the disclosure, or a designated portion thereof (e.g., nucleotides 58-2277 and 2320-4374 of SEQ ID NO:3), and a reference sequence, percent identity will be calculated by aligning dividing the number of matched nucleotides by the total number of nucleotides in the complete sequence of the optimized BDD- FVIII sequence, or a designated portion thereof, as recited herein.

[0078] As used herein, "nucleotides corresponding to nucleotides in the optimized BDDFVIII sequence of the disclosure" are identified by alignment of the optimized BDD FVIII sequence of the disclosure to maximize the identity to the reference FVIII sequence. The number used to identify an equivalent amino acid in a reference FVIII sequence is based on the number used to identify the corresponding amino acid in the optimized BDD FVIII sequence of the disclosure.

[0079] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide, or the amino acid sequences which normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide, e.g., fusion of a Factor VIII domain of the disclosure with an Ig Fc domain. A fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desiredrelationship. A chimeric protein can further comprises a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide bond or a non-covalent bond.

[0080] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or fragment, variant, or derivative thereof, which is immediately upstream of the position at which a heterologous moiety can be inserted. An "insertion site" is specified as a number, the number being the number of the amino acid in mature native FVIII (SEQ ID NO: 15; FIG. 1 1A) to which the insertion site corresponds, which is immediately N-terminal to the position of the insertion. For example, the phrase "a3 comprises a heterologous moiety at an insertion site which corresponds to amino acid 1656 of SEQ ID NO: 15" indicates that the heterologous moiety is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO: 15.

[0081] The phrase "immediately downstream of an amino acid" as used herein refers to position right next to the terminal carboxyl group of the amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to the position right next to the terminal amine group of the amino acid.

[0082] The terms "inserted," "is inserted," "inserted into" or grammatically related terms, as used herein refers to the position of a heterologous moiety in a recombinant FVIII polypeptide, relative to the analogous position in native mature human FVIII. As used herein the terms refer to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII, and do not indicate, imply or infer any methods or process by which the recombinant FVIII polypeptide was made.

[0083] As used herein, the term "half-life" refers to a biological half-life of a particular polypeptide in vivo. Half-life can be represented by the time required for half the quantity administered to a subject to be cleared from the circulation and / or other tissues in the animal. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid a-phase and longer b-phase. The a-phase typically represents an equilibration of the administered Fc polypeptide between the intra- and extra-vascular space and is, in part, determined by the size of the polypeptide. The b-phase typically represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, and thus do not have an alpha phase, but just the single beta phase. Therefore, in certain embodiments, the term half-life as used herein refers to the half- life of the polypeptide in the b-phase.

[0084] The term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence covalently or non-covalently joined to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly joined or juxtaposed to the second amino acid or nucleotide sequence or alternatively an interveningsequence can covalently join the first sequence to the second sequence. The term "linked" means not only a fusion of a first amino acid sequence to a second amino acid sequence at the C-terminus orthe N-terminus, but also includes insertion of the whole first amino acid sequence (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (or the first amino acid sequence, respectively). In one embodiment, the first amino acid sequence can be linked to a second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to a second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a nucleotide chain (for nucleotide chains) or any chemical moiety (for both polypeptide and polynucleotide chains). The term "linked" is also indicated by a hyphen (-).

[0085] As used herein the term "associated with" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" means a covalent, non-peptide bond or a non-covalent bond. This association can be indicated by a colon, i.e., (:). In another embodiment, it means a covalent bond except a peptide bond. For example, the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by a disulfide bond and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, a peptide bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond, a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non- covalent bond include an ionic bond (e.g., cation-pi bond or salt bond), a metal bond, an hydrogen bond (e.g., dihydrogen bond, dihydrogen complex, low-barrier hydrogen bond, or symmetric hydrogen bond), van der Walls force, London dispersion force, a mechanical bond, a halogen bond, aurophilicity, intercalation, stacking, entropic force, or chemical polarity.

[0086] The term "monomer-dimer hybrid" used herein refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain, which are associated with each other by a disulfide bond, wherein the first chain comprises a clotting factor, e.g., Factor VIII, and a first Fc region and the second chain comprises, consists essentially of, or consists of a second Fc region without the clotting factor. The monomer-dimer hybrid construct thus is a hybrid comprising a monomer aspect having only one clotting factor and a dimer aspect having two Fc regions.

[0087] Hemostasis, as used herein, means the stopping or slowing of bleeding or hemorrhage; or the stopping or slowing of blood flow through a blood vessel or body part.

[0088] Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to hemorrhage, either spontaneously or as a result of trauma, due to an impaired ability or inability to form a fibrin clot. Examples of such disorders include the hemophilias. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease") and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, e.g., von Willebrand disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency, deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor VII, Factor X or factor XIII, Bernard-Soulier syndrome, which is a defect or deficiency in GPIb. GPIb, the receptor for vWF, can be defective and lead to lack of primary clot formation (primary hemostasis) and increased bleeding tendency), and thrombasthenia of Glanzman and Naegeli (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of coagulation factors by the liver; this can increase bleeding risk.

[0089] The lentiviral vectors comprising the isolated nucleic acid molecule of the disclosure can be used prophylactically. As used herein the term "prophylactic treatment" refers to the administration of a molecule prior to a bleeding episode. In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. For example, a lentiviral vector of the disclosure can be administered prior to or after surgery as a prophylactic. The lentiviral vector of the disclosure can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, dental procedures, or stem cell transplantation.

[0090] The lentiviral vectors of the disclosure are also used for on-demand treatment. The term "on-demand treatment" refers to the administration of a lentiviral vector disclosed herein in response to symptoms of a bleeding episode or before an activity that can cause bleeding. In one aspect, the on-demand treatment can be given to a subject when bleeding starts, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, the on-demand treatment can be given prior to activities that increase the risk of bleeding, such as contact sports.

[0091] As used herein the term "acute bleeding" refers to a bleeding episode regardless of the underlying cause. For example, a subject can have trauma, uremia, a hereditary bleeding disorder (e.g., factor VII deficiency) a platelet disorder, or resistance owing to the development of antibodies to clotting factors.

[0092] Treat, treatment, treating, as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration of oneor more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition, or the prophylaxis of one or more symptoms associated with a disease or condition. In one embodiment, the term "treating" or "treatment" means maintaining a FVIII trough level at least about 1 ILI / dL, 2 ILI / dL, 3 ILI / dL, 4 lU / dL, 5 lU / dL, 6 lU / dL, 7 lU / dL, 8 lU / dL, 9 lU / dL, 10 lU / dL, 1 1 lU / dL, 12 lU / dL, 13 ILI / dL, 14 ILI / dL, 15 lU / dL, 16 lU / dL, 17 lU / dL, 18 lU / dL, 19 lU / dL, or 20 lU / dL in a subject by administering a lentiviral vector of the disclosure. In another embodiment, treating or treatment means maintaining a FVIII trough level between about 1 and about 20 ILI / dL, about 2 and about 20 ILI / dL, about 3 and about 20 ILI / dL, about 4 and about 20 ILI / dL, about 5 and about 20 ILI / dL, about 6 and about 20 ILI / dL, about 7 and about 20 ILI / dL, about 8 and about 20 ILI / dL, about 9 and about 20 ILI / dL, or about 10 and about 20 ILI / dL. Treatment or treating of a disease or condition can also include maintaining FVIII activity in a subject at a level comparable to at least about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophiliac subject. In one embodiment, the term "treating" or "treatment" means maintaining a FVIII trough level at least about 30 ILI / dL, 40 ILI / dL, 50 ILI / dL, 60 ILI / dL, 70 lU / dL, 80 lU / dL, 90 lU / dL, 100 lU / dL, 1 10 lU / dL, 120 lU / dL, 130 lU / dL, 140 lU / dL, or 150 ILI / dL in a subject by administering a lentiviral vector of the disclosure. In another embodiment, treating or treatment means maintaining a FVIII trough level between about 10 and about 20 ILI / dL, about 20 and about 23 ILI / dL, about 30 and about 40 ILI / dL, about 40 and about 50 ILI / dL, about 50 and about 60 ILI / dL, about 60 and about 70 ILI / dL, about 70 and about 80 ILI / dL, about 80 and about 90 ILI / dL, about 90 and about 100 ILI / dL, about 1 10 and about 120 ILI / dL, about 120 and about 130 ILI / dL, about 130 and about 140 ILI / dL, or about 140 and about 150 ILI / dL. Treatment or treating of a disease or condition can also include maintaining FVIII activity in a subject at a level comparable to at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 1 10%, 1 15%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the FVIII activity in a non-hemophiliac subject. The minimum trough level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each person.

[0093] "Administering," as used herein, means to give a pharmaceutically acceptableFactor Vlll-encoding nucleic acid molecule, Factor VIII polypeptide, or vector comprising a Factor Vlll-encoding nucleic acid molecule of the disclosure to a subject via a pharmaceutically acceptable route. Routes of administration can be intravenous, e.g., intravenous injection and intravenous infusion. Additional routes of administration include, e.g., subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The nucleic acid molecules,polypeptides, and vectors can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0094] As used herein, the phrase "subject in need thereof includes subjects, such as mammalian subjects, that would benefit from administration of a nucleic acid molecule, a polypeptide, or vector of the disclosure, e.g., to improve hemostasis. In one embodiment, the subjects include, but are not limited to, individuals with hemophilia. In another embodiment, the subjects include, but are not limited to, the individuals who have developed a FVIII inhibitor and thus are in need of a bypass therapy. The subject can be an adult or a minor (e.g., under 12 years old).

[0095] As used herein, the term "clotting factor," refers to molecules, or analogs thereof, naturally occurring or recombinantly produced which prevent or decrease the duration of a bleeding episode in a subject. In other words, it means molecules having pro-clotting activity, i.e., are responsible for the conversion of fibrinogen into a mesh of insoluble fibrin causing the blood to coagulate or clot. An "activatable clotting factor" is a clotting factor in an inactive form (e.g., in its zymogen form) that is capable of being converted to an active form.

[0096] Clotting activity, as used herein, means the ability to participate in a cascade of biochemical reactions that culminates in the formation of a fibrin clot and / or reduces the severity, duration or frequency of hemorrhage or bleeding episode.

[0097] As used herein the terms "heterologous" or "exogenous" refer to such molecules that are not normally found in a given context, e.g., in a cell or in a polypeptide. For example, an exogenous or heterologous molecule can be introduced into a cell and are only present after manipulation of the cell, e.g., by transfection or other forms of genetic engineering or a heterologous amino acid sequence can be present in a protein in which it is not naturally found.

[0098] As used herein, the term "heterologous nucleotide sequence" refers to a nucleotide sequence that does not naturally occur with a given polynucleotide sequence. In one embodiment, the heterologous nucleotide sequence encodes a polypeptide capable of extending the half-life of FVIII. In another embodiment, the heterologous nucleotide sequence encodes a polypeptide that increases the hydrodynamic radius of FVIII. In other embodiments, the heterologous nucleotide sequence encodes a polypeptide that improves one or more pharmacokinetic properties of FVIII without significantly affecting its biological activity or function (e.g., its procoagulant activity). In some embodiments, FVIII is linked or connected to the polypeptide encoded by the heterologous nucleotide sequence by a linker. Non-limiting examples of polypeptide moieties encoded by heterologous nucleotide sequences include an immunoglobulin constant region or a portion thereof, albumin or a fragment thereof, an albumin-binding moiety, a transferrin, the PAS polypeptides of U.S. Pat Application No. 20100292130, a HAP sequence, transferrin or a fragmentthereof, the C-terminal peptide (CTP) of the b subunit of human chorionic gonadotropin, albumin binding small molecule, an XTEN sequence, FcRn binding moieties (e.g., complete Fc regions or portions thereof which bind to FcRn), single chain Fc regions (ScFc regions, e.g., as described in US 2008 / 0260738, WO 2008 / 012543, or WO 2008 / 1439545), polyglycine linkers, polyserine linkers, peptides and short polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) with varying degrees of secondary structure from less than 50% to greater than 50%, amongst others, or two or more combinations thereof. In some embodiments, the polypeptide encoded by the heterologous nucleotide sequence is linked to a non-polypeptide moiety. Non-limiting examples of the non-polypeptide moieties include polyethylene glycol (PEG), albumin-binding small molecules, polysialic acid, hydroxyethyl starch (HES), a derivative thereof, or any combinations thereof.

[0099] As used herein, the term "Fc region" is defined as the portion of a polypeptide which corresponds to the Fc region of native Ig, i.e., as formed by the dimeric association of the respective Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, the term "genetically-fused Fc region" or "single-chain Fc region" (scFc region), as used herein, refers to a synthetic dimeric Fc region comprised of Fc domains genetically linked within a single polypeptide chain (i.e., encoded in a single contiguous genetic sequence).

[0100] In one embodiment, the "Fc region" refers to the portion of a single Ig heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e. residue 216 in IgG, taking the first residue of heavy chain constant region to be 1 14) and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0101] The Fc region of an Ig constant region, depending on the Ig isotype can include theCH2, CH3, and CH4 domains, as well as the hinge region. Chimeric proteins comprising an Fc region of an Ig bestow several desirable properties on a chimeric protein including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03 / 077834; US2003-0235536A1), which are incorporated herein by reference in their entireties.

[0102] A "reference nucleotide sequence," when used herein as a comparison to a nucleotide sequence of the disclosure, is a polynucleotide sequence essentially identical to the nucleotide sequence of the disclosure except that the portions corresponding to FVIII sequence are not optimized. For example, the reference nucleotide sequence for a nucleic acid molecule consisting of the codon optimized BDD FVIII of SEQ ID NO: 1 and a heterologous nucleotide sequence that encodes a single chain Fc region linked to SEQ ID NO: 1 at its 3' end is a nucleicacid molecule consisting of the original (or "parent") BDD FVIII of SEQ ID NO: 16 (FIG. 11) and the identical heterologous nucleotide sequence that encodes a single chain Fc region linked to SEQ ID NO: 16 at its 3' end.

[0103] A "codon adaptation index," as used herein, refers to a measure of codon usage bias. A codon adaptation index (CAI) measures the deviation of a given protein coding gene sequence with respect to a reference set of genes (Sharp PM and Li WH, Nucleic Acids Res. 15(3): 1281—95 (1987)). CAI is calculated by determining the geometric mean of the weight associated to each codon over the length of the gene sequence (measured in codons):

[0104] For each amino acid, the weight of each of its codons, in CAI, is computed as the ratio between the observed frequency of the codon (fi) and the frequency of the synonymous codon (fj) for that amino acid:

[0105] Formula 2:

[0106] As used herein, the term "optimized," with regard to nucleotide sequences, refers to a polynucleotide sequence that encodes a polypeptide, wherein the polynucleotide sequence has been mutated to enhance a property of that polynucleotide sequence. In some embodiments, the optimization is done to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease the binding of regulatory proteins such as general transcription factors, increase or decrease splicing, or increase the yield of the polypeptide produced by the polynucleotide sequence. Examples of changes that can be made to a polynucleotide sequence to optimize it include codon optimization, G / C content optimization, removal of repeat sequences, removal of AT rich elements, removal of cryptic splice sites, removal of cis-acting elements that repress transcription or translation, adding or removing poly-T or poly- A sequences, adding sequences around the transcription start site that enhance transcription, such as Kozak consensus sequences, removal of sequences that could form stem loop structures, removal of destabilizing sequences, and two or more combinations thereof.II. FVIII Lentiviral Gene Therapy

[0107] Somatic gene therapy has been explored as a possible treatment for bleeding disorders, and in particular, hemophilia A. Gene therapy is a particularly appealing treatment for hemophilia because of its potential to cure the disease through continuous endogenous production of FVIII following a single administration of a vector encoding FVIII. Haemophilia A is well suitedfor a gene replacement approach because its clinical manifestations are entirely attributable to the lack of a single gene product (FVIII) that circulates in minute amounts (200ng / ml) in the plasma.

[0108] Lentiviral vectors are gaining prominence as gene delivery vehicles due to their large capacity and ability to sustain transgene expression via integration. Lentiviral vectors have been evaluated in numerous ex-vivo cell therapy clinical programs with promising efficacy and safety profiles.

[0109] The present disclosure meets an important need in the art by providing lentiviral vectors comprising a codon optimized FVIII sequence that demonstrates increased expression in a subject and potentially results in greater therapeutic efficacy when used in gene therapy methods. Embodiments of the present disclosure are directed to lentiviral vectors comprising one or more codon optimized nucleic acid molecules encoding a polypeptide with FVIII activity described herein, host cells (e.g., hepatocytes) comprising the lentiviral vectors, and methods of use of the disclosed lentiviral vectors (e.g., treatments for bleeding disorders using the lentiviral vectors disclosed herein).

[0110] In general, the methods of treatment disclosed herein involve administration of a lentiviral vector comprising a nucleic acid molecule comprising at least one codon optimized nucleic acid sequence encoding a FVIII clotting factor, wherein the nucleic acid sequence encoding a FVIII clotting factor is operably linked to suitable expression control sequences, which in some embodiments are incorporated into the lentiviral vector (e.g., a replication-defective lentiviral viral vector).

[0111] The present disclosure provides methods of treating a bleeding disorder (e.g., hemophilia A) in a subject in need thereof comprising administering to the subject at least one dose of 5x1010or less transducing units / kg (TU / kg) (or 109or less TU / kg, or 10®or less TU / kg) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has:(i) at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1 ;(ii) at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2;(iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO:70;(iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO:71 ;(v) at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3;(vi) at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4;(vii) at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5;(viii) at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6; or(ix) or any combination of (i) to (viii).

[0112] The present disclosure also provides a method of treating a bleeding disorder (e.g., hemophilia A) in a subject in need thereof comprising administering to the subject at least one dose of 5x1010or less transducing units / kg (TU / kg) (or 109TU / kg or less, or 10®TU / kg or less) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a Factor VIII (FVIII) polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide;(a) wherein the first nucleic acid sequence has:(i) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 3;(ii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 4;(iii) at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 5; or(iv) at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 6;(b) wherein the second nucleotide sequence has:(i) at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 3;(ii) at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 4;(iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5; or(iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6; or(c) any combination of (a) and (b); andwherein the N-terminal portion and the C-terminal portion together have a FVI II polypeptide activity.

[0113] In some embodiments, the dose is about 5.0x1010TU / kg, about 4.9x1010TU / kg, about 4.8x1010TU / kg, about 4.7x1010TU / kg, about 4.6x1010TU / kg, about 4.5x1010TU / kg, about 4.4x1010TU / kg, about 4.3x1010TU / kg, about 4.2x1010TU / kg, about 4.1 x1010TU / kg, about 4.0x1010TU / kg, about 3.9x1010TU / kg, about 3.8x1010TU / kg, about 3.7x1010TU / kg, about 3.6x1010TU / kg, about 3.5x1010TU / kg, about 3.4x1010TU / kg, about 3.3x1010TU / kg, about 3.2x1010TU / kg, about 3.1x1010TU / kg, about 3.0x1010TU / kg, about 2.9x1010TU / kg, about 2.8x1010TU / kg, about 2.7x1010TU / kg, about 2.6x1010TU / kg, about 2.5x1010TU / kg, about 2.4x1010TU / kg, about 2.3x1010TU / kg, about 2.2x1010TU / kg, about 2.1x1010TU / kg, about 2.0x1010TU / kg, about 1 .9x1010TU / kg, about 1 .8x1010TU / kg, about 1 .7x1010TU / kg, about 1 .6x1010TU / kg, about 1 .5x1010TU / kg, about 1.4x1010TU / kg, about 1.3x1010TU / kg, about 1.2x1010TU / kg, about 1 .1x1010TU / kg, or about 1 .0x1010TU / kg.

[0114] In some embodiments, the dose is about 9.9x109TU / kg, about 9.8x109TU / kg, about 9.7x109TU / kg, about 9.6x109TU / kg, about 9.5x109TU / kg, about 9.4x109TU / kg, about 9.3x109TU / kg, about 9.2x109TU / kg, about 9.1x109TU / kg, about 9.0x109TU / kg, about 8.9x109TU / kg, about 8.8x109TU / kg, about 8.7x109TU / kg, about 8.6x109TU / kg, about 8.5x109TU / kg, about 8.4x109TU / kg, about 8.3x109TU / kg, about 8.2x109TU / kg, about 8.1x109TU / kg, about 8.0x109TU / kg, about 7.9x109TU / kg, about 7.8x109TU / kg, about 7.7x109TU / kg, about 7.6x109TU / kg, about 7.5x109TU / kg, about 7.4x109TU / kg, about 7.3x109TU / kg, about 7.2x109TU / kg, about 7.1x109TU / kg, about 7.0x109TU / kg, about 6.9x109TU / kg, about 6.8x109TU / kg, about 6.7x109TU / kg, about 6.6x109TU / kg, about 6.5x109TU / kg, about 6.4x109TU / kg, about 6.3x109TU / kg, about 6.2x109TU / kg, about 6.1x109TU / kg, about 6.0x109TU / kg, about 5.9x109TU / kg, about 5.8x109TU / kg, about 5.7x109TU / kg, about 5.6x109TU / kg, about 5.5x109TU / kg, about 5.4x109TU / kg, about 5.3x109TU / kg, about 5.2x109TU / kg, about 5.1x109TU / kg, about 5.0x109TU / kg, about 4.9x109TU / kg, about 4.8x109TU / kg, about 4.7x109TU / kg, about 4.6x109TU / kg, about 4.5x109TU / kg, about 4.4x109TU / kg, about 4.3x109TU / kg, about 4.2x109TU / kg, about 4.1x109TU / kg, about 4.0x109TU / kg, about 3.9x109TU / kg, about 3.8x109TU / kg, about 3.7x109TU / kg, about 3.6x109TU / kg, about 3.5x109TU / kg, about 3.4x109TU / kg, about 3.3x109TU / kg, about 3.2x109TU / kg, about 3.1x109TU / kg, about 3.0x109TU / kg, about 2.9x109TU / kg, about 2.8x109TU / kg, about 2.7x109TU / kg, about 2.6x109TU / kg, about 2.5x109TU / kg, about 2.4x109TU / kg, about 2.3x109TU / kg, about 2.2x109TU / kg, about 2.1x109TU / kg, about 2.0x109TU / kg, about 1 .9x109TU / kg, about 1.8x109TU / kg, about 1 .7x109TU / kg, about 1 .6x109TU / kg, about 1 .5x109TU / kg, about 1 .4x109TU / kg. about 1 .3x109TU / kg, about 1 .2x109TU / kg, about 1 .1x109TU / kg, or about 1 .0x109TU / kg.

[0115] In some embodiments, the dose is about 9.9x10®TU / kg, about 9.8x10®TU / kg, about 9.7x10®TU / kg, about 9.6x10®TU / kg, about 9.5x10®TU / kg, about 9.4x10®TU / kg, about 9.3x10®TU / kg, about 9.2x10®TU / kg, about 9.1x10®TU / kg, about 9.0x10®TU / kg, about 8.9x10®TU / kg, about 8.8x10®TU / kg, about 8.7x10®TU / kg, about 8.6x10®TU / kg, about 8.5x10®TU / kg, about 8.4x10®TU / kg, about 8.3x10®TU / kg, about 8.2x10®TU / kg, about 8.1x10®TU / kg, about 8.0x10®TU / kg, about 7.9x10®TU / kg, about 7.8x10®TU / kg, about 7.7x10®TU / kg, about 7.6x10®TU / kg, about 7.5x10®TU / kg, about 7.4x10®TU / kg, about 7.3x10®TU / kg, about 7.2x10®TU / kg, about 7.1x10®TU / kg, about 7.0x10®TU / kg, about 6.9x10®TU / kg, about 6.8x10®TU / kg, about 6.7x10®TU / kg, about 6.6x10®TU / kg, about 6.5x10®TU / kg, about 6.4x10®TU / kg, about 6.3x10®TU / kg, about 6.2x10®TU / kg, about 6.1x10®TU / kg, about 6.0x10®TU / kg, about 5.9x10®TU / kg, about 5.8x10®TU / kg, about 5.7x10®TU / kg, about 5.6x10®TU / kg, about 5.5x10®TU / kg, about 5.4x10®TU / kg, about 5.3x10®TU / kg, about 5.2x10®TU / kg, about 5.1x10®TU / kg, about 5.0x10®TU / kg, about 4.9x10®TU / kg, about 4.8x10®TU / kg, about 4.7x10®TU / kg, about 4.6x10®TU / kg, about 4.5x10®TU / kg, about 4.4x10®TU / kg, about 4.3x10®TU / kg, about 4.2x10®TU / kg, about 4.1x10®TU / kg, about 4.0x10®TU / kg, about 3.9x10®TU / kg, about 3.8x10®TU / kg, about 3.7x10®TU / kg, about 3.6x10®TU / kg, about 3.5x10®TU / kg, about 3.4x10®TU / kg, about 3.3x10®TU / kg, about 3.2x10®TU / kg, about 3.1x10®TU / kg, about 3.0x10®TU / kg, about 2.9x10®TU / kg, about 2.8x10®TU / kg, about 2.7x10®TU / kg, about 2.6x10®TU / kg, about 2.5x10®TU / kg, about 2.4x10®TU / kg, about 2.3x10®TU / kg, about 2.2x10®TU / kg, about 2.1x10®TU / kg, about 2.0x10®TU / kg, about 1 .9x10®TU / kg, about 1.8x10®TU / kg, about 1 .7x10®TU / kg, about 1 .6x10®TU / kg, about 1 .5x10®TU / kg, about 1 .4x10®TU / kg. about 1 .3x10®TU / kg, about 1 .2x10®TU / kg, about 1 .1x10®TU / kg, or about 1 .0x10®TU / kg.

[0116] In some embodiments, the dose is less than 5.0x1010TU / kg, less than 4.9x1010TU / kg, less than 4.8x1010TU / kg, less than 4.7x1010TU / kg, less than 4.6x1010TU / kg, less than 4.5x1010TU / kg, less than 4.4x1010TU / kg, less than 4.3x1010TU / kg, less than 4.2x1010TU / kg, less than 4.1x1010TU / kg, less than 4.0x1010TU / kg, less than 3.9x1010TU / kg, less than 3.8x1010TU / kg, less than 3.7x1010TU / kg, less than 3.6x1010TU / kg, less than 3.5x1010TU / kg, less than 3.4x1010TU / kg, less than 3.3x1010TU / kg, less than 3.2x1010TU / kg, less than 3.1x1010TU / kg, less than 3.0x1010TU / kg, less than 2.9x1010TU / kg, less than 2.8x1010TU / kg, less than 2.7x1010TU / kg, less than 2.6x1010TU / kg, less than 2.5x1010TU / kg, less than 2.4x1010TU / kg, less than 2.3x1010TU / kg, less than 2.2x1010TU / kg, less than 2.1x1010TU / kg, less than 2.0x1010TU / kg, less than 1 .9x1010TU / kg, less than 1 .8x1010TU / kg, less than 1.7x1010TU / kg, less than 1 .6x1010TU / kg, less than 1 .5x1010TU / kg, less than 1 .4x1010TU / kg, less than 1 .3x1010TU / kg, less than 1 .2x1010TU / kg, less than 1.1x1010TU / kg, or less than 1 .0x1010TU / kg.

[0117] In some embodiments, the dose is less than 9.9x109TU / kg, less than 9.8x109TU / kg, less than 9.7x109TU / kg, less than 9.6x109TU / kg, less than 9.5x109TU / kg, less than 9.4x109TU / kg, less than 9.3x109TU / kg, less than 9.2x109TU / kg, less than 9.1x109TU / kg, less than 9.0x109TU / kg, less than 8.9x109TU / kg, less than 8.8x109TU / kg, less than 8.7x109TU / kg, less than 8.6x109TU / kg, less than 8.5x109TU / kg, less than 8.4x109TU / kg, less than 8.3x109TU / kg, less than 8.2x109TU / kg, less than 8.1x109TU / kg, less than 8.0x109TU / kg, less than 7.9x109TU / kg, less than 7.8x109TU / kg, less than 7.7x109TU / kg, less than 7.6x109TU / kg, less than 7.5x109TU / kg, less than 7.4x109TU / kg, less than 7.3x109TU / kg, less than 7.2x109TU / kg, less than 7.1x109TU / kg, less than 7.0x109TU / kg, less than 6.9x109TU / kg, less than 6.8x109TU / kg, less than 6.7x109TU / kg, less than 6.6x109TU / kg, less than 6.5x109TU / kg, less than 6.4x109TU / kg, less than 6.3x109TU / kg, less than 6.2x109TU / kg, less than 6.1x109TU / kg, less than 6.0x109TU / kg, less than 5.9x109TU / kg, less than 5.8x109TU / kg, less than 5.7x109TU / kg, less than 5.6x109TU / kg, less than 5.5x109TU / kg, less than 5.4x109TU / kg, less than 5.3x109TU / kg, less than 5.2x109TU / kg, less than 5.1x109TU / kg, less than 5.0x109TU / kg, less than 4.9x109TU / kg, less than 4.8x109TU / kg, less than 4.7x109TU / kg, less than 4.6x109TU / kg, less than 4.5x109TU / kg, less than 4.4x109TU / kg, less than 4.3x109TU / kg, less than 4.2x109TU / kg, less than 4.1x109TU / kg, less than 4.0x109TU / kg, less than 3.9x109TU / kg, less than 3.8x109TU / kg, less than 3.7x109TU / kg, less than 3.6x109TU / kg, less than 3.5x109TU / kg, less than 3.4x109TU / kg, less than 3.3x109TU / kg, less than 3.2x109TU / kg, less than 3.1x109TU / kg, less than 3.0x109TU / kg, less than 2.9x109TU / kg, less than 2.8x109TU / kg, less than 2.7x109TU / kg, less than 2.6x109TU / kg, less than 2.5x109TU / kg, less than 2.4x109TU / kg, less than 2.3x109TU / kg, less than 2.2x109TU / kg, less than 2.1x109TU / kg, less than 2.0x109TU / kg, less than 1.9x109TU / kg, less than 1.8x109TU / kg, less than 1.7x109TU / kg, less than 1.6x109TU / kg, less than 1.5x109TU / kg, less than 1.4x109TU / kg, less than 1.3x109TU / kg, less than 1.2x109TU / kg, less than 1.1x109TU / kg, or less than 1.0x109TU / kg.

[0118] In some embodiments, the dose is less than 9.9x10®TU / kg, less than 9.8x10®TU / kg, less than 9.7x10®TU / kg, less than 9.6x10®TU / kg, less than 9.5x10®TU / kg, less than 9.4x10®TU / kg, less than 9.3x10®TU / kg, less than 9.2x10®TU / kg, less than 9.1x10®TU / kg, less than 9.0x10®TU / kg, less than 8.9x10®TU / kg, less than 8.8x10®TU / kg, less than 8.7x10®TU / kg, less than 8.6x10®TU / kg, less than 8.5x10®TU / kg, less than 8.4x10®TU / kg, less than 8.3x10®TU / kg, less than 8.2x10®TU / kg, less than 8.1x10®TU / kg, less than 8.0x10®TU / kg, less than 7.9x10®TU / kg, less than 7.8x10®TU / kg, less than 7.7x10®TU / kg, less than 7.6x10®TU / kg, less than 7.5x10®TU / kg, less than 7.4x10®TU / kg, less than 7.3x10®TU / kg, less than 7.2x10®TU / kg, less than 7.1x10®TU / kg, less than 7.0x10®TU / kg, less than 6.9x10®TU / kg, less than 6.8x10®TU / kg, less than 6.7x10®TU / kg, less than 6.6x10®TU / kg, less than 6.5x10®TU / kg, less than6.4x108TU / kg, less than 6.3x108TU / kg, less than 6.2x108TU / kg, less than 6.1x108TU / kg, less than 6.0x108TU / kg, less than 5.9x108TU / kg, less than 5.8x108TU / kg, less than 5.7x108TU / kg, less than 5.6x10®TU / kg, less than 5.5x10®TU / kg, less than 5.4x10®TU / kg, less than 5.3x10®TU / kg, less than 5.2x10®TU / kg, less than 5.1x10®TU / kg, less than 5.0x10®TU / kg, less than 4.9x10®TU / kg, less than 4.8x10®TU / kg, less than 4.7x10®TU / kg, less than 4.6x10®TU / kg, less than 4.5x10®TU / kg, less than 4.4x10®TU / kg, less than 4.3x10®TU / kg, less than 4.2x10®TU / kg, less than 4.1x10®TU / kg, less than 4.0x10®TU / kg, less than 3.9x10®TU / kg, less than 3.8x10®TU / kg, less than 3.7x10®TU / kg, less than 3.6x10®TU / kg, less than 3.5x10®TU / kg, less than 3.4x10®TU / kg, less than 3.3x10®TU / kg, less than 3.2x10®TU / kg, less than 3.1x10®TU / kg, less than 3.0x10®TU / kg, less than 2.9x10®TU / kg, less than 2.8x10®TU / kg, less than 2.7x10®TU / kg, less than 2.6x10®TU / kg, less than 2.5x10®TU / kg, less than 2.4x10®TU / kg, less than 2.3x10®TU / kg, less than 2.2x10®TU / kg, less than 2.1x10®TU / kg, less than 2.0x10®TU / kg, less than 1 .9x10®TU / kg, less than 1 .8x10®TU / kg, less than 1 .7x10®TU / kg, less than 1 .6x10®TU / kg, less than 1 .5x10®TU / kg, less than 1 .4x10®TU / kg, less than 1 .3x10®TU / kg, less than 1 .2x10®TU / kg, less than 1 .1x10®TU / kg, or less than 1 .0x10®TU / kg.

[0119] In some embodiments, the dose is between 1x10®TU / kg and 5x1010TU / kg, between 1 .5x10®TU / kg and 5x1010TU / kg, between 2x10®TU / kg and 5x1010TU / kg, between 2.5x10®TU / kg and 5x1010TU / kg, between 3x10®TU / kg and 5x1010TU / kg, between 3.5x10®TU / kg and 5x1010TU / kg, between 4x10®TU / kg and 5x1010TU / kg, between 4.5x10®TU / kg and 5x1010TU / kg, between 5x10®TU / kg and 5x1010TU / kg, between 5.5x10®TU / kg and 5x1010TU / kg, between 6x10®TU / kg and 5x1010TU / kg, between 6.5x10®TU / kg and 5x1010TU / kg, between 7x10®TU / kg and 5x1010TU / kg, between 7.5x10®TU / kg and 5x1010TU / kg, between 8x10®TU / kg and 5x1010TU / kg, between 8.5x10®TU / kg and 5x1010TU / kg, between 9x10®TU / kg and 5x1010TU / kg, between 9.5x10®TU / kg and 5x1010TU / kg, between 1x109TU / kg and 5x1010TU / kg, between 1 .5x109TU / kg and 5x1010TU / kg, between 2x109TU / kg and 5x1010TU / kg, between 2.5x109TU / kg and 5x1010TU / kg, between 3x109TU / kg and 5x1010TU / kg, between 3.5x109TU / kg and 5x1010TU / kg, between 4x109TU / kg and 5x1010TU / kg, between 4.5x109TU / kg and 5x1010TU / kg, between 5x109TU / kg and 5x1010TU / kg, between 5.5x109TU / kg and 5x1010TU / kg, between 6x109TU / kg and 5x1010TU / kg, between 6.5x109TU / kg and 5x1010TU / kg, between 7x109TU / kg and 5x1010TU / kg, between 7.5x109TU / kg and 5x1010TU / kg, between 8x109TU / kg and 5x1010TU / kg, between 8.5x109TU / kg and 5x1010TU / kg, between 9x109TU / kg and 5x1010TU / kg, between 9.5x109TU / kg and 5x1010TU / kg, between 1010TU / kg and 5x1010TU / kg, between 1 .5x1010TU / kg and 5x1010TU / kg, between 2x1010TU / kg and 5x1010TU / kg, between 2.5x1010TU / kg and 5x1010TU / kg, between 3x1010TU / kg and 5x1010TU / kg, between 3.5x1010TU / kg and 5x1010TU / kg, between 4x1010TU / kg and 5x1010TU / kg, or between 4.5x1010TU / kg and 5x1010TU / kg.

[0120] In some embodiments, the dose is between 1x10®TU / kg and 5x1010TU / kg, between 1x10®TU / kg and 4.5x1010TU / kg, between 1x10®TU / kg and 4x1010TU / kg, between 1x10®TU / kg and 3.5x1010TU / kg, between 1 x10®TU / kg and 3x1010TU / kg, between 1 x10®TU / kg and 2.5x1010TU / kg, between 1x10®TU / kg and 2x1010TU / kg, between 1x10®TU / kg and 1 .5x1010TU / kg, between 1x10®TU / kg and 1010TU / kg, between 1x10®TU / kg and 9x109TU / kg, between 1x10®TU / kg and 8.5x109TU / kg, between 1x10®TU / kg and 8x109TU / kg, between 1x10®TU / kg and 7.5x109TU / kg, between 1x10®TU / kg and 7x109TU / kg, between 1x10®TU / kg and 6.5x109TU / kg, between 1x10®TU / kg and 6x109TU / kg, between 1x10®TU / kg and 5.5x109TU / kg, between 1x10®TU / kg and 5x109TU / kg, between 1x10®TU / kg and 4.5x109TU / kg, between 1x10®TU / kg and 4x109TU / kg, between 1x10®TU / kg and 3.5x109TU / kg, between 1x10®TU / kg and 3x109TU / kg, between 1x10®TU / kg and 2.5x109TU / kg, between 1x10®TU / kg and 2x109, between 1x10®TU / kg and 1.5x109TU / kg, between 1x10®TU / kg and 1x109TU / kg, between 1x10®TU / kg and 9.5x10®TU / kg, between 1x10®TU / kg and 9x10®TU / kg, between 1x10®TU / kg and 8.5x10®TU / kg, between 1x10®TU / kg and 8x10®TU / kg, between 1x10®TU / kg and 7.5x10®TU / kg, between 1x10®TU / kg and 7x10®TU / kg, between 1x10®TU / kg and 6.5x10®TU / kg, between 1x10®TU / kg and 6x10®TU / kg, between 1x10®TU / kg and 5.5x10®TU / kg, between 1x10®TU / kg and 5x10®TU / kg, between 1x10®TU / kg and 4.5x10®TU / kg, between 1x10®TU / kg and 4x10®TU / kg, between 1x10®TU / kg and 3.5x10®TU / kg, between 1x10®TU / kg and 3x10®TU / kg, between 1x10®TU / kg and 2.5x10®TU / kg, between 1x10®TU / kg and 2x10®, or between 1x10®TU / kg and 1 .5x10®TU / kg,

[0121] In some embodiments, the dose is between 1x1010TU / kg and 2x1010TU / kg, between 1 .1x1010TU / kg and 1 .9x1010TU / kg, between 1 .2x1010TU / kg and 1 .8x1010TU / kg, between 1 .3x1010TU / kg and 1 .7x1010TU / kg, or between 1.4x1010TU / kg and 1 .6x1010TU / kg. In some embodiments, the dose is about 1 .5x1010TU / kg. In some embodiments, the dose is 1.5x1010TU / kg.

[0122] In some embodiments, the dose is between 1x109TU / kg and 2x109TU / kg, between1 .1 x109TU / kg and 1.9x109TU / kg, between 1 .2x109TU / kg and 1.8x109TU / kg, between 1.3x109TU / kg and 1 .7x109TU / kg, or between 1.4x109TU / kg and 1 .6x109TU / kg. In some embodiments, the dose is 1 .5x109TU / kg. In certain embodiments, the dose is about 3.0 x 109TU / kg.

[0123] In some embodiments, the dose is between 2.5x109TU / kg and 3.5x109TU / kg, between 2.6 x109TU / kg and 3.4x109TU / kg, between 2.7x109TU / kg and 3.3x109TU / kg, between 2.8x109TU / kg and 3.2x109TU / kg, or between 2.9x109TU / kg and 3.1x109TU / kg. In some embodiments, the dose is about 3.0x109TU / kg. In some embodiments, the dose is 3.0x109TU / kg.

[0124] In some embodiments, the dose is between 5.5x109TU / kg and 6.5x109TU / kg, between 5.6 x109TU / kg and 6.4x109TU / kg, between 5.7x109TU / kg and 6.3x109TU / kg, between5.8x109TU / kg and 6.2x109TU / kg, or between 5.9x109TU / kg and 6.1x109TU / kg. In some embodiments, the dose is about 6.0x109TU / kg. In some embodiments, the dose is 6.0x109TU / kg.

[0125] In some embodiments, plasma FVIII activity at 24 hours, 36 hours, or 48 hours post administration of the lentiviral vector of the present disclosure is increased relative to the plasma FVIII activity a subject administered a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16.

[0126] In some embodiments, plasma FVIII activity after 48 hours post administration of the lentiviral vector is increased relative to the plasma FVIII activity in a subject administered a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16.

[0127] In another embodiment, plasma FVIII activity is increased at about 21 days post administration of the lentiviral vector relative to a subject administered a reference nucleic acid molecule comprising SEQ ID NO: 16, a reference viral vector comprising the reference nucleic acid molecule, or a polypeptide encoded by the reference nucleic acid molecule.

[0128] In some embodiments, plasma FVIII activity is increased at about 6 hours, at about12 hours, at about 18 hours, at about 24 hours, at about 36 hours, at about 48 hours, at about 3 days, at about 4 days, at about 5 days, at about 6 days, at about 7 days, at about 8 days, at about 9 days, at about 10 days, at about 11 days, at about 12 days, at about 13 days, at about 14 days, at about 15 days, at about 16 days, at about 17 days, at about 18 days, at about 19 days, at about 20 days, at about 21 days, at about 22 days, at about 23 days, at about 24 days, at about 25 days, at about 26 days, at about 27 days, or at about 28 days post administration of the lentiviral vector relative to a subject administered a reference nucleic acid molecule comprising SEQ ID NO: 16, a reference viral vector comprising the reference nucleic acid molecule, or a polypeptide encoded by the reference nucleic acid molecule.

[0129] In some embodiments, the plasma FVIII activity in the subject is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 1 1-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45- fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, at least about 100-fold , at least about 1 10-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, or at least about 200-fold with respect to basal levels in the subject, relative to levels in a subject administered a reference nucleic acid molecule comprisingSEQ ID NO: 16, relative to levels in a subject administered a reference viral vector comprising the reference nucleic acid molecule, or relative to levels in a subject after administration of a polypeptide encoded by the reference nucleic acid molecule.

[0130] In some embodiments, the lentiviral vector is administered as a single dose or multiple doses. In some embodiments, the lentiviral vector dose is administered at once or divided into multiple sub-dose, e.g., two sub-doses, three sub-doses, four sub-doses, five sub-doses, six sub-doses, or more than six sub-doses. In some embodiments, more than one lentiviral vector is administered.

[0131] In some embodiments, the dose of lentiviral vector is administered repeated at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. In some embodiments, the lentiviral vector is administered via intravenous injection.

[0132] In some embodiments, the subject is a pediatric subject, whereas in other aspects, the subject is an adult subject.

[0133] In some embodiments, the lentiviral vector comprises at least one tissue specific promoter, i.e., a promoter that would regulate the expression of the polypeptide with FVIII activity in a particular tissue or cell type. In some embodiments, a tissue specific promoter in the lentiviral vector selectively enhances expression of the polypeptide with FVIII activity in a target liver cell. In some embodiments, the tissue specific promoter that selectively enhances expression of the polypeptide with FVIII activity in a target liver cell comprises an mTTR promoter. In some embodiments, the target liver cell is a hepatocyte.

[0134] Since the lentiviral vector can transduce all liver cell types, the expression of the transgene (e.g., FVIII) in different cell types can be controlled by using different promoters in the lentiviral vector. Thus, the lentiviral vector can comprise specific promoters which would control expression of the FVIII transgene in different tissues or cells types, such as different hepatic tissues or cell types. Thus, in some embodiments, the lentiviral vector can comprise an endothelial specific promoter which would control expression of the FVIII transgene in hepatic endothelial tissue, or a hepatocyte specific promoterwhich would control expression of the FVIII transgene in hepatocytes, or both.

[0135] In some embodiments, the lentiviral vector comprises a tissue-specific promoter or tissue-specific promoters that control the expression of the FVIII transgene in tissues other than liver. In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of the target cell or target tissue, for example, in the genome of a hepatocyte or in the genome of a hepatic endothelial cell.

[0136] In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity in the lentivirus vector of the present disclosure comprises, consists, or consists essentially of LV-coFVIII-6 (SEQ ID NO:71).

[0137] In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity in the lentivirus vector of the present disclosure comprises, consists, or consist essentially of LV-coFVIII-6-XTEN (SEQ ID NO:72).

[0138] In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity in the lentivirus vector of the present disclosure further comprises a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding a signal peptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to (i) nucleotides 1 to 57 of SEQ ID NO: 1 ; (ii) nucleotides 1 to 57 of SEQ ID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ IDNO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) nucleotides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71 ; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.

[0139] In some embodiments, the isolated nucleic acid molecule in a lentiviral vector of the present disclosure comprises in one or more property selected from the group consisting of: (a) the human codon adaptation index the nucleic acid molecule or a portion thereof is increased relative to SEQ ID NO: 16; (b) the frequency of optimal codons of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16; (d) the relative synonymous codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (e) the effective number of codons of the nucleotide sequence or a portion thereof is reduced relative SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21 and 22) relative to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) relative to SEQ ID NO: 16; and (h) any combination thereof.

[0140] In some embodiments, the isolated nucleic acid molecule in a lentiviral vector of the present disclosure further comprises a heterologous nucleotide sequence encoding a heterologous amino acid sequence (e.g., a half-life extender). In some embodiments, the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence. In some embodiments, the heterologous amino acid sequence is linked to the N-terminus or the C-terminus of the amino acid sequence encoded by the nucleotide sequence, or inserted between two amino acids in the amino acid sequence encoded by the nucleotide sequence at one or more insertion site selected from TABLE 3.

[0141] In some embodiments, the FVIII polypeptide is a full length FVIII or a B domain deleted FVIII.

[0142] The lentiviral vectors disclosed herein can be used at low dosages (e.g., 1010TU / kg or lower, 109TU / kg or lower, or 10®TU / kg or lower) in vivo in a mammal, e.g., a human patient, using a gene therapy approach to treatment of a bleeding disease or disorder selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath would be therapeutically beneficial. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A.

[0143] In some embodiments, target cells (e.g., hepatocytes) are treated in vitro with low doses (e.g., 1010TU / kg or lower, 109TU / kg or lower, or 10®TU / kg or lower) of the lentiviral vectors disclosed herein before being administered to the patient. In certain embodiments, target cells (e.g., hepatocytes) are treated in vitro with about 3.0 x 109TU / kg of the lentiviral vectors disclosed herein before being administered to the patient. In yet another embodiment, cells from the patient (e.g., hepatocytes) are treated ex vivo with low doses (e.g., 1010TU / kg or lower, 109TU / kg or lower, or 10®TU / kg or lower) of the lentiviral vectors disclosed herein before being administered to the patient.

[0144] In some embodiments, plasma FVIII activity post administration of a lentiviral vectors disclosed herein (administered, e.g., at 1010TU / kg or lower, 109TU / kg or lower, or 10®TU / kg or lower) is increased by at least about 100%, at least about 1 10%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300%, relative to physiologically normal circulating FVIII levels.

[0145] In one embodiment, the plasma FVIII activity post administration of a lentiviral vector of the present disclosure is increased by at least about 3,000% to about 5,000% relative to physiologically normal circulating FVIII levels. In some embodiments, at 21 days post administration of a lentiviral vector comprising a codon-optimized gene encoding polypeptides with Factor VIII (FVIII) activity described herein, plasma FVIII activity is increased by at least about 10- fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at leastabout 100-fold, at least about 1 10-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, or at least about 200-fold relative to a subject administered a corresponding lentiviral vector comprising a reference nucleic acid molecule comprising SEQ ID NO: 16.

[0146] The present disclosure also provides methods of treating, preventing. Or ameliorating a hemostatic disorder (e.g., a bleeding disorder such as hemophilia A) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the lentiviral vector is administered as at least one dose of 5x1010or less TU / kg, 109or less TU / kg, or 10®or less TU / kg.

[0147] The treatment, amelioration, and prevention by the lentiviral vector of the present disclosure can be a bypass therapy. The subject receiving bypass therapy can have already developed an inhibitor to a clotting factor, e.g., FVIII, or is subject to developing a clotting factor inhibitor.

[0148] The lentiviral vectors of the present disclosure treat or prevent a hemostatic disorder by promoting the formation of a fibrin clot. The polypeptide having FVIII activity encoded by the nucleic acid molecule of the disclosure can activate a member of a coagulation cascade. The clotting factor can be a participant in the extrinsic pathway, the intrinsic pathway or both.

[0149] The lentiviral vectors of the present disclosure can be used to treat hemostatic disorders known to be treatable with FVIII. The hemostatic disorders that can be treated using methods of the disclosure include, but are not limited to, hemophilia A, hemophilia B, von Willebrand's disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency, as well as deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor VII, Factor X, or Factor XIII, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intraabdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath.

[0150] Compositions for administration to a subject include lentiviral vectors comprising nucleic acid molecules which comprise an optimized nucleotide sequence of the disclosure encoding a FVIII clotting factor (for gene therapy applications) as well as FVIII polypeptide molecules. In some embodiments, the composition for administration is a cell contacted with a lentiviral vector of the present disclosure, either in vivo, in vitro, or ex vivo.

[0151] In some embodiments, the hemostatic disorder is an inherited disorder. In one embodiment, the subject has hemophilia A. In other embodiments, the hemostatic disorder is the result of a deficiency in FVIII. In other embodiments, the hemostatic disorder can be the result of a defective FVIII clotting factor.

[0152] In another embodiment, the hemostatic disorder can be an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, as an example, but not as a limitation, cancer, an autoimmune disease, or pregnancy. The acquired disorder can result from old age or from medication to treat an underlying secondary disorder (e.g., cancer chemotherapy).

[0153] The disclosure also relates to methods of treating a subject that does not have a hemostatic disorder or a secondary disease or condition resulting in acquisition of a hemostatic disorder. The disclosure thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of a lentiviral vector of the present disclosure. For example, in one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The lentiviral vector of the disclosure can be administered prior to or after surgery as a prophylactic.

[0154] The lentiviral vector of the disclosure can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, or stem cell transplantation.

[0155] In another embodiment, the lentiviral vector of the disclosure can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder. The acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding.

[0156] The lentiviral vector can be used to prophylactically treat a subject with a hemostatic disorder. The lentiviral vector can also be used to treat an acute bleeding episode in a subject with a hemostatic disorder.

[0157] In another embodiment, the administration of a lentiviral vector disclosed herein and / or subsequent expression of FVIII protein transgene does not induce an immune response in a subject. In some embodiments, the immune response comprises development of antibodies against FVIII. In some embodiments, the immune response comprises cytokine secretion. In some embodiments, the immune response comprises activation of B cells, T cells, or both B cells and T cells. In some embodiments, the immune response is an inhibitory immune response, wherein the immune response in the subject reduces the activity of the FVIII protein relative to the activity of the FVIII in a subject that has not developed an immune response. In certain embodiments, expression of FVIII protein by administering the lentiviral vector of the disclosure prevents aninhibitory immune response against the FVIII protein or the FVIII protein expressed from the isolated nucleic acid molecule or the lentiviral vector.

[0158] In some embodiments, a lentiviral vector of the disclosure is administered in combination with at least one other agent that promotes hemostasis. Said other agent that promotes hemostasis in a therapeutic with demonstrated clotting activity. As an example, but not as a limitation, the hemostatic agent can include Factor V, Factor VII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, prothrombin, or fibrinogen or activated forms of any of the preceding. The clotting factor or hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-amino- caproic acid, tranexamic acid.

[0159] In one embodiment of the disclosure, the composition (e.g., the lentiviral vector) is one in which the FVIII is present in activatable form when administered to a subject. Such an activatable molecule can be activated in vivo at the site of clotting after administration to a subject.

[0160] The lentiviral vector of the disclosure can be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, sublingually, nasally, rectally, vaginally or via pulmonary route. The lentiviral vector can be implanted within or linked to a biopolymer solid support that allows forthe slow release of the vector to the desired site.

[0161] In one embodiment, the route of administration of the lentiviral vectors is parenteral.The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous form of parenteral administration is preferred. While all these forms of administration are clearly contemplated as being within the scope of the disclosure, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection can comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods compatible with the teachings herein, the lentiviral vector can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0162] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0163] More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0164] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0165] In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., a polypeptide by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freezedrying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations can be packaged and sold in the form of a kit. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to clotting disorders.

[0166] The pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0167] Effective doses of the compositions of the present disclosure, for the treatment of conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0168] The lentiviral vector can be administered as a single dose or as multiple doses, wherein the multiple doses can be administered continuously or at specific timed intervals. In vitro assays can be employed to determine optimal dose ranges and / or schedules for administration. In vitro assays that measure clotting factor activity are known in the art. Additionally, effective doses can be extrapolated from dose-response curves obtained from animal models, e.g., a hemophiliac dog (Mount et al. 2002, Blood 99 (8): 2670).

[0169] Doses intermediate in the above ranges are also intended to be within the scope of the disclosure. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months.

[0170] The lentiviral vector of the disclosure can be administered on multiple occasions.Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of modified polypeptide or antigen in the patient. Dosage and frequency of the lentiviral vectors of the disclosure vary depending on the half-life of the FVIII polypeptide encoded by the transgene in the patient.

[0171] The dosage and frequency of administration of the lentiviral vectors of the disclosure can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the lentiviral vector of the disclosure are administered to a patient not already in the disease state to enhance the patient’s resistance or minimize effects of disease. Such an amount is defined to be a "prophylactic effective dose." A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.

[0172] The lentiviral vector of the disclosure can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).

[0173] As used herein, the administration of lentiviral vectors of the disclosure in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed polypeptides. Those skilled in the art will appreciate that the administration or application of the various components of the combined therapeutic regimen can be timed to enhance the overall effectiveness of the treatment. A skilled artisan (e.g., a physician) would be readily be able to discern effective combined therapeutic regimens without undue experimentation based on the selected adjunct therapy and the teachings of the instant specification.

[0174] It will further be appreciated that the lentiviral vectors of the disclosure can be used in conjunction or combination with an agent or agents (e.g. , to provide a combined therapeutic regimen). Exemplary agents with which a lentiviral vector of the instant disclosure can be combined include agents that represent the current standard of care for a particular disorder being treated. Such agents can be chemical or biologic in nature. The term "biologic" or "biologic agent" refers to any pharmaceutically active agent made from living organisms and / or their products which is intended for use as a therapeutic.

[0175] The amount of agent to be used in combination with the lentiviral vectors of the instant disclosure can vary by subject or can be administered according to what is known in the art. See, e.g., Bruce A Chabner et a!., Antineoplastic Agents, in GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 1233- 1287 ((Joel G. Hardman et at , eds., 9thed. 1996). In another embodiment, an amount of such an agent consistent with the standard of care is administered.

[0176] In certain embodiments, the lentiviral vectors of the present disclosure are administered in conjunction with an immunosuppressive, anti-allergic, or anti-inflammatory agent. These agents generally refer to substances that act to suppress or mask the immune system of the subject being treated herein. These agents include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines; azathioprine; cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde; anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone, methylprednisolone, and dexamethasone; cytokine or cytokine receptor antagonists including anti-interferon-g, -b, or -a antibodies, anti-tumor necrosis factor-a antibodies, anti-tumor necrosis factor-b antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti- LFA-1 antibodies, including anti-CD1 1a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies; soluble peptide containing a LFA-3binding domain; streptokinase; TGF-b; streptodornase; FK506; RS-61443; deoxyspergualin; and rapamycin. In certain embodiments, the agent is an antihistamine. An“antihistamine” as used herein is an agent that antagonizes the physiological effect of histamine. Examples of antihistamines are chlorpheniramine, diphenhydramine, promethazine, cromolyn sodium, astemizole, azatadine maleate, bropheniramine maleate, carbinoxamine maleate, cetirizine hydrochloride, clemastine fumarate, cyproheptadine hydrochloride, dexbrompheniramine maleate, dexchlorpheniramine maleate, dimenhydrinate, diphenhydramine hydrochloride, doxylamine succinate, fexofendadine hydrochloride, terphenadine hydrochloride, hydroxyzine hydrochloride, loratidine, meclizine hydrochloride, tripelannamine citrate, tripelennamine hydrochloride, and triprolidine hydrochloride.

[0177] Immunosuppressive, anti-allergic, or anti-inflammatory agents may be incorporated into the lentiviral vector administration regimen. For example, administration of immunosuppressive or anti-inflammatory agents may commence prior to administration of the disclosed lentiviral vectors, and may continue with one or more doses thereafter. In certain embodiments, the immunosuppressive or anti-inflammatory agents are administered as premedication to the lentiviral vectors.

[0178] As previously discussed, the lentiviral vectors of the present disclosure, can be administered in a pharmaceutically effective amount for the in vivo treatment of clotting disorders. In this regard, it will be appreciated that the lentiviral vectors of the disclosure can be formulated to facilitate administration and promote stability of the active agent. Preferably, pharmaceutical compositions in accordance with the present disclosure comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. Of course, the pharmaceutical compositions of the present disclosure can be administered in single or multiple doses to provide for a pharmaceutically effective amount of the polypeptide.

[0179] A number of tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assay, ROTEM®assay, prothrombin time (PT) test (also used to determine INR), fibrinogen testing (often by the Clauss method), platelet count, platelet function testing (often by PFA-100), TCT, bleeding time, mixing test (whether an abnormality corrects if the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphosholipid antibodies, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRWT), miscellaneous platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g, ROTEM®), or euglobulin lysis time (ELT).

[0180] The aPTT test is a performance indicator measuring the efficacy of both the"intrinsic" (also referred to the contact activation pathway) and the common coagulation pathways.This test is commonly used to measure clotting activity of commercially available recombinant clotting factors, e.g., FVIII or FIX. It is used in conjunction with prothrombin time (PT), which measures the extrinsic pathway.

[0181] ROTEM®analysis provides information on the whole kinetics of haemostasis: clotting time, clot formation, clot stability and lysis. The different parameters in thromboelastometry are dependent on the activity of the plasmatic coagulation system, platelet function, fibrinolysis, or many factors which influence these interactions. This assay can provide a complete view of secondary haemostasis.III. Lentiviral Vectors

[0182] Lentiviruses include members of the bovine lentivirus group, equine lentivirus group, feline lentivirus group, ovinecaprine lentivirus group, and primate lentivirus group. The development of lentivirus vectors for gene therapy has been reviewed in Klimatcheva et al. (1999) Frontiers in Bioscience 4:481-496. The design and use of lentiviral vectors suitable for gene therapy is described for example in U.S. Pat. Nos. 6,207,455 and 6,615,782. Examples of lentivirus include, but are not limited to, HIV-1 , HIV-2, HIV-1 / HIV-2 pseudotype, HIV-1 / SIV, FIV, caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus, and bovine immunodeficiency virus.

[0183] A schematic representation of a lentiviral vector of the present disclosure is presented in FIG. 19. In some embodiments, the lentiviral vector of the present disclosure is "third- generation" lentiviral vector. As used herein, the term "third-generation" lentiviral vector refers to a lentiviral packaging system that has the characteristics of a second-generation vector system, and that further lacks a functional tat gene, such as one from which the tat gene has been deleted or inactivated. Typically, the gene encoding rev is provided on a separate expression construct. See, e.g., Dull et al. (1998) J. Virol. 72: 8463-8471. As used herein, a "second-generation" lentiviral vector system refers to a lentiviral packaging system that lacks functional accessory genes, such as one from which the accessory genes vif, vpr, vpu, and nef have been deleted or inactivated. See, e.g., Zufferey et al. (1997) Nat. Biotechnol. 15:871-875. As used herein, "packaging system" refers to a set of viral constructs comprising genes that encode viral proteins involved in packaging a recombinant virus. Typically, the constructs of the packaging system will ultimately be incorporated into a packaging cell.

[0184] In some embodiments, the third-generation lentiviral vector of the present disclosure is a self-inactivating lentiviral vector. In some embodiments, the lentiviral vector is a VSV.G pseudo type lentiviral vector. In some embodiments, the lentiviral vector comprises a hepatocyte-specific promoter for transgene expression. In some embodiments, the hepatocyte- specific promoter is an enhanced transthyretin promoter. In some embodiments, the lentiviralvector comprises one or more target sequences for miR-142 to reduce immune response to the transgene product. In some embodiments, incorporating one or more target sequences for miR- 142 into a lentiviral vector of the present disclosure allows for a desired transgene expression profile. For example, incorporating one or more target sequences for miR-142 may suppress transgene expression in intravascular and extravascular hematopoietic lineages, whereas transgene expression is maintained in nonhematopoietic cells. No oncogenesis has been detected in tumor prone mice treated with the lentivirus vector system of the present disclosure. See Brown et al. (2007) Blood 1 10:4144-52, Brown at al. (2006) Nat. Ned. 12:585-91 , and Cantore et al. (2015) Sci. Transl. Med. 7(277):277ra28.

[0185] Lentiviral vectors of the disclosure include codon optimized polynucleotides encoding the BDD FVIII protein described herein. In one embodiment, the optimized coding sequences forthe BDD FVIII protein is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in such a way as to permit each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5' gene expression sequence results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0186] In certain embodiments, the lentiviral vector is a vector of a recombinant lentivirus capable of infecting non-dividing cells. In certain embodiments, the lentiviral vector is a vector of a recombinant lentivirus capable of infecting liver cells (e.g., hepatocytes). The lentiviral genome and the proviral DNA typically have the three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), a protease and an integrase; and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTR's serve to promote transcription and polyadenylation of the virion RNA's. The LTR contains all other cis-acting sequences necessary for viral replication.Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, net and vpx (in HIV-I, HIV-2 and / or SIV) .

[0187] Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA.

[0188] However, the resulting mutant remains capable of directing the synthesis of all virion proteins. The disclosure provides a method of producing a recombinant lentivirus capable of infecting a non-dividing cell comprising transfecting a suitable host cell with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat. As will be disclosed herein below, vectors lacking a functional tat gene are desirable for certain applications. Thus, for example, a first vector can provide a nucleic acid encoding a viral gag and a viral pol and another vector can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene, herein identified as a transfer vector, into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest.

[0189] According to the above-indicated configuration of vectors and foreign genes, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from nearly any suitable virus, including retroviruses. In some embodiments, the env protein is an amphotropic envelope protein which allows transduction of cells of human and other species.

[0190] Examples of retroviral-derived env genes include, but are not limited to: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). Other env genes such as Vesicular stomatitis virus (VSV) protein G (VSV G), that of hepatitis viruses and of influenza also can be used. In some embodiments, the viral env nucleic acid sequence is associated operably with regulatory sequences described elsewhere herein.

[0191] In certain embodiments, the lentiviral vector has the HIV virulence genes env, vif, vpr, vpu and nef deleted without compromising the ability of the vector to transduce non-dividing cells. In some embodiments, the lentiviral vector comprises a deletion of the U3 region of the 3' LTR. The deletion of the U3 region can be the complete deletion or a partial deletion.

[0192] In some embodiments, the lentiviral vector of the disclosure comprising the FVIII nucleotide sequence described herein can be transfected in a cell with (a) a first nucleotidesequence comprising a gag, a pol, or gag and pol genes and (b) a second nucleotide sequence comprising a heterologous env gene; wherein the lentiviral vector lacks a functional tat gene. In other embodiments, the cell is further transfected with a fourth nucleotide sequence comprising a rev gene. In certain embodiments, the lentiviral vector lacks functional genes selected from vif, vpr, vpu, vpx and nef, or a combination thereof.

[0193] In certain embodiments, a lentiviral vector of the instant disclosure comprises one or more nucleotide sequences encoding a gag protein, a Rev-response element, a central polypurine track (cPPT), or any combination thereof.

[0194] In some embodiments, the lentiviral vector expresses on its surface one or more polypeptides that improve the targeting and / or activity of the lentiviral vector or the encoded FVIII polypeptide. The one or more polypeptides can be encoded by the lentiviral vector or can be incorporated during budding of the lentiviral vector from a host cell. During lentiviral production, viral particles bud off from a producing host cell. During the budding process, the viral particle takes on a lipid coat, which is derived from the lipid membrane of the host cell. As a result, the lipid coat of the viral particle can include membrane bound polypeptides that were previously present on the surface of the host cell.

[0195] In some embodiments, the lentiviral vector expresses one or more polypeptides on its surface that inhibit an immune response to the lentiviral vector following administration to a human subject. In some embodiments, the surface of the lentiviral vector comprises one or more CD47 molecules. CD47 is a "marker of self protein, which is ubiquitously expressed on human cells. Surface expression of CD47 inhibits macrophage-induced phagocytosis of endogenous cells through the interaction of CD47 and macrophage expressed-SIRPa. Cells expressing high levels of CD47 are less likely to be targeted and destroyed by human macrophages in vivo.

[0196] In some embodiments, the lentiviral vector comprises a high concentration of CD47 polypeptide molecules on its surface. In some embodiments, the lentiviral vector is produced in a cell line that has a high expression level of CD47. In certain embodiments, the lentiviral vector is produced in a CD47hi9hcell, wherein the cell has high expression of CD47 on the cell membrane. In particular embodiments, the lentiviral vector is produced in a CD47highHEK 293T cell, wherein the HEK 293T is has high expression of CD47 on the cell membrane. In some embodiments, the HEK 293T cell is modified to have increased expression of CD47 relative to unmodified HEK 293T cells. In certain embodiments, the CD47 is human CD47.

[0197] In some embodiments, the lentiviral vector has little or no surface expression of major histocompatibility complex class I (MHC-I). Surface expressed MHC-I displays peptide fragments of “non-self proteins from within a cell, such as protein fragments indicative of an infection, facilitating an immune response against the cell. In some embodiments, the lentiviralvector is produced in a MHC-I|0Wcell, wherein the cell has reduced expression of MHC-I on the cell membrane. In some embodiments, the lentiviral vector is produced in an MHC-I- (or "MHC-Ifree", “MHC-1ne9” or "MHC-negative") cell, wherein the cell lacks expression of MHC-I.

[0198] In particular embodiments, the lentiviral vector comprises a lipid coat comprising a high concentration of CD47 polypeptides and lacking MHC-I polypeptides. In certain embodiments, the lentiviral vector is produced in a CD47hi9h / MHC-llowcell line, e.g., a CD47hi9h / MHC-llowHEK 293T cell line. In some embodiments, the lentiviral vector is produced in a CD47hi9h / MHC-lfreecell line, e.g., a CD47hi9h / MHC-lfreeHEK 293T cell line.

[0199] Examples of lentiviral vectors are disclosed in U.S. Patent No. 9,050,269 andInternational Publication Nos. W09931251 , W09712622, W09817815, W09817816, and W09818934, which are incorporated herein by reference in their entireties.

[0200] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3 or (ii) nucleotides 58-1791 of SEQ ID NO: 4; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

[0201] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO: 5; (ii) nucleotides 1792-4374 of SEQ ID NO: 6; (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5; or (iv) nucleotides 1792-2277 and 2320- 4374 of SEQ ID NO: 6; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

[0202] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at leastabout 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of SEQ ID NO: 1 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1 and is operably linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58-4374 of SEQ ID NO: 1 or (ii) nucleotides 58- 2277 and 2320-4374 of SEQ ID NO: 1.

[0203] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of SEQ ID NO: 2 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2 and is operably linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58-4374 of SEQ ID NO: 2 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2.

[0204] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 58-4374 of SEQ ID NO: 70 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70 and is operably linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58-4374 of SEQ ID NO: 70 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70.

[0205] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 58-4374 of SEQ ID NO: 71 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71 and is operably linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58-4374 of SEQ ID NO: 71 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71.

[0206] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at leastabout 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity (i) nucleotides 58- 4374 of SEQ ID NO: 3 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3 and is operably linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58-4374 of SEQ ID NO: 3 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3.

[0207] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of SEQ ID NO: 4 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4 and is operably linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58-4374 of SEQ ID NO: 4 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4.

[0208] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of SEQ ID NO: 5 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5 and is operably linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58- 4374 of SEQ ID NO: 5 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5.

[0209] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of SEQ ID NO: 6 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6 and is operably linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58- 4374 of SEQ ID NO: 6 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6.

[0210] The lentiviral vectors of the present disclosure are therapeutically effective when administered at doses of 5x1010TU / kg or lower, 109TU / kg or lower, or 10®TU / kg or lower. At suchdosages, the administration of the lentiviral vectors of the disclosure can result in an increase in plasma FVIII activity in a subject in need thereof at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 1 1-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35- fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, at least about 100-fold , at least about 1 10-fold, at least about 120- fold, at least about 130- fold, at least about 140-fold, at least about 150-fold, at least about 160- fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, or at least about 200- fold with respect to basal levels in the subject, relative to levels in a subject administered a reference nucleic acid molecule comprising SEQ ID NO: 16, relative to levels in a subject administered a reference viral vector comprising the reference nucleic acid molecule, or relative to levels in a subject after administration of a polypeptide encoded by the reference nucleic acid molecule.IV. Tissue Specific Expression

[0211] In certain embodiments, it will be useful to include within the lentiviral vector one or more miRNA target sequences which, for example, are operably linked to the optimized FVIII transgene. Thus, the disclosure also provides at least one miRNA sequence target operably linked to the optimized FVIII nucleotide sequence or otherwise inserted within a lentiviral vector. More than one copy of a miRNA target sequence included in the lentiviral vector can increase the effectiveness of the system.

[0212] Also included are different miRNA target sequences. For example, lentiviral vectors which express more than one transgene can have the transgene under control of more than one miRNA target sequence, which can be the same or different. The miRNA target sequences can be in tandem, but other arrangements are also included. The transgene expression cassette, containing miRNA target sequences, can also be inserted within the lentiviral vector in antisense orientation. Antisense orientation can be useful in the production of viral particles to avoid expression of gene products which can otherwise be toxic to the producer cells.

[0213] In other embodiments, the lentiviral vector comprises 1 , 2, 3, 4, 5 ,6, 7 or 8 copies of the same or different miRNA target sequence. However in certain other embodiments, the lentiviral vector will not include any miRNA target sequence. Choice of whether or not to include an miRNA target sequence (and how many) will be guided by known parameters such as the intended tissue target, the level of expression required, etc.

[0214] In one embodiment, the target sequence is an miR-223 target which has been reported to block expression most effectively in myeloid committed progenitors and at least partially in the more primitive HSPC. miR-223 target can block expression in differentiated myeloid cells including granulocytes, monocytes, macrophages, myeloid dendritic cells. miR-223 target can also be suitable for gene therapy applications relying on robust transgene expression in the lymphoid or erythroid lineage. miR-223 target can also block expression very effectively in human HSC.

[0215] In another embodiment, the target sequence is an miR142 target (tccataaagt aggaaacact aca (SEQ ID NO: 43)). In one embodiment, the lentiviral vector comprises 4 copies of miR-142 target sequences. In certain embodiments, the complementary sequence of hematopoietic-specific microRNAs, such as miR-142 (142T), is incorporated into the 3' untranslated region of a lentiviral vector, making the transgene-encoding transcript susceptible to miRNA-mediated down-regulation. By this method, transgene expression can be prevented in hematopoietic-lineage antigen presenting cells (APC), while being maintained in non- hematopoietic cells (Brown et al., Nat Med 2006). This strategy can imposes a stringent post- transcriptional control on transgene expression and thus enables stable delivery and long-term expression of transgenes. In some embodiments, miR-142 regulation prevents immune-mediated clearance of transduced cells and / or induce antigen-specific Regulatory T cells (T regs) and mediate robust immunological tolerance to the transgene-encoded antigen.

[0216] In some embodiments, the target sequence is an miR181 target. Chen C-Z andLodish H, Seminars in Immunology (2005) 17(2):155-165 discloses miR-181 , a miRNA specifically expressed in B cells within mouse bone marrow(Chen and Lodish, 2005). It also discloses that some human miRNAs are linked to leukemias.

[0217] The target sequence can be fully or partially complementary to the miRNA. The term "fully complementary" means that the target sequence has a nucleic acid sequence which is 100 % complementary to the sequence of the miRNA which recognizes it. The term "partially complementary" means that the target sequence is only in part complementary to the sequence of the miRNA which recognizes it, whereby the partially complementary sequence is still recognized by the miRNA. In other words, a partially complementary target sequence in the context of the present disclosure is effective in recognizing the corresponding miRNA and effecting prevention or reduction of transgene expression in cells expressing that miRNA. Examples of the miRNA target sequences are described at W02007 / 000668, W02004 / 094642, WO2010 / 055413, or WO2010 / 125471 , which are incorporated herein by reference in their entireties.V. Polynucleotide Sequence Encoding FVIII Protein

[0218] The present disclosure is directed to lentiviral gene therapies wherein the lentivirus vector comprises a codon optimized nucleic acid molecule comprising a polynucleotide (nucleicacid) sequence encoding a polypeptide with FVIII activity. In some embodiments, the codon optimized nucleic acid molecule encodes a full-length FVIII polypeptide. In other embodiments, the codon optimized nucleic acid molecule encodes a B domain-deleted (BDD) FVIII polypeptide, wherein all or a portion of the B domain of FVIII is deleted.

[0219] In one particular embodiment, the nucleic acid molecule encodes a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 17 (FIG. 1J) or a fragment thereof. In one embodiment, the nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 17 or a fragment thereof.

[0220] In some embodiments, the nucleic acid molecule encodes a FVIII polypeptide comprising a signal peptide or a fragment thereof. In other embodiments, the nucleic acid molecule encodes a FVIII polypeptide which lacks a signal peptide. In some embodiments, the signal peptide comprises amino acids 1-19 of SEQ ID NO: 17.

[0221] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 58- 1791 of SEQ ID NO: 3 or (ii) nucleotides 58-1791 of SEQ ID NO: 4; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

[0222] In one particular embodiment, the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 3. In another embodiment, the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 4. In other embodiments, the first nucleotide sequence comprises nucleotides 58-1791 of SEQ ID NO: 3 or nucleotides 58-1791 of SEQ ID NO: 4.

[0223] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1-1791 of SEQ ID NO: 3 or (ii) nucleotides 1-1791 of SEQ ID NO: 4; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

[0224] In one embodiment, the first nucleotide sequence comprises nucleotides 1-1791 ofSEQ ID NO: 3 or nucleotides 1-1791 of SEQ ID NO: 4. In another embodiment, the second nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO: 3 or 1792-4374 of SEQ ID NO: 4. In one particular embodiment, the second nucleotide sequence comprises nucleotides 1792-4374 of SEQ ID NO: 3 or 1792-4374 of SEQ ID NO: 4.

[0225] In still another embodiment, the second nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 3 or 1792- 2277 and 2320-4374 of SEQ ID NO: 4 (i.e., nucleotides 1792-4374 of SEQ ID NO: 3 or 1792-4374 of SEQ ID NO: 4 without the nucleotides encoding the B domain or B domain fragment).

[0226] In one particular embodiment, the second nucleotide sequence comprises nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 3 or 1792-2277 and 2320-4374 of SEQ ID NO: 4 (i.e., nucleotides 1792-4374 of SEQ ID NO: 3 or 1792-4374 of SEQ ID NO: 4 without the nucleotides encoding the B domain or B domain fragment).

[0227] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO: 5 or (ii) 1792-4374 of SEQ ID NO: 6; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

[0228] In certain embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO: 5. In other embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO: 6.

[0229] In one particular embodiment, the second nucleic acid sequence comprises nucleotides 1792-4374 of SEQ ID NO: 5 or 1792-4374 of SEQ ID NO: 6. In some embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 5 or nucleotides 58- 1791 of SEQ ID NO: 6.

[0230] In other embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1 - 1791 of SEQ ID NO: 5 or nucleotides 1-1791 of SEQ ID NO: 6.

[0231] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment) or (ii) 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment); and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

[0232] In certain embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment). In other embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment).

[0233] In one particular embodiment, the second nucleic acid sequence comprises nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 or 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 or 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment). In some embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 5 or nucleotides 58-1791 of SEQ ID NO: 6.

[0234] In other embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1 - 1791 of SEQ ID NO: 5 or nucleotides 1-1791 of SEQ ID NO: 6.

[0235] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 1 , (ii) nucleotides 58-1791 of SEQ ID NO: 2, (iii) nucleotides 58-1791 of SEQ ID NO: 70, or (iv) nucleotides 58-1791 of SEQ ID NO: 71 ; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity. In other embodiments, the first nucleotide sequence comprises nucleotides 58-1791 of SEQ ID NO: 1 , nucleotides 58-1791 of SEQ ID NO: 2, (iii) nucleotides 58-1791 of SEQ ID NO: 70, or (iv) nucleotides 58-1791 of SEQ ID NO: 71.

[0236] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1-1791 of SEQ ID NO: 1 , (ii) nucleotides 1-1791 of SEQ ID NO: 2, (iii) nucleotides 1-1791 of SEQ ID NO: 70, or (iv) nucleotides 1-1791 of SEQ ID NO: 71 ; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

[0237] In one embodiment, the first nucleotide sequence comprises nucleotides 1-1791 ofSEQ ID NO: 1 , nucleotides 1-1791 of SEQ ID NO: 2, (iii) nucleotides 1-1791 of SEQ ID NO: 70, or (iv) nucleotides 1-1791 of SEQ ID NO: 71. In another embodiment, the second nucleotide sequence linked to the first nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO: 1 , 1792-4374 of SEQ ID NO: 2, (iii) nucleotides 1792- 4374 of SEQ ID NO: 70, or (iv) nucleotides 1792-4374 of SEQ ID NO: 71.

[0238] In one particular embodiment, the second nucleotide sequence linked to the first nucleotide sequence comprises (i) nucleotides 1792-4374 of SEQ ID NO: 1 , (ii) nucleotides 1792- 4374 of SEQ ID NO: 2, (iii) nucleotides 1792-4374 of SEQ ID NO: 70, or (iv) nucleotides 1792- 4374 of SEQ ID NO: 71. In other embodiments, the second nucleotide sequence linked to the first nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792- 2277 and 2320-4374 of SEQ ID NO: 1 , (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 2, (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 70, or (iv) nucleotides 1792-2277 and2320-4374 of SEQ ID NO: 71. In one embodiment, the second nucleotide sequence comprises (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 1 , (ii) nucleotides 1792-2277 and 2320- 4374 of SEQ ID NO: 2, (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 70, or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 71.

[0239] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO: 1 , (ii) nucleotides 1792-4374 of SEQ ID NO: 2, (iii) nucleotides 1792-4374 of SEQ ID NO: 70, or (iv) nucleotides 1792-4374 of SEQ ID NO: 71 ; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

[0240] In one particular embodiment, the second nucleic acid sequence comprises (i) nucleotides 1792-4374 of SEQ ID NO: 1 , (ii) nucleotides 1792-4374 of SEQ ID NO: 2, (iii) nucleotides 1792-4374 of SEQ ID NO: 70, or (iv) nucleotides 1792-4374 of SEQ ID NO: 71. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 1 , (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 2, (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 70, or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 1792-4374 of SEQ ID NO: 1 , nucleotides 1792-4374 of SEQ ID NO: 2, nucleotides 1792-4374 of SEQ ID NO: 70, or nucleotides 1792-4374 of SEQ ID NO: 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the N-terminal portion and the C- terminal portion together have a FVIII polypeptide activity.

[0241] In one embodiment, the second nucleic acid sequence comprises (i) nucleotides1792-2277 and 2320-4374 of SEQ ID NO: 1 , (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 2, (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 70, or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 1792-4374 of SEQ ID NO: 1 , nucleotides 1792- 4374 of SEQ ID NO: 2, nucleotides 1792-4374 of SEQ ID NO: 70, or nucleotides 1792-4374 of SEQ ID NO: 71 without the nucleotides encoding the B domain or B domain fragment).

[0242] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 90%, at least 91 %, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 1.

[0243] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 without the nucleotides encoding the B domain or B domain fragment).

[0244] In other embodiments, the nucleic acid sequence has at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 58 to 4374 of SEQ ID NO: 1. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 1 (i.e., nucleotides 1-4374 of SEQ ID NO: 1 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 1 to 4374 of SEQ ID NO: 1.

[0245] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 2. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2.

[0246] In other embodiments, the nucleic acid sequence has at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2 (i.e., nucleotides 58-4374 of SEQ ID NO: 2 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 58 to 4374 of SEQ ID NO: 2. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 2 (i.e., nucleotides 1-4374 of SEQ ID NO: 2 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 1 to 4374 of SEQ ID NO: 2.

[0247] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 70.

[0248] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70 (i.e., nucleotides 58-4374 of SEQ ID NO:70 without the nucleotides encoding the B domain or B domain fragment).

[0249] In other embodiments, the nucleic acid sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 70.

[0250] In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70 (i.e., nucleotides 58-4374 of SEQ ID NO: 70 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 58 to 4374 of SEQ ID NO: 70. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 70 (i.e., nucleotides 1-4374 of SEQ ID NO: 70 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 1 to 4374 of SEQ ID NO: 70.

[0251] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 71.

[0252] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 58-4374 of SEQ ID NO:71 without the nucleotides encoding the B domain or B domain fragment). In other embodiments, the nucleic acid sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 71.

[0253] In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 71 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 58 to 4374 of SEQ ID NO: 71. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 1-4374 of SEQ ID NO: 71 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 1 to 4374 of SEQ ID NO: 71.

[0254] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 3. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3 (i.e., nucleotides 58-4374 of SEQ ID NO: 3 without the nucleotides encoding the B domain or B domain fragment).

[0255] In certain embodiments, the nucleic acid sequence has at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3 (i.e., nucleotides 58-4374 of SEQ ID NO: 3 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 58 to 4374 of SEQ ID NO: 3. In still other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3 (i.e., nucleotides 1-4374 of SEQ ID NO: 3 without the nucleotides encoding the B domain or B domain fragment)or nucleotides 1 to 4374 of SEQ ID NO: 3.

[0256] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 4.

[0257] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4 (i.e., nucleotides 58-4374 of SEQ ID NO: 4 without the nucleotides encoding the B domain or B domain fragment).

[0258] In other embodiments, the nucleic acid sequence has at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4 (i.e., nucleotides 58-4374 of SEQ ID NO: 4 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 58 to 4374 of SEQ ID NO: 4. In still other embodiments, the nucleotide sequence comprises nucleotides 1- 2277 and 2320-4374 of SEQ ID NO: 4 (i.e., nucleotides 1-4374 of SEQ ID NO: 4 without thenucleotides encoding the B domain or B domain fragment) or nucleotides 1 to 4374 of SEQ ID NO: 4.

[0259] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 5.

[0260] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 58-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment).

[0261] In certain embodiments, the nucleic acid sequence has at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 58-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 58 to 4374 of SEQ ID NO: 5.

[0262] In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 1 to 4374 of SEQ ID NO: 5.

[0263] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 6.

[0264] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 58-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment).

[0265] In certain embodiments, the nucleic acid sequence has at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

[0266] In some embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 58-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 58 to 4374 of SEQ ID NO: 6.

[0267] In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment) or nucleotides 1 to 4374 of SEQ ID NO: 6.

[0268] In some embodiments, the nucleotide sequence comprises a nucleic acid sequence encoding a signal peptide. In certain embodiments, the signal peptide is a FVIII signal peptide. In some embodiments, the nucleic acid sequence encoding a signal peptide is codon optimized.

[0269] In one particular embodiment, the nucleic acid sequence encoding a signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to (i) nucleotides 1 to 57 of SEQ ID NO: 1 ; (ii) nucleotides 1 to 57 of SEQ ID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ ID NO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) nucleotides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71 ; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.

[0270] SEQ ID NOs: 1-6, 70, and 71 are optimized versions of SEQ ID NO: 16, the starting or "parental" or "wild-type" FVIII nucleotide sequence. SEQ ID NO: 16 encodes a B domain-deleted human FVIII. While SEQ ID NOs: 1-6, 70, and 71 are derived from a specific B domain-deleted form of FVIII (SEQ ID NO: 16), it is to be understood that the lentiviral gene therapy methods of the present disclosure are also directed to optimized versions of nucleic acids encoding other versions of FVIII. For example, other versions of FVIII can include full length FVIII, other B-domain deletions of FVIII (described below), or other fragments of FVIII that retain FVIII activity.

[0271] "A polypeptide with FVIII activity" as used herein means a functional FVIII polypeptide in its normal role in coagulation, unless otherwise specified. The term a polypeptide with FVIII activity includes a functional fragment, variant, analog, or derivative thereof that retains the function of full-length wild-type Factor VIII in the coagulation pathway.

[0272] "A polypeptide with FVIII activity" is used interchangeably with FVIII protein, FVIII polypeptide, or FVIII. Examples of FVIII functions include, but are not limited to, an ability to activate coagulation, an ability to act as a cofactor for factor IX, or an ability to form a tenase complex with factor IX in the presence of Ca2+and phospholipids, which then converts Factor X to the activated form Xa.

[0273] In one embodiment, a polypeptide having FVIII activity comprises two polypeptide chains, the first chain having the FVIII heavy chain and the second chain having the FVIII light chain. In another embodiment, the polypeptide having FVIII activity is single chain FVIII. Singlechain FVIII can contain one or more mutation or substitutions at amino acid residue 1645 and / or 1648 corresponding to mature FVIII sequence. See International Application No. PCT / US2012 / 045784, incorporated herein by reference in its entirety. The FVIII protein can be the human, porcine, canine, rat, or murine FVIII protein. In addition, comparisons between FVIII from humans and other species have identified conserved residues that are likely to be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251 ,632).

[0274] The "B domain" of FVIII, as used herein, is the same as the B domain known in the art that is defined by internal amino acid sequence identity and sites of proteolytic cleavage by thrombin, e.g., residues Ser741-Arg1648 of full length human FVIII. The other human FVIII domains are defined by the following amino acid residues: A1 , residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-lle2032; C1 , residues Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is usually referred to as the FVIII light chain activation peptide. The locations of the boundaries for all of the domains, including the B domains, for porcine, mouse and canine FVIII are also known in the art. An example of a BDD FVIII is REFACTO® recombinant BDD FVIII (Wyeth Pharmaceuticals, Inc.).

[0275] A "B domain deleted FVIII" can have the full or partial deletions disclosed in U.S.Patent Nos. 6,316,226, 6,346,513, 7,041 ,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171 ,844, 5,1 12,950, 4,868,1 12, and 6,458,563, each of which is incorporated herein by reference in its entirety. In some embodiments, a B domain deleted FVIII sequence of the present disclosure comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and examples 1-5 of U.S. Patent No. 6,316,226 (also in US 6,346,513).

[0276] In some embodiments, a B domain deleted FVIII of the present disclosure has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S. Patent No. 5,789,203 (also US 6,060,447, US 5,595,886, and US 6,228,620). In some embodiments, a B domain deleted FVIII has a deletion described in col. 1 , lines 25 to col. 2, line 40 of US Patent No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Patent no. 6,048,720; col. 2, lines 17-46 of U.S. Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Patent no. 5,171 ,844; col. 2, lines 55-68, figure 2, and example 1 of U.S. Patent No. 5, 112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Patent No. 4,868, 112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 1 1 , line 5 to col. 13, line 39 of U.S. Patent no. 7,041 ,635; or col. 4, lines 25-53, of U.S. Patent No. 6,458,563.

[0277] In some embodiments, a B domain deleted FVIII has a deletion of most of the B domain, but still contains amino-terminal sequences of the B domain that are essential for in vivoproteolytic processing of the primary translation product into two polypeptide chain, as disclosed in WO 91 / 09122, which is incorporated herein by reference in its entirety. In some embodiments, a B domain deleted FVIII is constructed with a deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by reference in its entirety. A B domain deleted FVIII can also contain a deletion of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety.

[0278] Additional B domain deletions that are part of the disclosure include, e.g., : deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No. WO 87 / 04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 through 1648 (Pasek (PCT application No.88 / 00831)), 816 through 1598 or 741 through 1689 (Lagner ( Behring Inst. Mitt. (1988) No 82: 16-25, EP 295597)), each of which is incorporated herein by reference in its entirety. Each of the foregoing deletions can be made in any FVIII sequence.

[0279] A number of functional FVIII molecules, including B-domain deletions, are disclosed in the following patents US 6,316,226 and US 6,346,513, both assigned to Baxter; US 7,041 ,635 assigned to ln2Gen; US 5,789,203, US 6,060,447, US 5,595,886, and US 6,228,620 assigned to Chiron; US 5,972,885 and US 6,048,720 assigned to Biovitrum, US 5,543,502 and US 5,610,278 assigned to Novo Nordisk; US 5,171 ,844 assigned to Immuno Ag; US 5,1 12,950 assigned to Transgene S.A.; US 4,868, 1 12 assigned to Genetics Institute, each of which is incorporated herein by reference in its entirety.A. Codon Optimization

[0280] In one embodiment, the lentiviral vector of the disclosure comprises an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide with FVIII activity, wherein the nucleic acid sequence has been codon optimized. In another embodiment, the starting nucleic acid sequence that encodes a polypeptide with FVIII activity and that is subject to codon optimization is SEQ ID NO: 16. In some embodiments, the sequence that encodes a polypeptide with FVIII activity is codon optimized for human expression. In other embodiments, the sequence that encodes a polypeptide with FVIII activity is codon optimized for murine expression. SEQ ID NOs: 1-6, 70, and 71 are codon optimized versions of SEQ ID NO: 16, optimized for human expression.

[0281] The term "codon-optimized" as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

[0282] Deviations in the nucleotide sequence that comprises the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code" which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA. Table 1 : The Standard Genetic Code

[0283] Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference, or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0284] Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, the relative frequencies of codon usage have been calculated. Codon usage tables are available, for example, at the "Codon Usage Database" available at www.kazusa.or.jp / codon / (visited June 18, 2012). See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000).

[0285] Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs can be used to calculate an optimal sequence.

[0286] In some embodiments, the nucleic acid molecule comprises one or more properties:(a) the nucleic acid molecule or a portion thereof has an increased the human codon adaptation index relative to SEQ ID NO: 16; (b) the nucleotide sequence or a portion thereof has an increased frequency of optimal codons relative to SEQ ID NO: 16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16; (d) the nucleotide sequence or a portion thereof has an increased relative synonymous codon usage relative to SEQ ID NO: 16; (e) the nucleotide sequence or a portion thereof is a reduced effective number of codons relative SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21 and 22) relative to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) relative to SEQ ID NO: 16; (i) the nucleotide sequence does not contain a poly-T sequence, (j) the nucleotide sequence does not contain a poly-A sequence; or (k) any combination thereof. In some embodiments, the nucleic acid molecules contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten characteristics of (a) to (j).B. Codon Adaptation Index

[0287] In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the human codon adaptation index is increased relative to SEQ ID NO: 16. For example, the nucleotide sequence can have a human codon adaptation index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81 %), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%), at least about 0.88 (88%), at least about 0.89 (89%), at least about 0.90 (90%), at least about 0.91 (91 %), at least about 0.92 (92%), at least about 0.93 (93%), at least about 0.94 (94%), at least about 0.95 (95%), at least about 0.96 (96%), at least about 0.97 (97%), at least about 0.98 (98%), or at least about 0.99 (99%). In some embodiments, the nucleotide sequence has a human codon adaptation index that is at least about .88 (88%). In other embodiments, the nucleotide sequence has a human codon adaptation index that is at least about .91 (91 %). In other embodiments, the nucleotide sequence has a human codon adaptation index that is at least about .91 (97%).

[0288] In one particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the human codon adaptation index of the nucleotide sequence is increased relative to SEQ ID NO: 16.

[0289] In some embodiments, the nucleotide sequence has a human codon adaptation index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81 %), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%), at least about 0.88 (88%), at least about 0.89 (89%), at least about 0.90 (90%), or at least about .91 (91 %). In one particular the nucleotide sequence has a human codon adaptation index that is at least about .88 (88%). Inanother embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .91 (91 %).

[0290] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 or (ii) 1792-2277 and 2320- 4374 of SEQ ID NO: 6; wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the human codon adaptation index of the nucleotide sequence is increased relative to SEQ ID NO: 16.

[0291] In some embodiments, the nucleotide sequence has a human codon adaptation index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81 %), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%), or at least about 0.88 (88%). In one particular the nucleotide sequence has a human codon adaptation index that is at least about .83 (83%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .88 (88%).

[0292] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the human codon adaptation index of the nucleotide sequence is increased relative to SEQ ID NO: 16. In some embodiments, the nucleotide sequence has a human codon adaptation index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81 %), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%), or at least about 0.88 (88%).

[0293] In one particular the nucleotide sequence has a human codon adaptation index that is at least about .75 (75%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .83 (83%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .88 (88%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .91 (91 %). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .97 (97%).

[0294] In some embodiments, the isolated nucleic acid molecule has an increased frequency of optimal codons (FOP) relative to SEQ ID NO: 16. In certain embodiments, the FOP of the isolated nucleic acid molecule is at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 64, at least about 65, at least about 70, at least about 75, at least about 79, at least about 80, at least about 85, or at least about 90.

[0295] In other embodiments, the isolated nucleic acid molecule has an increased relative synonymous codon usage (RCSU) relative to SEQ ID NO: 16. In some embodiments, the RCSU of the isolated nucleic acid molecule is greater than 1.5. In other embodiments, the RCSU of the isolated nucleic acid molecule is greater than 2.0. In certain embodiments, the RCSU of the isolated nucleic acid molecule is at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.1 , at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, or at least about 2.7.

[0296] In still other embodiments, the isolated nucleic acid molecule has a decreased effective number of codons relative to SEQ ID NO: 16. In some embodiments, the isolated nucleic acid molecule has an effective number of codons of less than about 50, less than about 45, less than about 40, less than about 35, less than about 30, or less than about 25. In one particular embodiment, the isolated nucleic acid molecule has an effective number of codons of about 40, about 35, about 30, about 25, or about 20.C. G / C Content Optimization

[0297] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51 %, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, or at least about 60%.

[0298] In one particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16.

[0299] In some embodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity has a G / C content that is at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51 %, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, or at least about 58%. In one particular embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 58%.

[0300] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO: 5; (ii) nucleotides 1792-4374 of SEQ ID NO: 6; (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment), or (iv) 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment); wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16.

[0301] In other embodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity has a G / C content that is at least about 45%, at least about 46%, at least about 47%,at least about 48%, at least about 49%, at least about 50%, at least about 51 %, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, or at least about 57%. In one particular embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 52%. In another embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 55%. In another embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 57%.

[0302] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58- 4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 45%.

[0303] In one particular embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 52%. In another embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 55%. In another embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 57%. In another embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 58%. In still another embodiment, the nucleotide sequence that encodes a polypeptide with FVIII activity has a G / C content that is at least about 60%.

[0304] "G / C content" (or guanine-cytosine content), or "percentage of G / C nucleotides," refers to the percentage of nitrogenous bases in a DNA molecule that are either guanine or cytosine. G / C content can be calculated using the following formula: 100(III)

[0305] Human genes are highly heterogeneous in their G / C content, with some genes having a G / C content as low as 20%, and other genes having a G / C content as high as 95%. In general, G / C rich genes are more highly expressed. In fact, it has been demonstrated thatincreasing the G / C content of a gene can lead to increased expression of the gene, due mostly to an increase in transcription and higher steady state mRNA levels. See Kudla et a!., PLoS Biol., 4(6): e180 (2006).D. Matrix Attachment Region-Like Sequences

[0306] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the nucleotide sequence contains fewer MARS / ARS sequences relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 6, at most 5, at most 4, at most 3, or at most 2 MARS / ARS sequences. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 1 MARS / ARS sequence. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a MARS / ARS sequence.

[0307] In one particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer MARS / ARS sequences relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 6, at most 5, at most 4, at most 3, or at most 2 MARS / ARS sequences. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 1 MARS / ARS sequence. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a MARS / ARS sequence.

[0308] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequenceidentity to (i) nucleotides 1792-4374 of SEQ ID NO: 5; (ii) nucleotides 1792-4374 of SEQ ID NO: 6; (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment); wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer MARS / ARS sequences relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 6, at most 5, at most 4, at most 3, or at most 2 MARS / ARS sequences. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 1 MARS / ARS sequence. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a MARS / ARS sequence.

[0309] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, or 71 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, or 71 (i.e., nucleotides 58- 4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence contains fewer MARS / ARS sequences relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 6, at most 5, at most 4, at most 3, or at most 2 MARS / ARS sequences. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 1 MARS / ARS sequence. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a MARS / ARS sequence.

[0310] AT-rich elements in the human FVIII nucleotide sequence that share sequence similarity with Saccharomyces cerevisiae autonomously replicating sequences (ARSs) and nuclear-matrix attachment regions (MARs) have been identified (Fallux et a!., Mol. Cell. Biol. 16:4264-4272 (1996). One of these elements has been demonstrated to bind nuclear factors in vitro and to repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Id. It has been hypothesized that these sequences can contribute to the transcriptional repression of the human FVIII gene. Thus, in one embodiment, all MAR / ARS sequences are abolished in the FVIII gene of the present disclosure. There are four MAR / ARS ATATTT sequences (SEQ ID NO:21) and three MAR / ARS AAATAT sequences (SEQ ID NO: 22) in the parental FVIII sequence (SEQ ID NO: 16). All of these sites were mutated to destroy the MAR / ARS sequences in the optimized FVIII sequences (SEQ ID NOs: 1-6). The location of each of these elements, and the sequence of the corresponding nucleotides in the optimized sequences are shown in Table 2, below.Table 2: Summary of Changes to Repressive ElementsE. Destabilizing Sequences

[0311] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the nucleotide sequence contains fewer destabilizing elements relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 destabilizing elements. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 destabilizing elements. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a destabilizing element.

[0312] In one particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer destabilizing elements relative to SEQ ID NO: 16. In otherembodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 destabilizing elements. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 destabilizing elements. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a destabilizing element.

[0313] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO: 5; (ii) nucleotides 1792-4374 of SEQ ID NO: 6; (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment); wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer destabilizing elements relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 destabilizing elements. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 destabilizing elements. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a destabilizing element.

[0314] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence contains fewer destabilizing elements relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 destabilizing elements. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 destabilizing elements. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a destabilizing element.

[0315] There are ten destabilizing elements in the parental FVIII sequence (SEQ ID NO:16): six ATTTA sequences (SEQ ID NO: 23) and four TAAAT sequences (SEQ ID NO: 24). In one embodiment, sequences of these sites were mutated to destroy the destabilizing elements in optimized FVIII SEQ ID NOs: 1-6, 70, and 71. The location of each of these elements, and the sequence of the corresponding nucleotides in the optimized sequences are shown in Table 2.F. Potential Promoter Binding Sites

[0316] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the nucleotide sequence contains fewer potential promoter binding sites relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 potential promoter binding sites. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 potential promoter binding sites. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a potential promoter binding site.

[0317] In one particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer potential promoter binding sites relative to SEQ ID NO: 16.

[0318] In other embodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 potential promoter binding sites. In other embodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity contains at most 4, at most 3, at most 2, or at most 1 potential promoter binding sites. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a potential promoter binding site.

[0319] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO: 5; (ii) nucleotides 1792-4374 of SEQ ID NO: 6; (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment); wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer potential promoter binding sites relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 potential promoter binding sites. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 potential promoter binding sites. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a potential promoter binding site.

[0320] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence contains fewer potential promoter binding sites relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5potential promoter binding sites. In other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 potential promoter binding sites. In yet other embodiments, the nucleotide sequence that encodes a polypeptide with FVIII activity does not contain a potential promoter binding site.

[0321] TATA boxes are regulatory sequences often found in the promoter regions of eukaryotes. They serve as the binding site of TATA binding protein (TBP), a general transcription factor. TATA boxes usually comprise the sequence TATAA (SEQ ID NO: 28) or a close variant. TATA boxes within a coding sequence, however, can inhibit the translation of full-length protein. There are ten potential promoter binding sequences in the wild type BDD FVIII sequence (SEQ ID NO: 16): five TATAA sequences (SEQ ID NO: 28) and five TTATA sequences (SEQ ID NO: 29). In some embodiments, at least 1 , at least 2, at least 3, or at least 4 of the promoter binding sites are abolished in the FVIII genes of the present disclosure. In some embodiments, at least 5 of the promoter binding sites are abolished in the FVIII genes of the present disclosure. In other embodiments, at least 6, at least 7, or at least 8 of the promoter binding sites are abolished in the FVIII genes of the present disclosure. In one embodiment, at least 9 of the promoter binging sites are abolished in the FVIII genes of the present disclosure. In one particular embodiment, all promoter binding sites are abolished in the FVIII genes of the present disclosure. The location of each potential promoter binding site and the sequence of the corresponding nucleotides in the optimized sequences are shown in Table 2.G. Other Cis Acting Negative Regulatory Elements

[0322] In addition to the MAR / ARS sequences, destabilizing elements, and potential promoter sites described above, several additional potentially inhibitory sequences can be identified in the wild type BDD FVIII sequence (SEQ ID NO: 16). Two AU rich sequence elements (AREs) can be identified (ATTTTATT (SEQ ID NOs: 30); and ATTTTTAA (SEQ ID NO: 31), along with a poly-A site (AAAAAAA; SEQ ID NO: 26), a poly-T site (TTTTTT ; SEQ ID NO: 25), and a splice site (GGTGAT; SEQ ID NO: 27) in the non-optimized BDD FVIII sequence. One or more of these elements can be removed from the optimized FVIII sequences. The location of each of these sites and the sequence of the corresponding nucleotides in the optimized sequences are shown in Table 2.

[0323] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, for example, a splice site, a poly-T sequence, a poly-A sequence, an ARE sequence, or any combinations thereof.

[0324] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO: 5; (ii) nucleotides 1792-4374 of SEQ ID NO: 6; (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or B domain fragment); wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, for example, a splice site, a poly-T sequence, a poly-A sequence, an ARE sequence, or any combinations thereof.

[0325] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, for example, a splice site, a poly-T sequence, a poly-A sequence, an ARE sequence, or any combinations thereof.

[0326] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence does not contain the splice site GGTGAT (SEQ ID NO: 27).

[0327] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence does not contain a poly-T sequence (SEQ ID NO: 25).

[0328] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence does not contain a poly-A sequence (SEQ ID NO: 26).

[0329] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIIIpolypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO: 3; (ii) nucleotides 1-1791 of SEQ ID NO: 3; (iii) nucleotides 58-1791 of SEQ ID NO: 4; or (iv) nucleotides 1-1791 of SEQ ID NO: 4; wherein the N- terminal portion and the C-terminal portion together have a FVIII polypeptide activity; and wherein the nucleotide sequence does not contain an ARE element (SEQ ID NO: 30 or SEQ ID NO: 31).

[0330] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence does not contain the splice site GGTGAT (SEQ ID NO: 27).

[0331] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence does not contain a poly-T sequence (SEQ ID NO: 25).

[0332] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence does not contain a poly-A sequence (SEQ ID NO: 26).

[0333] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 58-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or B domain fragment); and wherein the nucleotide sequence does not contain an ARE element (SEQ ID NO: 30 or SEQ ID NO: 31).

[0334] In other embodiments, an optimized FVIII sequence of the disclosure does not comprise one or more of antiviral motifs, stem-loop structures, and repeat sequences.

[0335] In still other embodiments, the nucleotides surrounding the transcription start site are changed to a kozak consensus sequence (GCCGCCACCATGC (SEQ ID NO: 32), wherein the underlined nucleotides are the start codon). In other embodiments, restriction sites can be added or removed to facilitate the cloning process.H. Heterologous Nucleotide Sequences

[0336] In some embodiments, the isolated nucleic acid molecule further comprises a heterologous nucleotide sequence. In some embodiments, the isolated nucleic acid molecule further comprises at least one heterologous nucleotide sequence. The heterologous nucleotide sequence can be linked with the optimized BDD-FVIII nucleotide sequences of the disclosure at the 5' end, at the 3' end, or inserted into the middle of the optimized BDD-FVIII nucleotide sequence. Thus, in some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is linked to the N-terminus or the C-terminus of the FVIII amino acid sequence encoded by the nucleotide sequence or inserted between two amino acids in the FVIII amino acid sequence. In some embodiments, the heterologous amino acid sequence can be inserted between two amino acids at one or more insertion site selected from Table 3. In someembodiments, the heterologous amino acid sequence can be inserted within the FVIII polypeptide encoded by the nucleic acid molecule of the disclosure at any site disclosed in International Publication No. WO 2013 / 123457 A1 and WO 2015 / 106052 A1 or U.S. Publication No. 2015 / 0158929 A1 , which are herein incorporated by reference in their entirety.

[0337] In some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted within the B domain or a fragment thereof. In some embodiments, the heterologous amino acid sequence is inserted within the FVIII immediately downstream of an amino acid corresponding to amino acid 745 of mature human FVIII (SEQ ID NO:15). In one particular embodiment, the FVIII comprises a deletion of amino acids 746-1646, corresponding to mature human FVIII (SEQ ID NO: 15), and the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted immediately downstream of amino acid 745, corresponding to mature human FVIII (SEQ ID NO:15).TABLE 3: Heterologous Moiety Insertion SitesNote: Insertion sites indicate the amino acid position corresponding to an amino acid position of mature human FVIII (SEQ ID NO: 15).

[0338] In other embodiments, the isolated nucleic acid molecule further comprise two, three, four, five, six, seven, or eight heterologous nucleotide sequences. In some embodiments, all the heterologous nucleotide sequences are identical. In some embodiments, at least one heterologous nucleotide sequence is different from the other heterologous nucleotide sequences. In some embodiments, the disclosure can comprise two, three, four, five, six, or more than seven heterologous nucleotide sequences in tandem.

[0339] In some embodiments, the heterologous nucleotide sequence encodes an amino acid sequence. In some embodiments, the amino acid sequence encoded by the heterologous nucleotide sequence is a heterologous moiety that can increase the half-life (a "half-life extender") of a FVIII molecule.

[0340] In some embodiments, the heterologous moiety is a peptide or a polypeptide with either unstructured or structured characteristics that are associated with the prolongation of in vivo half-life when incorporated in a protein of the disclosure. Non-limiting examples include albumin, albumin fragments, Fc fragments of immunoglobulins, the C-terminal peptide (CTP) of the b subunit of human chorionic gonadotropin, a HAP sequence, an XTEN sequence, a transferrin or a fragment thereof, a PAS polypeptide, polyglycine linkers, polyserine linkers, albumin-binding moieties, or any fragments, derivatives, variants, or combinations of these polypeptides. In one particular embodiment, the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, transferrin, albumin, or a PAS sequence.

[0341] In some aspects, a heterologous moiety includes von Willebrand factor or a fragment thereof. In other related aspects a heterologous moiety can include an attachment site (e.g. , a cysteine amino acid) for a non-polypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations of these elements. In some aspects, a heterologous moiety comprises a cysteine amino acid that functions as an attachment site for a non-polypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations of these elements.

[0342] In one specific embodiment, a first heterologous nucleotide sequence encodes a first heterologous moiety that is a half-life extending molecule which is known in the art, and a second heterologous nucleotide sequence encodes a second heterologous moiety that can alsobe a half-life extending molecule which is known in the art. In certain embodiments, the first heterologous moiety (e.g., a first Fc moiety) and the second heterologous moiety (e.g., a second Fc moiety) are associated with each other to form a dimer. In one embodiment, the second heterologous moiety is a second Fc moiety, wherein the second Fc moiety is linked to or associated with the first heterologous moiety, e.g. , the first Fc moiety. For example, the second heterologous moiety (e.g. , the second Fc moiety) can be linked to the first heterologous moiety (e.g., the first Fc moiety) by a linker or associated with the first heterologous moiety by a covalent or non-covalent bond.

[0343] In some embodiments, the heterologous moiety is a polypeptide comprising, consisting essentially of, or consisting of at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1 100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2500, at least about 3000, or at least about 4000 amino acids.

[0344] In other embodiments, the heterologous moiety is a polypeptide comprising, consisting essentially of, or consisting of about 100 to about 200 amino acids, about 200 to about 300 amino acids, about 300 to about 400 amino acids, about 400 to about 500 amino acids, about 500 to about 600 amino acids, about 600 to about 700 amino acids, about 700 to about 800 amino acids, about 800 to about 900 amino acids, or about 900 to about 1000 amino acids.

[0345] In certain embodiments, a heterologous moiety improves one or more pharmacokinetic properties of the FVIII protein without significantly affecting its biological activity or function.

[0346] In certain embodiments, a heterologous moiety increases the in vivo and / or in vitro half-life of the FVIII protein of the disclosure. In other embodiments, a heterologous moiety facilitates visualization or localization of the FVIII protein of the disclosure or a fragment thereof (e.g., a fragment comprising a heterologous moiety after proteolytic cleavage of the FVIII protein). Visualization and / or location of the FVIII protein of the disclosure or a fragment thereof can be in vivo, in vitro, ex vivo, or combinations thereof.

[0347] In other embodiments, a heterologous moiety increases stability of the FVIII protein of the disclosure or a fragment thereof (e.g., a fragment comprising a heterologous moiety after proteolytic cleavage of the FVIII protein). As used herein, the term "stability" refers to an art- recognized measure of the maintenance of one or more physical properties of the FVIII protein in response to an environmental condition (e.g., an elevated or lowered temperature). In certain aspects, the physical property can be the maintenance of the covalent structure of the FVIII protein(e.g., the absence of proteolytic cleavage, unwanted oxidation or deamidation). In other aspects, the physical property can also be the presence of the FVIII protein in a properly folded state (e.g., the absence of soluble or insoluble aggregates or precipitates).

[0348] In one aspect, the stability of the FVIII protein is measured by assaying a biophysical property of the FVIII protein, for example thermal stability, pH unfolding profile, stable removal of glycosylation, solubility, biochemical function (e.g., ability to bind to a protein, receptor or ligand), etc., and / or combinations thereof. In another aspect, biochemical function is demonstrated by the binding affinity of the interaction. In one aspect, a measure of protein stability is thermal stability, i.e., resistance to thermal challenge. Stability can be measured using methods known in the art, such as, HPLC (high performance liquid chromatography), SEC (size exclusion chromatography), DLS (dynamic light scattering), etc. Methods to measure thermal stability include, but are not limited to differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), circular dichroism (CD), and thermal challenge assay.

[0349] In certain aspects, a FVIII protein encoded by the nucleic acid molecule of the disclosure comprises at least one half-life extender, i.e., a heterologous moiety which increases the in vivo half-life of the FVIII protein with respect to the in vivo half-life of the corresponding FVIII protein lacking such heterologous moiety. In vivo half-life of a FVIII protein can be determined by any methods known to those of skill in the art, e.g., activity assays (chromogenic assay or one stage clotting aPTT assay), ELISA, ROTEM™, etc.

[0350] In some embodiments, the presence of one or more half-life extenders results in the half-life of the FVIII protein to be increased compared to the half-life of the corresponding protein lacking such one or more half-life extenders. The half-life of the FVIII protein comprising a half-life extender is at least about 1 .5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 1 1 times, or at least about 12 times longer than the in vivo half-life of the corresponding FVIII protein lacking such half-life extender.

[0351] In one embodiment, the half-life of the FVIII protein comprising a half-life extender is about 1.5-fold to about 20-fold, about 1.5 fold to about 15 fold, or about 1.5 fold to about 10 fold longer than the in vivo half-life of the corresponding protein lacking such half-life extender. In another embodiment, the half-life of FVIII protein comprising a half-life extender is extended about 2-fold to about 10-fold, about 2-fold to about 9-fold, about 2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-fold to about 6-fold, about 2-fold to about 5-fold, about 2-fold to about 4-fold, about 2-fold to about 3-fold, about 2.5-fold to about 10-fold, about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about 2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about 2.5-foldto about 5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about 3-fold, about 3-fold to about 10-fold, about 3-fold to about 9-fold, about 3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-fold to about 6-fold, about 3-fold to about 5-fold, about 3-fold to about 4-fold, about 4-fold to about 6 fold, about 5-fold to about 7-fold, or about 6-fold to about 8 fold as compared to the in vivo half- life of the corresponding protein lacking such half-life extender.

[0352] In other embodiments, the half-life of the FVIII protein comprising a half-life extender is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0353] In still other embodiments, the half-life of the FVIII protein comprising a half-life extender is about 15 hours to about two weeks, about 16 hours to about one week, about 17 hours to about one week, about 18 hours to about one week, about 19 hours to about one week, about 20 hours to about one week, about 21 hours to about one week, about 22 hours to about one week, about 23 hours to about one week, about 24 hours to about one week, about 36 hours to about one week, about 48 hours to about one week, about 60 hours to about one week, about 24 hours to about six days, about 24 hours to about five days, about 24 hours to about four days, about 24 hours to about three days, or about 24 hours to about two days.

[0354] In some embodiments, the average half-life per subject of the FVIII protein comprising a half-life extender is about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days), about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours, about 96 hours (4 days), about 108 hours, about 120 hours (5 days), about six days, about seven days (one week), about eight days, about nine days, about 10 days, about 1 1 days, about 12 days, about 13 days, or about 14 days.

[0355] One or more half-life extenders can be fused to C-terminus or N-terminus of FVIII or inserted within FVIII.1. An Immunoglobulin Constant Region or a Portion Thereof

[0356] In another aspect, a heterologous moiety comprises one or more immunoglobulin constant regions or portions thereof (e.g., an Fc region). In one embodiment, an isolated nucleicacid molecule of the disclosure further comprises a heterologous nucleic acid sequence that encodes an immunoglobulin constant region or a portion thereof. In some embodiments, the immunoglobulin constant region or portion thereof is an Fc region.

[0357] An immunoglobulin constant region is comprised of domains denoted CH (constant heavy) domains (CH1 , CH2, etc.). Depending on the isotype, (i.e. IgG, IgM, IgA IgD, or IgE), the constant region can be comprised of three or four CH domains. Some isotypes (e.g. IgG) constant regions also contain a hinge region. See Janeway et al. 2001 , Immunobiology, Garland Publishing, N.Y., N.Y.

[0358] An immunoglobulin constant region or a portion thereof for producing the FVIII protein of the present disclosure can be obtained from a number of different sources. In one embodiment, an immunoglobulin constant region or a portion thereof is derived from a human immunoglobulin. It is understood, however, that the immunoglobulin constant region or a portion thereof can be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. , a mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Moreover, the immunoglobulin constant region or a portion thereof can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including lgG1 , lgG2, lgG3 and lgG4. In one embodiment, the human isotype lgG1 is used.

[0359] A variety of the immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains sequence can be selected having a particular effector function (or lacking a particular effector function) or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Ig constant region sequences (e.g., hinge, CH2, and / or CH3 sequences, or portions thereof) can be derived from these sequences using art recognized techniques. The genetic material obtained using any of the foregoing methods can then be altered or synthesized to obtain polypeptides of the present disclosure. It will further be appreciated that the scope of this disclosure encompasses alleles, variants and mutations of constant region DNA sequences.

[0360] The sequences of the immunoglobulin constant region or a portion thereof can be cloned, e.g., using the polymerase chain reaction and primers which are selected to amplify the domain of interest. To clone a sequence of the immunoglobulin constant region or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and antibody genes amplified by PCR. PCR amplification methods are described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800, 159; 4,965,188; and in, e.g., "PCR Protocols: A Guide to Methods and Applications" Innis et al. eds., Academic Press, SanDiego, CA (1990); Ho et al. 1989. Gene 77:51 ; Horton et al. 1993. Methods Enzymol. 217:270). PCR can be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. PCR also can be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries can be screened by consensus primers or larger homologous probes, such as mouse constant region probes. Numerous primer sets suitable for amplification of antibody genes are known in the art (e.g., 5’ primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7: 1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). The cloning of antibody sequences is further described in Newman et al., U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated by reference herein.

[0361] An immunoglobulin constant region used herein can include all domains and the hinge region or portions thereof. In one embodiment, the immunoglobulin constant region or a portion thereof comprises CH2 domain, CH3 domain, and a hinge region, i.e., an Fc region or an FcRn binding partner.

[0362] As used herein, the term "Fc region" is defined as the portion of a polypeptide which corresponds to the Fc region of native Ig, i.e., as formed by the dimeric association of the respective Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, the term "genetically-fused Fc region" or "single-chain Fc region" (scFc region), as used herein, refers to a synthetic dimeric Fc region comprised of Fc domains genetically linked within a single polypeptide chain (i.e., encoded in a single contiguous genetic sequence). See International Publication No. WO 2012 / 006635, incorporated herein by reference in its entirety.

[0363] In one embodiment, the "Fc region" refers to the portion of a single Ig heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e. residue 216 in IgG, taking the first residue of heavy chain constant region to be 1 14) and ending at the C-terminus of the antibody. Accordingly, a complete Fc region comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0364] An immunoglobulin constant region or a portion thereof can be an FcRn binding partner. FcRn is active in adult epithelial tissues and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). An FcRn binding partner is a portion of an immunoglobulin that binds to FcRn.

[0365] The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not otherimmunoglobulin classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Pat. Nos. 6,485,726, 6,030,613, 6,086,875; WO 03 / 077834; US2003-0235536A1) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.

[0366] FcRn binding partners useful in the present disclosure encompass molecules that can be specifically bound by the FcRn receptor including whole IgG, the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291 , 308-31 1 , and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991 , Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

[0367] Fc regions or FcRn binding partners bound to FcRn can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to systemically administer a desired therapeutic molecule. Additionally, fusion proteins comprising an Fc region or an FcRn binding partner are endocytosed by cells expressing the FcRn. But instead of being marked for degradation, these fusion proteins are recycled out into circulation again, thus increasing the in vivo half-life of these proteins. In certain embodiments, the portions of immunoglobulin constant regions are an Fc region or an FcRn binding partner that typically associates, via disulfide bonds and other non-specific interactions, with another Fc region or another FcRn binding partner to form dimers and higher order multimers.

[0368] Two FcRn receptors can bind a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds a single polypeptide of the Fc homodimer. In one embodiment, linking the FcRn binding partner, e.g., an Fc fragment of an IgG, to a biologically active molecule provides a means of delivering the biologically active molecule orally, buccally, sublingually, rectally, vaginally, as an aerosol administered nasally or via a pulmonary route, or via an ocularroute. In another embodiment, the FVIII protein can be administered invasively, e.g., subcutaneously, intravenously.

[0369] An FcRn binding partner region is a molecule or portion thereof that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the Fc region. Specifically bound refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA is higher than 106M1, or higher than 10®M1. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of the molecules, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g., serum albumin, milk casein), etc., can be optimized by a skilled artisan using routine techniques.

[0370] In certain embodiments, a FVIII protein encoded by the nucleic acid molecule of the disclosure comprises one or more truncated Fc regions that are nonetheless sufficient to confer Fc receptor (FcR) binding properties to the Fc region. For example, the portion of an Fc region that binds to FcRn (i.e., the FcRn binding portion) comprises from about amino acids 282-438 of lgG1 , EU numbering (with the primary contact sites being amino acids 248, 250-257, 272, 285, 288, 290- 291 , 308-31 1 , and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. Thus, an Fc region of the disclosure can comprise or consist of an FcRn binding portion. FcRn binding portions can be derived from heavy chains of any isotype, including IgGI, lgG2, lgG3 and lgG4. In one embodiment, an FcRn binding portion from an antibody of the human isotype lgG1 is used. In another embodiment, an FcRn binding portion from an antibody of the human isotype lgG4 is used.

[0371] The Fc region can be obtained from a number of different sources. In one embodiment, an Fc region of the polypeptide is derived from a human immunoglobulin. It is understood, however, that an Fc moiety can be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g., a mouse, rat, rabbit, guinea pig) or nonhuman primate (e.g., chimpanzee, macaque) species. Moreover, the polypeptide of the Fc domains or portions thereof can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including lgG1 , lgG2, lgG3 and lgG4. In another embodiment, the human isotype IgG 1 is used.

[0372] In certain embodiments, the Fc variant confers a change in at least one effector function imparted by an Fc moiety comprising said wild-type Fc domain (e.g., an improvement or reduction in the ability of the Fc region to bind to Fc receptors (e.g. FcyRI, FcyRII, or FcyRIII) orcomplement proteins (e.g. C1 q), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine residue.

[0373] The Fc region of the disclosure can employ art-recognized Fc variants which are known to impart a change (e.g., an enhancement or reduction) in effector function and / or FcR or FcRn binding. Specifically, an Fc region of the disclosure can include, for example, a change (e.g., a substitution) at one or more of the amino acid positions disclosed in International PCT Publications W088 / 07089A1 , W096 / 14339A1 , WO98 / 05787A1 , W098 / 23289A1 , W099 / 51642A1 , W099 / 58572A1 , WO00 / 09560A2, WOOO / 32767A1 , WO00 / 42072A2,WO02 / 44215A2, W002 / 060919A2, WO03 / 074569A2, W004 / 016750A2, W004 / 029207A2, WO04 / 035752A2, W004 / 063351 A2, WO04 / 074455A2, WO04 / 099249A2, W005 / 040217A2,WO04 / 044859, W005 / 070963A1 , W005 / 077981 A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1 , W006 / 047350A2, and WO06 / 085967A2; US Patent Publication Nos.US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767,US2007 / 0243188, US2007 / 0248603, US2007 / 0286859, US2008 / 0057056; or US Patents5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871 ; 6, 121 ,022; 6, 194,551 ; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821 ,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091 , each of which is incorporated by reference herein. In one embodiment, the specific change (e.g., the specific substitution of one or more amino acids disclosed in the art) can be made at one or more of the disclosed amino acid positions. In another embodiment, a different change at one or more of the disclosed amino acid positions (e.g., the different substitution of one or more amino acid position disclosed in the art) can be made.

[0374] The Fc region or FcRn binding partner of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example, the following single amino acid residues in human IgG 1 Fc (Fcy1) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q31 1A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391 F, K392A, L398A, S400A,D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wild type proline substituted by alanine at position number 238. As an example, a specific embodiment incorporates the N297A mutation, removing a highly conserved N-glycosylation site. In addition to alanine other amino acids can be substituted for the wild type amino acids at the positions specified above. Mutations can be introduced singly into Fc giving rise to more than one hundred Fc regions distinct from the native Fc. Additionally, combinations of two, three, or more of these individual mutations can be introduced together, giving rise to hundreds more Fc regions.

[0375] Certain of the above mutations can confer new functionality upon the Fc region orFcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half-life of the Fc region, and to render the Fc region incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As a further example of new functionality arising from mutations described above affinity for FcRn can be increased beyond that of wild type in some instances. This increased affinity can reflect an increased "on" rate, a decreased "off rate or both an increased "on" rate and a decreased "off rate. Examples of mutations believed to impart an increased affinity for FcRn include, but not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001 , J. Biol. Chem. 276:6591).

[0376] Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity can arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG 1 "ELLG" (SEQ ID NO: 45) to the corresponding sequence from lgG2 "PVA" (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII, which mediate various effector functions will not bind to IgG 1 when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

[0377] In another embodiment, the immunoglobulin constant region or a portion thereof comprises an amino acid sequence in the hinge region or a portion thereof that forms one or more disulfide bonds with a second immunoglobulin constant region or a portion thereof. The second immunoglobulin constant region or a portion thereof can be linked to a second polypeptide, bringing the FVIII protein and the second polypeptide together. In some embodiments, the second polypeptide is an enhancer moiety. As used herein, the term "enhancer moiety" refers to a molecule, fragment thereof or a component of a polypeptide which is capable of enhancing theprocoagulant activity of FVIII. The enhancer moiety can be a cofactor, such as soluble tissue factor (sTF), or a procoagulant peptide. Thus, upon activation of FVIII, the enhancer moiety is available to enhance FVIII activity.

[0378] In certain embodiments, a FVIII protein encoded by a nucleic acid molecule of the disclosure comprises an amino acid substitution to an immunoglobulin constant region or a portion thereof (e.g., Fc variants), which alters the antigen-independent effector functions of the Ig constant region, in particular the circulating half-life of the protein.2. scFc Regions

[0379] In another aspect, a heterologous moiety comprises a scFc (single chain Fc) region.In one embodiment, an isolated nucleic acid molecule of the disclosure further comprises a heterologous nucleic acid sequence that encodes a scFc region. The scFc region comprises at least two immunoglobulin constant regions or portions thereof (e.g., Fc moieties or domains (e.g. ,2. 3, 4, 5, 6, or more Fc moieties or domains)) within the same linear polypeptide chain that are capable of folding (e.g. , intramolecularly or intermolecularly folding) to form one functional scFc region which is linked by an Fc peptide linker. For example, in one embodiment, a polypeptide of the disclosure is capable of binding, via its scFc region, to at least one Fc receptor (e.g. , an FcRn, an FcyR receptor (e.g., FcyRIII), or a complement protein (e.g., C1 q)) in order to improve half-life or trigger an immune effector function (e.g., antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC) and / or to improve manufacturability).3. CTP

[0380] In another aspect, a heterologous moiety comprises one C-terminal peptide (CTP) of the b subunit of human chorionic gonadotropin or fragment, variant, or derivative thereof. One or more CTP peptides inserted into a recombinant protein is known to increase the in vivo half-life of that protein. See, e.g., U.S. Patent No. 5,712, 122, incorporated by reference herein in its entirety.

[0381] Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL(SEQ ID NO: 33) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 34). See, e.g., U.S. Patent Application Publication No. US 2009 / 008741 1 A1 , incorporated by reference.4. XTEN Sequence

[0382] In some embodiments, a heterologous moiety comprises one or more XTEN sequences, fragments, variants, or derivatives thereof. As used here "XTEN sequence" refers to extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. As a heterologous moiety,XTENs can serve as a half-life extension moiety. In addition, XTEN can provide desirable properties including but are not limited to enhanced pharmacokinetic parameters and solubility characteristics.

[0383] The incorporation of a heterologous moiety comprising an XTEN sequence into a protein of the disclosure can confer to the protein one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radii.

[0384] In certain aspects, an XTEN sequence can increase pharmacokinetic properties such as longer in vivo half-life or increased area under the curve (AUC), so that a protein of the disclosure stays in vivo and has procoagulant activity for an increased period of time compared to a protein with the same but without the XTEN heterologous moiety.

[0385] In some embodiments, the XTEN sequence useful for the disclosure is a peptide or a polypeptide having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, XTEN is a peptide or a polypeptide having greater than about 20 to about 3000 amino acid residues, greater than 30 to about 2500 residues, greater than 40 to about 2000 residues, greater than 50 to about 1500 residues, greater than 60 to about 1000 residues, greater than 70 to about 900 residues, greater than 80 to about 800 residues, greater than 90 to about 700 residues, greater than 100 to about 600 residues, greater than 110 to about 500 residues, or greater than 120 to about 400 residues. In one particular embodiment, the XTEN comprises an amino acid sequence of longer than 42 amino acids and shorter than 144 amino acids in length.

[0386] The XTEN sequence of the disclosure can comprise one or more sequence motif of 5 to 14 (e.g., 9 to 14) amino acid residues or an amino acid sequence at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence motif, wherein the motif comprises, consists essentially of, or consists of 4 to 6 types of amino acids (e.g., 5 amino acids) selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). See US 2010-0239554 A1.

[0387] In some embodiments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence consists of multiple units of non-overlapping sequences selected from a single motif family selected from Table 4, resulting in a family sequence.

[0388] As used herein, "family" means that the XTEN has motifs selected only from a single motif category from Table 4; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids in the XTEN not from a family motif are selected to achieve a needed property, such as to permit incorporation of a restriction site by the encoding nucleotides, incorporation of a cleavage sequence, orto achieve a better linkage to FVIII. In some embodiments of XTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or of the AE motif family, or of the AF motif family, or of the AG motif family, or of the AM motif family, or of the AQ motif family, or of the BC family, or of the BD family, with the resulting XTEN exhibiting the range of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families of Table 4.

[0389] These sequences can be selected to achieve desired physical / chemical characteristics, including such properties as net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness that are conferred by the amino acid composition of the motifs, described more fully below. In the embodiments hereinabove described in this paragraph, the motifs incorporated into the XTEN can be selected and assembled using the methods described herein to achieve an XTEN of about 36 to about 3000 amino acid residues.Table 4. XTEN Sequence Motifs of 12 Amino Acids and Motif Families* Denotes individual motif sequences that, when used together in various permutations, results in a "family sequence"

[0390] Examples of XTEN sequences that can be used as heterologous moieties in chimeric proteins of the disclosure are disclosed, e.g., in U.S. Patent Publication Nos. 2010 / 0239554 A1 , 2010 / 0323956 A1 , 201 1 / 0046060 A1 , 2011 / 0046061 A1 , 201 1 / 0077199 A1 , or2011 / 0172146 A1 , or International Patent Publication Nos. WO 2010 / 091 122 A1 , WO 2010 / 144502 A2, WO 2010 / 144508 A1 , WO 201 1 / 028228 A1 , WO 2011 / 028229 A1 , or WO 2011 / 028344 A2, each of which is incorporated by reference herein in its entirety.

[0391] XTEN can have varying lengths for insertion into or linkage to F VI 11. In one embodiment, the length of the XTEN sequence(s) is chosen based on the property or function to be achieved in the fusion protein. Depending on the intended property or function, XTEN can be short or intermediate length sequence or longer sequence that can serve as carriers. In certain embodiments, the XTEN includes short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to about 399 amino acid residues, and longer lengths of about 400 to about 1000 and up to about 3000 amino acid residues. Thus, the XTEN inserted into or linked to FVIII can have lengths of about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues in length. In other embodiments, the XTEN sequences is about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length.

[0392] The precise length of an XTEN inserted into or linked to FVIII can vary without adversely affecting the activity of the FVIII. In one embodiment, one or more of the XTENs used herein have 42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and can be selected from one or more of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD.

[0393] In some embodiments, the XTEN sequence used in the disclosure is at least 60%,70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG 108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG 324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM 1318, BC864, BD864, AE948, AE1044, AE1 140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG 1044, AG1 140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, AG2004, and any combination thereof. See US 2010-0239554 A1. In one particular embodiment, the XTEN comprises AE42, AE72, AE144, AE288, AE576, AE864, AG 42, AG72, AG 144, AG288, AG576, AG864, or any combination thereof.

[0394] Exemplary XTEN sequences that can be used as heterologous moieties in chimeric protein of the disclosure include XTEN AE42-4 (SEQ ID NO: 46, encoded by SEQ ID NO: 47; FIGs. 1 1 C and 1 1 D, respectively), XTEN 144-2A (SEQ ID NO: 48, encoded by SEQ ID NO: 49; FIGs. 1 1 E and 1 1 F, respectively), XTEN A144-3B (SEQ ID NO: 50, encoded by SEQ ID NO: 51 ; FIGs. 1 1 G and 11 H, respectively), XTEN AE144-4A (SEQ ID NO: 52, encoded by SEQ ID NO: 53; FIGs. 1 11 and 11 J, respectively), XTEN AE144-5A (SEQ ID NO: 54, encoded by SEQ ID NO: 55; FIGs. 1 1 K and 1 1 L, respectively), XTEN AE144-6B (SEQ ID NO: 56, encoded by SEQ ID NO: 57; FIGs. 1 1 M and 1 1 N, respectively), XTEN AG144-1 (SEQ ID NO: 58, encoded by SEQ ID NO: 59; FIGs. 1 10 and 11 P, respectively), XTEN AG144-A (SEQ ID NO: 60, encoded by SEQ ID NO: 61 ; FIGs. 1 1 Q and 11 R, respectively), XTEN AG144-B (SEQ ID NO: 62, encoded by SEQ ID NO: 63; FIGs. 1 1 S and 1 1T, respectively), XTEN AG144-C (SEQ ID NO: 64, encoded by SEQ ID NO: 65; FIGs. 1 1 U and 1 1V, resp...

Claims

WHAT IS CLAIMED IS:

1. A method of treating a bleeding disorder in a subject in need thereof comprising administering to the subject at least one dose of 5x1010or less transducing units / kg (TU / kg) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has:(i) at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1 ;(ii) at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2;(iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70;(iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71 ;(v) at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3;(vi) at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4;(vii) at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5;(viii) at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6; or(ix) or any combination of (i) to (viii).

2. A method of treating a bleeding disorder in a subject in need thereof comprising administering to the subject at least one dose of 5x1010or less transducing units / kg (TU / kg) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a Factor VIII(FVIII) polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide;(a) wherein the first nucleic acid sequence has:(i) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 3;(ii) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 4;(iii) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 5; or(iv) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 6;(b) wherein the second nucleotide sequence has:(i) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 3;(ii) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 4;(iii) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5; or(iv) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6; or(c) any combination of (a) and (b); andwherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity.

3. The method of claim 1 or 2, wherein the dose is about 5x1010TU / kg, about 4.5x1010TU / kg, about 4x1010TU / kg, about 3.5x1010TU / kg, about 3x1010TU / kg, about 2.5x1010TU / kg, about 2x1010TU / kg, about 1.5x1010TU / kg, about 1x1010TU / kg, about 9.5x109TU / kg, about 9x109TU / kg, about 8.5x109TU / kg, about 8x109TU / kg, about 7.5x109TU / kg, about 7x109TU / kg, about 6.5x109TU / kg, about 6x109TU / kg, about 5.5x109TU / kg, about 5x109TU / kg, about 4.5x109TU / kg, about 4x109TU / kg, about 3.5x109TU / kg, about 3x109TU / kg, about 2.5x109TU / kg, about 2x109TU / kg, about 1.5x109TU / kg, about 1x109TU / kg, about 9.5x10®TU / kg, about 9x10®TU / kg, about 8.5x10®TU / kg, about 8x10®TU / kg, about 7.5x10®TU / kg, about 7x10®TU / kg, about 6.5x10®TU / kg, about 6x10®TU / kg, about 5.5x10®TU / kg, about 5x10®TU / kg, about 4.5x10®TU / kg, about 4x10®TU / kg,about 3.5x108TU / kg, about 3x108TU / kg, about 2.5x108TU / kg, about 2x108TU / kg, about 1.5x108TU / kg, or about 1x108TU / kg.

4. The method of claim 1 or 2, wherein the dose is less than about 5x1010TU / kg, less than about 4.5x1010TU / kg, less than about 4x1010TU / kg, less than about 3.5x1010TU / kg, less than about 3x1010TU / kg, less than about 2.5x1010TU / kg, less than about 2x1010TU / kg, less than about 1 .5x1010TU / kg, less than about 1x1010TU / kg, less than about 9.5x109TU / kg, less than about 9x109TU / kg, less than about 8.5x109TU / kg, less than about 8x109TU / kg, less than about 7.5x109TU / kg, less than about 7x109TU / kg, less than about 6.5x109TU / kg, less than about 6x109TU / kg, less than about 5.5x109TU / kg, less than about 5x109TU / kg, less than about 4.5x109TU / kg, less than about 4x109TU / kg, less than about 3.5x109TU / kg, less than about 3x109TU / kg, less than about 2.5x109TU / kg, less than about 2x109TU / kg, less than about 1 .5x109TU / kg, less than about 1x109TU / kg, less than about 9.5x10®TU / kg, less than about 9x10®TU / kg, less than about 8.5x10®TU / kg, less than about 8x10®TU / kg, less than about 7.5x10®TU / kg, less than about 7x10®TU / kg, less than about 6.5x10®TU / kg, less than about 6x10®TU / kg, less than about 5.5x10®TU / kg, less than about 5x10®TU / kg, less than about 4.5x10®TU / kg, less than about 4x10®TU / kg, less than about 3.5x10®TU / kg, less than about 3x10®TU / kg, less than about 2.5x10®TU / kg, less than about 2x10®TU / kg, less than about 1 .5x10®TU / kg, or less than about 1x10®TU / kg.

5. The method of claim 1 or 2, wherein the dose is between 1x10®and 5x1010TU / kg, between 1x10®and 5x109TU / kg, between 1x10®and 1x109TU / kg, between 1x10®and 1x1010TU / kg, between 1x109and 5x1010TU / kg, between 2x109and 5x1010TU / kg, between 3x109and 5x1010TU / kg, between 4x109and 5x1010TU / kg, between 5x109and 5x1010TU / kg, between 6x109and 5x1010TU / kg, between 7x109and 5x1010TU / kg, 8x109and 5x1010TU / kg, between 9x109and 5x1010TU / kg, between 1010and 5x1010TU / kg, between 1 .5x1010and 5x1010TU / kg, between 2x1010and 5x1010TU / kg, between 2.5x1010and 5x1010TU / kg, between 3x1010and 5x1010TU / kg, between 3.5x1010and 5x1010TU / kg, between 4x1010and 5x1010TU / kg, or between 4.5x1010and 5x1010TU / kg6. The method of claim 1 or 2, wherein the dose is between 1x109and 5x1010TU / kg, between 1x109and 4.5x1010TU / kg, between 1x109and 4x1010TU / kg, between 1x109and 3.5x1010TU / kg, between 1x109and 3x1010TU / kg, between 1x109and 2.5x1010TU / kg, between 1x109and 2x1010TU / kg, between 1x109and 1.5x1010TU / kg, between 1x109and 1x1010TU / kg, between 1 x109and 9x109TU / kg, between 1x109and 8x109TU / kg, between 1x109and 7x109TU / kg, between 1x109and 6x109TU / kg, between 1x109and 5x109TU / kg, between 1x109and 4x109TU / kg, between 1x109and 3x109TU / kg, and between 1x109and 2x109TU / kg.

7. The method of claim 1 or 2, wherein the dose is between 1x1010and 2x1010TU / kg, between 1.1 x1010and 1.9x1010TU / kg, between 1.2x1010and 1.8x1010TU / kg, between 1.3x1010and 1.7x1010TU / kg, or between 1.4x1010and 1.6x1010TU / kg.

8. The method of claim 1 or 2, wherein the dose is about 1.5x1010TU / kg.

9. The method of claim 1 or 2, wherein the dose is about 1.0x109TU / kg10. The method of claim 1 or 2, wherein the dose is about 3.0x109TU / kg.1 1. The method of claim 1 or 2, wherein the dose is about 6.0x109TU / kg.

12. The method of claim 1 or 2, wherein the dose is about 1x10®TU / kg, about 8.3x10®TU / kg, about 1.5x109TU / kg, about 4.5x109TU / kg, or about 1.3x1010TU / kg.

13. The method of claim 1 or 2, wherein the dose is between 2.5x109TU / kg and 3.5x109TU / kg, between 2.6 x109TU / kg and 3.4x109TU / kg, between 2.7x109TU / kg and 3.3x109TU / kg, between 2.8x109TU / kg and 3.2x109TU / kg, or between 2.9x109TU / kg and 3.1x109TU / kg.

14. The method of claim 1 or 2, wherein the dose is between 5.5x109TU / kg and 6.5x109TU / kg, between 5.6 x109TU / kg and 6.4x109TU / kg, between 5.7x109TU / kg and 6.3x109TU / kg, between 5.8x109TU / kg and 6.2x109TU / kg, or between 5.9x109TU / kg and 6.1x109TU / kg.

15. The method of any one of claims 1 to 14, wherein plasma FVIII activity at 24 hours to 48 hours post administration of the lentiviral vector is increased relative to a subject administered a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16.

16. The method of claim 15, wherein the plasma FVIII activity is increased by at least about 2- fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 1 1- fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at leastabout 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 1 10-fold, at least about 120- fold, at least about 130- fold, at least about 140-fold, at least about 150-fold, at least about 160- fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, or at least about 200- fold.

17. The method of any one of claims 1 to 16, wherein the lentiviral vector is administered as a single dose or multiple doses.

18. The method of any one of claims 1 to 17, wherein the lentiviral vector is administered via intravenous injection.

19. The method of any one of claims 1 to 18, wherein the subject is a pediatric subject.

20. The method of any one of claims 1 to 18, wherein the subject is an adult subject.

21. The method of any one of claims 1 to 20, wherein the lentiviral vector comprises a tissue specific promoter.

22. The method of claim 21 , wherein the tissue specific promoter selectively enhances expression of the polypeptide with FVIII activity in a target liver cell.

23. The method of claim 22, wherein the tissue specific promoter that selectively enhances expression of the polypeptide with FVIII activity in a target liver cell comprises an mTTR promoter.

24. The method of claims 22 or 23, wherein the target liver cell is a hepatocyte.

25. The method of claim 24, wherein the isolated nucleic acid molecule is stably integrated into the genome of the hepatocyte.

26. The method of any one of claims 1 to 25, wherein the bleeding disorder is hemophilia A.

27. The method of any one of claims 1 to 26, wherein the isolated nucleic acid molecule comprises LV-coFVIII-6 (SEQ ID NO:71).

28. The method of any one of claims 1 to 26, wherein the isolated nucleic acid molecule comprises LV-coFVIII-6-XTEN (SEQ ID NO:72).

29. The method of any one of claims 1 to 28, wherein the dose of lentivirus vector is administered once or divided into at least two sub-doses.

30. The method of any one of claims 1 to 28, wherein the dose of lentivirus vector is repeated at least twice.

31. The method of any one of claims 1 to 30, wherein the nucleotide sequence encoding a polypeptide with FVIII activity further comprises a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding a signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to:(i) nucleotides 1 to 57 of SEQ ID NO: 1 ;(ii) nucleotides 1 to 57 of SEQ ID NO: 2;(iii) nucleotides 1 to 57 of SEQ ID NO: 3;(iv) nucleotides 1 to 57 of SEQ ID NO: 4;(v) nucleotides 1 to 57 of SEQ ID NO: 5;(vi) nucleotides 1 to 57 of SEQ ID NO: 6;(vii) nucleotides 1 to 57 of SEQ ID NO: 70;(viii) nucleotides 1 to 57 of SEQ ID NO: 71 ; or(ix) nucleotides 1 to 57 of SEQ ID NO: 68.

32. The method of any one of claims 1 to 31 , wherein a nucleotide sequence encoding a polypeptide with FVIII activity comprises one or more property selected from the group consisting of:(a) the human codon adaptation index the nucleic acid molecule or a portion thereof is increased relative to SEQ ID NO: 16;(b) the frequency of optimal codons of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO:16;(c) the nucleotide sequence or a portion thereof contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16;(d) the relative synonymous codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16;(e) the effective number of codons of the nucleotide sequence or a portion thereof is reduced relative SEQ ID NO: 16;(f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21 and 22) relative to SEQ ID NO: 16;(g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) relative to SEQ ID NO: 16; and(h) any combination thereof.

33. The method of any one of claims 1 to 32, wherein the nucleotide sequence encoding a polypeptide with FVIII activity further comprises a heterologous nucleotide sequence encoding a heterologous amino acid sequence.

34. The method of claim 33, wherein the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence.

35. The method of claim 33 or 34, wherein the heterologous amino acid sequence is linked to the N-terminus or the C-terminus of the amino acid sequence encoded by a nucleotide sequence encoding a polypeptide with FVIII activity or inserted between two amino acids in the amino acid sequence encoded by the nucleotide sequence at one or more insertion site selected from TABLE 3.

36. The method of any one of claims 1 to 35, wherein the FVIII polypeptide is a full length FVIII or a B domain deleted FVIII.

37. The method of any one of claims 1 to 36, wherein the lentiviral vector comprises a lipid coat.

38. The method of claim 37, wherein the lipid coat comprises one or more CD47 polypeptide.

39. The method of claim 38, wherein the CD47 polypeptide is a human CD47 polypeptide.

40. The method of any one of claims 37 to 39, wherein the lipid coat comprises a high concentration of CD47 polypeptides.

41. The method of any one of claims 37 to 40, wherein the lipid coat does not comprise an MHC-I polypeptide.

42. The method of any one of claims 37 to 41 , wherein the lipid coat comprises a high concentration of CD47 polypeptides and does not comprise an MHC-I polypeptide.

43. The method of any one of claims 1 to 42, wherein the lentiviral vector is produced in a host cell.

44. The method of claim 43, wherein the host cell expresses CD47.

45. The method of claim 43 or 44, wherein the host cell does not express MHC-I.

46. The method of any one of claims 43 to 45, wherein the host cell is CD47hi9h / MHC-l·.

47. The method of any one of claims 43 to 46, wherein the host cell is a CD47hi9h / MHC-|- HEK 293T cell.

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