Use of lentiviral vectors expressing factor viii

Abstract

To provide a pharmaceutical composition for use in a method for treating a bleeding disorder caused by factor VIII deficiency.SOLUTION: A pharmaceutical composition comprises lentiviral vector particles. A method comprises administering at least one dose of the lentiviral vector particles at 1×108 to 5×1010 transducing units per kg (TU / kg) to the subject. The lentiviral vector particles comprise: a lipid coat that comprises one or more CD47 polypeptides and does not comprise MHC-I polypeptides; and an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity. When the lentiviral vector particles are administered to the subject, the lentiviral vector particles express FVIII at a therapeutically beneficial level in the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application 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 by reference.

[0002] Reference to an electronically submitted sequence listing The contents of the Sequence Listing, an electronically submitted ASCII text file (Name: 609628_SA9_460PC_Sequence_Listing.txt; Size: 204,203 bytes; Created: January 31, 2019), are hereby incorporated by reference in their entirety. [Background technology]

[0003] The blood coagulation pathway involves the formation of an enzyme complex (Xase complex) of factor VIIIa (FVIIIa) and factor IXa (FIXa) 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 then interacts with factor Va (FVa) to cleave prothrombin to generate thrombin. Hemophilia A is a bleeding disorder caused by mutations and / or deletions in the FVIII (FVIII) gene, resulting in deficient FVIII activity (Non-Patent Document 1). In some cases, patients have reduced levels of FVIII due to the presence of FVIII inhibitors, such as anti-FVIII antibodies.

[0004] This disease can be treated with replacement therapy that targets the restoration of FVIII activity, preventing spontaneous bleeding. Plasma-derived recombinant FVIII products are available to treat bleeding episodes on demand or prevent the occurrence of bleeding episodes by prophylactic treatment. Based on the half-life of these products (10-12 hours) (Non-Patent Document 2; Non-Patent Document 3), treatment regimens require frequent intravenous administration, typically 2-3 times per week for prophylaxis and 1-3 times per day for on-demand treatment (Non-Patent Document 4). Such frequent administration is inconvenient and costly.

[0005] A major obstacle to providing patients with low-cost recombinant FVIII protein is the high cost of commercial production. FVIII protein is poorly expressed in heterologous expression systems, with expression levels two to three orders of magnitude lower than proteins of similar size (Non-Patent Document 5). Advances in our understanding of the biology of FVIII expression have led to the development of more potent FVIII variants. For example, biochemical studies have demonstrated that the FVIII B domain is unnecessary for FVIII cofactor activity. Deletion of the B domain resulted in a 17-fold increase in mRNA levels and a 30% increase in secreted protein relative to full-length wild-type FVIII (Non-Patent Document 6). Nevertheless, there remains a need in the art for FVIII sequences that are efficiently expressed in heterologous systems. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Peyvandi et al. 2006 [Non-patent document 2] White GC et al., Thromb. Hemost. 77:660-7 (1997) [Non-patent document 3] Morfini, M., Haemophilia 9 (Supplement 1): pp. 94-99; Discussion 100 (2003) [Non-patent document 4] Manco-Johnson, MJ et al., N.Engl.J.Med.357:535-544 (2007) [Non-Patent Document 5] Lynch et al., Hum. Gene. Ther.; 4:259-72 (1993) [Non-patent document 6] Toole et al., Proc Natl Acad Sci USA 83:5939-42 (1986) Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides a method of treating a bleeding disorder in a subject in need thereof, comprising administering 5×10 mAb of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity. 10 TU / kg transducing units / kg (TU / kg) or less (e.g., 5 x 10 9 Less than or equal to 10 8the method comprises administering to a subject at least one dose of 58-2277 and 2320-4374 of the nucleotide sequence of the present invention (e.g., 58-2277 and 2320-4374 of the nucleotide sequence of the present invention) ... (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 to 2277 and 2320 to 4374 of SEQ ID NO:70; (iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 99% sequence identity to nucleotides 58 to 2277 and 2320 to 4374 of SEQ ID NO:71 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 2277 and 2320 to 4374 of SEQ ID NO:3; (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 to 2277 and 2320 to 4374 of SEQ ID NO:3; (vi) 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 to 2277 and 2320 to 4374 of SEQ ID NO:4 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; (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 to 2277 and 2320 to 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 to 2277 and 2320 to 4374 of SEQ ID NO:6; or (ix) any combination of (i) through (viii);

[0008] The present disclosure provides a method of treating a bleeding disorder in a subject in need thereof, comprising administering 5×10 mAb of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence comprising 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 the FVIII polypeptide. 10 TU / kg or less (e.g., 5 x 10 9 Less than or equal to 10 8 TU / kg or less) for at least one dose (a) administering to a subject a first nucleic acid sequence having: (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; 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 58 to 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 to 1791 of SEQ ID NO:6; (b) a second nucleotide The 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 to 2277 and 2320 to 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 to 2277 and 2320 to 4374 of SEQ ID NO:4. (iii) 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 to 2277 and 2320 to 4374 of SEQ ID NO:5; or (iv) 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 to 2277 and 2320 to 4374 of SEQ ID NO:6;or (c) any combination of (a) and (b); wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity;

[0009] In some embodiments of the above-disclosed methods, the dose is about 9.5×10 8 TU / kg, approximately 9 x 10 8 TU / kg, approximately 8.5 x 10 8 TU / kg, approximately 8 x 10 8 TU / kg, approximately 7.5 x 10 8 TU / kg, approximately 7 x 10 8 TU / kg, approximately 6.5 x 10 8 TU / kg, approximately 6 x 10 8 TU / kg, approximately 5.5 x 10 8 TU / kg, approximately 5 x 10 8 TU / kg, approximately 4.5 x 10 8 TU / kg, approximately 4 x 10 8 TU / kg, approximately 3.5 x 10 8 TU / kg, approximately 3 x 10 8 TU / kg, approximately 2.5 x 10 8 TU / kg, approximately 2 x 10 8 TU / kg, approximately 1.5 x 10 8 TU / kg or approximately 1 x 10 8 TU / kg, approximately 5 x 10 10 TU / kg, approximately 4.5 x 10 10 TU / kg, approximately 4 x 10 10 TU / kg, approximately 3.5 x 10 10 TU / kg, approximately 3 x 10 10 TU / kg, approximately 2.5 x 10 10 TU / kg, approximately 2 x 10 10 TU / kg, approximately 1.5 x 10 10 TU / kg, approximately 1 x 10 10 TU / kg, approximately 9.5 x 10 9 TU / kg, approximately 9 x 10 9 TU / kg, approximately 8.5 x 10 9 TU / kg, approximately 8 x 10 9 TU / kg, approximately 7.5 x 10 9 TU / kg, approximately 7 x 10 9 TU / kg, approximately 6.5 x 10 9 TU / kg, approximately 6 x 109 TU / kg, approximately 5.5 x 10 9 TU / kg, approximately 5 x 10 9 TU / kg, approximately 4.5 x 10 9 TU / kg, approximately 4 x 10 9 TU / kg, approximately 3.5 x 10 9 TU / kg, approximately 3 x 10 9 TU / kg, approximately 2.5 x 10 9 TU / kg, approximately 2 x 10 9 TU / kg, approximately 1.5 x 10 9 TU / kg or approximately 1 x 10 9 It is TU / kg.

[0010] In some embodiments, the dose is about 9.5 x 10 8 Less than TU / kg, approximately 9 x 10 8 Less than TU / kg, approximately 8.5 x 10 8 Less than TU / kg, approximately 8 x 10 8 Less than TU / kg, approximately 7.5 x 10 8 Less than TU / kg, approximately 7 x 10 8 Less than TU / kg, approximately 6.5 x 10 8 Less than TU / kg, approximately 6 x 10 8 Less than TU / kg, approximately 5.5 x 10 8 Less than TU / kg, approximately 5 x 10 8 Less than TU / kg, approximately 4.5 x 10 8 Less than TU / kg, approximately 4 x 10 8 Less than TU / kg, approximately 3.5x 10 8 Less than TU / kg, approximately 3 x 10 8 Less than TU / kg, approximately 2.5 x 10 8 Less than TU / kg, approximately 2 x 10 8 Less than TU / kg, approximately 1.5 x 10 8 Less than TU / kg or approximately 1 x 10 8 Less than TU / kg, approximately 5 x 10 10 Less than TU / kg, approximately 4.5 x 10 10 Less than TU / kg, approximately 4 x 10 10 Less than TU / kg, approximately 3.5 x 10 10 Less than TU / kg, approximately 3 x 10 10 Less than TU / kg, approximately 2.5 x 10 10 Less than TU / kg, approximately 2 x 1010 Less than TU / kg, approximately 1.5 x 10 10 Less than TU / kg, approximately 1 x 10 10 Less than TU / kg, approximately 9.5 x 10 9 Less than TU / kg, approximately 9 x 10 9 Less than TU / kg, approximately 8.5 x 10 9 Less than TU / kg, approximately 8 x 10 9 Less than TU / kg, approximately 7.5 x 10 9 Less than TU / kg, approximately 7 x 10 9 Less than TU / kg, approximately 6.5 x 10 9 Less than TU / kg, approximately 6 x 10 9 Less than TU / kg, approximately 5.5 x 10 9 Less than TU / kg, approximately 5 x 10 9 Less than TU / kg, approximately 4.5 x 10 9 Less than TU / kg, approximately 4 x 10 9 Less than TU / kg, approximately 3.5 x 10 9 Less than TU / kg, approximately 3 x 10 9 Less than TU / kg, approximately 2.5 x 10 9 Less than TU / kg, approximately 2 x 10 9 Less than TU / kg, approximately 1.5 x 10 9 Less than TU / kg or approximately 1 x 10 9 Less than TU / kg.

[0011] In some embodiments, the dose is 1×10 8 ~5×10 10 TU / kg, 1 x 10 8 ~5×10 9 TU / kg, 1 x 10 8 ~1×10 9 TU / kg, 1 x 10 8 ~1×10 10 TU / kg, 1 x 10 9 ~5×10 10 TU / kg, 2 x 10 9 ~5×10 10 TU / kg, 3 x 10 9 ~5×10 10 TU / kg, 4 x 10 9 ~5×10 10 TU / kg, 5 x 10 9 ~5×10 10 TU / kg, 6 x 10 9~5×10 10 TU / kg, 7×10 9 ~5×10 10 TU / kg, 8×10 9 ~5×10 10 TU / kg, 9×10 9 ~5×10 10 TU / kg, 10 10 ~5×10 10 TU / kg, 1.5×10 10 ~5×10 10 TU / kg, 2×10 10 ~5×10 10 TU / kg, 2.5×10 10 ~5×10 10 TU / kg, 3×10 10 ~5×10 10 TU / kg, 3.5×10 10 ~5×10 10 TU / kg, 4×10 10 ~5×10 10 TU / kg, or 4.5×10 10 ~5×10 10 TU / kg. In some embodiments, the dosage is 1×10 9 ~5×10 10 TU / kg, 1×10[[ID=5​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​TU / kg, 1 x 10 9 ~6×10 9 TU / kg, 1 x 10 9 ~5×10 9 TU / kg, 1 x 10 9 ~4×10 9 TU / kg, 1 x 10 9 ~3×10 9 TU / kg, and 1 × 10 9 ~2×10 9 In some embodiments, the dose is 1 x 10 10 ~2×10 10 TU / kg, 1.1 x 10 10 ~1.9×10 10 TU / kg, 1.2 x 10 10 ~1.8×10 10 TU / kg, 1.3 x 10 10 ~1.7×10 10 TU / kg, or 1.4 x 10 10 ~1.6×10 10 In some embodiments, the dose is about 1.5 x 10 10 In some embodiments, the dose is 1.5 x 10 9 In some embodiments, the dose is 2.5 x 10 9 TU / kg ~ 3.5 x 10 9 TU / kg, 2.6 x 10 9 TU / kg ~ 3.4 x 10 9 TU / kg, 2.7 × 10 9 TU / kg ~ 3.3 x 10 9 TU / kg, 2.8 x 10 9 TU / kg ~ 3.2 x 10 9 TU / kg, or 2.9 x 10 9 TU / kg ~ 3.1 x 10 9 In some embodiments, the dose is about 3.0 x 10 9 In some embodiments, the dose is 5.5 x 10 9 TU / kg ~ 6.5 x 10 9 TU / kg, 5.6 x 10 9 TU / kg ~ 6.4 x 10 9 TU / kg, 5.7 x 10 9 TU / kg ~ 6.3 x 109 TU / kg, 5.8 x 10 9 TU / kg ~ 6.2 x 10 9 TU / kg, or 5.9 x 10 9 TU / kg ~ 6.1 x 10 9 In some embodiments, the dose is about 6.0 x 10 9 It is TU / kg.

[0012] In some embodiments of the methods disclosed above, the plasma FVIII activity 24 hours to 48 hours after administration of the lentiviral vector is measured using a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16. In some embodiments, 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 11-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 110-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.

[0013] In some embodiments of the methods disclosed above, the lentiviral vector is administered in 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.

[0014] In some embodiments, the lentiviral vector comprises a tissue-specific promoter. In some embodiments, the tissue-specific promoter selectively enhances the expression of a polypeptide having FVIII activity in target liver cells. In some embodiments, the tissue-specific promoter selectively enhances the expression of a polypeptide having FVIII activity in target liver cells comprises an mTTR promoter. In some embodiments, the target liver cells are hepatocytes. In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of hepatocytes. In some embodiments, the bleeding disorder is hemophilia A.

[0015] 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).

[0016] In some embodiments, the dose of the lentiviral vector is administered at once or divided into two, three, four, five, or six partial doses. In some embodiments, the administration of the lentiviral vector is repeated 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 at least ten times. In some embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity further comprises a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding the 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 1-57 of SEQ ID NO:1; (ii) nucleotides 1-57 of SEQ ID NO:2; (iii) nucleotides 1-57 of SEQ ID NO:3; (iv) nucleotides 1-57 of SEQ ID NO:4; (v) nucleotides 1-57 of SEQ ID NO:5; (vi) nucleotides 1-57 of SEQ ID NO:6; (vii) nucleotides 1-57 of SEQ ID NO:70; (viii) nucleotides 1-57 of SEQ ID NO:71; or (ix) nucleotides 1-57 of SEQ ID NO:68.

[0017] In some embodiments, the nucleic acid molecule (or nucleotide sequence encoding a polypeptide having FVIII activity) comprises one or more properties selected from the group consisting of: (a) the human codon compatibility index of the nucleic acid molecule or portion thereof is increased compared to SEQ ID NO: 16; (b) the optimal codon frequency of the nucleotide sequence or portion thereof is increased compared to SEQ ID NO: 16; (c) the nucleotide sequence or 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 frequency of the nucleotide sequence or a portion thereof is increased compared to SEQ ID NO: 16; (e) the effective number of codons of the nucleotide sequence or a portion thereof is reduced compared to SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21 and 22) compared to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) compared to SEQ ID NO: 16; and (h) any combination thereof.

[0018] In some embodiments, the nucleotide sequence encoding the polypeptide having 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 portion thereof, an XTEN, transferrin, albumin, or PAS sequence. In some embodiments, the heterologous amino acid sequence is linked to the N-terminus or 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 sites selected from Table 3. In some embodiments, the FVIII polypeptide is full-length FVIII or a B-domain deleted FVIII. [Brief explanation of the drawings]

[0019] [Figure 1-1]Figures 1A-1J provide codon-optimized nucleotide sequences encoding B-domain deleted factor VIII. Figure 1A shows the nucleotide sequence of coFVIII-3 (SEQ ID NO: 1). Figure 1B shows the nucleotide sequence of coFVIII-4 (SEQ ID NO: 2). Figure 1C shows the nucleotide sequence of coFVIII-5 (SEQ ID NO: 70). Figure 1D shows the nucleotide sequence of coFVIII-6 (SEQ ID NO: 71). Figure 1E shows the nucleotide sequence of coFVIII-52 (SEQ ID NO: 3). Figure 1F shows the nucleotide sequence of coFVIII-62 (SEQ ID NO: 4). Figure 1G shows the nucleotide sequence of coFVIII-25 (SEQ ID NO: 5). Figure 1H shows the nucleotide sequence of coFVIII-26 (SEQ ID NO: 6). Figures 1I 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). [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 1-5] Continued from Figure 1-4. [Figure 1-6] Continued from Figure 1-5. [Figure 1-7] Continued from Figure 1-6. [Figure 1-8] Continued from Figure 1-7. [Figure 1-9] Continued from Figure 1-8. [Figure 1-10] Continued from Figure 1-9. [Figure 2-1]Figures 2A-2J show codon usage bias adjustments in codon-optimized nucleotide sequences encoding BDD-FVIII. Figure 2A shows the relative frequencies of codons in a wild-type nucleotide sequence (before codon optimization) encoding BDD-FVIII, e.g., non-optimized BDD-FVIII. The human codon compatibility index (CAI) of the non-optimized BDD-FVIII sequence is 74%. Figure 2B shows the relative frequencies of codons in a coFVIII-1 variant sequence with a human CAI of 88%. Figure 2C shows the relative frequencies of codons in a coFVIII-3 variant sequence with a human CAI of 91%. Figure 2D shows the relative frequencies of codons in a coFVIII-4 variant sequence with a human CAI of 97%. Figure 2E shows the relative frequencies of codons in a coFVIII-5 variant sequence with a human CAI of 83%. Figure 2F shows the relative frequencies of codons in a coFVIII-6 variant sequence with a human CAI of 83%. Figure 2G shows the relative frequency of codons in the coFVIII-52 mutant sequence with 91% human CAI. Figure 2H shows the relative frequency of codons in the coFVIII-62 mutant sequence with 91% human CAI. Figure 2I shows the relative frequency of codons in the coFVIII-25 mutant sequence with 88% human CAI. Figure 2J shows the relative frequency of codons in the coFVIII-26 mutant sequence with 88% human CAI. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 2-5] Continued from Figure 2-4. [Figure 3] FIG. 1 provides a plasmid map of FVIII-303, which contains coFVIII-1 in a pcDNA3 backbone under the control of the ET-enhanced transthyretin promoter, located upstream of the coFVIII-1 translation start site and containing a synthetic enhancer, mTIR enhancer and mTIR promoter. [Figure 4]Figure 1 shows a graphical representation of FVIII plasma activity in HemA mice after hydrodynamic injection of 5 μg of FVIII-303 (coFVIII-1; circles) or 5 μg of FVIII-311 (BDD-FVIII; squares). Plasma FVIII activity was determined by a FVIII-specific chromogenic assay 24, 48, and 72 hours after injection. Relative activity levels after 72 hours, normalized to the expression level of FVIII-311, are shown. [Figure 5] FIG. 1 shows the plasmid map of pLV-coFVIII-52, which contains coFVIII-52 in a lentiviral plasmid under the control of the ET promoter, which is located upstream of the coFVIII-52 translation start site and contains a synthetic enhancer, the mTTR enhancer, and the mTTR promoter. [Figure 6-1]Figures 6A-6C show graphical representations of FVIII plasma activity in HemA mice after hydrodynamic injection of various FVIII-encoding nucleotides. FVIII plasma activity was determined by a FVIII-specific chromogenic assay 24, 48, and 72 hours after injection. Figure 6A shows FVIII plasma activity in HemA mice after hydrodynamic injection of 5 μg of LV-coFVIII-1 (closed circles), 5 μg of LV-coFVIII-3 (triangles), 5 μg of LV-coFVIII-4 (inverted triangles), 5 μg of LV-coFVIII-5 (diamonds), or 5 μg of LV-coFVIII-6 (open circles). Figure 6B shows the plasma activity of FVIII in HemA mice after hydrodynamic injection of 5 μg of LV-coFVIII-1 (circles), 5 μg of LV-coFVIII-25 (triangles), or 5 μg of LV-coFVIII-26 (inverted triangles). Figure 6C shows the plasma activity of FVIII in HemA mice after hydrodynamic injection of 20 μg of LV-2116 (non-codon-optimized (WT) BDD-FVIII nucleotide sequence; open circles), 20 μg of LV-coFVIII-1 (triangles), 20 μg of LV-coFVIII-52 (squares), or 20 μg of LV-coFVIII-62 (filled circles). Relative activity levels after 72 hours are shown for each plasmid, normalized to the expression levels of LV-coFVIII-1 (Figures 6A, 6B, and 6C) and / or LV-2116 (Figure 6C), as indicated. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 7] 1 shows plasma FVIII activity in HemA mice 24 days after injection with 1E8TU / mouse lentiviral vector containing coFVIII-1, coFVIII-5, coFVIII-52, coFVIII-6, or coFVIII-62, as measured by a FVIII-specific chromogenic assay, compared to the LV-2116 (BDD-FVIII) control. Error bars indicate standard deviation. [Figure 8-1]Figures 8A-8C provide various codon-optimized nucleotide sequences encoding BDD-FVIII fused to XTEN. Figure 8A shows the nucleotide sequence of coFVIII-52-XTEN (SEQ ID NO: 19), in which a nucleotide sequence encoding an XTEN having 144 amino acids ("XTEN144"; SEQ ID NO: 18; underlined) is inserted into the coFVIII-52 nucleotide sequence. Figure 8B shows the nucleotide sequence of coFVIII-1-XTEN (SEQ ID NO: 20), in which a nucleotide sequence encoding an XTEN having 144 amino acids ("XTEN144"; SEQ ID NO: 18; underlined) is inserted into the coFVIII-1 nucleotide sequence. Figure 8C shows the nucleotide sequence of coFVIII-6-XTEN (SEQ ID NO: 72), in which a nucleotide sequence encoding an XTEN having 144 amino acids ("XTEN144"; SEQ ID NO: 18; underlined) is inserted into the coFVIII-6 nucleotide sequence (e.g., amino acid residue 745, corresponding to the mature FVIII sequence). [Figure 8-2] Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 9] 1 provides a plasmid map of pLV-coFVIII-52-XTEN, which contains coFVIII-52-XTEN in a lentiviral vector under the control of an ET promoter. As described herein, lentiviral vectors containing each of the remaining codon-optimized nucleic acid molecules encoding polypeptides having FVIII activity were constructed similarly to pLV-coFVIII-52-XTEN, in which the same XTEN sequence was inserted to replace the B-domain of FVIII. [Figure 10-1]Figures 10A and 10B show FVIII activity in HemA mice after injection with plasmid DNA (Figure 10A) or lentiviral vectors (Figure 10B) containing various codon-optimized nucleotide sequences encoding BDD-FVIII. Figure 10A shows a graphical representation of FVIII plasma activity in HemA mice after hydrodynamic injection with 5 μg of FVIII-311 (non-codon-optimized, BDD-FVIII-encoding nucleotide sequence; squares), 5 μg of FVIII-303 (coFVIII-1; small circles), or FVIII-306 (coFVIII-1-XTEN144; large circles). Relative activity after 72 hours, normalized to FVIII-311, is shown for each plasmid. Figure 10B shows plasma FVIII activity in HemA mice 21 days after injection of 1E8TU / mouse of lentiviral vectors containing coFVIII-52 or coFVIII-52XTEN, as measured by a FVIII-specific chromogenic assay, compared to the LV-2116 (BDD-FVIII) control. Error bars indicate standard deviation. [Figure 10-2] Continued from Figure 10-1. [Figure 11-1]Figure 11A shows the amino acid sequence of full-length mature human factor VIII. Figure 11B shows the amino acid sequence of full-length human von Willebrand factor (SEQ ID NO: 44). Figures 11C and 11D 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 Figures 11E, 11G, 11I, 11K, 11M, 11O, 11Q, 11S, 11U, and 11W (SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, 62, 64, and 66, respectively), and the corresponding nucleotide sequences are shown in Figures 11F, 11H, 11J, 11L, 11N, 11P, 11R, 11T, 11V, and 11X (SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67, respectively). Figure 11Y shows the nucleotide sequence of the ET promoter (SEQ ID NO: 69). Figure 11Z shows the nucleotide sequence of coFVIII-1 (SEQ ID NO: 68). (See International Publication No. WO2014 / 127215, SEQ ID NO: 1). [Figure 11-2] Continued from Figure 11-1. [Figure 11-3] Continued from Figure 11-2. [Figure 11-4] Continued from Figure 11-3. [Figure 11-5] Continued from Figure 11-4. [Figure 11-6] Continued from Figure 11-5. [Figure 12-1]Figure 12A is a graphic representation of FVIII plasma activity (IU / mL) in 14-day-old HemA mice after IV administration of approximately 1.5 x 10 TU / kg of LV-wtBDD-FVIII (circles), LV-coFVIII-6 (squares), or LV-coFVIII-6XTEN (triangles). Figure 12B is a graphic representation of vector copy number (VCN) 150 days after treatment of 14-day-old HemA mice administered IV approximately 1.5 x 10 TU / 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. Figure 12C is a graphic representation of FVIII plasma activity (IU / mL) 21 days after treatment of 14-day-old HemA mice administered IV at approximately 1.5 x 10 TU / 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. [Figure 12-2] Continued from Figure 12-1. [Figure 13] Figures 13A and 13B are graphs illustrating plasma FVIII activity levels (Figure 13A) and anti-FVIII antibody levels (Figure 13B) in five HemA mice treated with lentivirus expressing coFVIII-5 mutants. Fourteen-day-old HemA littermates were administered approximately 1.5 x 10 TU / kg of lentivirus expressing coFVIII-5 mutants by intravenous injection. Each mouse is designated by a number (i.e., 1, 2, 3, 4, and 5; Figures 13A and 13B). [Figure 14]1 is a graphical representation of the correlation between LV-FVIII expression levels and the presence of anti-FVIII antibodies, as evidenced by FVIII plasma activity 21 days after lentivirus treatment. Each data point corresponds to a single HemA mouse. Each mouse received a dose of 1.5×10 TU / kg by intravenous injection of a lentivirus expressing one of the coFVIII variants disclosed herein. The horizontal line indicates the mean FVIII plasma activity. [Figure 15] 1 is a graphical representation of the correlation between vector copy number (VCN) and the presence of anti-FVIII antibodies 150 days after lentiviral treatment. Each data point corresponds to a single HemA mouse. Each mouse received a dose of 1.5×10 TU / kg by intravenous injection of a lentivirus expressing one of the coFVIII variants disclosed herein. The horizontal line indicates the mean VCN. [Figure 16] Figures 16A and 16B are graphs illustrating FVIII plasma activity levels (Figure 16A) and anti-FVIII antibody levels (Figure 16B) in two HemA mice treated with lentivirus expressing coFVIII-52 mutants (coFVIII-52-A and coFVIII-52-B). Fourteen-day-old HemA littermates were administered approximately 1.5 x 10 TU / kg of lentivirus expressing coFVIII-52 mutants by intravenous injection. Figures 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 (Figure 16C) and coFVIII-52-B (Figure 16D) mice in Figures 16A and 16B. [Figure 17]

[0023] Figure 1 is a graph showing long-term FVIII expression in HemA neonatal mice treated with lentivirus expressing wild-type B-domain deleted FVIII (wtBDD-FVIII; triangles), coFVIII-52-XTEN (circles), or coFVIII-6-XTEN (inverted triangle) mutants. Neonatal HemA mice were administered approximately 1.5 x 10 TU / kg of lentivirus expressing wtBDD-FVIII, coFVIII-52-XTEN, or coFVIII-6-XTEN by intravenous injection. FVIII plasma activity was measured over approximately 16 weeks. [Figure 18] 18A-18B are graphic representations of dose-response results for treatment of HemA mice with lentivirus expressing coFVIII-6 (FIG. 18A) or coFVIII-6-XTEN (FIG. 18B). [Figure 19] Schematic diagram of lentiviral vector for liver-targeted gene therapy. SD: splice donor site; SA: splice acceptor site; GA: truncated gag sequence; RRE: Rev response element; ET: enhanced transthyretin; FVIII: factor VIII; 142T: target sequence for miR-142; Wpre: mutated marmot hepatitis virus post-transcriptional regulatory element; Ψ (packaging signal). [Figure 20] Figures 20A-20B are graphic representations of peak circulating FVIII levels in male pigtail macaques administered 3 x 10 TU / kg lentivirus expressing coFVIII-6-XTEN produced from CD47 high / MHC-I free 293T cells, as measured by FVIII plasma activity (Figure 20A) and FVIII plasma antigen levels (Figure 20B). [Figure 21] 21A-21B are graphic representations of peak plasma levels of human FVIII activity (FIG. 21A) and human FVIII antigen levels (FIG. 21B) in male pigtail macaques administered 3×10 TU / kg or 6×10 TU / kg lentivirus expressing coFVIII-6. [Figure 22]Figures 22A-22B are graphic representations of peak plasma levels of human FVIII activity (Figure 22A) and mean human FVIII antigen levels (Figure 22B) in male pigtail macaques administered 1 x 10 or 3 x 10 TU / kg lentivirus expressing coFVIII-6-XTEN. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure describes liver-targeted lentiviral gene therapy using a codon-optimized gene encoding a polypeptide with Factor VIII (FVIII) activity. See International Publication WO2017136358, which is incorporated herein by reference in its entirety.

[0021] Thus, in some aspects, the present disclosure relates to gene therapy comprising administering a lentiviral vector comprising a codon-optimized nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having Factor VIII activity. In particular aspects, the present disclosure relates to a method for treating a bleeding disorder, such as hemophilia (e.g., hemophilia A), comprising administering to a subject a lentiviral vector comprising a codon-optimized Factor VIII nucleic acid sequence targeted to the liver (e.g., hepatocytes). The present disclosure fulfills an important need in the art through a gene therapy approach that results in stable integration of a transgene expression cassette comprising a codon-optimized Factor VIII nucleic acid sequence into the genome of targeted cells.

[0022] This system allows the delivery of lentiviral vectors up to 5 × 10 10 transducing units / kg (TU / kg) or less, e.g., about 1.5 x 10 10 TU / kg or less, or approximately 1.5 x 10 9 TU / kg or less, or about 10 8 When administered to a subject at least one dose of TU / kg or less, it demonstrates increased, long-term expression of Factor VIII in targeted cells (eg, hepatocytes).

[0023] In a specific embodiment, the lentiviral vector disclosed herein comprises a codon-optimized nucleic acid sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 71 (LV-coFVIII-6).

[0024] In some other specific embodiments, the lentiviral vectors disclosed herein comprise a codon-optimized nucleic acid sequence that comprises, consists of, or consists essentially of SEQ ID NO: 72 (LV-coFVIII-6-XTEN).

[0025] The liver-targeted lentiviral vectors disclosed herein allow for stable integration of a 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, and can be administered at low lentiviral vector doses (e.g., 5×10 10 For example, 10 9 TU / kg or less or 10 8 The disclosed lentiviral vectors achieve enhanced FVIII expression (e.g., 100-fold enhanced) at very low doses (e.g., 10 TU / kg or less). 9 TU / kg or less or 10 8 Achieving therapeutic levels of circulating FVIII (TU / kg or less) Because of this, these vectors can significantly reduce the potential acute toxicity associated with lentiviral vector treatment. Furthermore, the use of lentiviral vectors, especially third-generation vectors, can potentially result in lifelong integration into the subject's genome. The high capacity of lentiviral vectors (10 kb) relative to other gene delivery systems (e.g., AAV) allows for the inclusion of more regulatory elements into the transgene, such as promoters that will 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 in vivo, in vitro, or ex vivo treatments.

[0026] Exemplary constructs of the present disclosure are illustrated in the accompanying figures and sequence listing.

[0027] In order to provide a clear understanding of the specification and claims, the following definitions are provided below.

[0028] I. Definition It is noted that the terms "a" or "an" entity refer to one or more of that entity: for example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0029] The term "about" is used herein to mean approximately, roughly, around, or approximately. 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 numerical values ​​above and below the stated value by a variance of 10% above and below (high and low).

[0030] For purposes of this disclosure, the term "isolated" refers to biological material (cells, polypeptides, polynucleotides, or fragments, variants, or derivatives thereof) that has been removed from its original environment (the environment in which it naturally occurs). For example, a polynucleotide present in a natural state in a plant or animal is not isolated, but the same polynucleotide separated from the adjacent nucleic acids in which it naturally occurs is considered "isolated." No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated in this disclosure, as are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0031] The terms "nucleic acid," "nucleic acid molecule," "oligonucleotide," and "polynucleotide" are used interchangeably and refer to the phosphate polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any of their phosphate analogs, such as phosphorothioates and thioesters, in single-stranded or double-stranded helical form. Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, the sequences may be described herein by the usual convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having sequence homology to mRNA). A "nucleic acid molecule" is a DNA molecule that has undergone 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 present disclosure comprises one or more nucleic acids described herein.

[0032] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide consisting of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not generally translated into an amino acid, it can be considered part of the coding region, but any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region either. The boundaries of a coding region are generally determined by a start codon at the 5' end, which encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end, which encodes 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. Consequently, a single vector can contain only a single coding region, or can include two or more coding regions.

[0033] Certain proteins secreted by mammalian cells are associated with a secretory signal peptide that is cleaved from the mature protein upon initiation of translocation of the growing protein chain across the rough endoplasmic reticulum. Those skilled in the art will recognize that signal peptides are commonly fused to the N-terminus of a polypeptide and are cleaved from the complete or "full-length" polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide or a functional derivative of that sequence retains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, can be used.

[0034] The term "downstream" refers to the nucleotide sequence located 3' of the reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to the sequence following the start of transcription. For example, the translation start codon of a gene is located downstream of the start site of transcription.

[0035] The term "upstream" refers to the nucleotide sequence located 5' of the reference nucleotide sequence.In certain embodiments, the upstream nucleotide sequence relates to the sequence located 5' of the coding region or the transcription start site.For example, most promoters are located upstream of the transcription start site.

[0036] As used herein, the term "gene regulatory region" or "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region that influences 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 the 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.

[0037] A polynucleotide encoding a gene product, e.g., a polypeptide, can include a promoter and / or other expression (e.g., transcriptional or translational) control elements operably associated with one or more coding regions. In operably associated, the 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, the coding region and promoter are associated such that induction of promoter function is controlled by the coding region. A promoter is "operably associated" if it results in the transcription of mRNA encoding the encoded gene product, and if the nature of the linkage between the promoter and coding region does not interfere with the ability of the promoter to direct expression of the gene product or 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.

[0038] "Transcription control sequence" refers to a DNA regulatory sequence, such as a promoter, enhancer, terminator, etc., that enables expression of a coding sequence in a host cell. Various transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as promoter and enhancer segments from cytomegalovirus (immediate-early promoter with intron-A), simian virus 40 (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 β-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, and lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0039] Similarly, a variety of translation control elements are known to those skilled in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also called CITE sequences).

[0040] The term "expression," as used herein, refers to the process by which a polynucleotide produces a gene product, e.g., an RNA or polypeptide. It includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and translation of mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be a messenger RNA produced by transcription of a nucleic acid, e.g., a gene, or a polypeptide 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, lipid addition, conjugation to other protein subunits, or proteolytic cleavage. The term "yield," as used herein, refers to the amount of polypeptide produced by expression of a gene.

[0041] A "vector" refers to any vehicle for the cloning and / or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. A "replicon" is an in "Vector" refers to any genetic element (e.g., a plasmid, a phage, a cosmid, a chromosome, a 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 non-viral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous 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 an appropriate polynucleotide fragment into a selected vector with complementary cohesive termini.

[0042] Vectors can be engineered to encode selectable markers or reporters that allow for the selection or identification of cells that have incorporated the vector. Expression of the selectable marker or reporter allows for the 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 that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered reporters.

[0043] The term "selectable marker" refers to an identifying agent, usually an antibiotic or chemical resistance gene, that can be selected for based on the function of the marker gene, i.e., antibiotic resistance, herbicide resistance, colorimetric marker, enzyme, fluorescent marker, etc., where this function is used to track the inheritance of a nucleic acid of interest and / or to identify cells or organisms that have inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include: genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc.

[0044] The term "reporter gene" refers to a nucleic acid encoding an identifying factor that can be identified based on the action of the reporter gene, where this action is used to track the inheritance of the nucleic acid of interest, to identify cells or organisms that have inherited the nucleic acid of interest, and / or to measure the induction or transcription of gene expression. Examples of reporter genes known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable marker genes can also be considered reporter genes.

[0045] The terms "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. Generally, the coding sequence is located 3' from the promoter sequence. Promoters can be derived entirely from a native gene, composed of different elements derived from different promoters found in nature, or can include synthetic DNA segments. Those skilled in the art will appreciate 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. A promoter that causes a gene to be expressed in most cell types most of the time is generally referred to as a "constitutive promoter." A promoter that causes a gene to be expressed in a specific cell type is generally referred to as a "cell-specific promoter" or "tissue-specific promoter." A promoter that causes a gene to be expressed at a specific stage of development or cell differentiation is generally referred to as a "development-specific promoter" or "cell differentiation-specific promoter." A promoter that is induced to cause a gene to be expressed following exposure or treatment of cells with a promoter-inducing drug, biomolecule, chemical, ligand, light, etc. is generally referred to as an "inducible promoter" or "regulatable promoter." In most cases, the exact boundaries of regulatory sequences have not been completely defined; therefore, DNA fragments of different lengths can bind to identical promoters. It is further recognized that the agonist may have inhibitory activity.

[0046] A promoter sequence generally contains a transcription initiation site bounded at its 3' end and extends upstream (5') 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.

[0047] The terms "restriction endonucleases" and "restriction enzymes" are used interchangeably and refer to enzymes that bind and cut at specific nucleotide sequences in double-stranded DNA.

[0048] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of a cell and that are usually in the form of circular double-stranded DNA molecules. Such elements can be linear, circular, or supercoiled, autonomously replicating, genomic integrating, phage, or nucleotide sequences of single- or double-stranded DNA or RNA derived from any source, in which several nucleotide sequences are linked or recombined into a specific construct that is capable of introducing into a cell a promoter fragment and DNA sequence for a selected gene product, along with appropriate 3' untranslated sequences.

[0049] "Cloning vector" refers to a "replicon," a unit-length nucleic acid, such as a plasmid, phage, or cosmid, that replicates sequentially and contains an origin of replication, to which another nucleic acid segment can be attached so as to bring about replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria, and expression in another cell type, e.g., eukaryotic cells. Cloning vectors generally contain one or more sequences that can be used for selection of cells that contain the vector, and / or one or more multiple cloning sites for insertion of nucleic acid sequences of interest.

[0050] The term "expression vector" refers to a vehicle designed to enable expression of an inserted nucleic acid sequence after insertion into a host cell, the inserted nucleic acid sequence being placed in operable relationship with the regulatory regions described above.

[0051] Vectors are introduced into host cells by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or DNA vector transporters.

[0052] As used herein, "culture," "culturing," and "culturing" refer to incubating cells under in vitro conditions that allow the cells to grow or divide, or maintaining the cells in a viable state. As used herein, "cultured cells" refer to cells that have been grown in vitro.

[0053] As used herein, the term "polypeptide" encompasses the singular "polypeptide" as well as the 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, "proteins," "amino acid chains," 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" is used interchangeably with "polypeptide." The term "peptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" also refers to the products of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be produced by any method, including chemical synthesis.

[0054] 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 (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile 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 present disclosure and include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the techniques of Noren et al., Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these techniques involve chemically activating a suppressor tRNA with the non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA. Introduction of non-traditional amino acids can also be achieved using peptide chemistry known in the art. As used herein, the term "polar amino acid" includes amino acids that have a zero total charge but 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 and electrostatic interactions. As used herein, the term "charged amino acid" includes amino acids that can have a non-zero net charge on their side chains (e.g., R, K, H, E, D). These amino acids can participate in hydrophobic and electrostatic interactions.

[0055] Polypeptide fragments or variants, and any combinations thereof, are also included in the present disclosure. The term "fragment" or "variant," when referring to a polypeptide binding domain or binding molecule of the present disclosure, includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn-binding affinity for an FcRn-binding domain or Fc variant, clotting activity of an FVIII variant, or FVIII-binding activity to a VWF fragment). Polypeptide fragments include proteolytic fragments and deletion fragments, as well as specific antibody fragments discussed elsewhere herein, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of the polypeptide binding domain or binding molecule of the present disclosure also include the above-mentioned fragments, as well as polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0056] 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, such as a basic side chain (e.g., lysine, arginine, histidine), an acidic side chain (e.g., aspartic acid, glutamic acid), an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), or a nonpolar side chain. Families of amino acid residues having similar side chains have been defined in the art, including amino acid residues with similar 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, when an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, strings of amino acids can be conservatively replaced with structurally similar strings that differ in the order and / or composition of the side chain family members.

[0057] As known in the art, the term "percent identity" refers to 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 may be, as determined by the match between strings of such sequences. "Identity" can be easily calculated by known methods, including but not limited to those described in the following: Computational Molecular Biology (Lesk, AM, ed.), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, 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 for determining identity are designed to give the best match between the sequences tested. Methods for determining identity are codified in publicly available computer programs.Sequence alignments and percent identity calculations can be performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), the GCG program suite (Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA). Within the context of this application, when sequence analysis software is used for analysis, it is understood that the results of the analysis will be based on the "default values" of the referenced program unless otherwise specified. As used herein, "default values" refers to any set of values ​​or parameters that initially load with the software when first initialized. For purposes of determining percent identity between an optimized BDD FVIII sequence of the present disclosure and a reference sequence, only nucleotides of the reference sequence that correspond to nucleotides in the optimized BDD FVIII sequence of the present disclosure are used to calculate percent identity. For example, when comparing a full-length FVIII nucleotide sequence containing the B domain with an optimized B-domain deleted (BDD) FVIII nucleotide sequence of the present disclosure, the portion of the alignment including the A1, A2, A3, C1, and C2 domains is used to calculate percent identity. Nucleotides in the portion of the full-length FVIII sequence encoding the B domain (which results in a large "gap" in the alignment) are not counted as mismatches. Furthermore, in determining percent identity between an optimized BDD FVIII sequence of the present disclosure, or a specified portion thereof (e.g., nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3), and a reference sequence, percent identity is calculated based on the match. The total number of nucleotides in the optimized BDD-FVIII sequence is calculated by dividing the number of nucleotides in the optimized BDD-FVIII sequence by the total number of nucleotides in the complete sequence or a designated portion thereof listed herein.

[0058] As used herein, "nucleotides corresponding to nucleotides in the optimized BDD FVIII sequences of the present disclosure" are identified by aligning the optimized BDD FVIII sequences of the present disclosure to maximize identity with the reference FVIII sequence. The numbers used to identify equivalent amino acids in the reference FVIII sequence are based on the numbers used to identify corresponding amino acids in the optimized BDD FVIII sequences of the present disclosure.

[0059] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not originally naturally linked. Amino acid sequences normally present in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences normally present in the same protein can be placed in a new configuration in a fusion polypeptide, e.g., a fusion of the Factor VIII domain with the Ig Fc domain of the present disclosure. Fusion proteins are made, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Chimeric proteins can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide, or non-covalent bond.

[0060] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or a fragment, variant, or derivative thereof, immediately upstream of a position at which a heterologous moiety can be inserted. The "insertion site" is defined by a number, which is the number of the amino acid in mature, native FVIII (SEQ ID NO: 15, FIG. 11A) to which the insertion site corresponds, immediately N-terminal to the insertion site. For example, the phrase "a3 contains a heterologous moiety at an insertion site corresponding to amino acid 1656 of SEQ ID NO: 15" indicates that the heterologous moiety is located between the two amino acids corresponding to amino acids 1656 and 1657 of SEQ ID NO: 15.

[0061] As used herein, the phrase "immediately downstream of an amino acid" refers to a position immediately adjacent to the terminal carboxyl group of an amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to a position immediately adjacent to the terminal amine group of an amino acid.

[0062] As used herein, the terms "inserted," "inserted," "inserted into," or grammatically related terms refer to the location of a heterologous moiety in a recombinant FVIII polypeptide relative to the analogous location in native mature human FVIII. As used herein, these terms refer to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII and do not indicate, imply, or refer to any method or process by which the recombinant FVIII polypeptide was made.

[0063] As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. Half-life can be expressed as the time required for half of an administered dose to be cleared from the animal's circulation and / or other tissues. When the elimination curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic, with a rapid α-phase and a longer β-phase. The α-phase generally represents the equilibration of an administered Fc polypeptide between the intravascular and extravascular spaces and is determined, in part, by the size of the polypeptide. The β-phase generally represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, i.e., they have no alpha phase but only a single beta phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of a polypeptide in the β-phase.

[0064] As used herein, the term "linked" refers to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly linked or juxtaposed to the second amino acid or nucleotide sequence, or the first sequence can be covalently linked to the second sequence via an intervening sequence. The term "linked" not only refers to the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of any two amino acids of the second amino acid sequence (or of the first amino acid sequence, respectively) throughout the first amino acid sequence (or second amino acid sequence). In one embodiment, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (in the case of a polypeptide chain), a nucleotide or nucleotide chain (in the case of a nucleotide chain), or any chemical moiety (in the case of a polypeptide and a polynucleotide chain). The term "coupled" can also be indicated by a hyphen (-).

[0065] 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" refers to a covalent, non-peptide, or non-covalent bond. This association can be indicated by a colon, i.e., (:). In another embodiment, it refers to a covalent bond other than a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink 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 (positions 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidic bonds, ignorant bonds, bent bonds, dipole bonds, pi-back bonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, multidentate or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-pi bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, aurophilic, intercalation, stacking, entropic forces or chemical polarity.

[0066] As used herein, the term "monomer-dimer hybrid" refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain associated with each other by disulfide bonds, where 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. Thus, a monomer-dimer hybrid construct is a hybrid comprising a monomeric embodiment having only one clotting factor and a dimeric embodiment having two Fc regions.

[0067] As used herein, hemostasis means the stopping or slowing of bleeding or hemorrhage; or the stopping or slowing of blood flow through a blood vessel or body part.

[0068] Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to bleed, either spontaneously or as a result of trauma, due to an impaired or deficient ability to form fibrin clots. Examples of such disorders include hemophilia. 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 von Willebrand disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, and Bernard-Soulier syndrome, which is a defect or deficiency of GPIb. Deficiency of the vWF receptor, GPIb, can lead to loss of primary clot formation (primary hemostasis) and an increased bleeding tendency, as well as Glanzmann and Naegeli thrombasthenia (Glanzmann thrombasthenia). In acute and chronic liver failure, the liver produces insufficient clotting factors, which can increase the risk of bleeding.

[0069] The lentiviral vector comprising the isolated nucleic acid molecule of the present disclosure can be used prophylactically. As used herein, the term "prophylactic treatment" refers to the administration of the molecule before a bleeding episode. In one embodiment, the subject requiring a systemic hemostatic agent is undergoing or about to undergo surgery. For example, the lentiviral vector of the present disclosure can be administered before or after surgery as a prophylactic agent. The lentiviral vector of the present disclosure can be administered during or after surgery to control acute bleeding episodes. Surgery can include, but is not limited to, liver transplantation, liver resection, dental procedures, or stem cell transplantation.

[0070] The lentiviral vectors of the present disclosure can also be used for on-demand therapy. The term "on-demand therapy" refers to the administration of the lentiviral vectors disclosed herein in response to the symptoms of a bleeding episode or before an activity that may cause bleeding. In one aspect, on-demand therapy can be administered to a subject when bleeding begins, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, on-demand therapy can be administered before an activity that increases the risk of bleeding, such as contact sports.

[0071] As used herein, the term "acute bleeding" refers to a bleeding episode regardless of the underlying cause. For example, the subject may have trauma, uremia, a hereditary bleeding disorder (e.g., factor VII deficiency), a platelet disorder, or tolerance due to the development of antibodies against clotting factors.

[0072] As used herein, treat, therapy, or treatment refers to, for example, reducing the severity of a disease or condition; reducing the duration of a disease; ameliorating one or more symptoms associated with a disease or condition; or providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition or preventing one or more symptoms associated with the disease or condition. In one embodiment, the term "treating" or "treatment" refers to maintaining a FVIII trough level of at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL in a subject by administering a lentiviral vector of the disclosure. In another embodiment, treating or treatment means maintaining FVIII trough levels between about 1 to about 20 IU / dL, about 2 to about 20 IU / dL, about 3 to about 20 IU / dL, about 4 to about 20 IU / dL, about 5 to about 20 IU / dL, about 6 to about 20 IU / dL, about 7 to about 20 IU / dL, about 8 to about 20 IU / dL, about 9 to about 20 IU / dL, or about 10 to about 20 IU / dL. Treating or treating a disease or condition can also include maintaining FVIII activity in a subject at a level equivalent to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophilic subject. In one embodiment, the term "treating" or "treatment" refers to maintaining a disease or condition by administering a lentiviral vector of the present disclosure. By this, we mean maintaining a FVIII trough level of at least about 30 IU / dL, 40 IU / dL, 50 IU / dL, 60 IU / dL, 70 IU / dL, 80 IU / dL, 90 IU / dL, 100 IU / dL, 110 IU / dL, 120 IU / dL, 130 IU / dL, 140 IU / dL or 150 IU / dL in a subject. In another embodiment, treating or treatment means maintaining a FVIII trough level of about 10 to about 20 IU / dL, about 20 to about 23 IU / dL, about 30 to about 40 IU / dL, about 40 to about 50 IU / dL, about 50 to about 60 IU / dL, about 60 to about 70 IU / dL, about 70 to about 80 IU / dL, about 80 to about 90 IU / dL, about 90 to about 100 IU / dL, about 110 to about 120 IU / dL, about 120 to about 130 IU / dL, about 130 to about 140 IU / dL, or about 140 to about 150 IU / dL. Treating or treating a disease or condition can also include maintaining FVIII activity in a subject at a level equivalent to at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% of the FVIII activity in a non-hemophilic subject. The minimum trough level required for treatment can be determined by one or more known methods and can be adjusted (increased or decreased) for each individual.

[0073] As used herein, "administering" means providing a subject with a pharmaceutically acceptable Factor VIII-encoding nucleic acid molecule, Factor VIII polypeptide, or vector comprising a Factor VIII-encoding nucleic acid molecule of the present disclosure via a pharmaceutically acceptable route. The administration route may be intravenous, for example, intravenous injection and intravenous infusion. Additional administration routes include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The nucleic acid molecule, polypeptide, and vector may be administered as part of a pharmaceutical composition comprising at least one excipient.

[0074] As used herein, the phrase "subject in need thereof" includes subjects, such as mammalian subjects, who would benefit from administration of a nucleic acid molecule, polypeptide, or vector of the present disclosure, e.g., to improve hemostasis. In one embodiment, the subject includes, but is not limited to, an individual with hemophilia. In another embodiment, the subject includes, but is not limited to, an individual who has developed a FVIII inhibitor and therefore requires bypass therapy. The subject may be an adult or a minor (e.g., under 12 years of age).

[0075] As used herein, the term "clotting factor" refers to a naturally occurring or recombinantly produced molecule or analog thereof that prevents or shortens the duration of bleeding episodes in a subject. In other words, it refers to a molecule that has procoagulant activity, i.e., is responsible for the conversion of fibrinogen to an insoluble fibrin mesh that causes blood to clot or coagulate. An "activatable clotting factor" is a clotting factor in an inactive form (e.g., its proenzyme form) that can be converted to an active form.

[0076] As used herein, coagulation activity means the ability to participate in a cascade of biochemical reactions that complete the formation of a fibrin clot and / or reduce the severity, duration or frequency of hemorrhage or bleeding episodes.

[0077] As used herein, the terms "heterologous" or "exogenous" refer to a molecule that is not normally found in a given context, e.g., a cell or polypeptide. An exogenous or heterologous molecule can be introduced into a cell and is present only after manipulation of the cell, e.g., by transfection or other form of genetic engineering, or a heterologous amino acid sequence can be present in a protein in which it is not found in nature.

[0078] 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 that can extend 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 a heterologous nucleotide sequence include immunoglobulin constant regions or portions thereof, albumin or fragments thereof, albumin binding moieties, transferrin, the PAS polypeptide of U.S. Patent Application No. 20100292130, a HAP sequence, transferrin or fragments thereof, the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), albumin binding small molecules, XTEN sequences, FcRn binding moieties (e.g., a complete Fc region or portion thereof that binds to FcRn), single chain FcRn, and the like. Examples of heterologous nucleotide sequences include c-regions (ScFc regions, e.g., those described in US2008 / 0260738, WO2008 / 012543, or WO2008 / 1439545), polyglycine linkers, polyserine linkers, peptides, and short polypeptides of 6 to 40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), with varying degrees of secondary structure, particularly from less than 50% to more than 50%, or combinations of two or more thereof. In some embodiments, the polypeptide encoded by the heterologous nucleotide sequence is linked to a non-polypeptide moiety. Non-limiting examples of non-polypeptide moieties include polyethylene glycol (PEG), albumin-binding small molecules, polysialic acid, hydroxyethyl starch (HES), derivatives thereof, or any combination thereof.

[0079] As used herein, the term "Fc region" is defined as the portion of a polypeptide corresponding to the Fc region of a native Ig, i.e., formed by the dimeric association of the Fc domains of each of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, as used herein, the term "genetically fused Fc region" or "single-chain Fc region" (scFc region) refers to a synthetic dimeric Fc region composed of Fc domains genetically linked (i.e., encoded by a single contiguous gene sequence) in a single polypeptide chain.

[0080] In one embodiment, "Fc region" refers to that portion of a single Ig heavy chain beginning at the hinge region just upstream of the papain cleavage site (i.e., residue 216 of IgG, where the first residue of the heavy chain constant region is 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge, CH2, and CH3 domains.

[0081] The Fc region of an Ig constant region can comprise CH2, CH3, and CH4 domains, as well as a hinge region, depending on the Ig isotype. Chimeric proteins comprising the Fc region of an Ig confer several desirable properties to the chimeric protein, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO03 / 077834; US2003-0235536A1), which are incorporated herein by reference in their entireties.

[0082] As used herein in comparison with a nucleotide sequence of the present disclosure, a "reference nucleotide sequence" is a polynucleotide sequence that is substantially identical to the nucleotide sequence of the present disclosure, except that the portion corresponding to the FVIII sequence is not optimized. For example, SEQ ID NO: 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 encoding a single-chain Fc region linked at its 3' end to SEQ ID NO: 1 is a nucleic acid molecule consisting of the original (or "parent") BDD FVIII of SEQ ID NO: 16 and the same heterologous nucleotide sequence encoding a single-chain Fc region linked at its 3' end to SEQ ID NO: 16 (Figure 1I).

[0083] As used herein, "codon compatibility index" refers to a measure of codon usage bias. The codon compatibility index (CAI) measures the deviation of a given protein-coding gene sequence relative to a reference set of genes (Sharp PM and Li WH, Nucleic Acids Res. 15(3):1281-95 (1987)). CAI is calculated by taking the geometric mean of the weights associated with each codon over the entire length (measured in codons) of the gene sequence:

number

[0084] For each amino acid, the weight of each of its codons in the CAI is calculated as the ratio between the observed frequency of the codon (fi) and the frequency of the synonymous codon (fj) for that amino acid:

number

[0085] As used herein, the term "optimized" with respect to a nucleotide sequence refers to a polynucleotide sequence that encodes a polypeptide, wherein the polynucleotide sequence has been mutated to enhance the properties of the polynucleotide sequence. In some embodiments, optimization is performed to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease binding of regulatory proteins such as general transcription factors, increase or decrease splicing, or increase the yield of a polypeptide produced by the polynucleotide sequence. Examples of modifications that can be made to a polynucleotide sequence to optimize it include codon optimization, G / C content optimization, removal of repetitive 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 that enhance transcription, such as Kozak consensus sequences, around the transcription start site, removing sequences that may form stem-loop structures, removing destabilizing sequences, and combinations of two or more thereof.

[0086] II. FVIII lentiviral gene therapy Somatic gene therapy has been explored as a possible treatment for bleeding disorders, particularly hemophilia A. Gene therapy is a particularly attractive treatment for hemophilia due to its ability to treat hemophilia through continuous endogenous production of FVIII after a single administration of a vector encoding FVIII. Hemophilia A is well suited to a gene replacement approach because its clinical manifestations are entirely due to a deficiency of a single gene product (FVIII) that circulates in minute amounts (200 ng / ml) in plasma.

[0087] Lentiviral vectors have the potential to sustain transgene expression throughout integration. Due to their capacity and efficacy, lentiviral vectors have attracted attention as gene delivery vehicles. Lentiviral vectors have been evaluated in numerous ex-vivo cell therapy clinical programs, yielding promising efficacy and safety profiles.

[0088] The present disclosure fulfills an important need in the art by providing lentiviral vectors containing codon-optimized FVIII sequences that demonstrate increased expression in subjects and potentially result in greater therapeutic efficacy when used in gene therapy methods. Embodiments of the present disclosure relate to lentiviral vectors containing one or more codon-optimized nucleic acid molecules encoding polypeptides having FVIII activity as described herein, host cells (e.g., hepatocytes) containing the lentiviral vectors, and methods of using the disclosed lentiviral vectors (e.g., methods of treatment for bleeding disorders using the lentiviral vectors disclosed herein).

[0089] Generally, the methods of treatment disclosed herein involve the administration of a lentiviral vector comprising a nucleic acid molecule comprising at least one codon-optimized nucleic acid sequence encoding a FVIII coagulation factor, wherein the nucleic acid sequence encoding the FVIII coagulation factor is operably linked to a suitable expression control sequence, which, in some embodiments, is incorporated into a lentiviral vector (e.g., a replication-defective lentiviral vector).

[0090] The present disclosure provides a method of treating a bleeding disorder (e.g., hemophilia A) in a subject in need thereof, comprising administering 5×10 immunizations of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity. 10 Transducing units / kg (TU / kg) (or 10 9 TU / kg or less, or 10 8 administering to a subject at least one dose of: (i) at least about 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 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 4374 of SEQ ID NO: 6; or (ix) Any combination of (i) to (viii) It has.

[0091] The present disclosure provides a method of treating a bleeding disorder (e.g., hemophilia A) in a subject in need thereof, comprising administering 5×10 mAbs of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence comprising 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 the FVIII polypeptide. 10 Transducing units / kg (TU / kg) (or 10 9 TU / kg or less, or 10 8 Also provided are methods comprising administering to a subject at least one dose of 100 mg / kg or less of erythropoietin; (a) wherein the first nucleic acid sequence is (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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 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 to 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 to 1791 of SEQ ID NO:6. and (b) wherein the second nucleotide sequence is (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 to 2277 and 2320 to 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 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 4374 of SEQ ID NO:6. or (c) Any combination of (a) and (b) and Here, the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0092] In some embodiments, the dose is about 5.0 x 10 10 TU / kg, approximately 4.9 x 10 10 TU / kg, approximately 4.8 x 10 10 TU / kg, approximately 4.7 x 10 10 TU / kg, approximately 4.6 x 10 10 TU / kg, approximately 4.5 x 10 10 TU / kg, approximately 4.4 x 10 10 TU / kg, approximately 4.3 x 10 10 TU / kg, approximately 4.2 x 10 10 TU / kg, approximately 4.1 x 10 10 TU / kg, approximately 4.0 x 10 10 TU / kg, approximately 3.9 x 10 10 TU / kg, approximately 3.8 x 10 10 TU / kg, approximately 3.7 x 10 10 TU / kg, approximately 3.6 x 10 10 TU / kg, approximately 3.5 x 10 10 TU / kg, approximately 3.4 x 10 10TU / kg, approximately 3.3 x 10 10 TU / kg, approximately 3.2 x 10 10 TU / kg, approximately 3.1 x 10 10 TU / kg, approximately 3.0 x 10 10 TU / kg, approximately 2.9 x 10 10 TU / kg, approximately 2.8 x 10 10 TU / kg, approximately 2.7 x 10 10 TU / kg, approximately 2.6 x 10 10 TU / kg, approximately 2.5 x 10 10 TU / kg, approximately 2.4 x 10 10 TU / kg, approximately 2.3 x 10 10 TU / kg, approximately 2.2 x 10 10 TU / kg, approximately 2.1 x 10 10 TU / kg, approximately 2.0 x 10 10 TU / kg, Approximately 1.9×10 10 TU / kg, approximately 1.8 x 10 10 TU / kg, approximately 1.7 x 10 10 TU / kg, approximately 1.6 x 10 10 TU / kg, approximately 1.5 x 10 10 TU / kg, approximately 1.4 x 10 10 TU / kg, approximately 1.3 x 10 10 TU / kg, approximately 1.2 x 10 10 TU / kg, approximately 1.1 x 10 10 TU / kg or approximately 1.0 x 10 10 It is TU / kg.

[0093] In some embodiments, the dose is about 9.9 x 10 9 TU / kg, approximately 9.8 x 10 9 TU / kg, approximately 9.7 x 10 9 TU / kg, approximately 9.6 x 10 9 TU / kg, approximately 9.5 x 10 9 TU / kg, approximately 9.4 x 10 9 TU / kg, approximately 9.3 x 10 9 TU / kg, approximately 9.2 x 10 9 TU / kg, approximately 9.1 x 10 9 TU / kg, approximately 9.0 x 10 9 TU / kg, approximately 8.9 x 10 9 TU / kg, approximately 8.8 x 109 TU / kg, about 8.7×10 9 TU / kg, about 8.6×10 9 TU / kg, about 8.5×10 9 TU / kg, about 8.4×10 9 TU / kg, about 8.3×10 9 TU / kg, about 8.2×10 9 TU / kg, about 8.1×10 9 TU / kg, about 8.0×10 9 TU / kg, about 7.9×10 9 TU / kg, about 7.8×10 9 TU / kg, about 7.7×10 9 TU / kg, about 7.6×10 9 TU / kg, about 7.5×10 9 TU / kg, about 7.4×10 9 TU / kg, about 7.3×10 9 TU / kg, about 7.2×10 9 TU / kg, about 7.1×10 9 TU / kg, about 7.0×10 9 TU / kg, about 6.9×10 9 TU / kg, about 6.8×10 9 TU / kg, about 6.7×10 9 TU / kg, about 6.6×10 9 TU / kg, about 6.5×10 9 TU / kg, about 6.4×10 9 TU / kg, about 6.3×10 9 TU / kg, about 6.2×10 9 TU / kg, about 6.1×10 9 TU / kg, about 6.0×10 9 TU / kg, about 5.9×10 9 TU / kg, about 5.8×10 9 TU / kg, about 5.7×10 9 TU / kg, about 5.6×10 9 TU / kg, about 5.5×10 9 TU / kg, about 5.4×10 9 TU / kg, about 5.3×10 9 TU / kg, about 5.2×10 9 TU / kg, about 5.1×10[[ID=7历4]] 9 TU / kg, about 5.0×10 9TU / kg, approximately 4.9×10 9 TU / kg, approximately 4.8×10 9 TU / kg, approximately 4.7×10 9 TU / kg, approximately 4.6×10 9 TU / kg, approximately 4.5×10 9 TU / kg, approximately 4.4×10 9 TU / kg, approximately 4.3×10 9 TU / kg, approximately 4.2×10 9 TU / kg, approximately 4.1×10 9 TU / kg, approximately 4.0×10 9 TU / kg, approximately 3.9×10 9 TU / kg, approximately 3.8×10 9 TU / kg, approximately 3.7×10 9 TU / kg, approximately 3.6×10 9 TU / kg, approximately 3.5×10 9 TU / kg, approximately 3.4×10 9 TU / kg, approximately 3.3×10 9 TU / kg, approximately 3.2×10 9 TU / kg, approximately 3.1×10 9 TU / kg, approximately 3.0×10 9 TU / kg, approximately 2.9×10 9 TU / kg, approximately 2.8×10 9 TU / kg, approximately 2.7×10 9 TU / kg, approximately 2.6×10 9 TU / kg, approximately 2.5×10 9 TU / kg, approximately 2.4×10 9 TU / kg, approximately 2.3×10 9 TU / kg, approximately 2.2×10 9 TU / kg, approximately 2.1×10 9 TU / kg, approximately 2.0×10 9 TU / kg, approximately 1.9×10 9 TU / kg, approximately 1.8×10 9 TU / kg, approximately 1.7×10 9 TU / kg, approximately 1.6×z10 9 TU / kg, approximately 1.5×10 9 TU / kg, approximately 1.4×10 9 TU / kg, approximately 1.3×10 9 TU / kg, approximately 1.2×10 9 TU / kg, approximately 1.1×10 It should be noted that there seems to be a typo in the original text where "1.6×z10" is likely incorrect. It should probably be "1.6×10". This translation is based on the best understanding of the provided text with the assumption of the correction.9 TU / kg or approximately 1.0 x 10 9 It is TU / kg.

[0094] In some embodiments, the dose is about 9.9 x 10 8 TU / kg, approximately 9.8 x 10 8 TU / kg, approximately 9.7 x 10 8 TU / kg, approximately 9.6 x 10 8 TU / kg, approximately 9.5 x 10 8 TU / kg, approximately 9.4 x 10 8 TU / kg, approximately 9.3 x 10 8 TU / kg, approximately 9.2 x 10 8 TU / kg, approximately 9.1 x 10 8 TU / kg, approximately 9.0 x 10 8 TU / kg, approximately 8.9 x 10 8 TU / kg, approximately 8.8 x 10 8 TU / kg, approximately 8.7 x 10 8 TU / kg, approximately 8.6 x 10 8 TU / kg, approximately 8.5 x 10 8 TU / kg, approximately 8.4 x 10 8 TU / kg, approximately 8.3 x 10 8 TU / kg, approximately 8.2 x 10 8 TU / kg, approximately 8.1 x 10 8 TU / kg, approximately 8.0 x 10 8 TU / kg, approximately 7.9 x 10 8 TU / kg, approximately 7.8 x 10 8 TU / kg, approximately 7.7 x 10 8 TU / kg, approximately 7.6 x 10 8 TU / kg, approximately 7.5 x 10 8 TU / kg, approximately 7.4 x 10 8 TU / kg, approximately 7.3 x 10 8 TU / kg, approximately 7.2 x 10 8 TU / kg, approximately 7.1 x 10 8 TU / kg, approximately 7.0 x 10 8 TU / kg, approximately 6.9 x 10 8 TU / kg, approximately 6.8 x 10 8 TU / kg, approximately 6.7 x 10 8 TU / kg, approximately 6. 6×10 8TU / kg, approximately 6.5×10 8 TU / kg, approximately 6.4×10 8 TU / kg, approximately 6.3×10 8 TU / kg, approximately 6.2×10 8 TU / kg, approximately 6.1×10 8 TU / kg, approximately 6.0×10 8 TU / kg, approximately 5.9×10 8 TU / kg, approximately 5.8×10 8 TU / kg, approximately 5.7×10 8 TU / kg, approximately 5.6×10 8 TU / kg, approximately 5.5×10 8 TU / kg, approximately 5.4×10 8 TU / kg, approximately 5.3×10 8 TU / kg, approximately 5.2×10 8 TU / kg, approximately 5.1×10 8 TU / kg, approximately 5.0×10 8 TU / kg, approximately 4.9×10 8 TU / kg, approximately 4.8×10 8 TU / kg, approximately 4.7×10 8 TU / kg, approximately 4.6×10 8 TU / kg, approximately 4.5×10 8 TU / kg, approximately 4.4×10 8 TU / kg, approximately 4.3×10 8 TU / kg, approximately 4.2×10 8 TU / kg, approximately 4.1×10 8 TU / kg, approximately 4.0×10 8 TU / kg, approximately 3.9×10 8 TU / kg, approximately 3.8×10 8 TU / kg, approximately 3.7×10 8 TU / kg, approximately 3.6×10<00​​​​​​​​​​​​​​​​​8 TU / kg, approximately 2.6 x 10 8 TU / kg, approximately 2.5 x 10 8 TU / kg, approximately 2.4 x 10 8 TU / kg, approximately 2.3 x 10 8 TU / kg, approximately 2.2 x 10 8 TU / kg, approximately 2.1 x 10 8 TU / kg, approximately 2.0 x 10 8 TU / kg, approximately 1.9 x 10 8 TU / kg, approximately 1.8 x 10 8 TU / kg, approximately 1.7 x 10 8 TU / kg, approximately 1.6 x 10 8 TU / kg, approximately 1.5 x 10 8 TU / kg, approximately 1.4 x 10 8 TU / kg, approximately 1.3 x 10 8 TU / kg, approximately 1.2 x 10 8 TU / kg, approximately 1.1 x 10 8 TU / kg or approximately 1.0 x 10 8 It is TU / kg.

[0095] In some embodiments, the dose is 5.0 x 10 10 TU / kg, less than 4.9 × 10 10 TU / kg, less than 4.8 × 10 10 TU / kg, less than 4.7 × 10 10 TU / kg, less than 4.6 × 10 10 TU / kg, less than 4.5 × 10 10 TU / kg, less than 4.4 × 10 10 TU / kg, less than 4.3 × 10 10 TU / kg, less than 4.2 × 10 10 TU / kg, less than 4.1 × 10 10 TU / kg, less than 4.0 × 10 10 TU / kg, less than 3.9 × 10 10 TU / kg, less than 3.8 × 10 10 TU / kg, less than 3.7 × 10 10 TU / kg, less than 3.6 × 10 10 TU / kg, less than 3.5 × 10 10 TU / kg, less than 3.4 × 10 10 TU / kg, less than 3.3 × 10 10TU / kg, less than 3.2 × 10 10 TU / kg, less than 3.1 × 10 10 TU / kg, less than 3.0 × 10 10 TU / kg, less than 2.9 × 10 10 TU / kg, less than 2.8 × 10 10 TU / kg, less than 2.7 × 10 10 TU / kg, less than 2.6 × 10 10 TU / kg, less than 2.5 × 10 10 TU / kg, less than 2.4 × 10 10 TU / kg, less than 2.3 × 10 10 TU / kg, less than 2.2 × 10 10 TU / kg, less than 2.1 × 10 10 TU / kg, less than 2.0 × 10 10 TU / kg, less than 1.9 × 10 10 TU / kg, less than 1.8 × 10 10 TU / kg, less than 1.7 × 10 10 TU / kg, less than 1.6 × 10 10 TU / kg, less than 1.5 × 10 10 TU / kg, less than 1.4 × 10 10 TU / kg, less than 1.3 × 10 10 TU / kg, less than 1.2 × 10 10 TU / kg, less than 1.1 × 10 10 Less than TU / kg or 1.0 x 10 10 It is less than TU / kg.

[0096] In some embodiments, the dose is 9.9×10 9 TU / kg, less than 9.8 × 10 9 TU / kg, less than 9.7 × 10 9 TU / kg, less than 9.6 × 10 9 TU / kg, less than 9.5 × 10 9 TU / kg, less than 9.4 × 10 9 TU / kg, less than 9.3 × 10 9 TU / kg, less than 9.2 × 10 9 TU / kg, less than 9.1 × 10 9 TU / kg, less than 9.0 × 10 9 TU / kg, less than 8.9 × 10 9 TU / kg, less than 8.8 × 109 TU / kg, less than 8.7 × 10 9 TU / kg, less than 8.6 × 10 9 TU / kg, less than 8.5 × 10 9 TU / kg, less than 8.4 × 10 9 TU / kg, less than 8.3 × 10 9 TU / kg, less than 8.2 × 10 9 TU / kg, less than 8.1 × 10 9 TU / kg, less than 8.0 × 10 9 TU / kg, less than 7.9 × 10 9 TU / kg, less than 7.8 × 10 9 TU / kg, less than 7.7 × 10 9 TU / kg, less than 7.6 × 10 9 TU / kg, less than 7.5 × 10 9 TU / kg, less than 7.4 × 10 9 TU / kg, less than 7.3 × 10 9 TU / kg, less than 7.2 × 10 9 Less than TU / kg, 7.1 x 1 0 9 TU / kg, less than 7.0 × 10 9 TU / kg, less than 6.9 × 10 9 TU / kg, less than 6.8 × 10 9 TU / kg, less than 6.7 × 10 9 TU / kg, less than 6.6 × 10 9 TU / kg, less than 6.5 × 10 9 TU / kg, less than 6.4 × 10 9 TU / kg, less than 6.3 × 10 9 TU / kg, less than 6.2 × 10 9 TU / kg, less than 6.1 × 10 9 TU / kg, less than 6.0 × 10 9 TU / kg, less than 5.9 × 10 9 TU / kg, less than 5.8 × 10 9 TU / kg, less than 5.7 × 10 9 TU / kg, less than 5.6 × 10 9 TU / kg, less than 5.5 × 10 9 TU / kg, less than 5.4 × 10 9 TU / kg, less than 5.3 × 10 9 TU / kg, less than 5.2 × 10 9TU / kg, less than 5.1 × 10 9 TU / kg, less than 5.0 × 10 9 TU / kg, less than 4.9 × 10 9 TU / kg, less than 4.8 × 10 9 TU / kg, less than 4.7 × 10 9 TU / kg, less than 4.6 × 10 9 TU / kg, less than 4.5 × 10 9 TU / kg, less than 4.4 × 10 9 TU / kg, less than 4.3 × 10 9 TU / kg, less than 4.2 × 10 9 TU / kg, less than 4.1 × 10 9 TU / kg, less than 4.0 × 10 9 TU / kg, less than 3.9 × 10 9 TU / kg, less than 3.8 × 10 9 TU / kg, less than 3.7 × 10 9 TU / kg, less than 3.6 × 10 9 TU / kg, less than 3.5 × 10 9 TU / kg, less than 3.4 × 10 9 TU / kg, less than 3.3 × 10 9 TU / kg, less than 3.2 × 10 9 TU / kg, less than 3.1 × 10 9 TU / kg, less than 3.0 × 10 9 TU / kg, less than 2.9 × 10 9 TU / kg, less than 2.8 × 10 9 TU / kg, less than 2.7 × 10 9 TU / kg, less than 2.6 × 10 9 TU / kg, less than 2.5 × 10 9 TU / kg, less than 2.4 × 10 9 TU / kg, less than 2.3 × 10 9 TU / kg, less than 2.2 × 10 9 TU / kg, less than 2.1 × 10 9 TU / kg, less than 2.0 × 10 9 TU / kg, less than 1.9 × 10 9 TU / kg, less than 1.8 × 10 9 TU / kg, less than 1.7 × 10 9 TU / kg, less than 1.6 × 10 9 TU / kg, less than 1.5 × 10 9TU / kg, less than 1.4 × 10 9 TU / kg, less than 1.3 × 10 9 TU / kg, less than 1.2 × 10 9 TU / kg, less than 1.1 × 10 9 Less than TU / kg or 1.0 x 10 9 It is less than TU / kg.

[0097] In some embodiments, the dose is 9.9×10 8 TU / kg, less than 9.8 × 10 8 TU / kg, less than 9.7 × 10 8 TU / kg, less than 9.6 × 10 8 TU / kg, less than 9.5 × 10 8 TU / kg, less than 9.4 × 10 8 TU / kg, less than 9.3 × 10 8 TU / kg, less than 9.2 × 10 8 TU / kg, less than 9.1 × 10 8 TU / kg, less than 9.0 × 10 8 TU / kg, less than 8.9 × 10 8 TU / kg, less than 8.8 × 10 8 TU / kg, less than 8.7 × 10 8 TU / kg, less than 8.6 × 10 8 TU / kg, less than 8.5 × 10 8 TU / kg, less than 8.4 × 10 8 TU / kg, less than 8.3 × 10 8 TU / kg, less than 8.2 × 10 8 TU / kg, less than 8.1 × 10 8 TU / kg, less than 8.0 × 10 8 TU / kg, less than 7.9 × 10 8 TU / kg, less than 7.8 × 10 8 TU / kg, less than 7.7 × 10 8 TU / kg, less than 7.6 × 10 8 TU / kg, less than 7.5 × 10 8 TU / kg, less than 7.4 × 10 8 TU / kg, less than 7.3 × 10 8 TU / kg, less than 7.2 × 10 8 TU / kg, less than 7.1 × 10 8 TU / kg, less than 7.0 × 10 8TU / kg, less than 6.9 × 10 8 TU / kg, less than 6.8 × 10 8 TU / kg, less than 6.7 × 10 8 TU / kg, less than 6.6 × 10 8 TU / kg, less than 6.5 × 10 8 TU / kg, less than 6.4 × 10 8 TU / kg, less than 6.3 × 10 8 TU / kg, less than 6.2 × 10 8 TU / kg, less than 6.1 × 10 8 TU / kg, less than 6.0 × 10 8 TU / kg, less than 5.9 × 10 8 TU / kg, less than 5.8 × 10 8 TU / kg, less than 5.7 × 10 8 TU / kg, less than 5.6 × 10 8 TU / kg, less than 5.5 × 10 8 TU / kg, less than 5.4 × 10 8 TU / kg, less than 5.3 × 10 8 TU / kg, less than 5.2 × 10 8 TU / kg, less than 5.1 × 10 8 TU / kg, less than 5.0 × 10 8 TU / kg, less than 4.9 × 10 8 TU / kg, less than 4.8 × 10 8 TU / kg, less than 4.7 × 10 8 TU / kg, less than 4.6 × 10 8 TU / kg, less than 4.5 × 10 8 TU / kg, less than 4.4 × 10 8 TU / kg, less than 4.3 × 10 8 TU / kg, less than 4.2 × 10 8 TU / kg, less than 4.1 × 10 8 TU / kg, less than 4.0 × 10 8 TU / kg, less than 3.9 × 10 8 TU / kg, less than 3.8 × 10 8 TU / kg, less than 3.7 × 10 8 TU / kg, less than 3.6 × 10 8 TU / kg, less than 3.5 × 10 8 TU / kg, less than 3.4 × 10 8 TU / Less than kg, 3.3 x 108 TU / kg, less than 3.2 × 10 8 TU / kg, less than 3.1 × 10 8 TU / kg, less than 3.0 × 10 8 TU / kg, less than 2.9 × 10 8 TU / kg, less than 2.8 × 10 8 TU / kg, less than 2.7 × 10 8 TU / kg, less than 2.6 × 10 8 TU / kg, less than 2.5 × 10 8 TU / kg, less than 2.4 × 10 8 TU / kg, less than 2.3 × 10 8 TU / kg, less than 2.2 × 10 8 TU / kg, less than 2.1 × 10 8 TU / kg, less than 2.0 × 10 8 TU / kg, less than 1.9 × 10 8 TU / kg, less than 1.8 × 10 8 TU / kg, less than 1.7 × 10 8 TU / kg, less than 1.6 × 10 8 TU / kg, less than 1.5 × 10 8 TU / kg, less than 1.4 × 10 8 TU / kg, less than 1.3 × 10 8 TU / kg, less than 1.2 × 10 8 TU / kg, less than 1.1 × 10 8 Less than TU / kg or 1.0 x 10 8 It is less than TU / kg.

[0098] In some embodiments, the dose is 1×10 8 TU / kg~5×10 10 TU / kg, 1.5 x 10 8 TU / kg~5×10 10 TU / kg, 2 x 10 8 TU / kg~5×10 10 TU / kg, 2.5 x 10 8 TU / kg~5×10 10 TU / kg, 3 x 10 8 TU / kg~5×10 10 TU / kg, 3.5 x 10 8 TU / kg~5×10 10 TU / kg, 4 x 10 8TU / kg~5×10 10 TU / kg、4.5×10 8 TU / kg~5×10 10 TU / kg、5×10 8 TU / kg~5×10 10 TU / kg、5.5×10 8 TU / kg~5×10 10 TU / kg、6×10 8 TU / kg~5×10 10 TU / kg、6.5×10 8 TU / kg~5×10 10 TU / kg、7×10 8 TU / kg~5×10 10 TU / kg、7.5×10 8 TU / kg~5×10 10 TU / kg、8×10 8 TU / kg~5×10 10 TU / kg、8.5×10 8 TU / kg~5×10 10 TU / kg、9×10 8 TU / kg~5×10 10 TU / kg、9.5×10 8 TU / kg~5×10 10 TU / kg, 1×10 9 TU / kg~5×10 10 TU / kg、1.5×10 9 TU / kg~5×10 10 TU / kg、2×10 9 TU / kg~5×10 10 TU / kg、2.5×10 9 TU / kg~5×10 10 TU / kg、3×10 9 TU / kg~5×10 10 TU / kg、3.5×10 9 TU / kg~5×10 10 TU / kg、4×10 9 TU / kg~5×10 10 TU / kg、4.5×10 9 TU / kg~5×10 10 TU / kg、5×10 9 TU / kg~5×10 10 TU / kg、5.5×10 9 TU / kg~5×1010 TU / kg, 6×10 9 TU / kg to 5×10 10 TU / kg, 6.5×10 9 TU / kg to 5×10 10 TU / kg, 7×10 9 TU / kg to 5×10 10 TU / kg, 7.5×10 9 TU / kg to 5×10 10 TU / kg, 8×10 9 TU / kg to 5×10 10 TU / kg, 8.5×10 9 TU / kg to 5×10 10 TU / kg, 9×10 9 TU / kg to 5×10 10 TU / kg, 9.5×10 9 TU / kg to 5×10 10 TU / kg, 10 10 TU / kg to 5×10 10 TU / kg, 1.5×10 10 TU / kg to 5×10 10 TU / kg, 2×10 10 TU / kg to 5×10 10 TU / kg, 2.5×10 10 TU / kg to 5×10 10 TU / kg, 3×10 10 TU / kg to 5×10 10 TU / kg, 3.5×10<4000746>TU / kg to 5×10 10 TU / kg, 4×10 10 TU / kg to 5×10 10 TU / kg or 4.5×10 10 TU / kg to 5×10 10 TU / kg.

[0099] In some embodiments, the dosage is 1×10 8 TU / kg to 5×10 10 TU / kg, 1×10 8 TU / kg to 4.5×10 10 TU / kg, 1×10​​​​​​10 TU / kg, 1×10 8 TU / kg~3×10 10 TU / kg, 1×10 8 TU / kg~2.5×10 10 TU / kg, 1×10 8 TU / kg~2×10 10 TU / kg, 1×10 8 TU / kg~1.5×10 10 TU / kg, 1×10 8 TU / kg~10 10 TU / kg, 1×10 8 TU / kg~9×10 9 TU / kg, 1×10 8 TU / kg~8.5×10 9 TU / kg, 1×10 8 TU / kg~8×10 9 TU / kg, 1×10 8 TU / kg~7.5×10 9 TU / kg, 1×10 8 TU / kg~7×10 9 TU / kg, 1×10 8 TU / kg~6.5×10 9 TU / kg, 1×10 8 TU / kg~6×10 9 TU / kg, 1×10 8 TU / kg~5 .5×10 9 TU / kg, 1×10 8 TU / kg~5×10 9 TU / kg, 1×10 8 TU / kg~4.5×10 9 TU / kg, 1×10 8 TU / kg~4×10 9 TU / kg, 1×10 8 TU / kg~3.5×10 9 TU / kg, 1×10 8 TU / kg~3×10 9 TU / kg, 1×10 8 TU / kg~2.5×10 9 TU / kg, 1×10 8 TU / kg~2×10 9 TU / kg, 1×10 8 TU / kg~1.5×109 TU / kg, 1×10 8 TU / kg to 1×10 9 TU / kg, 1×10 8 TU / kg to 9.5×10 8 TU / kg, 1×10 8 TU / kg to 9×10 8 TU / kg, 1×10 8 TU / kg to 8.5×10 8 TU / kg, 1×10 8 TU / kg to 8×10 8 TU / kg, 1×10 8 TU / kg to 7.5×10 8 TU / kg, 1×10 8 TU / kg to 7×10 8 TU / kg, 1×10 8 TU / kg to 6.5×10 8 TU / kg, 1×10 8 TU / kg to 6×10 8 TU / kg, 1×10<000082;>TU / kg to 5.5×10 8 TU / kg, 1×10 8 TU / kg to 5×10 8 TU / kg, 同1×10 8 TU / kg to 4.5×10 8 TU / kg, 1×10 8 TU / kg to 4×10 8 TU / kg, 1×10 8 TU / kg to 3.5×10 8 TU / kg, 1×10 8 TU / kg to 3×10 8 TU / kg, 1×1(]] 8 TU / kg to 2.5×10 8 TU / kg, 1×10 8 TU / kg to 2×10 8 TU / kg, or 1×10 8 TU / kg to 1.5×10 8 TU / kg.

[0100] In some embodiments, the dosage is 1×10 10 TU / kg to 2×10 10 TU / kg, 1.1×10 10 TU / kg to 1.9×10 (注:原文中“同1×10”这里可能有误,我按照字面意思翻译了,你可根据实际情况调整。)10 TU / kg, 1.2 x 10 10 TU / kg ~ 1.8 x 10 10 TU / kg, 1.3 x 10 10 TU / kg ~ 1.7 x 10 10 TU / kg, or 1.4 x 10 10 TU / kg ~ 1.6 x 10 10 In some embodiments, the dose is about 1.5 x 10 10 In some embodiments, the dose is 1.5 x 10 10 It is TU / kg.

[0101] In some embodiments, the dose is 1×10 9 TU / kg~2×10 9 TU / kg, 1.1 x 10 9 TU / kg ~ 1.9 x 10 9 TU / kg, 1.2 x 10 9 TU / kg ~ 1.8 x 10 9 TU / kg, 1.3 x 10 9 TU / kg ~ 1.7 x 10 9 TU / kg, or 1.4 x 10 9 TU / kg ~ 1.6 x 10 9 In some embodiments, the dose is 1.5 x 10 9 In certain embodiments, the dose is about 3.0 x 10 9 It is TU / kg.

[0102] In some embodiments, the dose is 2.5×10 9 TU / kg ~ 3.5 x 10 9 TU / kg, 2.6 x 10 9 TU / kg ~ 3.4 x 10 9 TU / kg, 2.7 × 10 9 TU / kg ~ 3.3 x 10 9 TU / kg, 2.8 x 10 9 TU / kg ~ 3.2 x 10 9 TU / kg, or 2.9 x 10 9 TU / kg ~ 3.1 x 10 9 In some embodiments, the dose is about 3.0 x 10 9In some embodiments, the dose is 3.0 x 10 9 It is TU / kg.

[0103] In some embodiments, the dose is 5.5 x 10 9 TU / kg ~ 6.5 x 10 9 TU / kg, 5.6 x 10 9 TU / kg ~ 6.4 x 10 9 TU / kg, 5.7 x 10 9 TU / kg ~ 6.3 x 10 9 TU / kg, 5.8 x 10 9 TU / kg ~ 6.2 x 10 9 TU / kg, or 5.9 x 10 9 TU / kg ~ 6.1 x 10 9 In some embodiments, the dose is about 6.0 x 10 9 In some embodiments, the dose is 6.0 x 10 9 It is TU / kg.

[0104] In some embodiments, plasma FVIII activity 24 hours, 36 hours, or 48 hours after administration of a lentiviral vector of the present disclosure is increased compared to plasma FVIII activity in a subject administered a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16.

[0105] In some embodiments, plasma FVIII activity 48 hours after administration of the lentiviral vector. The activity is increased compared to plasma FVIII activity in subjects administered a reference vector comprising a nucleic acid molecule comprising SEQ ID NO:16.

[0106] In another embodiment, plasma FVIII activity is increased about 21 days after administration of the lentiviral vector compared 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.

[0107] In some embodiments, plasma FVIII activity is increased about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, or about 28 days after administration of the lentiviral vector compared 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.

[0108] In some embodiments, the plasma FVIII activity in the subject is 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 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 50-fold, at least about 51-fold, at least about 52-fold, at least about 53-fold, at least about 54-fold, at least about 55-fold, at least about 56-fold, at least about 57-fold, at An increase of 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 110-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.

[0109] In some embodiments, lentiviral vector is administered in a single dose or multiple doses.In some embodiments, the dose of lentiviral vector is administered at once or divided into multiple partial doses, for example, two partial doses, three partial doses, four partial doses, five partial doses, six partial doses or more than six partial doses.In some embodiments, more than one lentiviral vector is administered.

[0110] In some embodiments, the dose of the lentiviral vector is administered at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, the lentiviral vector is administered via intravenous injection.

[0111] In some embodiments, the subject is a pediatric subject, while in other aspects, the subject is an adult subject.

[0112] In some embodiments, the lentiviral vector comprises at least one tissue-specific promoter, i.e., a promoter that will regulate expression of a polypeptide having FVIII activity in a particular tissue or cell type. In some embodiments, the tissue-specific promoter in the lentiviral vector regulates expression of a polypeptide having FVIII activity in target liver cells. The expression of the polypeptide is selectively enhanced. In some embodiments, the tissue-specific promoter that selectively enhances the expression of the polypeptide having FVIII activity in target liver cells comprises an mTTR promoter. In some embodiments, the target liver cells are hepatocytes.

[0113] Because lentiviral vectors can transduce all liver cell types, expression of a transgene (e.g., FVIII) in different cell types can be controlled using different promoters in the lentiviral vector. Thus, lentiviral vectors can contain specific promoters that will control expression of a FVIII transgene in different tissues or cell types, such as different liver tissues or cell types. Thus, in some embodiments, lentiviral vectors can contain an endothelial-specific promoter that will control expression of a FVIII transgene in liver endothelial tissue, or a hepatocyte-specific promoter that controls expression of a FVIII transgene in hepatocytes, or both.

[0114] In some embodiments, the lentiviral vector comprises a tissue-specific promoter or multiple tissue-specific promoters that control expression of the FVIII transgene in tissues other than the liver. In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of a target cell or tissue, for example, the genome of a hepatocyte or the genome of an endothelial cell of the liver.

[0115] In some embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity in a lentiviral vector of the present disclosure comprises, consists of, or consists essentially of LV-coFVIII-6 (SEQ ID NO: 71).

[0116] In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity in the lentiviral vector of the present disclosure comprises, consists of, or consists essentially of LV-coFVIII-6-XTEN (SEQ ID NO: 72).

[0117] In some embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity in the lentiviral vector of the present disclosure further comprises a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding the 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 1-57 of SEQ ID NO:1; (ii) nucleotides 1-57 of SEQ ID NO:2; (iii) nucleotides 1-57 of SEQ ID NO:3; (iv) nucleotides 1-57 of SEQ ID NO:4; (v) nucleotides 1-57 of SEQ ID NO:5; (vi) nucleotides 1-57 of SEQ ID NO:6; (vii) nucleotides 1-57 of SEQ ID NO:70; (viii) nucleotides 1-57 of SEQ ID NO:71; or (ix) nucleotides 1-57 of SEQ ID NO:68.

[0118] In some embodiments, the isolated nucleic acid molecule in the lentiviral vector of the present disclosure comprises one or more properties selected from the group consisting of: (a) the human codon compatibility index of the nucleic acid molecule or portion thereof is increased compared to SEQ ID NO: 16; (b) the optimal codon frequency of the nucleotide sequence or portion thereof is increased compared to SEQ ID NO: 16; (c) the nucleotide sequence or 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 frequency of the nucleotide sequence or portion thereof is increased compared to SEQ ID NO: 16; (e) the effective number of codons of the nucleotide sequence or portion thereof is reduced compared to SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21 and 22) compared to SEQ ID NO: 16; (g) the nucleotide sequence is and (h) any combination thereof.

[0119] In some embodiments, the isolated nucleic acid molecule in the 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 portion thereof, an XTEN, transferrin, albumin, or PAS sequence. In some embodiments, the heterologous amino acid sequence is linked to the N-terminus or 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 sites selected from Table 3.

[0120] In some embodiments, the FVIII polypeptide is full-length FVIII or B-domain deleted FVIII.

[0121] The lentiviral vectors disclosed herein may be therapeutically beneficial in the treatment of bleeding diseases or disorders selected from the group consisting of bleeding coagulation disorders, hemorrhagic arthropathy, muscle bleed, mouth bleed, hemorrhage, bleeding into muscle, mouth bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. ... trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. The lentiviral vectors may be administered in vivo at low dosages (e.g., 10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A.

[0122] In some embodiments, target cells (e.g., hepatocytes) are administered a low dose (e.g., 10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8In certain embodiments, target cells (e.g., hepatocytes) are treated in vitro with approximately 3.0 x 10 TU / kg or less of the lentiviral vectors disclosed herein before administration to a patient. 9 In yet another embodiment, cells (e.g., hepatocytes) from a patient are treated in vitro with a low dose (e.g., 10 TU / kg) of a lentiviral vector disclosed herein before being administered to the patient. 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 TU / kg or less) ex vivo before being administered to patients.

[0123] In some embodiments, administration of a lentiviral vector disclosed herein (e.g., 10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 In some embodiments, plasma FVIII activity after administration of a dose of 100 mg / kg or less of FVIII per 100 mg of FVIII per day (administered at 100 mg / kg or less in TU / kg or less) is increased by at least about 100%, at least about 110%, 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% compared to physiologically normal circulating FVIII levels.

[0124] In one embodiment, plasma FVIII activity after administration of a lentiviral vector of the present disclosure is increased by at least about 3,000% to about 5,000% compared to physiologically normal circulating FVIII levels. In some embodiments, 21 days after administration of a lentiviral vector comprising a codon-optimized gene encoding a polypeptide having factor VIII (FVIII) activity described herein, the plasma FVIII activity is increased by at least about 3,000% to about 5,000% compared to physiologically normal circulating FVIII levels. In some embodiments, the plasma FVIII activity is increased by at least about 3,000% to about 5,000% compared to physiologically normal circulating FVIII levels. 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 least about 100-fold, at least about 110-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 increase compared to a subject administered the corresponding lentiviral vector.

[0125] The present disclosure provides a method 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 having FVIII activity, wherein the lentiviral vector is administered to a subject in a therapeutically effective amount of at least 5×10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 Also provided are methods wherein the antibody is administered in at least one dose of the following: TU / kg.

[0126] The treatment, amelioration, and prevention by the lentiviral vectors of the present disclosure can be a bypass therapy. A subject undergoing bypass therapy can already develop or be susceptible to developing inhibitors of clotting factors, such as FVIII.

[0127] The lentiviral vector of the present disclosure treats or prevents hemostatic disorders by promoting the formation of fibrin clots. The polypeptide having FVIII activity encoded by the nucleic acid molecule of the present disclosure can activate members of the coagulation cascade. The coagulation factors can be participants in the extrinsic pathway, the intrinsic pathway, or both.

[0128] The lentiviral vectors of the present disclosure can be used to treat hemostatic disorders known to be treatable with FVIII. Hemostatic disorders that can be treated using the methods of the present disclosure include, but are not limited to, hemophilia A, hemophilia B, von Willebrand's disease, factor XI deficiency (PTA deficiency), factor XII deficiency, and deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, hemarthrosis, muscle bleed, mouth bleed, bleeding, bleeding into muscle, mouth bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath.

[0129] The composition for administration to a subject comprises a lentiviral vector comprising a FVIII clotting factor (for gene therapy applications) and a nucleic acid molecule comprising an optimized nucleotide sequence of the present disclosure encoding a FVIII polypeptide molecule. In some embodiments, the composition for administration is a cell that has been contacted in vivo, in vitro, or ex vivo with a lentiviral vector of the present disclosure.

[0130] In some embodiments, the hemostatic disorder is a genetic disorder. In one embodiment, the subject has hemophilia A. In other embodiments, the hemostatic disorder is the result of a deficiency of FVIII. In other embodiments, the hemostatic disorder may be the result of a defective FVIII clotting factor.

[0131] In another embodiment, the hemostatic disorder may be an acquired disorder. The acquired disorder may result from an underlying secondary disease or condition. The unrelated condition may be, for example, but not limited to, cancer, autoimmune disease, or pregnancy. The acquired disorder may result from aging or from a medicine (e.g., cancer chemotherapy) used to treat the underlying secondary disorder.

[0132] The present disclosure relates to a method for treating hemostatic disorders, including the treatment of hemostatic disorders that involve secondary diseases or conditions that result in the acquisition of hemostatic disorders. The present disclosure also relates to a method for treating a subject who does not have a systemic hemostatic agent. Accordingly, the present disclosure relates to a method for treating a subject who needs a systemic hemostatic agent, the method comprising administering a therapeutically effective amount of a lentiviral vector of the present disclosure. For example, in one embodiment, the subject who needs a systemic hemostatic agent is undergoing or about to undergo surgery. The lentiviral vector of the present disclosure can be administered before or after surgery as a prophylactic agent.

[0133] The lentiviral vectors of the present disclosure can be administered during or after surgery to control acute bleeding episodes, which can include, but are not limited to, liver transplantation, liver resection, or stem cell transplantation.

[0134] In another embodiment, the lentiviral vectors of the present disclosure can be used to treat subjects with acute bleeding episodes who do not have hemostatic disorders. The acute bleeding episodes can result from severe trauma, such as surgery, car accidents, wounds, lacerations, gunshot wounds, or any other traumatic event that results in uncontrolled bleeding.

[0135] Lentiviral vectors can be used to prophylactically treat subjects with hemostatic disorders. Lentiviral vectors can also be used to treat acute bleeding episodes in subjects with hemostatic disorders.

[0136] In another embodiment, administration of the lentiviral vector disclosed herein and / or subsequent expression of the FVIII protein transgene does not induce an immune response in the subject. In some embodiments, the immune response includes the development of antibodies against FVIII. In some embodiments, the immune response includes cytokine secretion. In some embodiments, the immune response includes 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 compared to the activity of FVIII in a subject that did not mount an immune response. In certain embodiments, expression of the FVIII protein by administration of the lentiviral vector disclosed herein prevents an inhibitory immune response against the FVIII protein or the FVIII protein expressed from the isolated nucleic acid molecule or lentiviral vector.

[0137] In some embodiments, the lentiviral vector of the present disclosure is administered in combination with at least one other agent that promotes hemostasis. The other agent that promotes hemostasis is a therapeutic agent with demonstrated coagulation activity. By way of example, but not 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 foregoing. The coagulation factor or hemostatic agent can also include an antifibrinolytic agent, such as epsilon-aminocaproic acid or tranexamic acid.

[0138] In one embodiment of the present disclosure, the composition (e.g., lentiviral vector) is one in which FVIII is present in an activatable form when administered to a subject, and such an activatable molecule can be activated in vivo at the site of coagulation after administration to a subject.

[0139] The lentiviral vectors of the present disclosure can be administered intravenously, subcutaneously, intramuscularly, or through any mucosal surface, for example, orally, sublingually, buccally, sublingually, nasally, rectally, vaginally, or through the pulmonary route. The lentiviral vector can be implanted in or linked to a biopolymer solid support that allows for the slow release of the vector to the desired site.

[0140] In one embodiment, the route of administration of the lentiviral vector is parenteral. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. An intravenous form of parenteral administration is preferred. While all of these forms of administration are expressly contemplated within the scope of this disclosure, particularly for intravenous or intraarterial injection or infusion, the form for administration will be an injectable solution. Typically, suitable pharmaceutical compositions for injection may include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin). However, in other methods consistent with the teachings herein, lentiviral vectors can be delivered directly to the site of harmful cell populations, thereby increasing the exposure of affected tissues to the therapeutic agent.

[0141] 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 this disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, preferably 0.05 M, phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, 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 may be present, such as antimicrobials, antioxidants, chelating agents, and inert gases.

[0142] More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (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 fluid to the extent that easy syringability remains. It should be stable under the conditions of manufacture and storage, and preferably 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 suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0143] Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0144] In any case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., polypeptide itself or a combination with other active agents) in the required amount in a suitable solvent, together with one or a combination of the ingredients listed herein as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which gives a powder of the active ingredient and any additional desired ingredients from the solution previously sterilized by filtration. The preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions by methods known in the art. In addition, the preparations can be packaged in the form of a kit. Such articles of manufacture preferably have a label or package insert indicating that the associated composition is useful for treating a subject suffering from or predisposed to a clotting disorder.

[0145] Pharmaceutical compositions can also be formulated for rectal administration as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0146] The effective dose of the composition of the present disclosure for treating a condition varies depending on many different factors, including administration means, target site, the physiological condition of the patient, whether the patient is human or animal, other pharmaceuticals administered, and whether the treatment is preventive or therapeutic.Usually, the patient is human, but non-human mammals, including transgenic mammals, can also be treated.To optimize safety and efficacy, treatment dosage can be titrated using routine methods known to those skilled in the art.

[0147] The lentiviral vector can be administered as a single dose or as multiple doses, where the multiple doses can be administered consecutively or at specific time intervals. In vitro assays can be used to determine the optimal dose range and / or administration schedule. In vitro assays for measuring clotting factor activity are known in the art. Furthermore, effective doses can be determined using animal models, such as hemophilic dogs (Mount et al.). The dose-response curves can be estimated from those obtained from the Journal of Clinical Oncology, Vol. 2, No. 1, pp. 2670-2672 (2002).

[0148] The intermediate dose within the above range also falls within the scope of the present disclosure.Such dose can be administered to subject every day, every other day, every week or according to any other schedule that is determined by experimental analysis.Exemplary treatment requires long-term administration, for example, by multiple doses over at least 6 months.

[0149] The lentiviral vector of the present disclosure can be administered multiple times.The interval between single doses can be daily, weekly, monthly or yearly.The interval can also be irregular, as indicated by measuring the blood level of modified polypeptide or antigen in the patient.The dosage and frequency of the lentiviral vector of the present disclosure will depend on the half-life of the FVIII polypeptide encoded by the transgene in the patient.

[0150] The dosage and frequency of administration of the lentiviral vector of the present disclosure may vary depending on whether the treatment is preventive or therapeutic. In preventive applications, a composition containing the lentiviral vector of the present disclosure is administered to a patient who is not yet in a disease state to strengthen the patient's resistance or minimize the effects of the disease. Such an amount is defined as a "prophylactically 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.

[0151] The lentiviral vectors of the present disclosure can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (eg, prophylactic or therapeutic).

[0152] As used herein, administration of a lentiviral vector of the present disclosure together with or in combination with an adjunctive therapy refers to sequential, simultaneous, or concurrent administration of the therapy and the disclosed polypeptide. coexist "Combined" refers to concurrent, parallel, or simultaneous administration or application. Those skilled in the art will understand that the times of administration or application of the various components of a combined therapeutic regimen can be adjusted to enhance the overall effectiveness of the treatment. Those skilled in the art (e.g., physicians) will be able to determine effective combinations without undue experimentation based on the selected adjunctive therapy and the teachings herein. It will be easier to identify therapeutic regimens.

[0153] It is further understood that the lentiviral vectors of the present disclosure can be used together or in combination with a drug or drugs (e.g., to provide a combination therapy regimen). Exemplary drugs that can be combined with the lentiviral vectors of the present disclosure include drugs that represent the current standard of care for the particular disorder being treated. Such drugs can be chemical or biological in nature. The term "biological" or "biological agent" refers to any pharmaceutically active agent produced from living organisms and / or their products that is intended for use as a therapeutic agent.

[0154] The amount of drugs used in combination with the lentiviral vectors of the present disclosure can vary by subject or can be administered according to what is known in the art. See, for example, Bruce A. Chabner et al., Antineoplastic Agents, in GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, pp. 1233-1287 (Joel G. Hardman et al., eds., 9th ed. 1996). In another embodiment, an amount of such drug consistent with medical standards is administered.

[0155] In certain embodiments, the lentiviral vector of the present disclosure is administered together with immunosuppressants, anti-allergic or anti-inflammatory drugs.These drugs generally refer to substances that suppress or mask the immune system of the subject being treated herein.These drugs include substances that suppress cytokine production, downregulate or suppress autoantigen expression, or mask MHC antigens.Examples of such drugs include 2-amino-6-aryl-5-substituted pyrimidines; azathioprine; cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde; anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as glucocorticoids, for example, prednisone, methylprednisolone and dexamethasone; anti-interferon-γ, -β or -α antibodies, anti-tumor necrosis factor-α antibodies, anti-tumor necrosis factor The present invention relates to an antihistamine drug. ... Examples of antihistamines are chlorpheniramine, diphenhydramine, promethazine, cromolyn sodium, astemizole, azatadine maleate, brompheniramine maleate, carbinoxamine maleate, cetirizine hydrochloride, clemastine fumarate, cyproheptadine hydrochloride, d-brompheniramine maleate, d-chlorpheniramine maleate, dimenhydrinate, diphenhydramine hydrochloride, doxylamine succinate, fexofendazine hydrochloride, terfenadine hydrochloride, hydroxyzine hydrochloride, loratidine, meclizine hydrochloride, tripelennamine citrate, tripelennamine hydrochloride, and triprolidine hydrochloride.

[0156] Immunosuppressive, antiallergic or anti-inflammatory drugs can be incorporated into lentiviral vector administration regimen.For example, the administration of immunosuppressive or anti-inflammatory drugs can be started before the administration of the disclosed lentiviral vector, and can be continued by one or more administrations thereafter.In certain embodiments, immunosuppressive or anti-inflammatory drugs are administered as premedication of lentiviral vector.

[0157] As previously discussed, the lentiviral vectors of the present disclosure can be used to treat coagulation disorders in vivo. It can be administered in a pharmaceutically effective amount for treatment. In this regard, it is understood that the lentiviral vector of the present disclosure can be formulated to facilitate administration and promote the stability of the active agent. Preferably, the pharmaceutical composition according to the present disclosure comprises a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffer, preservative, etc. Of course, the pharmaceutical composition of the present disclosure can be administered in single or multiple doses to provide a pharmaceutically effective amount of the polypeptide.

[0158] To investigate the function of the coagulation system, several tests are available: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM® assay, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often by the Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (whether abnormalities are corrected when the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic testing (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), other platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0159] The aPTT test is a performance indicator that measures the efficacy of the "intrinsic" (also called the contact activation pathway) and common coagulation pathways. This test is commonly used to measure the clotting activity of commercially available recombinant coagulation factors, such as FVIII or FIX. It is used in conjunction with the prothrombin time (PT), which measures the extrinsic pathway.

[0160] The ROTEM® analysis provides information on the complete kinetics of hemostasis: clotting time, clot formation, clot stability, and lysis. The different parameters in thromboelastometry depend on many factors that affect the activity of the plasma coagulation system, platelet function, fibrinolysis, or their interactions. This assay can provide a complete picture of secondary hemostasis.

[0161] III. Lentiviral Vectors Lentiviruses include members of the bovine lentivirus group, equine lentivirus group, feline lentivirus group, ovine / caprine lentivirus group, and primate lentivirus group. The development of lentiviral vectors for gene therapy is reviewed in Klimatcheva et al. (1999) Frontiers in Bioscience 4:481-496. The design and use of lentiviral vectors suitable for gene therapy are described, for example, in U.S. Patent Nos. 6,207,455 and 6,615,782. Examples of lentiviruses include, but are not limited to, HIV-1, HIV-2, HIV-1 / HIV-2 pseudotypes, HIV-1 / SIV, FIV, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus, and bovine immunodeficiency virus.

[0162] A schematic diagram of a lentiviral vector of the present disclosure is provided in Figure 19. In some embodiments, the lentiviral vector of the present disclosure is a "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 further lacks a functional tat gene, e.g., one in which the tat gene is 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, e.g., The term "packaging system" refers to a set of viral constructs that contain genes encoding viral proteins involved in packaging of recombinant viruses. Generally, the constructs of the packaging system are ultimately incorporated into packaging cells.

[0163] 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 pseudotyped 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 lentiviral vector comprises one or more target sequences for miR-142 to reduce immune responses to the transgene product. In some embodiments, incorporating one or more target sequences for miR-142 into the lentiviral vector of the present disclosure enables a desired transgene expression profile. For example, incorporating one or more target sequences for miR-142 can suppress transgene expression in the intravascular and extravascular hematopoietic system, while maintaining transgene expression in non-hematopoietic cells. No carcinogenesis has been detected in tumor-prone mice treated with the lentiviral vector system of the present disclosure. See Brown et al. (2007) Blood 110:4144-52, Brown et al. (2006) Nat. Ned. 12:585-91 and Cantore et al. (2015) Sci. Transl. Med. 7(277):277ra28.

[0164] The lentiviral vector of the present disclosure comprises a codon-optimized polynucleotide encoding the BDD FVIII protein described herein. In one embodiment, the optimized coding sequence for the 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 a manner that allows each component nucleic acid sequence to retain its function. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in a manner that places the expression, 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 a 5' gene expression sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences does not (1) introduce frameshift mutations, (2) interfere with the ability of the promoter region to induce transcription of the coding sequence, or (3) interfere with the ability to translate the corresponding RNA transcript into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0165] In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of infecting non-dividing cells. In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of infecting liver cells (e.g., hepatocytes). Lentiviral genomes and proviral DNA generally contain three genes found in retroviruses: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-guided DNA polymerase (reverse transcriptase), protease, and integrase; and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNA. The TR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0166] Sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient encapsidation of viral RNA into particles (Psi site) flank the 5' LTR. If 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.

[0167] However, the resulting mutant remains capable of directing the synthesis of all virion proteins. The present disclosure provides a method for generating a recombinant lentivirus capable of infecting non-dividing cells, comprising transfecting a suitable host cell with two or more vectors carrying packaging functions, i.e., gag, pol, and env, and rev and tat. As disclosed herein below, vectors lacking a functional tat gene are desirable for certain applications. Thus, for example, one vector can provide nucleic acid encoding viral gag and viral pol, and another vector can provide nucleic acid encoding viral env to generate a packaging cell. Introducing a vector providing a heterologous gene, identified herein as a transfer vector, into the packaging cell results in a producer cell that releases infectious viral particles carrying the foreign gene of interest.

[0168] With the above configuration of vector and foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some embodiments, the env protein is an amphotropic envelope protein that allows transduction of cells of human and other species.

[0169] Examples of env genes from retroviruses include, but are not limited to, Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse 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), hepatitis virus, and influenza, can also be used. In some embodiments, the viral env nucleic acid sequence is operably associated with regulatory sequences described elsewhere herein.

[0170] In certain embodiments, the lentiviral vector is deleted from HIV pathogenicity genes env, vif, vpr, vpu and nef without impairing the vector's ability to transduce non-dividing cells.In some embodiments, the lentiviral vector comprises a deletion of the U3 region of 3'LTR.The deletion of the U3 region can be a complete deletion or a partial deletion.

[0171] In some embodiments, a lentiviral vector of the present disclosure comprising a FVIII nucleotide sequence described herein can be transfected into a cell with (a) a first nucleotide sequence comprising the gag, 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 the rev gene. In certain embodiments, the lentiviral vector comprises vif, vpr, vpu, vpx, and nef, or any of their equivalents. The combination lacks functional genes selected from the above.

[0172] In certain embodiments, the lentiviral vectors of the present disclosure comprise one or more nucleotide sequences encoding a gag protein, a Rev response element, a central polypurine tract (cPPT), or any combination thereof.

[0173] In some embodiments, the lentiviral vector expresses one or more polypeptides on its surface 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 the budding of the lentiviral vector from the host cell. During lentivirus production, viral particles bud from the producer host cell. During the budding process, the viral particles acquire 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-associated polypeptides that were previously present on the surface of the host cell.

[0174] In some embodiments, the lentiviral vector expresses one or more polypeptides on its surface that suppress immune responses to the lentiviral vector after 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 that is ubiquitously expressed on human cells. Surface expression of CD47 suppresses macrophage-induced phagocytosis of endogenous cells through the interaction of CD47 and SIRPα expressed by macrophages. Cells expressing high levels of CD47 are less likely to be targeted and destroyed by human macrophages in vivo.

[0175] In some embodiments, the lentiviral vector comprises a high concentration of CD47 polypeptide molecules on its surface. In some embodiments, the lentiviral vector is generated in a cell line with a high expression level of CD47. In certain embodiments, the lentiviral vector comprises a high concentration of CD47 polypeptide molecules on its surface. high In certain embodiments, the lentiviral vector is produced in cells that have high expression of CD47 on the cell membrane. high The CD47 is generated in HEK 293T cells, wherein the HEK 293T cells have high expression of CD47 on the cell membrane. In some embodiments, the HEK 293T cells are modified to have increased expression of CD47 compared to unmodified HEK 293T cells. In certain embodiments, the CD47 is human CD47.

[0176] In some embodiments, lentiviral vectors have little or no surface expression of major histocompatibility complex class I (MHC-I). Surface-expressed MHC-I presents peptide fragments of "non-self" proteins from within the cell, such as protein fragments indicative of infection, and promotes an immune response against the cell. In some embodiments, lentiviral vectors have little or no surface expression of MHC-I. low In some embodiments, the lentiviral vector is produced in a cell, wherein the cell has reduced expression of MHC-I on the cell membrane. - (or "MHC-I free ", "MHC-1 neg " or "MHC negative") cells, where the cells lack expression of MHC-I.

[0177] In certain embodiments, the lentiviral vector comprises a lipid coat that contains a high concentration of CD47 polypeptide and lacks MHC-I polypeptides. high / MHC-I low Cell lines, e.g., CD47 high / MHC-I low In some embodiments, the lentiviral vector is produced in a HEK 293T cell line. high / MHC-I free Cell lines, e.g., CD47 high / MHC-I free Produced in the HEK 293T cell line.

[0178] Examples of lentiviral vectors are disclosed in U.S. Pat. No. 9,050,269, as well as WO 9931251, WO 9712622, WO 9817815, WO 9817816 and WO 9818934, which are incorporated by reference in their entireties.

[0179] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence comprising: 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 FVIII polypeptide activity.

[0180] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence comprising 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 is selected from the group consisting of: (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 of SEQ ID NO:6. 77 and 2320-4374 have 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 the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0181] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having 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: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.

[0182] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having 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.

[0183] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having 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.

[0184] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having 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.

[0185] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having 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: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.

[0186] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having 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.

[0187] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence is at least about 89%, at least about 90%, at least about 91%, at least about 92%, or at least about 94% similar to (i) nucleotides 58 to 4374 of SEQ ID NO:5, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of SEQ ID NO:5. In another embodiment, the nucleic acid sequence comprises a nucleic acid sequence having 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 a promoter, a target sequence, or both, and is operably linked to a promoter, a target sequence, or both. In another embodiment, the nucleic acid sequence comprises (i) nucleotides 58 to 4374 of SEQ ID NO:5, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of SEQ ID NO:5.

[0188] In some embodiments, the present disclosure provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having 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.

[0189] The lentiviral vectors of the present disclosure are expressed in a concentration of 5×10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8At such dosages, administration of the lentiviral vector of the present disclosure can result in a subject in need thereof experiencing 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 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 50-fold, at least about 51-fold, at least about 52-fold, at least about 53-fold, at least about 54-fold, at least about 55-fold, at least about 56- It can result in an increase in plasma FVIII activity of 5-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 110-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.

[0190] IV. Tissue-specific expression In certain embodiments, it is beneficial to include one or more miRNA target sequences in the lentiviral vector, for example, operably linked to the optimized FVIII transgene. Thus, the present disclosure also provides at least one miRNA sequence target that is operably linked to the optimized FVIII nucleotide sequence or otherwise inserted into the lentiviral vector. More than one copy of the miRNA target sequence included in the lentiviral vector can increase the effectiveness of the system.

[0191] Different miRNA target sequences are also included. For example, a lentiviral vector expressing more than one transgene can have the transgene under the control of more than one miRNA target sequence, which may be the same or different. The miRNA target sequences can be tandem, although other arrangements are also included. A transgene expression cassette containing a miRNA target sequence can also be inserted into a lentiviral vector in an antisense orientation. The antisense orientation avoids expression of a gene product that might otherwise be toxic to the producing cell. These can be useful in the production of viral particles for

[0192] In other embodiments, the lentiviral vector contains one, two, three, four, five, six, seven, or eight copies of the same or different miRNA target sequences. However, in certain other embodiments, the lentiviral vector does not contain any miRNA target sequences. The choice of whether to include miRNA target sequences (and how many) is guided by known parameters such as the intended tissue target, the desired expression level, etc.

[0193] In one embodiment, the target sequence is the miR-223 target, which has been reported to be most effective in bone marrow-related precursors and at least partially block expression in earlier HSPCs.The miR-223 target can block expression in differentiated myeloid cells, including granulocytes, monocytes, macrophages, and myeloid dendritic cells.The miR-223 target may also be suitable for gene therapy applications that rely on robust transgene expression in lymphoid or erythroid lineages.The miR-223 target can also block expression very effectively in human HSCs.

[0194] In another embodiment, the target sequence is the miR142 target (tccataaagt aggaaacact aca (SEQ ID NO: 43)). In one embodiment, the lentiviral vector contains four copies of the miR-142 target sequence. In certain embodiments, the complementary sequence of a hematopoietic-specific microRNA, such as miR-142 (142T), is incorporated into the 3' untranslated region of the lentiviral vector, making the transgene-encoding transcript susceptible to miRNA-mediated downregulation. This method can block transgene expression in hematopoietic antigen-presenting cells (APCs), while maintaining it in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy can impose stringent post-transcriptional regulation on transgene expression, thus enabling stable delivery and long-term expression of the transgene. In some embodiments, miR-142 modulation prevents immune-mediated elimination of transduced cells and / or induces antigen-specific regulatory T cells (Tregs) and mediates robust immune tolerance to the antigen encoded by the transgene.

[0195] In some embodiments, the target sequence is a miR181 target. Chen CZ and Lodish H, Seminars in Immunology (2005) 17(2):155-165, discloses miR-181, a miRNA that is specifically expressed in B cells in mouse bone marrow (Chen and Lodish, 2005). It also discloses that some human miRNAs are associated with leukemia.

[0196] The target sequence may be fully or partially complementary to the miRNA. The term "fully complementary" means that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it. The term "partially complementary" means that the target sequence is only partially complementary to the sequence of the miRNA that recognizes it, whereby the partially complementary sequence is still recognized by the miRNA. In other words, in the context of the present disclosure, a partially complementary target sequence is effective in recognizing the corresponding miRNA and achieving inhibition or reduction of transgene expression in cells that express that miRNA. Examples of miRNA target sequences are described in WO2007 / 000668, WO2004 / 094642, WO2010 / 055413, or WO2010 / 125471, which are incorporated herein by reference in their entirety.

[0197] V. Polynucleotide sequences encoding FVIII proteins The present disclosure relates to lentiviral gene therapy, wherein the lentiviral vector comprises a codon-optimized nucleic acid molecule comprising a polynucleotide (nucleic acid) sequence encoding a polypeptide having 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, in which all or part of the B-domain of FVIII is deleted.

[0198] 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 (Figure 1), 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.

[0199] In some embodiments, the nucleic acid molecule encodes a FVIII polypeptide that includes a signal peptide or a fragment thereof. In other embodiments, the nucleic acid molecule encodes a FVIII polypeptide that lacks the signal peptide. In some embodiments, the signal peptide comprises amino acids 1-19 of SEQ ID NO: 17.

[0200] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 to 1791 of SEQ ID NO:3 or (ii) nucleotides 58 to 1791 of SEQ ID NO:4; and the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0201] 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 to 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 to 1791 of SEQ ID NO: 4. In other embodiments, the first nucleotide sequence comprises nucleotides 58 to 1791 of SEQ ID NO: 3 or nucleotides 58 to 1791 of SEQ ID NO: 4.

[0202] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0203] In one embodiment, the first nucleotide sequence comprises nucleotides 1 to 1791 of SEQ ID NO:3 or nucleotides 1 to 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 to 4374 of SEQ ID NO:3 or nucleotides 1792 to 4374 of SEQ ID NO:4. In a particular embodiment, the second nucleotide sequence comprises nucleotides 1792 to 4374 of SEQ ID NO:3 or nucleotides 1792 to 4374 of SEQ ID NO:4.

[0204] In yet 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 nucleotides 1792-4374 of SEQ ID NO:4 (i.e., nucleotides 1792-4374 of SEQ ID NO:3 or nucleotides 1792-4374 of SEQ ID NO:4 without the nucleotides encoding the B domain or B domain fragment).

[0205] In a specific embodiment, the second nucleotide sequence comprises nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:3 or nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:4 (i.e., nucleotides 1792-4374 of SEQ ID NO:3 or nucleotides 1792-4374 of SEQ ID NO:4 without the nucleotides encoding the B domain or B domain fragment).

[0206] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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) nucleotides 1792-4374 of SEQ ID NO:6; and the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0207] 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 to 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 to 4374 of SEQ ID NO: 6.

[0208] In a specific embodiment, the second nucleic acid sequence comprises nucleotides 1792 to 4374 of SEQ ID NO: 5 or nucleotides 1792 to 4374 of SEQ ID NO: 6. In some embodiments, the first nucleic acid sequence linked to said second nucleic acid 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 58 to 1791 of SEQ ID NO: 5 or nucleotides 58 to 1791 of SEQ ID NO: 6.

[0209] In other embodiments, the first nucleic acid sequence linked to the second nucleic acid 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 1 to 1791 of SEQ ID NO:5 or nucleotides 1 to 1791 of SEQ ID NO:6.

[0210] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 is selected from (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) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:6. 320-4374 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6, excluding any nucleotides encoding the B domain or B domain fragment); and the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0211] 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).

[0212] In a specific embodiment, the second nucleic acid sequence comprises nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:5 or nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:6 (i.e., nucleotides 1792-4374 of SEQ ID NO:5 or nucleotides 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 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.

[0213] In other embodiments, the first nucleic acid sequence linked to the second nucleic acid 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 1 to 1791 of SEQ ID NO:5 or nucleotides 1 to 1791 of SEQ ID NO:6.

[0214] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 the 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 the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity. In other embodiments, the first nucleotide sequence comprises (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.

[0215] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 is (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:3, (iv) nucleotides 1-1791 of SEQ ID NO:4, (v) nucleotides 1-1791 of SEQ ID NO:5, (vi) nucleotides 1-1791 of SEQ ID NO:6, (vii) nucleotides 1-1791 of SEQ ID NO:7, (v) nucleotides 1-1791 of SEQ ID NO:8, (v) nucleotides 1-1791 of SEQ ID NO:9, (v) nucleotides 1-1791 of SEQ ID NO:10, (v) nucleotides 1-1791 of SEQ ID NO:11, (v) nucleotides 1-1791 of SEQ ID NO:12, (v) nucleotides 1-1791 of SEQ ID NO:13, (v) nucleotides 1-1791 of SEQ ID NO:14, (v) nucleotides 1-1791 of SEQ ID NO:15, (v) nucleotides 1-1791 of SEQ ID NO:16, (v) nucleotides 1-1791 of SEQ ID NO:17, (v) nucleotides 1-1791 of SEQ ID NO:18, (v) nucleotides 1-1791 of SEQ ID NO:19, (v) nucleotides 1-1791 of SEQ ID NO:20, (v) nucleotides 1-1791 of SEQ ID NO:21 ii) nucleotides 1 to 1791 of SEQ ID NO: 70, or (iv) having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1 to 1791 of SEQ ID NO: 71; and the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0216] In one embodiment, the first nucleotide sequence comprises (i) nucleotides 1 to 1791 of SEQ ID NO: 1, (ii) nucleotides 1 to 1791 of SEQ ID NO: 2, (iii) nucleotides 1 to 1791 of SEQ ID NO: 70, or (iv) nucleotides 1 to 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 (i) nucleotides 1792 to 4374 of SEQ ID NO: 1, (ii) nucleotides 1792 to 4374 of SEQ ID NO: 2, (iii) nucleotides 1792 to 4374 of SEQ ID NO: 70, or (iv) nucleotides 1792 to 4374 of SEQ ID NO: 71.

[0217] In a particular embodiment, the second nucleotide sequence linked to the first nucleotide sequence comprises (i) nucleotides 1792 to 4374 of SEQ ID NO:1, (ii) nucleotides 1792 to 4374 of SEQ ID NO:2, (iii) nucleotides 1792 to 4374 of SEQ ID NO:70, or (iv) nucleotides 1792 to 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 and 2320-4374 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.

[0218] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 FVIII polypeptide activity.

[0219] In a 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 comprising 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; 74, (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 the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0220] In one embodiment, the second nucleic acid 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 (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).

[0221] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having 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 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:1.

[0222] 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).

[0223] 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 58-4374 of SEQ ID NO: 1. In yet 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 1-4374 of SEQ ID NO: 1.

[0224] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having 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 has 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 2277 and 2320 to 4374 of SEQ ID NO: 2. These include nucleic acid sequences having at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0225] 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 58-4374 of SEQ ID NO: 2. In yet 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 1-4374 of SEQ ID NO: 2.

[0226] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having 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:70.

[0227] 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).

[0228] 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.

[0229] 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 58-4374 of SEQ ID NO:70. In yet 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 1-4374 of SEQ ID NO:70.

[0230] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having 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.

[0231] In other embodiments, the nucleotide sequence has at least 8 amino acids greater than or equal 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.

[0232] 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 58-4374 of SEQ ID NO:71. In yet 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 1-4374 of SEQ ID NO:71.

[0233] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having 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 58-4374 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).

[0234] 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 58-4374 of SEQ ID NO: 3. In yet 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 1-4374 of SEQ ID NO: 3.

[0235] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having 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.

[0236] 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).

[0237] 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 58-4374 of SEQ ID NO: 4. In yet 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 the nucleotides encoding the B domain or B domain fragment), or nucleotides 1-4374 of SEQ ID NO: 4.

[0238] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having 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.

[0239] 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).

[0240] 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 58-4374 of SEQ ID NO: 5.

[0241] In yet 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 1-4374 of SEQ ID NO:5.

[0242] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having 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.

[0243] In other embodiments, the nucleotide sequence is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 98% identical 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). These include nucleic acid sequences that have at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with each other.

[0244] 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.

[0245] 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 58-4374 of SEQ ID NO:6.

[0246] In yet 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 1-4374 of SEQ ID NO:6.

[0247] 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 the signal peptide is codon-optimized.

[0248] In a particular embodiment, the nucleic acid sequence encoding the 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 1-57 of SEQ ID NO:1; (ii) nucleotides 1-57 of SEQ ID NO:2; (iii) nucleotides 1-57 of SEQ ID NO:3; (iv) nucleotides 1-57 of SEQ ID NO:4; (v) nucleotides 1-57 of SEQ ID NO:5; (vi) nucleotides 1-57 of SEQ ID NO:6; (vii) nucleotides 1-57 of SEQ ID NO:70; (viii) nucleotides 1-57 of SEQ ID NO:71; or (ix) nucleotides 1-57 of SEQ ID NO:68.

[0249] SEQ ID NOS: 1-6, 70, and 71 are optimized versions of SEQ ID NOS: 16, the starting or "parent" or "wild-type" FVIII nucleotide sequence. SEQ ID NOS: 1-6, 70, and 71 encode 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 NOS: 16), it should be understood that the lentiviral gene therapy methods of the present disclosure also relate 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.

[0250] Unless otherwise specified, as used herein, a "polypeptide having FVIII activity" refers to a functional FVIII polypeptide in its normal role in coagulation. The term polypeptide having FVIII activity includes functional fragments, variants, analogs, or derivatives thereof that retain the function of full-length wild-type factor VIII in the coagulation pathway.

[0251] "Polypeptide having FVIII activity" is used interchangeably with FVIII protein, FVIII polypeptide, or FVIII. Examples of FVIII functions include, but are not limited to, the ability to activate coagulation, act as a cofactor for factor IX, or bind to Ca. 2+ and the ability to form a tenase complex with factor IX in the presence of phospholipids, which subsequently converts factor X to the activated form, Xa.

[0252] In one embodiment, the polypeptide having FVIII activity comprises two polypeptide chains, the first chain comprising a FVIII heavy chain and the second chain comprising a FVIII light chain. In another embodiment, the polypeptide having FVIII activity is a single-chain FVIII. The single-chain FVIII can contain one or more mutations or substitutions at amino acid residues 1645 and / or 1648 corresponding to the mature FVIII sequence. See International Application PCT / US2012 / 045784, which is incorporated herein by reference in its entirety. The FVIII protein can be a human, porcine, canine, rat, or murine FVIII protein. Furthermore, comparisons between human-derived FVIII and FVIII from other species have identified conserved residues likely required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632).

[0253] As used herein, the "B domain" of FVIII is identical to B domains known in the art, defined by internal amino acid sequence identity and the site of proteolytic cleavage by thrombin, e.g., residues Ser741 to Arg1648 of full-length human FVIII. Other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1 to Arg372; A2, residues Ser373 to Arg740; A3, residues Ser1690 to Ile2032; C1, residues Arg2033 to Asn2172; and C2, residues Ser2173 to Tyr2332. The A3-C1-C2 sequence includes residues Ser1690 to Tyr2332. The remaining sequence, residues Glu1649 to Arg1689, is commonly referred to as the FVIII light chain activation peptide. The locations of the boundaries for all of the domains, including the B domain, for porcine, murine, and canine FVIII are also known in the art. One example of a BDD FVIII is REFACTO® recombinant BDD FVIII (Wyeth Pharmaceuticals, Inc.).

[0254] "B domain deleted FVIII" can have complete or partial deletions as 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, 5,112,950, 4,868,112 and 6,458,563, each of which is incorporated herein by reference in its entirety. In some embodiments, the B domain deleted FVIII sequences of the present disclosure comprise any one of the deletions disclosed in column 4, line 4 to column 5, line 28 and Examples 1-5 of U.S. Pat. No. 6,316,226 (also U.S. Pat. No. 6,346,513).

[0255] In some embodiments, the B domain deleted FVIII of the present disclosure has a deletion as disclosed in column 2, lines 26-51 and Examples 5-8 of U.S. Patent No. 5,789,203 (also U.S. Patent Nos. 6,060,447, 5,595,886, and 6,228,620). In some embodiments, the B domain deleted FVIII has a deletion as disclosed in column 1, line 25 to column 2, line 40 of U.S. Patent No. 5,972,885; column 6, lines 1-22 and Example 1 of U.S. Patent No. 6,048,720; column 2, lines 17-46 of U.S. Patent No. 5,543,502; column 4, line 22 to column 5, line 36 of U.S. Patent No. 5,171,844; column 2, lines 55-68 of U.S. Patent No. 5,112,950. 2 and Example 1; column 2, line 2 to column 19, line 21 and Table 2 of U.S. Pat. No. 4,868,112; column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39 of U.S. Pat. No. 7,041,635; or column 4, lines 25-53 of U.S. Pat. No. 6,458,563.

[0256] In some embodiments, as disclosed in International Publication No. 91 / 09122, which is incorporated herein by reference in its entirety, B-domain-deleted FVIII has most of the B domain deleted but still contains the amino-terminal sequence of the B domain, which is essential for in vivo proteolytic processing of the primary translation product into two polypeptide chains. In some embodiments, B-domain-deleted FVIII is constructed with a deletion of amino acids 747-1638, i.e., a virtually complete deletion of the B domain. Hoeben R.C. et al., J. Biol. Chem. 265(13):7318-7323 (1990), which is incorporated herein by reference in its entirety. 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), which is incorporated herein by reference in its entirety.

[0257] Additional B domain deletions that are part of the present disclosure include, for example, amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986) 83, 5939-5942), 797-1562 (Eaton et al., Biochemistry (1986) 25:8343-8347), 741-1646 (Kaufman (WO 87 / 04187)), 747-1560 (Sarver et al., DNA (1987) 6:553-564), 741-1648 (Pasek (PCT Application No. 88 / 00831)), 816-1598 or 741-1689 (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 above deletions can be made in any FVIII sequence.

[0258] Several functional FVIII molecules containing B-domain deletions are disclosed in the following patents: U.S. Pat. No. 6,316,226 and U.S. Pat. No. 6,346,513, both assigned to Baxter; U.S. Pat. No. 7,041,635, assigned to In2Gen; U.S. Pat. No. 5,789,203, U.S. Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620, assigned to Chiron; U.S. Pat. No. 5,972,885 and U.S. Pat. No. 6,048,720, assigned to Biovitrum; U.S. Pat. No. 5,543,502 and U.S. Pat. No. 5,610,278, assigned to Novo Nordisk; U.S. Pat. No. 5,171,844, assigned to Immuno Ag; U.S. Pat. No. 5,112,950, assigned to Transgene SA; and U.S. Pat. No. 5,112,950, assigned to Genetics. No. 4,868,112, assigned to the Institute, each of which is incorporated herein by reference in its entirety.

[0259] A. Codon Optimization In one embodiment, the lentiviral vector of the present disclosure comprises an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleic acid sequence is codon-optimized. In another embodiment, the starting nucleic acid sequence encoding the polypeptide having FVIII activity and subjected to codon optimization is SEQ ID NO: 16. In some embodiments, the sequence encoding the polypeptide having FVIII activity is codon-optimized for human expression. In other embodiments, the sequence encoding the polypeptide having FVIII activity is codon-optimized for mouse expression. SEQ ID NOs: 1-6, 70, and 71 are codon-optimized versions of SEQ ID NO: 16 optimized for human expression.

[0260] The term "codon optimization" when referring to genes or coding regions of a nucleic acid molecule for transformation into various hosts refers to the modification of the genes or coding regions of a nucleic acid molecule to reflect the general codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization refers to the alteration of codons in a gene. Such optimization involves 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.

[0261] Deviations in the nucleotide sequence, including the codons that code for the amino acids of any polypeptide chain, allow for variation in the sequence encoding a gene. Because each codon consists of three nucleotides and the nucleotides that make up DNA are limited to four specific bases, there are 64 possible combinations of nucleotides, 61 of which code for amino acids (the remaining three codons code for signals that terminate translation). The "genetic code," which shows which codons code for which amino acids, is reproduced herein as Table 1. As a result, many amino acids are specified by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, while tryptophan and methionine are coded for by only one triplet. This degeneracy allows DNA base composition to vary widely without altering the amino acid sequence of the protein coded for by the DNA.

[0262] [Table 1]

[0263] Many organisms exhibit biases regarding the use of specific codons to encode the insertion of specific amino acids in growing peptide chains. Codon preference or codon bias, the difference in codon usage among organisms, is conferred by the synonymy of the genetic code and is well documented for many organisms. Codon bias often correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend, among other things, on the properties of the codons translated in turn and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0264] Relative codon usage frequencies have been calculated taking into account the large number of gene sequences available for various animal, plant, and microbial species. Codon usage tables are available, for example, in the "Codon Usage Database," available at www.kazusa.or.jp / codon / (accessed June 18, 2012). See Nakamura, Y. et al., Nucl. Acids Res. 28:292 (2000).

[0265] Randomly assigning codons at optimal frequencies to encode a given polypeptide sequence can be performed manually by calculating the codon frequency for each amino acid and then randomly assigning codons to the polypeptide sequence. Additionally, various algorithms and computer software programs can be used to calculate optimal sequences.

[0266] In some embodiments, the nucleic acid molecule comprises one or more properties: (a) the nucleic acid molecule, or a portion thereof, has an increased human codon compatibility index compared to SEQ ID NO: 16; (b) the nucleotide sequence, or a portion thereof, has an increased optimal codon frequency compared 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 frequency compared to SEQ ID NO: 16; (e) the nucleotide sequence, or a portion thereof, has a reduced effective number of codons compared to SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21 and 22) compared to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) compared 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 molecule contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 features of (a) through (j).

[0267] B. Codon Conformity Index In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide having FVIII activity, wherein the human codon compatibility index is increased compared to SEQ ID NO: 16. For example, the nucleotide sequence may have a human codon compatibility index of 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 compatibility index that is at least about 0.88 (88%). In other embodiments, the nucleotide sequence has a human codon compatibility index that is at least about 0.91 (91%). In other embodiments, the nucleotide sequence has a human codon compatibility index that is at least about 0.97 (97%).

[0268] In one specific embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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; 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; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the human codon adaptation index of the nucleotide sequence is increased relative to SEQ ID NO:16.

[0269] 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 0.91 (91%). In a particular embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.88 (88%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.91 (91%).

[0270] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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; 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) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:6; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the human codon adaptation index of the nucleotide sequence is increased relative to SEQ ID NO:16.

[0271] In some embodiments, the nucleotide sequence has a nucleotide sequence of 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 ( In one particular embodiment, the nucleotide sequence has a human codon compatibility index that is 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 another embodiment, the nucleotide sequence has a human codon compatibility index that is at least about 0.83 (83%). In another embodiment, the nucleotide sequence has a human codon compatibility index that is at least about 0.88 (88%).

[0272] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence has 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 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71 without any nucleotides encoding the B domain or B domain fragment); and wherein the human codon compatibility index of the nucleotide sequence is increased compared 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%).

[0273] In one particular embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.75 (75%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.83 (83%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.88 (88%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.91 (91%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.97 (97%).

[0274] In some embodiments, the isolated nucleic acid molecule has an increased frequency of optimal codon (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.

[0275] 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.

[0276] In still other embodiments, the isolated nucleic acid molecule has a reduced effective number of codons relative to SEQ ID NO: 16. In some embodiments, the isolated nucleic acid molecule has an effective number of codons that is 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.

[0277] Optimization of CG / C content In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide having 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 encoding the polypeptide having 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%.

[0278] In one particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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; the first nucleic acid sequence is at least about 80%, at least about 85%, at least about 86%, 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%, at least about 99%, at least about 99%, at least about 99%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 1109%, at least about 1111, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 1211, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO:16.

[0279] In some embodiments, the nucleotide sequence encoding a polypeptide having 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%, or at least about 58%. In a specific embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content that is at least about 58%.

[0280] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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; the second nucleic acid sequence is selected from the group consisting of: (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., SEQ ID NO:5, excluding the nucleotides encoding the B domain or B domain fragment). nucleotides 1792 to 4374 of SEQ ID NO:6), or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:6 not containing the nucleotides encoding the B domain or B domain fragment). 6%, at least about 97%, at least about 98%, or at least about 99% sequence identity; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO:16.

[0281] In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content of 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 a specific embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content of at least about 52%. In another embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content of at least about 55%. In another embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content of at least about 57%.

[0282] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence has 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 to 4374 or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71 that do not contain nucleotides encoding the B domain or B domain fragment); and the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides of SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content that is at least about 45%.

[0283] In one specific embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content that is at least about 52%. In another embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content that is at least about 55%. In another embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content that is at least about 57%. In another embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content that is at least about 58%. In yet another embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content that is at least about 60%.

[0284] "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 is calculated using the following formula:

number

[0285] The G / C content of human genes is highly heterogeneous, with some genes having a G / C content as low as 20% and others as high as 95%. In general, G / C-rich genes are more highly expressed. In fact, it has been demonstrated that increasing the G / C content of a gene can lead to increased gene expression, which is mostly due to increased transcription and much more stable mRNA levels. See Kudla et al., PLoS Biol., 4(6):e180 (2006).

[0286] D. Matrix-binding domain-like sequence In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity as described herein, wherein the nucleotide sequence contains fewer MARS / ARS sequences relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains up to 6, up to 5, up to 4, up to 3, or up to 2 MARS / ARS sequences. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains up to 1 MARS / ARS sequence. In yet other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity does not contain a MARS / ARS sequence.

[0287] In one particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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; the first nucleic acid sequence is at least about 80%, at least about 85%, identical to (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4, The nucleotide sequence has 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; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains fewer MARS / ARS sequences than SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having 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 encoding a polypeptide having FVIII activity contains at most 1 MARS / ARS sequence. In yet other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity does not contain a MARS / ARS sequence.

[0288] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising: 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 is selected from the group consisting of: (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. 6, wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence has less MARS than SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains no more than six, no more than five, no more than four, no more than three, or no more than two MARS / ARS sequences. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains no more than one MARS / ARS sequence. In still other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity does not contain a MARS / ARS sequence.

[0289] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence has 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 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71 (i.e., nucleotides 58 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71 that do not contain nucleotides encoding the B domain or B domain fragment); and the nucleotide sequence contains fewer MARS / ARS sequences relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains up to 6, up to 5, up to 4, up to 3, or up to 2 MARS / ARS sequences. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains up to 1 MARS / ARS sequence. In yet other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity does not contain a MARS / ARS sequence.

[0290] AT-rich elements in the human FVIII nucleotide sequence have been identified that share sequence similarity with the autonomously replicating sequence (ARS) and nuclear-matrix-associated region (MAR) of Saccharomyces cerevisiae (Fallux et al., Mol. Cell. Biol. 16:4264-4272 (1996)). One of these elements has been demonstrated to bind to a nuclear factor in vitro and repress expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Ibid. It is hypothesized that these sequences may be responsible for transcriptional repression of the human FVIII gene. Thus, in one embodiment, all MAR / ARS sequences are absent in the FVIII gene of the present disclosure. In the parent FVIII sequence (SEQ ID NO: 16), four MAR / ARS ATATTT sequences (SEQ ID NO: 21) and three MAR / ARS AAATAT sequences (SEQ ID NO: 22) are present. All of these sites were mutated to disrupt the MAR / ARS sequences of the optimized FVIII sequence (SEQ ID NOs: 1-6). The location of each of these elements and the corresponding nucleotide sequence in the optimized sequence are shown in Table 2 below.

[0291] [Table 2] [Table 3]

[0292] E. Destabilizing Sequences In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein encoding a polypeptide having FVIII activity, wherein the nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 destabilizing elements. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains no more than 4, no more than 3, no more than 2, or no more than 1 destabilizing element. In still other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity contains no destabilizing elements.

[0293] In one specific embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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; 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; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains fewer destabilizing elements relative to SEQ ID NO:16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 9, up to 8, up to 7, up to 6, or up to 5 destabilizing elements. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 4, up to 3, up to 2, or up to 1 destabilizing element. In yet other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity does not contain a destabilizing element.

[0294] In another embodiment, the isolated nucleic acid molecule 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. the second nucleic acid sequence comprises a nucleotide sequence comprising: (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 without the nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:6 without the nucleotides encoding the B domain or B domain fragment). the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains fewer destabilizing elements relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 9, up to 8, up to 7, up to 6, or up to 5 destabilizing elements. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 4, up to 3, up to 2, or up to 1 destabilizing element. In yet other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no destabilizing elements.

[0295] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., a sequence that does not contain nucleotides encoding a B domain or a B domain fragment). The present invention also includes 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 58-4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71; wherein the nucleotide sequence contains fewer destabilizing elements than SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 9, up to 8, up to 7, up to 6, or up to 5 destabilizing elements. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 4, up to 3, up to 2, or up to 1 destabilizing element. In yet other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity does not contain a destabilizing element.

[0296] There are ten destabilizing elements in the parent FVIII sequence (SEQ ID NO: 16): six ATTTA sequences (SEQ ID NO: 23) and four TAAAT sequences (SEQ ID NO: 24). In one embodiment, the sequences at these sites were mutated to disrupt the destabilizing elements in optimized FVIII SEQ ID NOs: 1-6, 70, and 71. The location of each of these elements and the corresponding nucleotide sequence in the optimized sequence are shown in Table 2.

[0297] F. Potential promoter binding sites In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein encoding a polypeptide having FVIII activity, wherein the nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding the polypeptide having FVIII activity comprises a nucleotide sequence described herein encoding a polypeptide having FVIII activity, wherein the nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no more than two, no more than eight, no more than seven, no more than six, or no more than five potential promoter binding sites. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no more than four, no more than three, no more than two, or no more than one potential promoter binding site. In yet other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no potential promoter binding sites.

[0298] In one particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 is at least about 80%, at least about 85%, or at least about 85% identical to (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4. %, 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; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO:16.

[0299] In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 potential promoter binding sites. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no more than 4, no more than 3, no more than 2, or no more than 1 potential promoter binding site. In still other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no potential promoter binding sites.

[0300] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 is (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 without the nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (i.e., The nucleotide 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 SEQ ID NO: 6 (nucleotides 1792 to 4374), excluding any nucleotides encoding the B domain or B domain fragment; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 potential promoter binding sites. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no more than 4, no more than 3, no more than 2, or no more than 1 potential promoter binding site. In yet another embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity does not contain a potential promoter binding site.

[0301] In another embodiment, the isolated nucleic acid molecule encodes a polypeptide having FVIII activity. a nucleotide sequence encoding the B domain or B domain fragment, 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 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58 to 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 the nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 potential promoter binding sites. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no more than 4, no more than 3, no more than 2, or no more than 1 potential promoter binding site. In still other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains no potential promoter binding sites.

[0302] TATA boxes are regulatory sequences often found in eukaryotic promoter regions. They serve as binding sites for the general transcription factor TATA-binding protein (TBP). TATA boxes usually contain the sequence TATAA (SEQ ID NO: 28) or a close variant thereof. However, a TATA box within a coding sequence can inhibit translation of the full-length protein. The wild-type BDD FVIII sequence (SEQ ID NO: 16) contains 10 potential promoter binding sites: five TATAA sequences (SEQ ID NO: 28) and five TTATA sequences (SEQ ID NO: 29). In some embodiments, at least one, at least two, at least three, or at least four promoter binding sites are eliminated in the FVIII gene of the present disclosure. In some embodiments, at least five promoter binding sites are eliminated in the FVIII gene of the present disclosure. In other embodiments, at least six, at least seven, or at least eight promoter binding sites are eliminated in the FVIII gene of the present disclosure. In one embodiment, at least nine promoter binding sites are eliminated in the FVIII gene of the present disclosure. In one particular embodiment, all promoter binding sites are eliminated in the FVIII gene of the present disclosure. The location of each potential promoter binding site and the corresponding nucleotide sequence in the optimized sequence are shown in Table 3.

[0303] G. Other Cis-Acting Negative Regulatory Regions In addition to the MAR / ARS sequences, destabilizing elements, and potential promoter sites described above, several additional potential 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 NO: 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. No.No. 31). One or more of these elements can be removed from the optimized FVIII sequence. The location of each of these sites and the corresponding nucleotide sequence in the optimized sequence are shown in Table 2.

[0304] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 comprises: (i) a sequence (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) 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 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 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) 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 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 to 1791 of SEQ ID NO:4; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, such as a splice site, a poly-T sequence, a poly-A sequence, an ARE sequence, or any combination thereof.

[0305] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 is selected from the group consisting of: (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 without the nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 without the nucleotides encoding the B domain or B domain fragment). the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, such as a splice site, a poly-T sequence, a poly-A sequence, an ARE sequence, or any combination thereof.

[0306] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, or 71, excluding the nucleotides encoding the B domain or B domain fragment). nucleotide 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 the sequence of nucleotides 58 to 4374 of SEQ ID NO:1; the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, e.g., a splice site, a poly-T sequence, a poly-A sequence, an ARE sequence, or any combination thereof.

[0307] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 is 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%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 1109%, at least about 1111, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118% relative to (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4. have 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; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence does not contain the splice site GGTGAT (SEQ ID NO: 27).

[0308] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 FVIII polypeptide activity; and the nucleotide sequence does not contain a poly-T sequence (SEQ ID NO:25).

[0309] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 FVIII polypeptide activity; and the nucleotide sequence does not contain a polyA sequence (SEQ ID NO:26).

[0310] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising 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 is at least about 80%, at least about 80%, or at least about 80% identical to (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4. 5%, 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; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence does not contain an ARE element (SEQ ID NO: 30 or SEQ ID NO: 31).

[0311] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., The nucleic acid sequence has 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 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71, without any nucleotides encoding a B domain or B domain fragment; the nucleotide sequence does not contain the splice site GGTGAT (SEQ ID NO: 27).

[0312] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides encoding a B domain or a B domain fragment). the nucleotide sequence has 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 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71, without any other sequence identity; the nucleotide sequence does not contain a poly-T sequence (SEQ ID NO: 25).

[0313] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides encoding a B domain or a B domain fragment). The present invention also includes 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 SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71 (nucleotides 58 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71, excluding the polyA sequence); the nucleotide sequence does not contain a polyA sequence (SEQ ID NO: 26).

[0314] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., a sequence that does not include nucleotides encoding a B domain or a B domain fragment). 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 58-4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71; wherein the nucleotide sequence does not contain an ARE element (SEQ ID NO: 30 or SEQ ID NO: 31).

[0315] In other embodiments, the optimized FVIII sequences of the present disclosure do not include one or more antiviral motifs, stem-loop structures, and repeat sequences.

[0316] In yet other embodiments, the nucleotides surrounding the transcription start site include the Kozak consensus sequence (GCCGCCACC ATG C (SEQ ID NO: 32), in which the underlined nucleotide is the start codon.) In other embodiments, restriction sites are added or removed to facilitate the cloning process.

[0317] H. Heterologous Nucleotide Sequences 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 to the optimized BDD-FVIII nucleotide sequence of the present disclosure at the 5' end, 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 C-terminus of the FVIII amino acid sequence encoded by the nucleotide sequence, or inserted between two amino acids of 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 sites selected from Table 3. In some embodiments, the heterologous amino acid sequence can be inserted into a FVIII polypeptide encoded by a nucleic acid molecule of the present disclosure at any site disclosed in International Publication Nos. 2013 / 123457 A1 and 2015 / 106052 A1 or U.S. Publication No. 2015 / 0158929 A1, which are incorporated by reference in their entireties.

[0318] In some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted into the B domain or a fragment thereof. In some embodiments, the heterologous amino acid sequence is inserted into FVIII immediately downstream of the 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).

[0319] [Table 4]

[0320] In other embodiments, the isolated nucleic acid molecule further comprises two, three, four, five, six, seven or eight heterologous nucleotide sequences. In some embodiments, all heterologous nucleotide sequences are identical. In some embodiments, at least one heterologous nucleotide sequence is different from other heterologous nucleotide sequences. In some embodiments, the present disclosure can comprise more than two, three, four, five, six or seven heterologous nucleotide sequences in tandem.

[0321] 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 of the FVIII molecule (a "half-life extender").

[0322] In some embodiments, the heterologous moiety, when incorporated into a protein of the present disclosure, The heterologous amino acid sequence is a peptide or polypeptide having either non-structural or structural features associated with an increased half-life in vivo. Non-limiting examples include albumin, an albumin fragment, an Fc fragment of an immunoglobulin, the C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, a HAP sequence, an XTEN sequence, transferrin or a fragment thereof, a PAS polypeptide, a polyglycine linker, a polyserine linker, an albumin binding moiety, or any fragment, derivative, variant, or combination of these polypeptides. In a specific embodiment, the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, transferrin, albumin, or a PAS sequence.

[0323] In some embodiments, the heterologous moiety comprises von Willebrand factor or a fragment thereof. In other related embodiments, the heterologous moiety may comprise 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 derivative, variant, or combination of these elements. In some embodiments, the heterologous moiety comprises a cysteine ​​amino acid that serves as an attachment site for a non-polypeptide moiety, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivative, variant, or combination of these elements.

[0324] In a specific embodiment, the first heterologous nucleotide sequence encodes a first heterologous moiety that is a half-life extender known in the art, and the second heterologous nucleotide sequence encodes a second heterologous moiety that may also be a half-life extender 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) associate with each other to form a dimer. In one embodiment, the second heterologous moiety is a second Fc moiety, where 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 can be associated with the first heterologous moiety by a covalent or non-covalent bond.

[0325] 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 1100, 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.

[0326] 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.

[0327] In certain embodiments, the heterologous moiety improves one or more pharmacokinetic properties of the FVIII protein without significantly affecting its biological activity or function.

[0328] In certain embodiments, the heterologous moiety increases the in vivo and / or in vitro half-life of the FVIII protein of the present disclosure. In other embodiments, the heterologous moiety is a FVIII protein or fragment thereof (e.g., a protein of a FVIII protein) of the present disclosure. The visualization and / or localization of the FVIII protein or fragment thereof of the present disclosure can be achieved in vivo, in vivo, or in vivo. in vitro, ex vivo, or a combination thereof.

[0329] In other embodiments, the heterologous moiety increases the stability of the FVIII protein of the present disclosure or a fragment thereof (e.g., a fragment comprising the 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 a FVIII protein in response to environmental conditions (e.g., elevated or decreased temperature). In certain aspects, the physical property is the maintenance of the covalent structure of the FVIII protein (e.g., absence of proteolysis, undesired oxidation, or deamidation). In other aspects, the physical property is also the presence of the FVIII protein in a correctly folded state (e.g., absence of soluble or insoluble aggregation or precipitation).

[0330] In one embodiment, the stability of FVIII protein is measured by assaying the biophysical properties of FVIII protein, such as temperature stability, pH unfolding profile, stable removal of glycosylation, solubility, biochemical function (e.g., ability to bind to proteins, receptors, or ligands), and / or a combination thereof. In another embodiment, biochemical function is demonstrated by the binding affinity of an interaction. In one embodiment, a measure of protein stability is thermal stability, i.e., resistance to heat stress. 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 for measuring thermal stability include, but are not limited to, differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), circular dichroism (CD), and heat stress assay.

[0331] In certain embodiments, the FVIII protein encoded by the nucleic acid molecule of the present disclosure comprises at least one half-life extender, i.e., a heterologous moiety that increases the in vivo half-life of the FVIII protein relative to the in vivo half-life of a corresponding FVIII protein lacking the heterologous moiety. The in vivo half-life of a FVIII protein can be determined by any method known to those skilled in the art, such as an activity assay (e.g., chromosomal assay or one-stage clotting aPTT assay), ELISA, ROTEM®, etc.

[0332] In some embodiments, the presence of one or more half-life extenders increases the half-life of the FVIII protein compared to the half-life of a corresponding protein lacking such one or more half-life extenders, such that the half-life of a FVIII protein comprising a half-life extender is at least about 1.5-fold, at least about 2-fold, at least about 2.5-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 11-fold, or at least about 12-fold longer than the in vivo half-life of a corresponding FVIII protein lacking such half-life extenders.

[0333] In one embodiment, the half-life of a 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 a FVIII protein comprising a half-life extender is 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, or about 2.5-fold to about 9-fold longer than the in vivo half-life of the corresponding protein lacking such half-life extender. fold, about 2.5 times to about 8 times, about 2.5 times to about 7 times, about 2.5 times to about 6 times, about 2.5 times to about 5 times, about 2.5 times to about 4 times, about 2.5 times to about 3 times, about 3 times to about 10 times, about 3 times to about 9 times, about 3 times to about 8 times, about 3 times to about 7 times, about 3 times to about 6 times, about 3 times to about 5 times, about 3 times to about 4 times, about 4 times to about 6 times, about 5 times to about 7 times, or about 6 times to about 8 times.

[0334] In other embodiments, the half-life of the FVIII protein comprising the 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.

[0335] In still other embodiments, the half-life of the FVIII protein comprising the half-life extender is from about 15 hours to about 2 weeks, from about 16 hours to about 1 week, from about 17 hours to about 1 week, from about 18 hours to about 1 week, from about 19 hours to about 1 week, from about 20 hours to about 1 week, from about 21 hours to about 1 week, from about 22 hours to about 1 week, from about 23 hours to about 1 week, from about 24 hours to about 1 week, from about 36 hours to about 1 week, from about 48 hours to about 1 week, from about 60 hours to about 1 week, from about 24 hours to about 6 days, from about 24 hours to about 5 days, from about 24 hours to about 4 days, from about 24 hours to about 3 days, or from about 24 hours to about 2 days.

[0336] 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, or 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 6 days, about 7 days (1 week), about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0337] One or more half-life extenders can be fused to the C-terminus or N-terminus of FVIII or inserted within FVIII.

[0338] 1. Immunoglobulin constant region or a part thereof In another aspect, the heterologous moiety comprises one or more immunoglobulin constant regions or portions thereof (e.g., Fc regions). In one embodiment, the isolated nucleic acid molecule of the present disclosure further comprises a heterologous nucleic acid sequence encoding an immunoglobulin constant region or portion thereof. In some embodiments, the immunoglobulin constant region or portion thereof is an Fc region.

[0339] The immunoglobulin constant region is composed of domains designated CH (constant heavy chain) domains (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA IgD, or IgE), the constant region is composed of three or four CH domains. Some isotype (e.g., IgG) constant regions also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, NY, NY.

[0340] The immunoglobulin constant region or a portion thereof for producing the FVIII protein of the present disclosure can be obtained from several different sources. In one embodiment, the immunoglobulin constant region or a portion thereof is derived from a human immunoglobulin. However, it is understood that the immunoglobulin constant region or a portion thereof can also be derived from the immunoglobulin of another mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques). Furthermore, 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 IgG1, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgG1 is used.

[0341] Various immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domain sequences can be selected that have specific effector functions (or lack specific effector functions) or with specific modifications that 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 resulting genetic material, using any of the aforementioned methods, can then be modified or synthesized to obtain the polypeptides of the present disclosure. It will be further recognized that the scope of this disclosure encompasses alleles, variants, and mutations of constant region DNA sequences.

[0342] The sequence of immunoglobulin constant region or a part thereof can be cloned, for example, by using polymerase chain reaction and primers selected to amplify the domain of interest.To clone the sequence of immunoglobulin constant region or a part thereof from antibody, mRNA can be isolated from hybridoma, spleen or lymphocyte, reverse transcribed into DNA, and the antibody gene can be amplified by PCR.PCR amplification method is described in detail in U.S. Patent No. 4,683,195; U.S. Patent No. 4,683,202; U.S. Patent No. 4,800,159; U.S. Patent No. 4,965,188; and, for example, "PCR Protocols: A Guide to Methods and Applications" edited by Innis et al., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270. PCR can be initiated with consensus constant region primers or with more specific primers based on the published heavy and light chain DNA and amino acid sequences. PCR can also be used to isolate DNA clones encoding the light and heavy chains of an antibody. In this case, libraries can be screened with consensus primers or larger homologous probes, such as mouse constant region probes. Numerous primer sets suitable for amplifying 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). Cloning of antibody sequences is further described in U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated herein by reference).

[0343] As used herein, an immunoglobulin constant region can include all domains and hinge regions or portions thereof. In one embodiment, an immunoglobulin constant region or portion thereof includes a CH2 domain, a CH3 domain, and a hinge region, i.e., an Fc region. or an FcRn binding partner.

[0344] As used herein, the term "Fc region" is defined as the portion of a polypeptide corresponding to the Fc region of a native Ig, i.e., as formed by the dimeric association of the Fc domains of each 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 composed of Fc domains genetically linked (i.e., encoded by a single contiguous gene sequence) within a single polypeptide chain. See International Patent Application Publication No. WO 2012 / 006635, which is incorporated herein by reference in its entirety.

[0345] In one embodiment, "Fc region" refers to that portion of a single Ig heavy chain beginning with the hinge region just upstream of the papain cleavage site (i.e., residue 216 of IgG, with the first residue of the heavy chain constant region being 114) and ending at the C-terminus of the antibody. Thus, a complete Fc region includes at least the hinge, CH2, and CH3 domains.

[0346] The immunoglobulin constant region or a portion thereof may be an FcRn binding partner. FcRn is active in adult epithelial tissues and is expressed in the lumen of the intestine, the lung airways, the nasal cavity surface, the vaginal surface, the colon, and the rectal surface (U.S. Patent No. 6,485,726). An FcRn binding partner is a portion of an immunoglobulin that binds to FcRn.

[0347] FcRn receptors have been isolated from several mammalian species, including humans. The sequences of 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 other immunoglobulin classes such as IgA, IgM, IgD, and IgE) at a relatively low pH, actively transports IgG transcellularly in the lumen toward the serosal membrane, and then releases IgG at the relatively high pH found in interstitial fluid. It is expressed in adult epithelial tissues, 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), and nasal epithelium, vaginal surface, and biliary surface (U.S. Patent Nos. 6,485,726, 6,030,613, 6,086,875; WO03 / 077834; US2003-0235536).

[0348] FcRn binding partners useful in the present disclosure include molecules that are specifically bound by the FcRn receptor, including whole IgG, Fc fragments of IgG, and other fragments containing 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 main contact region of the Fc with FcRn is near the junction of the CH2 and CH3 domains. All Fc-FcRn contacts are within a single Ig heavy chain. FcRn binding partners include whole IgG, Fc fragments of IgG, and other fragments of IgG containing the complete binding region of the FcRn. Major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 in the CH2 domain, and amino acid residues 385-387, 428, and 433-436 in the CH3 domain. All references to the amino acid numbering of immunoglobulins or immunoglobulin fragments or regions are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, US Department of Public Health, Bethesda, Md.

[0349] Fc regions or FcRn-binding partners bound to FcRn can be efficiently transported across epithelial barriers by FcRn, providing a non-invasive means for systemic administration of desired therapeutic molecules. Furthermore, fusion proteins containing Fc regions or FcRn-binding partners are phagocytosed by cells expressing FcRn. However, these fusion proteins are not subject to degradation but are recycled and re-enter the blood circulation, thereby increasing the in vivo half-life of these proteins. In certain embodiments, the portion of the immunoglobulin constant region is an Fc region or FcRn-binding partner that typically associates with another Fc region or another FcRn-binding partner via disulfide bonds and other nonspecific interactions to form dimers and higher-order multimers.

[0350] Two FcRn receptors can bind to a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds to a single polypeptide of an Fc homodimer. In one embodiment, an FcRn binding partner, e.g., an Fc fragment of IgG, is linked to a biologically active molecule, providing a means for oral, buccal, sublingual, rectal, vaginal, nasal aerosol, or pulmonary delivery, or topical ocular delivery of the biologically active molecule. In another embodiment, the FVIII factor protein can be administered invasively, e.g., subcutaneously or intravenously.

[0351] An FcRn binding partner region is a molecule or moiety that specifically binds to the FcRn receptor, thereby allowing active transport of the Fc region by the FcRn receptor. Specific binding refers to two molecules forming a complex that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and low to moderate capacity, as distinguished from nonspecific binding, which typically has low affinity and moderate to high capacity. Typically, the affinity constant KA is greater than 10. 6 M -1 More than or equal to 10 8 M -1 Binding is considered specific when the binding affinity exceeds 0.05. If necessary, non-specific binding can be reduced by changing the binding conditions without substantially affecting the specific binding affinity. Those skilled in the art can optimize appropriate binding conditions, such as the concentration of the molecule, the ionic strength of the solution, temperature, binding time, and the concentration of the blocking agent (e.g., serum albumin, milk casein), using routine techniques.

[0352] In certain embodiments, the FVIII protein encoded by the nucleic acid molecule of the present disclosure comprises one or more truncated Fc regions that, despite being truncated, are sufficient to confer Fc receptor (FcR) binding properties to the Fc region. For example, the portion of the Fc region that binds to FcRn (i.e., the FcRn-binding portion) comprises amino acids approximately 282-438 of IgG1, according to EU numbering (major contact sites are amino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain). Thus, the Fc region of the present disclosure may comprise or consist of an FcRn-binding portion. The FcRn-binding portion may be derived from a heavy chain of any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, an FcRn-binding portion derived from an antibody of human isotype IgG1 is used. In another embodiment, an FcRn-binding portion derived from an antibody of human isotype IgG4 is used.

[0353] The Fc region can be obtained from several different sources. In one embodiment, the Fc region of the polypeptide is derived from a human immunoglobulin. However, the Fc portion may be derived from the immunoglobulin of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Furthermore, polypeptides of the Fc domain or portions thereof may be derived from immunoglobulins of IgM, IgG, IgM ... It may be from any immunoglobulin class, including IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In another embodiment, the human isotype IgG1 is used.

[0354] In certain embodiments, the Fc variant provides an alteration in at least one effector function conferred by the Fc portion comprising the wild-type Fc domain (e.g., an improved or decreased ability of the Fc region to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or to induce antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine ​​residue.

[0355] The Fc regions of the present disclosure may employ art-recognized Fc variants known to alter (e.g., enhance or decrease) effector function and / or FcR or FcRn binding.Specifically, the Fc regions of the present disclosure may be those described in, for example, International PCT Application Publication Nos. WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, and WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 0 Nos. 47350A2 and WO06 / 085967A2; U.S. Patent Application Publication Nos. US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US2007 / 0248603, US2007 / 0286859, US2008 / 0057056; or U.S. Patent Nos. 5,648,260, 5,739,277, 5,834, The present invention can include modifications (e.g., substitutions) at one or more of the amino acid positions disclosed in US Pat. Nos. 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.In one embodiment, a specific change (e.g., a specific substitution of one or more amino acids disclosed in the art) is made at one or more of the disclosed amino acid positions, while in another embodiment, a different change (e.g., a different substitution of one or more amino acid positions disclosed in the art) is made at one or more of the disclosed amino acid positions.

[0356] The Fc region of an IgG or an FcRn binding partner can be modified using well-recognized procedures, such as site-directed mutagenesis, to obtain modified IgG or Fc fragments or portions thereof that will bind FcRn. Such modifications include modifications at sites distant from the FcRn contact site as well as modifications within the contact site that retain or even enhance binding to FcRn. For example, the following single amino acid in human IgG1 Fc (Fcγ1) can be modified without significant loss of Fc binding affinity to FcRn: Acid residues can be substituted: 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, R 292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, 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, N361 A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y39 IF, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A (e.g., P238A represents a substitution of alanine for the wild-type proline at position 238). By way of example, in specific embodiments, an N297A mutation is incorporated to remove a highly conserved N-glycosylation site. In addition to alanine, other amino acids can be substituted for the wild-type amino acids at the above-identified positions. Mutations can be individually incorporated into an Fc, resulting in over 100 Fc regions that differ from the native Fc. Furthermore, combinations of two, three, or more of these individual mutations can be incorporated together, resulting in hundreds of additional Fc regions.

[0357] Certain of the above mutations can confer new functions to the Fc region or FcRn-binding partner. For example, in one embodiment, N297A is incorporated to remove a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby increasing the circulating half-life of the Fc region and rendering the Fc region unable to bind to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA 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 functions resulting from the above mutations, in some cases, affinity for FcRn can be increased compared to wild-type affinity. 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 that are thought to increase affinity for FcRn include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0358] Furthermore, at least three human Fc gamma receptors are thought to recognize a binding site on IgG within the downstream hinge region, generally amino acids 234-237. Therefore, another example of new function and potentially reduced immunogenicity could result from mutation of this region, for example, by substituting amino acids 233-236 "ELLG" (SEQ ID NO: 45) of human IgG1 with the corresponding sequence "PVA" (one amino acid deletion) of IgG2. When such mutations are introduced, FcγRI, FcγRII, and FcγRIII, which mediate various effector functions, have been shown to abolish binding to IgG1. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999 , Eur. J. Immunol. 29:2613.

[0359] In another embodiment, the immunoglobulin constant region or portion thereof comprises an amino acid sequence in the hinge region or portion thereof that forms one or more disulfide bonds with a second immunoglobulin constant region or portion thereof. The second immunoglobulin constant region or portion thereof can be linked to a second polypeptide to integrate the FVIII protein with the second polypeptide. In some embodiments, the second polypeptide is an enhancer moiety. As used herein, the term "enhancer moiety" refers to a molecule, fragment thereof, or polypeptide component that can enhance the procoagulant 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 the activity of FVIII.

[0360] In certain embodiments, the FVIII protein encoded by the nucleic acid molecule of the present disclosure comprises amino acid substitutions to an immunoglobulin constant region or portion thereof (e.g., an Fc variant) that alter the antigen-dependent effector function of the Ig constant region, particularly the circulating half-life of the protein.

[0361] 2.scFc region In another aspect, the heterologous moiety comprises an scFc (single-chain Fc) region. In one embodiment, the isolated nucleic acid molecule of the present disclosure further comprises a heterologous nucleic acid sequence encoding an ScFc region. The scFc region comprises at least two immunoglobulin constant regions or portions thereof (e.g., Fc moieties or Fc domains (e.g., 2, 3, 4, 5, 6, or more Fc moieties or domains)) that are capable of folding (e.g., intramolecularly or intermolecularly) within the same linear polypeptide chain to form a functional scFc region 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., FcRn, an FcγR receptor (e.g., FcγRIII), or a complement protein (e.g., C1q)) for purposes of improving half-life or eliciting immune effector function (e.g., antibod...

Claims

1. 1. A method of treating a bleeding disorder in a subject in need thereof, comprising administering 5 x 10 mAbs of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity. １０ administering to the subject at least one dose of the following transducing units / kg (TU / kg): (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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 4374 of SEQ ID NO:6; or (ix) Any combination of (i) to (viii) The method comprising:

2. 1. A method of treating a bleeding disorder in a subject in need thereof, comprising administering to a subject in need thereof 5×10 6 humanized cells of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence comprising 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 the FVIII polypeptide. １０ administering to the subject at least one dose of the following transducing units / kg (TU / kg): (a) the first nucleic acid sequence (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 to 2277 and 2320 to 1791 of SEQ ID NO:3; (ii) at least 85%, at least 90%, at least 95%, at least 96%, at least 100%, at least 110% relative to nucleotides 58 to 2277 and 2320 to 1791 of SEQ ID NO:4 97%, at least 98% or at least 99% sequence identity; (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 to 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 to 1791 of SEQ ID NO:

6. having (b) the second nucleic acid sequence is (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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 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 to 2277 and 2320 to 4374 of SEQ ID NO:

6. or (c) any combination of (a) and (b); The method, wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

3. The dosage is about 5×10 １０ TU / kg, about 4.5×10 １０ TU / kg, about 4×10 １０ TU / kg, about 3.5×10 １０ TU / kg, about 3×10 １０ TU / kg, about 2.5×10 １０ TU / kg, about 2×10 １０ TU / kg, about 1.5×10 １０ TU / kg, about 1×10 １０ TU / kg, about 9.5×10 ９ TU / kg, about 9×10 ９ TU / kg, about 8.5×10 ９ TU / kg, about 8×10 ９ TU / kg, about 7.5×10 ９ TU / kg, about 7×10 ９ TU / kg, about 6.5×10 ９ TU / kg, about 6×10 ９ TU / kg, about 5.5×10 ９ TU / kg, about 5×10 ９ TU / kg, about 4.5×(same number as the original)10 ９ TU / kg, about 4×10 ９ TU / kg, about 3.5×10 ９ TU / kg, about 3×10 ９ TU / kg, about 2.5×10 ９ TU / kg, about 2×10 ９ TU / kg, about 1.5×10 ９ TU / kg, about 1×10 ９ TU / kg, about 9.5×10 ８ TU / kg, about 9×10 ８ TU / kg, about 8.5×10 ８ TU / kg, about 8×10 ８ TU / kg, about 7.5×10 ８ TU / kg, about 7×10 ８ TU / kg, about 6.5×10<(same number as the original) ８ TU / kg, about 6×10 ８ TU / kg, about 5.5×10 ８ TU / kg, about 5×10 ８ TU / kg, about 4.5×10 ８ TU / kg, about 4×10 ８ TU / kg, about 3.5×10<00(This seems to be an incomplete tag, but keeping as is) ８ TU / kg, approximately 3×10 ８ TU / kg, approximately 2.5×10 ８ TU / kg, approximately 2×10 ８ TU / kg, approximately 1.5×10 ８ TU / kg or approximately 1 x 10 ８ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

4. The dose is approximately 5 x 10 １０ Less than TU / kg, approximately 4.5 x 10 １０ Less than TU / kg, approximately 4 x 10 １０ Less than TU / kg, approximately 3.5 x 10 １０ Less than TU / kg, approximately 3 x 10 １０ Less than TU / kg, approximately 2.5 x 10 １０ Less than TU / kg, approximately 2 x 10 １０ Less than TU / kg, approximately 1.5 x 10 １０ Less than TU / kg, approximately 1 x 10 １０ Less than TU / kg, approximately 9.5 x 10 ９ Less than TU / kg, approximately 9 x 10 ９ Less than TU / kg, approximately 8.5 x 10 ９ Less than TU / kg, approximately 8 x 10 ９ Less than TU / kg, approximately 7.5 x 10 ９ Less than TU / kg, approximately 7 x 10 ９ Less than TU / kg, approximately 6.5 x 10 ９ Less than TU / kg, approximately 6 x 10 ９ Less than TU / kg, approximately 5.5 x 10 ９ Less than TU / kg, approximately 5 x 10 ９ Less than TU / kg, approximately 4.5 x 10 ９ Less than TU / kg, approximately 4 x 10 ９ Less than TU / kg, approximately 3.5 x 10 ９ Less than TU / kg, approximately 3 x 10 ９ Less than TU / kg, approximately 2.5 x 10 ９ Less than TU / kg, approximately 2 x 10 ９ Less than TU / kg, approximately 1.5 x 10 ９ Less than TU / kg, approximately 1 x 10 ９ Less than TU / kg, approximately 9.5 x 10 ８ Less than TU / kg, approximately 9 x 10 ８ Less than TU / kg, approximately 8.5 x 10 ８ Less than TU / kg, approximately 8 x 10 ８ Less than TU / kg, approximately 7.5 x 10 ８ Less than TU / kg, approximately 7 x 10 ８ Less than TU / kg, approximately 6.5 x 10 ８ Less than TU / kg, approximately 6 x 10 ８ Less than TU / kg, approximately 5.5 x 10 ８ Less than TU / kg, approximately 5 x 10 ８ Less than TU / kg, approximately 4.5 x 10 ８ Less than TU / kg, approximately 4 x 10 ８ Less than TU / kg, approximately 3.5 x 10 ８ Less than TU / kg, approximately 3 x 10 ８ Less than TU / kg, approximately 2.5 x 10 ８ Less than TU / kg, approximately 2 x 10 ８ Less than TU / kg, approximately 1.5 x 10 ８ Less than or about 1 x 10 TU / kg ８ The method of claim 1 or 2, wherein the IL-10 is less than TU / kg.

5. The dosage is 1×10 ８ to 5×10 １０ TU / kg, 1×10 ８ to 5×10 ９ TU / kg, 1×10 ８ to 1×10 ９ TU / kg, 1×10 ８ to 1×10 １０ TU / kg, 1×10 ９ to 5×10 １０ TU / kg, 2×10 ９ to 5×10 １０ TU / kg, 3×10 ９ to 5×10 １０ TU / kg, 4×10 ９ to 5×10 １０ TU / kg, 5×10 ９ to 5×10 １０ TU / kg, 6×10 ９ to 5×10 １０ TU / kg, 7×10 ９ to 5×10 １０ TU / kg, 8×10 ９ to 5×10 １０ TU / kg, 9×10 ９ to 5×10 １０ TU / kg, 10 １０ to 5×10 １０ TU / kg, 1.5×10 １０ to 5×10 １０ TU / kg,​​​​​​​​​​​​​​​​​​​​​​​​

6. The dose is 1 x 10 ９ ~5 x 10 １０ TU / kg, 1 x 10 ９ ~4.5 x 10 １０ TU / kg, 1 x 10 ９ ~4 x 10 １０ TU / kg, 1 x 10 ９ ~3.5 x 10 １０ TU / kg, 1 x 10 ９ ~3 x 10 １０ TU / kg, 1 x 10 ９ ~2.5 x 10 １０ TU / kg, 1 x 10 ９ ~2 x 10 １０ TU / kg, 1 x 10 ９ ~1.5 x 10 １０ TU / kg, 1 x 10 ９ ~1 x 10 １０ TU / kg, 1 x 10 ９ ~9 x 10 ９ TU / kg, 1 x 10 ９ ~8 x 10 ９ TU / kg, 1 x 10 ９ ~7 x 10 ９ TU / kg, 1 x 10 ９ ~6 x 10 ９ TU / kg, 1 x 10 ９ ~5 x 10 ９ TU / kg, 1 x 10 ９ ~4 x 10 ９ TU / kg, 1 x 10 ９ ~3 x 10 ９ TU / kg, and 1 × 10 ９ ~2 x 10 ９ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

7. The dose is 1 x 10 １０ ~2 x 10 １０ TU / kg, 1.1×10 １０ ~1.9 x 10 １０ TU / kg, 1.2×10 １０ ~1.8 x 10 １０ TU / kg, 1.3×10 １０ ~1.7 × 10 １０ TU / kg, or 1.4 x 10 １０ ~1.6 x 10 １０ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

8. The dose is approximately 1.5 x 10 １０ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

9. The dose is approximately 1.0 x 10 ９ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

10. The dose is approximately 3.0 x 10 ９ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

11. The dose is approximately 6.0 x 10 ９ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

12. The dose is approximately 1 x 10 ８ TU / kg, approximately 8.3×10 ８ TU / kg, approximately 1.5×10 ９ T U / kg, approximately 4.5 x 10 ９ TU / kg, or approximately 1.3 x 10 １０ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

13. The dose is 2.5 x 10 ９ TU / kg~3.5×10 ９ TU / kg, 2.6×10 ９ TU / kg~3.4×10 ９ TU / kg, 2.7×10 ９ TU / kg~3.3×10 ９ TU / kg, 2.8×10 ９ TU / kg~3.2×10 ９ TU / kg, or 2.9 x 10 ９ TU / kg~3.1×10 ９ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

14. The dose is 5.5 x 10 ９ TU / kg~6.5×10 ９ TU / kg, 5.6×10 ９ TU / kg~6.4×10 ９ TU / kg, 5.7×10 ９ TU / kg~6.3×10 ９ TU / kg, 5.8×10 ９ TU / kg~6.2×10 ９ TU / kg, or 5.9 x 10 ９ TU / kg~6.1×10 ９ 3. The method of claim 1 or 2, wherein the saturation is TU / kg.

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

16.

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 11-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 110-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 in a single dose or in 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 having FVIII activity in target liver cells.

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

24. 24. The method of claim 22 or 23, wherein the target liver cells are hepatocytes.

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

26. 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. 27. 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 lentiviral 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 administration of the lentiviral vector is repeated at least twice.

31. The nucleotide sequence encoding the polypeptide having FVIII activity further comprises a nucleic acid sequence encoding a signal peptide, the nucleic acid sequence encoding the signal peptide comprising: (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 1-57 of SEQ ID NO: 71; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68 31. The method of any one of claims 1 to 30, wherein the 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%, at least 99% or 100% sequence identity to

32. The nucleotide sequence encoding the polypeptide having FVIII activity is (a) the human codon compatibility index of the nucleic acid molecule or portion thereof is increased compared to SEQ ID NO: 16; (b) the optimal codon frequency of said nucleotide sequence or portion thereof is increased compared to SEQ ID NO: 16; (c) the nucleotide sequence or 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 frequency of said nucleotide sequence or portion thereof is increased compared to SEQ ID NO: 16; (e) the effective number of codons of said nucleotide sequence or portion thereof is reduced compared to SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs:21 and 22) compared to SEQ ID NO:16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) compared to SEQ ID NO: 16; and (h) any combination thereof 32. The method of any one of claims 1 to 31, comprising one or more properties selected from the group consisting of:

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

34. 34. The method of claim 33, wherein the heterologous amino acid sequence is an immunoglobulin constant region or portion thereof, an 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 C-terminus of the amino acid sequence encoded by a nucleotide sequence encoding a polypeptide having FVIII activity, or is inserted between two amino acids in the amino acid sequence encoded by a nucleotide sequence of one or more insertion sites selected from Table 3.

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

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

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

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

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

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

42. 42. The method of any one of claims 37 to 41, wherein the lipid coat comprises a high concentration of CD47 polypeptide and is free of 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. 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. Host cells are CD47 ｈｉｇｈ / MHC-I － The method according to any one of claims 43 to 45, wherein

47. Host cells are CD47 ｈｉｇｈ / MHC-I － The method according to any one of claims 43 to 46, wherein the cells are HEK293T cells.

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