Lentiviral Vectors and Their Uses in the Treatment, Prevention, or Improvement of a Bleeding Disorder

Description

[0001] This application claims priority over Applications of Provisional Patents serial numbers US 62 / 625,145, filed February 1, 2018, US 62 / 671,915, filed May 15, 2018, and US 62 / 793,158, filed January 16, 2019, the descriptions of which are incorporated herein by reference. REFERENCE TO THE LIST OF SEQUENCES SENT ELECTRONICALLY

[0002] The content of the sequence listing sent electronically in the ASCII text file (Name: 609628_SA9_460PC_Sequence_Listing.txt; size: 204,203 bytes and creation date: January 31, 2019) is incorporated here by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] The blood coagulation pathway involves, in part, the formation of an enzymatic complex of Factor VIIIa (FVIIIa) and Factor IXa (FIXa) (Xase complex) on the surface of platelets. FIXa is a serine protease with relatively weak catalytic activity without its cofactor FVIIIa. The Xase complex cleaves Factor X (FX) into Factor Xa (FXa), which, in turn, interacts with Factor Va (FVa) to cleave prothrombin and generate thrombin. Hemophilia A is a bleeding disorder caused by mutations and / or deletions in the FVIII gene (FVIII), resulting in a deficiency of FVIII activity (Peyvandi et al. 2006). In some cases, patients have reduced levels of FVIII due to the presence of FVIII inhibitors, such as anti-FVIII antibodies. Petition 870260067982, dated 09 / 07 / 2026, page 21 / 275 2 / 233

[0004] The disease can be treated by means of replacement therapy that aims to restore FVIII activity to prevent spontaneous bleeding. There are plasma-derived and recombinant FVIII products available to treat bleeding episodes on demand or to prevent bleeding episodes from occurring through prophylactic treatment. Based on the half-life of these products (10-12 hours) (White GC et al., Thromb. Haemost. 77: 660-7 (1997); Morfini, M., Haemophilia 9 (suppl. 1): 94-99; Discussion 100 (2003)), treatment regimens require frequent intravenous administration, usually two to three times a week for prophylaxis and one to three times a day for on-demand treatment (Manco-Johnson, MJ et al., N. Engl. J. Med. 357: 535-544 (2007)). This frequent administration is inconvenient and expensive.

[0005] A major impediment in the supply of a protein The low-cost recombinant FVIII for patients is hampered by the high cost of commercial production. The FVIII protein is weakly expressed in heterologous expression systems, two to three orders of magnitude smaller than similarly sized proteins (Lynch et al., Hum. Gene. Ther.; 4: 259-72 (1993). 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 B domain of FVIII is dispensable for FVIII cofactor activity. Elimination of the B domain resulted in a 17-fold increase in mRNA levels relative to full-length wild-type FVIII and a 30% increase in secreted protein (Toole et al., Proc. Natl. Acad. Sci. USA 83: 5939-42 (1986)). SUMMARY OF THE INVENTION

[0006] The present invention provides methods for treating a Petition 870260067982, dated 09 / 07 / 2026, p. 22 / 275 3 / 233 bleeding disorder in an individual requiring the same, comprising administering to the individual at least one dose of 5 x 1010 TU / kg of transducer units / kg (TU / kg) or less (e.g., 5 x 109 or less or 108 TU / kg or less) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has (i) at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1; (ii) at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2;(iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70; (iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 23204374 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 with nucleotides 58-2277 and 23204374 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 with the; Petition 870260067982, dated 09 / 07 / 2026, page 23 / 275 4 / 233 nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4; (vii) at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5; (viii) at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 23204374 of SEQ ID NO: 6;or (ix) or any combination of (i) to (viii).

[0007] The present invention also provides methods of treating a bleeding disorder in an individual in need thereof comprising administering to the individual at least one dose of 5 x 1010TU / kg or less (for example, 5 x 109 or less or 108TU / kg or less) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a Factor VIII (FVIII) polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; (a) wherein the first nucleic acid sequence has: (i) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with 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 with nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 4; (iii) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with; Petition 870260067982, dated 09 / 07 / 2026, page 24 / 275 5 / 233 the nucleotides 58-1791 of SEQ ID NO: 5; or (iv) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the nucleotides 58-1791 of SEQ ID NO: 6; (b) wherein the second nucleotide sequence has: (i) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 3; (ii) at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 4;(iii) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5; or (iv) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6; or (c) any combination of (a) and (b);and in which the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0008] In some embodiments of the methods described above, the dose is approximately 9.5 x 10⁸TU / kg, approximately 9 x 10⁸TU / kg, approximately 8.5 x 10⁸TU / kg, approximately 8 x 10⁸TU / kg, approximately 7.5 x 10⁸TU / kg, approximately 7 x 10⁸TU / kg, approximately 6.5 x 10⁸TU / kg, approximately 6 x 10⁸TU / kg, approximately 5.5 x 10⁸TU / kg, approximately 5 x 10⁸TU / kg, approximately 4.5 x 10⁸TU / kg, approximately 4 x 10⁸TU / kg, approximately 3.5 x 10⁸TU / kg, approximately 3 x 10⁸TU / kg, approximately 2.5 x 10⁸TU / kg, approximately 2 x 10⁸TU / kg, approximately 1.5 x 10⁸TU / kg or approximately 1 x 10⁸TU / kg, approximately 5 x 10¹⁰TU / kg, approximately 4.5 x 10¹⁰TU / kg, approximately 4 x 10¹⁰TU / kg, approximately 3.5 x 10¹⁰TU / kg, approximately; Petition 870260067982, dated 09 / 07 / 2026, page 25 / 275 6 / 233 of 3 x 1010TU / kg, approximately 2.5 x 1010TU / kg, approximately 2 x 1010TU / kg, approximately 1.5 x 1010TU / kg, approximately 1 x 1010TU / kg, approximately 9.5 x 109TU / kg, approximately 9 x 109TU / kg, approximately 8.5 x 109TU / kg, approximately 8 x 109TU / kg, approximately 7.5 x 109TU / kg, approximately 7 x 109TU / kg, approximately 6.5 x 109TU / kg, approximately 6 x 109TU / kg, approximately 5.5 x 109TU / kg, approximately 5 x 109TU / kg, approximately 4.5 x 109TU / kg, approximately 4 x 109TU / kg, approximately 3.5 x 109TU / kg, approximately 3 x 109TU / kg, approximately 2.5 x 109TU / kg, approximately 2 x 109TU / kg, approximately 1.5 x 109TU / kg, or approximately 1 x 109TU / kg.

[0009] In some modalities, the dose is less than approximately 9.5 x 10⁸TU / kg, less than approximately 9 x 10⁸TU / kg, less than approximately 8.5 x 10⁸TU / kg, less than approximately 8 x 10⁸TU / kg, less than approximately 7.5 x 10⁸TU / kg, less than approximately 7 x 10⁸TU / kg, less than approximately 6.5 x 10⁸TU / kg, less than approximately 6 x 10⁸TU / kg, less than approximately 5.5 x 10⁸TU / kg, less than approximately 5 x 10⁸TU / kg, less than approximately 4.5 x 10⁸TU / kg, less than approximately 4 x 10⁸TU / kg, less than approximately 3.5 x 10⁸TU / kg, less than approximately 3 x 10⁸TU / kg, less than approximately 2.5 x 108TU / kg, less than about 2 x 108TU / kg, less than about 1.5 x 108TU / kg or less than about 1 x 108TU / kg, less than about 5 x 1010TU / kg, less than about 4.5 x 1010TU / kg, less than about 4 x 1010TU / kg, less than about 3.5 x 1010TU / kg, less than about x 1010TU / kg, less than about 2.5 x 1010TU / kg, less than about x 1010TU / kg, less than about 1.5 x 1010TU / kg, less than about x 1010TU / kg, less than about 9.5 x 109TU / kg, less than about x 109TU / kg,less than approximately 8.5 x 10⁹TU / kg, less than approximately x 10⁹TU / kg, less than approximately 7.5 x 10⁹TU / kg, less than approximately x 10⁹TU / kg, less than approximately 6.5 x 10⁹TU / kg, less than approximately x 10⁹TU / kg, less than approximately 5.5 x 10⁹TU / kg, less than approximately x 10⁹TU / kg, less than approximately 4.5 x 10⁹TU / kg, less than approximately x 10⁹TU / kg, less than approximately 3.5 x 10⁹TU / kg, less than approximately Petition 870260067982, dated 09 / 07 / 2026, page 26 / 275 7 / 233 x 109TU / kg, less than about 2.5 x 109TU / kg, less than about 2 x 109TU / kg, less than about 1.5 x 109TU / kg or less than about 1 x 109TU / kg.

[0010] In some forms, the dose is between 1 x 108 and 5 x 1010TU / kg, entre 1 x 108 e 5 x 109TU / kg, entre 1 x 108 e 1 x 109TU / kg, entre 1 x 108 e 1 x 1010TU / kg, entre 1 x 109e 5 x 1010TU / kg, entre 2 x 109e 5 x 1010TU / kg, entre 3 x 109e 5 x 1010TU / kg, entre 4 x 109e 5 x 1010TU / kg, entre 5 x 109e 5 x 1010TU / kg, entre 6 x 109e 5 x 1010TU / kg, entre 7 x 109e 5 x 1010TU / kg, 8 x 109e 5 x 1010TU / kg, entre 9 x 109e 5 x 1010TU / kg, entre 1010e 5 x 1010TU / kg, entre 1,5 x 1010e 5 x 1010TU / kg, entre 2 x 1010e 5 x 1010TU / kg, entre 2,5 x 1010e 5 x 1010TU / kg, entre 3 x 1010e 5 x 1010TU / kg, entre 3,5 x 1010e 5 x 1010TU / kg, entre 4 x 1010e 5 x 1010TU / kg ou entre 4,5 x 1010e 5 x 1010TU / kg.In some modalities, the dose ranges from 1 x 10⁹ to 5 x 10¹⁰TU / kg, from 1 x 10⁹ to 4.5 x 10¹⁰TU / kg, from 1 x 10⁹ to 4 x 10¹⁰TU / kg, from 1 x 10⁹ to 3.5 x 10¹⁰TU / kg, from 1 x 10⁹ to 3 x 10¹⁰TU / kg, from 1 x 10⁹ to 2.5 x 10¹⁰TU / kg, from 1 x 10⁹ to 2 x 10¹⁰TU / kg, from 1 x 10⁹ to 1.5 x 10¹⁰TU / kg, from 1 x 10⁹ to 10¹⁰TU / kg, from 1 x 10⁹ to 9 x 10⁹TU / kg, from 1 x 10⁹ to 8 x 10⁹TU / kg, from 1 x 109e 7 x 109TU / kg, between 1 x 109e 6 x 109TU / kg, between 1 x 109e 5 x 109TU / kg, between 1 x 109e 4 x 109TU / kg, between 1 x 109e 3 x 109TU / kg and between 1 x 109e 2 x 109. In some modalities, the dose is between 1 x 1010e 2 x 1010TU / kg, between 1.1 x 1010e 1.9 x 1010TU / kg, between 1.2 x 1010e 1.8 x 1010TU / kg, between 1.3 x 1010e 1.7 x 1010TU / kg or between 1.4 x 1010e 1.6 x 1010TU / kg. In some modalities, the dose is approximately 1.5 x 1010TU / kg. In some modalities, the dose is 1.5 x 109TU / kg.In some modalities, the dose is between 2.5 x 10⁹TU / kg and 3.5 x 10⁹TU / kg, between 2.6 x 10⁹TU / kg and 3.4 x 10⁹TU / kg, between 2.7 x 10⁹TU / kg and 3.3 x 10⁹TU / kg, between 2.8 x 10⁹TU / kg and 3.2 x 10⁹TU / kg, or between 2.9 x 10⁹TU / kg and 3.1 x 10⁹TU / kg. In some modalities, the dose is approximately 3.0 x 10⁹TU / kg. Petition 870260067982, dated 09 / 07 / 2026, page 27 / 275 8 / 233 In some modalities, the dose is between 5.5 x 109TU / kg and 6.5 x 109TU / kg, between 5.6 x 109TU / kg and 6.4 x 109TU / kg, between 5.7 x 109TU / kg and 6.3 x 109TU / kg, between 5.8 x 109TU / kg and 6.2 x 109TU / kg, or between 5.9 x 109TU / kg and 6.1 x 109TU / kg. In some modalities, the dose is approximately 6.0 x 109TU / kg.

[0011] In some embodiments of the methods described above, the plasma activity of FVIII 24 to 48 hours after administration of the lentiviral vector is increased relative to an individual who received a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16. In some embodiments, the plasma activity of FVIII is increased by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 50 times, at least about 60 times,at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, or at least about 200 times.

[0012] In some embodiments of the methods described above, the lentiviral vector is administered as a single dose or multiple doses. In some embodiments, the lentiviral vector is delivered via Petition 870260067982, dated 09 / 07 / 2026, p. 28 / 275 9 / 233 Intravenous injection. In some modalities, the individual is a pediatric individual. In some modalities, the individual is an adult individual.

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

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

[0015] In some embodiments, the lentiviral vector dose is administered at once or divided into two subdoses, three subdoses, four subdoses, five subdoses, or six subdoses. In some embodiments, the lentiviral vector dose is repeated at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity further comprises a nucleic acid sequence encoding a signaling peptide, wherein the nucleic acid sequence encoding a signaling peptide has at least 60%, at least 70%, at least 80%, at least 90% Petition 870260067982, dated 09 / 07 / 2026, p. 29 / 275 10 / 233%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with: (i) nucleotides 1 to 57 of SEQ ID NO: 1; (ii) nucleotides 1 to 57 of SEQ ID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ ID NO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) nucleotides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.

[0016] In some embodiments, the nucleic acid molecule (or the nucleotide sequence encoding a polypeptide with FVIII activity) comprises one or more selected properties from the group consisting of: (a) the human codon matching index of the nucleic acid molecule or a portion thereof is increased relative to SEQ ID NO: 16; (b) the ideal codon frequency of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16; (d) the relative equal codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (e) the effective number of codons in the nucleotide sequence or a portion thereof is reduced in relation 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.

[0017] In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity further comprises a heterologous nucleotide sequence encoding a sequence of Petition 870260067982, dated 09 / 07 / 2026, p. 30 / 275 11 / 233 heterologous amino acids (e.g., a half-life extender). In some embodiments, the heterologous amino acid sequence is a constant region of immunoglobulin or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence. In some embodiments, the heterologous amino acid sequence is attached to the N-terminal or C-terminal 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 a full-length FVIII or an FVIII with a deleted B domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1A-1J provide the nucleotide sequences with optimized codons encoding Factor VIII with the B domain deleted. Figure 1A shows the nucleotide sequence of coFVIII3 (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 nucleotide and amino acid sequences without optimized codons, respectively, of FVIII with the B domain deleted (BDD-FVIII) (SEQ ID NOs: 16 and 17, respectively).

[0019] Figures 2A-2J show adjustments in the codon usage trend in nucleotide sequences with optimized codons encoding BDD-FVIII. Figure 2A shows the relative frequency of Petition 870260067982, dated 09 / 07 / 2026, p. 31 / 275 12 / 233 codons in the wild-type nucleotide sequence (before codon optimization) encoding BDD-FVIII, for example, unoptimized BDD-FVIII. The human codon adaptation index (CAI) of the unoptimized BDD-FVIII sequence is 74%. Figure 2B shows the relative frequency of codons in the variant sequence coFVIII-1, which has a human CAI of 88%. Figure 2C shows the relative frequency of codons in the variant sequence coFVIII-3, which has a human CAI of 91%. Figure 2D shows the relative frequency of codons in the variant sequence coFVIII-4, which has a human CAI of 97%. Figure 2E shows the relative frequency of codons in the variant sequence coFVIII-5, which has a human CAI of 83%. Figure 2F shows the relative codon frequency in the variant sequence coFVIII-6, which has a human CAI of 83%. Figure 2G shows the relative codon frequency in the variant sequence coFVIII-52, which has a human CAI of 91%.Figure 2H shows the relative codon frequency in the variant sequence coFVIII-62, which has a human CAI of 91%. Figure 2I shows the relative codon frequency in the variant sequence coFVIII-25, which has a human CAI of 88%. Figure 2J shows the relative codon frequency in the variant sequence coFVIII-26, which has a human CAI of 88%.

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

[0021] Figure 4 shows a graphical representation of the plasma activity of FVIII in HemA mice after hydrodynamic injection of 5 pg of FVIII-303 (coFVIII-1; circles) or 5 pg of FVIII311 (BDD-FVIII; squares). The plasma activity of FVIII was Petition 870260067982, dated 09 / 07 / 2026, page 32 / 275 13 / 233 determined by a specific chromogenic assay for FVIII at 24, 48, and 72 hours post-injection. Relative activity levels at 72 hours normalized to the FVIII-311 expression level are shown.

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

[0023] Figures 6A-6C show graphical representations of plasma FVIII activity in HemA mice after hydrodynamic injection of various nucleotides encoding FVIII. Plasma FVIII activity was determined by means of an FVIII-specific chromogenic assay at 24, 48, and 72 hours post-injection. Figure 6A shows the plasma activity of FVIII in HemA mice after hydrodynamic injection of 5 pg of LV-coFVIII-1 (filled circles), 5 pg of LV-coFVIII-3 (triangles), 5 pg of LV-coFVIII-4 (inverted triangles), 5 pg of LV-coFVIII-5 (diamonds) or 5 pg of LV-coFVIII-6 (open circles).Figure 6B shows the plasma activity of FVIII in HemA mice after hydrodynamic injection of 5 pg of LV-coFVIII1 (circles), 5 pg of LV-coFVIII-25 (triangles), or 5 pg of LV-coFVIII26 (inverted triangles). Figure 6C shows the plasma activity of FVIII in HemA mice after hydrodynamic injection of 20 pg of LV-2116 (BDD-FVIII nucleotide sequence without optimized codons (WT); open circles), 20 pg of LV-coFVIII-1 (triangles), 20 pg of LV-coFVIII-52 (squares), or 20 pg of LV-coFVIII-62 (filled circles). The relative activity levels at 72 hours are shown for each plasmid, normalized to the expression levels of LV-coFVIII-1 (Figures 6A, 6B and 6C) and / or LV-2116 (Figure 6C), as indicated. Petition 870260067982, dated 09 / 07 / 2026, page 33 / 275 14 / 233

[0024] Figure 7 shows the plasma activity of FVIII in HemA mice 24 days after injection with 1E8 TU / mouse lentiviral vector comprising coFVIII-1, coFVIII-5, coFVIII-52, coFVIII-6 or coFVIII-62 when compared with LV-2116 (BDD-FVIII) and as measured by a specific chromogenic assay for FVIII. Error bars indicate standard deviations.

[0025] Figures 8A-8C provide the various nucleotide sequences with optimized codons encoding BDD-FVIII fused to an 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 within the nucleotide sequence of coFVIII-52. 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 (XTENW; SEQ ID NO: 18; underlined) is inserted within the nucleotide sequence of coFVIII-1. 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 (XTENW; SEQ ID NO: 18; underlined) is inserted into the nucleotide sequence of coFVIII-6 (e.g., amino acid residue 745 which corresponds to the mature FVIII sequence).

[0026] Figure 9 provides a plasmid map of pLV-coFVIII52-XTEN, which comprises coFVIII-52-XTEN in a lentiviral vector under the control of the ET promoter. The lentiviral vectors comprising each of the nucleic acid molecules with remaining optimized codons encoding a polypeptide with FVIII activity, as described herein, were constructed in the same manner as pLV-coFVIII-52-XTEN, in which the same XTEN sequence was inserted to replace the B domain of FVIII. Petition 870260067982, dated 09 / 07 / 2026, page 34 / 275 15 / 233

[0027] Figures 10A and 10B show the activity of FVIII in HemA mice after injection of plasmid DNA (Figure 10A) or lentiviral vector (Figure 10B) comprising the various nucleotide sequences with optimized codons encoding BDD-FVIII. Figure 10A shows a graphical representation of the plasma activity of FVIII in HemA mice after hydrodynamic injection with 5 pg of FVIII-311 (nucleotide sequence encoding BDD-FVIII without optimized codons; squares), 5 pg of FVIII-303 (coFVIII-1; small circles) or FVIII-306 (coFVIII-1-XTEN144; large circles). The relative activity at 72 hours, normalized to FVIII-311, is shown for each plasmid. Figure 10B shows the plasma activity of FVIII in HemA mice 21 days after injection with 1E8 TU / mouse of the lentiviral vector comprising coFVIII-52 or coFVIII-52-XTEN when compared with the control LV-2116 (BDDFVIII) and measured by means of a specific chromogenic assay for FVIII.Error bars indicate standard deviations.

[0028] Figure 11A shows the amino acid sequence of Factor VIII mature human complete. Figure 11B shows the amino acid sequence of the 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 that has 42 amino acids (XTEN AE42-4; SEQ ID NOs: 46 and 47, respectively). The amino acid sequences of several XTEN polypeptides that have 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 an ET promoter (SEQ ID Petition 870260067982, dated 09 / 07 / 2026, page 35 / 275 16 / 233 Figure 11Z shows the nucleotide sequence for coFVIII1 (SEQ ID NO: 68) (see International Publication No. WO 2014 / 127215, SEQ ID NO: 1).

[0029] Figure 12A is a graphical representation of plasma FVIII activity (IU / mL) in 14-day-old HemA mice after IV administration of approximately 1.5 x 1010TU / kg of LV-wtBDDFVIII (circles), LV-coFVIII-6 (squares), or LV-coFVIII-6XTEN (triangles). Figure 12B is a graphical representation of the vector copy number (VCN) 150 days after treatment of 14-day-old HemA mice that received approximately 1.5 x 1010TU / 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 graphical representation of plasma FVIII activity (IU / mL) 21 days after treatment of 14-day-old HemA mice that received IV approximately 1.5 x 1010TU / kg of lentiviral vectors expressing wtBDD-FVIII, coFVIII-1, coFVIII-3, coFVIII-4, coFVIII-5, coFVIII-6, coFVIII-52, coFVIII-62, coFVIII-25, or coFVIII-26.

[0030] Figures 13A and 13B are graphical representations illustrating the plasma activity levels of FVIII (Figure 13A) and the levels of anti-FVIII antibodies (Figure 13B) in five HemA mice treated with a lentivirus expressing the coFVIII-5 variant. Fourteen-day-old HemA littermates received approximately 1.5 x 1010TU / kg of a lentivirus expressing the coFVIII-5 variant via intravenous injection. Each mouse is designated by a number (i.e., 1, 2, 3, 4, and 5; Figures 13A and 13B).

[0031] Figure 14 is a graphical representation of the correlation between the level of LV-FVIII expression, as evidenced by plasma FVIII activity 21 days after lentiviral treatment, and the presence of anti-FVIII antibodies. Each data point corresponds to a single Petition 870260067982, dated 09 / 07 / 2026, p. 36 / 275 17 / 233 HemA mice. Each mouse received a dose of 1.5 x 1010TU / kg via intravenous injection of a lentivirus expressing one of the coFVIII variants described herein. Horizontal lines indicate the mean plasma activity of FVIII.

[0032] Figure 15 is a graphical representation of the correlation between vector copy number (VCN) per cell 150 days after lentiviral treatment and the presence of anti-FVIII antibodies. Each data point corresponds to a single HemA mouse. Each mouse received a dose of 1.5 x 1010TU / kg via intravenous injection of a lentivirus expressing one of the coFVIII variants described herein. Horizontal lines indicate the mean VCN.

[0033] Figures 16A and 16B are graphical representations illustrating the plasma activity levels of FVIII (Figure 16A) and the levels of anti-FVIII antibodies (Figure 16B) in two HemA mice (coFVIII-52-A and coFVIII-52-B) treated with a lentivirus expressing the coFVIII-52 variant. Fourteen-day-old HemA littermates received approximately 1.5 x 1010TU / kg of a lentivirus expressing the coFVIII-52 variant via intravenous injection. Figures 16C and 16D are images showing the RNA in situ hybridization staining for FVIII expression (dark staining) in liver tissue collected from coFVIII-52-A (Figure 16C) and coFVIII-52-B (Figure 16D) mice from Figures 16A and 16B.

[0034] Figure 17 is a graphical representation showing the long-term expression of FVIII in newborn HemA mice treated with a lentivirus expressing a wild-type FVIII with a deleted B domain (wtBDD-FVIII; triangles), the coFVIII-52-XTEN variant (circles), or the coFVIII-6-XTEN variant (inverted triangle). Newborn HemA mice received approximately 1.5 x 1010 TU / kg of a lentivirus via intravenous injection. Petition 870260067982, dated 09 / 07 / 2026, page 37 / 275 18 / 233 expressing wtBDD-FVIII, coFVIII-52-XTEN, or coFVIII-6-XTEN. Plasma FVIII activity was measured for approximately 16 weeks.

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

[0036] Figure 19 provides a schematic of a lentiviral vector for liver-targeted gene therapy. SD: splice donor site; SA: splice acceptor site; GA: truncated gag sequence; RRE: Rev responsive element; ET: transthyretin enhancer; FVIII: Factor VIII; 142T: miR-142 target sequence; Wpre: Woodchuck hepatitis virus post-transcriptional regulatory element; Ψ (packaging signal).

[0037] Figures 20A-20B are graphical representations of peak circulating FVIII levels in male Pigtail monkeys that received 3 x 109TU / kg of a lentivirus expressing coFVIII-6-XTEN produced from 293T CD47high / MHC-Ifree cells, as measured by plasma FVIII activity (Figure 20A) and plasma FVIII antigen levels (Figure 20B).

[0038] Figures 21A-21B are graphical representations of peak plasma levels of human FVIII activity (Figure 21A) and human FVIII antigen levels (Figure 21B) in pigtail monkeys that received 3 x 109TU / kg or 6 x 109TU / kg of a lentivirus expressing coFVIII-6.

[0039] Figures 22A-22B show a graphical representation of peak plasma levels of human FVIII activity (Figure 22A) and mean levels of human FVIII antigen (Figure 22B) in male pigtail monkeys that received 1 x 109 or 3 x 109TU / kg of coFVIII-6-XTEN lentivirus. Petition 870260067982, dated 09 / 07 / 2026, page 38 / 275 19 / 233 DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention describes liver-targeted lentiviral gene therapy using genes with optimized codons encoding polypeptides with Factor VIII (FVIII) activity. See, for example, International Publication WO2017136358, which is incorporated herein by reference in its entirety.

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

[0042] This system demonstrates an increase in long-term Factor VIII expression in target cells (e.g., hepatocytes) when the lentiviral vector is administered to the individual at a dose of at least 5 x 1010 transducer units / kg (TU / kg) or less, for example, about 1.5 x 1010TU / kg or less or about 1.5 x 109TU / kg or less or about 108TU / kg or less.

[0043] In specific embodiments, the lentiviral vectors described herein comprise a nucleic acid sequence with optimized codons comprising, consisting of, or essentially consisting of Petition 870260067982, dated 09 / 07 / 2026, p. 39 / 275 20 / 233 SEQ ID NO: 71 (LV-coFVIII-6).

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

[0045] The liver-targeted lentiviral vectors described herein allow for the stable integration of the transgenic expression cassette comprising an optimized codon-encoding nucleic acid FVIII into the genome of target cells (e.g., hepatocytes) from pediatric (e.g., newborns) or adult individuals, achieving an increase in FVIII expression (e.g., a 100-fold increase) at low doses of the lentiviral vector (e.g., 5 x 1010 or less, as well as 109 TU / kg or less or 108 TU / kg or less). Because the described lentiviral vectors can achieve therapeutic levels of circulating FVIII at very low doses (e.g., 109 TU / kg or less or 108 TU / kg or less), these vectors can significantly reduce the potential acute toxicity associated with lentivirus vector treatment.Furthermore, the use of lentiviral vectors, and in particular third-generation vectors, can lead to potentially lifelong integration into the individual's genome. The high capacity of lentiviral vectors (10 kb) compared to other gene delivery systems (e.g., AAV) allows for the inclusion of more regulatory elements in the transgene, for example, promoters that would control the expression of the FVIII transgene in different tissues (e.g., hepatocytes and hepatic endothelial cells). The lentiviral vectors described here can be used in in vivo, in vitro, or ex vivo treatments.

[0046] Exemplary constructions of the invention are illustrated in the attached figures and in the sequence listing.

[0047] In order to provide a clear understanding of the report Petition 870260067982, dated 09 / 07 / 2026, page 40 / 275 21 / 233 Description and claims, the following definitions are provided below. I. Definitions

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

[0049] The term about is used here to mean approximately, almost, around, or in the vicinity of. When the term about is used in conjunction with a numerical range, it modifies this range by extending the limits above and below the numerical values ​​presented. In general, the term about is used here to modify a numerical value above and below the value presented by a variation of 10 percent, upwards or downwards (greater or less).

[0050] The term isolate, for purposes of the present invention, designates a biological material (cell, polypeptide, polynucleotide or a fragment, variant or derivative) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or animal is not an isolate; however, the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present is considered an isolate. No specific level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolates for purposes of the invention, as are native or recombinant polypeptides that have been separated, fractionated or partially or substantially purified by means of Petition 870260067982, dated 09 / 07 / 2026, page 41 / 275 22 / 233 any suitable technique.

[0051] Nucleic acids, nucleic acid molecules, oligonucleotide, and polynucleotide are used interchangeably and refer to the polymeric phosphate ester form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; RNA molecules) or deoxyribonucleosides (deoxyadenosine, deoxyganosine, deoxythymidine, or deoxycytidine; DNA molecules) or any phosphoester analogs, such as phosphorothioates and thioesters, in single-stranded or double-stranded helix form. DNA-DNA, DNA-RNA, and RNA-RNA double-stranded helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any specific tertiary form. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes.When discussing the structure of particular double-stranded DNA molecules, the sequences may be described herein according to the normal convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand that has a sequence homologous to mRNA). A recombinant DNA 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 invention comprises one or more nucleic acids, as described herein.

[0052] 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 typically translated into an amino acid, it can be considered part of a coding region, but Petition 870260067982, dated 09 / 07 / 2026, page 42 / 275 23 / 233 Any flanking sequence, for example, promoters, ribosome binding sites, transcriptional terminators, introns, and so on, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' end, which codes for the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end that codes for the carboxyl terminus of the resulting polypeptide. Two or more coding regions may be present in a single polynucleotide construct, for example, in a single vector, or in separate polynucleotide constructs, for example, in separate (different) vectors. It follows, then, that a single vector may contain only a single coding region or comprise two or more coding regions.

[0053] Certain proteins secreted by mammalian cells are associated with a secretory signaling peptide that is cleaved from the mature protein after export of the growing protein chain through the coarse endoplasmic reticulum. Those skilled in the art know that signaling peptides are generally fused to the N-terminus of the polypeptide and are cleaved from the complete or full-length polypeptide to produce a secreted or mature form of the polypeptide. In certain embodiments, a signaling peptide is native or a functional derivative of this sequence that retains the ability to control the secretion of the polypeptide that is operatively associated with it. Alternatively, a heterologous mammalian signaling peptide may be used, for example, a human tissue plasminogen activator (TPA) or mouse β-glucuronidase signaling peptide or a functional derivative thereof.

[0054] The term downstream refers to a sequence of Petition 870260067982, dated 09 / 07 / 2026, page 43 / 275 24 / 233 nucleotides that are located 3' relative to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences refer to sequences that follow the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

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

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

[0057] A polynucleotide encoding a gene product, for example, a polypeptide, may include a promoter and / or other expression control elements (e.g., transcription or translation) operatively associated with one or more coding regions. In an operative association, a coding region Petition 870260067982, dated 09 / 07 / 2026, page 44 / 275 25 / 233 for a gene product, for example, a polypeptide, is associated with one or more regulatory regions in such a way as to place the expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are operatively associated if the induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region and if the nature of the linkage between the promoter and the coding region does not interfere with the promoter's ability to control gene product expression or interfere with the ability of the DNA template to be transcribed. Other expression control elements besides a promoter, for example, enhancers, operators, repressors, and transcription termination signals, may also be operatively associated with a coding region to control gene product expression.

[0058] Transcriptional control sequences refer to regulatory DNA sequences, such as promoters, enhancers, terminators, and so forth, that allow the expression of a coding sequence in a host cell. A variety of transcriptional control regions are known to those skilled in the art. This includes, without limitation, transcriptional control regions that function in vertebrate cells such as, without limitation, promoter and enhancer segments of cytomegalovirus (the immediate early promoter, together with intron A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as atin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells.Additional appropriate transcription control regions. Petition 870260067982, dated 09 / 07 / 2026, page 45 / 275 26 / 233 include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., interferon- or interleukin-inducible promoters).

[0059] Similarly, a variety of translation control elements are known to those skilled in the art. This includes, among others, ribosome binding sites, translation start and stop codons, and picornavirus-derived elements (particularly an internal ribosomal entry site, or IRES, also referred to as the CITE sequence).

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

[0061] A vector refers to any vehicle for cloning and / or transferring a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment is attached. Petition 870260067982, dated 09 / 07 / 2026, page 46 / 275 27 / 233 can be ligated in such a way as to trigger replication of the ligated segment. A replicon refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, that is, capable of replication under its own control. The term vector includes viral and nonviral vehicles for introducing nucleic acid into a cell in vitro, ex vivo, or in vivo. A large number of vectors are known and used in the technique, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. The insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0062] Vectors can be designed to encode selectable markers or reporters that allow the selection or identification of cells that have incorporated the vector. The expression of selectable markers or reporters allows the identification and / or selection of host cells that incorporate and express other coding regions contained in the vector. Examples of known selectable marker genes used in the technique include: genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialafos herbicide, sulfonamide, and so on; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and so on. Examples of known reporters used in the technique include: luciferase (Luc), green fluorescent protein (Green). Fluorescent Protein, GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and so on. Selectable markers may also be considered reporters.

[0063] The term selectable marker refers to an identifying factor, usually an antibiotic resistance gene or Petition 870260067982, dated 09 / 07 / 2026, page 47 / 275 28 / 233 chemical products, which can be selected based on the effect of the marker gene, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and so on, where the effect is used to trace the inheritance of a nucleic acid of interest and / or identify a cell or organism that has inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the technique include: genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, the herbicide bialafos, sulfonamide, and so on; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and so on.

[0064] The term reporter gene refers to a nucleic acid that encodes an identification factor that can be identified based on the effect of the reporter gene, where the effect is used to trace the inheritance of a nucleic acid of interest, identify a cell or organism that has inherited the nucleic acid of interest, and / or measure the induction or transcription of gene expression. Examples of known reporter genes used in the technique include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and so on. Selectable marker genes can also be considered reporter genes.

[0065] Promoter and promoter sequence are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' relative to a promoter sequence. Promoters can be derived entirely from a native gene or be composed of different elements derived from different promoters found in nature or even Petition 870260067982, dated 09 / 07 / 2026, page 48 / 275 29 / 233 understand synthetic DNA segments. It will be understood by those skilled in the art that different promoters can control the expression of a gene in different tissues or cell types or at different stages of development or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types most of the time are commonly referred to as constitutive promoters. Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as cell-specific promoters or tissue-specific promoters. Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as development-specific promoters or cell differentiation-specific promoters.Promoters that are induced and cause a gene to be expressed when the cell is exposed to or treated with an agent, biological molecule, chemical, ligand, light, or similar substance that induces the promoter are commonly referred to as inducible promoters or regulable promoters. It is also recognized that, in most cases, the exact boundaries of regulatory sequences have not been completely defined; DNA fragments of different lengths may have identical promoter activity.

[0066] The promoter sequence is typically bounded at its 3' end by the transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at detectable levels above the background. Within the promoter sequence, a transcription start site will be found (conveniently defined, for example, by mapping with S1 nuclease), as well as protein-binding domains (consensus sequences) responsible for Petition 870260067982, dated 09 / 07 / 2026, page 49 / 275 30 / 233 RNA polymerase binding.

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

[0068] The term plasmid refers to an extrachromosomal element that frequently carries a gene that is not part of the cell's central metabolism and is generally in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrator sequences, phage or nucleotide sequences, linear, circular or supercoiled single- or double-stranded DNA or RNA, derived from any source, in which several nucleotide sequences have been joined or recombined into a single construct capable of introducing a promoter fragment and DNA sequence for a selected gene product along with the appropriate 3' untranslated sequence into a cell.

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

[0070] The term expression vector refers to a vehicle Petition 870260067982, dated 09 / 07 / 2026, page 50 / 275 31 / 233 is designed to allow the expression of an inserted nucleic acid sequence upon insertion into a host cell. The inserted nucleic acid sequence is placed in operative association with regulatory regions as described above.

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

[0072] Culture and cultivate, as used herein, mean incubating cells under in vitro conditions that allow cell growth or division or maintain the cells in a living state. Cultured cells, as used herein, mean cells that are propagated in vitro.

[0073] As used herein, the term polypeptide is intended to encompass an individual polypeptide as well as multiple polypeptides and refers to a molecule composed of monomers (amino acids) linearly linked through 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 in the definition of polypeptide, and the term polypeptide may be used instead of or interchangeably with any of these terms.The term polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known guarding / blocking groups, proteolytic cleavage or. Petition 870260067982, dated 09 / 07 / 2026, page 51 / 275 32 / 233 modification of naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced through recombinant technology, but it is not necessarily translated from a designated nucleic acid sequence. It can be generated in either way, including through chemical synthesis.

[0074] The term amino acid includes alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (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 invention and include norleucine, omitine, norvaline, homoserine, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202: 301-336 (1991). To generate these non-naturally occurring amino acid residues, the procedures of Noren et al., Science 244: 182 (1989) and Ellman et al., supra, can be used.In summary, these procedures involve the chemical activation of a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA. The introduction of the non-traditional amino acid can also be achieved using peptide chemistries known in the art. As used herein, the term polar amino acid includes amino acids with a net zero charge but with non-zero partial charges in different parts of their side chains (e.g., M, F, W, S, Y, N, QC). 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. Petition 870260067982, dated 09 / 07 / 2026, p. 52 / 275 33 / 233 in their side chains (e.g., R, K, H, E, D). These amino acids can participate in hydrophobic and electrostatic interactions.

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

[0076] A conservative amino acid substitution is one in which the amino acid residue is replaced by an amino acid residue that has a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, Petition 870260067982, dated 09 / 07 / 2026, page 53 / 275 34 / 233 histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched beta side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, an amino acid chain can be conservatively replaced by a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0077] The term percentile identity, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the correspondence between the strands of such sequences. Identity can be easily calculated by means of known methods including, but not limited to, those described in: 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 a. Petition 870260067982, dated 09 / 07 / 2026, p. 54 / 275 35 / 233 best match between the tested sequences. Methods for determining identity are encoded in publicly available computer programs. Sequence alignments and percentage identity calculations can be performed using sequence analysis software, such as the Megalign program from the LASERGENE bioinformatics computing package (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, it will be understood that where sequence analysis software is used for analysis, the analysis results will be based on the default values ​​of the aforementioned program unless otherwise specified.As used herein, default values ​​means any set of values ​​or parameters that are originally loaded with the software when it is first initialized. For the purposes of determining the percentage of identity between an optimized BDD-FVIII sequence of the invention and a reference sequence, only the nucleotides in the reference sequence that correspond to the nucleotides in the optimized BDD-FVIII sequence of the invention are used to calculate the percentage of identity. For example, when comparing a complete FVIII nucleotide sequence containing the B domain with an optimized FVIII nucleotide sequence with the B domain deleted (BDD) of the invention, the portion of the alignment that includes the A1, A2, A3, C1, and C2 domains will be used to calculate the percentage of identity.The nucleotides in the full-length portion of the FVIII sequence that codes for domain B (which will result in a large gap in the alignment) will not be counted as a mismatch. Furthermore, in the determination... Petition 870260067982, dated 09 / 07 / 2026, p. 55 / 275 36 / 233 of the percentage of identity between an optimized BDD-FVIII sequence of the invention or a designated portion thereof (e.g., nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3) and a reference sequence, the percentage of identity will be calculated by aligning and dividing the number of matching nucleotides by the total number of nucleotides in the complete sequence of the optimized BDD-FVIII sequence or a designated portion thereof, as quoted herein.

[0078] As used herein, nucleotides that correspond to nucleotides in the optimized BDD-FVIII sequence of the invention are identified by aligning the optimized BDD-FVIII sequence of the invention to maximize identity with the reference FVIII sequence. The number used to identify an equivalent amino acid in a reference FVIII sequence is based on the number used to identify the corresponding amino acid in the optimized BDD-FVIII sequence of the invention.

[0079] A fusion or chimeric protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. Amino acid sequences that normally exist in separate proteins can be joined together in the fusion polypeptide, or amino acid sequences that normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide, for example, fusion of a Factor VIII domain of the invention with an Ig Fc domain. A fusion protein is created, for example, by chemical synthesis or by the creation and translation of a polynucleotide in which the peptide regions are encoded in the desired relationship. A chimeric protein may further comprise a second amino acid sequence associated with the first amino acid sequence through a covalent, non-peptide bond or a Petition 870260067982, dated 09 / 07 / 2026, page 56 / 275 37 / 233 non-covalent bond.

[0080] As used herein, the term insertion site refers to a position on an FVIII polypeptide or fragment, variant or derivative thereof, that is immediately upstream of the position at which a heterologous moiety may be inserted. An insertion site is specified as a number, the number being the amino acid number in the mature native FVIII (SEQ ID NO: 15; Figure 11A) to which the insertion site corresponds, which is immediately N-terminal to the insertion position. For example, the phrase a3 comprises 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 two amino acids that correspond to amino acid 1656 and amino acid 1657 of SEQ ID NO: 15.

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

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

[0083] As used herein, the term half-life refers to the biological half-life of a given polypeptide in vivo. The half-life can be represented by the time required for half of the amount administered to an individual to be removed from circulation. Petition 870260067982, dated 09 / 07 / 2026, page 57 / 275 38 / 233 and / or other tissues in the animal. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic, with a rapid α phase and a longer β phase. The α phase typically represents an equilibrium of the administered Fc polypeptide between the intra- and extravascular spaces and is partly determined by the size of the polypeptide. The β phase typically represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII proteins and chimeras comprising FVIII are monophasic and therefore do not have an alpha phase, but only a single beta phase. Therefore, in certain embodiments, the term half-life, as used herein, refers to the half-life of the polypeptide in the β phase.

[0084] The term linked, as used herein, refers to a first amino acid sequence or nucleotide sequence covalently or non-covalently joined to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence may be directly joined or juxtaposed to the second amino acid or nucleotide sequence or, alternatively, an intermediate sequence may covalently join the first sequence to the second sequence. The term linked means not only a fusion of a first amino acid sequence with a second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of the entire first amino acid sequence (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (or the first amino acid sequence, respectively).In one embodiment, the first amino acid sequence can be linked to a second amino acid sequence via a peptide bond or ligand. The first nucleotide sequence can be linked to a second sequence of... Petition 870260067982, dated 09 / 07 / 2026, p. 58 / 275 39 / 233 nucleotides through a phosphodiester bond or linker. The linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a nucleotide chain (for nucleotide chains) or any chemical moiety (for polypeptide and polynucleotide chains). The term linked is also indicated by a hyphen (-).

[0085] As used herein, the term "associated with" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" means a covalent, non-peptide bond or a non-covalent bond. This association may be indicated by a colon, i.e., (:). In another embodiment, it means a covalent bond other than a peptide bond. For example, the amino acid cysteine ​​comprises a thiol group that may form a disulfide bond or bridge with a thiol group on a second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated via a disulfide bond and the two heavy chains are associated via 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, a peptide bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, a double bond, a dipolar bond, a reverse pi bond, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bonds include an ionic bond (e.g., cationic pi bond or salt bond), a metallic bond, a hydrogen bond (e.g., bond). Petition 870260067982, dated 09 / 07 / 2026, page 59 / 275 40 / 233 dihydrogen, dihydrogen complex, low barrier hydrogen bond or symmetrical hydrogen bond), van der Waals force, London dispersion force, mechanical bond, halogen bond, aurophilicity, intercalation, stacking, entropic force or chemical polarity.

[0086] The term monomer-dimer hybrid used here refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain, which are linked together by a disulfide bond, wherein the first chain comprises a coagulation factor, for example, Factor VIII, and a first Fc region and the second chain comprises, consists essentially of, or consists of a second Fc region without the coagulation factor. The monomer-dimer hybrid construct is thus a hybrid comprising a monomeric aspect with only one coagulation factor and a dimeric aspect with two Fc regions.

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

[0088] Hemostatic disorder, as used herein, means an inherited or genetically acquired condition characterized by a tendency to bleed, spontaneously or as a result of trauma, due to a reduced ability or inability to form a fibrin clot. Examples of such disorders include hemophilias. The three main forms are hemophilia A (Factor VIII deficiency), hemophilia B (Factor IX deficiency or Christmas disease), and hemophilia C (Factor XI deficiency, mild tendency to bleed). Other hemostatic disorders include, for example, von Willebrand disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency, structural deficiencies or abnormalities in Petition 870260067982, dated 09 / 07 / 2026, page 60 / 275 41 / 233 fibrinogen, prothrombin, Factor V, Factor VII, Factor X, or Factor XIII, Bernard-Soulier syndrome, which is a defect or deficiency of GPIb. GPIb, the vWF receptor, may be defective and lead to a lack of primary clot formation (primary hemostasis) and an increased tendency to hemorrhage, and Glanzmann-Naegeli thrombosthenia (Glanzmann thrombosthenia). In liver failure (acute and chronic forms), there is insufficient production of coagulation factors by the liver; this can increase the risk of hemorrhage.

[0089] Lentiviral vectors comprising the isolated nucleic acid molecule of the invention can be used prophylactically. As used herein, the term prophylactic treatment refers to the administration of a molecule before a bleeding episode. In one embodiment, the individual requiring a hemostatic agent is generally undergoing or about to undergo surgery. For example, a lentiviral vector of the invention can be administered before or after surgery as a prophylactic. The lentiviral vector of the invention can be administered during or after surgery to control an acute bleeding episode. Surgery may include, but is not limited to, liver transplantation, hepatic resection, dental procedures, or stem cell transplantation.

[0090] The lentiviral vectors of the invention are also used for on-demand treatment. The term on-demand treatment refers to the administration of a lentiviral vector described herein in response to symptoms of a bleeding episode or before an activity that may cause bleeding. In one aspect, on-demand treatment may be administered to an individual when bleeding begins, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, on-demand treatment may be administered before activities that increase the risk of bleeding, such as contact sports. Petition 870260067982, dated 09 / 07 / 2026, page 61 / 275 42 / 233

[0091] As used herein, the term acute hemorrhage refers to an episode of bleeding, regardless of the underlying cause. For example, an individual may have trauma, uremia, a hereditary bleeding disorder (e.g., Factor VII deficiency), a platelet disorder, or resistance due to the development of antibodies to clotting factors.

[0092] Treating, treating, treatment, as used herein, refer, for example, to a reduction in the severity of a disease or condition; to a reduction in the duration of the course of a disease; to an improvement in one or more symptoms associated with a disease or condition; to the provision of beneficial effects to an individual who has a disease or condition, without necessarily curing the disease or condition; or to the prophylaxis of one or more symptoms associated with a disease or condition. In one embodiment, the term treat or treatment means maintaining a minimum FVIII 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 an individual by administering a lentiviral vector of the invention.In another interpretation, treatment means maintaining a minimum FVIII level between approximately 1 and approximately 20 IU / dL, approximately 2 and approximately 20 IU / dL, approximately 3 and approximately 20 IU / dL, approximately 4 and approximately 20 IU / dL, approximately 5 and approximately 20 IU / dL, approximately 6 and approximately 20 IU / dL, approximately 7 and approximately 20 IU / dL, approximately 8 and approximately 20 IU / dL, approximately 9 and approximately 20 IU / dL, or approximately 9 and approximately 20 IU / dL, or approximately 10 IU / dL 20 IU / dL. Treating or managing a disease or condition may also include maintaining FVIII activity in an individual at a level comparable 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-hemophiliac individual. Petition 870260067982, dated 09 / 07 / 2026, p. 62 / 275 43 / 233 In one embodiment, the term treat or treatment means maintaining a minimum FVIII 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 an individual when administering a lentiviral vector of the invention. In another interpretation, treatment means maintaining a minimum FVIII level between approximately 10 and approximately 20 IU / dL, approximately 20 and approximately 23 IU / dL, approximately 30 and approximately 40 IU / dL, approximately 40 and approximately 50 IU / dL, approximately 50 and approximately 60 IU / dL, approximately 60 and approximately 70 IU / dL, approximately 70 and approximately 80 IU / dL, approximately 80 and approximately 90 IU / dL, approximately 90 and approximately 100 IU / dL, approximately 90 and approximately 100 IU / dL, approximately 110 and 120 IU / dL, approximately 120 and approximately 130 IU / dL, approximately 130 and approximately 140 IU / dL, or approximately 140 and approximately 150 IU / dL. UI / dL.Treating or managing a disease or condition may also include maintaining FVIII activity in an individual at a level comparable to at least approximately 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 individual. The minimum level required for treatment can be measured using one or more known methods and can be adjusted (increased or decreased) for each person.

[0093] Administration, as used herein, means providing a pharmaceutically acceptable nucleic acid molecule encoding Factor VIII, Factor VIII polypeptide, or vector comprising a nucleic acid molecule encoding Factor VIII of the invention to an individual via a pharmaceutically acceptable route. Routes of administration may be intravenous, for example, intravenous injection and intravenous infusion. Additional routes of administration include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The molecules of Petition 870260067982, dated 09 / 07 / 2026, page 63 / 275 44 / 233 nucleic acids, polypeptides and vectors may be administered as part of a pharmaceutical composition comprising at least one excipient.

[0094] As used herein, the phrase "individual in need of the same" includes individuals, such as mammals, who would benefit from administration of a nucleic acid molecule, a polypeptide, or a vector of the invention, for example, to improve hemostasis. In one embodiment, individuals include, but are not limited to, individuals who have hemophilia. In another embodiment, individuals include, but are not limited to, individuals who have developed an FVIII inhibitor and therefore require bypass therapy. The individual may be an adult or a minor (e.g., under 12 years of age).

[0095] As used herein, the term coagulation factor refers to naturally occurring or recombinantly produced molecules or analogues thereof that prevent or shorten the duration of a bleeding episode in an individual. In other words, it means molecules that have pro-coagulation activity, that is, they are responsible for converting fibrinogen into an insoluble fibrin mesh, causing the blood to clot. An activatable coagulation factor is a coagulation factor in an inactive form (e.g., in its zymogen form) that is capable of being converted into an active form.

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

[0097] As used herein, the terms heterologous or exogenous refer to molecules that are not normally found in a given context, for example, in a cell or in a Petition 870260067982, dated 09 / 07 / 2026, page 64 / 275 45 / 233 polypeptide. For example, an exogenous or heterologous molecule can be introduced into a cell and is present only after manipulation of the cell, for example, through transfection or other forms of genetic engineering, or a heterologous amino acid sequence can be present in a protein where it is not naturally found.

[0098] As used herein, the term heterologous nucleotide sequence refers to a nucleotide sequence that does not occur naturally within a given polynucleotide sequence. In one embodiment, the heterologous nucleotide sequence encodes a polypeptide capable of prolonging 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 enhances 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 via a linker.Non-limiting examples of polypeptide moieties encoded by heterologous nucleotide sequences include an immunoglobulin constant region or a portion thereof, albumin or a fragment thereof, an albumin-binding moiety, transferrin, the PAS polypeptides of U.S. Patent Application No. 20100292130, a HAP sequence, transferrin or a fragment thereof, the C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, small albumin-binding molecule, an XTEN sequence, FcRn-binding moieties (e.g., complete Fc regions or portions thereof that bind to FcRn), single-stranded Fc regions (ScFc regions, for example, as described). Petition 870260067982, dated 09 / 07 / 2026, page 65 / 275 46 / 233 in documents US 2008 / 0260738, WO 2008 / 012543 or WO 2008 / 1439545), polyglycine linkers, polyserine linkers, peptides and short polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) with varying degrees of secondary structure from less than 50% to more than 50%, among others, or two or more combinations thereof. In some embodiments, the polypeptide encoded by the heterologous nucleotide sequence is linked to a non-polypeptide moiety. Non-limiting examples of non-polypeptide moieties include polyethylene glycol (PEG), small albumin-binding molecules, polysialic acid, hydroxyethylamide (HES), a derivative thereof, or any combination thereof.

[0099] As used herein, the term Fc region is defined as the portion of a polypeptide that corresponds to the Fc region of native Ig, that is, formed by the dimeric association of the respective Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. On the other hand, 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 genetically linked Fc domains within a single polypeptide chain (that is, encoded in a single contiguous genetic sequence).

[0100] In one embodiment, the Fc region refers to the portion of a single Ig heavy chain that begins at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, counting the first residue of the heavy chain constant region 114) and terminates at the C-terminal of the antibody. Consequently, a complete Fc domain comprises at least one hinge domain, one CH2 domain, and one CH3 domain. Petition 870260067982, dated 09 / 07 / 2026, page 66 / 275 47 / 233

[0101] The Fc region of an Ig constant region, depending on the Ig isotype, may include the CH2, CH3, and CH4 domains, as well as the hinge region. Chimeric proteins comprising an Ig Fc region confer several desirable properties to a chimeric protein, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337: 525), as well as binding to Fc receptors, such as the neonatal Fc receptor (FcRn) (U.S. Patents Nos. 6,086,875, 6,485,726, 6,030,613; documents WO 03 / 077834; US2003-0235536A1), which are incorporated herein by reference in full.

[0102] A reference nucleotide sequence, when used herein as a comparison with a nucleotide sequence of the invention, is a polynucleotide sequence essentially identical to the nucleotide sequence of the invention, except that the portions corresponding to the FVIII sequence are not optimized. For example, the reference nucleotide sequence for a nucleic acid molecule consisting of BDD-FVIII with optimized codons of SEQ ID NO: 1 and a heterologous nucleotide sequence encoding a single-stranded Fc region attached to SEQ ID NO: 1 at its 3' end is a nucleic acid molecule consisting of the original (or progenitor) BDD-FVIII of SEQ ID NO: 16 (Figure 1I) and the identical heterologous nucleotide sequence encoding a single-stranded Fc region attached to SEQ ID NO: 16 at its 3' end.

[0103] A codon adaptation index, as used herein, refers to a measure of codon usage tendency. A codon adaptation index (CAI) measures the deviation of a protein-coding gene sequence from a set of reference genes (Sharp PM and Li WH, Nucleic Acids Res. 15 (3): 1281-95 (1987)). The CAI is calculated by determining the geometric mean of the weight Petition 870260067982, dated 09 / 07 / 2026, p. 67 / 275 48 / 233 associated with each codon along the length of the gene sequence (measured in codons): CAI = exp(l / L (I)

[0104] For each amino acid, the weight of each of its codons, in CAI, is calculated as the ratio between the observed frequency of the codon (fi) and the frequency of the equal codon (fj) for this amino acid: Formula 2: Wi = 7—. ij € |[equal codons for amino acid] (II)

[0105] As used herein, the term optimized, in relation to nucleotide sequences, refers to a polynucleotide sequence encoding a polypeptide wherein the polynucleotide sequence has been mutated to improve a property of that polynucleotide sequence. In some embodiments, optimization is done to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease the binding of regulatory proteins such as general transcription factors, increase or decrease splicing, or increase the yield of the polypeptide produced by the polynucleotide sequence.Examples of changes that can be made to a polynucleotide sequence to optimize it include codon optimization, G / C ratio optimization, removal of repeated sequences, removal of AT-rich elements, removal of cryptic splicing sites, removal of cis-acting elements that repress transcription or translation, addition or removal of poly-T or poly-A sequences, addition of sequences around the transcription start site that increase transcription, such as Kozak consensus sequences, removal of sequences that could form loop and stem structures, and removal of destabilizing sequences. Petition 870260067982, dated 09 / 07 / 2026, p. 68 / 275 49 / 233 and two or more combinations thereof. II. FVIII Lentiviral Gene Therapy

[0106] Somatic gene therapy has been explored as a possible treatment for bleeding disorders and, in particular, hemophilia A. Gene therapy is a particularly attractive treatment for hemophilia because of its potential to cure the disease through the continuous endogenous production of FVIII after a single administration of a vector encoding FVIII. Hemophilia A is suitable for a gene replacement approach, since its clinical manifestations are entirely attributable to the lack of a single gene product (FVIII) that circulates in small quantities (200 ng / ml) in the plasma.

[0107] Lentiviral vectors are gaining prominence as gene delivery vehicles due to their high capacity and ability to sustain transgene expression through integration. Lentiviral vectors have been evaluated in several ex vivo cell therapy clinical programs with promising efficacy and safety profiles.

[0108] The present invention addresses an important need in the art by providing lentiviral vectors comprising an FVIII sequence with optimized codons that demonstrates higher expression in an individual and potentially results in greater therapeutic efficacy when used in gene therapy methods. Embodiments of the present invention are directed to lentiviral vectors containing one or more nucleic acid molecules with optimized codons encoding a polypeptide with FVIII activity described herein, host cells (e.g., hepatocytes) comprising the lentiviral vectors, and methods of using the lentiviral vectors described (e.g., treatments for bleeding disorders using the lentiviral vectors described herein).

[0109] In general, the treatment methods described here involve Petition 870260067982, dated 09 / 07 / 2026, page 69 / 275 50 / 233 the administration of a lentiviral vector comprising a nucleic acid molecule comprising at least one nucleic acid sequence with optimized codons encoding an FVIII coagulation factor, wherein the nucleic acid sequence encoding an FVIII coagulation factor is operatively linked to suitable expression control sequences which, in some embodiments, are incorporated into the lentiviral vector (e.g., a replication-defective lentiviral viral vector).

[0110] The present invention provides methods of treating a bleeding disorder (e.g., hemophilia A) in an individual in need thereof comprising administering to the individual a dose of at least 5 x 10¹⁰ or less transduction units / kg (TU / kg) (or 10⁹ or less TU / kg or 10⁸ or less TU / kg) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has: (i) at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1; (ii) at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2; (iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 Petition 870260067982, dated 09 / 07 / 2026, page 70 / 275 51 / 233 and 2320-4374 of SEQ ID NO: 70; (iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with 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 with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3; (vi) at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4; (vii) at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 23204374 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 with nucleotides 58-2277 and 23204374 of SEQ ID NO: 6; or (ix) or any combination of (i) to (viii).

[0111] The present invention also provides a method of treating a bleeding disorder (e.g., hemophilia A) in Petition 870260067982, dated 09 / 07 / 2026, p. 71 / 275 52 / 233 an individual who needs the same, which involves administering to the individual at least one dose of 5 x 1010 or less transducer units / kg (TU / kg) (or 109 TU / kg or less or 108 TU / kg or less) of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence which comprises a first nucleic acid sequence encoding an N-terminal portion of a Factor VIII (FVIII) polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; (a) in which the first nucleic acid sequence has: (i) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 3; (ii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-1791 of SEQ ID NO: 4; (iii) at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, by Petition 870260067982, dated 09 / 07 / 2026, page 72 / 275 53 / 233 less 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 with nucleotides 58-1791 of SEQ ID NO: 5;or (iv) at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 58-1791 of SEQ ID NO: 6; (b) where the second nucleotide sequence has: (i) at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of Petition 870260067982, dated 09 / 07 / 2026, page 73 / 275 54 / 233 sequence identity with nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 3; (ii) at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at 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 with nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 4; (iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 17922277 and 2320-4374 of SEQ ID NO: 5; or (iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 17922277 and 2320-4374 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 an FVIII polypeptide activity. Petition 870260067982, dated 09 / 07 / 2026, page 74 / 275 55 / 233

[0112] In some forms, the dose is approximately 5.0 x 1010 TU / kg, approximately 4.9 x 10¹⁰TU / kg, approximately 4.8 x 10¹⁰TU / kg, approximately 4.7 x 10¹⁰TU / kg, approximately 4.6 x 10¹⁰TU / kg, approximately 4.5 x 10¹⁰TU / kg, approximately 4.4 x 10¹⁰TU / kg, approximately 4.3 x 10¹⁰TU / kg, approximately 4.2 x 10¹⁰TU / kg, approximately 4.1 x 10¹⁰TU / kg, approximately 4.0 x 10¹⁰TU / kg, approximately 3.9 x 10¹⁰TU / kg, approximately 3.8 x 10¹⁰TU / kg, approximately 3.7 x 10¹⁰TU / kg, approximately 3.6 x 10¹⁰TU / kg, approximately 3.5 x 10¹⁰TU / kg, approximately 3.4 x 10¹⁰TU / kg, approximately 3.3 x 10¹⁰TU / kg, approximately 3.2 x 10¹⁰TU / kg, approximately 3.1 x 10¹⁰TU / kg, approximately 3.0 x 10¹⁰TU / kg, approximately 2.9 x 10¹⁰TU / kg, approximately 2.8 x 10¹⁰TU / kg, approximately 2.7 x 10¹⁰TU / kg, approximately 2.6 x 10¹⁰TU / kg, approximately 2.5 x 10¹⁰TU / kg, approximately 2.4 x 10¹⁰TU / kg, approximately 2.3 x 10¹⁰TU / kg, approximately 2.2 x 10¹⁰TU / kg, approximately 2.1 x 10¹⁰TU / kg, approximately 2.0 x 10¹⁰TU / kg, approximately 1.9 x 1010TU / kg, approximately 1.8 x 1010TU / kg, approximately 1.7 x 1010TU / kg, approximately 1.6 x 1010TU / kg, approximately 1.5 x 1010TU / kg, approximately 1.4 x 1010TU / kg, approximately 1.3 x 1010TU / kg, approximately 1.2 x 1010TU / kg,approximately 1.1 x 10¹⁰ TU / kg or approximately 1.0 x 10¹⁰ TU / kg. [0 113] In some modalities, the dose is approximately 9.9 x 10⁹TU / kg, approximately 9.8 x 10⁹TU / kg, approximately 9.7 x 10⁹TU / kg, approximately 9.6 x 10⁹TU / kg, approximately 9.5 x 10⁹TU / kg, approximately 9.4 x 10⁹TU / kg, approximately 9.3 x 10⁹TU / kg, approximately 9.2 x 10⁹TU / kg, approximately 9.1 x 10⁹TU / kg, approximately 9.0 x 10⁹TU / kg, approximately 8.9 x 10⁹TU / kg, approximately 8.8 x 10⁹TU / kg, approximately 8.7 x 10⁹TU / kg, approximately 8.6 x 10⁹TU / kg, approximately 8.5 x 10⁹TU / kg, approximately 8.4 x 109TU / kg, approximately 8.3 x 109TU / kg, approximately 8.2 x 109TU / kg, approximately 8.1 x 109TU / kg, approximately 8.0 x 109TU / kg, approximately 7.9 x 109TU / kg, approximately 7.8 x 109TU / kg, approximately 7.7 x 109TU / kg, approximately 7.6 x 109TU / kg, approximately 7.5 x 109TU / kg, approximately 7.4 x 109TU / kg, approximately 7.3 x 109TU / kg, approximately 7.2 x 109TU / kg, approximately 7.1 x 109TU / kg, approximately 7.0 x 109TU / kg, approximately 6.9 x 109TU / kg, approximately 6.8 x 109TU / kg, approximately 6.7 x 109TU / kg, approximately 6.6 x 109TU / kg, Petition 870260067982, dated 09 / 07 / 2026, p. 75 / 275 56 / 233 approximately 6.5 x 10⁹TU / kg, approximately 6.4 x 10⁹TU / kg, approximately 6.3 x 10⁹TU / kg, approximately 6.2 x 10⁹TU / kg, approximately 6.1 x 10⁹TU / kg, approximately 6.0 x 10⁹TU / kg, approximately 5.9 x 10⁹TU / kg, approximately 5.8 x 10⁹TU / kg, approximately 5.7 x 10⁹TU / kg, approximately 5.6 x 10⁹TU / kg, approximately 5.5 x 10⁹TU / kg, approximately 5.4 x 10⁹TU / kg, approximately 5.3 x 10⁹TU / kg, approximately 5.2 x 10⁹TU / kg, approximately 5.1 x 10⁹TU / kg, approximately 5.0 x 109TU / kg, approximately 4.9 x 109TU / kg, approximately 4.8 x 109TU / kg, approximately 4.7 x 109TU / kg, approximately 4.6 x 109TU / kg, approximately 4.5 x 109TU / kg, approximately 4.4 x 109TU / kg, approximately 4.3 x 109TU / kg, approximately 4.2 x 109TU / kg, approximately 4.1 x 109TU / kg, approximately 4.0 x 109TU / kg, approximately 3.9 x 109TU / kg, approximately 3.8 x 109TU / kg, approximately 3.7 x 109TU / kg, approximately 3.6 x 109TU / kg, approximately 3.5 x 109TU / kg, approximately 3.4 x 109TU / kg, approximately 3.3 x 109TU / kg, approximately 3.2 x 109TU / kg, approximately 3.1 x 109TU / kg, approximately 3.0 x 109TU / kg, approximately 2.9 x 109TU / kg, approximately 2.8 x 109TU / kg, approximately 2.7 x 109TU / kg, approximately 2,6 x 10⁹TU / kg, approximately 2.5 x 10⁹TU / kg, approximately 2.4 x 10⁹TU / kg, approximately 2.3 x 10⁹TU / kg, approximately 2.2 x 10⁹TU / kg, approximately 2.1 x 10⁹TU / kg, approximately 2.0 x 10⁹TU / kg, approximately 1.9 x 10⁹TU / kg, approximately 1.8 x 10⁹TU / kg, approximately 1.7 x 10⁹TU / kg, approximately 1.6 x 10⁹TU / kg, approximately 1.5 x 10⁹TU / kg, approximately 1.4 x 10⁹TU / kg, approximately 1.3 x 10⁹TU / kg, approximately 1.2 x 10⁹TU / kg, approximately 1.1 x 10⁹TU / kg or approximately 1.0 x 109TU / kg.,

[0114] In some modalities, the dose is approximately 9.9 x 10⁸TU / kg, approximately 9.8 x 10⁸TU / kg, approximately 9.7 x 10⁸TU / kg, approximately 9.6 x 10⁸TU / kg, approximately 9.5 x 10⁸TU / kg, approximately 9.4 x 10⁸TU / kg, approximately 9.3 x 10⁸TU / kg, approximately 9.2 x 10⁸TU / kg, approximately 9.1 x 10⁸TU / kg, approximately 9.0 x 10⁸TU / kg, approximately 8.9 x 10⁸TU / kg, approximately 8.8 x 10⁸TU / kg, approximately 8.7 x 10⁸TU / kg, approximately 8.6 x 10⁸TU / kg, approximately 8.5 x 10⁸TU / kg, approximately 8.4 x 108TU / kg, about 8.3 x 108TU / kg, about 8.2 x 108TU / kg, about 8.1 x 108TU / kg, about 8.0 x 108TU / kg, about 7.9 x 108TU / kg, about 7.8 x 108TU / kg, about 7.7 x 108TU / kg, Petition 870260067982, dated 09 / 07 / 2026, page 76 / 275 57 / 233 approximately 7.6 x 10⁸TU / kg, approximately 7.5 x 10⁸TU / kg, approximately 7.4 x 10⁸TU / kg, approximately 7.3 x 10⁸TU / kg, approximately 7.2 x 10⁸TU / kg, approximately 7.1 x 10⁸TU / kg, approximately 7.0 x 10⁸TU / kg, approximately 6.9 x 10⁸TU / kg, approximately 6.8 x 10⁸TU / kg, approximately 6.7 x 10⁸TU / kg, approximately 6.6 x 10⁸TU / kg, approximately 6.5 x 10⁸TU / kg, approximately 6.4 x 10⁸TU / kg, approximately 6.3 x 10⁸TU / kg, approximately 6.2 x 10⁸TU / kg, approximately 6.1 x 108TU / kg, approximately 6.0 x 108TU / kg, approximately 5.9 x 108TU / kg, approximately 5.8 x 108TU / kg, approximately 5.7 x 108TU / kg, approximately 5.6 x 108TU / kg, approximately 5.5 x 108TU / kg, approximately 5.4 x 108TU / kg, approximately 5.3 x 108TU / kg, approximately 5.2 x 108TU / kg, approximately 5.1 x 108TU / kg, approximately 5.0 x 108TU / kg, approximately 4.9 x 108TU / kg, approximately 4.8 x 108TU / kg, approximately 4.7 x 108TU / kg, approximately 4.6 x 108TU / kg, approximately 4.5 x 108TU / kg, approximately 4.4 x 108TU / kg, approximately 4.3 x 108TU / kg, approximately 4.2 x 108TU / kg, approximately 4.1 x 108TU / kg, approximately 4.0 x 108TU / kg, approximately 3.9 x 108TU / kg, approximately 3.8 x 108TU / kg, approximately 3,7 x 108TU / kg, about 3.6 x 108TU / kg, about 3.5 x 108TU / kg, about 3.4 x 108TU / kg, about 3.3 x 108TU / kg, about 3.2 x 108TU / kg, about 3.1 x 108TU / kg, about 3.0 x 108TU / kg, about of 2.9 x 108TU / kg, about 2.8 x 108TU / kg, about 2.7 x 108TU / kg, about 2.6 x 108TU / kg, about 2.5 x 108TU / kg, about 2.4 x 108TU / kg, about 2.3 x 108TU / kg, about 2.2 108TU / kg, about 2.1 x 108TU / kg, about 2.0 x 108TU / kg, about 1.9 x 108TU / kg, about 1.8 x 108TU / kg, about 1.7 x 108TU / kg, about 1.6 x 108TU / kg, about 1.5 x 108TU / kg, about 1.4 x 108TU / kg, about 1.3 x 108TU / kg, about 1.2 x 108TU / kg, about 1.1 x 108TU / kg or about 1.0 x 108TU / kg.,

[0115] In some forms, the dose is less than 5.0 x 1010 TU / kg, less than 4.9 x 1010TU / kg, less than 4.8 x 1010TU / kg, less than 4.7 x 1010TU / kg, less than 4.6 x 1010TU / kg, less than 4.5 x 1010TU / kg, less than 4.4 x 1010TU / kg, less than 4.3 x 1010TU / kg, less than 4.2 x 1010TU / kg, less than 4.1 x 1010TU / kg, less than 4.0 x 1010 Petition 870260067982, dated 09 / 07 / 2026, page 77 / 275 58 / 233 TU / kg, less than 3.9 x 10¹⁰TU / kg, less than 3.8 x 10¹⁰TU / kg, less than 3.7 x 10¹⁰TU / kg, less than 3.6 x 10¹⁰TU / kg, less than 3.5 x 10¹⁰TU / kg, less than 3.4 x 10¹⁰TU / kg, less than 3.3 x 10¹⁰TU / kg, less than 3.2 x 10¹⁰TU / kg, less than 3.1 x 10¹⁰TU / kg, less than 3.0 x 10¹⁰TU / kg, less than 2.9 x 10¹⁰TU / kg, less than 2.8 x 10¹⁰TU / kg, less than 2.7 x 10¹⁰TU / kg, less than 2.6 x 10¹⁰TU / kg, less than 2.5 x 10¹⁰TU / kg, less less than 2.4 x 1010TU / kg, less than 2.3 x 1010TU / kg, less than 2.2 x 1010TU / kg, less than 2.1 x 1010TU / kg, less than 2.0 x 1010TU / kg, less than 1.9 x 1010TU / kg, less than 1.8 x 1010TU / kg, less than 1.7 x 1010TU / kg, less than 1.6 x 1010TU / kg, less than 1.5 x 1010TU / kg, less than 1.4 x 1010TU / kg, less than 1.3 x 1010TU / kg, less than 1.2 x 1010TU / kg, less than 1.1 x 1010TU / kg or less than 1.0 x 1010TU / kg.

[0116] In some forms, the dose is less than 9.9 x 109 TU / kg, less than 9.8 x 10⁹TU / kg, less than 9.7 x 10⁹TU / kg, less than 9.6 x 10⁹TU / kg, less than 9.5 x 10⁹TU / kg, less than 9.4 x 10⁹TU / kg, less than 9.3 x 10⁹TU / kg, less than 9.2 x 10⁹TU / kg, less than 9.1 x 10⁹TU / kg, less than 9.0 x 10⁹TU / kg, less than 8.9 x 10⁹TU / kg, less than 8.8 x 10⁹TU / kg, less than 8.7 x 10⁹TU / kg, less than 8.6 x 10⁹TU / kg, less than 8.5 x 10⁹TU / kg, less than 8.4 x 10⁹TU / kg, less than 8.3 x 10⁹TU / kg, less less than 8.2 x 10⁹TU / kg, less than 8.1 x 10⁹TU / kg, less than 8.0 x 10⁹TU / kg, less than 7.9 x 10⁹TU / kg, less than 7.8 x 10⁹TU / kg, less than 7.7 x 10⁹TU / kg, less than 7.6 x 10⁹TU / kg, less than 7.5 x 10⁹TU / kg, less than 7.4 x 10⁹TU / kg, less than 7.3 x 10⁹TU / kg, less than 7.2 x 10⁹TU / kg, less than 7.1 x 10⁹TU / kg, less than 7.0 x 10⁹TU / kg, less than 6.9 x 10⁹TU / kg, less than 6.8 x 10⁹TU / kg, less than 6.7 x 10⁹TU / kg, less than 6.6 x 109TU / kg, less than 6.5 x 109TU / kg, less than 6.4 x 109TU / kg, less than 6.3 x 109TU / kg, less than 6.2 x 109TU / kg, less than 6.1 x 109TU / kg, less than 6.0 x 109TU / kg, less than 5,9 x 10⁹TU / kg, less than 5.8 x 10⁹TU / kg, less than 5.7 x 10⁹TU / kg, less, Petition 870260067982, dated 09 / 07 / 2026, p. 78 / 275 59 / 233 of 5.6 x 10⁹TU / kg, less than 5.5 x 10⁹TU / kg, less than 5.4 x 10⁹TU / kg, less than 5.3 x 10⁹TU / kg, less than 5.2 x 10⁹TU / kg, less than 5.1 x 10⁹TU / kg, less than 5.0 x 10⁹TU / kg, less than 4.9 x 10⁹TU / kg, less than 4.8 x 10⁹TU / kg, less than 4.7 x 10⁹TU / kg, less than 4.6 x 10⁹TU / kg, less than 4.5 x 10⁹TU / kg, less than 4.4 x 10⁹TU / kg, less than 4.3 x 10⁹TU / kg, less than 4.2 x 10⁹TU / kg, less than 4.1 x 10⁹TU / kg, less than 4.0 x 10⁹ TU / kg, less than 3.9 x 10⁹ TU / kg, less than 3.8 x 10⁹ TU / kg, less than 3.7 x 10⁹ TU / kg, less than 3.6 x 10⁹ TU / kg, less than 3.5 x 10⁹ TU / kg, less than 3.4 x 10⁹ TU / kg, less than 3.3 x 10⁹ TU / kg, less than 3.2 x 10⁹ TU / kg, less than 3.1 x 10⁹ TU / kg, less than 3.0 x 10⁹ TU / kg, less than 2.9 x 10⁹ TU / kg, less than 2.8 x 10⁹ TU / kg, less than 2.7 x 10⁹ TU / kg, less than 2.6 x 10⁹ TU / kg, less than 2.5 x 10⁹ TU / kg, less than 2.4 x 109TU / kg, less than 2.3 x 109TU / kg, less than 2.2 x 109TU / kg, less than 2.1 x 109TU / kg, less than 2.0 x 109TU / kg, less than 1.9 x 109TU / kg, less than 1.8 x 109TU / kg, less than 1.7 x 109TU / kg,less than 1.6 x 10⁹ TU / kg, less than 1.5 x 10⁹ TU / kg, less than 1.4 x 10⁹ TU / kg, less than 1.3 x 10⁹ TU / kg, less than 1.2 x 10⁹ TU / kg, less than 1.1 x 10⁹ TU / kg or less than 1.0 x 10⁹ TU / kg.

[0117] In some forms, the dose is less than 9.9 x 108 TU / kg, less than 9.8 x 108TU / kg, less than 9.7 x 108TU / kg, less than 9.6 x 108TU / kg, less than 9.5 x 108TU / kg, less than 9.4 x 108TU / kg, less than 9.3 x 108TU / kg, less than 9.2 x 108TU / kg, less than 9.1 108TU / kg, less than 9.0 x 108TU / kg, less than 8.9 x 108TU / kg, less than 8.8 x 108TU / kg, less than 8.7 x 108TU / kg, less than 8.6 x 108TU / kg, less than 8.5 x 108TU / kg, less than 8.4 x 108TU / kg, less than 8.3 x 108TU / kg, less of 8.2 x 108TU / kg, less than 8.1 x 108TU / kg, less than 8.0 x 108TU / kg, less than 7.9 x 108TU / kg, less than 7.8 x 108TU / kg, less than 7.7 x 108TU / kg, less than 7.6 x 108TU / kg, less than 7.5 x 108TU / kg, less than 7.4 x 108TU / kg, less than 7.3 x 108TU / kg, less than 7.2 x 108TU / kg, less than 7.1 x 108TU / kg, less than 7.0 x 108TU / kg, Petition 870260067982, dated 09 / 07 / 2026, p. 79 / 275 60 / 233 less than 6.9 x 10⁸TU / kg, less than 6.8 x 10⁸TU / kg, less than 6.7 x 10⁸TU / kg, less than 6.6 x 10⁸TU / kg, less than 6.5 x 10⁸TU / kg, less than 6.4 x 10⁸TU / kg, less than 6.3 x 10⁸TU / kg, less than 6.2 x 10⁸TU / kg, less than 6.1 x 10⁸TU / kg, less than 6.0 x 10⁸TU / kg, less than 5.9 x 10⁸TU / kg, less than 5.8 x 10⁸TU / kg, less than 5.7 x 10⁸TU / kg, less than 5.6 x 10⁸TU / kg, less than 5.5 x 10⁸TU / kg, less than 5.4 x 108TU / kg, less than 5.3 x 108TU / kg, less than 5.2 x 108TU / kg, less than 5.1 x 108TU / kg, less than 5.0 x 108TU / kg, less than 4.9 x 108TU / kg, less than 4.8 x 108TU / kg, less than 4.7 x 108TU / kg, less than 4.6 x 108TU / kg, less than 4.5 x 108TU / kg, less than 4.4 x 108TU / kg, less than 4.3 x 108TU / kg, less than 4.2 x 108TU / kg, less than 4.1 x 108TU / kg, less than 4.0 x 108TU / kg, less than 3.9 x 108TU / kg, less than 3.8 x 108TU / kg, less than 3.7 x 108TU / kg, less than 3.6 x 108TU / kg, less than 3.5 x 108TU / kg, less than 3.4 x 108TU / kg, less than 3.3 x 108TU / kg, less than 3.2 x 108TU / kg, less than 3.1 x 108TU / kg, less than 3,0 x 108TU / kg, less than 2.9 x 108TU / kg, less than 2.8 x 108TU / kg, less than 2.7 x 108TU / kg, less than 2.6 x 108TU / kg, less than 2.5 x 108TU / kg, less than 2.4 x 108TU / kg, less than 2.3 x 108TU / kg, less of 2.2 x 108TU / kg, less than 2.1 x 108TU / kg, less than 2.0 x 108TU / kg, less than 1.9 x 108TU / kg, less than 1.8 x 108TU / kg, less than 1.7 x 108TU / kg, less than 1.6 x 108TU / kg, less than 1.5 x 108TU / kg, less than 1.4x 108TU / kg, less than 1.3 x 108TU / kg, less than 1.2 x 108TU / kg, less than 1.1 x 108TU / kg or less than 1.0 x 108TU / kg.,

[0118] In some modalities, the dose is between 1 x 10⁸TU / kg and 10¹⁰TU / kg, between 1.5 x 10⁸TU / kg and 5 x 10¹⁰TU / kg, between 2 x 10⁸TU / kg and 5 x 10¹⁰TU / kg, between 2.5 x 10⁸TU / kg and 5 x 10¹⁰TU / kg, between 3 x 10⁸TU / kg and 5 x 10¹⁰TU / kg, between 3.5 x 10⁸TU / kg and 5 x 10¹⁰TU / kg, between 4 x 10⁸TU / kg and 5 x 10¹⁰TU / kg, between 4.5 x 10⁸TU / kg and 5 x 10¹⁰TU / kg, between 5 x 10⁸TU / kg and 5 x 10¹⁰TU / kg, between 5.5 x 108 TU / kg and 5 x 1010 TU / kg, between 6 x 108 TU / kg and 5 x 1010 TU / kg, between 6.5 x 108 TU / kg Petition 870260067982, dated 09 / 07 / 2026, p. 80 / 275 61 / 233 and 5 x 1010TU / kg, between 7 x 108TU / kg and 5 x 1010TU / kg, between 7.5 x 108TU / kg and 5 x 1010TU / kg, between 8 x 108TU / kg and 5 x 1010TU / kg, between 8.5 x 108TU / kg and 5 x 1010TU / kg, between 9 x 108TU / kg and 5 x 1010TU / kg, between 9.5 x 108TU / kg and 5 x 1010TU / kg, between 1 x 109TU / kg and 5 x 1010TU / kg, between 1.5 x 109TU / kg and 5 x 1010TU / kg, between 2 x 109TU / kg and 5 x 1010TU / kg, between 2.5 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 3 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 3.5 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 4 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 4.5 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 5 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 5.5 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 6 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 6.5 x 10⁹ TU / kg and 5 x 10¹⁰ TU / kg, between 7 x 10⁹ TU / kg and 5 x 1010TU / kg, between 7.5 x 109TU / kg and 5 x 1010TU / kg, between 8 x 109TU / kg and 5 x 1010TU / kg, between 8.5 x 109TU / kg and 5 x 1010TU / kg, between 9 x 109TU / kg and 5 x 1010TU / kg, between 9.5 x 109TU / kg and 5 x 1010TU / kg, between 1010TU / kg and 5 x 1010TU / kg, between 1.5 x 1010TU / kg and 5 x 1010TU / kg,between 2 x 1010TU / kg and 5 x 1010TU / kg, between 2.5 x 1010TU / kg and 5 x 1010TU / kg, between 3 x 1010TU / kg and 5 x 1010TU / kg, between 3.5 x 1010TU / kg and 5 x 1010TU / kg, between 4 x 1010TU / kg and 5 x 1010TU / kg, or between 4.5 x 1010TU / kg and 5 x 1010TU / kg.

[0119] In some modalities, the dose is between 1 x 10⁸TU / kg and 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 4.5 x 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 4 x 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 3.5 x 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 3 x 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 2.5 x 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 2 x 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 1.5 x 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 10¹⁰TU / kg, between 1 x 10⁸TU / kg and 9 x 109TU / kg, between 1 x 108TU / kg and 8.5 x 109TU / kg, between 1 x 108TU / kg and 8 x 109TU / kg, between 1 x 108TU / kg and 7.5 x 109TU / kg, between 1 x 108TU / kg and 7 x 109TU / kg, between 1 x 108TU / kg and 6.5 x 109TU / kg, between 1 x 108TU / kg and 6 x 109TU / kg, between 1 x 108TU / kg and 5.5 x 109TU / kg, between 1 x 108TU / kg and 5 x 109TU / kg, between 1 x 108TU / kg and 4.5 x 109TU / kg, between 1 x 108TU / kg and 4 x 109TU / kg, between 1 x 108TU / kg and 3.5 x 109TU / kg, between Petition 870260067982, dated 09 / 07 / 2026, page 81 / 275 62 / 233 x 10⁸TU / kg and 3 x 10⁹TU / kg, between 1 x 10⁸TU / kg and 2.5 x 10⁹TU / kg, between 1 x 10⁸TU / kg and 2 x 10⁹, between 1 x 10⁸TU / kg and 1.5 x 10⁹TU / kg, between 1 x 10⁸TU / kg and 1 x 10⁹TU / kg, between 1 x 10⁸TU / kg and 9.5 x 10⁸ TU / kg, between 1 x 10⁸ TU / kg and 9 x 10⁸ TU / kg, between 1 x 10⁸ TU / kg and 8.5 x 108TU / kg, between 1 x 108TU / kg and 8 x 108TU / kg, between 1 x 108TU / kg and 7.5 x 108TU / kg, between 1 x 108TU / kg and 7 x 108TU / kg, between 1 x 108TU / kg and 6.5 x 108TU / kg, between 1 x 108TU / kg and 6 x 108TU / kg, between 1 x 108TU / kg and 5.5 x 108TU / kg, between 1 x 108TU / kg and 5 x 108TU / kg, between 1 x 108TU / kg and 4.5 x 108TU / kg, between 1 x 108TU / kg and 4 x 108TU / kg, between 1 x 108TU / kg and 3.5 x 108TU / kg, between 1 x 108TU / kg and 3 x 108TU / kg, between 1 x 108TU / kg and 2.5 x 108TU / kg, between 1 x 108TU / kg and 2 x 108TU / kg, or between 1 x 108TU / kg and 1.5 x 108TU / kg,

[0120] In some modalities, the dose is between 1 x 1010TU / kg and 2 x 1010TU / kg, between 1.1 x 1010TU / kg and 1.9 x 1010TU / kg, between 1.2 x 1010TU / kg and 1.8 x 1010TU / kg, between 1.3 x 1010TU / kg and 1.7 x 1010TU / kg or between 1.4 x 1010TU / kg and 1.6 x 1010TU / kg. In some modalities, the dose is approximately 1.5 x 1010TU / kg. In some modalities, the dose is 1.5 x 1010TU / kg.

[0121] In some modalities, the dose is between 1 x 109TU / kg and 109TU / kg, between 1.1 x 109TU / kg and 1.9 x 109TU / kg, between 1.2 x 109TU / kg and 1.8 x 109TU / kg, between 1.3 x 109TU / kg and 1.7 x 109TU / kg or between 1.4 x 109TU / kg and 1.6 x 109TU / kg. In some modalities, the dose is 1.5 x 109TU / kg. In certain modalities, the dose is approximately 3.0 x 109TU / kg.

[0122] In some modalities, the dose is between 2.5 x 109TU / kg and 3.5 x 109TU / kg, between 2.6 x 109TU / kg and 3.4 x 109TU / kg, between 2.7 x 109TU / kg and 3.3 x 109TU / kg, between 2.8 x 109TU / kg and 3.2 x 109TU / kg or between 2.9 x 109TU / kg and 3.1 x 109TU / kg. In some modalities, the dose is approximately 3.0 x 109TU / kg. In some modalities, the dose is 3.0 x 109TU / kg. Petition 870260067982, dated 09 / 07 / 2026, page 82 / 275 63 / 233

[0123] In some modalities, the dose is between 5.5 x 109TU / kg and 6.5 x 109TU / kg, between 5.6 x 109TU / kg and 6.4 x 109TU / kg, between 5.7 x 109TU / kg and 6.3 x 109TU / kg, between 5.8 x 109TU / kg and 6.2 x 109TU / kg or between 5.9 x 109TU / kg and 6.1 x 109TU / kg. In some modalities, the dose is approximately 6.0 x 109TU / kg. In some modalities, the dose is 6.0 x 109TU / kg.

[0124] In some embodiments, the plasma activity of FVIII at 24 hours, 36 hours or 48 hours after administration of the lentiviral vector of the present invention is increased relative to the plasma activity of FVIII in an individual who received a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16.

[0125] In some embodiments, the plasma activity of FVIII after 48 hours of lentiviral vector administration is increased relative to the plasma activity of FVIII in an individual who received a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16.

[0126] In another embodiment, plasma FVIII activity is increased by approximately 21 days after administration of the lentiviral vector compared to an individual who received 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.

[0127] In some modalities, plasma FVIII activity is increased in about 6 hours, in about 12 hours, in about 18 hours, in about 24 hours, in about 36 hours, in about 48 hours, in about 3 days, in about 4 days, in about 5 days, in about 6 days, in about 7 days, in about 8 days, in about 9 days, in about 10 days, in about 11 days, in about 12 days, Petition 870260067982, dated 09 / 07 / 2026, p. 83 / 275 64 / 233 in approximately 13 days, in approximately 14 days, in approximately 15 days, in approximately 16 days, in approximately 17 days, in approximately 18 days, in approximately 19 days, in approximately 20 days, in approximately 21 days, in approximately 22 days, in approximately 23 days, in approximately 24 days, in approximately 25 days, in approximately 26 days, in approximately 27 days, or in approximately 28 days after administration of the lentiviral vector in relation to an individual who received a reference nucleic acid molecule comprising SEQ ID NO: 16, a reference viral vector comprising the reference nucleic acid molecule, or a polypeptide encoded by the reference nucleic acid molecule.

[0128] In some modalities, the plasma activity of FVIII in the individual is increased by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 95 times,at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, or at least about 200 times relative to the levels, Petition 870260067982, dated 09 / 07 / 2026, p. 84 / 275 65 / 233 baseline levels in the individual, relative to levels in an individual who received a reference nucleic acid molecule comprising SEQ ID NO: 16, relative to levels in an individual who received a reference viral vector comprising the reference nucleic acid molecule, or relative to levels in an individual after administration of a polypeptide encoded by the reference nucleic acid molecule.

[0129] In some embodiments, the lentiviral vector is administered as a single dose or multiple doses. In some embodiments, the lentiviral vector dose is administered at once or divided into multiple subdoses, for example, two subdoses, three subdoses, four subdoses, five subdoses, six subdoses, or more than six subdoses. In some embodiments, more than one lentiviral vector is administered.

[0130] In some embodiments, the lentiviral vector dose is administered repeatedly at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. In some embodiments, the lentiviral vector is administered via intravenous injection.

[0131] In some modalities, the individual is a pediatric individual while, in other aspects, the individual is an adult individual.

[0132] In some embodiments, the lentiviral vector comprises at least one tissue-specific promoter, that is, a promoter that would regulate the expression of the FVIII-active polypeptide in a particular tissue or cell type. In some embodiments, a tissue-specific promoter in the lentiviral vector selectively enhances the expression of the FVIII-active polypeptide in a target liver cell. In some embodiments, the tissue-specific promoter that selectively enhances the expression of Petition 870260067982, dated 09 / 07 / 2026, page 85 / 275 66 / 233 polypeptide with FVIII activity in a target liver cell comprises an mTTR promoter. In some embodiments, the target liver cell is a hepatocyte.

[0133] Since the lentiviral vector can transduce all types of liver cells, the expression of the transgene (e.g., FVIII) in different cell types can be controlled using different promoters in the lentiviral vector. Thus, the lentiviral vector may comprise specific promoters that would control the expression of the FVIII transgene in different tissue or cell types, such as different liver tissues or cell types. Thus, in some embodiments, the lentiviral vector may comprise an endothelium-specific promoter that would control the expression of the FVIII transgene in liver endothelial tissue or a hepatocyte-specific promoter that would control the expression of the FVIII transgene in hepatocytes, or both.

[0134] In some embodiments, the lentiviral vector comprises a tissue-specific promoter or tissue-specific promoters that control the expression of the FVIII transgene in tissues other than the liver. In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of the target cell or target tissue, for example, into the genome of a hepatocyte or into the genome of a liver endothelial cell.

[0135] In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity in the lentiviral vector of the present invention comprises, consists of, or essentially consists of LV-coFVIII-6 (SEQ ID NO: 71).

[0136] In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity in the lentiviral vector of the present invention comprises, consists of, or essentially consists of LV-coFVIII-6-XTEN (SEQ ID NO: 72). Petition 870260067982, dated 09 / 07 / 2026, page 86 / 275 67 / 233

[0137] In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity in the lentiviral vector of the present invention further comprises a nucleic acid sequence encoding a signaling peptide, wherein the nucleic acid sequence encoding a signaling 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 with (i) nucleotides 1 to 57 of SEQ ID NO: 1; (ii) nucleotides 1 to 57 of SEQ ID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ ID NO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) nucleotides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.

[0138] In some embodiments, the nucleic acid molecule isolated in a lentiviral vector of the present invention comprises one or more properties selected from the group consisting of: (a) the human codon matching index of the nucleic acid molecule or a portion thereof is increased relative to SEQ ID NO: 16; (b) the ideal codon frequency of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16; (d) the relative synonym codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (e) the effective number of codons in the nucleotide sequence or a portion thereof is reduced in relation to SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21) Petition 870260067982, dated 09 / 07 / 2026, p. 87 / 275 68 / 233 and 22) in relation to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) in relation to SEQ ID NO: 16; and (h) any combination thereof.

[0139] In some embodiments, the nucleic acid molecule isolated in a lentiviral vector of the present invention 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 a constant region of immunoglobulin or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence. In some embodiments, the heterologous amino acid sequence is linked to the N-terminal or C-terminal 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.

[0140] In some embodiments, the FVIII polypeptide is a full-length FVIII or an FVIII with a deleted B domain.

[0141] The lentiviral vectors described herein can be used in low doses (e.g., 1010TU / kg or less, 109TU / kg or less, or 108TU / kg or less) in vivo in a mammal, for example, a human patient, using a gene therapy approach for the treatment of a hemorrhagic disease or disorder selected from the group consisting of a hemorrhagic coagulation disorder, hemarthrosis, muscle bleeding, oral hemorrhage, hemorrhage, muscle hemorrhage, oral bleeding, trauma, capite trauma, gastrointestinal hemorrhage, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system hemorrhage, retropharyngeal space hemorrhage, retroperitoneal space hemorrhage, and iliopsoas sheath hemorrhage, which would be therapeutically beneficial. In Petition 870260067982, dated 09 / 07 / 2026, page 88 / 275 69 / 233 In one embodiment, the bleeding disorder or disease is hemophilia. In another embodiment, the bleeding disorder or disease is hemophilia A.

[0142] In some embodiments, target cells (e.g., hepatocytes) are treated in vitro with low doses (e.g., 1010 TU / kg or less, 109 TU / kg or less, or 108 TU / kg or less) of the lentiviral vectors described herein before being administered to the patient. In certain embodiments, target cells (e.g., hepatocytes) are treated in vitro with approximately 3.0 x 109 TU / kg of the lentiviral vectors described herein before being administered to the patient. In yet another embodiment, the patient's cells (e.g., hepatocytes) are treated ex vivo with low doses (e.g., 1010 TU / kg or less, 109 TU / kg or less, or 108 TU / kg or less) of the lentiviral vectors described herein before being administered to the patient.

[0143] In some embodiments, the plasma activity of FVIII after administration of a lentiviral vector described herein (administered, for example, at 1010 TU / kg or less, 109 TU / kg or less or 108 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 %, at least about 250%, at least about 260 %, at least about 270%, at least about 280%, at least about 290%, or at least about 300% relative to physiologically normal circulating FVIII levels.

[0144] In one embodiment, the plasma activity of FVIII after administration of a lentiviral vector of the present invention is increased by at least about 3,000% to about 5,000% compared to Petition 870260067982, dated 09 / 07 / 2026, p. 89 / 275 70 / 233 physiologically normal levels of FVIII in circulation. In some embodiments, 21 days after administration of a lentiviral vector comprising a polypeptide encoding a gene with Factor VIII (FVIII) activity-optimized codons described herein, plasma FVIII activity is increased by at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at 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 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold,at least approximately 190 times or at least approximately 200 times relative to an individual who received a lentiviral vector comprising a reference nucleic acid molecule comprising SEQ ID NO: 16.,

[0145] The present invention also provides methods of treating, preventing or improving a hemostatic disorder (for example, a bleeding disorder, such as hemophilia A) in an individual who needs the same, comprising administering to the individual a therapeutically effective amount of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the lentiviral vector is administered at a dose of at least 5 x 1010 or less TU / kg, 109TU / kg or less or 108TU / kg or less.

[0146] The lentiviral vector treatment, enhancement, and prevention of the present invention may be a bypass therapy. The individual receiving bypass therapy may already have developed an inhibitor to a Petition 870260067982, dated 09 / 07 / 2026, page 90 / 275 71 / 233 coagulation factor, for example, FVIII, or this individual is developing a coagulation factor inhibitor.

[0147] The lentiviral vectors of the present invention treat or prevent a hemostatic disorder by promoting the formation of a fibrin clot. The polypeptide containing FVIII activity encoded by the nucleic acid molecule of the invention can activate a member of a coagulation cascade. The coagulation factor can be a participant in the extrinsic pathway, the intrinsic pathway, or both.

[0148] The lentiviral vectors of the present invention can be used to treat hemostatic disorders known to be treatable with FVIII. Hemostatic disorders that can be treated using the methods of the invention include, but are not limited to, hemophilia A, hemophilia B, von Willebrand disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency, as well as deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor VII, Factor X or XIII, hemarthrosis, muscle hemorrhage, oral hemorrhage, hemorrhage, bleeding in muscles, oral bleeding, trauma, head trauma, gastrointestinal hemorrhage, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system hemorrhage, hemorrhage in the retropharyngeal space, hemorrhage in the retroperitoneal space and hemorrhage in the iliopsoas sheath.

[0149] Compositions for administration to an individual include lentiviral vectors comprising nucleic acid molecules comprising an optimized nucleotide sequence of the invention encoding a coagulation factor FVIII (for gene therapy applications), as well as FVIII polypeptide molecules. In some embodiments, the composition for administration is a cell in contact with a lentiviral vector of the present invention in vivo, in vitro or ex vivo. Petition 870260067982, dated 09 / 07 / 2026, page 91 / 275 72 / 233

[0150] In some forms, hemostatic disorder is an inherited disorder. In one form, the individual has hemophilia A. In other forms, hemostatic disorder is the result of an FVIII deficiency. In other forms, hemostatic disorder may be the result of a defective FVIII coagulation factor.

[0151] In another modality, hemostatic disorder can be an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, for example, but without limitation, cancer, autoimmune disease, or pregnancy. The acquired disorder can result from old age or medications to treat an underlying secondary disorder (e.g., chemotherapy for cancer).

[0152] The invention also relates to methods of treating an individual who does not have a hemostatic disorder or a secondary disease or condition that results in the acquisition of a hemostatic disorder. The invention thus relates to a method of treating an individual who needs a hemostatic agent in general, comprising administering a therapeutically effective amount of a lentiviral vector of the present invention. For example, in one embodiment, the individual who needs a hemostatic agent in general is undergoing or is about to undergo surgery. The lentiviral vector of the invention may be administered before or after surgery as a prophylactic.

[0153] The lentiviral vector of the invention can be administered during or after surgery to control an episode of acute bleeding. Surgery may include, among others, liver transplantation, hepatic resection, or stem cell transplantation.

[0154] In another embodiment, the lentiviral vector of the invention can be used to treat an individual who has an episode of acute bleeding who does not have a hemostatic disorder. The episode of Petition 870260067982, dated 09 / 07 / 2026, page 92 / 275 73 / 233 Acute hemorrhage can result from severe trauma, for example, surgery, car accident, injuries, gunshot wounds, laceration, or any other traumatic event that results in uncontrolled bleeding.

[0155] The lentiviral vector can be used to prophylactically treat an individual who has a hemostatic disorder. The lentiviral vector can also be used to treat an episode of acute bleeding in an individual who has a hemostatic disorder.

[0156] In another embodiment, administration of a lentiviral vector described herein and / or subsequent expression of the FVIII protein transgene does not induce an immune response in an individual. In some embodiments, the immune response comprises the development of antibodies against FVIII. In some embodiments, the immune response comprises the secretion of cytokines. In some embodiments, the immune response comprises the activation of B cells, T cells, or both B cells and T cells. In some embodiments, the immune response is an inhibitory immune response, in which the immune response in the individual reduces the activity of the FVIII protein relative to the activity of FVIII in an individual who did not develop an immune response. In certain embodiments, the expression of the FVIII protein by administration of the lentiviral vector of the invention prevents an inhibitory immune response against the FVIII protein or the FVIII protein expressed from the isolated nucleic acid molecule or the lentiviral vector.

[0157] In some embodiments, a lentiviral vector of the invention is administered in combination with at least one other agent that promotes hemostasis. Said other agent promotes hemostasis in a therapy with demonstrated coagulation activity. By way of example, but not as a limitation, the hemostatic agent may include Factor V, Factor VII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, prothrombin or fibrinogen or activated forms thereof. Petition 870260067982, dated 09 / 07 / 2026, page 93 / 275 74 / 233 one of the previous ones. The coagulation factor or hemostatic agent may also include antifibrinolytic drugs, for example, epsilon-aminocaproic acid, tranexamic acid.

[0158] In one embodiment of the invention, the composition (e.g., the lentiviral vector) is one in which FVIII is present in an activatable form when administered to an individual. This activatable molecule can be activated in vivo at the coagulation site after administration to an individual.

[0159] The lentiviral vector of the invention can be administered intravenously, subcutaneously, intramuscularly, or through any mucosal surface, for example, oral, sublingual, buccal, nasal, rectal, vaginal, or pulmonary. The lentiviral vector can be implanted within or attached to a solid biopolymer support that allows for the slow release of the vector at the desired location.

[0160] In one embodiment, the route of administration of the lentiviral vectors is parenteral. The term parenteral, as used herein, includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. The intravenous form of parenteral administration is preferred. Although all these forms of administration are clearly considered to be within the scope of the invention, one form of administration would be a solution for injection, in particular for intravenous or intra-arterial injection or infusion. Typically, a pharmaceutical composition suitable for injection may comprise a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizing agent (e.g., human albumin), etc.However, in other methods consistent with the teachings presented here, the lentiviral vector can be delivered directly to the site of the adverse cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent. Petition 870260067982, dated 09 / 07 / 2026, page 94 / 275 75 / 233

[0161] 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 solution and buffered media. In the present invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer, or 0.8% saline solution. Other common parenteral carriers include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous carriers include fluid and nutrient replacement solutions, electrolyte replacement solutions such as those based on Ringer's dextrose, and so on.Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and so on.

[0162] More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (where soluble in water) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In these cases, the composition must be sterile and flowable to the extent that it flows easily into a syringe. It must be stable under the manufacturing and storage conditions and preferably protected against contamination by microorganisms such as bacteria and fungi. The vehicle may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Adequate flowability may be maintained, for example, by the use of a coating such as Petition 870260067982, dated 09 / 07 / 2026, page 95 / 275 76 / 233 lecithin, due to the maintenance of the particle size required in the case of dispersions and the use of surfactants.

[0163] Prevention of microorganism action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and so on. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including an agent that retards absorption in the composition, for example, aluminum monostearate and gelatin.

[0164] In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., an isolated polypeptide or in combination with other active agents) in the required quantity into an appropriate solvent with one or a combination of the ingredients listed herein, as needed, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which produce a powder of an active ingredient plus any desired additional ingredient from a previously sterilized filtered solution.Injection preparations are processed, filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions according to methods known in the art. Additionally, the preparations may be packaged and sold in kit form. Such manufactured articles will preferably have labels or leaflets indicating the compositions. Petition 870260067982, dated 09 / 07 / 2026, page 96 / 275 77 / 233 combinations are useful for treating an individual who is suffering from or predisposed to coagulation disorders.

[0165] The pharmaceutical composition may also be formulated for rectal administration as a suppository or retention enema, for example, containing bases for conventional suppositories, such as cocoa butter or other glycerides.

[0166] The effective doses of the compositions of the present invention for the treatment of conditions vary according to many different factors, including means of administration, target site, physiological state of the patient, whether human or animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human mammal, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

[0167] The lentiviral vector can be administered as a single dose or as multiple doses, wherein the multiple doses can be administered continuously or at specific time intervals. In vitro assays can be employed to determine ideal dose ranges and / or administration schedules. In vitro assays measuring coagulation factor activity are known in the art. In addition, effective doses can be extrapolated from dose-response curves obtained from animal models, for example, a hemophilic dog (Mount et al. 2002, Blood 99 (8): 2670).

[0168] Intermediate doses in the above ranges are also intended to be within the scope of the invention. Individuals may receive these doses daily, on alternate days, weekly, or according to any other schedule determined through empirical analysis. An exemplary treatment involves Petition 870260067982, dated 09 / 07 / 2026, page 97 / 275 78 / 233 administration in multiple doses over a prolonged period, for example, at least six months.

[0169] The lentiviral vector of the invention can be administered on multiple occasions. The intervals between single doses can be daily, weekly, monthly, or yearly. The intervals can also be irregular, as indicated by measuring the blood levels of the modified polypeptide or antigen in the patient. The dosage and frequency of the lentiviral vectors of the invention vary depending on the half-life of the FVIII polypeptide encoded by the transgene in the patient.

[0170] The dosage and frequency of administration of the lentiviral vectors of the invention may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the lentiviral vector of the invention are administered to a patient who is not yet in the disease state to increase the patient's resistance or minimize the effects of the disease. This amount is defined as an effective prophylactic 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.

[0171] The lentiviral vector of the invention may optionally be administered in combination with other agents that are effective in treating the disorder or condition that needs treatment (e.g., prophylactic or therapeutic).

[0172] As used herein, the administration of lentiviral vectors of the invention in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant, or contemporaneous administration or application of the therapy and the polypeptides described. Those skilled in the art will appreciate that the administration or application of the various components of the regimen Petition 870260067982, dated 09 / 07 / 2026, page 98 / 275 79 / 233 Combined therapeutic regimens can be programmed to increase the overall effectiveness of treatment. Those skilled in the art (e.g., a physician) will be able to discern effective combined therapeutic regimens without undue experimentation, based on the selected adjunctive therapy and the teachings of this descriptive report.

[0173] It will also be appreciated that the lentiviral vectors of the invention can be used in conjunction with or in combination with an agent or agents (for example, to provide a combined therapeutic regimen). Exemplary agents with which a lentiviral vector of the present invention can be combined include agents that represent the current standard of care for a specific disorder being treated. These agents may be of a chemical or biological nature. The term biological or biological agent refers to any pharmaceutically active agent made from living organisms and / or their products that is intended for use as a therapeutic product.

[0174] The amount of agent to be used in combination with the lentiviral vectors of the present invention may vary according to the individual or may 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 1233-1287 (Joel G. Hardman et al., eds., 9th ed. 1996). In another embodiment, an amount of such agent according to the standard of care is administered.

[0175] In certain embodiments, the lentiviral vectors of the present invention are administered in conjunction with an immunosuppressive, antiallergic or anti-inflammatory agent. These agents generally refer to substances that act to suppress or mask the immune system of the individual being treated herein. These agents include substances that suppress cytokine production, Petition 870260067982, dated 09 / 07 / 2026, p. 99 / 275 80 / 233 regulate or suppress autoantigen expression or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines; azathioprine; cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde; anti-idiotypic antibodies to MHC antigens and fragments; cyclosporine A; steroids, such as glucocorticosteroids, for example, prednisone, methylprednisolone, and dexamethasone; cytokine or cytokine receptor antagonists, including anti-interferon-γ, -β, or -α antibodies, anti-tumor necrosis factor-α antibodies, anti-tumor necrosis factor-β antibodies, anti-interleukin-2 antibodies, and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous antilymphocyte globulin; pan-T antibodies; soluble peptide containing an LFA-3 binding domain; streptokinase; TGF-β; streptodornase; FK506; RS-61443; deoxyspergualin; and rapamycin.In certain modalities, the agent is an antihistamine. An antihistamine, as used herein, is an agent that antagonizes the physiological effect of histamine. Examples of antihistamines include chlorpheniramine, diphenhydramine, promethazine, sodium cromolyn, astemizole, azatadine maleate, bropheniramine maleate, carbinoxamine maleate, cetirizine hydrochloride, clemastine fumarate, cyproheptadine hydrochloride, dexbrompheniramine maleate, dexchlorpheniramine maleate, dimenhydrinate, diphenhydramine hydrochloride, doxylamine succinate, fexofenadine hydrochloride, terfenadine hydrochloride, hydroxyzine hydrochloride, loratadine, meclizine hydrochloride, tripelennamine citrate, tripelennamine hydrochloride, and triprolidine hydrochloride.

[0176] Immunosuppressive, antiallergic, or anti-inflammatory agents may be incorporated into the lentiviral vector administration regimen. For example, administration of immunosuppressive or anti-inflammatory agents may begin before Petition 870260067982, dated 09 / 07 / 2026, page 100 / 275 81 / 233 administration of the lentiviral vectors described and may continue with one or more doses subsequently. In certain modalities, immunosuppressive or anti-inflammatory agents are administered as premedication to the lentiviral vectors.

[0177] As discussed previously, the lentiviral vectors of the present invention can be administered in a pharmaceutically effective amount for the in vivo treatment of coagulation disorders. In this regard, it will be appreciated that the lentiviral vectors of the invention can be formulated to facilitate administration and promote the stability of the active agent. Preferably, the pharmaceutical compositions according to the present invention comprise a pharmaceutically acceptable, non-toxic, sterile vehicle, such as physiological saline solution, non-toxic buffers, preservatives, and so forth. Obviously, the pharmaceutical compositions of the present invention can be administered in single or multiple doses to provide a pharmaceutically effective amount of the polypeptide.

[0178] Several tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic test, ROTEM® test, prothrombin time (PT) test (also used to determine INR), fibrinogen test (usually by the Clauss method), platelet count, platelet function test (usually by PFA-100), TCT, bleeding time, mixing test (if an abnormality is corrected if the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., Factor V Leiden, prothrombin G20210A mutation), diluted Russell's viper venom time (dRVVT), various platelet function tests, Petition 870260067982, dated 09 / 07 / 2026, page 101 / 275 82 / 233 thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, for example, ROTEM®) or euglobulin lysis time (ELT).

[0179] The aPTT test is a performance indicator that measures the effectiveness of both the intrinsic coagulation pathway (also called the contact activation pathway) and the common coagulation pathways. This test is commonly used to measure the coagulation activity of commercially available recombinant coagulation factors, for example, FVIII or FIX. It is used in conjunction with prothrombin time (PT), which measures the extrinsic pathway.

[0180] ROTEM® analysis provides information on the entire kinetics of hemostasis: clotting time, clot formation, clot stability, and lysis. The different parameters in thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or many factors that influence these interactions. This assay can provide a complete overview of secondary hemostasis. III. Lentiviral Vectors

[0181] 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 lentivirus vectors for gene therapy was reviewed in Klimatcheva et al. (1999) Frontiers in Bioscience 4: 481-496. The design and use of suitable lentiviral vectors for gene therapy are described, for example, in U.S. Patents Nos. 6,207,455 and 6,615,782. Examples of lentiviruses include, but are not limited to, the pseudotypes HIV-1, HIV-2, HIV-1 / HIV-2, HIV-1 / SIV, FIV, caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus, and bovine immunodeficiency virus.

[0182] A schematic representation of a lentiviral vector of Petition 870260067982, dated 09 / 07 / 2026, page 102 / 275 83 / 233 The present invention is shown in Figure 19. In some embodiments, the lentiviral vector of the present invention is a third-generation lentiviral vector. As used herein, the term third-generation lentiviral vector refers to a lentiviral engulfment system that has the characteristics of a second-generation vector system and that does not yet have a functional tat gene, such as one from which the tat gene has been eliminated or inactivated. Typically, the rev coding gene is provided in a separate expression construct. See, for example, Dull et al. (1998) J. Virol. 72: 8463-8471. As used herein, a second-generation lentiviral vector system refers to a lentiviral engulfment system that lacks functional accessory genes, such as one from which the accessory genes vif, vpr, vpu, and nef have been eliminated or inactivated. See, for example, Zufferey et al. (1997) Nat. Biotechnol. 15: 871-875.As used herein, engulfing system refers to a set of viral constructs comprising genes encoding viral proteins involved in the engulfment of a recombinant virus. Typically, the constructs of the engulfing system will ultimately be incorporated into an engulfing cell.

[0183] In some embodiments, the third-generation lentiviral vector of the present invention is a self-inactivating lentiviral vector. In some embodiments, the lentiviral vector is a VSV.G pseudotype lentiviral vector. In some embodiments, the lentiviral vector comprises a hepatocyte-specific promoter for transgenic expression. In some embodiments, the hepatocyte-specific promoter is an enhanced transthyretin promoter. In some embodiments, the lentiviral vector comprises one or more miR-142 target sequences to reduce the immune response to the transgenic product. In some embodiments, the incorporation of one or more miR-142 target sequences into a lentiviral vector of the present invention allows for a profile Petition 870260067982, dated 09 / 07 / 2026, page 103 / 275 84 / 233 of desired transgenic expression. For example, incorporation of one or more target sequences for miR-142 can suppress transgene expression in intravascular and extravascular hematopoietic cell lines, while transgene expression is maintained in non-hematopoietic cells. No oncogenesis was detected in tumor-prone mice treated with the lentiviral vector system of the present invention. See Brown et al. (2007) Blood 110: 4144-52, Brown et al. (2006) Nat. Med. 12: 585-91 and Cantore et al. (2015) Sci. Transl. Med. 7 (277): 277ra28.

[0184] The lentiviral vectors of the invention include polynucleotides with optimized codons encoding the BDD-FVIII protein described herein. In one embodiment, the optimized coding sequences for the BDD-FVIII protein are operatively linked to an expression control sequence. As used herein, two nucleic acid sequences are operatively linked when they are covalently linked so as to allow each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operatively linked when they are covalently linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence.Two DNA sequences are said to be operatively linked if induction of a promoter in the 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 result in (1) the introduction of a frameshift mutation, (2) interference with the ability of the promoter region to control transcription of the coding sequence, or (3) interference with the ability of the corresponding RNA transcript to be translated into a protein. Thus, an expression sequence... Petition 870260067982, dated 09 / 07 / 2026, page 104 / 275 85 / 233 A gene would be operatively linked to a coding nucleic acid sequence if the gene expression sequence is capable of transcribing this coding nucleic acid sequence, so that the resulting transcript is translated into the desired protein or polypeptide.

[0185] In certain embodiments, the lentiviral vector is a recombinant lentivirus vector capable of infecting non-dividing cells. In certain embodiments, the lentiviral vector is a recombinant lentivirus vector capable of infecting liver cells (e.g., hepatocytes). The lentiviral genome and proviral DNA typically have the three genes found in retroviruses: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural proteins (matrix, capsid, and nucleocapsid); the pol gene encodes RNA-directed DNA polymerase (reverse transcriptase), a protease, and an integrase; and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAs. The LTR 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).

[0186] Adjacent to the 5' LTR are the sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site). If the sequences necessary for encapsulation (or engulfment of retroviral RNA into infectious virions) are absent in the viral genome, the cis defect prevents encapsulation of genomic RNA.

[0187] However, the resulting mutant remains capable of controlling the synthesis of all virionic proteins. The invention provides Petition 870260067982, dated 09 / 07 / 2026, p. 105 / 275 86 / 233 A method for producing a recombinant lentivirus capable of infecting an undivided cell comprising transfecting a suitable host cell with two or more vectors carrying engulfing functions, particularly gag, pol, and env, as well as rev and tat. As will be described below, vectors lacking a functional tat gene are desirable for certain applications. Thus, for example, a first vector may provide a nucleic acid encoding a viral env and a viral pol, and another vector may provide a nucleic acid encoding a viral env to produce an engulfing cell. The introduction of a vector providing a heterologous gene, identified here as a transfer vector, into this engulfing cell produces a producer cell that releases infectious viral particles carrying the foreign gene of interest.

[0188] According to the above-mentioned configuration of foreign vectors and genes, the second vector can provide a nucleic acid that encodes a viral envelope gene (env). The env gene can be derived from virtually any suitable virus, including retroviruses. In some embodiments, the env protein is an amphoteric envelope protein that allows transduction into cells of human and other species.

[0189] Examples of env genes derived from retroviruses include, among others: murine Moloney leukemia virus (MoMuLV or MMLV), murine Harvey sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon monkey leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes, such as the G protein of vesicular stomatitis virus (VSV) (VSV G), hepatitis virus, and influenza virus, can also be used. In some embodiments, the nucleic acid sequence of the viral env gene is operatively associated with the regulatory sequences described in Petition 870260067982, dated 09 / 07 / 2026, p. 106 / 275 87 / 233 other parts here.

[0190] In certain embodiments, the lentiviral vector has the HIV virulence genes env, vif, vpr, vpu, and nef eliminated without compromising the vector's ability to transduce non-dividing cells. In some embodiments, the lentiviral vector comprises an elimination of the U3 region of the LTR 3'. The elimination of the U3 region may be a complete or partial elimination.

[0191] In some embodiments, the lentiviral vector of the invention comprising the FVIII nucleotide sequence described herein may be transfected into a cell having (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 does not have a functional tat gene. In other embodiments, the cell is further transfected with a fourth nucleotide sequence comprising a rev gene. In certain embodiments, the lentiviral vector does not have functional genes selected from vif, vpr, vpu, vpx and nef or a combination thereof.

[0192] In certain embodiments, a lentiviral vector of the present invention comprises one or more nucleotide sequences encoding a gag protein, a Rev response element, a core polypurine track (cPPT), or any combination thereof.

[0193] In some embodiments, the lentiviral vector expresses on its surface one or more polypeptides that enhance the targeting and / or activity of the lentiviral vector or the encoded FVIII polypeptide. The one or more polypeptides may be encoded by the lentiviral vector or may be incorporated during budding of the lentiviral vector from a host cell. During lentiviral production, viral particles bud from a producing host cell. During the Petition 870260067982, dated 09 / 07 / 2026, page 107 / 275 88 / 233 budding process, the viral particle takes on a lipid coat, which is derived from the lipid membrane of the host cell. As a result, the lipid coat of the viral particle may include membrane-bound polypeptides that were previously present on the surface of the host cell.

[0194] In some embodiments, the lentiviral vector expresses one or more polypeptides on its surface that inhibit an immune response to the lentiviral vector after administration to a human. In some embodiments, the surface of the lentiviral vector comprises one or more CD47 molecules. CD47 is a self-marker protein that is abundantly expressed on human cells. CD47 expression on the surface inhibits macrophage-induced phagocytosis of endogenous cells through the interaction of CD47 and macrophage-expressed SIRPa. Cells expressing high levels of CD47 are less likely to be targeted and destroyed by human macrophages in vivo.

[0195] In some embodiments, the lentiviral vector comprises a high concentration of CD47 polypeptide molecules on its surface. In some embodiments, the lentiviral vector is produced in a cell line that has a high level of CD47 expression. In certain embodiments, the lentiviral vector is produced in a CD47high cell, where the cell has high CD47 expression on the cell membrane. In particular embodiments, the lentiviral vector is produced in a HEK 293T CD47high cell, where HEK 293T has high CD47 expression on the cell membrane. In some embodiments, the HEK 293T cell is modified to have increased CD47 expression compared to unmodified HEK 293T cells. In certain embodiments, the CD47 is human CD47.

[0196] In some forms, the lentiviral vector has little or no expression on the surface of the major complex of Petition 870260067982, dated 09 / 07 / 2026, page 108 / 275 89 / 233 histocompatibility class I (MHC-I). MHC-I expressed on the surface displays peptide fragments of non-self proteins of a cell, such as protein fragments indicative of an infection, facilitating an immune response against the cell. In some embodiments, the lentiviral vector is produced in an MHC-Ilow cell, in which the cell has reduced MHC-I expression on the cell membrane. In some embodiments, the lentiviral vector is produced in an MHC-I-free (or MHC-I-negative, MHC-I-negative) cell, in which the cell has no MHC-I expression.

[0197] In particular embodiments, the lentiviral vector comprises a lipid coating comprising a high concentration of CD47 polypeptides and no MHC-I polypeptide. In certain embodiments, the lentiviral vector is produced in a CD47high / MHC-Ilow cell line, for example, a HEK 293T CD47high / MHC-Ilow cell line. In some embodiments, the lentiviral vector is produced in a CD47high / MHC-Ifree cell line, for example, a HEK 293T CD47high / MHC-Ifree cell line.

[0198] Examples of lentiviral vectors are described in Patent North American Publication No. 9,050,269 and International Publications Nos. WO9931251, WO9712622, WO9817815, WO9817816 and WO9818934, which are incorporated herein by reference in full.

[0199] In some embodiments, the present invention 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 an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 Petition 870260067982, dated 09 / 07 / 2026, p. 109 / 275 90 / 233 approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98% or at least approximately 99% sequence identity with (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.

[0200] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with (i) nucleotides 1792-4374 of SEQ ID NO: 5; (ii) nucleotides 1792-4374 of SEQ ID NO: 6; (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5;or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6; and wherein the N-terminal portion and the C-terminal portion together have an FVIII polypeptide activity.

[0201] In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the sequence of Petition 870260067982, dated 09 / 07 / 2026, p. 110 / 275 91 / 233 nucleotides comprises a nucleic acid sequence that has 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 with (i) nucleotides 58-4374 of SEQ ID NO: 1 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1 and is operatively 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.

[0202] In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that has 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 with (i) nucleotides 58-4374 of SEQ ID NO: 2 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2 and is operatively 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.

[0203] In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that has at least 85%, at least 86%, at least 87%, at least 88 Petition 870260067982, dated 09 / 07 / 2026, page 111 / 275 92 / 233%, 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 with (i) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70 and is operatively 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.

[0204] In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with (i) nucleotides 584374 of SEQ ID NO: 71 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71 and is operatively 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.

[0205] In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that has 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. Petition 870260067982, dated 09 / 07 / 2026, p. 112 / 275 93 / 233 approximately 99% sequence identity with (i) nucleotides 584374 of SEQ ID NO: 3 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3 and is operatively 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.

[0206] In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with (i) nucleotides 58-4374 of SEQ ID NO: 4 or (ii) nucleotides 582277 and 2320-4374 of SEQ ID NO: 4 and is operatively 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.

[0207] In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that has 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% Petition 870260067982, dated 09 / 07 / 2026, p. 113 / 275 94 / 233 or at least about 99% sequence identity with (i) nucleotides 58-4374 of SEQ ID NO: 5 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5 and is operatively linked to a promoter, a target sequence, or both. In other embodiments, the nucleic acid sequence comprises (i) nucleotides 58-4374 of SEQ ID NO: 5 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5.

[0208] In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with (i) nucleotides 58-4374 of SEQ ID NO: 6 or (ii) nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6 and is operatively 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.

[0209] The lentiviral vectors of the present invention are therapeutically effective when administered at doses of 5 x 10¹⁰TU / kg or less, 10⁹TU / kg or less, or 10⁸ TU / kg or less. At such dosages, administration of the lentiviral vectors of the invention can result in an increase in plasma FVIII activity in an individual requiring it by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, by Petition 870260067982, dated 09 / 07 / 2026, p. 114 / 275 95 / 233 less about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 95 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, by less about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times,at least approximately 190 times or at least approximately 200 times relative to baseline levels in the individual, relative to levels in an individual who received a reference nucleic acid molecule comprising SEQ ID NO: 16, relative to levels in an individual who received a reference viral vector comprising the reference nucleic acid molecule, or relative to levels in an individual after administration of a polypeptide encoded by the reference nucleic acid molecule. IV. Specific Expression for Tissue

[0210] In certain embodiments, it will be useful to include in the lentiviral vector one or more miRNA target sequences which, for example, are operatively linked to the optimized FVIII transgene. Thus, the invention also provides at least one miRNA target sequence operatively linked to the optimized FVIII nucleotide sequence or otherwise inserted within a lentiviral vector. More than one copy of a miRNA target sequence Petition 870260067982, dated 09 / 07 / 2026, p. 115 / 275 96 / 233 included in the lentiviral vector may increase the effectiveness of the system.

[0211] Different miRNA target sequences are also included. For example, lentiviral vectors expressing more than one transgene may have the transgene under the control of more than one miRNA target sequence, which may be the same or different. The miRNA target sequences may be grouped together, but other arrangements are also included. The transgene expression cassette, which contains miRNA target sequences, may also be inserted into the lentiviral vector in an antisense orientation. The antisense orientation may be useful in the production of viral particles to avoid the expression of gene products that may be toxic to the producing cells.

[0212] In other forms, the lentiviral vector comprises 1, 2, 3, 4, 5, 6, 7, or 8 copies of it or of a different miRNA target sequence. However, in some other modalities, the lentiviral vector will not include any miRNA target sequence. The choice of whether or not to include a miRNA target sequence (and how many) will be guided by known parameters such as the intended tissue target, the required expression level, etc.

[0213] In one embodiment, the target sequence is a miR223 target that has been reported to block expression most effectively in myeloid-related progenitors and at least partially in the most primitive HSPC. Targeting miR-223 can block expression in differentiated myeloid cells, including granulocytes, monocytes, macrophages, and myeloid dendritic cells. Targeting miR223 may also be suitable for gene therapy applications that rely on robust transgene expression in the lymphoid or erythroid lineage. The miR-223 target can also block expression very effectively in human HSCs.

[0214] In another embodiment, the target sequence is a miR142 target (tccataaagt aggaaacact aca (SEQ ID NO: 43)). In one embodiment, the Petition 870260067982, dated 09 / 07 / 2026, p. 116 / 275 The 97 / 233 lentiviral vector comprises 4 copies of the miR-142 target sequences. In certain embodiments, the complementary sequence of hematopoietic-specific microRNAs, such as miR-142 (142T), is incorporated into the 3' untranslated region of a lentiviral vector, making the transgene-coding transcript susceptible to miRNA-mediated downregulation. Through this method, transgene expression can be avoided in hematopoietic lineage antigen-presenting cells (APCs) while being maintained in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy can impose tight post-transcriptional control on transgene expression and therefore allows for stable distribution and long-term expression of transgenes.In some embodiments, regulation of miR-142 prevents immune-mediated clearance of transduced cells and / or induces antigen-specific regulatory T cells (T regs) and mediates robust immune tolerance to the transgene-encoded antigen.

[0215] In some modalities, the target sequence is a miR181 target. Chen CZ and Lodish H., Seminars in Immunology (2005) 17 (2): 155-165 describe miR-181, a miRNA specifically expressed in B cells in the bone marrow of rats (Chen and Lodish, 2005). They also describe that some human miRNAs are related to leukemias.

[0216] The target sequence can be fully or partially complementary to the miRNA. The term fully complementary means that the target sequence has a nucleic acid sequence that is 100% complementary to the miRNA sequence that recognizes it. The term partially complementary means that the target sequence is only partially complementary to the miRNA sequence that recognizes it, so that the partially complementary sequence is still recognized by the miRNA. In other words, a partially complementary target sequence Petition 870260067982, dated 09 / 07 / 2026, p. 117 / 275 Complementary miRNA 98 / 233 in the context of the present invention is effective in recognizing the corresponding miRNA and in preventing or reducing the expression of the transgene in cells expressing this miRNA. Examples of miRNA target sequences are described in documents WO2007 / 000668, WO2004 / 094642, WO2010 / 055413 or WO2010 / 125471, which are incorporated herein by reference in full. V. Polynucleotide Sequence Encoding FVIII Protein

[0217] The present invention is directed to lentiviral gene therapies in which the lentivirus vector comprises a nucleic acid molecule with optimized codons comprising a polynucleotide (nucleic acid) sequence encoding a polypeptide with FVIII activity. In some embodiments, the nucleic acid molecule with optimized codons encodes a complete FVIII polypeptide. In other embodiments, the nucleic acid molecule with optimized codons encodes an FVIII polypeptide with a deleted B domain (BDD), in which all or part of the FVIII B domain is deleted.

[0218] In a particular embodiment, the nucleic acid molecule encodes a polypeptide comprising an amino acid sequence that 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 17 (Figure 1J) or a fragment thereof. In another embodiment, the nucleic acid molecule encodes a polypeptide that has the amino acid sequence of SEQ ID NO: 17 or a fragment thereof.

[0219] In some forms, the nucleic acid molecule Petition 870260067982, dated 09 / 07 / 2026, p. 118 / 275 99 / 233 encodes an FVIII polypeptide comprising a signal peptide or a fragment thereof. In other embodiments, the nucleic acid molecule encodes an FVIII polypeptide which lacks a signal peptide. In some embodiments, the signal peptide comprises amino acids 1-19 of SEQ ID NO: 17.

[0220] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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.

[0221] 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 with nucleotides 58-1791 of SEQ ID NO: 3. In another embodiment, the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 58-1791 of SEQ ID NO: 4. In other embodiments, the first nucleotide sequence comprises nucleotides 58-1791 of SEQ ID NO: 3 or nucleotides 58-1791 of SEQ ID NO: 4.

[0222] In other embodiments, the isolated nucleic acid molecule comprises a sequence of nucleotides which Petition 870260067982, dated 09 / 07 / 2026, p. 119 / 275 100 / 233 comprises a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (i) nucleotides 1-1791 of SEQ ID NO: 3 or (ii) nucleotides 1-1791 of SEQ ID NO: 4; and wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0223] In one embodiment, the first nucleotide sequence comprises nucleotides 1-1791 of SEQ ID NO: 3 or nucleotides 1-1791 of SEQ ID NO: 4. In another embodiment, the second nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 1792-4374 of SEQ ID NO: 3 or 1792-4374 of SEQ ID NO: 4. In a particular embodiment, the second nucleotide sequence comprises nucleotides 1792-4374 of SEQ ID NO: 3 or 1792-4374 SEQ ID NO: 4.

[0224] 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 with nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 3 or 17922277 and 2320-4374 of SEQ ID NO: 4 (i.e., nucleotides 1792-4374 of SEQ ID NO: 3 or 1792-4374 of SEQ ID NO: 4 without the nucleotides encoding domain B or a fragment of domain B).

[0225] In a particular embodiment, the second nucleotide sequence comprises nucleotides 1792-2277 and 2320-4374 of Petition 870260067982, dated 09 / 07 / 2026, page 120 / 275 101 / 233 SEQ ID NO: 3 or 1792-2277 and 2320-4374 of SEQ ID NO: 4 (that is, nucleotides 1792-4374 of SEQ ID NO: 3 or 1792-4374 of SEQ ID NO: 4 without the nucleotides encoding domain B or a fragment of domain B).

[0226] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (i) nucleotides 1792-4374 of SEQ ID NO: 5 or (ii) 1792-4374 of SEQ ID NO: 6; and wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0227] 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 with nucleotides 1792-4374 of SEQ ID NO: 5. In other embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 1792-4374 of SEQ ID NO: 6.

[0228] In a particular embodiment, the second nucleic acid sequence comprises nucleotides 1792-4374 of SEQ ID NO: 5 or 1792-4374 of SEQ ID NO: 6. In some embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, Petition 870260067982, dated 09 / 07 / 2026, pp. 121 / 275 102 / 233 at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-1791 of SEQ ID NO: 5 or nucleotides 58-1791 of SEQ ID NO: 6.

[0229] In other embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 1-1791 of SEQ ID NO: 5 or nucleotides 1-1791 of SEQ ID NO: 6.

[0230] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 a fragment of the B domain) or (ii) 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or a fragment of the B domain); and in which the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0231] 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 Petition 870260067982, dated 09 / 07 / 2026, page 122 / 275 103 / 233 minus 99% sequence identity with nucleotides 17922277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding domain B or a fragment of domain B). 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 with 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 domain B or a fragment of domain B).

[0232] In a particular embodiment, the second nucleic acid sequence comprises nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 or 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 or 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding domain B or a fragment of domain B). In some embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 58-1791 of SEQ ID NO: 5 or nucleotides 58-1791 of SEQ ID NO: 6.

[0233] In other embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 1-1791 of SEQ ID NO: 5 or nucleotides 1-1791 of SEQ ID NO: 6.

[0234] In some forms, the nucleic acid molecule Petition 870260067982, dated 09 / 07 / 2026, pp. 123 / 275 Isolated 104 / 233 comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 in which the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.In other embodiments, the first nucleotide sequence comprises nucleotides 58-1791 of SEQ ID NO: 1, nucleotides 58-1791 of SEQ ID NO: 2, (iii) nucleotides 58-1791 of SEQ ID NO: 70 or (iv) nucleotides 581791 of SEQ ID NO: 71.

[0235] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (i) nucleotides 1-1791 of SEQ ID NO: 1, (ii) nucleotides 1-1791 of SEQ ID NO: 2, (iii) nucleotides 1-1791 of SEQ ID NO: 70 or (iv) nucleotides 1-1791 of SEQ ID NO: 71; and in which the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0236] In one embodiment, the first nucleotide sequence Petition 870260067982, dated 09 / 07 / 2026, pp. 124 / 275 105 / 233 comprises nucleotides 1-1791 of SEQ ID NO: 1, nucleotides 11791 of SEQ ID NO: 2, (iii) nucleotides 1-1791 of SEQ ID NO: 70 or (iv) nucleotides 1-1791 of SEQ ID NO: 71. In another embodiment, the second nucleotide sequence attached 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 with nucleotides 1792-4374 of SEQ ID NO: 1, 1792-4374 of SEQ ID NO: 2, (iii) nucleotides 1792-4374 of SEQ ID NO: 70 or (iv) nucleotides 1792-4374 of SEQ ID NO: 71.

[0237] In a particular embodiment, the second nucleotide sequence attached to the first nucleotide sequence comprises (i) nucleotides 1792-4374 of SEQ ID NO: 1, (ii) nucleotides 1792-4374 of SEQ ID NO: 2, (iii) nucleotides 1792-4374 of SEQ ID NO: 70, or (iv) nucleotides 1792-4374 of SEQ ID NO: 71. In other embodiments, the second nucleotide sequence attached 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 with (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.

[0238] In another embodiment, the isolated nucleic acid molecule comprises a sequence of nucleotides which Petition 870260067982, dated 09 / 07 / 2026, pages 125 / 275 106 / 233 comprises a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 in which the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0239] 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 which comprises a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 os. Petition 870260067982, dated 09 / 07 / 2026, pp. 126 / 275 107 / 233 nucleotides encoding domain B or a fragment of domain B); and wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity.

[0240] 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 domain B or a fragment of domain B).

[0241] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58 to 4374 of SEQ ID NO: 1.

[0242] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence that 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 with 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 a fragment of the B domain).

[0243] In other embodiments, the nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least Petition 870260067982, dated 09 / 07 / 2026, pp. 127 / 275 108 / 233%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 domain B or a fragment of domain B) or nucleotides 58 to 4374 of SEQ ID NO: 1. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 1 (i.e., nucleotides 1-4374 of SEQ ID NO: 1 without the nucleotides encoding domain B or a fragment of domain B) or nucleotides 1 to 4374 of SEQ ID NO: 1.

[0244] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that has at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 58 to 4374 of SEQ ID NO: 2. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence that has at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2.

[0245] 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 with 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 domain B or a fragment of domain B) Petition 870260067982, dated 09 / 07 / 2026, page 128 / 275 109 / 233 or nucleotides 58 to 4374 of SEQ ID NO: 2. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 2 (i.e., nucleotides 1-4374 of SEQ ID NO: 2 without the nucleotides encoding domain B or a fragment of domain B) or nucleotides 1 to 4374 of SEQ ID NO: 2.

[0246] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58 to 4374 of SEQ ID NO: 70.

[0247] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58-2277 and 23204374 of SEQ ID NO: 70 (i.e., nucleotides 58-4374 of SEQ ID NO: 70 without the nucleotides encoding the B domain or a fragment of the B domain).

[0248] 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 with SEQ ID NO: 70.

[0249] In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70 Petition 870260067982, dated 09 / 07 / 2026, p. 129 / 275 110 / 233 (i.e., nucleotides 58-4374 of SEQ ID NO: 70 without the nucleotides encoding the B domain or a fragment of the B domain) or nucleotides 58 to 4374 of SEQ ID NO: 70. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 70 (i.e., nucleotides 1-4374 of SEQ ID NO: 70 without the nucleotides encoding the B domain or a fragment of the B domain) or nucleotides 1 to 4374 of SEQ ID NO: 70.

[0250] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the sequence of nucleotides comprise a nucleic acid sequence that 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 with nucleotides 58 to 4374 of SEQ ID NO: 71.

[0251] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58-2277 and 23204374 of SEQ ID NO: 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 71 without the nucleotides encoding the B domain or a fragment of the B domain). 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 with SEQ ID NO: 71.

[0252] In other forms, the nucleotide sequence Petition 870260067982, dated 09 / 07 / 2026, page 130 / 275 111 / 233 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 a fragment of the B domain) or nucleotides 58 to 4374 of SEQ ID NO: 71. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 1-4374 of SEQ ID NO: 71 without the nucleotides encoding the B domain or a fragment of the B domain) or nucleotides 1 to 4374 of SEQ ID NO: 71.

[0253] In some embodiments, the molecule an isolated nucleic acid sequence comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58 to 4374 of SEQ ID NO: 3.In other embodiments, the nucleotide sequence comprises a nucleic acid sequence that 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 with 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 a fragment of the B domain).

[0254] 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 with 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 that Petition 870260067982, dated 09 / 07 / 2026, page 131 / 275 112 / 233 encode domain B or a fragment of domain B) or nucleotides 58 to 4374 of SEQ ID NO: 3. In still other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3 (i.e., nucleotides 1-4374 of SEQ ID NO: 3 without the nucleotides encoding domain B or a fragment of domain B) or nucleotides 1 to 4374 of SEQ ID NO: 3.

[0255] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58 to 4374 of SEQ ID NO: 4.

[0256] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 582277 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 a fragment of the B domain).

[0257] 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 with 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 a fragment of the B domain) or nucleotides Petition 870260067982, dated 09 / 07 / 2026, page 132 / 275 113 / 233 to 4374 of SEQ ID NO: 4. In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 4 (i.e., nucleotides 1-4374 of SEQ ID NO: 4 without the nucleotides encoding domain B or a fragment of domain B) or nucleotides 1 to 4374 of SEQ ID NO: 4.

[0258] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58 to 4374 of SEQ ID NO: 5.

[0259] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence that 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 with 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 a fragment of the B domain).

[0260] 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 with 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 Petition 870260067982, dated 09 / 07 / 2026, page 133 / 275 114 / 233 SEQ ID NO: 5 without the nucleotides encoding domain B or a fragment of domain B) or nucleotides 58 to 4374 of SEQ ID NO: 5.

[0261] In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 1-4374 of SEQ ID NO: 5 without the nucleotides encoding domain B or a fragment of domain B) or nucleotides 1 to 4374 of SEQ ID NO: 5.

[0262] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58 to 4374 of SEQ ID NO: 6.

[0263] In other embodiments, the nucleotide sequence comprises a nucleic acid sequence that 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 with 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 domain B or a fragment of domain B).

[0264] 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 with SEQ ID NO: 6. Petition 870260067982, dated 09 / 07 / 2026, page 134 / 275 115 / 233

[0265] 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 domain B or a fragment of domain B) or nucleotides 58 to 4374 of SEQ ID NO: 6.

[0266] In still other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1-4374 of SEQ ID NO: 6 without the nucleotides encoding domain B or a fragment of domain B) or nucleotides 1 to 4374 of SEQ ID NO: 6.

[0267] In some embodiments, the nucleotide sequence comprises a nucleic acid sequence that encodes a signal peptide. In certain embodiments, the signal peptide is an FVIII signal peptide. In some embodiments, the nucleic acid sequence that encodes a signal peptide has optimized codons.

[0268] In a particular embodiment, the nucleic acid sequence encoding a signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with (i) nucleotides 1 to 57 of SEQ ID NO: 1; (ii) nucleotides 1 to 57 of SEQ ID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ ID NO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) nucleotides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.

[0269] SEQ ID Nos: 1-6, 70 and 71 are optimized versions of SEQ ID NO: 16, the nucleotide sequence of the initial or progenitor or wild-type FVIII. SEQ ID NO: 16 encodes a human FVIII with Petition 870260067982, dated 09 / 07 / 2026, pp. 135 / 275 116 / 233 B domain deleted. Although SEQ ID NOs: 1-6, 70 and 71 are derived from a specific form of FVIII with the B domain deleted (SEQ ID NO: 16), it should be understood that the lentiviral gene therapy methods of the present invention are also directed to optimized versions of nucleic acids encoding other versions of FVIII. For example, other versions of FVIII may include full-size FVIII, FVIII with other B domain deletions (described below), or other FVIII fragments that retain FVIII activity.

[0270] A polypeptide with FVIII activity, as used herein, means a functional FVIII polypeptide in its normal role in coagulation, unless otherwise specified. The term polypeptide with FVIII activity includes a functional fragment, variant, analog, or derivative that retains the function of full-length wild-type Factor VIII in the coagulation pathway.

[0271] A polypeptide with FVIII activity is used interchangeably with FVIII protein, FVIII polypeptide, or FVIII. Examples of FVIII functions include, among others, the ability to activate coagulation, the ability to act as a cofactor for Factor IX, or the ability to form a tenase complex with Factor IX in the presence of Ca2+ and phospholipids, which then convert Factor X into the activated form Xa.

[0272] In one embodiment, a polypeptide with FVIII activity comprises two polypeptide chains, the first chain with the FVIII heavy chain and the second chain with the FVIII light chain. In another embodiment, the polypeptide having FVIII activity is a single-chain FVIII. The single-chain FVIII may contain one or more mutations or substitutions at amino acid residue 1645 and / or 1648, which corresponds to the mature FVIII sequence. See International Application No. PCT / US2012 / 045784, incorporated herein by Petition 870260067982, dated 09 / 07 / 2026, page 136 / 275 117 / 233 full reference. The FVIII protein can be human, porcine, canine, rat, or murine protein. Furthermore, comparisons between FVIII from humans and other species have identified conserved residues that are likely required for function (Cameron et al., Thromb. Haemost. 79: 317-22 (1998); US document 6,251,632).

[0273] The FVIII B domain, as used herein, is the same as the B domain known in the art, which is defined by the internal identity of the amino acid sequence and the proteolytic cleavage sites by thrombin, for example, Ser741-Arg1648 residues of full-length human FVIII. The other human FVIII domains are defined by the following amino acid residues: A1, Ala1-Arg372 residues; A2, Ser373-Arg740 residues; A3, Ser1690Ile2032 residues; C1, Arg2033-Asn2172 residues; C2, Ser2173Tyr2332 residues. The A3-C1-C2 sequence includes Ser1690-Tyr2332 residues. The remaining sequence, Glu1649-Arg1689 residues, is generally referred to as the FVIII light chain activating peptide. The boundary locations for all domains, including the B domains, for porcine, mouse, and canine FVIII are also known in the art. An example of a BDD-FVIII is the recombinant BDD-FVIII REFACTO® (Wyeth Pharmaceuticals, Inc.).

[0274] An FVIII with domain B deleted may have the complete or partial deletions described in U.S. Patents 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, 5,612,844, 5,612,844, 5,612,944 and 6,458,563, each of which is incorporated herein by reference in its entirety. In some embodiments, an FVIII sequence with domain B deleted from the present invention comprises any of the deletions described in col. 4, line 4 to col. 5, line 28 and Examples 1-5 of U.S. Patent No. 6,316,226 (also U.S. document 6,346,513). Petition 870260067982, dated 09 / 07 / 2026, p. 137 / 275 118 / 233

[0275] In some embodiments, an FVIII with domain B eliminated from the present invention has an elimination described in col. 2, lines 26-51 and Examples 5-8 of U.S. Patent No. 5,789,203 (also U.S. documents 6,060,447, U.S. 5,595,886 and U.S. 6,228,620). In some embodiments, an FVIII with domain B eliminated has an elimination described in col. 1, lines 25 to col. 2, line 40 of U.S. Patent No. 5,972,885; col. 6, lines 1-22 and Example 1 of U.S. Patent No. 6,048,720; col. 2, lines 17-46 of U.S. Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Patent No. 5,171,844; col. 2, lines 55-68, Figure 2 and Example 1 of U.S. Patent No. 5,112,950; col. 2, line 2 to col. 19, line 21 and Table 2 of U.S. Patent No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26 and col. 11, line 5 to col. 13, line 39 of U.S. Patent No. 7,041.635; or col. 4, lines 25-53 of U.S. Patent No. 6,458,563.

[0276] In some embodiments, an FVIII with a deleted B domain has a deletion of most of the B domain, but still contains amino-terminal sequences of the B domain that are essential for the in vivo proteolytic processing of the primary translation product into two polypeptide chains, as described in WO 91 / 09122, which is incorporated herein by full reference. In some embodiments, an FVIII with a deleted B domain is constructed with a deletion of amino acids 747-1638, that is, virtually a complete deletion of the B domain. Hoeben RC et al., J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by full reference. The FVIII with a deleted B domain may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 from FVIII. Meulien P. et al., Protein Eng. 2 (4): 301-6 (1988), incorporated here by reference in full. Petition 870260067982, dated 09 / 07 / 2026, p. 138 / 275 119 / 233

[0277] Additional deletions of domain B that are part of the invention include, for example: deletion of amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986) 83, 5939-5942)), 797 to 1562 (Eaton, et al. Biochemistry (1986) 25: 8343-8347)), 741 to 1646 (Kaufman (PCT Published Application No. WO 87 / 04187)), 747 to 1560 (Sarver et al., DNA (1987) 6: 553-564)), 741 to 1648 (Pasek (PCT Application No. 88 / 00831)), 816 to 1598 or 741 to 1689 (Lagner (Behring Inst. Mitt. (1988)) 82: 16-25, document EP 295597)), each of which is incorporated herein by reference in full. Each of the foregoing deletions can be made in any sequence of FVIII.

[0278] A number of functional FVIII molecules, including B-domain deletions, are described in the following U.S. Patents: U.S. 6,316,226 and U.S. 6,346,513, both assigned to Baxter; U.S. 7,041,635 assigned to In2Gen; U.S. 5,789,203, U.S. 6,060,447, U.S. 5,595,886 and U.S. 6,228,620 assigned to Quiron; U.S. 5,972.885 and US 6,048,720 granted to Biovitrum; US 5,543,502 and US 5,610,278 granted to Novo Nordisk; US 5,171,844 granted to Immuno Ag; US 5,112,950 granted to Transgene SA; US 4,868,112 granted to the Genetics Institute, each of which is incorporated herein by reference in full. A. Codon Optimization

[0279] In one embodiment, the lentiviral vector of the invention comprises an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleic acid sequence has optimized codons. In another embodiment, the initial nucleic acid sequence encoding a polypeptide with FVIII activity and subject to codon optimization is SEQ ID NO: 16. In some embodiments, the sequence encoding a polypeptide with FVIII activity has codons optimized for expression in beings Petition 870260067982, dated 09 / 07 / 2026, p. 139 / 275 120 / 233 humans. In other forms, the sequence encoding a polypeptide with FVIII activity has codons optimized for murine expression. SEQ ID NOs: 1-6, 70, and 71 are codon-optimized versions of SEQ ID NO: 16, optimized for human expression.

[0280] The term codon-optimized, therefore, refers to genes or coding regions of nucleic acid molecules for multi-host transformation and refers to the alteration of codons in the gene or coding regions of nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. This optimization includes replacing at least one or more of one or a significant number of codons with one or more codons that are more frequently used in the genes of this organism.

[0281] Deviations in the nucleotide sequence comprising the codons that encode the amino acids of any polypeptide chain allow for variations in the sequence that encodes the gene. Since each codon consists of three nucleotides and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode the signals that terminate translation). The genetic code showing which codons encode which amino acids is reproduced here as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are encoded by four triplets, serine and arginine by six, while tryptophan and methionine are encoded by only one triplet. This degeneracy allows the DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA. Petition 870260067982, of 09 / 07 / 2026, p. 140 / 275 121 / 233 Table 1: The Pattern Genetic Code TCAGT TTT Phe (F) TTC TTA Leu (L) TTG TCT Ser (S) TCC TCA TCG TAT Tyr (Y) TAC TAA Terminal TAG Terminal TGT Cys (C) TGC TGA Terminal TGG Trp (W) c CTT Leu (L) CTC CTA CTG CCT Pro (PCG CCC CCA CAC His CA) (Q) CAG CGT Arg (R) CGC CGA CGG A ATT He (1) ATC ATA ATG Met (M) ACT Thr (T) ACC ACA ACG AAT Asn (N) AAC AAA Lys (K) AAG AGT Ser (S) AGC AGA Arg (R) AGG G GTT Vai (V) GTC GCC GTA GTA GCT GCA GCT Ala (V) (D) GAC GAAGIu (E) GAG GGT Gly (G) GGC GGA GGG

[0282] Many organisms exhibit a tendency to use specific codons to encode the insertion of a specific amino acid into a growing peptide chain. Codon preference or codon bias, differences in codon usage among organisms, is conferred by the degeneracy of the genetic code and is well documented among many organisms. Codon bias often correlates with the efficiency of messenger RNA (mRNA) translation, which, in turn, is believed to be dependent, inter alia, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Consequently, genes can be adapted for optimal gene expression in a Petition 870260067982, dated 09 / 07 / 2026, page 141 / 275 122 / 233 determined organism based on codon optimization.

[0283] Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, relative frequencies of codon usage have been calculated. Codon usage tables are available, for example, in the Codon Usage Database available at www.kazusa.or.jp / codon / (accessed 18 June 2012). See Nakamura, Y. et al., Nucl. Acids Res. 28: 292 (2000).

[0284] Random assignment of codons at a frequency optimized to encode a given polypeptide sequence can be done manually by calculating the codon frequencies for each amino acid and then randomly assigning the codons to the polypeptide sequence. In addition, various algorithms and computer software programs can be used to calculate an optimal sequence.

[0285] 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 matching index relative to SEQ ID NO: 16; (b) the nucleotide sequence or a portion thereof has an increased frequency of ideal codons relative to SEQ ID NO: 16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16; (d) the nucleotide sequence or a portion thereof has an increased relative use of identical codons relative to SEQ ID NO: 16; (e) the nucleotide sequence or a portion thereof has a reduced effective number of codons relative 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 Petition 870260067982, dated 09 / 07 / 2026, p. 142 / 275 123 / 233 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 two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten features from (a) to (j). B. Codon Adaptation Index

[0286] In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the human codon adaptation index is increased relative to SEQ ID NO: 16. For example, the nucleotide sequence may have a human codon adaptation index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81%), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), by less 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 fit index that is at least about 0.88 (88%). In other embodiments, the nucleotide sequence has a fit index. Petition 870260067982, dated 09 / 07 / 2026, pp. 143 / 275 124 / 233 of human codons which is at least about 0.91 (91%). In other embodiments, the nucleotide sequence has a human codon matching index which is at least about 0.91 (97%).

[0287] In a particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 an FVIII polypeptide activity; and wherein the human codon adaptation index of the nucleotide sequence is increased relative to SEQ ID NO: 16.

[0288] 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 Petition 870260067982, dated 09 / 07 / 2026, pp. 144 / 275 125 / 233 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 one 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%).

[0289] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 5 or (ii) 1792-2277 and 2320-4374 of SEQ ID NO: 6;wherein the N-terminal and C-terminal portions together have an FVIII polypeptide activity; and wherein the human codon adaptation index of the nucleotide sequence is increased relative to SEQ ID NO: 16.;

[0290] 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 Petition 870260067982, dated 09 / 07 / 2026, pp. 145 / 275 126 / 233 0.81 (81%), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%) or at least about 0.88 (88%). In one particular embodiment, the nucleotide sequence has a human codon matching index that is at least about 0.83 (83%). In another embodiment, the nucleotide sequence has a human codon matching index that is at least about 0.88 (88%).

[0291] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70 or 71 without the nucleotides encoding domain B or a fragment of domain B);and in which the human codon adaptation index of the nucleotide sequence is increased relative to SEQ ID NO: 16. In some embodiments, the nucleotide sequence has a human codon adaptation index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about; 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81%), at least about 0.82 (82%), at least about 0.83 (83%), at least about Petition 870260067982, dated 09 / 07 / 2026, pp. 146 / 275 127 / 233 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%).

[0292] 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%).

[0293] In some embodiments, the isolated nucleic acid molecule has an enhanced optimal codon frequency (OPF) relative to SEQ ID NO: 16. In certain embodiments, the OPF 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 less about 70, at least about 75, at least about 79, at least about 80, at least about 85, or at least about 90.

[0294] In other embodiments, the isolated nucleic acid molecule has an increased relative identical 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 Petition 870260067982, dated 09 / 07 / 2026, pp. 147 / 275 128 / 233 of 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.

[0295] 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 of less than about 50, less than about 45, less than about 40, less than about 35, less than about 30, or less than about 25. In one particular embodiment, the isolated nucleic acid molecule has an effective number of codons of about 40, about 35, about 30, about 25, or about 20. C. Optimization of G / C Content

[0296] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the nucleotide sequence contains a higher percentage of G / C nucleotides compared with the percentage of G / C nucleotides in SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C content that is at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, or at least about 60%.

[0297] In a particular embodiment, the acid molecule Petition 870260067982, dated 09 / 07 / 2026, pp. 148 / 275 129 / 233 isolated nucleic acids comprise a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 an FVIII polypeptide activity; and wherein the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16.;

[0298] In some embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio 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 particular embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio that is at least about 58%. Petition 870260067982, dated 09 / 07 / 2026, p. 149 / 275 130 / 233

[0299] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with (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 a fragment of the B domain) or (iv) 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or a fragment of the B domain); wherein the N-terminal portion and the C-terminal portion together have an FVIII polypeptide activity; and wherein the nucleotide sequence contains a higher percentage of G / C nucleotides compared with the percentage of G / C nucleotides in SEQ ID NO: 16.

[0300] In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio 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%, Petition 870260067982, dated 09 / 07 / 2026, pp. 150 / 275 131 / 233 at least about 55%, at least about 56%, or at least about 57%. In one particular embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio that is at least about 52%. In another embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio that is at least about 55%. In yet another embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio that is at least about 57%.

[0301] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with (i) nucleotides 584374 or (ii) nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 (that is, nucleotides 58-4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70 or 71 without the nucleotides encoding the B domain or a fragment of the B domain);and in which 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 a polypeptide with FVIII activity has a G / C content that is at least about 45%.

[0302] In a particular embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio. Petition 870260067982, dated 09 / 07 / 2026, pp. 151 / 275 132 / 233 which is at least about 52%. In another embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio that is at least about 55%. In another embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio that is at least about 57%. In another embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio that is at least about 58%. In yet another embodiment, the nucleotide sequence encoding a polypeptide with FVIII activity has a G / C ratio that is at least about 60%.

[0303] G / C ratio (or guanine-cytosine ratio) or G / C nucleotide percentage refers to the percentage of nitrogenous bases in a DNA molecule that are guanine or cytosine. The G / C ratio can be calculated using the following formula: x 100

[0304] Human genes are highly heterogeneous with respect to their G / C ratio, with some genes having a G / C ratio as low as 20% and other genes having a G / C ratio as high as 95%. In general, genes rich in G / C ratios are more highly expressed. In fact, it has been shown that increasing the G / C ratio of a gene can lead to increased gene expression primarily due to increased transcription and higher steady-state mRNA levels. See Kudla et al., PLoS BioL, 4(6): e180 (2006). D. Matrix Attachment Region Type Sequences

[0305] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the nucleotide sequence contains fewer MARS / ARS sequences relative to SEQ ID NO: 16. In other embodiments, the nucleotide sequence that Petition 870260067982, dated 09 / 07 / 2026, pp. 152 / 275 The nucleotide sequence 133 / 233 encodes a polypeptide with FVIII activity and 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 with FVIII activity contains at most 1 MARS / ARS sequence. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a MARS / ARS sequence.

[0306] In a particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 wherein the nucleotide sequence contains fewer MARS / ARS sequences compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 6, at most 5, at most 4, at most 3, or at most 2 MARS / ARS sequences. In other embodiments, the nucleotide sequence encoding a polypeptide; Petition 870260067982, dated 09 / 07 / 2026, pp. 153 / 275 134 / 233 with FVIII activity contains at most 1 MARS / ARS sequence. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a MARS / ARS sequence.

[0307] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with (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 domain B or a fragment of domain B); or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding domain B or a fragment of domain B); wherein the N-terminal portion and the C-terminal portion together have an FVIII polypeptide activity; and in which the nucleotide sequence contains fewer MARS / ARS sequences compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 6, at most 5, at most 4, at most 3, or at most 2 sequences; Petition 870260067982, dated 09 / 07 / 2026, pp. 154 / 275 135 / 233 MARS / ARS. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 1 MARS / ARS sequence. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a MARS / ARS sequence.

[0308] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with (i) the nucleotides 584374 of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, or 71 or (ii) the nucleotides 58-2277 and 2320-4374 of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 or 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70 or 71 without the nucleotides encoding domain B or a fragment of domain B);and in which the nucleotide sequence contains fewer MARS / ARS sequences compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 6, at most 5, at most 4, at most 3, or at most 2 MARS / ARS sequences. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 1 MARS / ARS sequence. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a single MARS / ARS sequence.

[0309] AT-rich elements in the nucleotide sequence of Human FVIII that share sequence similarity with Petition 870260067982, dated 09 / 07 / 2026, pp. 155 / 275 136 / 233 autonomously replicating sequences (ARSs) of Saccharomyces cerevisiae and matrix attachment regions (MARs) (Fallux et al., Mol. Cell. Biol. 16: 4264-4272, 1996). One of these elements has been shown to bind to nuclear factors in vitro and repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene. It has been hypothesized that these sequences may contribute to the transcriptional repression of the human FVIII gene. 16). All of these sites have been mutated to destroy MAR / ARS sequences in optimized FVIII sequences (SEQ ID NOs: 1-6).The location of each of these elements and the corresponding nucleotide sequences in the optimized sequences are shown in Table 2 below. Table 2: Summary of changes in repressive elements Location of the element Initial BDDFVIII sequence (SEQ ID NO: 16) Optimized BDD-FVIII sequence SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 70 SEQ ID NO: 71 Destabilizing sequences 639 ATTTA GTTT A GTTCA GTTC A GTTC A GTTC A GTTC A GTTC A GTTCA 1338 ATTTA GTTT A GTTCA CTTC A GTTC A GTTC A GTTC A CTTC A GTTCA 1449 ATTTA CTTT A CTTCA CTTC A CTTC A CTTC A CTTC A CTTC A CTTCA 1590 TAAA T TAAA T CAAGT CAAG T TAAG T CAAG T CAAG T CAAG T TAAGT 1623 TAAA T CAAA A GAAG A CTAA G CAAG A CAAG A CAAG A TAAG T CAAGA 2410 ATTTA ATCT ATCTA ATCT ATCT ATCT ATCT ATCT ATCTA Petition 870260067982, dated 09 / 07 / 2026, pp. 156 / 275 137 / 233 AAAAAA 2586 ATTTA GTTT A GTTCA GTTC A GTTC A GTTC A GTTC A GTTC A GTTCA 2630 TAAA T TGAA T TGAAC TGAA C TGAA C TCAA T TGAA C TCAA T TGAAC 3884 ATTCT G ATCT G ACCT G ACCT G 3887 TAAA T TGAA C TGAAC TGAA C TGAA C TGAA C TGAA C TGAA C TGAAC Promoter binding potentialis sites 641 TTATA TTAT C TCATC TCAT T TCAT C TCAT C TCAT C TCAT T TCACT T AACGTTA T ATACAAC 1275 A CTAC A CTAC A GTACA 1276 TTATA TATA A TACAA TACA A TACA A TACA A TACA A TACA A TACAA 1445 TTATA TCAT C TCATC TTAT C TCAT C TCAT C TCAT C TTAT C TCATC 1474 ATACA ATACA ACATAAC TACA A TACA A TACAA 1588 TATA A TATA A TACAA TATA A TATA A TACA A TACA A TACA A TATAA 2614 TTATA CTGT A CTGTA CTGT A CTGT A TTGT A CTGT A TTGT A CTGTA CTA CTA ATCCA CATC61 CA CATC A CATCC 3286 TATA A TATA A TACAA 3840 TTATA TTATA TTACT CTAC A CTAC A CTAC A CTAC T CTAC A CTACT Attachment type sequences tomatriz (MARS / ARS) 1287 ATATT T GTAT CT GTACC T GTAC CT GTAT CT GTAC CT GTAC CT GTAC CT GTATC T 1447 ATATT T ATCT TT ATCTT C ATCT TC ATCT TC ATCT TC ATCT TC ATCT TC ATCTT C 1577 AAAT AT AAAT CT AGATC T AAAT CT AAAT CT AGAT CT AGAT CT AAAT CT AAATC T 1585 AAAT AT AAGT AT AAGTA C AAGT AC AAGT AT AAGT AC AAGT AC AAGT AC AAGTA T 2231 ATATT T ACAT CA ATATC A ACAT CA ACAT CA ACAT CT ATAT CT ACAT CT ATATC T 3054 AAAT AT AAAC AT GAATA T GAAC AT GAAC AT GAAC AT GAAC AT GAAT AT GAAC AT GAATA T 3788 ATATT T ATAT CT ATATC T ACAT CT ACAT CT ACAT CT ACAT CT ACAT CT ACATC T Petition: 870260067982, on 07 / 09 / 2026, page. 157 / 275 138 / 233 AU-rich sequence elements (AREs) 2468 ATTTT ATT ACTT CATC ACTTC ATC ACTT CATT ACTT ACTT TATT ACTT TATC ACTT TATT ACTTT ATC 3790 ATTTT TAA ATCT TTAA ATCTT CAA ATCT TCAACT TCAAA AT TCAA ATCTT CAA Poly A / poly T sequences 3273 AAAA AAA GAAA AAA AAA GAAG AAG GAAG AAG GAAG AAG GAAG AAG CAAG AAG AAG GAAG AAG CAAGA AG 4195 TTTTT T TTCTT T TTCTT C TTCT TCti TCCTTC TTCTT of amendment 2203 GGTG AT GGGG AC GGCG AC GGGG AC GGGG AC GGAG AC GGAG AC GGAG AC GGAGA C E. Destabilizing Sequences

[0310] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that encodes a polypeptide with FVIII activity, wherein the nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 destabilizing elements. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 destabilizing elements. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a single destabilizing element.

[0311] In a particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80%, at least about 85%, Petition 870260067982, dated 09 / 07 / 2026, pp. 158 / 275 139 / 233 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 with (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 an FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 destabilizing elements.In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 destabilizing elements. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a destabilizing element.

[0312] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, Petition 870260067982, dated 09 / 07 / 2026, pp. 159 / 275 140 / 233 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 with (i) nucleotides 1792-4374 of SEQ ID NO: 5; (ii) nucleotides 1792-4374 of SEQ ID NO: 6; (iii) nucleotides 1792-2277 and 23204374 of SEQ ID NO: 5 (i.e., nucleotides 1792-4374 of SEQ ID NO: 5 without the nucleotides encoding domain B or a fragment of domain B); or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding domain B or a fragment of domain B); wherein the N-terminal portion and the C-terminal portion together have an FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16.In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 destabilizing elements. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 destabilizing elements. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a single destabilizing element.

[0313] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 Petition 870260067982, dated 09 / 07 / 2026, pp. 160 / 275 141 / 233 approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% sequence identity with (i) nucleotides 584374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 23204374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71 without the nucleotides encoding the B domain or a fragment of the B domain); and wherein the nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 destabilizing elements.In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 destabilizing elements. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a destabilizing element.

[0314] There are ten destabilizing elements in the FVIII progenitor 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 of these sites have been mutated to destroy the destabilizing elements at SEQ ID NOs: 1-6, 70, and 71 in the optimized FVIII. The location of each of these elements and the corresponding nucleotide sequences in the optimized sequences are shown in Table 2. F. Potential Promoter Link Sites

[0315] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence described herein that Petition 870260067982, dated 09 / 07 / 2026, pp. 161 / 275 142 / 233 encodes a polypeptide with 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 a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 potential promoter binding sites. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 potential promoter binding sites. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a potential promoter binding site.

[0316] In a particular embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 an FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer sites of; Petition 870260067982, dated 09 / 07 / 2026, pp. 162 / 275 143 / 233 potential promoter link regarding SEQ ID NO: 16.

[0317] In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 potential promoter binding sites. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 potential promoter binding sites. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a potential promoter binding site.

[0318] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with (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 domain B or a fragment of domain B); or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding domain B or a fragment of domain B; Petition 870260067982, dated 09 / 07 / 2026, pages 163 / 275 144 / 233 B); wherein the N-terminal and C-terminal portions together have FVIII polypeptide activity; and wherein the nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 potential promoter binding sites. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 potential promoter binding sites. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a potential promoter binding site.

[0319] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with (i) nucleotides 584374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 or (ii) nucleotides 58-2277 and 23204374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 (i.e., nucleotides 58-4374 from SEQ ID NO: 1, 2, 3, 4, 5, 6,70 or 71 without the nucleotides encoding domain B or a fragment of domain B); and where the nucleotide sequence contains fewer potential promoter binding sites in, Petition 870260067982, dated 09 / 07 / 2026, pp. 164 / 275 145 / 233 in relation to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 9, at most 8, at most 7, at most 6, or at most 5 potential promoter binding sites. In other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity contains at most 4, at most 3, at most 2, or at most 1 potential promoter binding sites. In still other embodiments, the nucleotide sequence encoding a polypeptide with FVIII activity does not contain a potential promoter binding site.

[0320] TATA boxes are regulatory sequences frequently found in promoter regions of eukaryotes. They serve as the binding site for TATA-binding protein (TBP), a general transcription factor. TATA boxes generally comprise the TATAA sequence (SEQ ID NO: 28) or a close variant. TATA boxes within a coding sequence, however, can inhibit full protein translation. There are ten potential promoter-binding sequences in the wild-type BDD-FVIII sequence (SEQ ID NO: 16): five TATAA sequences (SEQ ID NO: 28) and five TTATA sequences (SEQ ID NO: 29). In some embodiments, at least 1, at least 2, at least 3, or at least 4 of the promoter-binding sites are eliminated from the FVIII genes of the present invention. In some embodiments, at least 5 of the promoter-binding sites are eliminated from the FVIII genes of the present invention.In other embodiments, at least 6, at least 7, or at least 8 of the promoter binding sites are eliminated from the FVIII genes of the present invention. In one embodiment, at least 9 of the promoter binding sites are eliminated from the FVIII genes of the present invention. In a particular embodiment, all promoter binding sites are eliminated. Petition 870260067982, dated 09 / 07 / 2026, pp. 165 / 275 146 / 233 deleted from the FVIII genes of the present invention. The location of each potential promoter binding site and the corresponding nucleotide sequences in the optimized sequences are shown in Table 2. G. Other Negative Regulatory Elements of Cis Action

[0321] In addition to the MAR / ARS sequences, destabilizing elements, and potential promoter sites described above, several additional potentially inhibitory sequences can be identified in the wild-type BDD-FVIII sequence (SEQ ID NO: 16). Two AU-rich sequence elements (AREs) can be identified (ATTTTATT (SEQ ID 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) following non-optimized BDD-FVIII. One or more of these elements can be removed from optimized FVIII sequences. The location of each of these sites and the corresponding nucleotide sequences in the optimized sequences are shown in Table 2.

[0322] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 Petition 870260067982, dated 09 / 07 / 2026, pp. 166 / 275 147 / 233 approximately 98% or at least approximately 99% sequence identity with (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 wherein the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, for example, a splice site, a poly-T sequence, a poly-A sequence, an ARE sequence, or any combination thereof.

[0323] In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an FVIII polypeptide; wherein the second nucleic acid sequence has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with (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 a fragment of the B domain); or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 1792-4374 of SEQ ID NO: 6 without the nucleotides encoding the B domain or a fragment of the B domain); wherein the N-terminal portion and the C-terminal portion together have a; Petition 870260067982, dated 09 / 07 / 2026, pages 167 / 275 148 / 233 FVIII polypeptide activity; and wherein the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, for example, a splice site, a polyT sequence, a poly-A sequence, an ARE sequence, or any combination thereof.

[0324] In other embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with (i) nucleotides 584374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 or (ii) nucleotides 58-2277 and 23204374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 (i.e., nucleotides 58-4374 from SEQ ID NO: 1, 2, 3, 4, 5, 6,70 or 71 without the nucleotides encoding the B domain or a fragment of the B domain); and wherein the nucleotide sequence does not contain one or more cis-acting negative regulatory elements, for example, a splice site, a polyT sequence, a poly-A sequence, an ARE sequence, or any combination thereof.

[0325] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of Petition 870260067982, dated 09 / 07 / 2026, pp. 168 / 275 149 / 233 a polypeptide of FVIII; 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 with (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 an FVIII polypeptide activity; and wherein the nucleotide sequence does not contain the GGTGAT splice site (SEQ ID NO: 27).

[0326] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 portion; Petition 870260067982, dated 09 / 07 / 2026, pp. 169 / 275 The 150 / 233 terminal and the C-terminal portion together have an FVIII polypeptide activity; and where the nucleotide sequence does not contain a poly-T sequence (SEQ ID NO: 25).

[0327] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 with (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 an FVIII polypeptide activity; and wherein the nucleotide sequence does not contain a poly-A sequence (SEQ ID NO: 26).

[0328] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of an FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of an 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 Petition 870260067982, dated 09 / 07 / 2026, pp. 170 / 275 151 / 233 approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98% or at least approximately 99% sequence identity with (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 wherein the nucleotide sequence does not contain an ARE element (SEQ ID NO: 30 or SEQ ID NO: 31).

[0329] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with (i) nucleotides 584374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 or (ii) nucleotides 58-2277 and 23204374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 (i.e., nucleotides 58-4374 from SEQ ID NO: 1, 2, 3, 4, 5, 6,70 or 71 without the nucleotides encoding the B domain or a fragment of the B domain); and wherein the nucleotide sequence does not contain the GGTGAT splice site (SEQ ID NO: 27).

[0330] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a, Petition 870260067982, dated 09 / 07 / 2026, pp. 171 / 275 152 / 233 polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with (i) nucleotides 584374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 or (ii) nucleotides 58-2277 and 23204374 of a selected amino acid sequence from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 (i.e., nucleotides 58-4374 from SEQ ID NO: 1, 2, 3, 4, 5, 6, 70 or 71 without the nucleotides encoding the B domain or a fragment of the B domain); and where the nucleotide sequence does not contain a poly-T sequence (SEQ ID NO: 25).

[0331] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with (i) nucleotides 584374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 or (ii) nucleotides 58-2277 and 23204374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 (i.e., nucleotides 58-4374 from SEQ ID NO: 1, 2, 3, 4, 5, 6, 70 or 71 without the nucleotides encoding the Petition 870260067982, dated 09 / 07 / 2026, pages 172 / 275 153 / 233 domain B or a fragment of domain B); and wherein the nucleotide sequence does not contain a poly-A sequence (SEQ ID NO: 26).

[0332] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence that 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 with (i) nucleotides 584374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 or (ii) nucleotides 58-2277 and 23204374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70 and 71 (i.e., nucleotides 58-4374 from SEQ ID NO: 1, 2, 3, 4, 5, 6,70 or 71 without the nucleotides encoding domain B or a fragment of domain B); and where the nucleotide sequence does not contain an ARE element (SEQ ID NO: 30 or SEQ ID NO: 31).

[0333] In other embodiments, an optimized FVIII sequence of the invention does not comprise one or more antiviral motifs, stem-loop structures and repeated sequences.

[0334] In still other embodiments, the nucleotides surrounding the transcription start site are altered to a Kozak consensus sequence (GCCGCCACC ATG C (SEQ ID NO: 32), where the underlined nucleotides are the start codon). In other embodiments, restriction sites may be added or removed to facilitate the cloning process. H. Heterologous Nucleotide Sequences Petition 870260067982, dated 09 / 07 / 2026, pp. 173 / 275 154 / 233

[0335] 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 may be linked to optimized BDD-FVIII nucleotide sequences of the invention at the 5' end or inserted in 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-terminal or C-terminal of the FVIII amino acid sequence encoded by the nucleotide sequence or inserted between two amino acids in the FVIII amino acid sequence. In some embodiments, the heterologous amino acid sequence may 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 within the FVIII polypeptide encoded by the nucleic acid molecule of the invention at any location described in International Publications Nos. WO 2013 / 123457 A1 and WO 2015 / 106052 A1 or North American Publication No. 2015 / 0158929 A1, which are incorporated herein by full reference.

[0336] In some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted within domain B or a fragment thereof. In some embodiments, the heterologous amino acid sequence is inserted within FVIII immediately downstream of an amino acid that corresponds to amino acid 745 of mature human FVIII (SEQ ID NO: 15). In a particular embodiment, FVIII comprises a deletion of amino acids 746-1646 that correspond to mature human FVIII (SEQ ID NO: 15) and the encoded heterologous amino acid sequence Petition 870260067982, dated 09 / 07 / 2026, pages 174 / 275 155 / 233 by the heterologous nucleotide sequence is inserted immediately downstream of amino acid 745, which corresponds to mature human FVIII (SEQ ID NO: 15). TABLE 3: Heterologous Portion Insertion Sites Insertion Site Domain Insertion Site Domain 1 H H Site Insertion Domain 3 A1 375 A2 1 1 1749 A3 18 A1 378 A2 1 1 1796 A3 22 A1 399 A2 1 1 1802 A3 26 A1 403 A2 1 1 1827 A3 40 A1 409 A2 1 1 1861 A3 60 A1 416 A2 1 1 1896 A3 65 A1 442 A2 1 1 1900 A3 81 A1 487 A2 1 1 1904 A3 116 A1 490 A2 j 1905 A3 119 A1 494 A2 1 1 1910 A3 130 A1 500 A2 1 1 1937 A3 188 A1 518 A2 j 2019 A3 211 A1 599 A2 1 1 2068 C1 216 A1 603 A2 1 1 2111 C1 220 A1 713 A2 1 1 2120 C1 224 A1 745 B 1 1 2171 C2 230 A1 1656 Region a3 1 1 2188 C2 333 A1 1711 A3 1 1 2227 C2 336 A1 1720 A3 1 1 2332 CT 339 A1 1725 A3

[0337] Note: The insertion sites indicate the amino acid position that corresponds to an amino acid position of mature human FVIII (SEQ ID NO: 15).

[0338] In other forms, the nucleic acid molecule Petition 870260067982, dated 09 / 07 / 2026, pp. 175 / 275 Isolated 156 / 233 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 the other heterologous nucleotide sequences. In some embodiments, the disclosure may comprise two, three, four, five, six, or more than seven heterologous nucleotide sequences together.

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

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

[0341] In some respects, a heterologous moiety includes the von Willebrand factor or a fragment thereof. In other related respects, a heterologous moiety may include a binding site. Petition 870260067982, dated 09 / 07 / 2026, pp. 176 / 275 157 / 233 (for example, a cysteine ​​amino acid) to a non-polypeptide moiety, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations thereof. In some respects, a heterologous moiety comprises a cysteine ​​amino acid that functions as a binding site for a non-polypeptide moiety, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations thereof.

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

[0343] In some embodiments, the heterologous portion 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 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 Petition 870260067982, dated 09 / 07 / 2026, pp. 177 / 275 158 / 233 approximately 900, at least approximately 1000, at least approximately 1100, at least approximately 1200, at least approximately 1300, at least approximately 1400, at least approximately 1500, at least approximately 1600, at least approximately 1700, at least approximately 1800, at least approximately 1900, at least approximately 2000, at least approximately 2500, at least approximately 3000, or at least approximately 4000 amino acids.

[0344] In other embodiments, the heterologous portion 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 400 amino acids, about 400 to about 500 amino acids, about 500 to about 600 amino acids, about 600 to about 700 amino acids, about 700 to about 800 amino acids, about 800 to about 900 amino acids, or about 900 to about 1000 amino acids.

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

[0346] In certain embodiments, a heterologous moiety increases the in vivo and / or in vitro half-life of the FVIII protein of the invention. In other embodiments, a heterologous moiety facilitates the visualization or localization of the FVIII protein of the invention or a fragment thereof (for example, a fragment comprising a heterologous moiety after proteolytic cleavage of the FVIII protein). The visualization and / or localization of the FVIII protein of the invention or a fragment thereof may be in vivo, in vitro, ex vivo, or combinations thereof.

[0347] In other embodiments, a heterologous moiety increases the stability of the FVIII protein of the invention or a fragment thereof (for example, a fragment comprising a heterologous moiety). Petition 870260067982, dated 09 / 07 / 2026, pages 178 / 275 159 / 233 after proteolytic cleavage of protein FVIII). As used herein, the term stability refers to a measure recognized in the art of the maintenance of one or more physical properties of protein FVIII in response to an environmental condition (e.g., elevated or reduced temperature). In certain aspects, the physical property may be the maintenance of the covalent structure of protein FVIII (e.g., the absence of unwanted proteolytic cleavage, oxidation, or deamidation). In other aspects, the physical property may also be the presence of protein FVIII in an appropriately folded state (e.g., the absence of soluble or insoluble aggregates or precipitates).

[0348] In one aspect, the stability of the FVIII protein is measured by testing a biophysical property of the FVIII protein, for example: thermal stability, unfolding profile, pH, stable removal of glycosylation, solubility, biochemical function (e.g., ability to bind to a protein, receptor, or ligand), etc., and / or combinations thereof. In another aspect, biochemical function is demonstrated by the binding affinity of the interaction. In one aspect, a measure of protein stability is thermal stability, i.e., resistance to thermal challenge. Stability can be measured using methods known in the art, such as HPLC (high-performance liquid chromatography), SEC (size elimination chromatography), DLS (dynamic light scattering), etc. Methods for measuring thermal stability include, among others, differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), circular dichroism (CD), and thermal challenge assay.

[0349] In certain respects, an FVIII protein encoded by the nucleic acid molecule of the invention comprises at least one half-life extender, that is, a heterologous moiety that increases the in vivo half-life of the FVIII protein relative to the in vivo half-life of the Petition 870260067982, dated 09 / 07 / 2026, pages 179 / 275 160 / 233 corresponding FVIII protein without this heterologous portion. The in vivo half-life of an FVIII protein can be determined by any methods known to those skilled in the art, for example, activity assays (chromogenic assay or one-step aPTT coagulation assay), ELISA, ROTEMTM, etc.

[0350] In some embodiments, the presence of one or more half-life extenders results in an increased half-life of the FVIII protein when compared with the half-life of the corresponding protein that does not have one or more half-life extenders. The half-life of the FVIII protein comprising a half-life extender is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times greater than the in vivo half-life of the corresponding FVIII protein without this half-life extender.

[0351] In one embodiment, the half-life of the FVIII protein comprising a half-life extender is about 1.5 to 20 times, about 1.5 to 15 times, or 1.5 to 10 times longer than the in vivo half-life of the corresponding protein without this half-life extender. In another embodiment, the half-life of the FVIII protein comprising a half-life extender is extended approximately 2 times to approximately 10 times, approximately 2 times to approximately 9 times, approximately 2 times to approximately 8 times, approximately 2 times to approximately 7 times, approximately 2 times to approximately 6 times, approximately 2 times to approximately 5 times, approximately 2 times to approximately 4 times, approximately 2 times to approximately 3 times, approximately 2.5 times to approximately 10 times, approximately 2.5 times to approximately 9 times, approximately 2.5 times to approximately 8 times, approximately 2.5 times to approximately 7 times, approximately 2.5 times to approximately 6 times, approximately 2.5 times to approximately 5 times. Petition 870260067982, dated 09 / 07 / 2026, pages 180 / 275 161 / 233 times, approximately 2.5 times to approximately 4 times, approximately 2.5 times to approximately 3 times, approximately 3 times to approximately 10 times, approximately 3 times to approximately 9 times, approximately 3 times to approximately 8 times, approximately 3 times to approximately 7 times, approximately 3 times to approximately 6 times, approximately 3 times to approximately 5 times, approximately 3 times to approximately 4 times, approximately 4 times to approximately 6 times, approximately 5 times to approximately 7 times, or approximately 6 times to approximately 8 times when compared with the in vivo half-life of the corresponding protein without this half-life extender.

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

[0353] In still other embodiments, the half-life of the FVIII protein comprising a half-life extender is about 15 hours to about two weeks, about 16 hours to about one week, about 17 hours to about one week, about 18 hours to about one week, about 19 hours to about one week, about 20 hours to about one week, about 21 hours to about one week, about 22 hours to about one week, about 23 hours Petition 870260067982, dated 09 / 07 / 2026, pp. 181 / 275 162 / 233 approximately one week, approximately 24 hours approximately one week, approximately 36 hours approximately 48 hours approximately 60 hours approximately 24 hours approximately 24 hours approximately one week approximately one week approximately one week approximately six days approximately five days approximately 24 hours approximately four days, approximately hours approximately three days or approximately 24 hours approximately two days.

[0354] In some embodiments, the average half-life per individual of the FVIII protein comprising a half-life extender is approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours (1 day), approximately 25 hours, approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 31 hours, approximately 32 hours, approximately 33 hours, approximately 34 hours, approximately 35 hours, approximately 36 hours, approximately 40 hours, approximately 44 hours, approximately 48 hours (2 days), approximately 54 hours, approximately 60 hours, approximately 72 hours (3 days), approximately 84 hours, approximately 96 hours (4 days), approximately 108 hours, approximately 120 hours (5 days), approximately six days, approximately seven days (one week), approximately eight days, approximately nine days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, or approximately 14 days.

[0355] One or more half-life extenders may be fused to C-terminal or N-terminal of FVIII or inserted into FVIII. 1. A Constant Region of Immunoglobulin or a Portion Thereof

[0356] In another aspect, a heterologous portion comprises one or more constant regions of immunoglobulin or portions thereof (for example, an Fc region). In one embodiment, a molecule of Petition 870260067982, dated 09 / 07 / 2026, pp. 182 / 275 163 / 233 isolated nucleic acids of the invention further comprise a heterologous nucleic acid sequence encoding an immunoglobulin constant region or a portion thereof. In some embodiments, the immunoglobulin constant region or a portion thereof is an Fc region.

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

[0358] A constant region of immunoglobulin or a portion thereof for producing the FVIII protein of the present invention can be obtained from several different sources. In one embodiment, a constant region of immunoglobulin or a portion thereof is derived from a human immunoglobulin. It should be understood, however, that the constant region of immunoglobulin or a portion thereof can be derived from an immunoglobulin of another mammalian species including, for example, a rodent (e.g., a rat, mouse, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, monkey). Furthermore, the constant region of immunoglobulin or a portion thereof can be derived from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the human IgG1 isotype is used.

[0359] A variety of immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) are available in publicly accessible repositories. The constant region domain sequence can be Petition 870260067982, dated 09 / 07 / 2026, pages 183 / 275 164 / 233 selected with a specific effective function (or without a specific effective function) or with a specific modification to reduce immunogenicity. Many antibody sequences and antibody-coding 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 methods recognized in the art. The genetic material obtained using any of the foregoing methods can then be altered or synthesized to obtain the polypeptides of the present invention. It will be further appreciated that the scope of the present invention encompasses alleles, variants, and mutations of DNA sequences of the constant region.

[0360] Immunoglobulin constant region sequences or a portion thereof can be cloned, for example, using polymerase chain reaction and primers that are selected to amplify the domain of interest. To clone an immunoglobulin constant region sequence or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymphatic cells, reverse transcribed into DNA, and antibody genes amplified by PCR. PCR amplification methods are described in detail in U.S. Patents Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and, for example, in PCR Protocols: A Guide to Methods and Applications, Innis et al. eds., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Enzymol Methods. 217: 270).PCR can be initiated using consensus constant region primers or more specific primers based on published DNA and amino acid sequences of the heavy and light chains. PCR can also be used to isolate DNA clones encoding the light and heavy chains of the antibody. In this case, libraries can be screened. Petition 870260067982, dated 09 / 07 / 2026, pp. 184 / 275 165 / 233 by consensus primers or larger homologous probes, such as mouse constant region probes. Numerous sets of primers suitable for antibody gene amplification 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.

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

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

[0363] In one embodiment, the Fc region refers to the portion of a Petition 870260067982, dated 09 / 07 / 2026, pages 185 / 275 166 / 233 single heavy chain of Ig that begins in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, which carries the first residue of the constant region of heavy chain 114) and terminates at the C-terminal of the antibody. Consequently, a complete Fc region comprises at least one hinge domain, one CH2 domain, and one CH3 domain.

[0364] A constant region of immunoglobulin 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, pulmonary airways, nasal surfaces, vaginal surfaces, colon, and rectal surfaces (U.S. Patent No. 6,485,726). An FcRn binding partner is a portion of an immunoglobulin that binds to FcRn.

[0365] The FcRn receptor has been isolated from several mammalian species, including humans. The sequences of human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180: 2377). The FcRn receptor binds to IgG (but not to other classes of immunoglobulins, such as IgA, IgM, IgD, and IgE) at a relatively low pH, actively transports IgG transcellularly in the luminal direction to the serosa, and releases IgG at a relatively higher pH found in interstitial fluids. It is expressed in the epithelial tissue of adults (U.S. Patents Nos. 6,485,726, 6,030,613, 6,086,875; WO documents 03 / 077834; US20030235536A1), including the pulmonary and intestinal epithelia (Israel et al. 1997, Immunology 92:69), the renal proximal tubular epithelium (Kobayashi et al., 2002, Am. J. Physiol. Renal Physiol. 282: F358), as well as the nasal epithelium, vaginal surfaces, and biliary branch surfaces.

[0366] Useful FcRn binding partners in the present invention include molecules that can be specifically bound by the FcRn receptor, including whole IgG, the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. Petition 870260067982, dated 09 / 07 / 2026, pp. 186 / 275 167 / 233 The region of the Fc portion of IgG that binds to the FcRn receptor was described based on X-ray crystallography (Burmeister et al. 1994, Nature 372: 379). The main contact area of ​​Fc with FcRn is near the junction of the CH2 and CH3 domains. The Fc-FcRn contacts are all within a single Ig heavy chain. FcRn binding partners include whole IgG, the Fc fragment of IgG, and other IgG fragments that include the complete FcRn binding region. The main contact sites include amino acid residues 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. References to the amino acid numbering of immunoglobulins or immunoglobulin fragments or regions are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

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

[0368] Two FcRn receptors can bind to a single molecule. Fc. Crystallographic data suggest that each Fc...

Claims

1. Use of a lentiviral vector, characterized in that it is in the preparation of a pharmaceutical composition for the treatment, prevention or improvement of a bleeding disorder in an individual who needs it, comprising administering to the individual at least one dose of 5 x 1010 transducer units / kg (TU / kg) or less of a lentiviral vector, wherein: the lentiviral vector comprises a lipid coating comprising one or more CD47 polypeptides and does not comprise an MHC-I polypeptide; and the lentiviral vector comprises a nucleic acid comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has: (i) at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1;(ii) at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2; (iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70; (iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least Petition 870260067982, dated 09 / 07 / 2026, page 254 / 275 2 / 14 98% or at least 99% sequence identity with 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 with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3; (vi) at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4; (vii) at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 23204374 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 with nucleotides 58-2277 and 23204374 of SEQ ID NO: 6; or (ix) or any combination of (i) to (viii), wherein the dose results in the expression of FVIII in an individual at a beneficial therapeutic level.

2. Use of a lentiviral vector, characterized by the fact that it is in the preparation of a pharmaceutical composition for the treatment, prevention or improvement of a bleeding disorder in an individual who needs it, comprising administering to the individual at least one dose of 5 x 1010 transducer units / kg (TU / kg) or less of a lentiviral vector, wherein: the lentiviral vector comprises a lipid coating comprising one or more CD47 polypeptides and does not comprise an MHC-I polypeptide;and the lentiviral vector comprises a nucleic acid 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 an FVIII polypeptide, wherein: (a) the first nucleic acid sequence has: (i) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 of SEQ ID NO: 3; (ii) at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 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 with nucleotides 58-12277 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 with nucleotides 58-2277 of SEQ ID NO: 6; (b) wherein the second nucleotide sequence has: (i) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 2320-4374 of Petition 870260067982, dated 09 / 07 / 2026, page 256 / 275 4 / 14 SEQ ID NO: 3; (ii) at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with nucleotides 2320-4374 of SEQ ID NO: 4; (iii) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 2320-4374 of SEQ ID NO: 5;or (iv) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 2320-4374 of SEQ ID NO: 6; wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity, and wherein the dose results in FVIII expression in an individual at a beneficial therapeutic level.

3. Use according to claim 1 or 2, characterized in that: the dose is approximately 5 x 10¹⁰ TU / kg, approximately 4.5 x 10¹⁰ TU / kg, approximately 4 x 10¹⁰ TU / kg, approximately 3.5 x 10¹⁰ TU / kg, approximately 3 x 10¹⁰ TU / kg, approximately 2.5 x 10¹⁰ TU / kg, approximately 2 x 10¹⁰ TU / kg, approximately 1.5 x 10¹⁰ TU / kg, approximately 1 x 10¹⁰ TU / kg, approximately 9.5 x 10⁹ TU / kg, approximately 9 x 10⁹ TU / kg, approximately 8.5 x 10⁹ TU / kg, approximately 8 x 10⁹ TU / kg, approximately 7.5 x 10⁹ TU / kg, approximately 7 x 109 TU / kg, approx. 6.5 x 109 TU / kg, approx. 6 x 109 TU / kg, approx. 5.5 x 109 TU / kg, approx. 5 x 109 TU / kg, approx. TU / kg, approximately 3 x 109 TU / kg, approximately 2.5 x 109 TU / kg, approximately 2 x 109 TU / kg, approximately 1.5 x 109 TU / kg, approximately 1 x 109 TU / kg, approximately 9.5 108 TU / kg, approximately 8 x 108 TU / kg, approximately 7.5 x 108 TU / kg, approximately 7 x Petition 870260067982, dated 09 / 07 / 2026, page 257 / 275 5 / 14 108 TU / kg, approximately 6.5 x 108 TU / kg,approximately 6 x 10⁸ TU / kg, approximately 5.5 x 10⁸ TU / kg, approximately 5 x 10⁸ TU / kg, approximately 4.5 x 10⁸ TU / kg, approximately 4 x 10⁸ TU / kg, approximately 3.5 x 10⁸ TU / kg, approximately 3 x 10⁸ TU / kg, approximately 2.5 x 10⁸ TU / kg, approximately 2 x 10⁸ TU / kg, approximately 1.5 x 10⁸ TU / kg or approximately 1 x 10⁸ TU / kg, or the dose is less than approximately 5 x 10¹⁰ TU / kg, less than approximately 4.5 x 10¹⁰ TU / kg, less than approximately 4 x 10¹⁰ TU / kg, less than approximately 3.5 x 10¹⁰ TU / kg, less than approximately 3 x 10¹⁰ TU / kg, less than approximately 2.5 x 10¹⁰ TU / kg, less than approximately 2 x 10¹⁰ TU / kg, less than approximately 1.5 x 10¹⁰ TU / kg, less than approximately 1 x 10¹⁰ TU / kg, less than approximately 9.5 x 10⁹ TU / kg, less than approximately 9 x 10⁹ TU / kg, less than approximately 8.5 x 10⁹ TU / kg, less than approximately 8 x 10⁹ TU / kg, less than approximately 7.5 x 10⁹ TU / kg, less than approximately 7 x 10⁹ TU / kg, less than approximately 6.5 x 10⁹ TU / kg, less than approximately 6 x 10⁹ TU / kg, less than approximately 5.5 x 10⁹ TU / kg, less than approximately 5 x 10⁹ TU / kg, less than approximately 4.5 x 10⁹ TU / kgless than approximately 4 x 10⁹ TU / kg, less than approximately 3.5 x 10⁹ TU / kg, less than approximately 3 x 10⁹ TU / kg, less than approximately 2.5 x 10⁹ TU / kg, less than approximately 2 x 10⁹ TU / kg, less than approximately 1.5 x 10⁹ TU / kg, less than approximately 1 x 10⁹ TU / kg, less than approximately 9.5 x 10⁸ TU / kg, less than approximately 9 x 10⁸ TU / kg, less than approximately 8.5 x 10⁸ TU / kg, less than approximately 8 x 10⁸ TU / kg, less than approximately 7.5 x 10⁸ TU / kg, less than approximately 7 x 10⁸ TU / kg, less than approximately 6.5 x 10⁸ TU / kg, less than approximately 6 x 10⁸ TU / kg, less than approximately 5.5 x 10⁸ TU / kg, less than approximately 5 x 10⁸ TU / kg, less than approximately 4.5 x 10⁸ TU / kg, less than approximately 4 x 10⁸ TU / kg, less than approximately 3.5 x 10⁸ TU / kg, less than approximately 3 x 10⁸ TU / kg, less than approximately 2.5 x 10⁸ TU / kg, less than approximately 2 x 10⁸ TU / kg, less than approximately 1.5 x 10⁸ TU / kg or less than approximately 1 x 10⁸ TU / kg, or the dose is between 1 x 10⁸ and 5 x 10¹⁰ TU / kg, between 1 x 10⁸ and 5 x 10⁹ TU / kg, between 1 x 10⁸ and 1 x 10⁹ TU / kg, between 1 x 10⁸ and 1 x 10¹⁰ TU / kg,Petition 870260067982, dated 09 / 07 / 2026, page. 258 / 275 6 / 14 between 1 x 10⁹ and 5 x 10¹⁰ TU / kg, between 2 x 10⁹ and 5 x 10¹⁰ TU / kg, between 3 x 10⁹ and 5 x 10¹⁰ TU / kg, between 4 x 10⁹ and 5 x 10¹⁰ TU / kg, between 5 x 10⁹ and 5 x 10¹⁰ TU / kg, between 6 x 10⁹ and 5 x 10¹⁰ TU / kg, between 7 x 10⁹ and 5 x 10¹⁰ TU / kg, between 8 x 10⁹ and 5 x 10¹⁰ TU / kg, between 9 x 10⁹ and 5 x 10¹⁰ TU / kg, between 10¹⁰ and 5 x 10¹⁰ TU / kg, between 1.5 x 10¹⁰ and 5 x 1010 TU / kg, between 2 x 1010 and 5 x 1010 TU / kg, between 2.5 x 1010 and 5 x 1010 TU / kg, between 3 x 1010 and 5 x 1010 TU / kg, between 3.5 x 1010 and 5 x 1010 TU / kg, between 4 x 1010 and 5 x 1010 TU / kg or between 4.5 x 1010 and 5 x 1010 TU / kg, or the dose is between 1 x 109 and 5 x 1010 TU / kg, between 1 x 109 and 4.5 x 1010 TU / kg, between 1 x 109 and 4 x 1010 TU / kg, between 1 x 109 and 3.5 x 1010 TU / kg, between 1 x 10⁹ and 3 x 10¹⁰ TU / kg, between 1 x 10⁹ and 2.5 x 10¹⁰ TU / kg, between 1 x 10⁹ and 2 x 10¹⁰ TU / kg, between 1 x 10⁹ and 1.5 x 10¹⁰ TU / kg, between 1 x 10⁹ and 1 x 10¹⁰ TU / kg, between 1 x 10⁹ and 9 x 10⁹ TU / kg, between 1 x 10⁹ and 8 x 10⁹ TU / kg,between 1 x 10⁹ and 7 x 10⁹ TU / kg, between 1 x 10⁹ and 6 x 10⁹ TU / kg, between 1 x 10⁹ and 5 x 10⁹ TU / kg, between 1 x 10⁹ and 4 x 10⁹ TU / kg, between 1 x 10⁹ and 3 x 10⁹ TU / kg and between 1 x 10⁹ and 2 x 10⁹ TU / kg, or the dose is between 1 x 10¹⁰ and 2 x 10¹⁰ TU / kg, between 1.1 x 10¹⁰ and 1.9 x 10¹⁰ TU / kg, between 1.2 x 10¹⁰ and 1.8 x 10¹⁰ TU / kg, between 1.3 x 10¹⁰ and 1.7 x 10¹⁰ TU / kg or between 1.4 x 10¹⁰ and 1.6 x 10¹⁰ TU / kg, or the dose is approximately 1 x 10⁸ TU / kg, approximately 8.3 x 10⁸ TU / kg, approximately 1.5 x 10⁹ TU / kg, approximately 4.5 x 10⁹ TU / kg, or approximately 1.3 x 10¹⁰ TU / kg, or the dose is between 2.5 x 10⁹ TU / kg and 3.5 x 10⁹ TU / kg, between 2.6 x 10⁹ TU / kg and 3.4 x 10⁹ TU / kg, between 2.7 x 10⁹ TU / kg and 3.3 x 10⁹ TU / kg, between 2.8 x 10⁹ TU / kg and 3.2 x 10⁹ TU / kg, or between 2.9 x 10⁹ TU / kg and 3.1 x 10⁹ TU / kg, or the dose is between 5.5 x 10⁹ TU / kg and 6.5 x 10⁹ TU / kg, between 5.6 x 10⁹ TU / kg and 6.4 x 10⁹ TU / kg, between 5.7 x 10⁹ TU / kg and 6.3 x 10⁹ TU / kg, between 5.8 x 10⁹ TU / kg and 6.2 x 10⁹ TU / kg, or between 5.9 x 10⁹ TU / kg and 6.1 x 10⁹ TU / kg. Petition 870260067982.from 09 / 07 / 2026, page 259 / 275 7 / 14, 4. Use according to claim 1 or 2, characterized in that the dose is between 1.5 x 1010 TU / kg, approximately 1.0 x 109 TU / kg, approximately 3.0 x 109 TU / kg, or approximately 6.0 x 109 TU / kg.

5. Use, according to any one of claims 1 to 4, characterized in that plasma activity of FVIII 24 to 48 hours after administration of the lentiviral vector is increased relative to an individual who received a reference vector comprising a nucleic acid molecule comprising SEQ ID NO: 16, preferably in that plasma activity of FVIII is increased by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 50 times,at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, or at least about 200 times.

6. Use, according to any one of claims 1 to 5, characterized in that the lentiviral vector is administered as a single dose or multiple doses, preferably where the lentiviral vector is administered via intravenous injection. Petition 870260067982, dated 09 / 07 / 2026, pp. 260 / 275 8 / 14 7. Use, according to any one of claims 1 to 6, characterized in that the individual is a pediatric individual, or in that the individual is an adult individual.

8. Use, according to any one of claims 1 to 7, characterized in that the lentiviral vector comprises a tissue-specific promoter, preferably in that the tissue-specific promoter selectively enhances the expression of the FVIII-active polypeptide in a target liver cell, more preferably in that the tissue-specific promoter that selectively enhances the expression of the FVIII-active polypeptide in a target liver cell comprises an mTTR promoter.

9. Use according to claim 8, characterized in that the target liver cell is a hepatocyte, preferably wherein the isolated nucleic acid molecule is stably integrated into the hepatocyte genome.

10. Use, according to any one of claims 1 to 9, characterized in that the bleeding disorder is hemophilia A.

11. Use, according to any one of claims 1 to 10, characterized in that the isolated nucleic acid molecule comprises LV-coFVIII-6 (SEQ ID NO: 71) or LV-coFVIII-6XTEN (SEQ ID NO: 72).

12. Use, according to any one of claims 1 to 11, characterized in that the lentiviral vector dose is administered at once or divided into at least two subdoses.

13. Use, according to any one of claims 1 to 12, characterized in that the nucleotide sequence encoding a polypeptide with FVIII activity further comprises a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding a signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with: (i) nucleotides 1 to 57 of SEQ ID NO:1; (ii) nucleotides 1 to 57 of SEQ ID NO:2; (iii) nucleotides 1 to 57 of SEQ ID NO:3; (iv) nucleotides 1 to 57 of SEQ ID NO:4; (v) nucleotides 1 to 57 of SEQ ID NO:5; (vi) nucleotides 1 to 57 of SEQ ID NO:6; (vii) nucleotides 1 to 57 of SEQ ID NO:70; (viii) nucleotides 1 to 57 of SEQ ID NO:71; or (ix) nucleotides 1 to 57 of SEQ ID NO:

68.

14. Use, according to any one of claims 1 to 13, characterized in that the nucleotide sequence encoding a polypeptide with FVIII activity comprises one or more properties selected from the group consisting of: (a) the human codon matching index of the nucleic acid molecule or a portion thereof is increased relative to SEQ ID NO: 16; (b) the optimal codon frequency of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G / C nucleotides compared with the percentage of G / C nucleotides in SEQ ID NO: 16; (d) the relative synonym codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (e) the effective number of codons in the sequence of Petition 870260067982, dated 09 / 07 / 2026, page.262 / 275 10 / 14 nucleotides or a portion thereof is reduced relative to SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS / ARS sequences (SEQ ID NOs: 21 and 22) relative to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) relative to SEQ ID NO: 16; and (h) any combination thereof.

15. Use, according to any one of claims 1 to 14, characterized in that the nucleotide sequence encoding a polypeptide with FVIII activity further comprises a heterologous nucleotide sequence encoding a heterologous amino acid sequence, preferably wherein the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, XTEN, transferrin, albumin or a PAS sequence, and / or the heterologous amino acid sequence is linked to the N-terminal or C-terminal of the amino acid sequence encoded by a nucleotide sequence encoding a polypeptide with FVIII activity or inserted between two amino acids in the amino acid sequence encoded by the nucleotide sequence at one or more insertion sites selected from TABLE 3.

16. Use, according to any one of claims 1 to 15, characterized in that the FVIII polypeptide is either a full-length FVIII or an FVIII with a deleted B domain.

17. Use, according to claim 1, characterized in that the CD47 polypeptide is a human CD47 polypeptide, and / or in that the lipid envelope comprises a high concentration of CD47 polypeptides.

18. Use, according to any of the claims 1 Petition 870260067982, dated 09 / 07 / 2026, pp. 263 / 275 11 / 14 to 17, characterized in that the lentiviral vector is produced in a host cell, preferably in which: the host cell expresses CD47; and / or the host cell does not express MHC-I; and / or the host cell is CD47high / MHC-I-; and / or the host cell is a HEK 293T CD47high / MHC-I- cell.

19. Lentiviral vector for the treatment of a bleeding disorder in an individual in need thereof, comprising administering to the individual at least one dose of 5 x 1010 transducer units / kg (TU / kg) or less of a lentiviral vector, characterized in that: the lentiviral vector comprises a lipid coating comprising one or more CD47 polypeptides and does not comprise an MHC-I polypeptide; and the lentiviral vector comprises a nucleic acid comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has: (i) at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1; (ii) at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2;(iii) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least Petition 870260067982, dated 09 / 07 / 2026, page 264 / 275 12 / 14 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70; (iv) at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with 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 with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3;(vi) at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4; (vii) at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 23204374 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 with nucleotides 58-2277 and 23204374 of SEQ ID NO: 6;or (ix) any combination of (i) to (viii), wherein the dose results in the expression of FVIII in an individual at a beneficial therapeutic level. Petition 870260067982, dated 09 / 07 / 2026, pp. 265 / 275 13 / 14; 20. Lentiviral vector for the treatment of a bleeding disorder in an individual who needs it, comprising administering to the individual at least one dose of 5 x 1010 transducer units / kg (TU / kg) or less of a lentiviral vector, characterized in that: the lentiviral vector comprises a lipid coating comprising one or more CD47 polypeptides and does not comprise an MHC-I polypeptide;and the lentiviral vector comprises a nucleic acid 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 an FVIII polypeptide, wherein: (a) the first nucleic acid sequence has: (i) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 and 23201791 of SEQ ID NO: 3; (ii) at least 91%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 58-2277 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 with nucleotides 58-12277 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 with nucleotides 58-2277 of SEQ ID NO: 6; Petition 870260067982, dated 09 / 07 / 2026, p. 266 / 275 14 / 14 (b) wherein the second nucleotide sequence has: (i) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with nucleotides 2320-4374 of SEQ ID NO: 3; (ii) at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with nucleotides 2320-4374 of SEQ ID NO: 4; (iii) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 2320-4374 of SEQ ID NO: 5;or (iv) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with nucleotides 2320-4374 of SEQ ID NO: 6; wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity, and wherein the dose results in FVIII expression in an individual at a beneficial therapeutic level.

21. Use of a lentiviral vector, characterized by the fact that it is in the preparation of a pharmaceutical composition for the treatment, prevention or improvement of a disease and / or condition in an individual.

22. Kit, characterized in that it comprises: (a) the lentiviral vector, as defined in any of the preceding claims, or a pharmaceutical composition comprising the same; and (b) a label or package insert indicating that the associated compositions are useful for the treatment of an individual who is suffering from or is predisposed to coagulation disorders.

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