Use of lentiviral vectors expressing factor IX

Abstract

The present invention provides a pharmaceutical composition for treating bleeding disorders such as hemophilia (e.g., hemophilia B). [Solution] A pharmaceutical composition comprising a lentiviral vector for use in a method to reduce transduction into macrophages in a target that requires it, wherein the lentiviral vector comprises a specific nucleotide sequence encoding a polypeptide having factor IX (FIX) activity and a lipid coat containing the CD47 protein, and the method comprises 2 × 10 9 The pharmaceutical composition comprises administering a lentiviral vector to a subject in a dose of less than transduction units / kg (TU / kg), wherein the lentiviral vector is packaged in HEK293T cells overexpressing CD47 and contains a higher level of surface CD47 protein on its lipid coating than a control lentiviral vector produced in unmodified HEK293T cells.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 776,393, filed on 6 December 2018, the entirety of which is incorporated herein by reference.

[0002] References to electronically submitted sequence listings The contents of the sequence listing in the electronically submitted ASCII text file (name: SA9-468TW_SequenceListing_ST25; size: 28,470 bytes; creation date: December 2, 2019) are incorporated herein by reference in their entirety. [Background technology]

[0003] The blood coagulation pathway involves the formation of an enzyme complex (Xase complex) of factor VIIIa (FVIIIa) and factor IXa (FIXa) on the surface of platelets. FIXa is a serine protease that has relatively weak catalytic activity without its cofactor FVIIIa. The Xase complex cleaves factor X (FX) into factor Xa (FXa), which then interacts with factor Va (FVa) to cleave prothrombin and produce thrombin. Hemophilia B is a bleeding disorder caused by mutations and / or deletions in the FIX gene that result in a deficiency of FIX activity.

[0004] In hemophilia, blood clotting is impaired due to the deficiency of certain plasma blood clotting factors. Hemophilia B (also known as Christmas disease) is one of the most common hereditary bleeding disorders worldwide. Hemophilia B is caused by a deficiency of factor IX, resulting from either reduced synthesis of factor IX protein or an incomplete molecule with reduced activity. Hemophilia B results in reduced blood clotting activity in vivo and in vitro and requires extensive medical monitoring throughout the life of the affected individual. There is no effective preventive measure, and recurrent arthritis leads to the development of progressively impaired arthropathy and a decline in quality of life (Non-Patent Literature 1).

[0005] Treatment for hemophilia B involves replacing the deficient clotting factors with an exogenous factor concentrate containing a high concentration of factor IX. However, generating such concentrates from blood presents technical challenges. Therefore, there is a need in this field for FIX therapy to overcome the difficulties and limitations of current replacement therapies. Gene therapy represents a sustained treatment for hemophilia B as an effective approach, involving the stable integration of a transgene expression cassette containing nucleic acid sequences encoding polypeptides with FIX activity into the genomes of targeted cells. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Giangrande P., E×pert Opin Pharmacother.2005;6:1517~24 [Overview of the project] [Means for solving the problem]

[0007] This disclosure relates to a method for preventing or treating hemophilia in subjects requiring it, comprising administering an effective dose of a lentiviral vector comprising a nucleotide sequence encoding a polypeptide having factor IX (FIX) activity to subjects, wherein the lentiviral vector is administered to unmodified HEK293T cells (ATCC® CRL-11268®). The present invention provides a method for packaging CD47 into HEK293T cells that overexpress CD47, including higher levels of surface CD47 protein expression than the control lentiviral vector produced in the present invention, wherein the effective dose is lower compared to the control dose of the control lentiviral vector required to induce the same FIX activity as the lentiviral vector.

[0008] In some embodiments, the control lentiviral vector has a 1 μm surface.2 It contains 19 molecules of CD47 per hit. In some embodiments, the lentiviral vector has at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 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 more CD47 protein on its surface than the control lentiviral vector produced in HEK293T cells (ATCC® CRL-11268™).

[0009] In some embodiments, the effective dose is less than about 5×10 10 transducing units / kg (TU / kg), less than 4×10 10 TU / kg, less than 3×10 10 TU / kg, less than 2×10 10 TU / kg, less than 1×10 10 TU / kg, less than 9×10 9 TU / kg, less than 8×10 9 TU / kg, less than 7×10 9 TU / kg, less than 6×10 9 TU / kg, less than 5×10 9 TU / kg, less than 4×10 9 TU / kg, less than 3×10 9 TU / kg, less than 2×10 9 TU / kg, less than 1×10 9 TU / kg, less than about 9×10 8 TU / kg, or less than about 8×10 8 TU / kg.

[0010] In some embodiments, the subject exhibits the following characteristics after administration: (a) reduced transduction of the lentiviral vector into macrophages compared to a control lentiviral vector; (b) reduced allospecific immune response to the lentiviral vector compared to a control lentiviral vector; (c) at least 30% of the FIX activity compared to normal FIX activity at least 3 weeks after administration; (d) tissue-specific expression of the lentiviral vector in the liver, spleen, or both the liver and spleen; and (e) one or more combinations of any of (a) to (d).

[0011] In some embodiments, the allo-specific immune response includes the release of cytokines in response to the lentiviral vector. In some embodiments, the cytokines are selected from the group consisting of MIP-1a, MIP-1b, MCP-1, and any combination thereof. In some embodiments, subjects show a decrease in the expression level of MIP-1a after administration of the lentiviral vector compared to the expression of MIP-1a after administration of a control lentiviral vector. In some embodiments, subjects show a decrease in the expression level of MIP-1b after administration of the lentiviral vector compared to the expression of MIP-1b after administration of a control lentiviral vector. In some embodiments, subjects show a decrease in the expression level of MCP-1 after administration of the lentiviral vector compared to the expression of MCP-1 after administration of a control lentiviral vector.

[0012] In some embodiments, subjects exhibit at least approximately 75%, at least approximately 100%, at least approximately 125%, at least approximately 150%, at least approximately 175%, at least approximately 200%, at least approximately 225%, at least approximately 250%, at least approximately 275%, or at least approximately 300% FIX activity compared to normal FIX activity at least 3 weeks after administration of the lentiviral vector. In some embodiments, subjects exhibit at least approximately 150% FIX activity compared to normal FIX activity at least 3 weeks after administration of the lentiviral vector. It exhibits FIX activity. In some embodiments, plasma FIX activity 24 to 48 hours after administration of the lentiviral vector is increased compared to subjects who received a control dose of the control lentiviral vector. In some embodiments, plasma FIX activity increases after administration by at least approximately 2 times, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 6 times, at least approximately 7 times, at least approximately 8 times, at least approximately 9 times, at least approximately 10 times, at least approximately 11 times, at least approximately 12 times, at least approximately 13 times, at least approximately 14 times, at least approximately 15 times, at least approximately 20 times, at least approximately 25 times, at least approximately 30 times, at least approximately 35 times, at least approximately 40 times, at least approximately 50 times, at least approximately 60 times, at least approximately 70 times, at least approximately 80 times, at least approximately 90 times, at least approximately 100 times, at least approximately 110 times, at least approximately 120 times, at least approximately 130 times, at least approximately 140 times, at least approximately 150 times, at least approximately 160 times, at least approximately 170 times, at least approximately 180 times, at least approximately 190 times, or at least approximately 200 times compared to a subject who received a control dose of a control lentiviral vector.

[0013] In some embodiments, the subject shows increased localization of the lentiviral vector to the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of the subject. In some embodiments, the increased localization is 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 The increased localization is characterized by a 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, or at least about 200-fold greater vector copy number (VCN) of the lentiviral vector compared to organs other than the liver and spleen of the subject. In some embodiments, increased localization is characterized by at least 50-fold more VCNs of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of the target. In some embodiments, increased localization is characterized by at least 100-fold more VCNs of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of the target.

[0014] In some embodiments, CD47 is human CD47. In some embodiments, human CD47 contains an amino acid sequence that is at least 60%, at least about 70%, at least 70%, at least about 80%, at least 85%, at least about 90%, at least 95%, at least about 96%, at least 97%, at least about 98%, at least 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the lentiviral vector does not contain an MHC-I polypeptide. In some embodiments, the lentiviral vector is generated in host cells that express a higher concentration of CD47 compared to HEK293T cells (ATCC® CRL-11268®).

[0015] In some embodiments, the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 And it has sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0016] This disclosure relates to a method for preventing or treating hemophilia in a person requiring it, 5 × 10 10The present invention also provides a method comprising administering to a subject a lentiviral vector containing a nucleotide sequence encoding a factor IX (FIX) activity polypeptide in a transduction unit / kg (TU / kg) of less than 100%, wherein the lentiviral vector contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least 85% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least 85% sequence identity with respect to nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least 85% sequence identity with respect to nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence has at least 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7.

[0017] In some embodiments, the dose is approximately 5 × 10 10 TU / kg, approximately 4.5 x 10 10 TU / kg, approximately 4 x 10 10 TU / kg, approximately 3.5 x 10 10 TU / kg, approximately 3 x 10 10TU / kg, approximately 2.5×10 10 TU / kg, approximately 2×10 10 TU / kg, approximately 1.5×10 10 TU / kg, approximately 1×10 10 TU / kg, approximately 9.5×10 9 TU / kg, approximately 9×10 9 TU / kg, approximately 8.5×10 9 TU / kg, approximately 8×(10 9 TU / kg, approximately 7.5×10 9 TU / kg, approximately 7×10 9 TU / kg, approximately 6.5×10 9 TU / kg, approximately 6×10 9 TU / kg, approximately 5.5×10 9 TU / kg, approximately 5×10 9 TU / kg, approximately 4.5×10 9 TU / kg, approximately 4×10 9 TU / kg, approximately 3.5×10 9 TU / kg, approximately 3×10 9 TU / kg, approximately 2.5×10 9 TU / kg, approximately 2×10 9 TU / kg, approximately 1.5×10 9 TU / kg, approximately 1×10 9 TU / kg, approximately 9.5×10 8 TU / kg, approximately 9×10 8 TU / kg, approximately 8.5×10 8 TU / kg, approximately 8×(10 8 TU / kg, approximately 7.5×10 8 TU / kg, approximately 7×10 8 TU / kg, approximately 6.5×10 8 TU / kg, approximately 6×10 8 TU / kg, approximately 5.5×10 8 TU / kg, approximately 5×10 8 TU / kg, approximately 4.5×10​​​​​​​​​​​​​​​It is TU / kg. In some embodiments, the dosage is 5×10 10 less than TU / kg, 4.5×10 10 less than TU / kg, 4×10 10 less than TU / kg, 3.5×10 10 less than TU / kg, 3×10 10 less than TU / kg, 2.5×10 10 less than TU / kg, 2×10 10 less than TU / kg, 1.5×10 10 less than TU / kg, 1×10 10 less than TU / kg, 9.5×10 9 less than TU / kg, 9×10 9 less than TU / kg, 8.5×10 9 less than TU / kg, 8×10 9 less than TU / kg, 7.5×10 9 less than TU / kg, 7×10 9 less than TU / kg, 6.5×10 9 less than TU / kg, 6×10 9 less than TU / kg, 5.5×10 9 less than TU / kg, 5×10 9 less than TU / kg, 4.5×10 9 less than TU / kg, 4×10 9 less than TU / kg, 3.5×10 9 less than TU / kg, 3×10 9 less than TU / kg, 2.5×10 9 less than TU / kg, 2×10 9 less than TU / kg, 1.5×10 9 less than TU / kg, 1×10 9 less than TU / kg, about 9.5×10 8 less than TU / kg, about 9×10 8 less than TU / kg, about 8.5×10 8 less than TU / kg, about 8×10 8 less than TU / kg, about 7.5×10 8 less than TU / kg, about 7×10 8 less than TU / kg, about 6.5×10 8 less than TU / kg, about 6×10 8 less than TU / kg, about 5.5×10 8 less than TU / kg, about 5×10 8Less than TU / kg, approximately 4.5 × 10 8 Less than TU / kg, approximately 4 x 10 8 Less than TU / kg, approximately 3.5 × 10 8 Less than TU / kg, approximately 3 × 10 8 Less than TU / kg, approximately 2.5 × 10 8 Less than TU / kg, approximately 2 × 10 8 Less than TU / kg, approximately 1.5 × 10⁻⁶ 8 Less than TU / kg, or approximately 1 × 10⁻⁶ 8 It is less than TU / kg. In some embodiments, the dose is 1 × 10 8 ~5×10 10 TU / kg, 1 × 10 8 ~5×10 9 TU / kg, 1 × 10 8 ~1 × 10 9 TU / kg, 1 × 10 8 ~1 × 10 10 TU / kg, 1 × 10 9 ~5×10 10 TU / kg, 2 × 10 9 ~5×10 10 TU / kg, 3 × 10 9 ~5×10 10 TU / kg, 4×10 9 ~5×10 10 TU / kg, 5×10 9 ~5×10 10 TU / kg, 1 × 10 9 ~6×10 9 TU / kg, 2 × 10 9 ~6×10 9 TU / kg, 3 × 10 9 ~6×10 9 TU / kg, 4×10 9 ~6×10 9 TU / kg, 5×10 9 ~6×10 9 TU / kg, 6×10 9 ~5×10 10 TU / kg, 7×10 9 ~5×10 10 TU / kg, 8×10 9 ~5×10 10 TU / kg, 9×10 9 ~5×10 10 TU / kg, 10 10~5×10 10 TU / kg, 1.5×10 10 ~5×10 10 TU / kg, 2×10 10 ~5×10 10 TU / kg, 2.5×10 10 ~5×10 10 TU / kg, 3×10 10 ~5×10 10 TU / kg, 3.5×10 10 ~5×10 10 TU / kg, 4×10 10 ~5×10 10 TU / kg, or 4.5×10 10 ~5×10 10 TU / kg. In some embodiments, the dosage is 1×10 9 ~5×10 10 TU / kg, 1×10 9 ~4.5×10 10 TU / kg, 1×10 9 ~4×10 10 TU / kg, 1×10 9 ~3.5×10 10 TU / kg, 1×10 9 ~3×10 10 TU / kg, 1×10 9 ~2.5×10 10 TU / kg, 1×10 9 ~2×10 10 TU / kg, 1×10 9 ~1.5×10 10 TU / kg, 1×10 9 ~10 10 TU / kg, 1×10[[ID=6&7]] 9 ~9×10 9 TU / kg, 1×10 9 ~8×10 9 TU / kg, 1×10 9 ~7×10 9 TU / kg, 1×10 9 ~6×10[[ID=B1]] 9 TU / kg, 1×10 9 ~5×10 9 TU / kg, 1×10 9 ~4×10 9 TU / kg, 1×10 9 ~3×10 9TU / kg, and 1 × 10 9 ~2×10 9 The value is TU / kg. In some embodiments, the dose is 1 × 10⁻⁶ 10 ~2×10 10 TU / kg, 1.1 × 10 10 ~1.9×10 10 TU / kg, 1.2 × 10 10 ~1.8×10 10 TU / kg, 1.3 × 10 10 ~1.7×10 10 TU / kg, or 1.4 × 10 10 ~1.6×10 10 The value is TU / kg. In some embodiments, the dose is approximately 4 × 10 9 TU / kg ~ approximately 6 x 10 9 It is TU / kg.

[0018] In some embodiments, the lentiviral vector is administered in single or multiple doses. In some embodiments, the lentiviral vector is administered via intravenous injection. In some embodiments, the subjects are children. In some embodiments, the subjects are adults.

[0019] In some embodiments, the polypeptide having FIX activity includes an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 12.

[0020] In some embodiments, the lentiviral vector includes a tissue-specific promoter. In some embodiments, the tissue-specific promoter selectively enhances the expression of polypeptides having FIX activity in target liver cells. In some embodiments, in target liver cells Tissue-specific promoters that selectively enhance the expression of polypeptides with FIX activity include the APOA2 promoter, SERPINA1(hAAT) promoter, mTTR promoter, MIR122 promoter, or any combination thereof. In some embodiments, the target liver cells are hepatocytes. In some embodiments, the isolated nucleic acid molecule is stably incorporated into the genome of the hepatocytes.

[0021] In some embodiments, the lentiviral vector includes a splice donor site. In some embodiments, the lentiviral vector includes a splice acceptor site. In some embodiments, the lentiviral vector includes a gag sequence, a pol sequence, a rev sequence, a rev response element (RRE), or any combination thereof. In some embodiments, the gag sequence is a full-length or truncated gag sequence. In some embodiments, the lentiviral vector includes an enhancer, a target sequence for microRNA, a post-transcriptional regulatory element, a packaging signal, a polyA sequence, an intron sequence, or any combination thereof.

[0022] In some embodiments, the lentiviral vector dose is administered in a single dose or divided into at least two partial doses. In some embodiments, the lentiviral vector dose is repeated at least twice.

[0023] In some embodiments, the nucleotide sequence encoding the polypeptide having FIX activity further comprises a nucleic acid sequence encoding a signal peptide. In some embodiments, the nucleic acid sequence encoding the signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with (i) nucleotides 1-84 of SEQ ID NO: 2; (ii) nucleotides 1-84 of SEQ ID NO: 3; (iii) nucleotides 1-84 of SEQ ID NO: 4; (iv) nucleotides 1-84 of SEQ ID NO: 5; (v) nucleotides 1-84 of SEQ ID NO: 6; and (vi) nucleotides 1-84 of SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the polypeptide having FIX activity further comprises a nucleic acid sequence encoding a propeptide. In some embodiments, the nucleic acid sequence encoding the propeptide 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 85-138 of SEQ ID NO: 2; (ii) nucleotides 85-138 of SEQ ID NO: 3; (iii) nucleotides 85-138 of SEQ ID NO: 4; (iv) nucleotides 85-138 of SEQ ID NO: 5; (v) nucleotides 85-138 of SEQ ID NO: 6; or (vi) nucleotides 85-138 of SEQ ID NO: 7.

[0024] In some embodiments, the nucleotide sequence encoding the polypeptide having FIX activity further comprises a heteronucleotide sequence encoding a heteroamino acid sequence. In some embodiments, the heteroamino acid sequence is albumin, immunoglobulin Fc region, XTEN sequence, C-terminal peptide (CTP) of the β-subunit of human chorionic gonadotropin, PAS sequence, HAP sequence, CTP peptide sequence, transferrin, albumin-binding moiety, or any fragment, derivative, variant, or combination of these polypeptides. In some embodiments, the heteroamino acid sequence is ligated to the N-terminus or C-terminus of the amino acid sequence encoded by the nucleotide sequence encoding the polypeptide having FIX activity, or is inserted between two amino acids in the amino acid sequence. In some embodiments, the heterogeneous portion is amino acid 103 of SEQ ID NO: 2, amino acid 105 of SEQ ID NO: 2, amino acid 142 of SEQ ID NO: 2, amino acid 149 of SEQ ID NO: 2, amino acid 162 of SEQ ID NO: 2, amino acid 166 of SEQ ID NO: 2, amino acid 174 of SEQ ID NO: 2, amino acid 224 of SEQ ID NO: 2, amino acid 226 of SEQ ID NO: 2, amino acid 228 of SEQ ID NO: 2, amino acid 413 of SEQ ID NO: 2, or any of the others. The combination is inserted into a polypeptide having FIX activity immediately downstream of the corresponding amino acid. In some embodiments, the FIX polypeptide is the R338L variant FIX polypeptide.

[0025] In some embodiments, the lentiviral vector is generated within the host cell. In some embodiments, the host cell expresses CD47. In some embodiments, the host cell is modified to overexpress CD47. In some embodiments, the host cell does not express MHC-1. In some embodiments, the host cell expresses CD47 high / MHC-I - In some embodiments, the host cell is CD47 high / MHC-I - These are HEK293T cells.

[0026] The disclosure also provides a lentiviral vector comprising (i) a nucleotide sequence including a tissue-specific promoter, and (ii) a nucleic acid sequence such as that shown in SEQ ID NO: 1, wherein the tissue-specific promoter drives the expression of the nucleic acid sequence in liver cells.

[0027] The disclosure also provides a lentiviral vector comprising (i) a nucleotide sequence comprising a splice donor site; (ii) a splice acceptor site; (iii) a gag sequence; (iv) a Rev response element; (v) an enhancer; (vi) a post-transcriptional regulatory element; (vii) a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and (viii) a target sequence for microRNA.

[0028] In some embodiments, the nucleic acid sequence encodes a polypeptide having FIX activity, which includes an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the polypeptide having FIX activity includes the amino acid sequence shown in SEQ ID NO: 12.

[0029] In some embodiments, the surface of the lentiviral vector contains higher levels of CD47 protein than the control lentiviral vector produced in HEK293T cells (ATCC® CRL-11268®). In some embodiments, the surface of the lentiviral vector does not contain MHC-I.

[0030] This disclosure also relates to a method for treating hemophilia in a subject requiring such treatment, which includes administering an effective dose of the lentiviral vector disclosed herein to the subject. In some embodiments, the effective dose is approximately 5 × 10⁻⁶10 Less than 4 × 10⁻¹⁰ units / kg (TU / kg), 10 Less than TU / kg, 3 × 10 10 Less than TU / kg, 2 × 10 10 Less than TU / kg, 1 × 10 10 Less than TU / kg, 9 × 10 9 Less than TU / kg, 8 × 10 9 Less than TU / kg, 7 × 10 9 Less than TU / kg, 6 × 10 9 Less than TU / kg, 5 × 10 9 Less than TU / kg, 4 × 10 9 Less than TU / kg, 3 × 10 9 Less than TU / kg, 2 × 10 9 Less than TU / kg, 1 × 10 9 Less than TU / kg, approximately 9 x 10 8 Less than TU / kg, or approximately 8 × 10 8 It is less than TU / kg. In some embodiments, the effective dose is about 5 × 10 10 TU / kg, approximately 4.5 x 10 10 TU / kg, approximately 4 x 10 10 TU / kg, approximately 3.5 x 10 10 TU / kg, approximately 3 x 10 10 TU / kg, approximately 2.5 x 10 10 TU / kg, approximately 2 x 10 10 TU / kg, approximately 1.5 x 10 10 TU / kg, approximately 1 x 10 10 TU / kg, approximately 9.5 x 10 9 TU / kg, approximately 9 x 10 9 TU / kg, approximately 8.5 x 10 9 TU / kg, approximately 8 x 10 9 TU / kg, approximately 7.5 x 10 9 TU / kg, approximately 7 x 10 9 TU / kg, approximately 6.5 x 10 9 T U / kg, approximately 6 x 10 9 TU / kg, approximately 5.5 x 10 9 TU / kg, approximately 5 x 10 9 TU / kg, approximately 4.5 x 10 9 TU / kg, approximately 4 x 10 9 TU / kg, approximately 3.5 x 10 9 TU / kg, approximately 3 x 109 TU / kg, approximately 2.5 x 10 9 TU / kg, approximately 2 x 10 9 TU / kg, approximately 1.5 x 10 9 TU / kg, approximately 1 x 10 9 TU / kg, approximately 9.5 x 10 8 TU / kg, approximately 9 x 10 8 TU / kg, approximately 8.5 x 10 8 TU / kg, approximately 8 x 10 8 TU / kg, approximately 7.5 x 10 8 TU / kg, approximately 7 x 10 8 TU / kg, approximately 6.5 x 10 8 TU / kg, approximately 6 x 10 8 TU / kg, approximately 5.5 x 10 8 TU / kg, approximately 5 x 10 8 TU / kg, approximately 4.5 x 10 8 TU / kg, approximately 4 x 10 8 TU / kg, approximately 3.5 x 10 8 TU / kg, approximately 3 x 10 8 TU / kg, approximately 2.5 x 10 8 TU / kg, approximately 2 x 10 8 TU / kg, approximately 1.5 x 10 8 TU / kg, or approximately 1 × 10⁻⁶ 8 The value is TU / kg. In some embodiments, the effective dose is 5 × 10 10 Less than TU / kg, 4.5 × 10 10 Less than TU / kg, 4 × 10 10 Less than TU / kg, 3.5 × 10 10 Less than TU / kg, 3 × 10 10 Less than TU / kg, 2.5 × 10 10 Less than TU / kg, 2 × 10 10 Less than TU / kg, 1.5 × 10 10 Less than TU / kg, 1 × 10 10 Less than TU / kg, 9.5 × 10 9 Less than TU / kg, 9 × 10 9 Less than TU / kg, 8.5 × 10 9 Less than TU / kg, 8 × 10 9 Less than TU / kg, 7.5 × 10 9 Less than TU / kg, 7 × 10 9 Less than TU / kg, 6.5 × 10 9Less than TU / kg, 6 × 10 9 Less than TU / kg, 5.5 × 10 9 Less than TU / kg, 5 × 10 9 Less than TU / kg, 4.5 × 10 9 Less than TU / kg, 4 × 10 9 Less than TU / kg, 3.5 × 10 9 Less than TU / kg, 3 × 10 9 Less than TU / kg, 2.5 × 10 9 Less than TU / kg, 2 × 10 9 Less than TU / kg, 1.5 × 10 9 Less than TU / kg, 1 × 10 9 Less than TU / kg, approximately 9.5 × 10 8 Less than TU / kg, approximately 9 x 10 8 Less than TU / kg, approximately 8.5 × 10 8 Less than TU / kg, approximately 8 x 10 8 Less than TU / kg, approximately 7.5 × 10 8 Less than TU / kg, approximately 7 x 10 8 Less than TU / kg, approximately 6.5 × 10 8 Less than TU / kg, approximately 6 x 10 8 Less than TU / kg, approximately 5.5 × 10 8 Less than TU / kg, approximately 5 x 10 8 Less than TU / kg, approximately 4.5 × 10 8 Less than TU / kg, approximately 4 x 10 8 Less than TU / kg, approximately 3.5 × 10 8 Less than TU / kg, approximately 3 × 10 8 Less than TU / kg, approximately 2.5 × 10 8 Less than TU / kg, approximately 2 × 10 8 Less than TU / kg, approximately 1.5 × 10⁻⁶ 8 Less than TU / kg, or approximately 1 × 10⁻⁶ 8 It is less than TU / kg. In some embodiments, the effective dose is 1 × 10 8 ~5×10 10 TU / kg, 1 × 10 8 ~5×10 9 TU / kg, 1 × 10 8 ~1 × 10 9 TU / kg, 1 × 10 8 ~1 × 10 10 TU / kg, 1 × 10 9 ~5×10 10TU / kg, 2×10 9 ~5×10 10 TU / kg, 3×10 9 ~5×10 10 TU / kg, 4×10 9 ~5×10 10 TU / kg, 5×10 9 ~5×10 10 TU / kg, 1×10 9 ~6×10 9 TU / kg, 2×10 9 ~6×10 9 TU / kg, 3×10 9 ~6×10 9 TU / kg, 4×10 9 ~6×10 9 TU / kg, 5×10 9 ~6×10 9 TU / kg, 6×10 9 ~5×10 10 TU / kg, 7×10 9 ~5×10 10 TU / kg, 8×10 9 ~5×10 10 TU / kg, 9×10 9 ~5×10 10 TU / kg, 10 10 ~5×10 10 TU / kg, 1.5×10 10 ~5×10 10 TU / kg, 2×10 10 ~5×10 10 TU / kg, 2.5×10 10 ~5×10 10 TU / kg, 3×10 10 ~5×10 10 TU / kg, 3.5×10 10 ~5×10 10 TU / kg, 4×10 10 ~5×10 10 TU / kg, or 4.5×10 10 ~5×10 10 TU / kg. In some embodiments, the effective dose is 1×10 9 ~5×10 10 TU / kg, 1×10 9 ~4.5×10 10 TU / kg, 1×10 9 ~4×1010 TU / kg, 1 × 10 9 ~3.5×10 10 TU / kg, 1 × 10 9 ~3×10 10 TU / kg, 1 × 10 9 ~2.5×10 10 TU / kg, 1 × 10 9 ~2×10 10 TU / kg, 1 × 10 9 ~1.5×10 10 TU / kg, 1 × 10 9 ~10 10 TU / kg, 1 × 10 9 ~9×10 9 TU / kg, 1 × 10 9 ~8×10 9 TU / kg, 1 × 10 9 ~7×10 9 TU / kg, 1 × 10 9 ~6×10 9 TU / kg, 1 × 10 9 ~5×10 9 TU / kg, 1 × 10 9 ~4×10 9 TU / kg, 1 × 10 9 ~3×10 9 TU / kg, and 1 × 10 9 ~2×10 9 The value is TU / kg. In some embodiments, the effective dose is 1 × 10⁻⁶ 10 ~2×10 10 TU / kg, 1.1 × 10 10 ~1.9×10 10 TU / kg, 1.2 × 10 10 ~1.8×10 10 TU / kg, 1.3 × 10 10 ~1.7×10 10 TU / kg, or 1.4 × 1 0 10 ~1.6×10 10 The value is TU / kg. In some embodiments, the effective dose is approximately 4 × 10⁻⁶. 9 TU / kg ~ approximately 6 x 10 9 It is TU / kg.

[0031] In some embodiments, the lentiviral vector is administered in single or multiple doses. In some embodiments, the lentiviral vector is administered via intravenous injection. In some embodiments, the subjects are children. In some embodiments, the subjects are adults.

[0032] In some embodiments, a nucleotide sequence such as that shown in Sequence ID No. 1. This disclosure also relates to vectors comprising nucleic acid sequences disclosed herein. In some embodiments, the vector includes a tissue-specific promoter. In some embodiments, the tissue-specific promoter selectively enhances the expression of a polypeptide having FIX activity in target hepatocytes. In some embodiments, the tissue-specific promoter that selectively enhances the expression of a polypeptide having FIX activity in target hepatocytes includes the APOA2 promoter, the SERPINA1(hAAT) promoter, the mTTR promoter, the MIR122 promoter, or any combination thereof. In some embodiments, the target hepatocytes are hepatocytes.

[0033] In some embodiments, the vector includes a splice donor site. In some embodiments, the vector includes a splice acceptor site. In some embodiments, the vector includes a gag sequence, a pol sequence, a rev sequence, a rev response element (RRE), or any combination thereof. In some embodiments, the gag sequence is a full-length or truncated gag sequence. In some embodiments, the vector includes an enhancer, a target sequence for microRNA, a post-transcriptional regulatory element, a packaging signal, a polyA sequence, an intron sequence, or any combination thereof.

[0034] This disclosure also relates to cells containing nucleic acid sequences or vectors disclosed herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are CHO cells, HEK293 cells, BHK21 cells, PER.C6® cells, NS0 cells, and CAP cells. In some embodiments, the cells are human cells. In some embodiments, the cells express the CD47 protein. In some embodiments, the cells are modified to overexpress CD47. In some embodiments, the cells contain at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 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, and at least about 40 times more CD47 protein on their surface compared to control cells that have not been modified to overexpress CD47. In some embodiments, CD47 is human CD47. In some embodiments, the cells do not express MHC-I.

[0035] This disclosure also relates to a method for producing a lentiviral vector, which includes culturing the cells disclosed herein under preferred conditions. [Brief explanation of the drawing]

[0036] [Figure 1] This figure shows the vector map of a lentiviral vector ("LV-coFIX-1-R338L") containing nucleic acid encoding a polypeptide with FIX activity. [Figure 2]Figures 2A and 2B show graphs of plasma FIX activity in HemB mice after administration at 8 weeks of age of mice with a lentiviral vector containing a nucleic acid sequence encoding a polypeptide with FIX activity. Figure 2A shows the plasma FIX activity after administration of LV-coFIX-1-R338L via tail vein injection at doses of 3E9, 7.5E9, 2E10, or 6E10 TU / kg, and Figure 2B shows the corresponding dose-response curves. Error bars represent the standard deviation (Figure 2B). [Figure 3] Figures 3A and 3B show graphs of plasma FIX activity (Figure 3A) and plasma FIX antigen (Figure 3B) at various time points up to 6 months of age in 8-week-old HemB mice administered LV-coFIX-1-R338L via tail vein injection at doses of 7.5E9 (circles), 2E10 (squares), or 6E10 (triangles) TU / kg. Error bars represent the standard deviation (Figures 3A and 3B). [Figure 4A] This figure shows graphs of sustained FIX expression (Figure 4A) and LV-FIX dose response (Figure 4B) at various time points up to 6 months of age during the neonatal, adolescent, or adult stage. HemB mice were administered LV-coFIX-1-R338L at doses of 7.5E9, 2E10, or 6E10 TU / kg by intravenous injection at 8 weeks of age (square) or 2 days of age (circle); or, as shown, HemB mice were administered LV-cocoFIX-1-R338L at doses of 3E9, 7.5E9, or 2E10 TU / kg by intravenous injection at 2 weeks of age (triangle) (Figure 4A). Dose response was measured by FIX activity at various doses tested in Figure 4A in HemB mice at 8 weeks of age (square), 2 weeks of age (triangle), and 2 days of age (circle). Error bars represent the standard deviation (Figures 4A-4B). [Figure 4B]This figure shows graphs of sustained FIX expression (Figure 4A) and LV-FIX dose response (Figure 4B) at various time points up to 6 months of age during the neonatal, adolescent, or adult stage. HemB mice were administered LV-coFIX-1-R338L at doses of 7.5E9, 2E10, or 6E10 TU / kg by intravenous injection at 8 weeks of age (square) or 2 days of age (circle); or, as shown, HemB mice were administered LV-cocoFIX-1-R338L at doses of 3E9, 7.5E9, or 2E10 TU / kg by intravenous injection at 2 weeks of age (triangle) (Figure 4A). Dose response was measured by FIX activity at various doses tested in Figure 4A in HemB mice at 8 weeks of age (square), 2 weeks of age (triangle), and 2 days of age (circle). Error bars represent the standard deviation (Figures 4A-4B). [Figure 5] This figure shows plots of the vector copy number (VCN) of lentiviral vectors in macrophages of NOD mice after administration of a control lentiviral vector (LV; black circles) or a lentiviral vector with high surface levels of CD47 (CD47hi LV; gray circles), determining the number of lentiviral vector molecules present in the macrophages. VCN data from HEK293T cells are shown as a control. Error bars represent the standard deviation. [Figure 6-1] Figures 6A-6C show graphs of plasma FIX activity (Figure 6A), plasma FIX antigen (Figure 6B), and FIX function (expressed as APTT time; Figure 6C) in porcine-tailed monkeys after administration of a control lentiviral vector (LV-FIX; #1, #2, and #3), a lentiviral vector containing a nucleic acid sequence encoding a FIX-active polypeptide packaged in a lentiviral vector with high surface levels of CD47 (CD47hi LV-FIX; #4, #5, and #6), or a vehicle control (#7). [Figure 6-2] Continuation of Figure 6-1. [Figure 7]Figures 7A-7B show graphs of steady-state lentiviral vector-mediated FIX expression in pig-tailed monkeys after administration of a CD47high lentiviral vector containing a nucleic acid sequence encoding a polypeptide with FIX activity, administered at a dose of E9 TU / kg. Error bars represent the standard deviation (Figures 7A-7B). [Figure 8-1] Figures 8A-8D are graphs showing ALT levels (Figure 8A), AST levels (Figure 8B), lymph levels (Figure 8C), and body temperature (Figure 8D) in pig-tailed monkeys after administration of a vehicle (white circle), a control lentiviral vector (LV; black circle), or a lentiviral vector with high surface levels of CD47 (CD47hi LV; gray circle) (this lentiviral vector contains a lentiviral vector that includes a nucleic acid sequence encoding a polypeptide with FIX activity). [Figure 8-2] Continuation of Figure 8-1. [Figure 9-1] Figures 9A-9C are graphs showing the expression levels of MIP-1a (9A), MIP-1b (9B), and MCP-1 (9C) in pig-tailed monkeys after administration of a vehicle (black circle), a control lentiviral vector (LV; black square), or a lentiviral vector with high surface levels of CD47 (CD47hi LV; unfilled square) (this lentiviral vector contains a lentiviral vector containing a nucleic acid sequence encoding a polypeptide with FIX activity). [Figure 9-2] Continuation of Figure 9-1. [Figure 10] This figure shows a scatter plot illustrating the tissue-specific distribution (represented by VCN) of lentiviral vectors containing nucleic acid sequences encoding polypeptides with FIX activity after administration to pig-tailed macaques with a vehicle (triangle), a control lentiviral vector (inverted triangle), or CD47hi (LV; circle). Each dataset represents an individual pig-tailed macaque. [Figure 11]Figures 11A and 11B are graphs showing steady-state lentiviral vector-mediated FIX expression in pig-tailed monkeys after administration of a CD47high lentiviral vector containing a nucleic acid sequence encoding a polypeptide with FIX activity, administered at a dose of 2.5 E9TU / kg, as represented by plasma FIX activity (Figure 11A) and plasma FIX antigen (Figure 11B). [Modes for carrying out the invention]

[0037] This disclosure describes lentiviral vectors containing nucleic acids encoding polypeptides having FIX activity, and methods of using them. Accordingly, in some embodiments, this disclosure relates to gene therapy involving the administration of a lentiviral vector comprising a nucleic acid molecule containing a nucleic acid sequence encoding a polypeptide having FIX activity. In certain embodiments, this disclosure relates to a method for treating bleeding disorders such as hemophilia (e.g., hemophilia B), comprising targeting and administering a lentiviral vector containing a codon-optimized FIX nucleic acid sequence to the liver (e.g., hepatocytes). This disclosure addresses a critical need in the art through a gene therapy approach that results in the stable integration of an transgene expression cassette containing a nucleic acid sequence encoding a polypeptide having FIX activity into the genome of targeted cells.

[0038] This system uses lentiviral vectors 5 × 10 10 Less than or equal to the transduction unit / kg (TU / kg), for example, approximately 1.5 × 10⁻⁶ 10 Less than TU / kg, or approximately 1.5 × 10⁻⁶ 9 Less than TU / kg, or approximately 1 × 10⁻⁶ 8 When administered to subjects at least one dose of TU / kg or less, the study demonstrates increased long-term expression of polypeptides with FIX activity in targeted cells (e.g., hepatocytes).

[0039] Exemplary structures of this disclosure are illustrated in the accompanying drawings and sequence list.

[0040] To provide a clear understanding of the specification and claims, the following definitions are provided below.

[0041] I. Definition Note that the entity referred to as “a” or “an” refers to one or more of those entities: for example, “nucleotide sequence” is understood to represent one or more nucleotide sequences. Thus, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0042] In this specification, the term "approximately" is used to mean roughly, about, around, or nearly. The term "approximately" is used in conjunction with a numerical range to modify that range by extending the upper and lower boundaries of the given number. Generally, the term "approximately" is used herein to modify the numbers above and below the given value by a 10% variation (up or down).

[0043] For the purposes of this disclosure, the term “isolated” refers to a biomaterial (cell, polypeptide, polynucleotide or its fragments, variants, or derivatives) that has been removed from its original environment (the environment in which it naturally occurs). For example, a polynucleotide that occurs naturally in a plant or animal is not isolated, but the same polynucleotide that is separated from a naturally occurring adjacent nucleic acid is considered “isolated.” No specific level of purification is required. Recombinant polypeptides and proteins expressed in a host cell are considered isolated in this disclosure, as are natural or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any preferred technique.

[0044] The terms “nucleic acid,” “nucleic acid molecule,” “oligonucleotide,” and “polynucleotide” are used interchangeably and refer to phosphate ester polymer forms of ribonucleosides (adenosine, guanosine, uridine, or cytidine; “RNA molecule”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecule”), or any phosphate ester analogues thereof in single-stranded or double-stranded helical form, e.g., phosphorothioates and thioesters. Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, in particular DNA or RNA molecule, refers only to the primary and secondary structures of the molecule and does not limit it to any specific tertiary form. Thus, this term includes, among other things, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and double-stranded DNA found in chromosomes. In discussions of the structure of a particular double-stranded DNA molecule, the sequence may be described herein by the usual convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). “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. “Nucleic acid composition” in this disclosure comprises one or more nucleic acids described herein.

[0045] As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. A “stop codon” (TAG, TGA, or TAA) is not generally translated into an amino acid, but can be considered part of a coding region; however, any adjacent sequences, such as promoters, ribosome-binding sites, transcription terminators, or introns, are not part of a coding region. The boundaries of a coding region are generally determined by a 5' start codon encoding the amino terminus of the resulting polypeptide and a 3' translation stop codon encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions can exist, for example, in a single polynucleotide construct on a single vector, or in separate polynucleotide constructs on separate (different) vectors. Consequently, a single vector can then contain only one coding region or two or more coding regions.

[0046] Certain proteins secreted by mammalian cells are associated with secreted signal peptides, which are cleaved from mature proteins when the transition of the growing protein chain across the coarse endoplasmic reticulum begins. Those skilled in the art know that signal peptides are commonly fused to the N-terminus of polypeptides and that they are cleaved from the complete or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, or a poly(IU) operably associated with it, is used. Functional derivatives of the sequence that retain the ability to direct peptide secretion. Alternatively, signal peptides from other mammalian species, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or their functional derivatives can be used.

[0047] The term “FIX activity-possessive polypeptide” refers to a polypeptide having one or more activities related to coagulation factor IX, as used herein. Several tests are available to investigate the function of the coagulation system, including FIX: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM® assay, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often by the Krauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (whether abnormalities are corrected when the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimers, genetic testing (e.g., factor V Leiden, prothrombin mutation G20210A), diluted Russell's viper venom time (dRVVT), other platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0048] The aPTT test is a performance indicator that measures the efficacy of the "endogenous" (also known as the contact-activated pathway) and common coagulation pathways. This test is commonly used to measure the coagulation activity of commercially available recombinant coagulation factors, such as FIX. It is used in conjunction with prothrombin time (PT), which measures the exogenous pathway.

[0049] ROTEM® analysis provides complete kinetics of hemostasis: information on coagulation time, clot formation, clot stability, and lysis. Different parameters in thromboelastometry depend on many factors that affect the activity of the plasma coagulation system, platelet function, fibrinolysis, or their interactions. This assay can provide a complete picture of secondary hemostasis.

[0050] The term "downstream" refers to a nucleotide sequence located 3' to the reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to the sequence following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0051] The term "upstream" refers to a nucleotide sequence located 5' to the reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence refers to a sequence located 5' to the coding region or transcription start site. For example, most promoters are located upstream of the transcription start site.

[0052] As used herein, the term “gene regulatory region” or “regulatory region” refers to a nucleotide sequence located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding region that affects 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 stem-loop structures. When the coding region is intended for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequences are typically located on the 3' side of the coding sequence.

[0053] A gene product, such as a polynucleotide encoding a polypeptide, may include one or more coding regions and operably associated promoters and / or other expression (e.g., transcription or translation) regulatory elements. In operably associated regions, the coding region for the gene product, such as a polypeptide, may regulate the expression of the gene product. A coding region is associated with one or more regulatory regions in a manner that influences or controls them. For example, a coding region and a promoter are "operably associated" if the induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and the nature of the linkage between the promoter and the coding region does not interfere with the promoter's ability to direct gene product expression or the ability of the DNA template to be transcribed. Other expression regulatory elements other than promoters, such as enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[0054] A “transcriptional regulatory sequence” refers to a DNA regulatory sequence that enables the expression of a coding sequence in a host cell, such as a promoter, enhancer, or terminator. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as, but are not limited to, promoter and enhancer segments from cytomegalovirus (pre-early promoter with intron-A), Simian virus 40 (early promoter), and retroviruses (such as Roussarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of regulating gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters induceable by interferon or interleukin).

[0055] Similarly, various translational control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from picornaviruses (in particular, ribosome entry sites or IRESs, also known as CITE sequences).

[0056] As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or polypeptide. This includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interference RNA (siRNA) or any other RNA product, and the translation of mRNA into polypeptides. Expression produces a “gene product.” As used herein, a gene product may be a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from the transcript. Gene products described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, binding to other protein subunits, or proteolytic cleavage. As used herein, the term “yield” refers to the amount of polypeptide produced by gene expression.

[0057] "Vector" refers to any vehicle for cloning and / or transferring nucleic acids into host cells. A vector may be a replicon to which another nucleic acid segment can be attached to result in replication of the attached segment. "Replicon" is in The term "vector" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replicating under its own control. The term "vector" includes both viral and nonviral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors are known and used in the art, including plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of polynucleotides into a suitable vector is performed by selecting a vector with complementary sticky ends. This can be achieved by ligating a suitable polynucleotide fragment.

[0058] Vectors can be engineered to encode selectable markers or reporters that enable the selection or identification of cells incorporating the vector. Expression of a selectable marker or reporter incorporates other coding regions contained on the vector, enabling the identification and / or selection of host cells that express them. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyltransferase genes, etc. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. A selectable marker can also be considered a reporter.

[0059] The term “selectable marker” refers to identification factors, typically antibiotic or chemical resistance genes, that can be selected based on the action of a marker gene, i.e., antibiotic resistance, herbicide resistance, colorimetric markers, enzymes, fluorescent markers, etc., where this action is used to track the inheritance of a nucleic acid of interest and / or to identify cells or organisms that have inherited a nucleic acid of interest. Examples of selectable marker genes known and used in the art include: genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyltransferase genes, etc.

[0060] The term "reporter gene" refers to a nucleic acid that encodes an identification factor, which can be identified based on the action of the reporter gene, where this action is used to track the inheritance of a target nucleic acid, to identify cells or organisms that have inherited a target nucleic acid, and / or to measure the induction or transcription of gene expression. Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable marker genes can also be considered reporter genes.

[0061] The terms "promoter" and "promoter sequence" are used interchangeably and refer to DNA sequences capable of controlling the expression of coding sequences or functional RNA. Generally, coding sequences are located 3' to the promoter sequence. Promoters can be entirely derived from native genes, or they may consist of different elements derived from different naturally occurring promoters, or they may include synthetic DNA segments. Those skilled in the art understand that different promoters can direct gene expression in different tissues or cell types, at different stages of development, or in response to different environments or physiological conditions. Promoters that express a gene in most cell types and most of the time are generally called "constitutive promoters." Promoters that express a gene in a specific cell type are generally called "cell-specific promoters" or "tissue-specific promoters." Promoters that express a gene at a specific stage of development or cell differentiation are generally called "development-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced to express a gene after exposure or treatment of cells with promoter-inducing drugs, biomolecules, chemicals, ligands, light, etc., are generally called "inducible promoters." These are called "regulatory promoters" or "adjustable promoters." In most cases, the precise boundaries of regulatory sequences are not fully defined, so it is further recognized that DNA fragments of different lengths can have the same promoter activity.

[0062] A promoter sequence typically contains the minimum number of bases or elements necessary for the transcription start site to bind to its 3' end and extend upstream (5' direction) to initiate transcription at a detectable level above the background. Within the promoter sequence, the transcription start site (conveniently defined, for example, by mapping with nuclease S1) and the protein-binding domain (consensus sequence) responsible for RNA polymerase binding can be found.

[0063] The term "plasmid" refers to an extrachromosomal element that is not part of the cell's central metabolism and often contains genes, usually in the form of a circular double-stranded DNA molecule. Such elements may be linear, circular, or supercoiled self-replicating sequences, genomic integration sequences, phages, or nucleotide sequences of single-stranded or double-stranded DNA or RNA derived from any source, in which several nucleotide sequences are ligated or rearranged into a specific construct that allows for the introduction into the cell a promoter fragment and DNA sequence for a selected gene product, along with a suitable 3' untranslated sequence.

[0064] A "cloning vector" refers to a "replicon," which is a unit-length nucleic acid containing a replication origin, such as a plasmid, phage, or cosmid, that replicates sequentially and can have another nucleic acid segment attached to it to result in the replication of the attached segment. A particular cloning vector can replicate in one cell type, e.g., bacteria, and express in another cell type, eukaryotic cells. Cloning vectors typically contain one or more sequences that can be used for the selection of cells containing the vector, and / or one or more multiplexing sites for the insertion of the desired nucleic acid sequence.

[0065] The term "expression vector" refers to a vehicle designed to enable the expression of an inserted nucleic acid sequence after insertion into a host cell. The inserted nucleic acid sequence is placed in an operable relationship with the regulatory region described above.

[0066] The vector is introduced into a host cell by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter. “Lentiviral vector” as used herein refers to a replication-defect hybrid viral particle. In some contexts, lentiviral vector refers to a lentiviral vector particle and the encapsulated lentiviral genome. In some contexts, lentiviral vector refers to a lentiviral genome, including any modifications thereof.

[0067] As used herein, “culture,” “to culture,” and “to keep in culture” mean incubating cells under in vitro conditions that allow for cell proliferation or division, or maintaining cells in a viable state. As used herein, “cultured cells” means cells that have been grown in vitro.

[0068] As used herein, the term “polypeptide” encompasses both the singular and plural “polypeptides” and refers to a molecule composed of monomers (amino acids) linked linearly by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or number of chains of two or more amino acids, and its specific length This does not refer to the products of polypeptides. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to chains of two or more amino acids, and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" also refers to the products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with amino acids not present in nature. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. They can be produced by any method, including chemical synthesis.

[0069] 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 this disclosure and include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, such as those described by Ellman et al., Meth. Enzym. 202: pp. 301-336 (1991). To generate such naturally occurring amino acid residues, the methods of Noren et al., Science 244: p. 182 (1989) and Ellman et al. mentioned above can be used. Briefly, these methods involve chemically activating a suppressor tRNA with a naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA. The introduction of non-traditional amino acids can also be achieved using peptide chemistry known in the art. As used herein, the term “polar amino acids” includes amino acids that have zero total charge but have non-zero partial charge on different parts of their side chains (e.g., M, F, W, S, Y, N, Q, C). These amino acids can be involved in hydrophobic and electrostatic interactions. As used herein, the term “charged amino acids” includes amino acids that may have a non-zero total charge in their side chains (e.g., R, K, H, E, D). These amino acids can be involved in hydrophobic and electrostatic interactions.

[0070] Polypeptide fragments or variants and any combination thereof are also included in this disclosure. The terms “fragment” or “variant” as used to refer to polypeptide binding domains or binding molecules in this disclosure include any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity to an Fc variant or Fc binding domain, coagulation activity of a polypeptide having FIX activity). Polypeptide fragments include proteolytic and deletion fragments, in addition to specific antibody fragments discussed elsewhere in this specification, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of polypeptide binding domains or binding molecules in this disclosure also include the above-mentioned fragments, as well as polypeptides having altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may be naturally occurring or non-natural. Non-natural variants can be generated using mutagenic techniques known in the art. Variant polypeptides may include conserved or non-conserved amino acid substitutions, deletions, or additions.

[0071] "Conservative amino acid substitutions" are made by replacing amino acid residues with amino acid residues that have similar side chains. These are interchangeable. Families of amino acid residues having similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, when 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, a chain of amino acids can be conservatively replaced by a structurally similar chain with a different order and / or composition of side chain family members.

[0072] As is well known in the art, the term “identity percentage” is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by a comparison of sequences. In the art, “identity” also means the degree of sequence relevance between polypeptide or polynucleotide sequences, as determined by the agreement between the strings of such sequences. "Identity" can be readily calculated by known methods, including, but not limited to, those listed below: Computational Molecular Biology (edited by Lesk, AM), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (edited by Smith, DW), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (edited by von Heinje, G.), Academic Press (1987); and Sequence Analysis Primer (edited by Gribskov, M. and Develeux, J.), Stockton Press, New York (1991). Preferred methods for determining identity are designed to give the best match among the sequences being tested. Methods for determining identity have been systematized into publicly available computer programs.Sequence alignment and identity percentage calculations can be performed using sequence analysis software such as the Megaalign program (DNASTAR Inc., Madison, WI) from the LASERGENE Bioinformatics Computing Suite, 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, WI53715 USA).

[0073] Within the context of this application, when sequence analysis software is used for analysis, it is understood that, unless otherwise specified, the results of the analysis will be based on the “default values” of the referenced program. As used herein, “default values” means any set of values ​​or parameters that are initially loaded with the software when it is first initialized.

[0074] A "fusion" or "chimeric" protein contains a first amino acid sequence that is linked to a second amino acid sequence that is not naturally linked. Amino acid sequences normally present in separate proteins can be brought together into the fusion polypeptide, or amino acid sequences normally present in the same protein can be placed in a new configuration in the fusion polypeptide, for example, in the fusion with the Ig Fc domain of the FIX domain of this disclosure. The chimeric protein is produced, for example, by chemical synthesis, or by creating and translating polynucleotides in which the peptide region encodes in a desired relationship. The chimeric protein may further include a second amino acid sequence associated with the first amino acid sequence by covalent, non-peptide, or non-covalent bonds.

[0075] As used herein, the term “insertion site” refers to a location within a FIX-active polypeptide or its fragment, variant, or derivative immediately upstream of a site where a heterologous portion can be inserted. The “insertion site” is defined by a number, which, unless otherwise specified, is the amino acid number in the human FIX R338L variant (SEQ ID NOs. 11-12) to which the insertion site corresponds, immediately to the N-terminus of the insertion site.

[0076] As used herein, the phrase "immediately downstream of an amino acid" 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.

[0077] As used herein, the terms “inserted,” “to be inserted,” “to be inserted into,” or grammatically related terms refer to the position of a heterologous portion in the recombinant FIX polypeptide relative to a similar position in naturally mature human FIX. As used herein, these terms refer to the characteristics of the recombinant FIX polypeptide relative to naturally mature human FIX and do not indicate, imply, or mean any method or process by which the recombinant FIX polypeptide was produced.

[0078] As used herein, the term “half-life” refers to the biological half-life of a particular polypeptide in vivo. Half-life can be expressed as the time required for half of the amount administered to a subject to be eliminated from the animal’s circulation and / or other tissues. When the elimination curve of a given polypeptide is constructed as a function of time, the curve is typically biphasic, consisting of a rapid α-phase and a longer β-phase. The α-phase generally represents the equilibration of the administered Fc polypeptide between intravascular and extravascular spaces, and is partly determined by the size of the polypeptide. The β-phase generally represents the catabolism of the polypeptide in intravascular space. In some embodiments, FIX and chimeric proteins containing FIX are monophasic and therefore have no α-phase, but only a single β-phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the β-phase.

[0079] As used herein, the term “linked” refers to a first amino acid sequence or nucleotide sequence that is linked to a second amino acid sequence or nucleotide sequence, respectively, by covalent or noncovalent bonds. The first amino acid sequence or nucleotide sequence may be directly linked to or juxtaposed with the second amino acid sequence or covalently linked to the second sequence by an intervening sequence. The term “linked” includes not only the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also the insertion of the entire first amino acid sequence (or second amino acid sequence) into any two amino acids of the second amino acid sequence (or each of the first amino acid sequences). In one embodiment, the first amino acid sequence may be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence may be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker may be a peptide or polypeptide (in the case of a polypeptide chain), a nucleotide or nucleotide chain (in the case of a nucleotide chain), or any chemical moiety (in the case of polypeptides and polynucleotide chains). The term "linked" can also be indicated by a hyphen (-).

[0080] As used herein, the term “associated with” means a first amino acid chain and a second amino acid chain. This refers to covalent or non-covalent bonds formed between non-acid chains. In one embodiment, the term “associated with” means a covalent, non-peptide, or non-covalent bond. This association can 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 ​​contains a thiol group on the second cysteine ​​residue that can form a disulfide bond or bridge with the thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by a disulfide bond, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidic bonds, unknowable bonds, vent bonds, dipole bonds, pi-back bonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugations, hyperconjugations, aromatics, polydentates, or antibonds. Non-exclusive examples of non-covalent bonds include ionic bonds (e.g., cation-pi bonds or salt bonds), metallic bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds or symmetrical hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, gold affinity, intercalation, stacking, entropic forces, or chemical polarity.

[0081] As used herein, the term "monomer-dimer hybrid" refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain associated with each other by a disulfide bond, wherein the first chain comprises a coagulation factor, e.g., FIX, and a first Fc region, and the second chain comprises, essentially, or consists of, a second Fc region without a coagulation factor. Thus, a monomer-dimer hybrid construct is a hybrid comprising a monomeric form having only one coagulation factor and a dimeric form having two Fc regions.

[0082] As used herein, hemostasis means stopping or slowing down bleeding or heavy bleeding; or stopping or slowing down blood flow through a blood vessel or part of the body.

[0083] Hemostatic disorders, as used herein, mean genetically inherited or acquired conditions characterized by a bleeding tendency, either spontaneous or as a result of trauma, due to impaired or deficiency of the ability to form fibrin clots. Examples of such disorders include hemophilia. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease"), and hemophilia C (factor XI deficiency, a mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrandt disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiency or structural abnormality of fibrinogen, prothrombin, factor V, factor VII, factor X or XIII, and Bernard-Soulier syndrome, which is a GPIb deficiency or deficiency. Deficiency in the vWF receptor GPIb can lead to loss of primary clot formation (primary hemostasis) and increased bleeding tendency, as well as Glanzmann and Naegeli thrombasthenia (Glanzmann thrombasthenia). In hepatic failure (acute and chronic forms), the liver's production of coagulation factors is insufficient, which can increase the risk of bleeding.

[0084] Lentiviral vectors comprising isolated nucleic acid molecules of this disclosure can be used prophylactically. As used herein, the term “prophylactic treatment” refers to the administration of the molecule prior to a bleeding episode. In one embodiment, a subject requiring a systemic hemostatic agent is undergoing or about to undergo surgery. For example, the lentiviral vectors of this disclosure can be administered before or after surgery as a prophylactic agent. The lentiviral vectors of this disclosure can be administered during or after surgery to control an acute bleeding episode. Surgery may include, but is not limited to, liver transplantation, hepatectomy, dental procedures, or stem cell transplantation.

[0085] The lentiviral vectors of this disclosure may also be used for on-demand therapy. The term “on-demand therapy” refers to the administration of the lentiviral vectors disclosed herein in response to symptoms of a bleeding episode or before an activity that may cause bleeding. In one embodiment, on-demand therapy may be given to a subject when bleeding begins, such as after an injury, or when bleeding is anticipated, such as before surgery. In another embodiment, on-demand therapy may be given before an activity that increases the risk of bleeding, such as contact sports.

[0086] As used herein, the term “acute bleeding” refers to a bleeding episode regardless of the underlying cause. For example, the subject may have trauma, uremia, a hereditary bleeding disorder (e.g., FIX deficiency), platelet dysfunction, or resistance due to the development of antibodies against coagulation factors.

[0087] As used herein, treating, therapeutic, and therapeutic means, for example, reducing the severity of a disease or condition; reducing the duration of the disease; improving one or more symptoms associated with the disease or condition; or providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition or preventing one or more symptoms associated with the disease or condition. In one embodiment, the term “to treat” or “to treat” means that by administering the lentiviral vector of the Disclosure, a FIX trough level of at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL is maintained in a subject. In another embodiment, treating or maintaining the FIX trough level between approximately 1 to approximately 20 IU / dL, approximately 2 to approximately 20 IU / dL, approximately 3 to approximately 20 IU / dL, approximately 4 to approximately 20 IU / dL, approximately 5 to approximately 20 IU / dL, approximately 6 to approximately 20 IU / dL, approximately 7 to approximately 20 IU / dL, approximately 8 to approximately 20 IU / dL, approximately 9 to approximately 20 IU / dL, or approximately 10 to approximately 20 IU / dL. Treatment or management of a disease or condition may also include maintaining FIX activity in the subject at a level equivalent to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of FIX activity in a non-hemophilic subject. In one embodiment, the term “to treat” or “treatment” means maintaining a FIX trough level of at least about 30 IU / dL, 40 IU / dL, 50 IU / dL, 60 IU / dL, 70 IU / dL, 80 IU / dL, 90 IU / dL, 100 IU / dL, 110 IU / dL, 120 IU / dL, 130 IU / dL, 140 IU / dL, or 150 IU / dL in a subject by administering the lentiviral vector of the Disclosure.In another embodiment, taking or performing the treatment means maintaining a FIX trough level of approximately 10–20 IU / dL, approximately 20–23 IU / dL, approximately 30–40 IU / dL, approximately 40–50 IU / dL, approximately 50–60 IU / dL, approximately 60–70 IU / dL, approximately 70–80 IU / dL, approximately 80–90 IU / dL, approximately 90–100 IU / dL, approximately 110–120 IU / dL, approximately 120–130 IU / dL, approximately 130–140 IU / dL, or approximately 140–150 IU / dL. Treatment or management of a disease or condition may also include maintaining FIX activity in the subject at a level equivalent 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 FIX activity in non-hemophilic subjects. The minimum trough level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each individual.

[0088] As used herein, “administer” includes a pharmaceutically acceptable nucleic acid molecule encoding FIX, a FIX polypeptide, or a nucleic acid molecule encoding FIX as disclosed herein. This means administering the vector to the subject through a pharmaceutically acceptable route. The route of administration may be intravenous, for example, intravenous injection and intravenous infusion. Further routes of administration include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration. Nucleic acid molecules, polypeptides, and vectors can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0089] As used herein, the phrase “subjects requiring it” includes subjects such as mammalian subjects who would benefit from the administration of nucleic acid molecules, polypeptides, or vectors of this disclosure, for example, to improve hemostasis. In one embodiment, subjects include, but are not limited to, individuals with hemophilia. In another embodiment, subjects include, but are not limited to, individuals who develop FIX inhibitors and therefore require bypass therapy. Subjects are adults or minors (e.g., under 12 years of age). In some embodiments, subjects are female. In other embodiments, subjects are male.

[0090] As used herein, the term “coagulation factor” refers to a naturally occurring or recombinantly produced molecule or analogue that prevents or shortens the duration of a bleeding episode in a subject. In other words, it means a molecule that has procoagulation activity, i.e., plays a role in converting fibrinogen into a mesh of insoluble fibrin that causes blood agglutination or coagulation. “Activatable coagulation factor” is a coagulation factor that is in an inactive form (e.g., in the form of its enzyme precursor) and can be converted to an active form.

[0091] As used herein, coagulation activity means the ability to complete the formation of fibrin clots and / or participate in a cascade of biochemical reactions that reduce the severity, duration, or frequency of major bleeding or bleeding episodes.

[0092] As used herein, the terms “exogenous” or “non-exogenous” refer to molecules not normally found in a given context, e.g., a cell or polypeptide. Exogenous or non-exogenous molecules can be introduced into cells and may only be present after manipulation of the cell, e.g., by transfection or other forms of genetic engineering, or a non-exogenous amino acid sequence may be present in a protein that is not found naturally.

[0093] As used herein, the term “heteronucleotide sequence” refers to a nucleotide sequence that does not exist naturally together with a given polynucleotide sequence. In one embodiment, the heteronucleotide sequence encodes a polypeptide capable of extending the half-life of FIX. In another embodiment, the heteronucleotide sequence encodes a polypeptide that increases the hydrodynamic radius of FIX. In yet another embodiment, the heteronucleotide sequence encodes a polypeptide that improves one or more pharmacokinetic properties of FIX without significantly affecting its biological activity or function (e.g., its procoagulant activity). In some embodiments, FIX is linked or ligated by a linker to the polypeptide encoded by the heteronucleotide sequence. Non-limiting examples of polypeptide moieties encoded by heterologous nucleotide sequences include immunoglobulin constant regions or parts thereof, albumin or fragments thereof, albumin-binding moieties, transferrin, the PAS polypeptide of U.S. Patent Application No. 20100292130, HAP sequences, transferrin or fragments thereof, the C-terminal peptide (CTP) of the β-subunit of human chorionic gonadotropin, albumin-binding small molecules, XTEN sequences, FcRn-binding moieties (e.g., FcRn-binding moieties). A complete Fc region or a portion thereof, a single-chain Fc region (ScFc region, e.g., as described in US2008 / 0260738, WO2008 / 012543 or WO2008 / 1439545), a polyglycine linker, a polyserine linker, a peptide, and two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), having varying degrees of secondary structure from less than 50% to more than 50%. The embodiments include short polypeptides of 6 to 40 amino acids, or combinations of two or more thereof. In some embodiments, polypeptides encoded by heterologous nucleotide sequences are linked to a non-polypeptide moiety. Non-limiting examples of non-polypeptide moieties include polyethylene glycol (PEG), albumin-binding small molecules, polysialic acid (PAS), hydroxyethyl starch (HES), derivatives thereof, or any combination thereof.

[0094] As used herein, the term “Fc region” is defined as the polypeptide portion corresponding to the Fc region of natural Ig, i.e., formed by the dimerization of the respective Fc domains of its two heavy chains. Natural Fc regions form homodimers with other Fc regions. In contrast, as used herein, the term “gene fusion Fc region” or “single-chain Fc region” (scFc region) refers to a synthetic dimeric Fc region consisting of Fc domains that are genetically linked (i.e., encoded in a single continuous gene sequence) within a single polypeptide chain.

[0095] In one embodiment, the "Fc region" refers to a portion of a single Ig heavy chain that begins in the hinge region immediately upstream of the papain cleavage site (i.e., IgG residue 216, where 114 is the first residue in the heavy chain constant region) and ends at the C-terminus of the antibody. Therefore, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain.

[0096] The Fc region of the Ig constant region can include CH2, CH3, and CH4 domains, as well as a hinge region, depending on the Ig isotype. Chimeric proteins containing the Ig Fc region confer several desirable properties to the chimeric protein, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Patents 6,086,875, 6,485,726, and 6,030,613; WO03 / 077834; US2003-0235536A1), which are fully incorporated herein by reference.

[0097] As used herein, the term “optimized” in relation to nucleotide sequences refers to a polynucleotide sequence encoding a polypeptide, where the polynucleotide sequence has been mutated to enhance the properties of that polynucleotide sequence. In some embodiments, optimization is performed to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease binding affinity to regulatory proteins such as basic transcription factors, increase or decrease splicing, or increase the yield of polypeptides produced by the polynucleotide sequence. Examples of modifications that can be made to a polynucleotide sequence to optimize it include codon optimization, G / C content optimization, removal of repetitive sequences, removal of AT elements, removal of latent splice sites, removal of cis-acting elements that suppress transcription or translation, adding or removing poly-T or poly-A sequences, adding transcription-enhancing sequences such as Kozak consensus sequences around transcription start sites, removal of sequences that may form stem-loop structures, removal of destabilizing sequences, and two or more combinations thereof.

[0098] II. FIX Lentiviral Gene Therapy Somatic gene therapy has been explored as a possible treatment for bleeding disorders, particularly hemophilia. Gene therapy is a particularly attractive treatment for hemophilia due to its ability to treat hemophilia through the continuous endogenous generation of FIX after a single dose of a vector encoding FIX. Hemophilia B is particularly well-suited to the gene replacement approach because its clinical symptoms are due to reduced expression of functional FIX.

[0099] Lentiviral vectors are large tools that sustain transgene expression through integration. Due to their inclusiveness and capabilities, lentiviral vectors are attracting attention as gene delivery vehicles. They have been evaluated in numerous ex-vivo cell therapy clinical programs, demonstrating promising efficacy and safety profiles.

[0100] This disclosure provides a method for preventing or treating hemophilia in subjects requiring it, comprising administering an effective dose of a lentiviral vector comprising a nucleotide sequence encoding a polypeptide having FIX activity to the subject. In some embodiments, the lentiviral vector is packaged in lentiviral particles containing higher levels of surface CD47 protein expression than a control lentiviral vector, for example, a control lentiviral vector that does not have high levels of surface CD47 protein expression, i.e., a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®) where surface CD47 protein expression is at normal (naturally occurring) levels. In some embodiments, the effective dose is reduced compared to a control dose of the control lentiviral vector required to induce the same FIX activity as the lentiviral vector.

[0101] Other aspects of this disclosure are methods for preventing or treating hemophilia in subjects requiring it, 5 × 10 10 The present invention provides a method comprising administering to a subject a lentiviral vector containing a nucleotide sequence encoding a factor IX (FIX) activity polypeptide in a transduction unit / kg (TU / kg), wherein the lentiviral vector contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0102] In some embodiments, subjects show reduced macrophage transduction of the lentiviral vector after administration compared to a control lentiviral vector. In some embodiments, subjects show reduced allospecific immune response to the lentiviral vector after administration compared to a control lentiviral vector. In some embodiments, subjects show at least 30% FIX activity compared to normal FIX activity at least 3 weeks after administration. In some embodiments, subjects show tissue-specific expression of the lentiviral vector in the liver, spleen, or both liver and spleen after administration.

[0103] In some embodiments, the allo-specific response includes the release of cytokines in response to the administered lentiviral vector. In some embodiments, subjects show a decrease in cytokine expression associated with the allo-specific response after administration of a lentiviral vector compared to cytokine expression after administration of a control lentiviral vector. In some embodiments, the cytokines are inflammatory cytokines. In certain embodiments, the cytokines are selected from the group consisting of MIP-1a, MIP-1b, MCP-1, interleukin-2 (IL-2), interferon gamma, and any combination thereof. In certain embodiments, subjects show a decrease in the expression level of MIP-1a after administration of a lentiviral vector compared to the expression of MIP-1a after administration of a control lentiviral vector. In certain embodiments, subjects show a decrease in the expression level of MIP-1b after administration of a lentiviral vector compared to the expression of MIP-1b after administration of a control lentiviral vector. In certain embodiments, subjects show a decrease in the expression level of MCP-1 after administration of a lentiviral vector compared to the expression of MCP-1 after administration of a control lentiviral vector. In certain embodiments, subjects show a decrease in IL-2 expression levels after administration of a lentiviral vector compared to IL-2 expression after administration of a control lentiviral vector. In certain embodiments, subjects show a decrease in interferon-gamma expression after administration of a lentiviral vector compared to interferon-gamma expression after administration of a control lentiviral vector. This indicates a decrease in the expression level of [the substance].

[0104] In some embodiments, cytokine expression is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to cytokine expression after administration of a control lentiviral vector. In certain embodiments, cytokine expression is undetectable after administration of a lentiviral vector. In certain embodiments, the subject has only undetectable expression of cytokines selected from the group consisting of MIP-1a, MIP-1b, MCP-1, interleukin-2 (IL-2), interferon gamma, and any combination thereof after administration of a lentiviral vector.

[0105] In some embodiments, subjects show an increase in plasma FIX activity after administration of the lentiviral vector compared to plasma FIX activity before administration. In some embodiments, the increase is observed at least 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, or at least about 8 weeks after administration. In some embodiments, plasma FIX activity approximately 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 24 to 60 hours, 24 to 48 hours, 24 to 36 hours, 36 to 60 hours, 36 to 48 hours, or 48 to 60 hours after administration of the lentiviral vector is increased compared to subjects administered a control dose of the control lentiviral vector. In some embodiments, the FIX activity after administration of the lentiviral vector is at least approximately 75%, at least approximately 100%, at least approximately 125%, at least approximately 150%, at least approximately 175%, at least approximately 200%, at least approximately 225%, at least approximately 250%, at least approximately 275%, or at least approximately 300% compared to normal FIX activity at least 1 week, 2 weeks, or 3 weeks after administration of the lentiviral vector. In certain embodiments, the subject exhibits at least approximately 150% FIX activity compared to normal FIX activity at least 3 weeks after administration of the lentiviral vector. In certain embodiments, the subject exhibits at least approximately 200% FIX activity compared to normal FIX activity at least 3 weeks after administration of the lentiviral vector. In certain embodiments, the subject exhibits at least approximately 225% FIX activity compared to normal FIX activity at least 3 weeks after administration of the lentiviral vector.

[0106] In some embodiments, plasma FIX activity increases after administration by at least approximately 2 times, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 6 times, at least approximately 7 times, at least approximately 8 times, at least approximately 9 times, at least approximately 10 times, at least approximately 11 times, at least approximately 12 times, at least approximately 13 times, at least approximately 14 times, at least approximately 15 times, at least approximately 20 times, at least approximately 25 times, at least approximately 30 times, at least approximately 35 times, at least approximately 40 times, at least approximately 50 times, at least approximately 60 times, at least approximately 70 times, at least approximately 80 times, at least approximately 90 times, at least approximately 100 times, at least approximately 110 times, at least approximately 120 times, at least approximately 130 times, at least approximately 140 times, at least approximately 150 times, at least approximately 160 times, at least approximately 170 times, at least approximately 180 times, at least approximately 190 times, or at least approximately 200 times compared to a subject who received a control dose of a control lentiviral vector.

[0107] In some embodiments, after administration, the lentiviral vector specifically localizes to the liver, spleen, or both the liver and spleen of the subject, where higher concentrations of the lentiviral vector are observed in the liver, spleen, or both the liver and spleen than in other organs of the subject's body. In some embodiments, the other organs of the subject's body are selected from the group consisting of the testes, lymph nodes, muscles, thymus, kidneys, lungs, heart, frontal cortex, thalamus, caudate nucleus, colliculus, cerebellum, peripheral blood mononuclear cells (PBMCs), and any combination thereof. In some embodiments, the subject shows increased localization of the lentiviral vector to the liver, spleen, or both the liver and spleen compared to organs other than the liver and spleen of the subject after administration of the lentiviral vector. Lentiviral vector localization can be measured and / or expressed using any method and / or unit known in the art. In some embodiments, the increase in localization is 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, and at least about 40 times in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of the target. Characterized by a vector copy number (VCN) of a lentivirus vector that is at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 160 times, at least 170 times, at least 180 times, at least 190 times, at least 200 times, at least 250 times, at least 300 times, at least 400 times, or at least 500 times greater.

[0108] In certain embodiments, the subject exhibits increased localization of the lentiviral vector to the liver, spleen, or both the liver and spleen, characterized by at least 10 times more VCNs of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of the subject. In certain embodiments, the subject exhibits increased localization of the lentiviral vector to the liver, spleen, or both the liver and spleen, characterized by at least 50 times more VCNs of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of the subject. In certain embodiments, the subject exhibits increased localization of the lentiviral vector to the liver, spleen, or both the liver and spleen, characterized by at least 100 times more VCNs of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of the subject. In certain embodiments, the subjects exhibit increased localization of the lentiviral vector to the liver, spleen, or both the liver and spleen, and the increased localization is characterized by at least 150-fold greater amounts of lentiviral vector VCNs in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to other organs of the subject.

[0109] A. Inhibition of the immune response In some embodiments, a lentiviral vector, such as a lentiviral particle, contains one or more polypeptides on its surface that suppress the immune response to the lentiviral vector after administration to a human subject. Certain aspects of this disclosure relate to a lentiviral vector or a method of administering a lentiviral vector to a subject, wherein the lentiviral vector contains CD47 on its surface. In some embodiments, the surface of the lentiviral vector contains one or more CD47 molecules. CD47 is a "self-marker" CD47 is a protein that is ubiquitously expressed on human cells. Surface expression of CD47 suppresses macrophage-induced phagocytosis in endogenous cells through the interaction of CD47 and SIRPα, which is expressed by macrophages. Cells expressing high levels of CD47 are less likely to be targeted and destroyed by human macrophages in vivo.

[0110] In some embodiments, the lentiviral vector contains a high concentration of CD47 polypeptide molecules on its surface. In some embodiments, the lentiviral vector contains a heterologous polynucleotide encoding the CD47 protein, where the heterologous polynucleotide encoding CD47 is expressed. In some embodiments, the lentiviral vector further contains a heterologous polynucleotide encoding the CD47 protein, where the heterologous polynucleotide encoding CD47 is expressed.

[0111] In some embodiments, the lentiviral vector has high levels of CD47 protein because it is produced in a cell line that has high levels of CD47 expression. In certain embodiments, the lentiviral vector has high levels of CD47 high It is generated in cells, where the cells have high expression of CD47 on the cell membrane. In certain embodiments, the lentiviral vector is CD47 high HEK293T cells are generated, where HEK293T cells are modified to have increased CD47 expression compared to unmodified HEK293T cells. In some embodiments, HEK293T cells are modified to overexpress endogenous CD47 compared to unmodified HEK293T cells. In some embodiments, HEK293T cells are modified by transduction into HEK293T cells with a heterologous CD47 expression vector. In some embodiments, the heterologous CD47 expression vector includes a retroviral vector. In certain embodiments, the retroviral vector is a γ-retroviral vector. In some embodiments, the heterologous CD47 expression vector is not cross-packaged with a lentiviral vector.

[0112] In certain embodiments, the disclosure relates to a method for preventing or treating hemophilia in a subject in need, comprising administering to the subject an effective dose of a lentiviral vector comprising a nucleotide sequence encoding a polypeptide having FIX activity, wherein the lentiviral vector comprises higher levels of surface CD47 protein expression than a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®), and the effective dose is reduced compared to a control dose of the control lentiviral vector required to induce the same FIX activity as the lentiviral vector. While not constrained by any particular mechanism, CD47 expression on the surface of the lentiviral vector is thought to protect the lentiviral vector from degradation and / or removal by the subject's immune system and to inhibit and / or reduce the immune response to the lentiviral vector.

[0113] In some embodiments, CD47 is human CD47 (NCBI accession number NP_001768.1). In certain embodiments, CD47 contains an amino acid sequence that is at least 60%, at least about 70%, at least 70%, at least about 80%, at least 85%, at least about 90%, at least 95%, at least about 96%, at least 97%, at least about 98%, at least 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, human CD47 contains the amino acid sequence shown in SEQ ID NO: 14.

[0114] In some embodiments, CD47 is expressed by a lentiviral vector. In other embodiments, CD47 is not expressed by a lentiviral vector. In certain embodiments, the lentiviral vector is expressed in host cells, where the host cells The cells are modified to express CD47. In some embodiments, the host cells are modified to overexpress CD47. In certain embodiments, the lentiviral vector is generated in host cells expressing higher concentrations of CD47 compared to HEK293 cells (ATCC® CRL-1573®). In certain embodiments, the lentiviral vector is generated in HEK293T cells modified to overexpress CD47 compared to unmodified HEK293T cells.

[0115] In some embodiments, the lentiviral vector contains a higher level of surface CD47 protein expression than the control lentiviral vector. In a particular embodiment, the control lentiviral vector is 1 μm 2 It is known to generate 19 molecules per cell (see, for example, Figure S1(d) in Sosale et al., Methods & Clinical Development 3:16080 (2016)) in HEK293 cells (ATCC® CRL-1573®). In some embodiments, the control lentiviral vector is 1 μm on the surface of the control lentiviral vector. 2 Each contains fewer than 20 molecules of CD47.

[0116] In a particular embodiment, the lentiviral vector has at least about 2 to at least 100 times, at least about 2 to at least 75 times, at least about 2 to at least 50 times, at least about 2 to at least 40 times, at least about 2 to at least 30 times, at least about 2 to at least 20 times, and at least about 2 to at least 10 times on the surface of the lentiviral vector compared to the surface of the control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®). , containing at least about 10 to at least 100 times, at least about 10 to at least 75 times, at least about 10 to at least 50 times, at least about 10 to at least 40 times, at least about 10 to at least 30 times, at least about 10 to at least 20 times, at least about 20 to at least 100 times, at least about 20 to at least 75 times, at least about 20 to at least 50 times, at least about 20 to at least 40 times, or at least about 20 to at least 30 times more CD47 protein. In certain embodiments, the lentiviral vector contains at least about 10 to at least 30 times more CD47 protein on the surface of the lentiviral vector than on the surface of a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®).In some embodiments, the lentiviral vector has a surface on which the lentiviral vector is at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, and at least about 7.5 times compared to the surface of the control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®). Contains at least approximately 8.0 times, at least approximately 8.5 times, at least approximately 9.0 times, at least approximately 9.5 times, at least approximately 10 times, at least approximately 11 times, at least approximately 12 times, at least approximately 13 times, at least approximately 14 times, at least approximately 15 times, at least approximately 20 times, at least approximately 25 times, at least approximately 30 times, at least approximately 35 times, at least approximately 40 times, at least approximately 45 times, at least approximately 50 times more, at least approximately 60 times, at least approximately 70 times, at least approximately 80 times, at least approximately 90 times, or at least approximately 100 times more CD47 protein.

[0117] In certain embodiments, the lentiviral vector contains at least about 10 times more CD47 protein on its surface than on the surface of a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®). In certain embodiments, the lentiviral vector contains at least about 15 times more CD47 protein on its surface than on the surface of a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®). In certain embodiments, the lentiviral vector contains at least about 20 times more CD47 protein on its surface than on the surface of a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®). In certain embodiments, the lentiviral vector contains at least about 25 times more CD47 protein on its surface than on the surface of a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®). In certain embodiments, the lentiviral vector contains at least about 30 times more CD47 protein on its surface than on the surface of a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®).

[0118] In some embodiments, the lentiviral vector has a 1 μm layer on its surface. 2 At least 20 molecules per unit, 1 μm 2 At least approximately 25 molecules per 1 μm 2 At least approximately 30 molecules per 1 μm 2 At least approximately 35 molecules per 1 μm 2 At least approximately 40 molecules per 1 μm 2 At least approximately 45 molecules per 1 μm 2 At least approximately 50 molecules per 1 μm 2 At least approximately 55 molecules per 1 μm 2 At least approximately 60 molecules per 1 μm 2 At least approximately 65 molecules per 1 μm 2 At least approximately 70 molecules per 1 μm 2 At least approximately 75 molecules per 1 μm 2 At least approximately 80 molecules per 1 μm 2 At least approximately 85 molecules per 1 μm 2At least approximately 90 molecules per 1 μm 2 At least approximately 95 molecules per 1 μm 2 At least approximately 100 molecules per 1 μm 2 At least approximately 125 molecules per 1 μm 2 At least approximately 150 molecules per 1 μm 2 At least approximately 175 molecules per 1 μm 2 Each contains at least approximately 200 molecules of CD47 protein. In some embodiments, the lentiviral vector has a 1 μm layer on its surface. 2 At least approximately 225 molecules per 1 μm 2 At least approximately 250 molecules per 1 μm 2 At least approximately 275 molecules per 1 μm 2 At least approximately 300 molecules per 1 μm 2 At least approximately 325 molecules per 1 μm 2 At least approximately 350 molecules per 1 μm 2 At least approximately 375 molecules per 1 μm 2 At least approximately 400 molecules per 1 μm 2 At least approximately 425 molecules per 1 μm 2 At least approximately 450 molecules per 1 μm 2 At least approximately 475 molecules per 1 μm 2 At least approximately 500 molecules per 1 μm 2 At least approximately 525 molecules per 1 μm 2 At least approximately 550 molecules per 1 μm 2 At least approximately 575 molecules per 1 μm 2 At least approximately 600 molecules per 1 μm 2 At least approximately 625 molecules per 1 μm 2 At least approximately 650 molecules per 1 μm 2 At least approximately 675 molecules per 1 μm 2 At least approximately 700 molecules per 1 μm 2 At least approximately 725 molecules per 1 μm 2 At least approximately 750 molecules per 1 μm2 At least approximately 800 molecules per 1 μm 2 At least approximately 850 molecules per 1 μm 2 At least approximately 900 molecules per 1 μm 2 At least approximately 950 molecules per unit, or 1 μm 2 Each molecule contains at least approximately 1000 molecules of CD47 protein.

[0119] In one particular embodiment, the lentiviral vector has a 1 μm layer on its surface. 2 Each contains at least approximately 400 molecules of CD47 protein. In a particular embodiment, the lentiviral vector has a surface of 1 μm 2 Each contains at least approximately 450 molecules of CD47 protein. In a particular embodiment, the lentiviral vector has a surface of 1 μm 2 Each contains at least approximately 500 molecules of CD47 protein. In a particular embodiment, the lentiviral vector has a surface of 1 μm 2 Each contains at least approximately 600 molecules of CD47 protein. In certain embodiments, a lentiviral vector - is 1 μm on the surface of the lentiviral vector 2 Each contains at least approximately 700 molecules of CD47 protein. In a particular embodiment, the lentiviral vector has a surface of 1 μm 2 Each contains at least approximately 800 molecules of CD47 protein. In a particular embodiment, the lentiviral vector has a surface of 1 μm 2 Each contains at least approximately 900 molecules of CD47 protein. In a particular embodiment, the lentiviral vector has a surface of 1 μm 2 Each molecule contains at least approximately 1000 molecules of CD47 protein.

[0120] In some embodiments, the lentiviral vector is expressed in host cells that have been further modified to reduce the immunogenicity of the resulting lentiviral vector. In some embodiments, the lentiviral vector has little to no major histocompatibility complex class I (MHC-I) exposed on its surface. Surface-exposed MHC-I present peptide fragments of "non-self" proteins from within the cell, such as infection-inducing protein fragments, thereby promoting an immune response against the cell. In some embodiments, the lentiviral vector has MHC-I low It is generated in cells, where the cells have MHC-I exposed to a reduced surface on the cell membrane. In some embodiments, the lentiviral vector is MHC-I - (or, "MHC-I free "MHC-1 neg It is produced in cells (or "MHC-negative" cells), where the cells do not have MHC-I exposed on their surface.

[0121] MHC-I - or MHC-I low Cells can be generated using any means known in the art. In some embodiments, MHC-I - or MHC-I low Cells are generated by disrupting the expression of one or more genes encoding one or more proteins in the MHC. In some embodiments, MHC-I - or MHC-I low The cells are generated by interrupting the expression of the gene encoding beta-2-microglobulin (B2M; Ensembl ENSG00000166710; NCBI protein accession number ABB01003). In some embodiments, MHC-I - or MHC-I low The cells are generated by sustained interruption of the expression of the gene encoding beta-2-microglobulin (B2M). In some embodiments, MHC-I - or MHC-I lowThe cells are generated by blocking the expression of the gene encoding beta-2-microglobulin (B2M). In some embodiments, MHC-I - or MHC-I low The cells are generated by introducing a mutation into the gene encoding beta-2-microglobulin (B2M), where the mutation results in a loss of expression of the gene encoding B2M. In some embodiments, MHC-I - or MHC-I low The cells are generated by knocking out the gene encoding beta-2-microglobulin (B2M). In certain embodiments, MHC-I - or MHC-I low The cells are generated by modifying HECK 293T cells to block or reduce surface-exposed MHC-I. Unmodified HECK293T cells have surface-exposed MHC-I. See, for example, Dellgren et al., PLoS One 10(8):e0135385 (2015).

[0122] In some embodiments, cells are MHC-I - HEK293T cells, in which the cells do not have MHC-I exposed on their surface. In certain embodiments, MHC-I - The cells have less than 1% surface-exposed MHC-1 compared to unmodified HEK293T cells. In some embodiments, the cells have MHC-I low HEK293T cells, compared to unmodified HEK293T cells, are approximately less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, approximately Modified to have less than 1% of the surface exposed to MHC-I. In certain embodiments, MHC-I lowThe cells have less than 5% surface-exposed MHC-1 compared to unmodified HEK293T cells. In certain embodiments, MHC-I low The cells have less than 4% surface-exposed MHC-1 compared to unmodified HEK293T cells. In certain embodiments, MHC-I low The cells have less than 3% surface-exposed MHC-1 compared to unmodified HEK293T cells. In certain embodiments, MHC-I low The cells have less than 2% surface-exposed MHC-1 compared to unmodified HEK293T cells.

[0123] In certain embodiments, the lentiviral vector comprises a high concentration of CD47 polypeptide and a lipid coating lacking MHC-I exposed to the surface. In certain embodiments, the lentiviral vector comprises CD47 high / MHC-I low Cell lineage, for example, CD47 high / MHC-I low It is generated in the HEK293T cell line. In some embodiments, the lentiviral vector is CD47 high / MHC-I - Cell lineage, for example, CD47 high / MHC-I - It is generated in the HEK293T cell line.

[0124] In other embodiments, administration of the lentiviral vector disclosed herein and / or subsequent expression of the FIX protein transgene does not induce an immune response in the subject. In some embodiments, the immune response includes the development of antibodies against FIX. In some embodiments, the immune response includes cytokine secretion. In some embodiments, the immune response includes 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, where the immune response in the subject reduces the activity of the FIX protein compared to the activity of FIX in a subject that did not evoke an immune response. In certain embodiments, the expression of the FIX protein by administration of the lentiviral vector disclosed herein blocks an inhibitory immune response against the FIX protein or FIX protein expressed from an isolated nucleic acid molecule or lentiviral vector.

[0125] B. Administration The lentiviral vectors, lentiviral vector particles, and methods of use thereof of this disclosure enable the prophylaxis and / or treatment of hemophilia using a lower dose of lentiviral vector than that of a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573®). In some embodiments, the lentiviral vectors of this disclosure are effective at doses that are reduced compared to the control dose of a control lentiviral vector required to induce the same FIX activity as the lentiviral vector. In some embodiments, the dose of the lentiviral vector is at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% of the control dose of the control lentiviral vector. In some embodiments, the dose is at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of the control dose of the control lentiviral vector. In some embodiments, the dose is at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of the control dose of the control lentiviral vector. In some embodiments, the dose is at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the control dose of the control lentiviral vector.

[0126] In some embodiments, the dose of the lentiviral vector is approximately 5.0 × 10⁻⁶. 10 TU / kg, approximately 4.9 x 10 10 TU / kg, approximately 4.8 x 10 10 TU / kg, approximately 4.7 x 10 10 TU / kg, approximately 4.6 x 10 10 TU / kg, approximately 4.5 x 10 10 TU / kg, approximately 4.4 x 10 10 TU / kg, approximately 4.3 x 10 10 TU / kg, approximately 4.2 × 10 10 TU / kg, approximately 4.1 × 10 10 TU / kg, approximately 4.0 x 10 10 TU / kg, approximately 3.9 × 10 10 TU / kg, approximately 3.8 x 10 10TU / kg, approximately 3.7 × 10 10 TU / kg, approximately 3. 6×10 10 TU / kg, approximately 3.5 x 10 10 TU / kg, approximately 3.4 × 10 10 TU / kg, approximately 3.3 x 10 10 TU / kg, approximately 3.2 × 10 10 TU / kg, approximately 3.1 × 10 10 TU / kg, approximately 3.0 x 10 10 TU / kg, approximately 2.9 × 10 10 TU / kg, approximately 2.8 x 10 10 TU / kg, approximately 2.7 × 10 10 TU / kg, approximately 2.6 x 10 10 TU / kg, approximately 2.5 x 10 10 TU / kg, approximately 2.4 x 10 10 TU / kg, approximately 2.3 x 10 10 TU / kg, approximately 2.2 × 10 10 TU / kg, approximately 2.1 × 10 10 TU / kg, approximately 2.0 x 10 10 TU / kg, approximately 1.9 × 10 10 TU / kg, approximately 1.8 × 10 10 TU / kg, approximately 1.7 × 10 10 TU / kg, approximately 1.6 x 10 10 TU / kg, approximately 1.5 x 10 10 TU / kg, approximately 1.4 × 10 10 TU / kg, approximately 1.3 × 10 10 TU / kg, approximately 1.2 × 10 10 TU / kg, approximately 1.1 × 10 10 TU / kg, or approximately 1.0 × 10 10 It is TU / kg.

[0127] In some embodiments, the dose of the lentiviral vector is approximately 9.9 × 10⁻⁶. 9 TU / kg, approximately 9.8 x 10 9 TU / kg, approximately 9.7 x 10 9 TU / kg, approximately 9.6 x 10 9 TU / kg, approximately 9.5 x 10 9 TU / kg, approximately 9.4 x 10 9 TU / kg, approximately 9.3 x 10 9TU / kg, approximately 9.2×10 9 TU / kg, approximately 9.1×10 9 TU / kg, approximately 9.0×10 9 TU / kg, approximately 8.9×10 9 TU / kg, approximately 8.8×10 9 TU / kg, approximately 8.7×10 9 TU / kg, approximately 8.6×10 9 TU / kg, approximately 8.5×10 9 TU / kg, approximately 8.4×10 9 [[ID=A]]TU / kg, approximately 8.3×10 9 TU / kg, approximately 8.2×10 9 TU / kg, approximately 8.1×10 9 TU / kg, approximately 8.0×10 9 TU / kg, approximately 7.9×10 9 TU / kg, approximately 7.8×10 9 TU / kg, approximately 7.7×10 9 TU / kg, approximately 7.6×10 9 TU / kg, approximately 7.5×10 9 TU / kg, approximately 7.4×10 9 TU / kg, approximately 7.3×10 9 TU / kg, approximately 7.2×10 9 TU / kg, approximately 7.1×10 9 TU / kg, approximately 7.0×10 9 TU / kg, approximately 6.9×10 9 TU / kg, approximately 6.8×10 9 TU / kg, approximately 6.7×10 9 TU / kg, approximately 6.6×10 9 TU / kg, approximately 6.5×10 9 TU / kg, approximately 6.4×10 9 TU / kg, approximately 6.3×10 9 TU / kg, approximately 6.2×10 9 TU / kg, approximately 6.1×10 9 TU / kg, approximately 6.0×10 9 TU / kg, approximately 5.9×10 9 TU / kg, approximately 5.8×10 9 TU / kg, approximately 5.7×10 9 TU / kg, approximately 5.6×10 9 TU / kg, approximately 5.5×10 9 TU / kg, approximately 5.4×10 It should be noted that there seems to be an error in the original text where "TU / kg、約8.3×10 " is likely missing a digit after "10". I've translated it as "TU / kg, approximately 8.3×10" as is, but it might need to be corrected in the original for a more complete and accurate meaning. Also, there is an "[[ID=A]]" which seems to be an incorrect tag in the original sequence.9 TU / kg, about 5.3×10 9 TU / kg, about 5.2×10 9 TU / kg, about 5.1×10 9 TU / kg, about 5.0×10 9 TU / kg, about 4.9×10 9 TU / kg, about 4.8×10 9 TU / kg, about 4.7×10 9 TU / kg, about 4.6×10 9 TU / kg, about 4.5×10 9 TU / kg, about 4.4×10 9 TU / kg, about 4.3×10 9 TU / kg, about 4.2×10 9 TU / kg, about 4.1×10 9 TU / kg, about 4.0×10 9 TU / kg, about 3.9×10 9 TU / kg, about 3.8×10 9 TU / kg, about 3.7×10 9 TU / kg, about 3.6×10 9 TU / kg, about 3.5×10 9 TU / kg, about 3.4×10 9 TU / kg, about 3.3×10 9 TU / kg, about 3.2×10 9 TU / kg, about 3.1×10 9 TU / kg, about 3.0×10 9 TU / kg, about 2.9×10 9 TU / kg, about 2.8×10 9 TU / kg, about 2.7×10 9 TU / kg, about 2.6×10 9 TU / kg, about 2.5×10 9 TU / kg, about 2.4×10 9 TU / kg, about 2.3×10 9 TU / kg, about 2.2×10 9 TU / kg, about 2.1×10 9 TU / kg, about 2.0×10 9 TU / kg, about 1.9×10 9 TU / kg, about 1.8×10 9 TU / kg, about 1.7×10 9 TU / kg, about 1.6×10 9TU / kg, approximately 1.5 x 10 9 TU / kg, approximately 1.4 × 10 9 TU / kg, approximately 1.3 × 10 9 TU / kg, approximately 1.2 × 10 9 TU / kg, approximately 1.1 × 10 9 TU / kg or approximately 1.0 × 10 9 It is TU / kg.

[0128] In some embodiments, the dose of the lentiviral vector is approximately 9.9 × 10⁻⁶. 8 TU / kg, approximately 9.8 x 10 8 TU / kg, approximately 9.7 x 10 8 TU / kg, approximately 9.6 x 10 8 TU / kg, approximately 9.5 x 10 8 TU / kg, approximately 9.4 x 10 8 TU / kg, approximately 9.3 x 10 8 TU / kg, approximately 9.2 x 10 8 TU / kg, approximately 9.1 × 10 8 TU / kg, approximately 9.0 x 10 8 TU / kg, approximately 8.9 x 10 8 TU / kg, approximately 8.8 x 10 8 TU / kg, approximately 8.7 x 10 8 TU / kg, approximately 8.6 x 10 8 TU / kg, approximately 8.5 x 10 8 TU / kg, approximately 8.4 x 10 8 TU / kg, approximately 8.3 x 10 8 TU / kg, approximately 8.2 × 10 8 TU / kg, approximately 8.1 × 10 8 TU / kg, approximately 8.0 x 10 8 TU / kg, approximately 7.9 x 10 8 TU / kg, approximately 7.8 x 10 8 TU / kg, approximately 7.7 x 10 8 TU / kg, approximately 7.6 x 10 8 TU / kg, approximately 7.5 x 10 8 TU / kg, approximately 7.4 × 10 8 TU / kg, approximately 7.3 x 10 8 TU / kg, approximately 7.2 × 10 8 TU / kg, approximately 7.1 × 10 8TU / kg, about 7.0×10 8 TU / kg, about 6.9×10 8 TU / kg, about 6.8×10 8 TU / kg, about 6.7×10 8 TU / kg, about 6.6×10 8 TU / kg, about 6.5×10 8 TU / kg, about 6.4×10 8 TU / kg, about 6.3×10 8 TU / kg, about 6.2×10 8 TU / kg, about 6.1×10 8 TU / kg, about 6.0×10 8 TU / kg, about 5.9×10 8 TU / kg, about 5.8×10 8 TU / kg, about 5.7×10 8 TU / kg, about 5.6×10 8 TU / kg, about 5.5×10 8 TU / kg, about 5.4×10 8 TU / kg, about 5.3×10 8 TU / kg, about 5.2×10 8 TU / kg, about 5.1×10 8 TU / kg, about 5.0×10 8 TU / kg, about 4.9×10 8 TU / kg, about 4.8×10 8 TU / kg, about 4.7×10 8 TU / kg, about 4.6×10 8 TU / kg, about 4.5×10 8 TU / kg, about 4.4×10 8 TU / kg, about 4.3×10 8 TU / kg, about 4.2×10 8 TU / kg, about 4.1×10 8 TU / kg, about 4.0×10 8 TU / kg, about 3.9×10 8 TU / kg, about 3.8×10 8 TU / kg, about 3.7×10 8 TU / kg, about 3.6×10 8 TU / kg, about 3.5×10 8 TU / kg, about 3.4×10 8 TU / kg, about 3.3×10 8 TU / kg, about 3.2×108 TU / kg, approximately 3.1 × 10 8 TU / kg, approximately 3.0 x 10 8 TU / kg, approximately 2.9 × 10 8 TU / kg, approximately 2.8 x 10 8 TU / kg, approximately 2.7 × 10 8 TU / kg, approximately 2.6 x 10 8 TU / kg, approximately 2.5 x 10 8 TU / kg, approximately 2.4 x 10 8 TU / kg, approximately 2.3 x 10 8 TU / kg, approximately 2.2 × 10 8 TU / kg, approximately 2.1 × 10 8 TU / kg, approximately 2.0 x 10 8 TU / kg, approximately 1.9 × 10 8 TU / kg, approximately 1.8 × 10 8 TU / kg, approximately 1.7 × 10 8 TU / kg, approximately 1.6 x 10 8 TU / kg, approximately 1.5 x 10 8 TU / kg, approximately 1.4 × 10 8 TU / kg, approximately 1.3 × 10 8 TU / kg, approximately 1.2 × 10 8 TU / kg, approximately 1.1 × 10 8 TU / kg or approximately 1.0 × 10 8 It is TU / kg.

[0129] In some embodiments, the dose of the lentiviral vector is approximately 5.0 × 10⁻⁶. 10 Less than TU / kg, approximately 4.9 × 10 10 Less than TU / kg, approximately 4.8 × 10 10 Less than TU / kg, approximately 4.7 × 10 10 Less than TU / kg, approximately 4.6 × 10 10 Less than TU / kg, approximately 4.5 × 10 10 Less than TU / kg, approximately 4.4 × 10 10 Less than TU / kg, approximately 4.3 × 10 10 Less than TU / kg, approximately 4.2 × 10 10 Less than TU / kg, approximately 4.1 × 10 10 Less than TU / kg, approximately 4.0 × 10 10 Less than TU / kg, approximately 3.9 × 10 10Less than TU / kg, approximately 3.8 × 10 10 Less than TU / kg, approximately 3.7 × 10 10 Less than TU / kg, approximately 3.6 × 10 10 Less than TU / kg, approximately 3.5 × 10 10 Less than TU / kg, approximately 3.4 × 10 10 Less than TU / kg, approximately 3.3 × 10 10 Less than TU / kg, approximately 3.2 × 10 10 Less than TU / kg, approximately 3.1 × 10⁻⁶ 10 Less than TU / kg, approximately 3.0 × 10 10 Less than TU / kg, approximately 2.9 × 10 10 Less than TU / kg, approximately 2.8 × 10 10 Less than TU / kg, approximately 2.7 × 10⁻⁶ 10 Less than TU / kg, approximately 2.6 × 10⁻⁶ 10 Less than TU / kg, approximately 2.5 × 10 10 Less than TU / kg, approximately 2.4 × 10 10 Less than TU / kg, approximately 2.3 × 10⁻⁶ 10 Less than TU / kg, approximately 2.2 × 10⁻⁶ 10 Less than TU / kg, approximately 2.1 × 10⁻⁶ 10 Less than TU / kg, approximately 2.0 × 10 10 Less than TU / kg, approximately 1.9 × 10⁻⁶ 10 Less than TU / kg, approximately 1.8 × 10⁻⁶ 10 Less than TU / kg, approximately 1.7 × 10⁻⁶ 10 Less than TU / kg, approximately 1.6 × 10⁻⁶ 10 Less than TU / kg, approximately 1.5 × 10⁻⁶ 10 Less than TU / kg, approximately 1.4 × 10⁻⁶ 10 Less than TU / kg, approximately 1.3 × 10⁻⁶ 10 Less than TU / kg, approximately 1.2 × 10⁻⁶ 10 Less than TU / kg, approximately 1.1 × 10⁻⁶ 10 Less than TU / kg, or approximately 1.0 × 10⁻⁶ 10 It is less than TU / kg.

[0130] In some embodiments, the dose of the lentiviral vector is approximately 9.9 × 10⁻⁶. 9 Less than TU / kg, approximately 9.8 × 10 9 Less than TU / kg, approximately 9.7 × 10 9 Less than TU / kg, approximately 9.6 × 10 9Less than TU / kg, approximately 9.5 × 10 9 Less than TU / kg, approximately 9.4 × 10 9 Less than TU / kg, approximately 9.3 × 10 9 Less than TU / kg, approximately 9.2 × 10 9 Less than TU / kg, approximately 9.1 × 10⁻⁶ 9 Less than TU / kg, approximately 9.0 × 10 9 Less than TU / kg, approximately 8.9 × 10 9 Less than TU / kg, approximately 8.8 × 10 9 Less than TU / kg, approximately 8.7 × 10 9 Less than TU / kg, approximately 8.6 × 10 9 Less than TU / kg, approximately 8.5 × 10 9 Less than TU / kg, approximately 8.4 × 10 9 Less than TU / kg, approximately 8.3 × 10 9 Less than TU / kg, approximately 8.2 × 10 9 Less than TU / kg, approximately 8.1 × 10⁻⁶ 9 Less than TU / kg, approximately 8.0 × 10 9 Less than TU / kg, approximately 7.9 × 10 9 Less than TU / kg, approximately 7.8 × 10 9 Less than TU / kg, approximately 7.7 × 10 9 Less than TU / kg, approximately 7.6 × 10 9 Less than TU / kg, approximately 7.5 × 10 9 Less than TU / kg, approximately 7.4 × 10 9 Less than TU / kg, approximately 7.3 × 10 9 Less than TU / kg, approximately 7.2 × 10 9 Less than TU / kg, approximately 7.1 × 10⁻⁶ 9 Less than TU / kg, approximately 7.0 × 10 9 Less than TU / kg, approximately 6.9 × 10 9 Less than TU / kg, approximately 6.8 × 10 9 Less than TU / kg, approximately 6.7 × 10 9 Less than TU / kg, approximately 6.6 × 10 9 Less than TU / kg, approximately 6.5 × 10 9 Less than TU / kg, approximately 6.4 × 10 9 Less than TU / kg, approximately 6.3 × 10 9 Less than TU / kg, approximately 6.2 × 10 9 Less than TU / kg, approximately 6.1 × 10⁻⁶ 9 Less than TU / kg, approximately 6.0 × 109 Less than TU / kg, approximately 5.9 × 10 9 Less than TU / kg, approximately 5.8 × 10 9 Less than TU / kg, approximately 5.7 × 10 9 Less than TU / kg, approximately 5.6 × 10 9 Less than TU / kg, approximately 5.5 × 10 9 Less than TU / kg, approximately 5.4 × 10 9 Less than TU / kg, approximately 5.3 × 10 9 Less than TU / kg, approximately 5.2 × 10 9 Less than TU / kg, approximately 5.1 × 10⁻⁶ 9 Less than TU / kg, approximately 5.0 × 10 9 Less than TU / kg, approximately 4.9 × 10 9 Less than TU / kg, approximately 4.8 × 10 9 Less than TU / kg, approximately 4.7 × 10 9 Less than TU / kg, approximately 4.6 × 10 9 Less than TU / kg, approximately 4.5 × 10 9 Less than TU / kg, approximately 4.4 × 10 9 Less than TU / kg, approximately 4.3 × 10 9 Less than TU / kg, approximately 4.2 × 10 9 Less than TU / kg, approximately 4.1 × 10 9 Less than TU / kg, approximately 4.0 × 10 9 Less than TU / kg, approximately 3.9 × 10 9 Less than TU / kg, approximately 3.8 × 10 9 Less than TU / kg, approximately 3.7 × 10 9 Less than TU / kg, approximately 3.6 × 10 9 Less than TU / kg, approximately 3.5 × 10 9 Less than TU / kg, approximately 3.4 × 10 9 Less than TU / kg, approximately 3.3 × 10 9 Less than TU / kg, approximately 3.2 × 10 9 Less than TU / kg, approximately 3.1 × 10⁻⁶ 9 Less than TU / kg, approximately 3.0 × 10 9 Less than TU / kg, approximately 2.9 × 10 9 Less than TU / kg, approximately 2.8 × 10 9 Less than TU / kg, approximately 2.7 × 10⁻⁶ 9 Less than TU / kg, approximately 2.6 × 10⁻⁶ 9 Less than TU / kg, approximately 2.5 × 10 9Less than TU / kg, approximately 2.4 × 10 9 Less than TU / kg, approximately 2.3 × 10⁻⁶ 9 Less than TU / kg, approximately 2.2 × 10⁻⁶ 9 Less than TU / kg, approximately 2.1 × 10⁻⁶ 9 Less than TU / kg, approximately 2.0 × 10 9 Less than TU / kg, approximately 1.9 × 10⁻⁶ 9 Less than TU / kg, approximately 1.8 × 10⁻⁶ 9 Less than TU / kg, approximately 1.7 × 10⁻⁶ 9 Less than TU / kg, approximately 1.6 × 10⁻⁶ 9 Less than TU / kg, approximately 1.5 × 10⁻⁶ 9 Less than TU / kg, approximately 1.4 × 10⁻⁶ 9 Less than TU / kg, approximately 1.3 × 10⁻⁶ 9 Less than TU / kg, approximately 1.2 × 10⁻⁶ 9 Less than TU / kg, approximately 1.1 × 10⁻⁶ 9 Less than TU / kg or approximately 1.0 × 10⁻⁶ 9 It is less than TU / kg.

[0131] In some embodiments, the dose of the lentiviral vector is approximately 9.9 × 10⁻⁶. 8 Less than TU / kg, approximately 9.8 × 10 8 Less than TU / kg, approximately 9.7 × 10 8 Less than TU / kg, approximately 9.6 × 10 8 Less than TU / kg, approximately 9.5 × 10 8 Less than TU / kg, approximately 9.4 × 10 8 Less than TU / kg, approximately 9.3 × 10 8 Less than TU / kg, approximately 9.2 × 10 8 Less than TU / kg, approximately 9.1 × 10⁻⁶ 8 Less than TU / kg, approximately 9.0 × 10 8 Less than TU / kg, approximately 8.9 × 10 8 Less than TU / kg, approximately 8.8 × 10 8 Less than TU / kg, approximately 8.7 × 10 8 Less than TU / kg, approximately 8.6 × 10 8 Less than TU / kg, approximately 8.5 × 10 8 Less than TU / kg, approximately 8.4 × 10 8 Less than TU / kg, approximately 8.3 × 10 8 Less than TU / kg, approximately 8.2 × 10 8Less than TU / kg, about 8.1×10 8 Less than TU / kg, about 8.0×10 8 Less than TU / kg, about 7.9×10 8 Less than TU / kg, about 7.8×10 8 Less than TU / kg, about 7.7×10 8 Less than TU / kg, about 7.6×10 8 Less than TU / kg, about 7.5×10 8 Less than TU / kg, about 7.4×10 8 Less than TU / kg, about 7.3×10 8 Less than TU / kg, about 7.2×10 8 Less than TU / kg, about 7.1× 10 8 Less than TU / kg, about 7.0×10 8 Less than TU / kg, about 6.9×10 8 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Less than TU / kg, approximately 4.6 × 10 8 Less than TU / kg, approximately 4.5 × 10 8 Less than TU / kg, approximately 4.4 × 10 8 Less than TU / kg, approximately 4.3 × 10 8 Less than TU / kg, approximately 4.2 × 10 8 Less than TU / kg, approximately 4.1 × 10 8 Less than TU / kg, approximately 4.0 × 10 8 Less than TU / kg, approximately 3.9 × 10 8 Less than TU / kg, approximately 3.8 × 10 8 Less than TU / kg, approximately 3.7 × 10 8 Less than TU / kg, approximately 3.6 × 10 8 Less than TU / kg, approximately 3.5 × 10 8 Less than TU / kg, approximately 3.4 × 10 8 Less than TU / kg, approximately 3.3 × 10 8 Less than TU / kg, approximately 3.2 × 10 8 Less than TU / kg, approximately 3.1 × 10⁻⁶ 8 Less than TU / kg, approximately 3.0 × 10 8 Less than TU / kg, approximately 2.9 × 10 8 Less than TU / kg, approximately 2.8 × 10 8 Less than TU / kg, approximately 2.7 × 10⁻⁶ 8 Less than TU / kg, approximately 2.6 × 10⁻⁶ 8 Less than TU / kg, approximately 2.5 × 10 8 Less than TU / kg, approximately 2.4 × 10 8 Less than TU / kg, approximately 2.3 × 10⁻⁶ 8 Less than TU / kg, approximately 2.2 × 10⁻⁶ 8 Less than TU / kg, approximately 2.1 × 10⁻⁶ 8 Less than TU / kg, approximately 2.0 × 10 8 Less than TU / kg, approximately 1.9 × 10⁻⁶ 8 Less than TU / kg, approximately 1.8 × 10⁻⁶ 8 Less than TU / kg, approximately 1.7 × 10⁻⁶ 8 Less than TU / kg, approximately 1.6 × 10⁻⁶ 8 Less than TU / kg, approximately 1.5 × 10⁻⁶ 8 Less than TU / kg, approximately 1.4 × 10⁻⁶ 8 Less than TU / kg, approximately 1.3 × 10⁻⁶ 8 Less than TU / kg, approximately 1.2 × 10⁻⁶ 8 Less than TU / kg, approximately 1.1 × 10⁻⁶8 less than TU / kg or about 1.0×10 8 less than TU / kg.

[0132] In some embodiments, the dose of the lentiviral vector is about 1×10 8 TU / kg to about 5×10 10 TU / kg, about 1.5×10 8 TU / kg to about 5×10 10 TU / kg, about 2×10 8 TU / kg to about 5×10 10 TU / kg, about 2.5×10 8 TU / kg to about 5×10 10 TU / kg, about 3×10 8 TU / kg to about 5×10 10 TU / kg, about 3.5×10 8 TU / kg to about 5×10 10 TU / kg, about 4×10 8 TU / kg to about 5×10 10 TU / kg, about 4.5×10 8 TU / kg to about 5×10 10 TU / kg, about 5×10 8 TU / kg to about 5×10 10 TU / kg, about 5.5×10 8 TU / kg to about 5×10 10 TU / kg, about 6×10 8 TU / kg to about 5×10 10 TU / kg, about 6.5×10 8 TU / kg to about 5×10 10 TU / kg, about 7×10 8 TU / kg to about 5×10 10 TU / kg, about 7.5×10 8 TU / kg to about 5×10 10 TU / kg, about 8×10 8 TU / kg to about 5×10 10 TU / kg, about 8.5×10 8 TU / kg to about 5×10 10 TU / kg, about 9×10 8 TU / kg to about 5×10 10 TU / kg, about 9.5×10 8 TU / kg to about 5×10 10TU / kg, about 1×10 9 TU / kg~about 5×10 10 TU / kg, about 1.5×10 9 TU / kg~about 5×10 10 TU / kg, about 2×10 9 TU / kg~about 5×10 10 TU / kg, about 2.5×10 9 TU / kg~about 5×10 10 TU / kg, about 3×10 9 TU / kg~about 5×10 10 TU / kg, about 3.5×10 9 TU / kg~about 5×10 10 TU / kg, about 4×10 9 TU / kg~about 5×10 10 TU / kg, about 4.5×10 9 TU / kg~about 5×10 10 TU / kg, about 5×10 9 TU / kg~about 5×10 10 TU / kg, about 5.5×10 9 TU / kg~about 5×10 10 TU / kg, about 6×10 9 TU / kg~about 5×10 10 TU / kg, about 6.5×10 9 TU / kg~about 5×10 10 TU / kg, about 7×10 9 TU / kg~about 5×10 10 TU / kg, about 7.5×10[[ID=5th=3]] 9 TU / kg~about 5×10 10 TU / kg, about 8×10 9 TU / kg~about 5×10 10 TU / kg, about 8.5×10 9 TU / kg~about 5×10 10 TU / kg, about 9×10 9 TU / kg~about 5×10 10 TU / kg, about 9.5×10 9 TU / kg~about 5×10 10 TU / kg, about 1×10[[ID=z5]] 9 ~about 6×10 9 TU / kg, about 2×10 9 ~about 6×10 9 TU / kg, about 3×109 ~Approx. 6×10 9 TU / kg, approximately 4 x 10 9 ~Approx. 6×10 9 TU / kg, approximately 5 x 10 9 ~Approx. 6×10 9 TU / kg, approximately 10 10 TU / kg ~ approximately 5 x 10 10 TU / kg, approximately 1.5 x 10 10 TU / kg ~ approximately 5 x 10 10 TU / kg, approximately 2 x 10 10 TU / kg ~ approximately 5 x 10 10 TU / kg, approximately 2.5 x 10 10 TU / kg ~ approximately 5 x 10 10 TU / kg, approximately 3 x 10 10 TU / kg ~ approximately 5 x 10 10 TU / kg, approximately 3.5 x 10 10 TU / kg ~ approximately 5 x 10 10 TU / kg, approximately 4 x 10 10 TU / kg ~ approximately 5 x 10 10 TU / kg, or approximately 4.5 × 10 10 TU / kg ~ approximately 5 x 10 10 It is TU / kg.

[0133] In some embodiments, the dose of the lentiviral vector is approximately 1 × 10⁻⁶ 8 TU / kg ~ approximately 5 x 10 10 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 4.5 × 10 10 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 4 x 10 10 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 3.5 × 10 10 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 3 x 10 10 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 2.5 × 10 10 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 2 x 10 10 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 1.5 × 10 10 TU / kg, approximately 1 x 10 8TU / kg to approximately 10 10 TU / kg, approximately 1×10 8 TU / kg to approximately 9×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 8.5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 8×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 7.5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 7×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 6.5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 6×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 5.5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 4.5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 4×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 3.5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 3×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 2.5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 2×10 9 , approximately 1×10 8 TU / kg to approximately 1.5×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 1×10 9 TU / kg, approximately 1×10 8 TU / kg to approximately 9.5×10 8 TU / kg, approximately 1×10 8 TU / kg to approximately 9×10 8 TU / kg, approximately 1×x0 8 TU / kg to approximately 8.5×10 8TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 8 x 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 7.5 x 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 7 x 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 6.5 x 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approximately 6 x 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 5.5 × 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approximately 5 x 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 4.5 × 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 4 x 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 3.5 × 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 3 x 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 2.5 × 10 8 TU / kg, approximately 1 x 10 8 TU / kg ~ approx. 2 x 10 8 , or approximately 1 x 10 8 TU / kg ~ approx. 1.5 × 10 8 It is TU / kg.

[0134] In some embodiments, the dose of the lentiviral vector is approximately 1 × 10⁻⁶ 10 TU / kg ~ approx. 2 x 10 10 TU / kg, approximately 1.1 × 10 10 TU / kg ~ approx. 1.9 × 10 10 TU / kg, approximately 1.2 × 10 10 TU / kg ~ approx. 1.8 × 10 10 TU / kg, approximately 1.3 × 10 10 TU / kg ~ approx. 1.7 × 10 10 TU / kg, or approximately 1.4 × 10⁻⁶ 10 TU / kg ~ approx. 1.6 × 1010 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 1.5 × 10⁻⁶. 10 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is approximately 2 × 10⁻⁶. 10 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 2 × 10⁻⁶. 10 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is approximately 6 × 10⁻⁶. 10 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 6 × 10⁻⁶. 10 It is TU / kg.

[0135] In some embodiments, the dose of the lentiviral vector is approximately 1 × 10⁻⁶ 9 TU / kg ~ approx. 2 x 10 9 TU / kg, approximately 1.1 × 10 9 TU / kg ~ approx. 1.9 × 10 9 TU / kg, approximately 1.2 × 10 9 TU / kg ~ approx. 1.8 × 10 9 TU / kg, approximately 1.3 × 10 9 TU / kg ~ approx. 1.7 × 10 9 TU / kg, or approximately 1.4 × 10⁻⁶ 9 TU / kg ~ approx. 1.6 × 10 9 The value is TU / kg. In a particular embodiment, the dose of the lentiviral vector is approximately 4 × 10⁻⁶. 9 TU / kg ~ approximately 6 x 10 9 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 1.5 × 10⁻⁶. 9 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 4 × 10⁻⁶. 9 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 4.5 × 10⁻⁶. 9 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 5 × 10⁻⁶. 9 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 5.5 × 10⁻⁶. 9 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is approximately 6 × 10⁻⁶.9 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 6 × 10⁻⁶. 9 It is TU / kg.

[0136] In one particular embodiment, the dose of the lentiviral vector is approximately 2.5 × 10⁻⁶. 9 The value is TU / kg. In a particular embodiment, the dose of the lentiviral vector is 2.5 × 10⁻⁶. 9 The value is TU / kg. In a particular embodiment, the dose of the lentiviral vector is approximately 3.0 × 10⁻⁶. 9 The value is TU / kg. In a particular embodiment, the dose of the lentiviral vector is 3.0 × 10⁻⁶. 9 The value is TU / kg. In a particular embodiment, the dose of the lentiviral vector is approximately 7.5 × 10⁻⁶. 9 The value is TU / kg. In a particular embodiment, the dose of the lentiviral vector is 7.5 × 10⁻⁶. 9 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is approximately 2 × 10⁻⁶. 10 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 2 × 10⁻⁶. 10 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is approximately 6 × 10⁻⁶. 10 The value is TU / kg. In some embodiments, the dose of the lentiviral vector is 6 × 10⁻⁶. 10 It is TU / kg.

[0137] In some embodiments, the lentiviral vector is administered in a single dose or in multiple doses. In some embodiments, the dose of the lentiviral vector is administered at once or divided into multiple partial doses, for example, two partial doses, three partial doses, four partial doses, five partial doses, six partial doses, or more than six partial doses. In some embodiments, more than one lentiviral vector is administered.

[0138] In some embodiments, the lentiviral vector dose is administered at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the lentiviral vector dose is administered about once a week, about once every two weeks, about once every three weeks, or about once every four weeks. In some embodiments, the lentiviral vector dose is administered about once every 10 days, about once every 14 days, about once every two weeks, about once every 15 days, about once every three weeks, about once every 20 days, about once every four weeks, about once every month, about twice a month, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every two months, about once every nine weeks, about once every ten weeks, about once every eleven weeks, or about twelve weeks. It is administered once in between, approximately once every 3 months, approximately once every 13 weeks, approximately once every 14 weeks, approximately once every 15 weeks, approximately once every 16 weeks, approximately once every 4 months, approximately once every 17 weeks, approximately once every 18 weeks, approximately once every 19 weeks, approximately once every 20 weeks, approximately once every 5 months, approximately once every 21 weeks, approximately once every 22 weeks, approximately once every 23 weeks, approximately once every 24 weeks, approximately once every 25 weeks, approximately once every 26 weeks, and approximately once every 6 months.

[0139] In some embodiments, a first dose of the lentiviral vector is administered, the subject is monitored for transgene expression, and a second dose is administered to the subject if the subject has transgene expression below a predetermined threshold. In certain embodiments, the thresholds are approximately less than 100%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, and approximately 4 A second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than 0%, less than approximately 35%, less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, less than approximately 10%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 3%, or less than approximately 1% of the target cells express the transgene. In some embodiments, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than approximately 50% of the target cells express the transgene. In some embodiments, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than approximately 25% of the target cells express the transgene. In some embodiments, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than approximately 10% of the target cells express the transgene. In some embodiments, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than approximately 5% of the target cells express the transgene. In some embodiments, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than approximately 4% of the target cells express the transgene. In some embodiments, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than approximately 3% of the target cells express the transgene. In some embodiments, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than approximately 2% of the target cells express the transgene. In some embodiments, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the target when less than approximately 1% of the target cells express the transgene.

[0140] In some embodiments, the lentiviral vector is administered via intravenous injection. In some embodiments, the lentiviral vector is administered via non-intravenous injection (e.g., subcutaneous or intradermal).

[0141] In some embodiments, the subjects are children, while in other embodiments, the subjects are adults. In some embodiments, the subjects are male, while in other embodiments, the subjects are female.

[0142] The lentiviral vectors disclosed herein would be therapeutically beneficial to the treatment of hemorrhagic disorders or disorders selected from the group consisting of hemorrhagic coagulation disorders, hemorrhagic arthropathy, muscle bleeding, oral bleeding, bleeding, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intraperitoneal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system bleeding, hemorrhage in the retropharyngeal space, hemorrhage in the retroperitoneal space, and hemorrhage in the psoas sheath, using gene therapy approaches in mammals, e.g., human patients, at low or reduced doses (e.g., 10) in vivo. 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 It can be used at concentrations of TU / kg or less. In one embodiment, the hemorrhagic disorder or condition is hemophilia. In another embodiment, the hemorrhagic disorder or condition is hemophilia A.

[0143] In some embodiments, target cells (e.g., hepatocytes) are subjected to a low dose (e.g., 10) of the lentiviral vector disclosed herein. 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 The cells are treated in vitro before administration to the patient at a concentration of 3.0 × 10⁻¹⁶ TU / kg or less. In certain embodiments, target cells (e.g., hepatocytes) are treated with approximately 3.0 × 10⁻¹⁶ lentiviral vectors disclosed herein. 9 The cells are treated in vitro by TU / kg before being administered to the patient. In yet another embodiment, cells from the patient (e.g., hepatocytes) are administered to a low dose (e.g., 10) of the lentiviral vector disclosed herein. 10TU / kg or less, 10 9 TU / kg or less, or 10 8 The drug is processed ex vivo before administration to the patient (at a dose of TU / kg or less).

[0144] In some embodiments, the administration of the lentiviral vector disclosed herein (e.g., 10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 Plasma FIX activity after administration (at doses below TU / kg) increases by at least approximately 100%, at least approximately 110%, at least approximately 120%, at least approximately 130%, at least approximately 140%, at least approximately 150%, at least approximately 160%, at least approximately 170%, at least approximately 180%, at least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, or at least approximately 300% compared to physiologically normal circulating FIX levels.

[0145] In one embodiment, plasma FIX activity after administration of the lentiviral vector of the Disclosure increases by at least about 3,000% to about 5,000% compared to physiologically normal circulating FIX levels. In some embodiments, 21 days after administration of a lentiviral vector comprising a codon-optimized gene encoding a polypeptide having FIX activity as described herein, plasma FIX activity increases 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 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, or at least about 200-fold compared to subjects administered with the corresponding lentiviral vector comprising a reference nucleic acid molecule comprising SEQ ID NO: 8 or SEQ ID NO: 9.

[0146] This disclosure provides a method for treating, preventing or improving hemostatic disorders (e.g., bleeding disorders such as hemophilia A) in a subject requiring such treatment, comprising administering a therapeutically effective dose of a lentiviral vector to the subject, the lentiviral vector comprising an isolated nucleic acid molecule containing a nucleotide sequence encoding a polypeptide having FIX activity, wherein the lentiviral vector comprises 5 × 10⁻¹⁶ 10 The following TU / kg, 10 9 The following TU / kg, or 10 8 We also provide a method in which the drug is administered at least one dose of the following TU / kg.

[0147] Treatment, improvement, and prevention with the lentiviral vectors of this disclosure may be bypass therapy. Subjects receiving bypass therapy may already have inhibitors to coagulation factors, such as FIX, or may be prone to producing coagulation factor inhibitors.

[0148] The lentiviral vectors of this disclosure treat or prevent hemostatic disorders by promoting the formation of fibrin clots. Polypeptides having FIX activity encoded by the nucleic acid molecules of this disclosure can activate members of the coagulation cascade. The coagulation factors may be participants in the exogenous pathway, the endogenous pathway, or both.

[0149] The lentiviral vectors of this disclosure can be used to treat hemostatic disorders known to be treatable with FIX. Hemostatic disorders that can be treated using the methods of this disclosure 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 of fibrinogen, prothrombin, factor V, factor VII, factor X or XIII, hemorrhagic arthropathy, muscle bleeding, oral bleeding, bleeding, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intraperitoneal hemorrhage, intrathoracic hemorrhage, fractures, central nervous system bleeding, hemorrhage in the retropharyngeal space, hemorrhage in the retroperitoneal space and hemorrhage in the psoas sheath.

[0150] In some embodiments, the hemostatic disorder is a hereditary disorder. In one embodiment, the subject has hemophilia A. In other embodiments, the hemostatic disorder is a result of FIX deficiency. In the application method, impaired hemostasis may be a result of a defective FIX coagulation factor.

[0151] In another embodiment, the hemostatic disorder may be an acquired disorder. The acquired disorder may result from an underlying secondary disease or condition. Unrelated conditions may include, but are not limited to, cancer, autoimmune disease, or pregnancy. The acquired disorder may result from aging or from medications used to treat an underlying secondary disorder (e.g., cancer chemotherapy).

[0152] This disclosure also relates to methods for treating subjects who do not have hemostatic disorders or secondary diseases or conditions resulting in the acquisition of hemostatic disorders. Accordingly, this disclosure relates to methods for treating subjects requiring systemic hemostatic agents, comprising administering a therapeutically effective dose of the lentiviral vector of this disclosure. For example, in one embodiment, a subject requiring a systemic hemostatic agent is undergoing or about to undergo surgery. The lentiviral vector of this disclosure can be administered pre- or post-surgery as a prophylactic agent.

[0153] The lentiviral vectors of this disclosure may be administered during or after surgery to control acute bleeding symptoms. Surgery may include, but is not limited to, liver transplantation, hepatectomy, or stem cell transplantation.

[0154] In another embodiment, the lentiviral vector of the present disclosure can be used to treat subjects having acute bleeding symptoms without hemostatic impairment. Acute bleeding symptoms may result from severe trauma, such as surgery, a car accident, a wound, a laceration, a gunshot wound, or any other traumatic event resulting in uncontrolled bleeding.

[0155] Lentiviral vectors can be used to prophylactically treat subjects with hemostatic disorders. Lentiviral vectors can also be used to treat acute bleeding symptoms in subjects with hemostatic disorders.

[0156] In some embodiments, the lentiviral vector of this disclosure is administered in combination with at least one other agent that promotes hemostasis. The other agent that promotes hemostasis is a therapeutic agent having demonstrated coagulation activity. As an example, but not limited to, the hemostatic agent may include factor V, factor VII, factor VIII, factor X, factor XI, factor XII, factor XIII, prothrombin or fibrinogen, or any of the active forms thereof. The coagulation factor or hemostatic agent may also include antifibrinolytic agents, such as epsilon-aminocaproic acid or tranexamic acid.

[0157] In one embodiment of the present disclosure, the composition (e.g., a lentiviral vector) exists in a form that allows FIX to be activated when administered to a subject. Such an activatable molecule can be activated in vivo at a coagulation site after administration to the subject.

[0158] The lentiviral vectors of this disclosure can be administered intravenously, subcutaneously, intramuscularly, or through any mucosal surface, for example, orally, sublingually, oral cavity, sublingually, nasally, rectally, or vaginally, or via the pulmonary route. The lentiviral vectors can be implanted in or ligated to a biopolymer solid support that allows for sustained release of the vector to a desired site.

[0159] In one embodiment, the route of administration of the lentiviral vector 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. All of these forms of administration are disclosed herein. While it is clearly expected that the form of administration will be within the range, particularly for intravenous or intra-arterial injection or infusion, the form of administration will be an injectable solution. Typically, suitable pharmaceutical compositions for injection may include buffers (e.g., acetic acid, phosphoric acid, or citrate buffer), surfactants (e.g., polysorbate), and optionally stabilizers (e.g., human albumin). However, in other methods conforming to the teachings herein, lentiviral vectors can be delivered directly to the site of harmful cell populations, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0160] C. lentiviral vector Certain embodiments of this disclosure relate to lentiviral vectors, lentiviral vector particles, and / or methods of use thereof, wherein the lentiviral vector comprises a nucleotide sequence encoding a polypeptide having factor IX (FIX) activity. In some embodiments, the polypeptide having FIX activity comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the polypeptide having FIX activity comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the polypeptide having FIX activity is human FIX. In some embodiments, the polypeptide having FIX activity is a variant of human FIX. In certain embodiments, the polypeptide having FIX activity is the R338L variant of human FIX. In certain embodiments, the polypeptide having FIX activity is the Padua variant.

[0161] In some embodiments, the polypeptide having FIX activity is a monomer-dimer hybrid molecule containing FIX. As used herein, the term "monomer-dimer hybrid" refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain associated with each other by a disulfide bond, wherein the first chain comprises FIX and a first Fc region, and the second chain comprises, is essentially, or consists of a second Fc region that does not contain FIX. Thus, a monomer-dimer hybrid construct is a hybrid comprising a monomeric embodiment having only one coagulation factor and a dimeric embodiment having two Fc regions.

[0162] Nucleotide sequences encoding polypeptides with C.1.FIX activity In some embodiments, the nucleotide sequence encoding the FIX-active polypeptide is codon-optimized. In certain embodiments, the nucleotide sequence encoding the FIX-active polypeptide includes a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0163] In some embodiments, the nucleotide sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 85% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 90% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. The nucleotide sequence has at least about 91% sequence identity with respect to the creotide sequence. In some embodiments, the nucleotide sequence has at least about 92% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 93% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 94% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 95% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 96% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 97% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 98% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 99% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence is identical to the nucleotide sequence shown in SEQ ID NO: 1.

[0164] In some embodiments, the nucleotide sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 90% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 91% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 92% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 93% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 94% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 95% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 96% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 97% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the nucleotide sequence has at least about 98% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2.In some embodiments, the nucleotide sequence has at least about 99% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 2.

[0165] In some embodiments, the nucleotide sequence has sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% with respect to nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence is sequence The nucleotide sequence has at least approximately 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in sequence number 3. In some embodiments, the nucleotide sequence has at least approximately 90% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in sequence number 3. In some embodiments, the nucleotide sequence has at least approximately 91% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in sequence number 3. In some embodiments, the nucleotide sequence has at least approximately 92% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in sequence number 3. In some embodiments, the nucleotide sequence has at least approximately 93% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in sequence number 3. In some embodiments, the nucleotide sequence has at least approximately 94% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in sequence number 3. In some embodiments, the nucleotide sequence has at least approximately 95% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in sequence number 3. In some embodiments, the nucleotide sequence has at least about 96% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence has at least about 97% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence has at least about 98% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence has at least about 99% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 3.

[0166] In some embodiments, the nucleotide sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 90% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 91% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 92% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 93% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 94% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 95% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 96% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 97% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence has at least about 98% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4.In some embodiments, the nucleotide sequence has at least about 99% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 4. In the application form, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence shown in Sequence ID No. 4.

[0167] In some embodiments, the nucleotide sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 90% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 91% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 92% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 93% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 94% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 95% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 96% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 97% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence has at least about 98% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5.In some embodiments, the nucleotide sequence has at least about 99% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 5.

[0168] In some embodiments, the nucleotide sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 90% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 91% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 92% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 93% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 94% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 95% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 96% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 97% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 98% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence has at least about 99% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 6.

[0169] In some embodiments, the nucleotide sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 85% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 90% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 91% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 92% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 93% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 94% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 95% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 96% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 97% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the nucleotide sequence has at least about 98% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7.In some embodiments, the nucleotide sequence has at least about 99% sequence identity with nucleotides 139-1386 of the nucleotide sequence shown in SEQ ID NO: 7.

[0170] In certain embodiments, the nucleotide sequence encoding the polypeptide having FIX activity further comprises a nucleic acid sequence encoding the signal peptide. In some embodiments, the nucleic acid sequence encoding the signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with (i) nucleotides 1-84 of SEQ ID NO: 2; (ii) nucleotides 1-84 of SEQ ID NO: 3; (iii) nucleotides 1-84 of SEQ ID NO: 4; (iv) nucleotides 1-84 of SEQ ID NO: 5; (v) nucleotides 1-84 of SEQ ID NO: 6; or (vi) nucleotides 1-84 of SEQ ID NO: 7. In some embodiments, the nucleic acid sequence encoding the signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with (i) nucleotides 1-84 of SEQ ID NO: 2; (ii) nucleotides 1-84 of SEQ ID NO: 3 (iii) Consists of nucleotides 1-84 of SEQ ID NO: 4; (iv) Consists of nucleotides 1-84 of SEQ ID NO: 5; (v) Consists of nucleotides 1-84 of SEQ ID NO: 6; or (vi) Consists of nucleotides 1-84 of SEQ ID NO: 7.

[0171] In certain embodiments, the nucleotide sequence encoding the polypeptide having FIX activity further comprises a nucleic acid sequence encoding the propeptide. In some embodiments, the nucleic acid sequence encoding the propeptide 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 85-138 of SEQ ID NO: 2; (ii) nucleotides 85-138 of SEQ ID NO: 3; (iii) nucleotides 85-138 of SEQ ID NO: 4; (iv) nucleotides 85-138 of SEQ ID NO: 5; (v) nucleotides 85-138 of SEQ ID NO: 6; or (vi) nucleotides 85-138 of SEQ ID NO: 7. In some embodiments, the nucleic acid sequence encoding the propeptide includes the nucleotide sequence shown in (i) nucleotides 85-138 of SEQ ID NO: 2; (ii) nucleotides 85-138 of SEQ ID NO: 3; (iii) nucleotides 85-138 of SEQ ID NO: 4; (iv) nucleotides 85-138 of SEQ ID NO: 5; (v) nucleotides 85-138 of SEQ ID NO: 6; or (vi) nucleotides 85-138 of SEQ ID NO: 7.

[0172] C.1.a. Dissimilar parts In some embodiments, a nucleotide molecule encoding a polypeptide having FIX activity further comprises a nucleic acid sequence encoding at least one heterologous moiety. In some embodiments, the heterologous moiety is fused to the C-terminus or N-terminus of the polypeptide having FIX activity, where the polypeptide has procoagulation activity. In some embodiments, the heterologous moiety is inserted into one or more sites within the polypeptide having FIX activity, where the polypeptide has procoagulation activity. In some embodiments, the heterologous moiety is a heterologous polypeptide. In certain embodiments, the heterologous moiety is an XTEN. In some embodiments, the heterologous moiety comprises at least one XTEN inserted into one or more sites within the polypeptide having FIX activity. In other embodiments, the heterologous moiety is a half-life extension moiety (e.g., an in vivo half-life extension moiety) inserted into the polypeptide having FIX activity. In some embodiments, the heterologous moiety is inserted into the polypeptide having FIX activity at an insertion site disclosed in International Patent Application Publication WO2017 / 024060 (which is incorporated herein by reference in its entirety). In a particular embodiment, the heterogeneous portion is inserted into a polypeptide having FIX activity immediately downstream of the amino acid corresponding to amino acid 103, amino acid 105, amino acid 142, amino acid 149, amino acid 162, amino acid 166, amino acid 174, amino acid 224, amino acid 226, amino acid 228, amino acid 413, or any combination thereof.

[0173] In some embodiments, the heterogeneous portion is an FcRn binding partner (e.g., Fc and albumin, or a fragment thereof). In some embodiments, the heterogeneous portion is an FcRn binding partner fused to the C-terminus or N-terminus of a polypeptide having FIX activity.

[0174] Non-limiting examples of heterogeneous parts (e.g., half-life extension parts) include albumin, albumin fragments, Fc fragments of immunoglobulins, FcRn binding partners, C-terminal peptides (CTPs) of the β-subunit of human chorionic gonadotropins, HAP sequences, transferrin, PAS polypeptides of U.S. Patent Application No. 20100292130, polyglycine linkers, polyserine linkers, peptides, and in particular glycine (G), alanine (A), serine (S), threonine (T), glutamine having varying degrees of secondary structure from less than 50% to more than 50%. It contains short polypeptides of 6 to 40 amino acids, consisting of two types of amino acids selected from acid (E) and proline (P).

[0175] In certain embodiments, the heterologous moiety increases the in vivo or in vitro half-life of the FIX-active polypeptide produced from the lentiviral vector of the Disclosure. In other embodiments, the heterologous moiety facilitates the visualization or localization of the FIX-active polypeptide produced from the lentiviral vector of the Disclosure. Visualization and / or localization of the FIX-active polypeptide may be in vivo, in vitro, ex vivo, or a combination thereof. In other embodiments, the heterologous moiety increases the stability of the FIX-active polypeptide produced from the lentiviral vector of the Disclosure. As used herein, the term “stability” refers to a measure recognized in the Art of maintaining one or more physical properties of the FIX-active polypeptide in response to environmental conditions (e.g., rising or falling temperature). In certain embodiments, the physical property is the covalent maintenance of the structure of the FIX-active polypeptide (e.g., the absence of proteolysis, undesirable oxidation or deamidation). In other embodiments, the physical property is also the presence of the FIX-active polypeptide in a properly folded state (e.g., the absence of soluble or insoluble aggregation or precipitation).

[0176] In certain embodiments, the heterologous moiety that increases the half-life of the FIX fusion protein of this disclosure includes, but are not limited to, heterologous polypeptides such as albumin, immunoglobulin Fc region, XTEN sequence, C-terminal peptide (CTP) of the β-subunit of human chorionic gonadotropin, PAS sequence, HAP sequence, CTP peptide sequence, transferrin, albumin-binding moiety, or any fragment, derivative, variant, or combination of these polypeptides. In other relevant embodiments, the heterologous moiety includes a binding site to a non-polypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivative, variant, or combination of these moieties.

[0177] In certain embodiments, the FIX-active polypeptide of the present disclosure comprises one, two, three or more heterogeneous portions, each being the same or different molecules. In some embodiments, the lentiviral vector comprises one or more nucleotide sequences encoding XTEN. In other embodiments, the lentiviral vector comprises one or more nucleotide sequences encoding XTEN and one or more Fc domains. In a particular embodiment, the lentiviral vector comprises a nucleotide sequence encoding XTEN inserted into a nucleotide sequence encoding the FIX-active polypeptide and a nucleotide sequence encoding Fc fused to a portion of the nucleotide sequence encoding the C-terminus of the FIX-active polypeptide.

[0178] C.1.aiXTEN In some embodiments, at least one heterogeneous portion is XTEN. As used herein, “XTEN sequence” refers to an elongated polypeptide having a substantially non-repeating sequence that is not naturally occurring, is primarily composed of low molecular weight hydrophilic amino acids, and has little to no secondary or tertiary structure under physiological conditions. As a fusion protein partner, when ligated to the FIX sequence of this disclosure to create a fusion protein, XTEN acts as a carrier that confers certain desired pharmacokinetic, physicochemical, and pharmaceutical properties. Such desired properties include, but are not limited to, enhancements to pharmacokinetic parameters and solubility properties. As used herein, “XTEN” specifically excludes antibodies or antibody fragments such as light-chain or heavy-chain single-chain antibodies or Fc fragments.

[0179] In certain embodiments, the lentiviral vector of this disclosure is XTEN or a fragment thereof. The fusion polypeptide comprises at least one nucleotide sequence encoding a variant or derivative, wherein the nucleotide sequence encoding XTEN is inserted into a nucleotide sequence encoding a polypeptide having FIX activity, and the resulting fusion polypeptide has coagulation-promoting activity. In certain embodiments, two of the heterologous moieties are XTEN sequences. In some embodiments, three of the heterologous moieties are XTEN sequences. In some embodiments, four of the heterologous moieties are XTEN sequences. In some embodiments, five of the heterologous moieties are XTEN sequences. In some embodiments, six or more of the heterologous moieties are XTEN sequences.

[0180] In some embodiments, the XTEN sequence is a peptide or polypeptide having approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or more than 2000 amino acid residues. In certain embodiments, XTEN is a peptide or polypeptide having more than 20 to about 3000 amino acid residues, more than 30 to about 2500 residues, more than 40 to about 2000 residues, more than 50 to about 1500 residues, more than 60 to about 1000 residues, more than 70 to about 900 residues, more than 80 to about 800 residues, more than 90 to about 700 residues, more than 100 to about 600 residues, more than 110 to about 500 residues, or more than 120 to about 400 residues. In a particular embodiment, XTEN comprises an amino acid sequence whose length is longer than 42 amino acids and shorter than 144 amino acids.

[0181] An XTEN sequence may contain one or more sequence motifs of 5 to 14 (e.g., 9 to 14) amino acid residues, or an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence motif, the motif containing, essentially consisting of, or comprising 4 to 6 (e.g., 5 amino acids) selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P). See US2010-0239554A1.

[0182] Examples of XTEN sequences that can be used in accordance with this disclosure are, each incorporated herein by reference as a whole, U.S. Patent Application Publications 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, and This is disclosed in International Patent Application Publications WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, WO2011028344A2, WO2014 / 011819A2, WO2015 / 023891, or WO2017 / 024060.

[0183] C.1.a.ii.Fc region or FcRn binding partner In some embodiments, at least one heterogeneous portion is an Fc region (e.g., an FcRn binding partner) or a fragment thereof. In certain embodiments, the lentiviral vector of the present disclosure comprises at least one nucleotide sequence encoding an Fc region (e.g., an FcRn binding partner) which is inserted into a nucleotide sequence encoding a polypeptide having FIX activity, fused to a portion of a nucleotide sequence encoding the C-terminus of a polypeptide having FIX activity, or both, wherein the resulting fusion polypeptide has procoagulation activity. "Fc" or "Fc region" means, as used herein, a functional novel including an Fc domain, a variant thereof, or a fragment thereof, unless otherwise specified. These are live Fc receptor (FcRn) binding partners. An FcRn binding partner is any molecule to which the FcRn receptor specifically binds, and as a result, any FcRn binding partner, including albumin, can be actively transported by the FcRn receptor. Thus, the term Fc includes any variant of functional IgG Fc. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al., Nature 372:379 (1994) is incorporated herein in whole by reference). The main contact region of Fc with FcRn is near the junction of the CH2 and CH3 domains. All Fc-FcRn contacts are within a single Ig heavy chain. Examples of FcRn binding partners include, but are not limited to, whole IgG, the Fc fragment of IgG, and other fragments of IgG containing the complete FcRn binding region. Fc comprises the CH2 and CH3 domains of an immunoglobulin, with or without the hinge region of the immunoglobulin. Also included are Fc fragments, variants, or derivatives that maintain desired properties of the Fc region in the fusion protein, such as an increased half-life, e.g., an increased in vivo half-life. Numerous mutants, fragments, variants, and derivatives are described herein, for example, in PCT publications WO2011 / 069164A2, WO2012 / 006623A2, WO2012 / 006635A2, or WO2012 / 006633A2, all of which are incorporated herein by reference as a whole.

[0184] One or more nucleotide sequences encoding Fc domains are either inserted into a nucleotide sequence encoding a polypeptide having FIX activity, fused to a portion of the nucleotide sequence encoding the C-terminus of the polypeptide having FIX activity, or both. In some embodiments, the nucleotide sequence encoding the Fc domain is fused to the 5' end of the nucleotide sequence encoding the polypeptide having FIX activity. In some embodiments, the nucleotide sequence encoding the Fc domain is fused to the 3' end of the nucleotide sequence encoding the polypeptide having FIX activity. In some embodiments, the nucleotide sequence encoding the Fc domain is fused to a nucleotide sequence encoding another heterogeneous moiety, such as XTEN, either inserted into a nucleotide sequence encoding a polypeptide having FIX activity or fused to a portion of the nucleotide sequence encoding the C-terminus of the nucleotide sequence encoding XTEN. In some embodiments, the lentiviral vector includes a nucleotide sequence encoding a second Fc domain. The expressed second Fc domain can associate with the first Fc domain, for example, via one or more covalent bonds.

[0185] C.1.a.iii. Albumin In some embodiments, at least one heterogeneous portion is albumin, an albumin-binding domain, or an albumin-binding small molecule, or a variant, derivative, or fragment thereof. In certain embodiments, the lentiviral vector of the Disclosure comprises at least one nucleotide sequence encoding an albumin polypeptide or a fragment, variant, or derivative thereof, which is inserted into a nucleotide sequence encoding a polypeptide having FIX activity, fused to a portion of a nucleotide sequence encoding the C-terminus of a polypeptide having FIX activity, or both, wherein the resulting fusion polypeptide has procoagulation activity. Human serum albumin (HSA, or HA), a protein of 609 amino acids in its full-length form, is responsible for a significant portion of serum osmotic pressure and also functions as a carrier for endogenous and exogenous ligands. As used herein, the term “albumin” includes full-length albumin or its functional fragments, variants, derivatives, or analogs. Examples of albumin or its fragments or variants are incorporated herein by reference in their entirety by U.S. Patent Application Publications 2008 / 0194481 A1, 2008 / 0004206 A1, 2008 / 0161243 A1, 2008 / 0261877 A1, or 2008 / 0153751 A1, or PCT Patent Application Publications 2008 / 033413 A2, 2009 / 05 This is disclosed in document 8322 A1, or document 2007 / 021494 A2.

[0186] Albumin-binding polypeptides (ABPs) can impair bacterial albumin-binding domains, albumin-binding peptides, or albumin-binding antibody fragments capable of binding to albumin, without limitation. Domain 3 from streptococcal protein G is an example of a bacterial albumin-binding domain, as disclosed in Kraulis et al., FEBS Lett. 378:190-194 (1996) and Linhult et al., Protein Sci. 11:206-213 (2002). Examples of albumin-binding peptides include a series of peptides with the core sequence DICLPRWGCLW (SEQ ID NO: 15). See, for example, Dennis et al., J. Biol. Chem. 2002, 277:35035-35043 (2002). Examples of albumin-binding antibody fragments are disclosed in Muller and Kontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007); Roovers et al., Cancer Immunol. Immunother. 56:303-317 (2007); and Holt et al., Prot. Eng. Design Sci., 21:283-288 (2008), which are incorporated herein by reference in their entirety.

[0187] In certain embodiments, the lentiviral vector of the present disclosure comprises at least one nucleotide sequence encoding a binding site for an albumin-binding non-polypeptide small molecule, a variant thereof, or a derivative (e.g., an albumin-binding small molecule), which is inserted into a nucleotide sequence encoding a polypeptide having FIX activity, fused to a portion of a nucleotide sequence encoding the C-terminus of a polypeptide having FIX activity, or both, wherein the resulting fusion polypeptide has procoagulation activity. An example of such an albumin-binding site is Trussel et al., Bioconjugate This is 2-(3-maleimidopropanamide)-6-(4-(4-iodophenyl)butanamide)hexanoate ("Albu" tag) disclosed on pages 2286-2292 of Chem. 20 (2009).

[0188] In some embodiments, the albumin-binding polypeptide sequence in the expressed polypeptide is flanked at the C-terminus, N-terminus, or both ends by a Gly-Ser peptide linker sequence. In some embodiments, the Gly-Ser peptide linker is Gly4Ser (SEQ ID NO: 16). In other embodiments, the Gly-Ser peptide linker is (Gly4Ser)2 (SEQ ID NO: 17).

[0189] C.1.a.iv.CTP In some embodiments, at least one heterologous portion is the C-terminal peptide (CTP) of the β-subunit of human chorionic gonadotropin, or a fragment, variant, or derivative thereof. In certain embodiments, the lentiviral vector of the present disclosure comprises at least one nucleotide sequence encoding a CTP or a fragment, variant, or derivative thereof, which is inserted into a nucleotide sequence encoding a polypeptide having FIX activity, fused to a portion of a nucleotide sequence encoding the C-terminus of a polypeptide having FIX activity, or both, wherein the resulting fusion polypeptide has procoagulation activity. Insertion of one or more CTP peptides into a recombinant protein is known to increase the half-life of the protein. See, for example, U.S. Patent No. 5,712,122, which is incorporated herein by reference in its entirety. Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 18) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 19). For example, see U.S. Patent Application Publication No. 2009 / 0087411A1, incorporated by reference. In some embodiments, the expressed polypeptide The CTP sequence in this sequence is flanked at the C-terminus, N-terminus, or both ends by a Gly-Ser peptide linker sequence. In some embodiments, the Gly-Ser peptide linker is Gly4Ser (SEQ ID NO: 16). In other embodiments, the Gly-Ser peptide linker is (Gly4Ser)2 (SEQ ID NO: 17).

[0190] C.1.avPAS In some embodiments, at least one heterologous portion is a PAS peptide. In certain embodiments, the lentiviral vector of the present disclosure comprises at least one nucleotide sequence encoding a PAS peptide or a fragment, variant, or derivative thereof, which is inserted into a nucleotide sequence encoding a polypeptide having FIX activity, fused to a portion of a nucleotide sequence encoding the C-terminus of a polypeptide having FIX activity, or both, wherein the resulting fusion polypeptide has procoagulation activity. "PAS peptide" or "PAS sequence" means, as used herein, an amino acid sequence comprising primarily alanine and serine residues or primarily alanine, serine, and proline residues, which forms a random coil conformation under physiological conditions. Thus, a PAS sequence is a building block, amino acid polymer, or sequence cassette comprising, essentially consisting of, or comprising alanine, serine, and proline, which can be used as part of a heterologous portion in a fusion protein. However, if residues other than alanine, serine, and proline are added as trace components in the PAS sequence, the amino acid polymer may form a random coil conformation. "Trace components" means that amino acids other than alanine, serine, and proline can be added to the PAS sequence to a certain extent, for example, up to about 12% of the amino acids, i.e., about 12 out of 100 amino acids in the PAS sequence, up to about 10%, up to about 9%, up to about 8%, about 6%, about 5%, about 4%, about 3%, i.e., about 2%, or about 1%. Amino acids other than alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Under physiological conditions, the PAS peptide can form a random coil conformation, thereby mediating increased in vivo and / or in vitro stability.

[0191] Non-limiting examples of PAS peptides include ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 20), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 21), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 22), APSSPSPSAPSSPSPASPS (SEQ ID NO: 23), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 24), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 25), ASAAAPAAASAAASAPSAAA (SEQ ID NO: 26), or any variants, derivatives, fragments, or combinations thereof. Further examples of PAS sequences are known, for example, from U.S. Patent Application Publication 2010 / 0292130A1 and PCT Patent Application Publication WO2008 / 155134A1. European Patent EP2173890 is also included.

[0192] In some embodiments, the PAS sequence in the expressed polypeptide is flanked at the C-terminus, N-terminus, or both ends by a Gly-Ser peptide linker sequence. In some embodiments, the Gly-Ser peptide linker is Gly4Ser (SEQ ID NO: 16). In other embodiments, the Gly / Ser peptide linker is (Gly4Ser)2 (SEQ ID NO: 17).

[0193] C.1.a.vi.HAP In some embodiments, at least one heterogeneous portion is a homoamino acid polymer (HAP) peptide or a fragment, variant, or derivative thereof. In certain embodiments, The disclosed lentiviral vector comprises at least one nucleotide sequence encoding a homoamino acid polymer (HAP) peptide or a fragment, variant, or derivative thereof, which is inserted into a nucleotide sequence encoding a polypeptide having FIX activity, fused to a portion of a nucleotide sequence encoding the C-terminus of a polypeptide having FIX activity, or both, wherein the resulting fusion polypeptide has procoagulation activity. The HAP peptide comprises a glycine repeat sequence having an amino acid length of at least 50, at least 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 amino acids. The HAP sequence can extend the half-life of the portion fused or ligated to the HAP sequence. Non-limiting examples of HAP sequences include, but are not limited to, (Gly) n (Gly4Ser) n Or S (Gly4Ser) n Examples include (wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In one embodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. See, for example, Schlapschy M et al., Protein Eng. Design Selection, 20:273-284 (2007).

[0194] C.2. Adjustment Elements In some embodiments, the lentiviral vector includes gene expression regulatory elements. The gene expression regulatory sequences may be, for example, mammalian or viral promoters, such as constitutive or inducible promoters. Examples of constitutive promoters in mammals include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pirubate kinase, beta-actin promoter, and other constitutive promoters. Examples of viral promoters that function constitutively in eukaryotic cells include, for example, promoters from cytomegalovirus (CMV), Simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long-chain terminal repeats (LTRs), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus.

[0195] Other constitutive promoters may also be used. Inducible promoters are also useful as gene expression sequences in this disclosure. Inducible promoters are expressed in the presence of an inducer. For example, metallothionein promoters are induced in the presence of certain metal ions to promote transcription and translation. Other inducible promoters may be used.

[0196] In one embodiment, this disclosure involves expressing a transgene under the control of a tissue-specific promoter and / or enhancer. In another embodiment, the promoter or other expression regulatory sequence selectively enhances the expression of the transgene in hepatocytes. Examples of liver-specific promoters, but not limited to these, include the mouse tyretin promoter (mTTR), the endogenous human factor VIII promoter (F8), the human alpha-1 antitrypsin promoter (hAAT), the human albumin minimal promoter, and the mouse albumin promoter. In certain embodiments, the promoter includes the mTTR promoter. The mTTR promoter is described by RHCosta et al., 1986, Mol. Cell. Biol. 6:4697. The F8 promoter is described by Figueiredo and Brownlee, 1995, J. Biol. Chem. 2. This is described in 70:11828~11838. In some embodiments, the lentiviral vector includes at least one tissue-specific promoter, i.e., a promoter that will regulate the expression of a polypeptide having FIX activity in a particular tissue or cell type. In some embodiments, the tissue-specific promoter in the lentiviral vector selectively enhances the expression of a polypeptide having FIX activity in target liver cells. In some embodiments, the tissue-specific promoter that selectively enhances the expression of a polypeptide having FIX activity in target liver cells includes the APOA2 promoter, SERPINA1(hAAT) promoter, mTTR promoter, MIR122 promoter, ET promoter (GenBank No. AY661265; see also Vigna et al., Molecular Therapy 11(5):763 (2005)), or any combination thereof. In some embodiments, the target liver cells are hepatocytes.

[0197] One or more enhancers can be used to further increase expression levels and achieve therapeutic efficacy. One or more enhancers can be provided alone or in combination with one or more promoter elements. Typically, an expression regulatory sequence includes multiple enhancer elements and a tissue-specific promoter. In one embodiment, the enhancer includes one or more copies of an α1-microglobulin / bikunin enhancer (Rouet et al., 1992, J. Biol. Chem.). 267:pp. 20765-20773; Rouet et al., 1995, Nucleic Acids Res. 23:pp. 395-404; Rouet et al., 1998, Biochem. J. 334:pp. 577-584; Ill et al., 1997, Blood Coagulation Fibrinolysis 8:pp. S23-S30). In another embodiment, the enhancer is derived from liver-specific transcription factor binding sites such as EBP, DBP, HNF1, HNF3, HNF4, and HNF6, and Enh1 includes HNF1, (sense)-HNF3, (sense)-HNF4, (antisense)-HNF1, (antisense)-HNF6, (sense)-EBP, and (antisense)-HNF4 (antisense).

[0198] Examples of other suitable vectors and gene regulatory elements are described in WO02 / 092134, EP1395293, or U.S. Patent Nos. 6,808,905, 7,745,179, or 7,179,903, which are incorporated herein by reference in their entirety.

[0199] Generally, the gene expression regulatory sequence shall include, as necessary, 5' untranscribed and 5' untranslated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5' untranscribed sequences shall include a promoter region containing a promoter sequence for the transcriptional regulation of the operably linked coding nucleic acid. The gene expression sequence shall optionally include an enhancer sequence or an upstream activator sequence.

[0200] Since lentiviral vectors can transduce all liver cell types, the expression of a transgene (e.g., FIX) in different cell types can be controlled using different promoters within the lentiviral vector. Therefore, a lentiviral vector may contain specific promoters that will control the expression of the FIX transgene in different tissues or cell types, for example, different liver tissues or cell types. Thus, in some embodiments, a lentiviral vector may contain an endothelial-specific promoter that will control the expression of the FIX transgene in liver endothelial tissue, or a hepatocyte-specific promoter that will control the expression of the FIX transgene in hepatocytes, or both.

[0201] In some embodiments, lentiviral vectors deliver FIX to tissues other than the liver. The protocol includes one or more tissue-specific promoters that control gene expression. In some embodiments, the isolated nucleic acid molecule is stably incorporated into the genome of a target cell or target tissue, for example, the genome of a hepatocyte or the genome of liver endothelial cells.

[0202] In some embodiments, the lentiviral vector includes at least one splice donor site. In some embodiments, the lentiviral vector includes at least one splice acceptor site.

[0203] In some embodiments, the lentiviral vector includes a gag sequence, a pol sequence, a rev sequence, a rev response element (RRE), or any combination thereof. In certain embodiments, the lentiviral vector includes a full-length gag sequence. In some embodiments, the lentiviral vector includes a truncated gag sequence. In some embodiments, the lentiviral vector includes a full-length pol sequence. In some embodiments, the lentiviral vector includes a truncated pol sequence. In certain embodiments, the lentiviral vector includes a full-length rev sequence. In some embodiments, the lentiviral vector includes a truncated rev sequence. In certain embodiments, the lentiviral vector includes a full-length RRE sequence. In some embodiments, the lentiviral vector includes a truncated RRE sequence.

[0204] In some embodiments, the lentiviral vector comprises an enhancer, a promoter, a target sequence for microRNA (miRNA), a post-transcriptional regulatory element, a packaging signal, a poly(A) sequence, an intron sequence, or any combination thereof. In certain embodiments, the lentiviral vector comprises an enhancer that promotes the expression of a nucleotide sequence in liver cells.

[0205] In certain embodiments, it is beneficial to include one or more miRNA target sequences in the lentiviral vector that are operably ligated to, for example, the FIX transgene. Therefore, this disclosure also provides at least one miRNA sequence target that is operably ligated to the FIX nucleotide sequence or otherwise inserted into the lentiviral vector. More than one copy of the miRNA target sequence in the lentiviral vector can increase the effectiveness of the system.

[0206] Different miRNA target sequences are also included. For example, a lentiviral vector expressing more than one transgene may have more than one transgene under the control of miRNA target sequences, which may be the same or different. The miRNA target sequences can be arranged vertically, but other arrangements are also included. Transgene expression cassettes containing miRNA target sequences can also be inserted into a lentiviral vector in antisense orientation. Antisense orientation can be useful in the generation of viral particles to avoid the expression of gene products that may otherwise be toxic to the generating cells.

[0207] In other embodiments, the lentiviral vector contains one, two, three, four, five, six, seven, or eight copies of the same or different miRNA target sequences. However, in certain other embodiments, the lentiviral vector contains no miRNA target sequences at all. The choice of whether (and how many) to include miRNA target sequences is guided by parameters such as the intended tissue target and the required expression level.

[0208] In one embodiment, the target sequence is the miR-223 target, which has been reported to most effectively block expression in bone marrow-associated precursors and at least partially in earlier HSPCs. The miR-223 target is found in granulocytes, monocytes, macrophages, and bone marrow. It can block expression in differentiated bone marrow cells, including dendritic cells. miR-223 targets may also be suitable for gene therapy applications that rely on robust transgene expression in lymphoid or erythrocyte lineages. miR-223 targets can also block expression very effectively in human HSCs.

[0209] In another embodiment, the target sequence is the miR142 target (tccataaagt aggaaacact aca (SEQ ID NO: 27)). In one embodiment, the lentiviral vector contains at least one copy, at least two copies, at least three copies, at least four copies, at least five copies, or at least six copies of the miR-142 target sequence. In some embodiments, the lentiviral vector contains four copies of the miR-142 target sequence. In a particular embodiment, a complementary sequence of hematopoietic-specific microRNA, e.g., miR-142(142T) or "142-3pT", is incorporated into the 3' untranslated region of the lentiviral vector to make the transgene-coding transcript sensitive to miRNA-mediated downregulation. This method allows for the inhibition of transgene expression in hematopoietic antigen-presenting cells (APCs), while maintaining it in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy allows stringent post-transcriptional regulation to be imposed on transgene expression, thus enabling stable delivery and long-term expression of the transgene. In some embodiments, miR-142 regulation inhibits the immune-mediated elimination of transduced cells and / or induces antigen-specific regulatory T cells (Tregs) and mediates robust immune tolerance to the antigen encoded by the transgene.

[0210] In some embodiments, the target sequence is the miR181 target. Chen CZ and Lodish H, Seminars in Immunology (2005) 17(2): pp. 155-165 disclose miR-181, i.e., a miRNA specifically expressed in B cells in mouse bone marrow (Chen and Lodish, 2005). It also discloses that some human miRNAs are associated with leukemia.

[0211] The target sequence is fully or partially complementary to the miRNA. The term “fully complementary” means that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it. The term “partially complementary” means that the target sequence is only partially complementary to the sequence of the miRNA that recognizes it, thereby the partially complementary sequence is still recognized by the miRNA. In other words, in relation to this disclosure, a partially complementary target sequence is effective in achieving recognition of the corresponding miRNA and in blocking or reducing transgene expression in cells expressing that miRNA. Examples of miRNA target sequences are described in WO2007 / 000668, WO2004 / 094642, WO2010 / 055413, or WO2010 / 125471, which are fully incorporated herein by reference.

[0212] In other embodiments, the nucleotide sequence encoding the FIX-active polypeptide in the lentiviral vector of the present disclosure comprises, consists of, or is essentially derived from the lentiviral vector comprising coFIX-1-R338L (SEQ ID NO: 1).

[0213] C.3. Lentiviral vectors Lentiviruses include members of the bovine lentivirus group, horse lentivirus group, feline lentivirus group, sheep goat lentivirus group, and primate lentivirus group. The development of lentiviral vectors for gene therapy is reviewed in Klimatcheva et al. (1999) Frontiers in Bioscience 4: pp. 481-496. The design and use of suitable lentiviral vectors for gene therapy are described, for example, in U.S. Patent Nos. 6,207,455 and 6,615,782. Examples of lentiviruses include, but are not limited to, HIV-1, HIV-2, and HI This includes V-1 / HIV-2 pseudotype, HIV-1 / SIV, FIV, canine arthritis encephalitis virus (CAEV), equine infectious anemia virus, and bovine immunodeficiency virus.

[0214] A schematic diagram of the lentiviral vectors of this disclosure is shown in Figure 1. In some embodiments, the lentiviral vectors of this disclosure are “third-generation” lentiviral vectors. As used herein, the term “third-generation” lentiviral vector refers to a lentiviral packaging system that has the characteristics of a second-generation vector system and further lacks a functional tat gene, e.g., the tat gene is deleted or inactivated. Generally, the gene encoding rev is provided on a separate expression construct. See, for example, Dull et al. (1998) J. Virol. 72:8463–8471. As used herein, the “second-generation” lentiviral vector system refers to a lentiviral packaging system that lacks a functional accessory gene, e.g., the accessory genes vif, vpr, vpu, and nef are deleted or inactivated. See, for example, Zufferey et al. (1997) Nat. Biotechnol. 15:871–875. As used herein, “packaging system” refers to a set of viral constructs containing genes encoding viral proteins involved in the packaging of recombinant viruses. Generally, the constructs of a packaging system are ultimately incorporated into a packaging cell.

[0215] In some embodiments, the third-generation lentiviral vector of this disclosure is a self-inactivating lentiviral vector. In some embodiments, the lentiviral vector is a VSV.G pseudotyped lentiviral vector. In some embodiments, the lentiviral vector includes a mammalian-specific promoter for transgene expression. In some embodiments, the mammalian-specific promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the lentiviral vector includes a hepatocyte-specific promoter for transgene expression. In some embodiments, the hepatocyte-specific promoter is an enhanced trans tyretin promoter. In some embodiments, the lentiviral vector includes one or more target sequences for miR-142 to reduce the immune response to the transgene product. In some embodiments, incorporating one or more target sequences for miR-142 into the lentiviral vector of this disclosure enables a desired transgene expression profile. For example, incorporating one or more target sequences for miR-142 can suppress transgene expression in intravascular and extravascular hematopoietic systems, while transgene expression is maintained in non-hematopoietic cells. No cancer has been detected in tumor-prone mice treated with the lentiviral vector system described herein. See Brown et al. (2007) Blood 110:41 pp. 44-52, Brown et al. (2006) Nat. Ned. 12:58 pp. 585-91, and Cantore et al. (2015) Sci. Transl. Med. 7(277):277ra28.

[0216] The lentiviral vectors of this disclosure comprise a polynucleotide encoding a polypeptide having FIX activity as described herein. In one embodiment, the polypeptide having FIX activity is operably ligated to an expression regulatory sequence. As used herein, two nucleic acid sequences are operably ligated when they are covalently bonded in such a manner that each component nucleic acid sequence retains its function. A coding sequence and a gene expression regulatory sequence are said to be operably ligated when they are covalently bonded in such a manner that the expression or transcription and / or translation of the coding sequence is under the influence or control of the gene expression regulatory sequence. Two DNA sequences are operably ligated when the induction of a promoter in the 5' gene expression sequence results in the transcription of the coding sequence, and the nature of the ligation between the two DNA sequences is such that (1) it does not result in the introduction of a frameshift mutation, (2) it does not interfere with the ability of the promoter region to induce transcription of the coding sequence, or (3) it translates the corresponding RNA transcript into a protein. A gene expression sequence is said to be operably ligated if it does not interfere with the ability to do so. Therefore, a gene expression sequence will be operably ligated to a coding nucleic acid sequence if the gene expression sequence is capable of performing the transcription of that coding nucleic acid sequence so that the resulting transcript is translated into the desired protein or polypeptide.

[0217] In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of transducing into non-dividing cells. In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of transducing into liver cells (e.g., hepatocytes). The lentiviral genome and proviral DNA generally contain three genes found in retroviruses: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-induced DNA polymerase (reverse transcriptase), proteases, and integrases; and the env gene encodes the viral envelope glycoprotein. The 5' and 3' LTRs play a role in promoting virion RNA transcription and polyadenylation. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses possess additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0218] The sequences necessary for reverse transcription of the genome (tRNA primer binding sites) and efficient capsid formation of viral RNA into particles (Psi sites) are adjacent to the 5'LTR. If the sequences necessary for capsid formation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis-defect prevents capsid formation of the genomic RNA.

[0219] However, the resulting mutants remain capable of inducing the synthesis of all virion proteins. This disclosure provides a method for generating recombinant lentiviral vectors that can be transduced into non-dividing cells, comprising transfecting two or more vectors having packaging functions, i.e., gag, pol and env, and rev and tat, into a suitable host cell. As disclosed below herein, a vector lacking a functional tat gene is desirable for certain applications. Thus, for example, a first vector may provide nucleic acids encoding the viral gag and viral pol, and another vector may provide nucleic acids encoding the viral env to generate a packaging cell. Introducing a vector that provides heterologous genes, as specified herein, to its packaging cell gives a generating cell that releases infectious viral particles having the foreign genes of the choice.

[0220] With the above configuration of the vector and exogenous gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some embodiments, the env protein is a broad-host envelope protein that enables transduction of human and other species cells.

[0221] Examples of retrovirus-derived env genes include, but are not limited to, Moloney's mouse leukemia virus (MoMuLV or MMLV), Harvey's mouse sarcoma virus (HaMuSV or HSV), mouse mammary cancer virus (MuMTV or MMTV), gibbon leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes include, for example, vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis viruses, and insects. Those of influenza can also be used. In some embodiments, the env nucleic acid sequence of the virus operably associates with regulatory sequences described elsewhere herein.

[0222] In certain embodiments, the lentiviral vector has deletions of the HIV pathogenicity genes env, vif, vpr, vpu, and nef without impairing the vector's ability to transduce into non-dividing cells. In some embodiments, the lentiviral vector includes a deletion of the U3 region of the 3'LTR. The deletion of the U3 region may be a complete or partial deletion.

[0223] In some embodiments, the lentiviral vector of this disclosure, comprising a polypeptide having the FIX-active nucleotide sequence described herein, can be transfected in cells with (a) a first nucleotide sequence comprising the gag, pol, or gag and pol genes, and (b) a second nucleotide sequence comprising a heterologous env gene; where the lentiviral vector lacks a functional tat gene. In other embodiments, cells are further transfected with a fourth nucleotide sequence comprising the rev gene. In certain embodiments, the lentiviral vector lacks a functional gene selected from vif, vpr, vpu, vpx, and nef, or a combination thereof.

[0224] In certain embodiments, the lentiviral vector of the present disclosure comprises one or more nucleotide sequences encoding a gag protein, a Rev response element, a central polyprint lacte (cPPT), or any combination thereof.

[0225] In some embodiments, the lentiviral vector contains one or more polypeptides on its surface that target and / or enhance the activity of the lentiviral vector or the polypeptide having FIX activity encoded. One or more polypeptides can be incorporated during the budding of the lentiviral vector from the host cell. During lentivirus production, viral particles budding from the host cell. During the budding process, the viral particles acquire a lipid coat, which originates from the lipid membrane of the host cell. As a result, the lipid coat of the viral particles may contain membrane-bound polypeptides that were previously present on the surface of the host cell.

[0226] In some embodiments, the lentiviral vector expresses one or more polypeptides on its surface that suppress the immune response to the lentiviral vector after administration to a human subject. In some embodiments, the surface of the lentiviral vector contains one or more CD47 molecules. CD47 is a “self-marker” protein that is ubiquitously expressed on human cells. Surface expression of CD47 suppresses the macrophage-induced phagocytosis of endogenous cells through the interaction of CD47 and SIRPα expressed by macrophages. Cells expressing high levels of CD47 are less likely to be targeted and destroyed by human macrophages in vivo.

[0227] In some embodiments, the lentiviral vector contains a high concentration of CD47 polypeptide molecules on its surface. In some embodiments, the lentiviral vector is generated in a cell line having a high expression level of CD47. In a particular embodiment, the lentiviral vector is generated in CD47high cells, where the cells have high expression of CD47 on the cell membrane. In a particular embodiment, the lentiviral vector is generated in CD47high HEK293T cells, where HEK293T cells have high expression of CD47 on the cell membrane. In some embodiments, HEK293T cells are modified to have increased expression of CD47 compared to unmodified HEK293T cells. In a particular embodiment, CD47 is human CD47.

[0228] In some embodiments, the lentiviral vector comprises human CD47 having an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 14.

[0229] In some embodiments, the lentiviral vector has little to no surface expression of major histocompatibility complex class I (MHC-I). Surface-expressed MHC-I presents peptide fragments of "non-self" proteins from within the cell, such as infection-indicating protein fragments, thereby promoting an immune response against the cell. In some embodiments, the lentiviral vector is generated in MHC-I-low cells, where the cells have reduced MHC-I expression on the cell membrane. In some embodiments, the lentiviral vector is MHC-I-(or "MHC-I free "MHC-1 neg It is produced in cells (or "MHC-negative" cells), where the cells lack MHC-I expression.

[0230] In certain embodiments, the lentiviral vector comprises a high concentration of CD47 polypeptide and a lipid coat lacking MHC-I polypeptide. In certain embodiments, the lentiviral vector comprises CD47 high / MHC-I low Cell lineage, for example, CD47 high / MHC-I low It is generated in the HEK293T cell line. In some embodiments, the lentiviral vector is CD47 high / MHC-I free Cell lineage, for example, CD47 high / MHC-I free These are generated in the HEK293T cell line. Examples of lentiviral vectors are disclosed in U.S. Patent No. 9,050,269 and International Publication Nos. WO9931251, 9712622, 9817815, 9817816, and 9818934, which are incorporated herein by reference in their entirety.

[0231] Mai. Pharmaceutical composition The lentiviral vectors, nucleic acid molecules, polypeptides encoded by nucleic acid molecules, or compositions containing host cells of this disclosure may contain suitable pharmaceutically acceptable carriers. For example, they may contain excipients and / or adjuvants that facilitate the processing of the active compound into a preparation designed for delivery to the site of action.

[0232] In one embodiment, the disclosure relates to a pharmaceutical composition comprising (a) a nucleic acid molecule, lentiviral vector, polypeptide, or host cell as disclosed herein; and (b) a pharmaceutically acceptable excipient.

[0233] Pharmaceutical compositions can be formulated for parenteral administration by bolus injection (i.e., intravenous, subcutaneous, or intramuscular administration). Injectable formulations can be supplied in unit dosage forms, for example, in ampoules or multi-dose containers with preservatives added. Compositions may take the form of suspensions, liquids, or emulsions in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient may be in powder form composed of a suitable vehicle, such as pyrogen-free water.

[0234] In one embodiment, the route of administration of the lentiviral vector is parenteral. The term parenteral, as used herein, includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Intravenous parenteral administration is preferred. While all of these forms of administration are obviously expected to be within the scope of this disclosure, the form for administration will be an injectable solution, particularly for intravenous or intra-arterial injection or infusion. Typically, preferred pharmaceutical compositions for injection include buffers (e.g., acetic acid, phosphoric acid, or citrate buffer), surfactants (e.g., The lentiviral vector may contain polysorbates, and possibly stabilizers (e.g., human albumin). However, in other methods conforming to the teachings herein, the lentiviral vector can be delivered directly to the site of the harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0235] 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, alcohol / aqueous solutions, emulsions, or suspensions containing saline and a buffer medium. In this disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01–0.1 M, preferably 0.05 M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's glucose, glucose and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous vehicles include replacement fluids and nutrient replacement fluids, electrolyte replacement fluids, such as those based on Ringer's glucose. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may be present.

[0236] More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (water-soluble) or dispersions, and sterile powders for immediate-use preparations of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and fluid enough to allow for easy passage of an injection needle. It must be stable under manufacturing and storage conditions, preferably protected against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating material such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of a surfactant.

[0237] The prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. By including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition, prolonged absorption of the injectable composition can be achieved.

[0238] In any case, a sterile injectable solution can be prepared by incorporating the active compound (e.g., the polypeptide itself or a combination of other activators) in the required amount in a suitable solvent, along with one or a combination of the components listed herein as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired components from the pre-filtered solution. Preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes or vials and sealed under sterile conditions by methods known in the art. Furthermore, preparations can be packaged in kit form and sold. Such products preferably have a label or accompanying information indicating that the relevant composition is beneficial for treating subjects suffering from or predisposed to coagulation disorders.

[0239] Injectable depot formulations contain drugs in biodegradable polymers such as polylactide-polyglycolide. These formulations can be prepared by forming a microencapsulation matrix of substances. The drug release rate can be controlled depending on the ratio of drug to polymer and the properties of the polymer used. Other exemplary biodegradable polymers include polyorthoesters and polyanhydrides. Injectable depot formulations can also be prepared by capturing the drug in liposomes or microemulsions.

[0240] The pharmaceutical composition may also be formulated for rectal administration as a suppository or retained enema, for example, containing a conventional suppository base such as cocoa butter or other glycerides.

[0241] Complementary active compounds can be incorporated into the composition. In one embodiment, the nucleic acid molecules of this disclosure are formulated together with coagulation factors, or their variants, fragments, analogs, or derivatives. For example, coagulation factors include, but are not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor, or recombinant soluble tissue factor (rsTF) or activated forms of any of these. Coagulation factors of hemostatic agents may also include antifibrinolytic agents, such as epsilon-aminocaproic acid and tranexamic acid.

[0242] The administration regimen can be adjusted to achieve the optimal desired response. For example, a single bolus dose may be administered, or the dose may be divided into several doses over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and standardization of dosage, it is beneficial to formulate parenteral compositions in dose-unit form. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[0243] Intermediate doses within the above range are also within the scope of this disclosure. Such doses may be administered to subjects daily, every other day, weekly, or according to any other schedule determined by experimental analysis. Exemplary treatments require long-term administration, for example, multiple doses over at least six months.

[0244] The lentiviral vectors of this disclosure are administered to subjects at different developmental stages. For example, in humans, different developmental stages are classified as neonates (e.g., less than 1 month old), infants (1 month to 2 years old), children (2 to 12 years old), adolescents (12 to 16 years old), or adults (ages older than 16 years). In some embodiments, the lentiviral vectors of this disclosure are administered to human neonates. In some embodiments, the lentiviral vectors of this disclosure are administered to human subjects less than approximately 1 month old. In some embodiments, the lentiviral vectors of this disclosure are administered to human infants. In some embodiments, the lentiviral vectors of this disclosure are administered to human subjects from approximately 1 month old to approximately 2 years old. In some embodiments, the lentiviral vectors of this disclosure are administered to human children. In some embodiments, the lentiviral vectors of this disclosure are administered to human subjects from approximately 2 years old to approximately 12 years old. In some embodiments, the lentiviral vectors of this disclosure are administered to human adolescents. In some embodiments, the lentiviral vectors of this disclosure are administered to human subjects approximately 12 to 16 years of age. In some embodiments, the lentiviral vectors of this disclosure are administered to adult human subjects. In some embodiments, the lentiviral vectors of this disclosure are administered to human subjects older than approximately 16 years of age. Those skilled in the art can determine the developmental stage of other organisms. For example, those skilled in the art understand that a two-week-old mouse is in adolescence.

[0245] The dosage and frequency of the lentiviral vectors described herein vary depending on various factors known to those skilled in the art.

[0246] The lentiviral vectors of this disclosure may, depending on the circumstances, be administered in combination with other agents that are effective in treating a disorder or condition requiring treatment (e.g., prophylactic or therapeutic).

[0247] As used herein, administration of the lentiviral vectors of this disclosure together with or in combination with adjuvant therapy means sequential, simultaneous, coexisting, parallel, concurrent, or concurrent administration or application of the therapy and the disclosed polypeptides. Those skilled in the art will understand that the timing of administration or application of the various components of the combined therapeutic regimen can be adjusted to enhance the overall effect of the treatment. Those skilled in the art (e.g., physicians) will be able to easily determine an effective combination therapy regimen without disproportionate experimentation based on the selected adjuvant therapy and the teachings herein.

[0248] It will be further understood that the lentiviral vectors of this disclosure may be used together or in combination with a drug or multiple drugs (for example, to provide a combination therapy regimen). Exemplary drugs that may be combined with the lentiviral vectors of this disclosure include drugs that correspond to current medical standards for the specific disorders to be treated. Such drugs may be chemical or biological in nature. The terms “biological” or “biological drug” refer to any pharmaceutically active drug made from living organisms and / or their products, intended for use as a therapeutic agent.

[0249] The amount of the drug used in combination with the lentiviral vector of this disclosure may vary depending on the subject or may be administered by means of those known in the art. See, for example, Bruce A Chabner et al., Antineoplastic Agents (edited by Joel G. Hardman et al., 9th edition, 1996), pp. 1233–1287, GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS. In another embodiment, such a drug amount consistent with medical standards is administered.

[0250] In certain embodiments, the lentiviral vectors of this disclosure are administered together with immunosuppressants, antiallergic agents, or anti-inflammatory agents. These agents generally refer to substances that suppress or mask the immune system of the target being treated herein. These agents include substances that suppress cytokine production, downregulate 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-idiotype antibodies for MHC antigens and MHC fragments; cyclosporine A; steroids such as glucocorticoids, e.g., prednisone, methylprednisolone, and dexamethasone; anti-interferon-γ, -β, or -α antibodies, anti-tumor necrosis factor-α antibodies, anti-tumor necrosis factor-β These include antibodies, cytokines or cytokine receptor antagonists including 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 anti-lymphocyte globulins; pan-T antibodies; soluble peptides containing LFA-3 binding domains; streptokinase; TGF-β; streptodolase; FK506; RS-61443; deoxysperguarin; and rapamycin. In certain embodiments, the agent is an antihistamine. As used herein, “antihistamine” is an agent that antagonizes the physiological effects of histamine. Examples of antihistamines include chlorpheniramine, diphenhydramine, promethazine, cromolyn sodium, astemizole, azatadine maleate, brompheniramine maleate, carbinoxamine maleate, cetirizine hydrochloride, clemastine fumarate, cyproheptadine hydrochloride, d-brompheniramine maleate, d-chlorpheniramine maleate, and dimethazine. These include hydrinate, diphenhydramine hydrochloride, doxylamine succinate, fexofendazine hydrochloride, terfenadine hydrochloride, hydroxyzine hydrochloride, loratidine, meclizine hydrochloride, triperanamine citrate, triperenamine hydrochloride, and triprolidine hydrochloride.

[0251] Immunosuppressants, anti-allergic agents, or anti-inflammatory agents can be incorporated into lentiviral vector administration regimens. For example, administration of an immunosuppressant or anti-inflammatory agent can be initiated before administration of the disclosed lentiviral vector, and can be continued in a single or multiple doses thereafter. In certain embodiments, the immunosuppressant or anti-inflammatory agent is administered as premedication for the lentiviral vector.

[0252] As previously discussed, the lentiviral vectors of this disclosure can be administered in pharmaceutically effective amounts for the in vivo treatment of coagulation disorders. In this regard, it is understood that the lentiviral vectors of this disclosure can be formulated to facilitate administration and enhance the stability of the activator. Preferably, the pharmaceutical compositions of this disclosure include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, a non-toxic buffer, and a preservative. Naturally, the pharmaceutical compositions of this disclosure can be administered in single or multiple doses to provide a pharmaceutically effective amount of polypeptide.

[0253] In addition to the active compound, the liquid dosage form may contain inert components such as water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil, glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan.

[0254] Non-limiting examples of suitable pharmaceutical carriers are also described in Remington's Pharmaceutical Sciences by E.W. Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerin monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene glycol, water, and ethanol. The composition may also contain pH buffering agents and wetting or emulsifying agents.

[0255] In some embodiments, the composition is administered via a route selected from the group consisting of topical administration, intraocular administration, intrathecal administration, and subdural administration. Parenteral administration may be intravenous or subcutaneous.

[0256] In some embodiments, the composition is used to treat hemorrhagic disorders or conditions in subjects requiring it. These hemorrhagic disorders or conditions are selected from the group consisting of hemorrhagic blood coagulation disorders, arthritis, muscle bleeding, oral bleeding, massive bleeding, massive intramuscular bleeding, massive oral bleeding, trauma, head trauma, gastrointestinal bleeding, massive intracranial bleeding, massive intraperitoneal bleeding, massive intrathoracic bleeding, fractures, central nervous system bleeding, retropharyngeal space bleeding, retroperitoneal space bleeding, intracystosal tuberosity bleeding, and any combination thereof. In yet other embodiments, the subject is scheduled to undergo surgery. In yet other embodiments, the treatment is prophylactic or on demand.

[0257] Several tests are available to investigate the function of the coagulation system: activated partial thromboplastin time (aPTT) test, colorimetric assay, ROTEM® assay, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often by the Krauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (to see if abnormalities are corrected when the patient's plasma is mixed with normal plasma), coagulation factor assay, antiphospholipid antibody, D-dimer, Genetic testing (e.g., factor V Leiden, prothrombin mutation G20210A), diluted Russell's viper venom time (dRVVT), other platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0258] The aPTT test is a performance indicator that measures the efficacy of the "endogenous" (also known as the contact activation pathway) and common coagulation pathways. This test is commonly used to measure the coagulation activity of commercially available recombinant coagulation factors, such as FVIII or FIX. It is used in conjunction with prothrombin time (PT), which measures the exogenous pathway.

[0259] ROTEM® analysis provides complete kinetics of hemostasis: information on coagulation time, clot formation, clot stability, and lysis. Different parameters in thromboelastometry depend on many factors that affect the activity of the plasma coagulation system, platelet function, fibrinolysis, or their interactions. This assay can provide a complete picture of secondary hemostasis.

[0260] IV. Nucleic acid molecules The disclosure also provides a series of isolated nucleic acid molecules encoding polypeptides having FIX activity. In certain embodiments, the isolated nucleic acid molecules include a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1.

[0261] In some embodiments, the isolated nucleic acid molecule contains a nucleotide sequence having at least about 85% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 90% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 91% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 92% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 93% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 94% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 95% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 96% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 97% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 98% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence is identical to the nucleotide sequence shown in SEQ ID NO: 1.

[0262] In certain embodiments, the isolated nucleic acid molecule further comprises a nucleic acid sequence encoding a signal peptide. In some embodiments, the nucleic acid sequence encoding the signal peptide is (i) nucleotides 1-84 of SEQ ID NO: 2; (ii) nucleotides 1-84 of SEQ ID NO: 3; (iii) nucleotides 1-84 of SEQ ID NO: 4; (iv) nucleotides 1-84 of SEQ ID NO: 5; (v) nucleotides 1-84 of SEQ ID NO: 6; or (vi) nucleotides of SEQ ID NO: 7 The sequence identity with respect to nucleotides 1-84 is 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%. In some embodiments, the nucleic acid sequence encoding the signal peptide includes the nucleotide sequences shown in (i) nucleotides 1-84 of SEQ ID NO: 2; (ii) nucleotides 1-84 of SEQ ID NO: 3; (iii) nucleotides 1-84 of SEQ ID NO: 4; (iv) nucleotides 1-84 of SEQ ID NO: 5; (v) nucleotides 1-84 of SEQ ID NO: 6; or (vi) nucleotides 1-84 of SEQ ID NO: 7.

[0263] In certain embodiments, the isolated nucleic acid molecule further comprises a nucleic acid sequence encoding a propeptide. In some embodiments, the nucleic acid sequence encoding the propeptide 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 85-138 of SEQ ID NO: 2; (ii) nucleotides 85-138 of SEQ ID NO: 3; (iii) nucleotides 85-138 of SEQ ID NO: 4; (iv) nucleotides 85-138 of SEQ ID NO: 5; (v) nucleotides 85-138 of SEQ ID NO: 6; or (vi) nucleotides 85-138 of SEQ ID NO: 7. In some embodiments, the nucleic acid sequence encoding the propeptide includes the nucleotide sequence shown in (i) nucleotides 85-138 of SEQ ID NO: 2; (ii) nucleotides 85-138 of SEQ ID NO: 3; (iii) nucleotides 85-138 of SEQ ID NO: 4; (iv) nucleotides 85-138 of SEQ ID NO: 5; (v) nucleotides 85-138 of SEQ ID NO: 6; or (vi) nucleotides 85-138 of SEQ ID NO: 7.

[0264] This disclosure also provides vectors comprising nucleic acid molecules described herein. In some embodiments, the vector is a lentiviral vector, for example, any lentiviral vector disclosed herein. In certain embodiments, the vector comprises a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0265] In some embodiments, the vector contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector contains a nucleotide sequence having at least about 85% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector contains a nucleotide sequence having at least about 90% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector contains a nucleotide sequence having at least about 91% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector contains a nucleotide sequence having at least about 92% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector contains a nucleotide sequence having at least about 93% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector includes a nucleotide sequence having at least about 94% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector includes a nucleotide sequence having at least about 95% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector includes a nucleotide sequence having at least about 96% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. The vector contains a rheotide sequence. In some embodiments, the vector contains a nucleotide sequence having at least about 97% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector contains a nucleotide sequence having at least about 98% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector contains a nucleotide sequence having at least about 99% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the vector contains a nucleotide sequence that is identical to the nucleotide sequence shown in SEQ ID NO: 1.

[0266] In some embodiments, the vector further comprises one or more regulatory elements described herein. In certain embodiments, the vector comprises a tissue-specific promoter. In certain embodiments, the tissue-specific promoter selectively enhances the expression of a polypeptide having FIX activity in target hepatocytes. In certain embodiments, the tissue-specific promoter that selectively enhances the expression of a polypeptide having FIX activity in target hepatocytes includes the APOA2 promoter, the SERPINA1(hAAT) promoter, the mTTR promoter, the MIR122 promoter, or any combination thereof. In some embodiments, the target hepatocytes are hepatocytes.

[0267] V. Tissue-specific expression In certain embodiments, it is beneficial to include one or more miRNA target sequences in the lentiviral vector that are operably ligated to, for example, the optimized FIX transgene. Therefore, this disclosure also provides at least one miRNA sequence target that is operably ligated to the optimized FIX nucleotide sequence or otherwise inserted into the lentiviral vector. More than one copy of the miRNA target sequence in the lentiviral vector can increase the effectiveness of the system.

[0268] This also includes different miRNA target sequences. For example, a lentiviral vector expressing more than one transgene may have transgenes under the control of more than one miRNA target sequence, which may be the same or different. The miRNA target sequences can be arranged vertically, but other arrangements are also included. Transgene expression cassettes containing miRNA target sequences can also be inserted into a lentiviral vector in antisense orientation. Antisense orientation can be useful in the generation of viral particles to avoid the expression of gene products that might otherwise be toxic to the generating cells.

[0269] In other embodiments, the lentiviral vector contains one, two, three, four, five, six, seven, or eight copies of the same or different miRNA target sequence. However, in certain other embodiments, the lentiviral vector contains no miRNA target sequence at all. The choice of whether (and how many) to include miRNA target sequences is guided by known parameters such as the intended tissue target and the required expression level.

[0270] In one embodiment, the target sequence is the miR-223 target, which has been reported to most effectively block expression in bone marrow-associated precursors and at least partially in earlier HSPCs. The miR-223 target can block expression in differentiated bone marrow cells, including granulocytes, monocytes, macrophages, and bone marrow dendritic cells. The miR-223 target may also be suitable for gene therapy applications that rely on robust transgene expression in lymphoid or erythrocyte lineages. The miR-223 target can also block expression very effectively in human HSCs.

[0271] In another embodiment, the target sequence is the miR142 target (tccataaagt aggaaacact aca (SEQ ID NO: 27)). In one embodiment, the lentiviral vector contains four copies of the miR-142 target sequence. In a particular embodiment, hematopoietic specific The complementary sequence of the target microRNA, e.g., miR-142(142T), is incorporated into the 3' untranslated region of the lentiviral vector, making the transgene-coding transcript sensitive to miRNA-mediated downregulation. This method allows for the inhibition of transgene expression in hematopoietic antigen-presenting cells (APCs), while maintaining it in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy allows for stringent post-transcriptional regulation of transgene expression, thus enabling stable delivery and long-term expression of the transgene. In some embodiments, miR-142 regulation inhibits the immune-mediated elimination of transduced cells and / or induces antigen-specific regulatory T cells (Tregs) and mediates robust immune tolerance to the antigen encoded by the transgene.

[0272] In some embodiments, the target sequence is the miR181 target. Chen CZ and Lodish H, Seminars in Immunology (2005) 17(2): pp. 155-165 disclose miR-181, i.e., a miRNA specifically expressed in B cells in mouse bone marrow (Chen and Lodish, 2005). It also discloses that some human miRNAs are associated with leukemia.

[0273] The target sequence is fully or partially complementary to the miRNA. The term “fully complementary” means that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it. The term “partially complementary” means that the target sequence is only partially complementary to the sequence of the miRNA that recognizes it, thereby the partially complementary sequence is still recognized by the miRNA. In other words, in relation to this disclosure, a partially complementary target sequence is effective in achieving recognition of the corresponding miRNA and in blocking or reducing transgene expression in cells expressing that miRNA. Examples of miRNA target sequences are described in WO2007 / 000668, WO2004 / 094642, WO2010 / 055413, or WO2010 / 125471, which are fully incorporated herein by reference.

[0274] VI. Host cells and methods for producing them This disclosure also provides host cells containing nucleic acid molecules or lentiviral vectors of this disclosure. Certain aspects of this disclosure relate to the preparation or production of lentiviral vectors, including transfecting and / or transforming host cells with lentiviral vectors disclosed herein. As used herein, the term “transformation” is used in a broad sense to refer to the introduction of DNA into a recipient host cell that alters its genotype and consequently brings about a change in the recipient cell.

[0275] "Host cell" refers to a cell transformed with a lentiviral vector disclosed herein. The host cells of this disclosure are preferably of mammalian origin; most preferably of human or mouse origin. Those skilled in the art will be able to preferentially determine the particular host cell line best suited to their purposes. Exemplary host cell lines, though not limited to these, include CHO, DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR-negative), HELA (human cervical cancer), CVI (monkey kidney cell line), COS (a derivative of CVI with the SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), PER.C6®, NS0, CAP, BHK21, and HEK293 (human kidney). In a particular embodiment, the host cells are selected from the group consisting of: CHO cells, HEK293 cells (e.g., HEK293T cells), BHK21 cells, PER.C6® cells, NS0 cells, CAP cells, and any combination thereof. In some embodiments, the host cells of this disclosure are of insect origin. In a particular embodiment, the host cells are SF9 cells. The host cell line is, Generally, it is available from the commercial service American Tissue Culture Collection or from publicly available literature.

[0276] The introduction of the nucleic acid molecules or vectors of this disclosure into host cells can be achieved by various techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with envelope DNA, microinjection, and infection with intact viruses. See Ridgway, AAG, "Mammalian E×pression Vectors," Chapter 24.2, pp. 470-472, Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). Most preferably, plasmid introduction into the host is by electroporation. Transformed cells are grown under conditions suitable for generating light and heavy chains and assayed for heavy and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), and immunohistochemistry.

[0277] Host cells containing isolated nucleic acid molecules or lentiviral vectors of this disclosure are grown in a suitable growth medium. As used herein, the term “suitable growth medium” means a medium containing nutrients necessary for cell growth. Nutrients necessary for cell growth include carbon sources, nitrogen sources, essential amino acids, vitamins, minerals, and growth factors. The medium may contain one or more selective factors. The medium may contain calf serum or fetal bovine serum (FCS). In one embodiment, the medium is substantially IgG-free. Growth media are generally selected for cells containing DNA constructs, for example, by drug selection or deficiency of essential nutrients supplemented by selectable markers on the DNA construct or cotransfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM / F12). In one embodiment, the medium is CDoptiCHO (Invitrogen, Carlsbad, CA.). In another embodiment, the culture medium is CD17 (Invitrogen, Carlsbad, CA). The selection of a suitable culture medium for the specific cell line used is within the realm of those skilled in the art.

[0278] In some embodiments, the host cells are further modified as described herein. For example, the host cells may be modified to overexpress CD47 as described herein. In some embodiments, the host cells are modified to lack surface-exposed MHC-I. In some embodiments, the host cells are modified to have reduced surface-exposed MHC-I compared to unmodified host cells. In certain embodiments, the host cells are modified to overexpress CD47 high / MHC-I low These are HEK293T cells.

[0279] Certain aspects of this disclosure relate to a method for producing a lentiviral vector, comprising culturing host cells described herein under preferred conditions and isolating the lentiviral vector. In certain aspects, this disclosure relates to a method for producing a lentiviral vector disclosed herein, comprising culturing host cells described herein under preferred conditions and isolating the lentiviral vector.

[0280] All of the various aspects, embodiments, and options described herein can be combined in any and all of the variations.

[0281] All publications, patents, and patent applications referred to herein are subject to the terms of individual publications, patents, and patents. or incorporated herein by reference to the same extent as a patent application is specifically and individually indicated as being incorporated by reference.

[0282] While this disclosure has been given a general overview, a deeper understanding can be gained by referring to the examples provided herein. These examples are for illustrative purposes only and are not intended to limit the scope of this disclosure. [Examples]

[0283] Long-term FIX expression and dose response mediated by LV-coFIX-1-R338L in adult HemB mice A codon-optimized nucleotide sequence encoding a human FIX variant with the R338L ("Padua") substitution (coFIX-1-R338L; SEQ ID NO: 1) was cloned into a lentiviral vector to create LV-coFIX-1-R338L (Figure 1). To determine the dose-response profile of LV-FIX in animal models, LV-coFIX-1-R338L generated in 293T cells was evaluated in adult HemB mice. Eight-week-old HemB mice were treated with LV-coFIX-1-R338L via tail vein injection at doses of 3E9, 7.5E9, 2E10, or 6E10 TU / kg (n=2-10 animals / dose level). Plasma FIX activity and antigen levels mediated by LV-FIX were monitored using FIX dye sources and ELISA assays. The steady-state plasma FIX levels for each animal are shown in Figure 2A, and the LV-coFIX-1-R338L dose-response curves are shown in Figure 2B. In the HemB mouse model, LV-coFIX-1-R338L demonstrated a log-log dose-response profile, and it was determined that the LV-coFIX-1-R338L dose level required to achieve normal circulating FIX activity of 10-200% was in the range of 5E9-2E10 TU / kg in HemB mice.

[0284] The long-term FIX expression profiles of animals treated with LV-coFIX-1-R338L in three higher dose levels were monitored for 6 months after LV treatment. Levels of circulating FIX activity (Figure 3A) and antigen (Figure 3B) were plotted. Consistent levels of LV-mediated FIX expression were observed in all experimental animals, and no loss of FIX expression was detected during the study period, demonstrating the long-term stability of the incorporated gene therapy treatment. Furthermore, lower percentages of normal FIX antigen levels were observed compared to those with FIX activity (Figure 3B) (Figure 3A), which reflects the use of acquiring a functional R338L mutation in the FIX transgene. [Examples]

[0285] LV-coFIX-1-R338L exhibits similar transduction efficiency in adult and neonatal animals. Lentiviral vectors can be integrated into the host genome to mediate long-term transgene expression. Unlike AAV-mediated transgene expression, which is rapidly lost after neonatal treatment, lentiviral-mediated transgene expression is expected to maintain a sustained transgene expression profile not only in adult animals treated with LV-FIX but also in neonatal animals. To investigate the transduction efficiency and transgene expression profile of lentiviral FIX after neonatal treatment, 2-day-old HemB pups were treated with LV-coFIX-1-R338L via temporal vein injection at doses of 7.5E9, 2E10, and 6E10 TU / kg. Compared to adult treatment (administered at 8 weeks), systemically administered LV-coFIX-1-R338L mediated sustained and similar levels of FIX expression over a 6-month study period at each dose level, suggesting that lentiviral FIX can efficiently treat both adult and pediatric patients. Treatment of adolescent mice with LV-coFIX-1-R338L via temporal vein injection at doses of 3E9, 7.5E9, or 2E10 TU / kg (administered over 2 weeks) We also investigated this. The level of FIX expression at each dose level was higher than the corresponding dose in mice treated at 8 weeks or 2 days (n=6 animals / dose level / days (weeks) age; Figure 4A). FIX activity was measured and the dose-response of LV-coFIX-1-R338L determined in HemB mice administered via temporal vein injection at doses of 7.5E9, 2E10, and 6E10 TU / kg at 8 weeks and 2 days, and at doses of 3E9, 7.5E9, or 2E10 TU / kg at 2 weeks. Consistent with the long-term data in Figure 4A, mice treated at 2 weeks (adolescent mice) showed higher FIX activity compared to mice treated at 2 days or 8 weeks (Figure 4B). [Examples]

[0286] Evaluation of CD47high LV-coFIX-1-R338L in non-human primates To modulate the immunological properties of lentiviral vectors, we created a HEK293T cell line overexpressing human CD47. Lentiviral vector particles with high surface levels of human CD47 showed less Kupffer cell uptake and more hepatocyte transduction in NOD mice (NOD mice can recognize human CD47). In addition, compared to a control lentiviral vector that did not overexpress CD47, only lentiviral vector particles with lower surface human CD47 expression were taken up by macrophages (Figure 5).

[0287] To further evaluate the effect of high-surface levels of human CD47 on transduction to the liver in vivo, in non-human primates (NHPs) after intravenous administration of CD47 at a dose of 7.5E9TU / kg, n=3 / treatment group, high LV-coFIX-1-R338L was compared to LV-coFIX-1-R338L. Pig-tailed monkeys were used to circumvent the limitations of lentiviral vectors in post-treatment NHP.

[0288] Circulating human FIX levels after treatment with lentiviral vectors were measured by human FIX-specific activity (Figure 6A) and antigen assay (Figure 6B). CD47 high LV-coFIX-1-R338 conferred three times higher human FIX expression after treatment with a lentiviral vector compared to LV-coFIX-1-R338L, representing 200-300% and 50-150% of normal FIX activity levels, respectively (Figure 6A). CD47 high The use of LV-coFIX-1-R338L may reduce LV-FIX and potentially decrease acute toxicity associated with lentiviral vector treatment.

[0289] In addition to human FIX expression levels, homeostasis in treated animals was also monitored by APTT assay (Figure 6C). While APTT times remained within the same range in vehicle-treated animals, significantly shorter APTT times were observed in all LV-FIX-treated animals (Figure 6C), indicating that the human FIX protein obtained by lentiviral vector treatment is functionally active. [Examples]

[0290] Dose response of CD47high LV-coFIX-1-R338L in non-human primates CD47 in NHP high To determine the dose-response profile of LV-coFIX-1-R338L, two low doses of CD47 were administered: 1.5E9 and 3E9 TU / kg (n=3 / dose level). high LV-coFIX-1-R338L was tested. Human FIX expression mediated by the lentiviral vector was monitored by analyzing the FIX-specific activity (Figure 7A) and antigen levels (Figure 7B) of humans circulating under steady state.

[0291] Consistent with the results observed in HemB mice, a log / log dose-response curve was also observed with NHP's LV-coFIX-1-R338L. CD47 required to achieve 0-100% high The dose range for LV-coFIX-1-R338L is lower than that in HemB mice, at 3.5–6E9TU / kg. This shift in the therapeutic dose range is due to 5–10 times higher human FIX expression levels in NHP compared to HemB mice at the same dose level, likely due to species differences and recognition of human CD47 (human CD47 is not recognized in HemB mice).

[0292] Animals administered LV-coFIX-1-R338L showed a very mild acute immune response, as indicated by post-administration ALT levels (Figure 8A), AST levels (Figure 8B), lymphocyte levels (Figure 8C), and body temperature (Figure 8D). CD47 high A decrease in the cytokine response was observed after administration of LV-coFIX-1-R338L compared to the LV control vector (Figures 9A-9C). While a moderate increase in MIP-1a, MIP-1b, and MCP-1 was observed after administration of control LV, MIP-1a, MIP-1b, and MCP-1, CD47 high Following administration of LV-coFIX-1-R338L, expression decreased and was undetectable in some cases. As expected, LV-coFIX-1-R338L was primarily localized in the liver and spleen, where it had more than 100 times the vector copy number (VCN) compared to other organs (Figure 10). These data were used to support CD47 high This suggests that LV-coFIX-1-R338L induces a decrease in allospecific immune responses, increases resistance to phagocytosis, and improves gene transfer to hepatocytes. [Examples]

[0293] Additional studies on the dose-response relationship of CD47high LV-coFIX-1-R338L in non-human primates. CD47 at a dose of 2.5E9TU / kg high Further porcine-tailed monkeys were treated with LV-coFIX-1-R338L via intravenous administration. Lentiviral vector-mediated human FIX expression was monitored by analyzing steady-state circulating human FIX-specific activity (Figure 11A) and antigen levels (Figure 11B). After LV treatment, steady-state circulating human FIX activity was approximately 33% of normal, and the circulating human FIX antigen level was 700 ng / mL, which correlated with 14% of normal FIX antigen levels.

[0294] The foregoing descriptions of specific embodiments sufficiently illustrate the general nature of the disclosure so that others can easily modify such specific embodiments and / or apply various applications thereto by applying their knowledge of the art without excessive experimentation and without departing from the general concepts of the disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and equivalents of the embodiments of the disclosure, based on the teachings and guidance presented herein. The expressions and terminology used herein are for illustrative purposes only, not limiting purposes, and as a result, it should be understood that the terms and expressions used herein should be interpreted by those skilled in the art in consideration of the teachings and guidance.

[0295] Other embodiments of this disclosure will be apparent to those skilled in the art through consideration of this specification and the practices of this disclosure disclosed herein. This specification and the examples are for illustrative purposes only, and the true scope and spirit of this disclosure are intended to be shown by the following claims.

[0296] All patents and publications cited herein are incorporated herein in their entirety by reference.

Claims

1. A method for preventing or treating hemophilia in a subject requiring such treatment, comprising administering to the subject an effective dose of a lentiviral vector comprising a nucleotide sequence encoding a polypeptide having factor IX (FIX) activity, wherein the lentiviral vector is packaged in HEK293T cells that overexpress CD47, including higher levels of surface CD47 protein expression than a control lentiviral vector produced in unmodified HEK293T cells (ATCC® CRL-11268™), and the effective dose is reduced compared to a control dose of the control lentiviral vector required to induce the same FIX activity as the lentiviral vector.

2. The control lentiviral vector has a 1 μm layer on its surface. ２ The method according to claim 1, comprising 19 molecules of CD47 per unit.

3. The method according to claim 1 or 2, wherein the lentiviral vector contains at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 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, and at least about 40 times more CD47 protein on the surface of the lentiviral vector than a control lentiviral vector produced in HEK293 T cells (ATCC® CRL-11268®).

4. The effective dose is less than about 5 × 10 １０ transducing units / kg (TU / kg), less than 4 × 10 １０ TU / kg, less than 3 × 10 １０ TU / kg, less than 2 × 10 １０ TU / kg, less than 1 × 10 １０ TU / kg, less than 9 × 10 ９ TU / kg, less than 8 × 10 ９ TU / kg, less than 7 × 10 ９ TU / kg, less than 6 × 10 ９ TU / kg, less than 5 × 10 ９ TU / kg, less than 4 × 10 ９ TU / kg, less than 3 × 10 ９ TU / kg, less than 2 × 10 ９ TU / kg, less than 1 × 10 ９ TU / kg, less than about 9 × 10 ８ TU / kg, or less than about 8 × 10 ８ TU / kg, the method according to any one of claims 1 to 3.

5. The target group is those exhibiting the following characteristics after administration: (a) Reduced transduction of lentiviral vectors into macrophages compared to control lentiviral vectors; (b) Reduced allo-specific immune response to lentiviral vectors compared to control lentiviral vectors; (c) FIX activity of at least 30% compared to normal FIX activity at least 3 weeks after administration; (d) Tissue-specific expression of lentiviral vectors in the liver, spleen, or both the liver and spleen; and (e) Any combination of (a) to (d) The method according to any one of claims 1 to 4, wherein one or more of the following are represented.

6. The method according to claim 5, wherein the allospecific immune response includes the release of cytokines in response to a lentiviral vector.

7. Cytokines include MIP-1a, MIP-1b, MCP-1, and any combination thereof. The method according to claim 6, selected from the group consisting of the following.

8. The method according to any one of claims 1 to 7, which shows a decrease in the expression level of MIP-1a after administration of a lentiviral vector compared to the expression level of MIP-1a after administration of a control lentiviral vector.

9. The method according to any one of claims 1 to 8, which shows a decrease in the expression level of MIP-1b after administration of a lentiviral vector compared to the expression level of MIP-1b after administration of a control lentiviral vector.

10. The subject is the method according to any one of claims 1 to 9, which shows a decrease in the expression level of MIP-1 after administration of a lentiviral vector compared to the expression of MIP-1 after administration of a control lentiviral vector.

11. The method according to any one of claims 1 to 10, wherein the subject exhibits FIX activity of at least about 75%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% compared to normal FIX activity at least three weeks after administration of a lentiviral vector.

12. The subject is the method according to any one of claims 1 to 11, which exhibits at least about 150% of the FIX activity compared to normal FIX activity at least three weeks after administration of a lentiviral vector.

13. The method according to any one of claims 1 to 12, wherein plasma FIX activity 24 to 48 hours after administration of a lentiviral vector is increased compared to subjects who were administered a control dose of a control lentiviral vector.

14. The method according to claim 13, wherein the plasma FIX activity increases after administration by at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, 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, or at least about 200 times compared to a subject who received a control dose of a control lentiviral vector.

15. The method according to any one of claims 1 to 14, wherein the subject exhibits increased localization of the lentiviral vector to the liver, spleen, or both the liver and spleen, compared to other organs of the subject, after administration of the lentiviral vector.

16. The increased localization is 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, and at least about 3 times, in the liver, spleen, or both of the liver and spleen, compared to organs other than the target liver and spleen, after administration of the lentiviral vector. The method according to claim 15, characterized by a vector copy number (VCN) of the lentivirus vector that is 5 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 greater.

17. The method according to claim 15 or 16, wherein the increased localization is characterized by at least 10 times more VCNs of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of interest.

18. The method according to claim 15 or 16, wherein the increased localization is characterized by at least 50 times more VCNs of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of interest.

19. The method according to any one of claims 15 to 18, wherein the increased localization is characterized by at least 100 times more VCNs of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration of the lentiviral vector compared to organs other than the liver and spleen of the subject.

20. The method according to any one of claims 1 to 19, wherein CD47 is human CD47.

21. The method according to claim 20, wherein human CD47 comprises an amino acid sequence that is at least 60%, at least about 70%, at least 70%, at least about 80%, at least 85%, at least about 90%, at least 95%, at least about 96%, at least 97%, at least about 98%, at least 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO:

14.

22. The method according to any one of claims 1 to 21, wherein the lentiviral vector does not contain an MHC-I polypeptide.

23. The method according to any one of claims 1 to 22, wherein the lentiviral vector is produced in host cells that express a higher concentration of CD47 compared to HEK293 T cells (ATCC® CRL-11268®).

24. The method according to any one of claims 1 to 23, wherein the nucleotide sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7.

25. A method for preventing or treating hemophilia in a person in need thereof, 5 x 10 １０ The method involves administering to a subject a lentiviral vector containing a nucleotide sequence encoding a polypeptide having factor IX (FIX) activity at a transduction unit / kg (TU / kg) level of less than 70%, at least about 75%, at least about 80%, and at least about 8% of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7. The method comprising a nucleotide sequence having sequence identity of 5%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

26. The method according to any one of claims 1 to 25, wherein the nucleotide sequence has at least 85% sequence identity with respect to the nucleotide sequence shown in Sequence ID No.

1.

27. The method according to any one of claims 1 to 26, wherein the nucleotide sequence has at least 85% sequence identity with nucleotides 139 to 1386 of the nucleotide sequence shown in Sequence ID No.

2.

28. The method according to any one of claims 1 to 27, wherein the nucleotide sequence has at least 85% sequence identity with nucleotides 139 to 1386 of the nucleotide sequence shown in Sequence ID No.

3.

29. The method according to any one of claims 1 to 28, wherein the nucleotide sequence has at least 85% sequence identity with nucleotides 139 to 1386 of the nucleotide sequence shown in Sequence ID No.

4.

30. The method according to any one of claims 1 to 29, wherein the nucleotide sequence has at least 85% sequence identity with nucleotides 139 to 1386 of the nucleotide sequence shown in Sequence ID No.

5.

31. The method according to any one of claims 1 to 30, wherein the nucleotide sequence has at least 85% sequence identity with nucleotides 139 to 1386 of the nucleotide sequence shown in Sequence ID No.

6.

32. The method according to any one of claims 1 to 31, wherein the nucleotide sequence has at least 85% sequence identity with nucleotides 139 to 1386 of the nucleotide sequence shown in Sequence ID No.

7.

33. The dosage is about 5×10 １０ TU / kg, about 4.5×10 １０ TU / kg, about 4×10 １０ TU / kg, about 3.5×10 １０ TU / kg, about 3×10 １０ TU / kg, about 2.5×10 １０ TU / kg, about 2×10 １０ TU / kg, about 1.5×10 １０ TU / kg, about 1×10 １０ TU / kg, about 9.5×10 ９ TU / kg, about 9×10 ９ TU / kg, about 8.5×10 ９ TU / kg, about 8×10 ９ TU / kg, about 7.5×10 ９ [[ID=2,8]]TU / kg, about 7×10 ９ TU / kg, about 6.5×10 ９ TU / kg, about 6×10 ９ TU / kg, about 5.5×10 ９ TU / kg, about 5×10 ９ TU / kg, about 4.5×10 ９ TU / kg, about 4×10 ９ TU / kg, about 3.5×10 ９ TU / kg, about 3×10 ９ TU / kg, about 2.5×10 ９ TU / kg, about 2×10 ９ TU / kg, about 1.5×10 ９ TU / kg, about 1×10 ９ TU / kg, about 9.5×10 ８ [[ID=5,6]]TU / kg, about 9×10 ８ TU / kg, about 8.5×10 ８ TU / kg, about 8×10 ８ TU / kg, about 7.5×10 ８ TU / kg, about 7×10 ８ TU / kg, about 6.5×10 ８ TU / kg, about 6×10 ８ TU / kg, about 5.5×10 ８ TU / kg, about 5×10 ８ TU / kg, about 4.5×10 ８ TU / kg, about 4×10 ８ TU / kg, about 3.5×10 ８ TU / kg, approximately 3×10 ８ TU / kg, approximately 2.5×10 ８ TU / kg, approximately 2×10 ８ TU / kg, approximately 1.5×10 ８ TU / kg or approximately 1 × 10⁻⁶ ８ The method according to any one of claims 1 to 32, wherein the value is TU / kg.

34. The dosage is 5 x 10 １０ Less than TU / kg, 4.5 x 10 １０ Less than TU / kg, 4 x 10 １０ Less than TU / kg, 3.5 × 10 １０ Less than TU / kg, 3 x 10 １０ Less than TU / kg, 2 less than 0.5×10 １０ TU / kg, less than 2×10 １０ TU / kg, less than 1.5×10 １０ TU / kg, less than 1×10 １０ TU / kg, less than 9.5×10 ９ TU / kg, less than 9×10 ９ TU / kg, less than 8.5×10 ９ TU / kg, less than 8×10 ９ TU / kg, less than 7.5×10 ９ TU / kg, less than 7×10 ９ TU / kg, less than 6.5×10 ９ TU / kg, less than 6×10 ９ TU / kg, less than 5.5×10 ９ TU / kg, less than 5×10 ９ TU / kg, less than 4.5×10 ９ TU / kg, less than 4×10 ９ TU / kg, less than 3.5×10 ９ TU / kg, less than 3×10 ９ TU / kg, less than 2.5×10 ９ TU / kg, less than 2×10 ９ TU / kg, less than 1.5×10 ９ TU / kg, less than 1×10 ９ TU / kg, less than about 9.5×10 ８ TU / kg, less than about 9×10 ８ TU / kg, less than about 8.5×10 ８ TU / kg, less than about 8×10 ８ TU / kg, less than about 7.5×10 ８ TU / kg, less than about 7×10 ８ TU / kg, less than about 6.5×10 ８ TU / kg, less than about 6×10 ８ TU / kg, less than about 5.5×10 ８ TU / kg, less than about 5×10 ８ TU / kg, less than about 4.5×10 ８ TU / kg, less than about 4×10 ８ TU / kg, less than about 3.5×10 ８ TU / kg, less than about 3×10 ８ TU / kg, less than about 2.5×10 ８ TU / kg, less than about 2×10 ８ Less than TU / kg, approximately 1.5 × 10⁻⁶ ８ Less than TU / kg, or approximately 1 × 10⁻⁶ ８ The method according to any one of claims 1 to 32, wherein the TU / kg is less than TU.

35. The dosage is 1×10 ８ to 5×10 １０ TU / kg, 1×10 ８ to 5×10 ９ TU / kg, 1×10 ８ to 1×10 ９ TU / kg, 1×10 ８ to 1×10 １０ TU / kg, 1×10 ９ to 5×10 １０ TU / kg, 2×10 ９ to 5×10 １０ TU / kg, 3×10 ９ to 5×10 １０ TU / kg, 4×10 ９ to 5×10 １０ TU / kg, 5×10 ９ to 5×10 １０ TU / kg, 1×10 ９ to 6×10 ９ TU / kg, 2×10 ９ to 6×10 ９ TU / kg, 3×10 ９ to 6×10 ９ TU / kg, 4×10 ９ to 6×10 ９ TU / kg, 5×10 ９ to 6×10 ９ TU / kg, 6×10 ９ to 5×10 １０ TU / kg, 7×10 ９ to 5×10 １０ TU / kg, 8×10 ９ to 5×10 １０ TU / kg, 9×10 ９ to 5×10 １０ TU / kg, 10 １０ to 5×10 １０ TU / kg, 1.5×10 １０ to 5×10 １０ TU / kg, 2×10 １０ to 5×10 １０ TU / kg, 2.5×10 １０ to 5×10 １０ TU / kg, 3×10 １０ to 5×10 １０ TU / kg, 3.5×10 １０ to 5×10 １０ TU / kg, 4×10 １０ ~5 x 10 １０ TU / kg, or 4.5 × 10 １０ ~5 x 10 １０ The method according to any one of claims 1 to 32, wherein the value is TU / kg.

36. The dosage is 1 x 10 ９ ~5 x 10 １０ TU / kg, 1 × 10 ９ ~4.5 x 10 １０ TU / kg, 1 × 10 ９ ~4 x 10 １０ TU / kg, 1 × 10 ９ ~3.5 x 10 １０ TU / kg, 1 × 10 ９ ~3 x 10 １０ TU / kg, 1 × 10 ９ ~2.5 x 10 １０ TU / kg, 1 × 10 ９ ~2 x 10 １０ TU / kg, 1 × 10 ９ ~1.5 x 10 １０ TU / kg, 1 × 10 ９ ~1 x 10 １０ TU / kg, 1 × 10 ９ ~9 x 10 ９ TU / kg, 1 × 10 ９ ~8 x 10 ９ TU / kg, 1 × 10 ９ ~7 x 10 ９ TU / kg, 1 × 10 ９ ~6 x 10 ９ TU / kg, 1 × 10 ９ ~5 x 10 ９ TU / kg, 1 × 10 ９ ~4 x 10 ９ TU / kg, 1 × 10 ９ ~3 x 10 ９ TU / kg, and 1 × 10⁻⁶ ９ ~2 x 10 ９ The method according to any one of claims 1 to 32, wherein the value is TU / kg.

37. The dosage is 1 x 10 １０ ~2 x 10 １０ TU / kg, 1.1×10 １０ ~1.9 x 10 １０ TU / kg, 1.2×10 １０ ~1.8 x 10 １０ TU / kg, 1.3×10 １０ ~1.7 x 10 １０ TU / kg, or 1.4 × 10⁻⁶ １０ ~1.6 x 10 １０ The method according to any one of claims 1 to 32, wherein the value is TU / kg.

38. The dosage is approximately 4 x 10 ９ TU / kg ~ approx. 6×10 ９ The method according to any one of claims 1 to 37, wherein the value is TU / kg.

39. The lentiviral vector is administered in a single dose or multiple doses, according to claims 1 to 38. The method described in any one of the items.

40. The method according to any one of claims 1 to 39, wherein the lentiviral vector is administered by intravenous injection.

41. The method according to any one of claims 1 to 40, wherein the target is children.

42. The method according to any one of claims 1 to 40, wherein the target is an adult.

43. The method according to any one of claims 1 to 40, the target is young people.

44. The method according to any one of claims 1 to 43, wherein the polypeptide having FIX activity comprises an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:

12.

45. The method according to any one of claims 1 to 44, wherein the polypeptide having FIX activity comprises the amino acid sequence shown in SEQ ID NO:

12.

46. The method according to any one of claims 1 to 45, wherein the lentiviral vector comprises a tissue-specific promoter.

47. The method according to claim 46, wherein the tissue-specific promoter selectively enhances the expression of a polypeptide having FIX activity in target liver cells.

48. The method according to claim 47, wherein a tissue-specific promoter for selectively enhancing the expression of a polypeptide having FIX activity in target liver cells includes the APOA2 promoter, the SERPINA1 (hAAT) promoter, the mTTR promoter, the MIR122 promoter, or any combination thereof.

49. The method according to claim 47 or 48, wherein the target liver cells are hepatocytes.

50. The method according to claim 49, wherein the isolated nucleic acid molecule is stably incorporated into the genome of liver cells.

51. The lentiviral vector comprising a splice donor site, according to any one of claims 1 to 50.

52. The lentiviral vector comprising a splice acceptor site, according to any one of claims 1 to 51.

53. The method according to any one of claims 1 to 52, wherein the lentiviral vector comprises a gag sequence, a pol sequence, a rev sequence, a rev response element (RRE), or any combination thereof.

54. The method according to claim 53, wherein the gag sequence is a full-length or truncated gag sequence.

55. The method according to any one of claims 1 to 54, wherein the lentiviral vector comprises an enhancer, a target sequence for a microRNA, a post-transcriptional regulatory element, a packaging signal, a polyA sequence, an intron sequence, or any combination thereof.

56. The method according to any one of claims 1 to 55, wherein the dose of the lentiviral vector is administered at one time or divided into at least two partial doses.

57. The method according to any one of claims 1 to 55, wherein the dose of the lentiviral vector is repeated at least twice.

58. The method according to any one of claims 1 to 57, wherein the nucleotide sequence encoding a polypeptide having FIX activity further comprises a nucleic acid sequence encoding a signal peptide.

59. The nucleic acid sequence encoding the signal peptide is, (i) Nucleotides 1-84 of Sequence ID No. 2; (ii) Nucleotides 1-84 of Sequence ID No. 3; (iii) Nucleotides 1-84 of Sequence ID No. 4; (iv) Nucleotides 1-84 of Sequence ID No. 5; (v) Nucleotides 1-84 of SEQ ID NO: 6; or (vi) Nucleotides 1-84 of Sequence ID No. 7 The method according to claim 58, wherein the sequence identity is 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%.

60. The method according to any one of claims 1 to 59, wherein the nucleotide sequence encoding a polypeptide having FIX activity further comprises a nucleic acid sequence encoding a propeptide.

61. The nucleic acid sequence encoding the propeptide is, (i) Nucleotides 85-138 of Sequence ID No. 2; (ii) Nucleotides 85-138 of Sequence ID No. 3; (iii) Nucleotides 85-138 of Sequence ID No. 4; (iv) Nucleotides 85-138 of Sequence ID No. 5; (v) Nucleotides 85-138 of SEQ ID NO: 6; or (vi) Nucleotides 85-138 of Sequence ID No. 7 The method according to claim 60, wherein the sequence identity is 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%.

62. The method according to any one of claims 1 to 61, wherein the nucleotide sequence encoding a polypeptide having FIX activity further comprises a heteronucleotide sequence encoding a heterogeneous amino acid sequence.

63. The method according to claim 62, wherein the heterologous amino acid sequence is albumin, immunoglobulin Fc region, XTEN sequence, C-terminal peptide (CTP) of the β-subunit of human chorionic gonadotropin, PAS sequence, HAP sequence, CTP peptide sequence, transferrin, albumin-binding moiety, or any fragment, derivative, variant, or combination of these polypeptides.

64. The method according to claim 62 or 63, wherein the heterologous amino acid sequence is ligated to the N-terminus or C-terminus of an amino acid sequence encoded by a nucleotide sequence encoding a polypeptide having FIX activity, or is inserted between two amino acids of the amino acid sequence.

65. The heterogeneous parts are amino acid 103 of SEQ ID NO: 2, amino acid 105 of SEQ ID NO: 2 The method according to any one of claims 62 to 64, wherein an amino acid corresponding to amino acid 142 of SEQ ID NO: 2, amino acid 149 of SEQ ID NO: 2, amino acid 162 of SEQ ID NO: 2, amino acid 166 of SEQ ID NO: 2, amino acid 174 of SEQ ID NO: 2, amino acid 224 of SEQ ID NO: 2, amino acid 226 of SEQ ID NO: 2, amino acid 228 of SEQ ID NO: 2, amino acid 413 of SEQ ID NO: 2, or any combination thereof, is inserted into a polypeptide having FIX activity immediately downstream.

66. The method according to any one of claims 1 to 65, wherein the FIX polypeptide is the R338L variant FIX polypeptide.

67. The lentiviral vector is produced in a host cell, according to any one of claims 1 to 66.

68. The method according to claim 67, wherein the host cell expresses CD47.

69. The method according to claim 68, wherein the host cell is modified to overexpress CD47.

70. The method according to any one of claims 67 to 69, wherein the host cells do not express MHC-I.

71. The host cell is CD47 ｈｉｇｈ / MHC-I － The method according to any one of claims 67 to 70.

72. The host cell is CD47 ｈｉｇｈ / MHC-I － The method according to any one of claims 69 to 71, wherein the cells are HEK293T cells.

73. (i) a nucleotide sequence containing a tissue-specific promoter, and (ii) a nucleic acid sequence as shown in Sequence ID No. 1, wherein the tissue-specific promoter drives the expression of the nucleic acid sequence in liver cells.

74. (i) Splice donor site; (ii) Splice acceptor site; (iii) gag array; (iv) Rev response element; (v) enhancer; (vi) Nucleotide sequence containing post-transcriptional regulatory elements, (vii) Nucleic acid sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 100% sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and (viiii) A lentiviral vector containing a target sequence for microRNA.

75. The lentiviral vector according to claim 73 or 74, wherein the nucleic acid sequence encodes a polypeptide having FIX activity, comprising an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:

12.

76. The lentiviral vector according to claim 75, wherein the polypeptide having FIX activity comprises the amino acid sequence shown in SEQ ID NO:

12.

77. The lentiviral vector according to any one of claims 74 to 76, wherein the surface of the lentiviral vector contains a higher level of CD47 protein than a control lentiviral vector produced in HEK293 T cells (ATCC® CRL-11268®).

78. The lentiviral vector according to claim 77, wherein the surface of the lentiviral vector does not contain MHC-I.

79. A method for treating hemophilia in a subject requiring such treatment, comprising administering to the subject an effective dose of a lentiviral vector according to any one of claims 73 to 78.

80. The effective dose is approximately 5 x 10 １０ Less than 4 x 10 units / kg (TU / kg), １０ Less than TU / kg, 3 x 10 １０ Less than TU / kg, 2 x 10 １０ Less than TU / kg, 1 x 10 １０ Less than TU / kg, 9 x 10 ９ Less than TU / kg, 8 x 10 ９ Less than TU / kg, 7 x 10 ９ Less than TU / kg, 6 x 10 ９ Less than TU / kg, 5 x 10 ９ Less than TU / kg, 4 x 10 ９ Less than TU / kg, 3 × 10 ９ Less than TU / kg, 2 x 10 ９ Less than TU / kg, 1 x 10 ９ Less than TU / kg, approximately 9 x 10 ８ Less than TU / kg, or approximately 8 x 10⁻⁶ ８ The method according to claim 79, wherein TU / kg is less than TU.

81. The effective dosage is about 5×10 １０ TU / kg, about 4.5×10 １０ TU / kg, about 4×10 １０ TU / kg, about 3.5×10 １０ TU / kg, about 3×10 １０ TU / kg, about 2.5×10 １０ TU / kg, about 2×10 １０ TU / kg, about 1.5×10 １０ TU / kg, about 1×10 １０ TU / kg, about 9.5×10 ９ TU / kg, about 9×10 ９ TU / kg, about 8.5×10 ９ TU / kg, about 8×10 ９ TU / kg, about 7.5×10 ９ TU / kg, about 7×10 ９ TU / kg, about 6.5×10 ９ TU / kg, about 6×10 ９ TU / kg, about 5.5×10 ９ TU / kg, about 5×10 ９ TU / kg, about 4.5×10 ９ TU / kg, about 4×10 ９ TU / kg, about 3.5×10 ９ TU / kg, about 3×10 ９ TU / kg, about 2.5×10 ９ TU / kg, about 2×10 ９ TU / kg, about 1.5×10 ９ TU / kg, about 1×10 ９ TU / kg, about 9.5×10 ８ TU / kg, about 9×10 ８ TU / kg, about 8.5×10 ８ TU / kg, about 8×10 ８ TU / kg, about 7.5×10 ８ TU / kg, about 7×10 ８ TU / kg, about 6.5×10 ８ TU / kg, about 6×10 ８ TU / kg, about 5.5×10 ８ TU / kg, about 5×10 ８ TU / kg, about 4.5×10 ８ TU / kg, about 4×10 ８ TU / kg, about 3.5×10 ８ TU / kg, approximately 3×10 ８ TU / kg, approximately 2.5×10 ８ TU / kg, approximately 2×10 ８ TU / kg, approximately 1.5×10 ８ TU / kg, or approximately 1 × 10⁻⁶ ８ The method according to claim 79 or 80, wherein the value is TU / kg.

82. The effective dose is 5 x 10 １０ Less than TU / kg, 4.5 x 10 １０ Less than TU / kg, 4 x 10 １０ Less than TU / kg, 3.5 × 10 １０ Less than TU / kg, 3 x 10 １０ Less than TU / kg, 2.5 × 10 １０ Less than TU / kg, 2 x 10 １０ Less than TU / kg, 1.5 × 10 １０ Less than TU / kg, 1 x 10 １０ Less than TU / kg, 9.5 x 10 ９ Less than TU / kg, 9 x 10 ９ Less than TU / kg, 8.5 x 10 ９ Less than TU / kg, 8 x 10 ９ Less than TU / kg, 7.5 x 10 ９ Less than TU / kg, 7 x 10 ９ Less than TU / kg, 6.5 x 10 ９ Less than TU / kg, 6 x 10 ９ Less than TU / kg, 5.5 x 10 ９ Less than TU / kg, 5 x 10 ９ Less than TU / kg, 4.5 x 10 ９ Less than TU / kg, 4 x 10 ９ Less than TU / kg, 3.5 × 10 ９ Less than TU / kg, 3 x 10 ９ Less than TU / kg, 2.5 × 10 ９ Less than TU / kg, 2 x 10 ９ Less than TU / kg, 1.5 × 10 ９ Less than TU / kg, 1 x 10 ９ Less than TU / kg, approximately 9.5 × 10⁻⁶ ８ Less than TU / kg, approximately 9 x 10 ８ Less than TU / kg, approximately 8.5 × 10⁻⁶ ８ Less than TU / kg, approximately 8 x 10 ８ Less than TU / kg, approximately 7.5 × 10⁻⁶ ８ Less than TU / kg, approximately 7 x 10 ８ Less than TU / kg, approximately 6.5 × 10⁻⁶ ８ Less than TU / kg, approximately 6 x 10 ８ Less than TU / kg, approximately 5.5 x 10 ８ Less than TU / kg, approximately 5 x 10 ８ Less than TU / kg, approximately 4.5 × 10⁻⁶ ８ Less than TU / kg, approximately 4 x 10 ８ Less than TU / kg, approximately 3.5 × 10⁻⁶ ８ Less than TU / kg, approximately 3 x 10⁻⁶ ８ Less than TU / kg, approximately 2.5 × 10⁻⁶ ８ Less than TU / kg, approximately 2 x 10⁻⁶ ８ Less than TU / kg, approximately 1.5 × 10⁻⁶ ８ Less than TU / kg, or approximately 1 × 10⁻⁶ ８ The method according to claim 79 or 80, wherein the TU / kg is less than TU.

83. The effective dose is 1×10 ８ to 5×10 １０ TU / kg, 1×10 ８ to 5×10 ９ TU / kg, 1×10 ８ to 1×10 ９ TU / kg, 1×10 ８ to 1×10 １０ TU / kg, 1×10 ９ to 5×10 １０ TU / kg, 2×10 ９ to 5×10 １０ TU / kg, 3×10 ９ to 5×10 １０ TU / kg, 4×10 ９ to 5×10 １０ TU / kg, 5×10 ９ to 5×10 １０ TU / kg, 1×10 ９ to 6×10 ９ TU / kg, 2×10 ９ to 6×10 ９ TU / kg, 3×10 ９ to 6×10 ９ TU / kg, 4×10 ９ to 6×10 ９ TU / kg, 5×10 ９ to 6×10 ９ TU / kg, 6×10 ９ to 5×10 １０ TU / kg, 7×10 ９ to 5×10 １０ TU / kg, 8×10 ９ to 5×10 １０ TU / kg, 9×10 ９ to 5×10 １０ TU / kg, 10 １０ to 5×10 １０ TU / kg, 1.5×10 １０ to 5×10 １０ TU / kg, 2×10 １０ to 5×10 １０ TU / kg, 2.5×10 １０ to 5×10 １０ TU / kg, 3×10 １０ to 5×10 １０ TU / kg, 3.5×10 １０ to 5×10 １０ TU / kg, 4×10 １０ ~5 x 10 １０ TU / kg, or 4.5 × 10 １０ ~5 x 10 １０ The method according to claim 79 or 80, wherein the value is TU / kg.

84. The effective dose is 1 x 10 ９ ~5 x 10 １０ TU / kg, 1 × 10 ９ ~4.5 x 10 １０ TU / kg, 1 × 10 ９ ~4 x 10 １０ TU / kg, 1 × 10 ９ ~3.5 x 10 １０ TU / kg, 1 × 10 ９ ~3 x 10 １０ TU / kg, 1 × 10 ９ ~2.5 x 10 １０ TU / kg, 1 × 10 ９ ~2 x 10 １０ TU / kg, 1 × 10 ９ ~1.5 x 10 １０ TU / kg, 1 × 10 ９ ~10 １０ TU / kg, 1 × 10 ９ ~9 x 10 ９ TU / kg, 1 × 10 ９ ~8 x 10 ９ TU / kg, 1 × 10 ９ ~7 x 10 ９ TU / kg, 1 × 10 ９ ~6 x 10 ９ TU / kg, 1 × 10 ９ ~5 x 10 ９ TU / kg, 1 × 10 ９ ~4 x 10 ９ TU / kg, 1 × 10 ９ ~3 x 10 ９ TU / kg, and 1 × 10⁻⁶ ９ ~2 x 10 ９ The method according to claim 79 or 80, wherein the value is TU / kg.

85. The effective dose is 1 x 10 １０ ~2 x 10 １０ TU / kg, 1.1×10 １０ ~1.9 x 10 １０ TU / kg, 1.2×10 １０ ~1.8 x 10 １０ TU / kg, 1.3×10 １０ ~1.7 x 10 １０ TU / kg, or 1.4 × 10⁻⁶ １０ ~1.6 x 10 １０ The method according to claim 79 or 80, wherein the value is TU / kg.

86. The effective dose is approximately 4 x 10 ９ TU / kg ~ approx. 6×10 ９ The method according to any one of claims 79 to 85, wherein the value is TU / kg.

87. The method according to any one of claims 79 to 86, wherein the lentiviral vector is administered in a single dose or multiple doses.

88. The method according to any one of claims 79 to 87, wherein the lentiviral vector is administered by intravenous injection.

89. The method according to any one of claims 79 to 88, the target subject is children.

90. The method according to any one of claims 79 to 88, wherein the target is an adult.

91. A nucleic acid sequence containing a nucleotide sequence as shown in Sequence ID No.

1.

92. A vector comprising the nucleic acid sequence described in claim 91.

93. The vector according to claim 92, comprising a tissue-specific promoter.

94. The vector according to claim 93, wherein the tissue-specific promoter selectively enhances the expression of a polypeptide having FIX activity in target liver cells.

95. The vector according to claim 94, wherein the tissue-specific promoter for selectively enhancing the expression of a polypeptide having FIX activity in target liver cells includes the APOA2 promoter, the SERPINA1 (hAAT) promoter, the mTTR promoter, the MIR122 promoter, or any combination thereof.

96. The vector according to claim 94 or 95, wherein the target liver cells are hepatocytes.

97. A vector according to any one of claims 94 to 96, comprising a splice donor site.

98. A vector according to any one of claims 94 to 97, comprising a splice acceptor region.

99. A vector according to any one of claims 94 to 98, comprising a gag sequence, a pol sequence, a rev sequence, a rev response element (RRE), or any combination thereof.

100. The vector according to claim 99, wherein the gag sequence is a full-length or truncated gag sequence.

101. A vector according to any one of claims 92 to 100, comprising an enhancer, a target sequence for microRNA, a post-transcriptional regulatory element, a packaging signal, a polyA sequence, an intron sequence, or any combination thereof.

102. A cell comprising the nucleic acid sequence described in claim 91 or the vector described in any one of claims 92 to 101.

103. The cell according to claim 102, which is a mammalian cell.

104. The cells according to claim 102, which are CHO cells, HEK293 cells, BHK21 cells, PER. C6® cells, NS0 cells, and CAP cells.

105. The cell according to claim 102 or 103, which is a human cell.

106. A cell expressing the CD47 protein, according to any one of claims 102 to 105.

107. The cell according to claim 106, which is modified to overexpress CD47.

108. Compared to control cells that have not been modified to overexpress CD47 on the cell surface, the amount of CD47 was at least approximately 1.5 times, at least approximately 2.0 times, at least approximately 2.5 times, at least approximately 3.0 times, at least approximately 3.5 times, at least approximately 4.0 times, at least approximately 4.5 times, at least approximately 5.0 times, at least approximately 5.5 times, at least approximately 6.0 times, at least approximately 6.5 times, at least approximately 7.0 times, at least approximately 7.5 times, at least approximately 8.0 times, at least approximately 8.5 times, at least approximately 9.0 times, at least approximately 9.5 times, at least approximately 10 times, at least approximately 11 times, at least approximately 12 times, at least approximately 13 times, at least approximately 14 times, and less The cell according to claim 107, which contains approximately 15 times, at least approximately 20 times, at least approximately 25 times, at least approximately 30 times, at least approximately 35 times, and at least approximately 40 times more CD47 protein.

109. The cell according to any one of claims 106 to 108, wherein CD47 is human CD47.

110. A cell according to any one of claims 102 to 109, which does not express MHC-I.

111. A method for producing a lentiviral vector, comprising culturing the cells described in any one of claims 102 to 110 under preferred conditions.

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