Use of lentiviral vectors expressing factor IX

Abstract

The present disclosure provides a lentiviral vector comprising a nucleic acid sequence encoding a polypeptide having factor IX (FIX) activity, and a method for using such a lentiviral vector. The liver-targeted lentiviral vector disclosed herein can be used for gene therapy, where lentiviral gene delivery allows stable integration of a transgene expression cassette into the genome of targeted cells (e.g., hepatocytes) of children (e.g., newborns) or adult subjects, achieving improved FIX expression at a low lentiviral vector dose. The present disclosure also provides a method for treating bleeding disorders, such as hemophilia (e.g., hemophilia B), comprising administering a liver-targeted lentiviral vector comprising a nucleic acid sequence encoding a polypeptide having a FIX activity sequence at a low dosage to a subject in need thereof.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 776,393, filed December 6, 2018, the entire contents of which are incorporated herein by reference.

[0002] Reference to an Electronically Submitted Sequence Listing The contents of the Sequence Listing in the electronically submitted ASCII text file (Name: SA9-468TW_SequenceListing_ST25; Size: 28,470 bytes; Created: December 2, 2019) are hereby incorporated by reference in their entirety. [Background technology]

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

[0004] In hemophilia, blood clotting is disturbed by the lack of certain plasma blood clotting factors. Hemophilia B (also known as Christmas disease) is one of the most common inherited bleeding disorders worldwide. Hemophilia B is caused by a deficiency of factor IX due to either a reduced synthesis of the factor IX protein or a defective molecule with reduced activity. Hemophilia B causes reduced blood clotting activity in vivo and in vitro and requires extensive medical monitoring throughout the life of the affected individual. Without effective prophylaxis, recurrent hemarthrosis leads to the development of progressively disabling arthropathy and reduced quality of life (Non-Patent Document 1).

[0005] Hemophilia B is treated by replacing the missing coagulation factor with exogenous factor concentrates that are highly enriched in factor IX. However, producing such concentrates from blood is technically difficult. Therefore, there is a need in the art for a FIX therapy that overcomes the difficulties and limitations of current replacement therapies. Gene therapy represents an effective approach to the sustained treatment of hemophilia B by the stable integration of a transgene expression cassette that contains a nucleic acid sequence that codes for a polypeptide with FIX activity into the genome of targeted cells. [Prior art documents] [Non-patent literature]

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

[0007] The present disclosure provides a method of preventing or treating hemophilia in a subject in need thereof, 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 into HEK293T cells that overexpress CD47, comprising a higher level of surface CD47 protein expression than a control lentiviral vector generated 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.

[0008] In some embodiments, the control lentiviral vector is 1 μm thick on the surface of the control lentiviral vector. 2Each cell contains 19 molecules of CD47. In some embodiments, the lentiviral vector comprises at least about 1.5 fold, at least about 2.0 fold, at least about 2.5 fold, at least about 3.0 fold, at least about 3.5 fold, at least about 4.0 fold, at least about 4.5 fold, at least about 5.0 fold, at least about 5.5 fold, at least about 6.0 fold, at least about 6.5 fold, at least about 7.0 fold, at least about 7.5 fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold more CD47 protein on the surface of the lentiviral vector than a control lentiviral vector produced in HEK293T cells (ATCC® CRL-11268™).

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

[0010] In some embodiments, the subject exhibits one or more of the following characteristics following administration: (a) reduced transduction of macrophages by the lentiviral vector 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% 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) any combination of (a)-(d).

[0011] In some embodiments, the allospecific immune response comprises 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, the subject exhibits a reduced 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, the subject exhibits a reduced 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, the subject exhibits a reduced 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, the subject exhibits 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% FIX activity compared to normal FIX activity at least 3 weeks after administration of the lentiviral vector. In some embodiments, the subject exhibits at least about 150% FIX activity compared to normal FIX activity at least 3 weeks after administration of the lentiviral vector. In some embodiments, plasma FIX activity is increased 24 hours to 48 hours after administration of the lentiviral vector compared to subjects administered a control dose of a control lentiviral vector. In some embodiments, plasma FIX activity is increased following administration by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, or at least about 200-fold, as compared to a subject administered a control dose of a control lentiviral vector.

[0013] In some embodiments, the subject exhibits 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-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about The vector copy number (VCN) of the lentiviral vector is characterized by 30 times, at least about 35 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, at least about 180 times, at least about 190 times, or at least about 200 times greater. In some embodiments, the increased localization is characterized by at least 10 times greater VCN 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 some embodiments, the increased localization is characterized by at least 50 times more VCN of the lentiviral vector in the liver, spleen, or both liver and spleen after administration of the lentiviral vector, compared to the organs other than the liver and spleen of the subject.In some embodiments, the increased localization is characterized by at least 100 times more VCN of the lentiviral vector in the liver, spleen, or both liver and spleen after administration of the lentiviral vector, compared to the organs other than the liver and spleen of the subject.

[0014] In some embodiments, the CD47 is human CD47. In some embodiments, the 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 set forth in SEQ ID NO: 14. In some embodiments, the lentiviral vector does not comprise an MHC-I polypeptide. In some embodiments, the lentiviral vector is generated in a host cell that expresses a high concentration of CD47 compared to HEK293T cells (ATCC® CRL-11268™).

[0015] 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 to the nucleotide sequence set forth 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.

[0016] The present disclosure provides a method of preventing or treating hemophilia in a subject in need thereof, comprising administering to a subject a dose of 5×10 10Also provided are methods comprising administering to a subject a lentiviral vector comprising a nucleotide sequence encoding a polypeptide having factor IX (FIX) activity at less than transducing units / kg (TU / kg), wherein the lentiviral vector comprises 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 to the nucleotide sequence set forth 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 to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7.

[0017] In some embodiments, the 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 10TU / 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, about 3×10 8 TU / kg, about 2.5×10 8 TU / kg, about 2×10 8 TU / kg, about 1.5×10 8 TU / kg, or about 1×10 8In some embodiments, the dose is 5×10 10 Less than TU / kg, 4.5 x 10 10 Less than TU / kg, 4 x 10 10 Less than TU / kg, 3.5 x 10 10 Less than TU / kg, 3 x 10 10 Less than 2.5 x 10 TU / kg 10 Less than 2 x 10 TU / kg 10 Less than 1.5 x 10 TU / kg 10 Less than 1×10 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 8 x 10 TU / kg 9 TU / kg, less than 7.5 x 10 9 Less than 7 x 10 TU / kg 9 Less than 6.5 x 10 TU / kg 9 Less than 6 x 10 TU / kg 9 Less than TU / kg, 5.5 x 10 9 Less than 5 x 10 TU / kg 9 Less than TU / kg, 4.5 x 10 9 Less than TU / kg, 4 x 10 9 Less than TU / kg, 3.5 x 10 9 Less than TU / kg, 3 x 10 9 Less than 2.5 x 10 TU / kg 9 Less than 2 x 10 TU / kg 9 Less than 1.5 x 10 TU / kg 9 Less than 1×10 9 Less than TU / kg, approximately 9.5 x 10 8 Less than TU / kg, approximately 9 x 10 8 Less than TU / kg, approximately 8.5 x 10 8 Less than TU / kg, approximately 8 x 10 8 Less than TU / kg, approximately 7.5 x 10 8 Less than TU / kg, approximately 7 × 10 8 Less than TU / kg, approximately 6.5 x 10 8 Less than TU / kg, approximately 6 x 10 8 Less than TU / kg, approximately 5.5 x 10 8 Less than TU / kg, approximately 5 × 10 8 Less than TU / kg, approximately 4.5 x 108 Less than TU / kg, approximately 4 x 10 8 Less than TU / kg, approximately 3.5 x 10 8 Less than TU / kg, approximately 3 × 10 8 Less than TU / kg, approximately 2.5 x 10 8 Less than TU / kg, approximately 2 x 10 8 Less than TU / kg, approximately 1.5 x 10 8 Less than TU / kg, or about 1 × 10 8 In some embodiments, the dose is less than 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 x 10 9 ~5×10 10 TU / kg, 3 x 10 9 ~5×10 10 TU / kg, 4 x 10 9 ~5×10 10 TU / kg, 5 x 10 9 ~5×10 10 TU / kg, 1×10 9 ~6×10 9 TU / kg, 2 x 10 9 ~6×10 9 TU / kg, 3 x 10 9 ~6×10 9 TU / kg, 4 x 10 9 ~6×10 9 TU / kg, 5 x 10 9 ~6×10 9 TU / kg, 6 x 10 9 ~5×10 10 TU / kg, 7 x 10 9 ~5×10 10 TU / kg, 8 x 10 9 ~5×10 10 TU / kg, 9 x 10 9 ~5×10 10 TU / kg, 10 10 ~5×10 10TU / 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 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 In some embodiments, the dose is 1×10 10 ~2×10 10 TU / kg, 1.1 x 10 10 ~1.9×10 10 TU / kg, 1.2 x 10 10 ~1.8×10 10 TU / kg, 1.3 x 10 10 ~1.7×10 10 TU / kg, or 1.4 x 10 10 ~1.6×10 10 In some embodiments, the dose is about 4×10 9 TU / kg ~ approx. 6 x 10 9 It is TU / kg.

[0018] In some embodiments, the lentiviral vector is administered in a single dose or multiple doses. In some embodiments, the lentiviral vector is administered via intravenous injection. In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is an adult subject.

[0019] In some embodiments, a polypeptide having FIX activity comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, a polypeptide having FIX activity comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0020] In some embodiments, the lentiviral vector comprises a tissue-specific promoter. In some embodiments, the tissue-specific promoter selectively enhances the expression of the polypeptide having FIX activity in target liver cells. In some embodiments, the tissue-specific promoter selectively enhances the expression of the polypeptide having FIX activity in target liver cells comprises APOA2 promoter, SERPINA1 (hAAT) promoter, mTTR promoter, MIR122 promoter, or any combination thereof. In some embodiments, the target liver cell is a hepatocyte. In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of hepatocyte.

[0021] In some embodiments, the lentiviral vector comprises a splice donor site. In some embodiments, the lentiviral vector comprises a splice acceptor site. In some embodiments, the lentiviral vector comprises 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 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.

[0022] In some embodiments, the dose of the lentiviral vector is administered once or divided into at least two partial doses. In some embodiments, the dose of the lentiviral vector 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 to (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 to (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 heterologous nucleotide sequence encoding a heterologous amino acid sequence. In some embodiments, 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 portion, or any fragment, derivative, variant, or combination of these polypeptides. In some embodiments, the heterologous amino acid sequence is linked to the N-terminus or C-terminus of the amino acid sequence encoded by the nucleotide sequence encoding the polypeptide having FIX activity, or is inserted between two amino acids of the amino acid sequence. In some embodiments, the heterologous moiety is inserted into the polypeptide having FIX activity immediately downstream of an amino acid corresponding to 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 combination thereof. In some embodiments, the FIX polypeptide is a R338L variant FIX polypeptide.

[0025] In some embodiments, the lentiviral vector is produced in a 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 does not express CD47. high / MHC-I - In some embodiments, the host cell is CD47 high / MHC-I - HEK293T cells.

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

[0027] The present disclosure also provides a lentiviral vector comprising: (i) 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 set forth 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 a microRNA.

[0028] In some embodiments, the nucleic acid sequence encodes a polypeptide having FIX activity comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the polypeptide having FIX activity comprises the amino acid sequence set forth in SEQ ID NO: 12.

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

[0030] The present disclosure also relates to a method of treating hemophilia in a subject in need thereof, comprising administering to the subject an effective dose of a lentiviral vector disclosed herein. In some embodiments, the effective dose is about 5×1010 Transducing units / kg (TU / kg) less than 4 x 10 10 Less than TU / kg, 3 x 10 10 Less than 2 x 10 TU / kg 10 Less than 1×10 10 Less than TU / kg, 9 x 10 9 Less than 8 x 10 TU / kg 9 Less than 7 x 10 TU / kg 9 Less than 6 x 10 TU / kg 9 Less than 5 x 10 TU / kg 9 Less than TU / kg, 4 x 10 9 Less than TU / kg, 3 x 10 9 Less than 2 x 10 TU / kg 9 Less than 1×10 9 Less than TU / kg, approximately 9 x 10 8 Less than TU / kg, or about 8 × 10 8 In some embodiments, the effective dose is less than 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 TU / 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 10 9TU / 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 8 In some embodiments, the effective dose is 5×10 10 Less than TU / kg, 4.5 x 10 10 Less than TU / kg, 4 x 10 10 Less than TU / kg, 3.5 x 10 10 Less than TU / kg, 3 x 10 10 Less than 2.5 x 10 TU / kg 10 Less than 2 x 10 TU / kg 10 Less than 1.5 x 10 TU / kg 10 Less than 1×10 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 8 x 10 TU / kg 9 TU / kg, less than 7.5 x 10 9 Less than 7 x 10 TU / kg 9 Less than 6.5 x 10 TU / kg 9 Less than 6 x 10 TU / kg9 Less than TU / kg, 5.5 x 10 9 Less than 5 x 10 TU / kg 9 Less than TU / kg, 4.5 x 10 9 Less than TU / kg, 4 x 10 9 Less than TU / kg, 3.5 x 10 9 Less than TU / kg, 3 x 10 9 Less than 2.5 x 10 TU / kg 9 Less than 2 x 10 TU / kg 9 Less than 1.5 x 10 TU / kg 9 Less than 1×10 9 Less than TU / kg, approximately 9.5 x 10 8 Less than TU / kg, approximately 9 x 10 8 Less than TU / kg, approximately 8.5 x 10 8 Less than TU / kg, approximately 8 x 10 8 Less than TU / kg, approximately 7.5 x 10 8 Less than TU / kg, approximately 7 × 10 8 Less than TU / kg, approximately 6.5 x 10 8 Less than TU / kg, approximately 6 x 10 8 Less than TU / kg, approximately 5.5 x 10 8 Less than TU / kg, approximately 5 × 10 8 Less than TU / kg, approximately 4.5 x 10 8 Less than TU / kg, approximately 4 x 10 8 Less than TU / kg, approximately 3.5 x 10 8 Less than TU / kg, approximately 3 × 10 8 Less than TU / kg, approximately 2.5 x 10 8 Less than TU / kg, approximately 2 x 10 8 Less than TU / kg, approximately 1.5 x 10 8 Less than TU / kg, or about 1 × 10 8 In some embodiments, the effective dose is less than 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 x 109 ~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 ~4×10 10TU / 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 In some embodiments, the effective dose is 1×10 10 ~2×10 10 TU / kg, 1.1 x 10 10 ~1.9×10 10 TU / kg, 1.2 x 10 10 ~1.8×10 10 TU / kg, 1.3 x 10 10 ~1.7×10 10 TU / kg, or 1.4 x 10 10 ~1.6×10 10 In some embodiments, the effective dose is about 4×10 9 TU / kg ~ approx. 6 x 10 9 It is TU / kg.

[0031] In some embodiments, the lentiviral vector is administered in a single dose or multiple doses. In some embodiments, the lentiviral vector is administered via intravenous injection. In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is an adult subject.

[0032] In some embodiments, the nucleotide sequence is as shown in SEQ ID NO: 1. The present disclosure also relates to a vector comprising the nucleic acid sequence disclosed herein. In some embodiments, the vector comprises a tissue-specific promoter. In some embodiments, the tissue-specific promoter selectively enhances the expression of the polypeptide having FIX activity in the target liver cell. In some embodiments, the tissue-specific promoter selectively enhances the expression of the polypeptide having FIX activity in the target liver cell comprises APOA2 promoter, SERPINA1 (hAAT) promoter, mTTR promoter, MIR122 promoter, or any combination thereof. In some embodiments, the target liver cell is a hepatocyte.

[0033] In some embodiments, the vector comprises a splice donor site. In some embodiments, the vector comprises a splice acceptor site. In some embodiments, the vector comprises 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 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.

[0034] The present disclosure also relates to a cell comprising the nucleic acid sequence or vector disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a CHO cell, a HEK293 cell, a BHK21 cell, a PER.C6® cell, a NS0 cell, and a CAP cell. In some embodiments, the cell is a human cell. In some embodiments, the cell expresses CD47 protein. In some embodiments, the cell is 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, at least about 40 times more CD47 protein on the surface of the cells compared to a control cell that is not modified to overexpress CD47. In some embodiments, the CD47 is human CD47. In some embodiments, the cells do not express MHC-I.

[0035] The present disclosure also relates to a method of producing a lentiviral vector comprising culturing under suitable conditions the cells disclosed herein. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 shows a vector map of a lentiviral vector ("LV-coFIX-1-R338L") containing a nucleic acid encoding a polypeptide having FIX activity. [Diagram 2]Figures 2A-2B show graphical representations of plasma FIX activity for HemB mice after administration at 8 weeks of age of a lentiviral vector containing a nucleic acid sequence encoding a polypeptide with FIX activity. Figure 2A shows plasma FIX activity following 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 curve. Error bars represent standard deviation (Figure 2B). [Diagram 3] Figures 3A-3B show graphical representations of plasma FIX activity (Figure 3A) and plasma FIX antigen (Figure 3B) at various time points up to 6 months for 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 standard deviation (Figures 3A-3B). [Figure 4A] Graphical representation of sustained FIX expression (Figure 4A) and LV-FIX dose response at similar doses (Figure 4B) in neonatal, adolescent, or adulthood at various time points up to 6 months. 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 (squares) or 2 days of age (circles); or HemB mice were administered LV-coFIX-1-R338L at doses of 3E9, 7.5E9, or 2E10 TU / kg by intravenous injection at 2 weeks of age (triangles) as indicated (Figure 4A). Dose response was measured by FIX activity at various doses tested in Figure 4A in 8-week-old (squares), 2-week-old (triangles), and 2-day-old (circles) HemB mice. Error bars represent standard deviation (Figures 4A-4B). [Figure 4B]Graphical representation of sustained FIX expression (Figure 4A) and LV-FIX dose response at similar doses (Figure 4B) in neonatal, adolescent, or adulthood at various time points up to 6 months. 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 (squares) or 2 days of age (circles); or HemB mice were administered LV-coFIX-1-R338L at doses of 3E9, 7.5E9, or 2E10 TU / kg by intravenous injection at 2 weeks of age (triangles) as indicated (Figure 4A). Dose response was measured by FIX activity at various doses tested in Figure 4A in 8-week-old (squares), 2-week-old (triangles), and 2-day-old (circles) HemB mice. Error bars represent standard deviation (Figures 4A-4B). [Diagram 5] FIG. 1 shows a plot depicting the vector copy number (VCN) of lentiviral vectors in macrophages of NOD mice following administration of a control lentiviral vector (LV; black circles) or a lentiviral vector with high surface levels of CD47 (CD47hi LV; gray circles), which determined the number of lentiviral vector molecules present in the macrophages. VCN data for HEK293T cells is shown as a control. Error bars represent standard deviation. [Figure 6-1] Figures 6A-6C show graphical representations of plasma FIX activity (Figure 6A), plasma FIX antigen (Figure 6B), and FIX function (expressed as APTT time; Figure 6C) in pigtailed macaques following administration of a control lentiviral vector (LV-FIX; #1, #2, and #3), a lentiviral vector containing a nucleic acid sequence encoding a polypeptide having FIX activity 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] Continued from Figure 6-1. [Figure 7]7A-7B show graphical representations of steady-state lentiviral vector-mediated FIX expression in pigtailed macaques following administration of a CD47high lentiviral vector containing a lentiviral vector containing a nucleic acid sequence encoding a polypeptide having FIX activity, administered at a dose of E9TU / kg, as represented by plasma FIX activity (FIG. 7A) and plasma FIX antigen (FIG. 7B). Error bars represent standard deviation (FIG. 7A-7B). [Figure 8-1] Figures 8A-8D show graphical representations of ALT levels (Figure 8A), AST levels (Figure 8B), lymph levels (Figure 8C), and body temperature (Figure 8D) in pigtailed macaques following administration of vehicle (white circles), control lentiviral vector (LV; black circles) or lentiviral vector with high surface levels of CD47 (CD47hi LV; gray circles), which comprises a lentiviral vector that includes a nucleic acid sequence encoding a polypeptide with FIX activity. [Figure 8-2] Continued from Figure 8-1. [Figure 9-1] 9A-9C show graphical representations of expression levels of MIP-1a (9A), MIP-1b (9B), and MCP-1 (9C) in pigtailed macaques following administration of vehicle (closed circles), a control lentiviral vector (LV; closed squares), or a lentiviral vector with high surface levels of CD47 (CD47hi LV; open squares), which includes a lentiviral vector that includes a nucleic acid sequence encoding a polypeptide with FIX activity. [Figure 9-2] Continued from Figure 9-1. [Figure 10] 1 shows a scatter plot depicting the tissue-specific distribution (represented by VCN) of lentiviral vectors comprising a nucleic acid sequence encoding a polypeptide having FIX activity following administration of vehicle (triangles), a control lentiviral vector (inverted triangles) or CD47hi (LV; circles) to pigtailed macaques. Each data set represents an individual pigtailed macaque. [Figure 11]Figures 11A and 11B show graphical representations of steady-state lentiviral vector-mediated FIX expression in pigtailed macaques following administration of a CD47high lentiviral vector comprising a lentiviral vector containing a nucleic acid sequence encoding a polypeptide having 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). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure describes a lentiviral vector comprising a nucleic acid encoding a polypeptide having FIX activity, and a method of using the same.Thus, in some aspects, the present disclosure relates to a gene therapy comprising administering a lentiviral vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having FIX activity.In certain aspects, the present disclosure relates to a method for treating bleeding disorders, such as hemophilia (e.g., hemophilia B), comprising administering to a subject a lentiviral vector comprising a codon-optimized FIX nucleic acid sequence targeted to the liver (e.g., hepatocytes).The present disclosure meets an important need in the art through a gene therapy approach that results in the stable integration of a transgene expression cassette comprising a nucleic acid sequence encoding a polypeptide having FIX activity into the genome of targeted cells.

[0038] This system delivers lentiviral vectors at 5 × 10 10 Transducing units / kg (TU / kg) or less, e.g., about 1.5 x 10 10 TU / kg or less, or about 1.5 x 10 9 TU / kg or less, or about 1 x 10 8 When administered to a subject at least one dose of TU / kg or less, it demonstrates increased and long-term expression of a polypeptide having FIX activity in targeted cells (eg, hepatocytes).

[0039] Exemplary constructs of the present disclosure are illustrated in the accompanying figures and sequence listing.

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

[0041] I. Definition It is noted that the term "a" or "an" entity refers to one or more of that entity: for example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0042] The term "about" is used herein to mean approximately, roughly, around, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10% above and below (high and low).

[0043] For purposes of this disclosure, the term "isolated" refers to a biological material (cell, polypeptide, polynucleotide or fragment, variant or derivative thereof) 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 the adjacent nucleic acids in which it naturally occurs is considered to be "isolated". No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells are considered to be isolated in this disclosure, as are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0044] "Nucleic acid", "nucleic acid molecule", "oligonucleotide", and "polynucleotide" are used interchangeably and refer to the phosphate polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecule"), or any of their phosphate analogs, such as phosphorothioates and thioesters, in single-stranded or double-stranded helical form. Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein by the usual convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having sequence homology to mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the present 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 translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not generally translated into an amino acid, it can be considered to be part of the coding region, but neither can any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., be part of the coding region. The boundaries of a coding region are generally determined by a start codon at the 5' end, which encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end, which encodes the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. As a result, then, a single vector can contain only a single coding region, or can include two or more coding regions.

[0046] Certain proteins secreted by mammalian cells are associated with secretory signal peptides that are cleaved from the mature protein once the translocation of the growing protein chain across the rough endoplasmic reticulum is initiated. Those skilled in the art know that signal peptides are generally fused to the N-terminus of a polypeptide and are cleaved from the complete or "full-length" polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide or a functional derivative of its sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide can be used, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof.

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

[0048] The aPTT test is a performance indicator that measures the efficacy of the "intrinsic" (also called 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 FIX. It is used together with the prothrombin time (PT), which measures the extrinsic pathway.

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

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

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

[0052] As used herein, the term "gene regulatory region" or "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region that influences the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, polyadenylation signals and transcription termination sequences are usually located 3' to the coding sequence.

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

[0054] "Transcriptional control sequence" refers to a DNA regulatory sequence, such as a promoter, enhancer, terminator, etc., that allows the expression of a coding sequence in a host cell. A variety of transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but are not limited to, promoters and enhancer segments from cytomegalovirus (immediate early promoter with intron-A), simian virus 40 (early promoter) and retroviruses (such as Rous sarcoma virus). Other transcriptional control 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 that can control gene expression in eukaryotic cells. Further suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0055] Similarly, a variety of translation control elements are known to those of skill in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also called CITE sequences).

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

[0057] "Vector" refers to any vehicle for cloning and / or transferring a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment can be attached to effect replication of the attached segment. "Replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and non-viral vehicles for introducing a nucleic acid into a cell in vitro, ex vivo, or in vivo. Numerous vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector with complementary cohesive termini.

[0058] Vectors can be engineered to encode selectable markers or reporters that allow for the selection or identification of cells that have incorporated the vector. Expression of the selectable markers or reporters allows for the identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered as reporters.

[0059] The term "selectable marker" refers to an identifying agent, usually an antibiotic or chemical resistance gene, that can be selected based on the function of the marker gene, i.e., antibiotic resistance, herbicide resistance, colorimetric marker, enzyme, fluorescent marker, etc., where this function is used to track the inheritance of the nucleic acid of interest and / or to identify cells or organisms that have inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include: genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc.

[0060] The term "reporter gene" refers to a nucleic acid encoding an identifying factor that can be identified based on the action of the reporter gene, where this action is used to track the inheritance of the nucleic acid of interest, to identify cells or organisms that have inherited the nucleic acid of interest, and / or to measure 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] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. Promoters can be derived entirely from a native gene, be composed of different elements derived from different promoters found in nature, or can also include synthetic DNA segments. Those skilled in the art will appreciate that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed in most cell types at most times is generally referred to as a "constitutive promoter." A promoter that causes a gene to be expressed in a specific cell type is generally referred to as a "cell-specific promoter" or "tissue-specific promoter." A promoter that causes a gene to be expressed at a specific stage of development or cell differentiation is generally referred to as a "development-specific promoter" or "cell differentiation-specific promoter." A promoter that causes a gene to be expressed after exposure or treatment of the cell with a drug, biomolecule, chemical, ligand, light, etc. that induces the promoter is generally referred to as an "inducible promoter" or "regulatable promoter." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0062] A promoter sequence generally includes a transcription initiation site bounded at its 3' end and extending upstream (in the 5' direction) the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0063] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell and are usually in the form of a circular double-stranded DNA molecule. Such elements can be linear, circular or supercoiled, autonomously replicating sequences, genomic integration sequences, phages or nucleotide sequences of single- or double-stranded DNA or RNA derived from any source, in which several nucleotide sequences are linked or recombined into a specific construct that is capable of introducing into a cell a promoter fragment and a DNA sequence for a selected gene product along with appropriate 3' non-translated sequences.

[0064] "Cloning vector" refers to a "replicon" that is a unit-length nucleic acid, such as a plasmid, phage, or cosmid, that replicates sequentially and contains an origin of replication, to which another nucleic acid segment can be attached so as to effect replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria, and expression in another cell type, e.g., eukaryotic cells. Cloning vectors generally contain one or more sequences that can be used for selection of cells that contain the vector, and / or one or more multiple cloning sites for insertion of a nucleic acid sequence of interest.

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

[0066] The vector is introduced into the host cell by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), using gene gun, or DNA vector transporter. "Lentiviral vector" as used herein refers to replication-defective hybrid virus particle. In some contexts, lentiviral vector refers to lentiviral vector particle and encapsulated lentiviral genome. In some contexts, lentiviral vector refers to lentiviral genome including any modification thereof.

[0067] As used herein, "culture," "culturing," and "culturing" refer to incubating cells under in vitro conditions that permit the cells to grow or divide, or to maintaining the cells in a viable state. As used herein, "cultured cells" refer to cells that have been grown in vitro.

[0068] As used herein, the term "polypeptide" encompasses the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains" or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" also refers to the product of post-expression modification of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any manner, 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 the present disclosure, including norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the techniques of Noren et al., Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these techniques involve chemical activation of a suppressor tRNA with the non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA. Introduction of non-traditional amino acids can also be achieved using peptide chemistry known in the art. As used herein, the term "polar amino acid" includes amino acids that have zero total charge but non-zero partial charges at different portions of their side chains (e.g., M, F, W, S, Y, N, Q, C). These amino acids can participate in hydrophobic and electrostatic interactions. As used herein, the term "charged amino acid" includes amino acids that can have a nonzero total charge on their side chains (e.g., R, K, H, E, D). These amino acids can participate in hydrophobic and electrostatic interactions.

[0070] Polypeptide fragments or variants and any combinations thereof are also included in the present disclosure. The term "fragment" or "variant" when referring to a polypeptide binding domain or binding molecule of the present disclosure includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity for FcRn binding domains or Fc variants, coagulation activity for polypeptides with FIX activity). Polypeptide fragments include proteolytic fragments as well as deletion fragments in addition to specific antibody fragments discussed elsewhere herein, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of the polypeptide binding domains or binding molecules of the present disclosure also include the above fragments, and polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0071] "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. The art defines a family of amino acid residues with similar side chains, 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), non-polar 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 with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, the amino acid string can be conservatively replaced with a structurally similar string that differs in the order and / or composition of side chain family members.

[0072] As known in the art, the term "percent identity" refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be easily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods for determining identity are codified in publicly available computer programs.Sequence alignments and percent identity calculations can be performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), the GCG suite of programs (Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)) and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA).

[0073] Within the context of this application, when sequence analysis software is used for analysis, it is understood that the results of the analysis will be based on the "default values" of the referenced program, unless otherwise specified. As used herein, "default values" refers to any set of values ​​or parameters that originally load with the software when it is first initialized.

[0074] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked. Amino acid sequences that are normally present in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that are normally present in the same protein can be placed in a new configuration in a fusion polypeptide, such as the fusion of the FIX domain of the present disclosure with the Ig Fc domain. Fusion proteins are made, for example, by chemical synthesis, or by making and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Chimeric proteins can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide or non-covalent bond.

[0075] As used herein, the term "insertion site" refers to a position in a polypeptide having FIX activity or a fragment, variant or derivative thereof that is immediately upstream of a position at which a heterologous moiety can be inserted. An "insertion site" is defined by a number, which, unless otherwise specified, is the number of the amino acid in human FIX R338L variant (SEQ ID NOs: 11-12) to which the insertion site corresponds, immediately N-terminal to the insertion site.

[0076] As used herein, the phrase "immediately downstream of an amino acid" refers to a position immediately adjacent to the terminal carboxyl group of an amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to a position immediately adjacent to the terminal amine group of an amino acid.

[0077] As used herein, the terms "inserted," "inserted," "inserted into," or grammatically related terms refer to the location of a heterologous moiety in a recombinant FIX polypeptide relative to the analogous location in native mature human FIX. As used herein, these terms refer to the characteristics of a recombinant FIX polypeptide relative to native mature human FIX and do not indicate, imply, or imply any method or process by which the recombinant FIX polypeptide was made.

[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 cleared from the circulation and / or other tissues of the animal. When the clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid alpha phase and a longer beta phase. The alpha phase generally represents the equilibration of the administered Fc polypeptide between the intravascular and extravascular spaces and is determined, in part, by the size of the polypeptide. The beta phase generally represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FIX and chimeric proteins comprising FIX are monophasic, thus having no alpha phase, only a single beta phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the beta phase.

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

[0080] As used herein, the term "associated with" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" refers to a covalent, non-peptide or non-covalent bond. This association can be indicated by a colon, i.e. (:). In another embodiment, it refers to a covalent bond other than a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with the thiol group on the second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by disulfide bonds, 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, ignorable bonds, bent bonds, dipole bonds, pi-back bonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, hexaplex bonds, conjugation, hyperconjugation, aromaticity, multidentate or antibonds. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-pi bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low barrier hydrogen bonds or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, aurophilic, intercalation, stacking, entropic forces or chemical polarity.

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

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

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

[0084] The lentiviral vector comprising the isolated nucleic acid molecule of the present disclosure can be used prophylactically. As used herein, the term "prophylactic treatment" refers to the administration of the molecule before a bleeding episode. In one embodiment, the subject who needs a systemic hemostatic agent is undergoing or about to undergo surgery. For example, the lentiviral vector of the present disclosure can be administered before or after surgery as a prophylactic agent. The lentiviral vector of the present disclosure can be administered during or after surgery to control acute bleeding episodes. Surgery can include, but is not limited to, liver transplantation, liver resection, dental procedure, or stem cell transplantation.

[0085] The lentiviral vector of the present disclosure can also be used for on-demand treatment.The term "on-demand treatment" refers to the administration of the lentiviral vector disclosed herein in response to the symptoms of a bleeding episode or before an activity that may cause bleeding.In one aspect, on-demand treatment can be given to a subject when bleeding begins, such as after an injury, or when bleeding is expected, such as before surgery.In another aspect, on-demand treatment can 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, a subject may have trauma, uremia, inherited bleeding disorder (e.g., FIX deficiency), platelet disorder, or tolerance due to the development of antibodies against clotting factors.

[0087] As used herein, treat, treatment, or treating refers to, for example, reducing the severity of a disease or condition; reducing the duration of a disease; ameliorating one or more symptoms associated with a disease or condition; or providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition or preventing one or more symptoms associated with the disease or condition. In one embodiment, the term "treating" or "treatment" means maintaining 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 in a subject by administering a lentiviral vector of the disclosure. In another embodiment, treating or treatment means maintaining FIX trough levels between about 1 to about 20 IU / dL, about 2 to about 20 IU / dL, about 3 to about 20 IU / dL, about 4 to about 20 IU / dL, about 5 to about 20 IU / dL, about 6 to about 20 IU / dL, about 7 to about 20 IU / dL, about 8 to about 20 IU / dL, about 9 to about 20 IU / dL, or about 10 to about 20 IU / dL. Treating or treating a disease or condition can also include maintaining FIX activity in a subject at a level equivalent to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the FIX activity in a non-hemophilic subject. In one embodiment, the term "treating" 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 a lentiviral vector of the disclosure.In another embodiment, treating or treatment means maintaining a FIX trough level of about 10 to about 20 IU / dL, about 20 to about 23 IU / dL, about 30 to about 40 IU / dL, about 40 to about 50 IU / dL, about 50 to about 60 IU / dL, about 60 to about 70 IU / dL, about 70 to about 80 IU / dL, about 80 to about 90 IU / dL, about 90 to about 100 IU / dL, about 110 to about 120 IU / dL, about 120 to about 130 IU / dL, about 130 to about 140 IU / dL, or about 140 to about 150 IU / dL. Treating or treating a disease or condition can also include maintaining FIX activity in a subject at a level equivalent to at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of FIX activity in a non-hemophilic subject. The minimum trough level required for treatment can be determined by one or more known methods and can be adjusted (increased or decreased) for each individual.

[0088] As used herein, "administering" means providing a subject with a pharma- ceutically acceptable FIX-encoding nucleic acid molecule, FIX polypeptide, or vector comprising a nucleic acid molecule encoding FIX of the present disclosure through a pharma- ceutically acceptable route. The administration route can be intravenous, for example, intravenous injection and intravenous infusion. Additional administration routes include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The nucleic acid molecule, polypeptide, and vector can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0089] As used herein, the phrase "subject in need thereof" includes subjects, such as mammalian subjects, who would benefit from administration of a nucleic acid molecule, polypeptide, or vector of the present disclosure, for example, to improve hemostasis. In one embodiment, the subject includes, but is not limited to, an individual with hemophilia. In another embodiment, the subject includes, but is not limited to, an individual who has developed a FIX inhibitor and thus requires bypass therapy. The subject is an adult or a minor (e.g., under the age of 12). In some embodiments, the subject is a female. In other embodiments, the subject is a male.

[0090] As used herein, the term "clotting factor" refers to a naturally occurring or recombinantly produced molecule or analog thereof that prevents or shortens the duration of bleeding episodes in a subject. In other words, it refers to a molecule that has procoagulant activity, i.e., is responsible for the conversion of fibrinogen into a mesh of insoluble fibrin that causes blood to clot or coagulate. An "activatable clotting factor" is a clotting factor in an inactive form (e.g., in the form of its proenzyme) that can be converted to an active form.

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

[0092] As used herein, the term "heterologous" or "exogenous" refers to a molecule that is not normally found in a given context, e.g., a cell or polypeptide. An exogenous or heterologous molecule can be introduced into a cell and is present only after manipulation of the cell, e.g., by transfection or other forms of genetic engineering, or a heterologous amino acid sequence can be present in a protein in which it is not found in nature.

[0093] As used herein, the term "heterologous nucleotide sequence" refers to a nucleotide sequence that does not naturally occur with a given polynucleotide sequence. In one embodiment, the heterologous nucleotide sequence encodes a polypeptide capable of extending the half-life of FIX. In another embodiment, the heterologous nucleotide sequence encodes a polypeptide that increases the hydrodynamic radius of FIX. In other embodiments, the heterologous nucleotide 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 connected to the polypeptide encoded by the heterologous nucleotide sequence by a linker. Non-limiting examples of polypeptide moieties encoded by a heterologous nucleotide sequence include immunoglobulin constant regions or portions thereof, albumin or fragments thereof, albumin binding moieties, transferrin, the PAS polypeptide of U.S. Patent Application No. 20100292130, HAP sequences, transferrin or fragments thereof, the C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, albumin binding small molecules, XTEN sequences, FcRn binding moieties (e.g., a complete Fc region or portion thereof that binds to FcRn), single chain FcRn, or a combination thereof, such as a ribozyme, ... c-region (ScFc region, e.g., as described in US2008 / 0260738, WO2008 / 012543 or WO2008 / 1439545), polyglycine linkers, polyserine linkers, peptides and short polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P), especially with varying degrees of secondary structure from less than 50% to more than 50%, or combinations of two or more thereof. In some embodiments, the polypeptide encoded by the heterologous nucleotide sequence is linked to a non-polypeptide moiety. Non-limiting examples of non-polypeptide moieties include polyethylene glycol (PEG), albumin-binding small molecules, polysialic acid (PAS), hydroxyethyl starch (HES), derivatives thereof or any combination thereof.

[0094] As used herein, the term "Fc region" is defined as that portion of a polypeptide that corresponds to the Fc region of a native Ig, i.e., formed by the dimeric association of the Fc domains of each of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, as used herein, the term "genetically fused Fc region" or "single-chain Fc region" (scFc region) refers to a synthetic dimeric Fc region composed of Fc domains that are genetically linked (i.e., encoded in a single contiguous gene sequence) in a single polypeptide chain.

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

[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 that include the Fc region of an Ig confer several desirable properties to the chimeric protein, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors, such as the neonatal Fc receptor (FcRn) (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO03 / 077834; US2003-0235536A1), which are incorporated herein by reference in their entirety.

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

[0098] II.FIX Lentiviral Gene Therapy Somatic cell 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 continuous endogenous production of FIX after a single administration of a vector encoding FIX. Hemophilia B is highly amenable to a gene replacement approach because its clinical symptoms are due to reduced expression of functional FIX.

[0099] Lentiviral vectors have attracted attention as gene delivery vehicles due to their large capacity and ability to sustain transgene expression throughout integration. Lentiviral vectors have been evaluated in numerous ex-vivo cell therapy clinical programs, resulting in promising efficacy and safety profiles.

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

[0101] Another aspect of the present disclosure is a method of preventing or treating hemophilia in a subject in need thereof, comprising administering to a subject a dose of 5×10 10 The method includes administering to a subject a lentiviral vector comprising a nucleotide sequence encoding a polypeptide having Factor IX (FIX) activity at less than transducing units / kg (TU / kg), wherein the lentiviral vector comprises 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 to the nucleotide sequence set forth 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, the subject exhibits reduced macrophage transduction of the lentiviral vector after administration compared to a control lentiviral vector. In some embodiments, the subject exhibits reduced allospecific immune response to the lentiviral vector after administration compared to a control lentiviral vector. In some embodiments, the subject exhibits at least 30% FIX activity compared to normal FIX activity at least 3 weeks after administration. In some embodiments, the subject exhibits tissue-specific expression of the lentiviral vector in the liver, spleen, or both the liver and spleen after administration.

[0103] In some embodiments, the allospecific response comprises release of cytokines in response to the administered lentiviral vector. In some embodiments, the subject exhibits a decrease in expression of a cytokine associated with an allospecific response after administration of the lentiviral vector, compared to the expression of the cytokine after administration of a control lentiviral vector. In some embodiments, the cytokine is a proinflammatory cytokine. In certain embodiments, the cytokine is 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, the subject exhibits a decrease in expression level of MIP-1a after administration of the lentiviral vector, compared to the expression of MIP-1a after administration of the control lentiviral vector. In certain embodiments, the subject exhibits a decrease in expression level of MIP-1b after administration of the lentiviral vector, compared to the expression of MIP-1b after administration of the control lentiviral vector. In certain embodiments, the subject exhibits a decrease in expression level of MCP-1 after administration of the lentiviral vector, compared to the expression of MCP-1 after administration of the control lentiviral vector. In certain embodiments, the subject shows a reduced expression level of IL-2 after administration of the lentiviral vector, compared to the expression of IL-2 after administration of a control lentiviral vector.In certain embodiments, the subject shows a reduced expression level of interferon gamma after administration of the lentiviral vector, compared to the expression of interferon gamma after administration of a control lentiviral vector.

[0104] In some embodiments, the expression of the cytokine 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 the expression of the cytokine after administration of a control lentiviral vector.In certain embodiments, the expression of the cytokine is undetectable after administration of the lentiviral vector.In certain embodiments, the subject has only undetectable expression of a cytokine 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 the lentiviral vector.

[0105] In some embodiments, the subject shows an increase in plasma FIX activity after administration of lentiviral vector compared to the 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 at about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 12 hours to about 36 hours, about 12 hours to about 24 hours, about 24 hours to about 60 hours, about 24 hours to about 48 hours, about 24 hours to about 36 hours, about 36 hours to about 60 hours, about 36 hours to about 48 hours, or about 48 hours to about 60 hours after administration of the lentiviral vector is increased compared to a subject administered a control dose of a control lentiviral vector. In some embodiments, the FIX activity after administration of the lentiviral vector is 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 1 week, 2 weeks, or 3 weeks after administration of the lentiviral vector. In certain embodiments, the subject shows at least about 150% FIX activity compared to normal FIX activity at least 3 weeks after administration of lentiviral vector.In certain embodiments, the subject shows at least about 200% FIX activity compared to normal FIX activity at least 3 weeks after administration of lentiviral vector.In certain embodiments, the subject shows at least about 225% FIX activity compared to normal FIX activity at least 3 weeks after administration of lentiviral vector.

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

[0107] In some embodiments, after administration, the lentiviral vector is specifically localized in the liver, spleen, or both liver and spleen of the subject, where a higher concentration of the lentiviral vector is found in the liver, spleen, or both liver and spleen than in another organ of the subject's body. In some embodiments, the other organ of the subject's body is selected from the group consisting of testis, lymph node, muscle, thymus, kidney, lung, 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 in the liver, spleen, or both liver and spleen, compared to organs other than the liver and spleen of the subject after administration of the lentiviral vector. The localization of the lentiviral vector 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-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-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 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 10 ... 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, at least about 200 fold, at least about 250 fold, at least about 300 fold, at least about 400 fold, or at least about 500 fold greater vector copy number (VCN) of the lentiviral vector.

[0108] In certain embodiments, the subject shows increased localization of the lentiviral vector to the liver, spleen, or both liver and spleen, and the increased localization is characterized by at least 10 times more VCN of the lentiviral vector in the liver, spleen, or both liver and spleen after administration of the lentiviral vector, compared to the subject's organs other than the liver and spleen.In certain embodiments, the subject shows increased localization of the lentiviral vector to the liver, spleen, or both liver and spleen, and the increased localization is characterized by at least 50 times more VCN of the lentiviral vector in the liver, spleen, or both liver and spleen after administration of the lentiviral vector, compared to the subject's organs other than the liver and spleen.In certain embodiments, the subject shows increased localization of the lentiviral vector to the liver, spleen, or both liver and spleen, and the increased localization is characterized by at least 100 times more VCN of the lentiviral vector in the liver, spleen, or both liver and spleen after administration of the lentiviral vector, compared to the subject's organs other than the liver and spleen. In certain embodiments, the subject exhibits increased localization of the lentiviral vector to the liver, spleen, or both the liver and spleen, wherein the increased localization is characterized by at least 150-fold more VCN of the lentiviral vector in the liver, spleen, or both the liver and spleen following administration of the lentiviral vector, compared to organs other than the liver and spleen of the subject.

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

[0110] In some embodiments, lentiviral vector comprises high concentration of CD47 polypeptide molecules on its surface.In some embodiments, lentiviral vector comprises heterologous polynucleotide encoding CD47 protein, wherein the heterologous polynucleotide encoding CD47 is expressed.In some embodiments, lentiviral vector further comprises heterologous polynucleotide encoding CD47 protein, wherein 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 expression levels of CD47. high In certain embodiments, the lentiviral vector is produced in a cell, where the cell has high expression of CD47 on the cell membrane. highThe present invention is produced in HEK293T cells, wherein the HEK293T cells are modified to have increased expression of CD47 relative to expression of CD47 in unmodified HEK293T cells. In some embodiments, the HEK293T cells are modified to overexpress endogenous CD47 compared to unmodified HEK293T cells. In some embodiments, the HEK293T cells are modified by transducing the HEK293T cells with a heterologous CD47 expression vector. In some embodiments, the heterologous CD47 expression vector comprises a retroviral vector. In certain embodiments, the retroviral vector is a gamma-retroviral vector. In some embodiments, the heterologous CD47 expression vector is incapable of being cross-packaged by a lentiviral vector.

[0112] In certain aspects, the present disclosure relates to a method of preventing or treating hemophilia in a subject in need thereof, 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 a higher level 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. Without being bound by any mechanism, it is believed that CD47 expression on the surface of the lentiviral vector protects the lentiviral vector from degradation and / or removal by the subject's immune system and prevents and / or reduces immune responses against the lentiviral vector.

[0113] In some embodiments, CD47 is human CD47 (NCBI accession number NP_001768.1). In certain embodiments, 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 set forth in SEQ ID NO: 14. In certain embodiments, human CD47 comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0114] In some embodiments, CD47 is expressed by the lentiviral vector. In other embodiments, CD47 is not expressed by the lentiviral vector. In certain embodiments, the lentiviral vector is expressed in a host cell, where the host cell is modified to express CD47. In some embodiments, the host cell is modified to overexpress CD47. In certain embodiments, the lentiviral vector is generated in a host cell that expresses a high concentration of CD47 compared to HEK293 cells (ATCC® CRL-1573™). In certain embodiments, the lentiviral vector is generated in a HEK293T cell that is modified to overexpress CD47 compared to unmodified HEK293T cells.

[0115] In some embodiments, the lentiviral vector comprises a higher level of surface CD47 protein expression than a control lentiviral vector. 2 In some embodiments, the control lentiviral vector is produced in HEK293 cells (ATCC® CRL-1573™), which are known to produce 19 molecules per 1 μm (see, e.g., Figure S1(d) in Sosale et al., Methods & Clinical Development 3:16080 (2016)). ... 2 Contains fewer than 20 molecules of CD47 per cell.

[0116] In certain embodiments, the lentiviral vector has a surface that is at least about 2-fold to at least about 100-fold, at least about 2-fold to at least about 75-fold, at least about 2-fold to at least about 50-fold, at least about 2-fold to at least about 40-fold, at least about 2-fold to at least about 30-fold, at least about 2-fold to at least about 20-fold, at least about 2-fold to at least about 10-fold higher than the surface of a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573™). , at least about 10-fold to at least about 100-fold, at least about 10-fold to at least about 75-fold, at least about 10-fold to at least about 50-fold, at least about 10-fold to at least about 40-fold, at least about 10-fold to at least about 30-fold, at least about 10-fold to at least about 20-fold, at least about 20-fold to at least about 100-fold, at least about 20-fold to at least about 75-fold, at least about 20-fold to at least about 50-fold, at least about 20-fold to at least about 40-fold, or at least about 20-fold to at least about 30-fold 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 that is at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5 ... fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold more, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, or at least about 100 fold more CD47 protein.

[0117] In certain embodiments, the lentiviral vector comprises at least about 10 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 certain embodiments, the lentiviral vector comprises at least about 15 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 certain embodiments, the lentiviral vector comprises at least about 20 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 certain embodiments, the lentiviral vector comprises at least about 25 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 certain embodiments, the lentiviral vector contains at least about 30-fold 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™).

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

[0119] In certain embodiments, the lentiviral vector comprises a 1 μm 2 In certain embodiments, the lentiviral vector comprises at least about 400 molecules of CD47 protein per μm. 2 In certain embodiments, the lentiviral vector comprises at least about 450 molecules of CD47 protein per μm. 2 In certain embodiments, the lentiviral vector comprises at least about 500 molecules of CD47 protein per μm. 2 In certain embodiments, the lentiviral vector comprises at least about 600 molecules of CD47 protein per μm. 2 In certain embodiments, the lentiviral vector comprises at least about 700 molecules of CD47 protein per 1 μm. 2 In certain embodiments, the lentiviral vector comprises at least about 800 molecules of CD47 protein per μm. 2In certain embodiments, the lentiviral vector comprises at least about 900 molecules of CD47 protein per μm. 2 Each antibody contains at least about 1000 molecules of CD47 protein.

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

[0121] MHC-I - or MHC-I low The cells can be generated using any means known in the art. In some embodiments, the cells are - or MHC-I low The cells are generated by disrupting expression of one or more genes encoding one or more proteins in the MHC. In some embodiments, the MHC-I - or MHC-I low The cells are generated by disrupting expression of the gene encoding beta-2-microglobulin (B2M; Ensembl ENSG00000166710; NCBI protein accession no. ABB01003). -or MHC-I low The cells are generated by chronically disrupting expression of the gene encoding beta-2-microglobulin (B2M). - or MHC-I low The cells are generated by blocking expression of the gene encoding beta-2-microglobulin (B2M). - or MHC-I low The cells are generated by introducing a mutation into a gene encoding beta-2-microglobulin (B2M), where the mutation results in loss of expression of the gene encoding B2M. In some embodiments, the MHC-I - or MHC-I low The cells are generated by knocking out the gene encoding beta-2-microglobulin (B2M). In certain embodiments, the MHC-I - or MHC-I low The cells are generated by modifying HECK 293T cells to block or reduce MHC-I exposed on the surface. Unmodified HEK293T cells have MHC-I exposed on the surface. See, for example, Dellgren et al., PLoS One 10(8):e0135385 (2015).

[0122] In some embodiments, the cells are MHC-I - In certain embodiments, the cells are selected from the group consisting of HEK293T cells, wherein the cells do not have MHC-I exposed on the surface. - The cells have less than 1% of the surface-exposed MHC-I compared to unmodified HEK293T cells. In some embodiments, the cells have less than 1% of the surface-exposed MHC-I lowHEK293T cells, wherein the HEK293T cells are modified to have less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of MHC-I exposed on the surface compared to unmodified HEK293T cells. low The cells have less than 5% of the surface-exposed MHC-I compared to unmodified HEK293T cells. In certain embodiments, the MHC-I low The cells have less than 4% of the surface-exposed MHC-I compared to unmodified HEK293T cells. In certain embodiments, the MHC-I low The cells have less than 3% of the surface-exposed MHC-I compared to unmodified HEK293T cells. In certain embodiments, the MHC-I low The cells have less than 2% of the surface-exposed MHC-1 compared to unmodified HEK293T cells.

[0123] In certain embodiments, the lentiviral vector comprises a lipid coat that contains a high concentration of CD47 polypeptide and lacks surface-exposed MHC-I. high / MHC-I low Cell lines, e.g., CD47 high / MHC-I low In some embodiments, the lentiviral vector is generated in a HEK293T cell line. high / MHC-I - Cell lines, e.g., CD47 high / MHC-I - Produced in the HEK293T cell line.

[0124] In another embodiment, 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 development of antibodies against FIX. In some embodiments, the immune response includes cytokine secretion. In some embodiments, the immune response includes activation of B cells, T cells, or both B cells and T cells. In some embodiments, the immune response is an inhibitory immune response, where the immune response in the subject reduces the activity of FIX protein compared to the activity of FIX in a subject that did not mount an immune response. In certain embodiments, expression of FIX protein by administration of the lentiviral vector of the present disclosure prevents an inhibitory immune response against FIX protein or FIX protein expressed from the isolated nucleic acid molecule or lentiviral vector.

[0125] B. Administration The lentiviral vectors, lentiviral vector particles, and methods of use of the present disclosure allow for the prevention and / or treatment of hemophilia using lower doses of lentiviral vector than a control lentiviral vector produced in HEK293 cells (ATCC® CRL-1573™). In some embodiments, the lentiviral vectors of the present disclosure are effective at doses that are reduced compared to the control dose of the 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 a control lentiviral vector.

[0126] In some embodiments, the dose of lentiviral vector is about 5.0×10 10 TU / kg, approximately 4.9 x 10 10 TU / kg, approximately 4.8 x 10 10 TU / kg, approximately 4.7 x 10 10 TU / kg, approximately 4.6 x 10 10 TU / kg, approximately 4.5 x 10 10 TU / kg, approximately 4.4 x 10 10 TU / kg, approximately 4.3 x 10 10 TU / kg, approximately 4.2 x 10 10 TU / kg, approximately 4.1 x 10 10 TU / kg, approx. 4.0 x 10 10 TU / kg, approximately 3.9 x 10 10 TU / kg, approximately 3.8 x 10 10TU / kg, approximately 3.7 x 10 10 TU / kg, approximately 3.6 x 10 10 TU / kg, approximately 3.5 x 10 10 TU / kg, approximately 3.4 x 10 10 TU / kg, approximately 3.3 x 10 10 TU / kg, approximately 3.2 x 10 10 TU / kg, approximately 3.1 x 10 10 TU / kg, approx. 3.0 x 10 10 TU / kg, approximately 2.9 x 10 10 TU / kg, approximately 2.8 x 10 10 TU / kg, approximately 2.7 x 10 10 TU / kg, approximately 2.6 x 10 10 TU / kg, approximately 2.5 x 10 10 TU / kg, approximately 2.4 x 10 10 TU / kg, approximately 2.3 x 10 10 TU / kg, approximately 2.2 x 10 10 TU / kg, approximately 2.1 x 10 10 TU / kg, approximately 2.0 x 10 10 TU / kg, approximately 1.9 x 10 10 TU / kg, approximately 1.8 x 10 10 TU / kg, approximately 1.7 x 10 10 TU / kg, approximately 1.6 x 10 10 TU / kg, approximately 1.5 x 10 10 TU / kg, approximately 1.4 x 10 10 TU / kg, approximately 1.3 x 10 10 TU / kg, approximately 1.2 x 10 10 TU / kg, approximately 1.1 x 10 10 TU / kg, or approximately 1.0 x 10 10 It is TU / kg.

[0127] In some embodiments, the dose of lentiviral vector is about 9.9×10 9 TU / kg, approximately 9.8 x 10 9 TU / kg, approximately 9.7 x 10 9 TU / kg, approximately 9.6 x 10 9 TU / kg, approximately 9.5 x 10 9 TU / kg, approximately 9.4 x 10 9 TU / kg, approximately 9.3 x 10 9 TU / kg, approximately 9.2 x 109 TU / kg, 9.1×10 9 TU / kg, 9.0×10 9 TU / kg, 8.9×10 9 TU / kg, 8.8×10 9 TU / kg, 8.7×10 9 TU / kg, 8.6×10 9 TU / kg, 8.5×10 9 TU / kg, 8.4×10 9 TU / kg, 8.3×10 9 TU / kg, 8.2×10 9 TU / kg, 8.1×10 9 TU / kg, 8.0×10 9 TU / kg, 7.9×10 9 TU / kg, 7.8×10 9 TU / kg, 7.7×10 9 TU / kg, 7.6×10 9 TU / kg, 7.5×10 9 TU / kg, 7.4×10 9 TU / kg, 7.3×10 9 TU / kg, 7.2×10 9 TU / kg, 7.1×10 9 TU / kg, 7.0×10 9 TU / kg, 6.9×10 9 TU / kg, 6.8×10 9 TU / kg, 6.7×10 9 TU / kg, 6.6×10 9 TU / kg, 6.5×10 9 TU / kg, 6.4×10 9 TU / kg, 6.3×10 9 TU / kg, 6.2×10 9 TU / kg, 6.1×10 9 TU / kg, 6.0×10 9 TU / kg, 5.9×10 9 TU / kg, 5.8×10 9 TU / kg, 5.7×10 9 TU / kg, 5.6×10 9 TU / kg, 5.5×10 9 TU / kg, 5.4×10 9TU / kg, 5.3×10 9 TU / kg, 5.2×10 9 TU / kg, 5.1×10 9 TU / kg, 5.0×10 9 TU / kg, 4.9×10 9 TU / kg, 4.8×10 9 TU / kg, 4.7×10 9 TU / kg, 4.6×10 9 TU / kg, 4.5×10 9 TU / kg, 4.4×10 9 TU / kg, 4.3×10 9 TU / kg, 4.2×10 9 TU / kg, 4.1×10 9 TU / kg, 4.0×10 9 TU / kg, 3.9×10 9 TU / kg, 3.8×10 9 TU / kg, 3.7×10 9 TU / kg, 3.6×10 9 TU / kg, 3.5×10 9 TU / kg, 3.4×10 9 TU / kg, 3.3×10 9 TU / kg, 3.2×10 9 TU / kg, 3.1×10 9 TU / kg, 3.0×10 9 TU / kg, 2.9×10 9 TU / kg, 2.8×10 9 TU / kg, 2.7×10 9 TU / kg, 2.6×10 9 TU / kg, 2.5×10 9 TU / kg, 2.4×10 9 TU / kg, 2.3×10 9 TU / kg, 2.2×10 9 TU / kg, 2.1×10 9 TU / kg, 2.0×10 9 TU / kg, 1.9×10 9 TU / kg, 1.8×10 9 TU / kg, 1.7×10 9 TU / kg, 1.6×10 9 TU / kg, 1.5×109 TU / kg, approximately 1.4 x 10 9 TU / kg, approximately 1.3 x 10 9 TU / kg, approximately 1.2 x 10 9 TU / kg, approximately 1.1 x 10 9 TU / kg or approximately 1.0 x 10 9 It is TU / kg.

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

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

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

[0131] In some embodiments, the dose of lentiviral vector is about 9.9×10 8 Less than TU / kg, approximately 9.8 x 10 8 Less than TU / kg, approximately 9.7 x 10 8 Less than TU / kg, approximately 9.6 x 10 8 Less than TU / kg, approximately 9.5 x 10 8 Less than TU / kg, approximately 9.4 x 10 8 Less than TU / kg, approximately 9.3 x 10 8 Less than TU / kg, approximately 9.2 x 10 8 Less than TU / kg, approximately 9.1 x 10 8 Less than TU / kg, approximately 9.0 x 10 8 Less than TU / kg, approximately 8.9 x 10 8 Less than TU / kg, approximately 8.8 x 10 8 Less than TU / kg, approximately 8.7 x 10 8 Less than TU / kg, approximately 8.6 x 10 8 Less than TU / kg, approximately 8.5 x 10 8 Less than TU / kg, approximately 8.4 x 10 8 Less than TU / kg, approximately 8.3 x 10 8 Less than TU / kg, approximately 8.2 x 10 8 Less than TU / kg, approximately 8.1 x 10 8Less than TU / kg, approximately 8.0 x 10 8 Less than TU / kg, approximately 7.9 x 10 8 Less than TU / kg, approximately 7.8 x 10 8 Less than TU / kg, approximately 7.7 x 10 8 Less than TU / kg, approximately 7.6 x 10 8 Less than TU / kg, approximately 7.5 x 10 8 Less than TU / kg, approximately 7.4 x 10 8 Less than TU / kg, approximately 7.3 x 10 8 Less than TU / kg, approximately 7.2 x 10 8 Less than TU / kg, approximately 7.1 x 10 8 Less than TU / kg, approximately 7.0 x 10 8 Less than TU / kg, approximately 6.9 x 10 8 Less than TU / kg, approximately 6.8 x 10 8 Less than TU / kg, approximately 6.7 x 10 8 Less than TU / kg, approximately 6.6 x 10 8 Less than TU / kg, approximately 6.5 x 10 8 Less than TU / kg, approximately 6.4 x 10 8 Less than TU / kg, approximately 6.3 x 10 8 Less than TU / kg, approximately 6.2 x 10 8 Less than TU / kg, approximately 6.1 x 10 8 Less than TU / kg, approximately 6.0 x 10 8 Less than TU / kg, approximately 5.9 x 10 8 Less than TU / kg, approximately 5.8 x 10 8 Less than TU / kg, approximately 5.7 x 10 8 Less than TU / kg, approximately 5.6 x 10 8 Less than TU / kg, approximately 5.5 x 10 8 Less than TU / kg, approximately 5.4 x 10 8 Less than TU / kg, approximately 5.3 x 10 8 Less than TU / kg, approximately 5.2 x 10 8 Less than TU / kg, approximately 5.1 x 10 8 Less than TU / kg, approximately 5.0 x 10 8 Less than TU / kg, approximately 4.9 x 10 8 Less than TU / kg, approximately 4.8 x 10 8 Less than TU / kg, approximately 4.7 x 10 8 Less than TU / kg, approximately 4.6 x 10 8 Less than TU / kg, approximately 4.5 x 108 Less than TU / kg, approximately 4.4 x 10 8 Less than TU / kg, approximately 4.3 x 10 8 Less than TU / kg, approximately 4.2 x 10 8 Less than TU / kg, approximately 4.1 x 10 8 Less than TU / kg, approximately 4.0 x 10 8 Less than TU / kg, approximately 3.9 x 10 8 Less than TU / kg, approximately 3.8 x 10 8 Less than TU / kg, approximately 3.7 x 10 8 Less than TU / kg, approximately 3.6 x 10 8 Less than TU / kg, approximately 3.5 x 10 8 Less than TU / kg, approximately 3.4 x 10 8 Less than TU / kg, approximately 3.3 x 10 8 Less than TU / kg, approximately 3.2 x 10 8 Less than TU / kg, approximately 3.1 x 10 8 Less than TU / kg, approximately 3.0 x 10 8 Less than TU / kg, approximately 2.9 x 10 8 Less than TU / kg, approximately 2.8 x 10 8 Less than TU / kg, approximately 2.7 x 10 8 Less than TU / kg, approximately 2.6 x 10 8 Less than TU / kg, approximately 2.5 x 10 8 Less than TU / kg, approximately 2.4 x 10 8 Less than TU / kg, approximately 2.3 x 10 8 Less than TU / kg, approximately 2.2 x 10 8 Less than TU / kg, approximately 2.1 x 10 8 Less than TU / kg, approximately 2.0 x 10 8 Less than TU / kg, approximately 1.9 x 10 8 Less than TU / kg, approximately 1.8 x 10 8 Less than TU / kg, approximately 1.7 x 10 8 Less than TU / kg, approximately 1.6 x 10 8 Less than TU / kg, approximately 1.5 x 10 8 Less than TU / kg, approximately 1.4 x 10 8 Less than TU / kg, approximately 1.3 x 10 8 Less than TU / kg, approximately 1.2 x 10 8 Less than TU / kg, approximately 1.1 x 10 8 Less than TU / kg or about 1.0 × 10 8It is less than TU / kg.

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

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

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

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

[0136] In certain embodiments, the dose of lentiviral vector is about 2.5×10 9 In certain embodiments, the dose of the lentiviral vector is 2.5×10 9 In certain embodiments, the dose of the lentiviral vector is about 3.0×10 9 In certain embodiments, the dose of the lentiviral vector is 3.0×10 9 In certain embodiments, the dose of the lentiviral vector is about 7.5×10 9 In certain embodiments, the dose of the lentiviral vector is 7.5×10 9 In some embodiments, the dose of the lentiviral vector is about 2×10 10 In some embodiments, the dose of the lentiviral vector is 2×10 10 In some embodiments, the dose of the lentiviral vector is about 6×10 10 In some embodiments, the dose of the lentiviral vector is 6×10 10 It is TU / kg.

[0137] In some embodiments, lentiviral vector is administered in a single dose or multiple doses.In some embodiments, the dose of lentiviral vector is administered at once or divided into multiple partial doses, for example, 2 partial doses, 3 partial doses, 4 partial doses, 5 partial doses, 6 partial doses or more than 6 partial doses.In some embodiments, more than one lentiviral vector is administered.

[0138] In some embodiments, the dose of the lentiviral vector is administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. In some embodiments, the dose of the lentiviral vector 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 dose of the lentiviral vector 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 a 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, about once every twelve weeks, or about once every twenty weeks. It is administered once every approximately 3 months, once every approximately 13 weeks, once every approximately 14 weeks, once every approximately 15 weeks, once every approximately 16 weeks, once every approximately 4 months, once every approximately 17 weeks, once every approximately 18 weeks, once every approximately 19 weeks, once every approximately 20 weeks, once every approximately 5 months, once every approximately 21 weeks, once every approximately 22 weeks, once every approximately 23 weeks, once every approximately 24 weeks, once every approximately 25 weeks, once every approximately 26 weeks, or once every approximately 6 months.

[0139] In some embodiments, a first dose of lentiviral vector is administered, the subject is monitored for transgene expression, and a second dose is administered to the subject when the subject has transgene expression below a predetermined threshold.In certain embodiments, when less than about 100%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 3%, or less than about 1% of target cells express transgene, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of lentiviral vector is administered to the subject. In some embodiments, the second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the subject when less than about 50% of the target cells express the transgene. In some embodiments, the second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the subject when less than about 25% of the target cells express the transgene. In some embodiments, the second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the subject when less than about 10% of the target cells express the transgene. In some embodiments, the second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the subject when less than about 5% of the target cells express the transgene. In some embodiments, the second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the subject when less than about 4% of the target cells express the transgene. In some embodiments, the second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the subject when less than about 3% of the target cells express the transgene. In some embodiments, the second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the subject when less than about 2% of the target cells express the transgene.In some embodiments, when less than about 1% of target cells express the transgene, a second (third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth) dose of the lentiviral vector is administered to the subject.

[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., subcutaneously or intradermally).

[0141] In some embodiments, the subject is a pediatric subject, while in other aspects, the subject is an adult subject. In some embodiments, the subject is male. In other embodiments, the subject is female.

[0142] The lentiviral vectors disclosed herein can be administered in vivo at low or reduced dosages (e.g., 10 to 20 mg / kg / day) in a mammal, e.g., human patient, using a gene therapy approach that would be therapeutically beneficial for the treatment of a bleeding disease or disorder selected from the group consisting of bleeding coagulation disorders, hemorrhagic arthropathy, muscle bleed, mouth bleed, bleeding, bleeding into muscle, mouth bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A.

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

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

[0145] In one embodiment, plasma FIX activity after administration of the lentiviral vector of the present disclosure is increased 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 is increased by at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 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 a subject administered a corresponding lentiviral vector comprising a reference nucleic acid molecule comprising SEQ ID NO:8 or SEQ ID NO:9.

[0146] The present disclosure provides a method of treating, preventing, or ameliorating a hemostatic disorder (e.g., a bleeding disorder such as hemophilia A) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FIX activity, wherein the lentiviral vector is at least 5×10 10 TU / kg or less, 10 9 Less than 10 TU / kg or 8 Also provided are methods in which the antibody is administered in at least one dose of:

[0147] The treatment, amelioration and prevention by the lentiviral vector of the present disclosure may be a bypass therapy. The subject undergoing the bypass therapy may already have an inhibitor of a clotting factor, such as FIX, or may be susceptible to an inhibitor of a clotting factor.

[0148] The lentiviral vector of the present disclosure promotes the formation of fibrin clots, thereby treating or preventing hemostatic disorders. The polypeptide with FIX activity encoded by the nucleic acid molecule of the present disclosure can activate members of the coagulation cascade. The coagulation factor can be a participant in the extrinsic pathway, the intrinsic pathway, or both.

[0149] The lentiviral vector of the present disclosure can be used to treat hemostatic disorders that are known to be treatable with FIX.Hemostatic disorders that can be treated using the method of the present disclosure include, but are not limited to, hemophilia A, hemophilia B, von Willebrand's disease, factor XI deficiency (PTA deficiency), factor XII deficiency, and deficiency or structural abnormality of fibrinogen, prothrombin, factor V, factor VII, factor X or factor XIII, hemorrhagic joint disease, muscle bleed, mouth bleed, bleeding, bleeding into muscle, mouth bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space and bleeding in the iliopsoas sheath.

[0150] In some embodiments, the hemostatic disorder is an inherited disorder. In one embodiment, the subject has hemophilia A. In other embodiments, the hemostatic disorder is the result of a deficiency of FIX. In other embodiments, the hemostatic disorder may be the result of a defective FIX clotting factor.

[0151] In another embodiment, the hemostatic disorder can be an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, by way of example but not limited to, cancer, autoimmune disease or pregnancy. The acquired disorder can result from old age or from medicines (e.g., cancer chemotherapy) for treating the underlying secondary disorder.

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

[0153] The lentiviral vectors of the present disclosure can be administered during or after surgery to control acute bleeding episodes, which can include, but are not limited to, liver transplantation, liver resection, or stem cell transplantation.

[0154] In another embodiment, the lentiviral vector of the present disclosure can be used to treat subjects with acute bleeding episodes who do not have hemostatic disorders.Acute bleeding episodes can result from severe trauma, such as surgery, car accidents, wounds, lacerations, gunshot wounds, or any other traumatic events that result 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 episodes in subjects with hemostatic disorders.

[0156] In some embodiments, the lentiviral vector of the present disclosure is administered in combination with at least one other agent that promotes hemostasis. Said other agent that promotes hemostasis is a therapeutic agent with demonstrated coagulation activity. By way of example, but not by way of limitation, the hemostatic agent can include Factor V, Factor VII, Factor VIII, Factor X, Factor XI, Factor XII, Factor XIII, prothrombin or fibrinogen, or any of the previous activated forms. The coagulation factor or hemostatic agent can also include an antifibrinolytic agent, such as epsilon-aminocaproic acid, tranexamic acid.

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

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

[0159] In one embodiment, the route of administration of the lentiviral vector is parenteral. The term parenteral, as used herein, includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. An intravenous form of parenteral administration is preferred. Although all these forms of administration are clearly anticipated to be within the scope of the present disclosure, particularly for intravenous or intraarterial injection or infusion, the form for administration will be an injectable solution. Typically, suitable pharmaceutical compositions for injection can include buffers (e.g., acetate, phosphate or citrate buffers), surfactants (e.g., polysorbates), optionally stabilizers (e.g., human albumin), and the like. However, in other methods consistent with the teachings of the present specification, the lentiviral vector 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 Vectors Certain aspects of the present 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 to the amino acid sequence set forth 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 that contains FIX. The term "monomer-dimer hybrid" as used herein refers to a chimeric protein that includes a first polypeptide chain and a second polypeptide chain that are associated with each other by disulfide bonds, where the first chain includes FIX and a first Fc region, and the second chain includes, consists essentially of, or consists of a second Fc region that does not include FIX. Thus, the monomer-dimer hybrid construct is a hybrid that includes a monomeric embodiment with only one coagulation factor and a dimeric embodiment with two Fc regions.

[0162] C.1. Nucleotide Sequences Encoding Polypeptides Having FIX Activity In some embodiments, the nucleotide sequence encoding the polypeptide having FIX activity is codon-optimized.In certain embodiments, the nucleotide sequence encoding the polypeptide having FIX activity 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 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 to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 85% sequence identity to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 90% sequence identity to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 91% sequence identity to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 92% sequence identity to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 93% sequence identity to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 94% sequence identity to the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 96% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 98% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence is identical to the nucleotide sequence set forth 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 to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 90% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 91% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 92% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 93% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 94% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 95% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 96% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 97% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence has at least about 98% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2.In some embodiments, the nucleotide sequence has at least about 99% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:2.

[0165] 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 to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 90% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 91% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 92% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 93% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 94% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 95% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 96% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 97% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the nucleotide sequence has at least about 98% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:3.In some embodiments, the nucleotide sequence has at least about 99% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence set forth 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 to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 90% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 91% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 92% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 93% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 94% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 95% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 96% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 97% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence has at least about 98% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:4.In some embodiments, the nucleotide sequence has at least about 99% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ 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 to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 90% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 91% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 92% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 93% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 94% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 95% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 96% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 97% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the nucleotide sequence has at least about 98% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:5.In some embodiments, the nucleotide sequence has at least about 99% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence set forth 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 to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 90% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 91% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 92% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 93% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 94% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 95% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 96% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 97% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence has at least about 98% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:6.In some embodiments, the nucleotide sequence has at least about 99% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence set forth 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 to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 85% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 90% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 91% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 92% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 93% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 94% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 95% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 96% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 97% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the nucleotide sequence has at least about 98% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO:7.In some embodiments, the nucleotide sequence has at least about 99% sequence identity to nucleotides 139-1386 of the nucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleotide sequence is identical to nucleotides 139-1386 of the nucleotide sequence set forth 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 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 to (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 comprises the nucleotide sequence set forth 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.

[0171] In certain 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 to (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 comprises the nucleotide sequence set forth 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, the nucleotide molecule encoding the 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 procoagulant activity. In some embodiments, the heterologous moiety is inserted into one or more sites in the polypeptide having FIX activity, where the polypeptide has procoagulant activity. In some embodiments, the heterologous moiety is a heterologous polypeptide. In certain aspects, the heterologous moiety is an XTEN. In some aspects, the heterologous moiety comprises at least one XTEN inserted into one or more sites in the polypeptide having FIX activity. In other aspects, the heterologous moiety is a half-life extending moiety (e.g., an in vivo half-life extending 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 Application Publication No. WO2017 / 024060, which is incorporated herein by reference in its entirety. In certain embodiments, the heterologous moiety is inserted into the polypeptide having FIX activity immediately downstream of an amino acid corresponding to 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 combination thereof.

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

[0174] Non-limiting examples of heterologous moieties (e.g., half-life extending moieties) include albumin, albumin fragments, Fc fragments of immunoglobulins, FcRn binding partners, C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, HAP sequences, transferrin, the PAS polypeptides of US Patent Application No. 20100292130, polyglycine linkers, polyserine linkers, peptides and short polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P), among others, with varying degrees of secondary structure from less than 50% to more than 50%.

[0175] In certain embodiments, the heterologous moiety increases the in vivo or in vitro half-life of the polypeptide having FIX activity produced from the lentiviral vector of the present disclosure. In other embodiments, the heterologous moiety facilitates visualization or localization of the polypeptide having FIX activity produced from the lentiviral vector of the present disclosure. The visualization and / or localization of the polypeptide having FIX activity is in vivo, in vitro, ex vivo, or a combination thereof. In other embodiments, the heterologous moiety increases the stability of the polypeptide having FIX activity produced from the lentiviral vector of the present disclosure. As used herein, the term "stability" refers to an art-recognized measure of the maintenance of one or more physical properties of a polypeptide having FIX activity in response to environmental conditions (e.g., elevated or decreased temperature). In certain embodiments, the physical property is the maintenance of the covalent structure of the polypeptide having FIX activity (e.g., no proteolysis, no undesired oxidation or deamidation). In other embodiments, the physical property is also the presence of the polypeptide having FIX activity in a correctly folded state (e.g., no soluble or insoluble aggregation or precipitation).

[0176] In certain embodiments, heterologous moieties that increase the half-life of the FIX fusion proteins of the present disclosure include, but are not limited to, heterologous polypeptides such as albumin, immunoglobulin Fc regions, XTEN sequences, C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), PAS sequences, HAP sequences, CTP peptide sequences, transferrin, albumin binding moieties, or any fragments, derivatives, variants, or combinations of these polypeptides. In other related embodiments, the heterologous moiety comprises a binding site for a non-polypeptide moiety, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations of these moieties.

[0177] In certain aspects, the polypeptide with FIX activity of the present disclosure comprises one, two, three or more heterologous moieties, each of which is 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 specific embodiment, the lentiviral vector comprises the nucleotide sequence encoding XTEN inserted into the nucleotide sequence encoding the polypeptide with FIX activity and the nucleotide sequence encoding Fc fused to the part of the nucleotide sequence encoding the C-terminus of the polypeptide with FIX activity.

[0178] C.1.aiXTEN In some embodiments, at least one heterologous moiety is an XTEN. As used herein, "XTEN sequence" refers to an extended polypeptide having a non-naturally occurring, substantially non-repetitive sequence that is composed primarily of small hydrophilic amino acids and has little or no secondary or tertiary structure under physiological conditions. As a fusion protein partner, XTEN acts as a carrier that imparts certain desired pharmacokinetic, physicochemical and pharmaceutical properties when linked to the FIX sequence of the present disclosure to create a fusion protein. Such desired properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility properties. As used herein, "XTEN" specifically excludes antibodies or antibody fragments, such as single-chain antibodies or Fc fragments of light or heavy chains.

[0179] In certain embodiments, the lentiviral vector of the present disclosure comprises at least one nucleotide sequence encoding XTEN or its fragment, variant, or derivative, wherein the nucleotide sequence encoding XTEN is inserted into the nucleotide sequence encoding a polypeptide having FIX activity, and the resulting fusion polypeptide has procoagulant 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 more than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, the XTEN is a peptide or polypeptide having from about 20 to about 3000 amino acid residues, from about 30 to about 2500 residues, from about 40 to about 2000 residues, from about 50 to about 1500 residues, from about 60 to about 1000 residues, from about 70 to about 900 residues, from about 80 to about 800 residues, from about 90 to about 700 residues, from about 100 to about 600 residues, from about 110 to about 500 residues, or from about 120 to about 400 residues. In one particular embodiment, the XTEN comprises an amino acid sequence longer than 42 amino acids and shorter than 144 amino acids in length.

[0181] The XTEN sequence can include 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 a sequence motif, which motif comprises, consists essentially of, or consists of 4 to 6 amino acids (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 the present disclosure are described in U.S. Patent Application Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, each of which is incorporated by reference in its entirety. are disclosed in International Patent Application Publication Nos. 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 heterologous moiety is an Fc region (e.g., an FcRn binding partner) or a fragment thereof. In certain aspects, the lentiviral vector of the present disclosure comprises at least one nucleotide sequence encoding an Fc region (e.g., an FcRn binding partner) that 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, where the resulting fusion polypeptide has procoagulant activity. "Fc" or "Fc region," as used herein, unless otherwise specified, refers to a functional neonatal Fc receptor (FcRn) binding partner that includes an Fc domain, a variant thereof, or a fragment thereof. An FcRn binding partner is any molecule that the FcRn receptor specifically binds to, and thus can actively transport an FcRn binding partner, including, but not limited to, albumin, by the FcRn receptor. Thus, the term Fc includes any variant of IgG Fc that is functional. 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), incorporated herein by reference in its entirety). 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. FcRn binding partners include, but are not limited to, whole IgG, Fc fragments of IgG, and other fragments of IgG that contain the complete binding region of FcRn. Fc comprises the CH2 and CH3 domains of immunoglobulins 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, e.g., increased half-life, e.g., increased in vivo half-life. Numerous mutants, fragments, variants, and derivatives are described, for example, in PCT Publication Nos. WO2011 / 069164A2, WO2012 / 006623A2, WO2012 / 006635A2, or WO2012 / 006633A2, all of which are incorporated by reference in their entirety herein.

[0184] The nucleotide sequence encoding one or more Fc domains is inserted into the nucleotide sequence encoding a polypeptide having FIX activity, or 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 heterologous moiety, such as XTEN, which is inserted into the nucleotide sequence encoding the 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 comprises a nucleotide sequence encoding a second Fc domain. The expressed second Fc domain can be associated 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 heterologous moiety is albumin, an albumin binding domain, or an albumin binding small molecule, or a variant, derivative, or fragment thereof. In certain aspects, the lentiviral vector of the present 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, or 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 procoagulant activity. Human serum albumin (HSA, or HA), a 609 amino acid protein in full-length form, is responsible for a significant portion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. As used herein, the term "albumin" includes full-length albumin or its functional fragment, variant, derivative, or analog. Examples of albumin or fragments or variants thereof are disclosed in U.S. Patent Application Publication Nos. 2008 / 0194481 A1, 2008 / 0004206 A1, 2008 / 0161243 A1, 2008 / 0261877 A1, or 2008 / 0153751 A1, or PCT Patent Application Publication Nos. 2008 / 033413 A2, 2009 / 058322 A1, or 2007 / 021494 A2, which are incorporated by reference in their entireties.

[0186] Albumin binding polypeptides (ABPs) may include, but are not limited to, bacterial albumin binding domains, albumin binding peptides, or albumin binding antibody fragments capable of binding to albumin. 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 having 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 by reference in their entireties.

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

[0188] In some embodiments, the albumin-binding polypeptide sequence in the expressed polypeptide is flanked at the C-terminus, N-terminus, or both termini 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 moiety is a C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, or a fragment, variant, or derivative thereof. In certain aspects, the lentiviral vector of the present disclosure comprises at least one nucleotide sequence encoding CTP or a fragment, variant, or derivative thereof, 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 procoagulant activity. It is known that the insertion of one or more CTP peptides into a recombinant protein increases 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). See, for example, US Patent Application Publication No. 2009 / 0087411A1, which is incorporated by reference. In some embodiments, the CTP sequence in the expressed polypeptide is flanked at the C-terminus, N-terminus, or both termini 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 moiety is a PAS peptide. In certain aspects, 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 procoagulant activity. "PAS peptide" or "PAS sequence" as used herein means an amino acid sequence that contains primarily alanine and serine residues or contains primarily alanine, serine, and proline residues, and forms a random coil conformation under physiological conditions. Thus, a PAS sequence is a building block, amino acid polymer, or sequence cassette that contains, consists essentially of, or consists of alanine, serine, and proline that can be used as part of a heterologous moiety in a fusion protein. However, when residues other than alanine, serine, and proline are added as minor components in the PAS sequence, the amino acid polymer can form a random coil conformation. "Minor 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 of the 100 amino acids of 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%. The amino acids different from 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 forms a random coil conformation, which can mediate increased in vivo and / or in vitro stability.

[0191] Non-limiting examples of PAS peptides include ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 20), AAPASPAPAAPSAPAAPS (SEQ ID NO: 21), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 22), APSSPSPSAPSSPSPASPS (SEQ ID NO: 23), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 24), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 25), ASAAAAPAAASAAASAPSAAA (SEQ ID NO: 26) or any variant, derivative, fragment, or combination thereof. Further examples of PAS sequences are known, for example, from US Patent Application Publication No. 2010 / 0292130A1 and PCT Patent Application Publication No. WO2008 / 155134A1. European issued patent EP2173890.

[0192] In some embodiments, the PAS sequence in the expressed polypeptide is flanked at the C-terminus, N-terminus, or both termini 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 heterologous moiety is a homoamino acid polymer (HAP) peptide or a fragment, variant, or derivative thereof. In certain aspects, the lentiviral vector of the present disclosure 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 procoagulant activity. The HAP peptide comprises a glycine repeat sequence having a length of at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids. The HAP sequence can extend the half-life of the moiety fused or linked to the HAP sequence. Non-limiting examples of HAP sequences include, but are not limited to, (Gly) n , (Gly4Ser) n or S(Gly4Ser) n 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, e.g., Schlapschy M et al., Protein Eng. Design Selection, 20:273-284 (2007).

[0194] C.2. Regulatory Elements In some embodiments, the lentiviral vector comprises a gene expression control element. The gene expression control sequence can be, for example, a mammalian or viral promoter, for example, a constitutive or inducible promoter. Mammalian constitutive promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary 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 terminal repeat (LTR), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus.

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

[0196] In one embodiment, the disclosure includes expressing the transgene under the control of a tissue-specific promoter and / or enhancer. In another embodiment, the promoter or other expression control sequence selectively enhances expression of the transgene in hepatocytes. Examples of liver-specific promoters include, but are not limited to, the mouse thyretin 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 comprises the mTTR promoter. The mTTR promoter is described in RH Costa et al., 1986, Mol. Cell. Biol. 6:4697. The F8 promoter is described in Figueiredo and Brownlee, 1995, J. Biol. Chem. 270:11828-11838. In some embodiments, the lentiviral vector comprises at least one tissue-specific promoter, i.e., a promoter that will regulate expression of a polypeptide having 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 the polypeptide having FIX activity in target liver cells.In some embodiments, the tissue-specific promoter selectively enhances the expression of the polypeptide having FIX activity in target liver cells comprises APOA2 promoter, SERPINA1 (hAAT) promoter, mTTR promoter, MIR122 promoter, ET promoter (GenBank No. AY661265; also see Vigna et al., Molecular Therapy 11(5):763 (2005)), or any combination thereof.In some embodiments, the target liver cell is a hepatocyte.

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

[0198] Other examples of suitable vectors and gene regulatory elements are described in WO02 / 092134, EP1395293, or US Pat. Nos. 6,808,905, 7,745,179, or 7,179,903, which are incorporated herein by reference in their entirety.

[0199] In general, expression control sequences will include, as appropriate, 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, such as the TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed sequences include a promoter region that includes a promoter sequence for transcriptional control of an operably linked coding nucleic acid. Gene expression sequences optionally include enhancer sequences or upstream activator sequences, as appropriate.

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

[0201] In some embodiments, the lentiviral vector comprises a tissue-specific promoter or multiple tissue-specific promoters that control the expression of FIX transgene in tissues other than the liver.In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of target cell or target tissue, for example, the genome of hepatocyte or the genome of liver endothelial cell.

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

[0203] In some embodiments, the lentiviral vector comprises a gag sequence, a pol sequence, a rev sequence, a rev response element (RRE), or any combination thereof. In certain embodiments, the lentiviral vector comprises a full-length gag sequence. In some embodiments, the lentiviral vector comprises a truncated gag sequence. In some embodiments, the lentiviral vector comprises a full-length pol sequence. In some embodiments, the lentiviral vector comprises a truncated pol sequence. In certain embodiments, the lentiviral vector comprises a full-length rev sequence. In some embodiments, the lentiviral vector comprises a truncated rev sequence. In certain embodiments, the lentiviral vector comprises a full-length RRE sequence. In some embodiments, the lentiviral vector comprises 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 polyA sequence, an intron sequence, or any combination thereof.In certain embodiments, the lentiviral vector comprises an enhancer that promotes the expression of nucleotide sequence in liver cells.

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

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

[0207] In other embodiments, lentiviral vector comprises 1, 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequence.However, in certain other embodiments, lentiviral vector does not comprise any miRNA target sequence.Whether to include miRNA target sequence (and its number) is selected by parameters such as intended tissue target, required expression level, etc.

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

[0209] In another embodiment, the target sequence is miR142 target (tccataaagt aggaaacact aca (SEQ ID NO: 27)). In one embodiment, the lentiviral vector comprises 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 comprises four copies of the miR-142 target sequence. In certain embodiments, the complementary sequence of a hematopoietic-specific microRNA, for example, miR-142 (142T) or "142-3pT", is incorporated into the 3' untranslated region of the lentiviral vector, making the transgene-encoded transcript susceptible to miRNA-mediated downregulation. This method can block transgene expression in hematopoietic lineage antigen-presenting cells (APCs), while maintaining it in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy can impose stringent post-transcriptional regulation on transgene expression, thus allowing stable delivery and long-term expression of the transgene. In some embodiments, miR-142 regulation prevents immune-mediated elimination of transduced cells and / or induces antigen-specific regulatory T cells (Tregs) and mediates robust immune tolerance to the antigen encoded by the transgene.

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

[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, whereby the partially complementary sequence is still recognized by the miRNA. In other words, in the context of the present disclosure, the partially complementary target sequence is effective in recognizing the corresponding miRNA and achieving inhibition or reduction of transgene expression in cells that express that miRNA. Examples of miRNA target sequences are described in WO2007 / 000668, WO2004 / 094642, WO2010 / 055413, or WO2010 / 125471, which are incorporated herein by reference in their entirety.

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

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

[0214] A schematic diagram of the lentiviral vector of the present disclosure is presented in FIG. 1. In some embodiments, the lentiviral vector of the present disclosure is a "third generation" lentiviral vector. As used herein, the term "third generation" lentiviral vector refers to a lentiviral packaging system that has the characteristics of a second generation vector system and further lacks a functional tat gene, e.g., the tat gene is deleted or inactivated. Generally, the gene encoding rev is provided on a separate expression construct. See, e.g., Dull et al. (1998) J. Virol. 72:8463-8471. As used herein, 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, e.g., Zufferey et al. (1997) Nat. Biotechnol. 15:871-875. As used herein, "packaging system" refers to a set of viral constructs that contain genes encoding viral proteins involved in packaging of recombinant virus. Generally, the constructs of a packaging system are ultimately integrated into a packaging cell.

[0215] In some embodiments, the third generation lentiviral vector of the present disclosure is a self-inactivating lentiviral vector. In some embodiments, the lentiviral vector is a VSV.G pseudotyped lentiviral vector. In some embodiments, the lentiviral vector comprises a mammalian specific promoter for transgene expression. In some embodiments, the mammalian specific promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the lentiviral vector comprises a hepatocyte specific promoter for transgene expression. In some embodiments, the hepatocyte specific promoter is an enhanced transthyretin promoter. In some embodiments, the lentiviral vector comprises one or more target sequences for miR-142 to reduce immune responses to the transgene product. In some embodiments, incorporating one or more target sequences for miR-142 into the lentiviral vector of the present disclosure allows for a desired transgene expression profile. For example, incorporating one or more target sequences for miR-142 can suppress transgene expression in the intravascular and extravascular hematopoietic system, while transgene expression is maintained in non-hematopoietic cells. No carcinogenesis has been detected in tumor-prone mice treated with the lentiviral vector system of the present disclosure. See Brown et al. (2007) Blood 110:4144-52, Brown et al. (2006) Nat. Ned. 12:585-91, and Cantore et al. (2015) Sci. Transl. Med. 7(277):277ra28.

[0216] The lentiviral vector of the present disclosure comprises a polynucleotide encoding a polypeptide having FIX activity as described herein. In one embodiment, the polypeptide having FIX activity is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that allows each component nucleic acid sequence to retain its function. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in a manner that places the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of the promoter in the 5' gene expression sequence results in transcription of the coding sequence, and if the nature of the link between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to induce transcription of the coding sequence, or (3) interfere with the ability to translate the corresponding RNA transcript into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0217] In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of transducing non-dividing cells. In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of transducing liver cells (e.g., hepatocytes). Lentiviral genomes and proviral DNA generally have three genes found in retroviruses: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene codes for internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene codes for RNA-guided DNA polymerase (reverse transcriptase), protease, and integrase; the env gene codes for viral envelope glycoproteins. The 5' and 3' LTRs serve to facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences required for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx (as in HIV-1, HIV-2 and / or SIV).

[0218] Sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient encapsidation of viral RNA into particles (Psi site) flank the 5'LTR. If sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA.

[0219] However, the resulting mutant remains capable of inducing the synthesis of all virion proteins. The present disclosure provides a method for generating a recombinant lentiviral vector capable of transducing non-dividing cells, comprising transfecting a suitable host cell with two or more vectors having packaging functions, i.e., gag, pol and env, and rev and tat. As disclosed herein below, for certain applications, vectors lacking a functional tat gene are desirable. Thus, for example, a first vector can provide nucleic acid encoding viral gag and viral pol, and another vector can provide nucleic acid encoding viral env to generate a packaging cell. Introducing a vector providing a heterologous gene, identified herein as a transfer vector, into the packaging cell provides a producer cell that releases infectious viral particles carrying the foreign gene of interest.

[0220] According to the above configuration of vector and foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retrovirus. In some embodiments, the env protein is an amphotropic envelope protein that allows transduction of cells of human and other species.

[0221] Examples of env genes from retroviruses include, but are not limited to, Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes, such as vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis virus, and influenza, can also be used. In some embodiments, the viral env nucleic acid sequence is operably associated with the regulatory sequences described elsewhere herein.

[0222] In certain embodiments, lentiviral vector is deleted of HIV pathogenicity genes env, vif, vpr, vpu and nef without impairing the vector's ability to transduce non-dividing cells.In some embodiments, lentiviral vector comprises deletion of U3 region of 3'LTR.Deletion of U3 region can be complete deletion or partial deletion.

[0223] In some embodiments, the lentiviral vector of the present disclosure, which comprises a polypeptide having the FIX activity nucleotide sequence described herein, can be transfected into a cell with (a) a first nucleotide sequence comprising gag, pol, or gag and pol genes, and (b) a second nucleotide sequence comprising a heterologous env gene; wherein the lentiviral vector lacks a functional tat gene.In other embodiments, the cell is further transfected with a fourth nucleotide sequence comprising a 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, a lentiviral vector of the present disclosure comprises one or more nucleotide sequences encoding a gag protein, a Rev response element, a central polypurine tract (cPPT), or any combination thereof.

[0225] In some embodiments, the lentiviral vector contains one or more polypeptides on its surface that improve the targeting and / or activity of the lentiviral vector or the polypeptide with FIX activity encoded therein. One or more polypeptides can be incorporated during the budding of the lentiviral vector from the host cell. During lentivirus production, the viral particles bud from the production 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 particle can include membrane-associated 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 immune response to the lentiviral vector after administration to a human subject. In some embodiments, the surface of the lentiviral vector comprises one or more CD47 molecules. CD47 is a "marker of self" protein that is ubiquitously expressed on human cells. Surface expression of CD47 suppresses macrophage-induced phagocytosis of endogenous cells through the interaction of CD47 and SIRPα expressed by macrophages. Cells that express high levels of CD47 are less likely to be targeted and destroyed by human macrophages in vivo.

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

[0228] In some embodiments, the lentiviral vector comprises a human CD47 comprising 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 set forth in SEQ ID NO:14.

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

[0230] In certain embodiments, the lentiviral vector comprises a lipid coat that contains a high concentration of CD47 polypeptide and lacks MHC-I polypeptides. high / MHC-I low Cell lines, e.g., CD47 high / MHC-I low In some embodiments, the lentiviral vector is generated in a HEK293T cell line. high / MHC-I free Cell lines, e.g., CD47 high / MHC-I free Produced in 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] III. Pharmaceutical Compositions The compositions containing the lentiviral vectors, nucleic acid molecules, polypeptides encoded by the nucleic acid molecules, or host cells of the present disclosure can contain suitable pharma- ceutically acceptable carriers.For example, they can contain excipients and / or auxiliary agents that facilitate the processing of the active compounds into preparations designed for delivery to the site of action.

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

[0233] The pharmaceutical composition can be formulated for parenteral administration (i.e., intravenous, subcutaneous, or intramuscular administration) by bolus injection. The preparation for injection can be supplied in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of preservatives. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient can be in powder form, which is made up of a suitable vehicle, for example, 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, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. An intravenous form of parenteral administration is preferred. Although all these forms of administration are clearly anticipated to be within the scope of the present disclosure, particularly for intravenous or intraarterial injection or infusion, the form for administration will be an injectable solution. Typically, suitable pharmaceutical compositions for injection can include buffers (e.g., acetate, phosphate or citrate buffers), surfactants (e.g., polysorbates), optionally stabilizers (e.g., human albumin), and the like. However, in other methods consistent with the teachings of the present specification, the lentiviral vector can be delivered directly to the site of harmful cell populations, 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, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In this disclosure, pharma- ceutically acceptable carriers include, but are not limited to, 0.01-0.1M, preferably 0.05M, phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may be present, such as antimicrobials, antioxidants, chelating agents, and inert gases.

[0236] More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability remains. It should be stable under the conditions of manufacture and storage, and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0237] Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0238] In any case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., the polypeptide itself or a combination with other active agents) in the required amount in a suitable solvent, together with one or a combination of the components listed herein as necessary, followed by filtration sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains 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, the preferred preparation method is vacuum drying and freeze-drying, which gives a powder of the active ingredient and any additional desired ingredients from its solution that has previously been sterilized by filtration. The preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions by methods known in the art. In addition, the preparations can be packaged and sold in the form of a kit. Such products preferably have a label or package insert indicating that the relevant composition is useful for treating subjects suffering from or predisposed to coagulation disorders.

[0239] Injectable depot preparations can be made by forming microencapsulated matrices of drugs in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer and the nature of the polymer used, the rate of drug release can be controlled.Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides.Injectable depot preparations can also be prepared by entrapping drugs in liposomes or microemulsions.

[0240] Pharmaceutical compositions can also be formulated for rectal administration as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0241] Supplementary active compounds can be incorporated into the composition.In one embodiment, the nucleic acid molecule of the present disclosure is formulated with coagulation factors, or variants, fragments, analogs or derivatives thereof.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 form of any of these.Hemostatic coagulation factors can also include antifibrinolytic agents, such as epsilon-aminocaproic acid, tranexamic acid.

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

[0243] The intermediate doses within the above range are also within the scope of the present disclosure.Such doses can be administered to subjects daily, every other day, weekly or according to any other schedule that is determined by experimental analysis.Exemplary treatment requires administration of multiple doses over a long period, for example, at least 6 months.

[0244] The lentiviral vectors of the present disclosure are administered to subjects at different developmental stages. For example, in humans, the 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 (greater than 16 years old). In some embodiments, the lentiviral vectors of the present disclosure are administered to human neonates. In some embodiments, the lentiviral vectors of the present disclosure are administered to human subjects less than about 1 month old. In some embodiments, the lentiviral vectors of the present disclosure are administered to human infants. In some embodiments, the lentiviral vectors of the present disclosure are administered to human subjects about 1 month old to about 2 years old. In some embodiments, the lentiviral vectors of the present disclosure are administered to human children. In some embodiments, the lentiviral vectors of the present disclosure are administered to human subjects about 2 years old to about 12 years old. In some embodiments, the lentiviral vectors of the present disclosure are administered to human adolescents. In some embodiments, the lentiviral vector of the present disclosure is administered to a human subject between about 12 and about 16 years of age. In some embodiments, the lentiviral vector of the present disclosure is administered to a human adult. In some embodiments, the lentiviral vector of the present disclosure is administered to a human subject greater than about 16 years of age. One of skill in the art can determine the developmental stages of other organisms. For example, one of skill in the art would understand that a 2-week-old mouse is an adolescent.

[0245] The dosage and frequency of administration of the lentiviral vectors of the present disclosure will vary depending on a variety of factors known to those of skill in the art.

[0246] The lentiviral vectors of the present disclosure can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (eg, prophylactic or therapeutic).

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

[0248] It is further understood that the lentiviral vector of the present disclosure can be used together or in combination with a drug or drugs (e.g., to provide a combination therapy regimen). Exemplary drugs that can be combined with the lentiviral vector of the present disclosure include drugs that represent the current medical standard for the particular disorder to be treated. Such drugs can be chemical or biological in nature. The term "biological" or "biological agent" refers to any pharmacologic active agent that is made from living organisms and / or their products and is intended for use as a therapeutic agent.

[0249] The amount of drugs used in combination with the lentiviral vectors of the present disclosure can vary from subject to subject or can be administered according to what is known in the art. See, for example, Bruce A Chabner et al., Antineoplastic Agents (Joel G. Hardman et al., eds., 9th ed. 1996), in GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, pp. 1233-1287. In another embodiment, the amount of such drugs is administered consistent with medical standards.

[0250] In certain embodiments, the lentiviral vector of the present disclosure is administered together with an immunosuppressant, antiallergic, or anti-inflammatory agent. These agents generally refer to substances that act to suppress or mask the immune system of the subject being treated herein. These agents include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines; azathioprine; cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde; anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as glucocorticoids, for example, prednisone, methylprednisolone, and dexamethasone; anti-interferon-gamma, -beta, or -alpha antibodies, anti-tumor necrosis factor-alpha antibodies, anti-tumor necrosis factor-beta Antibodies include cytokine 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; streptodornase; FK506; RS-61443; deoxyspergualin; and rapamycin. In certain embodiments, the drug is an antihistamine. As used herein, "antihistamine" refers to a drug that antagonizes the physiological action of histamine. Examples of antihistamines are chlorpheniramine, diphenhydramine, promethazine, cromolyn sodium, astemizole, azatadine maleate, brompheniramine maleate, carbinoxamine maleate, cetirizine hydrochloride, clemastine fumarate, cyproheptadine hydrochloride, d-brompheniramine maleate, d-chlorpheniramine maleate, dimenhydrinate, diphenhydramine hydrochloride, doxylamine succinate, fexofendazine hydrochloride, terfenadine hydrochloride, hydroxyzine hydrochloride, loratidine, meclizine hydrochloride, tripelennamine citrate, tripelennamine hydrochloride and triprolidine hydrochloride.

[0251] Immunosuppressant, antiallergic or anti-inflammatory agent can be incorporated into lentiviral vector administration regimen.For example, administration of immunosuppressant or anti-inflammatory agent can be started before administration of the disclosed lentiviral vector, and can be continued for one or more times after administration.In certain embodiments, immunosuppressant or anti-inflammatory agent is administered as a premedication of lentiviral vector.

[0252] As previously discussed, the lentiviral vector of the present disclosure can be administered in a pharmacologic effective amount for the in vivo treatment of coagulation disorders.In this regard, it is understood that the lentiviral vector of the present disclosure can be formulated to facilitate administration and promote the stability of the active agent.Preferably, the pharmaceutical composition according to the present disclosure comprises a pharmacologic acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffer, preservative, etc.Of course, the pharmaceutical composition of the present disclosure can be administered in a single or multiple doses to provide a pharmacologic effective amount of polypeptide.

[0253] In addition to the active compound, liquid dosage forms may contain inactive ingredients such as water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, 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 EW Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerin monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, etc. The composition can also contain pH buffering agents, and wetting or emulsifying agents.

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

[0256] In some embodiments, the composition is used to treat a bleeding disease or condition in a subject in need thereof. The bleeding disease or condition is selected from the group consisting of bleeding coagulation disorders, hemarthrosis, muscle bleeding, oral bleeding, major bleeding, major bleeding into muscles, major oral bleeding, trauma, head trauma, gastrointestinal bleeding, major intracranial bleeding, major intraperitoneal bleeding, major intrathoracic bleeding, bone fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, iliopsoas external bleed, and any combination thereof. In still other embodiments, the subject is scheduled to undergo surgery. In still other embodiments, the treatment is prophylactic or on demand.

[0257] To investigate the function of the coagulation system, several tests are available: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM® assay, prothrombin time (PT) test (also used to determine the INR), fibrinogen test (often by the Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (whether abnormalities are corrected when the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimers, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), other platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0258] The aPTT test is a performance indicator that measures the efficacy of the "intrinsic" (also called 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 together with the prothrombin time (PT), which measures the extrinsic pathway.

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

[0260] IV. Nucleic acid molecules The present disclosure also provides an isolated nucleic acid molecule sequence encoding a polypeptide having FIX activity. In certain embodiments, the isolated nucleic acid molecule 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 to the nucleotide sequence set forth in SEQ ID NO:1.

[0261] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 85% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 91% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 92% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 93% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 94% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 96% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence has at least about 98% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleotide sequence is identical to the nucleotide sequence set forth 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 has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to (i) nucleotides 1-84 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 comprises the nucleotide sequence set forth 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 to (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 comprises the nucleotide sequence set forth 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] The present disclosure also provides a vector comprising the nucleic acid molecule described herein.In some embodiments, the vector is a lentiviral vector, for example, any lentiviral vector disclosed herein.In certain embodiments, the vector comprises the nucleic acid sequence with 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 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 comprises 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 to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 85% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 91% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 92% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 93% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 94% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 96% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 98% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence having at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the vector comprises a nucleotide sequence that is identical to the nucleotide sequence set forth 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 the polypeptide having FIX activity in target liver cells.In certain embodiments, the tissue-specific promoter selectively enhances the expression of the polypeptide having FIX activity in target liver cells comprises APOA2 promoter, SERPINA1 (hAAT) promoter, mTTR promoter, MIR122 promoter, or any combination thereof.In some embodiments, the target liver cell is a hepatocyte.

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

[0268] Different miRNA target sequences are also included.For example, lentiviral vectors expressing more than one transgene can have more than one transgene under the control of miRNA target sequences, which can be the same or different.miRNA target sequences can be arranged in tandem, but other arrangements are also included.Transgene expression cassettes containing miRNA target sequences can also be inserted into lentiviral vectors in antisense orientation.Antisense orientation can be useful in the production of viral particles to avoid the expression of gene products that may otherwise be toxic to the production cell.

[0269] In other embodiments, lentiviral vector comprises 1, 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequence.However, in certain other embodiments, lentiviral vector does not comprise any miRNA target sequence.Whether to include miRNA target sequence (and its number) is selected by known parameters such as intended tissue target, required expression level, etc.

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

[0271] In another embodiment, the target sequence is miR142 target (tccataaagt aggaaacact aca (SEQ ID NO: 27)). In one embodiment, the lentiviral vector contains 4 copies of the miR-142 target sequence. In a particular embodiment, the complementary sequence of hematopoietic specific microRNA, for example miR-142 (142T), is incorporated into the 3' untranslated region of the lentiviral vector, making the transgene coding transcript susceptible to miRNA-mediated downregulation. This method can block transgene expression in hematopoietic lineage antigen presenting cells (APCs), while maintaining it in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy can impose stringent post-transcriptional regulation on transgene expression, thus allowing stable delivery and long-term expression of the transgene. In some embodiments, miR-142 modulation prevents immune-mediated elimination of transduced cells and / or induces antigen-specific regulatory T cells (Tregs) and mediates robust immune tolerance to the antigen encoded by the transgene.

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

[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, whereby the partially complementary sequence is still recognized by the miRNA. In other words, in the context of the present disclosure, the partially complementary target sequence is effective in recognizing the corresponding miRNA and achieving inhibition or reduction of transgene expression in cells that express that miRNA. Examples of miRNA target sequences are described in WO2007 / 000668, WO2004 / 094642, WO2010 / 055413, or WO2010 / 125471, which are incorporated herein by reference in their entirety.

[0274] VI. Host Cells and Methods of Making The present disclosure also provides a host cell comprising the nucleic acid molecule or lentiviral vector of the present disclosure.A certain aspect of the present disclosure relates to making or generating lentiviral vector, comprising transfecting and / or transforming host cell with the lentiviral vector disclosed herein.As used herein, the term "transformation" is used in a broad sense to refer to the introduction of DNA into recipient host cell, which changes genotype and results in change in the recipient cell.

[0275] "Host cell" refers to a cell transformed with the lentiviral vector disclosed herein. The host cell of the present disclosure is 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 that is most suitable for their purpose. Exemplary host cell lines include, but are not limited to, CHO, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with the SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), PER.C6®, NS0, CAP, BHK21, and HEK293 (human kidney). In a particular embodiment, the host cell is 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 the present disclosure are of insect origin. In a particular embodiment, the host cell is an SF9 cell. Host cell lines are generally available from commercial services American Tissue Culture Collection or from published literature.

[0276] Introduction of the nucleic acid molecules or vectors of the present disclosure into host cells can be accomplished by a variety of techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG, Mammalian Expression Vectors, Chapter 24.2, pp. 470-472, Vectors, eds. Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). Most preferably, introduction of the plasmid into the host is by electroporation. The transformed cells are grown under conditions appropriate to produce the 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), immunohistochemistry, and the like.

[0277] The host cells containing the isolated nucleic acid molecule or lentiviral vector of the present disclosure are grown in a suitable growth medium. As used herein, the term "suitable growth medium" refers to a medium containing nutrients required for cell growth. Nutrients required for cell growth include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium can contain one or more selection factors. Optionally, the medium can contain calf serum or fetal calf serum (FCS). In one embodiment, the medium is substantially free of IgG. The growth medium generally selects for cells containing the DNA construct, for example, by drug selection or by depletion of an essential nutrient that is supplemented by a selectable marker on the DNA construct or co-transfected 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, Calif.). In another embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.) Selection of an appropriate medium for the particular cell line used is within the level of ordinary skill in the art.

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

[0279] Certain aspects of the present disclosure relate to methods of producing a lentiviral vector comprising culturing a host cell described herein under suitable conditions and isolating the lentiviral vector. In certain aspects, the present disclosure relates to methods of producing a lentiviral vector disclosed herein comprising culturing a host cell described herein under suitable conditions and isolating the lentiviral vector.

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

[0281] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0282] Having generally described the present disclosure, a better understanding can be obtained by reference to the examples provided herein, which are for illustrative purposes only and are not intended to be limiting. 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 an R338L ("Padua") substitution (coFIX-1-R338L; SEQ ID NO: 1) was cloned into a lentiviral vector to generate LV-coFIX-1-R338L (Figure 1). To determine the dose-response profile of LV-FIX in an animal model, LV-coFIX-1-R338L produced 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). LV-FIX-mediated plasma FIX activity and antigen levels were monitored by FIX chromogen and ELISA assays. Steady-state FIX plasma levels for each animal are shown in Figure 2A, and the LV-coFIX-1-R338L dose-response curve is 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 LV-coFIX-1-R338L dose levels required to achieve 10-200% normal circulating FIX activity ranged from 5E9-2E10 TU / kg in HemB mice.

[0284] The long-term FIX expression profile of animals treated with LV-coFIX-1-R338L in the three higher dose level groups was monitored for 6 months after LV treatment. Circulating FIX activity (Figure 3A) and antigen (Figure 3B) levels were plotted. Consistent levels of LV-mediated FIX expression were observed in all experimental animals, and no loss of FIX expression was detected within the study period, demonstrating the long-term stability of the integrated gene therapy treatment. Furthermore, a lower percentage of normal FIX antigen levels was observed (Figure 3A) compared to those of FIX activity (Figure 3B), reflecting the use of the gain of functional R338L mutation in the FIX transgene. EXAMPLES

[0285] LV-coFIX-1-R338L has similar transduction efficiency in adult and newborn animals. Lentiviral vectors can integrate into the host genome to mediate long-term transgene expression, and unlike the rapid loss of AAV-mediated transgene expression 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 7.5E9, 2E10 and 6E10 TU / kg. Compared with adult treatment (administered at 8 weeks), systemically administered LV-coFIX-1-R338L mediated sustained similar levels of FIX expression over the 6-month study period at each dose level, suggesting that both adult and pediatric patients can be efficiently treated by administration of lentiviral FIX. Treatment of adolescent mice with LV-coFIX-1-R338L via temporal vein injection at doses of 3E9, 7.5E9, or 2E10 TU / kg (administered at 2 weeks) was also investigated. The levels of FIX expression at each dose level were higher than the corresponding doses in mice administered treatment at 8 weeks or 2 days (n=6 animals / dose level / day (weeks) of age; Figure 4A). FIX activity was measured and the dose response of LV-coFIX-1-R338L determined in HemB mice administered 7.5E9, 2E10, and 6E10 TU / kg doses at 8 weeks and 2 days via temporal vein injection, and 3E9, 7.5E9, or 2E10 TU / kg doses at 2 weeks. Consistent with the longitudinal data in Figure 4A, mice administered treatment at 2 weeks (adolescent mice) showed higher FIX activity compared to mice administered treatment at 2 days or 8 weeks (Figure 4B). EXAMPLES

[0286] Evaluation of CD47high LV-coFIX-1-R338L in non-human primates To modulate the immune properties of lentiviral vectors, we generated a HEK293T cell line that overexpresses 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, lentiviral vector particles with high surface human CD47 expression were taken up by macrophages less than control lentiviral vectors that do not overexpress CD47 (Figure 5).

[0287] To further evaluate the effect of high surface levels of human CD47 on hepatic transduction in vivo, CD47 was expressed in non-human primates (NHPs) after intravenous administration at a dose of 7.5E9TU / kg, n=3 / treatment group. high LV-coFIX-1-R338L was compared with LV-coFIX-1-R338L using pigtailed macaques to circumvent lentiviral vector limitations in NHPs following treatment.

[0288] Circulating human FIX levels after treatment with lentiviral vectors were measured by human FIX specific activity (Figure 6A) and antigen assays (Figure 6B). high LV-coFIX-1-R338 conferred 3-fold higher human FIX expression after treatment with lentiviral vectors compared to LV-coFIX-1-R338L, which are 200-300% and 50-150% of normal FIX activity levels, respectively (Figure 6A). high The use of LV-coFIX-1-R338L could potentially reduce LV-FIX and the acute toxicity associated with lentiviral vector treatment.

[0289] In addition to human FIX expression levels, the homeostasis of treated animals was also monitored by APTT assay (Figure 6C).While the APTT times of vehicle-treated animals remained within the same range, 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 was functionally active. EXAMPLES

[0290] Dose response of CD47high LV-coFIX-1-R338L in non-human primates CD47 in NHPs high To determine the dose-response profile of LV-coFIX-1-R338L, two low doses of CD47 1.5E9 and 3E9TU / kg (n=3 / dose level) were administered. high LV-coFIX-1-R338L was tested. Lentiviral vector-mediated human FIX expression was monitored by analyzing steady-state circulating human FIX-specific activity (Figure 7A) and antigen levels (Figure 7B).

[0291] Consistent with the results observed in HemB mice, we also observed a log / log dose-response curve with LV-coFIX-1-R338L in NHPs. high The dose range of LV-coFIX-1-R338L is 3.5-6E9TU / kg, lower than that in HemB mice. The shift in therapeutic dose range is due to 5-10-fold higher human FIX expression levels in NHPs compared to HemB mice at the same dose levels, which may be due to the change in animal species and recognition of human CD47 (human CD47 is not recognized in HemB mice).

[0292] Animals treated with LV-coFIX-1-R338L showed a very mild acute immune response, as shown by ALT levels (Figure 8A), AST levels (Figure 8B), lymph levels (Figure 8C) and body temperature (Figure 8D) after treatment.high A reduced cytokine response was observed following administration of LV-coFIX-1-R338L compared to the LV control vector (Figures 9A-9C). Modest increases in MIP-1a, MIP-1b, and MCP-1 were observed following administration of control LV, MIP-1a, MIP-1b, and MCP-1, whereas CD47 high After administration of LV-coFIX-1-R338L, expression was reduced and in some cases undetectable. As expected, LV-coFIX-1-R338L was primarily localized to the liver and spleen, which had over 100-fold higher vector copy numbers (VCN) than other organs (Figure 10). These data support the conclusion that CD47 high These results suggest that LV-coFIX-1-R338L induces a decrease in the allospecific immune response, concomitantly increasing resistance to phagocytosis and improving gene transfer into hepatocytes. EXAMPLES

[0293] Further study of dose response of CD47high LV-coFIX-1-R338L in non-human primates CD47 at a dose of 2.5E9TU / kg high Additional pigtailed macaques were treated with LV-coFIX-1-R338L by intravenous administration. Lentiviral vector-mediated human FIX expression was monitored by analyzing steady-state circulating human FIX specific activity (FIG. 11A) and antigen levels (FIG. 11B). After LV treatment, steady-state circulating human FIX activity was approximately 33% of normal, and circulating human FIX antigen levels were 700 ng / mL, correlating with 14% of normal FIX antigen levels.

[0294] The above description of the specific embodiments sufficiently reveals the general nature of the present disclosure, so that others can easily modify such specific embodiments and / or apply various applications thereto by applying knowledge of the art without undue experimentation and without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the embodiments of the present disclosure, based on the teaching and guidance presented herein. It should be understood that the phraseology or terminology used herein is for the purpose of description and not for the purpose of limitation, and thus the terminology or terminology used herein should be interpreted by those skilled in the art in light of the teaching and guidance.

[0295] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

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

Claims

1. 1. A pharmaceutical composition comprising a lentiviral vector for use in a method for preventing or treating a disorder caused by a deficiency of factor IX in a subject in need thereof, comprising: The lentiviral vector comprises: A nucleotide sequence of SEQ ID NO:1, which encodes a polypeptide having factor IX (FIX) activity, and A lipid coat comprising the CD47 protein, Including, The method is carried out at 1×10 9 and 6×10 9 administering to the subject an effective dose of the lentiviral vector in between transducing units / kg (TU / kg); the lentiviral vector is packaged in HEK293T cells that overexpress CD47 and contains higher levels of surface CD47 protein on its lipid coat than a control lentiviral vector produced in unmodified HEK293T cells; 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; and The lentiviral vector at an effective dose induces circulating FIX activity up to 100% of normal levels that persists for at least 60 days after administration. The pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, wherein the lentiviral vector comprises at least 1.5 times, at least 2.0 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times more CD47 protein on the surface of the lentiviral vector than a control lentiviral vector produced in unmodified HEK293T cells.

3. The effective dose is 6×10 9 Less than TU / kg, 5 x 10 9 Less than TU / kg, 4 x 10 9 Less than TU / kg, 3 x 10 9 Less than TU / kg or 2 x 10 9 The pharmaceutical composition of claim 1 or 2, wherein the dose is less than TU / kg.

4. The subject exhibits the following characteristics after administration of the pharmaceutical composition: (a) reduced transduction of macrophages by lentiviral vectors compared to a control lentiviral vector; (b) a reduced allospecific immune response to the lentiviral vector compared to a control lentiviral vector, preferably the allospecific immune response comprises the release of a cytokine in response to the lentiviral vector, preferably the cytokine is selected from the group consisting of MIP-1a, MIP-1b, MCP-1, and any combination thereof; (c) at least 30% 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) any combination of (a) to (d) The pharmaceutical composition according to any one of claims 1 to 3, which exhibits one or more of the following:

5. The lentiviral vector results in an increase in plasma FIX activity in a subject 24 to 48 hours after administration of the lentiviral vector. The pharmaceutical composition according to any one of claims 1 to 4.

6. the subject exhibits, following administration of the lentiviral vector, increased localization of the lentiviral vector to the liver, spleen, or both the liver and the spleen relative to organs other than the liver and spleen of the subject; Preferably, the increased localization is characterized by a vector copy number (VCN) of the lentiviral vector that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 110-fold, at least 120-fold, at least 130-fold, at least 140-fold, at least 150-fold, at least 160-fold, at least 170-fold, at least 180-fold, at least 190-fold, or at least 200-fold greater in the liver, spleen, or both the liver and the spleen following administration of the lentiviral vector relative to organs other than the liver and spleen of the subject. The pharmaceutical composition according to any one of claims 1 to 5.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the polypeptide having FIX activity preferably comprises the amino acid sequence shown in SEQ ID NO:

12.

8. The lentiviral vector comprises a tissue-specific promoter, Preferably, the tissue-specific promoter selectively enhances expression of a polypeptide having FIX activity in a target liver cell, preferably the target liver cell is a hepatocyte; More preferably, the tissue-specific promoter comprises an APOA2 promoter, a SERPINA1 (hAAT) promoter, a mTTR promoter, an MIR122 promoter, or any combination thereof.

9. the lentiviral vector comprises a splice acceptor site; the lentiviral vector comprises a gag sequence, a pol sequence, a rev sequence, a rev response element (RRE), or any combination thereof, preferably the gag sequence is a full-length or truncated gag sequence; and / or 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; The pharmaceutical composition according to any one of claims 1 to 8.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the FIX polypeptide is a R338L variant FIX polypeptide.

11. The lentiviral vector does not contain an MHC-I polypeptide and / or is produced in a host cell that does not express MHC-I. The pharmaceutical composition according to any one of claims 1 to 10.

12. The lentiviral vector is 1 μm 2 20 to 1 μm per 2 containing 1000 CD47 polypeptides per The pharmaceutical composition according to any one of claims 1 to 11.

13. The lentiviral vector is 1 μm 2 at least 225 CD47 polypeptides per 13. The pharmaceutical composition of claim 12.

14. The lentiviral vector is 1 μm 2 At least 400 CD47 polypeptides per 13. The pharmaceutical composition of claim 12.

15. the nucleotide sequence encoding a polypeptide having factor IX (FIX) activity further comprises a heterologous nucleotide sequence encoding a heterologous amino acid sequence; the heterologous amino acid sequence is albumin, an immunoglobulin Fc region, an XTEN sequence, a C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), a PAS sequence, a HAP sequence, a CTP peptide sequence, transferrin, an albumin binding portion, or any fragment, derivative, variant, or combination of these polypeptides; The heterologous amino acid sequence is linked 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 of the amino acid sequence. The pharmaceutical composition according to any one of claims 1 to 14.

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