Bioproduction methods for lentiviral vectors

Abstract

To provide methods for making stable producer cell lines that produce SIN-lentivirus vectors at scales to support clinical trials.SOLUTION: From a lentiviral vector transfer plasmid, and from a second plasmid such as an antibiotic resistance cassette plasmid, a DNA fragment is generated, and the DNA fragment is used to form a concatemeric array. Then, the concatemeric array is introduced into a packaging cell line by, for example, transfection.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 161,133, filed May 13, 2015, and further claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 161,152, filed May 13, 2015, the disclosures of which are hereby incorporated herein by reference in their entirety.

[0002] Field of the Disclosure The present disclosure generally relates to the fields of molecular biology and virology. In particular, the present disclosure relates to the bioproduction of lentiviral vectors and lentiviral transfer plasmids.

[0003] Description of Industrial Applicability The present disclosure has industrial applicability in the fields of gene therapy and biomanufacturing.

Background Art

[0004] Background of the Disclosure HIV - 1 is the causative pathogen of acquired immunodeficiency syndrome (AIDS), which infects approximately 30 million people worldwide. HIV impairs the immune system and increases the probability of death from opportunistic infections. As is evident from its designation as a pandemic by the World Health Organization, HIV infection is a major global health problem. Many people infected with HIV, especially in developing countries, ultimately develop AIDS, and more than one million people are killed every year.

[0005] HIV-1 is a retroviral virus with an envelope, whose genome consists of two single-stranded RNA molecules (ssRNA). The primary target of HIV-1 is CD4+ expressing cells, such as CD4+ T cells. The HIV-1 viral glycoprotein interacts with the CD4 molecule on the target cell and with the chemokine co-receptor CCRS or CXCR4 on the target cell surface. After fusion and entry into the target cell, the nucleocapsid containing the viral genome dissociates, releasing the viral contents, including ssRNA, into the cytoplasm. The HIV-1 reverse transcription (RT) enzyme synthesizes double-stranded viral DNA (dsDNA) from the ssRNA genome. After the synthesis of the double-stranded HIV-1 DNA molecule, the HIV-1 DNA is integrated into the host genome.

[0006] The integrated HIV-1 DNA is flanked by identical 5' and 3' terminal repeat sequences (LTRs), from which HIV-1 can initiate transcription of the integrated HIV-1 genome. Transcription of viral DNA requires transcription factors such as NF-κB, which are upregulated in activated T cells. As a result, viral transcription is most active in activated T cells, such as during infection. The viral RNA resulting from the transcription of the integrated HIV-1 genome is subsequently translated and packaged into viral particles, which then leave the cell and become infectious viruses.

[0007] Treatment for HIV-1 infection includes combination antiretroviral therapy (cART). cART involves a combination of nucleoside analog reverse transcriptase inhibitors, protease inhibitors, non-nucleoside reverse transcriptase inhibitors, integrases, and fusion inhibitors to slow the progression of HIV. This, in turn, dramatically reduces HIV / AIDS morbidity and mortality in the regions of the world where the treatment is available. However, cART does not cure or completely eliminate all symptoms of HIV / AIDS. Furthermore, cART therapy can be jeopardized by drug-resistant mutations and has a variety of side effects that appear serious and cumulative. Additionally, discontinuation of cART therapy has been shown to almost without exception lead to a relapse of detectable viral replication and progression to AIDS, and is associated with an increased incidence of death from any cause and serious non-AIDS events. For these reasons, as well as the high cost and strict adherence to medication required for cART, this treatment may be relatively ineffective for many patients.

[0008] HIV-based lentiviral vectors are rapidly becoming the retroviral vector system of choice for research and clinical applications of gene delivery. Improved ability of lentiviral vectors to transduce both quiescent stem cells and non-mitotic terminal cells has led to the development of a wide range of therapeutic gene delivery vectors, as well as promising research tools such as short hairpin RNA (shRNA) gene knockdown libraries and vectors for inducing pluripotency in terminal cells. Early gamma retroviral clinical gene therapy vectors restored immune function in patients with X-linked severe combined immunodeficiency (SCID-X1), but they were later found to cause proliferative disorders through the transactivation of proto-oncogenes. Newer lentiviral vector designs may greatly reduce their risks, and they are awaiting clinical trials for final confirmation of their predicted safety. The field remains fluid, and the results of its clinical trials are unpredictable.

[0009] Producing SIN-lentiviral vectors on a scale that supports clinical trials is a critical challenge in this field. While gamma retroviral vectors can be produced by either transient transfection or the creation of stable producing cell lines, lentiviruses require the expression of multiple cytotoxic accessory genes, which complicates the creation of producing cells (Greene et al., Transduction of Human CD34+ Repopulating Cells with a Self-Inactivating Lentiviral Vector for SCID-X1 Produced at Clinical Scale by a Stable Cell Line, HGTM, 23, 297-308 (October 2012) (Non-Patent Literature 1) is incorporated herein by reference as a whole). Transient transfection is rather a recent technique for pilot production of LVs and is not practical for ultra-large-scale applications from the standpoint of safety, cost, and reproducibility. In fact, this technology is expensive, difficult to standardize and scale up, and suffers from batch-to-batch variability and low reverse transcriptase fidelity (Stornaiuolo et al., RD2-MolPack-Chim3, a Packaging Cell Line for Stable Production of Lentiviral Vectors for Anti-HIV Gene Therapy, HGTM, 24:228-240 (August 2013) (Non-Patent Literature 2) is incorporated herein by reference as a whole). [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Greene et al., Transduction of Human CD34+ Repopulating Cells with a Self - Inactivating Lentiviral Vector for SCID-X1 Produced at Clinical Scale by a Stable Cell Line, HGTM, 23, 297-308 (October 2012) [Non-Patent Document 2] Stornaiuolo et al., RD2-MolPack-Chim3, a Packaging Cell Line for Stable Production of Lentiviral Vectors for Anti-HIV Gene Therapy, HGTM, 24:228-240 (August 2013) [Overview of the Initiative]

[0011] Summary of this disclosure One aspect of this disclosure is a plasmid containing a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence has at least 95% identity to the sequence of SEQ ID NO: 1. In some embodiments, a lentiviral vector is derived from the plasmid. In some embodiments, this derived lentiviral vector contains one or more additional sequences encoding short hairpin RNA and / or HIV-1 fusion inhibitors for downregulation of the HIV-1 coreceptor. In some embodiments, the derived lentiviral vector is LVsh5 / C46 (as defined herein).

[0012] Another aspect of this disclosure is a plasmid containing the nucleotide sequence of SEQ ID NO: 1. In some embodiments, a lentiviral vector is derived from the plasmid. In some embodiments, this derived lentiviral vector includes one or more additional sequences encoding short hairpin RNA and / or an HIV-1 fusion inhibitor (e.g., C46) for downregulation of an HIV-1 coreceptor (e.g., CCR5). Information regarding CCR5 and C46, ​​including their nucleotide sequences, is further described in U.S. Patent Application Publication No. US2012 / 0201794, the disclosure of which is incorporated herein by reference in whole.

[0013] Another aspect of this disclosure is a plasmid containing a nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO: 2. In some embodiments, the plasmid contains a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 2. In some embodiments, the plasmid contains a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 2. In some embodiments, the plasmid contains a nucleotide sequence having at least 97% identity to the sequence of SEQ ID NO: 2. In some embodiments, the plasmid contains the nucleotide sequence of SEQ ID NO: 2. In some embodiments, a lentiviral vector is derived from the plasmid. In some embodiments, this derived lentiviral vector contains one or more additional sequences encoding short hairpin RNA and / or an HIV-1 fusion inhibitor (e.g., C46) for downregulation of an HIV-1 coreceptor (e.g., CCR5).

[0014] In another aspect of this disclosure, there are plasmids having a sequence that differs from the sequence shown at SEQ ID NO: 1 (e.g., discontinuous or continuous) by 500 nucleotides or less. In another aspect of this disclosure, there are plasmids having a sequence that differs from the sequence shown at SEQ ID NO: 1 (e.g., discontinuous or continuous) by 250 nucleotides or less. In another aspect of this disclosure, there are plasmids having a sequence that differs from the sequence shown at SEQ ID NO: 1 (e.g., discontinuous or continuous) by 150 nucleotides or less. In another aspect of this disclosure, there are plasmids having a sequence that differs from the sequence shown at SEQ ID NO: 1 (e.g., discontinuous or continuous) by 100 nucleotides or less. In some embodiments, the sequence differs from the sequence shown at SEQ ID NO: 1 (e.g., discontinuous or continuous) by 50 nucleotides or less.

[0015] Another aspect of this disclosure is a plasmid containing approximately 6,500 to 6,750 nucleotides, wherein the plasmid contains a sequence or fragment thereof that has at least 90% identity with the sequence of SEQ ID NO: 2. In some embodiments, the plasmid contains approximately 6,600 to 6,700 nucleotides. In some embodiments, the plasmid contains approximately 6,611 nucleotides.

[0016] Another aspect of this disclosure is a plasmid shown in Figure 11 as pUC57-TL20c. In some embodiments, a lentiviral vector is derived from the plasmid. In some embodiments, this derived lentiviral vector includes one or more additional sequences encoding short hairpin RNA and / or HIV-1 fusion inhibitors for downregulation of the HIV-1 coreceptor.

[0017] Another aspect of this disclosure is a plasmid comprising a multicloning site essentially consisting of BstBI, MluI, NotI, and ClaI restriction endonuclease sites. In some embodiments, the plasmid further comprises: a nucleotide sequence encoding a packaging signal; a nucleotide sequence encoding a central polypurine tract; a nucleotide sequence encoding a Rev response element; and a nucleotide sequence encoding a self-inactivating terminal repeat. In other embodiments, the plasmid comprises a multicloning site consisting of BstBI, MluI, NotI, and ClaI restriction endonuclease sites. In some embodiments, a lentiviral vector is derived from the plasmid. In some embodiments, this derived lentiviral vector comprises one or more additional sequences encoding short hairpin RNA and / or HIV-1 fusion inhibitors for downregulation of the HIV-1 coreceptor.

[0018] Another aspect of this disclosure is a plasmid comprising: (a) a nucleotide sequence encoding a packaging signal; (b) a nucleotide sequence encoding a central polyprint lactate (cPPT); (c) a nucleotide sequence encoding a Rev response element; (d) a nucleotide sequence encoding a self-inactivating terminal repeat; and (e) a nucleotide sequence encoding a multi-cloning site having restriction sites for the enzymes BstBI, MluI, NotI, and ClaI. In some embodiments, the nucleotide sequence encoding the packaging signal comprises the sequence with SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the central polyprint lactate (cPPT) comprises the sequence with SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the Rev response element comprises the sequence with SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the self-inactivating terminal repeat comprises the sequence with SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the multi-cloning site comprises the sequence with SEQ ID NO: 7.

[0019] Another aspect of this disclosure is a plasmid comprising: (a) a packaging sequence located at approximately nucleotides 762 to 1104 of the plasmid nucleotide sequence; (b) a central polyprint lactate located at approximately nucleotides 1121 to 1597 of the plasmid nucleotide sequence; (c) a Rev response element located at approximately nucleotides 1598 to 2366 of the plasmid nucleotide sequence; (d) a self-inactivating terminal repeat sequence located at approximately nucleotides 409 to 589 of the plasmid nucleotide sequence; and (e) a multicloning site located at approximately nucleotides 2376 to 2400 of the plasmid nucleotide sequence. In some embodiments, the plasmid nucleotide sequence comprises a sequence having at least 90% identity to the sequence with SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the packaging signal comprises the sequence with SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the central polyprint lactate (cPPT) comprises the sequence with SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the Rev response element includes the sequence with SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the self-inactivating terminal repeat sequence includes the sequence with SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the multicloning site includes the sequence with SEQ ID NO: 7.

[0020] Another aspect of this disclosure is a plasmid comprising a multicloning site including BstBI, MluI, NotI, and ClaI restriction endonuclease sites, wherein the plasmid contains a nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO: 1. In some embodiments, a lentiviral vector is derived from the plasmid. In some embodiments, this derived lentiviral vector contains one or more additional sequences encoding short hairpin RNA and / or HIV-1 fusion inhibitors for downregulation of the HIV-1 coreceptor.

[0021] Another aspect of the present disclosure is a plasmid comprising a nucleotide sequence encoding a vector backbone having at least 95% identity to the sequence of SEQ ID NO: 2, wherein the vector backbone is adjacent to at least two additional restriction endonuclease sites, the at least two additional restriction endonuclease sites independently selected from the group consisting of sfiI and Bsu36I. In some embodiments, the lentiviral vector is derived from the plasmid. In some embodiments, the derived lentiviral vector comprises one or more additional sequences encoding short hairpin RNA and / or HIV-1 fusion inhibitors for downregulation of the HIV-1 coreceptor.

[0022] Another aspect of the disclosure is a plasmid comprising a nucleotide sequence encoding a vector skeleton having at least 90% identity to the sequence of SEQ ID NO: 2, the vector skeleton comprising a multicloning site having BstBI, MluI, NotI, and ClaI restriction endonuclease sites, and the plasmid further comprising a tetracycline repressive promoter upstream of the vector skeleton. Yet another aspect of the disclosure is a plasmid comprising a nucleotide sequence encoding a vector skeleton having at least 85% identity to the sequence of SEQ ID NO: 2, the vector skeleton comprising a multicloning site having BstBI, MluI, NotI, and ClaI restriction endonuclease sites.

[0023] Another aspect of the present disclosure includes cells containing the plasmids described herein or lentiviral vectors derived therefrom. In some embodiments, the cells are hematopoietic progenitor cells / stem cells, monocytes, macrophages, peripheral blood mononuclear cells, CD4+ T lymphocytes, CD8+ T lymphocytes, or dendritic cells. Another aspect of the invention includes a kit containing hematopoietic progenitor cells / stem cells in a first container and the plasmid of FIG. 11 or a lentiviral vector derived therefrom.

[0024] Another aspect of the present disclosure includes: (a) synthesizing a lentiviral vector by cloning one or more genes into a plasmid described herein, for example pUC57-TL20c; (b) generating a DNA fragment from the synthesized lentiviral vector; (c) forming a concatemer array from the generated DNA fragment of the synthesized lentiviral vector and a DNA fragment derived from an antibiotic resistance cassette plasmid; (d) transfecting the formed concatemer array into a GPR, GPRG, GPRT, GPRGT, or GPRT-G packaging cell line or a derivative thereof; and (e) isolating one or more stable producer cell line clones. In some embodiments, the method further includes inducing the stable producer cell line to produce a lentiviral vector.

[0025] Another aspect of the disclosure includes: (a) synthesizing a lentiviral vector encoding a short hairpin RNA for downregulation of an HIV-1 co-receptor and encoding an HIV-1 fusion inhibitor, wherein the lentiviral vector is synthesized by cloning into a plasmid described herein a cDNA encoding both the short hairpin RNA and the fusion inhibitor; (b) generating a DNA fragment from the synthesized lentiviral vector; (c) forming a concatemer array from the generated DNA fragment derived from the synthesized lentiviral vector and from a DNA fragment derived from an antibiotic resistance cassette plasmid; (d) transfecting the formed concatemer array into a GPR, GPRG, GPRT, GPRGT, or GPRT-G packaging cell line or a derivative thereof; and (e) isolating one or more stable producer cell line clones. In some embodiments, the method further includes inducing the stable producer cell line to produce a lentiviral vector (LVsh5 / C46) encoding a short hairpin RNA for downregulation of an HIV-1 co-receptor and encoding an HIV-1 fusion inhibitor.

[0026] Another aspect of the disclosure includes a method of recovering vector supernatant from a stable producer cell line, the method comprising recovering the vector supernatant approximately every 48 hours. Another aspect of the disclosure includes a method of recovering vector supernatant from a stable producer cell line, the method comprising recovering the vector supernatant every 40 - 56 hours.

[0027] Another aspect of the disclosure includes a method of recovering a vector supernatant comprising the LVsh5 / C46 lentiviral vector, the method comprising recovering the vector supernatant approximately every 48 hours.

[0028] Another aspect of this disclosure is a stable producing cell line suitable for producing LVsh5 / C46. In some embodiments, the stable producing cell line is based on a GPRG packaging cell line. In some embodiments, the stable producing cell line is based on a GPRT packaging cell line. In some embodiments, the stable producing cell line is based on a GPR packaging cell line. In some embodiments, the stable producing cell line is based on a GPRT-G packaging cell line.

[0029] Another aspect of this disclosure is the existence of a stable producing cell line suitable for producing a self-inactivating lentiviral vector having at least 90% identity to the sequence of SEQ ID NO: 8. In some embodiments, the stable producing cell line is based on a GPRG packaging cell line. In some embodiments, the stable producing cell line is based on a GPRT packaging cell line. In some embodiments, the stable producing cell line is based on a GPR packaging cell line. In some embodiments, the stable producing cell line is based on a GPRT-G packaging cell line.

[0030] Another aspect of this disclosure is a concatemer array containing DNA fragments derived from a first plasmid and a second plasmid; the first plasmid is derived from pUC57-TL20c; the second plasmid contains a bleomycin antibiotic-resistant cassette; the ratio of DNA fragments from the first plasmid to those from the second plasmid is in the range of about 50:1 to about 1:50. In some embodiments, the ratio of DNA fragments from the first plasmid to those from the second plasmid is in the range of about 25:1 to about 1:25. In some embodiments, the ratio of DNA fragments from the first plasmid to those from the second plasmid is in the range of about 15:1 to about 1:15.

[0031] Another aspect of this disclosure is a stable-producing cell line, which is prepared by transfecting a packaging cell line selected from the group consisting of GPR, GPRG, GPRT, GPRT-G, and their derivatives with a concatemer array, the concatemer array comprising DNA fragments derived from a first plasmid and a second plasmid; the first plasmid being derived from pUC57-TL20c; the second plasmid comprising a bleomycin antibiotic-resistant cassette; and the ratio of DNA fragments from the first plasmid to those from the second plasmid being in the range of approximately 25:1 to approximately 1:25. In some embodiments, the stable-producing cell line produces LVsh5 / C46. In some embodiments, LVsh5 / C46 can be harvested approximately every 48 hours.

[0032] Another aspect of this disclosure is an isolated vector having a plasmid map in Figure 11, which includes a multicloning site containing the nucleotide sequence shown in SEQ ID NO: 7.

[0033] Another aspect of this disclosure is a kit comprising (1) a plasmid described herein and (2) a bleomycin-resistant (ble) cassette. In some embodiments, the kit further includes instructions for preparing a lentiviral vector and / or concatemer array, for example, in accordance with the procedures described herein.

[0034] Another aspect of this disclosure is a kit comprising (a) a lentiviral transfer vector plasmid as described herein and (b) packaging cells. In some embodiments, the packaging cells are selected from the group consisting of GPR, GPRG, GPRT, GPRTG, and their derivatives. In some embodiments, the kit further comprises a bleomycin-resistant (ble) cassette. In some embodiments, the kit further comprises instructions for preparing a lentiviral vector and / or concatemer array, for example, in accordance with the procedures described herein.

[0035] Another aspect of this disclosure is a lentiviral vector derived from a plasmid described herein. In some embodiments, the lentiviral vector comprises at least one additional nucleotide sequence. In some embodiments, the at least one additional nucleotide sequence is selected from the group consisting of nucleotide sequences encoding short hairpin RNA for downregulation of the HIV-1 coreceptor and nucleotide sequences encoding an HIV-1 fusion inhibitor. In some embodiments, the lentiviral vector is LVsh5 / C46. In some embodiments, the lentiviral vector comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 8.

[0036] Another aspect of this disclosure is a pharmaceutical composition comprising the above-described lentiviral vector and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier includes solvents, buffers, solutions, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are acceptable for formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. Methods for formulating compounds using pharmaceutically acceptable carriers are known in the art and are described, for example, in Remington's Pharmaceutical Science, (17th edition, Mack Publishing Company, Easton, Pa. 1985); and Goodman & Gillman's: The Pharmacological Basis of Therapeutics (11th edition, McGraw-Hill Professional, 2005); the contents of each of these disclosures are incorporated herein by reference in whole. [Invention 1001] A recombinant plasmid containing a nucleotide sequence that has at least 90% identity with sequence SEQ ID NO: 1. [Invention 1002] The recombinant plasmid of the present invention 1001, wherein the nucleotide sequence has at least 95% identity with the sequence of SEQ ID NO: 1. [Invention 1003] A recombinant plasmid containing the nucleotide sequence of SEQ ID NO: 1. [Invention 1004] A recombinant plasmid containing a nucleotide sequence that has at least 90% identity with the sequence of SEQ ID NO: 2. [Invention 1005] A recombinant plasmid whose sequence differs from the sequence shown in SEQ ID NO: 1 by only 100 or fewer nucleotides. [Invention 1006] The recombinant plasmid of the present invention 1005, wherein the sequence differs from the sequence shown in SEQ ID NO: 1 by only 50 nucleotides or less. [Invention 1007] A recombinant plasmid containing approximately 6500 to 6750 nucleotides, and having at least 90% identity with the sequence of SEQ ID NO: 2. [Invention 1008] A recombinant plasmid of the present invention 1007, containing approximately 6600 to 6700 nucleotides. [Invention 1009] A recombinant plasmid of the present invention 1007, containing approximately 6611 nucleotides. [Invention 1010] The recombinant plasmid shown as pUC57-TL20c in Figure 11. [Invention 1011] A recombinant plasmid containing multiple cloning sites essentially derived from BstBI, MluI, NotI, and ClaI restriction endonuclease sites. [Invention 1012] A recombinant plasmid according to the present invention 1011, further comprising: a nucleotide sequence encoding a packaging signal; a nucleotide sequence encoding a central polyprint lacte; a nucleotide sequence encoding a Rev response element; and a nucleotide sequence encoding a self-inactivating terminal repeat sequence. [Invention 1013] A recombinant plasmid comprising: (a) a nucleotide sequence encoding a packaging signal; (b) a nucleotide sequence encoding a central polyprint lacte (cPPT); (c) a nucleotide sequence encoding a Rev response element; (d) a nucleotide sequence encoding a self-inactivating terminal repeat sequence; and (e) a nucleotide sequence encoding a multicloning site having restriction sites for the enzymes BstBI, MluI, NotI, and ClaI. [Invention 1014] The recombinant plasmid of the present invention 1013, wherein the nucleotide sequence encoding the packaging signal includes the sequence of SEQ ID NO: 3. [Invention 1015] The recombinant plasmid of the present invention 1013, wherein the nucleotide sequence encoding the central polyprint lacte (cPPT) includes the sequence with SEQ ID NO: 4. [Invention 1016] A recombinant plasmid according to the present invention 1013, wherein the nucleotide sequence encoding the Rev response element includes the sequence of SEQ ID NO: 5. [Invention 1017] A recombinant plasmid of the present invention 1013, wherein the nucleotide sequence encoding the self-inactivating terminal repeat sequence includes the sequence of SEQ ID NO: 6. [Invention 1018] The recombinant plasmid of the present invention 1013, wherein the nucleotide sequence encoding the multicloning site includes the sequence of SEQ ID NO: 7. [Invention 1019] A recombinant plasmid comprising: (a) a packaging sequence located between approximately nucleotides 762 and 1104 of the plasmid nucleotide sequence; (b) a central polyprint lactate located between approximately nucleotides 1121 and 1597 of the plasmid nucleotide sequence; (c) a Rev response element located between approximately nucleotides 1598 and 2366 of the plasmid nucleotide sequence; (d) a self-inactivating terminal repeat sequence located between approximately nucleotides 409 and 589 of the plasmid nucleotide sequence; and (e) a multicloning site located between approximately nucleotides 2376 and 2400 of the plasmid nucleotide sequence. [Invention 1020] The recombinant plasmid of the present invention 1019, wherein the plasmid nucleotide sequence contains a sequence having at least 90% identity with the sequence of SEQ ID NO: 1. [Invention 1021] The recombinant plasmid of the present invention 1019, wherein the nucleotide sequence encoding the packaging signal includes the sequence of SEQ ID NO: 3. [Invention 1022] The recombinant plasmid of the present invention 1019, wherein the nucleotide sequence encoding the central polyprint lacte (cPPT) includes the sequence with SEQ ID NO: 4. [Invention 1023] A recombinant plasmid according to the present invention 1019, wherein the nucleotide sequence encoding the Rev response element includes the sequence of SEQ ID NO: 5. [Invention 1024] The recombinant plasmid of the present invention 1019, wherein the nucleotide sequence encoding the self-inactivating terminal repeat sequence includes the sequence of SEQ ID NO: 6. [Invention 1025] The recombinant plasmid of the present invention 1019, wherein the nucleotide sequence encoding the multicloning site includes the sequence of SEQ ID NO: 7. [Invention 1026] A recombinant plasmid comprising multiple cloning sites including BstBI, MluI, NotI, and ClaI restriction endonuclease sites, and containing a nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO: 1. [Invention 1027] A recombinant plasmid comprising a nucleotide sequence encoding a vector cassette having at least 95% identity with the sequence of SEQ ID NO: 2, wherein the vector cassette is adjacent to at least two restriction endonuclease sites, and the at least two restriction endonuclease sites are independently selected from the group consisting of sfiI and Bsu36I. [Invention 1028] A recombinant plasmid comprising a nucleotide sequence encoding a vector cassette having at least 90% identity with the sequence of SEQ ID NO: 2, wherein the vector cassette comprises a multicloning site having BstBI, MluI, NotI, and ClaI restriction endonuclease sites, and further comprising a tetracycline-repressive promoter upstream of the vector cassette. [Invention 1029] A cell containing any plasmid from Invention 1001 to 1028. [Invention 1030] A kit comprising any plasmid from Invention 1001 to 1028 and a bleomycin-resistant (ble) cassette. [Invention 1031] A method for producing a stable producing cell line, comprising the steps of: (a) synthesizing a lentiviral vector by cloning one or more genes into a plasmid of any of the present invention 1001 to 1028; (b) generating DNA fragments from the synthesized lentiviral vector; (c) forming a concatemer array from the generated DNA fragments derived from the synthesized lentiviral vector and from DNA fragments derived from an antibiotic-resistant cassette plasmid; (d) transfecting a GPR, GPRG, GPRT, GPRGT, or GPRT-G packaging cell line or a derivative thereof with the formed concatemer array; and (e) isolating a stable producing cell line. [Invention 1032] (a) a lentiviral vector encoding a short hairpin RNA for downregulation of the HIV-1 coreceptor and an HIV-1 fusion inhibitor, wherein the lentiviral vector is synthesized by cloning cDNA encoding both the short hairpin RNA and the fusion inhibitor into a plasmid of any of the inventions 1001 to 1028; (b) generating DNA fragments from the synthesized lentiviral vector; (c) forming a concatemer array from the generated DNA fragments derived from the synthesized lentiviral vector and from DNA fragments derived from an antibiotic-resistant cassette plasmid; (d) transfecting a GPR, GPRG, GPRT, GPRGT, or GPRT-G packaging cell line or a derivative thereof with the formed concatemer array; and (e) isolating a stable producing cell line, comprising these steps. [Invention 1033] A method for recovering vector supernatant from a stable producing cell line, wherein the vector supernatant is collected approximately every 40 to 56 hours. [Invention 1034] A method for recovering a vector supernatant containing an LVsh5 / C46 lentivirus vector, wherein the vector supernatant is collected approximately every 40 to 56 hours. [Invention 1035] A stable cell line suitable for producing LVsh5 / C46. [Invention 1036] A stable producing cell line of the present invention 1035, based on a GPRG packaging cell line. [Invention 1037] A stable producing cell line of the present invention 1035, based on a GPRT packaging cell line. [Invention 1038] A stable producing cell line of the present invention 1035, based on a GPR packaging cell line. [Invention 1039] A stable cell line suitable for producing a self-inactivating lentiviral vector, wherein the lentiviral vector contains a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO: 8. [Invention 1040] A stable producing cell line of the present invention 1039, based on a GPRG packaging cell line. [Invention 1041] A stable producing cell line of the present invention 1039, based on a GPRT packaging cell line. [Invention 1042] A stable producing cell line of the present invention 1039, based on a GPR packaging cell line. [Invention 1043] A concatemer array comprising DNA fragments derived from a first plasmid and a second plasmid, wherein the first plasmid is derived from pUC57-TL20c, the second plasmid comprises a bleomycin antibiotic-resistant cassette, and the ratio of DNA fragments derived from the first plasmid to DNA fragments derived from the second plasmid is in the range of about 25:1 to about 1:25. [Invention 1044] A stable producing cell line is created by transfecting a packaging cell line selected from the group consisting of GPR, GPRG, GPRT, GPRGT, and GPRT-G packaging cell lines or their derivatives with the concatemer array of the present invention 1043. [Invention 1045] A stable cell line of the present invention 1044 that produces LVsh5 / C46. [Invention 1046] A stable producing cell line of the present invention 1045, which allows for the collection of the aforementioned LVsh5 / C46 approximately every 40 to 56 hours. [Invention 1047] An isolated vector having the plasmid map shown in Figure 11, including a multi-cloning site containing the nucleotide sequence shown in SEQ ID NO: 7. [Invention 1048] A method for preparing a plasmid vector, comprising the steps of cleaving a plasmid according to any of invention 1001 to 1028, and ligating the end of the cleaved plasmid to a compatible end of a polynucleotide to be introduced. [Invention 1049] The method of the present invention 1048, wherein the introduced polynucleotide encodes at least one short hairpin RNA for downregulation of the HIV-1 coreceptor, or encodes an HIV-1 fusion inhibitor. [Invention 1050] A kit comprising (a) a lentiviral transfer vector plasmid of any of the invention 1001 to 1028; and (b) cells of a packaging cell line. [Invention 1051] The kit of the present invention 1050, wherein the cells of the packaging cell line are selected from the group consisting of GPR, GPRG, GPRT, GPRTG, and their derivatives. [Invention 1052] A kit of the present invention 1050 further comprising a bleomycin-resistant (ble) cassette. [Invention 1053] A lentiviral vector derived from any plasmid 1001 to 1028 of the present invention. [Invention 1054] A lentiviral vector according to the present invention 1053, comprising at least one additional nucleotide sequence. [Invention 1055] The lentiviral vector of the present invention 1054, wherein the at least one additional nucleotide sequence is selected from the group consisting of nucleotide sequences encoding short hairpin RNA for downregulation of the HIV-1 coreceptor and nucleotide sequences encoding an HIV-1 fusion inhibitor. [Invention 1056] A lentiviral vector according to the present invention 1055, which is LVsh5 / C46. [Invention 1057] A lentiviral vector according to the present invention 1055, comprising a sequence having at least 95% identity with the sequence of SEQ ID NO: 8. [Invention 1058] A cell containing any lentiviral vector according to invention 1053 to 1057. [Invention 1059] A pharmaceutical composition comprising a lentiviral vector and a carrier according to any of invention 1053 to 1057. [Brief explanation of the drawing]

[0037] [Figure 1]This demonstrates that the same lentiviral vector was repeatedly produced by either transient transfection of HEK293T / 17 cells using an established procedure or by using a stable, GPRG-based producing cell line. The vector-containing medium (VCM) was concentrated 100-fold by ultracentrifugation, and lentiviral (LV) titers were measured by a gene transfection assay. [Figure 2] This flowchart shows a method for creating a stable lentiviral cell line and recovering the lentiviral vector produced from the created cell line. [Figure 3] This report describes the evaluation of the stability of two different cell lines, MWCB, that produce LV over a continuous 3-month passaging period. LV was induced at regular intervals by tetracycline (TET) removal, and LV titer in VCM was evaluated by a gene transfection assay. Both cell lines were stable and were able to produce LV at a level exceeding 1 e6 / ml for over 25 passaging cycles over the 3-month period. [Figure 4] The dynamics of lentiviral vector production after TET removal are shown. Vector titer was evaluated in VCM by gene transfection assay. In all cases, stable GPRG-based producing cell lines were able to maintain LV production at levels above 1e6 TU / ml (unenriched) for at least 5 days after induction. [Figure 5] This study illustrates the dynamics of lentiviral vector production from stable cell lines. (A) During vector production, the culture medium was replaced daily (■) or every two days (□). (B) The total amount of LV in the collected medium was measured using 293T cells. The data shown are mean ± SD (N=2). TU: Transduction unit. [Figure 6] (A) shows GPRG and 293T cells induced in doxycycline (Dox)-free medium. The induced cells were stained with anti-VSVG antibody to detect VSVG expression and measured by flow cytometry. (B) shows the LV production capacity of GPRG as evaluated after long-term culture. [Figure 7]This shows lentivirus production under different culture conditions. (A) Culture / production in serum-containing medium. (B, left) Culture / production in serum-containing medium; (B, right) Culture / production in serum-free medium. D10: 500 mL DMEM / GlutaMAX (trademark), 50 mL FBS (10% w / v), 5 mL penicillin / streptomycin; SFM: serum-free medium. [Figure 8] FACS analysis of 293T cells or TF-1a cells incubated in fresh medium (without vector) or with the LVsh5 / C46 vector is shown. [Figure 9] This section describes the quantification of lentiviral vector copy numbers in infected cells. Using C46 qPCR, the number of vector copies per host genome after transduction with two doses (MOI = 1 or 0.3) was measured. [Figure 10] Ghost-CCR5 cells were transduced with the LVsh5 / C46 vector. The decrease in CCR5 expression levels was measured by FACS. [Figure 11] A schematic diagram of pUC57-TL20 is shown. [Figure 12] The present disclosure describes several aspects of HIV-1-based lentiviral transfer vectors. These particular transfer vectors encode short hairpin RNA (shRNA) for downregulation of the HIV-1 coreceptor CCR5, in combination with an HIV-1 fusion inhibitor (C46). [Figure 13] This document demonstrates lentiviral induction using the methods disclosed herein, both with and without serum. Cells cultured in serum-free medium produced approximately the same amount of virus as cells cultured in 10% PBS. The methods disclosed herein appear to be adaptable to serum-free culture environments. [Figure 14] This flowchart shows the method for generating DNA fragments. [Figure 15] This flowchart shows a method for synthesizing concatemer arrays. [Figure 16] This flowchart shows a method for introducing concatemer arrays into packaging cell lines. [Figure 17] This flowchart shows how to select a transfected clone. [Figure 18] This is a flowchart showing a method for isolating a single colony. [Figure 19] This flowchart shows a method for evaluating virus production. [Figure 20A] Generally, we describe the producing cells for synthesizing the TL20-Cal1-wpre and TL20-Unc-GFP vectors. Figure 20A shows flow cytometry analysis of 293T cells incubated with either fresh medium (left: without vector) or TL20-Cal1-WPRE (right) recovered from the most potent producing cell clone. [Figure 20B] Generally, we describe the producing cells for synthesizing the TL20-Cal1-wpre and TL20-Unc-GFP vectors. Figure 20B shows flow cytometry analysis of 293T cells incubated with either fresh medium (dark gray bars: no vector) or TL20-UbcGFP (light gray bars) recovered from the most potent producing cell clones. Symbols: Ubc: ubiquitin C promoter; GFP: highly sensitive green fluorescent protein. [Figure 20C] Generally, the producing cells for synthesizing TL20-Cal1-wpre and TL20-Unc-GFP vectors are described. Figure 20C shows the distribution of measured vector titers in the supernatants obtained from independent producing cell clones for preparing the TL20-Cal1-WPRE vector (left) or the TL20-UbcGFP vector (right). The vectors were titrated in 293T cells and analyzed by flow cytometry. The highest titer achieved for vectors prepared using polyclonal producing cells (before single clone selection) is shown by the dashed line. Symbols: Ubc: Ubiquitin C promoter; GFP: High-sensitivity green fluorescent protein. [Modes for carrying out the invention]

[0038] Detailed explanation Generally, this disclosure provides a method for producing stable, producible cell lines. The production of stable, producible cell lines, as provided in this invention, enhances the reproducibility and ease of generating high-titer lentiviral stocks while mitigating biosafety concerns, and the affinity of the produced virus is determined by changes in the expressed envelope protein. This disclosure also provides novel lentiviral transfer vector plasmids.

[0039] As used herein, the singular forms "a," "an," and "the" refer to plural objects unless the context explicitly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context explicitly indicates otherwise.

[0040] The terms “comprising,” “including,” and “having” are interchangeable and have the same meaning. Similarly, “comprises,” “includes,” and “has” are interchangeable and have the same meaning. Specifically, each of these terms is defined in accordance with the common U.S. Patent Law definition of “comprising,” and is therefore interpreted as an open term meaning “at least the following,” and not to exclude any additional features, limitations, aspects, etc. Thus, for example, “a device having parts a, b, and c” means that the device includes at least parts a, b, and c. Similarly, the phrase “a method including steps a, b, and c” means that the method includes at least steps a, b, and c. Furthermore, although the steps and processes are described herein in a specific order, those skilled in the art will recognize that the ordering of the steps and processes may vary.

[0041] As used herein, the term "cloning" refers to the process of ligating a nucleic acid molecule onto a plasmid and introducing it into a suitable host cell for replication during host growth.

[0042] As used herein, the term "HIV" includes not only HIV-1 but also various HIV-1 strains (e.g., the BaL strain or the SF162 strain) and various HIV-1 subtypes (e.g., subtypes A, B, C, D, F, G, H, J, and K).

[0043] As used herein, the term “multicloning site” (MCS) refers to a nucleotide sequence containing restriction sites for cloning nucleic acid fragments into a cloning vector plasmid. MCSs, also known as polylinkers or polycloning sites, are collections of cloning sites within which multiple restriction enzymes can act. In some embodiments, cloning sites are known sequences on which restriction enzymes act to linearize or cleave the plasmid.

[0044] As used herein, the term "producing cell" refers to a cell that contains all the elements necessary for the production of lentiviral vector particles.

[0045] As used herein, the term “packaging cell” refers to a cell containing elements necessary for the production of an infectious recombinant virus that are missing from a recombinant viral vector or lentiviral transfer vector plasmid. Typically, such packaging cells contain one or more expression cassettes capable of expressing viral structural proteins (e.g., gag, pol, and env), but they do not contain packaging signals.

[0046] As used herein, the term “restriction endonuclease” or “restriction enzyme” refers to a member of a class of catalytic molecules that bind to a cognitive sequence in a nucleic acid molecule (e.g., DNA) and cleave it at a precise location within that sequence.

[0047] The terms “self-inactivating” or “SIN,” as used interchangeably herein, refer to a modified vector whose ability to recruit is significantly reduced once it has been incorporated into the recipient’s genome, thereby increasing the safety of its use as a gene delivery vector.

[0048] As used herein, the term "vector" refers to a nucleic acid molecule capable of mediating the entry, for example, introduction, or transport of another nucleic acid molecule into a cell. The nucleic acid to be introduced is generally ligated, for example, inserted, into the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication or a sequence sufficient to enable integration into host cell DNA. As will be apparent to those skilled in the art, a viral vector may contain various viral components in addition to the nucleic acid that mediates the entry of the nucleic acid to be introduced.

[0049] Overview of the method Lentiviral vectors (LVs) are important tools for gene delivery due to their efficiency and ability to reliably transduce both dividing and non-dividing cells. As a result, researchers use them as gene delivery vehicles in a wide range of clinical applications. Nevertheless, large-scale clinical production using current good manufacturing practice (cGMP) methods comes with a set of challenges that must be considered when clinical trials using lentiviral vectors receive regulatory approval. One of the key considerations when designing a cGMP-compliant process is that regulatory considerations must be incorporated into a manufacturing process that is capable of producing consistent lentiviruses for multiplex cGMP production. The majority of lentiviral vectors used clinically have been produced by transient transfection. However, production based on transient transfection is often labor-intensive and variable. For this reason, several stable packaging cell line systems have recently been developed. While using these cell lines for LV biofabrication is particularly attractive in terms of both scalability and consistency, the development of such cell lines is time-consuming, and a well-established regulatory pathway for the cGMP use of these cell lines has not yet been found.

[0050] With this in mind, this disclosure describes a process for the clinical production of a self-inactivating lentiviral vector (SIN-LV). It is believed that a stable producing cell line can be constructed to enable the production of a self-inactivating lentiviral vector (e.g., LVsh5 / C46) by using a novel lentiviral transfer vector plasmid in conjunction with a GPR, GPRG, GPRT, GPRGT, or GPRT-G packaging cell line (or a derivative or analogue packaging cell line derived therefrom). While certain embodiments and examples described herein refer to the production of LVsh5 / C46, a self-inactivating lentiviral vector encoding a short hairpin RNA (shRNA) for downregulation of the HIV-1 coreceptor CCR5 in combination with an HIV-1 fusion inhibitor (i.e., C46), those skilled in the art will recognize that the methods described herein are suitable for constructing a stable producing cell line capable of producing SIN-LV containing any desired or client-provided gene or sequence.

[0051] The applicant has demonstrated that, compared to SIN-LV produced by transient transfection, the method of the present disclosure can (i) produce SIN-LV of similar quality and quantity; (ii) produce LV that may have better efficacy; and (ii) significantly reduce the variation between preparations seen in transient transfection while maintaining yield.

[0052] pUC57-TL20c One aspect of this disclosure is a third-generation self-inactivating (SIN) lentiviral transfer vector plasmid based on human immunodeficiency virus type 1 (HIV-1) that includes a novel, versatile multicloning site (MCS) (hereinafter referred to as "pUC57-TL20") (see Figure 11).

[0053] In some embodiments, the lentiviral vector transfer plasmid contains a vector skeleton ("TL20c") that does not contain an internal promoter (and is therefore "promoterless"). In some embodiments, the lentiviral vector transfer plasmid contains a single promoter upstream of the vector skeleton, such as a tetracycline repressive promoter (see Figure 12). While we do not wish to be bound by any particular theory, the promoterless design of the vector skeleton is thought to enable the creation of lentiviral transfer vector plasmids that allow for the delivery of the gene of interest and subsequent expression from a user-determined promoter.

[0054] Figure 11 shows a gene map illustrating the constituent elements of a lentiviral vector transfer plasmid. In some embodiments, the lentiviral vector transfer plasmid contains approximately 6,500 to 6,750 nucleotides. In other embodiments, the lentiviral vector transfer plasmid contains approximately 6,600 to 6,700 nucleotides. In some embodiments, the vector backbone of the lentiviral vector transfer plasmid contains approximately 3,850 to 3,950 nucleotides. In some embodiments, the vector backbone of the lentiviral vector transfer plasmid contains approximately 3,901 nucleotides.

[0055] As shown in Figure 11, the plasmid comprises a 5' flanking HIV LTR, a packaging signal or ψ+, a central polyprint lacte (cPPT), a Rev response element (RRE), a multicloning site (MCS), and a 3' flanking HIV LTR. The LTR region further comprises U3 and U5 regions and an R region.

[0056] According to certain aspects of this disclosure, the transfer plasmid comprises a self-inactivated (SIN) LTR. As is known in the art, during the life cycle of a retrovirus, the U3 region of the 3'LTR replicates to form the corresponding region of the 5'LTR during the process of reverse transcription and viral DNA synthesis. The creation of a SIN LTR is achieved by inactivating the U3 region of the 3'LTR (preferably by partial deletion, e.g., removal of the TATA sequence). This modification is transferred to the 5'LTR after reverse transcription, thus eliminating the transcription unit of the LTR in the provirus, which is thought to prevent recruitment by a replicable virus. Further safety improvements are achieved by replacing the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production.

[0057] In some embodiments, the packaging signal includes approximately 361 base pairs of the Gag sequence and approximately 448 base pairs of the Pol sequence of wild-type HIV (e.g., HIV01 HXB2_LAI_IIIB). In some embodiments, the cPPT includes approximately 85 base pairs of the Vif sequence of wild-type HIV. In some embodiments, the HIV polyprint lact (pPu) includes approximately 106 base pairs of the Nef sequence of wild-type HIV. In some embodiments, the RRE includes approximately 26 base pairs of the Rev sequence, approximately 25 base pairs of the tat sequence, and approximately 769 base pairs of the Env sequence of wild-type HIV. In some embodiments, the transfer plasmid includes a chromatin insulator and / or a β-globulin polyadenylation signal.

[0058] In some embodiments, the nucleotide sequence encoding the packaging signal includes the sequence of SEQ ID NO: 3, or a sequence having at least 85% identity to the sequence of SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the packaging signal includes the sequence of SEQ ID NO: 3, or a sequence having at least 90% identity to the sequence of SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the packaging signal includes the sequence of SEQ ID NO: 3, or a sequence having at least 95% identity to the sequence of SEQ ID NO: 3.

[0059] In some embodiments, the nucleotide sequence encoding the central polyprint lactate (cPPT) includes the sequence with SEQ ID NO: 4, or a sequence having at least 85% identity to the sequence with SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the central polyprint lactate (cPPT) includes the sequence with SEQ ID NO: 4, or a sequence having at least 90% identity to the sequence with SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the central polyprint lactate (cPPT) includes the sequence with SEQ ID NO: 4, or a sequence having at least 95% identity to the sequence with SEQ ID NO: 4.

[0060] In some embodiments, the nucleotide sequence encoding the Rev response element includes the sequence of SEQ ID NO: 5, or a sequence having at least 85% identity to the sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the Rev response element includes the sequence of SEQ ID NO: 5, or a sequence having at least 90% identity to the sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the Rev response element includes the sequence of SEQ ID NO: 5, or a sequence having at least 95% identity to the sequence of SEQ ID NO: 5.

[0061] In some embodiments, the nucleotide sequence encoding the self-inactivating terminal repeat sequence includes the sequence with SEQ ID NO: 6, or a sequence having at least 85% identity to the sequence with SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the self-inactivating terminal repeat sequence includes the sequence with SEQ ID NO: 6, or a sequence having at least 90% identity to the sequence with SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the self-inactivating terminal repeat sequence includes the sequence with SEQ ID NO: 6, or a sequence having at least 95% identity to the sequence with SEQ ID NO: 6.

[0062] In some embodiments, the plasmid includes a nucleotide sequence encoding a doxycycline repressive promoter having at least 85% identity with the sequence of SEQ ID NO: 10. In some embodiments, the plasmid includes a nucleotide sequence encoding a doxycycline repressive promoter having at least 90% identity with the sequence of SEQ ID NO: 10. In some embodiments, the plasmid includes a nucleotide sequence encoding a doxycycline repressive promoter having at least 95% identity with the sequence of SEQ ID NO: 10.

[0063] In some embodiments, the plasmid includes a nucleotide sequence encoding an HIV LTR R5 region having at least 85% identity with the sequence of SEQ ID NO: 11. In some embodiments, the plasmid includes a nucleotide sequence encoding an HIV LTR R5 region having at least 90% identity with the sequence of SEQ ID NO: 11. In some embodiments, the plasmid includes a nucleotide sequence encoding an HIV LTR R5 region having at least 95% identity with the sequence of SEQ ID NO: 11.

[0064] In some embodiments, the plasmid includes a nucleotide sequence encoding an HIV LTR U5 region having at least 85% identity with the sequence of SEQ ID NO: 12. In some embodiments, the plasmid includes a nucleotide sequence encoding an HIV LTR U5 region having at least 90% identity with the sequence of SEQ ID NO: 12. In some embodiments, the plasmid includes a nucleotide sequence encoding an HIV LTR U5 region having at least 95% identity with the sequence of SEQ ID NO: 12.

[0065] In some embodiments, the plasmid includes a nucleotide sequence encoding a chromatin insulator having at least 85% identity to the sequence of SEQ ID NO: 13. In some embodiments, the plasmid includes a nucleotide sequence encoding a chromatin insulator having at least 90% identity to the sequence of SEQ ID NO: 13. In some embodiments, the plasmid includes a nucleotide sequence encoding a chromatin insulator having at least 95% identity to the sequence of SEQ ID NO: 13.

[0066] In some embodiments, the plasmid includes a nucleotide sequence encoding a β-globin polyadenylation signal having at least 85% identity with the sequence of SEQ ID NO: 14. In some embodiments, the plasmid includes a nucleotide sequence encoding a β-globin polyadenylation signal having at least 90% identity with the sequence of SEQ ID NO: 14. In some embodiments, the plasmid includes a nucleotide sequence encoding a β-globin polyadenylation signal having at least 95% identity with the sequence of SEQ ID NO: 14.

[0067] In some embodiments, the plasmid contains a nucleotide sequence having at least 85% identity with the sequence of SEQ ID NO: 15. In some embodiments, the plasmid contains a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO: 15. In some embodiments, the plasmid contains a nucleotide sequence having at least 95% identity with the sequence of SEQ ID NO: 15.

[0068] This disclosure provides lentiviral transfer vector plasmids incorporating MCSs for various different restriction enzymes. According to certain embodiments of this disclosure, the MCS comprises a sequence having approximately 20 to 40 nucleotides. In some embodiments, the MCS of the plasmid of this disclosure comprises at least two restriction enzyme cleavage sites. In other embodiments, the MCS of the plasmid of this disclosure comprises at least three restriction enzyme cleavage sites. In yet another embodiment, the MCS of the plasmid of this disclosure comprises approximately 2 to approximately 10 restriction sites. In some embodiments, the restriction sites within the MCS are selected from the group consisting of BstBI, MluI, NotI, ClaI, ApaI, XhoI, XbaI, HpaI, NheI, PacI, NsiI, SphI, Sma / Xma, AccI, BamHI, and SphI, or any derivatives or analogues thereof.

[0069] In some embodiments, the MCS region of the lentiviral transfer vector plasmid has four distinct restriction enzyme cleavage sites that are thought to facilitate the easy subcloning of the desired transgene cassette. In some embodiments, the multicloning site includes BstBI, MluI, NotI, and ClaI restriction endonuclease sites. In some embodiments, the nucleotide sequence encoding the multicloning site includes the sequence of SEQ ID NO: 7, or a sequence having at least 90% identity to the sequence of SEQ ID NO: 7. These restriction sites may be arranged in any order.

[0070] In some embodiments, the transfer plasmid includes one or more additional restriction enzyme cleavage sites adjacent to the vector backbone (see Figure 11). While we do not wish to be bound by any particular theory, the additional flanking restriction enzyme cleavage sites are thought to enable the construction of a unidirectional (head-to-tail) concatemer array. In some embodiments, the restriction enzyme cleavage sites are selected from Sfil and Bsu36I. In some embodiments, the lentiviral vector containing one or more genes is derived from the plasmid.

[0071] In some embodiments, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 80% identity with the sequence of SEQ ID NO: 1. In other embodiments, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 85% identity with the sequence of SEQ ID NO: 1. In yet another embodiment, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO: 1. In yet another embodiment, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 95% identity with the sequence of SEQ ID NO: 1. In yet another embodiment, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 97% identity with the sequence of SEQ ID NO: 1. In some embodiments, the lentiviral vector transfer plasmid contains the sequence of SEQ ID NO: 1. In some embodiments, the lentiviral vector transfer plasmid has a sequence that differs from the sequence shown in SEQ ID NO: 1 by no more than 100 nucleotides.

[0072] In some embodiments, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 80% identity with the sequence of SEQ ID NO: 2. In other embodiments, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 85% identity with the sequence of SEQ ID NO: 2. In yet another embodiment, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 90% identity with the sequence of SEQ ID NO: 2. In yet another embodiment, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 95% identity with the sequence of SEQ ID NO: 2. In yet another embodiment, the lentiviral vector transfer plasmid contains a nucleotide sequence having at least 97% identity with the sequence of SEQ ID NO: 2. In some embodiments, the lentiviral vector transfer plasmid contains the sequence of SEQ ID NO: 2. In some embodiments, the lentiviral vector transfer plasmid has a sequence that differs from the sequence shown in SEQ ID NO: 2 by no more than 100 nucleotides.

[0073] In some embodiments, lentiviral transfer vector plasmids are synthesized according to methods known to those skilled in the art. For example, such plasmids may be synthesized using traditional restriction digestion and ligation techniques known to those skilled in the art. For instance, a donor plasmid containing the TL20c vector backbone can be subcloned into a pU57C recipient plasmid (e.g., one commercially available from Genescript) using standard digestion and ligation methods known to those skilled in the art (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor, NY, the disclosure of which is incorporated herein by reference in whole).

[0074] This disclosure also includes a method for constructing a lentiviral vector, such as LVsh5 / C46. In one embodiment, the method includes the steps of synthesizing a cDNA of a gene and cloning the synthesized cDNA to a restriction site in a plasmid such as pUC57-TL20c. The gene can be inserted into a suitable cloning site using techniques known to those skilled in the art. For example, the gene can be amplified by PCR and then cloned into a plasmid containing a desired promoter or gene expression regulatory element.

[0075] In some embodiments, and merely as examples, the method includes the steps of synthesizing a cDNA of a gene that expresses a protein capable of preventing HIV fusion or replication into cells; and subsequently cloning the synthesized cDNA to a restriction site in a plasmid disclosed herein.

[0076] Production of stable lentiviral cell lines and recovery of lentiviral vectors produced therefrom. Some aspects of this disclosure are methods for producing a stable producing cell line and recovering a lentiviral vector produced from the produced stable producing cell line. Referring to Figure 2, the first step in producing a stable producing cell line is to generate DNA fragments from, for example, a lentiviral vector transfer plasmid and a second plasmid such as an antibiotic resistance cassette plasmid (10). Following the generation of DNA fragments (10), a concatemer array is formed using the DNA (20). The concatemer array is then introduced into a packaging cell line (e.g., a GPR, GPRG, GPRT, GPRG, GPRT-G, or a packaging cell line of the same derivatives) by, for example, transfection (30). Following the introduction of the array (30) and subsequent transfection, clones are selected (40) and separated (50) to produce a stable producing cell line (60). The vector supernatant containing the lentiviral vector can then be recovered.

[0077] Formation and purification of concatemer arrays A “concatemer” or “concatemer array” (as used interchangeably herein) (a long, continuous DNA molecule containing multiple copies of the same DNA sequence linked directly or indirectly in series) is constructed and used in the transfection of packaging cell lines. In some embodiments, the concatemer is a large array of linked vector genome expression cassettes interspersed with antibiotic resistance cassettes.

[0078] Referring to Figure 14, DNA fragments are generated from a lentiviral transfer vector plasmid (step 100) and an antibiotic-resistant cassette plasmid (step 110) to form a concatemer array. In some embodiments, the DNA fragments are prepared by digesting each plasmid according to protocols known to those skilled in the art, and then ligating the digested fragments. In some embodiments, the desired DNA fragments are obtained using electrophoresis and an agarose gel (step 120). In some embodiments, the concentration of the DNA fragments is measured using a NanoDrop spectrophotometer (step 130). Various strategies for ligating the DNA fragments are available, the selection of which depends on the properties of the ends of the DNA fragments, and the selection can be easily made by those skilled in the art.

[0079] In some embodiments, the lentiviral transfer vector plasmid is based on pUC57-TL20c. In some embodiments, the antibiotic-resistant cassette plasmid is driven by a PGK promoter. In some embodiments, the antibiotic-resistant cassette plasmid contains a flanking site for concatemerization with the lentiviral cassette in the lentiviral transfer vector plasmid. In some embodiments, the antibiotic-resistant cassette plasmid is PGK-ble (bleomycin-resistant). In some embodiments, the PGK-ble plasmid contains a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 9. In some embodiments, the concatemer array is formed by in vitro ligation of DNA fragments derived from the lentiviral transfer vector plasmid and the PGK-ble plasmid.

[0080] Figure 15 outlines the typical steps used to form the concatemer array. In step 200, the generated DNA fragments are mixed to maximize the volume of fragments in the ligation reaction product and maintain the desired ratio (step 210). In some embodiments, the ratio of lentiviral transfer vector plasmid DNA to antibiotic-resistant cassette plasmid DNA is in the range of approximately 100:1 to approximately 1:100. In other embodiments, the ratio of lentiviral transfer vector plasmid DNA to antibiotic-resistant cassette plasmid DNA is in the range of approximately 50:1 to approximately 1:50. In yet another embodiment, the ratio of lentiviral transfer vector plasmid DNA to antibiotic-resistant cassette plasmid DNA is in the range of approximately 25:1 to approximately 1:25. In yet another embodiment, the ratio of lentiviral transfer vector plasmid DNA to antibiotic-resistant cassette plasmid DNA is in the range of approximately 10:1 to approximately 1:10.

[0081] In some embodiments, the concatemer reaction mixture is incubated overnight at room temperature (step 220). Subsequently, the DNA fragment concentration of each sample can be measured using a NanoDrop spectrophotometer (step 230).

[0082] In some embodiments, a unidirectional concatemer array is formed and used in the transfection of packaging cell lines. In some embodiments, the formation of the directional array is achieved by using restriction enzyme sites in a lentiviral transfer vector plasmid adjacent to the lentiviral vector backbone. In some embodiments, restriction digestion is performed using restriction enzyme sites adjacent to the TL20c vector cassette, enabling the formation of nucleotide non-palindromic overhangs that can be used solely for head-to-tail ligation. In some embodiments, unidirectional ligation according to the method described herein enables the preparation of a concatemer array primarily containing head-to-tail DNA products.

[0083] In some embodiments, concatemer arrays are formed according to the method described in Example 3 herein. Naturally, those skilled in the art will understand that the procedure provided in Example 3 is adaptable to the formation of concatemer arrays with different ratios of the first plasmid to the second plasmid, and to transfer plasmids other than LVsh5 / C46.

[0084] In some embodiments, concatemer arrays are purified by phenol extraction and ethanol precipitation prior to transfection into packaging cell lines. While this conventional technique is inexpensive and effective, the procedure is time-consuming and may not yield reproducible results. Furthermore, there is a perceived risk of phenol / chloroform carryover to the final sample. Additionally, this process is thought to involve hazardous chemicals and may generate toxic waste that must be carefully disposed of in accordance with hazardous waste guidelines.

[0085] Alternatively, in other embodiments, a silica-based method is used to purify the newly synthesized concatemer array after ligation. This method is considered to provide a simple, reliable, rapid, and convenient method for separating high-quality transfection-grade concatemer arrays. In some embodiments, the concatemer array is purified using a DNeasy Mini spin column available from Qiagen, for example, using the procedure described in Example 6.

[0086] Transfection / Single Clone Isolation After purification of the concatemer array, the array is used to transfect cells of a packaging cell line. As used herein, the terms “transformation” and “transfection” refer to various techniques recognized in the art for introducing foreign nucleic acids (e.g., DNA or RNA) into cells. As will be apparent from the examples provided herein, when a host cell tolerant to lentiviral particle production is transfected with the prepared concatemer array, the cell becomes a producing cell, i.e., a cell that produces infectious lentiviral particles.

[0087] Generally, concatemer arrays or directional concatemer arrays can be introduced into cells by conventional transfection techniques. Referring to Figure 16, in some embodiments, cells are harvested and seeded 20–24 hours before transfection (step 300), and then transfected with the synthesized concatemer array (step 310) (step 320). A procedure for transfecting cells of a packaging cell line is provided herein as Example 4.

[0088] One packaging cell line suitable for transfection with the formed concatemer array is the GPR packaging cell line. The GPR line is an HIV-1 based packaging cell line derived from 293T / 17 cells containing the necessary viral components gagpol and rev (see Throm et al., Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection. Blood 113: 5104-5110, the disclosure of which is incorporated herein by reference in whole).

[0089] Another packaging cell line suitable for transfection with the formed concatemer array is the GPRG packaging cell line. In some embodiments, the GPRG packaging cell line includes gagpol, rev, and VSV-G.

[0090] Another packaging cell line suitable for transfection with the formed concatemer array is the GPRT packaging cell line (gagpol, rev, and tat). GPRG and GPRT packaging cell lines, as well as methods for their preparation, have also been disclosed by Throm et al., and their disclosure is again incorporated herein by reference in its entirety. Other suitable packaging cell lines (e.g., GPRT-G) are described in Wielgosz et al. "Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy," Molecular Therapy-Methods & Clinical Development 2, Article number: 14063 (2015), and their disclosure is again incorporated herein by reference in its entirety.

[0091] Those skilled in the art will understand that other packaging cell lines suitable for use in the methods disclosed herein may also be used. In some embodiments, other packaging cell lines may be derived from GPR, GPRG, GPRT, or GPRT-G packaging cell lines. While we do not wish to be bound by any particular theory, GPRT-G cell lines are thought to have higher transduction efficiency in CD34+ cells (see Wielgosz). "Derived from" means a population of cells clonally derived from individual cells that possess several selected properties, such as the ability to produce active proteins at a given titer or the ability to proliferate to a particular density.

[0092] Figure 17 shows a general procedure for selecting transfected cells. In some embodiments, GPRG cells are cultured in a selection medium (zeosin and doxycycline) approximately 72 hours after transfection (step 400). The cells are then supplied with the selection medium (zeosin and doxycycline) every 3-4 days until cell focus is confirmed (step 410). Subsequently, the cell line is grown and evaluated (step 420).

[0093] In some embodiments, a single-focus selection / screening step is used after transfection to identify single-cell clones with good productivity. According to this method, in some embodiments, selected cells are sparsely seeded in a 150 × 25 mm culture dish and grown for 2-3 weeks to form visible colonies. Individual colonies can then be transferred to a smaller culture vessel for monoclonal growth. This method is considered cost-effective and frequently employed; however, due to the inherent limitations of single-focus selection techniques, achieving monoclonality of good productive cell lines with a high probability can be difficult.

[0094] Figure 18 shows the isolation of single colonies. In step 500, single-cell sorting is prepared using flow cytometry. The cells are then seeded in conditioned medium (step 510) and grown (step 520).

[0095] In other embodiments, fluorescence-activated cell sorting (FACS) is used to isolate single clones to generate stable cell lines producing high-titer lentiviral vectors (see, for example, Figure 8). Furthermore, conditioned media, such as zeosin (50 μg / mL) and doxycycline (1 ng / mL), may be added during the sorting process to enhance cell adhesion and viability and promote colony formation. The use of conditioned growth media and the high processing capacity of the FACS system are thought to enable screening of a large number of clones, and therefore increase the probability of finding high-titer lentiviral vector-producing cell clones.

[0096] In some embodiments, clones with good growth rate and virus production ability are tested for stability over approximately 20 passages.

[0097] Induction of virus-producing cell lines Following clone selection and proliferation, the selected clones are induced to produce vector supernatant, which can be performed according to procedures known to those skilled in the art. To produce lentiviral vectors from the induced stable producing cell lines, the culture supernatant is harvested daily for up to seven days in some embodiments. This production protocol can be readily used to produce various test vectors on a small scale. This repeated virus harvest protocol can also increase the final yield of viral vectors. However, daily harvesting and medium changes are often uneconomical, and a new two-day harvest protocol has been devised as an alternative to daily harvesting. This new viral vector production protocol, described below, can produce the same amount of viral vector with less medium consumption.

[0098] Figure 19 further illustrates the induction and evaluation steps. In step 600, the viral vector is induced, and then 293T spinoculation is performed to measure the transduction efficiency (step 610). The top three clones are screened (step 620) and propagated (step 630). The clones are then stored (e.g., under liquid nitrogen) (step 640).

[0099] Collection every two days The applicant unexpectedly found that two-day recovery allows for the production of nearly the same amount of viral vector as the more traditional daily recovery method, while simultaneously offering the advantage of requiring less culture medium.

[0100] One embodiment of the present invention is a first method for generating a viral vector from a 2-day recovery, which includes the following steps. (1) Remove as much of the old culture medium as possible from the culture dish of the producing cell line, and wash the cells with 1×PBS. (2) Add TrypLE® Express enzyme (1x) to the culture dish (available from ThermoFisher Scientific). (3) Place in a 37°C incubator for 2 minutes. (4) Wash the cells by adding D10 medium (without drugs), and dissociate the cell clusters into single cells by pipetting up and down (D10 medium: Dulbecco's modified Eagle medium / high glucose, GlutaMAX® supplement, 10% (w / v) FBS and 1% (w / v) penicillin / streptomycin). (5) Centrifuge the cells at 4°C and 1200 rpm for 5 minutes. (6) Aspirate the culture medium and gently suspend the pellet in fresh D10 medium (without drugs). (7) Seed the cells into a culture dish when they are approximately 95% confluent (by seeding approximately 4 × 10⁶ cells / 6 mm culture dish; vector induction). (8) The seeded cells were supplemented with pre-warmed fresh D10 medium 24 hours later (day 1 after induction). (9) The viral vector can be first collected from the cells 48 hours after the first culture medium change (3 days after induction). (10) Add fresh culture medium that has been warmed in advance to the culture dish. (11) Forty-eight hours after the second culture medium change (fifth day after induction), the second viral vector is collected. (12) Add fresh culture medium that has been warmed in advance to the culture dish. (13) Forty-eight hours after the third culture medium change (7 days after induction), the third viral vector recovery is performed.

[0101] The applicant found that viral titers could be obtained by collecting the viral vector a second time, on the 4th and 5th days after induction, compared to a more conventional method that allows collection of the viral vector for 7 days after induction.

[0102] Another embodiment is a second method according to the present invention for generating a viral vector from a 2-day recovery, the first method comprising the following steps: (1) Remove the culture medium from the culture dish of the producing cell line as completely as possible, and wash the cells with 1×PBS. (2) Gently pipette 1×TrypLE Express onto the washed cell monolayer, using 3 mL for a 100 mm culture dish. (3) Rotate the flask using TrypLE Express so that the single layer is covered. (4) Return the flask to the incubator and leave it for 2 minutes. (5) Gently tap one side of the flask to detach any remaining cells that are attached. (6) Resuspend the cells in 2 mL of fresh D10 medium (without antibiotics) and transfer to a 15 mL conical centrifuge tube. (7) Centrifuge the cells at 1200 rpm for 5 minutes. (8) Aspirate the culture medium and gently suspend the pellet in 5 mL of fresh D10 medium (without antibiotics). (9) The number of cells is measured using the TC10 (trademark) fully automated cell counter. (10) Seed the cells into a culture dish when they are >95% confluent (by seeding 4 × 10⁶ live cells into a 60 mm culture dish). (11) The seeded cells were replenished daily (every 24 hours) with fresh D10 medium that had been warmed beforehand.

[0103] The applicant found that the viral vector could be recovered from cells 48 hours after induction, and that the highest viral titer was obtained 72 hours after induction. Unexpectedly, the applicant also found that the viral vector could be recovered 2 to 4 days after induction.

[0104] In some embodiments, the recovered vector is purified by filtration. In some embodiments, the recovered vector is characterized by measuring the viral titer, viral copy number per cell genome, and p24 concentration.

[0105] A daily comparison between daily collection and bi-day collection is shown in Figure 5. [Examples]

[0106] Example 1 - Detailed comparison of a self-inactivated lentiviral vector produced by transient transfection and the vector produced by the disclosed stable cell line method. Using the method described herein, a stable cell line was created to produce LVsh5 / C46, a self-inactivating lentiviral vector (SIN-LV) encoding short hairpin RNA (shRNA) for downregulation of the HIV-1 coreceptor CCR5, combined with the HIV-1 fusion inhibitor C46. This LV, produced by transient transfection, is currently being evaluated in clinical trials in HIV-infected individuals. Here, the inventors perform a comparative analysis of LVsh5 / C46 produced by transient transfection and LVsh5 / C46 produced using the method described herein to support the applicability of this system to the clinical production of LVsh5 / C46 and other SIN-LVs.

[0107] Lentiviral vectors (LVs) were produced in 293T cells by calcium phosphate transfection using a four-plasmid system (one transfer vector, two packaging vectors, and one envelope vector). Virus-containing medium (VCM) was collected 48 hours after transfection and concentrated by ultracentrifugation through a 20% sucrose cushion.

[0108] For cell line production, the producing cells were induced in doxycycline (Dox)-free medium, and the VCM was collected after 72 hours and concentrated by ultracentrifugation. As shown in Table 1 and Figures 8 and 9, the LVs produced by each method were compared based on particle titer and using three independent assays for gene transduction efficacy against 293T and TF-1a T cell lines. These included FACS assays for cell surface C46 expression and shRNA-mediated CCR5 expression knockdown, as well as a qPCR assay for vector copy number (VCN) per host cell genome. In all assays, titer was measured over a range of vector dilutions to clarify linear relationships. In the qPCR assays, genomic DNA extracted from transduced cells was used to detect C46 transgenes and sequences derived from the endogenous β-globin gene. In this way, the C46 VCN could be normalized relative to the cell genome.

[0109] (Table 1) Stable viral vector production compared to transient viral vector production TIFF0007833493000001.tif571511. Abbreviations: TU, transduction unit; VCM, virus-containing culture medium. 2. The VCM was concentrated 100-fold by passing it through a 20% sucrose cushion using ultracentrifugation.

[0110] Compared to transient transfection, higher concentrations of p24 were observed in VCM produced by the producible cell line. However, the yield and potency of LVsh5 / C46 produced using the two different systems were similar. First, the vectors were evaluated for C46 titer by FACS using equivolute VCM. Vectors produced by transient transfection showed a slightly increased titer, but when the C46 titer was normalized and the vector preparations were assayed for gene transduction using qPCR assay or functional knockdown of CCR5, vectors produced by the stable producible cell line showed greater potency (see Table 2). Downregulation of CCR5 expression and genomic C46 transgenes (VCNs) were significantly higher in target cells treated with LVsh5 / C46 produced by the method disclosed herein than in target cells treated with vectors produced by transient transfection (see Table 3 and Figure 10).

[0111] (Table 2) Analysis of C46 in transduced cells by qPCR TIFF0007833493000002.tif721361. Abbreviations: ND, Not Detected; MOI, Multiple Infections. 2. LVsh5 / C46 single-copy cell line. 3. MOI based on C46 trait induction titer.

[0112] (Table 3) Analysis of C46 in transduced cells by qPCR TIFF0007833493000003.tif731431. Abbreviations: ND, Not Detected; MOI, Multiple Infections. 2. LVsh5 / C46 single-copy cell line. 3. MOI based on C46 trait induction titer.

[0113] Based on three independent assays, the method described herein is demonstrated to provide a stable LV production system capable of producing SIN-LV of comparable quality and quantity compared to transient transfection methods. Higher CCR5 downregulatory potency and C46 VCN (normalized against C46 titer) in transduced cells indicate that LVsh5 / C46 produced by the producing cells has superior potency to vectors produced using conventional 4-plasmid transient transfection. While we do not wish to be tied to any particular theory by eliminating the lengthy transient transfection step, this production system is considered to be easily adaptable to cGMP conditions for producing clinical-grade material for human use.

[0114] Example 2 - Development and characterization of GPRG-based producing cell lines for bioproduction of lentiviral vectors for HIV gene therapy. The GPRG cell line system has been previously established for the clinical production of self-inactivating lentiviral vectors (SIN-LVs). Here, we sought to establish a GPRG-based producing cell line for producing LVsh5 / C46, a SIN-LV currently being evaluated in clinical settings for the treatment of HIV-infected individuals. This vector encodes two viral entry inhibitors: sh5, a short hairpin RNA targeting the HIV coreceptor CCR5, and C46, ​​a viral fusion inhibitor. We also sought to clarify the GPRG packaging cell line, the GRPG-based LVsh5 / C46 producing cell line, and the stability of LVsh5 / C46 production after tetracycline induction, which are necessary for regulatory approval and clinical application of the GPRG system for the bioproduction of LVsh5 / C46.

[0115] GPRG cells were cultured in D10 medium containing doxycycline (Dox) and puromycin (Puro). To generate LVsh5 / C46-producing cells, GPRG cells were transfected with the transfer plasmid TL20-LVsh5 / C46 and the zeosin resistance plasmid as concatemer arrays. Individual clones were evaluated for their ability to produce the LVsh5 / C46 vector and maintained in D10 medium containing Dox, Puro, and zeosin. To assess the stability of the parental GPRG cell line for LV production, GPRG cells were transfected with the transfer vector every 10 passages for 3 months (over 50 passages in total) (see Figures 3A and 3B). Virus-containing medium (VCM) was collected 48 hours after transfection, and vector titers were evaluated by complementary gene transfer assays. To assess the stability of LV production from stable-producing cell clones, cells were induced in Dox-free D10 medium. VCMs were collected 72 hours after induction, and titers were similarly evaluated across a range of vector dilutions. To analyze the stability of post-induction VSV-G expression after long-term passage, GPRG cells were induced by discontinuing Dox use, then stained with biotin-conjugated anti-VSV-G antibody, followed by secondary staining with streptavidin-phycoerythrin.

[0116] GPRG cells exhibit strictly tetracycline-controlled VSV-G expression. This packaging cell line was able to produce up to 10⁷ LV transduction units (TU) / mL after transfection with an LV transfer vector and maintained high levels of LV production for over 50 passages in continuous culture (see Figures 6A and 6B). By using concatemer array transfection, we demonstrate the efficient construction of a GPRG-based producing cell line for LVsh5C46 production. This cell line consistently yielded titers exceeding 10⁶ TU / mL. Further increases in titer are expected to be achieved by recloning and selection of secondary producing cell lines. Titer peaked 2–5 days after induction. We also showed that the established stable producing cell line could consistently maintain LVsh5 / C46 production at titers exceeding 10⁶ TU / mL during continuous culture for over 25 passages.

[0117] The GPRG cell line efficiently expressed VSV-G on its cell surface after Dox removal. It was also able to produce high-titer LV after transfection with a transfer vector plasmid. Furthermore, this cell line enabled the derivation of a high-titer SIN-LV producing cell line. The producing cell line showed stable vector production during long-term culture, and its adaptability to serum-free and suspension culture systems was evaluated (see Figures 7A and 7B).

[0118] Example 3 - Protocol for the preparation of concatemer arrays Stage 1 500 mL of 1 × TAE running buffer is prepared by combining 490 mL of deionized water with 10 mL of 50 × TAE ((Tris-acetate-EDTA) buffer). Prepare a 1% agarose gel by placing 1 g of agarose and 100 mL of 1 × TAE buffer (2 mL of 50 × TAE with 98 mL of autoclaved sterile water added) in a beaker, and microwave this mixture until no solid particles or bubbles remain (approximately 2.5 minutes). The mixture is allowed to cool for 3 minutes. Add 10 μL of GelRed™ to the agarose gel mixture and stir (available from Biotium). Assemble the gel caster and gel comb. Pour the mixture into the gel mold and let it cool for 30 minutes (capacity for a large comb: 60 μL). Once the gel has cooled, fill the box with 1x TAE buffer until the gel is completely submerged. A digestion reaction mixture is prepared at room temperature to linearize the DNA. Digest 25 μg of the vector plasmid with restriction enzyme SfiI. In a separate reaction, digest the resistance cassette plasmid PGK-ble with PflMI (10 μg is more than enough). TIFF0007833493000004.tif101128

[0119] Mix lightly, then heat and incubate at 37°C for 15 minutes. Add 10 μL of GeneRuler 1kb plus DNA ladder mixture (2 μL of DNA ladder + 8 μL of nuclease-free water) and 50 μL of sample mixture to an available slot. Turn on the electrophoresis apparatus and run it at a voltage of 150V for 1 hour. The gel is transferred to a UVP PhotoDoc-It imaging system, and the resulting image is acquired. Download gel images from the Eye-Fi website. The DNA concentration of each sample is measured using a NanoDrop 2000 spectrophotometer.

[0120] Stage 2 DNA bands are extracted from the agarose gel. Add three times the volume of Buffer QG to one volume of gel (generally 500 μL of QG). After the gel slices have completely dissolved, incubate at 50°C for 10 minutes. Apply the sample to a QIAquick column and centrifuge at 17,900 rpm for 1 minute (available from Qiagen). Discard the flow-through and return the QIAquick column to the same collection tube. Add 0.5 ml of Buffer QG to the QIAquick column and centrifuge for 1 minute. Add 0.75 ml of Buffer PE to the QIAquick column and centrifuge for 1 minute. Discard the flow-through and centrifuge the QIAquick column at 17,900 rpm for another minute. Place the QIAquick column into a clean 1.5 ml microcentrifuge tube. To elute the DNA, add 35 μL of Buffer EB to the center of the QIAquick membrane and centrifuge the column at 17,900 rpm for 1 minute (Buffer EB is 10 mM Tris-Cl, pH 8.5). The concentration of DNA fragments was measured using a NanoDrop 2000 spectrophotometer (Table 1; EB buffer was used for the blank measurement).

[0121] Stage 3 Set up the ligation reaction in a 1.7 mL Eppendorf microcentrifuge tube on ice. Using a pre-built spreadsheet (Concatemeric Ligations.xlsx), calculate the volume of each fragment that needs to be mixed so that the molar ratio of vector to PGK-ble is approximately 25:1. To maximize the volume of fragments in the ligation reaction and maintain the desired molar ratio. The T4 DNA ligase buffer should be thawed and resuspended at room temperature (the T4 DNA ligase buffer contains the following components: 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5). The ligation reaction solution is dispensed using a pipette. In the example above, 90 μL of DNA mixture was used by adding 10 μL of 10× ligation buffer (NEB Quick Ligation Kit) and 0.5 μL of ligase enzyme (available from New England BioLabs). Prepare a reaction mixture containing the following components at room temperature. TIFF0007833493000005.tif68138 Mix gently by pipetting up and down. Incubate at room temperature overnight.

[0122] Stage 4 The concatemer array was recovered and purified using a silica-based membrane (DNeasy Blood & Tissue Kit) before transfection with GPRG cells. Pipette the concatemer array mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge at 8000 x g for 1 minute. Discard the flow-through and collection tube. Place the DNeasy Mini spin column into a new 2 ml collection tube (equipped) (available from Qiagen). Add 500 μL of Buffer AW1 and centrifuge at 8000 × g for 1 minute. Discard the flow-through tube and collection tube. Place the DNeasy Mini spin column into a new 2 ml collection tube (equipped). Add 500 μL of Buffer AW2 and centrifuge at 20,000 × g for 3 minutes to dry the DNeasy membrane. Discard the flow-through tube and collection tube. Place the DNeasy Mini spin column into a clean 1.7 ml Eppendorf microcentrifuge tube. 200 μL of Buffer AE is added directly to the DNeasy membrane. Incubate at room temperature for 4 minutes. Centrifuge at 8000 x g for 1 minute to elute the DNA mixture. Repeat the elution process once. The concatemer DNA concentration is measured using a NanoDrop Lite spectrophotometer.

[0123] Example 4 - Protocol for creating a producing cell line using a concatemer array Before using the cells to produce viral vectors, passage them at least four times after thawing. Before vector induction, trypan blue is used to confirm that the cells are healthy and that more than 95% are viable (trypan blue is commonly used in dye exclusion methods for counting viable cells. This method is based on the principle that living (viable) cells do not take up a specific dye, while dead (unviable) cells do. Staining facilitates the visualization of cell morphology). Culture the desired amount of GPRG cells. Before seeding the cells, perform secondary cultures at least twice by daily subculturing. On day 1, remove the culture medium from the GPRG cell line culture dish and wash the cells with 1×PBS. Gently pipette 1×TrypLE Express onto a washed cell monolayer, using 3 ml for a T75 flask or 1 ml for a T25 flask. Rotate the flask using the SteppLE Express so that the single layer is covered. Return the flask to the incubator and leave it for 2 minutes. Gently tap one side of the flask to detach any remaining cells. Resuspend the cells in 2 mL of fresh D10 medium and transfer them to a 15 mL conical centrifuge tube. Centrifuge the cells at 1200 rpm for 5 minutes. Aspirate the culture medium and gently suspend the pellet in 5 mL of fresh D10 medium containing doxycycline (1 ng / mL). The number of cells is measured using the TC10 (trademark) fully automated cell counter (Table 5). The cells were seeded in a culture dish at 80% confluence 20–24 hours before transfection (by seeding 3.2 × 10⁶ live cells in a 60 mm culture dish containing doxycycline; Table 9). Prepare for the formation of the concatemer array (see, for example, Example 3). On the second day, return the CalPhos® Mammalian Transfection Kit to room temperature prior to transfection (Table 7) (available from ClonTech). The concatemer DNA is purified and its concentration is measured (the concatemer array can be purified according to the method described herein). Prepare a transfection plasmid DNA table (Table 8; 4 mL, 60 mm culture dish). For each transfection, prepare solution A and solution B in separate 15 mL conical centrifuge tubes. Using a pipette, bubble solution B (2×HBS) and add solution A (DNA mixture) drop by drop. Incubate the transfection solution at room temperature for 15 minutes. Gently add the transfection solution to the culture dish. Gently move the plate back and forth to distribute the transfection solution evenly. Incubate the plate in a CO2 incubator at 37°C for 4 hours. Warm 5 mL of fresh D10 medium per 60 mm culture dish in a 37°C CO2 incubator. After 4 hours, wash with 1 mL of pre-warmed D10 medium and replace with 4 mL of pre-warmed fresh D10 medium. Incubate in a 5% CO2 incubator at 37°C. 48 hours after concatemer transfection, the transfected GPRG cells are harvested (using cells intended for secondary culture). The cells are re-seed in a T150 flask or a 30 mL, 150 mm culture dish using fresh D10 medium containing zeosin (50 μg / ml) and doxycycline (1 ng / mL). The cells are supplied with selective medium containing doxycycline (1 ng / mL) (zeosin, 50 μg / mL) every 3-4 days until cell focus is observed (usually within 1-2 weeks).

[0124] Example 5 - Description of the cell line and sequence used to prepare the GPRG-packaged cell line HEK-293T / 17 is a subclone of HEK-293T. These cells stably express the SV-40 T antigen, and specific clones were selected, particularly for their high transfection ability. A master cell bank based on HEK-293T / 17 (HEK-293T / 17 MCB) was created.

[0125] SFG-IC-HIVgp-Ppac2 is a puromycin-resistant gamma retroviral vector that expresses codon-optimized HIV gagpol under the control of the CMV promoter. The plasmid used to construct this vector (pSFG-IC-HIVgp-Ppac2) was constructed using the following components: (1) pSFG tcLuc ECT3 is a derivative of a retroviral vector scaffold plasmid (SFG) suitable for regulated gene expression using a tetracycline-regulated promoter system (Lindemann, D., Patriquin, E., Feng, S., & Mulligan, RC Versatile retrovirus vector systems for regulated gene expression in vitro and in vivo. Mol. Med. 3, 466-476 (1997)). (2) CMV enhancer / promoter-driven codon-optimized HIV NL4-3 gagpol gene; (3) PGK promoter-driven puromycin resistance gene derived from pMSCVpac (Hawley, RG, Lieu, FH, Fong, AZ, & Hawley, TS Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1, 136-138 (1994)).

[0126] Infection of HEK-293T / 17 MCB with the SFG-IC-HIVgp-Ppac2 retroviral vector resulted in GP cell lines.

[0127] SFG-tc-revco is a gamma retroviral vector that expresses codon-optimized HIV rev under the control of a tetracycline-responsive promoter. The plasmid (pSFG-tc-revco) used to construct this vector was constructed using the following components: (1) HIV rev gene based on the sequence of the NL4-3 strain as described above; and (2) pSFG tcLuc ECT3 (as above).

[0128] SFG-tTA is a gamma retrovirus vector that expresses a chimeric transcriptional transactivator under the control of the retroviral LTR (Lindemann, D., Patriquin, E., Feng, S., and Mulligan, RC Versatile retrovirus vector systems for regulated gene expression in vitro and in vivo. Mol. Med. 3, 466-476 (1997)). It is based on the SFG retrovirus vector and incorporates a Tet promoter element derived from plasmid pUHD15-1 (Gossen M, and Bujard, H. (1992) PNAS 89 12:5547-5551).

[0129] Infection of GP cell lines with SFG-tc-revco and SFG-tTA resulted in the development of GPR cell lines.

[0130] SFG-tc-VSVG is a gamma retrovirus vector that expresses VSV glycoprotein G under the control of a tetracycline-regulated promoter. The plasmid used to construct this vector (pSFG-tc-VSVG) was prepared using the same pSFGtcLucECT3 backbone as other vectors, and plasmid pMD.G as the source of the VSVG envelope protein (see Ory, DS, Neugeboren, BA, and Mulligan, RC A stable human-derived packaging cell line for production of high titer retrovirus / vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. USA 93, 11400-11406 (1996) and Rose, JK & Gallione, C. (1981) J. Virol. 39, 519-528).

[0131] Infection of GPR cell lines with SFG-tc-VSVG resulted in the development of GPRG cell lines.

[0132] The infection of GPR cell lines with Retro-SVGmu for the creation of GPRS cell lines is described in Lee, Chi-Lin et al. "Construction of Stable Producer Cells to Make High-Titer Lentiviral Vectors for Dendritic Cell-Based Vaccination." Biotechnology and Bioengineering 109.6 (2012): 1551-1560. PMC. Web. 14 Apr. 2016.

[0133] Example 6 - Purification of concatemer array Prior to transfection with GPRG cells, concatemers were collected and purified using a silica-based membrane (DNeasy Blood & Tissue Kit). Transfer the concatemer array mixture to a DNeasy Mini spin column in a 2 mL collection tube using a pipette. Centrifuge at 6000 x g for 1 minute. Discard the flow-through and collection tube. Place the DNeasy Mini spin column into a new 2 mL collection tube (equipped). Add 500 μL of Buffer AW1 and centrifuge at 6000 × g for 1 minute. Discard the flow-through tube and collection tube. Place the DNeasy Mini spin column into a new 2 ml collection tube (equipped). Add 500 μL of Buffer AW2 and centrifuge at 20,000 × g for 3 minutes to dry the DNeasy membrane. Discard the flow-through tube and collection tube. Place the DNeasy Mini spin column into a clean 1.7 mL Eppendorf microcentrifuge tube. 200 μL of Buffer AE is added directly to the DNeasy membrane. Incubate at room temperature for 4 minutes. Centrifuge at 6000 x g for 1 minute to elute the DNA mixture. Repeat the elution process once (add new elution buffer). The concatemer DNA concentration is measured using a NanoDrop Lite spectrophotometer.

[0134] Example 7 - TL20-Ubc-GFP and TL20-Cal1-WPRE producing cell lines The following table summarizes two producing cell lines synthesized according to the method described herein. Data for the TL20-Cal1-wpre and TL20-Unc-GFP vectors are further shown in Figures 20A, 20B, and 20C.

[0135] TIFF0007833493000006.tif1031681. Abbreviation: TU, trait introduction unit. 2. Single-cell sorting is performed using a flow cytometer at the USC Flow Cytometry Core Facility. 3. Conditioning medium: GlutaMax-containing DMEM; FBS (10% w / v); penicillin / streptomycin (1% w / v); doxycycline (1 ng / mL).

[0136] All publications referenced herein are incorporated herein by reference in their entirety. Those skilled in the art will understand that many variations and / or modifications can be made to this disclosure, as shown in particular embodiments, without departing from the spirit or scope of the extensively described disclosure. Therefore, these embodiments should be considered in all respects to be illustrative and not restrictive.

[0137] While the disclosures herein are described in relation to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of this disclosure. Therefore, it should be understood that many modifications can be made to the exemplary embodiments, and other configurations can be conceived without departing from the spirit and scope of this disclosure as defined by the appended claims.

[0138] Sequence information SEQUENCE LISTING <110> CSL BEHRING GENE THERAPY, INC. <120> BIO-PRODUCTION OF LENTIVIRAL VECTORS <150> US 62 / 161,133 <151> 2015-05-13 <150> US 62 / 161,152 <151> 2015-05-13 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 6565 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: PUC57-TL20C <400> 1 ggccgcctcg gccaaacagc ccttgagttt accactccct atcagtgata gagaaaagtg 60 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact 120 ccctatcagt gatagaagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga 180 aaagtgaaag tcgagtttac cagtccctat cagtgataga gaaaagtgaa agtcgagttt 240 accactccct atcagtgata gagaaagtg aaagtcgagt ttaccactcc ctatcagtga 300 tagagaaaag tgaaagtcga gctcgccatg ggaggcgtgg cctgggcggg actggggagt 360 ggcgagccct cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg 420 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 480 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 540 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga 600 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaatttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cgtattaag cgggggagaa 780 ttagatcgcg atgggaaaa attcggttaa ggccaggggg aaagaaaaaa tataattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatact ggcctgttag aaacatcaga aggctgtaga caatactgg accatccctt cagacaggat cagaagaact tagatcatta fathercag tagcaacct ctattgtgtg catcaaagga 1080. 1080. 1080. 1080. 1080. 1080. 1080. 1080. 1080 agaaaaaagc acagcaagca gcaggatctt cagacctgga aattccctac aatccccaaa gtcaaggagt agtagaatct atgaataaag aattaaaga aattatagga caggtaagag atcaggctga acatcttaag acagcagtac aaatggcagt atcatccac aattttaaaa gaaaaggggg gattgggggg snow snow snow snow snow acatacaac windowaaaaaaaaaaaat tcaaattt cgggttttatt 1380 acagggacag cagaaatcca ctttggaag gaccagcaa gctcctctgg aaggtgaag 1440 gggcagtagt atacagat atagtgaca taaaagtagt gccaaga aaagchaaga 1500 1560 aggattagaa catggaaaag tttagaaaa caccataagg aggatag agggaaatt 1620 ggagaagtga atataataa tataaagtag taaaaattga accattagga gtagcacca 1680 ccaaggcaaa gagagagtg gtgcagagag aaaaagagc agtgggaata ggagctttgt 1740 tccttgggtt cttgggagca gcaggaagca ctatggggcgc agcgtcaatg acgctgacgg 1800 tacaggccag acattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcactca cagtctgggg catcagcag ctccaggcag 1920 gatcctggc tgtggaaaga tacctaagg atcacagct cctggggatt tggggttgct 1980 ctggaaaact cattgcacc actgctgtgc cttggaatgc tagttggatt aaaatctc 2040 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca 2100 caagcttaat acactcctta attgaagaat cgcaaacca gcaagaaaag aatgaacaag 2160 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc 2220 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt 2280 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga 2340 cccacctccc aaccccgagg ggaccgagct caagcttcga acgcgtgcgg ccgcatcgat 2400 gccgtagtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta 2460 aaagaaaagg ggggactgga agggctaatt cactcccaaa gaagacaaga tccctgcagg 2520 cattcaaggc caggctggat gtggctctgg gcagcctggg ctgctggttg atgaccctgc 2580 acatagcagg gggttggatc tggatgagca ctgtgctcct ttgcaaccca ggccgttcta 2640 tgattctgtc attctaaatc tctctttcag cctaaagctt tttccccgta tccccccagg 2700 tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc gtgccacctt 2760 ccccgtgcccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg agcgccggac 2820 cggagcggag ccccgggcgg ctcgctgctg cccctagcg ggggagggac gtaattacat 2880 ccctgggggc tttggggggg ggctgtcccc gtgagctccc cagatctgct ttttgcctgt 2940 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac 3000 ccactgctta agcctcaata aagcttcagc tgctcgagct agcagatctt tttccctctg 3060 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc tataaagga 3120 aatttatttt cattgcaata gtgtgttgga atttttgtg tctctcactc ggaaggacat 3180 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat 3240 atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga 3300 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt 3360 tttttatatt ttgttttgtg ttattttt cttaacatc cctaaaattt tccttacatg 3420 tttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctctc 3480 tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt 3540 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 3600 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 3660 cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa 3720 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 3780 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct 3840 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 3900 tgtcgactgc agaggcctgc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt 3960 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 4020 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 4080 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 4140 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 4200 cgtcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4260 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4320 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4380 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4440 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta cggatacct 4500 gtccgcctt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 4560 cagttcggtg tagtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4620 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4680 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4740 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 4800 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagt ggtagctctt gatccggcaa 4860 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4920 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4980 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 5040 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 5100 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 5160 catagttgcc tgactccccg tcgtgtagat aactacgata cgggaggct taccatctgg 5220 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5280 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5340 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5400 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5460 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5520 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5580 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5640 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 5700 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 5760 gctcatcatt ggaaaacgtt cttcggggcg aaactctca aggatcttac cgctgttgag 5820 atccagtcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5880 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5940 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 6000 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aaaatagg 6060 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 6120 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gttcggtga 6180 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 6240 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 6300 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 6360 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgcattcgc cattcaggct 6420 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 6480 aggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttcc agtcacgacg 6540 ttgtaaaacg acggccagtg aattc 6565 <210> 2 <211> 3901 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: TL20C vector backbone <400> 2 ggccgcctcg gccaaacagc ccttgagttt accactccct atcagtgata gagaaaagtg 60 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact 120 ccctatcagt gatagaagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga 180 aaagtgaaag tcgagtttac cagtccctat cagtgataga gaaaagtgaa agtcgagttt 240 accactccct atcagtgata gagaaagtg aaagtcgagt ttaccactcc ctatcagtga 300 tagagaaaag tgaaagtcga gctcgccatg ggaggcgtgg cctgggcggg actggggagt 360 ggcgagccct cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg 420 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 480 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 540 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga 600 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg 660 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga 720 ctagcggagg ctagaaggag agagatgggt gcgagagcgt সাতাতাতাস cggggagaa 780 ttagatcgcg atgggaaaaa attcggttaa ggccagggg aaagaaaaaa tataaattaa 840 aacatagat atgggcaagc agggagctag aacgattcgc agttaatact ggcctgttag 900 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat 960 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga 1020 tagagataaa agacaccaag gaagctttag acaagataga ggagagcaa aacaaaagta 1080 agaaaaaagc acagcaagca gcaggatctt cagacctgga aattccctac aatccccaaa 1140 gtcaggagt agtagaatct atgaataag aattaaaga aattatagga caggtaagg 1200 atcaggctga acatcttaag acagcagtac aaatggcagt atcatccac aattttaaaa 1260 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata tagcacag 1320 acatacaac windowaaaaaaaaaaaat tcaaattt cgggttttatt 1380 acagggacag cagaaatcca ctttggaag gaccagcaa gctcctctgg aaggtgaag 1440 gggcagtagt atacagat atagtgaca taaaagtagt gccaaga aaagchaaga 1500 1560 aggattagaa catggaaaag tttagaaaa caccataagg aggatag agggaaatt 1620 ggagaagtga atataataa tataaagtag taaaaattga accattagga gtagcacca 1680 ccaaggcaaa gagagagtg gtgcagagag aaaaagagc agtgggaata ggagctttgt 1740 tccttgggtt cttgggagca gcaggaagca ctatggggcgc agcgtcaatg acgctgacgg 1800 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta 1860 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa 1920 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct 1980 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc 2040 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca 2100 caagcttaat acactcctta attgaagaat cgcaaacca gcaagaaaag aatgaacaag 2160 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc 2220 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt 2280 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga 2340 cccacctccc aaccccgagg ggaccgagct caagcttcga acgcgtgcgg ccgcatcgat 2400 gccgtagtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta 2460 aaagaaaagg ggggactgga agggctaatt cactcccaaa gaagacaaga tccctgcagg 2520 cattcaaggc caggctggat gtggctctgg gcagcctggg ctgctggttg atgaccctgc 2580 acatagcagg gggttggatc tggatgagca ctgtgctcct ttgcaaccca ggccgttcta 2640 tgattctgtc attctaaatc tctctttcag cctaaagctt tttccccgta tccccccagg 2700 tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc gtgccacctt 2760 ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg agcgccggac 2820 cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac gtaattacat 2880 ccctgggggc tttggggggg ggctgtcccc gtgagctccc cagatctgct ttttgcctgt 2940 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac 3000 ccactgctta agcctcaata aagcttcagc tgctcgagct agcagatctt tttccctctg 3060 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga 3120 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat 3180 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat 3240 atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga 3300 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt 3360 tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg 3420 ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc 3480 tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt 3540 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 3600 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 3660 cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa 3720 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 3780 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct 3840 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 3900 t 3901 <210> 3 <211> 343 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: nucleotide sequence encoding the packing signal <400> 3 agtattaagc gggggagaat tegtcgcga tgggaaaaa ttcggttaag gccaggggga 60 aagaaaaaat aataatttaa acatagta tggcagca gggagctga acgattcgca 120 gttaatactg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa 180 ccatcccttc agacaggatc agagaactt agatcattat atatacagt agcaccctc 240 tattgtgtgc atcaaggat agagaataaa gatcaagg aagctttaga agatagag 300 gagaaaaaaaaaaaaaaaaaaaaaaaaaagg fromg 343 <210> 4 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: Central polypurine tract (cppt) <400> 4 aattccctac aatccccaaa gtcaggagt agtagaatct atgaataag attaaagaa 60 aattatagga caggtaagg atcaggctga acatcttaag acagcagtac aaatggcagt 120 attcatccac aattttaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat 180 attachaaaaaaaaaaac window aaaaaaaaaaaaat 240 tcaaatttt cgggttttatt acaggcag cagaaatcca ctttggaaag gaccagcaaa 300 gctcctctgg aaagtgaag gggcagtagt atacagat atagtgaca tAAagtagt 360 gccaagaga aaagchaaga tcattaggga ttatggaaa cagatggcag gtgatgattg 420 tgtggcaagt agacaggatg aggattagaa catggaaag tttagtaaaa caccata 477 <210> 5 <211> 769 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: nucleotide sequence encoding the rev response element <400> 5 yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yay yeah tgaaccatta ggagtagcac ccaccaggc aaagagaga gtggtgcaga gagaaaaaag 120 agcagtggga ataggactt tgttccttgg gttcttggga gcagcaggaa gcactatggg 180 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca 240 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg 300 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca 360 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa 420 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg 480 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa 540 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa 600 ttggtttaac ataacaaatt ggctgtggta tataaatta ttcataatga tagtaggagg 660 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg 720 atattcacca ttatcgtttc agacccacct cccaaccccg aggggaccg 769 <210> 6 <211> 181 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: nucleotide sequence encoding the self-inactivating long terminal repeat <400> 6 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180 a 181 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: nucleotide sequence encoding the multiple cloning site <400> 7 ttcgaacgcg tgcggccgca tcgat 25 <210> 8 <211> 1978 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: LVSH5 / C46 <400> 8 gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa 60 cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc 120 tgcaattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg gatttgggaa tcttataagt tctgtatgag accacggatc cccgagcaag ctcagtttac 240 accttgtccg acggtgtaaa ctgagcttgc tctttttgag acgagtcctc gagccataaa gatggttaat taacccaccc aagatctggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct cctcacggcg agcgctgcca cgtcagacga agggcgcagc gagcgtcctg 420 atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac 480 cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg gcactggttt 540 tctttccaga gagcggaaca ggcgaggaa agtagtccct tctcggcgat tctgcggagg gatctccgtg gggcggtga cgccgatgat father cgcgccgggt gtggcacagc 660 tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc 720 acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg 780 tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt 840 gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg agtcttgaat 900 ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg catggtgggc 960 ggcaagaacc caaggtcttg aggccttcgc taatgcggga aagctcttat tcgggtgaga 1020 tgggctgggg caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg 1080 tttgtcgtct gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc 1140 tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca 1200 gggtggggcc acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt 1260 tcgggcctag ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga 1320 taagtgaggc gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag 1380 ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca 1440 ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc agtgttagac tagtaaattg 1500 tccgctaaat tctggccgtt tttggctttt ttgttagacg aagcttggta ccgagctcgg atccgccacc atgggagcag gagcaaccgg aagggcaatg gacggacca gattgttact tctgctcctg ctaggcgtga gcctgggagg agcaaggagc tggatggagt gggacaggga 1680 gatcaacaac tacaccagcc tgatccacag cctgatcgag gagagccaga accagcagga gaagaacgag caggagctgc tggagctgga caagtgggcc agcctgtgga actggttccg gagcgagcgg aagtgctgcg tggagtgccc accatgccca gcaccaccag tggcaggacc 1860. cctgatcgca ctggtgacca gcggagccct gctggccgtg ctgggcatca caggctactt cctgatgaac aggaggagct ggagcccaac cggagagcgg ctggagctgg agccatga <210> 9 <211> 1113 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: PGK-BLE SEQUENCE <400> 9 ccagcctttg gaattcctgc aggatgggat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctggggcac ttggcgctac acaagtggcc 120 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg 180 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttcccccccc gccccgcagc 240 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg 300 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt 360 gctccttgc tttctgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg 420 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg 480 ggcctttcga cctggatcct ccagcacgtg ttgacaatta atcatcggca tagtatatcg 540 catatagtata atacgactca ctatagggagg gccaccatgg ccaagttgac cagtgccgtt 600 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg 660 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg 720 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg 780 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac 840 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg 900 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg atgctttatt 960 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 1020 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt 1080 taaactagtg agtcgtatta cccagccttt ggg 1113 <210> 10 <211> 288 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: (7tetO): doxycycline repressible promoter <400> 10 tttaccactc cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag 60 tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa 120 gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccagtccc 180 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa 240 gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaa 288 <210> 11 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: HIV LTR R5 region <400> 11 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60 ctgcttaagc ctcaataaag cttgccttga gtgcttca 98 <210> 12 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: HIV LTR U5 region <400> 12 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 60 gtcagtgtgg aaaatctcta gca 83 <210> 13 <211> 412 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: chicken HS4 400 bp chromatin insulator <400> 13 atccctgcag gcattcaagg ccaggctgga tgtggctctg ggcagcctgg gctgctggtt 60 gatgaccctg cacatagcag ggggttggat ctggatgagc actgtgctcc tttgcaaccc 120 aggccgttct atgattctgt cattctaaat ctctctttca gcctaaagct ttttccccgt 180 atccccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc 240 cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg 300 gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga 360 cgtaattaca tccctggggg ctttgggggg gggctgtccc cgtgagctcc cc 412 <210> 14 <211> 449 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: rabbit beta-globin polyadenylation signal <400> 14 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 60 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 120 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt 180 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga 240 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg 300 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta 360 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc 420 atagctgtcc ctcttctctt atggagatc 449 <210> 15 <211> 2664 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: pUC57 Plasmid portion <400> 15 gtcgactgca gaggcctgca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg 60 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 120 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 180 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 240 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 300 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 360 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 420 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 480 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataagata ccaggcgttt 540 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 600 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 660 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 720 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 780 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 840 acagagttct tgaagtggtg gcctaactac ggctacacta gaaacagt atttggtatc 900 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 960 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 1020 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 1080 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 1140 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 1200 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 1260 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 1320 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 1380 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 1440 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 1500 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 1560 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 1620 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 1680 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 1740 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 1800 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 1860 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 1920 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 1980 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 2040 acacggaaat gttgaatact catactcttc cttttcaat attattgaag catttatcag 2100 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 2160 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 2220 acattaacct ataaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat 2280 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 2340 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 2400 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 2460 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg 2520 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 2580 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 2640 tgtaaaacga cggccagtga attc 2664

Claims

1. A method for recovering the vector supernatant, wherein the method is A step of producing cells of a stable producing cell line, wherein the cells of the stable producing cell line are derived from one of the following: GPRT, GPRGT, or GPRT-G packaging cell lines or their derivatives. The steps include inducing viral vector production from cells of the prepared stable production cell line, A step comprising the repeated recovery of the viral vector from the cells of the induced, prepared, stable-producing cell line every 40 to 56 hours after the initial recovery of the viral vector, wherein each repeated recovery includes adding fresh culture medium to the cells of the induced, prepared, stable-producing cell line without introducing additional cells of the prepared, stable-producing cell line, Includes, The stable producing cell line created is produced by the steps of (a) synthesizing a lentiviral vector by cloning one or more genes into a recombinant plasmid, (b) forming a concatemer array from (i) lentiviral transfer vector DNA excised from the synthesized lentiviral vector and (ii) an expression cassette obtained from an antibiotic resistance cassette plasmid, (c) transfecting the formed concatemer array into one of GPRT, GPRGT, or GPRT-G packaging cell lines, and (d) isolating the stable producing cell line created, wherein the recombinant plasmid contains a nucleotide sequence having at least 85% identity with the sequence of SEQ ID NO:

1. method.

2. The method according to claim 1, wherein the initial recovery is performed 40 to 56 hours after induction.

3. The method according to claim 1, wherein the repeated collection is performed at least twice, and the first collection is performed less than 48 hours after induction.

4. The method according to claim 1, wherein the repeated collection is performed every 44 to 52 hours.

5. The method according to claim 1, wherein the repeated collection is performed at least twice, and the repeated collection is performed every 48 hours.

6. After induction, for at least 5 days, at least 1 x 10 6 The method according to claim 1, wherein a viral titer of TU / mL is produced.

7. Through at least 8 passages, at least 1 x 10 6 The method according to claim 1, wherein a viral titer of TU / mL is produced.

8. Through at least 21 passages, 1 x 10 6 TU / mL ~ 10 x 10 6 The method according to claim 1, wherein a viral titer of TU / m is produced.

9. The method according to claim 1, wherein the culture medium to be recovered is replaced after each repeated recovery, and no additional culture medium to be recovered is introduced into the prepared stable producing cell line during viral vector production.

10. The method according to claim 1, wherein the stable producing cell line is derived from a GPRT packaging cell line.

11. The method according to claim 1, wherein the antibiotic-resistant cassette plasmid is a bleomycin antibiotic-resistant cassette.

12. The method according to claim 11, wherein the molar ratio of the expression cassette excised from the synthesized vector and the expression cassette obtained from the pre-bleomycin antibiotic resistance cassette is in the range of 50:1 to 1:

50.

13. The method according to claim 12, wherein the molar ratio is in the range of 25:1 to 1:

25.

14. The method according to claim 12, wherein the molar ratio is in the range of 15:1 to 1:

15.

15. The method according to claim 1, wherein the recombinant plasmid comprises a nucleotide sequence having at least 90% identity with respect to the sequence of SEQ ID NO:

1.

16. The method according to claim 1, wherein the recombinant plasmid comprises a vector cassette having the sequence SEQ ID NO:

2.

17. The method according to claim 1, wherein the recombinant plasmid comprises a multicloning site having BstBI, MluI, NotI, and ClaI restriction endonuclease sites.

18. The method according to claim 17, wherein the nucleotide sequence encoding the multicloning site has at least 90% sequence identity with respect to the sequence of SEQ ID NO:

7.

19. The method according to claim 17, wherein the recombinant plasmid further comprises a nucleotide sequence encoding a packaging signal, a nucleotide sequence encoding a central polyprint lacte, a nucleotide sequence encoding a Rev response element, and a nucleotide sequence encoding a self-inactivating terminal repeat.

20. The method according to claim 19, further comprising a vector cassette having the sequence SEQ ID NO:

2.

21. The method according to claim 20, wherein the vector cassette is adjacent to at least two additional restriction endonuclease sites, and the at least two additional restriction endonuclease sites are independently selected from the group consisting of sfiI and Bsu36I.

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