Novel recombinant vector and its uses

Abstract

The present invention relates to a recombinant vector, and a gene transfer system, recombinant virus, and transformant comprising the same. The recombinant vector according to the present invention is designed to express one target gene by one promoter, and by transferring two or more target genes into a cell, one vector can stably express up to three target genes simultaneously. Therefore, such a recombinant vector can be usefully used in an efficient gene transfer system.

Description

[Technical field]

[0001] The present invention relates to a recombinant vector, and a gene delivery system, a recombinant virus and a transformant comprising the same. [Background technology]

[0002] Gene therapy is a generic name for a treatment technique that uses genes, such as inserting new genes into the patient's cells, eliminating malfunctioning genes, or replacing mutated genes with normal genes. This type of gene therapy can be targeted immediately by identifying only the genes that cause the disease, and can be produced more quickly and cheaply than other antibodies or compound therapeutics, increasing the possibility of treating diseases that are difficult to treat with existing drug therapeutics.

[0003] For effective gene therapy, it is first necessary to find a target gene that is important for treating a disease, and then it is essential to develop a technology to develop a vector that can effectively deliver the gene into cells without side effects. Vectors are gene carriers and are broadly divided into viral carriers and non-viral carriers. Viral carriers are produced by removing most or some of the essential genes of a virus to prevent it from replicating on its own, and then inserting a therapeutic gene into the virus instead. Although they can deliver genes into cells with high efficiency, there are problems such as difficulty in mass production, induction of immune responses, toxicity, and emergence of replicable viruses depending on the type of virus. Currently, the main viral carriers used in the development of gene therapy agents include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and pox viruses. On the other hand, non-viral carriers, such as liposomes and plasmids, do not induce immune responses, are less toxic and are easy to mass-produce, but they have low gene transfer efficiency and their expression is temporary, which limits the development of gene therapy agents.

[0004] In the development of gene therapy drugs, vector technology using adeno-associated viruses and retroviruses is the mainstream. Lentivirus, a type of retrovirus, is capable of gene transfer regardless of cell division, and the chromosomes where the gene to be transferred is inserted are distributed in areas with a relatively low risk of oncogenic insertional mutagenesis. It has the advantage of inducing self-inactivation by deleting the U3 portion from the 3'LTR (long terminal repeat), fundamentally blocking the risk of tumorigenicity that can be induced by LTR.

[0005] However, the size of a target gene that can be introduced into a viral vector is limited, and the larger the size of the target gene, the greater the possibility that the target gene will be lost due to homologous recombination. In addition, multiple target genes contained in one vector may be non-uniformly silenced depending on the type and state of the cell, and such silencing phenomena are difficult to predict or prevent with current technology. Therefore, it is not easy to construct a vector that expresses two or more types of target genes uniformly without silencing in cells (e.g., mesenchymal stem cells), and research on this issue is urgently needed. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent No. 10-1885438 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a recombinant vector capable of transferring two or more target genes into cells and stably expressing them.

[0008] Another object of the present invention is to provide a gene transfer system comprising the recombinant vector.

[0009] Another object of the present invention is to provide a recombinant virus comprising the recombinant vector.

[0010] A further object of the present invention is to provide a transformant into which the recombinant vector or recombinant virus has been introduced. [Means for solving the problem]

[0011] One aspect of the present invention provides a recombinant vector comprising a CTE, a first gene of interest, a first promoter, a second promoter and a second gene of interest, wherein the first gene of interest and the first promoter are in opposite orientations.

[0012] The term "vector" as used herein refers to an expression vector capable of expressing a gene of interest in a suitable host cell, and refers to a genetic construct that contains essential regulatory elements operably linked to express a gene insert contained in the vector. Here, "operably linked" refers to a functional linkage between a nucleic acid expression regulatory sequence that performs a general function and a nucleic acid sequence that encodes a gene of interest.

[0013] The vector according to the present invention includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression regulatory elements such as a promoter, an operator, a start codon, a stop codon, a polyadenylation signal, and an enhancer, and can be prepared in a variety of ways depending on the purpose. The promoter of the vector may be constitutive or inducible. In addition, the expression vector may include a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, may include a replication origin. Such a vector may be self-replicating or integrated into a host DNA. Examples of the vector include a plasmid vector, a cosmid vector, a bacteriophage vector, and a virus vector.

[0014] A recombinant vector comprising such a CTE, a first gene of interest, a first promoter, a second promoter and a second gene of interest contains two genes of interest and two promoters that regulate the expression of each of the two genes of interest, thereby enabling two genes of interest to be expressed simultaneously in a single vector.

[0015] Furthermore, the recombinant vector according to the present invention can additionally contain a gene of interest and a promoter regulating its expression, thereby enabling three genes of interest to be expressed simultaneously in one vector.

[0016] According to one embodiment of the present invention, the recombinant vector may further comprise a third promoter and a third gene of interest.

[0017] The term "constitutive transport element (CTE)" used in the present invention refers to a factor that promotes the nuclear export of incompletely spliced ​​mRNA. It is known that the closer the CTE is located to the poly A tail, which is important for the nuclear export, translation and stability of mRNA, the more its function is improved and the more the expression of the target gene is increased (MR Mautino, et al., Gene Therapy. July 2000, pp. 1421-1424).

[0018] As used herein, the term "target gene" refers to a gene whose expression is induced by a promoter.

[0019] The term "promoter" used in the present invention refers to a portion of DNA that is involved in the binding of RNA polymerase to initiate transcription. Generally, it is located adjacent to and upstream of a gene of interest, and is a site to which RNA polymerase or a transcription factor, a protein that induces RNA polymerase, binds, and can induce the enzyme or protein to be located at the correct transcription start site. That is, it is located at the 5' site of the gene to be transcribed on the sense strand, and RNA polymerase binds to this site directly or via a transcription factor to induce the initiation of mRNA synthesis for the gene of interest, and has a specific gene sequence.

[0020] The elements of the recombinant vector, ie, the CTE, the gene of interest and the promoter, can affect the expression of the gene of interest depending on the ligation direction.

[0021] According to one embodiment of the present invention, the CTE may be linked to the 3' end of a poly A tail.

[0022] According to one embodiment of the present invention, the CTE may include a base sequence represented by SEQ ID NO:1.

[0023] According to one embodiment of the present invention, the CTE may be normal or reverse.

[0024] As used herein, "forward orientation" means that the sequence encoded by each gene within the viral genome is in a sense orientation (5'→3'), and "reverse orientation" means that the sequence encoded by each gene within the viral genome is in an anti-sense orientation (3'→5').

[0025] In order to simultaneously express two or more genes of interest, the recombinant vector according to the present invention comprises two or more promoters that induce the expression of each gene of interest, wherein the two or more promoters may be of the same type or different types.

[0026] In such a recombinant vector, the second gene of interest and the second promoter, and / or the third gene of interest and the third promoter, except for the first gene of interest and the first promoter, which are in the reverse orientation, can be ligated in the forward orientation.

[0027] According to one embodiment of the present invention, the promoter may be one or more selected from the group consisting of a Simian virus 40 (SV40) promoter, a cytomegalovirus (CMV) promoter, a minimal CMV promoter, a human ubiquitin C promoter (UBC) promoter, a human elongation factor 1a (EF1A) promoter, a phosphoglycerate kinase 1 (PGK) promoter, and a cytomegalovirus immediate-early enhancer / chicken β-actin (CAG) promoter.

[0028] The first promoter, the second promoter and the third promoter according to the present invention are preferably different from each other in order to enhance the expression of each target gene.

[0029] As an example, the first promoter, the second promoter and the third promoter may be selected from the group consisting of a CAG promoter (sequence number 2), a minimal CMV (hereinafter referred to as "mCMV") (sequence number 3), a PGK promoter (sequence number 4) and SV40 (promoter 5).

[0030] More specifically, the first promoter and the second promoter may be a CAG promoter and an mCMV promoter, a mCMV promoter and a CAG promoter, a PGK promoter and an mCMV promoter, or a mCMV promoter and a PGK promoter, respectively, where the first promoter is linked in the reverse direction.

[0031] According to one embodiment of the present invention, the target gene may be one or more selected from the group consisting of a disease therapeutic gene, a reporter gene, a selection marker gene, and a cell marker gene.

[0032] The recombinant vector according to the present invention is capable of simultaneously expressing two or more genes of interest, and can therefore express either a disease therapeutic gene, a reporter gene or a selectable marker gene, or can express all of a disease therapeutic gene, a reporter gene and a selectable marker gene.

[0033] The term "disease-treating gene" as used herein means a polynucleotide sequence or a base sequence that encodes a polypeptide that has a therapeutic effect on cells that abnormally express the gene, such as cancer cells.

[0034] According to one embodiment of the present invention, the disease treatment gene may be one or more selected from the group consisting of drug sensitivity genes, cell death genes, cell proliferation inhibitory genes, cell growth genes, cytotoxicity genes, tumor suppressor genes, antigenic genes, cytokine genes, neurogenesis genes, anti-angiogenesis genes, and hormone genes.

[0035] The drug-sensitizing gene is a gene that encodes an enzyme that converts a non-toxic prodrug into a toxic substance, and is also called a suicide gene because the cell into which the gene is introduced is killed. That is, when a precursor that is not toxic to normal cells is administered locally or systemically, the precursor is converted into a toxic metabolite only in the target cell, and the target cell can be destroyed by changing the drug sensitivity. Examples of such drug-sensitizing genes include, but are not limited to, the HSV-TK (Herpes simplex virus-thymidine kinase) gene, which uses ganciclovir or valganciclovir as a precursor, and the cytosine deaminase of E. coli, which uses 5-fluorocytosine (5-FC) as a precursor.

[0036] The proapoptotic gene is a nucleotide sequence that induces programmed cell death when expressed. Examples of such proapoptotic genes include, but are not limited to, p53, adenovirus E3-11.6K (from Ad2 and Ad5) or adenovirus E3-10.5K (from Ad), adenovirus E4 genes, p53 pathway genes, and genes encoding caspases.

[0037] The cytostatic gene is a nucleotide sequence that is expressed in a cell to stop the cell cycle midway through the cell cycle, and examples thereof include, but are not limited to, p21, the retinoblastoma gene, the E2F-Rb fusion protein gene, genes encoding cyclin-dependent kinase inhibitors (e.g., p16, p15, p18, and p19), and the growth arrest specific homeobox (GAX) gene.

[0038] The cell growth gene is a nucleotide sequence that promotes the division, growth, and differentiation of stem cells or cells that have already completed differentiation. Examples of such cell growth genes include, but are not limited to, hepatocyte growth factor, stem cell factor, insulin-like growth factor, epidermal growth factor, fibroblastic growth factor, nerve growth factor, transforming growth factor, platelet-derived growth factor, bone-derived growth factor, and colony stimulation factor.

[0039] The cytotoxic gene is a nucleotide sequence that is expressed in a cell to exert a toxic effect, and examples thereof include, but are not limited to, nucleotide sequences encoding Pseudomonas exotoxin, ricin toxin, diphtheria toxin, etc.

[0040] The tumor suppressor gene refers to a nucleotide sequence that can be expressed in a target cell to suppress a tumor phenotype or induce cell death. Examples of such tumor suppressor genes include, but are not limited to, tumor necrosis factor-α gene, p53 gene, APC gene, DPC-4 / Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, retinoblastoma gene, MMAC-1 gene, MMSC-2 gene, NF-1 gene, nasopharyngeal tumor suppressor gene located on chromosome 3p21.3, MTS1 gene, CDK4 gene, NF-1 gene, NF-2 gene, VHL gene, and PD-1 (programmed death-1) gene.

[0041] The antigenic gene is a nucleotide sequence that is expressed in target cells to produce a cell surface antigenic protein that can be recognized by the immune system, examples of which include, but are not limited to, carcinoembryonic antigen and p53.

[0042] The cytokine gene refers to a nucleotide sequence that is expressed in a cell to produce a cytokine, examples of which include, but are not limited to, GMCSF, interleukins (e.g., IL-1, IL-2, IL-4, IL-6, IL-12, IL-10, IL-15, IL-19, IL-20), and interferons (e.g., α, β, γ).

[0043] The neurogenic gene is a nucleotide sequence that promotes the generation and development of nerves, examples of which include, but are not limited to, brain-derived neurotrophic factor and nerve growth factor.

[0044] The neuronal differentiation gene is a nucleotide sequence involved in the division of neuroepithelial cells into neuroblasts and neuroglia from stem cell division, and examples thereof include, but are not limited to, neurogenin, NeuroD, and ASCL1 (Achaete-scute homolog 1).

[0045] The anti-angiogenic gene refers to a nucleotide sequence that is expressed to release an anti-angiogenic factor extracellularly, and examples thereof may include, but are not limited to, angiostatin, a vascular endothelial growth factor (VEGF) inhibitor, and endostatin.

[0046] The hormone gene refers to a nucleotide sequence that produces a hormone or a precursor thereof, examples of which include, but are not limited to, insulin, growth hormone, thyroid stimulating hormone, adrenocorticotropic hormone, gonadotropic hormone, prolactin, luteotropic hormone, melanocyte stimulating hormone, vasopressin, and oxytocin.

[0047] In addition, any gene that has a therapeutic effect can be used without restriction as a gene for treating a disease.

[0048] The "reporter gene" used in the present invention is a gene used to monitor the presence or absence of introduction of a recombinant vector or the expression efficiency of a target gene, and any gene that can be monitored without damaging the infected cells or tissues can be used without limitation.

[0049] According to one embodiment of the present invention, the reporter gene may be one or more selected from the group consisting of TdTomato, luciferase, copGFP isolated from Pontellina plumata, green fluorescent protein (GFP) isolated from Aequorea victoria, modified green fluorescent protein (mGFP), enhanced green fluorescent protein (eGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (eRFP), blue fluorescent protein (BFP), modified blue fluorescent protein (mBFP), enhanced blue fluorescent protein (eBFP), yellow fluorescent protein (YFP), modified yellow fluorescent protein (mYFP), enhanced yellow fluorescent protein (eYFP), cyan fluorescent protein (CFP), modified cyan fluorescent protein (mCFP) and enhanced cyan fluorescent protein (eCFP).

[0050] The "selection marker gene" used in the present invention is an antibiotic resistance gene used to select cells into which a gene has been introduced, and since only cells expressing the selection marker gene, i.e., the antibiotic resistance gene, can survive in a medium treated with an antibiotic, it is possible to select cells into which a gene has been introduced. As the selection marker gene, any antibiotic resistance gene known in the art can be used without limitation.

[0051] According to one embodiment of the present invention, the selection marker gene may be one or more selected from the group consisting of beta-lactamase, puromycin N-acetyltransferase, aminoglycoside phosphotransferase, and hygromycin B phosphotransferase, but is not limited thereto.

[0052] As used herein, the term "cell marker gene" refers to a polynucleotide sequence or a base sequence that encodes a molecule, such as a polypeptide, protein, lipid, glycolipid, glycoprotein, sugar, etc., that is specifically expressed in the cell membrane or cell surface.

[0053] According to one embodiment of the present invention, the cell marker gene may be one or more selected from the group consisting of sodium / iodide symporter, Thy-1 cell surface antigen (CD90), CD3, CD4, CD8 and CD25, but is not limited thereto.

[0054] Such a gene of interest may be one that is optimized for human codons. Here, "optimized for human codons" means that when DNA is transcribed and translated into a protein in a host cell, there are codons that are highly preferred depending on the host among the codons that specify amino acids, but by substituting them with human codons, the expression efficiency of the amino acid or protein encoded by the nucleic acid is increased.

[0055] As an example, the first gene of interest, the second gene of interest and the third gene of interest may be selected from the group consisting of the HGF gene (SEQ ID NO: 6), the Ngn1 gene (SEQ ID NO: 7 or 8), the HSV-TK gene (SEQ ID NO: 9 or 10) and the puromycin resistance gene (SEQ ID NO: 11).

[0056] The HGF gene is a gene encoding a heparin-binding glycoprotein known as hepatocyte growth factor, scatter factor or hepatopoietin-A. The HGF is produced by various mesenchymal cells and is known to promote cell proliferation, regulate the growth of endothelial cells and the migration of vascular smooth muscle cells, and induce angiogenesis.

[0057] The Ngn1 gene is a gene encoding Neurogenin1, which is specifically involved in neural differentiation. Neurogenin1 plays a role in inducing neural differentiation by regulating bone-morphogenetic protein and leukemia inhibitory factor. The Ngn1 gene may include the base sequence represented by SEQ ID NO: 7, and when a flag is attached to the 5' end of the Ngn1 gene, it may be the base sequence represented by SEQ ID NO: 8.

[0058] The HSV-TK gene is a gene encoding thymidine kinase (TK) of herpes simplex virus (HSV). The TK is an enzyme that catalyzes the reaction of generating thymidylic acid by binding the phosphate at the γ-position of ATP to thymidine, and is known to phosphorylate thymidine as well as antiviral drugs such as ganciclovir, valganciclovir, acyclovir, and valacyclovir, and the phosphorylated products disrupt DNA replication, thereby inducing cell death.

[0059] The puromycin resistance gene (PuroR) is a gene that encodes puromycin N-acetyltransferase. The puromycin resistance gene is used to select cells into which the gene has been introduced by inactivating puromycin, which inhibits protein translation by being similar to aminoacyl t-RNA, and blocking the function of puromycin to induce resistance.

[0060] According to one embodiment of the present invention, the recombinant vector may be one in which one or more of the first gene of interest, the second gene of interest and the third gene of interest contain a Kozak sequence.

[0061] The Kozak sequence is the translation initiation site in eukaryotes, equivalent to the Shine-Dalgarno base sequence in prokaryotes, and is located immediately before the AUG codon that codes for methionine in the mRNA base sequence, to which the ribosome binds.

[0062] According to one embodiment of the present invention, the Kozak sequence may comprise CCACC or CCGCC.

[0063] As an example, the Kozak sequence may include the base sequence represented by SEQ ID NO:12.

[0064] The recombinant vector of the present invention is capable of regulating the expression of two or more target genes loaded therein by two or more promoters, and is designed so that one target gene is expressed by one promoter by operably linking the promoters to the target genes to be expressed thereby, and up to three target proteins can be translated simultaneously with one vector.

[0065] Another aspect of the present invention provides a gene delivery system comprising the recombinant vector described above.

[0066] The term "gene delivery system" as used herein means a system that can increase the efficiency of delivery of a target gene and / or nucleic acid sequence into a cell and thereby increase the expression efficiency, and can be classified into viral-mediated systems and non-viral systems.

[0067] The virus-mediated system uses a viral vector such as a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a poxvirus vector, etc., and is known to have a higher intracellular gene transfer efficiency than a non-viral system by utilizing a mechanism of intracellular entry inherent to viruses that infect eukaryotic cells such as human cells. In addition, after entering the cell, non-viral vectors have a problem in that genes are degraded in endolysosomes after endosomes fuse with lysosomes, whereas viral vectors have the advantage of less gene loss and higher gene transfer efficiency due to a mechanism of immediately transferring genes into the nucleus without passing through lysosomes.

[0068] According to one embodiment of the invention, the gene delivery system may use a viral vector.

[0069] More specifically, the viral vector may be derived from one or more viruses selected from the group consisting of retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and poxviruses, and is preferably a lentivirus.

[0070] Such viral vectors can be incorporated into viral particles and then introduced into cells by transduction methods such as infection.

[0071] The gene delivery system according to the present invention functions to deliver a recombinant vector expressing two or more genes of interest to a target cell, so that the two or more genes of interest of the recombinant vector delivered into the cell can be expressed by an intracellular transcription system.

[0072] Another aspect of the present invention provides a recombinant virus comprising the recombinant vector described above.

[0073] According to one embodiment of the invention, said recombinant virus may be of lentiviral origin.

[0074] Such recombinant viruses may target any dividing cell, and in particular immune cells or stem cells.

[0075] According to one embodiment of the present invention, the immune cells may be selected from or derived from the group consisting of neutrophils, eosinophils, basophils, macrophages, mast cells, dendritic cells, B lymphocytes, T lymphocytes and natural killer cells (NK cells).

[0076] According to one embodiment of the present invention, the stem cells may be one or more selected from the group consisting of embryonic stem cells, fetal stem cells, adult stem cells, amniotic stem cells, cord blood stem cells, induced pluripotent stem cells, mesenchymal stem cells (MSCs), neural stem cells, hematopoietic stem cells, and cancer stem cells.

[0077] Another aspect of the present invention provides a transformant into which the above-mentioned recombinant vector or recombinant virus has been introduced.

[0078] The term "transformant" as used herein refers to a cell produced by inserting an exogenous gene into a host cell, which is transformed (transfected) with a recombinant vector or transduced (transduced) with a recombinant virus, and is also called a recombinant host cell, recombinant cell, or recombinant microorganism.

[0079] As used herein, "host cell" refers to a cell which is transformed, transduced or introduced with a recombinant vector or virus.

[0080] The host cell may be any host cell known in the art, including, but not limited to, NS / O myeloma cells, human 293T cells, Chinese hamster ovary cells (CHO cells), HeLa cells, human amniotic fluid-derived cells (CapT cells), COS cells, Canine D17 cells, Feline PG4 cells, stem cells, etc.

[0081] The transformation can be performed by introducing a gene into a cell by a biological, chemical or physical method known in the art. The transfection can be performed by any one or more methods selected from the group consisting of lipofectamine method, microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, DEAE-dextran treatment method, polybrene treatment method, polyethyleneamine treatment method and gene bombardment.

[0082] The transformant can be cultured using a medium commonly used for culturing animal cells. For example, the medium may be any one or more selected from the group consisting of Eagle's MEM, a-MEM, Iscove's MEM, 199 medium, CMRL1066, RPMI1640, F12, F10, DMEM, a mixture of DMEM and F12, Way-mouth's MB752 / 1, McCoy's 5A, and MCDB series media.

[0083] According to one embodiment of the present invention, the transformant may be an immune cell or a mesenchymal stem cell.

[0084] The immune cells are cells that are differentiated from blood stem cells and are involved in immunity, and may be selected from the group consisting of neutrophils, eosinophils, basophils, macrophages, mast cells, dendritic cells, B lymphocytes, T lymphocytes, and NK cells (natural killer cells), or may be derived therefrom.

[0085] The mesenchymal stem cells are cells that differentiate from mesenchyme into muscle cells, fat cells, osteoblasts, chondrocytes, and the like during division of a fertilized egg.

[0086] The mesenchymal stem cells maintain stemness and self renewal, and have the plasticity to differentiate into various mesenchymal tissues, and can be extracted from bone marrow, adipose tissue, umbilical cord blood, synovial membrane, trabecular bone, infrapatellar fat pad, placenta, etc. Such mesenchymal stem cells have the immunoregulatory ability to 1) suppress the activity and proliferation of T lymphocytes and B lymphocytes, 2) suppress the activity of natural killer cells, and 3) regulate the functions of dendritic cells and macrophages, and therefore can be used for allotransplantation and xenotransplantation and can be used to treat damaged articular cartilage and nerves.

[0087] More specifically, the transformant may be a mesenchymal stem cell derived from any one selected from the group consisting of bone marrow, adipose tissue, umbilical cord blood, amniotic membrane, synovial membrane, trabecular bone, and infrapatellar fat pad, but is not limited thereto. Effect of the Invention

[0088] The recombinant vector according to the present invention is designed to express one target gene under one promoter, and by transferring two or more target genes into a cell, up to three target genes can be stably expressed simultaneously with one vector. Therefore, such a recombinant vector can be usefully utilized as an efficient gene transfer system. [Brief description of the drawings]

[0089] [Figure 1] FIG. 1 is a schematic diagram showing a series of processes in which a plasmid is prepared according to one embodiment of the present invention, a virus expressing the plasmid is produced, and a target cell is infected with the virus. [Diagram 2] The structure of a plasmid prepared according to one embodiment of the present invention includes mCMV, CAG, PGK and / or SV40 promoter as a promoter and RFP, copGFP and PuroR as a target gene (see Figs. 2 to 5 below, the mCMV promoter is linked in the reverse direction to the CAG or PGK promoter). [Diagram 3] 1 shows the structure of a plasmid prepared according to one embodiment of the present invention, which contains mCMV, CAG and SV40 promoter as promoters, RFP, copGFP and PuroR as target genes, and which may or may not contain CTE and SV40 poly A. [Figure 4] This shows the structure of a plasmid prepared according to one embodiment of the present invention, which contains mCMV, CAG, PGK and / or SV40 promoter as a promoter, RFP, copGFP, PuroR as a target gene, and which may or may not contain CTE and SV40 poly A. [Diagram 5] This shows the structure of a plasmid prepared according to one embodiment of the present invention, which contains mCMV, CAG and SV40 promoter as promoters, and RFP, copGFP, PuroR, Ngn1, HGF and / or HSV-TK, CTE and SV40 poly A as target genes. (Hereinafter, the target genes are indicated in the figures and omitted in the description of the figures.) [Figure 6] FIG. 1 shows, according to one embodiment of the present invention, (A) the structure of pLenti-GIII-CMV-GFP-2A-Puro, and (B) the results of a comparison of the viral packaging efficiency of pLenti-GIII-CMV-GFP-2A-Puro (represented as "CMV" in the figure) with that of mCMV and the CAG promoter (represented as "CMV+CAG" in the figure). [Figure 7]1 shows the results of comparing the orientation of mCMV and CAG promoters and the efficiency of viral packaging depending on CTE according to one embodiment of the present invention. [Figure 8] 1 shows the results of comparing viral packaging efficiency depending on the orientation, position, or number of CTEs according to one embodiment of the present invention. [Figure 9] 1 shows the results of comparing the orientation of the PGK promoter and the efficiency of viral packaging depending on CTE according to one embodiment of the present invention. [Figure 10] 1 shows the results of comparing viral packaging efficiency between the CAG promoter and the PGK promoter according to one embodiment of the present invention. [Figure 11] 1 shows the results of comparing the expression rates of target genes copGFP and RFP between the CAG promoter and the PGK promoter according to one embodiment of the present invention. [Figure 12] FIG. 2 is a schematic diagram showing a schedule for producing a lentivirus and then infecting and culturing mesenchymal stem cells according to an embodiment of the present invention. [Figure 13] 1 shows the results of a comparison of the sustained expression of target genes copGFP and RFP depending on the direction of the promoter and CTE, using a fluorescence microscope, according to one embodiment of the present invention. [Figure 14] 1 shows the results of a comparison by FACS analysis of the sustained expression of target genes copGFP and RFP depending on the orientation of the promoter and CTE according to one embodiment of the present invention. [Figure 15] 15 shows the results of comparing the viral infection rate of a vector containing a dual promoter in each cell by FACS analysis according to one embodiment of the present invention. In Fig. 15, PBNK (peripheral blood natural killer cell) is NK cell, Single cells means a single cell group passed through FACS, and Live cells means a living cell in the single cell group. [Figure 16] 1 shows a comparison of viral infection rates of dual promoter-containing vectors in each cell line by FACS analysis according to one embodiment of the present invention. [Figure 17]13 shows optical (BF) and fluorescent (GFP and RFP) images comparing viral infectivity of a vector containing a dual promoter to NK cells, according to one embodiment of the present invention. [Figure 18] 1 shows a comparison of viral infectivity of a vector containing a dual promoter in Jurkat T cells by FACS analysis according to one embodiment of the present invention. [Figure 19] 1 shows a comparison of viral infectivity of a vector containing a dual promoter in Jurkat T cells by FACS analysis according to one embodiment of the present invention. [Figure 20] 13 shows optical (BF) and fluorescent (GFP and RFP) images comparing viral infectivity of a vector containing dual promoters to Jurkat T cells according to one embodiment of the present invention. [Figure 21] 1 shows (A) fluorescence (GFP and RFP) images and (B) survival rate results for cells infected with a viral vector containing the suicide-inducing gene HSV-TK according to one embodiment of the present invention. [Figure 22] FIG. 1 shows (A) a tumor photograph and (B) the results of changes in tumor size and body weight in an animal model infected with a viral vector containing the suicide-inducing gene HSV-TK according to one embodiment of the present invention. [Figure 23] According to one embodiment of the present invention, (A) the mRNA levels of HGF, Ngn1, and TK, and (B) the HGF expression rate are shown after MSC passaging. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] The present invention will be described in more detail below. However, such description is merely provided as an example for understanding the present invention, and the scope of the present invention is not limited by such exemplary description.

[0091] 1. Experimental Method 1-1. Preparation of plasmids A plasmid was prepared using SV40 poly(A), CTE (sequence number 1), CAG (sequence number 2), mCMV (sequence number 3), PGK (sequence number 4) and SV40 (sequence number 5) as promoters, HGF (sequence number 6), Ngn1 (sequence number 7 or 8), HSV-TK (sequence number 9 or 10) as target genes, puromycin resistance gene (sequence number 11), copGFP (sequence number 13) and RFP (sequence number 14), LTR (Truncated) (sequence number 15), Ψ (sequence number 16), RRE (sequence number 17) and WPRE (sequence number 18).

[0092] For the preparation of all plasmids except for #1 plasmid, PCR was performed by initial denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 60 seconds, annealing at 57°C for 30 seconds, and polymerization at 72°C for 60 seconds / kb, repeated 25 times, and final polymerization at 72°C for 5 minutes. Digestion with restriction enzymes BamHI, FspAI, EcoRI, and PmeI was performed at 37°C for 2 hours. Ligation was performed at room temperature (RT) for 150 seconds and on ice for 10 minutes.

[0093] The primers used to construct the plasmids are shown in Table 1 below.

[0094] [Table 1]

[0095] #1 Plasmid Using the Lenti-Bi-Cistronic vector (Cat. No. LV037, abm) as a backbone, the CAG-mCMV Dual Promoter and RFP from Mir19 Tracer (Jinju Han et al., 2016, Neuron) and dscGFP (copGFP) from pGreenFier1-mCMV (Cat. No. TR010PA-P, SBI) were inserted. Finally, a plasmid (#1, pL1.4P-GR) was constructed that contains CAG and copGFP in the reverse orientation and mCMV, RFP, sv40, and the puromycin resistance gene (PuroR) in the forward orientation.

[0096] #2 Plasmid Using the #1 plasmid as a backbone, a fragment containing BamHI and FspAI in mRFP1 was generated in the forward direction by PCR using primers. The CAG-mCMV fragment was then removed from the fragment generated by enzyme digestion (BamHI and FspAI), and the two fragments were religated in the forward direction to generate a plasmid (#2) containing mCMV-CAG.

[0097] Plasmids #3 and #4 A CAG-deleted fragment was prepared in the same manner as the preparation of the #2 plasmid. Using the pLenti-Bi-Cistronic-CopGFP plasmid (abm) as a backbone, a fragment containing restriction enzyme sites (BamHI and FspAI), PGK, and mCMV was prepared by PCR using primers. The CAG-deleted fragment and the fragment containing PGK and mCMV were ligated in the forward or reverse direction to prepare a plasmid containing PGK-mCMV (#3) or mCMV-PGK (#4).

[0098] Plasmids #9 and #10 Using the pLK2.4T-HN plasmid (see "Plasmid #25" below) as a backbone, a fragment containing CTE and SV40 poly(A) was generated in the forward or reverse orientation by PCR using a primer containing EcoRI. The generated fragment was then switched with copGFP in the #1 plasmid to generate a plasmid containing SV40 poly(A)-CTE (#9) or SV40 poly(A)-CTE (#10).

[0099] Plasmids #11 and #12 Using the pLK2.4T-HN plasmid as a backbone, a fragment containing CTE and SV40 poly(A) was generated in the forward or reverse direction by PCR using a primer containing PmeI. The generated fragment was then inserted into the PmeI site in the #1 plasmid to generate a plasmid containing CTE-SV40 poly(A) (#11) or CTE-SV40 poly(A) (#12).

[0100] Plasmids #13 and #14 A fragment containing CTE and SV40 poly(A) was prepared in the same manner as in the preparation of plasmids #11 and #12. The prepared fragment was then inserted in the forward or reverse direction into the PmeI site of plasmid #9 to prepare plasmids containing two CTEs and SV40 poly(A) (#13 and #14).

[0101] Plasmids #15 and #16 A fragment containing CTE and SV40 poly(A) was prepared in the same manner as in the preparation of plasmids #11 and #12. The prepared fragment was then inserted in the forward or reverse direction into the PmeI site of plasmid #10 to prepare plasmids containing two CTEs and SV40 poly(A) (#15 and #16).

[0102] Plasmids #17 and #18 A fragment containing CTE and SV40 poly(A) was prepared in the same manner as in the preparation of plasmids #9 and #10. The prepared fragment was then inserted into the EcoRI site of plasmid #2 to prepare plasmids containing SV40 poly(A)-CTE (#17) or SV40 poly(A)-CTE (#18).

[0103] Plasmids #19 and #20 A fragment containing CTE and SV40 poly(A) was prepared in the same manner as in the preparation of plasmids #9 and #10. The prepared fragment was then inserted into the EcoRI site of plasmid #3 to prepare a plasmid containing SV40 poly(A)-CTE (#19) or SV40 poly(A)-CTE (#20).

[0104] Plasmids #21 and #22 A fragment containing CTE and SV40 poly(A) was prepared in the same manner as in the preparation of plasmids #9 and #10. The prepared fragment was then inserted into the EcoRI site of plasmid #4 to prepare plasmids containing SV40 poly(A)-CTE (#21) or SV40 poly(A)-CTE (#22).

[0105] Plasmids #23 and #24 The pL2.4P-RG plasmid was first prepared by combining the CAG-mCMV Dual Promoter and RFP from Mir19 Tracer (Jinju Han et al., 2016, Neuron), dscGFP (copGFP) from pGreenFire1-mCMV (Cat. No. TR010PA-P, SBI), and CTEf from pLVX-TetOne-Puro Vector (Cat. No. 124797, Addgene) with the Lenti-Bi-Cistronic vector (Cat. No. LV037, abm) as the backbone.

[0106] Then, using pAL119-TK (Cat. No. 21911, addgene) as a backbone, HSV-TK was amplified by PCR to generate a fragment containing HSV-TK. The third gene of interest was digested from the pL2.4P-RG plasmid with PmeI and Acc65I, and a fragment containing HSV-TK (#23) or mutant HSV-TK (#24) was inserted to generate the respective plasmids.

[0107] The mutant HSV-TK has a substitution of histidine for the 168th alanine in the amino acid sequence constituting the protein, and it has been reported that its kinase activity is about four times higher than that of wild-type HSV-TK (Jan Balzarini et al., 2006, J Biol Chem.). A single amino acid mutation (A168H) was introduced using the over-lapping PCR technique.

[0108] #25 Plasmid A DNA fragment containing SV40, PuroR, and WPRE elements was obtained from the Lenti-Bi-Cistronic vector (Cat. No. LV037, abm) by PCR and inserted into pGEM (registered trademark)-T Easy Vector Systems (Cat. No. A1360, Promega) by TA Cloning. This was named pTA1. After obtaining the backbone from Mir19 Tracer (Jinju Han et., al., 2016, Neuron) using NheI and Acc65I enzymes, pTA1 was cut with the same enzymes to obtain the SV40 promoter, PuroR, and WPRE fragment and insert it into the backbone plasmid. This was named pCB1.

[0109] The Neurogein1 gene was obtained by PCR from human origin cells using primers containing both 5' and 3' EcoRI sites and Flag, and inserted into pCB1 after EcoRI cleavage. In a similar manner, the HGF gene was obtained by PCR using primers containing NheI on the 5' side and XbaI cleavage site on the 3' side, and inserted into pCB1 after XbaI and NheI cleavage. This was named pCB7.

[0110] CAG+mCMV was obtained by PCR from Mir19 Tracer (Jinju Han et al., 2016, Neuron) using primers containing both 5' and 3' NheI cleavage sites, and inserted into pCB7 after digestion with NheI. This was named pL1.3P-NH.

[0111] The CTE obtained from pLVX-TetOne-Puro Vector (Cat. No. 124797, Addgene) was inserted into pL1.3P-NH, and the HSV-TK portion was obtained by digestion with PmeI and MauBI in the #24 plasmid and inserted. This was named pL2.3T-NH.

[0112] pL2.3T-NH was digested with AgeI and PmeI and then religated to obtain a clone in which the CAG promoter was inserted in the opposite direction, which was named pLK2.4T-HN plasmid (#25).

[0113] 1-2.Virus Packaging 5 × 10 Lenti-X-293T cells (Cat. No. 632180, Takara) were cultured in a 6-well plate using PEI (Cat. No. 101000033, Polyplus). 5The cells / well were transfected with each of the plasmids prepared in Example 1 or the pLenti-GIII-CMV-GFP-2A-Puro vector (Cat. No. LV180162, abm) (0.6 μg) and 3.6 μl (1.8 μg) of pPACKH1 HIV Lentivector Packaging Kit (Systembio, LV500A-1). After culturing in an incubator under conditions of 37°C and 5% CO2 for 48 hours, the medium was obtained, filtered through a 0.45 μm syringe filter, and immediately stored at -80°C until further use.

[0114] 1-3. Transduction and Virus Titration Lenti-X 293T cells were dispensed at 50,000 cells / well in a 24-well plate and cultured at 37°C and 5% CO2 for 24 hours. After removing the culture medium, virus dilutions (2, 5 or 25 times) diluted in DMEM containing 10% FBS and 1% P / S in a final volume of 250 μl were added to the cells in the presence of polybrene (8 μg / ml) (Cat. No. TR 1003-G, Sigma-Aldrich). The cells were cultured at 37°C and 5% CO2 for 24 hours. The transduction medium was removed and 500 μl of DMEM containing 10% FBS and 1% P / S was added to each well, followed by culture at 37°C and 5% CO2 for 48 hours. After removing the medium, the cells were washed with DPBS (Cat. No. LB001-02, WELGENE). The transduced cells were obtained using 0.25% trypsin-EDTA (Cat. No. 25200056, Thermo Fisher Scientific). After washing the cells with DPBS, they were transferred to eBioscience TM The cells were resuspended in Flow Cytometry Staining Buffer (Cat. No. 00422226, Invitrogen). GFP-positive cells were stained with Attune TM Sorting was performed using an Attune NxT Acoustic Focusing Cytometer (Invitrogen) equipped with NxT software.

[0115] Viral titers were determined using the following formula: Functional titer (IFU / mL) = [(total cell number at transduction) x (% of cells transduced) x (dilution factor)] / (transduction volume (mL) added to cells). Titer values ​​outside the linear range were not used to determine the final titer.

[0116] For viral vectors encoding Ngn1, HGF, and HSV-TK, intracellular HGF staining was followed by FACS analysis to determine the percentage of transduced cells. Briefly, transduced cells were obtained using 0.25% trypsin-EDTA. Thereafter, cells were washed with DPBS, resuspended in 500 μl of DPBS, mixed with 500 μl of IC fixation buffer (Cat. No. 00-8222-49, Invitrogen), and incubated at room temperature for 30 min. Cells were washed three times with 5 ml of 1X permeabilization buffer (Cat. No. 00-8333-56, Invitrogen) by centrifugation at 500 g for 5 min each at room temperature. Before treatment with secondary antibodies, cells were blocked with blocking solution (1% Albumin from BSA (Cat. No. A7906-100G, Sigma) in 1X permeabilization buffer) for 30 min at room temperature. After removing the blocking solution, the cells were incubated with HGF antibody (Cat. No. AB-294-NA, R&D systems) (1:200 dilution in blocking buffer) at room temperature for 1 hour. As a control, Goat IgG antibody (Cat. No. I9140, Sigma-Aldrich) (1:2000 dilution in blocking buffer) was used. The primary antibody was washed three times with 5 ml of 1X permeabilization buffer by centrifugation at 500 g for 5 minutes at room temperature. Thereafter, Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 1000 mAb, and 1000 mAb were used as secondary antibodies. TM The cells were treated with 568 (Cat. No. A-11057, Invitrogen) in the dark at room temperature for 1 hour. The secondary antibody was washed twice with 5 ml of 1X permeabilization buffer, and finally with 5 ml of DPBS. TMThe cells were resuspended in 250 μl of Flow Cytometry Staining Buffer (Cat. No. 00-4222-26, Invitrogen). HGF-positive (Alexa 568 positive) cells were counted by FACS, and the virus titer was measured as described above.

[0117] 1-4.MSC transduction Mesenchymal stem cells (MSCs) were isolated from human bone marrow and subcultured in MSC culture medium (DMEM (Cat. No. LM001-05, Welgene) containing 10% FBS (Cat. No. 16000-044, Gibco), 100 U / ml penicillin and 100 μg / ml streptomycin (Cat. No. 15140-122, Gibco), and 10 ng / ml b-FGF (Cat. No. 100-18B, PeproTech). MSCs that had been passaged 5 times were cultured at 200,000 cells / well in a 6-well plate with MSC culture medium for 24 h. MSCs were transduced with viral vectors at 30 MOI in a final transduction volume of 1 ml in the presence of 4 μg / ml polybrene for 8 h in a 37°C incubator supplemented with 95% O2 + 5% CO2. When the cells reached 80-90% cell confluency, GFP, RFP fluorescent images and optical (bright-field, BF) images were acquired using an EVOS M5000 imaging system (Thermo Fisher Scientific). Transduced cells were detached with 0.25% trypsin-EDTA and washed with DPBS. After resuspending in 1 ml DPBS, the cells were imaged using a Nucleoview Imaging System (Nucleoview Imaging Systems) according to the manufacturer's guidelines. TM NC-250 TM The cell number was calculated using a nucleocounter with software (Chemetec). The cells were cultured at 1,000 cells / cm for the next passage. 2 All cell culture media was replaced with fresh media every 2–3 days. The remaining cells were used for GFP / RFP FACS analysis as previously described.

[0118] 1-5. Measurement of in vitro cell death (cellular suicide) U87 cells, a human brain cell line, were infected with lentivirus produced via the #23 or #24 plasmid to produce U87+#23 or U87+#24 cells. Each cell was cultured in a 12-well plate at 1 × 10 4 After 24 hours of culture, the cells were treated with Ganciclovir (GCV, Cat. No. G2536, Sigma) at the required concentration. On the fourth day, the cells were treated with 0.5 mg / mL of MTT (Cat. No. M2128, Sigma). After 2 hours of culture, 500 μl of DMSO was added to each well to induce the production of MTT-formazan. To confirm the degree of cell death, the absorbance at 540 nm of each well plate was measured.

[0119] 1-6. Measurement of in vivo cell death Six-week-old Balb / c nude mice were cultured with 10 U87+#23 or U87+#24 cells. 6 The animal model was prepared by intraperitoneally injecting 100uL of PBS containing 100 cells and 20% Corning® Matrigel® Growth Factor Deduced (GFR) Basement Membrane Matrix LDEV-free (Cat. No. 354230, Corning). After 6 weeks, tumors were grown to an appropriate size (100-300mm). 3 When the tumors grew to 100 mm in size, valganciclovir (vGCV, Cat. No. V0158, Tokyo Chemical Industry) was administered orally at a concentration of 50 or 200 mg / kg in normal saline every day for 14 days. The tumor size was measured every 2 days.

[0120] 2.Results Referring to Figure 1, the third generation Lentivirus Packaging System was used, and Lenti-X-293T (Cat. No. 632180, Takara) was used as the virus-producing cell line to produce lentiviruses expressing each plasmid. The infectivity of the produced lentiviruses was measured using Lenti-X-293T, and the lentiviruses were infected into mesenchymal stem cells (MSCs) to verify the expression level and persistence of the target gene, with the results being as follows:

[0121] 2-1. Comparison of viral packaging efficiency using CAG promoter We compared the lentivirus packaging efficiency between the vector pLenti-GIII-CMV-GFP-2A-Puro (Cat. No. LV180162, abm), which contains the CMV promoter and is mainly used for conventional lentivirus production, and the vector (#1 plasmid) containing the mCMV and CAG promoters.

[0122] As a result, as shown in FIG. 6, the mCMV+CAG vector had a higher viral titer than the vector having only the CMV promoter, confirming that the mCMV+CAG vector can stably perform lentivirus packaging.

[0123] 2-2. Comparison of viral packaging efficiency depending on the orientation of the CAG promoter and the presence or absence of CTE We compared the viral packaging efficiency when the CAG promoter was in the forward or reverse orientation, and also examined the effect of CTE, which is known to have the function of RNA export, on the expression of target proteins.

[0124] As shown in Figure 7, the virus titer was higher when the CAG promoter was in the reverse orientation (#1) than when it was in the forward orientation (#2). Furthermore, when the CTE was included (#9 and #10), the virus titer was higher.

[0125] 2-3. Comparison of viral packaging efficiency depending on the orientation, position, and number of CTEs The lentivirus packaging efficiency was compared depending on the orientation, position, and number of CTEs in a vector in which the CAG promoter was positioned in an inverted orientation.

[0126] As a result, as shown in Figure 8, when one CTE was located at the 5' end of the CAG promoter in the forward or reverse direction, the virus titer was high (#9 and #10). When the CTE was located at the 3' end of the CAG promoter, the virus could not be produced properly (#11 to #16).

[0127] 2-4. Comparison of viral packaging efficiency depending on the orientation of the PGK promoter and the presence or absence of CTE The orientation of the PGK promoter and the efficiency of viral packaging by CTE in vectors containing the mCMV promoter and the PGK promoter instead of the CAG promoter were compared.

[0128] As a result, as shown in FIG. 9, the PGK promoter produced constant viral titers regardless of orientation (#3 and #4), whereas the viral titers were increased more in the presence of CTE (#21 and #22).

[0129] 2-5. Comparison of viral packaging efficiency between CAG promoter and PGK promoter The lentivirus packaging efficiency was compared between a vector containing a CAG promoter and a vector containing an mCMV and PGK promoter depending on the orientation of the promoter and CTE.

[0130] As a result, as shown in Figure 10, when the CTE was included, regardless of the promoter orientation, the viral titer increased, and was particularly increased when the CTE was in the positive direction. When the PGK promoter was included in the positive direction, the viral titer differed by up to 3-4 times depending on the presence or absence of the CTE.

[0131] 2-6. Comparison of target gene expression rates depending on promoter orientation between CAG promoter and PGK promoter To compare the expression rate of the target gene between the vector containing mCMV and CAG promoter and the vector containing mCMV and PGK promoter, the GFP and RFP expression rates were measured. Due to the characteristics of bidirectionally expressed promoters, the expression rate of the same target gene is different. In the case of the vector containing mCMV and CAG promoter, the CAG promoter part has a strong expression rate, and in the case of the vector containing mCMV and PGK promoter, the PGK promoter part has a strong expression rate, and the target gene downstream of such a promoter was named the "stronger side". In both vectors, the target gene downstream of the mCMV promoter had a weak expression rate, and such a target gene was named the "weaker side".

[0132] As a result, as shown in Figure 11, in the case of the PGK promoter, the expression rate of the target gene at the weak site differed depending on the direction of the promoter, and in particular, when the PGK promoter was in the reverse direction, the expression rate of the target gene at the weak site was very low. However, in the case of the CAG promoter, the target gene at the weak site was expressed equally regardless of the direction.

[0133] 2-7. Comparison of expression rate of target gene depending on promoter and CTE orientation Referring to Figure 12, lentivirus was generated and then infected into target cells, mesenchymal stem cells (MSCs). At each passage, fluorescent images were taken to confirm the expression of the target gene (GFP or RFP), and FACS experiments were performed to confirm the infection rate.

[0134] We also compared the persistence of target gene expression depending on the direction of the promoter and CTE in vectors containing mCMV and CAG promoters and vectors containing mCMV and PGK promoters. In the case of the reverse CAG promoter, the strong site expressed the GFP gene and the weak site expressed the RFP gene, while in the case of the forward mCMV+CAG promoter and mCMV+PGK promoter, the strong site expressed the RFP gene and the weak site expressed the GFP gene.

[0135] As a result, the infection rate (%) of lentivirus was higher for vectors containing mCMV and PGK promoter than for vectors containing mCMV and CAG, as shown in Figures 13 and 14. The infection rate of each vector candidate group was measured for the target gene at the strong site, and the infection rate of the target gene at the weak site was also measured.

[0136] The infection rates of A and B in Figure 14 were not the same, and even when infected with a lentivirus, when the target gene at the strong site was expressed, the target gene at the opposite weak site was not expressed. It was found that such a disadvantage of the bidirectional promoter was compensated for by the increase in the infection rate of the target gene at the weak site in the plasmid containing the forward or reverse CTE.

[0137] In general, when target cells were cultured for a long time, the expression rate of the target gene at the weak site of the bidirectional promoter decreased (Figure 14B, #1). When the vector containing the reverse CAG contained a CTE, the expression of the target gene was more sustained (Figure 14B, #1-#10), and the vector containing the forward CAG or the vector containing the forward mCMV+PGK overcame the disadvantage of the weak side only when the vector contained the reverse CTE (Figure 14B, #17 or #21).

[0138] This result suggests that the bidirectional plasmid candidates may show various differences depending on the target cell. It is necessary to select the optimal bidirectional promoter for the target cell, taking into consideration the difference in the expression rate of the target gene that was not observed in cells such as 293T, the decrease in the persistence of expression, etc.

[0139] 2-8. Comparison of target gene expression rates of vectors containing mCMV+CAG or mCMV+PGK in different target cells Since the expression rate of the target gene may differ depending on the type of target cell, natural killer (NK) cells and Jurkat T cells were used to examine whether the lentiviral plasmid candidates could be applied to various target cells.

[0140] As a result, as shown in Figures 15 to 17, the infection rate of NK cells was confirmed by FACS and the fluorescence expression rate.

[0141] When checking for fluorescent expression, more cells were found to be infected with vectors containing mCMV+PGK (#21 or #22) than with vectors containing mCMV+CAG (#9 or #10). Based on these results, it is expected that the expression of the target gene is possible in NK cells when vectors containing mCMV+PGK are used.

[0142] Similarly, as shown in Figures 18 to 20, in Jurkat T cells, the expression of the target gene was found to be higher in the case of a vector containing mCMV+CAG than in the case of a vector containing mCMV+PGK.

[0143] 2-9. Target gene co-expression plasmid application model In order to apply the plasmids expressing two or more genes of interest simultaneously to practical research, we prepared plasmids (#23 and #24) containing a suicide-inducing gene (third gene of interest), infected them into U87 cells, and measured the expression rate.

[0144] As a result, as shown in Figure 21, U87 cells were infected and normal expression of the first target gene (RFP) and the second target gene (copGFP) was confirmed by fluorescence, and expression of the third target gene (suicide-inducing gene, HSV-TK) was confirmed by an MTT assay using Ganciclovir.

[0145] U87 cells infected with such lentiviruses were injected into actual animals to create a tumor formation model, and valganciclovir was administered orally to induce suicide of the U87 cells.

[0146] As a result, as shown in FIG. 22, it was confirmed that the tumor size significantly decreased over time compared to the control group.

[0147] This result suggests that gene co-expression plasmids can be modified with different genes of interest and applied to experimental and industrial populations.

[0148] 2-10. Confirmation of expression after target gene modification In the above experiments, gene expression was confirmed using GFP, RFP or HSV-TK as the target gene, and whether the same gene expression occurred for other target genes was confirmed. For this purpose, the first target gene was changed to HGF, the second target gene to Neurogenin1, and the third target gene to HSV-TK, and normal expression was confirmed.

[0149] As a result, as shown in FIG. 23, it was confirmed that even when the target gene was changed to HGF, Ngn1, or TK, mRNA transcription occurred normally, and the expression rate was not decreased by MSC passaging.

[0150] The present invention has been described above with a focus on its preferred embodiments. Those skilled in the art will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an explanatory perspective, not a restrictive one. The scope of the present invention is defined in the claims, not the above description, and all differences within the scope of the equivalents should be interpreted as being included in the present invention.

Claims

1. CTE, a first gene of interest, a first promoter, a second promoter and a second gene of interest, A recombinant vector, wherein the first gene of interest and the first promoter are in opposite orientations.

2. The recombinant vector of claim 1 , further comprising a third promoter and a third gene of interest.

3. The recombinant vector according to claim 1 , wherein the CTE is a polynucleotide having the base sequence of SEQ ID NO:

2.

4. The recombinant vector of claim 1 , wherein the CTE is forward or reverse.

5. The promoters include simian virus 40 (SV40) promoter, cytomegalovirus (CMV) promoter, minimal CMV promoter, human ubiquitin C promoter (UBC) promoter, human elongation factor 1a (EF1A) promoter, phosphoglycerate kinase 1 (PGK) promoter, and chicken beta actin (cytomegalovirus immediate-early enhancer / chicken 3. The recombinant vector according to claim 1, wherein the promoter is one or more selected from the group consisting of β-actin, β-actin, and CAG promoters.

6. 3. The recombinant vector according to claim 1, wherein the target gene is one or more selected from the group consisting of a disease therapeutic gene, a reporter gene, a selection marker gene and a cell marker gene.

7. The recombinant vector of claim 6, wherein the disease treatment gene is one or more selected from the group consisting of drug sensitivity genes, cell death genes, cell proliferation inhibitory genes, cell growth genes, cytotoxic genes, tumor suppressor genes, antigenic genes, cytokine genes, neurogenesis genes, anti-angiogenesis genes and hormone genes.

8. The recombinant vector of claim 6, wherein the reporter gene is at least one selected from the group consisting of TdTomato, luciferase, copGFP isolated from Pontellina plumata, green fluorescent protein (GFP) isolated from Aequorea victoria, modified green fluorescent protein (mGFP), enhanced green fluorescent protein (eGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (eRFP), blue fluorescent protein (BFP), modified blue fluorescent protein (mBFP), enhanced blue fluorescent protein (eBFP), yellow fluorescent protein (YFP), modified yellow fluorescent protein (mYFP), enhanced yellow fluorescent protein (eYFP), cyan fluorescent protein (CFP), modified cyan fluorescent protein (mCFP) and enhanced cyan fluorescent protein (eCFP).

9. The recombinant vector according to claim 6, wherein the selectable marker gene is at least one selected from the group consisting of beta-lactamase, puromycin N-acetyltransferase, hygromycin B phosphotransferase, and aminoglycoside phosphotransferase.

10. The recombinant vector according to claim 6, wherein the cell marker gene is one or more selected from the group consisting of sodium / iodide symporter, Thy-1 cell surface antigen (CD90), CD3, CD4, CD8 and CD25.

11. The recombinant vector according to claim 1 or 2, wherein at least one of the first target gene, the second target gene and the third target gene contains a Kozak sequence.

12. A gene delivery system comprising the recombinant vector of claim 1 or 2.

13. A recombinant virus comprising the recombinant vector of claim 1 or 2.

14. The recombinant virus of claim 13, wherein the recombinant virus is derived from a Lentivirus.

15. A transformant comprising the recombinant vector according to claim 1 or 2, or the recombinant virus according to claim 13.

16. The transformant according to claim 15 , which is an immune cell or a mesenchymal stem cell.

17. The transformant according to claim 16, wherein the immune cell is selected from the group consisting of neutrophils, eosinophils, basophils, macrophages, mast cells, dendritic cells, B lymphocytes, T lymphocytes and NK cells or is derived therefrom.

18. 17. The transformant according to claim 16, wherein the mesenchymal stem cells are derived from any one selected from the group consisting of bone marrow, adipose tissue, umbilical cord blood, amniotic membrane, synovial membrane, trabecular bone, and infrapatellar fat pad.

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