Novel recombinant vectors and their applications

Abstract

The present invention aims to provide a recombinant vector capable of stably expressing two or more target genes through intracellular transmission, as well as a gene transmission system, recombinant virus, and transformant containing the same. [Solution] The recombinant vector of the present invention is designed so that one target gene is expressed by one promoter, and by transmitting two or more target genes into cells, up to three target genes can be stably expressed simultaneously with a single vector. Therefore, such recombinant vectors can be usefully utilized in efficient gene transfer systems.

Description

[Technical Field]

[0001] The present invention relates to recombinant vectors, gene transfer systems containing the same, recombinant viruses, and transformants. [Background technology]

[0002] Gene therapy is a general term for therapeutic techniques that use genes, such as incorporating new genes into a patient's cells, eliminating malfunctioning genes, or replacing mutated genes with normal ones. Such gene therapies can be targeted immediately by identifying only the gene causing the disease, and can be manufactured more quickly and inexpensively than other antibody or compound therapies, thus increasing the possibility of treating diseases that are difficult to treat with existing drug therapies.

[0003] For effective gene therapy, it is essential to first identify target genes crucial for treating the disease, and then develop vectors that efficiently transmit these genes into cells without side effects. Vectors are gene carriers and are broadly divided into viral and nonviral carriers. Viral carriers are manufactured by removing most or some of the essential genes from a viral gene to prevent it from replicating itself, and then inserting the therapeutic gene in its place. While they can efficiently transmit genes into cells, problems exist depending on the type of virus, such as difficulty in mass production, induction of immune responses, toxicity, and the emergence of replicable viruses. Currently, major viral carriers used in the development of gene therapy drugs include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and pox viruses. On the other hand, non-viral transduction media, such as liposomes and plasmids, do not induce an immune response, are less toxic, and are easy to mass-produce. However, their gene transfer efficiency is low and their expression is temporary, which limits the development of gene therapy drugs.

[0004] In the development of gene therapy drugs, vector technology using adenoviruses and retroviruses is the mainstream. Lentiviruses, a type of retrovirus, have the advantage of being able to transmit genes regardless of cell division, and the location on the chromosome where the transmitted gene is inserted is distributed in a place where the risk of "oncogenic insertional mutagenesis (tumor development due to gene insertion)" is relatively low. Furthermore, by inducing self-inactivation by deleting the U3 portion from the 3'LTR (long terminal repeat), the risk of tumorigenicity that can be induced by the LTR can be fundamentally blocked.

[0005] However, the size of the target gene that can be introduced into a viral vector is limited, and the larger the target gene, the higher the likelihood of it being lost due to homologous recombination. Furthermore, multiple target genes contained in a single vector can be silencing unevenly depending on the cell type and state, and such silencing phenomena are currently difficult to predict or prevent with current technology. Therefore, constructing a vector that uniformly expresses two or more target genes without silencing within cells (e.g., mesenchymal stem cells) is not easy, and research on this is urgently needed. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Korean Registered Patent No. 10-1885438 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a recombinant vector that can transmit two or more target genes intracellularly and express them stably.

[0008] Furthermore, the present invention aims to provide a gene transfer system that includes the recombinant vector.

[0009] Furthermore, the present invention aims to provide a recombinant virus containing the recombinant vector.

[0010] Furthermore, the present invention aims 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 target gene, a first promoter, a second promoter, and a second target gene, wherein the first target gene and the first promoter are in the reverse direction.

[0012] The "vector" used in this invention is an expression vector capable of expressing a target gene in a suitable host cell, and refers to a gene structure containing essential regulatory elements that are operably linked so that the gene insert contained within the vector is expressed. Here, "operably linked" means that a nucleic acid expression regulatory sequence that performs a general function and a nucleic acid sequence that encodes the target gene are functionally linked.

[0013] The vectors according to the present invention include expression regulatory elements such as promoters, operators, start codons, termination codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be manufactured in diverse ways depending on the purpose. The promoter of the vector may be constitutive or inductive. The expression vector also includes a selectivity marker for selecting host cells containing the vector, and in the case of a replicable expression vector, it may include a replication origin. Such vectors may self-replicate or integrate into host DNA. Examples of such vectors include plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors.

[0014] Such a recombinant vector containing a CTE, a first target gene, a first promoter, a second promoter, and a second target gene can express two target genes simultaneously in a single vector by including two target genes and two promoters that regulate the expression of each target gene.

[0015] Furthermore, the recombinant vector according to the present invention includes a promoter that additionally regulates the target gene and its expression, thereby enabling the simultaneous expression of three target genes with a single vector.

[0016] According to one specific example of the present invention, the recombinant vector may further include a third promoter and a third target gene.

[0017] The "CTE (constitutive transport element)" used in this invention refers to a factor that promotes the nuclear export of incomplete splice mRNA. It is known that the closer a CTE is located to the poly A tail, which is important for mRNA nuclear export, translation, and stability, the better its function and the greater the expression of the target gene (MR Mautino, et al., Gene Therapy. 2000. 7, 1421-1424).

[0018] The "target gene" used in the present invention means a gene whose expression is induced by a promoter.

[0019] The "promoter" used in the present invention is a part of DNA that is involved in the binding of RNA polymerase so as to initiate transcription. Generally, it is located upstream adjacent to the target gene and is a site to which a transcription factor, which is RNA polymerase or 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 position directly or via a transcription factor to induce the initiation of mRNA synthesis for the target gene, and has a specific gene sequence.

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

[0021] According to one specific example of the present invention, the CTE may be ligated to the 3' end of the poly A tail.

[0022] According to one specific example of the present invention, the CTE may contain the nucleotide sequence represented by SEQ ID NO: 1.

[0023] According to one specific example of the present invention, the CTE may be in the forward or reverse direction.

[0024] In this invention, "forward orientation" means that the sequence encoded by each gene within the viral gene is in the sense orientation (5'→3'), and "reverse orientation" means that the sequence encoded by each gene within the viral gene is in the antisense orientation (3'→5').

[0025] The recombinant vector according to the present invention includes two or more promoters that induce the expression of each target gene in order to simultaneously express two or more target genes, wherein the two or more promoters may be of the same type or may be different from each other.

[0026] In such recombinant vectors, the second target gene and second promoter, and / or the third target gene and third promoter, are ligable in the forward direction, excluding the first target gene and first promoter in the reverse direction.

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

[0028] The first promoter, second promoter, and 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, second promoter, and third promoter may be selected from the group consisting of the CAG promoter (SEQ ID NO: 2), minimal CMV (hereinafter referred to as "mCMV") (SEQ ID NO: 3), PGK promoter (SEQ ID NO: 4), and SV40 (promoter 5).

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

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

[0032] The recombinant vector according to the present invention can express two or more target genes simultaneously, so it can express a disease treatment gene, a reporter gene, or a selection marker gene individually, or it can express all of the disease treatment gene, reporter gene, and selection marker gene.

[0033] The term "disease-treating gene" used in this invention refers to a polynucleotide sequence or base sequence that encodes a polypeptide that exhibits therapeutic effects on cells that abnormally express genes, such as cancer cells.

[0034] According to one specific example 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, cytotoxic genes, tumor suppressor genes, antigenic genes, cytokine genes, neurogenesis genes, anti-neoangiogenesis genes, and hormone genes.

[0035] The aforementioned drug-sensitizing gene is a gene that encodes an enzyme that converts a non-toxic precursor (prodrug) into a toxic substance. Since cells into which this gene is introduced die, it is also called a suicide gene. In other words, 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 target cells, altering drug sensitivity and destroying the target cells. 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 E. coli cytosine deaminase, which uses 5-fluorocytosine (5-FC) as a precursor.

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

[0037] The aforementioned cytostatic genes are nucleotide sequences that are expressed within cells and cause the cell cycle to be stopped midway through the cycle. Examples 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 aforementioned cell growth genes are nucleotide sequences that promote the division, growth, and differentiation of stem cells or already differentiated cells. 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 aforementioned cytotoxic gene is a nucleotide sequence that is expressed within a cell and exhibits toxic effects. Examples include, but are not limited to, the nucleotide sequences that encode Pseudomonas exotoxin, lysine toxin, and diphtheria toxin.

[0040] The aforementioned tumor suppressor gene refers to a nucleotide sequence that, when expressed in target cells, can suppress the tumor phenotype or induce cell death. Examples of such tumor suppressor genes include, but are not limited to, the 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 aforementioned antigenic gene is a nucleotide sequence that is expressed in target cells and produces cell surface antigenic proteins that can be recognized by the immune system. Examples include carcinoembryonic antigen and p53, but are not limited to these.

[0042] The aforementioned cytokine gene refers to a nucleotide sequence that is expressed within a cell to produce cytokines. Examples 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 aforementioned neurogenic genes are nucleotide sequences that promote the generation and development of nerves. Examples include, but are not limited to, brain-derived neurotrophic factor and nerve growth factor.

[0044] The aforementioned neuronal differentiation genes are nucleotide sequences involved in the division of neuroepithelial cells from stem cell-like cell division to neuroblasts and neuroglial cells. Examples include, but are not limited to, neurogenin, NeuroD, and ASCL1 (Achaete-scute homolog1).

[0045] The aforementioned anti-angiogenic gene refers to a nucleotide sequence that, when expressed, releases anti-angiogenic factors extracellularly. Examples of such genes include, but are not limited to, angiostatin, a vascular endothelial growth factor (VEGF) inhibitor, and endostatin.

[0046] The aforementioned hormone gene refers to a nucleotide sequence that produces a hormone or its precursor. Examples 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] Furthermore, any gene that has therapeutic effects can be used without restriction as a gene for disease treatment.

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

[0049] According to one specific example 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), but is not limited thereto.

[0050] The "selection marker gene" used in this invention is an antibiotic resistance gene used to select cells into which the gene has been introduced. In a culture medium treated with an antibiotic, only cells expressing the selection marker gene, i.e., the antibiotic resistance gene, can survive, thus enabling the selection of cells into which the gene has been introduced. Any antibiotic resistance gene known in the industry can be used without restriction as the selection marker gene.

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

[0052] The term "cell marker gene" used in this invention refers to a polynucleotide sequence or base sequence that encodes molecules such as polypeptides, proteins, lipids, glycolipids, glycoproteins, and sugars that are specifically expressed on the cell membrane or cell surface.

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

[0054] Such target genes may be optimized for human codons. Here, "optimized for human codons" means that when DNA is transcribed and translated into protein in a host cell, there are codons between the codons that specify amino acids that have a higher preference depending on the host, but by substituting them with human codons, the expression efficiency of the amino acids or proteins encoded by that nucleic acid is increased.

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

[0056] The aforementioned HGF gene is a hepatocyte growth factor, and it is the gene that encodes a heparin-binding glycoprotein known as scatter factor or hepatopoietin-A. 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 that encodes neurogenin 1, which is specifically involved in neurogenic differentiation. Neurogenin 1 plays a role in inducing neurogenic differentiation by regulating bone morphogenetic protein and leukemia inhibitory factor. The Ngn1 gene may contain the nucleotide sequence represented by Sequence ID No. 7, and may also contain the nucleotide sequence of Sequence ID No. 8 if a flag is attached to the 5' end of the Ngn1 gene.

[0058] The aforementioned HSV-TK gene is the gene that encodes the thymidine kinase (TK) of the herpes simplex virus (HSV). This TK is an enzyme that catalyzes the thymidylate production reaction by binding the phosphate group at the gamma position of ATP to thymidine. In addition to thymidine, it is known to phosphorylate antiviral drugs such as ganciclovir, valganciclovir, acyclovir, and valacyclovir, and that the phosphorylated products disrupt DNA replication, thereby inducing cell death.

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

[0060] According to one specific example of the present invention, the recombinant vector may contain a Kozak sequence in one or more of the first target gene, second target gene, and third target gene.

[0061] The aforementioned Kozak sequence is the translation initiation site in eukaryotes, corresponding to the Shine & Dalgarno sequence in prokaryotes. It is located at the very beginning of the AUG codon, which encodes methionine in the mRNA sequence, and to which a ribosome attaches.

[0062] According to one specific example of the present invention, the Kozak sequence may include CCACC or CCGCC.

[0063] As an example, the Kossack sequence may include the nucleotide sequence represented by Sequence ID No. 12.

[0064] The recombinant vector according to the present invention can regulate the expression of two or more target genes loaded thereon by two or more promoters. By operably linking the promoters to the target genes expressed thereon, the vector is designed so that one promoter expresses one target gene, and up to three target proteins can be translated simultaneously from a single vector.

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

[0066] The "gene delivery system" used in this invention refers to a system that can increase the efficiency of gene and / or nucleic acid sequence transmission into cells and thereby increase expression efficiency, and can be classified into virus-mediated systems and non-viral systems.

[0067] The aforementioned virus-mediated systems utilize viral vectors such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, and poxvirus vectors. They are known to have relatively higher intracellular gene transfer efficiency than nonviral systems by utilizing the unique intracellular entry mechanisms of viruses that cause infection in eukaryotic cells such as human cells. Furthermore, while nonviral vectors have the problem of gene degradation in endolysosomes after the endosome fuses with the lysosome, viral vectors have the advantage of less gene loss and higher gene transfer efficiency because they transmit genes directly into the nucleus without passing through lysosomes.

[0068] According to one specific example of the present invention, the gene transfer 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, after being incorporated into viral particles, can be introduced into cells via transduction methods such as infection.

[0071] The gene transfer system according to the present invention functions to transfer a recombinant vector expressing two or more target genes to target cells, thereby enabling the expression of the two or more target genes of the recombinant vector transferred into the cells by the intracellular transcription system.

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

[0073] According to one specific example of the present invention, the recombinant virus may be of lentivirus origin.

[0074] Such recombinant viruses can target any cell that is dividing, which may specifically be immune cells or stem cells.

[0075] According to one specific example 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 NK cells (natural killer cells).

[0076] According to one specific example 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 transformants into which the aforementioned recombinant vector or recombinant virus has been introduced.

[0078] The term "transformed organism" used in this invention refers to a cell produced by inserting an external gene into a host cell, and is a cell transformed (transfection) with a recombinant vector or transduction (transduction) with a recombinant virus. It is also called a recombinant host cell, recombinant cell, or recombinant microorganism.

[0079] In this invention, "host cell" means a cell into which a recombinant vector or recombinant virus is transformed, transduced, or introduced.

[0080] The host cells may be host cells known in the art. Examples of such host cells include, but are 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, and stem cells.

[0081] The transformation can be carried out by introducing genes into cells by biological, chemical, or physical methods known in the art. Examples of such phenotypic infection may be one or more methods selected from the group consisting of lipofectamine method, microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated phenotypic infection method, DEAE-dextran treatment method, polybrene treatment method, polyethyleneamine treatment method, and gene bombardment.

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

[0083] According to one specific example of the present invention, the transformed organism may be an immune cell or a mesenchymal stem cell.

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

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

[0086] The aforementioned mesenchymal stem cells are cells that maintain stem cell function and self-renewal ability, and possess the plasticity to differentiate into various mesenchymal tissues. They 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 immunomodulatory capabilities that 1) suppress the activity and proliferation of T lymphocytes and B lymphocytes, 2) suppress the activity of natural killer cells, and 3) regulate the function of dendritic cells and macrophages. Therefore, they can be used in allotransplantation and xenotransplantation to treat damaged articular cartilage and nerves.

[0087] More specifically, the transformed organism may be, but is not limited to, 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. [Effects of the Invention]

[0088] The recombinant vector according to the present invention is designed so that one target gene is expressed by one promoter, and by transmitting two or more target genes into cells, up to three target genes can be stably expressed simultaneously with a single vector. Therefore, such recombinant vectors can be usefully utilized in efficient gene transfer systems. [Brief explanation of the drawing]

[0089] [Figure 1] This is a schematic diagram illustrating a series of steps taken to produce a plasmid according to one embodiment of the present invention, followed by the production of a virus that expresses the plasmid and infecting target cells. [Figure 2] The structure of a plasmid containing the mCMV, CAG, PGK, and / or SV40 promoters as promoters, and RFP, copGFP, and PuroR as target genes, is shown below. (Figures 2 to 5 below show the mCMV promoter ligated in the opposite direction to the CAG or PGK promoter.) [Figure 3] The plasmid structure, prepared according to one embodiment of the present invention, contains the mCMV, CAG, and SV40 promoters as promoters, and RFP, copGFP, and PuroR as target genes, and may or may not contain CTE and SV40 poly A. [Figure 4] The plasmid structure, prepared according to one embodiment of the present invention, contains the mCMV, CAG, PGK, and / or SV40 promoters as promoters, and RFP, copGFP, and PuroR as target genes, and may or may not contain CTE and SV40 poly A. [Figure 5] The structure of a plasmid containing the mCMV, CAG, and SV40 promoters as promoters, and RFP, copGFP, PuroR, Ngn1, HGF, and / or HSV-TK, CTE, and SV40 poly A as target genes, is shown in one embodiment of the present invention. (Hereafter, the target genes are indicated in the figures but omitted in the descriptions of the figures.) [Figure 6] This figure shows the structure of (A) pLenti-GIII-CMV-GFP-2A-Puro and the results of comparing the viral packaging efficiency of (B) pLenti-GIII-CMV-GFP-2A-Puro (referred to as "CMV" in the figure) with mCMV and a CAG promoter (referred to as "CMV+CAG" in the figure) according to one embodiment of the present invention. [Figure 7]This is a result of comparing the directionality of mCMV and CAG promoters and the viral packaging efficiency by CTE according to one embodiment of the present invention. [Figure 8] This is a result of comparing the virus packaging efficiency based on the orientation, position, or number of CTEs according to one embodiment of the present invention. [Figure 9] This is a comparison of the directionality of the PGK promoter and the virus packaging efficiency by CTE according to one embodiment of the present invention. [Figure 10] This is the result of comparing the viral packaging efficiency between a CAG promoter and a PGK promoter according to one embodiment of the present invention. [Figure 11] This is a result of comparing the expression rates of the target genes copGFP and RFP between the CAG promoter and the PGK promoter according to one embodiment of the present invention. [Figure 12] This is a schematic diagram illustrating the process of generating a lentivirus according to one embodiment of the present invention, and then infecting and culturing mesenchymal stem cells with it. [Figure 13] This is a result of comparing the expression persistence of target genes copGFP and RFP, oriented by promoter and CTE directionality, using fluorescence microscopy, according to one embodiment of the present invention. [Figure 14] This is a result of comparing the expression persistence of target genes copGFP and RFP based on promoter and CTE directionality using FACS analysis, according to one embodiment of the present invention. [Figure 15] Figure 15 shows the results of comparing the viral infection rates of vectors containing dual promoters for each cell type using FACS analysis, according to one embodiment of the present invention. In Figure 15, PBNK (peripheral blood natural killer cell) refers to NK cells, Single cells refers to a group of single cells that passed through FACS, and Live cells refers to living cells within the group of single cells. [Figure 16] This is the result of comparing the viral infection rates of vectors containing a dual promoter for each cell type using FACS analysis, according to one embodiment of the present invention. [Figure 17]These are optical (BF) and fluorescence (GFP and RFP) images comparing the viral infection rates of vectors containing dual promoters against NK cells according to one embodiment of the present invention. [Figure 18] This is a comparison of the viral infection rates of vectors containing a dual promoter against Jurkat T cells using FACS analysis, according to one embodiment of the present invention. [Figure 19] This is a comparison of the viral infection rates of vectors containing a dual promoter against Jurkat T cells using FACS analysis, according to one embodiment of the present invention. [Figure 20] These are optical (BF) and fluorescence (GFP and RFP) images comparing the viral infection rates of vectors containing dual promoters against Jurkat T cells according to one embodiment of the present invention. [Figure 21] According to one embodiment of the present invention, (A) fluorescence (GFP and RFP) images and (B) survival rate results of cells infected with a viral vector containing the suicide-inducing gene HSV-TK are shown. [Figure 22] According to one embodiment of the present invention, (A) a tumor photograph and (B) the results of tumor size and weight changes in an animal model infected with a viral vector containing the suicide-inducing gene HSV-TK. [Figure 23] According to one embodiment of the present invention, the following are the results of (A) mRNA levels of HGF, Ngn1, and TK and (B) HGF expression rate obtained by MSC passaging. [Modes for carrying out the invention]

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

[0091] 1. Experimental Method 1-1. Plasmid construction Plasmids were constructed using SV40 poly(A), CTE (SEQ ID NO: 1), CAG (SEQ ID NO: 2), mCMV (SEQ ID NO: 3), PGK (SEQ ID NO: 4), and SV40 (SEQ ID NO: 5) as promoters, and HGF (SEQ ID NO: 6), Ngn1 (SEQ ID NO: 7 or 8), HSV-TK (SEQ ID NO: 9 or 10), puromycin resistance gene (SEQ ID NO: 11), copGFP (SEQ ID NO: 13), RFP (SEQ ID NO: 14), LTR (Truncated) (SEQ ID NO: 15), Ψ (SEQ ID NO: 16), RRE (SEQ ID NO: 17), and WPRE (SEQ ID NO: 18) as target genes.

[0092] For the preparation of all plasmids except plasmid #1, 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, with the final polymerization performed at 72°C for 5 minutes. Restriction enzyme digestion was performed using BamHI, FspAI, EcoRI, and PmeI, all reacted at 37°C for 2 hours. Ligation was performed at room temperature (RT) for 150 seconds, followed by 10 minutes on ice.

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

[0094] [Table 1]

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

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

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

[0098] Plasmids #9 and #10 Using the pLK2.4T-HN plasmid (see "#25 plasmid" below) as the backbone, fragments containing CTE and SV40 poly(A) were generated in either the forward or reverse direction by PCR using primers containing EcoRI. Subsequently, the generated fragments were repositioned (switched) with copGFP in plasmid #1 to generate plasmids containing SV40 poly(A)-CTE(#9) or SV40 poly(A)-CTE(#10).

[0099] Plasmids #11 and #12 Using the pLK2.4T-HN plasmid as the backbone, fragments containing CTE and SV40 poly(A) were generated in either the forward or reverse direction by PCR using primers containing PmeI. Subsequently, the generated fragments were inserted into the PmeI site within plasmid #1 to generate plasmids containing CTE-SV40 poly(A)(#11) or CTE-SV40 poly(A)(#12).

[0100] Plasmids #13 and #14 Fragments containing CTE and SV40 poly(A) were prepared using the same method as for the preparation of plasmids #11 and #12. Subsequently, the prepared fragments were inserted into the PmeI region of plasmid #9 in either the forward or reverse direction to prepare plasmids containing two CTEs and SV40 poly(A) (#13 and #14).

[0101] Plasmids #15 and #16 Fragments containing CTE and SV40 poly(A) were prepared using the same method as for the preparation of plasmids #11 and #12. Subsequently, the prepared fragments were inserted into the PmeI region of plasmid #10 in either the forward or reverse direction to prepare plasmids containing two CTEs and SV40 poly(A) (#15 and #16).

[0102] Plasmids #17 and #18 Fragments containing CTE and SV40 poly(A) were prepared using the same method as for the preparation of plasmids #9 and #10. Subsequently, the prepared fragments were 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 Fragments containing CTE and SV40 poly(A) were prepared using the same method as for the preparation of plasmids #9 and #10. Subsequently, the prepared fragments were inserted into the EcoRI site of plasmid #3 to prepare plasmids containing SV40 poly(A)-CTE(#19) or SV40 poly(A)-CTE(#20).

[0104] Plasmids #21 and #22 Fragments containing CTE and SV40 poly(A) were prepared using the same method as for the preparation of plasmids #9 and #10. Subsequently, the prepared fragments were 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 Using a Lenti-Bi-Cistronic vector (Cat.No.LV037, abm) as the backbone, the pL2.4P-RG plasmid was first constructed by combining 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 obtained from pLVX-TetOne-Puro Vector (Cat.No.124797, Addgene).

[0106] Subsequently, using pAL119-TK (Cat. No. 21911, addgene) as the backbone, HSV-TK was amplified by PCR to produce fragments containing HSV-TK. After cleaving the third target gene from the pL2.4P-RG plasmid with PmeI Acc65I, fragments containing HSV-TK (#23) or mutant HSV-TK (#24) were inserted to produce the respective plasmids.

[0107] Mutant HSV-TK has a substitution of histidine at position 168 of the amino acid sequence that makes up 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.). Therefore, we introduced a single amino acid mutation (A168H) using the over-lapping PCR method.

[0108] #25 plasmid DNA sections containing SV40, PuroR, and WPRE elements were obtained from a Lenti-Bi-Cistronic vector (Cat.No.LV037, abm) by PCR and inserted into pGEM®-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 the enzymes NheI and Acc65I, pTA1 was cleaved with the same enzymes to obtain the SV40 promoter, PuroR, and WPRE sections, which were then inserted into the backbone plasmid. This was named pCB1.

[0109] The Neurogein1 gene was obtained from human-derived cells by PCR using primers containing both the 5' and 3' EcoRI sites and a Flag, and then inserted into pCB1 after EcoRI cleavage. Similarly, the HGF gene was obtained by PCR using primers containing NheI cleavage sites at the 5' end and XbaI cleavage sites at the 3' end, and then inserted after XbaI and NheI cleavage. This was named pCB7.

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

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

[0112] We obtained a clone of pL2.3T-NH by cleaving it via AgeI and PmeI and then rejoining it, resulting in a clone with the CAG promoter inserted in the opposite direction. This clone was named pLK2.4T-HN plasmid (#25).

[0113] 1-2. Virus Packaging Using PEI (Cat. No. 101000033, Polyplus), 5 × 10¹ Lenti-X-293T cells (Cat. No. 632180, Takara) were placed in a 6-well plate. 5Cells / wells were transfused with either the plasmid 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 the pPACKH1 HIV Lentivector Packaging Kit (Systembio, LV500A-1). Subsequently, the cells were cultured in an incubator at 37°C and 5% CO2, and after 48 hours, the culture medium was obtained. After filtering through a 0.45 μm syringe filter, the medium was immediately stored at -80°C before next use.

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

[0115] The viral titer was determined using the following formula: Functional titer (IFU / mL) = [(Total number of cells at transduction) × (Percentage of transduced cells) × (Dilution factor)] / (Transduction volume added to cells (mL)). Titer values ​​exceeding 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 proportion of transduced cells. Briefly, transduced cells were obtained using 0.25% trypsin-EDTA. Subsequently, the 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 cultured at room temperature for 30 minutes. The cells were washed three times by centrifugation at 500 g for 5 minutes each with 5 ml of 1X permeabilization buffer (Cat. No. 00-8333-56, Invitrogen) at room temperature. Before processing with secondary antibodies, the cells were blocked with blocking solution (1% Albumin from BSA (Cat. No. A7906-100G, Sigma) in 1X permeabilization buffer) at room temperature for 30 minutes. After removing the blocking solution, cells were cultured at room temperature for 1 hour with HGF antibody (Cat. No. AB-294-NA, R&D systems) (1:200 dilution in blocking buffer). Goat IgG antibody (Cat. No. I9140, Sigma-Aldrich) (1:2000 dilution in blocking buffer) was used as the control group. The primary antibody was washed three times by centrifugation at 500g in 5ml of 1X permeabilization buffer at room temperature for 5 minutes each. Subsequently, Donkey anti-Goat IgG(H+L) Cross-Adsorbed Secondary Antibody and Alexa Fluor were used as secondary antibodies. TM Cell 568 (Cat. No. A-11057, Invitrogen) was treated at room temperature in the dark for 1 hour. The secondary antibody was washed twice with 5 ml of 1X permeabilization buffer, and finally washed with 5 ml of DPBS. Cells were taken from eBioscience 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 measured by FACS, and viral titers were measured as previously described.

[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 subcultured five times were cultured for 24 hours at a rate of 200,000 cells / well in a 6-well plate containing MSC culture medium. MSCs were transduced with a 30 MOI viral vector to a final transduction volume of 1 ml in the presence of 4 μg / ml polyblen in a 37°C incubator supplemented with 95% O2 + 5% CO2 for 8 hours. When the cells had grown to approximately 80-90% (cell confluency), GFP, RFP fluorescence images and optical (Bright-field, BF) images were acquired using the EVOS M5000 imaging system (Thermo Fisher Scientific). Transduced cells were separated with 0.25% trypsin-EDTA and washed with DPBS. After resuspending in 1 ml of DPBS, the cells were processed according to the manufacturer's guidelines using Nucleoview. TM NC-250 TM The number of cells was calculated using nucleocounter with software (Chemetec). The cells were 1,000 cells / cm³ for the next passage. 2 The cells were plated. All cell culture media were replaced with fresh medium every 2-3 days. The remaining cells were used for GFP / RFP FACS analysis, as described earlier.

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

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

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

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

[0122] As a result, as shown in Figure 6, the mCMV+CAG vector showed a higher viral titer compared to vectors containing only the CMV promoter, confirming that the mCMV+CAG vector can stably perform lentiviral packaging.

[0123] 2-2. Comparison of virus packaging efficiency with and without CAG promoter directionality and CTE We compared the viral packaging efficiency using the forward and reverse directions of the CAG promoter. We also examined the effect of CTEs, known to have RNA export function, on target protein expression.

[0124] As shown in Figure 7, viral titers were higher when the CAG promoter was positioned in the reverse direction (#1) compared to when it was positioned in the forward direction (#2). Furthermore, even higher viral titers were observed when CTEs were present (#9 and #10).

[0125] 2-3. Comparison of virus packaging efficiency based on CTE directionality, location, and number. We compared the lentiviral packaging efficiency based on the orientation, location, and number of CTEs in vectors where the CAG promoter was positioned in the opposite direction.

[0126] As a result, as shown in Figure 8, viral titers were high when a single CTE was located at the 5' end of the CAG promoter in either the forward or reverse direction (#9 and #10). When the CTE was located at the 3' end of the CAG promoter, proper virus production was not possible (#11-#16).

[0127] 2-4. Comparison of virus packaging efficiency with and without PGK promoter directionality and CTE Instead of the CAG promoter, we compared the directionality of the PGK promoter and the viral packaging efficiency by CTE in vectors containing both the mCMV promoter and the PGK promoter.

[0128] As a result, as shown in Figure 9, the PGK promoter resulted in a constant viral titer regardless of orientation (#3 and #4), while the presence of CTEs led to a greater increase in viral titer (#21 and #22).

[0129] 2-5. Comparison of viral packaging efficiency between CAG promoter and PGK promoter We compared the lentiviral packaging efficiency based on promoter and CTE orientation in vectors containing the CAG promoter and vectors containing the mCMV and PGK promoters.

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

[0131] 2-6. Comparison of target gene expression rates based on promoter orientation between CAG promoter and PGK promoter. To compare the expression rates of target genes between vectors containing mCMV and CAG promoters and vectors containing mCMV and PGK promoters, GFP and RFP expression rates were measured. Due to the bidirectional nature of the promoters, the expression rates of the same target gene differed. In the case of vectors containing mCMV and CAG promoters, the CAG promoter region showed a strong expression rate, and in the case of vectors containing mCMV and PGK promoters, the PGK promoter region showed a strong expression rate. These subordinate target genes of such promoters were named the "stronger side." In both vectors, the subordinate target genes of the mCMV promoter showed a weak expression rate, and these target genes were 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 in the weak region differed depending on the direction of the promoter, and in particular, when the PGK promoter was positioned in the opposite direction, the expression rate of the target gene in the weak region was very low. However, in the case of the CAG promoter, the target gene in the weak region was expressed identically regardless of the direction.

[0133] 2-7. Comparison of target gene expression rates based on promoter and CTE directionality As shown in Figure 12, after generating the lentivirus, it was used to infect the target cells, mesenchymal stem cells (MSCs). For each passage, fluorescence imaging was performed to confirm the expression of the target gene (GFP or RFP), and FACS experiments were conducted to confirm the infection rate.

[0134] Furthermore, the expression persistence of target genes was compared based on the directionality of the promoters and CTEs in vectors containing mCMV and CAG promoters, and vectors containing mCMV and PGK promoters. In the case of the reversed CAG promoter, the strong region was expressed in the GFP gene and the weak region was expressed in the RFP gene. In the case of the forward-directed mCMV+CAG promoter and mCMV+PGK promoter, the strong region was expressed in the RFP gene and the weak region was expressed in the GFP gene.

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

[0136] The infection rates for A and B in Figure 14 were not identical, and even after lentivirus infection, when the target gene in the strong region was expressed, the target gene in the weaker region (the opposite region) was not expressed. This disadvantage of bidirectional promoters was compensated for by the increased infection rate of the target gene in the weaker region in plasmids containing forward or reverse CTEs.

[0137] Furthermore, in general, the expression of target genes with bidirectional promoters showed decreased expression rates at weak sites when target cells were cultured for extended periods (Figure 14B, #1). Vectors containing reverse-directed CAG showed increased persistence of target gene expression when CTE was also present (Figure 14B, #1-#10), while vectors containing forward-directed CAG or forward-directed mCMV+PGK overcame the weakness at the weak side only when CTE was also present in the reverse direction (Figure 14B, #17 or #21).

[0138] These results suggest that candidate bidirectional plasmids may exhibit diverse differences depending on the target cell type. It is necessary to select the optimal bidirectional promoter for the target cell, taking into account differences in the expression rate of the target gene (which may not have been observed in cells like 293T) and decreased expression persistence.

[0139] 2-8. Comparison of target gene expression rates for vectors containing mCMV+CAG or mCMV+PGK in different target cell types. Since the expression rate of target genes can differ depending on the type of target cell, we investigated whether the candidate lentiviral plasmids are applicable to a diverse range of target cells by testing them in natural killer (NK) cells and Jurkat T cells.

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

[0141] When fluorescence expression was confirmed, more cells were found 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 target gene expression can be achieved even in NK cells using vectors containing mCMV+PGK.

[0142] Similarly, as shown in Figures 18-20, it was revealed that in Jurkat T cells, the expression of the target gene was higher with a vector containing mCMV+CAG than with a vector containing mCMV+PGK.

[0143] 2-9. Plasmid Application Model for Simultaneous Expression of Target Genes To apply plasmids that simultaneously express two or more target genes to actual research, plasmids containing suicide-inducing genes (third target genes) (#23 and #24) were constructed, and then U87 cells were infected with these plasmids and their expression rates were measured.

[0144] As a result, as shown in Figure 21, the normal expression of the first target gene (RFP) and the second target gene (copGFP) was confirmed by fluorescence after infecting U87 cells, and the expression of the third target gene (suicide induction gene, HSV-TK) was confirmed by a ganciclovir-based MTT assay.

[0145] We created a tumorigenesis model by injecting U87 cells infected with such lentiviruses into actual animals, and then induced cell suicide of the U87 cells by orally administering valganciclovir.

[0146] As a result, as shown in Figure 22, we confirmed that the size of the tumors decreased significantly over time compared to the control group.

[0147] These results suggest that the target gene in a gene co-expression plasmid can be modified for application in experiments and industrial settings.

[0148] 2-10. Confirmation of gene expression after target gene modification. In the above experiment, gene expression was confirmed using GFP, RFP, or HSV-TK as the target gene, and it was confirmed whether the same gene expression occurred for other target genes. 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 it was confirmed whether they were expressed normally.

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

[0150] The present invention has been described so far, focusing on preferred embodiments. Those with ordinary skill in the art to which the present invention pertains will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive view. The scope of the present invention is shown in the claims, not in the above description, and all differences within an equivalent scope should be interpreted as being included in the present invention.

Claims

1. It includes CTE, first target gene, first promoter, second promoter, and second target gene, The first target gene is HGF, and the second target gene is Ngn1. The first target gene and the first promoter are in the reverse direction of the recombinant vector.

2. The recombinant vector according to claim 1, further comprising a third promoter and a third target gene.

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 according to claim 1, wherein the CTE is in the forward or reverse direction.

5. The promoters include the Simian virus 40 (SV40) promoter, the cytomegalovirus (CMV) promoter, the minimal CMV promoter, the human ubiquitin C (UBC) promoter, the human elongation factor 1a (EF1A) promoter, the phosphoglycerate kinase 1 (PGK) promoter, and chicken beta-actin conjugated with the cytomegalovirus early enhancer. The recombinant vector according to claim 1 or 2, which is one or more selected from the group consisting of β-actin and CAG promoter.

6. The recombinant vector according to claim 2, wherein the third target gene is one or more selected from the group consisting of disease treatment genes, reporter genes, selection marker genes, and cell marker genes.

7. The recombinant vector according to 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-neoangiogenesis genes, and hormone genes.

8. The recombinant vector according to claim 6, wherein the reporter gene is one or more selected from the group consisting of TdTomato, luciferase, copGFP isolated from Pontelina 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 selected marker gene is one or more 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 Na / I co-transporter (sodium / iodide symboler), Thy-1 cell surface antigen (CD90), CD3, CD4, CD8, and CD25.

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

12. A gene transfer system comprising a recombinant vector according to claim 1 or 2.

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

14. The recombinant virus according to claim 13, wherein the recombinant virus is derived from a lentivirus.

15. A recombinant vector according to claim 1 or 2, or a transformant into which a recombinant virus containing the recombinant vector has been introduced.

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

17. The transformed organism according to claim 16, wherein the immune cells are selected from or derived from the group consisting of neutrophils, eosinophils, halophils, macrophages, mast cells, dendritic cells, B lymphocytes, T lymphocytes, and NK cells.

18. 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.

Images