Novel recombinant vectors and uses thereof

Abstract

The present invention relates to a recombinant vector, and a gene delivery system, a recombinant virus and a transformant comprising the same, the recombinant vector according to the present invention being designed such that one target gene is expressed by one promoter, and by delivering two or more target genes into a cell, it is possible to stably express at most three target genes at the same time in one vector, and to a gene delivery system, a recombinant virus and a transformant comprising the same. Therefore, the recombinant vector can be usefully applied to an efficient gene delivery 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 Art

[0002] Gene therapy refers to therapeutic techniques that utilize genes, such as inserting new genes into a patient's cells, removing genes that are not working properly, or replacing mutated genes with normal genes. Such gene therapy can be targeted immediately when only the gene causing the disease is identified, and can be manufactured quickly and cheaply compared to other antibody or compound therapeutics, which can increase the possibility of treating diseases that are difficult to treat with existing drug therapeutics.

[0003] In order to achieve effective gene therapy, the first step is to find a target gene that is important for disease treatment, and it is necessary to develop a vector technology that can deliver it well into cells without side effects. Vectors are gene delivery bodies, which can be roughly divided into viral vectors and non-viral vectors. Viral delivery bodies are made by removing most or part of the essential genes of viral genes, making them unable to replicate on their own, and inserting therapeutic genes into them. Although they can deliver genes into cells with high efficiency and low efficiency, there are problems such as difficulty in mass production, inducing immune response, toxicity, and the emergence of replicable viruses depending on the type of virus. At present, the main viral delivery bodies used for the development of gene therapy agents include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, pox viruses, etc. On the other hand, non-viral delivery bodies refer to liposomes, plasmids, etc., which do not induce immune response, have low toxicity, and are easy to mass produce, but have low gene delivery efficiency and their expression is temporary, which limits the development of gene therapy agents.

[0004] In the development of gene therapy agents, vector technologies using adeno-associated viruses or retroviruses are the mainstream. Lentivirus, a type of retrovirus, can deliver genes regardless of how cells divide, and the location of the chromosome where the delivered gene is inserted is distributed in an area with a relatively low risk of "oncogenic insertional mutagenesis". It induces self-inactivation by deleting the U3 part of the 3'LTR (long terminal repeat), which has the advantage of fundamentally blocking the risk of tumorigenicity caused by LTR.

[0005] However, the size of the target gene that can be introduced into the viral vector is limited, and as the size of the target gene increases, the possibility of the target gene disappearing due to homologous recombination increases. In addition, depending on the type and state of the cell, most of the target genes contained in a vector may be silencing unevenly, and such silencing phenomenon is difficult to predict or prevent with existing technology. Therefore, it is not easy to construct a vector that uniformly expresses two or more target genes in cells (such as mesenchymal stem cells) without silencing, and it is urgently needed to study it.

[0006] Prior art literature

[0007] Patent Literature

[0008] Korean Patent No. 10-1885438 Summary of the invention

[0009] The object of the present invention is to provide a recombinant vector that can deliver two or more target genes into cells and stably express them.

[0010] Another object of the present invention is to provide a gene delivery system comprising the above-mentioned recombinant vector.

[0011] Another object of the present invention is to provide a recombinant virus comprising the above-mentioned recombinant vector.

[0012] Another object of the present invention is to provide a transformant into which the above-mentioned recombinant vector or recombinant virus has been introduced.

[0013] One embodiment of the present invention provides a recombinant vector comprising 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 reverse orientation.

[0014] The "vector" used in the present invention is an expression vector that can express the target gene in a suitable host cell, and refers to a gene product that includes the necessary regulatory elements that are operably linked in order to express the gene insert contained in the vector. Here, "operably linked" refers to the functional linkage (functional linkage) of a nucleic acid expression regulatory sequence that performs a general function and a nucleic acid sequence that encodes the target gene.

[0015] The vector according to the present invention includes, in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal, enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and can be manufactured in a variety of ways according to the purpose. The promoter of the vector can be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and when it is a reproducible expression vector, a replication origin can be included. Such a vector can self-replicate or be integrated into the host DNA. As an example of the above-mentioned vector, there are plasmid vectors, cosmid vectors, phage vectors, viral vectors, etc.

[0016] Such a recombinant vector including CTE, the first target gene, the first promoter, the second promoter and the second target gene can simultaneously express the two target genes in one vector by containing two target genes and two promoters that regulate the expression of the respective target genes.

[0017] In addition, the recombinant vector according to the present invention can simultaneously express three target genes in one vector by further comprising a target gene and a promoter that regulates the expression thereof.

[0018] According to a specific example of the present invention, the above-mentioned recombinant vector may further include a third promoter and a third target gene.

[0019] The "CTE (constitutive transport element)" used in the present invention refers to a factor that promotes the nuclear export of incompletely spliced ​​mRNA. It is known that the closer it is to the poly A tail that is important for the nuclear export, translation and stability of mRNA, the more its function is enhanced, thereby increasing the expression of the target gene (MR Mautino, et al., Gene Therapy. 2000.7, 1421-1424).

[0020] The "target gene" used in the present invention refers to a gene whose expression is induced by a promoter.

[0021] The "promoter" used in the present invention is involved in the binding of RNA polymerase, so that it can start transcription as a part of DNA. It is usually adjacent to the target gene and located upstream thereof, and serves as a site for inducing RNA polymerase or RNA polymerase protein, i.e., transcription factor, to bind, and can induce the above 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 induces RNA polymerase to bind to the corresponding site directly or through a transcription factor to start the mRNA synthesis of the target gene, and has a specific gene sequence.

[0022] The elements of the above-mentioned recombinant vector, namely, CTE, target gene and promoter, may affect the expression of the target gene depending on the connection direction.

[0023] According to one embodiment of the present invention, the CTE can be connected to the 3' end of the poly A tail.

[0024] According to a specific example of the present invention, the CTE may include a base sequence represented by SEQ ID NO:1.

[0025] According to a specific example of the present invention, the CTE can be positive or negative.

[0026] As used in the present invention, "forward orientation" means that the sequence encoded by each gene in the viral gene is in the sense orientation (5'→3'), and "reverse orientation" means that the sequence encoded by each gene in the viral gene is in the anti-sense orientation (3'→5').

[0027] In order to simultaneously express two or more target genes, the recombinant vector according to the present invention comprises two or more promoters for inducing the expression of the respective target genes. Here, the types of the two or more promoters may be the same or different from each other.

[0028] In such a recombinant vector, in addition to the first target gene and the first promoter in the reverse direction, the second target gene and the second promoter and / or the third target gene and the third promoter may be linked in the forward direction.

[0029] According to a specific example of the present invention, the above-mentioned promoter can be one or more selected from the group consisting of Simian virus 40 (SV40) promoter, cytomegalovirus (CMV) promoter, minimal CMV promoter, human ubiquitin C (UBC) promoter, human elongation factor 1a (EF1A) promoter, phosphoglycerate kinase 1 (PGK) promoter and cytomegalovirus immediate-early enhancer / chicken β-actin (CAG) hybrid promoter.

[0030] 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.

[0031] As an example, the first promoter, the second promoter and the third promoter can be selected from CAG promoter (SEQ ID NO: 2), minimal (mimimal) CMV (hereinafter referred to as "mCMV") (SEQ ID NO: 3), PGK promoter (SEQ ID NO: 4) and SV40 (promoter 5).

[0032] More specifically, the first promoter and the second promoter may be CAG promoter and mCMV promoter, mCMV promoter and CAG promoter, PGK promoter and mCMV promoter, or mCMV promoter and PGK promoter, respectively. Here, the first promoter is connected in reverse.

[0033] According to a specific example 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.

[0034] The recombinant vector according to the present invention can express two or more target genes simultaneously, so the disease therapeutic gene, reporter gene or selection marker gene can be expressed separately or all of the disease therapeutic gene, reporter gene and selection marker gene can be expressed.

[0035] The "disease therapeutic gene" used in the present invention refers to a polynucleotide sequence or base sequence encoding a polypeptide that exhibits a therapeutic effect on cells that abnormally express the gene, such as cancer.

[0036] According to a specific example of the present invention, the above-mentioned disease treatment gene can be one or more selected from drug sensitivity genes, apoptosis genes, cell proliferation inhibition genes, cell growth genes, cytotoxicity genes, tumor suppressor genes, antigenic genes, cytokine genes, neurogenesis genes, anti-angiogenesis genes and hormone genes.

[0037] The above-mentioned drug-sensitizing gene is a gene encoding an enzyme that converts a non-toxic precursor (prodrug) into a toxic substance. Since the cell into which the gene is introduced undergoes apoptosis, it is also called a suicide gene. That is, when a precursor that is non-toxic to normal cells is administered locally or systemically, the precursor can be converted into a toxic metabolite only in the target cell, thereby changing the drug sensitivity and destroying the target cell. As an example of such a drug-sensitizing gene, there is the HSV-TK (Herpes simplex virus-thymidine kinase) gene using ganciclovir or valganciclovir as a precursor, the cytosine deaminase (cytosine deaminase) of Escherichia coli using 5-fluorocytosine (5-FC) as a precursor, etc., but it is not limited to this.

[0038] The above-mentioned proapoptotic gene is a nucleotide sequence that induces programmed cell apoptosis when expressed. Examples of such proapoptotic genes include 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 gene encoding caspase, but are not limited thereto.

[0039] The above-mentioned cytostatic gene is a nucleotide sequence that is expressed in cells and arrests the cell cycle during the cell cycle. Examples thereof include p21, retinoblastoma gene, E2F-Rb fusion protein gene, genes encoding cyclin-dependent kinase inhibitors (such as p16, p15, p18 and p19), growth arrest specific homeobox (GAX) gene, etc., but are not limited to these.

[0040] The cell growth gene is a nucleotide sequence that promotes the division, growth and differentiation of stem cells or cells that have completed differentiation. Examples of such cell growth genes include hepatocyte growth factor, stem cell factor, insulin-like growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, transforming growth factor, platelet-derived growth factor, bone-derived growth factor, colony stimulating factor, etc., but are not limited thereto.

[0041] The cytotoxic gene is a nucleotide sequence that is expressed in cells and exhibits a toxic effect. Examples thereof include nucleotide sequences encoding Pseudomonas exotoxin, ricin, diphtheria toxin, etc., but the present invention is not limited thereto.

[0042] The tumor suppressor gene is a nucleotide sequence that is expressed in target cells and can suppress tumor phenotypes or induce apoptosis. Examples of such tumor suppressor genes include 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, PD-1 (programmed death-1) gene, etc., but are not limited thereto.

[0043] The 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 thereof include carcinoembryonic antigen and p53, but are not limited thereto.

[0044] The above-mentioned cytokine gene refers to a nucleotide sequence that is expressed in cells and produces cytokines. An example thereof includes GMCSF, interleukins (such as IL-1, IL-2, IL-4, IL-6, IL-12, IL-10, IL-15, IL-19, IL-20), interferons (such as α, β, γ), etc., but is not limited to these.

[0045] The neurogenic gene is a nucleotide sequence that promotes the generation and development of nerves. Examples thereof include brain-derived neurotrophic factor and nerve growth factor, but are not limited thereto.

[0046] The above-mentioned neural differentiation gene is a nucleotide sequence that participates in the division of neuroepithelial cells into neuroblasts and neuroglia during stem cell division, and examples thereof include neurogenin, NeuroD, ASCL1 (Achaete-scute homolog 1), etc., but are not limited to these.

[0047] The anti-angiogenic gene refers to a nucleotide sequence that expresses and releases an anti-angiogenic factor to the outside of cells, and an example thereof may be angiostatin, an inhibitor of vascular endothelial growth factor (VEGF), endostatin, etc., but is not limited thereto.

[0048] The above-mentioned hormone gene refers to a nucleotide sequence that produces a hormone or its precursor, and examples thereof include insulin, growth hormone, thyroid stimulating hormone, adrenocorticotropic hormone, gonadotropic hormone, prolactin, luteotropic hormone, melanocyte stimulating hormone, vasopressin and oxytocin, but are not limited to these.

[0049] In addition, any gene that has a therapeutic effect can be used regardless of whether it is a gene for treating a disease.

[0050] The "reporter gene" used in the present invention is a gene suitable for detecting whether a recombinant vector has been introduced or the expression efficiency of a target gene, and any gene can be used without limitation as long as it can be detected without damaging infected cells or tissues.

[0051] According to a specific example of the present invention, the reporter gene may be selected from 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). At least one of the following is not limited thereto.

[0052] The "selection marker gene" used in the present invention is an antibiotic resistance gene used to select cells into which the gene has been introduced. Since only cells expressing the selection marker gene, i.e., the antibiotic resistance gene, can survive in a culture medium treated with antibiotics, cells into which the gene has been introduced can be screened. As the above-mentioned selection marker gene, any antibiotic resistance gene known in the art can be used without limitation.

[0053] According to a specific example of the present invention, the above-mentioned selection marker gene can be one or more selected from β-lactamase, puromycin N-acetyltransferase, aminoglycoside phosphotransferase, and hygromycin B-phosphotransferase, but is not limited thereto.

[0054] The "cell marker gene" used in the present invention is a polynucleotide sequence or base sequence encoding a molecule such as a polypeptide, protein, lipid, glycolipid, glycoprotein, sugar, etc. that is specifically expressed on the cell membrane or cell surface.

[0055] According to a specific example 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.

[0056] Such target genes can be optimized with human codons. "Optimized with human codons" means that when DNA is transcribed and translated into protein in a host cell, there are codons with high preference depending on the host among the codons for specifying amino acids, which means that the expression efficiency of the amino acids or proteins encoded by the nucleic acid is increased by replacing them with human codons.

[0057] As an example, the first target gene, the second target gene and the third target gene can be selected from 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).

[0058] The HGF gene is a hepatocyte growth factor, and is a gene encoding a heparin-binding glycoprotein known as scatter factor or hepatopoietin-A. It is known that the HGF is produced by various mesenchymal cells, promotes cell proliferation, regulates the growth of endothelial cells and the migration of vascular smooth muscle cells, and induces angiogenesis.

[0059] The Ngn1 gene is a gene encoding neurogenin 1 that specifically participates in neural differentiation. The neurogenin 1 plays a role in regulating bone-morphogenetic-protein and leukemia inhibitory factor to induce neural differentiation. The Ngn1 gene may include a 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 a base sequence of SEQ ID NO: 8.

[0060] The HSV-TK gene is a gene encoding thymidine kinase (TK) of herpes simplex virus (HSV). The TK is an enzyme that binds the γ-position phosphoric acid of ATP to thymidine to promote the thymidylate production reaction. In addition to thymidine, antiviral agents such as ganciclovir, valganciclovir, acyclovir, and valacyclovir are known to be phosphorylated, and the phosphorylated product disrupts DNA replication, thereby inducing cell apoptosis.

[0061] The puromycin resistance gene (PuroR) is a gene encoding puromycin N-acetyltransferase. The puromycin resistance gene is similar to amynoacyl t-RNA and blocks the function of puromycin by inactivating puromycin that interferes with protein translation, thereby inducing resistance and screening cells into which the gene has been introduced.

[0062] According to a specific example of the present invention, at least one of the first target gene, the second target gene and the third target gene of the above-mentioned recombinant vector may contain a Kozak sequence.

[0063] The Kozak sequence is a translation initiation determining site of eukaryotes corresponding to the Shine & Dalgarno base sequence of prokaryotes, and is located before the codon AUG encoding methionine in the base sequence of mRNA, and a ribosome is attached thereto.

[0064] According to a specific example of the present invention, the Kozak sequence may include CCACC or CCG CC.

[0065] As an example, the above-mentioned Kozak sequence may include the base sequence represented by SEQ ID NO:12.

[0066] The recombinant vector according to the present invention can regulate the expression of more than two target genes loaded thereon through more than two promoters, and is designed to express one target gene through one promoter by operably connecting the promoter and the target gene expressed thereby, so that up to three target proteins can be translated simultaneously in one vector.

[0067] Another aspect of the present invention provides a gene delivery system comprising the above-mentioned recombinant vector.

[0068] The "gene delivery system" used in the present invention refers to a system that can increase the expression efficiency by improving the efficiency of delivering the target gene and / or nucleic acid sequence into the cell, and can be divided into virus-mediated cooperative and non-viral systems.

[0069] It is known that the above-mentioned viral-mediated systems use viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex virus vectors, poxvirus vectors, etc., and utilize the intracellular penetration mechanism inherent to viruses that cause infection of eukaryotic cells such as human cells, and the intracellular gene delivery efficiency is relatively high compared to non-viral systems. In addition, the problem of non-viral vectors after entering the cell is that the gene is decomposed in the endosome after the fusion of the endosome and the lysosome, but the viral vector has the mechanism of delivering the gene directly to the nucleus without passing through the lysosome, and the advantage is that the loss of the gene is small and the gene delivery efficiency is high.

[0070] According to one embodiment of the present invention, the gene delivery system may use a viral vector.

[0071] More specifically, the viral vector may be derived from one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus and poxvirus, and is preferably a lentivirus.

[0072] After such viral vectors are assembled into viral particles, they can be introduced into cells through transduction methods such as infection.

[0073] The gene delivery system according to the present invention performs the function of delivering a recombinant vector expressing two or more target genes to a target cell, so that the two or more target genes of the recombinant vector delivered into the cell can be expressed through the intracellular transcription system.

[0074] Another aspect of the present invention provides a recombinant virus comprising the above-mentioned recombinant vector.

[0075] According to one embodiment of the present invention, the recombinant virus may be derived from a lentivirus.

[0076] Such recombinant viruses can target all dividing cells, specifically, immune cells or stem cells.

[0077] According to a specific example of the present invention, the above-mentioned immune cells can be selected from or derived from neutrophils, eosinophils, basophils, macrophages, mast cells, dendritic cells, B lymphocytes, T lymphocytes and NK cells (natural killer cells).

[0078] According to a specific example of the present invention, the above-mentioned stem cells can be one or more selected from embryonic stem cells, fetal stem cells, adult stem cells, amniotic embryonic stem cells, cord blood stem cells, induced pluripotent stem cells, mesenchymal stem cells (MSC), neural stem cells, hematopoietic stem cells and cancer stem cells.

[0079] Another aspect of the present invention provides a transformant introduced with the above-mentioned recombinant vector or recombinant virus.

[0080] The "transformant" used in the present invention refers to a cell produced by inserting a foreign gene into a host cell, transformed with a recombinant vector (transfection) or transduced with a recombinant virus (transduction), and is also called a recombinant host cell, a recombinant cell or a recombinant microorganism.

[0081] The "host cell" used in the present invention refers to a cell transformed, transduced or introduced with a recombinant vector or a recombinant virus.

[0082] The host cell may be a host cell known in the art. As an example of the host cell, it may be NS / O myeloma cell, human 293T cell, Chinese hamster ovary cell (CHO cell), HeLa cell, human amniotic fluid-derived cell (CapT cell), COS cell, canine D17 cell, feline PG4 cell, stem cell, etc., but is not limited thereto.

[0083] The above transformation can be carried out by introducing genes into cells according to biological, chemical or physical methods known in the art. As an example of the above transfection, it can be carried out by any one or more methods selected from liposome method, microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, DEAE-dextran treatment method, polybrene treatment method, polyethyleneimine treatment method and gene bombardment method.

[0084] The transformant can be cultured using a medium commonly used in animal cell culture. As an example, the medium can be any one or more selected from Eagles' MEM, a-MEM, Iscove'sMEM, 199 medium, CMRL 1066, RPMI1640, F12, F10, DMEM, a mixed medium of DMEM and F12, Way-mouth's MB752 / 1, McCoy's 5A, and MCDB series medium.

[0085] According to one embodiment of the present invention, the transformant may be an immune cell or a mesenchymal stem cell.

[0086] The above-mentioned immune cells are cells differentiated from hematopoietic stem cells and involved in immunity, and can be selected from or derived from neutrophils, eosinophils, basophils, macrophages, mast cells, dendritic cells, B lymphocytes, T lymphocytes and NK cells (natural killer cells).

[0087] The mesenchymal stem cells are cells that differentiate from the mesenchyme into muscle cells, fat cells, osteoblasts, chondrocytes, and the like when the fertilized egg divides.

[0088] The above-mentioned mesenchymal stem cells are cells that maintain stemness and self-renewal ability and have the ability to differentiate into various mesenchymal tissues (plasticity), and can be extracted from bone marrow, adipose tissue, umbilical cord blood, synovial membrane, bone tissue (trabecular bone), infrapatellar fat pad, placenta, etc. Such mesenchymal stem cells have the following immunoregulatory abilities: 1) inhibiting the activity and proliferation of T lymphocytes and B lymphocytes, 2) inhibiting the activity of natural killer cells, and 3) regulating the functions of dendritic cells and macrophages, so that allotransplantation and xenotransplantation can be achieved, and they can be used to treat damaged articular cartilage or nerves.

[0089] 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, bone tissue (trabecular bone) and infrapatellar fat pad, but is not limited thereto.

[0090] The recombinant vector according to the present invention is designed to express one target gene from one promoter, and by delivering two or more target genes into cells, up to three target genes can be stably expressed simultaneously in one vector. Therefore, such a recombinant vector can be usefully applied to an efficient gene delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Schematic diagram showing a series of processes of manufacturing a plasmid, generating a virus expressing the plasmid, and infecting a target cell according to an embodiment of the present invention.

[0092] Figure 2 The structure of a plasmid comprising mCMV, CAG, PGK and / or SV40 promoter as a promoter and RFP, copGFP and PuroR as target genes manufactured according to one embodiment of the present invention. (In the figure below, the mCMV promoter is reversely connected to the CAG or PGK promoter)

[0093] Figure 3 The structure of a plasmid comprising mCMV, CAG and SV40 promoters as promoters and RFP, copGFP and PuroR as target genes and comprising or not comprising CTE and SV40 poly A is prepared according to one embodiment of the present invention.

[0094] Figure 4 The plasmid structure is prepared according to one embodiment of the present invention and contains mCMV, CAG, PGK and / or SV40 promoter as a promoter and contains RFP, copGFP, PuroR as a target gene and contains or does not contain CTE and SV40 poly A.

[0095] Figure 5The structure of a plasmid comprising 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, prepared according to one embodiment of the present invention. (In the figure below, the target gene is marked in the figure and omitted from the description of the figure)

[0096] Figure 6 (A) The structure of pLenti-GIII-CMV-GFP-2A-Puro and (B) Comparison results of viral packaging efficiency between pLenti-GIII-CMV-GFP-2A-Puro (labeled as "CMV") and mCMV and CAG promoter (labeled as "CMV+CAG") according to an embodiment of the present invention.

[0097] Figure 7 1 is a comparison result of the viral packaging efficiency under the influence of the directionality and CTE of the mCMV and CAG promoters according to an embodiment of the present invention.

[0098] Figure 8 This is a comparison result of virus packaging efficiency under the influence of the directionality, site or number of CTE according to an embodiment of the present invention.

[0099] Fig. 9 1 is a comparison result of the viral packaging efficiency under the influence of the directionality of the PGK promoter and CTE according to an embodiment of the present invention.

[0100] Fig.10 FIG. 5 is a comparison result of the virus packaging efficiency between the CAG promoter and the PGK promoter according to an embodiment of the present invention.

[0101] Fig.11 It is a comparison result of the expression rates of the target genes, namely, copGFP and RFP, between the CAG promoter and the PGK promoter according to one embodiment of the present invention.

[0102] Fig.12 FIG. 1 is a schematic diagram of a timetable for producing lentivirus and then infecting mesenchymal stem cells for culturing according to an embodiment of the present invention.

[0103] Fig.13 The results of comparing the expression persistence of the target genes copGFP and RFP under the influence of the directionality of the promoter and CTE using a fluorescence microscope according to one embodiment of the present invention.

[0104] Fig.14 The results of comparing the expression persistence of the target genes copGFP and RFP under the influence of the directionality of the promoter and CTE by FACS analysis according to one embodiment of the present invention.

[0105] Fig.15 and 16 The results of comparing the viral infection rate of the vector containing the dual promoter of each cell by FACS analysis according to one embodiment of the present invention. Fig.15 In the present invention, PBNK (peripheral blood natural killer cell) refers to NK cells, single cells refer to a single cell population detected by FACS, and live cells refer to live cells in a single cell population.

[0106] Fig.17 1 is an optical (BF) and fluorescent (GFP and RFP) image comparing the viral infection rate of a vector containing a dual promoter of NK cells according to an embodiment of the present invention.

[0107] Fig.18 and 19 The figure is the result of comparing the virus infection rate of the vector containing dual promoters in Jurket T cells by FACS analysis according to one embodiment of the present invention.

[0108] Fig. 20 are optical (BF) and fluorescent (GFP and RFP) images comparing the viral infection rates of Jurket T cells with vectors containing dual promoters according to an embodiment of the present invention.

[0109] Fig.21 1 and 2 are (A) fluorescence (GFP and RFP) images and (B) survival rate results of cells infected with a virus containing a vector of the suicide-inducing gene HSV-TK according to an embodiment of the present invention.

[0110] Fig. 22 (A) Tumor photos and (B) results of tumor size and body weight changes of an animal model infected with a virus containing a vector of the suicide-inducing gene HSV-TK according to one embodiment of the present invention.

[0111] Fig.23 1 and 2 are the results of (A) mRNA levels of HGF, Ngn1 and TK and (B) HGF expression rate under different MSC passaging according to one embodiment of the present invention. DETAILED DESCRIPTION

[0112] The present invention will be described in more detail below, but such description is provided for illustrative purposes only to help understand the present invention, and the scope of the present invention is not limited to such illustrative description.

[0113] 1. Experimental Methods

[0114] 1-1. Plasmid preparation

[0115] A plasmid was prepared 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, 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) and 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.

[0116] When making all plasmids except plasmid #1, PCR was completed by repeating 25 times of initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 60 seconds, annealing at 57°C for 30 seconds, and polymerization at 72°C for 60 seconds / kb, and finally polymerization at 72°C for 5 minutes. Digestion using restriction enzymes used BamHI, FspAI.EcoRI and PmeI, all at 37°C for 2 hours. Ligation was performed at room temperature (RT) for 150 seconds and on ice for 10 minutes.

[0117] The primers used for plasmid preparation are shown in Table 1 below.

[0118] [Table 1]

[0119]

[0120]

[0121] Plasmid #1 used Lenti-Bi-Cistronic vector (vector) (Cat.No.LV037, abm) as the backbone, inserted CAG-mCMV dual promoter (CAG-mCMV Dual Promoter) and RFP in Mir19 tracer (Tracer) (Jinju Han et., al., 2016, Neuron), and inserted dscGFP (copGFP) in pGreenFier1-mCMV (Cat.No.TR010PA-P, SBI). Finally, a plasmid (#1, pL1.4P-GR) containing reverse CAG and copGFP and forward mCMV, RFP, sv40 and puromycin resistance gene (PuroR) was made.

[0122] #2 Plasmid

[0123] Using plasmid #1 as the backbone, a fragment containing BamH and FspAI contained in mRFP1 was prepared by PCR in the forward direction using primers. Then, the CAG-mCMV fragment was removed from the prepared fragment using enzyme digestion (BamHI and FspAI), and the two fragments were reconnected in the forward direction to prepare a plasmid containing mCMV-CAG (#2).

[0124] Plasmids #3 and #4

[0125] The fragment with CAG removed was prepared by the same method as the preparation of plasmid #2. The pLenti-Bi-Cistronic-CopGFP plasmid (abm) was used as the backbone, and a fragment containing restriction enzyme sites (BamHI and FspAI), PGK, and mCMV was prepared by PCR using primers. The fragment with CAG removed 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).

[0126] Plasmids #9 and #10

[0127] Using pLK2.4T-HN plasmid (see "Plasmid #25" below) as a backbone, a fragment containing CTE and SV40 poly(A) was prepared by PCR in the forward or reverse direction using primers containing EcoRI. The prepared fragment was then positionally swapped with copGFP in plasmid #1 to prepare a plasmid containing SV40 poly(A)-CTE (#9) or SV40 poly(A)-CTE (#10).

[0128] Plasmids #11 and #12

[0129] Using the pLK2.4T-HN plasmid as the backbone, a fragment containing CTE and SV40 poly(A) was produced by PCR in the forward or reverse direction using primers containing PmeI. The produced fragment was then inserted into the PmeI site in the #1 plasmid to produce a plasmid containing CTE-SV40 poly(A) (#11) or CTE-SV40 poly(A) (#12).

[0130] Plasmids #13 and #14

[0131] A fragment containing CTE and SV40 poly (A) was prepared by the same method as the preparation of plasmids #11 and #12. Then, the prepared fragment was inserted into the PmeI site of plasmid #9 in the forward or reverse direction to prepare plasmids (#13 and #14) containing two CTEs and SV40 poly (A).

[0132] Plasmids #15 and #16

[0133] A fragment containing CTE and SV40 poly (A) was prepared by the same method as the preparation of plasmids #11 and #12. Then, the prepared fragment was inserted into the PmeI site of plasmid #10 in the forward or reverse direction, thereby preparing plasmids (#15 and #16) containing two CTE and SV40 poly (A).

[0134] Plasmids #17 and #18

[0135] A fragment containing CTE and SV40 poly(A) was prepared by the same method as that used to prepare plasmids #9 and #10. The prepared fragment was then inserted into the EcoRI site of plasmid #2 to prepare a plasmid containing SV40 poly(A)-CTE (#17) or SV40 poly(A)-CTE (#18).

[0136] Plasmids #19 and #20

[0137] A fragment containing CTE and SV40 poly(A) was prepared by the same method as that used to prepare 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).

[0138] Plasmids #21 and #22

[0139] A fragment containing CTE and SV40 poly(A) was prepared by the same method as that used to prepare plasmids #9 and #10. The prepared fragment was then inserted into the EcoRI site of plasmid #4 to prepare a plasmid containing SV40 poly(A)-CTE (#21) or SV40 poly(A)-CTE (#22).

[0140] Plasmids #23 and #24

[0141] First, the Lenti-Bi-Cistronic vector (vector) (Cat. No. LV037, abm) was used as the backbone, and the CAG-mCMV dual promoter and RFP from the Mir19 tracer (Tracer) (Jinju Han et., al., 2016, Neuron), dscGFP (copGFP) from pGreenFire1-mCMV (Cat. No. TR010PA-P, SBI), and CTEf from the pLVX-TetOne-Puro vector (vector) (Cat. No. 124797, Addgene) were combined to produce the pL2.4P-RG plasmid.

[0142] Then, pAL119-TK (Cat. No. 21911, addgene) was used as the backbone, and HSV-TK was PCR amplified to produce a fragment containing HSV-TK. The third target gene was cut out from the pL2.4P-RG plasmid with PmeI and Acc65I, and a fragment containing HSV-TK (#23) or variant HSV-TK (#24) was inserted to produce each plasmid.

[0143] In the mutant HSV-TK, alanine at position 168 in the amino acid sequence constituting the protein was replaced by histidine. It was reported that the kinase activity was about 4 times higher than that of the wild-type HSV-TK (Jan Balzariniet., al., 2006, J Biol Chem.), so a single amino acid mutation (A168H) was introduced by over-lapping PCR technology.

[0144] #25 plasmid

[0145] In the Lenti-Bi-Cistronic vector (vector) (Cat. No. LV037, abm), a DNA fragment containing the SV40, PuroR and WPRE elements was ensured by PCR, cloned by TA and cloned by Easyvector Systems (Cat. No. A1360, Promega) was used for TA cloning and inserted. It was named pTA1. In Mir19 tracer (Tracer) (Jinju Han et., al., 2016, Neuron), NheI and Acc65I enzymes were used to ensure the backbone, and pTA1 was cut with the same enzymes to obtain SV40 promoter, PuroR and WPRE sections, and inserted into the backbone plasmid. It was named pCB1.

[0146] In human cells, Neurogein1 gene was obtained by PCR using primers containing EcoRI cleavage sites and flags on both 5' and 3' sides, and then inserted into pCB1 after EcoRI cleavage. In a similar way, HGF gene was obtained by PCR using primers containing NheI cleavage sites on the 5' side and XbaI cleavage sites on the 3' side, and then inserted after XbaI and NheI cleavage. It was named pCB7.

[0147] In Mir19 tracer (Tracer) (Jinju Han et al., 2016, Neuron), CAG+mCMV was obtained by PCR using primers containing NheI cleavage sites on both 5' and 3' sides, and inserted into pCB7 after cutting NheI. It was named pL1.3P-NH.

[0148] CTE obtained from pLVX-TetOne-Puro vector (Cat. No. 124797, Addgene) was inserted into pL1.3P-NH, and the HSV-TK part was obtained by cutting the PmeI and MauBI sites of the #24 plasmid and inserted. It was named pL2.3T-NH.

[0149] pL2.3T-NH was cut with AgeI and PmeI and then religated to obtain a clone with the CAG promoter inserted in the reverse direction, which was named pLK2.4T-HN plasmid (#25).

[0150] 1-2. Virus Packaging

[0151] Lenti-X-293T cells (Cat. No. 632180, TaKaRa) were transfected in 6-well plates using PEI (Cat. No. 101000033, Polyplus) for 5×10 5 1.5 μg) of each plasmid or pLenti-GIII-CMV-GFP-2A-Puro vector (Cat. No. LV180162, abm) prepared in Example 1 and 3.6 μl (1.8 μg) of pPACKH1 HIV lentiviral vector packaging kit (Lentivector Packaging Kit) (Systembio, LV500A-1). Then, after culturing in a culture medium at 37°C and 5% CO2 for 48 hours, the culture medium was obtained, filtered with a 0.45 μm syringe filter, and immediately stored at -80°C until the next use.

[0152] 1-3. Transduction and Virus Titration

[0153] Lenti-X 293T cells were dispensed into a 24-well plate at 50,000 cells / well and cultured at 37°C, 5% CO2 for 24 hours. After removing the medium, a final volume of 250 μl of virus dilution (2, 5 or 25 times) diluted with DMEM containing 10% FBS and 1% P / S was 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, 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, and cultured at 37°C, 5% CO2 for 48 hours. After removing the medium, the cells were washed with DPBS (Cat. No. LB 001-02, WELGENE). The transduced cells were obtained using 0.25% trypsin-EDTA (Cat. No. 25200056, Thermo Fisher Scientific). Then, the cells were washed with DPBS and purified by eBioscience. TM The cells were resuspended in Flow Cytometry Staining Buffer (Cat. No. 00422226, Invitrogen). GFP-positive cells were stained with Attune TMSorting was performed using the Attune NxT Acoustic Focusing Cytometer (Invitrogen) with NxT software.

[0154] The titer of the virus was determined using the following formula: Functional titer (IFU / mL) = [(total number of cells at transduction) x (% of cells transduced) x (dilution factor)] / (transduction volume added to cells (mL)). Titer values ​​outside the linear range were not used to determine the final titer.

[0155] For viral vectors encoding Ngn1, HGF and HSV-TK, intracellular HGF combustion was performed and FACS analysis was performed to determine the ratio of transduced cells. In short, transduced cells were obtained using 0.25% trypsin-EDTA. Then, the cells were washed with DPBS, resuspended in 500μl of DPBS, mixed with 500μl of IC fixation buffer (Cat.No.00-8222-49, Invitrogen), and incubated at room temperature for 30 minutes. The cells were washed three times by centrifugation at 500g for 5 minutes each time at room temperature with 5ml of 1X permeabilization buffer (Cat.No.00-8333-56, Invitrogen). Prior to secondary antibody treatment, cells were blocked with blocking solution (1% BSA albumin (Albumin from BSA) (Cat.No.A7906-100G, Sigma) dissolved in 1x permeabilization buffer) at room temperature for 30 minutes. After removing the blocking solution, 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 group, 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 at room temperature at 500g, 5 minutes each time. Then, as the secondary antibody, donkey anti-goat IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor TM 568 (Cat. No. A-11057, Invitrogen) was treated for 1 hour at room temperature in a dark place. The secondary antibody was washed twice with 5 ml of 1X permeabilization buffer, and finally washed with 5 ml of DPBS. The cells were resuspended in 250 μl of eBioscience TMFlow Cytometry Staining Buffer (Cat. No. 00-4222-26, Invitrogen) was used to stain HGF-positive (Alexa 568-positive) cells by FACS, and the virus titer was determined as described above in the dark.

[0156] 1-4. MSC transduction

[0157] Mesenchymal stem cells (MSCs) were isolated from human bone marrow and subcultured in MSC culture medium (DMEM (Cat. No. LM 001-05, 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 in a 6-well plate with MSC culture medium at 200,000 cells / well for 24 hours. MSCs were transduced with viral vectors at 30 MOI for 8 hours in a 37°C culture medium supplemented with 95% O2 + 5% CO2 in the presence of 4 μg / ml polybrene, with a final transduction volume of 1 ml. When the cells grew to about 80-90% (cell confluency), GFP, RFP fluorescence images and optical (Bright-field, BF) images were obtained using the EVOS M5000 imaging system (Thermo Fisher Scientific). The transduced cells were separated with 0.25% trypsin-EDTA and washed with DPBS. After resuspending in 1 ml of DPBS, Nucleoview was used according to the manufacturer's instructions. TM NC-250 TM The number of cells was calculated using the NucleoCounter software (Chemetec). 2 Plated for next passage. All cell culture media were replaced with fresh media every 2-3 days. The remaining cells were used for GFP / RFP FACS analysis as described above.

[0158] 1-5. In vitro apoptosis (cellular suicide) assay

[0159] The lentivirus produced by plasmid #23 or #24 was used to infect human brain cell line U87 cells, thereby producing U87+#23 or U87+#24 cells. Each cell was cultured at 1×10 4 Cells were plated in 12-well plates at a concentration of 10 cells / mL. After 24 hours of culture, cells were treated with Ganciclovir (GCV, Cat. No. G2536, Sigma) at the desired concentration. On the fourth day, MTT (Cat. No. M2128, Sigma) was treated at 0.5 mg / mL. After 2 hours of culture, each well was treated with 500 μl of DMSO to induce the production of MTT-formazan. In order to confirm the degree of apoptosis, the absorbance at 540 nm of each well plate was measured.

[0160] 1-6. In vivo cell apoptosis assay

[0161] Six-week-old Balb / c nude mice were intraperitoneally injected with 10 μg of U87+#23 or U87+#24 cells. 6 cells and The animal model was established by adding 100uL of PBS containing growth factor deduced (GFR) basement membrane matrix-LDEV-free (Cat. No. 354230, Corning). After 6 weeks, when the tumor grew to an appropriate size (100-300mm 3 ), valganciclovir (vGCV, Cat. No. V0158, Tokyo Chemical Industry) at a concentration of 50 or 200 mg / kg dissolved in normal saline was orally administered daily for 14 days. The size of the tumor was measured every two days.

[0162] 2. Results

[0163] Reference Figure 1, using the third-generation virus packaging system (Lentivirus Packaging System), and using Lenti-X-293T (Cat.No.632180, Takara) as a virus production cell, to produce lentivirus expressing each plasmid. The infectivity of the produced lentivirus was measured by Lenti-X-293T, and the lentivirus was infected with mesenchymal stem cells (MSC) to verify the expression level and persistence of the target gene, and the results are shown below.

[0164] 2-1. Comparison of viral packaging efficiency under the influence of CAG promoter

[0165] The lentiviral packaging efficiency was compared between the vector pLenti-GIII-CMV-GFP-2A-Puro (Cat. No. LV180162, abm) containing the CMV promoter, which has been mainly used for lentiviral production in the past, and the vector containing mCMV and CAG promoters (plasmid #1).

[0166] The results are as follows Figure 6 As shown, 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 lentiviral packaging.

[0167] 2-2. Comparison of viral packaging efficiency under the influence of CAG promoter directionality and CTE

[0168] The virus packaging efficiency was compared according to the forward or reverse orientation of the CAG promoter. In addition, the effect of CTE, which is known to have a function of RNA nuclear export, on the expression of the target protein was confirmed.

[0169] like Figure 7 As shown, when the CAG promoter was in the reverse orientation (#1), the viral titer was higher than when it was in the forward orientation (#2). In addition, when CTE was included (#9 and #10), higher viral titers were shown.

[0170] 2-3. Comparison of virus packaging efficiency under the influence of CTE directionality, position and number

[0171] The lentiviral packaging efficiency was compared under the influence of the directionality, position and number of CTEs in vectors with the CAG promoter located in the reverse direction.

[0172] The results are as follows Figure 8 As shown, 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 normally (#11 to #16).

[0173] 2-4. Comparison of viral packaging efficiency under the influence of PGK promoter directionality and CTE

[0174] The directionality of the PGK promoter and the influence of CTE on viral packaging efficiency were compared in vectors containing the mCMV promoter and the PGK promoter instead of the CAG promoter.

[0175] The results are as follows Fig. 9 As shown, viral titers were constant regardless of the directionality of the PGK promoter (#3 and #4) and further increased when CTE was present (#21 and #22).

[0176] 2-5. Comparison of viral packaging efficiency between CAG promoter and PGK promoter

[0177] The lentiviral packaging efficiency under the influence of promoter and CTE directionality in vectors containing CAG promoter and vectors containing mCMV and PGK promoters was compared.

[0178] The results are as follows Fig.10 As shown, regardless of the directionality of the promoter, the inclusion of CTE increased the viral titer, particularly when CTE was in the positive direction. When the PGK promoter was included in the positive direction, the viral titer showed a maximum difference of 3 to 4 times under the influence of the presence or absence of CTE.

[0179] 2-6. Comparison of target gene expression by CAG promoter and PGK promoter according to promoter directionality

[0180] In order 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 expression rate of GFP and RFP was determined. Based on the characteristics of the bidirectional expression of the promoter, the expression rate of the same target gene is different. In the vector containing mCMV and CAG promoter, the CAG promoter part has a strong expression rate, and in the vector containing mCMV and PGK promoter, the PGK promoter part has a strong expression rate. The downstream target gene of such a promoter is named "strong site (stronger side)". The downstream target gene of the mCMV promoter in both vectors has a weak expression rate, and such a target gene is named "weak site (weaker side)".

[0181] The results are as follows Fig.11 As shown in the figure, in the case of the PGK promoter, the expression rate of the target gene at the weak site varies depending on the directionality, and in particular, when the PGK promoter is in the reverse direction, the expression rate of the target gene at the weak site is very low. However, in the case of the CAG promoter, the target gene at the weak site is expressed equally regardless of the directionality.

[0182] 2-7. Comparison of target gene expression rates under the influence of promoter and CTE directionality

[0183] Reference Fig.12 After the lentivirus is produced, it is infected with the target cells, namely mesenchymal stem cells (MSC). Fluorescence imaging for confirming the expression of the target gene (GFP or RFP) and FACS experiments for confirming the infection rate are performed at each passage.

[0184] In addition, the expression persistence of the target gene under the influence of the directionality of the promoter and CTE in the vector containing the mCMV and CAG promoter and the vector containing the mCMV and PGK promoter was compared. In the case of the reverse CAG promoter, the strong site was expressed by GFP and the weak site was expressed by the RFP gene, and in the case of the forward mCMV+CAG promoter and mCMV+PGK promoter, the strong site was expressed by RFP and the weak site was expressed by the GFP gene.

[0185] The results are as follows Fig.13 and 14 As shown, the infection rate (%) of the lentivirus containing the mCMV and PGK promoter is higher than that of the vector containing mCMV and CAG. The infection rate of each candidate vector group was measured using the target gene at the strong site, and the infection rate was also measured using the target gene at the weak site.

[0186] Fig.14 The infection rates of A and B are not the same. Even if the lentivirus is infected, when the target gene at the strong site is expressed, the target gene at the opposite part, that is, the weak site, cannot be expressed. It can be seen that the disadvantage of such a bidirectional promoter is compensated by the increase in the infection rate of the target gene at the weak site in the plasmid containing the forward and reverse CTEs.

[0187] In addition, usually, the expression rate of the target gene of the bidirectional promoter is reduced when the target cells are cultured for a long time ( Fig.14 B, #1). This shows that the expression of the target gene is increased consistently when the vector containing the inverted CAG contains the CTE ( Fig.14 B, #1 to #10), while when the vector containing the forward CAG contains CTE, in the case of the vector containing the weak mCMV+PGK, the disadvantage is only compensated when the CTE enters in the reverse direction ( Fig.14 B, #17 or #21).

[0188] Such results indicate that the candidate set of bidirectional plasmids can show various differences depending on the target cell. Considering the difference in expression rate of the target gene that is not expressed in cells such as 293T, the decrease in expression persistence, etc., it is necessary to select the best bidirectional promoter for the target cell.

[0189] 2-8. Comparison of target gene expression rates of vectors containing mCMV+CAG or mCMV+PGK in different target cells

[0190] The expression rate of the target gene may vary depending on the type of target cell. In order to determine whether the candidate lentiviral plasmid group can be applied to various target cells, it was confirmed in natural killer (NK) cells and Jurket T cells.

[0191] The results are as follows Figures 15 to 17 As shown, the infection rate of NK cells was confirmed by FACS and fluorescence expression rate.

[0192] When confirming the fluorescence expression, it was found that more cells were infected in the vector containing mCMV+PGK (#21 or #22) than in the vector containing mCMV+CAG (#9 or #10). Considering this result, it is expected that the target gene can also be expressed in NK cells when using the vector containing mCMV+PGK.

[0193] Similarly, if Figures 18 to 20 As shown, it was shown that in Jurket T cells, the expression of the target gene was higher in the case of the vector containing mCMV+CAG than in the case of the vector containing mCMV+PGK.

[0194] 2-9. Application model of target gene co-expression plasmid

[0195] In order to use plasmids that co-express two or more target genes for actual research, plasmids (#23 and #24) containing a suicide-inducing gene (third target gene) were prepared and infected into U87 cells, and the expression rate was measured.

[0196] The results are as follows Fig.21 As shown, the normal expression of the first target gene (RFP) and the second target gene (copGFP) was confirmed by infecting U87 cells and by fluorescence, and the expression of the third target gene (suicide-inducing gene, HSV-TK) was confirmed by MTT assay using Ganciclovir.

[0197] A tumor formation model was prepared by injecting U87 cells infected with such lentivirus into actual animals, and the suicide of U87 cells was induced by oral administration of valganciclovir.

[0198] The results are as follows Fig. 22 As shown, it was confirmed that the size of the tumor decreased significantly over time compared with the control group.

[0199] Such results indicate that by changing the target gene of the gene co-expression plasmid, it can be suitable for experimental and industrial groups.

[0200] 2-10. Confirmation of expression of target gene after modification

[0201] In the previous experiment, gene expression was confirmed using GFP, RFP or HSV-TK as the target gene, and the other target genes were also confirmed to achieve the same gene expression. Therefore, the first target gene was changed to HGF, the second target gene was changed to Neurogenin1, and the third target gene was changed to HSV-TK, and normal expression was confirmed.

[0202] The results are as follows Fig.23 As shown, it was confirmed that even when the target gene was changed to HGF, Ngn1, and TK, mRNA transcription occurred normally and the expression rate did not decrease with MSC passaging.

[0203] So far, the present invention has been studied around its preferred embodiments. It will be appreciated by those skilled in the art that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative perspective rather than a restrictive perspective. The scope of the present invention is shown in the claims rather than in the above description, and should be interpreted as all differences within the scope of their equivalents are included in the present invention.

Claims

1. A recombinant vector comprising CTE, a first target gene, a first promoter, a second promoter and a second target gene, The first target gene and the first promoter are in reverse orientation.

2. The recombinant vector according to claim 1, in, The recombinant vector also includes a third promoter and a third target gene.

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

2.

4. The recombinant vector according to claim 1, in, The CTE is either positive or negative.

5. The recombinant vector according to claim 1 or 2, in, The promoter is one or more selected from the group consisting of the simian virus 40 promoter, i.e., SV40 promoter, the cytomegalovirus promoter, i.e., CMV promoter, the minimal CMV promoter, the human ubiquitin C promoter, i.e., UBC promoter, the human elongation factor 1a promoter, i.e., EF1A promoter, the phosphoglycerate kinase 1 promoter, i.e., PGK promoter, and the cytomegalovirus very early enhancer / chicken β-actin promoter, i.e., CAG promoter.

6. The recombinant vector according to claim 1 or 2, in, The target gene is one or more selected from the group consisting of disease therapeutic genes, reporter genes, selection marker genes and cell marker genes.

7. The recombinant vector according to claim 6, in, The disease treating gene is one or more selected from drug sensitivity genes, apoptosis genes, cell proliferation inhibition genes, cell growth genes, cytotoxicity genes, tumor suppressor genes, antigenicity genes, cytokine genes, neurogenesis genes, anti-angiogenesis genes and hormone genes.

8. The recombinant vector according to claim 6, in, The reporter gene is selected from TdTomato, luciferase, copGFP isolated from Pontellina plumata, green fluorescent protein (GFP) isolated from Aequoreavictoria, 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). One or more.

9. The recombinant vector according to claim 6, in, The selection marker gene is one or more selected from the group consisting of β-lactamase, puromycin N-acetyltransferase, hygromycin B-phosphotransferase and aminoglycoside phosphotransferase.

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

11. The recombinant vector according to claim 1 or 2, in, At least one of the first target gene, the second target gene and the third target gene of the recombinant vector comprises a Kozak base sequence.

12. A gene delivery system comprising the 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, in, The recombinant virus is derived from a lentivirus.

15. A transformant into which the recombinant vector according to claim 1 or 2 or the recombinant virus according to claim 13 is introduced.

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

17. The transformant according to claim 16, in, The immune cells are selected from or derived from neutrophils, eosinophils, basophils, macrophages, mast cells, dendritic cells, B lymphocytes, T lymphocytes and NK cells.

18. The transformant according to claim 16, in, The mesenchymal stem cells are derived from any one selected from the group consisting of bone marrow, adipose tissue, umbilical cord blood, amnion, synovium, bone tissue and infrapatellar fat pad.

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