WO2020171269A1 - Prc1 promoter for regulating cancer cell-specific gene expression and recombinant vector containing same - Google Patents

Abstract

The present invention relates to a PRC1 promoter, a recombinant vector comprising same, and various applications thereof. The PRC1 promoter of the present invention can effectively overexpress foreign genes more specifically in cancer cells than conventionally known promoters, and thus can be used for the treatment, prevention, and diagnosis of cancer by overexpressing genes related to the treatment and death of cancer. In addition, the PRC1 promoter can be used in a method for screening an anti-cancer agent comprising the PRC1 promoter of the present invention, and also in a method for diagnosing cancer and predicting metastasis, and thus can be effectively used in related pharmaceutical and medical fields.

Description

PRC1 promoter regulating cancer cell-specific gene expression and recombinant vector comprising the same

The present invention relates to a PRC1 promoter, a recombinant vector comprising the same, and various applications thereof.

Gene therapy is a technology that seeks to treat a disease by introducing a gene with a function that can eliminate the cause of the disease directly into the patient's body. Representative examples include a method that restores the lost function by inserting a wild-species gene into a gene that causes a disease due to a loss of function due to a mutation, and a gene that can selectively kill a cell that causes a disease such as cancer is a specific target cell. There are only methods of delivery and expression. In the latter case, there have been many attempts to develop a promoter that specifically overexpresses a target gene for gene therapy only in cancer cells. Promoters that have been widely developed in this field so far include BIRC5 (baculoviral IAP repeatcontaining 5, survivin) and telomerase reverse transcriptase (TERT). It was confirmed that the two genes were expressed specifically for cancer, but it was reported that TERT has strong cancer specificity but weak transcriptional activity. Therefore, to compensate for this, a modified promoter was developed in which the binding nucleotide sequence of HIF-1α (Hypoxiainducible factor 1), a transcription factor activated in an anoxic state, which is a specific environment of cancer cells, is inserted into the TERT promoter (Shin et al., Oncology Reports, 10:2063, 2003; Yin et al., J. Lab. Cli. Med., 144:302, 2004).

The protein regulator of cytokinesis 1 (PRC1) is a protein that binds to a spindle essential for cytokinesis in mammalian cells, and its expression is reported to be inhibited by the p53 protein ( Li et al., Oncogene, 23:9336, 2004). Considering that p53 inhibits cancer and loses normal function beyond expression of p53 in the majority of cancer cells, these reports suggest that PRC1 may be overexpressed in cancer cells. In this regard, the use of PRC1 as a diagnostic marker for pancreatic cancer has been previously reported (WO 2004/078205A1). In addition, cancer cell-specific expression vector containing the promoter of the PRC1 gene and the gene for killing cancer cells, the promoter of the ribonucleotide reductase subunit 2 (RRM2) gene and the specific expression of cancer cells containing the gene for killing cancer cells A vector and a cancer cell-specific expression vector further containing an ER binding sequence (estrogen response element, ERE) to which an estrogen receptor (ER), a transcription factor frequently expressed in breast cancer cells, binds to the expression vector has been reported. Yes (Korean Patent Registration No. 10-0799626). However, the previously developed cancer cell-specific expression vectors have poor expression capacity in cancer cells, so there is a need to improve them.

An object of the present invention is to provide a protein regulator of cytokinesis 1 (PRC1) promoter.

An object of the present invention is to provide a recombinant expression vector in which a foreign gene is operably linked to the promoter.

An object of the present invention is to provide a transformant transformed with the recombinant expression vector.

An object of the present invention is to provide a pharmaceutical composition for anticancer.

An object of the present invention is to provide a composition for diagnosis of cancer.

An object of the present invention is to provide a kit for diagnosis of cancer.

An object of the present invention is to provide an anticancer agent screening method.

An object of the present invention is to provide a method of providing information for diagnosing cancer or predicting the risk of metastasis of cancer.

An object of the present invention is to provide a method for preventing and treating cancer comprising administering to an individual a recombinant expression vector in which a foreign gene is operably linked to the above promoter in a pharmaceutically effective amount.

In order to achieve the above object, the present invention binds one or more transcription factors selected to the group consisting of LCR-F1, Nkx2-5, ARP-1, STAT3, N-Myc, POU2F1, CART-1, E2F-1 and E47 Provides a protein regulator of cytokinesis 1 (PRC1) promoter containing the site.

In addition, the present invention provides a recombinant expression vector in which a foreign gene is operably linked to the promoter.

In addition, the present invention provides a transformant transformed with the recombinant expression vector.

In addition, the present invention provides a pharmaceutical composition for anticancer, comprising a recombinant expression vector operably linked to the promoter to the foreign gene as an active ingredient.

In addition, the present invention provides a composition for diagnosis of cancer, comprising a recombinant expression vector operably linked to the promoter with a foreign gene.

In addition, the present invention provides a kit for diagnosis of cancer comprising a recombinant expression vector operably linked to the promoter with a foreign gene.

In addition, the present invention comprises the steps of (1) treating and culturing a test substance on the transformant; And (2) a foreign gene in the cultured transformant of step (1); Or it provides an anticancer agent screening method comprising; measuring the expression or inhibition of the protein encoded by the gene.

In addition, the present invention includes the steps of: (1) infecting a biological sample and a control sample of a patient suspected of cancer with a recombinant expression vector operably linked to the promoter with a foreign gene; And (2) comparing the foreign gene expression level of the biological sample of the patient and the control sample of the step (1); It provides a method for providing information for diagnosis of cancer or predicting the risk of metastasis of cancer comprising.

In addition, the present invention provides a method for preventing and treating cancer comprising administering to an individual a recombinant expression vector operably linked to a foreign gene to the promoter in a pharmaceutically effective amount.

The PRC1 promoter of the present invention can effectively overexpress a foreign gene in cancer cells more effectively than a conventionally known promoter, so it can be used for treatment, prevention, and diagnosis of cancer related to it by overexpressing genes related to the treatment and death of cancer. . In addition, it can be used for an anticancer agent screening method including the PRC1 promoter of the present invention, a method for diagnosing cancer and predicting metastasis, and thus can be usefully used in related fields of pharmacy and medicine.

1 shows the entire PRC1 gene promoter (Full), a conventional promoter fragment (M3) as a control, and the novel M4 + STAT3 promoter fragment of the present invention, M3 + N-Myc promoter fragment, M3 + N-Myc + PU2F1 promoter It is a diagram schematically showing the fragment and the M2 + CART-1 promoter fragment.

2 is a diagram showing the results of measuring the activity of luciferase after introducing a recombinant vector expressing each promoter into a normal lung cell (pneumocyte) by an infection method.

3 is a diagram showing the results of measuring the activity of luciferase after introducing a recombinant vector expressing each promoter into an A549 lung cancer cell line (ATCC, CCL-185) by an infection method.

The present invention is a cytokinesis comprising at least one transcription factor binding site selected from the group consisting of LCR-F1, Nkx2-5, ARP-1, STAT3, N-Myc, POU2F1, CART-1, E2F-1 and E47 It provides a protein regulator of cytokinesis 1 (PRC1) promoter.

In the present invention, the term "transcription factor" refers to a transcriptional regulatory protein that specifically binds to the transcriptional regulatory region DNA of a specific gene and activates or inhibits the transcription of the gene, and the activity of RNA polymerase Control gene transcription. In the present invention, the transcription factor binding site (transcription factor binding site) is mainly expressed in cancer LCR-F1, Nkx2-5, ARP-1, STAT3, N-Myc, POU2F1, CART-1, E2F-1 and E47 The transcription factors were classified, and a binding site capable of binding the transcription factors to the PRC1 promoter was searched for.

In addition, each of the transcription factor binding sites of LCR-F1, Nkx2-5, ARP-1, STAT3, N-Myc, POU2F1, CART-1, E2F-1 and E47 of the present invention may have multiple and consensus sequences. However, preferably, each GCCTGC (SEQ ID NO: 2), GCGGCAG (SEQ ID NO: 3), TGCCGGGAA (SEQ ID NO: 4), CACGTTAT (SEQ ID NO: 5), ATGCTTAT (SEQ ID NO: 6), TAATTA (SEQ ID NO: 7), TTAATTG (SEQ ID NO: 8), CAGGTG (SEQ ID NO: 9) and GGGATTA (SEQ ID NO: 10), but are not limited thereto.

The promoter is from 700 to 1500 bp of the full length PRC1 promoter represented by SEQ ID NO: 1.

The promoter is a nucleotide sequence represented by one or more selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, preferably SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or a nucleotide sequence represented by SEQ ID NO: 27, more preferably a nucleotide sequence represented by SEQ ID NO: 26 or a promoter sequence for achieving the purpose of specifically overexpressing a foreign gene in cancer cells, limited thereto. It doesn't work.

The nucleotide sequence of the present invention includes functional equivalents thereof that perform functionally the same function as these, and the functional equivalents include deletion, substitution, and insertion of some nucleotide sequences due to artificial modification. Or it may include variants modified by a combination thereof or functional fragments of the nucleotide sequence performing the same action.

Those skilled in the art will fall within the scope of the present invention as an equivalent to the nucleotide sequence of the present invention as long as the nucleotide sequence that maintains 70% or more homology by such artificial modification expresses the target gene in the present invention. You will be able to understand easily.

The promoter is one or more transcription factors selected from the group consisting of LCR-F1, Nkx2-5, ARP-1, STAT3, N-Myc, POU2F1, CART-1, E2F-1 and E47 that regulate the expression of the PRC1 gene and However, as long as it binds to achieve the purpose of specifically overexpressing a foreign gene in cancer cells, it is not limited thereto.

The promoter specifically overexpresses a foreign gene in cancer cells, and the foreign gene is a cancer cell death gene, a fluorescent protein gene, a cancer cell suppressor gene, an antigenic gene, a cytotoxic gene, a cell proliferation inhibitory gene, a cytokine gene, a parent- It may include at least one gene selected from the group consisting of a pro-apoptotic gene and an anti-angiogenic gene.

The cancer cell death gene is a BCL-2 lineage pro-apoptotic gene or a suicide receptor/ligand gene, and the BCL-2 (B-cell leukemia/lymphoma 2) lineage cell death promotion ( pro-apoptotic) gene may include Bax (BCL2-associated X) or Bad (BCL2-antagonist of cell death). In addition, the suicide receptor/ligand gene is a tumor necrosis factor-α, TNF-α or a Fas ligand (FasL), or a target that can be specifically overexpressed in cancer cells. If it is a cancer cell death gene to achieve, it is not limited thereto.

The fluorescent protein gene is luciferase, enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein. (Red Fluorescent Protein, RFP) and blue fluorescent protein (cyan fluorescent protein, CFP) one or more selected from the group consisting of, but is not limited thereto.

The cancer cell suppressor gene is p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, MMAC-1 gene, MMSC-2 gene, NF-1 gene, MTS1 Gene, CDK4 gene, NF-1 gene, NF-2 gene, and at least one selected from the group consisting of VHL gene, and if it is a cancer cell suppressor gene for achieving the purpose of being specifically overexpressed in cancer cells, it is not limited thereto. Does not.

In addition, in the present invention, the term "antigenic gene" refers to a nucleotide sequence that is expressed in a target cell to produce a cell surface antigenic protein that can be recognized by the immune system. Examples of such antigenic genes include carcinoembryonic antigen (CEA) or p53 (Levine, A., International Patent Application Publication No. WO 94/02167). In order for the immune system to readily recognize, the antigenic gene can be bound to the MHC type I antigen.

In addition, in the present invention, the term "cytotoxic gene" refers to a nucleotide sequence that is expressed in cells and exhibits a toxic effect. Examples of such cytotoxic genes include nucleotide sequences encoding Pseudomonas exotoxin, lysine toxin, diphtheria toxin, and the like.

In addition, in the present invention, the term "cytostatic gene" refers to a nucleotide sequence that is expressed in a cell to stop the cell cycle during the cell cycle. Examples of such cell proliferation inhibitory genes include p21, retinoblastoma gene, E2F-Rb fusion protein gene, genes encoding cyclin-dependent kinase inhibitors (e.g., p16, p15, p18 and p19), growth arrest specific homeo Box (growth arrest specific homeobox, GAX) gene (International Patent Application Publication No. WO 97/16459 and WO 96/30385), and the like, but are not limited thereto.

In addition, in the present invention, the term "cytokine gene" refers to a nucleotide sequence that is expressed in a cell to generate a cytokine. Examples of such cytokines include GM-CSF, interleukin (especially IL-1, IL-2, IL-4, IL-12, IL-10, IL-19, IL-20), interferon α, β, γ (In particular, interferon α-2b) and fusions such as interferon α-2α-1 are included.

In addition, in the present invention, the term "pro-apoptotic gene" refers to a nucleotide sequence that is expressed and induces programmed cell death. Examples of such pro-apoptotic 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 caspase encoding. It includes genes that do.

In addition, in the present invention, the term "anti-angiogenic gene" refers to a nucleotide sequence that is expressed and releases an anti-angiogenic factor to the outside of the cell. Anti-angiogenic factors include angiostatin, an inhibitor of vascular endothelial growth factor (VEGF) such as Tie 2 (PNAS (USA), 1998, 95,8795-800), endostatin, and the like.

The cancer is lung cancer, blood cancer, colon cancer, rectal cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, fallopian tube carcinoma, ovarian cancer, vaginal carcinoma, vulvar carcinoma, liver cancer, gastric cancer, esophageal cancer, small intestine cancer, pancreatic cancer, gallbladder cancer, kidney Cancer, bladder cancer, urethral cancer, penile cancer, prostate cancer, testicular cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, non-small cell lung cancer, bone cancer, skin cancer, head or neck cancer, melanoma in the skin or eye, Hodgkin's disease, Endocrine adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, central nervous system tumor, spinal cord tumor, brainstem glioma and pituitary adenoma, but is not limited to one or more selected from the group consisting of.

In addition, the present invention provides a recombinant expression vector in which a foreign gene is operably linked to the promoter.

Since the foreign gene includes a foreign gene overexpressed by the above-described promoter, the description thereof is omitted in order to avoid excessive complexity of the present specification due to the description of duplicate contents.

In the present invention, the term "vector" refers to a DNA preparation containing a polynucleotide of a gene operably linked to a suitable regulatory sequence so that the desired gene can be expressed in a suitable host, and the regulatory sequence can initiate transcription. Promoters, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate termination of transcription and translation.

In the present invention, "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the coding sequence. The operative linkage with the recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage use enzymes generally known in the art.

The vector of the present invention may include a promoter, an initiation codon, an end codon, an expression control element such as a polyadenylation signal and an enhancer, a secretion signal, and the like, and may be variously prepared according to the purpose. The start and stop codons must exhibit an action in the subject when the genetic construct is administered and must be in frame with the coding sequence.

The vector used in the present invention is not particularly limited as long as it is replicable among hosts, and any vector known in the art may be used. For example, non-viral vectors or viral vectors can be used.

Plasmids are representative of the non-viral vectors. Plasmid expression vectors are a method of delivering plasmid DNA directly to human cells by an FDA approved gene delivery method that can be used in humans, and plasmid DNA has the advantage of being homogeneously purified unlike viral vectors. As a plasmid expression vector that can be used in the present invention, a mammalian expression plasmid known in the art can be used. For example, although not limited thereto, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91/08291), and pVL1392 (PharMingen) are representative.

In addition, a viral vector can be used as the expression vector of the present invention. Viral vectors include, for example, retrovirus, adenovirus, adenovirus-associated virus, herpes virus, and abipoxvirus. The viral vector must meet the following criteria: (1) it must be able to infect the cell of interest, and accordingly, a viral vector with an appropriate host range must be selected, and (2) the transferred gene must be preserved in the cell for an appropriate period of time. And be able to be expressed, and (3) the vector is safe for the host.

The retroviral vector is constructed so that all of the viral genes have been removed or altered so that a non-viral protein is made in the infected cells by the viral vector. The main advantages of retroviral vectors for gene therapy are that they deliver large amounts of genes into cloned cells, accurately integrate the transferred genes into cellular DNA, and do not cause continuous infection after gene transfection. The retroviral vector certified by the FDA was prepared using PA317 amphoteric retrovirus packaged cells (Miller, A.D. and Buttimore, C., Molec. Cell Biol., 6:2895-2902, 1986).

Non-retroviral vectors include adenoviruses as mentioned above. The main advantage of adenoviruses is that they carry large amounts of DNA fragments (36 kb genome) and have the ability to infect non-replicating cells with very high titers. In addition, Herpis virus can also be usefully used for human gene therapy (Wolfe, J.H., et al., Nature Genetics, 1:379-384, 1992). In addition, known suitable viral vectors can be used.

Other viral vectors that can be used for gene transfer into cells include murine leukemia virus (MLV), JC, SV40, polyoma, Epstein-Barr virus papilloma virus, vaccinia, poliovirus, herpes virus, Sindbis virus, and Lenti. Viruses and other human and animal viruses may be included.

The expression vector according to the present invention can be introduced into cells using a method known in the art. For example, but not limited thereto, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known for introducing nucleic acids into cells It can be introduced into cells by the method of (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988). .

In addition, the vector of the present invention may further include a selection marker (selection market). Selectable markers are used to select cells transformed with a vector, that is, to confirm the insertion of a target gene, and give a selectable phenotype such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface proteins. Markers can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit different phenotypic traits, and thus transformed cells can be selected.

In addition, the present invention provides a transformant transformed with the recombinant expression vector.

The transformant of the present invention can be constructed by introducing a vector into a host cell in a manner in which a promoter can act.

In the present invention, the "transformation" means that DNA is introduced into a host so that DNA can be replicated as an extrachromosomal factor or by chromosomal integration completion. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and as known in the art can be performed by selecting a suitable standard technique according to the host cell. These methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and acetic acid. Lithium-DMSO method and the like may be included, but are not limited thereto.

Depending on the host cell transformed with the expression vector, the expression level and modification of the protein are different, so you can select and use the most suitable host cell for the purpose. Host cells that can be used in the present invention may be insect cell lines, yeast, fungi, bacteria or algae.

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In addition, the present invention provides a pharmaceutical composition for anticancer, comprising a recombinant expression vector operably linked to the promoter to the foreign gene as an active ingredient.

Since the foreign gene includes a foreign gene overexpressed by the above-described promoter, the description thereof is omitted in order to avoid excessive complexity of the present specification due to the description of duplicate contents.

The pharmaceutical composition of the present invention may further include an adjuvant. Any of the adjuvants known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase its immunity.

The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated in a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited thereto, but lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. .

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, in the active ingredient. It can be prepared by mixing and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and other excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used diluents. I can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual. Serine degrading enzyme inhibitory peptide contained in the pharmaceutical composition; Alternatively, it is obvious that the concentration of the expression vector can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. When the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg/ml, toxicity to humans may occur.

In addition, the present invention provides a composition for diagnosis of cancer, comprising a recombinant expression vector operably linked to the promoter with a foreign gene.

Since the foreign gene includes a foreign gene overexpressed by the above-described promoter, the description thereof is omitted in order to avoid excessive complexity of the present specification due to the description of duplicate contents.

In the present invention, "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether or not cancer has developed or is likely to develop.

In addition, the present invention provides a kit for diagnosis of cancer comprising a recombinant expression vector operably linked to the promoter with a foreign gene.

Since the foreign gene includes a foreign gene overexpressed by the above-described promoter, the description thereof is omitted in order to avoid excessive complexity of the present specification due to the description of duplicate contents.

In the present invention, the term "kit" refers to a tool capable of diagnosing the presence of cancer, and the PRC1 promoter of the present invention effectively overexpresses a foreign gene in cancer cells, and thus, for example, a fluorescent protein gene as a foreign gene. When used, cancer can be diagnosed according to the expression of the fluorescent protein gene. The cancer diagnostic kit of the present invention may contain one or more other component compositions, solutions, or devices suitable for analysis methods.

In addition, the present invention comprises the steps of (1) treating and culturing a test substance on the transformant; And (2) a foreign gene in the cultured transformant of step (1); Or it provides an anticancer agent screening method comprising; measuring the expression or inhibition of the protein encoded by the gene.

Since the PRC1 promoter of the present invention effectively overexpresses a foreign gene specifically in cancer cells, for example, when a fluorescent protein gene is used as a foreign gene, it can be determined that the effect as an anticancer agent is high when the expression of the fluorescent protein gene is decreased. However, when the fluorescent protein gene is expressed, it can be determined that the effect as an anticancer agent is low.

The measurement in step (2) may be measured using two-dimensional electrophoresis, western blot, RT-PCR, real-time PCR, or a biochip array, and the biochip is preferably a protein chip or a nucleic acid array. In addition, methods for measuring using antibodies that can specifically bind to proteins include Western blot, ELISA (enzymelinked immunosorbent assay), colorimetric method, electrochemical method, fluorescence method, and luminescence. It may be at least one selected from the group consisting of a luminometry, a particle counting method, a visual assessment, and a scintillation counting method.

In addition, the present invention includes the steps of: (1) infecting a biological sample and a control sample of a patient suspected of cancer with a recombinant expression vector operably linked to the promoter with a foreign gene; And (2) comparing the foreign gene expression level of the biological sample of the patient and the control sample of the step (1); It provides a method for providing information for diagnosis of cancer or predicting the risk of metastasis of cancer comprising.

Since the PRC1 promoter of the present invention effectively overexpresses foreign genes specifically in cancer cells, for example, when a fluorescent protein gene is used as a foreign gene, it can be predicted that the possibility of cancer or cancer metastasis is low when the expression of the fluorescent protein gene is decreased. However, it can be predicted that the possibility of cancer or cancer metastasis is high when the fluorescent protein gene is expressed.

The biological sample may be any biological sample in which target cells for diagnosis of cancer or prediction of cancer metastasis risk may exist, for example, a biopsy sample, a tissue sample, a cell suspension in which the isolated cells are suspended in a liquid medium , Cell cultures and combinations thereof may be selected from the group consisting of. The sample may be at least one selected from the group consisting of blood, bone marrow fluid, saliva, tear fluid, urine, semen, and mucous membrane fluid.

In the present invention, the term "predicting the risk of metastasis" means to determine in advance the possibility of metastasis to tissues other than the tissue in which the cancer has occurred. More preferably, the metastasis risk prediction in the present invention means that a patient with cancer metastasis receives cancer treatment using surgical therapy, radiation therapy, chemotherapy, etc., and then determines the possibility of recurrence and metastasis in the treated tissue in advance. Can mean that. In another aspect, in the present invention, metastasis risk prediction may mean preliminarily determining the possibility of metastasis of a cancer patient by diagnosing a cancer sample that has not been metastasized and a sample of a patient at risk of metastasis.

In addition, the present invention provides a pharmaceutical composition for anticancer, comprising a recombinant expression vector operably linked to the promoter to the foreign gene as an active ingredient.

Since the foreign gene includes a foreign gene overexpressed by the above-described promoter, the description thereof is omitted in order to avoid excessive complexity of the present specification due to the description of duplicate contents.

The pharmaceutical composition of the present invention may further include an adjuvant. Any of the adjuvants known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase its immunity.

The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated in a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited thereto, but lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. .

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, in the active ingredient. It can be prepared by mixing and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and other excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used diluents. I can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual. Serine degrading enzyme inhibitory peptide contained in the pharmaceutical composition; Alternatively, it is obvious that the concentration of the expression vector can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. When the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg/ml, toxicity to humans may occur.

In addition, the present invention provides a composition for diagnosis of cancer, comprising a recombinant expression vector operably linked to the promoter with a foreign gene.

Since the foreign gene includes a foreign gene overexpressed by the above-described promoter, the description thereof is omitted in order to avoid excessive complexity of the present specification due to the description of duplicate contents.

In the present invention, "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether or not cancer has developed or is likely to develop.

In addition, the present invention provides a kit for diagnosis of cancer comprising a recombinant expression vector operably linked to the promoter with a foreign gene.

Since the foreign gene includes a foreign gene overexpressed by the above-described promoter, the description thereof is omitted in order to avoid excessive complexity of the present specification due to the description of duplicate contents.

In the present invention, the term "kit" refers to a tool capable of diagnosing the presence of cancer, and the PRC1 promoter of the present invention effectively overexpresses a foreign gene in cancer cells, and thus, for example, a fluorescent protein gene as a foreign gene. When used, cancer can be diagnosed according to the expression of the fluorescent protein gene. The cancer diagnostic kit of the present invention may contain one or more other component compositions, solutions, or devices suitable for analysis methods.

In addition, the present invention comprises the steps of (1) treating and culturing a test substance on the transformant; And (2) a foreign gene in the cultured transformant of step (1); Or it provides an anticancer agent screening method comprising; measuring the expression or inhibition of the protein encoded by the gene.

Since the PRC1 promoter of the present invention effectively overexpresses a foreign gene specifically in cancer cells, for example, when a fluorescent protein gene is used as a foreign gene, it can be determined that the effect as an anticancer agent is high when the expression of the fluorescent protein gene is decreased. However, when the fluorescent protein gene is expressed, it can be determined that the effect as an anticancer agent is low.

The measurement in step (2) may be measured using two-dimensional electrophoresis, western blot, RT-PCR, real-time PCR, or a biochip array, and the biochip is preferably a protein chip or a nucleic acid array. In addition, methods for measuring using antibodies that can specifically bind to proteins include Western blot, ELISA (enzymelinked immunosorbent assay), colorimetric method, electrochemical method, fluorescence method, and luminescence. It may be at least one selected from the group consisting of a luminometry, a particle counting method, a visual assessment, and a scintillation counting method.

In addition, the present invention includes the steps of: (1) infecting a biological sample and a control sample of a patient suspected of cancer with a recombinant expression vector operably linked to the promoter with a foreign gene; And (2) comparing the foreign gene expression level of the biological sample of the patient and the control sample of the step (1); It provides a method for providing information for diagnosis of cancer or predicting the risk of metastasis of cancer comprising.

Since the PRC1 promoter of the present invention effectively overexpresses foreign genes specifically in cancer cells, for example, when a fluorescent protein gene is used as a foreign gene, it can be predicted that the possibility of cancer or cancer metastasis is low when the expression of the fluorescent protein gene is decreased. However, it can be predicted that the possibility of cancer or cancer metastasis is high when the fluorescent protein gene is expressed.

The biological sample may be any biological sample in which target cells for diagnosis of cancer or prediction of cancer metastasis risk may exist, for example, a biopsy sample, a tissue sample, a cell suspension in which the isolated cells are suspended in a liquid medium , Cell cultures and combinations thereof may be selected from the group consisting of. The sample may be at least one selected from the group consisting of blood, bone marrow fluid, saliva, tear fluid, urine, semen, and mucous membrane fluid.

In the present invention, the term "predicting the risk of metastasis" means to determine in advance the possibility of metastasis to tissues other than the tissue in which the cancer has occurred. More preferably, the metastasis risk prediction in the present invention means that a patient with cancer metastasis receives cancer treatment using surgical therapy, radiation therapy, chemotherapy, etc., and then determines the possibility of recurrence and metastasis in the treated tissue in advance. Can mean that. In another aspect, in the present invention, metastasis risk prediction may mean preliminarily determining the possibility of metastasis of a cancer patient by diagnosing a cancer sample that has not been metastasized and a sample of a patient at risk of metastasis.

In addition, the present invention provides a method for preventing and treating cancer comprising administering to an individual a recombinant expression vector operably linked to a foreign gene to the promoter in a pharmaceutically effective amount.

The pharmaceutical composition of the present invention is administered in a therapeutically effective amount or in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, age, sex, activity of the drug, and Sensitivity, administration time, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention and the present invention is not limited thereto.

Example 1. Classification of cancer expression transcription factors and identification of transcription factor control site sequences

LCR-F1, E2F-1, STAT3, N-Myc, POU2F1, CART-1, Nkx2-5, E47 and ARP-1, which are transcription factors mainly expressed in cancer, were classified. Then, the sequence position of each transcription factor control site that can bind on the protein regulator of cytokinesis 1 (PRC1) gene promoter, wherein each of the transcription factors is specifically overexpressed in cancer cells, is determined in the JARSPAR database. It was searched using. As a result, transcription factors related to cancer inhibition are LCR-F1, Nkx2-5 and ARP-1, transcription factors related to cancer expression are STAT3, N-Myc, POU2F1 and CART-1, and Controversal transcription factors are It was confirmed that they were E2F-1 and E47. Each of the transcription factors was identified on the sequence of the human PRC1 gene (NCBI GenBank AC068831), as shown in Table 1 below. In addition, the entire sequence of the PRC1 promoter corresponding to 83254-85000bp among the sequences of the PRC1 gene is shown in SEQ ID NO: 1.

[Table 1]

Example 2. Construction of a novel PRC1 gene promoter segment

A portion of the PRC1 gene promoter upstream region was deleted, and a novel PRC1 gene promoter fragment including each transcription factor regulatory region identified in Example 1 was constructed.

Specifically, in the prior invention (Korean Patent Registration No. 10-1204895), the promoter fragment of the PRC1 gene promoter (83254-85000bp in NCBI GenBank AC068831, SEQ ID NO: 1) from which part of the upstream has been deleted [(84178-83254 bp in NCBI GenBank AC068831) , M2); [(Corresponding to 83818-83254 bp in NCBI GenBank AC068831, M3); (83716-83254 bp corresponding in NCBI GenBank AC068831, M4)] was confirmed. In order to produce a more improved promoter fragment from the conventional promoter fragment, the transcription factor regulatory site searched for in Example 1 was included and the restriction enzyme recognition sequences of XmeI and NcoI were included. A promoter segment (Full) comprising the entire PRC1 gene promoter sequence (SEQ ID NO: 1); And M3, which is the most active in the conventional invention, was set as a control. The novel PRC1 gene promoter fragment of the present invention includes a conventional promoter fragment (M4) and a promoter fragment (M4 + STAT3) comprising a STAT3 transcription factor regulatory site sequence; A promoter segment (M3 + N-Myc) comprising a conventional promoter segment (M3) and an N-Myc transcription factor regulatory region sequence; A conventional promoter fragment (M3) and a promoter fragment (M3 + N-Myc + PU2F1) comprising a sequence of a N-Myc, PU2F1 transcription factor regulatory site; Or a conventional promoter fragment (M2) and a promoter fragment (M2 + CART-1) comprising a CART-1 transcription factor regulatory site sequence; a total of four new fragments were prepared. The primers for the two control groups and four new fragments are shown in Table 2 below (the underline of Forward indicates Xma1, and the underline of Reverse indicates Nco1). The primers were amplified under PCR conditions of 94° 1 minute, 58° 1 minute, 72° 1 minute and 25 cycles, and the sizes of each promoter segment are shown in Table 2 below. In addition, the nucleotide sequence of the entire PRC1 gene promoter (Full) is SEQ ID NO: 23, the M4 + STAT3 promoter fragment is SEQ ID NO: 24, the M3 + N-Myc promoter fragment is SEQ ID NO: 25, M3 + N-Myc + PU2F1 promoter The fragment is shown in SEQ ID NO: 26, the M2 + CART-1 promoter fragment is shown in SEQ ID NO: 27, and the conventional promoter fragment (M3) is shown in SEQ ID NO: 28. A schematic diagram thereof is shown in FIG. 1.

[Table 2]

Example 3. Construction of a recombinant vector containing a novel PRC1 promoter segment

A recombinant vector containing the novel PRC1 promoter fragment prepared in Example 2 was constructed.

Specifically, two promoter fragments as controls and four novel PRC1 promoter fragments were cloned into a luciferase reporter vector (pBV-Luc, Addgene, 16539). The vector was cut with XmaI/NcoI, and the insertion of each fragment of Example 2 amplified through a primer with each enzyme site attached to the end (see Table 2) was ligated using a ligase (NEB, USA cat #M0202). .

As a result, a recombinant vector (rBV_Full_luc) containing the entire PRC1 gene promoter (Full), a recombinant vector containing a conventional promoter segment (M3) (rBV_M3_luc), M4 + a recombinant vector containing the STAT3 promoter segment (rBV_M4_STAT3_luc), M3 + Recombinant vector containing the N-Myc promoter fragment (rBV_ M3_N-Myc_luc), a recombinant vector containing the M3 + N-Myc + PU2F1 promoter fragment (rBV_ M3_N-Myc_PU2F1_luc) and a recombinant vector containing M2 + CART-1 promoter fragment (rBV_M2_CART-1_luc) was prepared.

Example 4. of a vector containing a novel PRC1 gene promoter segment in vitro Luciferase activity measurement

Each recombinant vector prepared in Example 3 was introduced into A549 lung cancer cell lines (ATCC, CCL-185) and lung normal cells (pneumocyte) by an infection method to measure the activity of luciferase, thereby analyzing the novel promoter activity of the present invention. I did.

4-1. Preparation of lung normal cells (pneumocyte)

Specifically, in order to prepare normal cells in the lungs, after anesthetizing the animal, a syringe was intubated into a place where the chest was cut and the lungs were ventilated. A catheter was inserted into the right ventricle of the heart and the lungs were washed with 0.15 M NaCl solution. Thereafter, the lungs were placed in a digestion buffer containing 0.25% trypsin (142 mM NaCl, 6.7 mM KCl, 10 mM Hepes, 1.29 mM MgSO ₄ , 89 mM CaCl ₂ and 1 mg/mL glucose adjusted to pH 7.4) at 37 degrees. Treated for 20 minutes. Then, the lung tissue was chopped and shaken to add FCS to stop the enzyme reaction. After filtration through two polyamide nets (150 μM and 30 μM mesh width), the cell suspension was separated into a discontinuous Percoll gradient (hard gradient 1.040, medium gradient 1.089). The cells precipitated in two gradients were collected and plated in a 90 mm Petri dish for 1 hour at 37 degrees so that the remaining macrophages adhere to the plate. Thereafter, after obtaining normal lung cells (Pneumocytes), the activity of the cells was measured by a trypan blue exclusion technique, and the normal lung cells were inoculated at 2.5 × 10 ⁵ cells per 35 mm Petri dish. After 5 days, normal lung cells were used.

4-2. Measurement of the activity of luciferase

A549 lung cancer cell line (ATCC, CCL-185) and normal lung cells were plated on a 12-well plate at a specified number (2.5 × 10 ⁵ cells/well), and then each vector prepared in Example 3 was prepared with lipofectamine 3000 (Invitrogen , USA) was used for transfection. After incubation for an additional 24 hours, luciferase activity was measured with a luciferase assay kit (Promega, E1500). The results are shown in FIGS. 2 and 3.

As shown in Figure 2, in normal lung cells (pneumocyte), it was confirmed that the promoter activity of the recombinant vector including the novel promoter fragment of the present invention, including the recombinant vector containing the conventional promoter fragment (M3) as a control, is generally low. Thus, it was confirmed that in normal cells, the PRC1 promoter, which specifically induces overexpression in cancer cells, did not exhibit high activity, and thus was not expressed in cells other than cancer cells.

As shown in Figure 3, the expression of luciferase in the recombinant vector (rBV_ M3_N-Myc_PU2F1_luc) containing the M3 + N-Myc + PU2F1 promoter fragment of the present invention in the A549 lung cancer cell line is conventionally characterized by including the promoter fragment (M3). It was confirmed that the expression of luciferase in the recombinant vector (rBV_M3_luc) was increased by about 2.5 times or more, and it was confirmed to be superior to the conventional promoter. In addition, it was confirmed that the activity of the recombinant vector (rBV_ M3_N-Myc_PU2F1_luc) containing the M3 + N-Myc + PU2F1 promoter fragment of the present invention is about 10 times higher than that of the PRC1 gene full promoter (Full). Therefore, it was confirmed that the novel PRC1 promoter of the present invention effectively regulates overexpression of foreign genes.

Accordingly, it was confirmed that the specific strong or low expression of the foreign gene in cancer cells was due to deletion and selection of partial sequences of the entire promoter of the PRC1 gene. Accordingly, it was confirmed that the M3 + N-Myc + PU2F1 promoter of the present invention can effectively regulate foreign genes by specifically overexpressing foreign genes only in cancer cells.

Claims (18)

1. LCR-F1 (Nuclear factor erythroid 2-related factor 1), Nkx2-5 (NK2 homeobox 5), ARP-1 (arginine rich protein-1), STAT3 (Signal transducer and activator of transcription 3), N-Myc (N -myc proto-oncogene protein), POU2F1 (POU domain, class 2, transcription factor 1), CART-1 (Cocaine and amphetamine regulated transcript-1), E2F-1 (E2F transcription factor 1) and E47 (fucosyltransferase) Protein regulator of cytokinesis 1 (PRC1) promoter comprising at least one transcription factor binding site selected from the group.
2. The method of claim 1, wherein the promoter is 700 to 1500 bp of a full-length PRC1 promoter represented by SEQ ID NO: 1 Characterized in that the deletion, PRC1 promoter.
3. The PRC1 promoter according to claim 1, wherein the promoter is a nucleotide sequence represented by at least one selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
4. The method of claim 1, wherein the promoter is selected from the group consisting of LCR-F1, Nkx2-5, ARP-1, STAT3, N-Myc, POU2F1, CART-1, E2F-1 and E47 that regulate the expression of the PRC1 gene. PRC1 promoter, characterized in that binding to one or more kinds of transcription factors.
5. The PRC1 promoter according to claim 1, wherein the promoter specifically overexpresses a foreign gene in cancer cells.
6. The method of claim 5, wherein the foreign gene is a cancer cell death gene, a fluorescent protein gene, a cancer cell suppressor gene, an antigenic gene, a cytotoxic gene, a cell proliferation inhibitory gene, a cytokine gene, a pro-apoptotic gene. ) And anti-angiogenic gene (anti-angiogenic gene), characterized in that at least one gene selected from the group consisting of, PRC1 promoter.
7. The PRC1 promoter according to claim 6, wherein the cancer cell death gene is a BCL-2 lineage pro-apoptotic gene or a suicide receptor/ligand gene.
8. The method of claim 6, wherein the fluorescent protein gene is luciferase, enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), yellow fluorescent protein, YFP), red fluorescent protein (Red Fluorescent Protein, RFP), and blue fluorescent protein (cyan fluorescent protein, CFP), characterized in that at least one selected from the group consisting of, PRC1 promoter.
9. The method of claim 6, wherein the cancer cell suppressor gene is a p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, MMAC-1 gene, MMSC-2 gene, PRC1 promoter, characterized in that at least one selected from the group consisting of NF-1 gene, MTS1 gene, CDK4 gene, NF-1 gene, NF-2 gene and VHL gene.
10. The method of claim 6, wherein the cancer is lung cancer, blood cancer, colon cancer, rectal cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, fallopian tube carcinoma, ovarian cancer, vaginal carcinoma, vulvar carcinoma, liver cancer, gastric cancer, esophageal cancer, small intestine cancer. , Pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, urethral cancer, penile cancer, prostate cancer, testicular cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, non-small cell lung cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular blackness Species, Hodgkin's disease, endocrine adenocarcinoma, chronic or acute leukemia, lymphocyte lymphoma, central nervous system tumor, spinal cord tumor, brainstem glioma and pituitary adenoma, characterized in that at least one selected from the group consisting of, PRC1 promoter.
11. A recombinant expression vector in which a foreign gene is operably linked to the promoter of claim 1.
12. A transformant transformed with the recombinant expression vector of claim 11.
13. A pharmaceutical composition for anticancer, comprising a recombinant expression vector operably linked to the promoter of claim 1 as an active ingredient.
14. A composition for diagnosis of cancer comprising a recombinant expression vector in which a foreign gene is operably linked to the promoter of claim 1.
15. A kit for cancer diagnosis comprising a recombinant expression vector operably linked to the promoter of claim 1.
16. (1) treating and culturing the test material on the transformant of claim 12; And

    (2) a foreign gene in the cultured transformant of step (1); Or measuring the expression or inhibition of the protein encoded by the gene; anticancer agent screening method comprising.
17. (1) infecting a biological sample and a control sample of a patient suspected of cancer with a recombinant expression vector operably linked to the promoter of claim 1 with a foreign gene; And

    (2) Comparing the foreign gene expression level of the biological sample of the patient and the control sample of the step (1); a method for providing information for diagnosis of cancer or predicting the risk of metastasis of cancer comprising.
18. A method for preventing and treating cancer, comprising administering to an individual a recombinant expression vector operably linked with a foreign gene to the promoter of claim 1 in a pharmaceutically effective amount.

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