KR100486878B1 - Expression vector specifically expressing target protein at the endothelial cells - Google Patents

Abstract

The present invention relates to a vascular endothelial cell-specific expression vector, and more specifically, to a cancer or cardiovascular disease comprising a vector specifically expressing a target protein in endothelial cells, including an endothelial cell-specific promoter. It relates to a pharmaceutical composition. Since the expression vector of the present invention specifically expresses endothelial cells and increases its expression level in a hypoxic state, the expression vector may be usefully used to treat cancer or cardiovascular disease depending on angiogenesis.

Description

Expression vector specifically expressing target protein at the endothelial cells

The present invention relates to an endothelial cell specific expression vector, and more particularly to a vector that specifically expresses a target protein in endothelial cells, including an endothelial cell specific promoter.

Around six million cancer (or tumor) cases occur worldwide annually, and cancer ranks second in heart mortality in the US, following heart disease, making cancer a major health problem. Fortunately, most cancers are diagnosed at an early stage. Surgery and radiotherapy are used to remove malignant tumors. However, the remaining tumors are spread by metastasis and only chemotherapy is used as the last treatment. . In addition, since resistance develops due to repeated treatment, only 5-10% of metastatic cancers can be treated. In order to treat cancer, which can be called incurable or incurable, advanced countries are actively conducting various researches to combat cancer with enormous budgets, and the cancer treatment rate has improved a lot, but cancer is still one of the most serious diseases of mankind with AIDS. In the United States, about half a million patients die of cancer each year. In the case of anticancer drugs, no significant special drugs have been developed so far, but when all the gene functions are revealed in the 21st century, it is expected to develop gene therapeutics using excellent anticancer genes as cutting-edge new drugs using genetic information.

Since gene therapy was first applied to the ADA-SCID in 1990 (Rosenberg SA et al ., New Engl. J. Med., 1990 , 323, 570-578), the rapid development of the protocol resulted in more than 370 clinical trials. There are 3,200 people. Gene therapy was originally thought to be the ideal treatment for hereditary diseases, especially monogeneic diseases caused by the breakdown of one gene, but the main targets of ongoing gene therapy clinical studies include cancer, Hereditary disease, AIDS. About two-thirds of ongoing gene therapies target cancer patients, with solid cancers predominantly more than hematologic tumors such as leukemia, but all other cancers are subject to clinical research.

The core technologies of gene therapy can be divided into three types: therapeutic genes that actually show a therapeutic effect, mediators (vectors) that deliver genes to the human body, and technologies that deliver genes to the human body. The most important of the three fields of gene therapy is the gene transfer vector technology, which includes nonviral plasmid vectors and viral plasmid vectors. To date, gene therapy has not been popularized and industrialized due to issues of safety and efficiency of gene delivery, in particular, proteins are not produced in large enough quantities to treat disease, gene expression time is shorter than expected, and stability The fact that this is not established is pointed out as a big problem.

Nonviral plasmid vectors have several advantages that are easy to manufacture and mass produce, do not induce an immune response, have no possibility of replication and recombination, and have no size limitations on the genes to be delivered. Although non-viral plasmid vectors have a disadvantage of low gene transfer efficiency or transient gene expression, their use is limited, but their applications are rapidly increasing in recent years, and sufficient gene expression is induced in vivo. There is no non-viral vector that can do this, but efforts are being made to use it more widely.

Several studies are currently underway to compensate for the low expression and low duration of genes, one of the disadvantages of nonviral vectors. For example, since the gene expression efficiency of gene transfer plasmids depends on the promoter used, there is a method of increasing gene expression using a strong promoter. Promoters include viral promoters, promoters of housekeeping genes that always induce gene expression in eukaryotic cells, and tissue-specific promoters that regulate gene expression in specific tissues (Smith RL et al ., J Virol , et al . 2000 , 74 (23), 11254-61).

However, since viral promoters are nucleotide sequences derived from heterologous genes, they may induce antigen and antibody responses. In the case of housekeeping genes, gene expression rates are low and expression is not tissue specific. Developing a vector that properly expresses the therapeutic gene in the patient's required area using a tissue-specific promoter can develop a safe and effective therapeutic agent to treat intractable diseases such as cancer, so that it can be specifically targeted to the target cells desired for gene therapy. Strong expression is required, and it is moving toward developing a vector in which the required amount of gene expression is made in response to the environment.

In addition, since the growth of cancer cells depends on the supply of blood vessels, cancer cells are highly dependent on endothelial cells that form blood vessels, and thus endothelial cells are suitable as target cells for cancer treatment. Anti-angiogenic drugs such as Angiostatin and Endostatin can inhibit the growth of cancer cells and also endogenous inhibitors of angiogenesis can inhibit cancer. . Therefore, endothelial cell-specific vectors are useful in the use of therapeutic genes for cancer treatment and may be useful in the treatment of cardiovascular diseases. In the United States, endothelial cell-specific vectors are currently being developed around retroviral vectors (Mavria G et al ., Gene Therapy , 2000 , 7, 368-376; Martin SG et al ., Advanced Drug Reviews , 2000 , 41, 223 -233).

Thus, the inventors of the present invention have developed a vector that specifically expresses endothelial cells while developing a vector for expressing a therapeutic gene more efficiently in gene therapy, which is a safe and efficient alternative for treating cancer. The present invention has been completed by revealing that it can be usefully used to treat cancer or cardiovascular disease depending on formation.

It is an object of the present invention to provide a vector that specifically expresses a target protein in endothelial cells, including an endothelial cell specific promoter.

In order to achieve the above object, the present invention provides an expression vector that specifically expresses a target protein in endothelial cells, including an endothelial cell specific promoter, and a pharmaceutical composition for treating cancer or cardiovascular disease comprising the vector. .

Hereinafter, the present invention will be described in detail.

The present invention provides an expression vector that specifically expresses a target protein in endothelial cells, including an endothelial cell specific promoter.

The endothelial cell specific promoter of the present invention is further characterized by a promoter of the ppET-1 gene, which is a human endothelial cell gene, and an untranslated exon 1 downstream of the promoter.

The vector of the present invention specifically expresses the gene of interest in endothelial cells and increases the expression of the gene in the hypoxic state. In a preferred embodiment of the present invention, a gene having the sequence set forth in SEQ ID NO: 5 comprising a promoter of the ppET-1 gene, which is a human endothelial cell gene, and an untranslated exon downstream of the promoter gene, is cloned into a pcDNA 3.1 vector (Invitrogen). This was named 'pcDNA3-PPET-2'. After transducing pcDNA3-PPET-2 vector to cells, luciferase activity was measured to measure promoter activity. As a result, the expression level was highest in endothelial CPAE cells (see FIG. 5 ). As a result of measuring the promoter activity by culturing cells in a steady state or hypoxic state, it was found that the expression level was higher in the hypoxic state than in the normal state (see FIG. 6 ).

The present inventors transfected with E. coli , which was specifically expressed in endothelial cells and confirmed high expression in hypoxic state, and deposited it at Korea Microorganism Conservation Center on October 15, 2002 (Accession No. KCCM-10436).

Since the vector of the present invention is a non-viral vector, it can be safely used in gene therapy, and inserting a therapeutic gene into the vector of the present invention can express the therapeutic gene specifically in endothelial cells, and in the hypoxic state, It can increase expression and can be useful for treating cancer by attacking growing cancer cells by receiving blood vessels from endothelial cells. In addition, it can be usefully used for the treatment of cardiovascular diseases such as cancer and hypertension, arteriosclerosis or ischemic diseases using the vector of the present invention. Therapeutic genes that may be included in the vector of the present invention include, but are not limited to, VEGF (vascular endothelial growth factor), EPO (erythropoietin) or VEGF antisense oligomer.

The present invention also provides a pharmaceutical composition for treating cancer or cardiovascular disease, containing the vector as an active ingredient.

The vector of the present invention includes a promoter specifically expressed in endothelial cells, so that the protein of interest may be overexpressed in endothelial cells. Therefore, an expression vector further comprising a therapeutic gene may be prepared, and then a pharmaceutical composition containing the same as an active ingredient may be used to treat cancer or cardiovascular disease.

The pharmaceutical composition of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

In other words, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used Or using excipients. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, water Prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of the pharmaceutical composition for treating cancer or cardiovascular disease comprising the vector of the present invention is 0.01 to 1 mg / kg, preferably 0.05 to 0.5 mg / kg, and may be administered 1 to 3 times a day.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Cloning of the ppET-1 Gene Promoter

The present inventors are expressed in endothelial cells to produce a vector expressing a target gene specifically in endothelial cells and are well-characterized in vitro, and have a hypoxia response element (HRE), resulting in a hypoxic state. Promoters of human endothelial cell gene-1 (human preproendothelin-1, hereinafter referred to as 'ppET-1') known to increase expression of genes (Yamashita K et al ., J. Biol. Chem ., 2001 , 20; 276 (16), 12645-53) were cloned into pcDNA3.1 vector ( FIG. 1 ).

<1-1> Amplification of the ppET-1 Promoter

In order to enhance the gene expression efficiency of the promoter, the inventors of the present invention describe the untranslated exon and intron sites known to greatly increase the activity of the promoter as well as the promoter at the time of preparing the promoter of the ppET-1 gene (Lee YJ et al ., Biochem. Biophys Res. Commun., 2000 , 272 (1), 230-5) were also inserted and amplified. Specifically, amplifying a gene containing only the ppET-1 promoter sequence by amplifying a gene from -177 to +33 of the ppET-1 promoter gene through PCR from human genomic DNA, and then -177 to +267 (starting codon). Genes up to just before were amplified by inserting an untranslated exon 1 site into the ppET-1 promoter sequence.

First, a PCR reaction was performed to amplify a promoter including a portion of the untranslated exon 1 (+1 to +33) as well as a promoter region of the ppET-1 promoter sequence -177 to -133. PCR reactions were performed on human genomic DNA with a primer as set out in SEQ ID NO: 1 containing a Mlu I restriction enzyme sequence, a primer as set out in SEQ ID NO: 2 comprising a Hind III restriction enzyme sequence, dNTP, 10 x PCR buffer and Taq polymerase. The reaction was performed at 94 DEG C for 5 minutes, 30 seconds at 94 DEG C, 1 minute at 60 DEG C, and 30 seconds at 72 DEG C for 30 minutes, followed by 5 minutes at 72 DEG C and 10 minutes at 4 DEG C. I was. As a result of electrophoresis of the reaction product obtained after the PCR reaction, it was confirmed that the DNA fragment was about 210 bp including the gene sequence from the ppET-1 promoter sequence -177 to +33 and the restriction enzyme sequence of the primer ( FIG. 2 ). . The DNA fragment of about 210 bp identified above was inserted into the pGEMTeasy vector (promega) and subjected to sequencing using the T7 / SP6 primer position. As a result, a sequence including the ppET-1 promoter sequence and a part of exon 1 was obtained. It was confirmed that it has the nucleotide sequence described in SEQ ID NO: 3 it was named 'PPET-1'.

Next, amplify the gene containing the entirety of exon 1 (+1 to +267) that is not translated into the ppET-1 promoter (-177 to -1) with the genes from the ppET-1 promoter sequence -177 to +267. PCR was performed. PCR reactions were performed on human genomic DNA with a primer as set forth in SEQ ID NO: 1 containing the Mlu I restriction enzyme sequence, a primer set forth in SEQ ID NO: 4 containing the Hind III restriction enzyme sequence, dNTP, 10 x PCR buffer and Taq polymerase. The reaction was performed at 94 DEG C for 5 minutes, 30 seconds at 94 DEG C, 1 minute at 60 DEG C, and 30 seconds at 72 DEG C for 30 seconds, followed by 5 minutes at 72 DEG C and 10 minutes at 4 DEG C. I was. The reaction product obtained after the PCR reaction was inserted into pGEMTeasy vector (promega), and then digested with EcoR I and subjected to electrophoresis. About 460 including the restriction enzyme sequence of the gene and primers from the ppET-1 promoter sequence -177 to +267 It was confirmed that the size of the bp ( Fig. 3 ). In addition, as a result of sequencing using the T7 / SP6 primer position of the pGEM.Teasy vector into which the PCR product of about 460 bp was inserted, SEQ ID NO: 5 including the ppET-1 promoter sequence and the entire sequence of exon 1 It was confirmed that it has a base sequence described as 'PPET-2' was named.

<1-2> Cloning of the ppET-1 Promoter

The present inventors cloned the PPET-1 or PPET-2 promoter gene amplified in Example <1-1> into a pcDNA 3.1 vector (Invitrogen). Specifically, each of the PPET-1 or PPET-2 DNA fragments amplified in Example <1-1> was cleaved with Mlu I and Hind III and then inserted into the Mlu I / Hind III position of the pCDNA 3.1 vector to pcDNA 3.1 vector. In order to replace the CMV promoter of and to confirm the efficacy of the vector, a luciferase gene was inserted into the Hind III / Xba I position as a reporter gene to prepare an expression vector into which the PPET-1 or PPET-2 promoter was inserted. These were named 'pcDNA3-PPET-1' and 'pcDNA3-PPET-2', respectively.

In addition, in order to compare the efficiency of the expression vector, a control vector having a luciferase gene inserted into the pcDNA3.1 vector was prepared and named 'pcDNA3-Luciferase'.

Example 2 Efficiency Measurement of Endothelial Cell Expression Vector

In order to measure the efficiency of the endothelial cell expression vector, the present inventors transduced the endothelial cell expression vector prepared in Example 1 and measured the expression level of the reporter gene.

<2-1> cell culture

The present inventors have used CPAE cells (bovine pulmonary endothelium) of bovine pulmonary endothelium (Korea Cell Line Bank, 10209), human umbilical vascular endothelial cells (Human umbilical vein) to transduce vectors pcDNA3-PPET-1 or pcDNA3-PPET-2. HUVEC cells (ATCC), endothelial cells), BAEC cells (Kim HP et al ., Biochemical and Biophysical Research Communications , 1999 , 263, 257-262), bovine aortic endothelial cells, and COS, monkey kidney cells Cells (Korea Cell Line Bank) were used. HUVEC cells were cultured using an EBM kit (Bio Whittaker, Inc), BAEC cells were cultured with 10% FBS added to DMEM medium, COS cells were cultured with 10% FBS added to DMEM medium, and CPAE cells Cultures were added by adding 20% FBS to RPMI medium.

<2-2> Transduction

The present inventors transduced the vector prepared in Example 1 to each cell cultured in Example <2-1> by the calcium phosphate coprecipitation method. Specifically, cells were plated the day before transduction and replaced with fresh medium when the cell density reached 50% the next day. A DNA-CaCl ₂ mixture was prepared by mixing 2 M CaCl ₂ and water to each of pcDNA3-PPET-1, pcDNA3-PPET-2 or pcDNA3-Luciferase vector DNA. HBS (28 mM NaCl, 10 mM KCl, 1.5 mM Na ₂ HPO ₄ , 12 mM dextrose and 50 mM HEPES) was added dropwise to the mixture. The mixed solution was well sprayed on the cells and replaced with fresh medium 24 hours later.

<2-3> Luciferase Activity Measurement

We transduced a pcDNA3-PPET-1, pcDNA3-PPET-2 or pcDNA3-Luciferase vector into a COS cell line, washed the cells with PBS and lysed with lysis buffer (125 mM Tris pH 7.8, 10 mM EDTA, 10 mM DTT). , 50% glycerol and 5% Triton X-100) were added to destroy the cells, and the supernatant was obtained. The amount of protein was measured using a Bradford assay. Luciferase activity was determined by assay buffer (20 mM Tricine, 1.07 mM (MgCO ₃ ) ₄ Mg (OH) ₂ .5H ₂ O, 2.67 mM MgSO ₄ , 0.1 mM EDTA, 33.3 mM DTT, 270 μM coenzyme A (lithium salt), 470 μM luciferin and 530 μM ATP) were added and measured using luminoskan (luminoskan ascent, Labysystems). And, to analyze the activity of the promoter, the luciferase activity of the cells into which the pcDNA3-PPET-1 or pcDNA3-PPET-2 vector was introduced was compared to the luciferase activity of the cells into which the pcDNA3-Luciferase vector was introduced.

As a result, the luciferase activity of pcDNA3-PPET-2 was 2.5 times higher than the luciferase activity of pcDNA3-PPET-1 in the COS cell line ( FIG. 4 ). Therefore, the distance from the start of transcription to the start codon was found to be important in the expression of the gene.

Next, after transducing vector pcDNA3-PPET-2, which was found to have excellent luciferase activity to COS cells, BAEC cells, HUVEC cells, and CPAE cells, and comparing luciferase activity, lucifer of pcDNA3-PPET-2 was compared. Raise activity was about 2 times higher for HUVEC cells, 1.8 times for BAEC cells, and about 8 times higher for CPAE cells, which are endothelial cells ( Figure 5 ). Therefore, it was found that the expression vector pcDNA3-PPET-2 of the present invention specifically expressed in endothelial cells.

In addition, in order to confirm whether the expression level of pcDNA3-PPET-2, which is an endothelial cell-specific expression vector, is increased in the hypoxia state, transduced pcDNA3-PPET-2 to CPEA cells and then normal state (normoxia). Luciferase activity was measured after incubation in hypoxia (hypoxia, cobalt chloride 100 μM, 24 hours). As a result, the expression rate of pcDNA3-PPET-2 was about 1.6 times higher in the hypoxic state than in the normal state ( FIG. 6 ). Therefore, the endothelial cell expression vector of the present invention was found to increase the expression of the target gene in the hypoxic state.

The present inventors expressed the target gene specifically in endothelial cells and transduced high-expression pcDNA3-PPET-2 vector into E. coli and deposited it in Korea Microorganism Conservation Center on October 15, 2002. (Accession No .: KCCM-10436).

As described above, the expression vector of the present invention expresses a target gene specifically in vascular endothelial cells, and in particular, its expression is increased in a hypoxic state, and thus may be useful for the treatment of cancer or cardiovascular diseases depending on vascular supply. have.

1 is a schematic diagram showing a process of confirming the efficiency of the vector by cloning the expression vector of the present invention,

2 is a photoelectrophoresis of the PCR amplified PPET-1 fragment,

Lane 1: size marker, lane 2: PPET-1

3 is a photograph showing electrophoresis of a fragment cut into EcoR I after insertion of PPET-2 into the EcoR I position of the pGEMTeasy vector,

Lane 1: size marker,

Lanes 2, 3, 4, 5, 6, 7: PPET-2 / EcoR I segments of each clone

4 is a graph measuring luciferase activity after transduction of pcDNA3-PPET-1 or pcDNA3-PPET-2 into COS cells,

5 is a graph measuring luciferase activity after transduction of pcDNA3-PPET-2 in COS, BAEC, HUVEC and CPAE cells,

Figure 6 is a graph measuring the luciferase activity after transduction of pcDNA3-PPET-2 in CPAE cells and cultured in a steady state or hypoxic state.

<110> LEE, YoungJoo <120> Expression vector specifically expressing target protein at the endothelial cells <130> 2p-10-03 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for ppET-1 promoter (-177) <400> 1 acgcgttctg aagttagcag tgat 24 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer for ppET-1 promoter (+33) <400> 2 aagcttccgt tcgcctggcg ca 22 <210> 3 <211> 204 <212> DNA <213> Homo sapiens <220> <221> promoter (222) (-177) .. (27) <223> DNA comprising ppET-1 promoter and partial RBS <400> 3 tctgaagtta gcagtgattt cctttcgggc ctggcttatc tccggctgca cgttgcctgt -118 tggtgactaa taacacaata acattgtctg gggctggaat aaagtcggag ctgtttaccc -58 ccactctaat aggggttcaa tataaaaagc cggcagagag ctgtccaagt cagacgcgcc 3 tctgcatctg cgccaggcga acgg 27 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for ppET-1 promoter (+267) <400> 4 aagctttctg aaaaaaaggg atca 24 <210> 5 <211> 439 <212> DNA <213> Homo sapiens <220> <221> promoter (222) (-177) .. (262) <223> DNA comprising ppET-1 promoter and total RBS <400> 5 tctgaagtta gcagtgattt cctttcgggc ctggcttatc tccggctgca cgttgcctgt -118 tggtgactaa taacacaata acattgtctg gggctggaat aaagtcggag ctgtttaccc -58 ccactctaat aggggttcaa tataaaaagc cggcagagag ctgtccaagt cagacgcgcc 3 tctgcatctg cgccaggcga acgggtcctg cgcctcctgc agtcccagct ctccaccgcc 63 gcgtgcgcct gcagacgctc cgctcgctgc cttctctcct ggcaggcgct gccttttctc 123 cccgttaaag ggcacttggg ctgaaggatc gctttgagat ctgaggaacc cgcagcgctt 183 tgagggacct gaagctgttt ttcttcgttt tcctttgggt tcagtttgaa cgggaggttt 243 ttgatccctt tttttcaga 262

Claims (4)

An endothelial cell specific nonviral expression vector represented by FIG. 1, comprising the sequence set forth in SEQ ID NO: 5 consisting of a promoter of human preproendothelin-1 (ppET-1) gene and untranslated exon 1 downstream of said promoter. delete The endothelial cell specific nonviral expression vector according to claim 1, which is pcDNA3-PPET-2 (Accession No .: KCCM-10436). delete