KR20000056226A - Hepatomacellular carcinoma specific cytotoxic recombinant adenovirus - Google Patents

Abstract

PURPOSE: Recombinant adenovirus having hepatoma specific cytotoxicity and a small size of hepatoma specific promoter are provided. The recombinant adenovirus is nontoxic to normal hepatic cells but kills specifically hepatoma cell. Therefore, it is useful for treating hepatoma effectively as a therapeutic agent. CONSTITUTION: Adenovirus has broad cell specificity and high infection rate. Promoter region of alpha-fetoprotein including enhancer and silencer from -4.9kb to +29bb is introduced into adenovirus shuttle vector containing thymidine kinase of herpes simplex virus. Promoter region of alpha-fetoprotein is too big to pack into the infectious virus particle, so E1 and E3 regions are removed from the promoter region. The removing of E3 gene induces active host immune response because of loss of activity of E3 protein. Reporter gene such as chloramphenicol acetyl transferase (CAT) is joined with the promoter region of alpha-fetoprotein and deletion experiment is performed to find proper size of promoter region for the construction of adenovirus vector. Hepatoma specific enhancer of HindIII/EcoRI fragment from -3.7kb to -3.1kb and basic promoter region of EcoRI/HindII fragment from -0.9kb to +29bp are ligated and used for construction of recombinant adenovirus.

Description

Liver cancer specific cell killing recombinant adenovirus {HEPATOMACELLULAR CARCINOMA SPECIFIC CYTOTOXIC RECOMBINANT ADENOVIRUS}

[Industrial use]

The present invention relates to a liver cancer specific recombinant adenovirus, and more particularly, to a recombinant adenovirus useful as a therapeutic agent that can effectively treat liver cancer.

[Prior art]

Liver cancer kills more than 1 million people annually worldwide, and is a very frequent tumor in Korea. However, this tumor is extremely difficult to treat, and only a few cases can be treated by surgical removal or transplantation of liver. In particular, when cancer cells are metastasized remotely or cancerous tumors are found in various focis, there is no satisfactory treatment method, and thus, the development of new therapies is urgently needed.

Gene therapy, a method of treating cancer, is a method of removing cancer cells by injecting specific genes into cancer cells.

There are many gene therapies for cancer treatment, but the cancer treatment method that directly kills cancer cells uses the thymidine kinase gene of Herpes simplex virus, a suicide gene that activates an inhibitor. There is this. This method involves injecting herpes simplex virus thymidine kinase into cancer cells and administering a prodrug, ganciclovir, which is currently a brain tumor, cervic cancer, etc. In the case of clinical trials. The herpes simplex virus thymidine kinase converts gancyclovir, a protoxic nucleside analogue, into toxic phosphorylated gancyclovir to stop DNA synthesis or act as an inhibitor of DNA synthase to promote cleavage Mitogenic state plays a role in inducing the killing of cells. In solid cancers such as gastric carcinoma, malignant mesothelioma, and metastatic colon cancer, even if the herpes simplex virus thymidine canase gene is transmitted to only a part of the carcinoma through animal experiments, The bystander effect is attracting attention as a method for maximizing the assassination effect by gene transfer considering the practical limitation that there is no way to deliver genes to all cancer cells.

Recently, gene therapy using several cancer-specific properties is being developed as a next-generation strategy to selectively remove only cancer cells while minimizing side effects on normal cells of patients with the development of gene therapy. These carcinoma or tissue specific cell killing gene therapy methods include osteosarcoma gene therapy using an osteocalcin promoter and prostate cancer gene therapy using a prostate specific antigen promoter. It has been developed by the method and is known for preparation for clinical trials. In the case of liver cancer, alpha-fetoprotein is expressed only in hepatocellular carcinoma cells, not normal hepatocytes of liver cancer patients. This is due to the base sequence characteristics of the transcriptional regulatory region of the gene [Refs. 1,2,3]. , 4,5,6,7]. To date, it has been reported that several cis- or trans-activation sites exist in the human alpha-fetoprotein gene, among which the hepatocyte-specific enhancer site is alpha-fetoprotein. It was found to be located at -4.0 kb and -3.3 kb from the start of transcription, and a hepatocyte-specific silencer was also present between the enhancer and the promoter so that the alpha-fetoprotein was not found in surrounding cells or normal hepatocytes. It has been reported to regulate expression only in liver cancer cells (Refs. 1,2,3,4,5,6,7). Therefore, if the characteristics of the electronic regulatory region can be used to induce selective killing of liver cancer cells, many groups have been actively studied in the world.

Several recent gene therapy studies of liver cancer have shown that adenoviruses expressing the herpes simplex virus thymidine kinase gene through the blood vessels directly through the liver by the cytomegalovirus promoter without liver cancer tissue specificity are severe. Hepatotoxicity has been reported [1,2,3,4,5,6,7]. Adenovirus must be injected directly into the liver through blood vessels under the premise of treating liver cancer patients. Therefore, in order to avoid such serious hepatotoxicity, it is thought that the application of cytotoxic gene therapy to liver cancer is possible only by using a liver cancer tissue specific promoter. Therefore, there are reports of recombinant adenoviruses containing alpha-fetoprotein promoter and herpes simplex virus thymidine kinase as such liver cancer tissue specific promoters (Fumihiko Kanai, et al., Gene Therapy for α-Fetoprotein-Production Human Hepatoma). Cells by Adenovirus-Mediated Transfer of the Herpes Simplex Virus Thymidine Kinase Gene, Hepatology June, 1996, pp 359-360). However, the alpha-fetoprotein promoter, a liver cancer tissue specific promoter used in the recombinant adenovirus, has a problem that the size of the alpha-fetoprotein promoter is too large to obtain a recombinant adenovirus obtained by removing only the E1 gene, which is a specific region of the initial gene of the adenovirus.

In addition, to date, animal experiments have been carried out by injecting human liver cancer cells subcutaneous to nude mice (subcutaneous) to produce a carcinoma, herpes simplex genetically engineered to regulate expression by the alpha-fetoprotein transcriptional regulatory region Rex virus thymidine kinase gene was injected with liver cyclovir to confirm the assassination effect, but the results on liver toxicity have not been confirmed.

It is an object of the present invention to provide a liver cancer specific killing recombinant adenovirus useful for effectively treating liver cancer by reducing the size of the liver cancer specific promoter while minimizing toxicity to the liver.

Another object of the present invention is to provide a liver cancer specific killing recombinant adenovirus that can be produced simply.

1 is a genetic map of the liver cancer specific killing recombinant adenovirus of the present invention.

Figure 2 is a genetic map of the alpha-fetopropane promoter used in the present invention.

Figure 3 is a genetic map of the shuttle vector (shuttle vector) used in the present invention.

Figure 4 is a genetic map of the rescue vector (rescue vector) used in the present invention.

Figure 5 is a gene map of the CAT (construct) used for the CAT (chlroamphenicol acetyl transferase) in the present invention

[Means for solving the problem]

In order to achieve the above object, the present invention provides a liver cancer specific killing recombinant adenovirus containing the herpes simplex virus thymidine kinase comprising the alpha-fetoprotein genes of SEQ ID NOs: 1 and 2.

[SEQ ID NO: 1]

[SEQ ID NO: 2]

Hereinafter, the present invention will be described in more detail.

The recombinant adenovirus of the present invention is genetically recombined so that one of the suicide genes, herpes simplex virus thymidine kinase, is expressed by alpha-fetoprotein. A genetic map of the recombinant adenovirus of the present invention is shown in FIG. As the recombinant adenovirus of the present invention, the alpha-fetoprotein promoter was used in which the unnecessary portion was removed to reduce its size. After infecting the recombinant adenovirus to a patient's liver cancer, liver cyclovir injection can effectively treat liver cancer. In the present invention, the appearance of alpha-phytoprotein in the blood by liver cancer cells is observed in most liver cancer patients, and this phenomenon is known to be correlated with the growth of liver cancer, and by using the liver cancer specific expression properties of the alpha-fetoprotein gene The present invention relates to a gene therapy material that kills liver cancer cells while minimizing toxicity to liver for liver cancer.

Such a recombinant adenovirus of the present invention is prepared by the following method.

1. Plasmid Construction

Adenoviruses are the second most popular viral vectors after retroviruses because they have a wide range of cells for gene delivery and mass production of viruses, and therefore have high virus-to-host cell infection rates. In this invention, this adenovirus is used.

Gene map of alpha-fetoprotein is shown in FIG. In order to induce liver cancer specific expression of such alpha-fetoprotein, both an enhancer and a silencer site are required. Thus, the alpha-fetoprotein promoter portion corresponding to +29 bp at -4.9 kb is injected into an adenovirus shuttle vector containing the herpes simplex virus thymidine kinase gene. A genetic map of an adenovirus shuttle vector comprising the herpes simplex virus thymidine kinase gene is shown in FIG. 3.

The promoter portion of alpha-fetoprotein must then be analyzed to reduce its size. In order to make an infectious adenovirus for practical use, the adenovirus is packaged by recombining the entire liver cancer specific electronic regulatory region (-4.9 kb / + 29 bp) of the alpha-fetoprotein gene with the rescue vector (Fig. 4). Since the size of the genome can be exceeded, the promoter region may be further reduced, or the rescue vector may be recombined using the E1 and E3 genes, which are the specific regions of the early genes of the adenovirus genome. If the rescue vector uses adenoviruses with the E1 and E3 genes removed, there is a possibility that even if a recombinant adenovirus is obtained, the host immune response is more severe because there is no E3 gene site that can suppress the host immune system.

Therefore, the size of the alpha-fetoprotein promoter should be reduced to enable the use of, for example, the pJM17 vector without E3 removal as a rescue vector. To this end, the alpha-fetoprotein promoter is subcloned in a vector for promoter analysis including a chloramphenicol acetyl thransferase (CAT) reporter gene to analyze the alpha-fetoprotein promoter to find the most appropriate structure. In more detail, this experiment is shown in FIG. In the alpha-fetoprotein gene, the CAT construct shown in FIG. 5 was made and then transfected into liver cancer cell lines to express the gene to reconfirm the strength and specificity of the promoter.

The alpha-fetoprotein promoter site used in the present invention obtained in this experiment has a gene sequence as shown in SEQ ID NOS: 1 and 2 below. In more detail, the alpha-fetoprotein promoter region of the present invention is a hepatoma specific enhancer portion from -3.7kb cut with a restriction enzyme HindIII to -3.1kb cut with EcoRI (Sequence 1 below). ) And a hepatoma specific recombinant promoter construct prepared by ligation of the basic promoter portion from -0.9kb cut with EcoRI to + 29bp cut with HindIII.

[SEQ ID NO: 1]

[SEQ ID NO: 2]

A plasmid that is an adenovirus shuttle vector comprising such an alpha-fetoprotein specific recombinant promoter construct is obtained.

2. Promoter / Enhancer Analysis

Using the plasmid pSV-CAT as a positive control, only the promoter region of this plasmid is replaced with the alpha-fetoprotein enhancer / promoter region and compared with the plasmid which is the adenovirus shuttle vector prepared above.

Human positive hepatoma cell lines SK-Hep1, HepG2, Hep3B were transfected with calcium phosphate with 10 μg of the positive control and plasmid DNA of the present invention and then assayed for CAT. This method is described in detail as follows.

20 μl of phosphate was added to 1 mL HBS solution (0.885 mL distilled water + 0.1 mL 10X HBS + 100 μl Ca solution), and 1 mL DNA solution (860 μl distilled water + 20 μl DNA (corresponding to 20 μg DNA) + 20 μl) Mix Ca solution + 100 μl Ca solution) and use 0.5 ml per 60-mm dish. Add a drop of Ca solution to this DNA solution. Allow to stand at room temperature for 20 minutes, release again, and add 0.5 ml per 5 ml of medium to the culture dish containing cells.

Cell lines are cultured in the medium for 2 days, collected, frozen and thawed repeatedly in 100 μl of 0.25M Tris-HCl buffer (pH 7.5) several times to completely crush the cells and centrifuged at 15,000 rpm for 5 minutes. Collect the supernatant and use it for CAT assay. When incubating for 1 hour or more during the reaction, ⅛ volume of 8 mM acetyl-CoA is added every hour. [14C] chloramphenicol ([14C] chloramphenicol) and acetylated chloramphenicol derivatives were extracted with ethyl acetate and analyzed by thin layer chromatography (TLC).

3. Cytotoxic Assays Following Gancyclovir Administration

Subsequently, the effect of the adenovirus shuttle vector of the present invention is measured by experimenting with the liver cancer treatment effect according to the administration of liver cyclovir between the plasmid which is the adenovirus shuttle vector of the present invention and the control plasmid.

First, 5,000 liver cancer cell lines SK-Hep1, HepG2, and Hep3B per well were plated in RPMI medium (Gibco-BRL, USA) (10% FBS (fetal bovine serum) + penicillin / streptomycin), and then 2 One day later, the control and the plasmid of the present invention are infected. One day after infection, the medium is removed and the liver cyclovir is diluted by concentration (0-100 µg / ml) and treated with each liver cancer cell line in 96-well culture plates and cultured. Every two days, the cells were left a week later by changing to a new GCV-containing medium. Quantify by).

The MTT assay adds 25 μl of MTT to 5 mg / ml in 100 μl of medium per well incubated for one week. After 4 hours of incubation in a 37 ° C CO ₂ incubator, 100 μl of a solution of 1N HCl and isopropanol was added to the wells by shaking for at least 1 hour, and then the absorbance A ₅₇₀ -A ₆₃₀ was measured. Calculate% survival rate.

4. Recombinant Adenovirus Production

AdAFPtk (adenovirus vector containing herpes simplex thymidine kinase induced by AFP enhancer / promoter: adenovirus vector containing herpes simplex virus thymidine kinase driven by AFP enhancer / promoter) Co-transfection of the shuttle vector and the adenoviral rescue vector of MicoBix company to 293 cells extracts the virus when in vivo genetic recombination cytolysis occurs. . The properties of adenoviruses infect the host cells, allow them to multiply well and eventually lead to the cytology of the host cells. Therefore, when the shuttle vector and the rescue vector, which are part of the genes constituting the adenovirus, are introduced into the host cell cells and are genetically recombined to form adenoviruses, cytotoxicity is induced. Therefore, if genetic recombination does not occur, it can be expected that cytorisis cannot be observed. Sometimes, the adenovirus occurs even though cytotoxicity occurs because the desired gene may be recombined to form adenovirus. Whether there is a desired gene (AFPTK) in the later steps need to check.

1) 293 cell preparation

One day prior to transfection, cells are plated to 70-80% confluent in a 60 mm dish. Cells are mixed with penicillin / streptomycin and 10% fetal bovine serum (FBS) or 5% HICS (heat inactivated calf serum) in MEM (minimal eagles medium).

2) Transfection

20 μl of phosphate was added to 1 mL HBS solution (0.885 mL distilled water + 0.1 mL 10X HBS + 15 μl 1N NaOH) and 1 mL DNA solution (860 μl distilled water + 20 μL DNA (corresponding to 20 μg DNA) + 20 μL Ca) Solution + 100 μl Ca solution) and use 0.5 ml per 60-mm dish. Add a drop of Ca solution to this DNA solution. Allow to stand at room temperature for 20 minutes, release again, and add 0.5 ml per 5 ml of medium to the culture dish containing cells. After incubation for 4.5-18 hours, the medium is removed and covered with 0.5% agarose (45 ° C., 2X MEMF11 (Gibco-BRL, USA) + 10% HICS + 45 ° C., 1% agarose).

3) screening plaque isolates

After a few days, the plaque begins to pick up and release the plaque in a solution of 1 ml PBS (phosphate buffered saline) and 10% glycerol. 0.2 ml of the virus solution was removed from the 293 cells prepared as above, followed by releasing the medium and allowed to attach at room temperature for 30 minutes, followed by culture with 5 ml medium. (cytopathic lysis) can be observed.

4) Collection of viruses

The petri dish is left in the hood for 20-30 minutes, then the medium is removed slightly and 4 ml is stored in a -70 ° C. freezer by adding 0.5 ml sterile glycerol to sterile glass vials. Remaining medium is removed under reduced pressure. 0.5 ml of pronase (0.5 mg / ml pronase + 0.5% SDS (sodium dodecyl sulfate)) is added to completely dissolve the cells at 37 ° C. for 3-4 hours. Viscose lysate obtained after complete decomposition is transferred to a 1.5 ml tube and phenol extraction is performed. Add 50 μl of 30% sodium acetate with 1 ml ethanol to collect viral DNA and wash twice in 95% ethanol. After drying, it is dissolved in 50 µl of 0.1X SSC (a solution prepared by dissolving 175.3 g sodium chloride and 88.2 g sodium citrate in 1 L of H ₂ O to pH 7.0) for several hours.

To determine whether the resulting adenovirus is an unwanted replication competent virus or a desired recombinant adenovirus, the polymerase is determined using oligomers corresponding to the adenovirus E1 gene segment and the herpes simplex virus thymidine kinase gene segment. A polymerase chain reaction (PCR) is performed.

After PCR and restriction enzyme treatment, electrophoresis confirms that the desired recombinant adenovirus is followed by mass culture, ultracentrifugation using CsCl density gradient, purification, and residual CsCl removed via dialysis.

5) Investigation of cytotoxicity by the bystander effect in vitro

Liver cancer cell lines such as HepG2 or Hep3B, which are known to express alpha-fetoprotein, and liver cell lines known to lack alpha-fetoprotein, such as SK-Hep-1 or Chang liver cell, After delivery of AdAFPtk, cytotoxicity was compared by administering various concentrations of gancyclovir up to 50 μg / ml. In each cell line, the bystander effect is confirmed by varying the ratio of cells to which the alpha-fetoprotein gene is delivered and the number of cells that are not delivered.

6) Investigation of anticancer effect through animal experiment

When the specific cell killing effect was confirmed in liver cancer cells expressing alpha-fetoprotein, the control group was injected with Hep3B and SK-Hep-1 cell line, which is known to express alpha-fetoprotein in nude mice, as a control. do. Carcinoma is formed in the liver by injecting cancer cells into the spleen to perform animal experiments closest to the Orthotopic model with carcinoma at the normal or correct site of the body. The adenovirus gene transfer method is a method through which the liver is delivered to the hepatic artery in clinical trials to minimize systemic toxicity and dilution caused by the spread of the virus. Select the delivery method.

Specifically, 30 of 60 7 week old atherosclerosis (athymic nude mice) are injected with about 5 × 10 ⁵ Hep3B cells and the other 30 are injected with about 5 × 10 ⁵ SK-Hep1 cells to form a carcinoma. Each of Hep3B and SK-Hep1 were divided into 6 groups according to the following treatment method, and the group injected with AdAFPHSVtk and administered with cyclocyclovir (n = 10), and the group injected with AdAFPHSVtk and not treated with cyclocyclovir (n = 10). In addition, hepatic cancer cells were divided into groups administered with only cyclocyclovir (n = 10), and 7 days after the injection of liver cancer cells, 1 × 10 ⁹ PFU (plaque forming unit) adenovirus was injected continuously for 2 days at 100 μl carcinoma sites. Twenty-four hours after injection, gancyclocyb is administered daily at 14 mg / kg for intraperitoneal injection (ip) for 14 days. Two weeks later, the number of tumor nodules formed in the liver is counted for confirmation of carcinoma growth. Statistical checks for carcinoma specific killing effects.

Repeat the same experiment to verify reproducibility.

EXAMPLE

Hereinafter, preferred embodiments of the present invention will be described. However, the following examples are only preferred embodiments of the present invention and the present invention is not limited to the following examples.

1. Plasmid Construction

An adenovirus shuttle vector was injected by injecting an alpha-fetoprotein promoter portion (obtained from Professor Taiki Tamoki, University of Calgary, Canada) from -4.9kb to an adenovirus vector containing the thymidine kinase gene. Phosphorus plasmid was prepared.

Subsequently, the alpha-fetoprotein promoter was subcloned into a vector for promoter analysis including the CAT reporter inheritance to remove unnecessary portions of the alpha-fetoprotein promoter. The alpha-fetoprotein promoter region obtained by removal was a hepatoma specific enhancer portion from -3.7kb cut with the restriction enzyme HindIII to -3.1kb cut with EcoRI and + 0.9kb cut with HindIII cut with EcoRI. Hepatoma specific recombinant promoter construct prepared by ligation of the basic promoter portion up to 29 bp.

A plasmid which is an adenovirus shuttle vector containing such an alpha-fetoprotein specific recombinant promoter construct was obtained.

2. Promoter / Enhancer Analysis

Using the plasmid pSV-CAT as a positive control, only the promoter region of this plasmid was substituted with the alpha-fetoprotein enhancer / promoter region and compared with the plasmid which is the adenovirus shuttle vector prepared above.

Add 20 μl of phosphate to 1 mL HBS solution (0.885 mL distilled water + 0.1 mL 10X HBS + 100 μL Ca solution), and add 1 mL DNA solution (860 μl distilled water + 20 μL DNA + 20 μL Ca solution + 100 μL Ca solution). ) Was used to mix 0.5 ml per 60-mm dish. Ca solution was added dropwise to this DNA solution. After standing at room temperature for 20 minutes, it was released again and 0.5 ml of 5 ml of medium was added to the culture dish containing cells.

Cell lines were incubated in the medium for 2 days, collected, frozen and thawed repeatedly in 100 μl of 0.25M Tris-HCl buffer (pH 7.5) several times to completely crush the cells and centrifuged at 15,000 rpm for 5 minutes. The supernatant was collected and used for CAT assay. When incubated for 1 hour or more during the reaction, a volume of 8 mM acetyl-CoA was added every hour. [14C] chloramphenicol ([14C] chloramphenicol) and acetylated chloramphenicol derivatives were extracted with ethyl acetate and analyzed by thin layer chromatography (TLC).

3. Cytotoxic Assays Following Gancyclovir Administration

First, 5,000 liver cancer cell lines SK-Hep1, HepG2, Hep3B per well were plated in RPMI medium (10% FBS + penicillin / streptomycin) and then infected with the plasmid of the control and the present invention 2 days later. One day after infection, the medium was removed and the liver cyclovir was diluted by concentration (0-100 µg / ml) and treated with each liver cancer cell line in 96-well culture plates and cultured. The number of cells remaining one week later was quantified by MTT assay in the following manner while changing to a new GCV-containing medium every two days.

The MTT assay adds 25 μl of MTT to 5 mg / ml in 100 μl of medium per well incubated for one week. After 4 hours of incubation in a 37 ° C CO ₂ incubator, 100 μl of a solution of 1N HCl and isopropanol was added to the wells for 1 hour to shake for more than 1 hour, followed by measurement of absorbance A ₅₇₀ -A ₆₃₀ . % Survival rate was calculated. The results are shown in Table 1 below.

Cell line type ^* MOI ^** = 1 GCV = 25 μm MOI = 10GCV = 1 μm Chang liver cell 89 ± 4% 85 ± 2% SK-Hep-1 90 ± 4% 90 ± 10% HepG2 25 ± 5% 35 ± 5% Hep3B 55 ± 4% 40 ± 5%

*: Changed liver cells and SK-Hep-1 did not express AFP and were used as controls. That is, it should not show cytotoxicity (toxicity) even after administration of GDV.

**: MOI (Multiplicity of Infection). That is, the number of adenoviruses infected per host cell (liver cancer cell) is shown.

4. Recombinant Adenovirus Production

AdAFPtk (adenovirus vector containing HSVtk driven by AFP enhancer / promoter) induced by AFP enhancer / promoter is adenovirus rescue vector of the present invention prepared by the above method and adenovirus rescue vector of MicoBix company. The vector was co-transfected into 293 cells and the virus was extracted when genetic recombination cytology occurred in vivo.

1) 293 cell preparation

One day prior to transfection, cells were plated to a concentration of 70-80% in a 60 mm dish. Cells were mixed with penicillin / streptomycin and 10% FBS in MEM.

2) Transfection

Add 20 μl of phosphate to 1 mL HBS solution (0.885 mL distilled water + 0.1 mL 10X HBS + 15 μl 1N NaOH) and add 1 mL DNA solution (860 μl distilled water + 20 μL DNA + 20 μL Ca solution + 100 μL Ca solution) Were mixed and used 0.5 ml per 60-mm dish. Ca solution was added dropwise to this DNA solution. After standing at room temperature for 20 minutes, it was released again and 0.5 ml of 5 ml of medium was added to the culture dish containing cells. After incubation for 6 hours, the medium is removed and covered with 0.5% agarose (45 ° C., 2 × MEMF11).

3) Plaque Screening

A few days later, when plaque began to appear, the plaque was selected and released in a solution containing 1 ml of PBS and 10% glycerol. 0.2 ml of the virus solution was removed from the 293 cells prepared as above, followed by releasing the medium and allowed to attach at room temperature for 30 minutes, followed by culture with 5 ml medium. (cytopathic lysis) could be observed.

4) Virus Collection

The petri dish was left in the hood for 20-30 minutes, then the medium was gently removed and 4 ml was added to 0.5 ml sterile glycerol in sterile glass vials and stored in a -70 ° C. freezer. The remaining medium was removed under reduced pressure. 0.5 ml of pronase (0.5 mg / ml pronase + 0.5% SDS (sodium dodecyl sulfate)) was added to completely dissolve the cells at 37 ° C. for 3 hours. The viscose lysate obtained after complete decomposition was transferred to a 1.5 ml tube and phenol extraction was performed. 50 μl of 30% sodium acetate was added together with 1 ml ethanol to collect viral DNA and washed twice with 95% ethanol. After drying, it was dissolved in 50 μl of 0.1X SSC over several hours and used.

To determine whether the resulting adenovirus is an unwanted replication competent virus or a desired recombinant adenovirus, the polymerase is determined using oligomers corresponding to the adenovirus E1 gene segment and the herpes simplex virus thymidine kinase gene segment. Polymerase chain reaction (PCR) was performed.

After PCR and restriction enzyme treatment, if the desired recombinant adenovirus was confirmed by electrophoresis, mass culture, ultracentrifugation using CsCl density gradient, purification and residual CsCl were removed by dialysis.

5) Examination of cytotoxic effect in vitro

AdAFPtk made in the above was used to target liver cancer cell lines such as HepG2 or Hep3B, which are known to express alpha-fetoprotein, and cell lines known to not express alpha-fetoprotein, such as SK-Hep-1 or Chang liver cell. Cytotoxicity was compared by administering various concentrations of gancyclovir up to 50 μg / ml after delivery. In each cell line, the effect of cell killing was confirmed by varying the ratio of cells to which the alpha-fetoprotein gene was delivered and the number of cells to which it was not delivered.

6) Investigation of anticancer effect through animal experiment

Of the sixty seven week-old athymic nude mice, 30 were injected with 2 × 10 Hep3B cells through the spleen and the other 30 were injected with 2 × 10 ⁶ SK-Hep1 cells through the spleen for carcinoma. Formed. Each of Hep3B and SK-Hep1 was divided into 6 groups according to the following treatment method, and the group injected with AdAFPHSVtk and administered with cyclocyclovir (n = 5), and the group injected with AdAFPHSVtk and not treated with cyclocyclovir (n = 5). In addition, liver cancer cells were injected into the group administered with liver cyclovir only (n = 5), and 12 days later, 1 × 10 ⁹ PFU of adenovirus was injected into 100 µl carcinoma sites. Twenty four hours after injection, gancyclocyb was administered daily at 14 mg / kg for 14 days. Two weeks later, tumor nodules formed in the liver were counted for confirmation of carcinoma growth. Statistically confirmed whether there is a carcinoma specific killing effect. As a result, Chang liver cells and SK-Hep-1 showed no killing effect.

The same experiment was repeated once to confirm reproducibility.

As described above, the recombinant adenovirus of the present invention can selectively induce herpes simplex thymidine kinase gene in liver cancer cells to induce cancer killing without affecting surrounding normal liver cells. Therefore, this method can effectively remove only liver cancer cells while minimizing cytotoxicity to normal cells, which is very effective in treating liver cancer.

References in the present specification are as follows, and reference numbers are indicated as [Reference X] in the present specification.

1.A position-dependent silence plays a major role in repressing alpha-fetoprotein expression in human hepatoma. Mol Cell Biol 11-5885-93, 1991.

Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro. Cancer Research, 46: 1341-1345, 1996.

3.Abelev GI Alphafetoprotein in oncogenesis and its association with malignant tumors. Adv Cancer Res 14: 295-358, 1971.

Nakabayashi H, Watanabe K, Saito A, Otsuru A, Sawadaishi K, Tamaoki T. Transcriptional regulation of alpha-fetoprotein expression by dexamethasone in human hepatoma cell. J Bio Chem 264: 266-271, 1989.

5. Sawadaishi K, Morinaga T, Tamaoki T. Interaction of a hepatoma-specific nuclear factor with transcription-regulatory sequences of the human alpha-fetoprotein and albumin genes. Mol Cell Biol 8: 5179-87, 1988.

6. Watanabe K, Saito A, Tamaoki T. Cell-specific enhancer activity in a far upstream region of the human alpha-fetoprotein gene. J Bio Chem 262: 4812-8, 1987.

Nakata K, Motomura M, Nakabayashi H, Ido A, Tamaoki T. A possinle mechanism of inverse developmental regulation of alpha-fetoprotein and albumin genes. J Bio Chem 267: 1331-4, 1992.

Claims (2)

A recombinant adenovirus containing the herpes simplex virus thymidine kinase comprising the alpha-fetoprotein genes of SEQ ID NOs: 1 and 2. [SEQ ID NO: 1] [SEQ ID NO: 2] The method of claim 1, wherein the alpha-fetoprotein gene is ligated between -3.7kb to -3.1kb hepatoma specific enhancer portion and -0.9kb to + 29bp basic promoter portion cut by HindIII and EcoRI restriction enzyme Recombinant adenovirus, which is a prepared hepatoma specific recombinant promoter.