KR100732248B1 - - / Recombinant Adeno-associated Virus Comprising VEGF Antisense cDNA and Gene Therapeutic Agent Specific to Large Intestine Cancer and/or Lung Cancer Comprising the Same - Google Patents

Abstract

The present invention relates to gene therapy using VEGF antisense cDNA and adeno-associated virus (AAV) system, and more particularly, rAAV vector containing VEGF-B antisense cDNA, rAAV vector containing VEGF-C antisense cDNA and It relates to a colorectal cancer and / or lung cancer-specific gene therapy comprising the two vectors and rAAV vectors containing VEGF-A antisense cDNA.

Gene therapy agents according to the present invention can be effectively used to treat cancer at the genetic level by inhibiting the expression of VEGF involved in angiogenesis essential for tumor growth and metastasis, thereby reducing tumor growth.

Angiogenesis, gene therapy, VEGF, VEGF receptor, adeno-associated virus

Description

Recombinant Adeno-associated Virus Comprising VEGF Antisense cDNA and Gene Therapeutic Agent Specific to Large Intestine Cancer and / or Oral Recombinant Adeno-associated Virus Containing VEE Antisense CDNA Lung Cancer Comprising the Same}

Figure 1 shows a standard curve for particle titration of rAAV-AShVEGF-A vector.

Figure 2 shows a standard curve for particle titration of rAAV-AShVEGF-A-IRES-EGFP vector.

Figure 3 shows a standard curve for particle titration of rAAV-AShVEGF-B-IRES-EGFP vector.

Figure 4 shows the standard curve for particle titration of rAAV-AShVEGF-C-IRES-EGFP vector.

Figure 5 shows the standard curve for particle titration of rAAV-IRES-EGFP vector.

Figure 6 shows the standard curve for titration of rAAV-EGFP vector.

7 is a confocal micrograph of the transduction efficiency of rAAV-AShVEGF-A.

8 is a confocal micrograph of the transduction efficiency of rAAV-AShVEGF-B.

9 is a confocal micrograph of the transduction efficiency of rAAV-AShVEGF-C.

10 is a confocal micrograph of the transduction efficiency of rAAV-IRES-EGFP.

11 is a graph showing the effect of reducing the amount of VEGF of rAAV-AShVEGF-A vector in each cancer cell line.

12 is a graph showing the change in the volume of the tumor in the tumor model nude mice injected with the rAAV vectors of the present invention.

Field of invention

The present invention relates to gene therapy using VEGF antisense cDNA and adeno-associated virus (AAV) system, and more particularly, rAAV vector containing VEGF-B antisense cDNA, rAAV vector containing VEGF-C antisense cDNA and It relates to a colorectal cancer and / or lung cancer-specific gene therapy comprising the two vectors and rAAV vectors containing VEGF-A antisense cDNA.

Background of the Invention

The mortality rate from cancer is increasing year by year, both at home and abroad. Cancer is one of the leading causes of death in the world, along with infectious and cardiovascular diseases. According to the World Health Report of the World Health Organization (WHO), 7,121,000 people, 12.5% of all deaths in 2004, died of cancer and cancer populations in the next 25 years due to changes in eating habits, the environment and life expectancy. Is expected to increase to about 30 million people annually, with 20 million people dying from cancer.

Currently, the methods used for the treatment of cancer are largely surgical surgery, radiation treatment and drug treatment, these methods are used independently for the treatment of cancer or two or more methods are used in combination.

In general, surgical procedures can be applied at various stages of cancer. Early stage cancers can usually be treated by surgical surgery, but if the cancer is advanced or metastasized, it is difficult to perform surgery alone.

Radiation therapy treats cancer by irradiating the cancer cells with X-rays or γ-rays from external radiation or radioactive substances administered into the body.

Drug therapy is a method of destroying or inhibiting DNA or related enzymes necessary for the proliferation of cancer cells by administering oral or injection anticancer drugs. The advantage of pharmacotherapy over other methods is that the drug can reach any cancer in any part of the body and treat the metastasized cancer. Drug therapy is currently used as a standard therapy for the treatment of metastatic cancer. Of course, metastatic cancer cannot be cured by pharmacotherapy, but it plays an important role in prolonging lifespan by alleviating symptoms. However, chemotherapeutic agents that are the mainstay of such drug therapy have problems such as side effects and anticancer drug resistance.

However, thanks to remarkable developments in the life sciences, biotherapeutics are growing rapidly. Biotherapeutics are the therapeutic basis for preventing cancer progression by weakening the activity of cancer cells by restoring or increasing the body's natural immune function. Cancer cells can be effectively killed when the body's immune system is functioning, but otherwise cancer cells can easily proliferate or other pathogens can easily attack. It may be used with other therapies such as surgical surgery, radiotherapy, chemotherapy and the like to compensate for the above disadvantages of biotherapeutics.

Biotherapeutics currently of interest in the life sciences include antisense anticancer agents and angiogenesis inhibitors. Antisense anticancer agents use DNA fragments capable of complementarily binding to specific mRNAs of cancer cells, and induce cancer cell death by inhibiting the processing of mRNA or expression of proteins. As a result of the Human Genome Project, over 30,000 gene sequences were read and over 100,000 mRNA sequences were available. As a result, a large amount of information about mRNA candidate groups associated with cancer cells is secured, and antisense anticancer agents for genes related to signaling systems, apoptosis and cell proliferation are screened for clinical trials.

Tumor growth is only possible with the creation of new blood vessels that provide the oxygen and nutrients needed for this. In general, as cancer cells proliferate, the inside of the tumor becomes hypoxic and the tissues become necrotic. In addition, the hypoxia worsens because blood vessels are destroyed by the pressure of the tumor itself. To overcome this hypoxia, tumors express factors related to neovascularization (VEGF, bFGF, IL-8, PDGF, PD-EGF, etc.) to promote neovascularization. In other words, neovascularization is an essential process for tumor growth.

Neovascularization inhibitors aim to treat cancer by interfering with neovascularization of such tumors and inhibiting tumor growth. Direct neovascularization inhibitors interfere with the proliferation and migration of vascular endothelial cells or inhibit neovascular production by inhibiting the response to neovascular generating factors. Direct neovascularization inhibitors have the advantage of less inducing acquired drug resistance.

Indirect neovascularization inhibitors inhibit neovascularization by inhibiting protein expression in tumors that activate neovascularization or by blocking binding between the tumor protein and vascular endothelial cell surface receptors.

As described above, looking at the changes in the field of treatment, it can be seen that the change from the artificial chemicals to using the natural in vivo material. In particular, as genome research projects in various fields progress, genes that act as etiologies for various diseases are being discovered. In order to link these findings with clinical treatment or preventive effects, researches on gene transfer technology that normalizes functions or activates therapeutic functions by introducing genes for correcting lost or modified genes in the body, In other words, research is being actively conducted in the field of gene therapy. The field of gene therapy has been rapidly increasing in recent years with a lot of attention and research.

Gene therapy is a method of treating diseases by gene transfer and expression. Unlike drug therapy, gene therapy is a method of correcting genetic defects targeting a specific gene with a disorder. The ultimate goal of gene therapy is to genetically modify living cells to obtain a beneficial therapeutic effect. These therapies have the advantages of accurate delivery of the gene to the disease site, complete degradation in vivo, absence of toxic and immunogenic antigens, and long-term stable expression of the gene, thus providing the best possible treatment for the disease. It is in the spotlight as a treatment.

The main research field of gene therapy is to introduce genes that have a therapeutic effect on specific diseases, enhance resistance of normal cells to show resistance to anticancer drugs, or replace modified or missing genes in various genetic diseases. Can be summarized.

Gene therapy is largely divided into in vivo and in vitro . In vivo gene therapy involves injecting therapeutic genes directly into the body, and in vitro gene therapy primarily involves culturing a target cell in vitro, introducing the gene into these cells, and then regenerating the genetically modified cells. It is injected into the body. Currently, gene therapy research uses in vitro gene therapy rather than in vivo gene therapy.

Gene delivery techniques are largely based on viral vector-based transfer methods, non-viral delivery methods using synthetic phospholipids or synthetic cationic polymers, and transient electrical stimulation on cell membranes. The present invention can be classified into physical methods such as electroporation to introduce genes.

Among the delivery techniques, the method of using a viral transporter is a preferred method for gene therapy because the transfer of genes can be efficiently carried out as a vector lacking the replication ability of some or all of the genes replaced with the therapeutic genes. to be. Viruses used as viral transporters or viral vectors include RNA viral vectors (retroviral vectors, lentivirus vectors, etc.) and DNA viral vectors (adenovirus vectors, adeno-associated virus vectors, etc.), as well as herpes simplex virus vectors. (herpes simplex viral vector) and alpha viral vector. Particularly active studies are retroviruses and adenoviruses.

The characteristics of retroviruses that integrate into the genome of host cells are harmless to the human body, but they can inhibit the function of normal cells during integration, infect various cells, easily proliferate, and range from 1 to 7 kb. Is able to accept the external genes of the genes and to generate replication-deficient viruses. However, it is difficult to infect cells after mitosis, it is difficult to transfer genes in vivo , and somatic cell tissues must be proliferated in vitro at all times. Retroviruses can also be integrated into the proto-oncogene, which is a risk of mutations and can also kill cells.

Adenoviruses, on the other hand, have a number of advantages as cloning vectors, which can be replicated in the nucleus of a medium size, are clinically non-toxic, and stable even when external genes are inserted. Eukaryotes can be transformed without rearrangement or loss of genes, and are expressed at stable and high levels even when integrated into host cell chromosomes. Good host cells for adenoviruses are the cells responsible for human hematopoiesis, lymph, and myeloma. However, because it is linear DNA, it is difficult to proliferate, it is not easy to recover the infected virus, and the infection rate of the virus is low. In addition, the expression of the delivered gene is greatest after 1 to 2 weeks, and in some cells, expression is maintained for only 3 to 4 weeks. Also problematic is the high immunogenicity.

Adeno-associated virus (AAV) has recently been preferred because it can complement the above problems and has many advantages as a gene therapy agent. AAV is a single-stranded provirus, which requires an auxiliary virus to replicate, and the AAV genome is 4,680 bp and can be inserted into a specific region of chromosome 19 of an infected cell. Transgene (trans -gene) is inserted in the plasmid DNA (plasmid DNA) is connected by two inverted terminal repeats of 145bp (inverted terminal repeat, ITR) sequence and partial signal sequence (signal sequence) part, respectively. Transfection is performed with other plasmid DNA expressing the AAV rep and cap portions and adenovirus is added as a co-virus. AAV has a wide range of host cells that deliver genes, fewer immune side effects when repeated administration, and long gene expression periods. Moreover, it is safe for the AAV genome to integrate into the chromosome of the host cell and does not modify or rearrange the gene expression of the host.

Since 1994, AAV vectors containing CFTR genes have been approved by the NIH for the treatment of cystic fibrosis, and have been used for clinical treatment of various diseases. AAV vectors containing a factor IX gene, which is a coagulation factor, are used for the treatment of hemophilia B (hemophilia B), and development of a hemophilia A (hemophilia A) therapeutic agent using the AAV vector is in progress. In addition, AAV vectors containing various types of anticancer genes have been certified for use as tumor vaccines.

Gene therapy with VEGF can be exemplified by the recombinant defective adenovirus (Korean patent application: 10-2001-7013633) comprising a nucleic acid encoding an angiogenic factor to treat pulmonary circulatory boost, but the therapy is a VEGF sense base. Because the sequence is used, it can be used only for the treatment of ischemic diseases, and cannot be used for the treatment of neovascularization diseases, especially cancer. Moreover, adenoviruses are not suitable for therapeutic use because they have high immunogenicity, low infectivity to host cells, and short duration of expression.

Although there have been many studies on polypeptides for treating cancer, most of the substances belong to chemotherapeutic agents. In addition, although research on polynucleotides has been continuously conducted, the results are insufficient in connection with a method of efficiently moving in vivo through a viral vector or the like.

Accordingly, the present inventors have prepared rAAV-AShVEGF prepared to insert VEGF antisense cDNA into the rAAV vector having high anticancer effect, and confirmed that rAAV-AShVEGF has a tumor suppressor effect in vivo and completed the present invention.

It is an object of the present invention to provide rAAV vectors containing gene therapy, VEGF-B antisense cDNA or VEGF-C antisense cDNA.

Another object of the present invention is to provide a colorectal cancer and / or lung cancer specific gene therapy containing rAAV vectors comprising VEGF antisense cDNA.

In order to achieve the above object, the present invention comprises the steps of: (a) transfecting an animal cell line with a pAAV vector containing the VEGF-B antisense cDNA of SEQ ID NO: 4, AAV rep-cap plasmid DNA and adenovirus helper plasmid; (b) culturing the transfected animal cell line; And (c) crushing the cultured cell line, and then separating and purifying the recombinant rAAV particles, to provide an rAAV vector containing the VEGF-B antisense cDNA of SEQ ID NO: 4.

The present invention also provides a method of transfecting an animal cell line, comprising: (a) transfecting an animal cell line with a pAAV vector, an AAV rep-cap plasmid DNA and an adenovirus helper plasmid containing the VEGF-C antisense cDNA of SEQ ID NO: 7; (b) culturing the transfected animal cell line; And (c) crushing the cultured cell line, and then separating and purifying the recombinant rAAV particles to provide an rAAV vector containing the VEGF-C antisense cDNA of SEQ ID NO: 7.

The invention also relates to a colorectal cancer comprising the rAAV vector containing the VEGF-B antisense cDNA, the rAAV vector containing the VEGF-C antisense cDNA and the rAAV vector containing the VEGF-A antisense cDNA of SEQ ID NO: 1 and / or Lung cancer-specific gene therapy is provided.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 pAAV Plasmid Cloning Containing Angiogenesis Inhibitory Genes

VEGF-A (NCBI accession # NM_003376 for human), VEGF-B (NCBI accession # NM_003377 for human) and VEGF-C (NCBI accession # NM_005429 for pAAV-FIX cis plasmid DNA (US 6,093,292) according to the cloning method In order to insert cDNA of human) into pAAV-FIX cis plasmid DNA in the antisense direction, the factor IX cDNA gene contained in pAAV-FIX cis plasmid DNA was replaced with the antisense cDNA of each of the above VEGF isoforms. Trans-gene constructs of pAAV-AShVEGF-B and pAAV-AShVEGF-C were constructed. In addition, trans-gene constructs of pAAV-AShVEGF-A-IRES-EGFP, pAAV-AShVEGF-B-IRES-EGFP, and pAAV-AShVEGF-C-IRES-EGFP, each VEGF isoform antisense cDNA linked to IRES-EGFP, were prepared. . The transgene was always inserted into plasmid DNA linked by two 145 bp, two inverted terminal repeat sequences (ITR), a human cytomegalovirus (CMV) immediate early promoter, and an SV40 early mRNA polyadenylation signal sequence.

(1) Construction of pAAV-AShVEGF-A

RNA was extracted from HUVEC (Cambrex Bio Science Walkersville, Inc., USA) cells to synthesize cDNA, and 429 bp human VEGF-A isoform antisense cDNA (SEQ ID NO: 1) by RT-PCR method using the following AShVEGF-A primer. ) (Positions 1025-1453). The amplification fragment was restriction enzyme Kpn I and Treatment with Xho I ligated with pAAV-FIX cis plasmid DNA digested with the same restriction enzyme to prepare pAAV-AShVEGF-A.

AShVEGF-A F2: GG GGTACC GTCTTGCTCTATCTTTC (SEQ ID NO: 2)

Kpn I

AShVEGF-A R1: CC CTCGAG GGCCTCCGAAACCATGAACT (SEQ ID NO: 3)

Xho I

(2) Construction of pAAV-AShVEGF-B

RNA was extracted from HUVEC (Cambrex Bio Science Walkersville, Inc., USA) cells to synthesize cDNA, and 466bp of human VEGF-B isoform antisense cDNA (SEQ ID NO: 4) by RT-PCR method using the following AShVEGF-B primer. (Positions 41-506) were amplified. Amplification fragments restriction enzyme EcoRV and Treatment with Xho I ligated with pAAV-FIX cis plasmid DNA digested with the same restriction enzyme to prepare pAAV-AShVEGF-B.

AShVEGF-B F2: A GATATC CAGAGTCCCAGCCCGGAACAGA (SEQ ID NO: 5)

Eco RV

AShVEGF-B R2: CC CTCGAG ATGAGCCCTCTGCTCCG (SEQ ID NO: 6)

Xho I

(3) Construction of pAAV-AShVEGF-C

RNA was extracted from HUVEC cells to synthesize cDNA, and 383bp of human VEGF-C isoform antisense cDNA (SEQ ID NO: 7) (positions 536-918) was amplified by RT-PCR method using the following AShVEGF-C primers. The amplified fragment with restriction enzymes Kpn I and Xho I by treatment with, to the ligated DNA and plasmid pAAV-FIX cis Lai cut with the same restriction enzymes, to prepare the pAAV-AShVEGF-C.

AShVEGF-C F1: GG GGTACC ACATCTGTAGACGGACACACA (SEQ ID NO: 8)

Kpn I

AShVEGF-C R3: CC CTCGAG CTCGACCTCTCGGACGC (SEQ ID NO: 9)

Xho I

(4) Construction of pAAV-AShVEGF-A-IRES-EGFP

pEGFP-N1 plasmid DNA (Clontech, USA) was treated with restriction enzymes Kpn I and Not I to prepare a linearized insert, and pAAV-FIX cis plasmid DNA used in Example 1 was treated with restriction enzymes Kpn I and Not I. pAAV-EGFP was constructed by ligation with insert.

pIRES2-EGFP plasmid DNA (Clontech, USA) was treated with restriction enzyme Nhe I, blunted with Klenow fragment, and then with restriction enzyme Not I to prepare a linearized insert, and the pAAV-EGFP plasmid DNA with restriction enzyme Kpn I PAAV-IRES-EGFP was prepared by ligating with a linearized vector treated with a restriction enzyme Not I, blunted with T4 DNA polymerase.

The pAAV-IRES-EGFP plasmid was treated with restriction enzyme Bam HI, and the previously constructed pAAV-AShVEGF-A plasmid DNA was treated with restriction enzyme Bam HI to ligate the two DNA fragments to pAAV-AShVEGF-A-IRES-. EGFP was produced.

(5) Construction of pAAV-AShVEGF-B-IRES-EGFP

pIRES2-EGFP plasmid DNA (Clontech, USA) was treated with restriction enzymes Xho I and Not I to prepare an IRES-EGFP cDNA, and the pAAV-AShVEGF-B plasmid prepared in this Example was used as Xho I and Bam HI restriction enzymes. After treatment, the cDNA and linearized plasmid were ligated to prepare pAAV-AShVEGF-B-IRES-EGFP.

(6) Construction of pAAV-AShVEGF-C-IRES-EGFP

pIRES2-EGFP plasmid DNA restriction enzymes Xho I and IRES-EGFP cDNA was prepared by treatment with Not I, and the pAAV-AShVEGF-C plasmid DNA prepared in (3) of this Example was treated with restriction enzymes Xho I and Bam HI, followed by ligation of the cDNA and linearized plasmid. PAAV-AShVEGF-C-IRES-EGFP was prepared.

Example 2 Construction of rAAV Vectors Used as Angiogenesis Inhibitory Gene Therapy

Recombinant to be used in gene therapy AAV (rAAV) in order to produce a vector of Example 1, each of pAAV plasmid produced from DNA in addition to AAV rep-cap plasmid DNA (pAAV-RC plasmid expressing AAV rep portion and a cap portion, Stratagene Co , USA) and adenovirus helper plasmids (pHelper plasmid, Stratagene Co., USA). All three kinds of plasmid DNA were simultaneously transfected into HEK293 (human embryonic kidney 293; ATCC CRL-1573) cells, and then cultured for 96 hours, HEK293 cells were collected and ultrasonically disrupted, and recombinant AAV (rAAV) particles were obtained. CsCl density gradient centrifugation was repeated three times, and a portion of RI (Refractive Index) of 1.37 to 1.41 g / ml was collected and separated.

The titration of the isolated rAAV-AShVEGF-A, rAAV-AShVEGF-B, and rAAV-AShVEGF-C virus particles was carried out by PCR primers of CMV promoter sites (CMV F1: 5'-GGG CGT GGA TAG CGG TTT GAC TC -3 '(SEQ ID NO: 10), CMV R1: 5'-CGG GGC GGG GTT ATT ACG ACA TT-3' (SEQ ID NO: 11)) was prepared and titrated by Quantitative PCR method. At this time, each of the pAAV plasmid DNA having a known concentration was used as a reference material, and the recombinant rAAV vector produced and separated by the above method was confirmed to obtain a rAAV particle titer of 10 ¹² to 10 ¹³ viral particles / ml.

The particle titration results of each rAAV are shown below.

(1) Particle titration of rAAV-AShVEGF-A

The concentration of pAAV-AShVEGF-A DNA measured by spectrophotometer was continuously diluted and subjected to PCR for standard curve. The results of electrophoresis at each concentration are shown in the left panel of FIG. 1A. From the left lanes, samples of 10 ⁴ to 10 ⁸ molecules were shown, and the intensity of the amplified DNA bands of each lane was measured using an imager densitometer (Alpha Innotech, USA).

The purely separated rAAV-AShVEGF-A vector was serially diluted to perform particle titration PCR, and the results of electrophoresis at each concentration are shown in the right panel of FIG. 1A. Samples of 10 ⁻³ to 10 ⁰ dilutions were shown in turn from the right lanes, and the amplified DNA band intensities of each lane were measured using an imager densitometer.

The standard curve in the right panel was made using the PCR result for the standard curve, and the titer of 5 × 10 ¹¹ viral particles / ml was obtained by applying the PCR result for particle titration to the standard curve (FIG. 1B).

(2) Particle Titration of rAAV-AShVEGF-A-IRES-EGFP

PAAV-AShVEGF-A-IRES-EGFP DNA was continuously diluted by spectrophotometer, and PCR for standard curve was performed. The result of electrophoresis at each concentration is shown in the left panel of FIG. 2A. From the left lanes, samples of 10 ³ to 10 ⁹ molecules were shown, and the intensity of the amplified DNA bands of each lane was measured using an imager densitometer (Alpha Innotech, USA).

The purely separated rAAV-AShVEGF-A-IRES-EGFP vector was serially diluted to perform particle titration PCR, and the results of electrophoresis at each concentration are shown in the right panel of FIG. 2A. Samples of dilution multiples of 10 ⁻⁴ to 10 ^{0 were shown in} turn from the right lane, and the amplified DNA band intensities of each lane were measured using an imager densitometer.

The standard curve in the right panel was made using the PCR result for the standard curve, and the titration value of 1.55 × 10 ¹¹ viral particles / ml was obtained by applying the PCR result for particle titration to the standard curve (FIG. 2B).

(3) Particle Titration of rAAV-AShVEGF-B-IRES-EGFP

PAAV-AShVEGF-B-IRES-EGFP DNA was continuously diluted by spectrophotometer, and PCR for standard curve was performed. The result of electrophoresis at each concentration is shown in the left panel of FIG. 3A. From the left lanes, samples of 10 ³ to 10 ⁸ molecules were shown in turn, and the intensity of the amplified DNA bands of each lane was measured using an imager densitometer (Alpha Innotech, USA).

The purely separated rAAV-AShVEGF-B-IRES-EGFP vector was serially diluted to perform particle titration PCR, and the results of electrophoresis at each concentration are shown in the right panel of FIG. 3A. Samples of 10 ⁻⁴ to 10 ⁰ dilutions were shown in turn from the left lanes, and the amplified DNA band intensities of each lane were measured using an imager densitometer.

The standard curve in the right panel was made using the PCR result for the standard curve, and the titration value of 5.30 × 10 ¹⁰ viral particles / ml was obtained by applying the PCR result for particle titration to the standard curve (FIG. 3B).

(4) Particle Titration of rAAV-AShVEGF-C-IRES-EGFP

PAAV-AShVEGF-C-IRES-EGFP DNA was continuously diluted by spectrophotometer and subjected to PCR for standard curve, and the result of electrophoresis at each concentration is shown in the left panel of FIG. 4A. From the left lanes, samples of 10 ³ to 10 ⁸ molecules were shown in turn, and the intensity of the amplified DNA bands of each lane was measured using an imager densitometer (Alpha Innotech, USA).

The purely separated rAAV-AShVEGF-B-IRES-EGFP vector was serially diluted to perform particle titration PCR, and the results of electrophoresis at each concentration are shown in the right panel of FIG. 4A. Samples of 10 ⁻⁴ to 10 ⁰ dilutions were shown in turn from the left lanes, and the amplified DNA band intensities of each lane were measured using an imager densitometer.

The standard curve on the right panel was made using the PCR result for the standard curve, and the titration value of 1.29 × 10 ¹¹ viral particles / ml was obtained by applying the PCR result for particle titration to the standard curve (FIG. 4B).

(5) Particle titration of rAAV-IRES-EGFP

PAAV-IRES-EGFP DNA serially measured with a spectrophotometer was continuously diluted and subjected to standard curve PCR, and the results of electrophoresis for each concentration are shown in the left panel of FIG. 5A. From the left lanes, samples of 10 ³ to 10 ⁸ molecules were shown in turn, and the intensity of the amplified DNA bands of each lane was measured using an imager densitometer (Alpha Innotech, USA).

Serially diluted rAAV-IRES-EGFP vectors were diluted in the above to perform particle titration PCR, and the results of electrophoresis at each concentration are shown in the right panel of FIG. 5A. Samples of dilution multiples of 10 ⁻⁴ to 10 ^{0 were shown in} turn from the right lane, and the amplified DNA band intensities of each lane were measured using an imager densitometer.

The standard curve in the right panel was made using the PCR result for the standard curve, and the titration value of 8.91 × 10 ¹¹ viral particles / ml was obtained by applying the PCR result for particle titration to the standard curve (FIG. 5B).

(6) Particle titration of rAAV-EGFP

PAAV-EGFP DNA was measured by the spectrophotometer serially dilute to perform the standard curve PCR electrophoresis for each concentration is shown in the left panel of Figure 6a. From the left lane, samples of 10 ⁵ to 10 ⁹ molecules were shown, and the intensity of the amplified DNA bands of each lane was measured using an imager densitometer (Alpha Innotech, USA).

Serially diluted rAAV-EGFP vectors were isolated from the above, and particle titration PCR was performed. The results of electrophoresis at each concentration are shown in the right panel of FIG. 6A. Samples of 10 ⁻³ to 10 ⁰ dilutions were shown in turn from the right lanes, and the amplified DNA band intensities of each lane were measured using an imager densitometer.

The standard curve in the right panel was made using the PCR result for the standard curve, and a titer of 5.36 × 10 ⁹ viral particles / ml was obtained by applying the PCR result for particle titration to the standard curve (FIG. 6B). .

Example 3. Cancer Cell Line Cultures and rAAV Vector Gene Therapy Treatment

Human colon cancer cells, to study the anticancer effects of preventing the growth and metastases of cancer on the in vitro state (T84: ATCC CCL-248) , human lung cancer cell line (# LCSC 1: WO 02/061069) and a human gastric cancer cell line (NUGC3, Five cancer cell lines of MKN45, MKN74) were cultured under the optimal conditions of each cell line. In order to examine the anticancer effect of the cancer cell line of the recombinant rAAV prepared in Example 2, cells were divided into 6 well plates by 10 ⁵ cell / well and grown for 24 hours, and various combinations of angiogenesis inhibitor rAAV vectors were collected. MOI was added to 10 ⁵ and incubated for 24 hours. The negative control group of the present Example was used in the same conditions as the recombinant rAAV prepared in Example 2 using rAAV vectors expressing only the same cell group without rAAV and GFP without VEGF antisense. Cells treated with the rAAV vector were washed twice with 1 × PBS and incubated for 72 hours with the addition of fresh medium.

Example 4. Measurement of Transduction Efficiency of Cancer Cell Lines

 Transduction efficiency of the cultured cells was confirmed by monitoring GFP protein expression through confocal microscopy.

The rAAV expressing GFP among the rAAV vectors prepared in Example 2 was used in each of the five cancer cell lines (T84, LCSC # 1, MKN45, MKN74, NUGC3) of Example 3, and the total MOI was 10 ^5. After 48 hours, the transduction efficiency was measured by monitoring GFP protein expression by confocal microscopy (excitation 488 nm; emission> 500 nm). Three types of rAAV vectors, rAAV-AShVEGF-A-IRES-EGFP, rAAV-AShVEGF-B-IRES-EGFP, and rAAV-AShVEGF-C-IRES-EGFP, are expected to be able to independently express GFP proteins Bicistronic. Infection efficiency can be measured by monitoring protein expression. Meanwhile, GFP protein expression was monitored by including both negative control cells not treated with the rAAV vector and control cells treated with the rAAV-IRES-EGFP vector.

As a result, as shown in Figures 7 to 10, when the four types of rAAV vectors infected with three or five cancer cell lines, the transduction efficiency was determined to be very high to almost 100%, anti-cancer genes RAAV was found to be very suitable as a therapeutic carrier.

Example 5 Confirmation of Angiogenesis Inhibitory Effect of rAAV Vectors in Cancer Cell Lines Using ELISA

(1) Measurement of VEGF Amount Reduction Effect of rAAV-AShVEGF-A Vector

rAAV-AShVEGF-A vector was treated 24 hours with total MOI of 10 ⁵ in 3 human gastric cancer cell lines (NUGC3, MKN74, MKN45), 1 human lung cancer cell line (LCSC # 1) and 1 human colorectal cancer cell line (T84) After 72 hours, the cells were disrupted and cell lysate was prepared. As a control group, two cell groups that were not treated with the rAAV vector and a group that were treated with the rAAV-GFP vector were used. Protein quantification of each cell lysates was performed, ELISA was performed using lysates containing 10 μg of total protein, and the change in VEGF amount by rAAV-AShVEGF-A vector treatment was measured three times. In the ELISA experiment for VEGF-A quantification, the amount of VEGF contained in each cell line was measured using an ELISA kit (RPN2779, Amersham Biosciences, USA). Cell lysate containing 10 μg of total protein was added to the plate coated with anti-human VEGF antibody and allowed to react for 2 hours at room temperature. The plate was washed three times, and the secondary antibody was added to the plate for 1 hour. After rinsing, streptavidin-HRP was added and treated again for 1 hour. Then, the substrate solution of HRP enzyme was added thereto, and the amount of VEGF generated by measuring the OD at 450 nm with a spectrophotometer was measured.

As a result, as shown in FIG. 11 and Table 1, in the colon cancer cell line T84, the amount of VEGF in the cell group treated with the rAAV-AShVEGF-A vector was reduced by more than 55% compared to the control group treated with the rAAV-EGFP vector. In the lung cancer cell line, LCSC # 1, the amount of VEGF was reduced by 45% compared to the control cell group. In contrast, three gastric cancer cell lines showed little change in VEGF levels.

Changes in VEGF Levels in Cancer Cell Lines Treated with rAAV-AShVEGF-A T84 MKN74 MKN45 NUGC3 LCSC # 1 negative 20.32 ± 0.28 2.95 ± 0.37 19.13 ± 0.67 16.13 ± 0.95 4.78 ± 0.18 Gfp 26.62 ± 0.71 3.69 ± 0.31 11.92 ± 0.72 13.55 ± 0.55 5.27 ± 0.24 AShVEGF-A 11.62 ± 1.74 2.65 ± 0.10 12.5 ± 0.37 12.91 ± 0.09 2.92 ± 0.36

Example 6 Angiogenesis Inhibition in vivo Anticancer effect study

Subcutaneous injection of 5 x 10 ⁶ cancer cells of the NCI-H460 human lung cancer cell line (ATCC HTB-177) into BABL / c nude mice (BABL / c nu / nu mouse, Central Laboratory Animal, Japan SLC, Inc.) The tumor model was made by injection.

The tumor volume of the tumor cells by BABL / c nude mice injected with reaches ^{200~300 mm 3 10 11 ~10 12 viral} particles / 1 object to rAAV vector (rAAV-AShVEGF-A in various combinations and rAAV-AShVEGF-A + rAAV-AShVEGF-B + rAAV-AShVEGF-C) was transduced by tail vein injection method. After 4 weeks of transduction, the general symptoms (normal state, motility, appearance, autonomic nerves, and death) of the animals were observed, the body weight was measured, and tumors were obtained using an electronic caliper (Absolute digimatic, Mitutoyo Corp., Japan). Volume was measured. After 28 days of transduction, the rats were autopsied and solid tumors were isolated from the rats. Tumor volume was measured using a digital plethysmometer (LE7500, Letica, Spain). Each organ (liver, spleen, kidney, heart, lung, testes and epididymis) and tumors are fixed with 10% neutral formalin and Bouin Solution (tumor, testicles, epididymis) after extraction, and then paraffin embedded sections to create immune tissue. Chemical and histological analyzes were performed. In order to measure endothelial cell density, immunohistochemical analysis was performed using anti-CD34 antibody, and histological analysis was performed by hematoxylin / eosin staining.

As a result, as shown in Figure 12, compared to the negative control group injected with HEPES, tumor volume in the experimental group injected with rAAV-AShVEGF-A and rAAV-AShVEGF-A + rAAV-AShVEGF-B + rAAV-AShVEGF-C It was confirmed that the decrease, especially in the experimental group injected with rAAV-AShVEGF-A + rAAV-AShVEGF-B + rAAV-AShVEGF-C showed a significant decrease in tumor volume.

Having described the specific parts of the present invention in detail, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be.

The present invention has the effect of providing a rAAV vector containing VEGF-B antisense cDNA or VEGF-C antisense cDNA for gene therapy. The invention also relates to a colorectal cancer and / or lung cancer specific gene comprising both rAAV vectors containing VEGF-A antisense cDNA, rAAV vectors containing VEGF-B antisense cDNA and rAAV vectors containing VEGF-C antisense cDNA. It has the effect of providing a therapeutic.

In accordance with the present invention, gene therapeutic agents can be effectively used to treat cancer at the genetic level by inhibiting the expression of VEGF involved in angiogenesis essential for tumor growth and metastasis, thereby reducing tumor growth.

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Claims (3)

delete delete Colon cancer comprising a rAAV vector containing a VEGF-B antisense cDNA of SEQ ID NO: 4, a rAAV vector containing a VEGF-C antisense cDNA of SEQ ID NO: 7 and a rAAV vector containing a VEGF-A antisense cDNA of SEQ ID NO: 1 And / or lung cancer-specific gene therapy.

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