WO2007026973A1

WO2007026973A1 - Recombinant adeno-associated virus containing vegfr truncated cdna and gene therapeutic agent for cancer comprising the same

Info

Publication number: WO2007026973A1
Application number: PCT/KR2005/002881
Authority: WO
Inventors: Keerang Park; Wun-Jae Kim; Young-Hwa Cho
Original assignee: Juseong College Industry Academy Cooperation Group
Priority date: 2005-08-31
Filing date: 2005-08-31
Publication date: 2007-03-08

Abstract

The present invention relates to a gene therapeutic agent using truncated cDNA of VEGF receptor protein (VEGFR) and adeno-associated virus(AVV) system, and more particularly, relates to a gene therapeutic agent specific to cancer, comprising a rAAV vector containing truncated cDNA of VEGFR-I, a rAAV vector containing truncated cDNA of VEGFR-2 and said vector. The gene therapeutic agent according to the present invention reduces the growth of tumors by inhibiting the expression and function of VEGF involved in angiogenesis necessary for the proliferation and metastasis of tumors. Thus, the inventive gene therapeutic agent can be effectively used to treat cancer at a gene level.

Description

Recombinant Adeno-associated Virus Containing VEGFR Truncated cDNA and Gene Therapeutic Agent for Cancer Comprising the Same

TECHNICAL FIELD

The present invention relates to a gene therapeutic agent using truncated cDNA of VEGF receptor protein (VEGFR) and adeno-associated virus (AVV) system, and more particularly, relates to a gene therapeutic agent specific to cancer, comprising a rAAV vector containing truncated cDNA of VEGFR- 1 , a rAAV vector containing truncated cDNA of VEGFR-2 and said vector.

BACKGROUND ART

Mortality caused by cancer is on the rise annually worldwide. Cancer is one of the leading causes of death in the world together with infectious diseases and cardiovascular diseases. According to the World Health Report of WHO, 7,121,000 people that account for 12.5% of the total number of deaths in 2004 died of cancer, and it is expected that, due to eating habits, changes in environment and an increase in life span, the population with cancer incidence will increase to about 30,000,000 people every year within 25 years into the future, and a population of 20,000,000 people of which will die of cancer.

Currently, methods used for the treatment of cancer can be broadly divided into surgical operation, radiotherapy and drug therapy, which are used alone or in combination of two or more thereof for the treatment of cancer. Generally, the surgical operation can be applied in various cancer stages. Cancer in an early stage can be treated by the surgical operation, but in the case where cancer is significantly progressed or metastasized, the surgical operation is used in combination with radiotherapy or drug therapy because it is difficult to achieve the treatment of the cancer only by the surgical operation. The radiotherapy is a method of treating cancer by external radiation or by irradiating cancer cells with X-rays or γ-rays from radioactive substances administered in vivo. The drug therapy is a method of administering an anticancer agent orally or by injection to destroy or suppress the DNA or related enzymes, required for the proliferation of cancer cells. In comparison with other methods, the advantages of the drug therapy are that it allows a drug to be delivered to cancer occurred in any site of the body and can treat metastasized cancer. Currently, the drug therapy is used as a standard therapy for the treatment of metastatic cancer. Although it is, of course, impossible to completely cure metastasized cancer by the drug therapy, the drug therapy plays an important role in increasing life span by alleviating symptoms. However, chemical therapeutic agents used in this chemical therapy have problems, such as side effects and anticancer drug resistance.

However, by virtue of the remarkable development of the bioscience field, biotherapeutic agents are rapidly being developed. The biotherapeutic agents are used for the basic purpose of restoring or increasing the natural immune function of the body to weaken the activity of cancer cells, thus preventing the progression of cancer. If the immune system of the body exhibits its own function, the death of cancer cells can be effectively induced, but if not, cancer cells will be easily proliferated or our body gets easily attacked by other germs. To reduce this disadvantage, the biotherapeutic agents are sometimes used in combination with other therapies, such as surgical therapy, radiotherapy and chemotherapy.

Currently, biotherapeutic agents receiving attention in the bioscience field may include anti-sense anticancer agents and angiogenesis inhibitors. The anti-sense anticancer agents are strategies of using DNA fragments capable of complementally binding to cancer cell-specific mRNAs, to inhibit the processing of mRNAs or the expression of proteins, thus inducing the death of cancer cells. As a result of the human genome project, about 30,000 gene sequences were interpreted and about 100,000 mRNA sequences could be identified. With this, as a large amount of information for cancer cell-associated mRNA candidates are established, genes associated with signaling pathways and genes associated with apoptosis and cell proliferation are screened and now in clinical trials.

The growth of tumors is possible only by angiogenesis supplying oxygen and nutrients required therefor. Generally, as cancer cells proliferate, the inside of tumors will become a low-oxygen state, and tissue necrosis will occur. Also, blood vessels will be broken by the pressure of tumors themselves to make the low- oxygen state more severe. To overcome this low-oxygen state, tumors express angiogenesis-associated factors (e.g., VEGF, bFGF, IL-8, PDGF, PD-EGF, etc.), thus stimulate angiogenesis. Namely, angiogenesis is a necessary process for the growth of tumors.

Angiogenesis inhibitors are used to interfere with angiogenesis caused by tumors so as to suppress the growth of tumors, thus treat cancer. Direct angiogenesis inhibitors interfere with angiogenesis by inhibiting the proliferation or migration of vascular endothelial cells or by inhibiting reactions to angiogenesis factors. The direct angiogenesis inhibitors have an advantage in that they cause less acquired drug resistance. Indirect angiogenesis inhibitors suppress angiogenesis by inhibiting the expression of proteins in tumors activating angiogenesis or by blocking the binding between tumor proteins and vascular endothelial cell surface receptors.

As described above, it can be seen that the therapeutic substances in this field has been changed from the use of artificial chemical substances to the use of natural substances in vivo. Particularly, as genome research projects in various fields are conducted, genes acting as the cause of various diseases are found. In order to apply such research results to clinical treatment or prevention, studies on the gene transfer technology of introducing genes for correcting genes with lost or modified function into the body to normalize function or activate therapeutic function, i.e., studies on the gene therapy field, are actively being conducted. As interests and studies are concentrated in the gene therapy field for recent several years, the results therefrom are rapidly increasing.

The gene therapy, which is a method of treating diseases by gene transfer and expression, is used to correct the genetic defect of a certain disordered gene, unlike the drug therapy. The ultimate purpose of the gene therapy is to obtain useful therapeutic effects by genetically modifying a living gene. The gene therapy has various advantages, such as the accurate transfer of a genetic factor into a disease site, the complete decomposition of the genetic factor in vivo, the absence of toxicity and immune antigenicity, and the long-term stable expression of the genetic factor and thus is being spotlighted as the best therapy for the treatment of diseases.

The main research field for gene therapy can be summarized in three fields of introducing a gene showing a therapeutic effect on a certain disease, increasing the resistant function of a normal cell so as to show resistance to an anticancer agent and the like, or substituting for a modified or deleted gene in patients with various genetic diseases.

The gene therapies are broadly classified into two categories, i.e., in vivo and in vitro therapies. The in vivo gene therapy comprises introducing a therapeutic gene directly into the body, and the in vitro gene therapy comprises culturing a target cell in vitro, introducing a gene into the cell, and then, introducing the genetically modified cell into the body. Currently, in the research field for gene therapy, the in vitro therapy is more frequently used than the in vivo therapy.

The gene transfer technologies are broadly divided into a viral vector-based transfer method using virus as a carrier, a non-viral delivery method using synthetic phospholipid or synthetic cationic polymer, and a physical method, such as electroporation of introducing a gene by applying temporary electrical stimulation to a cell membrane.

Among the gene transfer technologies, the viral vector-based transfer method is considered to be preferable for the gene therapy because the transfer of a genetic factor can be efficiently made with a vector with the loss of a portion or whole of replicative ability, which has a gene having a therapeutic gene substituted. Examples of virus used as the virus carrier or vector include RNA virus vectors (retrovirus vectors, lentivirus vector, etc.), and DNA virus vectors (adenovirus vectors, adeno-associated virus vectors, etc.). In addition, its examples include herpes simplex viral vectors, alpha viral vectors, etc. Among them, retrovirus and adenovirus vectors are being particularly actively studied.

The characteristics of retrovirus acting to integrate into the genome of host cells are that it is harmless to the human body, but can inhibit the function of normal cells upon integration, also it infects various cells, proliferates easily, can receive about 1-7 kb of foreign genes, and is capable of producing replication-deficient virus. However, it has disadvantages in that it is hard to infect cells after mitosis, it is difficult to transfer a gene in vivo, and the somatic cell tissue needs to be proliferated always in vitro. In addition, since it can be integrated into a proto- oncogene, it has the risk of mutation and can cause cell necrosis.

Meanwhile, adenovirus has various advantages for use as a cloning vector; it has moderate size, can be replicated within a cell nucleus, and is clinically nontoxic. Also, it is stable even when inserted with a foreign gene, and does not cause the rearrangement or loss of genes, can transform eucaryotes, and is stably expressed at a high level even when it is integrated into the chromosome of host cells. Good host cells for adenovirus are cells causing human hematosis, lymphoma and myeloma. However, these cells are difficult to proliferate because they are linear DNAs, it is not easy to recover infected virus, and they have low virus infection rate. Also, the expression of a transferred gene is the highest after 1-2 weeks, and in some cells, the expression is kept only for about 3-4 weeks. In addition, these have the problem of high immune antigenicity.

Adeno-associated virus (AAV) can overcome the above-described problems, at the same time, has many advantages for use as a gene therapeutic agent and thus is recently considered to be preferable. AAV, which is single-strand provirus, requires an assistant virus for replication, and the AAV genome is 4,680 bp in size and can be inserted into a certain site of chromosome 19 of infected cells. A trans-gene is inserted into plasmid DNA linked with 145 bp of each of two inverted terminal repeat sequence (ITR) and a signal sequence. This gene is transfected with another plasmid DNA expressing AAV rep and cap genes, and adenovirus is added as an assistant virus. AAV has advantages in that the range of its host cells to be transferred with a gene is wide, immune side effects due to repeated administration are little, and the gene expression time is long. Furthermore, it is stable even when the AAV genome is integrated into the chromosome of a host cell, and it does not cause the modification or rearrangement of gene expression in host cells.

Since an AAV vector containing a CFTR gene was approved by NIH for the treatment of cystic fibrosis in 1994, it has been used for the clinical treatment of various diseases. An AAV vector containing a factor IX gene, which is a blood coagulation factor, is used for the treatment of hemophilia B, and the development of a therapeutic agent for hemophilia A using the AAV vector is currently being conducted. Also, AAV vectors containing various kinds of anticancer genes were certified for use as tumor vaccines.

Gene therapies using VEGF can be exemplified by a recombinant deficient adenovirus containing a nucleic acid encoding an angiogenesis factor for the treatment of pulmonary hypertension (Korean patent application No. 10-2001- 7013633). However, because this therapy uses a VEGF sense base sequence, it can be used only for the treatment of ischemic diseases, and is unsuitable for the treatment of diseases caused by angiogenesis, particularly cancer. Furthermore, adenovirus is unsuitable to use for treatment, because it has the disadvantages of high immune antigenicity, low infectivity towards host cells, and short gene expression time.

Studies on polypeptides for the treatment of cancer have been made in the prior art, but most of these substances belong to chemical therapeutic agents. Also, studies on polynucleotides have been continually made, but the results thereof are insufficient in connection with a method of effectively transferring the polynucleotides in vivo by virus vectors, and the like.

Meanwhile, the present inventors have found that a rAAV-ASh VEGF-A vector containing the anti-sense cDNA of VEGF-A (Korean patent application No. 10- 2004-54043), and a rAAV-AShVEGF-ABC vector containing the anti-sense cDNAs of VEGF-A, VEGF-B and VEGF-C (PCT/KR2005/002435), are effective as gene therapeutic agents for cancer. In addition, the present inventors filed a patent application relating to a gene therapeutic agent for cancer, comprising a rAAV- AShVEGF-A vector containing the anti-sense cDNA of VEGF-A, a rAAV- TShVEGFR-I vector containing the truncated soluble cDNA of VEGFR-I, and a rAAV-TShVEGFR-2 containing the truncated soluble cDNA of VEGFR-2 (Korean patent application No. 10-2005-67661).

The present inventors have made extensive efforts to develop a more effective therapeutic agent for cancer, as a result, constructed a rAAV-TRh VEGFR-I vector and a rAAV-TRh VEGFR-2 vector containing truncated soluble cDNA of VEGFR, and found that said vectors inhibit the function of VEGF and also show excellent tumor inhibitory effects in vivo, thereby completing the present invention.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a rAAV vector containing truncated cDNA of VEGFR-I and a rAAV vector containing truncated cDNA of VEGFR-2, for gene therapy.

It is another object of the present invention to provide a gene therapeutic agent specific to cancer, comprising the above rAAV vector containing truncated cDNA of VEGFR-I and/or the above rAAV vector containing truncated cDNA of VEGFR-2.

To achieve the above object, the present invention comprises following steps: (a) transfecting a pAAV vector containing truncated cDNA of VEGFR-I, AAV rep-cap plasmid DNA and an adeno-virus helper plasmid into an animal cell line; (b) culturing the transfected animal cell line; and (c) isolating and purifying recombinant rAAV particles after disrupting the cultured cell line; and provides a rAAV vector containing truncated cDNA of VEGFR- 1.

The present invention also comprises following steps: (a) transfecting a pAAV vector containing truncated cDNA of VEGFR-2, AAV rep-cap plasmid DNA and an adeno-virus helper plasmid into an animal cell line; (b) culturing the transfected animal cell line; and (c) isolating and purifying recombinant rAAV particles after disrupting the cultured cell line; and provides a rAAV vector containing truncated cDNA of VEGFR-2.

The present invention also provides a gene therapeutic agent specific to cancer, comprising the above rAAV vector containing truncated cDNA of VEGFR-I and the above rAAV vector containing truncated cDNA of VEGFR-2.

In the present invention, the truncated cDNA of VEGFR-I and truncated cDNA of VEGFR-2 preferably have SEQ ID NOs: 1 and 4, respectively. The gene therapeutic agent according to the present invention is preferably specific to large intestine cancer, bladder cancer and lung cancer.

Other features and embodiments of the present invention will be more fully apparent from the following detailed description and appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a gene map of p A AV-TRh VEGFR-I according to the present invention.

FIG. 2 is a gene map of pAAV-TRhVEGFR-2 according to the present invention.

FIG. 3A is a result showing the analysis of wound healing effect of rAAV- TRhVEGF-I and/or rAAV-TRhVEGF-2 according to the present invention, FIG. 3B is a microscopic photograph showing wound healing effect of rAAV- TRhVEGF-I and/or rAAV-TRhVEGF-2 according to the present invention.

FIG. 4 is a gene map of rAAV-AShVEGF-A.

FIG. 5 shows the change in tumor volume of nude mouse intra-abdominally injected with the rAAV vector according to the present invention. DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS THEREOF

Hereinafter, the present invention will be described in more detail by specific examples. However, the present invention is not limited to these examples, and it is obvious to those who are skilled in the art that numerous variations or modifications could be made within the spirit and scope of the present invention.

Example 1. Cloning of pAAV plasmid containing a gene having angiogenesis inhibitory function

(1) Construction of pA AV-TRhVEGFR-I

cDNA was synthesized by the extraction of RNA from HUVEC (Cambrex Bio Science Walkersville, Inc., USA) cells, and 2,427bρ of truncated human VEGFR-I receptor cDNA (SEQ ID NO: 1) was amplified by RT-PCR using the following primers, TRhVEGFR-I Fl and TRhVEGFR-I Rl. The amplified fragment was digested with restriction enzymes Kpnl and Xhol and ligated with a pAAV-FIX cis plasmid DNA (US 6,093,292) digested with the same restriction enzymes, thus constructing pAAV-TRhVEGFR- 1 (FIG. 1).

TRhVEGFR-I Fl (SEQ ID NO: 2):

AAQg7}4CC6iCG4CCΑTGGTCAGCTACTGGGACA Kpnl Kozak

TRhVEGFR-I Rl (SEQ ID NO: 3):

CCC7£^46C7MCTGCTCATCCAAAGGAACTTCA Xhol stop c o don

(2) Construction of pAAV-TRhVEGFR-2 cDNA was synthesized by the extraction of RNA from cancer cell line LCSC#1 (WO 02/061069), and 2,430bp of truncated human VEGFR-2 receptor cDNA (SEQ ID NO: 4) was amplified by RT-PCR using the following primers, TRhVEGFR-2 Fl and TRhVEGFR-2 Rl. The amplified fragment was digested with restriction enzymes Kpnl and Xhol, and ligated with pAAV-FIX cis plasmid DNA digested with the same restriction enzymes, thus constructing pA AV-TRh VEGFR-2 (FIG. 2).

TRhVEGFR-2 Fl (SEQ ID NO: 5): GGGGTACCGCCACCATGGAGAGCAAGGTGCT

Kpnl Kozak TRhVEGFR-2 Rl (SEQ ID NO: 6):

CC CTCGAGCTATTCATCTGGATCCATGAC

Xhol stop codon

Example 2. Construction of rAAV vector for use as gene therapeutic agent for inhibition of angiogenesis

To construct a recombinant AAV (rAAV-TRhVEGFR-1) vector for use in gene therapy, AAV rep-cap plasmid DNA (pAAV-RC plasmid; Stratagene Co., USA) expressing AAV rep and cap genes and an adenovirus helper plasmid (pHelper plasmid; Stratagene Co., USA) are required in addition to the pAAV-TRhVEGFR-1 plasmid DNA constructed in Example 1. These three plasmid DNAs (pAAV- TRhVEGFR-I, pAAV-RC and pHelper) were all transfected into HEK293 (human embryonic kidney 293; ATCC CRL- 1573) cells, and cultured for 96 hours. The cultured HEK293 cells were collected and disrupted by sonication, and the recombinant AAV (rAAV) particles were subjected to CsCl density gradient centrifugation three times, so as to collect a pure fraction with a RI (Refractive Index) of 1.37-1.41 g/ml, thus obtaining a rAAV-TRhVEGFR-1. A rAAV-TRhVEGFR-2 was obtained in the same manner as described above except that pAAV-TRhVEGFR-2 was used in place of p AAV-TRhVEGFR-I.

The titration of the obtained rAAV-TRhVEGFR-1 and rAAV-TRhVEGFR-2 particles was performed by quantitative PCR using the following PCR primers constructed for a CMV promoter region:

CMV Fl (SEQ ID NO: 7): 5'-GGG CGT GGA TAG CGG TTT GAC TC-3' CMV Rl (SEQ ID NO: 8): 5'-CGG GGC GGG GTT ATT ACG ACA TT-3'. At this time, each of pAAV plasmid DNAs with known concentrations was used as a standard substance, and it was found that the recombinant rAAV produced and isolated as described above generally had a rAAV particle titer of 10¹²~10¹³ viral particles/ml.

Example 3: Wound healing assay of the inventive rAAV vector

HUVEC cells cultured in a complete medium (EGM-2 BulletKit, Cat# CC-3162, Cambrex Bio Science Walkersville, Inc., USA) were treated with each or both of rAAV-TRhVEGFR-1 and rAAV-TRhVEGFR-2 to an M.O.I of 2 x 10⁵ for 24 hours. After 48 hours, a plate where the cells have been grown was wounded with a cell scraper. The wounded plate was washed two times with collection medium [EBM-2 (Cat# CC-3156, Cambrex Bio Science Walkersville, Inc., USA) + 1% FBS] to remove detached cells. The plate was treated with 10 ng/ml of a VEGF protein (Calbiochem, Cat. #676472), and 24 hours later, migrated cells were photographed and analyzed.

As a result, as shown in Table 1, FIG. 3 A and FIG. 3B, the number of the migrated cells was remarkably lower in the case of treatment with rAAV- TRh VEGFR-I and/or rAAV-TRhVEGFR-2 than a control group and a group treated with the VEGF protein alone. This suggests that treatment with r AAV-TRhVEGFR-I and/or rAAV-TRhVEGFR-2 inhibits the function of VEGF, thus delaying wound-healing. From this result, it could be seen that the inventive r AAV-TRhVEGFR-I vector and rAAV- TRhVEGFR-2 vector inhibited VEGF from causing angiogenesis by the migration of vascular endothelial cells, so as to prevent the proliferation of tumors, thus showing anticancer effects.

Table 1 : Delaying effect of wound-healing by treatment of rAAV vector according to this invention

Example 4; Anticancer effect of rAAV vector (Xenograft assay)

In this Example, the anticancer effect of the case administered with [rAAV- TRhVEGFR-I + rAAV-TRhVEGFR-2] having anticancer effects proven in the wound healing migration assay of Example 3, was examined in vivo.

First, tumor model was prepared by injecting 5 x 10 NCI-H460 human lung cancer cell line (ATCC HTB- 177) to BABL/c nude mice (BABL/c nu/nu mouse, Central Experiment Animal Inc., Japan SLC, Inc.) by hypodermic injection.

When the tumor volume of BABL/c nude mouse, injected with the above cancer cell, reaches 70~80mm³, 10^π~10¹² viral particles/mouse of rAAV vector (rAAV- AShVEGF-A and rAAV-TRhVEGFR-l+rAAV-TRhVEGFR-2 was transducted by tail vein injection. After the transduction, general symptoms of animals (general condition, motility, appearance, autonomic nerve and death) were observed for 3 weeks, weight was measured, and the tumor volume was measured using electric calipers (Absolute digimatic, Mitutoyo Corp., Japan).

The rAAV- ASh VEGF-A vector (FIG. 4) used as a control group in this Example was constructed by following method. cDNA was synthesized by the extraction of RNA from HUVEC (Cambrex Bio Science Walkersville, Inc., USA) cells, and 429bp of human VEGF-A isoform antisense cDNA (1025-1453 region) was amplified by RT-PCR using the following AShVEGF-A primer. The amplified fragment was digested with restriction enzymes Kpnl and Xhol and ligated with pAAV-FIX cis plasmid DNA digested with the same restriction enzymes, thus constructing pAAV-AShVEGF-A.

AShVEGF-A F2 (SEQ ID NO: 9): GGGGΣ4CCGTCTTGCTCTATCTTTC Kpnl

AShVEGF-A Rl (SEQ ID NO: 10):CCCJC£4(7GGCCTCCGAAACCATGAACT

Xhol

Meanwhile, using taxol (paclitaxel; Cat# Tl 912, Sigma, USA) currently widely being used, which is an anticancer agent, as a control group, xenograft experiment was performed simultaneously. When the tumor volume of BABL/c nude mice, injected with cancer cell, reaches 70~80mm³, 200μg/mouse was intraperitoneally injected, and from this point on, the same dose was injected once every week during the experiment period. Conditions of the animals were observed, and bodyweight and tumor volume of the animals were measured in the same way as the other test groups. After taxol (paclitaxel) was dissolved in DMSO to a concentration of 50 mg/ml, taxol was diluted to a concentration of 1 mg/ml with HEPES buffer solution before injection, and 0.2ml (200 μg/mouse dose) was administered to each mouse by intraperitoneal injection. Bodyweight of all the animals was measured just before administration, and at an interval of 3 days after administration of the test substance, the results showed a low increase in bodyweight, and there was no correlation between cancer therapeutic effect and a change in bodyweight of the test animals.

Meanwhile, the volume of tumors was measured at an interval of 3 days with Vernier calipers for 3 weeks. As a result, as shown in FIG. 5, it could be seen that the group administered with [rAAV-TRhVEGFR-1 + rAAV-TRhVEGFR-2], which is a gene therapeutic agent according to the present invention, showed excellent tumor inhibitory effect as compared to the group administered with rAAV- AShVEGF-A and the group administered with taxol.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

INDUSTRIALAPPLICABILITY

As described in detail above, the present invention provides the rAAV vector for gene therapy containing truncated cDNA of VEGFR-I and truncated cDNA of VEGFR-2. The present invention also provides a gene therapeutic agent specific to cancer, comprising the rAAV vector containing truncated cDNA of VEGFR-I and/or the rAAV vector containing truncated cDNA of VEGFR-2. The gene therapeutic agent according to the present invention reduces the growth of tumors by inhibiting the expression and function of VEGF involved in angiogenesis necessary for the proliferation and metastasis of tumors. Thus, the inventive gene therapeutic agent can be effectively used to treat cancer at a gene level.

Claims

THE CLAIMS

What is claimed is:

L A rAAV vector containing truncated cDNA of VEGFR-I, which is constructed by the following steps:

(a) transfecting a pAAV vector containing truncated cDNA of VEGFR-I, AAV rep-cap plasmid DNA and an adeno- virus helper plasmid into an animal cell line; (b) culturing the transfected animal cell line; and

(c) isolating and purifying recombinant rAAV particles after disrupting the cultured cell line.

2. The rAAV vector according to claim 1, wherein the truncated cDNA of VEGFR- 1 has a DNA sequence of SEQ ID NO: 1.

3. A rAAV vector containing truncated cDNA of VEGFR-2, which is constructed by the following steps:

(a) transfecting a pAAV vector containing truncated cDNA of VEGFR-2, AAV rep-cap plasmid DNA and an adeno-virus helper plasmid into an animal cell line;

(b) culturing the transfected animal cell line; and

4. The rAAV vector according to claim 3, wherein the truncated cDNA of VEGFR- 2 has a DNA sequence of SEQ ID NO: 4.

5. A gene therapeutic agent specific to cancer, comprising the rAAV vector of claim 1 or claim 2 and the rAAV vector of claim 3 or claim 4.