KR20040077600A - Alphavirus-based expression vectors expressing tumor suppressor proteins, the preparation and the use thereof - Google Patents

Abstract

PURPOSE: Alphavirus-based expression vectors expressing tumor suppressor proteins, a method for preparing the same and the use thereof are provided, thereby simultaneously expressing various tumor suppressor proteins, and consequently suppressing tumor effectively. CONSTITUTION: The alphavirus-based expression vector expressing tumor suppressor proteins comprises a first tumor suppressing gene or angiogenesis suppressing gene which is operably linked to a first subgenomic promoter; a second tumor suppressing gene or angiogenesis suppressing gene which is operably linked to a second subgenomic promoter; and a third tumor suppressing gene or angiogenesis suppressing gene which is operably linked to a third subgenomic promoter, wherein the first, second and third tumor suppressing genes or angiogenesis suppressing genes are the same or different; the tumor suppressing gene is p53 gene or PTEN gene; the antigiogenesis suppressing gene is angiostatin gene; and the alphavirus-based expression vector expresses tumor suppressor proteins Semliki forest virus(SFV) and Sindbis virus.

Description

Alphavirus-based expression vector expressing cancer suppressor protein, preparation method thereof and use thereof {ALPHAVIRUS-BASED EXPRESSION VECTORS EXPRESSING TUMOR SUPPRESSOR PROTEINS, THE PREPARATION AND THE USE THEREOF}

The present invention relates to a method of treating cancer using cancer suppression genes. More specifically, the present invention relates to an alphavirus expression vector, preferably a Semliki Forest Virus (hereinafter abbreviated as SFV) -based expression vector expressing a foreign cancer suppressor protein, a method and a preparation thereof. will be.

In many malignant tumor cells, specific chromosomal deletions have been found. Oncogenesis of normal cells when certain genes are missing means that these genes maintain normal cell function and at the same time prevent cell cancer, and in fact most of these missing genes are tumor suppressors known as gene.

Surgical treatment, drug therapy, and radiation therapy are mainly used to treat malignant tumors. In addition, gene therapy is introduced by introducing foreign genes into cancer cells, thereby treating cancer, or gene therapy. Recently developed.

An example of cloning a tumor suppressor gene into a recombinant vector for gene therapy is when p53 gene is inserted into a recombinant adenovirus and applied to human ovarian cancer, head cancer, neck cancer, chest cancer, and lung cancer cells [ Kalpana Mujoo. et al., Oncogene, 12, 1617-1623 (1996); Ta-Jen Liu, et al., Cancer Research, 15, 3662-3667 (1994); Prem Seth, et al., Cancer Research, 15, 1346-1351 (1996); US Patent No. 6,143,290]. The literature also reports examples of cloning the PTEN gene to recombinant adenovirus or retrovirus and applying it to human brain or ovarian cancer cells [Da-Ming Li, et al., Proc. Natl. Acad. Sci. USA, 95, 15406-15411 (1998); L. Wayne Cheney, et al., Cancer Research, 58, 2331-2334 (1998); Takeo Minaguchi, et al., Cancer Research, 59, 6063-6067 (1999).

In some cases, two different cancer suppressor genes were simultaneously expressed in cancer cells. James et al. Produced recombinant adenovirus vectors containing p53 and p21, respectively, and applied them to cancer cells simultaneously. Volkner et al. Virus has been cloned and applied to human liver, chest, neck, and colon cancer cells [James A. et al., Cancer Research, 55, 5151-5155 (1995); Volker Sandig. et al., Nature Medicine 3, 313-318 (1997).

Angiogenesis inhibitor proteins that normally exist in the body and regulate homeostasis include angiostatin, endostatin, thrombopoietin, interferon, platelet factor IV, interleukin-12, proliferlin-related proteins, and protease inhibitors. There are also examples of gene therapy using these proteins. When the angiostatin gene was inserted into a recombinant adenovirus [Frank Griscell, et al., Proc. Natl. Acad. Sci. USA, 95, 6367-6372 (1998)], when angiostatin was inserted into a retrovirus [Toshihide Tanaka, et al., Cancer Research, 58, 3362-3369 (1998)], and angiostatin and endostatin genes were recombinant adenosine, respectively. For example, when inserted into a virus and applied to human cancer cells at the same time (Josephine T. et al., Cancer Research, 58, 5673-5677 (1998)).

However, all of the methods performed above were when one cancer suppressor protein was cloned into one vector. That is, until now, a recombinant vector expressing one cancer suppressor protein was applied to a cell, or two kinds of recombinant vectors expressing one cancer suppressor protein were simultaneously applied to a cell, but more than one cancer suppressor was applied to one vector. No successful cloning of the protein has been reported.

The present inventors have succeeded in making an expression vector expressing two or more cancer suppressor proteins using alphaviruses, and further confirm that certain combinations of cancer suppressor proteins show synergistic effects in the cell and complete the present invention. Reached.

The present invention relates to a method of treating cancer using an alphavirus-based expression vector, its RNA transcript or recombinant virus.

The present invention provides an alphavirus-based expression vector expressing a cancer suppressor gene and / or an angiogenesis inhibitor gene.

The present invention provides a method for preparing cancer suppressor or angiogenesis inhibiting protein, comprising introducing and expressing the expression vector, its RNA transcript or recombinant virus into a host cell.

The present invention provides a method of treating cancer by introducing a vector comprising at least two genes selected from p53, PTEN, and angiostatin, an RNA transcript thereof, or a recombinant virus into a cell.

The present invention provides a pharmaceutical composition comprising as an active ingredient a vector comprising two or more genes selected from p53, PTEN and angiostatin, an RNA transcript thereof or a recombinant virus containing the same.

1 is a schematic diagram showing the manufacturing process of pcSFV and pc-helper.

Fig. 2 is a schematic diagram showing the manufacturing process of pcSFV-p53.

3 is a schematic diagram showing the manufacturing process of pcSFV-Agt and pcSFV-PTEN.

4 is a schematic diagram showing a manufacturing process of pcSFV-PTEN / p53.

5 is a schematic diagram showing a manufacturing process of pcSFV-Agt / p53.

6 is a schematic diagram illustrating a manufacturing process of pcSFV-Agt / p53 / PTEN.

FIG. 7 shows the expression of p53 (A), PTEN (B) and angiostatin (C) by transfection (column 2) or infection of BHK-21 cells with RNA and recombinant virus (column 3) by immunocytochemical method. This is a confirmed picture.

8 is a photograph confirmed by Western immunoblotting the expression of p53 (A), PTEN (B) and angiostatin (C) by infecting BHK-21 cells with recombinant virus.

9 is a photograph confirming the expression of p53 by immunocytochemistry by infecting U87MG cells with a recombinant virus.

10 is a photograph confirming the expression of p53 by immunocytochemistry by infecting U251MG cells with a recombinant virus.

11 is a photograph confirming the expression of p53 by Western immunoblotting by infecting U87MG and U251MG cells with a recombinant virus.

12 is a schematic diagram confirming the expression of PTEN by immunocytochemical method by infecting U87MG and U251MG cells with a recombinant virus.

Figure 13 is a schematic diagram confirming the expression of PTEN by Western immunoblotting by infecting U251MG cells with recombinant virus.

14 is a photograph confirming the expression of angiostatin by immunocytochemistry by infecting U251MG cells with a recombinant virus.

15 is a photograph confirming the expression of angiostatin by infecting U251MG cells with a recombinant virus in cells (A) or in extracellular medium (B), by Western immunoblotting.

16 is a chart measuring the viability of U87MG and U251MG cells infected with recombinant virus.

Figure 17 is a chart confirming apoptosis in U251MG cells infected with recombinant virus by FACScan.

18 is a chart confirming the inhibition of cancer by intratumorally administering a recombinant virus to nude mice with U251MG cells.

19 is a chart confirming the inhibition of cancer by intratumorally administering a recombinant virus to nude mice with U251MG cells.

20 is a photograph confirming the expression of p53 (A), PTEN (B) and angiostatin (C) by transfecting BHK-21 cells with the expression vector DNA of the present invention by immunocytochemical method.

21 is a photograph confirming the expression of p53 (A), PTEN (B) and angiostatin (C) by Western immunoblotting by transfecting BHK-21 cells with the expression vector DNA of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides an alphavirus-based expression vector expressing a cancer suppressor gene and / or an angiogenesis inhibitor gene.

"Alphavirus" refers to viral species and subtypes commonly classified in the art as alpha viruses, for example Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Encephalitis virus (WEE), Sindbis virus, South African Arbovirus No. 86, Girdwood S.A. virus, Ockelbo virus, Semliki Forest virus, Middleburg virus, Chikungunya virus, O'Nyong-Nyong virus, Ross River virus, Barmah Forest virus, Mayaro virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus This includes what is classified as an alpha virus by the International Committee on Taxonomy of Viruses, Whataroa virus, Babanki virus, Kyaylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, Buggy Creek virus and other virus scientific names. .

Alphavirus is a virus belonging to Togaviridae, a positive-strand RNA virus with a coating, and can infect a wide range of cells (insects, birds, mammals). It is an expression system in which RNA replication and transcription are efficiently performed because the RNA genome of a virus encodes an RNA replicaase itself (Liljestrom, P. and H. Garoff, Biotechnology, 9, 1356-1361 (1991)).

Alphaviruses of the present invention are preferably "Semliki Forest Virus (SFV)" and "Sindbis Virus". Alphaviruses of the invention are more preferably SFV. The SFV-based expression vector pSFV-1 used in the embodiment of the present invention inserts the cDNA genome of SFV into a plasmid having an SP6 promoter, and is capable of in vitro transcription using SP6 polymerase, and is inserted in place of a structural gene. It is an expression vector transformed so that a gene can be expressed by a 26S subgenomic promoter.

In order to introduce recombinant RNA into cells by infection, it is necessary to use an in vivo packaging system. In vitro packaging systems may use helper-plasmids and packaging cell lines. The structure of the SFV-based heter plasmid used in the embodiment of the present invention is shown in FIG.

The inventors have surprisingly found that the gene is stably expressed even when a foreign gene of at least about 5 kb is inserted into the alphavirus-based vector, and the alphavirus-based recombinant vector expressing at least one cancer suppressor gene and / or angiogenesis suppressor gene. Was prepared.

By the definition of the present invention, "tumor suppressor genes" refers to genes encoding proteins having an activity of inhibiting the cancerization of normal cells and / or an activity of inhibiting the proliferation of cancer cells. Examples of cancer suppressor genes include the following; pRb / p110 or its analogs p107 and p120, p53 protein, p21 protein (WAF-1), bak, BRCA1, BRCA2, PTEN, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN -II, zac1, p73, VHL, FCC and MCC.

Particularly preferred as the cancer suppressor gene of the present invention is p53. The p53 gene is a cancer suppressor gene that is defective in most cancer cells of human cancer cells. Located in band 13 of human chromosome 17 and encodes a nucleophosphoprotein (53 kDa) consisting of 393 amino acids. p53 protein is a multifunctional protein that acts as a transcriptional activator and signaling mediator. It is known to regulate specific gene expression, inhibit cell development during DNA replication during the cell cycle, and induce cell death or programmed cell death. Nigro, J. et al., Nature, 342, 705-708 (1989); Takahashi, T. et al., Science, 246, 491-494 (1989)].

Also preferred as the cancer suppressor gene of the present invention is the PTEN gene. PTEN (phosphatase and tensin homolog deleted from chromosomal 10) gene is a cancer suppressor gene with defects in cancer cells such as external cancer, chest cancer, prostate cancer and melanoma. Located on human chromosome 10q23 encodes a protein consisting of 403 amino acids (47 kDa). PTEN, also known as MMAC1 (mutated in multiple advanced cancers) or TEP1 (TGFβ-regulated and epithelial cell-enrich phosphatase), is independent of the PTEN gene and the p53 gene in cells. PTEN proteins have lipid and protein phosphatase activity and are known to be involved in the proliferation / survival and migration / adherence of cells [Cantley, L. C et al., Proc. Natl. Acad. Sci. USA, 96, 4240-4245 (1999); Antonio, D. C. et al., Cell, 100, 387-390 (2000).

By the definition of the present invention "anti-angiogenic genes" refers to genes that encode proteins with activity that inhibits the formation of blood vessels. Examples of angiogenesis inhibitor genes include the following; PAI-1, PAI-2, PAI-3, angiostatin, endostatin, interferon-alpha, interferon-beta, interferon-gamma, platelet factor IV, IL-12, TIMP-1, TIMP-2, TIMP-3, LIF ( leukemia inhibitory factor, proliferlin-related protein, protease inhibitor, protamine, prolactin fragment, laminin, thrombospondin 1.

Angiogenesis is the process of creating new blood vessels and is necessary for the growth and metastasis of cancer cells. Angiostatin is a representative protein that inhibits angiogenesis. Angiostatin was extracted from serum and urine of mice with Lewis lung carcinoma as a tumor-induced angiogenesis inhibitor, and is isolated from the domain of plasminogen by protease and functions as a 38 kDa protein. Do it. It is known to inhibit the differentiation and metastasis of cells by specifically acting on endothelial cells, inhibit the angiogenesis of tumor sites and inhibit the growth of cancer cells while circulating body fluids [O'Reilly, MS et al., Cell, 79 , 315-328 (1994).

By the definition of the present invention an "expression vector" is a vector capable of expressing mRNA or protein in a suitable host cell, which facilitates DNA sequences and other genetic manipulations linked to operable to control sequences capable of controlling the expression of the protein. DNA constructs that contain sequences necessary for maximizing the expression of proteins, optimizing the expression of proteins, or replicating vectors. Such regulatory sequences include promoters for regulating transcription, operators optionally added to modulate transcription, appropriate mRNA ribosomal binding sites, and sequences regulating termination of transcription / translation.

One embodiment of the "alphavirus-based expression vector expressing cancer suppressor genes and / or angiogenesis suppressor genes" within the scope of the present invention comprises cancer suppressor genes and / or angiogenesis suppressor genes in one sub-genome promoter, Preferably, it is a Semiki forest virus (SFV) based expression vector operably linked under a 26S subgenomic promoter. The cancer suppressor gene and the angiogenesis inhibitory gene may be inserted into the expression vector of the present invention alone or in combination of two or more.

The expression vector of the present invention may be introduced into the cell by making the RNA transcript by the SP6 promoter inserted into the vector, or by inserting a structure that enables transcription in the cell directly. Examples of such DNA constructs are the immediate-early expression (IE) enhancer / promoter of cytomegalovirus (CMV), the SV40 early promoter and the long terminal gene of the Raus sacoma virus.

In one embodiment, the present invention provides an alphavirus-based expression vector, preferably an SFV-based expression vector, wherein the wild-type p53 gene is operably linked under one subgenomic promoter, preferably the 26S subgenomic promoter. In another aspect, the present invention provides an alphavirus-based expression vector, preferably an SFV-based expression vector, wherein the PTEN gene is operably linked under one subgenomic promoter, preferably the 26S subgenomic promoter. In another aspect, the present invention provides an alphavirus-based expression vector, preferably an SFV-based expression vector, wherein the angiostatin gene is operably linked under one subgenomic promoter, preferably the 26S subgenomic promoter.

The present invention further provides an alphavirus-based expression vector expressing two cancer suppressor proteins and / or angiogenesis inhibiting protein genes that are identical (eg A-A) or different (eg A-B). Two cancer suppressor proteins and / or angiogenesis inhibitor protein genes are each linked under a subgenomic promoter, and the genes can be inserted in any combination.

In an embodiment, the PTEN gene is operably linked under a first subgenomic promoter, preferably a 26S subgenomic promoter, and the wild type p53 gene is operable under a second subgenomic promoter, preferably a 26S subgenomic promoter. Linked alphavirus based expression vectors, preferably SFV expression vectors, are provided. In another aspect, the present invention provides for linkage of the angiostatin gene to be operable under a first subgenomic promoter, preferably a 26S subgenomic promoter, and the wild type p53 gene under a second subgenomic promoter, preferably a 26S subgenomic promoter. Alphavirus-based expression vectors, preferably SFV expression vectors, which are operably linked are provided. In another embodiment, the invention links angiostatin genes operably under a first subgenomic promoter, preferably the 26S subgenomic promoter, and the PTEN gene operates under a second subgenomic promoter, preferably 26S subgenomic promoter. Alphavirus-based expression vectors, preferably SFV expression vectors, are provided that are linked as possible.

The present invention further provides alphavirus-based expression vectors expressing three cancer suppressor proteins and / or angiogenesis inhibitory protein genes that are identical (eg A-A-A) or different (eg A-B-C, A-B-A, A-A-B). Three cancer suppressor proteins and / or angiogenesis inhibiting protein genes are each linked under a subgenomic promoter, and the genes may be inserted in any combination. In an embodiment, the invention connects the angiostatin gene to be operable under a first subgenomic promoter, preferably a 26S subgenomic promoter, and the p53 gene is operable under a second subgenomic promoter, preferably a 26S subgenomic promoter. Linkage and then operably linked the PTEN gene under a third subgenomic promoter, preferably the 26S subgenomic promoter, to provide an SFV expression vector.

The alphavirus of the present invention is a sequence that enables transcription in animal cells, in order to enable gene therapy by directly applying DNA itself directly to cancer cells without using RNA system and making RNA in vitro transcripts more conveniently. For example, the SV40 promoter or CMV / IE gene can be inserted. The pcSFV of the present invention is an example of the vector (FIG. 1). Although the expression vector was used as a base vector through the following examples, the present invention is not limited to this expression vector.

The expression vector pSFV-1 used in the preparation of the basic vector pcSFV for illustrating the present invention has a SP6 promoter as a product of GIBCO BRL (Cat. No.:18448-019), and also provides a replication enzyme necessary for virus replication. It has a gene encoding a non-structural protein to be included, a multicloning site into which a foreign gene can be inserted, and a subgenomic promoter for expressing a foreign gene. In addition to pSFV-1, plasmid pSFV-helper (GIBCO BRL, Cat. No. 10180-016), which encodes the structural protein of SFV, was used to prepare infectious viruses.

The present invention provides a recombinant viral expression vector that can be used for cancer therapy by inserting a gene expressing a cancer suppressor protein and an angiogenesis inhibiting protein into a basic expression vector pcSFV. PcSFV-p53, pcSFV-PTEN, pcSFV-Agt of the present invention are examples of expression vectors in which one gene is inserted (FIGS. 2 and 3). PcSFV-PTEN / p53 and pcSFV-Agt / p53 of the present invention are examples of expression vectors expressing two cancer suppressor genes and / or angiogenesis suppressor genes (FIGS. 4 and 5). PcSFV-Agt / p53 / PTEN made using the pcSFV base vector is an example of an expression vector expressing three cancer suppressor genes and / or angiogenesis suppressor genes (FIG. 6). The thus prepared pcSFV-Agt / p53 / PTEN was deposited as accession number KCCM-10249 (deposited February 5, 2001 at the Korea Microbiological Conservation Center, Seoul, Korea).

The present invention provides a host cell transformed with the expression vector or its RNA transcript and a method for producing cancer suppressor or angiogenesis inhibiting protein comprising expressing the expression vector in the host cell.

Alpha virus expression vectors or RNA transcripts thereof are preferably transfected methods such as DEAE-dextran, calcium phosphate method, microinjection method, DNA-containing liposome method, lipofectamine-DNA complex method and the like. By electroporation it is possible to introduce into the host cells of alphaviruses, preferably animal cells, more preferably BHK cells, CHO cells, COS cells and the following cancer cells. Such transfection methods are known in the art [Molecular Cloning, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press (1989)].

Examples of cancer cells are as follows; Brain cancer cells (U87MG, U251MG, 534T, 132T, U373MG, 134T, DBTRG-05MG, U178, etc.), Ovarian cancer cells (BCE, HUVE, MA148, SK-OV-3, Cao-3, ES-2, MDAH2774, NIH : OVCAR-3, OV-1063, SW626, SNU-8, SNU-119, Caov-4, etc., chest cancer cells (MDA-MB-231, MDA-MB-157, MDA-MB-453, MCF-7) , MDA-MB-468, BT549, SK-BR-3, SW613, Hs578T, ZR-75-1, etc.), prostate cancer cells (DU145, LNCaP, TSU, PC-3, LNCaP, etc.), lung cancer cells (NCI- H23, NCI-H522, NCI-H1435, NCI-H1793, SK-LU-1, NCI-H835, NCI-H727, A-549, A-427, etc.), liver cancer cells (Hep3B, Hep3B, Huh-7, PLC / PRF5, SNU-398, SNU-423, SNU-475, SNU-182, SNU-387, SNU-354, SNU-368, etc., Colon cancer cells (SW480, LS174T, Lovo, NCI-H854, WiDr, Colo 320DM , Colo 205, DLD-1, Colo 201, HCT-15, NCI-H548, HT-29, SNU-C1, SNU-C2A, SNU-C4, SNU-C5, NCI-H716, NCI-H508, NCI-H498 , HCT-8, HCT-116, etc., melanoma cells (UM449, WN35, A375P, A375SM, DX3, DM-4, G361, TXM18, TXM40, TXM13, SK-MEL-31, Malme-3M, RPMI-7951 , SK-MEL-1, SK-MEL-2, SK-MEL-3, SK-MEL-5, etc.), cervical cancer or cervical cancer (SNU-682, SNU-523, SNU-703, SNU-1160, SNU-17, SNU-778, SNU-902, SNU-1005, SNU-1299, SiHa, HeLa, HeLa 229, HeLa S3, etc.), stomach cancer Cells (SNU-1, MKN-45, AGS, SNU-5, SNU-16, SNU-216, SNU-484, SNU-520, SNU-719, MKN-28, MKN-74, etc.), kidney cancer cells ( A-704, A-498, SN12C, SN12PM6, Caki-2, etc.), bladder cancer cells (253J, 253J-BV, HT-1197, 5637, J82, T24, HT-1376, etc.), epidermal cancer cells (A-431 , Hep-2. KB, PANC-1, A-253, etc.) and lymphoma cells (LBRM-33-1A5, RPMI 6666, Daudi, H9, NCI-H78; Hut 78, NCI-H102; Hut 102).

Transformation of cells increases their expression efficiency when infected with infectious viral particles. The present invention provides a method for producing a recombinant protein by infecting a host cell with a recombinant virus particle (Recombinant Virus Particle), which is an infectious virus particle containing an RNA transcript according to the present invention made using a helper plasmid.

Cancer suppressor proteins or angiogenesis inhibitor proteins produced by the vectors, transcripts or recombinant viruses of the invention can be isolated by a variety of methods. The gene can be expressed in a suitable host cell to make the cell lysate, and then the recombinant protein can be purified by protein isolation methods known in the art. Usually, in order to remove cell debris and the like, the cell lysate or the result of in vitro translation is centrifuged, followed by precipitation, dialysis, various column chromatography, and the like. Examples are ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, preparative SDS-PAGE, affinity columns and the like.

The present invention provides a method of treating cancer by introducing a vector comprising two or more genes selected from p53, PTEN, and angiostatin into a cell. Even if two or more genes are expressed in one cell at the same time, it is difficult to see that the two proteins are always synergistic in cells with multiple bioactive networks. Most cancer suppressor proteins and angiogenesis inhibitor proteins are multifunctional proteins and are complicated to regulate cell physiology, and thus, their synergistic effects are hard to predict. In the case of simultaneous application of p53, a cancer suppressor protein, and interleukin-2, a cytokine, the growth inhibitory effect of subcutaneous C6 glioma was similar to that of each of them. , I. et al., Anticancer Res., 20, 1337-1342 (2000).

However, the present inventors surprisingly, when p53 and angiostatin and PTEN and p53 are expressed at the same time, the effect of inhibiting cancer is increased than when each protein is expressed, and when both p53 and PTEN and angiostatin are simultaneously expressed, a better cancer It was confirmed experimentally that the inhibitory effect was obtained (see FIGS. 16 to 19 and Table 1). This was particularly surprising in in vivo experiments, which is difficult to expect in an in vivo system where homeostasis is maintained and the signal transduction system is entangled.

When genetic material is introduced into a cell to treat cancer, it is not easy to introduce it into the cell with high efficiency even if it is predicted that the combination of household specific genes has a synergistic effect. Therefore, cloning genes known to have a synergistic effect into one vector is a springboard for effective gene therapy. We not only confirmed the synergistic effects of p53 and PTEN and angiostatin in vitro, ex vivo and in vivo, but also succeeded in cloning multiple anticancer genes in one vector to deliver them with high efficiency. Accordingly, the present invention provides a method for treating cancer, in particular gene therapy, using the expression vector, its RNA transcripts and recombinant virus. It is known to introduce expression vectors and the like of the present invention for gene therapy.

The present invention further provides a pharmaceutical composition for treating cancer comprising the expression vector, an RNA transcript of the vector or the recombinant virus in an amount sufficient to treat cancer, and a method for preventing cancer by administering the same. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Carriers include solvents, dispersion media, absorption retardants, and the like commonly used in the pharmaceutical art. The pharmaceutical composition of the present invention may be administered via any general route as long as it can reach the desired tissue. Thus, the compositions of the present invention can be administered topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally, and formulated into solutions, suspensions, tablets, pills, capsules, sustained release formulations, and the like. can do. Preferred formulations are injections. Dosage is determined in consideration of the skill of the art depending on the type and extent of the disease, age, sex, etc. of the patient.

The pharmaceutical composition of the present invention can be used to treat diseases such as brain cancer, ovarian cancer, chest cancer, prostate cancer, lung cancer, liver cancer, colon cancer, melanoma, cervical cancer, uterine cancer, gastric cancer, kidney cancer, bladder cancer, epidermal cancer and lymphoma Can be.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the examples.

<Example 1>

Construction of DNA-based Expression Vector pcSFV

RNA-based expression plasmid pSFV-1 (GIBCO BRL, Cat. No: 18448-019) was used to prepare DNA-based expression vectors using SFV as a substrate. First, primers 1 and 2 (with SphI recognition sites) were used as primers to obtain genes encoding cytomegalovirus (CMV) immediate-early (IE) enhancer / promoter. polymerase chain reaction (PCR) was performed. As template, pCMV-Script (STRATAGENE, Cat. No .: 212220) with cytomegalovirus (CMV) immediate early expression (IE) enhancer / promoter was used [Shimotohno, K. et al., Journal of virology, 73, 4713-4720 (1999).

In plasmid pCMV-Script (100 ng / μl), 4 μl 2.5 mM dNTP (Boehringer Mannheim, Germany), 5 μl sense primer (10 pmol / μl), 5 μl antisense primer (10 pmol / μl), Taq polymerase 10 × buffer 5 After adding μl and 37 μl of distilled water to make a mixed solution and modifying the mixture at 95 ° C. for 5 minutes, 1 μl of Taq polymerase was added, followed by a series of 50 seconds at 94 ° C., 50 seconds at 54 ° C. and 2 minutes at 72 ° C. The reaction was repeated 30 times.

Primer 1: Sense (SEQ ID NO: 1)

5'-ACATGCATGCGTCCGTTACATAACTTAC-3 '

Primer 2: antisense (SEQ ID NO: 2)

5'-ACATGCATGCGTCCGGAGGCTGGATCGG-3 '

CMV-IE Enhancer / Promoter gene region (686bp-1279bp) was amplified. The CMV-IE enhancer / promoter gene was cut with SphI, a restriction enzyme, and inserted into the SphI site of pSFV-1 to construct a DNA-based SFV expression vector (11754bp), which was designated as the basic expression vector pcSFV. The produced expression vector pcSFV is shown in FIG.

<Example 2>

Construction of cancer suppressor gene expression vector pcSFV-p53

Using the pcSFV prepared in Example 1 was prepared an expression vector pcSFV-p53 capable of expressing wild-type human p53 cancer suppressor gene. To introduce the human p53 cancer suppressor gene into the vector pcSFV, p53 gene (1182bp) cloned into the universal mammalian expression vector pCEP4 (Invitrogen, Cat. No: V044-50) was used as a template, primer 3 and primer 4 The polymerase chain reaction was performed as in Example 1 using (the presence of the recognition site of BamH1) as the primer.

Primer 3: sense (SEQ ID NO: 3)

5'-CCCGGATCCATGGAGGAGCCGCAG-3 '

Primer 4: antisense (SEQ ID NO: 4)

5'-CCCGGATCCTAACCTCAGTCTGAGTC-3 '

p53 gene (406bp-1588bp) was amplified. The amplified p53 gene was treated with the restriction enzyme BamHI and inserted into the BamH1 region of pcSFV to prepare an expression vector pcSFV-p53 (12936bp) expressing a human p53 gene. Thus produced expression vector pcSFV-p53 is shown in FIG.

<Example 3>

Construction of cancer suppressor gene expression vector pcSFV-PTEN

Using the pcSFV prepared in Example 1 was prepared an expression vector pcSFV-PTEN that can express wild-type human PTEN cancer suppressor genes. In order to introduce the human wild type PTEN gene into the vector pcSFV, the PTEN gene (1212 bp) cloned into the universal mammalian expression vector pcDNA-3 (Invitrogen, Cat. No: V790-20) was used as a template, primer 5 and primer The polymerase chain reaction was carried out as in Example 1 using 6 (the presence of the recognition site of BamHI) as the primer.

Primer 5: Sense (SEQ ID NO: 5)

5'-CCCGGATCCATGACAGCCATCATCAAAGAG-3 '

Primer 6: antisense (SEQ ID NO: 6)

5'-CCCGGATCCAATCTTCAGACTTTTGTAATTTGTG-3 '

PTEN genes (908bp-2120bp) were amplified. The amplified p53 gene was treated with the restriction enzyme BamHI and inserted into the BamHI site of pcSFV to prepare an expression vector pcSFV-PTEN (12966 bp) expressing the human wild-type PTEN gene. The expression vector pcSFV-PTEN thus prepared is shown in FIG. 3.

<Example 4>

Construction of cancer suppressor gene expression vector pcSFV-Agt

Using the pcSFV prepared in Example 1 was prepared an expression vector pcSFV-Agt that can express and secrete angiostatin gene having the effect of inhibiting cancer and inhibiting angiogenesis. To introduce the angiostatin gene into the vector pcSFV, pSecTaq2A (Invitrogen, Cat. No: V900-20), a universal mammalian expression vector containing angiostatin gene (1220 bp), was used as a template, and primer 7 and primer 8 (BglII recognition). Polymerase chain reaction was carried out as in Example 1 with the site present) as the primer.

Primer 7: Sense (SEQ ID NO: 7)

5'-CCCAGATCTATGGAGACAGACACACTC-3 '

Primer 8: antisense (SEQ ID NO: 8)

5'-CCCAGATCTTAGAAGGCACAGTCGAGGC-3 '

Mouse Ig κ chain leader (905bp-967bp) gene for secretion of expression protein before angiostatin gene and C-myc epitope (1082bp-1114bp) and 6 series of histidine (1127bp) for identification and purification of expression protein after angiostatin gene Genes (905-2381 bp; hereinafter Agt gene) including the -1144 bp) gene were amplified. The amplified gene is 1476 bp. The amplified Agt gene was inserted into the BglII site of pcSFV to prepare an expression vector pcSFV-Agt expressing the human angiostatin gene. The expression vector pcSFV-Agt thus prepared is shown in FIG. 3.

Example 5

Construction of cancer suppressor gene expression vector pcSFV-PTEN / p53

The expression vector pcSFV-PTEN / p53 capable of simultaneously expressing the PTEN gene and the p53 gene was prepared using the pcSFV prepared in Example 1. In order to introduce the human p53 gene into the expression vector pcSFV-PTEN prepared in Example 3, the expression vector pcSFV-p53 prepared in Example 2 was used as a template, and primer 9 and primer 10 were present (SmaI recognition site). As the primer, polymerase chain reaction was performed as in Example 1.

Primer 9: Sense (SEQ ID NO: 9)

5'-TCCCCCGGGACCTCTACGGCGGTCCTA-3 '

Primer 10: antisense (SEQ ID NO: 10)

5'-TTACCCGGGCCCTCGCGAGCGGCCGCTTAACCTCAGTCTGAGTC-3 '

The reaction amplified the p53 gene (7350bp-8582bp) region containing the 26S subgenomic promoter. In the lower part of the p53 gene, restriction enzyme sites such as NotI, NruI, and ApaI were artificially inserted to create a multicloning site. The amplified gene region was 1262 bp in size. The amplified p53 gene region was treated with restriction enzyme SmaI and inserted into the SmaI region of pcSFV-PTEN to prepare an expression vector pcSFV-PTEN / p53 (14228bp). The expression vector pcSFV-PTEN / p53 thus prepared is shown in FIG. 4.

<Example 6>

Construction of cancer suppressor gene expression vector pcSFV-Agt / p53

An expression vector pcSFV-Agt / p53 capable of simultaneously expressing angiostatin gene and p53 gene was prepared using pcSFV prepared in Example 1. To introduce the p53 gene into the expression vector pcSFV-Agt expressing the angiostatin gene, the p53 gene region (1262 bp) including the promoter and multiple cloning sites was amplified as in Example 5.

The amplified p53 gene region was treated with restriction enzyme SmaI and inserted into the SmaI region of the expression vector pcSFV-Agt to prepare an expression vector pcSFV-Agt / p53 (14492bp) expressing angiostatin and p53 gene. The expression vector pcSFV-Agt / p53 thus prepared is shown in FIG. 5.

<Example 7>

Construction of cancer suppressor gene expression vector pcSFV-Agt / p53 / PTEN

An expression vector pcSFV-Agt / p53 / PTEN capable of simultaneously expressing angiostatin, p53, and PTEN was prepared using the pcSFV prepared in Example 1. In order to introduce the PTEN gene into the expression vector pcSFV-Agt / p53, a 1212 bp PTEN gene cloned into the expression vector pcSFV-PTEN produced in Example 3 was used as a template, and primers 11 and 12 (NruI recognition sites were present). Polymerase chain reaction was carried out as in Example 1 with the primer.

Primer 11: sense (SEQ ID NO: 11)

5'-CTAGTCGCGAACCTCTACGGCGGTCCTA-3 '

Primer 12: antisense (SEQ ID NO: 12)

5'-CTAGTCGCGACAGACTTTGTAATTTGTG-3 '

The reaction amplified the PTEN gene (7350bp-8624bp) of size 1274 bp including the 26S subgenomic promoter of the SFV vector. The amplified PTEN gene was treated with the restriction enzyme NruI and inserted into the NruI site of the expression vector pcSFV-Agt / p53 to prepare an expression vector pcSFV-Agt / p53 / PTEN (15766bp) expressing angiostatin, human p53 and PTEN. . The expression vector pcSFV-Agt / p53 / PTEN thus prepared is shown in FIG. 6.

<Example 8>

RNA expression of cancer suppressor gene p53

In vitro transcripts of the plasmid and pc-helper plasmid were prepared to confirm the expression of p53 protein by the expression vectors pcSFV-p53, pcSFV-Agt / p53, pcSFV-PTEN / p53, pcSFV-Agt / p53 / PTEN, After transfection into BHK-21 cells, their expression was confirmed by immunocytochemistry. Specifically, each DNA containing pc-helper was treated with restriction enzyme SpeI to make linear DNA, followed by treatment with phenol / chloroform / isoaminoalcohol, and then 10 μl SP × buffer (TaKaRa) was added to 10 μl of the obtained DNA. 2 μl 10 mM m7G (5 ′) ppp (5 ′) G (BM), 1 μl 100 mM DTT, rNTP mixture (10 mM ATP, 10 mM CTP, 10 mM UTP, 5 mM GTP, Beoringer Manheim) 2 μl, RNasin (Beoringer Manheim) 2 µl and 2 µl of SP6 RNA polymerase (TaKaRa) were added to form a mixture, followed by reaction at 37 ° C. for 1 hour 30 minutes.

MRNA transcribed into 800 μl of BHK-21 cells (10 ⁷ cells / ml) suspended in MEM (containing 5% bovine serum) (pcSFV-p53, pcSFV-Agt / p53, pcSFV in 10 μl pc-helper mRNA transcript) MRNA mixture of PTEN / p53 and pcSFV-Agt / p53 / PTEN mRNA transcripts added in 20 μl each, followed by pulses twice at 0.4 cm electroporation cuvette at 850 V / 25 μs. Infection was performed (Bio-Rad Gene Pulser).

48 hours after transfection, immunocytochemistry was performed as follows to confirm p53 protein expression in each cell.

First, cells were fixed with precooled methanol and then blocked with PBS buffer containing 1% gelatin. After reacting with the first antibody p53 monoclonal antibody (DAKO) for about 3 hours, washed three times with PBS buffer, the second antibody with biotin-labeled mouse IgG for about 1 hour and then washed three times with PBS buffer It was reacted with avidin-biotin solution for about 30 minutes. After washing, color development was performed using a solution of DAB substrate kit (product of VECTOR Laboratories, composition containing buffer, hydrogen peroxide enzyme solution, 3,3'-diaminobenzidine [DAB] solution, nickel solution) and transfected with mRNA of each expression vector. P53 protein expression in the cells was confirmed by light microscopy. P53 protein was expressed almost similarly in all vectors. Expression with pcSFV-Agt / p53 / PTEN is shown in the second column of FIGS. 7A.

7A: immunocytochemical picture; Column 1: negative control, column 2: transfection with pcSFV-Agt / p53 / PTEN, column 3: infection with recombinant virus from pcSFV-Agt / p53 / PTEN]

After transfecting the media of cells 48 hours after transfection with the mRNA and pc-helper mRNA of each expression vector for 15 minutes at 3,000 rpm for 4 hours at 35,000 rpm with an ultracentrifuge (SW 41 rotor, BECKMAN) Recombinant SFV particles secreted from the cells into the medium by centrifugation were concentrated. Each pellet containing recombinant SFV particles was suspended in TNE [50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5 mM EDTA] buffer and stored at −70 ° C. until the experiment. Cells were infected with recombinant SFV particles suspended in TNE buffer to confirm p53 expression by immunocytochemistry and Western immunoblotting.

Specifically, 5 µl of chymotrypsin (chymotrypsin (10 mg / ml) and _2.5 µl of 50 mM CaCl ₂ were added to 100 µl of the virus particles suspended in TNE buffer solution, and reacted on ice for 30 minutes. Aprotinin (2 mg / 45 μl) was added to activate virus particles and then suspended in MEM (1% bovine serum) medium to infect 70% monolayer BHK-21 cells. Infection was performed in a 5% CO 2 incubator at 37 ° C. for about 1 hour and 30 minutes, after which fresh medium was added and incubated for 24 to 48 hours. Cells 24 to 48 hours after infection were observed according to the immunocytochemical method above. As a result, it is shown in column 3 of FIG. 7 that all BHK-21 cells express p53, angiostatin and PTEN, respectively, after infection with the recombinant virus obtained from pcSFV-Agt / p53 / PTEN.

Western blotting was performed as follows. Cells were lysed with Cell Lysis Buffer [1% NP40, 50 mM Tris-HCl (pH7.6), 150 mM NaCl, 2 mM EDTA, 1 μg / μl PMSF] and sampled from the same cell concentration as SDS-polyacrylic. After amide gel electrophoresis, the fractionated proteins were transferred to PVDF membrane. PVDF membranes to which proteins were transferred were shaken for 1 hour in blocking buffer (50 mM Tris-HCl, 5% Scheme Milk, pH 8.0) and then reacted with monoclonal primary antibody (1000-fold dilution) to p53 protein for 16 hours. Membranes were washed with wash buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20) and reacted with biotin-labeled mouse IgG secondary antibody (1000-fold dilution) for 4 hours, then washed again with wash buffer and avidin-biotin. After reacting with (avidin-biotin) reagent for 1 hour, it was washed again. The washed membrane was developed with a solution in DAB substrate kit (VECTORlaboratories).

From this western blot, it was confirmed that p53 protein was expressed in BHK-21 cells by plasmids pcSFV-p53, pcSFV-Agt / p53, pcSFV-PTEN / p53, pcSFV-Agt / p53 / PTEN, respectively (FIG. 8A). 8A: Western blot after recombinant virus infection; Lane 1: BHK-21, lane 2: pcSFV-p53, lane 3: pcSFV-PTEN / p53, lane 4: pcSFV-Agt / p53, lane 5 is pcSFV-Agt / p53 / PTEN]

The concentrated recombinant SFV particles obtained above were infected with 70% of monolayer brain cancer cells (U87MG: PTEN mutant, U251MG: PTEN and p53 mutant) in the same manner as described above. The immunocytochemical method was performed in the same manner as described above to confirm the expression of p53 protein in each cell after 48 hours. As a result, it was confirmed that p53 protein was expressed in brain cancer cells (U87MG and U251MG), which are cancer cells (FIGS. 9 and 10).

9 and 10: immunocytochemistry pictures; 9 shows U87MG cells, FIG. 6 shows U251MG cells; A: negative control, B: pcSFV-p53, C: pcSFV-Agt / p53, D: pcSFV-PTEN / p53, E: pcSFV-Agt / p53 / PTEN]

In addition, Western blotting of U87MG and U251MG brain cancer cells infected with enriched recombinant SFV particles using a monoclonal antibody against p53 confirmed that p53 protein was produced (FIG. 11). FIG. 11: Western Blot; (A: U87MG cells, B: U251MG cells); Lane 1: negative control, lane 2: pcSFV-p53, lane 3: pcSFV-PTEN / p53, lane 4: pcSFV-Agt / p53, lane 5: pcSFV-Agt / p53 / PTEN]

From the above results, RNA transcripts of pcSFV-p53, pcSFV-Agt / p53, pcSFV-PTEN / p53, pcSFV-Agt / p53 / PTEN vectors express p53 protein in BHK-21 cells and brain cancer cells (U87MG, U251MG). In addition, the recombinant SFV particles obtained from each of the above expression vector and pc-helper were found to be filling the p53 gene.

Example 9

Expression of the Cancer Suppressor Gene PTEN by RNA System

In this example, the experiment was performed according to the method of Example 8 to confirm the expression of the PTEN protein with the expression vectors pcSFV-PTEN, pcSFV-PTEN / p53, pcSFV-Agt / p53 / PTEN plasmid. Immunocytochemical methods and Western blots used monoclonal antibodies (Santa Cruz Biotechnology) against PTEN.

As a result of immunocytochemistry, recombinant viruses obtained from RNA transcripts of pc-helper and pcSFV-PTEN, pcSFV-PTEN / p53, and pcSFV-Agt / p53 / PTEN plasmid expressed PTEN protein in BHK-21 cells. The PTEN protein was expressed almost similarly in all vectors. In addition, Western immunoblotting confirmed that all of the proteins expressing about 55 KDa specifically bind to monoclonal antibodies against PTEN. Expression by pcSFV-Agt / p53 / PTEN plasmid is shown in B of FIG. 7 and B of FIG. 8.

7B: immunocytochemical picture; Column 1: negative control, column 2: transfection with pcSFV-Agt / p53 / PTEN, column 3: infection with recombinant virus from pcSFV-Agt / p53 / PTEN]

8B: Western blot after recombinant virus infection; Lane 1: BHK-21, lane 2: pcSFV-PTEN, lane 3: pcSFV-PTEN / p53, lane 4: pcSFV-Agt / p53 / PTEN]

Recombinant SFV particles produced and secreted in the BHK-21 cells were infected with U87MG and U251MG brain cancer cells to perform immunocytochemical methods, and Western blotting of each cell sample was performed. PTEN protein was expressed by pcSFV-PTEN, pcSFV-PTEN / p53, pcSFV-Agt / p53 / PTEN plasmid in U87MG and U251MG brain cancer cells (FIG. 12), confirming that the expressed protein is a PTEN protein of about 55 KDa (FIG. 13).

12: immunocytochemistry pictures; (A to D: U87MG cells, E to H: U251MG cells); (A, E): negative control, (B, F): pcSFV-PTEN, (C, G): pcSFV-PTEN / p53, (D, H): pcSFV-Agt / p53 / PTEN]

13: Western blot after recombinant virus infection; Lane 1: negative control, lane 2: pcSFV-PTEN, lane 3: pcSFV-PTEN / p53, lane 4: pcSFV-Agt / p53 / PTEN]

The experimental results indicate that in the case of pcSFV-PTEN / p53 and pcSFV-Agt / p53 / PTEN together with the results of Example 8, p53 and PTEN proteins are expressed in one cell. It also indicates that the PTEN is charging.

<Example 10>

RNA expression of cancer suppressor gene angiostatin

In this Example, the experiment was carried out according to the method of Example 8 to confirm the expression of angiostatin protein with the expression vectors pcSFV-Agt, pcSFV-Agt / p53, pcSFV-Agt / p53 / PTEN plasmid. Immunocytochemical methods and Western immunoblotting used monoclonal antibodies against c-myc (Invitrogen).

Immunocytochemistry confirmed that recombinant viruses obtained from RNA transcripts of pc-helper and pcSFV-Agt, pcSFV-Agt / p53, and pcSFV-Agt / p53 / PTEN plasmid all expressed similar angiostatin proteins in BHK-21 cells. In addition, Western immunoblotting confirmed that all of the recombinant angiostatin proteins of about 63 KDa that specifically bind to monoclonal antibodies against C-myc were expressed. Expression by pcSFV-Agt / p53 / PTEN plasmid is shown in C of FIG. 7 and C of FIG. 8.

Figure C: immunocytochemical picture; Column 1: negative control, column 2: transfection with pcSFV-Agt / p53 / PTEN, column 3: infection with recombinant virus from pcSFV-Agt / p53 / PTEN]

8C: Western blot after recombinant virus infection; Lane 1: BHK-21, lane 2: pcSFV-Agt, lane 3: pcSFV-Agt / p53, lane 4: pcSFV-Agt / p53 / PTEN]

Recombinant SFV particles produced and secreted from the BHK-21 cells were infected with U251MG brain cancer cells to perform immunocytochemical methods, and each cell sample was western blotted. Angiostatin protein was expressed by all of pcSFV-Agt, pcSFV-Agt / p53, pcSFV-Agt / p53 / PTEN plasmid in U251MG cells (FIG. 14), and the protein expressed by Western blotting was about 63 kDa recombinant angiostatin. (FIG. 15A). Recombinant angiostatin was also detected in U251MG brain cancer cell culture medium (FIG. 15B).

14: immunocytochemical picture; A: negative control (U251MG), B: pcSFV-Agt, C: pcSFV-Agt / p53, D: pcSFV-Agt / p53 / PTEN]

15: Western blot; (A: intracellular expression, B: secreted into extracellular medium) Lane 1: negative control (U251MG), lane 2: pcSFV-Agt, lane 3: pcSFV-Agt / p53 lane 4: pcSFV-Agt / p53 / PTEN]

From the above results, it can be seen that angiostatin was expressed in one cell together with p53 in pcSFV-Agt / p53, and angiostatin in combination with p53 and PTEN in pcSFV-Agt / p53 / PTEN. The experiment further shows that the recombinant SFV particles by pcSFV-Agt / p53 / PTEN are charged with angiostatin together with the p53 and PTEN genes.

<Example 11>

Inhibition of cancer cell proliferation in vitro by recombinant SFV particles

In this example, mRNAs of the expression vectors pcSFV-p53, pcSFV-PTEN, pcSFV-Agt, pcSFV-PTEN / p53, pcSFV-Agt / p53, pcSFV-Agt / p53 / PTEN prepared in Examples 2 to 5 Recombinant SFV particles (containing one, two, or three p53, PTEN, or Agt genes, respectively) produced by helper mRNA and secreted into the medium from cells are infected with brain cancer cells (U87MG and U251MG), which are cancer cells, and exclude trypan blue Inhibition of cancer cell proliferation was confirmed by the method.

Specifically, brain cells (U87MG and U251MG), 1 × 10 ⁶ cancer cells, were infected with each recombinant SFV particle 5 × 10 ⁶ , replaced with fresh medium after 3 hours, and then incubated for 48 hours. The cells were collected and stained with trypan blue, and the number thereof was measured by a hemocytometer (FIG. 16). 16: Cancer cell inhibitory activity experiment; None: negative control, A: pcSFV-lacZ, B: pcSFV-p53, C: pcSFV-PTEN, D: pcSFV-Agt, E: pcSFV-Agt / p53, F: pcSFV-PTEN / p53, G: pcSFV-Agt / p53 / PTEN]. As shown in FIG. 16, U87MG (white bars) and U251MG (hatched bars) cancer cells of the negative control group continued to grow and differentiate and were initially infected with pSFV-lacZ recombinant virus (recombinant SFV particles). About 10% reduction, when infected with recombinant viruses of each of the expression vectors pcSFV-p53, pcSFV-PTEN, pcSFV-Agt, showed a 50% reduction in the initial cell number, confirming the inhibition of cancer cells. When infected with the recombinant viruses of pcSFV-PTEN / p53 and pcSFV-Agt / p53, the initial cell number was reduced by about 60 to 70%, and the inhibition of cancer cells was confirmed. A 90% reduction showed clear inhibition of cancer cells.

As a result of the above example, recombinant SFV particles charged with or expressing p53, PTEN and angiostatin genes, respectively, or two or three genes are infected with brain cancer cells (U87MG and U251MG), which are cancer cells, thereby synergistically differentiating and growing cells. It can be seen that.

<Example 12>

Induction of apoptosis of cancer cells by recombinant SFV particles

In the present embodiment, the expression vector pcSFV-p53, pcSFV-PTEN, pcSFV-Agt, pcSFV-PTEN / p53, pcSFV-Agt / p53, pcSFV-Agt / p53 / PTEN is made of mRNA and pc-helper mRNA Recombinant SFV particles containing, respectively, two or three p53, PTEN and angiostatin genes secreted into the medium from the cancer cell brain cancer cells (U87MG and U251MG) to inhibit the proliferation is due to the induction of apoptosis of cancer cells It was confirmed by fluorescein (Promega) which is an apoptosis detection system. Fluorescein (Promega), an apoptosis detection system, was measured by TUNEL staining and propidium iodide (PI) labeling of fragmented DNA of apoptosis cells.

Tunnel analysis of apoptosis of U251MG cells showed no fluorescence indicating apoptosis in uninfected U251MG cells as a negative control, but pcSFV-p53, pcSFV-PTEN, pcSFV-Agt, pcSFV-Agt / p53, When infected with the recombinant viruses of pcSFV-PTEN / p53 and pcSFV-Agt / p53 / PTEN, fluorescence was shown to indicate cell death. The number of apoptotic bodies was increased when infected with recombinant SFV particles containing two and three genes more than the p53, PTEN and angiostatin genes alone.

The cell death rate was measured using the FACScan flow cytometry (Becton Dickinson) for a more quantitative comparison. As a negative control, apoptosis rate was low among the uninfected U251MG cells, pcSFV-lacZ, pcSFV-p53, pcSFV-Agt / p53, pcSFV-PTEN / p53, pcSFV-Agt / p53 / PTEN. It was confirmed quantitatively the highest in cells infected with recombinant virus of pcSFV-Agt / p53 / PTEN, a plasmid containing dog genes (FIG. 17).

In conclusion, when p53, PTEN and angiostatin genes are co-expressed, it is interpreted that the differentiation and growth of cancer cells is more markedly inhibited by the expression of each protein.

Example 13

Inhibition of cancer cell proliferation in vitro by recombinant SFV particles

In the present embodiment, the expression vector pcSFV-p53, pcSFV-PTEN, pcSFV-Agt, pcSFV-PTEN / p53, pcSFV-Agt / p53, pcSFV-Agt / p53 / PTEN is made of mRNA and pc-helper mRNA P53, PTEN, and angiostatin genes secreted in the medium were infected with recombinant SFV particles containing two or three genes to cancerous brain cancer cells (U251MG), and then infected cancer cells were subcutaneously injected into nude mice. By injection, the proliferation inhibition of cancer cells was confirmed by the size of cancer cells.

Specifically, 1 x 10 ⁶ brain cancer cells (U251MG) were infected with each recombinant SFV particle 5 x 10 ⁷ , and after 3 hours, the cells were trypsinized and collected and stained with trypan blue, and the cells were stained with live 2 x 10 ⁶ . Cells were injected subcutaneously in 4 nude mice per group. Thirty days after the infusion, the size of the cancer grown in each rat was measured by a caliper, and then calculated using the formula 0.5 × longest diameter of cancer x (shortest diameter of cancer) ² for the size of the cancer per group. Mean and standard deviation were measured (Table 1).

As shown in Table 1, the control group U251MG cancer cells continued to grow and differentiate, and the size of the cancer grew to 3124 () ± 801 (sd) and injected with cells infected with the recombinant virus (recombinant SFV particles) of pSFV-lacZ. Nude rats were approximately 52% of the control size, and nude mice injected with cells infected with the recombinant virus of each of the expression vectors pcSFV-p53, pcSFV-PTEN, and pcSFV-Agt were about 20 to about the size of the control. 25% size. Nude mice injected with cells infected with the pcSFV-PTEN / p53 and pcSFV-Agt / p53 recombinant viruses were suppressed to about 10-15% of the control size, and in the case of pcSFV-Agt / p53 / PTEN. It was confirmed that inhibiting cancer cells to a size of about 5% or less of the control size.

In this embodiment, p53 / PTEN, PTEN / angiostatin, p53 / angiostatin, and p53 / PTEN / angiostatin proteins express synergistic effects on cancer suppression in ex vivo as in vitro than in protein expression, respectively. To show.

Tumor Proliferation in Nude Mice Following In Vitro Injection of U251MG Cells Infected with Recombinant Virus in Nude Mice Treatment Average volume (㎣ ± s.d.) Mock group (n = 4) 3124 ± 801 SFV-lacZ (n = 4) 1634 ± 754 SFV-p53 (n = 4) 726 ± 194 SFV-PTEN (n = 4) 751 ± 367 SFV-Agt (n = 4) 692 ± 259 SFV-Agt / p53 (n = 4) 312 ± 103 SFV-PTEN / p53 (n = 4) 479 ± 179 SFV-Agt / p53 / PTEN (n = 4) 156 ± 72 Cells were infected with recombinant virus for 4-5 hours, then washed and subsequently injected subcutaneously into nude mice. Tumor volume was measured after 4 weeks. The results are expressed as mean size ± standard deviation of tumors.

<Example 14>

Inhibition of cancer cell proliferation in vivo by recombinant SFV particles

In the present embodiment, the cells of the expression vectors pcSFV-p53, pcSFV-PTEN, pcSFV-Agt, pcSFV-PTEN / p53, pcSFV-Agt / p53, pcSFV-Agt / p53 / PTEN are made of mRNA and pc-helper mRNA. Recombinant SFV particles containing p53, PTEN or angiostatin, respectively, or two and three genes secreted into the medium were injected subcutaneously in nude mice to confirm the inhibitory activity of brain cancer cells (U251MG), which are cancer cells, in the size of cancer cells. .

Specifically, brain cells (U251MG), a cancer cell of 1 × 10 ⁷ , were administered subcutaneously in nude rats, and the volume of cancer tissue reached about 20 mm ^3. Each recombinant SFV particle suspended in 100 μl of TNE buffer 1 × 10 ⁹ were divided into 4 rats per group and injected directly into the subcutaneous cancer tissues of each group of rats. Three days after the injection, the same amount of recombinant SFV particles were administered to the same cancer tissues again. Every 5 days after infusion, the size of cancers grown in each rat was measured with a caliper and measured again after 30 days, and calculated according to the formula of 0.5 × longest diameter of cancer x (shortest diameter of cancer) ² The mean and standard deviation for the size of were measured (FIGS. 18 and 19).

As shown in FIG. 20, the control group of U251MG cancer cells continued to grow and differentiate, and the size of the cancer grew to 7432 (mm ³ ) ± 669 (sd) and injected with cells infected with the recombinant virus (○) of pSFV-lacZ. In nude mice, the size of the cancer cells was about 65% of the control size, and the cells infected with the recombinant viruses of the respective expression vectors pcSFV-p53 (■), pcSFV-PTEN (▽) and pcSFV-Agt (▼) For injected nude mice, the size of the cancer cells was about 40-45% of the control size. As shown in FIG. 21, in nude mice injected with cells infected with the recombinant viruses of pcSFV-PTEN / p53 (▽) and pcSFV-Agt / p53 (▼), the size of the cancer cells was about 20% or less of the control size. In the case of pcSFV-Agt / p53 / PTEN (■), it was observed to be less than about 5% of the control size, confirming the inhibition of cancer cells.

This example is significant in that the results of Examples 11 to 13 were also confirmed in vivo.

<Example 15>

DNA expression of cancer suppressor gene p53

In this example, each of the pcSFV-p53, pcSFV-Agt / p53, pcSFV-PTEN / p53, pcSFV-Agt / p53 / PTEN plasmids was purified to BHK-21 cells without making an in vitro transcript of the expression vector. 6 reagents were used to transfect. Expression of p53 was confirmed by immunocytochemistry and Western immunoblotting. Immunocytochemistry and Western blotting were performed according to the method of Example 8.

A large amount of p53 was expressed in cells transfected with the DNA of the expression vectors pcSFV-p53, pcSFV-Agt / p53, pcSFV-PTEN / p53 and pcSFV-Agt / p53 / PTEN of the present invention. The case of pcSFV-Agt / p53 / PTEN DNA is shown in A of FIG. 20. Figure 20 A: immunocytochemical picture; Left column: negative control, right column: pcSFV-Agt / p53 / PTEN]

Furthermore, unlike the negative control (BHK-21: lane 1), the western blot result shown in FIG. 21A also shows that pcSFV-p53 (lane 2), pcSFV-PTEN / p53 (lane 3), pcSFV-Agt / p53 / The presence of p53 expressed in cells transfected with DNA of PTEN (lane 4).

<Example 16>

Expression of the cancer suppressor gene PTEN by the DNA system

This Example was carried out in the same manner as in Example 15 to confirm the expression of the PTEN protein by the expression vectors pcSFV-PTEN, pcSFV-PTEN / p53, pcSFV-Agt / p53 / PTEN plasmid. Immunocytochemical methods and Western immunoblotting used monoclonal antibodies against PTEN.

It was confirmed that a large amount of PTEN was expressed in cells transfected with DNA of the expression vectors pcSFV-PTEN, pcSFV-PTEN / p53, and pcSFV-Agt / P53 / PTEN. The case of pcSFV-Agt / p53 / PTEN DNA is shown in B of FIG. 20. FIG. 20B: immunocytochemical picture; Left column: negative control, right column: pcSFV-Agt / p53 / PTEN]

In addition, in the western blot experiment shown in FIG. 21B, unlike the negative control (BHK-21: lane 1), pcSFV-PTEN (lane 2), pcSFV-PTEN / p53 (lane 3), pcSFV-Agt / p53 / PTEN ( The presence of PTEN expressed in the cells transfected with the DNA of lane 4) was confirmed.

<Example 17>

Expression by the DNA System of Cancer Inhibitory Gene Angiostatin

In this Example, the experiment was carried out in the same manner as in Example 15 to confirm the expression of angiostatin protein by the expression vectors pcSFV-Agt, pcSFV-Agt / p53, pcSFV-Agt / p53 / PTEN plasmid. Immunocytochemical methods and Western immunoblotting used monoclonal antibodies against C-myc.

The immunocytochemical method confirmed that angiostatin was expressed in a large amount of cells transfected with DNA of the expression vectors pcSFV-Agt, pcSFV-Agt / p53, and pcSFV-Agt / P53 / PTEN of the present invention, unlike the negative control group. The case of pcSFV-Agt / p53 / PTEN DNA is shown in C of FIG. 20. FIG. 20C: immunocytochemical picture; Left column: negative control, right column: pcSFV-Agt / p53 / PTEN]

As shown in FIG. 21C, an angiostatin protein band of about 63 KDa expressed by the expression plasmid pcSFV-Agt (lane 2) was identified.

The alphavirus-based expression vector of the present invention and the recombinant alphavirus particles prepared therefrom, specifically, the SFV expression vector and the recombinant SFV virus particles, may express one or two or more foreign cancer suppressor proteins or angiogenesis inhibitor genes, Proliferation of cancer cells can be inhibited by directly administering the expression vector as a DNA system to the mammalian cells, by administering the mRNA transcript thereof, or by infecting the viral particles. In addition, expression vectors comprising angiostatin, p53 and PTEN genes disclosed in the present invention and recombinant SFVs thereof confirmed that each gene provided an increased anticancer effect than when only one or two species were inserted. Therefore, the vaccine composition comprising the alphavirus expression vector, its RNA transcript or recombinant alphavirus particles can be usefully used as a therapeutic agent for inhibiting or preventing the proliferation of cancer cells.

<110> KIM, Chul Jung <120> Alphavirus-Based Expression Vectors Expressing Tumor Suppressor          Proteins, the Preparation And the Use Thereof <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 acatgcatgc gtccgttaca taacttac 28 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acatgcatgc gtccggaggc tggatcgg 28 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cccggatcca tggaggagcc gcag 24 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cccggatcct aacctcagtc tgagtc 26 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cccggatcca tgacagccat catcaaagag 30 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cccggatcca atcttcagac ttttgtaatt tgtg 34 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cccagatcta tggagacaga cacactc 27 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cccagatctt agaaggcaca gtcgaggc 28 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tcccccggga cctctacggc ggtccta 27 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ttacccgggc cctcgcgagc ggccgcttaa cctcagtctg agtc 44 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctagtcgcga acctctacgg cggtccta 28 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ctagtcgcga cagactttgt aatttgtg 28

Claims (5)

A first cancer suppressor gene or angiogenesis suppressor gene is operably linked under a first subgenomic promoter, a second cancer suppressor gene or angiogenesis inhibitor gene is operably linked under a second subgenomic promoter, A third cancer suppressor gene or angiogenesis suppressor gene is operably linked under a third subgenomic promoter, wherein the first to third cancer suppressor genes or angiogenesis inhibitor genes are the same or different, and the cancer suppressor gene Is a p53 gene or a PTEN gene and the angiogenesis suppressor gene is an angiostatin gene, causing an immune response to the Semiki Forest Virus (SFV) or Sindbis-virus expression vector, which simultaneously expresses three cancer suppressor genes or angiogenesis suppressor genes. Anticancer pharmaceutical composition, characterized in that it comprises a sufficient amount. A first cancer suppressor gene or angiogenesis inhibitor gene is operably linked under a first subgenomic promoter, and a second cancer suppressor gene or angiogenesis inhibitor gene is operably linked under a second subgenomic promoter and Two cancer suppressor genes or angiogenesis inhibitors, wherein the first and second cancer suppressor genes or angiogenesis inhibitor genes are the same or different, and the cancer suppressor genes are p53 genes or PTEN genes and the angiogenesis inhibitor genes are angiostatin genes. A pharmaceutical composition for anticancer treatment, comprising Semiki forest virus (SFV) or Sindbis-virus expression vectors that simultaneously express genes in an amount sufficient to cause an immune response. A first cancer suppressor gene or angiogenesis suppressor gene is operably linked under a first subgenomic promoter, a second cancer suppressor gene or angiogenesis inhibitor gene is operably linked under a second subgenomic promoter, A third cancer suppressor gene or angiogenesis suppressor gene is operably linked under a third subgenomic promoter, wherein the first to third cancer suppressor genes or angiogenesis inhibitor genes are the same or different, and the cancer suppressor gene Animal cells are either Semiki forest virus (SFV) or Sindbis-virus expression vectors and helper vectors that simultaneously express three cancer suppressor genes or angiogenesis inhibitor genes, wherein p53 gene or PTEN gene and angiogenesis inhibitor gene is angiostatin gene. Cancer suppressor genes or angiogenesis isolated from culture medium by transfection The recombinant virus is charged gene. A first cancer suppressor gene or angiogenesis inhibitor gene is operably linked under a first subgenomic promoter, and a second cancer suppressor gene or angiogenesis inhibitor gene is operably linked under a second subgenomic promoter and Two cancer suppressor genes or angiogenesis inhibitors, wherein the first and second cancer suppressor genes or angiogenesis inhibitor genes are the same or different, and the cancer suppressor genes are p53 genes or PTEN genes and the angiogenesis inhibitor genes are angiostatin genes. A recombinant virus filled with a cancer suppressor gene or an angiogenesis suppressor gene isolated from a culture medium by transfecting an animal cell with a Sempriki forest virus (SFV) or a Sindbis-virus expression vector and a helper vector that simultaneously express genes. A pharmaceutical composition for anticancer treatment comprising the recombinant virus of claim 3 or 4 in an amount sufficient to cause an immune response.