KR20140140356A - A bacterium salmonella harboring siRNA against syntrophin beta and antitumoral composition thereof - Google Patents

Abstract

The present invention relates to a bacterium salmonella which is transformed into a recombinant expression vector including a gene which transcribes small interfering RNA (siRNA) which inhibits expression of the syntrophin beta2 gene, and to an antitumoral composition comprising the same. According to the organization of the present invention, the present invention induces cancer inhibition or anti-tumor immunities in order to minimize side effects while treating cancer.

Description

[0001] The present invention relates to a Salmonella strain expressing a sirturopin beta 2 siRNA and an anticancer composition containing the siRNA against a syntrophin beta and an antitumoral composition,

The present invention relates to a Salmonella strain expressing a syntrophin beta2 siRNA and a composition comprising the same. More particularly, the present invention relates to a Salmonella strain expressing a syntrophin beta2 siRNA which is capable of expressing a syntrophin beta 2 siRNA capable of enhancing anticancer immunity Vector and an anticancer composition containing the same.

Cancer, which is the number one cause of death in Korea, is on the rise due to aging. Currently, chemotherapy and radiation therapy are mainly used for chemotherapy.

In the case of chemotherapy and radiotherapy, except for surgical operations, various preparations and radiation are used, but their application is limited to the extent that the human body can afford. The limited application of these chemotherapy and radiation therapies has the disadvantage of limiting the range of application and limiting efficacy in clinical trials no matter how good the efficacy is in animal tests. This is due to adverse effects caused by the appearance of cancer cells with anticancer drug resistance or cytotoxicity of each therapy.

For example, cisplatin (cis-diamminedichloroplatinum; CDDP) is one of the most effective anti-cancer agents among the 30 types of cancer drugs currently used in clinical practice. , And cervical cancer (Teni Boulikas, Oncology Reports, 10: 1663-1682, 2003). However, recently, many cancers resistant to cisplatin have been reported, and cancer cells having such resistance are currently being challenged, and there is a risk of recurrence after cancer treatment, so that a combination therapy of cisplatin with other chemicals or cisplatin and intracellular expressed protein (Tito Fojo, Oncogene, 22: 7512-7523, 2003). In the present study,

In addition, radiotherapy is an anti-cancer therapy for treating various cancers occurring in the human body by irradiating an affected part with an appropriate amount of radiation. Such a radiation therapy lowers the hematopoietic function of the human body and suppresses the immune system, There is a problem of lowering. Furthermore, repeated irradiation of radiation causes cancer cells to acquire resistance, which limits radiation therapy.

The side effects of chemotherapy and radiotherapy in such chemotherapy treatments can lead to poor quality of life of the patients, and furthermore, a fatal disease, apart from the success of chemotherapy.

One of these fatal side effects is bone marrow suppression, which can lead to anemia due to erythropoiesis, immunosuppression due to leukocytopenia, and bleeding due to thrombocytopenia. Another side effect is side effects on the liver and kidney, and the decomposition and excretion of anticancer drugs in the liver is inhibited, and other side effects of anticancer drugs are often severe. If the renal function that excretes waste of the body is dropped by the anticancer agent, cancer or other factors, the excretion of the anticancer agent may be delayed, and the vicious circle in which the side effect of the anticancer agent is maximized may be repeated. May appear. There are also many known side effects that affect the heart, reproductive system, and nervous system depending on the individual characteristics of the chemotherapeutic agent or radiation therapy.

Therefore, in patients requiring chemotherapy, management of the side effects will be as important as chemotherapy itself, unlike other diseases.

Salmonella, on the other hand, is a pathogenic microorganism that causes food poisoning in most cases. However, Yale University researchers report in a paper published in the European Journal of Cancer that they have been very successful in treating melanomas by using a combination of gene-manipulated salmonella and radiation therapy (Antitumour effects of genetically engineered Salmonella in combination with radiation. Journal of Cancer, 18,23972402 J. Platts.). These researchers have previously found that salmonella has the property of attacking mouse tumors. Genomic manipulation of Salmonella can remove toxicity while maintaining tumor-attacking properties, and the injection of the genetically engineered Salmonella The tumor can be suppressed through the. Currently, the bacteria that attack cancer cells are in clinical trials targeting humans in the US and Europe.

In addition, according to the above-mentioned article, when the radiation therapy or the salmonella therapy is administered alone, the tumor suppression effect is only maintained for 2-3 weeks in the mouse, but when the two methods are used in parallel, the effect is doubled It is said. However, even with this method, the tumor did not disappear completely, and there was a limitation that extended the life of the mouse even longer.

The present inventors have found that a Salmonella typhimurium strain expressing an interferon gamma protein that induces an anti-cancer effect and a composition for chemotherapy therewith (Korean Patent No. 10-0818144 (Mar. 31, 2008)) and a tumor Salmonella strains expressing necrotic factor alpha and compositions for chemotherapeutic treatment containing them (Korean Patent No. 10-0790376 (2008.01.02.)) Have been patented for a composition for treating cancer using Salmonella strains. In addition, the present inventors have found that, by transfecting a plasmid capable of expressing Tumor Necrosis Factor Alpha (TNF-α) in eukaryotic cells in an attenuated Salmonella strain having no pathogenicity, it can exhibit a far superior anticancer effect than conventional Salmonella In addition, a Salmonella strain expressing TNF-α capable of solving the toxicity problem of Salmonella which expresses TNF-α in the past has been developed (Korean Patent No. 10-0852687 (2008.08.19.)).

On the other hand, it has been known that the sirtrophin beta 2 protein is involved in the regulation of insulin secretion and is involved in cell structure formation. Korean Patent No. 10-1046344 (Jul. 2011) uses SNGT2 polymorphism and its monoclonal antibody as a biomarker A method for diagnosing stroke is described. However, the antitumor effect of Sintropin beta 2 has not been elucidated. The present inventors have confirmed the relationship between the sirtrophin beta 2 protein and cancer development, and tried to use siRNA that inhibits the sirtrophin beta 2 gene for cancer suppression or anti-cancer immunity enhancement.

Numerous cited documents and patent documents are referred to and cited throughout this specification. The disclosures of the cited documents and patents are incorporated herein by reference in their entirety to more clearly describe the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent No. 10-0818144 (Mar. 31, 2008) Korean Patent No. 10-0790376 (2008.01.02.) Korean Patent No. 10-0852687 (Aug. 19, 2008) Korean Patent No. 10-1046344 (2011.07.05)

The inventors of the present invention have confirmed that the overexpression of the sirturopin beta2 protein is associated with the development of cancer and completed the present invention. Accordingly, an object of the present invention is to provide a Salmonella strain which expresses a syn-rotin beta 2 siRNA that induces cytotoxicity that inhibits the sirtrophin beta 2 gene and activates a cell suicide program to induce cancer inhibition or anti-cancer immunity There is.

Another object of the present invention is to provide an anticancer agent composition containing the Salmonella strain.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a recombinant expression vector comprising a gene transcribing a small interfering RNA (siRNA) inhibiting the expression of a syntrophin beta2 gene, which is transformed with a recombinant expression vector Salmonella strains are provided.

According to another aspect of the present invention, there is provided an anticancer composition comprising the Salmonella strain.

According to still another aspect of the present invention, there is provided a recombinant vector comprising a recombinant expression vector comprising a gene for transcribing a small interfering RNA (siRNA) having a nucleotide sequence of SEQ ID NO: 1 inhibiting the expression of a syndropin beta2 gene Thereby providing an anticancer composition.

Hereinafter, the present invention will be described in detail.

In the present invention, functional microorganisms were designed to express siRNA in Salmonella strains in order to inhibit cancer and enhance the anti-cancer immune response, confirming that overexpression of the sirtrophin beta2 protein is associated with cancer development (see Figs. 1A and 1B) Respectively.

Thus, according to one aspect of the present invention, the present invention is characterized in that a recombinant expression vector comprising a gene that transcribes a small interfering RNA (siRNA) that inhibits the expression of a syntrophin beta2 gene is characterized Gt; Salmonella < / RTI >

In the present invention, 'siRNA (small interfering RNA)' refers to a short double stranded RNA capable of inducing RNA interference through cleavage of a specific mRNA. siRNAs are not limited to pairs of double-stranded RNA portions that are paired with each other, but are paired by mismatch (corresponding bases are not complementary), bulge (no bases corresponding to one chain) May be included. The paired base length may preferably be 50 to 80 bases. The siRNA terminal structure can be either a smooth end or a protruding end if it can inhibit the expression of the target gene by RNA interference. The sticky end structure can have both a 3 'end protruding structure and a 5' end protruding structure.

The siRNA of the human syntrophin beta2 gene of the present invention may preferably be a double-stranded siRNA comprising the nucleotide sequence of SEQ ID NO: 1 and a sequence complementary thereto.

SiRNAs of the invention include variants having one or more substitutions, insertions, deletions, and combinations thereof that do not degrade their activity. Such modifications may have a sequence homology of 80% or more, preferably 90%, more preferably 95% or more, with the sequence of the above siRNA.

The siRNA of the present invention can be synthesized directly (Sui G et al., (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells, Proc Natl Acad Sci USA 99: 5515-5520) (Brummelkamp TR et al., (2002) A system for stable expression of short interfering RNAs in mammalian cells, Science 296: 550-553) using a transcription transcription method .

The siRNA of the present invention is basically a form in which two strands of RNA are paired to form a double-stranded siRNA, but a structure having a short hairpin so that it can be used for transfection by a plasmid-based shRNA vector and a PCR expression cassette It may also be in the form of

The Salmonella strain of the present invention belongs to the group D Salmonella causing typhoid fever. In the present invention, an attenuated Salmonella strain was used to eliminate the pathogenicity of the Salmonella strain. The attenuated Salmonella strain is characterized in that it is an attenuated vaccine for typhoid prevention. The term " attenuation "refers to artificially weakening the toxicity of a living pathogen. It means mutating a gene involved in essential metabolism of a pathogen and inducing the immune system by stimulating only the immune system without causing disease in the body .

Salmonella strains used in specific embodiments of the invention are Salmonella typhimurium head Titanium (Salmonella typhimurium BRD509, an attenuated strain without pathogenicity.

A siRNA polynucleotide sequence according to the present invention may be operably linked to an expression control sequence, wherein the operably linked gene sequence and expression control sequence comprise an expression < RTI ID = 0.0 > Vector. Said "operably linked" may be a gene and expression control sequence linked in such a way as to enable gene expression when the appropriate molecule binds to the expression control sequence. The expression control sequence refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, sequences controlling the termination of transcription and translation.

In the present invention, the recombinant vector may be any expression vector having a promoter capable of expressing the siRNA and a multicloning site for cloning. In a specific example of the present invention, an expression vector that secretes siRNA transduced into the strain was prepared by adding a syntrophin beta 2 siRNA sequence to pcDNATM 6.2-GW / EmGFP-miR (INVITROGEN, USA) 2a).

Salmonella strains containing the syntrophin beta 2 siRNA inducing the anticancer effect in the present invention can be produced by a transfusion method commonly used in the art, and the prepared Salmonella strains are Salmonella strains resistant to Spectinomycin , Which can secrete the synrofun beta 2 siRNA when introduced into the body.

According to another aspect of the present invention, there is provided an anticancer composition comprising the Salmonella strain.

In a specific example of the present invention, the salmonella of the present invention is prepared by transfecting the attenuated Salmonella strain with a recombinant vector expressing an siRNA that inhibits the sirtrophin beta 2 gene, and the above-described transduction The effect of the Salmonella on the prevention and treatment of cancer by administering Salmonella was confirmed by comparison with the effect of the control Salmonella itself which did not transduce the cancer. As a result of the analysis, as shown in Figs. 2 (a) to 4 (b), unlike the case where only salmonella without transfection or only PBS solution was treated, the recombinant vector expressing the syntrophin beta 2 siRNA according to the present invention was transfected with salmonella Was effective in the treatment of cancer.

According to still another aspect of the present invention, there is provided a recombinant vector comprising a recombinant expression vector comprising a gene for transcribing a small interfering RNA (siRNA) having a nucleotide sequence of SEQ ID NO: 1 inhibiting the expression of a syndropin beta2 gene Thereby providing an anticancer composition.

Because of the overexpression of the syn-rotin beta 2 protein upon cancer development, a recombinant expression vector containing a gene that transcribes small interfering RNA (siRNA) having the nucleotide sequence of SEQ ID NO: 1, which inhibits the expression of the syndropin beta 2 gene, By inhibiting the expression of the sirtotropium beta 2, the therapeutic effect of cancer can be achieved. Since the siRNA and the recombinant expression vector are as described above, detailed description will be omitted.

In the present invention, the kind of cancer is not particularly limited, but it may preferably be skin cancer or colon cancer.

The anticancer composition of the present invention may be prepared by a method known in the pharmaceutical art to be used as a pharmaceutical agent, and may be prepared by mixing with a pharmaceutically acceptable carrier, excipient, diluent or the like to prepare powder, granule, tablet, capsule, And the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995). They may also be administered parenterally (e. G., Intravenously, subcutaneously, intraperitoneally or topically) or orally.

The anticancer composition of the present invention is administered in a therapeutically effective amount. The term "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and includes the age, sex, weight, , The time of administration, and the method of administration. For example, the dose may be 10 ⁵ cells to 10 ⁹ cells once per adult.

As described above, according to the Salmonella strain expressing the sirturopin beta 2 siRNA according to the present invention, the cytotoxicity that activates the apoptosis program by inhibiting the sirtrophin beta 2 gene induces the cytotoxicity to induce cancer inhibition or anti-cancer immune response And thus exhibits a more excellent anticancer effect than the existing Salmonella strains. Especially skin cancer and colorectal cancer, and can solve the drawbacks of administering only the syn-rotin beta 2 siRNA, which is a very useful invention in the pharmaceutical industry related to chemotherapy and adjuvant prophylactic agents.

FIGS. 1A and 1B are views showing the expression level of the SNTB2 protein in colorectal cancer patients by immunohistochemical staining, and comparing the expression levels of SNTB2 protein with that of normal tissues.
2A is a schematic diagram of Salmonella strains expressing siRNA inhibiting SNTB2 protein.
FIG. 2B is a graph showing the expression of the macrophage line (RAW264.7 (a)) obtained by treating the macrophage line (RAW264.7 CELL) with Salmonella (a), PBS solution (b) and Salmonella strain CELL) of the siRNA vector.
FIGS. 3A and 3B are photographs showing the killing effect of each Salmonella strain including the synoptophil beta 2 siRNA vector in a skin cancer cell line (B16F10) or a colon cancer cell line (CT26) and plotted using LDH ASSAY Respectively.
4A and 4B are graphs showing the degree of cancer expression by the respective Salmonella strains containing the synoptophin beta 2 siRNA vector in a skin cancer or a colon cancer-induced mouse in terms of cancer volume.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

< Example  1: In clinical patient organizations Syntropin Better 2  Identification of protein expression>

Twenty patients in an ancient hospital cancer were screened for their cancer The degree of expression of the sirtrophin beta2 protein was examined by immunohistochemistry and over 70% of the normal tissue was overexpressed (FIGS. 1A and 1B).

< Example  2: Syntropin Beta 2 siRNA RTI ID = 0.0 > Salmonella &lt; / RTI &gt;

First, the attenuated Salmonella strain BRD509 ( Salmonella typhimurium BRD509) was cultured at 37 占 폚 in Luria Bertani medium. Transfection of the cultured Salmonella strains with the expression plasmids and pcDNA3 vectors of the syntrophin beta 2 siRNA (TGC TGA GGA GAA TGT AGC TCT ATC AAG TTT TGG CCA CTG ACT GAC TTG ATA GAT ACA TTC TCC T; , The Salmonella strain was again cultured in an amount of 100 mL for 24 hours, and the bacteria were centrifuged at a culturing concentration of 5500 rpm at an optical absorbance (OD600) of 0.7 at a wavelength of 600 nm. After centrifugation, only the bacteria that settled on the bottom were collected and diluted with cold distilled water of the same volume as the culture volume.

The diluted bacteria were again centrifuged in the same manner as described above, diluted with 2 mL of cold 20% glycerol distilled water, and centrifuged again to dilute the obtained bacteria into 100 μl of 20% glycerol. 30 ((2 ㎍) of the mixed solution obtained in the above procedure was placed in a mini-centrifuge, and 3 ((2)) of pcDNA ™ 6.2-GW / EmGFP-miR vector, which is a plasmid of siRNA expression vector Followed by electrowinning at 275-325V (Fig. 2a). In order to confirm the gene expression effect of the recombinant Salmonella strain produced, 10 ⁶ cells were cultured in the macrophage cell line (Raw 264.7 cell), Salmonella strain was added at a concentration of 10 ⁸ CFU, and after 1 hour of culture, And cultured for 3 days.

The GFP gene was designed to be expressed when gene expression was induced in the cultured cell line. The cultured cell line was observed under a fluorescence microscope to confirm the effect of siRNA expression Salmonella strains. As a result, as shown in Fig. 2b, the expression of GFP by Salmonella strains expressing the sirturopin beta2 siRNA was confirmed to be expressed by salmonella, as compared with the control group.

< Example  3: Confirmation of anticancer effect in the cell line of Salmonella strain transformed by the present invention>

B16F10 (skin cancer cell line, Korean cell line bank) and CT26 (colorectal cancer cell line and Korean cell line bank), which are skin cancer cells, were cultured in DMEM medium and used as cancer cells for confirming anticancer effect. In the presence of about 70% cells cultured in a 100 mm culture dish, the Salmonella strain expressing the sirtotropin beta 2 siRNA prepared in Example 1 was cultured, and 10 ⁶ colonies were diluted in 100 μl of PBS solution. After 24-36 hours, the cytotoxicity after culturing was examined using a microscope and an LDH cytotoxicity detection kit (Promega, USA) (FIGS. 3A and 3B).

< Example  4: Confirmation of anticancer effect in an animal model of Salmonella strain transformed by the present invention>

After diluting the skin cancer cells B16F10 (skin cancer cell line, Korean cell line bank) and CT26 (colorectal cancer cell line and Korean cell line bank) to 100 μl of PBS solution to contain 10 ⁵ cells, C57 / BL6 mouse (SLC, Japan) was selected and 100 쨉 l of the diluted solution was subcutaneously injected into the back region of a mouse to transplant the cancer. 7 days to 10 days after the injection, it was confirmed that cancer cells proliferated on the cancer graft site. The size of the cancer was confirmed by measuring the length and length of the cancer grown with the vernier caliper. The syntrophin beta2 siRNA-expressing salmonella strain prepared in Example 1 was cultured and 10 ⁶ colonies were diluted in 100 μl of PBS solution.

The PBS solution was injected subcutaneously into the cancer transplantation site of C57 / BL6 mice at one week after the cancer occurrence was confirmed. Thereafter, the size of the cancer cells and the survival rate of the mice were measured to confirm the anti-cancer effect of the Salmonella strain expressing the sirtotropin beta 2 siRNA according to the present invention. At this time, only the PBS solution was treated in the other rats as a control group, and BRD509 Salmonella strains not transfected with the sirtotropin beta 2 siRNA were treated in another rat, and the experiment was carried out in the same manner as above.

The results are shown in Figs. 4A and 4B. In FIGS. 4A and 4B, the first bar shows the degree of cancer expression in normal cancer model mice treated with only PBS solution without any Salmonella strains, and the second bar shows the Salmonella strain without the control plasmid Lt; RTI ID = 0.0 &gt; cancer &lt; / RTI &gt; Thirdly, the tumor size of the mice treated with Salmonella strains expressing the sirturopin beta 2 siRNA of the present invention can be suppressed as compared with the mice treated with the PBS solution treatment model or the plasmid control salmonella.

As is apparent from the above examples, the present invention provides a sirturopin beta 2 siRNA-expressing Salmonella strain, which is produced by transfecting a syntrophin beta 2 siRNA expression vector into an attenuated Salmonella strain which is not pathogenic, Especially Salmonella. In particular, it solves the disadvantages of administering only the syn-rotopin beta 2 siRNA, and particularly provides a more excellent therapeutic effect in skin cancer and colon cancer. Therefore, It is a very useful invention in the related pharmaceutical industry.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> A bacterium salmonella harboring siRNA against syntrophin beta          and antitumoral composition thereof <130> P11-130426-01 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 64 <212> DNA <213> Homo sapiens <400> 1 tgctgaggag aatgtagctc tatcaagttt tggccactga ctgacttgat agatacattc 60 tcct 64

Claims (5)

Wherein the recombinant expression vector is transformed with a recombinant expression vector comprising a gene transcribing a small interfering RNA (siRNA) that inhibits the expression of the syntrophin beta2 gene. 2. The Salmonella sp. Strain according to claim 1, wherein the siRNA is an siRNA having the nucleotide sequence of SEQ ID NO: 1. An anticancer composition comprising the Salmonella strain according to claim 1 or 2. The anticancer composition according to claim 3, wherein the type of cancer is skin cancer or colon cancer. 1. An anticancer composition comprising a recombinant expression vector comprising a gene that transcribes a small interfering RNA (siRNA) having a nucleotide sequence of SEQ ID NO: 1 which inhibits the expression of a syndropin beta 2 gene.

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