KR101069101B1 - mTOR targeting siRNA with cross-species activity, and expression vector and pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to siRNA targeting mTOR having cross-linking activity, a recombinant vector comprising the same, and a pharmaceutical composition containing the same as an active ingredient.The mTOR target siRNA has a sequence complementary to a part of the mTOR gene, Because it can degrade mRNA or inhibit translation of mTOR gene, it is composed of various diseases that target mTOR, such as cancer, neurodegenerative disease, immune disease, infectious disease, aging, heart disease, liver disease and Crohn's disease. Diseases selected from the group, in particular cancer diseases, can be prevented or treated.

mTOR, siRNA, recombinant vector, pharmaceutical composition, cancer disease

Description

MIR targeting siRNA with cross-species activity, and expression vector and pharmaceutical composition comprising the same}

The present invention is to prevent or treat a variety of diseases targeting mTOR, for example, cancer, neurodegenerative diseases, immune diseases, infectious diseases, aging, heart disease, liver disease and Crohn's disease, in particular cancer disease The present invention relates to siRNAs targeting mTOR having cross-linking activity, a recombinant vector comprising the same, and a pharmaceutical composition containing the same as an active ingredient.

mTOR (mammalian Target of rapamycin) is a cytokine-stimulated cell proliferation, translation of mRNA for several important proteins that regulate G1 phase of the cell cycle, and interleukin-2 (IL-2) ) Is an important enzyme in a variety of signal transduction pathways, including induced transcription.

Inhibition of mTOR causes inhibition of progression from G1 to S in the cell cycle. The antibiotic rapamycin (commercially available as Sirolimus ^™ ) manufactured by Streptomyces hygroscopicus has been found to be an important mTOR inhibitor.

Since mTOR inhibitors exhibit immunosuppressive, antiproliferative and anticancer activity, mTOR is targeted for the treatment of these diseases (Current Opinion in Lipidology, 16: 317-323, 2005). In addition, mTOR is an important factor in regulating autophage, and targets mTOR that regulates the autophagy pathway, thereby targeting various diseases such as cancer, neurodegenerative diseases, heart disease, aging, immune diseases, infectious diseases and Crohn's disease. Diseases and the like can be treated (Immunology, 7: 767-777; Nature 451: 1069-1075, 2008).

On the other hand, ribonucleic acid mediated interference phenomenon (RNAi) is a small interference ribonucleic acid having a double-helix structure of 21-25 nucleotides, specifically binding to a transcript having a complementary sequence (mRNA transcript) to degrade the transcript. This is the phenomenon of inhibiting the expression of a specific protein. In recent years, studies on the use of ribonucleic acid-mediated interference phenomena in the development of chemical synthetic medicines have been conducted to selectively inhibit the expression of specific proteins at the transcript level and to use them in the development of various therapeutic drugs, especially tumor therapies. It is becoming.

Small molecule chemical drugs require long development time and development costs to be optimized for specific protein targets, while the biggest advantage of siRNA medicine using ribonucleic acid mediated interference is that The development of optimized read compounds for all protein targets, including the target material, can proceed quickly.

While protein and antibody drugs suffer from a complicated manufacturing process, siRNA is relatively easy to mass-produce due to its ease of synthesis and separation and purification, and has a higher storage stability than protein drugs due to the characteristics of nucleic acid materials. In addition, unlike conventional drugs, it is emerging as a new drug candidate based on several advantages, such as being able to antagonize specific molecular targets only.

The present inventors have a sequence complementary to a portion of the mTOR gene known as targets of various diseases, thereby completing the present invention by developing siRNA having cross-linking activity that can degrade mRNA or inhibit translation of mTOR gene. .

Accordingly, an object of the present invention is to provide a mTOR target siRNA, a recombinant vector comprising the siRNA and a pharmaceutical composition comprising the siRNA as an active ingredient having an interstitial cross-linking activity that can suppress the expression of mTOR genes known as targets for various diseases. To provide.

In addition, another object of the present invention to provide a pharmaceutical composition for the treatment and prevention of cancer diseases comprising mTOR target siRNA having an interspecies cross-activity that can inhibit the expression of mTOR gene as an active ingredient.

In order to achieve the above object, the present invention provides an siRNA having any one nucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 4.

SiRNA targeting mTOR in the present invention has a sequence 100% complementary to a part of the mTOR gene of humans, monkeys, and rats, and can degrade mRNA or inhibit translation of the mTOR gene. Complementarity of 80-90% can inhibit the translation of mRNA, and 100% can degrade mRNA.

In addition, siRNA targeting mTOR according to the present invention comprises a nucleotide sequence having a homology of 80%, preferably 90%, more preferably 100% with the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 4 can do. Each base sequence may be linked to each other by a loop region of 5 to 15bp to form a hairpin structure.

The siRNA of the present invention can be prepared according to the preparation method of RNA molecules known in the art. As a method for preparing RNA molecules, chemical synthesis methods and enzymatic methods can be used. For example, the chemical synthesis of RNA molecules can use the methods described in the literature (Verma and Eckstein, Annu. Rev. Biochem. 67, 99-134, 1999), and the enzymatic synthesis of RNA molecules is T7, T3. And phage RNA polymerases such as SP6 RNA polymerase are disclosed in the literature (Milligan and Uhlenbeck, Methods Enzymol. 180: 51-62, 1989).

The present invention also provides a recombinant vector comprising an siRNA having any one base sequence selected from SEQ ID NO: 1 to SEQ ID NO: 4.

The recombinant vector shows a cleavage map shown in Figure 1, preferably pSP72-scAAV-GFP-mTOR.

Recombinant vectors of the present invention can be prepared by recombinant DNA methods known in the art.

Viral or viral vectors useful for delivering siRNA to mTOR in the present invention include baculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae, Poxviridiae, adenoviridiae, and the like, but are not limited thereto.

In the present invention, it is most preferable to use an Adeno Associated Virus (AAU). Adeno-associated viruses rarely cause immune responses and cytotoxicity. In particular, adeno-associated virus serotype 2 can efficiently deliver genes to neurons of the CNS, and also can efficiently express transgenes in the nervous system for a long time.

In addition, non-viral vectors useful for delivering siRNA for mTOR in the present invention include all vectors commonly used for gene therapy, except for the aforementioned viral vectors, for example, various plasmids and liposomes that can be expressed in eukaryotic cells. Etc.

Meanwhile, in the present invention, siRNA targeting mTOR is preferably operably linked to at least a promoter in order to be properly transcribed in delivered cells. The promoter may be any promoter capable of functioning in eukaryotic cells, but a human H1 polymerase-III promoter is more preferable. For efficient transcription of siRNA targeting mTOR it may further comprise regulatory sequences, including leader sequence, polyadenylation sequence, promoter, enhancer, upstream activation sequence, signal peptide sequence and transcription terminator.

The present invention also provides a pharmaceutical composition containing siRNA having any one of nucleotide sequences selected from SEQ ID NO: 1 to SEQ ID NO: 4 as an active ingredient.

The pharmaceutical composition may prevent or treat a disease selected from the group consisting of cancer, neurodegenerative disease, immune disease, infectious disease, aging, heart disease, liver disease and Crohn's disease.

The present invention also provides a pharmaceutical composition for the treatment and prevention of cancer diseases, containing as an active ingredient siRNA having any one selected from SEQ ID NO: 1 to SEQ ID NO: 4.

The cancer diseases include liver cancer, lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, breast cancer, Fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma Abnormal cancer disease can be prevented or treated.

When the mTOR target siRNA according to the present invention is used as a pharmaceutical composition, it may further include a suitable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

The compositions can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose ( Prepare by mixing sucrose or lactose, gelatin, etc.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of the composition may vary depending on the age, sex, and weight of the patient, but the amount of 0.1 to 2.0 mg / kg may be administered once to several times daily.

In addition, the dosage of such compositions may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The siRNA targeting mTOR according to the present invention has a sequence complementary to a portion of mTOR genes of various species, for example, humans, monkeys, and rats, thereby degrading mRNA or inhibiting translation of mTOR genes. It can prevent or treat a variety of diseases, for example cancer, neurodegenerative diseases, immune diseases, infectious diseases, aging, heart disease, liver disease and Crohn's disease, especially cancer diseases.

Preferred examples are provided below to aid in the understanding of the present invention. However, these examples are only provided to more easily understand the present invention, the present invention is not limited to the following examples.

Example 1 Design of siRNA

Target sequences were selected from a completely conserved squence pattern in various species of mTOR. At this time, the species used include human (LOCUS: NM_004958), monkey (XR_014791), rat (NM_019906) and mouse (NM_020009).

For the design of effective siRNAs, siRNA design software developed in-house, called MWG Biotech AG software (www.mwgbiotech.com) or CAPSID (Convenient Application Program for siRNA Design) was used.

After determining the siRNA sequence, nonspecific control siRNA was purchased from Bioneer (Seoul, Korea). siRNA double stranded oligonucleotides were resuspended in nuclease-free water as directed by the manufacturer. Nonspecific siRNAs labeled with Cy3-fluorescent dye were used as controls to investigate transfection efficiency into cells. The sequences of all siRNAs used in this example are shown in Table 1.

siRNA Target sequence (5 '→ 3') start siRNA-score SEQ ID NO: mTOR-1 GGAGUCUACUCGCUUCUAU 253 9.5 One mTOR-2 GAAGAAGGUCACUGAGGAU 5677 9 2 mTOR-3 ACAACCUCCAGGAUACACU 5772 8.5 3 mTOR-4 GAAUGUUGACCAAUGCUAU 7221 13 4

Example 2 Cell Culture and Transfection

HeLa, Vero and H9C2 cells were purchased from the American Type Culture Collection (ATCC). The cells were supplemented with 10% FBS (fetal bovine serum), glutamax-1 (2 mM), penicillin (100 IU / ml) and streptomycin (50 μg / ml) at 37 ° C. in a 5% CO ₂ incubator. (Dulbecco's modified Eagle's medium; Gibco BRL, Carlsbad, Calif.) Medium.

For transient transfection, cells were incubated with 100 nM siRNA for 4 hours using oligofectamine reagent (Invitrogen) in the serum-free state, followed by addition of fresh medium containing 10% serum. Cells were harvested 24 hours after transfection for further experiments.

Example 3 Real-time RT-PCR

Total RNA was extracted from HeLa, Vero and H9C2 cells using Trizol reagent (Invitrogen, Carlsbad, Calif.). Reverse transcription (RT) was performed with Superscript III (Invitrogen, Carlsbad, Calif.) At 55 ° C. using oligo-dT primer (Invitrogen). Synthesized cDNA was amplified by real-time RT-PCR using iQ SYBR Green Supermix (Bio-rad, Hercules, CA).

The reaction consisted of 39 cycles of 3 minutes at 95 ° C. for polymerase activation and [15 seconds at 95 ° C. (denature), 30 seconds at 60 ° C. (annealing), 30 seconds at 72 ° C. (extended)]. . β-actin was used as a normal control to assess the relative degree of gene expression.

At this time, the primers used were rat-mTOR-sense and antisense primers (SEQ ID NOs 5 and 6), human-mTOR-sense and antisense primers (SEQ ID NOs 7 and 8), general β-actin-sense and antisense primers (SEQ ID NO: 9 and 10), and monkey mTOR primers were identical to those of humans.

Relative gene expression was assessed by GeneXpression Macro chromo4 software (Bio-Rad). As a result, all four siRNAs suppressed mTOR mRNA expression in HeLa cell lines as shown in the upper part of FIG. 1, and also suppressed mTOR mRNA expression in Vero cell lines and H9C2 cell lines as shown in FIGS. 3A and 3B.

Example 4 Western Blot Analysis

The cells were recovered and lysed with 100 μl of lysis buffer (Intron, Seoul, Korea).

It was. The lysate was boiled with 5X sample buffer at 100 ° C for 5 minutes and the denatured protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then semi-drier (Bio-Rad) was used. Transfer to PVDF membrane at 20V for 30 minutes.

After treatment with block solution (0.4% Tween 20 and 5% dry milk in PBS) for 1 hour at room temperature, the membrane was treated with primary antibodies: mTOR (1: 1000, Cell signaling, Boston, MA), β-actin (1: 5000). , Sigma, St. Louis, MI) and GFP (1: 1000, Santa Cruz) overnight. After washing with PBST or TBST, a secondary antibody (Jackson laboratory) conjugated with horse radish peroxidase was applied and the blot was expressed with an improved chemiluminescent reagent (Pierce Biotechnology, Rockford, IL).

As a result, all four siRNAs suppressed mTOR protein expression in HeLa cell lines as shown in the lower part of FIG. 1, and also inhibited mTOR protein expression in Vero cell lines and H9C2 cell lines as shown in FIGS. 3A and 3B.

Example 5 Flow Cytometry Analysis

Flow cytometric analysis was performed to analyze mTOR expression by methods other than Western blot analysis. For flow cytometry, cells were pooled and fixed in 4% paraformaldehyde dissolved in PBS. After permeation with 0.05% TritonX-100, cells were incubated with mTOR primers (Cell Signaling, Boston, Mass.) And FITC-conjugated secondary antibody (in 1% BSA). Marker cells were then analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, Calif.).

As a result, as shown in Figure 2 all four siRNA knocked down the mTOR gene.

Example 6 Construction of Recombinant Vector Expressing mTOR shRNA

6-1: Construction of pSP72-pH1

Human H1 polymerase-III promoter (pH1) as a promoter for shRNA expression was amplified by PCR based methods. That is, the sequence of pH1 is primer 5'-CCA TGG AAT TCG AAC GCT GA-3 '(SEQ ID NO: 11) and 5'-GGG AAA GAG TGG TCT CAT AC-3 from human genomic DNA isolated from HeLa cells. (SEQ ID NO: 12) was amplified by PCR. The PCR product thus amplified was used as a template for sense primer (EcoRI linker) 5'-ATC GAA TTC ATA TTT GCA TGT CGC TAT GTG-3 '(SEQ ID NO: 13) and antisense primer (BamHI linker) 5'-ATC GGA TCC GAG Secondary PCR was performed using TGG TCT CAT ACA GAAC-3 '(SEQ ID NO: 14). PCR products were digested with EcoRI / BamHI and isolated from agarose gels. PSP72-pH1 was then made by cloning to the EcoRI / BamHI position of the cloning vector pSP72 (Promega, Madison, Wis.).

6-2: Construction of pSP72-pH1-shmTOR # 1 or pSP72-pH1-shmTOR # 4

SiRNA 'mTOR sh # 1' or 'mTOR sh # 4' targeting mTOR of SEQ ID NO: 1 or SEQ ID NO: 4 was constructed to include BamHI and HindIII 5'- or 3'- overhangs and then added to the pSP72-pH1 vector Ligation to prepare siRNA 'mTOR sh # 1' or 'mTOR sh # 4'. The sense primer sequence with BamHI linker for mTOR sh # 1 is 5'-GATCC GGAGTCTACTCGCTTCTAT TTCAAGAGA ATAGAAGCGAGTAGACTCC TTTTTT GGAAA-3 '(SEQ ID NO: 15), and the antisense primer sequence with HindIII linker is 5'-AGCTT TTCC AAAATCTC ATAGAAGCGAGTAGACTCC G-3 '(SEQ ID NO .: 16). In addition, the sense primer sequence with BamHI linker for mTOR sh # 4 is 5'-GATCC GAATGTTGACCAATGCTAT TTCAAGAGA ATAGCATTGGTCAACATTC TTTTTT GGAAA-3 '(SEQ ID NO: 17), and the antisense primer sequence with HindIII linker is 5'-AGCTT TTCC AAAAAA GAATGTTGACCAATGCTAT TCTCTTGAA ATAGCATTGGTCTTCTTTC G-3 '(SEQ ID NO: 18). Nucleotide specific for the mTOR gene in the sequence is underlined and the loop structure is shown in bold.

6-3: Construction of pSP72-XP-rAAV-MCS

PHpa-Trs-SK, the original backbone of self-complementary AAV, was provided by McCarty (Ohio State University). Modifications were made to new vectors with multi-cloning sites. First, a pSP72-XP-rAAV vector was constructed. This was constructed by cleaving essential regions of AAV, including internal repeat (ITR), CMV promoter, reporter gene GFP, and SV40 poly A signal from pHpa-Trs-SK using XhoI and PvuII, and then ligating with pSP72 vector. .

Next, a linker with multiple cloning positions was produced. This linker is 5'- CGG GAG ATC TTC CTG CAG GAT ATC TGG ATC CAC GAA GCT TCC CAC CGG TTC TAG AGC G-3 '(SEQ ID NO: 19), which is a sense oligonucleotide sequence having a Kpn I restriction enzyme site; 5'-TCG ACG CTC TAG AAC CGG TGG GAA GCT TCG TGG ATC CAG ATA TCC TGC AGG AAG ATC TCC CGG TAC-3 '(SEQ ID NO: 20). The linker was constructed from the previously prepared pSP72-XP-rAAV with KpnI and SalI, and then the linker was ligated to prepare pSP72-XP-rAAV-MCS.

6-4: Construction of rAAV-shmTOR # 1 or rAAV-shmTOR # 4

CMV promoter from pSP72-XP-rAAV-MCS was removed by KpnI and XbaI digestion.

And then blunt ends were made with DNA polymerase (New England BioLabs, Beverly, Mass.). pH1-shmTOR # 1 or pH1-shmTOR # 4 were allowed to lie adjacent to the BGH poly A position (present in pSP72-XP-rAAV-MCS). The pSP72-XP-rAAV-MCS vector then cleaved the poly A tail with Sal I.

Inserts for expressing shmTOR # 1 or shmTOR # 4, ie pH1-shmTOR # 1 or pH1-shmTOR # 4, were obtained by digesting with EcoRV and XhoI from pSP72-pH1-shmTOR # 1 or pSP72-pH1-shmTOR # 4 vectors. The vector and insert were then blunted with DNA polymerase (New England BioLabs, Beverly, Mass.).

The thus prepared vector and insert were blunt ligated. It does not express GFP and is designed to express only shmTOR # 1 or shmTOR # 4. This was named pSP72-XP-rAAV-pH1-shmTOR # 1 or pSP72-XP-rAAV-pH1-shmTOR # 4 for plasmids and rAAV-shmTOR # 1 or rAAV-shmTOR # 4 for viruses. .

6-5: Construction of rAAV-GFP-shmTOR # 1 or rAAV-GFP-shmTOR # 4

Dual genes of GFP and shmTOR # 1 or shmTOR # 4 containing CMV promoter for expression of GFP expression cassette finally independent of pH1 for expression of shmTOR # 1 or shmTOR # 4 to detect cells into which the vector has been introduced Expression rAAV vectors were constructed. The cloning strategy is as follows: The pSP72-XP-rAAV-MCS vector (also called the pSP72-XP-rAAV-GFP vector) was digested with EcoRI and the insert pH1-shmTOR # 1 or pH1-shmTOR # 4 was pSP72- Obtained by digestion with EcoRV and XhoI from pH1-shmTOR # 1 or pSP72-pH1-shmTOR # 4 vectors. The vectors and inserts were then blunted with DNA polymerase (New England BioLabs, Beverly, Mass.). This vector may express shmTOR # 1 or shmTOR # 4 and GFP.

This vector is named pSP72-scAAV-GFP-shmTOR # 1 or pSP72-scAAV-GFP-shmTOR # 4 for plasmids (FIGS. 4A and 4B) and scAAV-GFP-shmTOR # 1 or scAAV for viruses. It was named -GFP-shmTOR # 4. The pSP72-scAAV-GFP vector was also named scAAV-GFP in the case of viruses.

<Example 7> Examination of mTOR expression inhibitory effect of mTOR shRNA using real-time RT-PCR

Total RNA was extracted from HeLa cells using Trizol reagent (Invitrogen, Carlsbad, CA) as in Example 3. Reverse transcription (RT) and real-time RT-PCR were also performed in the same manner as in Example 3. The primers used were human-mTOR-sense and antisense primers (SEQ ID NOS: 7 and 8) and general β-actin-sense and antisense primers (SEQ ID NOs: 9 and 10).

Relative gene expression was also evaluated by GeneXpression Macro chromo4 software (Bio-Rad) as in Example 3. As a result, as shown in FIG. 5, mTOR shRNA # 1 or mTOR shRNA # 4 inhibited mTOR mRNA expression in HeLa cell lines with a similar or better effect than siRNA.

Example 8 Analysis of Changes in Human Cancer Cell Cycle by mTOR shRNA

In order to analyze the change in the cancer cell cycle by mTOR shRNA, propidium iodide staining was performed to use flow cytometry. The cancer cell line used was A549, a human lung cancer cell line, and sk-hep-1, a human liver cancer cell line.

Each cancer cell line was infected with rAAV2-mTOR shRNA and four days later, the cells were collected and fixed dropwise by dropping cold 75% ethanol dissolved in PBS. Thereafter, propidium iodide and RNAse were added at 2 µg / ml and 10 µg / ml, respectively, and incubated in a 37 ° C incubator for 30 minutes. Thereafter, cell cycles, namely G1, S and G2 phases, were measured by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, Calif.).

As a result, as shown in Figures 6a and 6b it was confirmed that each cell causes G1 arrest by reducing the S phase by mTOR shRNA.

Example 9 Observation of Intracellular Morphology by mTOR shRNA

Autophagy, one of the phenomena caused by mTOR inhibition by mTOR shRNA, has been reported to affect cancer cell death. Autophagy is a mechanism of cell death, in which the vesicles with double membranes in the cytoplasm increase.

Transmission electron microscopy (TEM) was used to observe morphological changes such as increase of autophagic vesicles in cytoplasm by mTOR shRNA. For this, rAAV2-scramble shRNA and rAAV2-mTOR shRNA were infected with the A549 and sk-hep-1 cell lines, and the cells were collected four days, and the cells were observed with a transmission electron microscope.

As a result, the cells infected with rAAV2-mTOR shRNA, as shown in Figure 7a and 7b it can be seen that the autophagic vesicle (biphalic vesicle) having a double membrane in the cytoplasm significantly compared to the cells infected with rAAV2-scramble shRNA. In addition, it was confirmed that the shape of the nucleus was also abnormally aggregated. This proves that the survival of cancer cells is threatened by mTOR shRNA.

Figure 1 relates to the inhibitory effect of mTOR gene expression by four mTOR-specific siRNA in the human cell line (HeLa), the top of the RNA level of the endogenous mTOR measured using real-time RT-PCR and the bottom of the Western blot analysis Shows the protein level of the endogenous mTOR measured using,

Figure 2 shows the flow cytometry analysis results specific for VP1,

3a and 3b show the inhibitory effect of mTOR gene expression by mTOR-4 siRNA in monkey cell line (Vero) and rat cell line (H9C2),

4a and 4b show a cleavage map of the recombinant vector pSP72-scAAV-GFP-mTOR according to the present invention,

Figure 5 shows the inhibitory effect of mTOR expression of mTOR shRNA in human cell line (HeLa),

6a and 6b analyze the human cancer cell cycle change by mTOR shRNA,

7A and 7B show morphological changes in human cancer cells by mTOR shRNA.

<110> UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERATION <120> mTOR targeting siRNA, and expression vector and pharmaceutical          composition comprising the same <130> dp-2009-0558 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> mTOR-1 <400> 1 ggagucuacu cgcuucuau 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> mTOR-2 <400> 2 gaagaagguc acugaggau 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> mTOR-3 <400> 3 acaaccucca ggauacacu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> mTOR-4 <400> 4 gaauguugac caaugcuau 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat-mTOR-sense primer <400> 5 ccactgtgcc agaatccatc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat-mTOR-antisense primer <400> 6 gagaaatccc gaccagtgag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human-mTOR-sense primer <400> 7 ccacagtgcc agaatctatt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human-mTOR-antisense primer <400> 8 gagaagtccc gaccagtgag 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Universal beta-actin-sense primer <400> 9 tgaagatcaa gatcattgct c 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Universal beta-actin-antisense primer <400> 10 tgaagatcaa gatcattgct c 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pH1-sense primer <400> 11 ccatggaatt cgaacgctga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pH1-antisense primer <400> 12 gggaaagagt ggtctcatac 20 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pH1-sense-EcoRI primer <400> 13 atcgaattca tatttgcatg tcgctatgtg 30 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> pH1-antisense-BamHI primer <400> 14 atcggatccg agtggtctca tacagaac 28 <210> 15 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> mTOR sh # 1-sense-BamHI primer <400> 15 gatccggagt ctactcgctt ctatttcaag agaatagaag cgagtagact ccttttttgg 60 aaa 63 <210> 16 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> mTOR sh # 1-antisense-HindIII primer <400> 16 agcttttcca aaaaaggagt ctactcgctt ctattctctt gaaatagaag cgagtagact 60 cc 62 <210> 17 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> mTOR sh # 4-sense-BamHI primer <400> 17 gatccgaatg ttgaccaatg ctatttcaag agaatagcat tggtcaacat tcttttttgg 60 aaa 63 <210> 18 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> mTOR sh # 4-antisense-HindIII primer <400> 18 agcttttcca aaaaagaatg ttgaccaatg ctattctctt gaaatagcat tggtcttctt 60 tcg 63 <210> 19 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> AAV-sense-KpnI primer <400> 19 cgggagatct tcctgcagga tatctggatc cacgaagctt cccaccggtt ctagagcg 58 <210> 20 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> AAV-antisense-SalI primer <400> 20 tcgacgctct agaaccggtg ggaagcttcg tggatccaga tatcctgcag gaagatctcc 60 cggtac 66  

Claims (9)

SiRNA having a cross-linking activity, characterized in that having one of the nucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 4, and binds to mTOR to inhibit the expression. delete Recombinant vector comprising siRNA according to claim 1. The recombinant vector according to claim 3, wherein the recombinant vector has a cleavage map shown in FIG. 4A or 4B. The recombinant vector according to claim 3 or 4, wherein the recombinant vector is pSP72-scAAV-GFP-shmTOR # 1 or pSP72-scAAV-GFP-shmTOR # 4. delete delete A pharmaceutical composition for treating and preventing cancer diseases containing siRNA according to claim 1 as an active ingredient. The method of claim 8, wherein the cancer disease is liver cancer, lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal muscle cancer , Colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra Cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma A pharmaceutical composition for treating and preventing cancer diseases, which is at least one cancer disease selected from the group consisting of:

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