WO2018143626A1 - Mtor 유전자 및 stat3 유전자의 발현을 동시에 억제하는 핵산 - Google Patents
Mtor 유전자 및 stat3 유전자의 발현을 동시에 억제하는 핵산 Download PDFInfo
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Definitions
- the present invention relates to a nucleic acid molecule that simultaneously inhibits the expression of the mTOR gene and the STAT3 gene, and an anticancer pharmaceutical composition comprising the same.
- Cancer is one of the world's most fatalities, and the development of innovative cancer treatments can reduce the cost of treatment and create high added value.
- molecular therapies that can overcome existing anticancer drug resistance accounted for $ 17.5 billion in seven major countries (US, Japan, France, Germany, Italy, Spain, UK) and about 2018 It has a market size of about $ 45 billion and is expected to grow 9.5% over 2008.
- the treatment of cancer is divided into surgery, radiation therapy, chemotherapy, and biological therapy.
- chemotherapy is a chemical substance that inhibits or kills the proliferation of cancer cells.
- the anticancer drug is effective, the resistance is lost after a certain period of time, but the development of an anticancer drug that selectively acts on cancer cells and does not develop is urgently needed. 2004. 6 (19).
- RNA interference interference RNA
- mRNA transcript a transcript with complementary sequences of small interfering ribonucleic acid short interfering RNA (siRNA) with a double-stranded structure of 21-25 nucleotides in size. It is a phenomenon that suppresses expression of a specific protein by decomposing a transcript.
- RNA double strand is processed by an endonuclease called Dicer and converted into 21-23 base pair (bp) siRNAs, which bind to an RNA-induced silencing complex (RISC).
- RISC RNA-induced silencing complex
- siRNA against the same target gene is superior to antisense oligonucleotides (ASOs) in inhibiting mRNA expression in vitro and in vivo, and the effect lasts for a long time.
- ASOs antisense oligonucleotides
- the market for therapeutic drugs based on RNAi technology, including siRNA, is estimated to form more than 12 trillion won in the global market by 2020. It is evaluated as the next generation gene therapy technology that can cure diseases that are difficult to treat.
- siRNA binds complementarily with the target mRNA and regulates the expression of the target gene in a sequence-specific manner, allowing long time for conventional antibody-based drugs or small molecule drugs to be optimized for specific protein targets.
- the applicable target can be significantly expanded and the development period can be shortened, so that lead compounds optimized for all protein targets including non-pharmaceutical target substances can be developed.
- siRNA therapeutics have the advantage of having a clear target and predictable side effects.However, in the case of tumors, which are caused by problems of various genes, these target specificities do not have high therapeutic effects. It can also be a cause.
- 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. 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).
- 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).
- STAT3 signal transducer and activator of transcription 3 is a transcription factor that promotes transcription by transmitting signals of various growth factors and cytokines outside the cell to the nucleus. Phosphorylated tyrosine residues in the transactivation domain are activated and introduced into the nucleus (STAT3 inhibitors for cancer therapy: Have all roads been explored Jak-Stat. 2013; 1; 2 (1): e22882). Phosphorylated STAT3 (p-STAT3) binds to the DNA of the nucleus and induces the expression of a broad range of target genes involved in tumorigenesis, such as proliferation and differentiation of cells. It is constantly active in about 70% (Role of STAT3 in cancer metastasis and translational advances. BioMed research international.
- Stat3 and mTOR are the major cancer-related genes whose prognosis is determined in lung cancer, prostate cancer, head and neck cancer, etc. according to the expression level, but they are targeted for the development of anticancer drugs. This is impossible, and cystemic side effects are expected to deliver the drug, it is difficult to develop a new drug to suppress them.
- An object of the present invention to provide a nucleic acid that inhibits the expression of mTOR gene and STAT3 gene at the same time, in order to overcome the disadvantage that the therapeutic effect due to the target specificity of siRNA in the present invention, mTOR gene and STAT3 gene associated with cancer SiRNA and shRNA were produced to inhibit the expression of the same, and its anticancer activity and synergistic anticancer activity with anticancer drugs to confirm the purpose of using it as a pharmaceutical composition for preventing or treating cancer.
- the present invention provides a nucleic acid molecule that simultaneously inhibits the expression of mTOR and STAT3 genes.
- the present invention also provides a recombinant expression vector comprising the nucleic acid molecule.
- the present invention also provides a recombinant microorganism incorporating the recombinant expression vector.
- the present invention provides a pharmaceutical composition for anticancer, comprising the nucleic acid molecule as an active ingredient.
- the present invention also provides a method for preventing and treating cancer, comprising administering to a subject a pharmaceutically effective amount of the nucleic acid molecule of claim 1.
- the double-stranded siRNA or shRNA of the present invention inhibits the expression of the mTOR gene and the antisense strand inhibits the expression of the STAT3 gene, thereby simultaneously inhibiting the two genes without treating each siRNA or shRNA separately.
- FIG. 1 is a diagram showing a map of a vector for expressing shRNAs in a cell comprising a double strand siRNA sequence of the present invention together with a loop sequence in one strand.
- Figure 2 is a diagram confirming the inhibitory effect of mTOR or STAT3 gene expression by double-stranded siRNA of the double target of the present invention.
- siRNA for the mTOR gene or STAT3 gene (nc2 is a control siRNA; simTOR is siRNA targeting only mTOR; siSTAT3 is siRNA targeting only STAT3; simTOR & STAT3 is mTOR targeting siRNA and STAT3 targeting siRNA in combination).
- Figure 4 is a diagram confirming the cell survival rate of human lung cancer cell line A549 cells when the dual target siRNA of the present invention simultaneously inhibited mTOR and STAT3.
- FIG. 5 is a diagram confirming the cell survival rate of human lung cancer cell line A549 cells when mTOR and STAT3 are simultaneously suppressed by the double target siRNA of the present invention after cisplatin treatment.
- Figure 7 is a diagram confirming the cell survival rate of human lung cancer cell line A549 cells when 5-FU (5-fluorouracil) treatment, when mTOR and STAT3 are simultaneously suppressed by the dual target siRNA of the present invention.
- FIG. 8 is a diagram showing the expression amount of mTOR and STAT3 by the vector containing the TTGGATCCAA loop shRNA sequence of SEQ ID NO: 20 or the TTCAAGAGAG loop shRNA of SEQ ID NO: 21 according to the DNA amount of shRNA.
- the present invention provides nucleic acid molecules that simultaneously inhibit the expression of mTOR and STAT3 genes.
- the nucleic acid molecules include SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 5 and 6; SEQ ID NOs: 7 and 8; SEQ ID NOs: 9 and 10; SEQ ID NOs: 11 and 12; SEQ ID NOs: 13 and 14; SEQ ID NOs: 15 and 16; Or the nucleotide sequences of SEQ ID NOs: 17 and 18;
- the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 and 17 inhibits mTOR gene expression by RNA interference
- SEQ ID NO: 2, 4 The base sequences represented by 6, 8, 10, 12, 14, 16, and 18 can inhibit the expression of the STAT3 gene by RNA interference, and the nucleic acid molecule of the present invention simultaneously inhibits the expression of the mTOR gene and the STAT3 gene. can do.
- SEQ ID NO: 1 is SEQ ID NO: 2
- SEQ ID NO: 3 is SEQ ID NO: 4
- SEQ ID NO: 5 is SEQ ID NO: 6
- SEQ ID NO: 7 is SEQ ID NO: 8
- SEQ ID NO: 9 is SEQ ID NO: 10
- SEQ ID NO: 11 is SEQ ID NO: 12
- SEQ ID NO: 13 is SEQ ID NO: 14
- SEQ ID NO: 15 is SEQ ID NO: 16
- SEQ ID NO: 17 is partially complementary to the strand strand (double strand) ) siRNAs were constructed, and double stranded siRNAs were identified as double target siRNA sets by targeting mTOR and STAT3 genes to inhibit expression.
- siRNA targeting mTOR or STAT3 has a sequence 100% complementary to a part of the mTOR gene or STAT3 gene of human ( Homo sapiens ), and can degrade mRNA or inhibit translation of mTOR gene or STAT3 gene. have.
- the term “inhibition of expression” means to cause the expression or translational degradation of the target gene, preferably by means that the target gene expression becomes undetectable or present at an insignificant level.
- siRNA small interfering RNA
- siRNA refers to a short double-chain RNA that can induce RNA interference (RNAi) phenomenon through cleavage of a specific mRNA.
- siRNA is composed of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto
- the double-stranded siRNA of the present invention is the sense RNA strand SEQ ID NO: 1, 3, 5, SiRNA (antisense strand to mTOR gene) represented by the nucleotide sequences of 7, 9, 11, 13, 15 and 17, and the antisense RNA strand is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
- the double-stranded siRNA can suppress the expression of mTOR and STAT3 gene at the same time, respectively, as an efficient gene knock-down method or gene It is provided by
- the siRNA of SEQ ID NOS: 1 and 2 of set 1 is 17mer of 21mer
- the siRNA of SEQ ID NOs: 3 and 4 of set 2 16mer of 20mer
- the siRNA of SEQ ID NOs: 5 and 6 of set 3 is 19mer 15mer of the pairs
- siRNAs of SEQ ID NOs: 7 and 8 of set 4 14mer of 18mers
- 16mers of the 17mers of SEQ ID NOs: 9 and 10 of Set 5 bind complementarily.
- siRNAs of SEQ ID NOs: 11 and 12 of Set 6 were 17mers in 20mers
- the siRNAs of SEQ ID NOs: 13 and 14 of Set 7 were 16mers of 19mers
- the siRNAs of SEQ ID NOs: 15 and 16 of Set 8 were 15mers of 18mers
- the siRNA of SEQ ID NOs: 17 and 18 in set 9 is complementary to 15mer of 17mer.
- nucleic acid molecules that simultaneously inhibit the expression of mTOR and STAT3 genes of the present invention may have deletion, substitution or insertion of functional equivalents of the nucleic acid molecules constituting them, for example, some of the nucleotide sequences of the nucleic acid molecules.
- the gene is a base having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with each of the base sequences of SEQ ID NOs: 1-18. Sequences may be included.
- the "% sequence homology" for a nucleic acid molecule is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the nucleic acid molecule sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
- the present invention also provides a recombinant expression vector comprising the nucleic acid molecule.
- shRNA comprising the same, in particular, the nucleotide sequence of SEQ ID NOS: 1 to 18, may be capable of at least activating at least a promoter. It is preferred to be connected.
- the promoter may be any promoter capable of functioning in eukaryotic cells. Additional regulatory sequences, including leader sequences, polyadenylation sequences, promoters, enhancers, upstream activation sequences, signal peptide sequences, and transcription terminators, as needed, for efficient transcription of double stranded siRNAs or shRNAs targeting mTOR and STAT3 It may also include.
- the shRNA may be represented by the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 21.
- shRNA short hairpin RNA
- shRNA short hairpin RNA
- the length of the double-stranded structure is not particularly limited, but is preferably 10 nucleotides or more, more preferably. Is at least 20 nucleotides.
- the shRNA may be included in a vector.
- vector refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Phagemid vector; Cosmid vector; And viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated virus vectors, and the like.
- the genes in the vectors of the invention are operatively linked with the promoter.
- operably linked means a functional binding between a gene expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another gene sequence, thereby The regulatory sequence will control transcription and / or translation of the other gene sequence.
- a gene expression control sequence eg, a promoter, signal sequence, or array of transcriptional regulator binding sites
- the vector system of the present invention may be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, This document is incorporated herein by reference.
- Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression.
- the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.
- powerful promoters capable of promoting transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoters, recA promoters, SP6 promoters, trp promoters and T7 promoters, etc.
- ribosome binding sites for initiation of translation e.g., ribosome binding sites for initiation of translation
- transcription / detox termination sequences e.g., ribosome binding sites for initiation of translation, and transcription / detox termination sequences.
- coli eg, HB101, BL21, DH5 ⁇ , etc.
- E. coli tryptophan biosynthesis pathway Yanofsky, C. (1984), J. Bacteriol., 158: 1018-). 1024
- a phage ⁇ left promoter pL ⁇ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14: 399-445) can be used as regulatory sites.
- vectors that can be used in the present invention are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14).
- plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14).
- pGEX series, pET series and pUC19, etc.) phagemids (eg pComb3X), phage or virus (eg SV40, etc.) can be engineered.
- the vector of the present invention may be fused with other sequences as needed to facilitate purification of the protein, and the sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA). ), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA) and the like can be used, but are not limited thereto.
- the expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selectable label, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin Resistance genes to neomycin and tetracycline.
- the present invention also provides a recombinant microorganism incorporating the recombinant expression vector.
- Host cells capable of stably and continuously cloning and expressing the vectors of the present invention can use any host cell known in the art, such as Escherichia coli, Bacillus subtilis and Bacillus thuringin Bacillus strains such as cis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g.
- prokaryotic host cells such as, but not limited to, Staphylocus carnosus.
- the host cell is preferably E. coli and more preferably E. coli ER2537, E. coli ER2738, E. coli XL-1 Blue, E. coli BL21 (DE3), E. coli JM109, E. coli DH Series, E. coli TOP10, E. coli TG1 and E. coli HB101.
- the method of carrying the vector of the present invention into a host cell is performed by CaCl 2 method (Cohen, SN et al. (1973), Proc. Natl. Acac. Sci. USA, 9: 2110-2114), one method (Cohen, SN et al. (1973), Proc. Natl. Acac. Sci. USA, 9: 2110-2114; and Hanahan, D. (1983), J. Mol. Biol., 166: 557-580) and electroporation methods (Dower, WJ et al. (1988), Nucleic. Acids Res., 16: 6127-6145) and the like.
- the present invention also provides an anticancer pharmaceutical composition comprising the nucleic acid molecule as an active ingredient.
- the nucleic acid molecule may further comprise an anticancer agent, for example, acibaicin, aclarubicin, acodazole, acronycin, adozelesin, alanosine, aldesleukin, allopurinol sodium, altre Tamine, Aminoglutetimide, Amonaphide, Ampligen, Amsacrine, Androgens, Anguidine, Apidicholine glycinate, Asari, Asparaginase, 5-Azacytidine, Azathioprine, Bacillus Calmette-Guerin (BCG), Bakers Antipol, Beta-2-Dioxythioguanosine, Bisanthrene HCl, Bleomycin Sulfate, Bullspanine, Butionine Suloximine, BWA 773U82, BW 502U83 / HCl, BW 7U85 mesylate, cerasemide, carbetimer, carboplatin, carmustine, chlor
- the cancer is colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, gastric cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal cancer, small intestine cancer , Anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head cancer, cervical cancer, skin melanoma , Intraocular melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, glioblastoma multiforme and pituitary gland It may be any one
- the pharmaceutical composition of the present invention may further comprise an adjuvant in addition to the single domain antibody.
- the adjuvant may be used without any limitation as long as it is known in the art, but may further include the Freund's complete adjuvant or incomplete adjuvant to increase its effect.
- compositions according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical art.
- Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
- compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. .
- Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient in the active ingredient, for example starch, calcium carbonate, sucrose, lactose, gelatin It can be prepared by mixing.
- lubricants such as magnesium stearate, talc can also be used.
- Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be.
- Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories.
- 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, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
- composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
- the dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the individual. It is apparent that the concentration of a single domain antibody included in the pharmaceutical composition can be variously selected according to a subject, and preferably, the pharmaceutical composition is included in a concentration of 0.01 to 5,000 ⁇ g / ml. If the concentration is less than 0.01 ⁇ g / ml, the pharmaceutical activity may not appear, and when the concentration exceeds 5,000 ⁇ g / ml, the human body may be toxic.
- the pharmaceutical composition of the present invention may be used for the prevention or treatment of cancer and its complications, and may also be used as an anticancer adjuvant.
- the present invention also provides a method for preventing and treating cancer, comprising administering to a subject a pharmaceutically effective amount of the nucleic acid molecule of claim 1.
- the pharmaceutical composition of the present invention is administered in a therapeutically effective amount or in a pharmaceutically effective amount.
- pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, drug Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.
- a double target siRNA (double strand) capable of simultaneously inhibiting STAT3 (signal transducer and activator of transcription 3) and mTOR (mammalian target of rapamycin) was prepared in the sequence of Table 1 below (Bioneer, Daejeon, Korea).
- the siRNA of SEQ ID NO: 1 and 2 of the set 1 is 17mer of 21mer
- the siRNA of SEQ ID NO: 3 and 4 of the set 2 is 16mer of 20mer
- the siRNA of SEQ ID NO: 5 and 6 of set 3 is 15mer of 19mer
- the siRNAs of SEQ ID NOs: 7 and 8 of 4 bind 14mer in 18mer
- the siRNAs of SEQ ID NOs: 9 and 10 of Set 5 bind complementarily of 16mers in 17mer.
- siRNAs of SEQ ID NOs: 11 and 12 of Set 6 were 17mers in 20mers
- the siRNAs of SEQ ID NOs: 13 and 14 of Set 7 were 16mers of 19mers
- the siRNAs of SEQ ID NOs: 15 and 16 of Set 8 were 15mers of 18mers
- the siRNAs of SEQ ID NOs: 17 and 18 in set 9 are complementary to 14mers of 17mers.
- the siRNA of the antisense_mTOR of each set of mTOR mRNA (gi
- each set of antisense_STAT3 siRNAs targets STAT mRNA (gi
- the shRNA comprising the sequence of the siRNA double strand and the loop sequence (TTGGATCCAA loop shRNA and TTCAAGAGAGAG) Loop shRNA) was constructed (Table 2).
- the prepared shRNAs were placed to be after the U7 promoter (SEQ ID NO: 19) at the restriction enzymes Pst I and Eco RV cleavage positions of the pE3.1 vector (FIG. 1), respectively, to include a dual target siRNA targeting mTOR and STAT3.
- Recombinant expression vectors were constructed that can express two species of shRNA in cells.
- the cells were incubated at 37 ° C. and 5% CO 2 in RPMI medium (Hyclone) to which 10% FBS (Hyclone) was added until the cell confluent became 50%. Thereafter, the cells were transfected with lipofectamine3000 (Invitrogen, Carlsbad, Calif., USA) using the dual target siRNA prepared in Example 1 to simultaneously drop down Bcl1, BI1, AR, mTOR and STAT3. 48 hours after transfection, cells were disrupted and total RNA was extracted with GeneJET RNA Purification Kit (Invitrogen).
- Reverse transcription reaction was performed with RevoScriptTM RT PreMix (iNtRON BIOTECHNOLOGY) using the extracted total RNA as a template.
- RevoScriptTM RT PreMix iNtRON BIOTECHNOLOGY
- mTOR Hs00234522_m1
- STAT3 Hs01047580_m1
- GAPDH Hs02758991_g1
- the reaction was performed using a PRISM 7700 Sequence Detection System and QS3 Real-time PCR (Biosystems).
- Real-time PCR reaction conditions were performed in a total of 40 cycles [two step cycles of 2 minutes at 50 ° C., 10 minutes at 95 ° C., and 15 seconds at 95 ° C. and 60 seconds at 60 ° C.]. All reactions were repeated three times and averaged. The results obtained were normalized to the mRNA value of GAPDH, a housekeeping gene.
- mTOR and STAT3 were found to have a residual expression of about 20 to 40% compared to the control by the dual target siRNAs of sets 1-9, indicating that the dual target siRNAs inhibited expression of both genes simultaneously. (FIG. 2).
- human lung cancer cell line A549 cells were dispensed at 5 ⁇ 10 3 cells / well in 96-well plates, and then double-targeted with lipofectamine 3000.
- siRNA current knockdown of mTOR and STAT3
- cells were treated with 5 mg / mL MTT (Promega, Ltd.) for an additional 24 hours and incubated for 4 hours.
- the medium was then removed, treated with 150 ⁇ l of solubilization solution and stop solution and incubated at 37 ° C. for 4 hours.
- the absorbance of the reaction solution was measured at 570 nm and cell viability was calculated using the following equation.
- Human lung cancer cell line A549 cells were dispensed in 96-well plates at 5 ⁇ 10 3 cells / well, followed by transfection of cells with lipofectamine3000 to double target siRNA (mTOR and STAT3 simultaneous knockdown), respectively. 48 hours after transfection, 5 ⁇ M cisplatin was treated and incubated for 10 hours. Thereafter, MTT reaction was performed as in Experimental Example 3, and its absorbance was measured at 570 nm to calculate cell viability.
- Human lung cancer cell line A549 cells were dispensed in 96-well plates at 5 ⁇ 10 3 cells / well and then transfected with lipofectamine3000 to double target siRNA (mTOR and STAT3 simultaneous knockdown), respectively. After 48 hours of transfection, 1 ⁇ M of 5-fluorouracil was treated and incubated for 10 hours. Thereafter, MTT reaction was performed as in Experimental Example 3, and its absorbance was measured at 570 nm to calculate cell viability.
- Vectors containing the TTGGATCCAA loop shRNA sequence of SEQ ID NO: 20 or the TTCAAGAGAG loop shRNA of SEQ ID NO: 21 prepared in Example 2 were transfected 0, 1 and 2 ⁇ g into A549 cells using lipofectamine3000, respectively. After 48 hours of transfection, the degree of gene expression reduction of mTOR and STAT3 was confirmed using the Real time PCR analysis method described in Experimental Example 1.
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Abstract
Description
Claims (13)
- mTOR(mammalian target of rapamycin) 유전자 및 STAT3(signal transducer and activator of transcription 3) 유전자의 발현을 동시에 억제하는 핵산 분자.
- 제1항에 있어서, 상기 핵산 분자는 서열번호 1 및 2; 서열번호 3 및 4; 서열번호 5 및 6; 서열번호 7 및 8; 서열번호 9 및 10; 서열번호 11 및 12; 서열번호 13 및 14; 서열번호 15 및 16; 또는 서열번호 17 및 18;의 염기서열을 포함하는 핵산 분자.
- 제2항에 있어서, 상기 서열번호 1, 3, 5, 7, 9, 11, 13, 15 및 17로 표시되는 염기서열은 RNA 간섭에 의해 mTOR유전자 발현을 억제하는 것을 특징으로 하는, 핵산 분자.
- 제2항에 있어서, 상기 서열번호 2, 4, 6, 8, 10, 12, 14, 16, 및 18로 표시되는 염기서열은 RNA 간섭에 의해 STAT3 유전자의 발현을 억제하는 것을 특징으로 하는, 핵산 분자.
- 제2항에 있어서, 상기 서열번호 1은 서열번호 2와, 서열번호 3은 서열번호 4와, 서열번호 5는 서열번호 6과, 서열번호 7은 서열번호 8과, 서열번호 9는 서열번호 10과, 서열번호 11은 서열번호 12와, 서열번호 13은 서열번호 14와, 서열번호 15는 서열번호 16과, 서열번호 17은 서열번호 18과 부분적으로 상보적 결합을 이루고 있는 이중 가닥(double strand) siRNA인 것을 특징으로 하는, 핵산 분자.
- 제1항에 있어서, 상기 핵산 분자는 shRNA(short hairpin RNA)를 더 포함하는 것을 특징으로 하는, 핵산 분자.
- 제 6항에 있어서, 상기 shRNA는 서열번호 20 또는 서열번호 21의 염기서열로 표시되는 것을 특징으로 하는, 핵산 분자.
- 제1항의 핵산 분자를 포함하는 재조합 발현 벡터.
- 제8항의 재조합 발현 벡터를 도입한 재조합 미생물.
- 제1항의 핵산 분자를 유효성분으로 포함하는, 항암용 약학적 조성물.
- 제10항에 있어서, 상기 핵산 분자는 항암제를 추가로 포함하는 것을 특징으로 하는, 항암용 약학적 조성물.
- 제11항에 있어서, 상기 항암제는 시스플라틴, 파클리탁셀, 5-FU(5-fluorouracil), 메토트렉세이트, 독소루비신, 다우노루비신, 사이토신아라비노시드, 에토포시드, 멜파란, 클로람부실, 사이클로포스파마이드, 빈데신, 마이토마이신, 블레오마이신, 타목시펜 및 탁솔로 구성된 군에서 선택된 1종 이상의 항암제인 것을 특징으로 하는, 항암용 약학적 조성물.
- 약학적으로 유효한 양의 제1항의 핵산분자를 개체에 투여하는 단계를 포함하는 암의 예방 및 치료방법.
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AU2018216509A AU2018216509B2 (en) | 2017-01-31 | 2018-01-29 | Nucleic acid simultaneously inhibiting expression of mTOR gene and STAT3 gene |
CN201880009393.9A CN110234764B (zh) | 2017-01-31 | 2018-01-29 | 同时抑制mTOR基因及STAT3基因表达的核酸 |
EP18747648.6A EP3578655B1 (en) | 2017-01-31 | 2018-01-29 | Nucleic acid simultaneously inhibiting expression of mtor gene and stat3 gene |
JP2019562531A JP6962600B2 (ja) | 2017-01-31 | 2018-01-29 | Mtor遺伝子およびstat3遺伝子の発現を同時に抑制する核酸 |
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WO2023086927A1 (en) * | 2021-11-11 | 2023-05-19 | Vanderbilt University | Combined targeting of stat3 and ulk1 to treat glioblastoma |
WO2023116607A1 (zh) * | 2021-12-21 | 2023-06-29 | 苏州瑞博生物技术股份有限公司 | 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 |
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