WO2010064851A2 - Arn interférant court à réaction croisée interspécifique, vecteur de recombinaison contenant ce dernier et composition pharmaceutique contenant ce dernier - Google Patents

Arn interférant court à réaction croisée interspécifique, vecteur de recombinaison contenant ce dernier et composition pharmaceutique contenant ce dernier Download PDF

Info

Publication number
WO2010064851A2
WO2010064851A2 PCT/KR2009/007175 KR2009007175W WO2010064851A2 WO 2010064851 A2 WO2010064851 A2 WO 2010064851A2 KR 2009007175 W KR2009007175 W KR 2009007175W WO 2010064851 A2 WO2010064851 A2 WO 2010064851A2
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
mtor
seq
disease
sirna
Prior art date
Application number
PCT/KR2009/007175
Other languages
English (en)
Korean (ko)
Other versions
WO2010064851A3 (fr
Inventor
이희란
김승후
안정현
김성진
이휘선
이윤선
Original Assignee
울산대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020090110639A external-priority patent/KR101069101B1/ko
Application filed by 울산대학교 산학협력단 filed Critical 울산대학교 산학협력단
Publication of WO2010064851A2 publication Critical patent/WO2010064851A2/fr
Publication of WO2010064851A3 publication Critical patent/WO2010064851A3/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01037—Protein kinase (2.7.1.37)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • 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
  • 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 mimmalian Target of rapamycin
  • IL-2 interleukin-2
  • Inhibition of mTOR results in inhibition of progression from G1 to S in the cell cycle.
  • the antibiotic rapamycin (commercially available as Sirolimus TM ) produced by Streptomyces hygroscopicus has been found to be an important mTOR inhibitor.
  • 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).
  • RNAi ribonucleic acid mediated interference phenomenon
  • mRNA transcript complementary sequence
  • 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 read compounds optimized for all protein targets, including the target material, can proceed quickly.
  • 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.
  • siRNA 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. .
  • 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.
  • 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.
  • the present invention provides an siRNA having any one nucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 4.
  • 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 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 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 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.
  • diseases for example cancer, neurodegenerative diseases, immune diseases, infectious diseases, aging, heart disease, liver disease and Crohn's disease, especially cancer diseases.
  • 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,
  • FIG. 2 shows the flow cytometry analysis results specific for VP1
  • 3 and 4 show the inhibitory effect of mTOR gene expression by mTOR-4 siRNA in monkey cell line (Vero) and rat cell line (H9C2),
  • 5 and 6 show a cleavage map of the recombinant vector pSP72-scAAV-GFP-mTOR according to the present invention
  • Figure 7 shows the inhibitory effect of mTOR expression of mTOR shRNA in human cell line (HeLa).
  • 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.
  • siRNA targeting mTOR 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.
  • a method for preparing RNA molecules chemical synthesis methods and enzymatic methods can be used.
  • 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.
  • 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.
  • AAU Adeno Associated Virus
  • Adeno-associated viruses rarely cause immune responses and cytotoxicity.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mTOR target siRNA according to the present invention when 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.
  • 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.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient, for example, starch, calcium carbonate, sucrose ( Prepare by mixing sucrose or lactose, gelatin, etc.
  • 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.
  • 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.
  • 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.
  • 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. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
  • 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.
  • Target sequences were selected from a completely conserved squence pattern in various species of mTOR.
  • the species used include human (LOCUS: NM_004958), monkey (XR_014791), rat (NM_019906) and mouse (NM_020009).
  • siRNA design software developed in-house, called MWG Biotech AG software (www.mwgbiotech.com) or CAPSID (Convenient Application Program for siRNA Design) was used.
  • 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
  • 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 ⁇ g / ml) at 37 ° C. in a 5% CO 2 incubator. (Dulbecco's modified Eagle's medium; Gibco BRL, Carlsbad, Calif.) Medium.
  • FBS fetal bovine serum
  • glutamax-1 2 mM
  • penicillin 100 IU / ml
  • streptomycin 50 ⁇ g / ml
  • 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)]. .
  • ⁇ -actin was used as a normal control to assess the relative degree of gene expression.
  • 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 ⁇ -actin-sense and antisense primers (SEQ ID NO: 9 and 10), and monkey mTOR primers were identical to those of humans.
  • the cells were recovered and lysed with 100 ⁇ l of lysis buffer (Intron, Seoul, Korea).
  • 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.
  • mTOR 1000, Cell signaling, Boston, MA
  • ⁇ -actin 1: 5000
  • GFP 1000, Santa Cruz
  • Flow cytometric analysis was performed to analyze mTOR expression by methods other than Western blot analysis.
  • cells were pooled and fixed in 4% paraformaldehyde dissolved in PBS. After permeation with 0.05% TritonX-100, cells were incubated with mTOR primary (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.).
  • 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.
  • 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.).
  • 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, followed by 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).
  • the sense primer sequence with BamHI linker for mTOR sh # 4 is 5'-GATCC GAATGTTGACCAATGCTAT TTCAAGAGA ATAGCATTGGTCAACATTC TTTTTT GGAAA-3 '(SEQ ID NO: 17)
  • 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.
  • 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.
  • 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. .
  • 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.
  • CMV promoter from pSP72-XP-rAAV-MCS was removed by KpnI and XbaI digestion.
  • 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. .
  • 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-XP-rAAV-GFP-pH1-shmTOR # 1 or plasmid pSP72-XP-rAAV-GFP-pH1-shmTOR # 4 for plasmids and rAAV-GFP-shmTOR # 1 or for viruses. It was named rAAV-GFP-shmTOR # 4 (FIGS. 5 and 6).
  • the pSP72-XPrAAV-GFP vector was also named rAAV-GFP in the case of viruses.
  • the primers used were human-mTOR-sense and antisense primers (SEQ ID NOS: 7 and 8) and general ⁇ -actin-sense and antisense primers (SEQ ID NOs: 9 and 10).
  • mTOR shRNA # 1 or mTOR shRNA # 4 as shown in Figure 7 inhibited mTOR mRNA expression in HeLa cell line with a similar or better effect than siRNA.
  • 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 ⁇ g / ml and 10 ⁇ g / 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.).
  • 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.
  • TEM Transmission electron microscopy
  • the cells infected with rAAV2-mTOR shRNA showed a significant increase in autophagic vesicles with a double membrane in the cytoplasm compared with cells infected with rAAV2-scramble shRNA.
  • the shape of the nucleus was also abnormally aggregated. This proves that the survival of cancer cells is threatened by mTOR shRNA.
  • SEQ ID NO: 1 to 4 shows the sequence of the siRNA used in one embodiment of the present invention
  • SEQ ID NOs: 5-10 show the sequence of each primer set of rat, human and normal ⁇ -actin used for Real time RT-PCR
  • SEQ ID NOS: 11-14 show the sequence of each primer set for the first amplification of pH1 and the second amplification of the amplified pH1 PCR product
  • SEQ ID NO: 15 shows a sense primer sequence with a BamHI linker for mTOR sh # 1,
  • SEQ ID NO: 16 shows an antisense primer sequence with a HindIII linker for mTOR sh # 1,
  • SEQ ID NO: 17 shows a sense primer sequence with a BamHI linker for mTOR sh # 4,
  • SEQ ID NO: 18 shows an antisense primer sequence with a HindIII linker for mTOR sh # 4,
  • SEQ ID NO: 19 shows a sense oligonucleotide sequence having a Kpn I restriction enzyme site
  • SEQ ID NO: 20 shows an antisense oligonucleotide sequence having Sal I restriction enzyme sites.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un ARNic ciblé sur mTOR ayant une réaction croisée interspécifique, un vecteur de recombinaison contenant ce dernier et une composition pharmaceutique contenant ce dernier en tant qu'ingrédient actif. L'ARNic ciblé sur mTOR a une séquence complémentaire respectivement à une partie du gène mTOR et peut par conséquent décomposer l'ARNm du gène mTOR ou inhiber la traduction de l'ARNm. Par conséquent, l'ARNic ciblé sur mTOR selon la présente invention peut prévenir ou traiter une grande diversité de maladies liées à mTOR telles que le cancer, les maladies neurodégénératives, les maladies autoimmunes, les maladies infectieuses, les maladies cardiaques, la maladie de Crohn, peut prévenir le vieillissement et se trouve particulièrement utile dans la prévention ou le traitement du cancer.
PCT/KR2009/007175 2008-12-02 2009-12-02 Arn interférant court à réaction croisée interspécifique, vecteur de recombinaison contenant ce dernier et composition pharmaceutique contenant ce dernier WO2010064851A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20080121507 2008-12-02
KR10-2008-0121507 2008-12-02
KR10-2009-0110639 2009-11-17
KR1020090110639A KR101069101B1 (ko) 2008-12-02 2009-11-17 종간 교차활성을 지닌 ｍＴＯＲ을 표적으로 하는 ｓｉＲＮＡ, 이를 포함하는 재조합벡터 및 이를 함유하는 약학조성물

Publications (2)

Publication Number Publication Date
WO2010064851A2 true WO2010064851A2 (fr) 2010-06-10
WO2010064851A3 WO2010064851A3 (fr) 2010-10-07

Family

ID=42233740

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2009/007175 WO2010064851A2 (fr) 2008-12-02 2009-12-02 Arn interférant court à réaction croisée interspécifique, vecteur de recombinaison contenant ce dernier et composition pharmaceutique contenant ce dernier

Country Status (1)

Country Link
WO (1) WO2010064851A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180028890A (ko) * 2016-09-09 2018-03-19 (주) 씨드모젠 mTOR 저해제를 함유하는 황반변성 치료용 의약조성물
WO2018048046A3 (fr) * 2016-09-09 2018-09-07 주식회사 케이알진바이오 Composition pharmaceutique contenant un inhibiteur de mtor pour le traitement de la dégénérescence maculaire
WO2023116607A1 (fr) * 2021-12-21 2023-06-29 苏州瑞博生物技术股份有限公司 Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation associés

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060094674A1 (en) * 2002-07-05 2006-05-04 Neel Benjamin G Combination of mtor inhibitor and a tyrosine kinase inhibitor for the treatment of neoplasms
US20070039072A1 (en) * 2002-11-14 2007-02-15 Dharmacon Inc. Functional and hyperfunctional siRNA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060094674A1 (en) * 2002-07-05 2006-05-04 Neel Benjamin G Combination of mtor inhibitor and a tyrosine kinase inhibitor for the treatment of neoplasms
US20070039072A1 (en) * 2002-11-14 2007-02-15 Dharmacon Inc. Functional and hyperfunctional siRNA

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 12 October 2008 'Homo sapiens FK506 binding protein 12-rapamycin associated protein 1 (FRAP 1), mRNA' Database accession no. NM_ 004958 *
FUMITAKA TAKESHITA ET AL.: 'Therapeutic potential of RNA interference against cancer' CANCER SCI vol. 97, 13 June 2006, pages 689 - 696 *
KATRIN TIEMANN ET AL.: 'RNAi-based therapeutics-current status, challenges and prospects' EMBO MOL MED vol. 1, 30 June 2009, pages 142 - 151 *
KYUNG SONG ET AL.: 'Novel roles of Akt and mTOR in suppressing TGF-b/ALK5-mediated Smad3 activation' EMBO J vol. 25, no. 1, 15 December 2005, pages 58 - 69 *
XINXIN BU ET AL.: 'Coupled down-regulation of mTOR and telomerase activity during fluorouracil induced apoptosis of hepatocarcinoma Cells' BMC CANCER vol. 7, 12 November 2007, page 208 *
YUKI TAKAHASHI ET AL.: 'Nonviral vector-mediated RNA interference: Its gene silencing characteristics and important factors to achieve RNAi-based gene therapy' ADVANCED DRUG DELIVERY REVIEWS vol. 61, 20 April 2009, pages 760 - 766 *
ZHOU P. ET AL.: 'siRNA targeting mammalian target of rapamycin (mTOR) attenuates experi-mental proliferative vitreoretinopathy' CURR EYE RES. vol. 32, no. 11, 30 November 2007, pages 973 - 984 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180028890A (ko) * 2016-09-09 2018-03-19 (주) 씨드모젠 mTOR 저해제를 함유하는 황반변성 치료용 의약조성물
WO2018048046A3 (fr) * 2016-09-09 2018-09-07 주식회사 케이알진바이오 Composition pharmaceutique contenant un inhibiteur de mtor pour le traitement de la dégénérescence maculaire
KR101951787B1 (ko) * 2016-09-09 2019-03-05 주식회사 큐로진생명과학 mTOR 저해제를 함유하는 황반변성 치료용 의약조성물
CN109937053A (zh) * 2016-09-09 2019-06-25 奎罗津生命科学 用于治疗黄斑变性的含有mTOR抑制剂的药物组合物
JP2019530675A (ja) * 2016-09-09 2019-10-24 クロジーン・ライフ・サイエンシズ・カンパニー・リミテッド ｍＴＯＲ阻害剤を含有する黄斑変性治療用薬学組成物
US10583150B2 (en) 2016-09-09 2020-03-10 Curogene Life Sciences Co., Ltd. Pharmaceutical composition containing mTOR inhibitor for treating macular degeneration
EP3517133A4 (fr) * 2016-09-09 2020-04-08 CuroGene Life Sciences Co., Ltd. Composition pharmaceutique contenant un inhibiteur de mtor pour le traitement de la dégénérescence maculaire
AU2017323898B2 (en) * 2016-09-09 2021-02-25 Cdmogen Co., Ltd. Pharmaceutical composition containing mTOR inhibitor for treating macular degeneration
CN109937053B (zh) * 2016-09-09 2023-09-26 赛德摩金有限公司 用于治疗黄斑变性的含有mTOR抑制剂的药物组合物
WO2023116607A1 (fr) * 2021-12-21 2023-06-29 苏州瑞博生物技术股份有限公司 Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation associés

Also Published As

Publication number Publication date
WO2010064851A3 (fr) 2010-10-07

Similar Documents

Publication Publication Date Title
RU2394041C2 (ru) Полимерные олигонуклеотидные пролекарства
JP2005518452A (ja) 肝搬送のため、修飾オリゴヌクレオチドを用いる方法
WO2013103249A1 (fr) Structure d'oligo-arn double hélice de type nanoparticule à efficacité élevée et son procédé de préparation
WO2018143626A1 (fr) Acide nucléique inhibant simultanément l'expression du gène mtor et du gène stat3
WO2020245233A1 (fr) Oligonucléotides antisens ciblant l'atxn3
WO2010064851A2 (fr) Arn interférant court à réaction croisée interspécifique, vecteur de recombinaison contenant ce dernier et composition pharmaceutique contenant ce dernier
WO2015005669A1 (fr) Arnsi spécifique de gènes liés au cancer du foie, molécules d'oligo-arn double brin comprenant l'arnsi, et composition de prévention ou de traitement du cancer le comprenant
CN110201172B (zh) Yy1表达抑制剂在制备治疗乳腺癌药物中的应用
WO2024053933A1 (fr) Construction comprenant une séquence utr améliorant la stabilité intracellulaire et la biosynthèse d'arnm et son utilisation
KR101069101B1 (ko) 종간 교차활성을 지닌 ｍＴＯＲ을 표적으로 하는 ｓｉＲＮＡ, 이를 포함하는 재조합벡터 및 이를 함유하는 약학조성물
WO2018155890A1 (fr) Petit arni asymétrique permettant d'inhiber l'expression d'un gène cible de perte de cheveux masculine
WO2021194179A1 (fr) Virus oncolytique comprenant une séquence d'acide nucléique ciblant stat3 et mtor de manière bispécifique
WO2018110814A1 (fr) Parni spécifique du gène de fusion (ss18-ssx), et composition pharmaceutique destinée à prévenir ou à traiter le cancer
WO2021256855A1 (fr) Composition pour le traitement d'un cancer solide métastatique, comprenant un inhibiteur de tsg6
US9932585B2 (en) Manipulating microRNA for the management of neurological diseases or conditions and compositions related thereto
WO2010095879A2 (fr) Petit arn interferent a lier a des transcrits de genes associes a l'apoptose et compositions de traitement du cancer contenant ce petit arni
WO2013165061A1 (fr) Composition comprenant une matière pour inhiber un facteur de cellules souches (fcs) ou son récepteur pour traiter ou prévenir des maladies associées à une perméabilité vasculaire
WO2022191661A1 (fr) Acides nucléiques inhibant simultanément l'expression du gène c-met et du gène pd-l1
WO2023014192A1 (fr) Ligand d'acide nucléique non naturel, ses utilisations, et composition pharmaceutique pour la prévention ou le traitement du cancer comprenant celui-ci en tant que principe actif
KR102489708B1 (ko) GDF11 유전자의 발현을 조절하는 PCBP2와 microRNA를 함유하는 조성물
WO2006003804A1 (fr) Oligonucléotide inhibant la prolifération de cellules de tumeur et procédé pour cela
KR102371833B1 (ko) GDF11 유전자의 발현을 조절하는 microRNA 및 이를 함유하는 조성물
WO2021194181A1 (fr) Virus anticancéreux comprenant une molécule d'acide nucléique ciblant ar et mtor de manière bispécifique
Allaman-Pillet et al. The 5′ repeat elements of the mouse Xist gene inhibit the transcription of X-linked genes
EP3724333A2 (fr) Oligonucléotide pour la modulation d'expression du fndc3b

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09830589

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09830589

Country of ref document: EP

Kind code of ref document: A2