WO2013115579A1 - Shrna for suppressing tgf-β2 expression - Google Patents

Shrna for suppressing tgf-β2 expression Download PDF

Info

Publication number
WO2013115579A1
WO2013115579A1 PCT/KR2013/000791 KR2013000791W WO2013115579A1 WO 2013115579 A1 WO2013115579 A1 WO 2013115579A1 KR 2013000791 W KR2013000791 W KR 2013000791W WO 2013115579 A1 WO2013115579 A1 WO 2013115579A1
Authority
WO
WIPO (PCT)
Prior art keywords
shrna
tgf
seq
expression
adenovirus
Prior art date
Application number
PCT/KR2013/000791
Other languages
French (fr)
Korean (ko)
Inventor
송재진
김주항
오세은
Original Assignee
연세대학교 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 KR1020130010233A external-priority patent/KR101420564B1/en
Application filed by 연세대학교 산학협력단 filed Critical 연세대학교 산학협력단
Priority to CN201380007620.1A priority Critical patent/CN104245936B/en
Priority to JP2014555484A priority patent/JP2015506696A/en
Publication of WO2013115579A1 publication Critical patent/WO2013115579A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
    • 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 relates to shRNAs that inhibit TGF- ⁇ 2 expression and antitumor compositions comprising them.
  • TGF- ⁇ 2 like TGF- ⁇ 1, inhibits the proliferation and differentiation of cytotoxic T cells, natural killer cells, and macrophages, thereby inhibiting immune search for growing tumors.
  • TGF- ⁇ 2 is a multifunctional secretory protein that plays various roles such as proliferation suppression, replication, invasion, metastasis, apoptosis, immunoassay, and angiogenesis, depending on the type and timing of the cells, as well as TGF- ⁇ 2 like TGF- ⁇ 1.
  • TGF- ⁇ 2 plays a role in further developing tumors when tumors develop in the late stages of tumor progression, resulting in resistance to TGF- ⁇ 2 proliferation inhibition due to inactivation of signal pathways or abnormal cell cycle regulation. Done.
  • TGF- ⁇ 2 acts clearly differently from TGF- ⁇ 1 as follows.
  • TGF- ⁇ 2 induces Foxp3 to significantly induce immunosuppression, and also affects tumor metastasis, neovascularization and proliferation, leading to tumor progression to malignancy.
  • Non-Patent Document 2 (Chenyu Zhang et al, Transforming growth factor- ⁇ 2 is a molecular determinant for site-specific melanoma metastasis in the brain, Cancer Res. 2009 February 1; 69 (3): 828-35.)
  • shRNA for ⁇ 2 was produced with TGCTGTTGACAGTGAGCGCGGTGTATAAATCGAGACCAAATTAGTGTGAAGCCACAGATGTATTTGGTCTCGATTTATACACCTTGCCCCTACTGCCTCGGA (target), and a lentiviral (lentivirus) that produces TGF- ⁇ 2 shRNA was also produced by chromosome incorporation. There is a big problem with the side effects are delivered.
  • the present inventors have made efforts to solve the above problems, and as a result, by selecting a target that effectively induces silencing of human TGF- ⁇ 2 or mouse TGF- ⁇ 2 to produce shRNA, and mounted it in adenovirus
  • the present invention has been completed by drastically improving the ability to deliver shRNAs by existing non-viral agents.
  • an object of the present invention is to provide a shRNA that suppresses TGF- ⁇ 2 expression using a nucleotide sequence represented by SEQ ID NO: 1 or 2 as a target sequence.
  • Another object of the present invention is to provide an anti-tumor composition containing the shRNA as an active ingredient.
  • the present invention also has another object to provide a recombinant expression vector expressing the shRNA.
  • the present invention also has another object to provide an anti-tumor composition comprising the recombinant expression vector as an active ingredient.
  • Another object of the present invention is to provide an adenovirus into which the recombinant expression vector is introduced.
  • the present invention as a means for solving the above problems
  • an shRNA that suppresses TGF- ⁇ 2 expression is provided.
  • the present invention is another means for solving the above problems
  • the present invention is another means for solving the above problems
  • the present invention is another means for solving the above problems
  • composition comprising the recombinant expression vector as an active ingredient.
  • the present invention is another means for solving the above problems
  • It provides an adenovirus introduced with the recombinant expression vector.
  • the present invention produced a novel shRNA that inhibits TGF- ⁇ 2 expression, and significantly increased specificity, delivery capacity and expression suppression ability compared to the prior art by increasing the infection rate using adenovirus as a gene carrier.
  • the present invention provides an anti-tumor composition containing shRNA that inhibits TGF- ⁇ 2 expression.
  • shRNA that inhibits TGF- ⁇ 2 expression.
  • most cancer cells can be applied to all cancers by assigning a target to adenovirus having excellent delivery efficiency.
  • 1 shows the pSP72 ⁇ E3 / si-negative vector, which is an E3 shuttle vector.
  • FIG. 2 is a schematic diagram of the process of homologous recombination with the adenovirus backbone dl324 after subcloning (TGF- ⁇ 2 shRNA) into the shuttle vector.
  • FIG. 3 shows screening results of E3 region PCR of adenoviruses
  • (b) shows IX gene region PCR results of adenoviruses for selection of homologously recombined colonies in bacteria that are easily recombinant.
  • (c) shows the result of the appearance of fragment DNA after PacI cleavage confirming whether transfection of homologously recombined adenovirus genomic DNA is possible.
  • Figure 4 shows the final recombinant colonies screened in HindIII digestion pattern (digestion pattern).
  • Figure 5 shows the results confirmed by the sequence analysis whether the selected colonies of Figure 4 has shRNA hTGF- ⁇ 2 nucleotide sequence.
  • Figure 6 confirms the ability to inhibit TGF- ⁇ 2 expression by adenovirus expressing human TGF- ⁇ 2 shRNA of Example 2 by real-time PCR.
  • Figure 7 confirms the ability to inhibit TGF- ⁇ 2 expression by adenovirus expressing the mouse TGF- ⁇ 2 shRNA of Example 2 by real-time PCR.
  • Figure 8 confirms the inhibition of TGF- ⁇ 2 expression by the shuttle vector expressing the mouse TGF- ⁇ 2 shRNA of Example 2 by real-time PCR.
  • Figure 9 confirms the ability to inhibit TGF- ⁇ 2 expression by adenovirus expressing human TGF- ⁇ 2 shRNA of Example 2 by ELISA.
  • Figure 10 shows the pBSKII-3484 vector (a), pCA14-3484 vector (b), pCA14-CMV-3484 vector (c) and pCA14-CMV-3484- ⁇ E1B55 vector (d).
  • Figure 11 is a schematic diagram showing the process of producing dl324-CMV-3484-shTGF- ⁇ 2 adenovirus from dl324 adenovirus.
  • Figure 12 shows the homologous recombination process containing mouse shTGF- ⁇ 2, screening confirmed homologous recombination colonies by E3 screening (a), clones (1, 2, 4) homologous recombination by the HindIII cleavage pattern When screening and confirming (b), when the DNA of clones 1, 2 and 4 were cut with PacI, only clone 1 was correctly homologously recombined (c).
  • C control, dl324- ⁇ E3-sh-mTGF ⁇ 2]
  • S shuttle vector pCA14-CMV-3484- ⁇ E1B55
  • 1-6 homologous recombined colony].
  • Figure 13 shows the homologous recombination process containing human shTGF- ⁇ 2, screening confirmation homologous recombination colonies in the HindIII (digestion pattern) (a), final screening confirmation homologous recombination colonies after PacI cleavage (b) [C: control, dl324- ⁇ E3-sh-mTGF ⁇ 2], 1-3: homologous recombination colonies.
  • Figure 14 will confirm cell hemolysis in cancer cells of the tumor selective replicable adenovirus of Example 4.
  • Figure 15 compares the ability to inhibit hTGF- ⁇ 1,2,3 expression by adenovirus expressing human TGF- ⁇ 2 shRNA and adenovirus expressing human TGF- ⁇ 1 shRNA in real-time PCR results.
  • FIG. 16 compares adenovirus expressing human TGF- ⁇ 2 shRNA with adenovirus expressing human TGF- ⁇ 1 shRNA and inhibits TGF- ⁇ 1,2,3 expression by ELISA results.
  • RNA interference is a natural mechanism that selectively inhibits expression of target genes.
  • Mediators of sequence specific mRNA degradation are small interfering RNAs of 19-23 nucleotides produced by cleavage of ribonuclease III from longer ds RNA.
  • the cytoplasmic RNA-induced silencing complex directs the degradation of mRNA comprising a sequence that binds to a siRNA and is complementary to one strand of the siRNA.
  • RISC cytoplasmic RNA-induced silencing complex
  • siRNA interference in mammals has the efficacy of therapeutic gene silencing.
  • siRNAs have limitations in clinical applications in that they must be prepared in vitro and knockdown genes are typically delivered by transient transfection for 6-10 days.
  • the small-hairpin RNA (shRNA) expression system of the present invention can solve the aforementioned disadvantages.
  • a shRNA is a molecule of about 20 bases or more that includes a double-stranded base sequence in single-stranded RNA, which has a double-stranded structure in the molecule and becomes a hairpin-like structure.
  • the present invention relates to shRNA that inhibits TGF- ⁇ 2 expression, characterized in that the following sequence is used as a target sequence.
  • Human target sequence 5'- GGATTGAGCTATATCAGATTCTCAA -3 '[SEQ ID NO: 2]
  • shRNA that inhibits TGF- ⁇ 2 expression has a sequence complementary to a part of the TGF- ⁇ 2 gene, and may degrade or inhibit translation of the mRNA of the TGF- ⁇ 2 gene. Complementarity of 80-90% can inhibit the translation of mRNA, and 100% can degrade mRNA.
  • the shRNA that inhibits TGF- ⁇ 2 expression is 80% or more, preferably 90% or more, to the 494-518 nucleotides of the mouse mRNA and the complementary sequence to the 578-602 nucleotides of the human mRNA. More preferably, it may include a base sequence having 100% homology.
  • the mouse shRNA consists of the nucleotide sequence shown in SEQ ID NO: 1 (target sequence) and its complementary nucleotide sequence
  • the human shRNA consists of the nucleotide sequence shown in SEQ ID NO: 2 (target sequence) and its complementary nucleotide sequence Can be.
  • Each base sequence and its complementary base sequence may be linked palindrom by a loop region of 4 to 10 bp to form a hairpin structure.
  • shRNAs of the invention may include the following sequences:
  • ShRNA as the mouse target sequence of SEQ ID NO: 5'-GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC-3 '[SEQ ID NO: 3]
  • shRNA short hairpin RNA
  • shRNA short hairpin RNA
  • RNAi The substance which inhibits the expression of TGF- ⁇ 2 by RNAi may be artificially chemically synthesized, and the DNA of the hairpin structure in which the DNA sequences of the sense strand and the antisense strand are connected in the reverse direction is subjected to laboratory conditions by T7 RNA polymerase.
  • RNA may be synthesized in vitro.
  • T7 RNA polymerase and T7 promoter can be used to synthesize antisense and sense RNA from template DNA. After annealing them in laboratory conditions, introduction into cells induces RNAi, leading to degradation of TGF- ⁇ 2 mRNA.
  • Introduction into a cell can be performed by the method using the calcium phosphate method or various transfection reagents (for example, oligofectamine, lipofectamine, lipofection, etc.).
  • an expression vector containing shRNA or DNA may be used, or a cell containing the expression vector may be used.
  • the said expression vector and the kind of cell are not specifically limited, The expression vector and cell which are already used as a medicine are preferable.
  • shRNA having a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as a target sequence can be used.
  • the present invention includes the recombinant expression vector for shRNA expression.
  • the recombinant vector of the present invention can be constructed by recombinant DNA methods known in the art.
  • Viruses (or viral vectors) useful for delivering shRNAs in the present invention include adenoviruses, retroviruses, lentiviruses, adenoviruses, and the like, and adenoviruses are preferred for reasons such as tumor induction.
  • the following DNA can be prepared based on the shRNA sequence.
  • non-viral vector useful for delivering shRNA in the present invention means all the vectors commonly used in gene therapy, except for the aforementioned viral vector, and examples thereof include various plasmids and liposomes that can be expressed in eukaryotic cells. .
  • shRNA that inhibits TGF- ⁇ 2 expression is preferably operably linked to at least a promoter in order to be properly transcribed in the delivered cells.
  • the promoter may be any promoter capable of functioning in eukaryotic cells, but the U6 promoter is particularly preferable for the advantage of producing small size RNA as RNA polymerase III.
  • regulatory sequences including leader sequences, polyadenylation sequences, promoters, enhancers, upstream activation sequences, signal peptide sequences, and transcription terminators may be used as needed. It may also be included.
  • operably linked means that the binding between nucleic acid sequences is functionally related.
  • the case where any nucleic acid sequence is operably linked is when any nucleic acid sequence is positioned to be functionally related to another nucleic acid sequence.
  • any transcriptional regulatory sequence affects the transcription of shRNA, said transcriptional regulatory sequence is said to be operably linked to the shRNA.
  • the present invention comprises a shRNA that inhibits the expression of TGF- ⁇ 2 of SEQ ID NO: 3 or 4, a top strand represented by SEQ ID NO: 5, and a bottom strand represented by SEQ ID NO: 6
  • a shRNA that inhibits the expression of TGF- ⁇ 2 of SEQ ID NO: 3 or 4
  • a top strand represented by SEQ ID NO: 5 and a bottom strand represented by SEQ ID NO: 6
  • An anti-tumor composition comprising DNA or a top strand represented by SEQ ID NO: 7 and a bottom strand represented by SEQ ID NO: 8 or a recombinant expression vector expressing the same as an active ingredient will be.
  • the route of administration of the anti-tumor composition of the present invention is not particularly limited, and oral or parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, It may be administered by any one of the administration routes of topical administration, skin administration, etc. to a patient.
  • oral or parenteral administration for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, It may be administered by any one of the administration routes of topical administration, skin administration, etc. to a patient.
  • the form of a formulation suitable for oral administration may be in the form of a solid or liquid, and the form of a suitable formulation for parenteral administration may be in the form of injections, drops, suppositories, external preparations, eye drops, nasal drops, and the like.
  • the anti-tumor composition of the present invention may contain a pharmaceutically acceptable additive,
  • pharmaceutically acceptable additives include, for example, excipients, binders, disintegrants, glidants, antioxidants, preservatives, stabilizers, tonicity agents, colorants, copulating agents, diluents, emulsifiers, suspending agents, solvents, fillers Extenders, buffers, delivery carriers, carriers, excipients and / or pharmaceutical adjuvants.
  • an excipient is added to the active ingredient, and an additive for preparation such as a binder, a disintegrant, a lubricant, a colorant, or a copper is added, if necessary. Then, it can prepare as a tablet, a granule, a powder, a capsule in accordance with a conventional method.
  • an anti-tumor composition of the present invention in the form of an oral liquid formulation, one or two or more additives for the preparation, such as a copulation agent, a stabilizer, or a preservative, are added to the active ingredient, and according to a conventional method, an oral solution agent and a syrup agent , Elixirs and the like can be prepared.
  • aqueous or non-aqueous may be sufficient.
  • Liquid formulations can be prepared by methods well known in the art.
  • the injection can be prepared by dissolving in a solvent such as physiological saline, a buffer such as PBS, and sterile water, followed by filtration and sterilization with a filter paper or the like, followed by filling into a sterile container (for example, an ampoule).
  • This injection may contain the usual pharmaceutical carrier as needed.
  • the administration method using a non-invasive catheter may be used.
  • the carrier that can be used in the present invention include neutral, buffered saline, or saline containing serum albumin.
  • the method is not particularly limited as long as the shRNA or shRNA expression vector that inhibits TGF- ⁇ 2 expression is expressed in the cell to be applied. It is possible to use gene introduction using viral vectors, liposomes.
  • a viral vector animal viruses, such as a retrovirus, a vaccinia virus, an adenovirus, and a sinrinseliki virus, are mentioned, for example.
  • Substances that inhibit TGF- ⁇ 2 expression by RNAi may be injected directly into cells.
  • the active ingredient of the anti-tumor composition of the present invention is used in a therapeutically effective amount, and the dosage of the composition is the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as the active ingredient. It can be determined by those skilled in the art in consideration of the above. For example, about 1 ⁇ 10 10 particles to 1 ⁇ 10 12 particles per kg of adult as an active ingredient.
  • the frequency of administration of the anti-tumor composition of the present invention may be, for example, once a day to once a few months.
  • the pharmaceutical compositions of the present invention can be used in various diseases or disorders associated with tumors, such as cancer, specifically brain cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, non-human It can be used for the prevention and treatment of head cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer, colon cancer, colon cancer and cervical cancer.
  • treatment refers to (i) prevention of tumor cell formation; (ii) inhibiting a disease or condition associated with the tumor following removal of the tumor cells; And (iii) alleviation of a disease or disorder associated with a tumor following removal of tumor cells.
  • therapeutically effective amount as used herein means an amount sufficient to achieve the above pharmacological effect.
  • a shRNA based on sense 25 mer / antisense 25 mer (including a 4-base loop in the middle) is constructed and expressed in a shuttle vector for expression in adenovirus. Introduction and homologous recombination virus was made.
  • a shRNA having an effect of inhibiting at least 75% of TGF- ⁇ 2 mRNA in mice at least 10 nM of TGF- ⁇ 2 shRNA was obtained through a real-time PCR method.
  • mouse shRNA was transfected into skin cancer cells B16F10 and examined after 24 hours.
  • the experimental method is as follows.
  • the forward primer was 5'-GTGAATGGCTCTCCTTCGAC-3 '[SEQ ID NO: 9] and the reverse primer was 5'-CCTCGAGCTCTTCGCTTTTA-3' [SEQ ID NO: 10], and the reaction conditions were performed as follows. .
  • Stage 1 Reverse transcription (42 ° C. 5 min, 95 ° C. 10 sec),
  • Step 2 PCR reaction (95 °C 5 sec, 60 °C 20 sec) 50 cycles
  • Step 3 The separation was carried out (60 ° C-> 95 ° C).
  • the shRNAs having 25/25 +4 loops were synthesized with respect to the target sequences, and their inhibitory effects on the target sequences were confirmed by real-time PCR.
  • Reverse primer 5'- ATATAAGCTCAGGACCCTGCTG-3 '[SEQ ID NO: 12]
  • reaction conditions were 1 step: reverse transcription (42 ° C 5 min, 95 ° C 10 sec), 2 step: PCR reaction (95 ° C 5 sec, 60 ° C 20 sec) 50 cycles, 3 step: separation (60 ° C-> 95 ° C) Was carried out.
  • Human target sequence 5'- GGATTGAGCTATATCAGATTCTCAA -3 '[SEQ ID NO: 2]
  • the shRNAs having 25/25 +4 loops were synthesized with respect to the target sequences, and their inhibitory effects on the target sequences were confirmed by real-time PCR.
  • ShRNA for human target sequence (SEQ ID NO: 2): 5'- GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC - 3 '[SEQ ID NO: 4]
  • Example 2 Construction of non-replicating adenovirus vectors expressing shRNA against a target sequence
  • oligonucleotide consisting of bases with BamHI and HindIII restriction enzyme sequences at both ends, with the sense and antisense sequences positioned between tctc or tctctc with the most effective inhibitory shRNA sequences identified by real-time RT-PCR. And oligonucleotides complementary to each other were synthesized and annealed, and then the adenovirus E3L (26591-28588) and E3R (30504-) were added to the pSP72 ⁇ E3 / si-negative vector (Fig. 1, pSP72 cloning vector (Promega)).
  • the final recombinant was selected by the HindIII digestion pattern (Fig. 4).
  • the dl324 / IX lanes are the dl324 backbones;
  • the shuttle lane is pSP72-sh-hTGF- ⁇ 2.
  • Lanes 1 to 10 show a result of amplifying the E3 region of a plasmid obtained from a bacterial clone after homologous recombination between the dl324 backbone and pSP72-sh-hTGF- ⁇ 2, and a band corresponding to about 2 kb is positive.
  • lane dl324 / IX is the dl324 backbone;
  • the shuttle vector is pSP72-hTGF- ⁇ 2. Homologous by PCR results of reselecting clones containing genomic DNA from among clones containing sh-hTGF- ⁇ 2 (# 1, 2, 5, 6, 7, 8, 9) identified in FIG. (A) of FIG. 3 confirming that the recombination was performed, which means that the positive clones on both sides were homologous recombination.
  • PCR was performed on the IX gene region, homologous recombination was confirmed using the difference between the dl324 backbone having the IX gene and the shuttle vector having no IX gene. As a result, only # 1, 2, 6 and 7 were reselected.
  • Figure 3 (c) is finally confirmed whether the homologous recombination according to the difference in the pattern when the cut (cut) the HindIII of the backbone and the sample.
  • Lanes 1 to 3 are DNAs derived from the # 1 clone
  • lanes 4 to 6 are clones # 2
  • lanes 7 to 9 are DNAs obtained from a clone cell # 6 again in a competent cell called DH5a.
  • Progeny clones obtained from the parental clones of each of the three DNA from the # 1 clone only showed a HindIII pattern different from the existing dl324-IX (leftmost first lane). This means that the backbone adenovirus DNA has homologous recombination with the shuttle vector, and therefore the present invention is based on the # 1 clone.
  • the E3 shuttle vectors prepared by the above method were treated with Xmn I restriction enzymes to form single strands, and then treated with Spe I restriction enzymes and co-transfected with E. coli BJ5183 together with dl324, a non-replicable adenovirus. Conversion led to gene homologous recombination. Homologously recombined plasmid DNA was obtained and treated with Hind III restriction enzyme to confirm the change of DNA pattern, and finally sequenced to confirm homologous recombination. The identified plasmids were cut with Pac I and transformed into 293 cell lines. A nonreplicating adenovirus expressing shRNA TGF- ⁇ 2 was constructed.
  • a shRNA When a shRNA is produced in a replicable adenovirus, an inhibitor of a shRNA and a cell lysis effect are mixed, so that only an inhibitory effect is difficult to be clearly identified.
  • the adenovirus was grown in 293 cell lines and concentrated to CsCl gradients to determine the titer of the virus by limiting dilution or plaque assay.
  • the final virus titer was 2.5 ⁇ 10 9 pfu / ml by limiting dilution titration.
  • TGF- ⁇ 2 Expression inhibition was confirmed in humans, DU-145, a human prostate cancer cell, infected with adenovirus 1 to 100 moi of Example 2, and after 2 days, the cells were lysed with Trizol and chloroform TGF- ⁇ 2 after harvesting RNA by continuously treating with isopropanol, ethanol, etc. The degree of mRNA expression inhibition was confirmed by real-time PCR.
  • mice B16F10, a mouse melanoma cell, was infected with the adenovirus 100, 500, and 1000 moi of Example 2, and the procedure thereafter was carried out in the same manner as in humans.
  • RNA-to-Ct 1step kit 0.2 ⁇ l RT enzyme mix (125X), 12.5 ⁇ l RT-PCR Mix (2x), 0.5 ⁇ l Forward Primer (100 pM), 0.5 ⁇ l reverse primer (100 pM), RNA (10ng) / ⁇ l) 5 ⁇ l, Nuclease-free water 6.3 ⁇ l total volume was 25 ⁇ l, the reaction conditions are shown in Table 3.
  • RNA-to-Ct 1step kit 0.2 ⁇ l RT enzyme mix (125X), 12.5 ⁇ l RT-PCR Mix (2x), 0.5 ⁇ l Forward Primer (100 pM), 0.5 ⁇ l reverse primer (100 pM), RNA (10ng) / ⁇ l) 5 ⁇ l, Nuclease-free water 6.3 ⁇ l total volume was 25 ⁇ l, the reaction conditions are shown in Table 3.
  • TGF- ⁇ 2 secreted in serum-free medium was measured in the last 24 hours while incubating in the prostate cancer cells of human Example 2 for 2 days after the adenovirus 1, 5, 10, 50 moi infection.
  • an adenovirus was produced that expresses this shRNA and selectively kills cells.
  • various enzyme sites include E1A and E1B55kDa genes in the pBSKII plasmid [Stratagene, USA].
  • PBSKII-3484 synthetic gene was prepared including [FIG. 10 (a)].
  • pBSKII-3484 was PCR-treated with restriction enzymes using Fsp I and then blunt end with a blunting enzyme. was prepared and treated with Bam HI again.
  • pCA14 [Microbix BiosystemsInc, Canada] was Ssp were cut using a restriction enzyme to I, create a blunt end using a block reonting enzyme, which processes the Bgl II same transmission restriction enzyme (Isoschizomer) Bam HI and Bgl II and ends
  • a shuttle vector pCA14-3484 was constructed by inserting the synthesized gene through the blunt end of [Fig. 10 (b)].
  • the shuttle vector pCA14-CMV-3484- ⁇ E1B55 was cut and linearized by XmnI, and then dl324-BstBI-human shTGF- ⁇ 2 (or mouse shTGF- ⁇ 2) without IX gene was cut with BstBI and transformed simultaneously in E. coli BJ5183. Recombination was induced.
  • Homologously recombined plasmid DNA was obtained and treated with Hind III restriction enzyme to confirm the change of DNA pattern, and finally sequenced to confirm homologous recombination.
  • the identified plasmids were cut with Pac I and transformed into 293 cell lines.
  • a dl324-CMV-3484-shTGF- ⁇ 2 adenovirus was produced that selectively killed tumors and inhibited the expression of human (or mouse) TGF- ⁇ 2 (FIG. 11).
  • Figure 12 shows the homologous recombination process for the production of tumor-selective replicable adenovirus containing the actual mouse shTGF- ⁇ 2, as a result of E3 screening, 1, 2, 4, 5, 6 clones were first selected as a positive clone ( a), the homologous recombination colonies were selected by the HindIII digestion pattern, and colonies 1, 2, and 4 were compared with the control, and confirmed to be recombinant (b), 1, 2, and 4 colonies after PacI cleavage.
  • Figure 13 shows the homologous recombination process for the production of tumor-selective replicable adenovirus containing human shTGF- ⁇ 2, clones (1, 2, 3) homologous recombination by the HindIII cleavage pattern was selected (a), When the DNAs of the clones 1, 2 and 3 were cut with PacI, the clone DNAs 1,2 and 3 were all cut and confirmed to have a band of about 2 kb, which is dl324-CMV- in which the DNA of the 1,2,3 colonies was homologously recombined. DNA of 3484- ⁇ E1B55- ⁇ E3-sh-hTGF- ⁇ 2 was confirmed (b).
  • each cell was sorted according to the size of the cells from 4 ⁇ 10 4 to 1 ⁇ 10 5 in a 24-well plate, and then divided and cultured. The next day, the cloned adenovirus with the survivin promoter and CMV promoter was infected by MOI, and when the cells were all killed by the virus at the lowest MOI in the positive control 293A cell line, the experiment was terminated. Stained with. The cells were fixed with 3.7% paraformaldehyde at room temperature for 5 minutes and then stained with 0.05% crystal violet for 30 minutes at room temperature and washed with water to observe the stained cells. As a result of comparing the tumor killing effect of the virus with two types of tumor killing viruses, the difference in killing effect according to the promoter was not significant and both showed excellent tumor selectivity.
  • FIG. 14 shows tumor-selective replicable adenoviruses (dl324-CMV-3484 and surviving promoter not expressing CMV promoter and E1B 55KDa and dl324-hSurvivin-3484 expressing 55KDa) are normal cells (BJ cells). In Esau, cloning does not occur, whereas cloning occurs in various types of human cancer cells, demonstrating that cell hemolysis occurs.
  • TGF- ⁇ 1 present in cells after infection with non-replicating adenovirus of Example 2 expressing human sh-TGF- ⁇ 1 or sh-TGF- ⁇ 2 in an A375 melanoma cell line with 1, 5, 10, 50, 100 moi , TGF- ⁇ 2, TGF- ⁇ 3 mRNA levels were performed by real-time PCR method.
  • TGF- ⁇ 1 shRNA decreased intracellular TGF- ⁇ 1 mRNA but increased TGF- ⁇ 2 mRNA or TGF- ⁇ 3 mRNA.
  • TGF- ⁇ 2 shRNA was found to reduce TGF- ⁇ 2 mRNA or TGF- ⁇ 3 mRNA simultaneously while effectively reducing intracellular TGF- ⁇ 2 mRNA [FIG. 15]. This is an advantage that the reduction in efficacy due to the compensation effect in the cell at least when expressing the TGF- ⁇ 2 shRNA not only does not exhibit this phenomenon but also has the side effect of inhibiting other isotype of TGF- ⁇ . Similar results showed that the expression pattern of the reduced expression of TGF- ⁇ protein using ELISA was similar to that of real-time PCR [FIG. 16].

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Endocrinology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention relates to shRNA for suppressing TGF-β2 expression. The present invention can provide an antitumour composition using shRNA for suppressing TGF-β2 expression.

Description

ＴＧＦ－β２ 발현을 억제하는 ｓｈＲＮＡＳｈＲＮＡ Suppresses the expression of TVF-β2
본 발명은 TGF-β2 발현을 억제하는 shRNA 및 이를 포함하는 항종양 조성물에 관한 것이다.The present invention relates to shRNAs that inhibit TGF-β2 expression and antitumor compositions comprising them.
TGF-β2는 TGF-β1과 마찬가지로 세포독성 T 세포, 자연살해 세포, 그리고 마크로파아지 등의 증식과 분화를 억제하여 점점 자라나고 있는 종양에 대한 면역탐색을 저해. 뿐만 아니라 다기능의 분비단백질로 세포의 형태(type)와 시기에 따라 증식억제, 복제, 침윤, 전이, 세포사멸, 면역탐색, 그리고 혈관생성 등 다양한 역할을 수행뿐만 아니라 TGF-β1처럼 TGF-β2도 신호경로를 비활성화시키거나 혹은 세포주기의 비정상적 조절 등에 기인하여 TGF-β2에 의한 증식억제 작용에 저항성이 생기게 되는 종양이 진행(progression)되는 후기에 이르게 되면 TGF-β2는 종양을 더욱 발전시키는 역할을 하게 된다. 따라서 인체의 면역체계를 극복하고 증식한 종양세포들은 TGF-β2를 분비함으로써 면역감시로부터 자유로워지면서 동시에 증식과 침윤 전이 및 혈관생성에 플러스 요인으로 작용하게 된다. TGF-β2가 TGF-β1과 명백하게 다르게 작용하는 점은 다음과 같다. TGF-β2는 Foxp3를 유도하여 면역억제 유도를 현저히 진행시킨다는 점과, 종양의 전이, 신생혈관형성 그리고 증식 등에도 영향을 미쳐 악성으로의 종양 진행을 유도하는 점이다. TGF-β2, like TGF-β1, inhibits the proliferation and differentiation of cytotoxic T cells, natural killer cells, and macrophages, thereby inhibiting immune search for growing tumors. In addition, it is a multifunctional secretory protein that plays various roles such as proliferation suppression, replication, invasion, metastasis, apoptosis, immunoassay, and angiogenesis, depending on the type and timing of the cells, as well as TGF-β2 like TGF-β1. TGF-β2 plays a role in further developing tumors when tumors develop in the late stages of tumor progression, resulting in resistance to TGF-β2 proliferation inhibition due to inactivation of signal pathways or abnormal cell cycle regulation. Done. Therefore, tumor cells that proliferate after overcoming the immune system of the human body release TGF-β2 and are free from immune surveillance, while at the same time acting as a positive factor for proliferation, invasion metastasis and angiogenesis. TGF-β2 acts clearly differently from TGF-β1 as follows. TGF-β2 induces Foxp3 to significantly induce immunosuppression, and also affects tumor metastasis, neovascularization and proliferation, leading to tumor progression to malignancy.
TGF-β2 관련 선행 연구로, 비특허문헌 1(Schlingensiepen et al, Transforming growth factor-beta2 gene silencing with trabedersen (AP 12009) in pancreatic cancer, Cancer Sci 102: 1193-1200, 2011.)에는 인간 TGF-β2의 코딩 서열에 대한 합성 18-mer 포스포티오레이트 안티센스 올리고뉴클레오티드(phosphothioate antisense oligonucleotide)를 사용하고, 종양의 면역억제 제거, 종양 크기 감소, 림프절로의 전이 및 혈관형성 감소 등이 관찰되었으나, 그 효과는 미비하였다.Prior studies related to TGF-β2 include non-patent literature 1 (Schlingensiepen et al, Transforming growth factor-beta2 gene silencing with trabedersen (AP 12009) in pancreatic cancer, Cancer Sci 102: 1193-1200, 2011.). Synthetic 18-mer phosphothioate antisense oligonucleotides were used for the coding sequence of, and the tumor immunosuppression, tumor size reduction, lymph node metastasis and angiogenesis were observed. Was incomplete.
비특허문헌 2(Chenyu Zhang et al, Transforming growth factor-β2 is a molecular determinant for site-specific melanoma metastasis in the brain, Cancer Res. 2009 February 1; 69(3): 828-35.)에는 murine의 TGF-β2에 대한 shRNA를 TGCTGTTGACAGTGAGCGCGGTGTATAAATCGAGACCAAATTAGTGTGAAGCCACAGATGTATTTGGTCTCGATTTATACACCTTGCCCCTACTGCCTCGGA(target)으로 제작하고, 또한, TGF-β2 shRNA 를 생성하는 렌티바이러스(lentivirus)를 제작하였으나, 렌티바이러스가 염색체에 인테그레이션(integration)되어 암이 소실된 이후에라도 정상세포에는 계속 전달되어 부작용이 큰 문제가 있다.Non-Patent Document 2 (Chenyu Zhang et al, Transforming growth factor-β2 is a molecular determinant for site-specific melanoma metastasis in the brain, Cancer Res. 2009 February 1; 69 (3): 828-35.) shRNA for β2 was produced with TGCTGTTGACAGTGAGCGCGGTGTATAAATCGAGACCAAATTAGTGTGAAGCCACAGATGTATTTGGTCTCGATTTATACACCTTGCCCCTACTGCCTCGGA (target), and a lentiviral (lentivirus) that produces TGF-β2 shRNA was also produced by chromosome incorporation. There is a big problem with the side effects are delivered.
이에, 본 발명자들은 상기와 같은 문제점을 해결하기 위하여 연구 노력한 결과, 인간 TGF-β2 또는 마우스 TGF-β2의 침묵(silencing)을 효과적으로 유도하는 타겟을 선정하여 shRNA를 제작하고, 이를 아데노바이러스에 탑재시켜 기존 비바이러스성 제제에 의한 shRNA의 전달능력을 획기적으로 개선시킴으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have made efforts to solve the above problems, and as a result, by selecting a target that effectively induces silencing of human TGF-β2 or mouse TGF-β2 to produce shRNA, and mounted it in adenovirus The present invention has been completed by drastically improving the ability to deliver shRNAs by existing non-viral agents.
따라서, 본 발명은 서열번호 1 또는 2로 표시되는 염기서열을 표적서열로 하고, TGF-β2 발현을 억제하는 shRNA을 제공하는데 그 목적이 있다.Accordingly, an object of the present invention is to provide a shRNA that suppresses TGF-β2 expression using a nucleotide sequence represented by SEQ ID NO: 1 or 2 as a target sequence.
본 발명은 또한, 상기 shRNA를 유효성분으로 함유하는 항종양 조성물을 제공하는데 다른 목적이 있다.Another object of the present invention is to provide an anti-tumor composition containing the shRNA as an active ingredient.
본 발명은 또한, 상기 shRNA 발현하는 재조합 발현벡터를 제공하는데 또 다른 목적이 있다.The present invention also has another object to provide a recombinant expression vector expressing the shRNA.
본 발명은 또한, 상기 재조합 발현벡터를 유효성분으로 하는 항종양 조성물을 제공하는데 또 다른 목적이 있다. The present invention also has another object to provide an anti-tumor composition comprising the recombinant expression vector as an active ingredient.
본 발명은 또한, 상기 재조합 발현벡터가 도입된 아데노바이러스를 제공하는데 또 다른 목적이 있다.Another object of the present invention is to provide an adenovirus into which the recombinant expression vector is introduced.
본 발명은 상기 과제를 해결하기 위한 수단으로서, The present invention as a means for solving the above problems,
서열번호 1 또는 2로 표시되는 염기서열을 표적서열로 하고, TGF-β2 발현을 억제하는 shRNA을 제공한다.Using the nucleotide sequence shown in SEQ ID NO: 1 or 2 as a target sequence, an shRNA that suppresses TGF-β2 expression is provided.
본 발명은 상기 과제를 해결하기 위한 다른 수단으로서, The present invention is another means for solving the above problems,
상기 shRNA를 유효성분으로 함유하는 항종양 조성물을 제공한다.It provides an anti-tumor composition containing the shRNA as an active ingredient.
본 발명은 상기 과제를 해결하기 위한 또 다른 수단으로서, The present invention is another means for solving the above problems,
상기 shRNA 발현용 재조합 발현벡터를 제공한다.It provides a recombinant expression vector for the shRNA expression.
본 발명은 상기 과제를 해결하기 위한 또 다른 수단으로서, The present invention is another means for solving the above problems,
상기 재조합 발현벡터를 유효성분으로 포함하는 항종양 조성물을 제공한다.It provides an anti-tumor composition comprising the recombinant expression vector as an active ingredient.
본 발명은 상기 과제를 해결하기 위한 또 다른 수단으로서, The present invention is another means for solving the above problems,
상기 재조합 발현벡터가 도입된 아데노바이러스를 제공한다.It provides an adenovirus introduced with the recombinant expression vector.
본 발명은 TGF-β2 발현을 억제하는 새로운 shRNA을 제작하고, 유전자 전달체로 아데노바이러스를 사용하여 감염율을 증가시킴으로써 종래 기술에 비해 특이성, 전달능 및 발현억제능을 크게 향상시켰다.The present invention produced a novel shRNA that inhibits TGF-β2 expression, and significantly increased specificity, delivery capacity and expression suppression ability compared to the prior art by increasing the infection rate using adenovirus as a gene carrier.
즉, 본 발명에 의해 TGF-β2 발현을 억제하는 shRNA를 함유하는 항종양 조성물이 제공된다. 특히, 대부분의 암세포에서 전달효율성이 뛰어난 아데노바이러스에 표적성을 부여함으로써 모든 암에 적용 가능하다.That is, the present invention provides an anti-tumor composition containing shRNA that inhibits TGF-β2 expression. In particular, most cancer cells can be applied to all cancers by assigning a target to adenovirus having excellent delivery efficiency.
도 1은 E3 셔틀 벡터인 pSP72ΔE3/si-negative 벡터를 나타낸 것이다.1 shows the pSP72ΔE3 / si-negative vector, which is an E3 shuttle vector.
도 2는 TGF-β2 shRNA가 셔틀 벡터로 서브클로닝(subcloning)된 후 아데노바이러스 백본인 dl324와 상동 재조합하는 과정의 모식도이다.Figure 2 is a schematic diagram of the process of homologous recombination with the adenovirus backbone dl324 after subcloning (TGF-β2 shRNA) into the shuttle vector.
도 3은 실제로 재조합이 용이한 박테리아에서 상동 재조합된 콜로니의 선별을 위하여, (a)는 아데노바이러스의 E3 부위 PCR 결과를 나타낸 것이고, (b)는 아데노바이러스의 IX 유전자 부위 PCR 결과를 나타낸 것이며, (c)는 상동 재조합된 아데노바이러스 게놈(genomic) DNA의 트랜스펙션(transfection) 가능 여부를 확인하는 PacI 절단 후 단편(fragment) DNA가 출현하는 결과를 나타낸 것이다.FIG. 3 shows screening results of E3 region PCR of adenoviruses, and (b) shows IX gene region PCR results of adenoviruses for selection of homologously recombined colonies in bacteria that are easily recombinant. (c) shows the result of the appearance of fragment DNA after PacI cleavage confirming whether transfection of homologously recombined adenovirus genomic DNA is possible.
도 4는 HindⅢ 절단 패턴(digestion pattern)으로 최종 재조합된 콜로니를 선별 확인한 것이다.Figure 4 shows the final recombinant colonies screened in HindIII digestion pattern (digestion pattern).
도 5는 도 4의 선별된 콜로니가 shRNA hTGF-β2 염기서열을 가지고 있는지를 서열 분석으로 확인한 결과이다.Figure 5 shows the results confirmed by the sequence analysis whether the selected colonies of Figure 4 has shRNA hTGF-β2 nucleotide sequence.
도 6은 실시예 2의 인간 TGF-β2 shRNA를 발현하는 아데노바이러스에 의한 TGF-β2 발현 억제능을 실시간-PCR로 확인한 것이다.Figure 6 confirms the ability to inhibit TGF-β2 expression by adenovirus expressing human TGF-β2 shRNA of Example 2 by real-time PCR.
도 7은 실시예 2의 마우스 TGF-β2 shRNA를 발현하는 아데노바이러스에 의한 TGF-β2 발현 억제능을 실시간-PCR로 확인한 것이다.Figure 7 confirms the ability to inhibit TGF-β2 expression by adenovirus expressing the mouse TGF-β2 shRNA of Example 2 by real-time PCR.
도 8은 실시예 2의 마우스 TGF-β2 shRNA를 발현하는 셔틀 벡터에 의한 TGF-β2 발현 억제능을 실시간-PCR으로 확인한 것이다.Figure 8 confirms the inhibition of TGF-β2 expression by the shuttle vector expressing the mouse TGF-β2 shRNA of Example 2 by real-time PCR.
도 9는 실시예 2의 인간 TGF-β2 shRNA를 발현하는 아데노바이러스에 의한 TGF-β2 발현 억제능을 ELISA로 확인한 것이다.Figure 9 confirms the ability to inhibit TGF-β2 expression by adenovirus expressing human TGF-β2 shRNA of Example 2 by ELISA.
도 10은 pBSKⅡ-3484 벡터(a), pCA14-3484 벡터(b), pCA14-CMV-3484 벡터(c)와 pCA14-CMV-3484-ΔE1B55 벡터(d)를 나타낸 것이다.Figure 10 shows the pBSKII-3484 vector (a), pCA14-3484 vector (b), pCA14-CMV-3484 vector (c) and pCA14-CMV-3484-ΔE1B55 vector (d).
도 11은 dl324 아데노바이러스에서 dl324-CMV-3484-shTGF-β2 아데노바이러스를 제작하는 과정을 나타낸 모식도이다.Figure 11 is a schematic diagram showing the process of producing dl324-CMV-3484-shTGF-β2 adenovirus from dl324 adenovirus.
도 12는 마우스 shTGF-β2가 포함된 상동 재조합 과정을 나타낸 것으로, E3 스크리닝으로 상동 재조합된 콜로니를 선별 확인하고(a), HindⅢ 절단 패턴에 의한 상동 재조합이 된 클론(1, 2, 4)을 선별 확인하고(b), 상기 클론 1, 2, 4의 DNA를 PacI으로 잘라 보았을 때 클론1 만이 제대로 상동 재조합된 것을 확인(c)한 것이다[C: 대조군, dl324-△E3-sh-mTGFβ2], S: 셔틀 벡터 pCA14-CMV-3484-△E1B55, 1~6: 상동재조합된 콜로니].Figure 12 shows the homologous recombination process containing mouse shTGF-β2, screening confirmed homologous recombination colonies by E3 screening (a), clones (1, 2, 4) homologous recombination by the HindIII cleavage pattern When screening and confirming (b), when the DNA of clones 1, 2 and 4 were cut with PacI, only clone 1 was correctly homologously recombined (c). [C: control, dl324-ΔE3-sh-mTGFβ2] , S: shuttle vector pCA14-CMV-3484-ΔE1B55, 1-6: homologous recombined colony].
도 13은 인간 shTGF-β2가 포함된 상동 재조합 과정을 나타낸 것으로, HindⅢ 절단 패턴(digestion pattern)으로 상동 재조합된 콜로니를 선별 확인(a), PacI 절단 후 상동 재조합된 콜로니를 최종 선별 확인(b)한 것이다[C: 대조군, dl324-△E3-sh-mTGFβ2], 1~3: 상동재조합된 콜로니].Figure 13 shows the homologous recombination process containing human shTGF-β2, screening confirmation homologous recombination colonies in the HindIII (digestion pattern) (a), final screening confirmation homologous recombination colonies after PacI cleavage (b) [C: control, dl324-ΔE3-sh-mTGFβ2], 1-3: homologous recombination colonies.
도 14는 실시예 4의 종양선택적 복제 가능 아데노바이러스의 암세포에서 세포 용혈을 확인 것이다.Figure 14 will confirm cell hemolysis in cancer cells of the tumor selective replicable adenovirus of Example 4.
도 15는 인간 TGF-β2 shRNA를 발현하는 아데노바이러스와 인간 TGF-β1 shRNA를 발현하는 아데노바이러스에 의한 hTGF-β1,2,3 발현 억제능을 실시간-PCR 결과로 비교한 것이다. Figure 15 compares the ability to inhibit hTGF-β1,2,3 expression by adenovirus expressing human TGF-β2 shRNA and adenovirus expressing human TGF-β1 shRNA in real-time PCR results.
도 16은 인간 TGF-β2 shRNA를 발현하는 아데노바이러스와 인간 TGF-β1 shRNA를 발현하는 아데노바이러스에 의한 TGF-β1,2,3 발현 억제능을 ELISA 결과로 비교한 것이다.FIG. 16 compares adenovirus expressing human TGF-β2 shRNA with adenovirus expressing human TGF-β1 shRNA and inhibits TGF-β1,2,3 expression by ELISA results.
RNA 간섭(RNA interference, RNAi)은 표적 유전자의 발현을 선택적으로 억제하는 천연의 매커니즘이다. 서열 특이적 mRNA 분해의 매개자는 보다 긴 ds RNA로부터 리보뉴클레아제 Ⅲ의 절단에 의해 생산된 19~23 뉴클레오타이드의 작은 간섭 RNA이다. 세포질의 RISC(RNA-induced silencing complex)는 siRNA에 결합하고 그 siRNA 중 한 가닥에 상보적인 서열을 포함하는 mRNA의 분해를 지시한다. 포유동물에서 RNA 간섭의 적용은 치료 유전자 침묵(silencing)의 효능을 가지고 있다. siRNA의 장점에도 불구하고 siRNA는 시험관 내에서 제조되어야 하고 녹다운 유전자를 통상적으로 6 내지 10일 동안 일시적 형질감염에 의해 전달되어야 한다는 점에서 임상에 적용하는데 제한을 가지고 있다. 본 발명의 shRNA(small-hairpin RNA) 발현 시스템이 전술된 단점을 해결할 수 있다.RNA interference (RNAi) is a natural mechanism that selectively inhibits expression of target genes. Mediators of sequence specific mRNA degradation are small interfering RNAs of 19-23 nucleotides produced by cleavage of ribonuclease III from longer ds RNA. The cytoplasmic RNA-induced silencing complex (RISC) directs the degradation of mRNA comprising a sequence that binds to a siRNA and is complementary to one strand of the siRNA. Application of RNA interference in mammals has the efficacy of therapeutic gene silencing. Despite the advantages of siRNAs, siRNAs have limitations in clinical applications in that they must be prepared in vitro and knockdown genes are typically delivered by transient transfection for 6-10 days. The small-hairpin RNA (shRNA) expression system of the present invention can solve the aforementioned disadvantages.
shRNA는 1본쇄 RNA에서 부분적으로 회문상의 염기서열을 포함함으로써, 분자 내에서 2본쇄 구조를 가지고 헤어핀과 같은 구조가 되는 약 20염기 이상의 분자이다.A shRNA is a molecule of about 20 bases or more that includes a double-stranded base sequence in single-stranded RNA, which has a double-stranded structure in the molecule and becomes a hairpin-like structure.
본 발명은 TGF-β2 발현을 억제하는 shRNA에 관한 것으로, 하기 서열을 표적서열로 하는 것을 특징으로 한다.The present invention relates to shRNA that inhibits TGF-β2 expression, characterized in that the following sequence is used as a target sequence.
마우스 표적서열: 5'- GGATTGAACTGTATCAGATCCTTAA - 3' [서열번호 1]Mouse target sequence: 5'- GGATTGAACTGTATCAGATCCTTAA-3 '[SEQ ID NO: 1]
인간 표적서열: 5'- GGATTGAGCTATATCAGATTCTCAA -3' [서열번호 2]Human target sequence: 5'- GGATTGAGCTATATCAGATTCTCAA -3 '[SEQ ID NO: 2]
본 발명에서 TGF-β2 발현을 억제하는 shRNA는 TGF-β2 유전자의 일부에 상보적인 서열을 가지고, TGF-β2 유전자의 mRNA를 분해하거나, 번역을 억제할 수 있다. 상보성이 80-90%인 경우에는 mRNA의 번역을 억제할 수 있고, 100%인 경우에는 mRNA를 분해시킬 수 있다.In the present invention, shRNA that inhibits TGF-β2 expression has a sequence complementary to a part of the TGF-β2 gene, and may degrade or inhibit translation of the mRNA of the TGF-β2 gene. Complementarity of 80-90% can inhibit the translation of mRNA, and 100% can degrade mRNA.
따라서, 본 발명에서 TGF-β2 발현을 억제하는 shRNA는 마우스 mRNA의 494~518번째 뉴클레오타이드에, 인간 mRNA의 578~602번째 뉴클레오타이드에 대한 상보적인 서열에 대하여 80% 이상, 바람직하게는 90% 이상, 보다 바람직하게는 100% 상동성을 갖는 염기서열을 포함할 수 있다.Therefore, in the present invention, the shRNA that inhibits TGF-β2 expression is 80% or more, preferably 90% or more, to the 494-518 nucleotides of the mouse mRNA and the complementary sequence to the 578-602 nucleotides of the human mRNA. More preferably, it may include a base sequence having 100% homology.
한 양태로서, 마우스 shRNA는 서열번호 1(표적서열)에 나타낸 염기서열과 그의 상보적인 염기서열로 이루어지고, 인간 shRNA는 서열번호 2(표적서열)에 나타낸 염기서열과 그의 상보적인 염기서열로 이루어질 수 있다. 상기 각각의 염기서열과 그의 상보적인 염기서열은 4 내지 10 bp의 루프 영역에 의해 회문적으로(palindrom) 연결되어 헤어핀 구조를 형성하는 것일 수 있다.In one embodiment, the mouse shRNA consists of the nucleotide sequence shown in SEQ ID NO: 1 (target sequence) and its complementary nucleotide sequence, and the human shRNA consists of the nucleotide sequence shown in SEQ ID NO: 2 (target sequence) and its complementary nucleotide sequence Can be. Each base sequence and its complementary base sequence may be linked palindrom by a loop region of 4 to 10 bp to form a hairpin structure.
본 발명의 shRNA의 구체적인 예로는 하기 서열을 포함할 수 있다:Specific examples of shRNAs of the invention may include the following sequences:
서열번호 1의 마우스 표적서열으로 하는 shRNA: 5'-GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC-3' [서열번호 3]ShRNA as the mouse target sequence of SEQ ID NO: 5'-GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC-3 '[SEQ ID NO: 3]
서열번호 2의 인간 표적서열을 위한 shRNA: 5'- GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC-3' 서열번호 4].ShRNA for human target sequence of SEQ ID NO: 5′-GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC-3 ′ SEQ ID NO: 4].
RNAi에 의해 TGF-β2의 발현을 억제하는 물질로서는, 3'말단에 돌출부를 가지는 짧은 헤어핀 구조로 구성된 shRNA(short hairpin RNA)를 사용할 수도 있다. As a substance which inhibits the expression of TGF-β2 by RNAi, shRNA (short hairpin RNA) composed of a short hairpin structure having a protrusion at the 3 'end can also be used.
RNAi에 의해 TGF-β2의 발현을 억제하는 물질은, 인공적으로 화학 합성하여도 좋고, 센스 가닥 및 안티센스 가닥의 DNA 서열을 역방향으로 연결한 헤어핀 구조의 DNA를 T7 RNA 폴리머라제에 의해 실험실 조건(in vitro)에서 RNA를 합성하여 제작하여도 무방하다. 실험실 조건에서 합성하는 경우, T7 RNA 폴리머라제 및 T7 프로모터를 이용하여, 주형 DNA로부터 안티센스 및 센스 RNA를 합성할 수 있다. 이들을 실험실 조건에서 어닐링한 후, 세포에 도입하면 RNAi가 유발되어, TGF-β2 mRNA의 분해를 유도한다. 세포에의 도입은 예를 들면, 인산칼슘법, 또는 각종 트랜스펙션 시약(예를 들면, oligofectamine, lipofectamine 및 lipofection 등)을 이용한 방법에 의해 행할 수 있다.The substance which inhibits the expression of TGF-β2 by RNAi may be artificially chemically synthesized, and the DNA of the hairpin structure in which the DNA sequences of the sense strand and the antisense strand are connected in the reverse direction is subjected to laboratory conditions by T7 RNA polymerase. RNA may be synthesized in vitro. When synthesized in laboratory conditions, T7 RNA polymerase and T7 promoter can be used to synthesize antisense and sense RNA from template DNA. After annealing them in laboratory conditions, introduction into cells induces RNAi, leading to degradation of TGF-β2 mRNA. Introduction into a cell can be performed by the method using the calcium phosphate method or various transfection reagents (for example, oligofectamine, lipofectamine, lipofection, etc.).
RNAi에 의해 TGF-β2의 발현을 억제하는 물질로서는, shRNA 또는 상기 DNA을 포함하는 발현벡터를 이용하여도 좋고, 상기 발현벡터를 함유하는 세포를 이용하여도 좋다. 상기 발현벡터나 세포의 종류는 특별히 한정되지 않으나, 이미 의약으로서 사용되고 있는 발현벡터나 세포가 바람직하다.As a substance which inhibits the expression of TGF-β2 by RNAi, an expression vector containing shRNA or DNA may be used, or a cell containing the expression vector may be used. Although the said expression vector and the kind of cell are not specifically limited, The expression vector and cell which are already used as a medicine are preferable.
본 발명에서는 서열번호 1 또는 서열번호 2로 표시되는 염기서열을 표적서열로 하는 shRNA를 이용할 수 있다. In the present invention, shRNA having a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as a target sequence can be used.
따라서, 본 발명은 상기 shRNA 발현용 재조합 발현벡터를 포함한다. Therefore, the present invention includes the recombinant expression vector for shRNA expression.
본 발명의 재조합 벡터는 당해 분야에 공지된 재조합 DNA 방법에 의해 구성될 수 있다.The recombinant vector of the present invention can be constructed by recombinant DNA methods known in the art.
본 발명에서 shRNA를 전달하기에 유용한 바이러스 (또는 바이러스 벡터)로는 아데노바이러스, 레트로바이러스, 렌티바이러스, 아데노부속바이러스 등이 있으며, 종양에서와 같이 한시적인 발현 유도가 필요한 이유로 아데노바이러스가 바람직하다. Viruses (or viral vectors) useful for delivering shRNAs in the present invention include adenoviruses, retroviruses, lentiviruses, adenoviruses, and the like, and adenoviruses are preferred for reasons such as tumor induction.
아데노바이러스에 상기 shRNA를 도입하기 위하여, shRNA 서열을 근거로 하여 하기 DNA를 제작할 수 있다. In order to introduce the shRNA into the adenovirus, the following DNA can be prepared based on the shRNA sequence.
<마우스 표적서열에 대한 DNA><DNA for Mouse Target Sequence>
탑 스트랜드: 5'- gatcc GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC tttt a - 3' [서열번호 5]Top Strand: 5'- gatcc GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC tttt a-3 '[SEQ ID NO: 5]
바텀 스트랜드: 5'- agctt aaaa GGATTGAACTGTATCAGATCCTTAA gaga TTAAGGATCTGATACAGTTCAATCC g - 3' [서열번호 6]Bottom strand: 5'- agctt aaaa GGATTGAACTGTATCAGATCCTTAA gaga TTAAGGATCTGATACAGTTCAATCC g-3 '[SEQ ID NO: 6]
<인간 표적서열에 대한 DNA>DNA for human target sequence
탑 스트랜드: 5'- gatcc GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC tttt a - 3' [서열번호 7]Top Strand: 5'- gatcc GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC tttt a-3 '[SEQ ID NO: 7]
바텀 스트랜드: 5'- agctt aaaa GGATTGAGCTATATCAGATTCTCAA gaga TTGAGAATCTGATATAGCTCAATCC g - 3' [서열번호 8]Bottom strand: 5'- agctt aaaa GGATTGAGCTATATCAGATTCTCAA gaga TTGAGAATCTGATATAGCTCAATCC g-3 '[SEQ ID NO: 8]
또한, 본 발명에서 shRNA를 전달하기에 유용한 비바이러스 벡터로는 전술한 바이러스 벡터를 제외한 통상적으로 유전자 요법에 사용되는 모든 벡터를 의미하며, 그러한 예로는 진핵세포에서 발현 가능한 다양한 플라스미드 및 리포좀 등이 있다.In addition, the non-viral vector useful for delivering shRNA in the present invention means all the vectors commonly used in gene therapy, except for the aforementioned viral vector, and examples thereof include various plasmids and liposomes that can be expressed in eukaryotic cells. .
한편, 본 발명에서 TGF-β2 발현을 억제하는 shRNA는 전달된 세포에서 적절히 전사되기 위하여 적어도 프로모터에 작동 가능하게 연결되는 것이 바람직하다. 상기 프로모터는 진핵세포에서 기능할 수 있는 프로모터라면 어떤 것이든지 무방하나, U6 프로모터가 RNA 중합효소 Ⅲ로서 small size RNA를 생성하는데 유리한 이유로 특히 바람직하다. TGF-β2 발현을 억제하는 shRNA의 효율적인 전사를 위하여 필요에 따라 리더 서열, 폴리아데닐화 서열, 프로모터, 인핸서(enhancer), 업스트림(upstream) 활성화 서열, 시그날 펩타이드 서열 및 전사 종결인자를 비롯한 조절서열을 추가로 포함할 수도 있다.On the other hand, in the present invention, shRNA that inhibits TGF-β2 expression is preferably operably linked to at least a promoter in order to be properly transcribed in the delivered cells. The promoter may be any promoter capable of functioning in eukaryotic cells, but the U6 promoter is particularly preferable for the advantage of producing small size RNA as RNA polymerase III. For efficient transcription of shRNAs that inhibit TGF-β2 expression, regulatory sequences including leader sequences, polyadenylation sequences, promoters, enhancers, upstream activation sequences, signal peptide sequences, and transcription terminators may be used as needed. It may also be included.
여기서, 이용된 용어 "작동 가능하게 연결된"이란 핵산 서열간의 결합이 기능적으로 연관되어 있는 것을 의미한다. 임의의 핵산서열이 작동 가능하게 연결된 경우는 임의의 핵산서열이 다른 핵산서열과 기능적으로 관련성을 가지도록 위치해 있는 경우이다. 본 발명에 있어서, 임의의 전사 조절서열이 shRNA의 전사에 영향을 미치는 경우, 상기 전사 조절서열이 상기 shRNA와 작동 가능하게 연결되어 있다고 말한다.As used herein, the term “operably linked” means that the binding between nucleic acid sequences is functionally related. The case where any nucleic acid sequence is operably linked is when any nucleic acid sequence is positioned to be functionally related to another nucleic acid sequence. In the present invention, when any transcriptional regulatory sequence affects the transcription of shRNA, said transcriptional regulatory sequence is said to be operably linked to the shRNA.
또한, 본 발명은 상기 서열번호 3 또는 4의 TGF-β2 발현을 억제하는 shRNA, 서열번호 5로 표시되는 탑 스트랜드(top strand)와, 서열번호 6으로 표시되는 바텀 스트랜드(bottom strand)를 포함하는 DNA 또는 서열번호 7로 표시되는 탑 스트랜드(top strand)와, 서열번호 8로 표시되는 바텀 스트랜드(bottom strand)를 포함하는 DNA 또는 이를 발현하는 재조합 발현벡터를 유효성분으로 포함하는 항종양 조성물에 관한 것이다.In addition, the present invention comprises a shRNA that inhibits the expression of TGF-β2 of SEQ ID NO: 3 or 4, a top strand represented by SEQ ID NO: 5, and a bottom strand represented by SEQ ID NO: 6 An anti-tumor composition comprising DNA or a top strand represented by SEQ ID NO: 7 and a bottom strand represented by SEQ ID NO: 8 or a recombinant expression vector expressing the same as an active ingredient will be.
본 발명의 항종양 조성물의 투여경로는 특별히 한정되지 않고, 경구 투여 또는 비경구 투여(예를 들면, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 점막 투여, 직장내 투여, 질내 투여, 환자에의 국소 투여, 피부투여 등)의 어느 하나의 투여경로에 의해 투여하여도 좋다. 경구 투여에 적당한 제제 형태로서는 고형 또는 액체의 형태가 가능하고, 비경구 투여의 적당한 제제 형태로서는 주사제, 점적제, 좌제, 외용제, 점안제, 점비제 등의 형태가 가능하다. 본 발명의 항종양 조성물은 그 제제형태에 의해, 필요에 따라 약학적으로 허용 가능한 첨가제를 함유하여도 좋다. 약학적으로 허용 가능한 첨가제의 구체적인 예로서는, 예를 들면, 부형제, 결합제, 붕해제, 활택제, 항산화제, 보존제, 안정화제, 등장화제, 착색제, 교미제, 희석제, 유화제, 현탁화제, 용매, 충진제, 증량제, 완충제, 송달 담체, 캐리어, 부형제 및/또는 약학적 어쥬번트 등을 들 수 있다.The route of administration of the anti-tumor composition of the present invention is not particularly limited, and oral or parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, It may be administered by any one of the administration routes of topical administration, skin administration, etc. to a patient. The form of a formulation suitable for oral administration may be in the form of a solid or liquid, and the form of a suitable formulation for parenteral administration may be in the form of injections, drops, suppositories, external preparations, eye drops, nasal drops, and the like. The anti-tumor composition of the present invention may contain a pharmaceutically acceptable additive, if necessary, in the form of its formulation. Specific examples of pharmaceutically acceptable additives include, for example, excipients, binders, disintegrants, glidants, antioxidants, preservatives, stabilizers, tonicity agents, colorants, copulating agents, diluents, emulsifiers, suspending agents, solvents, fillers Extenders, buffers, delivery carriers, carriers, excipients and / or pharmaceutical adjuvants.
경구용 고형제제 형태의 본 발명의 항종양 조성물로서는, 예를 들면, 유효성분에 부형제를 가하고, 아울러, 필요에 따라서, 결합제, 붕해제, 활택제, 착색제 또는 교미제 등의 제제용 첨가물을 가한 후, 통상의 방법에 따라, 정제, 과립제, 산제, 캡슐제로서 조제할 수 있다. 경구용 액체 제제형태의 본 발명의 항종양 조성물로서는, 유효성분에, 교미제, 안정화제, 또는 보존제 등의 제제용 첨가물 1종 또는 2종 이상을 가하고, 통상의 방법에 따라, 내복액제, 시럽제, 엘릭실제 등으로서 조제할 수 있다.As an anti-tumor composition of the present invention in the form of an oral solid preparation, for example, an excipient is added to the active ingredient, and an additive for preparation such as a binder, a disintegrant, a lubricant, a colorant, or a copper is added, if necessary. Then, it can prepare as a tablet, a granule, a powder, a capsule in accordance with a conventional method. As an anti-tumor composition of the present invention in the form of an oral liquid formulation, one or two or more additives for the preparation, such as a copulation agent, a stabilizer, or a preservative, are added to the active ingredient, and according to a conventional method, an oral solution agent and a syrup agent , Elixirs and the like can be prepared.
본 발명의 항종양 조성물을 액체 제제로서 처방하기 위하여 사용되는 용매로서는, 수성 또는 비수성의 어느 것도 무방하다. 액체제제는 당해 분야에 주지된 방법에 의해 조제할 수 있다. 예를 들면, 주사제는 생리식염수, PBS와 같은 완충액, 멸균수 등의 용제에 용해시킨 후, 여과지 등으로 여과멸균하고, 이어서 멸균용기(예를 들면, 앰플 등)에 충진하여 조제할 수 있다. 이 주사제에는 필요에 따라, 관용의 약학적 캐리어를 포함하여도 무방하다.As a solvent used in order to prescribe the anti-tumor composition of this invention as a liquid formulation, either aqueous or non-aqueous may be sufficient. Liquid formulations can be prepared by methods well known in the art. For example, the injection can be prepared by dissolving in a solvent such as physiological saline, a buffer such as PBS, and sterile water, followed by filtration and sterilization with a filter paper or the like, followed by filling into a sterile container (for example, an ampoule). This injection may contain the usual pharmaceutical carrier as needed.
또한, 비침습적인 카테터를 이용하는 투여방법을 사용하여도 좋다. 본 발명에서 사용할 수 있는 캐리어로서는, 중성, 완충화 생리식염수, 또는 혈청알부민을 포함하는 생리식염수 등을 들 수 있다.Moreover, the administration method using a non-invasive catheter may be used. Examples of the carrier that can be used in the present invention include neutral, buffered saline, or saline containing serum albumin.
TGF-β2 발현을 억제하는 shRNA 발현벡터 등 유전자 송달에 관해서는, 적용되는 세포 내에서 TGF-β2 발현을 억제하는 shRNA 또는 shRNA 발현벡터를 발현시키는 한, 특별히 방법은 한정되지 않으나, 예를 들면, 바이러스 벡터, 리포좀을 이용한 유전자 도입을 이용하는 것이 가능하다. 바이러스 벡터로서는, 예를 들면, 레트로바이러스, 백시니아 바이러스, 아데노바이러스, 신린셈리키 바이러스 등의 동물바이러스를 들 수 있다.Regarding gene delivery such as a shRNA expression vector that inhibits TGF-β2 expression, the method is not particularly limited as long as the shRNA or shRNA expression vector that inhibits TGF-β2 expression is expressed in the cell to be applied. It is possible to use gene introduction using viral vectors, liposomes. As a viral vector, animal viruses, such as a retrovirus, a vaccinia virus, an adenovirus, and a sinrinseliki virus, are mentioned, for example.
RNAi에 의해 TGF-β2 발현을 억제하는 물질은 세포에 직접 주입하여도 무방하다.Substances that inhibit TGF-β2 expression by RNAi may be injected directly into cells.
본 발명의 항종양 조성물의 유효성분은 치료학적 유효량으로 사용하며, 상기 조성물의 투여량은 사용 목적, 질환의 중독도, 환자의 연령, 체중, 성별, 기왕력, 또는 유효성분으로서 사용되는 물질의 종류 등을 고려하여 당업자가 결정할 수 있다. 예를 들면, 유효성분으로서 성인 1 kg 당 약 1x1010 particles 내지 1x1012 particles 이다. 본 발명의 항종양 조성물의 투여빈도는, 예를 들면, 1일 1회 내지 수개월에 1회이면 좋다.The active ingredient of the anti-tumor composition of the present invention is used in a therapeutically effective amount, and the dosage of the composition is the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as the active ingredient. It can be determined by those skilled in the art in consideration of the above. For example, about 1 × 10 10 particles to 1 × 10 12 particles per kg of adult as an active ingredient. The frequency of administration of the anti-tumor composition of the present invention may be, for example, once a day to once a few months.
본 발명의 shRNA는 TGF-β2 발현을 억제하므로, 본 발명의 약제학적 조성물은 종양과 관련된 다양한 질병 또는 질환, 예컨대 암, 구체적으로 뇌암, 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 식도암, 췌장암, 방광암, 전립선암, 대장암, 결장암 및 자궁경부암 등의 예방 및 치료에 이용될 수 있다. 본 명세서에서 용어 "치료"는 (i) 종양 세포 형성의 예방; (ii) 종양 세포의 제거에 따른 종양과 관련된 질병 또는 질환의 억제; 및 (iii) 종양 세포의 제거에 따른 종양과 관련된 질병 또는 질환의 경감을 의미한다. 따라서, 본 명세서에서 용어 "치료학적 유효량"은 상기한 약리학적 효과를 달성하는 데 충분한 양을 의미한다.Since the shRNA of the present invention inhibits TGF-β2 expression, the pharmaceutical compositions of the present invention can be used in various diseases or disorders associated with tumors, such as cancer, specifically brain cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, non-human It can be used for the prevention and treatment of head cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer, colon cancer, colon cancer and cervical cancer. As used herein, the term "treatment" refers to (i) prevention of tumor cell formation; (ii) inhibiting a disease or condition associated with the tumor following removal of the tumor cells; And (iii) alleviation of a disease or disorder associated with a tumor following removal of tumor cells. Thus, the term "therapeutically effective amount" as used herein means an amount sufficient to achieve the above pharmacological effect.
이하, 본 발명에 따르는 실시예를 통하여 본 발명을 보다 상세히 설명하나, 본 발명의 범위가 하기 제시된 실시예에 의해 제한되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited to the examples given below.
실시예 1: shRNA 제조-TGF-β2의 침묵(silencing)을 효과적으로 유도하는 타겟 선정Example 1: shRNA Preparation-Target Selection That Induces Silencing of TGF-β2
본 발명은 TGF-β2의 침묵(silencing)을 유도하기 위하여, 센스 25 mer/안티센스 25 mer(4 개 염기를 가지는 루프를 가운데에 포함)에 근거한 shRNA를 제작하고 아데노바이러스에서 발현시키기 위해 셔틀벡터에 도입하고 상동 재조합(homologous recombination) 바이러스를 제작하였다. In order to induce silencing of TGF-β2, a shRNA based on sense 25 mer / antisense 25 mer (including a 4-base loop in the middle) is constructed and expressed in a shuttle vector for expression in adenovirus. Introduction and homologous recombination virus was made.
shRNA TGF-β2의 특이성 검증을 위하여, 스크램블드(scrambled) shRNA를 가지는 셔틀벡터도 동시에 제작하였다. 종래 방법에 비하여 특이성과 발현억제능이 크게 향상되었다. In order to verify the specificity of shRNA TGF-β2, a shuttle vector having a scrambled shRNA was also prepared. Compared with the conventional method, specificity and expression suppression ability are greatly improved.
이를 위해, TGF-β2의 shRNA 최소 10 nM에서 마우스의 TGF-β2 mRNA를 75% 이상 억제하는 효과가 있는 shRNA를 실시간 PCR 방법을 통해 확보하였다. To this end, a shRNA having an effect of inhibiting at least 75% of TGF-β2 mRNA in mice at least 10 nM of TGF-β2 shRNA was obtained through a real-time PCR method.
이를 위해 마우스용 shRNA는 피부암세포인 B16F10에 트랜스펙션시키고 24시간이 지난 후 감소되는 정도를 조사하였다. To this end, the mouse shRNA was transfected into skin cancer cells B16F10 and examined after 24 hours.
실험방법은 다음과 같다. The experimental method is as follows.
실시간 RT-PCR로 여러 가지 종류의 후보 shRNA 10nM을 30% B16F10에 트랜스펙션하고 24시간 배양 후, 확인(validation)을 통하여 마우스 TGF-β2에 대해 5개의 shRNA 스크리닝 결과 타겟에 해당되는 shRNA에서 73.75% 침묵(silencing) 효과를 확인하였다. Real-time RT-PCR transfected several types of candidate shRNA 10nM to 30% B16F10 and incubated for 24 hours, screening five shRNAs against mouse TGF-β2 via validation, 73.75 in shRNAs corresponding to targets % Silencing effect was confirmed.
실시간 RT-PCR을 위하여, 정방향 프라이머로는 5'-GTGAATGGCTCTCCTTCGAC-3' [서열번호 9]과 역방향 프라이머로는 5'-CCTCGAGCTCTTCGCTTTTA-3' [서열번호 10]이며, 반응조건은 다음과 같이 수행하였다. For real-time RT-PCR, the forward primer was 5'-GTGAATGGCTCTCCTTCGAC-3 '[SEQ ID NO: 9] and the reverse primer was 5'-CCTCGAGCTCTTCGCTTTTA-3' [SEQ ID NO: 10], and the reaction conditions were performed as follows. .
1 단계: 역전사(42 ℃ 5 min, 95 ℃ 10 sec), Stage 1: Reverse transcription (42 ° C. 5 min, 95 ° C. 10 sec),
2 단계: PCR 반응 (95 ℃ 5 sec, 60 ℃ 20 sec) 50 cycles, Step 2: PCR reaction (95 ℃ 5 sec, 60 ℃ 20 sec) 50 cycles,
3 단계: 분리(60 ℃-> 95 ℃)로 수행하였다. Step 3: The separation was carried out (60 ° C-> 95 ° C).
확인(validation) 실험 결과, 10개의 표적서열 후보 중 하기 표 1에 근거하여 하기 타겟을 선정하였다.As a result of validation experiment, the following targets were selected based on Table 1 below among 10 target sequence candidates.
마우스 표적서열: 5'- GGATTGAACTGTATCAGATCCTTAA - 3' [서열번호 1]Mouse target sequence: 5'- GGATTGAACTGTATCAGATCCTTAA-3 '[SEQ ID NO: 1]
표 1
Figure PCTKR2013000791-appb-T000001
 Table 1
Figure PCTKR2013000791-appb-T000001
상기 표적서열에 대하여 25/25 +4 루프를 가지는 shRNA를 합성하고 이들의 표적서열에 대한 억제 효과를 실시간 PCR로 확인하였다. The shRNAs having 25/25 +4 loops were synthesized with respect to the target sequences, and their inhibitory effects on the target sequences were confirmed by real-time PCR.
마우스 표적서열(서열번호 1)으로 하는 shRNA: 5'- GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC-3' [서열번호 3]ShRNA with mouse target sequence (SEQ ID NO: 1): 5'-GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC-3 '[SEQ ID NO: 3]
앞서 설명한 실시간 PCR에 의해 선정된 서열번호 3의 염기서열을 아데노바이러스에서 발현시키기 위해 양 끝단에 BamHI과 HindⅢ 염기 사이트를 삽입하고 중간에 tctc의 4개의 염기를 가지는 루프(loop)를 가지게끔 제작하였다. 즉, 마우스 shRNA의 기본적인 구조는 5'-25 mer-루프(4 mer)-25mer-3'으로 구성되어 있다. In order to express the base sequence of SEQ ID NO: 3 selected by the real-time PCR described above in adenovirus, BamHI and HindIII base sites were inserted at both ends, and a loop having four bases of tctc was prepared in the middle. . That is, the basic structure of mouse shRNA is composed of 5'-25 mer-loop (4 mer) -25mer-3 '.
이에 근거하여 아데노바이러스에 도입시키기 위한 하기 2가닥의 DNA 스트랜드(strand)를 제작하여 shRNA 생성을 유도하였다.Based on this, the following two strands of DNA strand for incorporation into adenoviruses were produced to induce shRNA production.
탑 스트랜드: 5'- gatcc GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC tttt a - 3' [서열번호 5]Top Strand: 5'- gatcc GGATTGAACTGTATCAGATCCTTAA tctc TTAAGGATCTGATACAGTTCAATCC tttt a-3 '[SEQ ID NO: 5]
바텀 스트랜드: 5'- agctt aaaa GGATTGAACTGTATCAGATCCTTAA gaga TTAAGGATCTGATACAGTTCAATCC g - 3' [서열번호 6]Bottom strand: 5'- agctt aaaa GGATTGAACTGTATCAGATCCTTAA gaga TTAAGGATCTGATACAGTTCAATCC g-3 '[SEQ ID NO: 6]
인간 TGF-β2 억제를 위한 실시간 PCR용 프라이머는 다음과 같다.Primers for real-time PCR for human TGF-β2 inhibition are as follows.
정방향 프라이머: 5'- GCTGCCTACGTCCACTTTACAT - 3' [서열번호 11]Forward primer: 5'- GCTGCCTACGTCCACTTTACAT-3 '[SEQ ID NO: 11]
역방향 프라이머: 5'- ATATAAGCTCAGGACCCTGCTG - 3' [서열번호 12]Reverse primer: 5'- ATATAAGCTCAGGACCCTGCTG-3 '[SEQ ID NO: 12]
반응조건은 1 단계: 역전사(42 ℃ 5 min, 95 ℃ 10 sec), 2 단계: PCR 반응 (95 ℃ 5 sec, 60 ℃ 20 sec) 50 cycles, 3 단계: 분리(60 ℃-> 95 ℃)로 수행하였다. The reaction conditions were 1 step: reverse transcription (42 ° C 5 min, 95 ° C 10 sec), 2 step: PCR reaction (95 ° C 5 sec, 60 ° C 20 sec) 50 cycles, 3 step: separation (60 ° C-> 95 ° C) Was carried out.
확인(validation) 실험 결과, 3개의 표적서열 후보 중 하기 표 2에 근거하여 하기 타겟을 선정하였다.As a result of validation experiment, the following targets were selected based on Table 2 among three target sequence candidates.
인간 표적서열: 5'- GGATTGAGCTATATCAGATTCTCAA -3' [서열번호 2]Human target sequence: 5'- GGATTGAGCTATATCAGATTCTCAA -3 '[SEQ ID NO: 2]
표 2
Figure PCTKR2013000791-appb-T000002
 TABLE 2
Figure PCTKR2013000791-appb-T000002
상기 표적서열에 대하여 25/25 +4 루프를 가지는 shRNA를 합성하고 이들의 표적서열에 대한 억제 효과를 실시간 PCR로 확인하였다. The shRNAs having 25/25 +4 loops were synthesized with respect to the target sequences, and their inhibitory effects on the target sequences were confirmed by real-time PCR.
인간 표적서열(서열번호 2)을 위한 shRNA: 5'- GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC-3' [서열번호 4]ShRNA for human target sequence (SEQ ID NO: 2): 5'- GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC-3 '[SEQ ID NO: 4]
앞서 설명한 실시간 PCR에 의해 선정된 서열번호 4의 염기서열을 아데노바이러스에서 발현시키기 위해 양 끝단에 BamHI과 HindⅢ 염기사이트를 삽입하고 중간에 tctc의 4개의 염기를 가지는 루프를 가지게끔 제작하였다. 즉, 인간 shRNA의 기본 구조는 5'-25 mer-루프(4 mer)-25mer-3'으로 구성되어 있다.In order to express the base sequence of SEQ ID NO: 4 selected by the real-time PCR described above in adenovirus, BamHI and HindIII base sites were inserted at both ends, and a loop having four bases of tctc was prepared in the middle. That is, the basic structure of human shRNA is composed of 5'-25 mer-loop (4 mer) -25mer-3 '.
이에 근거하여 아데노바이러스에 도입시키기 위한 하기 2가닥의 DNA를 제작하였다.Based on this, the following two strands of DNA for introduction into the adenovirus were produced.
탑 스트랜드: 5'- gatcc GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC tttt a - 3' [서열번호 7]Top Strand: 5'- gatcc GGATTGAGCTATATCAGATTCTCAA tctc TTGAGAATCTGATATAGCTCAATCC tttt a-3 '[SEQ ID NO: 7]
바텀 스트랜드: 5'- agctt aaaa GGATTGAGCTATATCAGATTCTCAA gaga TTGAGAATCTGATATAGCTCAATCC g - 3' [서열번호 8]Bottom strand: 5'- agctt aaaa GGATTGAGCTATATCAGATTCTCAA gaga TTGAGAATCTGATATAGCTCAATCC g-3 '[SEQ ID NO: 8]
실시예 2: 타겟 서열에 대한 shRNA 발현하는 복제 불능 아데노바이러스 벡터 제작Example 2: Construction of non-replicating adenovirus vectors expressing shRNA against a target sequence
실시간 RT-PCR을 통하여 확인된 가장 효과적으로 발현을 억제하는 shRNA 염기서열을 센스와 안티센스 서열이 tctc 혹은 tctctc를 사이에 두고 위치하게 하고, 양 끝에 BamHI과 HindⅢ 제한효소 염기서열을 가진 염기로 구성된 올리고뉴클레오티드와 상보적인 올리고뉴클레오티드를 각각 합성하여 합체(annealing)시킨 뒤, E3 셔틀 벡터인 pSP72ΔE3/si-negative 벡터[도 1, pSP72 cloning 벡터(Promega)에 아데노바이러스 E3L(26591-28588)과 E3R(30504-31057)을 삽입하고 Ambion사의 psilencer 2.1-U6 hygro에서 -EcoRI-U6 promoter + -BamHI-nonsense shRNA 용 염기서열인 actaccgttgttataggtgttcaagagacacctataacaacggtagttttttggaa-HindⅢ가 들어간 형태의 pSP72ΔE3/si-negative (scrambled)]를 제작하였다. An oligonucleotide consisting of bases with BamHI and HindIII restriction enzyme sequences at both ends, with the sense and antisense sequences positioned between tctc or tctctc with the most effective inhibitory shRNA sequences identified by real-time RT-PCR. And oligonucleotides complementary to each other were synthesized and annealed, and then the adenovirus E3L (26591-28588) and E3R (30504-) were added to the pSP72ΔE3 / si-negative vector (Fig. 1, pSP72 cloning vector (Promega)). 31057) was inserted and pSP72ΔE3 / si-negative (scrambled) containing a form of actaccgttgttataggttcaagagacacctataacaacggtagttttttggaa-HindIII, which is a base sequence for -EcoRI-U6 promoter + -BamHI-nonsense shRNA, was prepared from Ambion's psilencer 2.1-U6 hygro.
인간 또는 마우스의 shRNA TGF-β2 도입을 위하여, 먼저 상기한 pSP72ΔE3/si-negative 플라스미드를 BamHI과 HindⅢ를 처리한 후, 인간 또는 마우스의 shRNA TGF-β2를 삽입시켜 pSP72ΔE3-sh-human TGF-β2 또는 pSP72ΔE3-sh-mouse TGF-β2를 제작하였다[도 2]. 음성 대조군 아데노바이러스로는 양끝에 BamHI과 HindⅢ를 가지게 하고 스크램블드(scrambled) 염기서열(actaccgttgttataggtg)과 loop(ttcaagaga) 제작하였다. In order to introduce shRNA TGF-β2 into human or mouse, the above-described pSP72ΔE3 / si-negative plasmid was first treated with Bam HI and Hind III, followed by inserting human or mouse shRNA TGF-β2 into pSP72ΔE3-sh-human TGF- β2 or pSP72ΔE3-sh-mouse TGF-β2 was constructed [FIG. 2]. Negative control adenoviruses had BamHI and HindIII at both ends, and scrambled sequences (actaccgttgttataggtg) and loop (ttcaagaga) were prepared.
아데노바이러스의 E3 부위 PCR로 양성 클론(#1, 2, 5, 6, 7, 8, 9)만을 선별한 후[도 3의 (a): dl324/IX 아데노바이러스 백본(backbone) 게놈(genomic) DNA와 pSP72-sh-hTGF-β2 셔틀 벡터와의 상동 재조합 후 sh-hTGF-β2가 포함된 클론들을 선별하는 PCR 결과, 도 3의 (b): dl324/IX 아데노바이러스 백본 게놈 DNA와 pSP72-sh-hTGF-β2 셔틀 벡터와의 상동재조합 후 IX 유전자 유무를 통하여 도 3의 (a)에서 확인된 sh-hTGF-β2가 포함된 클론 중에서 게놈 DNA도 포함된 클론들을 재차 선별하는 PCR 결과], 도 3의 (c)에서 보듯이 HindⅢ 절단 패턴(digestion pattern)으로 최종 재조합체를 선별하였다[도 4]. After screening only positive clones (# 1, 2, 5, 6, 7, 8, 9) by E3 site PCR of adenovirus [FIG. 3 (a): dl324 / IX adenovirus backbone genomic PCR results of screening clones containing sh-hTGF-β2 after homologous recombination between DNA and pSP72-sh-hTGF-β2 shuttle vector, Figure 3 (b): dl324 / IX adenovirus backbone genomic DNA and pSP72-sh PCR result of reselecting clones containing genomic DNA from among clones containing sh-hTGF-β2 identified in FIG. 3 (a) after homologous recombination with -hTGF-β2 shuttle vector] As shown in (c) of 3, the final recombinant was selected by the HindIII digestion pattern (Fig. 4).
도 3과 도 4를 구체적으로 설명하면 다음과 같다.3 and 4 will be described in detail.
도 3의 (a)에서, dl324/IX 레인은 dl324 백본이고; 셔틀 레인은 pSP72-sh-hTGF-β2이다. 레인 1~10은 dl324 백본과 pSP72-sh-hTGF-β2 간의 상동 재조합 후 박테리아 클론(bacterial clone)으로부터 얻은 플라스미드를 E3 부위 증폭시킨 결과를 보여주는 것으로 약 2 kb에 해당하는 밴드가 나타나야 positive이다. E3 부분을 PCR을 하였을 때 E3 부분이 없는 dl324 백본에서는 2 kb에 해당하는 밴드가 나타나지 않지만, E3부분에 U6 프로모터와 sh-hTGF-β2의 sh 컨스트럭트(construct)가 삽입된 셔틀 벡터의 경우 PCR하면 2 kb의 산물(product)의 크기(size)가 나타나는 것을 통하여 상동 재조합되었는지 확인할 수 있다. In FIG. 3A, the dl324 / IX lanes are the dl324 backbones; The shuttle lane is pSP72-sh-hTGF-β2. Lanes 1 to 10 show a result of amplifying the E3 region of a plasmid obtained from a bacterial clone after homologous recombination between the dl324 backbone and pSP72-sh-hTGF-β2, and a band corresponding to about 2 kb is positive. In the dl324 backbone without the E3 portion when PCR was performed, the band corresponding to 2 kb did not appear, but the shuttle vector with the U6 promoter and sh-hTGF-β2 sh construct inserted into the E3 region was used. PCR confirms homologous recombination through the appearance of a product of 2 kb.
도 3의 (b)에서, dl324/IX 레인은 dl324 백본이고; 셔틀 벡터는 pSP72-hTGF-β2이다. 도 3의 (a)에서 확인된 sh-hTGF-β2가 포함된 클론 중(#1, 2, 5, 6, 7, 8, 9)에서 게놈 DNA도 포함된 클론들을 재차 선별하는 PCR 결과로 상동 재조합이 되었다는 것을 확인하는 도 3의 (a)에 이은 연속적인 선별 실험으로 양쪽에서 positive한 클론들이 상동 재조합이 되었음을 의미한다. IX 유전자 부분을 PCR을 하였을 때 IX 유전자를 가지고 있는 dl324 백본과 IX 유전자를 가지고 있지 않은 셔틀 벡터의 차이를 이용하여 상동 재조합이 되었는지를 확인하였다. 그 결과 #1, 2, 6, 7 만이 다시 선별되었다.In Figure 3 (b), lane dl324 / IX is the dl324 backbone; The shuttle vector is pSP72-hTGF-β2. Homologous by PCR results of reselecting clones containing genomic DNA from among clones containing sh-hTGF-β2 (# 1, 2, 5, 6, 7, 8, 9) identified in FIG. (A) of FIG. 3 confirming that the recombination was performed, which means that the positive clones on both sides were homologous recombination. When PCR was performed on the IX gene region, homologous recombination was confirmed using the difference between the dl324 backbone having the IX gene and the shuttle vector having no IX gene. As a result, only # 1, 2, 6 and 7 were reselected.
도 3의 (c)는, 백본과 샘플과의 HindⅢ로 컷팅(cutting)하였을 때 달라지는 패턴의 차이에 따라 상동 재조합되었는지를 최종적으로 확인한 것이다. 레인1~3은 상기 #1 클론 유래 DNA이고, 레인 4~6은 #2 클론, 레인 7~9는 #6 클론에서 얻은 DNA를 DH5a라는 컴피턴트 셀(competent cell)에 재차 트랜스포메이션(transformation)하여 얻은 자손(progeny) 클론들로 각 모체(parental) 클론들에서 유래된 각각 3개의 DNA들 중 #1 클론만이 기존의 dl324-IX(맨 왼쪽 첫 번째 lane)과는 다른 HindⅢ 패턴을 보였다. 이는 백본 아데노바이러스 DNA가 셔틀 벡터와 상동 재조합을 이루었음을 의미하며, 따라서 본 발명은 #1 클론에 기초로 하고 있다.Figure 3 (c) is finally confirmed whether the homologous recombination according to the difference in the pattern when the cut (cut) the HindIII of the backbone and the sample. Lanes 1 to 3 are DNAs derived from the # 1 clone, lanes 4 to 6 are clones # 2, and lanes 7 to 9 are DNAs obtained from a clone cell # 6 again in a competent cell called DH5a. Progeny clones obtained from the parental clones of each of the three DNA from the # 1 clone only showed a HindIII pattern different from the existing dl324-IX (leftmost first lane). This means that the backbone adenovirus DNA has homologous recombination with the shuttle vector, and therefore the present invention is based on the # 1 clone.
도 4는, pPoly2라는 플라스미드에 PacI 부위에 삽입되어있는 Ad-dl324-IX-sh-hTGF-β2를 PacI으로 절단하여 pPoly2가 제대로 절단되는지 확인함으로써 바이러스 생산에 요구되는 최종 컨스트럭트(construct)를 결정하는 실험이다. 도 3의 (c)에서 확인한 #1 클론에 속하는 3개의 DNA들은 PacI으로 절단 시 전부 약 2 kb에 해당하는 pPoly2 백본 DNA가 모두 빠져나왔다. 이들이 각각 shRNA hTGF-β2 염기서열을 가지고 있는지를 서열 분석하여 확인한 결과 모든 클론들에서 동일한 shRNA hTGF-β2 염기서열을 가지고 있음을 확인하였다(도 5). 그런 다음, 이들을 PacI 절단 후 함께 293A 세포에 트랜스펙션(transfection)하여 아데노바이러스를 생산하였다.4 is a final construct required for virus production by cutting Ad-dl324-IX-sh-hTGF-β2 inserted into the PacI site into a plasmid called pPoly2 with PacI to confirm that pPoly2 is correctly cleaved. It is an experiment to determine. Three DNAs belonging to the # 1 clone identified in (c) of FIG. 3 were all pPoly2 backbone DNA corresponding to about 2 kb when cut out with PacI. As a result of sequencing of each of them having shRNA hTGF-β2 sequences, it was confirmed that all clones had the same shRNA hTGF-β2 sequences (FIG. 5). These were then transfected together to 293A cells after PacI cleavage to produce adenoviruses.
즉, 상기의 방법으로 제작된 E3 셔틀 벡터들을 각각 XmnI 제한 효소로 처리하여 단일가닥으로 만든 다음, SpeI 제한효소를 처리하여 단일가닥이 된 복제 불능 아데노바이러스인 dl324와 함께 대장균 BJ5183에서 동시에 형질전환시켜 유전자 상동 재조합을 유도하였다. 상동 재조합된 플라스미드 DNA를 수득하여 HindⅢ 제한효소로 처리하여 DNA 패턴의 변화를 확인하고 최종적으로 서열 분석하여 상동 재조합 유무를 확인한 후, 확인된 플라스미드들을 PacI으로 절단한 뒤 293 세포주에 형질전환하여 shRNA TGF-β2를 발현하는 복제불능 아데노바이러스를 제작하였다. (복제 가능 아데노바이러스에 shRNA를 제작하는 경우에는 shRNA에 의한 억제 효과와 세포 라이시스(lysis) 효과가 혼재되어 있어 억제 효과만을 명확하게 확인하기 어렵기 때문에 복제 불능 아데노바이러스를 제작하였다). 이 아데노바이러스는 293 세포주에서 증식시켜 CsCl 변화도(gradient)로 농축하여 한계 희석배양법(limiting dilution) 또는 용균반검사(plaque assay)로 바이러스의 역가를 결정하였다.In other words, the E3 shuttle vectors prepared by the above method were treated with Xmn I restriction enzymes to form single strands, and then treated with Spe I restriction enzymes and co-transfected with E. coli BJ5183 together with dl324, a non-replicable adenovirus. Conversion led to gene homologous recombination. Homologously recombined plasmid DNA was obtained and treated with Hind III restriction enzyme to confirm the change of DNA pattern, and finally sequenced to confirm homologous recombination. The identified plasmids were cut with Pac I and transformed into 293 cell lines. A nonreplicating adenovirus expressing shRNA TGF-β2 was constructed. (When a shRNA is produced in a replicable adenovirus, an inhibitor of a shRNA and a cell lysis effect are mixed, so that only an inhibitory effect is difficult to be clearly identified. The adenovirus was grown in 293 cell lines and concentrated to CsCl gradients to determine the titer of the virus by limiting dilution or plaque assay.
최종 바이러스 역가(virus titer)는 한계 희석 적정법(limiting dilution titration)에 의해 2.5 × 109 pfu/ml 이였다.The final virus titer was 2.5 × 10 9 pfu / ml by limiting dilution titration.
실시에 3: 암세포에서의 효과 확인-shRNA 발현하는 아데노바이러스에 의한 TGF-β2 발현 억제 확인Example 3 Confirmation of Effects in Cancer Cells- Confirmation of Inhibition of TGF-β2 Expression by Adenovirus Expressing shRNA
1) 실시간 RT-PCR로 확인 1) Check by real-time RT-PCR
TGF-β2 발현 억제 확인은 인간의 경우, 인간 전립선암 세포인 DU-145에 실시예 2의 아데노바이러스 1 내지 100 moi로 감염시켜 2일 후, 트리졸(Trizol)로 세포를 라이시스(lysis)시키고, 클로로포름, 이소프로판올, 에탄올 등을 연속적으로 처리하여 RNA를 수확한 후 TGF-β2 mRNA 발현 억제 정도를 실시간 PCR로 확인하였다.TGF-β2 Expression inhibition was confirmed in humans, DU-145, a human prostate cancer cell, infected with adenovirus 1 to 100 moi of Example 2, and after 2 days, the cells were lysed with Trizol and chloroform TGF-β2 after harvesting RNA by continuously treating with isopropanol, ethanol, etc. The degree of mRNA expression inhibition was confirmed by real-time PCR.
마우스인 경우, 마우스 흑색종세포인 B16F10에 실시예 2의 아데노바이러스 100, 500, 1000 moi로 감염시키고 그 이후의 과정은 인간과 동일하게 실시하였다. In the case of mice, B16F10, a mouse melanoma cell, was infected with the adenovirus 100, 500, and 1000 moi of Example 2, and the procedure thereafter was carried out in the same manner as in humans.
인간 TGF-β2 억제를 위한 실시간 PCR용 프라이머는 정방향 프라이머: 5'-GCTGCCTACGTCCACTTTACAT-3' [서열번호 11]과 역방향 프라이머: 5'-ATATAAGCTCAGGACCCTGCTG-3' [서열번호 12]를 사용하였으며, AB powerSYBR Green RNA-to-Ct 1step kit를 사용하여 RT enzyme mix (125X) 0.2㎕, RT-PCR Mix(2x) 12.5 ㎕, Forward Primer(100 pM) 0.5 ㎕, reverse Primer(100 pM) 0.5 ㎕, RNA (10ng/㎕) 5 ㎕, Nuclease-free water 6.3 ㎕로 총 부피는 25 ㎕가 되게 하였고, 반응 조건은 다음 표 3과 같다.Real-time PCR primers for human TGF-β2 inhibition were used as forward primer: 5'-GCTGCCTACGTCCACTTTACAT-3 '[SEQ ID NO: 11] and reverse primer: 5'-ATATAAGCTCAGGACCCTGCTG-3' [SEQ ID NO: 12], AB powerSYBR Green Using the RNA-to-Ct 1step kit, 0.2 μl RT enzyme mix (125X), 12.5 μl RT-PCR Mix (2x), 0.5 μl Forward Primer (100 pM), 0.5 μl reverse primer (100 pM), RNA (10ng) / Μl) 5 μl, Nuclease-free water 6.3 μl total volume was 25 μl, the reaction conditions are shown in Table 3.
마우스 TGF-β2 억제를 위한 실시간 PCR용 프라이머는 정방향 프라이머: 5'-GTGAATGGCTCTCCTTCGAC-3' [서열번호 9]과 역방향 프라이머: 5'-CCTCGAGCTCTTCGCTTTTA-3' [서열번호 10]를 사용하였으며, AB powerSYBR Green RNA-to-Ct 1step kit를 사용하여 RT enzyme mix (125X) 0.2㎕, RT-PCR Mix(2x) 12.5 ㎕, Forward Primer(100 pM) 0.5 ㎕, reverse Primer(100 pM) 0.5 ㎕, RNA (10ng/㎕) 5 ㎕, Nuclease-free water 6.3 ㎕로 총 부피는 25 ㎕가 되게 하였고, 반응 조건은 다음 표 3과 같다.Real-time PCR primers for mouse TGF-β2 inhibition were used as forward primer: 5'-GTGAATGGCTCTCCTTCGAC-3 '[SEQ ID NO: 9] and reverse primer: 5'-CCTCGAGCTCTTCGCTTTTA-3' [SEQ ID NO: 10], AB powerSYBR Green Using the RNA-to-Ct 1step kit, 0.2 μl RT enzyme mix (125X), 12.5 μl RT-PCR Mix (2x), 0.5 μl Forward Primer (100 pM), 0.5 μl reverse primer (100 pM), RNA (10ng) / Μl) 5 μl, Nuclease-free water 6.3 μl total volume was 25 μl, the reaction conditions are shown in Table 3.
표 3
Figure PCTKR2013000791-appb-T000003
 TABLE 3
Figure PCTKR2013000791-appb-T000003
인간 TGF-β2의 shRNA 확인 결과, 1 moi의 아데노바이러스에서 73%의 침묵(silencing) 효과를 보이는 등, 50 moi의 아데노바이러스에서 90% 이상의 TGF-β2 발현 억제를 관찰하였다[표 4, 도 6].As a result of shRNA confirmation of human TGF-β2, over 90% inhibition of TGF-β2 expression was observed in 50 moi of adenovirus, showing 73% silencing effect in 1 moi of adenovirus [Table 4, FIG. 6. ].
표 4
Figure PCTKR2013000791-appb-T000004
 Table 4
Figure PCTKR2013000791-appb-T000004
마우스 TGF-β2의 shRNA 확인 결과, 1000 moi의 아데노바이러스에서 50 %의 침묵(silencing) 효과를 보였다[도 7]. 인간에 비해 상대적으로 낮은 억제율은 마우스세포에 대한 아데노바이러스의 낮은 감염율에 기인한 것으로 보인다. 이에 대한 확인은 마우스 shTGF-β2가 발현하는 플라스미드를 트랜스펙션(transfection)함으로써 발현된 shRNA가 효과적으로 TGF-β2 mRNA를 억제하는 것으로 확인하였다[도 8]. As a result of shRNA confirmation of mouse TGF-β2, a 50% silencing effect was observed in 1000 moi of adenovirus [FIG. 7]. The relatively low inhibition rate compared to humans seems to be due to the low infection rate of adenovirus against mouse cells. Confirmation of this was confirmed by transfection of the plasmid expressing mouse shTGF-β2 and the expressed shRNA effectively inhibited TGF-β2 mRNA [FIG. 8].
2) ELISA로 확인 2) Confirmation by ELISA
상기한 아데노바이러스 1, 5, 10, 50 moi 감염 후 2일 동안 인간 실시예 2의 전립선암세포에 배양하면서 마지막 24 시간은 무혈청 배지로 분비된 TGF-β2 양을 측정하였다.The amount of TGF-β2 secreted in serum-free medium was measured in the last 24 hours while incubating in the prostate cancer cells of human Example 2 for 2 days after the adenovirus 1, 5, 10, 50 moi infection.
TGF-β2의 shRNA를 발현하는 아데노바이러스를 1 moi만 감염되도 하루 동안 분비되는 TGF-β2는 거의 검출되지 않았다[표 5, 도 9]. 이는 TGF-β2의 shRNA가 매우 효과적으로 TGF-β2의 mRNA를 분해하고 있음을 의미한다.Even when only 1 moi of adenovirus expressing the shRNA of TGF-β2 was secreted, almost no TGF-β2 was detected during the day [Table 5, FIG. 9] . This means that the shRNA of TGF-β2 degrades the mRNA of TGF-β2 very effectively.
표 5
Figure PCTKR2013000791-appb-T000005
 Table 5
Figure PCTKR2013000791-appb-T000005
실시예 4: 종양 선택적 살상 아데노바이러스 제작 Example 4 Tumor Selective Killing Adenovirus Construction
복제 불능한 아데노바이러스에서 TGF-β2에 대한 shRNA의 효과를 확인한 후 이 shRNA를 발현하면서 종양 선택적으로 세포를 살상하는 아데노바이러스를 제작하였다. 종양 선택적 살상 복제 가능한 아데노바이러스를 만들기에 앞서 아데노바이러스의 E1A부분에 여러 유전자를 넣을 수 있는 셔틀벡터를 제작하고자 pBSKII 플라스미드[Stratagene, USA]에E1A와 E1B55kDa 유전자를 포함하며 다양한 효소 부위(Enzyme site)를 포함하는 pBSKⅡ-3484 합성 유전자를 제작하였다[도 10의 (a)]. 합성된 유전자를 상동 재조합 확인을 용이하게 하기 위한 pCA14 셔틀벡터에 도입하기 위한 형태로 바꾸기 위하여 pBSKⅡ-3484를 PCR하여 FspⅠ으로 제한효소를 처리한 뒤 블런팅(blunting) 효소로 블런트 엔드(blunt end)를 만들고 다시 BamHⅠ으로 처리하였다. pCA14[Microbix BiosystemsInc, Canada]는 SspⅠ으로 제한효소를 이용하여 자른 후 블런팅 효소를 이용하여 블런트 엔드를 만든 후, BglⅡ를 처리하여 동일전달제한효소(Isoschizomer)인 BamHⅠ과 BglⅡ 그리고 양끝의 블런트 엔드를 통해 합성된 유전자를 삽입하여 셔틀 벡터 pCA14-3484를 제작하였다[도 10의 (b)]. 그 후 CMV 프로모터 유전자를 KpnⅠ과 XhoⅠ으로 pCA14-3484에 삽입하여 pCA14-CMV-3484를 제작하였다[도 10의 (c)]. 그리고 pCA14-CMV-3484에서 EcoRI과 SalI 제한효소의 의해 E1B55kDa 부분을 자르고 블런팅(blunting)한 후 다시 연결(ligation)된 pCA14-CMV-3484-ΔE1B55를 얻었다[도 10의 (d)]. 제작된 셔틀 벡터 pCA14-CMV-3484-ΔE1B55를 XmnI으로 잘라 linearization시킨 후 IX 유전자가 없는 dl324-BstBⅠ-human shTGF-β2(또는 mouse shTGF-β2)를 BstBI으로 자른 후 대장균 BJ5183 에서 동시에 형질전환시켜 상동 재조합을 유도하였다. After confirming the effect of shRNA on TGF-β2 in non-replicating adenoviruses, an adenovirus was produced that expresses this shRNA and selectively kills cells. In order to construct a shuttle vector that can insert several genes into the E1A portion of the adenovirus prior to making adenovirus capable of tumor selective killing, various enzyme sites include E1A and E1B55kDa genes in the pBSKII plasmid [Stratagene, USA]. PBSKII-3484 synthetic gene was prepared including [FIG. 10 (a)]. To convert the synthesized gene into a form for introduction into a pCA14 shuttle vector for facilitating homologous recombination, pBSKII-3484 was PCR-treated with restriction enzymes using Fsp I and then blunt end with a blunting enzyme. Was prepared and treated with Bam HI again. pCA14 [Microbix BiosystemsInc, Canada] was Ssp were cut using a restriction enzyme to Ⅰ, create a blunt end using a block reonting enzyme, which processes the Bgl Ⅱ same transmission restriction enzyme (Isoschizomer) Bam HⅠ and Bgl Ⅱ and ends A shuttle vector pCA14-3484 was constructed by inserting the synthesized gene through the blunt end of [Fig. 10 (b)]. Then by inserting the gene into Kpn pCA14-3484 the CMV promoter and the Xho Ⅰ Ⅰ was prepared pCA14-CMV-3484 [(c ) of Fig. And pCA14-CMV-3484 was cut and blunted the E1B55kDa portion by EcoRI and SalI restriction enzyme and then re-linked to obtain pCA14-CMV-3484-ΔE1B55 (Fig. 10 (d)). The shuttle vector pCA14-CMV-3484-ΔE1B55 was cut and linearized by XmnI, and then dl324-BstBI-human shTGF-β2 (or mouse shTGF-β2) without IX gene was cut with BstBI and transformed simultaneously in E. coli BJ5183. Recombination was induced.
상동 재조합된 플라스미드 DNA를 수득하여 HindⅢ 제한효소로 처리하여 DNA 패턴의 변화를 확인하고 최종적으로 서열 분석하여 상동 재조합 유무를 확인한 후, 확인된 플라스미드들을 PacI으로 절단한 뒤 293 세포주에 형질전환하여 종양을 선택적으로 살상하면서 인간(또는 마우스) TGF-β2의 발현을 억제하는 dl324-CMV-3484-shTGF-β2 아데노바이러스를 제작하였다[도 11]. Homologously recombined plasmid DNA was obtained and treated with Hind III restriction enzyme to confirm the change of DNA pattern, and finally sequenced to confirm homologous recombination. The identified plasmids were cut with Pac I and transformed into 293 cell lines. A dl324-CMV-3484-shTGF-β2 adenovirus was produced that selectively killed tumors and inhibited the expression of human (or mouse) TGF-β2 (FIG. 11).
도 12는 실제 마우스 shTGF-β2가 포함된 종양선택적 복제 가능 아데노바이러스 제작을 위한 상동 재조합 과정을 나타낸 것으로, E3 스크리닝 결과, 1, 2, 4, 5, 6 클론이 positive clone으로 1차 선별되었고(a), HindⅢ 절단 패턴(digestion pattern)으로 상동 재조합된 콜로니를 선별하고 1,2,4번 콜로니 모두 대조구와 패턴 비교한 결과 재조합된 것으로 확인하였고(b), PacI 절단 후 1, 2, 4 콜로니 중 1번만 PacI으로 절단되어 2kb 정도의 밴드를 확인함으로써 1번 콜로니의 DNA가 상동 재조합된 dl324-CMV-3484-△E1B55-△E3-sh-mTGF-β2의 DNA임을 확인할 수 있었다(c) Figure 12 shows the homologous recombination process for the production of tumor-selective replicable adenovirus containing the actual mouse shTGF-β2, as a result of E3 screening, 1, 2, 4, 5, 6 clones were first selected as a positive clone ( a), the homologous recombination colonies were selected by the HindIII digestion pattern, and colonies 1, 2, and 4 were compared with the control, and confirmed to be recombinant (b), 1, 2, and 4 colonies after PacI cleavage. The DNA of colony No. 1 was homologously recombined dl324-CMV-3484-ΔE1B55-ΔE3-sh-mTGF-β2 by confirming the band of about 2 kb, which was cleaved with PacI only once (c).
도 13은 인간 shTGF-β2가 포함된 종양선택적 복제 가능 아데노바이러스 제작을 위한 상동 재조합 과정을 나타낸 것으로, HindⅢ 절단 패턴에 의한 상동 재조합이 된 클론(1, 2, 3)이 선별되었으며(a), 상기 클론 1, 2, 3의 DNA를 PacI으로 잘라 보았을 때 클론 DNA 1,2,3 모두 절단되어 2 kb 정도의 밴드 확인하였으며, 이는 1,2,3 콜로니의 DNA가 상동 재조합된 dl324-CMV-3484-ΔE1B55-ΔE3-sh-hTGF-β2의 DNA 확인할 수 있었다(b).Figure 13 shows the homologous recombination process for the production of tumor-selective replicable adenovirus containing human shTGF-β2, clones (1, 2, 3) homologous recombination by the HindIII cleavage pattern was selected (a), When the DNAs of the clones 1, 2 and 3 were cut with PacI, the clone DNAs 1,2 and 3 were all cut and confirmed to have a band of about 2 kb, which is dl324-CMV- in which the DNA of the 1,2,3 colonies was homologously recombined. DNA of 3484-ΔE1B55-ΔE3-sh-hTGF-β2 was confirmed (b).
실시예 5: 세포용혈 확인Example 5 Cell Hemolysis
복제 가능한 아데노바이러스의 세포 살상능을 확인하기 위하여 24 웰 플레이트(well plate)에 각 종류의 세포를 4×104에서 1×105까지 세포의 크기에 따라 세포수를 정한 뒤 분주하여 배양한 후 다음날 survivin promoter와 CMV 프로모터를 가진 복제 가능한 아데노바이러스를 MOI별로 감염시켜 양성 대조군인 293A 세포주에서 가장 낮은 MOI에서 세포가 바이러스에 의해 다 죽을 때 실험을 종료하여 각 플레이트에 죽지 않고 살아있던 세포를 Crystal violet으로 염색하였다. 3.7% 파라포름알데히드(Paraformaldehyde)로 세포를 5분간 상온에서 고정시킨 후 0.05% Crystal violet으로 상온에서 30분간 염색시킨 후 물로 세척하여 염색된 세포를 관찰하였다. 두 종류의 종양 살상 바이러스로 바이러스의 종양 살상 효과를 비교한 결과 프로모터에 따른 살상효과의 차이는 크지 않은 것으로 나타났으며 둘 다 종양 선택성이 뛰어남을 확인하였다. In order to confirm the cell killing ability of the replicable adenovirus, each cell was sorted according to the size of the cells from 4 × 10 4 to 1 × 10 5 in a 24-well plate, and then divided and cultured. The next day, the cloned adenovirus with the survivin promoter and CMV promoter was infected by MOI, and when the cells were all killed by the virus at the lowest MOI in the positive control 293A cell line, the experiment was terminated. Stained with. The cells were fixed with 3.7% paraformaldehyde at room temperature for 5 minutes and then stained with 0.05% crystal violet for 30 minutes at room temperature and washed with water to observe the stained cells. As a result of comparing the tumor killing effect of the virus with two types of tumor killing viruses, the difference in killing effect according to the promoter was not significant and both showed excellent tumor selectivity.
도 14는 종양선택적 복제 가능 아데노바이러스(CMV promoter와 E1B 55KDa가 발현되지 못하는 dl324-CMV-3484과 surviving promoter에 의해 선택성을 부여하고 55KDa가 발현되는 dl324-hSurvivin-3484)들이 정상세포(BJ 세포)에서는 복제가 일어나지 않는데 반해 여러 종류의 인간암세포에서는 복제가 일어나 세포의 용혈이 일어나는 것을 보여준 것이다.FIG. 14 shows tumor-selective replicable adenoviruses (dl324-CMV-3484 and surviving promoter not expressing CMV promoter and E1B 55KDa and dl324-hSurvivin-3484 expressing 55KDa) are normal cells (BJ cells). In Esau, cloning does not occur, whereas cloning occurs in various types of human cancer cells, demonstrating that cell hemolysis occurs.
실시예 6: 암세포에서의 효과 확인Example 6: Confirmation of Effects in Cancer Cells
A375 멜라노마 세포주에서 인간 sh-TGF-β1 또는 sh-TGF-β2를 발현하는 실시예 2의 복제 불능 아데노바이러스를 1, 5, 10, 50, 100 moi로 감염시키고 나서 세포내에 존재하는 TGF-β1, TGF-β2, TGF-β3 mRNA의 수준을 실시간 PCR 방법으로 실행하였다. TGF-β1 present in cells after infection with non-replicating adenovirus of Example 2 expressing human sh-TGF-β1 or sh-TGF-β2 in an A375 melanoma cell line with 1, 5, 10, 50, 100 moi , TGF-β2, TGF-β3 mRNA levels were performed by real-time PCR method.
그 결과, TGF-β1의 shRNA를 발현하는 경우 세포내 TGF-β1 mRNA를 감소시키기는 하나 TGF-β2 mRNA나 TGF-β3 mRNA가 증가하는 경향으로 나타났다. 반면에 TGF-β2의 shRNA를 발현하는 경우 세포내 TGF-β2 mRNA를 효과적으로 감소시키면서 동시에 TGF-β2 mRNA나 TGF-β3 mRNA도 감소하는 경향으로 나타났다[도 15]. 이는 세포 내의 보상 효과 측면에 의한 효능 감소 우려가 적어도 TGF-β2의 shRNA를 발현하는 경우에는 이런 현상이 나타나지 않았을 뿐만 아니라 다른 TGF-β의 isotype도 억제시키는 부수 효과도 가질 수 있는 장점이 된다. 이와 비슷한 결과가 ELISA를 사용하여 TGF-β 단백질의 발현 감소 효과 패턴도 실시간 PCR에서와 유사하게 나타났다[도 16]. As a result, the expression of TGF-β1 shRNA decreased intracellular TGF-β1 mRNA but increased TGF-β2 mRNA or TGF-β3 mRNA. On the other hand, the expression of TGF-β2 shRNA was found to reduce TGF-β2 mRNA or TGF-β3 mRNA simultaneously while effectively reducing intracellular TGF-β2 mRNA [FIG. 15]. This is an advantage that the reduction in efficacy due to the compensation effect in the cell at least when expressing the TGF-β2 shRNA not only does not exhibit this phenomenon but also has the side effect of inhibiting other isotype of TGF-β. Similar results showed that the expression pattern of the reduced expression of TGF-β protein using ELISA was similar to that of real-time PCR [FIG. 16].
이는 TGF-β2에 대한 shRNA 발현하는 아데노바이러스가 TGF-β1에 대한 shRNA 발현하는 아데노바이러스 보다 발현 억제 효과가 상대적으로 우수함을 확인한 것이다.This confirms that the shRNA expressing adenovirus for TGF-β2 has a relatively superior expression inhibitory effect than the shRNA expressing adenovirus for TGF-β1.

Claims (10)

  1. 서열번호 1 또는 2로 표시되는 염기서열을 표적서열로 하고, TGF-β2 발현을 억제하는 shRNA.A shRNA that inhibits TGF-β2 expression using a nucleotide sequence represented by SEQ ID NO: 1 or 2 as a target sequence.
  2. 제 1 항에 있어서,The method of claim 1,
    서열번호 3 또는 서열번호 4로 표시되는 shRNA.ShRNA represented by SEQ ID NO: 3 or SEQ ID NO: 4.
  3. 제 1 항의 shRNA를 유효성분으로 함유하는 항종양 조성물.An anti-tumor composition comprising the shRNA of claim 1 as an active ingredient.
  4. 제 1 항의 shRNA 발현하는 재조합 발현벡터. The recombinant expression vector expressing shRNA of claim 1.
  5. 제 4 항에 있어서,The method of claim 4, wherein
    서열번호 5로 표시되는 탑 스트랜드(top strand)와, 서열번호 6으로 표시되는 바텀 스트랜드(bottom strand)를 포함하는 DNA를 포함하는 재조합 발현벡터.A recombinant expression vector comprising a DNA comprising a top strand represented by SEQ ID NO: 5 and a bottom strand represented by SEQ ID NO: 6.
  6. 제 4 항에 있어서,The method of claim 4, wherein
    서열번호 7로 표시되는 탑 스트랜드(top strand)와, 서열번호 8로 표시되는 바텀 스트랜드(bottom strand)를 포함하는 DNA를 포함하는 재조합 발현벡터.A recombinant expression vector comprising a DNA comprising a top strand represented by SEQ ID NO: 7 and a bottom strand represented by SEQ ID NO: 8. 8.
  7. 제 4 항에 있어서, The method of claim 4, wherein
    U6 프로모터를 함유하는 벡터에 DNA를 재조합시켜 수득하는 재조합 발현벡터.Recombinant expression vector obtained by recombining DNA into a vector containing the U6 promoter.
  8. 제 4 항에 있어서, The method of claim 4, wherein
    pSP72△E3-sh-human TGF-β2 또는 pSP72△E3-sh-mouse TGF-β2인 재조합 발현벡터.A recombinant expression vector of pSP72ΔE3-sh-human TGF-β2 or pSP72ΔE3-sh-mouse TGF-β2.
  9. 제 4 항의 재조합 발현벡터를 유효성분으로 하는 항종양 조성물.An anti-tumor composition comprising the recombinant expression vector of claim 4 as an active ingredient.
  10. 제 4 항의 재조합 발현벡터를 도입한 아데노바이러스.Adenovirus introduced with a recombinant expression vector of claim 4.
PCT/KR2013/000791 2012-01-31 2013-01-31 Shrna for suppressing tgf-β2 expression WO2013115579A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201380007620.1A CN104245936B (en) 2012-01-31 2013-01-31 For the shRNA suppressing TGF-β 2 to express
JP2014555484A JP2015506696A (en) 2012-01-31 2013-01-31 ShRNA that suppresses TGF-β2 expression

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20120009811 2012-01-31
KR10-2012-0009811 2012-01-31
KR1020130010233A KR101420564B1 (en) 2012-01-31 2013-01-30 The shRNA downregulating TGF-β2 for treatment of tumor
KR10-2013-0010233 2013-01-30

Publications (1)

Publication Number Publication Date
WO2013115579A1 true WO2013115579A1 (en) 2013-08-08

Family

ID=48905548

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2013/000791 WO2013115579A1 (en) 2012-01-31 2013-01-31 Shrna for suppressing tgf-β2 expression

Country Status (1)

Country Link
WO (1) WO2013115579A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210154329A1 (en) * 2015-12-08 2021-05-27 University-Industry Foundation, Yonsei University ANTI-TUMOR COMPOSITION COMPRISING GM-CSF GENE, Flt3L-TRAIL FUSION GENE, shRNA INHIBITING TGF-ß EXPRESSION, AND shRNA INHIBITING HSP EXPRESSION

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060031596A (en) * 2003-04-01 2006-04-12 인트라디그엠 코오포레이션 Targets for tumor growth inhibition
KR20100066429A (en) * 2007-06-20 2010-06-17 독립행정법인 산업기술종합연구소 Cancer remedy containing mortalin sirna
US20110262408A1 (en) * 2009-12-23 2011-10-27 Gradalis, Inc. Furin-knockdown and gm-csf-augmented (fang) cancer vaccine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060031596A (en) * 2003-04-01 2006-04-12 인트라디그엠 코오포레이션 Targets for tumor growth inhibition
KR20100066429A (en) * 2007-06-20 2010-06-17 독립행정법인 산업기술종합연구소 Cancer remedy containing mortalin sirna
US20110262408A1 (en) * 2009-12-23 2011-10-27 Gradalis, Inc. Furin-knockdown and gm-csf-augmented (fang) cancer vaccine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YOO, JI YOUNG ET AL.: "VEGF specific short hairpin RNA expressing oncolytic adenovirus elicits potent inhibition of angiogenesis and tumor growth", MOLECULAR THERAPY, vol. 15, no. 2, February 2007 (2007-02-01), pages 295 - 302, XP055081246 *
ZHANG, CHENYU ET AL.: "Transforming growth factor-beta2 is a molecular determinant for site- specific melanoma metastasis in the brain", CANCER RESEARCH, vol. 29, no. 3, 13 January 2009 (2009-01-13), pages 828 - 835, XP055081244 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210154329A1 (en) * 2015-12-08 2021-05-27 University-Industry Foundation, Yonsei University ANTI-TUMOR COMPOSITION COMPRISING GM-CSF GENE, Flt3L-TRAIL FUSION GENE, shRNA INHIBITING TGF-ß EXPRESSION, AND shRNA INHIBITING HSP EXPRESSION

Similar Documents

Publication Publication Date Title
UA126860C2 (en) Cytomegalovirus vectors eliciting t cells restricted by major histocompatibility complex e molecules
WO2018143626A1 (en) Nucleic acid simultaneously inhibiting expression of mtor gene and stat3 gene
CA3185387A1 (en) Nucleic acid artificial mini-proteome libraries
WO2020149702A1 (en) Asymmetric sirna for inhibiting expression of neural retina leucine zipper (nrl)
US9173964B2 (en) Diphtheria toxin first open reading frame operably linked to an H19 promoter and a diphtheria toxin second open reading frame operably linked to an IGF-II promoter as nucleic acid construct
US20050048466A1 (en) Specific proliferation in tumour cell which can express antioncogene with high efficiency and the use of it
KR101420564B1 (en) The shRNA downregulating TGF-β2 for treatment of tumor
WO2013115579A1 (en) Shrna for suppressing tgf-β2 expression
WO2018155890A1 (en) Asymmetric sirna for inhibiting expression of male pattern hair loss target gene
KR101286053B1 (en) The shRNA downregulating TGF-β1 for treatment of tumor
CN111763669B (en) Double-stranded RNA molecule and application thereof
WO2021194179A1 (en) Oncolytic virus comprising nucleic acid sequence that bispecifically targets stat3 and mtor
RU2636003C2 (en) Composition for treatment of cancer associated with hpv infection
JPWO2006038608A1 (en) Oligo double-stranded RNA and pharmaceutical composition
WO2005035718A2 (en) Biderectional promoters for small rna expression
EP2128261A1 (en) A recombinant adenovirus comprising recombinant khp50 gene and preparation method and uses thereof
WO2010064851A2 (en) Mtor-targeted sirna having an interspecific cross reaction, recombination vector containing same, and pharmaceutical composition containing same
WO2020122534A1 (en) Asymmetric sirna for inhibiting expression of snai1
WO2022131398A1 (en) Nucleic acid construct enabling expression and inhibition of gene simultaneously
KR101374585B1 (en) The shRNA downregulating HSP27 for treatment of tumor
WO2010095879A2 (en) Sirna to be bound to transcripts of apoptosis-related genes, and compositions for treating cancer using same
CN113583977A (en) Isolated recombinant oncolytic poxvirus regulated by microRNA and application thereof
WO2019235771A1 (en) Mesenchymal stem cell line in which infection and replication of adenovirus are enabled
WO2019203317A1 (en) Pharmaceutical composition for treating proliferative diseases
JP2002544236A (en) Organ, tissue and cell-specific immunotherapeutic agents for hepatic chronic viral infections and inflammatory, degenerative and proliferative diseases and cancer, especially based on recombinant parapoxvirus

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201380007620.1

Country of ref document: CN

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

Ref document number: 13743966

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2014555484

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13743966

Country of ref document: EP

Kind code of ref document: A1