WO2006035974A1 - オリゴリボヌクレオチド - Google Patents
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- WO2006035974A1 WO2006035974A1 PCT/JP2005/018244 JP2005018244W WO2006035974A1 WO 2006035974 A1 WO2006035974 A1 WO 2006035974A1 JP 2005018244 W JP2005018244 W JP 2005018244W WO 2006035974 A1 WO2006035974 A1 WO 2006035974A1
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- 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
- C12N15/1131—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 viruses
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Definitions
- the present invention relates to a specific oligoribonucleotide that can be used for gene-targeted therapy against human papillomavirus (hereinafter referred to as HPV) type 18 positive cervical cancer, and an antiviral agent against HPV and a child containing the same.
- HPV human papillomavirus
- the present invention relates to a therapeutic agent for cervical cancer.
- RNA interference (hereinafter referred to as RNAi) has been discovered as a new method for specifically inhibiting gene expression.
- This method is a revolutionary method that effectively inhibits target gene expression by introducing RNA double short strands (hereinafter referred to as siRNA) complementary to the target gene. It is expected to bring about change in these fields.
- siRNA RNA double short strands
- Cervical cancer is one of the major malignant tumors in gynecology, and its main cause is HPV, especially type 16 (HPV16) and type 18 (HPV18). Expression of the E6 and E7 oncogenes of these viruses has been shown to be essential for cell carcinogenesis and maintenance of canceration traits.
- HPV genome integration is observed in most cancer cells, and the above E6 and E7 genes bind to and inactivate the tumor suppressor gene products P53 and Rb, respectively. It is known to be deeply involved in the development and proliferation of
- Non-patent Document 1 reports that siRNA on E7 is more prominent in growth inhibition than siRNA on HPV E6.
- the present inventor confirmed in the previously reported Non-Patent Document 2 that a sufficient inhibitory effect was obtained using siRNA on HPVE6. From these results, it was not clear what sequence would be the most effective inhibitory effect.
- Non-Patent Document 1 M. Jiang and J. Milner, “Selective silencing of viral gene expression in HPV-positive chick chick cancer cells treated with siRNA and RNA interfering primers ( Selective silencing of viral gene expression in HPV— positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference.) ”Oncogene 2002, 21st, p.6041-6048
- Non-Patent Document 2 M. Yoshinouchi et al., “In vitro and in vivo growth suppression of E6 siRNA in vitro and in vivo growth of human papillomavirus (HPV) 16-positive cervical cancer cells. human papilloma virus (HPV) 16- positive cervical cancer cells by E6 siRNA) ”Molecule ⁇ ⁇ * Cefpy (Molecular Therapy) 2003, No. 8, No. 5, p.762-768 Disclosure of the Invention
- HPV-positive cervical cancers Approximately two-thirds of HPV-positive cervical cancers are type 6 and type 18, so if siRNA therapy for these two types of E6 oncogenes can be established, it will be an effective and less invasive treatment for many cervical cancer patients. Become. However, for HPV18 type, a target sequence that exhibits sufficient growth inhibition has not yet been clarified.
- siRNA The effects of siRNA are known to vary greatly depending on the target site. It is very difficult to predict the effects.
- the present inventor created siRNA targeting HPV18-E6 from the first exon and splicing donor site and biologically produced it. A comparative analysis of the effect was carried out to determine a target sequence having a better growth inhibitory effect, thereby completing the present invention.
- the present invention provides the following (1) to (9).
- nucleotide sequence shown in SEQ ID NO: 2 or 4 it hybridizes under stringent conditions with DNA consisting of a sequence in which uracil is replaced with thymine, and suppresses the expression of the E6 gene of human papillomavirus type 18.
- oligoribonucleotide consisting of 19 bases on the 5 ′ side of the base sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, and is a human papilloma Oligoribonucleotide having a base length of 21 to 23 bases capable of suppressing the expression of the virus type 18 E6 gene.
- oligoribonucleotide according to any one of (1) to (4), wherein the oligonucleotide is a double strand.
- An expression vector comprising a nucleotide sequence obtained by converting 19 nucleotides on the 5 ′ side of the nucleotide sequence represented by SEQ ID NO: 2 into DNA.
- An expression vector comprising a base sequence obtained by converting 19 bases on the 5 ′ side of the base sequence represented by SEQ ID NO: 3 into DNA.
- An antiviral agent comprising the oligoribonucleotide according to any one of (1) to (4) above or the expression vector according to (6) or (7) above.
- a therapeutic agent for cervical cancer comprising the oligoribonucleotide according to any one of (1) to (4) above or the expression vector according to (6) or (7) above.
- FIG. 1 shows the structure of the 3XFLAG-18E6 expression vector used in the present invention.
- FIG. 2 shows the suppressive effect of the siRNA of the present invention on the expression of human papillomavirus (HPV) type 18 E6 gene.
- FIG. 3 shows suppression of lamin A / C expression by siRNA in HeLa cells and SW756 cells.
- FIG. 4 shows the suppressive effect of the siRNA of the present invention on the expression of E6 and E7 mRNA in HeLa cells.
- Figure 5 shows the effect of siRNA on p53 and E7 protein expression in HeLa and SW756 cells.
- FIG. 6 shows the suppressive effect of siRNA on monolayer growth and anchorage independent growth of HeLa cells and SW756 cells.
- Figure 7 shows the results of confirming the induction of apoptosis in HeLa cells by siRNA using flow cytometry.
- FIG. 8 shows graphically the induction of apoptosis in HeLa cells by siRNA.
- the present invention is based on the base sequence of HPV 18 type E6 gene and can suppress its expression. W
- W In order to obtain effective s iRNA, a large number of siRNA candidates were prepared and their effects were examined.
- the genome sequence information of HPV type 18 is registered in databases such as GenBank as registration numbers X04354 and Y18491, and siRNA candidates can be created based on such information.
- siRNA that recognizes the base sequence represented by catagaaata acctgtgtat attgcaagac agtattggaa cttacagagg tatttgaatt tgcatttaaa gattt effectively suppresses the expression of the HPV 18 type E6 gene, and completed the present invention.
- the present invention provides an oligoribonucleotide having a base length of 19 bases that can hybridize with a DNA comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions and suppress the expression of the E6 gene of human papillomavirus type 18, Alternatively, an oligoribonucleotide having a base length of 21 to 23 bases containing the same is provided.
- stringent conditions include, but are not limited to, for example, hybridization at 68 ° C in SSC / 5 X Denhardt's solution / 1.0% SDS, and 0.2 X SSC / Includes cleaning at 42 ° C in 0.1% SDS or cleaning at 68 ° C in 0.1 X SSC / 0.1% SDS.
- these conditions for example, Sambrook et al., “Molecular Cloning: A Laboratory Manual, old Spring Harbor La Oratory Press, New York, US, 1989” and Ausubel et al. See Current Protocols in Molecular Biology, Third Edition, John Wiley & Sons, Inc., 1995).
- oligoribonucleotide of the present invention for example, in the base sequence represented by SEQ ID NO: 2 or 3, it hybridizes with a DNA comprising a sequence in which uracil is substituted with thymine under stringent conditions, and is treated with a human papillomavirus.
- examples thereof include oligoribonucleotides having a base length of 21 to 23 bases that can suppress the expression of type 18 E6 gene.
- oligoribonucleotide of the present invention 90% or more sequence identity with an oligoribonucleotide consisting of 19 bases on the 5 ′ side of the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 21 to 23 base oligoribonucleotides comprising a base sequence having the sequence E6 gene of human papillomavirus type 18 What can suppress the expression of is mentioned.
- oligoribonucleotide of the present invention is SEQ ID NO:
- an oligoribonucleotide having the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 is exemplified.
- the above oligoribonucleotide can effectively suppress the expression of HPV 18 E6 gene and E7 gene.
- These oligoribonucleotides can be used in a single strand, but are preferably double-stranded.
- the oligoribonucleotide represented by SEQ ID NO: 2 and the oligoribonucleotide represented by SEQ ID NO: 3 were hybridized, and 3 '
- the two terminal thymine bases protrude (overhang) to form a duplex that acts as the siRNA described herein.
- the oligoribonucleotide represented by SEQ ID NO: 2 and the oligoribonucleotide represented by SEQ ID NO: 3 formed a double strand
- the oligoribonucleotide represented by SEQ ID NO: 4 is Sp-18E6
- the oligoribonucleotide represented by SEQ ID NO: 5 are referred to as EX-18E6, respectively.
- Inhibition of the expression of HPV18 type E6 gene and E7 gene by the oligoribonucleotide of the present invention can be observed as degradation of mRNA, suppression of expression, and suppression of protein expression due to these.
- “suppression” of expression means that the expression level is reduced to 50% or less compared to the control expression in the absence of oligoribonucleotide.
- HPV-related cancer cell lines have low expression levels of E6 and E7 proteins, and there is no method or antibody for detecting E6 protein expression.
- the present inventors have developed 3 X FLAG-tagged E6 and E7 expression plasmids in order to detect siRNA suppression effects with high sensitivity and ease. The configuration is shown in Fig. 1 (E6).
- This plasmid is introduced into C0S-1 cultured cells. After 24 hours of culture, C0S-1 cell proteins are extracted, and Western plotting using FLAG antibody is performed to measure the expression of E6 and E7 proteins. can do. If this plasmid is introduced into C0S-1 cultured cells together with the siRNA to be evaluated, the inhibition rate of E6 and E7 protein expression can be measured. By using this method, not only synthetic double-stranded siRNA, but also RNAi activity of siRNA expression plasmids and shRNA expression plasmids with lower introduction efficiency can be easily measured.
- siRNA having RNAi activity may be introduced into HeLa cells, and expression suppression at the RNA level encoding E6 and E7 may be evaluated by RT-PCR.
- the optimal siRNA for HPV18-type cervical cancer used in the present invention is an oligoribonucleotide (Sp-18E6) in which the oligoribonucleotide represented by SEQ ID NO: 2 and the oligoribonucleotide represented by SEQ ID NO: 3 form a double strand. It is. This is characterized by a sequence containing a splicing donor site in the E6 gene.
- siRNA may be procured by any method such as a culture method, an artificial synthesis method or a gene manipulation method.
- E6-E7 gene transcribes E6ZE7 mRNA and spliced E6 * / E7 mRNA. Both EX-18E6 and Sp-18E6 suppressed E6 mRNA expression almost completely, but E7 mRNA expression was more completely suppressed by Ex-18E6. This was also true for the expression level of E7 protein, which resulted in an increase in the total amount of Rb protein and an increase in non-phosphorylated Rb protein. On the other hand, Sp-18E6 increased P53 more strongly. As a biological effect, Sp-18E6 was overwhelmingly superior in suppressing anchorage-dependent and independent growth, and Sp-18E6 induced apoptosis more strongly.
- the siRNA of the present invention can be used alone in cells that express E6 protein. Alternatively, it can be incorporated into an expression vector.
- an expression vector comprising a nucleotide sequence obtained by converting 19 bases on the 5 ′ side into DNA of the nucleotide sequence represented by SEQ ID NO: 2 and the nucleotide sequence represented by SEQ ID NO: 3
- an expression vector containing a base sequence in which the 5 base 19 bases are converted to DNA.
- nucleotide sequence represented by SEQ ID NO: 2 and the nucleotide sequence represented by SEQ ID NO: 3 or the nucleotide sequence represented by SEQ ID NO: 4 so that the respective oligonucleotides forming the double strand are transcribed together. It is also preferable to construct an expression vector containing both the base sequence shown by No. 5. Any vector that is known to be used for expression of siRNA can be used as the vector.
- the present invention also provides an antiviral agent containing the above-described oligoribonucleotide of the present invention or the above-described expression vector of the present invention.
- the present invention further provides a therapeutic agent for cervical cancer containing the oligoribonucleotide of the present invention or the expression vector of the present invention.
- the antiviral agent and cervical cancer therapeutic agent in the present invention contain an effective amount of the oligoribonucleotide of the present invention or the expression vector of the present invention, and are usually used as a buffering agent, excipient, carrier, etc. as necessary. Components can be contained as appropriate.
- the dose is appropriately determined according to the patient's age, weight, cancer progression, symptoms, etc., and is not particularly limited, but is usually in the range of 1 ng lOOtngZ days. ,.
- the administration method may be any administration route commonly used in the art, and may be either oral administration or parenteral administration including injection such as intravenous injection.
- s iRNA is attracting attention as a new knockdown method for gene expression. It has a lower concentration than antisense oligo DNA and ribozyme, and suppresses intracellular gene expression for a long period of time. There is great expectation as a cancer drug.
- HPV is greatly involved in the development of cervical cancer, and there is no doubt that suppression of E6 and E7 oncogene expression of these viruses will lead to treatment of cervical cancer.
- siRNA targeting HPV an exogenous virus, does not act on cells other than HPV-positive cells due to its sequence specificity, it can be said to be an ideal gene-targeted therapy with no side effects.
- control double-stranded RNA includes the following non-specific double-stranded XII (31% GC content,
- the polymer was used after being treated for 1 hour.
- COS-1 cells were seeded 80,000 per 24-well plate, and 24 hours later, 500 ng of 3 X FLAG / 18E6 expression vector (Fig. 1) and 20 pmole of siRNA were transfected.
- Fig. 1 3 X FLAG / 18E6 expression vector
- Lipofectamine2000 (Lif eTechnologies) was used and the method in the package insert was followed.
- the plate was washed with PBS, directly dissolved in ⁇ SDS-gel loading buffer, treated at 95 ° C for 5 minutes, and Western blotted.
- the antibody used was an anti-FLAG monoclonal antibody (M2) Sigma (SaintLouis, MZ).
- M2 anti-FLAG monoclonal antibody
- anti- ⁇ -vimentin antibody Oncogene, Boston, MA was also reacted.
- HPV18 genome was donated by Dr. Hiroyuki Sakai of the Institute for Virus Research, Kyoto University. After the E6 and E7 coding regions were amplified and cloned into a plasmid by PCR using this genome as a cage, the nucleotide sequence was determined.
- RNAi can be induced by siRNA administration in HPV18 positive cervical cancer cell lines HeLa and SW756
- a laminA / C suppression experiment was conducted.
- HeLa and SW756 cells 10% FBS-containing DMEM medium, and cultured in 5% C0 2, 37 ° C .
- the laminA / cs iRNA sequence used was
- HeLa and SW756 cells respectively 5 X 10 4 cells per 24-well plate, and 7 X 10 4 cells by Maki, administered laminA / C s iRNA after 24 hours, washed with PBS yet introduced after 48 hours, Iotaomikuron'omikuron mu Dissolve directly in 1 SDS-gel loading buffer, 95. After treatment with C for 5 minutes, a Western plot was performed. Use anti-lamin A / C antibody (Cell Signaling Technology, Beverly, MU).
- siRNA was introduced as follows. 3 x 1 Oligofuectamin (Life Technology) was mixed with 12 1 OPTI-MEM I medium (Life Technology). On the other hand, 3 ⁇ 1 siRNA (20 ⁇ ⁇ ) was mixed with 50 ⁇ 1 ⁇ - ⁇ I medium and allowed to stand at room temperature for 5 minutes. The two were mixed and allowed to stand at room temperature for 20 minutes, and then a total of 100 ⁇ 1 containing 32 1 OPTI MEM I medium was added to 500 ⁇ 1 medium. The final concentration of siRNA is 100 nM. The introduction in the absence of serum was performed in a 500 ⁇ l serum-free medium, and 60 ⁇ l of serum was added 4 hours after siRNA administration. As a result, as shown in FIG. 3, laminA / C was efficiently suppressed in all cells, and administration of siRNA in the presence of serum was particularly efficient.
- HeLa cells were administered with the two s iRNAs (Sp-18E6 and Ex_18E6) used in Example 1,
- HeLa and SW756 cells were administered 60 pmol e Sp_18E6 or Ex-18E6 siRNA, and the effect on p53 / Rb / E7 protein level after 72 hours was examined.
- siRNA administration and Western blotting were performed in the same manner as in Example 2.
- the antibodies used were anti-p53 antibody (Bp53-12), ⁇ / l HPV18-E7 (Santa Cruz Biotechnology Incorporation (Santa Cruz, CA), and anti-vimentin antibody (Oncogene, Boston, MA).
- both Sp-18E6 and Ex-18E6 increased the p53 protein, and the effect was stronger in Sp-18E6.
- the expression of E7 protein was moderately suppressed by Sp-18E6 and almost completely suppressed by EX-18E6.
- Coloni formation ability (scaffold-independent growth) was examined as follows. s Collect cells 24 hours after iRNA introduction, 10 ° /. FCS, 0.8ml 0.5 ° /. Agar noble (Difco) 10% FCS-containing DMEM medium 1.2 ml of 0.3% agar noble 10% FCS-containing DMEM medium is overlaid at 500 cells / ml (HeLa), 2000 cells / ml (SW756). It was. Lml growth medium was placed on the agar medium and changed every 3 days. After 10-20 days, colonies of 50 cells or more were counted. All experiments were performed in triplicate three times.
- annexin V-positive cells were identified by flow cytometry. Annexin V-positive cells were identified 72 hours after siRNA administration using the Annexin V-FITC apoptosis detection kit (Oncogene) according to the method described in the attached manual. As a result, as shown in FIGS. 7 and 8, it was confirmed that both Sp-18E6 and EX-18E6 induced apoptosis, and Sp-18E6 induced apoptosis more effectively than EX-18E6.
- RNAi is a phenomenon that should be called the self-defense system of the cell itself, and its effect is characterized by being tens of times more efficient than gene expression suppression by antisense DNA and extremely high gene sequence specificity. This means that the risk of side effects is low when RNAi is applied to gene therapy. Furthermore, since the gene expression can be suppressed only by administration of 21-23 base synthetic RNA double short chain, there is a great advantage that it is not necessary to consider the effect of the plasmid genome on the human body.
- the present invention can specifically and effectively suppress the expression of oncogenes by siRNA, and is highly expected as a new gene therapy strategy.
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US20100062051A1 (en) * | 2006-11-03 | 2010-03-11 | Young Kee Shin | Composition for treatment of cervix cancer |
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WO2003008573A2 (en) * | 2001-07-17 | 2003-01-30 | Anne Josephine Milner | Silencing of gene expression by sirna |
EP1439227A1 (en) * | 2003-01-15 | 2004-07-21 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | HPV-E6-specific siRNAs for the treatment of HPV-related tumors |
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WO2003008573A2 (en) * | 2001-07-17 | 2003-01-30 | Anne Josephine Milner | Silencing of gene expression by sirna |
EP1439227A1 (en) * | 2003-01-15 | 2004-07-21 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | HPV-E6-specific siRNAs for the treatment of HPV-related tumors |
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Cited By (2)
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US20100062051A1 (en) * | 2006-11-03 | 2010-03-11 | Young Kee Shin | Composition for treatment of cervix cancer |
US8377899B2 (en) * | 2006-11-03 | 2013-02-19 | Snu R&Db Foundation | Composition for treatment of cervix cancer |
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