WO2010061881A1 - C型肝炎ウイルスの働きを阻害するオリゴリボヌクレオチドまたはペプチド核酸 - Google Patents
C型肝炎ウイルスの働きを阻害するオリゴリボヌクレオチドまたはペプチド核酸 Download PDFInfo
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- 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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Definitions
- the present invention relates to an oligoribonucleotide or peptide nucleic acid that inhibits the action of hepatitis C virus, a vector that expresses the oligonucleotide, a therapeutic agent for hepatitis C containing these as active ingredients, and the oligoribonucleotide or peptide nucleic acid as C
- the present invention relates to a method for inhibiting viral replication by binding to hepatitis B virus RNA.
- HCV Hepatitis C virus
- Hepatitis caused by HCV infection is characterized by chronicity for a long time, and it is known that it causes chronic hepatitis and then has a very high rate of liver cirrhosis and further transition to liver cancer. Treatment is an important issue.
- Interferon (IFN) therapy is widely used for the treatment of chronic hepatitis C, but the effective rate is about 30%, side effects such as fever are frequently induced, and the drug price is high. Problems such as that exist.
- the type of IFN, usage, and dose have been studied, and the effective rate is expected to improve due to the development of consensus IFN.
- Treatment with a combination of IFN and antiviral agents such as ribavirin has also been attempted. However, none of them has reached a reliable treatment.
- RNA interference RNA interference
- dsRNA double-stranded RNA
- IRES Internal Ribosomal Entry Site
- HCV has multiple HCVs with different genotypes.
- examples of such HCV include HCJ6, HCJ8, HCV-1, HCV-BK, HCV-J, HCVSHIMO, JCH1, JCH3, JFH1, R24, R6, S14J, and the like.
- IRES region having high identity among a plurality of HCV sequences having different genotypes.
- the IRES region plays a function of translation initiation due to its higher-order structure, so it has a complex structure, and it is difficult to identify siRNA sequences that exhibit highly efficient RNAi activity with existing siRNA sequence identification algorithms. there were.
- Non-patent Document 13 siRNA sequences exhibiting highly efficient RNAi activity
- siRNA sequences exhibiting more effective RNAi activity against hepatitis C virus RNA The identification of was demanded.
- the present invention has been made in view of the above circumstances, and an object thereof is an oligoribonucleotide or peptide that inhibits the action of hepatitis C virus that exhibits more effective RNAi activity than conventionally identified oligoribonucleotides.
- Another object of the present invention is to provide a method for designing siRNA exhibiting more effective RNAi activity.
- the present inventors centered on siE sequences that have been shown to exhibit RNAi activity against HCV viral RNA so far, mainly D5-50 present in the IRES region. As a result of selecting the D5-197 region and proceeding with the analysis, the present inventors have succeeded in identifying an siRNA sequence exhibiting more effective RNAi activity against hepatitis C virus RNA, thereby completing the present invention. Furthermore, when the HCV proliferation inhibitory effect in an in vivo system was examined, it became clear that the said siRNA showed a significant HCV proliferation inhibitory effect also in an in vivo system.
- the present invention relates to the following [1] to [16].
- [6] A therapeutic agent for hepatitis C comprising the oligoribonucleotide or peptide nucleic acid according to any one of [1] to [4] or the vector according to [5] as an active ingredient.
- [7] A method of inhibiting the replication ability of HCV by binding the oligoribonucleotide or peptide nucleic acid according to any one of [1] to [4] to HCV RNA.
- a method for designing siRNA having an efficient RNAi activity against a target gene comprising the following steps; i) cleaving RNA corresponding to the target gene or a fragment thereof with dicer ii) identifying the cleavage site of the RNA iii) A step of selecting a sequence comprising the above-mentioned cleavage site and comprising 18 to 23 bases of the RNA. iv) A step of designing an siRNA having the base sequence selected in iii).
- the design method according to [8], wherein the target gene is a host cell gene.
- the design method according to [8], wherein the target gene is an animal cell gene.
- the design method according to [8], wherein the target gene is a viral gene.
- the design method according to [11], wherein the viral gene is an RNA viral gene.
- the design method according to [11] or [12], wherein the RNA corresponding to the target gene or a fragment thereof is an RNA having a higher-order structure and comprising a sequence that is conserved by 80 to 90% or more among strains .
- RNA sequence in which an RNA corresponding to the target gene or a fragment thereof has a higher order structure and is conserved by 80 to 90% or more among strains contains an internal ribosome entry site (IRES region)
- the virus is HCV, HIV, influenza virus, HBV, dengue virus, or measles virus, norovirus, SARS virus, rubella virus, poliovirus, RS virus, Marburg virus, Ebola virus, Crimea-Congo hemorrhagic fever virus,
- siRNA produced by this method shows the strongest inhibitory activity. It is the figure which showed the secondary structure of 5 'untranslated region (5'-UTR) of HCV. It can be seen that it has a complex high-order structure. It is a figure which shows the HCV proliferation inhibitory effect of siRNA in an in-vivo system
- A-1 to A-3 A group of mice administered with siSB-Cont # 3, 1 mg / kg 180 days after induction of poly IC.
- B-1 to B-3 shows a group of mice administered with si197- # 1,1 mg / kg 180 days after induction of poly IC.
- the oligo RNA that binds to HCV-RNA of the present invention in a sequence-specific manner is an oligonucleotide having ribose as a sugar, and as a base, in addition to adenine, guanine, cytosine, uracil existing in natural RNA, Those containing thymine and other modified bases are also included.
- the oligo RNA of the present invention is not particularly limited as long as it is an oligo RNA that can bind to HCV-RNA in a sequence-specific manner, but is preferably an oligo RNA that inhibits the replication ability of HCV.
- siRNA which is a preferred embodiment of the present invention, hybridizes to a target gene in a cell and cleaves the target gene via a dicer. The target gene is thought to be cleaved to a length of 19-23 bases.
- the antisense nucleic acid which is another aspect of the present invention is considered to degrade the target gene by hybridizing to the target gene, inducing IFN, and activating RNase.
- the binding causes a structural change of the target RNA to inhibit translation.
- the sequence of HCV-RNA may be either the sequence of HCV genomic RNA (-strand) or the sequence of mRNA (+ strand) transcribed from genomic RNA, preferably the sequence of + strand It is.
- siRNA refers to oligo RNA having a length of 19 to 23 bases (19 to 23 bp). When siRNA forms a double strand, one or both may have a protruding end.
- high identity means identity of 70% or more, preferably identity of 80% or more, more preferably identity of 90% or more (for example, identity of 95% or more).
- wordlength 3.
- Hybridization techniques are well known to those skilled in the art (eg, Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab.press, 1989, etc.)
- Gentle conditions can also be appropriately selected by those skilled in the art.
- stringent conditions include, for example, conditions of 42 ° C., 5 ⁇ SSC, 0.1% SDS in washing after hybridization, preferably 50 ° C., 5 ⁇ SSC, 0.1% SDS. More preferably, the conditions are 65 ° C., 0.1 ⁇ SSC and 0.1% SDS.
- a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. .
- the oligo RNA of the present invention may be single-stranded or double-stranded, and further may be formed from a plurality of two or more strands, but is preferably double-stranded. .
- the double strand may be formed of two independent strands, or may be a double strand formed in a self-complementary single-stranded RNA.
- a loop structure can be formed.
- the oligo RNA When the oligo RNA is double stranded, it may form double stranded in all regions, or some regions (such as both ends or one end) form other structures such as single stranded You may do it.
- the length of the oligo RNA of the present invention is not limited as long as it has a sequence-specific binding ability to HCV-RNA.
- the length of the oligo RNA of the present invention is, for example, 5 to 1000 bases (5 to 1000 bp in the case of a double strand), preferably 10 to 100 bases (10 to 10 in the case of a double strand). 100 bp), more preferably 15 to 25 bases (in the case of double strands, 15 to 25 bp), particularly preferably 19 to 23 bases (in the case of double strands, 19 to 23 bp) .
- oligo RNAs in the present invention are oligo RNAs having the nucleotide sequences shown in SEQ ID NOs: 1 to 20, and particularly preferred are those shown in SEQ ID NOs: 11, 12, 19 and 20 (si197- # 1, si197- # 6).
- An oligo RNA having a nucleotide sequence can be mentioned.
- Other preferred oligo RNAs in the present invention include oligo RNAs represented by nucleotide sequences consisting of 19 to 23 consecutive nucleotides in the nucleotide sequences shown in SEQ ID NOs: 24-29.
- RNA of the present invention can be expressed from an antisense coding DNA that encodes an antisense RNA to any region of the target gene mRNA and a sense code DNA that encodes a sense RNA of any region of the target gene mRNA. Moreover, dsRNA can also be produced from these antisense RNA and sense RNA.
- Examples of the combination of antisense RNA and sense RNA include a combination of oligoribonucleotides having the nucleotide sequence shown in SEQ ID NO: SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, sequence No: 15 and 16, SEQ ID NO: 17 and 18, and SEQ ID NO: 19 and 20.
- the part of the double-stranded RNA in which the RNAs in dsRNA are paired is not limited to a perfect pair, but mismatch (corresponding base is not complementary), bulge (no base corresponding to one strand) ) Or the like may include an unpaired portion.
- the terminal structure of the siRNA of the present invention may be either a blunt end or a sticky (protruding) end as long as the expression of the HCV virus gene can be suppressed by the RNAi effect.
- the sticky (protruding) terminal structure can include not only a structure in which the 3 ′ terminal side protrudes but also a structure in which the 5 ′ terminal side protrudes as long as the RNAi effect can be induced.
- the number of protruding bases is not limited to a few reported bases, and can be the number of bases that can induce the RNAi effect.
- the number of bases can be 1 to 8 bases, preferably 2 to 4 bases.
- the protruding sequence portion since the protruding sequence portion has low specificity with the transcript of the HCV virus gene, it is complementary (antisense) sequence to the target HCV virus gene transcript sequence or the same (sense) sequence. There is no necessity.
- an HCV RNA region having a sequence complementary to the oligo RNA having the nucleotide sequence shown in SEQ ID NOs: 1 to 20 above, or hybridizing with the oligo RNA under stringent conditions Complementary to an oligo RNA that hybridizes with an RNA region of soybean HCV under stringent conditions and an oligo RNA represented by a nucleotide sequence consisting of 19 to 23 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NOs: 24-29 above
- An HCV RNA region having a sequence, or an oligo RNA that hybridizes with the oligoribonucleotide under stringent conditions with an HCV RNA region An HCV RNA region having a sequence, or an oligo RNA that hybridizes with the oligoribonucleotide under stringent conditions with an HCV RNA region.
- RNA region of HCV that hybridizes with these oligo RNAs under stringent conditions in any HCV species.
- these oligo RNAs for example, in the nucleotide sequence shown in the above SEQ ID Nos. 1 to 20, or in the nucleotide sequence consisting of 19 to 23 bases in the nucleotide sequence shown in the above SEQ ID Nos. 24 to 29, 7 or less, preferably Examples thereof include those having a nucleotide sequence in which 5 or less, more preferably 3 or less nucleotides are deleted, substituted or added, and capable of inhibiting HCV replication by hybridizing with HCV RNA.
- peptide nucleic acids that can be suitably used in the present invention include peptide nucleic acids having a base sequence corresponding to oligo RNA that can be suitably used in the present invention.
- HCV RNA consists of an approximately 340 nucleotide 5 ′ untranslated region (5 ′ untranslated region), an open reading frame (ORF) consisting of approximately 9400 nucleotides, and a 3 ′ untranslated region consisting of approximately 50 nucleotides. (3 ′ untranslated region).
- ORF open reading frame
- 3 ′ untranslated region consisting of approximately 50 nucleotides.
- the site targeted by the oligo RNA of the present invention is not particularly limited and may be any site, but is preferably 5 ′ untranslated region to 5 ′ end region of ORF, 3 ′ untranslated region, Particularly preferred is a 5 ′ untranslated region.
- FIG. 7 shows a general secondary structure in the 5 'untranslated region of HCV-RNA.
- HCV HCV with different genotypes.
- examples of such HCV include HCJ6, HCJ8, HCV-1, HCV-BK, HCV-J, JCH1, JCH3, JFH1, R24, R6, S14J, pH77J6S (GenBank Accession no. AF177039), HCJ6CH, 2b_AB030907, etc. Is mentioned.
- a region having a high identity among a plurality of HCV gene sequences having different genotypes refers to a plurality of types of HCV RNA sequences having an identity of 80% or more, preferably 90% or more, Preferably, it is a region having 95% or more identity.
- Such a region preferably has a length of 10 bases or more, more preferably 15 bases or more, and particularly preferably 20 bases or more.
- the plurality of types of HCV generally means 3 or more types of HCV, preferably 5 types or more, particularly preferably 10 types or more.
- the identity of gene sequences can be calculated by comparing the sequences of a plurality of target genes and using the algorithm described above.
- the oligoribonucleotide used in the present invention is not limited to those having the structure of a normal RNA that is not modified, but can also be modified RNA having a modified phosphodiester moiety or sugar moiety, and the like. It is not something.
- the oligo RNA of the present invention may contain a molecule that is not a ribonucleotide, such as deoxyribonucleotide, in a part of the oligo RNA.
- PNA peptide nucleic acid
- oligo RNA RNA
- PNA peptide nucleic acid
- Those capable of binding to HCV-RNA in a sequence-specific manner can be produced.
- the length of the peptide nucleic acid suitable in the present invention is, for example, 5 to 1000 bases (5 to 1000 bp in the case of a double strand), preferably 10 to 100 bases (in the case of a double strand, 10 to 100 bp), more preferably 15 to 25 bases (15 to 25 bp in the case of double strands), particularly preferably 19 to 23 bases (19 to 23 bp in the case of double strands). It is.
- the oligo RNA or peptide nucleic acid of the present invention can be prepared by methods known to those skilled in the art.
- a vector that expresses the oligo RNA of the present invention may be prepared.
- Vectors can be prepared by methods known to those skilled in the art. For example, it can be prepared by introducing a gene encoding the oligo RNA of the present invention into a known vector such as those described in Nature Biotech (2002) 19, 497-500.
- Suitable promoters for expression of the oligo RNA of the present invention include, but are not limited to, T7 promoter, tRNA promoter, U6 promoter and the like.
- the siRNA of the present invention can be expressed in cells using a DNA encoding the antisense RNA strand (hereinafter referred to as antisense code DNA) and a DNA encoding the sense RNA strand (hereinafter referred to as sense code DNA).
- antisense code DNA a DNA encoding the antisense RNA strand
- sense code DNA a DNA encoding the sense RNA strand
- the antisense code DNA and the sense code DNA are abbreviated as the DNA of the present invention.
- the above-mentioned “antisense-coding DNA” and “sense-coding DNA” can be directly introduced into a chromosome in a cell together with a promoter to express antisense RNA and sense RNA in the cell to form siRNA. In order to introduce cells or the like, it is preferable to hold the siRNA expression system in a vector.
- the “vector” that can be used here can be selected according to the cell or the like to be introduced.
- retrovirus vectors for example, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, lentivirus vectors, herpes virus vectors, alphavirus vectors, EB virus vectors, papilloma virus vectors, foamy virus vectors, etc.
- Virus vectors, cationic liposomes, ligand DNA complexes, gene guns and other non-viral vectors Y. Niitsu et al., Molecular Medicine 35: 1385-1395 (1998)), but are not limited thereto. .
- dumbbell-shaped DNA Zanta (MA et al.,. Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc Natl Acad Sci U S A. 1999 Jan 5; 96 ( 1): 91-6), modified DNA with nuclease resistance, or naked plasmid can also be suitably used (Liu F, Huang L. Iproving plasmid DNA-mediated liver gene transfer by prolonging its retention in the hepatic vasculature. J. Gene Med. 2001 Nov-Dec; 3 (6): 569-76).
- an antisense RNA strand and a sense RNA strand can be expressed from the same vector as an antisense coding DNA and an antisense in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the sense coding DNA. It can be constructed by constructing a sense RNA expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction.
- an expression system in which the antisense code DNA and the sense code DNA are arranged in opposite directions so as to face each other on different strands.
- one double-stranded DNA in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA from each strand are provided on both sides thereof.
- a promoter is provided oppositely so that it can be expressed.
- a terminator is provided at the 3 ′ end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide.
- this terminator a sequence in which four or more A (adenine) bases are continued can be used.
- the types of the two promoters are different.
- RNA having a hairpin structure is inserted by inserting an appropriate sequence (preferably an intron sequence) between inverted repeats of the target sequence.
- '(RNA (hpRNA) constructs can also be used.
- antisense RNA and sense RNA from different vectors for example, antisense coding DNA and an antisense in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the sense coding DNA.
- An RNA expression cassette and a sense RNA expression cassette can be constructed, and these cassettes can be held in different vectors.
- the “DNA encoding siRNA (double-stranded RNA)” in the present invention is a combination of two DNAs encoding each strand, even one DNA encoding both strands of siRNA. Also good.
- a vector in which a DNA encoding siRNA (double-stranded RNA) is inserted is a single vector that expresses each strand of siRNA as two transcripts. One vector may be expressed as one transcript, or two vectors may be used to express each strand of siRNA.
- the DNA used for RNAi need not be completely identical to the target gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more (for example, 96,97, 98,99% or higher) sequence identity.
- the identity of the base sequence can be determined by the algorithm BLAST described above.
- the oligo RNA of the present invention is useful as a therapeutic agent for hepatitis C because it can inhibit HCV replication and suppress HCV proliferation.
- treatment can be performed without identifying the type of virus that the patient is infected in the clinical setting, and multiple types of oligoribonucleotides Or it is preferable because it is not necessary to mix and use peptide nucleic acids.
- oligo RNA or peptide nucleic acid When used for treatment, it can be administered in a form that can function as it is in the cell.
- the length of the oligo RNA or peptide nucleic acid is optimally about 19 to 23 bases.
- oligo RNA or peptide nucleic acid having a longer sequence including the target sequence can be administered.
- the double-stranded RNA (dsRNA) taken up into the cell is decomposed to about 21 mer by an enzyme called Dicer and becomes siRNA (short-interfering RNA), and a complex called RISC (RNA-induced Silence complex) is formed. And destroying RNA having a specific base sequence transcribed from the genome (Bernstein, E. et al., Nature, 409: 363-366, 2001; Hammond, SM, et al., Nature, 404: 293-296) 2000).
- siRNA can be prepared in advance in vitro using a commercially available dicer.
- RNA can be introduced directly into the cell.
- Various physical methods such as administration by microinjection, are generally used in such cases.
- Other methods for cell delivery include permeabilization and electroporation of cell membranes in the presence of siRNA, liposome-mediated transfection, or transfection using chemicals such as calcium phosphate.
- a number of established gene therapy techniques may be used to introduce siRNA into cells.
- the therapeutic agent for hepatitis C containing the oligo RNA or peptide nucleic acid of the present invention as an active ingredient is pharmaceutically acceptable excipient, isotonic agent, solubilizing agent, stabilizer, preservative, It can be prepared as a pharmaceutical composition such as tablets, powders, granules, capsules, liposome capsules, injections, solutions, nasal drops, etc. by adding a soothing agent, etc., and further can be a lyophilized agent. . These can be prepared according to conventional methods. It is also possible to administer a vector that expresses the oligo RNA of the present invention.
- the administration route of the oligo RNA or peptide nucleic acid of the present invention is not particularly limited, but it is preferably applied directly to the affected area of the patient or administered intravascularly so that the affected area can be reached as a result. Apply to patients. Furthermore, an encapsulating material that enhances durability and membrane permeability can also be used. For example, liposome, poly-L-lysine, lipid, cholesterol, lipofectin, or derivatives thereof can be mentioned.
- the dosage of the oligo RNA or peptide nucleic acid of the present invention can be appropriately adjusted according to the patient's condition, and a preferable amount can be used.
- it can be administered in the range of 0.001 to 100 mg / kg, preferably 0.1 to 10 mg / kg, but is not particularly limited.
- the present invention further provides a method for inhibiting the replication ability of HCV by binding the oligo RNA or peptide nucleic acid of the present invention to HCV RNA.
- the methods of the invention comprise contacting a sample that contains or may contain HCV both in vivo and in vitro with an oligo RNA or peptide nucleic acid of the invention.
- the presence or absence of inhibition of HCV replication ability can be detected by a method commonly used in the art.
- the present invention further discloses a method capable of efficiently selecting and designing an siRNA sequence having high RNAi activity against a target gene.
- various algorithms are known for siRNA design methods, and various siRNAs are designed based on the algorithms. For example, it is also known that a target RNA is cleaved with Dicer and the excised sequence is used as siRNA.
- the siRNA group found by these methods cannot be said to have significantly high RNAi activity, or the ratio of those having high activity is low.
- the present inventors specify the cleavage site by Dicer in the target RNA sequence, and select 19 to 23 nucleotide sequences of the target RNA containing the specific site, not based on the sequence cut out by Dicer.
- the present inventors have also found that siRNA having higher RNAi activity can be designed more efficiently than before by selecting the siRNA sequence based on the sequence.
- siRNA having higher activity than before can be designed by the design method of the present invention.
- the length of the target RNA when cleaved with Dicer is not particularly limited, but is preferably 20 to 400 bases in length.
- siRNA when targeting the IRES region of HCV, siRNA can be designed based on a sequence containing a cleavage site when a target RNA having a length of about 50 to about 200 bases is cleaved with Dicer. preferable.
- the cleavage site of the corresponding target gene is preferably present in the vicinity of the center of the designed siRNA sequence, specifically, 5 to 5 ′ around the cleavage site.
- 12 to 3 bases or 5 to 12 bases are present on the 3 ′ side, more preferably 8 to 12 bases on the 5 ′ side, or 8 to 12 bases on the 3 ′ side.
- siRNA for the target gene is designed by cleaving the mRNA or fragment thereof with Dicer and specifying the cleavage site. can do.
- the RNA sequence corresponding to the viral gene has a higher-order structure and is 80% or more, preferably 90% among strains.
- An siRNA for a target virus gene can be designed by cleaving an RNA fragment of a target gene containing a sequence that is conserved in% or more with Dicer and specifying the cleavage site.
- sequences having the above-mentioned higher-order structure and conserved between strains of 80% or more, preferably 90% or more include, for example, internal ribosome entry sites (IRES sequences) in RNA viruses such as HCV ), Highly conserved regions of HIV (the best conserved regions; Retrovirology, 2007, 4:80, Yuki Naito et al), highly conserved regions at the 5 'end of influenza viruses, and the like.
- IRS sequences internal ribosome entry sites in RNA viruses such as HCV
- Highly conserved regions of HIV the best conserved regions; Retrovirology, 2007, 4:80, Yuki Naito et al
- highly conserved regions at the 5 'end of influenza viruses and the like.
- the target virus in the design method of the present invention is not particularly limited as long as it has a higher-order structure and has a base sequence that is conserved by 80% or more among strains, but is preferably a DNA virus (Poxviridae) , Herpesviridae, Adenoviridae, Papovaviridae, Parvoviridae, or Pepadnaviridae) and RNA viruses (Arenaviridae, Orthomyxoviridae, Caliciviridae, Coronaviridae, Togaviridae, Nodaviridae) , Paramyxoviridae, Picornaviridae, Ferroviridae, Bunyaviridae, Rhabdoviridae, Reoviridae, Retroviridae).
- DNA virus Poxviridae
- Herpesviridae Herpesviridae, Adenoviridae, Papovaviridae, Parvoviridae, or Pepadnavirida
- target virus examples include HCV, HIV, influenza virus, HBV, dengue virus, measles virus, norovirus, SARS virus, rubella virus, poliovirus, RS virus, Marburg virus, Ebola virus, Crimea and Congo hemorrhagic fever.
- Virus, yellow fever virus, dengue virus, hepatitis G virus, rabies virus, human T lymphophilic virus, etc. preferably HCV, HIV, influenza virus, HBV, dengue virus, measles virus, most preferably Is HCV.
- all prior art documents cited in the present specification are incorporated herein by reference.
- Example 1 Analysis of HCV replicon RNA cleavage site of Diced-siRNA Diced-siRNA transfection ⁇ Transfection method using Lipofectamine 2000>
- HCV replicon-retaining cells div. Blank 3 were seeded in 6-well plates (BECTON DICKINSON, cat. # 353046) at 250,000 / 2 ml / well.
- DMEM + GlutaMAX-I invitrogen, cat. # 10569-044
- FCS invitrogen, cat.
- a 300 nM Diced-siRNA solution and a 3% Lipofectamine 2000 solution were mixed in equal amounts and left at room temperature for 20 minutes.
- the siRNA-Lipofectamine mixed solution was added to the 6-well plate at 500 ⁇ L / well to the div.
- the cells were cultured in a 37 ° C., 5% CO 2 incubator, and samples were collected 6 hours after the start of transfection.
- siRNA (siE-R5) (FIG. 3) was transfected into div. Blank 3 using Lipofectamine RNAiMAX Reagent (invitrogen, cat. # 13778-015) to a final concentration of 30 nM.
- the method (method according to the product manual) is shown below.
- Transfection siRNA (siE-R5) was diluted to 180 nM with 0.23% NaHCO 3 / OPTI-MEM I. The diluted siRNA was dispensed into a 6-well plate at 500 ⁇ L / well, and Lipofectamine RNAiMAX Reagent was added at 2.5 ⁇ L / well.
- Div.blan3 cells were seeded in a 6-well plate containing siRNA-Lipofectamine mixed solution at 380,000 cells / 2.5 ml / well. At this time, DMEM + GlutaMAX-I containing 10% deactivated FCS was used. The cells were cultured in a 37 ° C., 5% CO 2 incubator, and samples were collected 6 hours after the start of transfection.
- RNA extraction method Six hours after the start of transfection, the medium was aspirated and discarded, and 5 M GTC solution (5 M Guanidine thiocyanate (Fluka, cat.) Containing 1.4% 2-Mercaptoethanol (nacalai tesque, cat. # 21438-82). # 50980), 37.5 mM Sodium Citrate (pH 7.0) (WAKO, cat. # 191-01785), 0.75% Sarkosyl; N-Lauroylsarcosine Sodium Salt (nacalai tesque, cat. # 201-17) was added at 1300 ⁇ L / well And completely dissolved by pipetting.
- 5 M GTC solution 5 M Guanidine thiocyanate (Fluka, cat.) Containing 1.4% 2-Mercaptoethanol (nacalai tesque, cat. # 21438-82). # 50980), 37.5 mM Sodium Citrate (pH 7.0) (WAKO, cat. # 191-01785), 0.75%
- the mixture was centrifuged at 15000 rpm and 4 ° C. for 20 minutes, the upper layer was transferred to a new tube, and an equal volume of 2-propanol (Wako, 166-04836) was added and mixed.
- the mixture was treated at ⁇ 20 ° C. for 2 hours and centrifuged at 15000 rpm and 4 ° C. for 10 minutes to precipitate RNA.
- the RNA pellet was washed with 80% ethanol (Wako, cat. # 057-00451) cooled at ⁇ 20 ° C. and centrifuged at 15000 rpm at 4 ° C. for 5 minutes. This washing operation was repeated 4 times.
- RNA / DW 10 mM DTT (Fluka, cat. # 43815), 200 U / ml Ribonuclease Inhibitor (TaKaRa, cat. # 2310A)), and the RNA concentration was quantified.
- Identification method 1 Identification of HCV replicon RNA cleavage site by adapter method This was performed using a part of GeneRacer Kit (invitrogen, cat. # L1502-01) (in accordance with the protocol after “Addition of RNA Oligo” in the product manual) ).
- RNA Oligo to HCV RNA sample 1-5 ⁇ g of RNA sample was adjusted to a total volume of 7 ⁇ L, added to the freeze-dried GeneRacer RNA Oligo tube, and pipetted several times to mix with RNA Oligo. It was treated at 65 ° C. for 5 minutes and rapidly cooled on ice. 1 ⁇ L each of ligation reagents (10 ⁇ Ligase Buffer, 10 mM ATP, RNase OUT, T4 RNA Ligase) were added and treated at 37 ° C. for 1 hour. After adding 90 ⁇ L of Nuclease-Free Water (Ambion, cat.
- the mixture was mixed with an equal volume of phenol / chloroform solution and centrifuged, and the supernatant was transferred to a new tube.
- 2 ⁇ L of 10 mg / ml mussel glycogen and 10 ⁇ L of 3 M Na-Acetate (pH 5.2) were added and mixed, and 220 ⁇ L of EtOH was added and mixed.
- the mixture was cooled at ⁇ 80 ° C. for 15 minutes and centrifuged at 15000 rpm and 4 ° C. for 20 minutes. The pellet was washed with 70% EtOH and lightly air dried. Dissolved in 9 ⁇ L of Nuclease-Free Water.
- RNA Superscript III RT Module included with GeneRacer Kit was used. 1 ⁇ L of 100 ⁇ M Gene Specific Reverse Primer (R6-876-R20) (SEQ ID NO: 21, see Table 1) is added to RNA to which RNA Oliogo has been added, and 10 mM dNTP Mixture solution (GE Healthcare, cat. # 28- 1 ⁇ L of 4065-51) was added. It was treated at 70 ° C. for 3 minutes and rapidly cooled on ice. 2 ⁇ L of 10 ⁇ RT Buffer, 4 ⁇ L of 25 mM MgCl 2 , 2 ⁇ L of 0.1 M DTT, and 1 ⁇ L of RNase OUT were added and mixed by pipetting.
- R6-876-R20 Gene Specific Reverse Primer
- the mixture was treated at 25 ° C. for 2 minutes, 1 ⁇ L of SuperScript III RT was added and mixed. Thereafter, treatment was performed at 25 ° C. for 10 minutes, 50 ° C. for 30 minutes, 55 ° C. for 30 minutes, and 85 ° C. for 5 minutes, and left on ice. 1 ⁇ L of RNase H was added and treated at 37 ° C. for 20 minutes. 1st PCR was performed using 2 ⁇ L of this reaction solution as a template.
- Oligo-added HCV RNA 1st PCR Phusion DNA Polymerase (Finnzymes, cat. # F-530L) was used. 2 ⁇ L of template cDNA, 28.2 ⁇ L of ddH 2 O, 10 ⁇ L of 5 x Phusion HF Buffer, 1 ⁇ L of 10 mM dNTPs, 5 ⁇ L of 10 ⁇ M GeneRacer 5 'Primer, 10 ⁇ M R6 610-R24 reverse primer (PCR reaction was performed with 3.3 ⁇ L of SEQ ID NO: 22 (see Table 1) and 0.5 ⁇ L of Phusion DNA Polymerase in a total of 50 ⁇ L.
- the PCR program was 98 ° C for 2 minutes, followed by 20 cycles of 98 ° C for 10 seconds ⁇ 72 ° C for 30 seconds, treated at 72 ° C for 5 minutes, and then rapidly cooled to 4 ° C. Using this 1st PCR product as a template, 2nd PCR was performed.
- Oligo-added HCV RNA 2nd PCR AmpliTaq Gold with GeneAmp (Applied Biosystems, cat. # N888-0249) was used. 5 ⁇ L of 1st PCR DNA sample, 24.2 ⁇ L of ddH 2 O, 5 ⁇ L of 10 x PCR Buffer II, 6 ⁇ L of 25 mM MgCl 2 , 1 ⁇ L of 10 mM dNTPs, 5 ⁇ L of 10 ⁇ M GeneRacer 5 'Primer , 10 ⁇ M R6 536-R20 reverse primer (SEQ ID NO: 23, see Table 1) and 3.3 ⁇ L Taq Gold DNA Polymerase were added, and 3.3 ⁇ L Taq Gold DNA Polymerase were added, and 3.3 ⁇ L Taq Gold DNA Polymerase were added, and 3.3 ⁇ L Taq Gold DNA Polymerase were added, and 3.3 ⁇ L Taq Gold DNA Polymerase were added, and 3.3 ⁇ L Taq Gold DNA Polymerase were added, and 3.3
- Identification method 2 Identification of HCV replicon RNA cleavage site by c-tailing method Using part of 5 'Race System (invitrogen, cat. # 18374-058) (Used in accordance with product manual) .
- HCV RNA 0.5 ⁇ l Gene Specific Reverse Primer R6-876-R20; see SEQ ID NO: 21, see Table 1
- HCV RNA 1-5 ⁇ g of the sample was added, and the total volume was adjusted to 15.5 ⁇ L with Nuclease-Free Water (Ambion, cat. # 9932). It was treated at 70 ° C. for 10 minutes and rapidly cooled on ice.
- Excision and purification of amplified fragment A fragment of the PCR product amplified by 2nd PCR was excised and purified. From the results of 2nd PCR, siE-R5 and siD5-50 were confirmed to be between 200bp-300bp, and siD5-197 was confirmed to be between 150bp-400bp. Using (ATTO, Model; AE-6580), electrophoresis was performed at 50 V for 1 hour to elute the DNA. The eluted DNA sample was treated with TE saturated phenol and chloroform, followed by ethanol precipitation. The precipitated DNA sample was used for ligation.
- Cloning ⁇ Chemical transform method> (Performed according to the product manual) 4 ⁇ L of the sample after ligation reaction was added to the one-shot TOP10 competent cell (invitrogen, cat. # C4040-10) (50 ⁇ L / tube) dissolved in ice, mixed gently, and placed on ice for 30 minutes. . It was treated at 42 ° C. for 30 seconds and quenched with ice water. 250 ⁇ L of Hi-Competence Broth (Nippon Gene, cat. # 319-01343) was added and cultured with shaking at 37 ° C. for 1 hour. Thereafter, 150 ⁇ L of the culture solution was spread on an LB plate (A) that had been warmed at 37 ° C.
- ⁇ Miniprep 1 ml of the bacterial solution in 10 ml of shake cultured Rich LB medium was placed in a 1.5 ml tube and centrifuged at 15 krpm and 4 ° C. for 1 minute. Discard the supernatant and vortex, then add 300 ⁇ L of TENS solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 N NaOH, 0.5% SDS (Sodium Dodecyl Sulfate; Wako, 191-07145)) Mix gently.
- TENS solution 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 N NaOH, 0.5% SDS (Sodium Dodecyl Sulfate; Wako, 191-07145)
- the reaction conditions were 95 ° C. for 1 minute, 95 ° C. for 30 seconds ⁇ 58 ° C. for 20 seconds ⁇ 60 ° C. for 3 minutes, 30 cycles, and then rapidly cooled to 4 ° C.
- a Sephadex column was produced.
- the boundary between the HCV homologous sequence and the RNA Oligo sequence or the continuous sequence of C indicates the 5 'end of the cleaved HCV sequence, that is, it is considered to be the cleavage site of HCV RNA by Diced siRNA.
- FIG. 3 The number of clones having the same HCV RNA cleavage site was counted, and siRNAs were designed around the HCV RNA cleavage sites with a large number of clones.
- the design method was designed with a total of 21 bases consisting of 9 bases at the 5 ′ end and 12 bases at the 3 ′ end centered on the HCV RNA cleavage site as the sense strand of siRNA.
- the antisense strand of siRNA has a total of 21 bases, 11 bases on the 3 ′ end side and 10 bases on the 5 ′ end side, centering on the HCV RNA cleavage site.
- the sequence of the designed siRNA is shown in Table 2. The synthesis of siRNA was commissioned to Invitrogen custom siRNA synthesis and Dhamacon custom siRNA synthesis.
- Example 2 Examination of HCV replicon replication inhibitory activity of designed siRNA ⁇ Reverse transfection of siRNA>
- Each siRNA sample was diluted to 0.108 ⁇ M with opti-MEM containing 0.23% NaHCO 3 , and 10-fold dilution series was prepared by 3-fold dilution.
- Samples of each dilution series were placed in the cell culture multiplate 96FII (white) for luciferase assay (Sumitomo Bakelite, cat. # MS-8096W) and the cytotoxicity measurement 96-well plate (BD, cat. # 353072), respectively.
- n 3 was added to a 96-well plate at 10 ⁇ L / well.
- Lipofectamine RNAiMAX Reagent (invitrogen, cat. # 13778-150) diluted to 1% with OPTI-MEM I containing 0.23% NaHCO 3 and added to 96-well plate at 10 ⁇ L / well to wells where siRNA was dispensed After thorough mixing, the mixture was incubated at room temperature for 20 minutes to prepare a siRNA-Lipofectamene complex. A well containing only Lipofectamine RNAiMAX was used as a control. In this Example, siRNA assay was performed using R6 FLR41-14 cells, FLR3-1 cells, and JFH luc 3-13 cells as HCV replicon cells containing a luciferase gene.
- each cell and suspend in DMEM + GlutaMax-I containing 10% inactivated FCS For R6 FLR41-14 and FLR3-1 cells, use 5200cells / 100 ⁇ L / well. In the case of -13 cells, the cells were seeded in a 96-well plate at 6000 cells / 100 ⁇ L / well. The final siRNA concentrations were 0.5, 1.4, 4.1, 12.3, 37.0, 111.1, 333.3, 1000, 3000, and 9000 pM. The cells were incubated at 37 ° C. and 5% CO 2 , and 72 hours later, luciferase assay and cytotoxicity test using WST-8 were performed.
- Bright-Glo Luciferase Assay System (Promega, cat. # E2620) was used for luciferase activity measurement. The medium in all wells of the 96-well plate for luciferase assay was discarded, and the medium was changed by adding DMEM + GlutaMax-I containing 5% inactivated FCS at 75 ⁇ L / well. Bright-Glo Luciferase Assay System was added at 75 ⁇ L / well and shaken for 1 minute with Mithras LB 940 (Berthold), and then luciferase activity was measured.
- Cell counting kit-8 (Dojindo, cat. # 343-07623) was used for cytotoxicity measurement.
- Cell Counting Kit-8 solution was diluted to 7% with DMEM + GlutaMax-I containing 5% inactivated FCS to prepare an assay solution.
- the medium in all wells of a 96-well plate for cytotoxicity test was discarded, and the above-mentioned assay solution was added at 100 ⁇ L / well and incubated at 37 ° C. and 5% CO 2 for 1 hour.
- the wavelength 450 nm reference wavelength 655 nm
- si197- # 1 and si197- # 6 showed the strongest HCV replicon replication inhibitory activity, and were found to have minimal cytotoxicity (FIGS. 4a, b, and c).
- Example 3 Measurement of HCV protein synthesis inhibitory activity of si50 and si197 by Western blot analysis ⁇ Reverse transfection of siRNA>
- siRNA sample was diluted to 18 nM with opti-MEM containing 0.23% NaHCO 3 and added to a 6-well plate at 500 ⁇ L / well.
- Lipofectamine RNAiMAX was added to the wells into which siRNA was dispensed at 2.5 ⁇ L / well in a 6-well plate and mixed well, and incubated at room temperature for 20 minutes.
- the plate was washed 3 times with 0.1% Tween / TBS solution for 10 minutes, and detected with ECL Western Blotting Detection Reagents (GE Healthcare, cat. # RPN2106).
- ECL Western Blotting Detection Reagents GE Healthcare, cat. # RPN2106
- the primary antibody was Monoclonal Anti- ⁇ -Actin antibody produced in mouse (SIGMA, cat. # A5441) at a 5000-fold dilution
- the secondary antibody was ECL Anti-Mouse IgG, HRP-Linked Whole Detection was performed using Ab (GE Healthcare, cat. # NA931).
- SIGMA Monoclonal Anti- ⁇ -Actin antibody produced in mouse
- ECL Anti-Mouse IgG HRP-Linked Whole Detection was performed using Ab (GE Healthcare, cat. # NA931).
- Example 4 Examination of HCV growth inhibitory effect of siRNA in in vivo system ⁇ Creation of HCV expressing mice> MxCre / CN2-29 mice that express hepatitis C virus (Satoshi Sekiguchi, Yoshimi Tobita, Ohara Michi method: Mechanism of persistent infection and pathogenicity of hepatitis C virus Medical Science Digest. 35 (6): 14-17 2009) Poly IC (GE, USA) was administered at 300 ⁇ g / animal three times every 48 hours and then reared for 180 days.
- Invivofectamin (Invitrogen) was used for liposome introduction of siRNA. That is, 100 ⁇ L of Invivofectamin was added to 100 ⁇ g of siRNA and mixed gently. The mixture of Invivofectamin and siRNA was shaken at room temperature for 30 minutes (using an orbital shaker). 14 times the amount of 5% butter sugar (1400 ⁇ L) was added and mixed. Centrifugation was carried out with Amicon Ultra-15 (Millipore Cat. UFC900308) at 5,000 ⁇ G at 4 ° C. at room temperature. The concentrated portion was collected and made up to 2 mL with butter sugar.
- SiRNA was mixed with 0.2 mL of liposome administration solution per mouse and administered once intravenously on the first day. After collecting the liver on the second day, the HCV core quantification kit (Ortho Clinical Diagnostics Co., Ltd.) was used. The amount of HCV in the liver was quantified.
- oligoribonucleotides or peptide nucleic acids that bind to sequences more efficiently and inhibit the action of HCV than those conventionally identified for HCV-RNA, and C containing these as active ingredients
- a therapeutic agent for hepatitis B has been provided, and it has become possible to provide a novel and reliable treatment method for HCV.
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Abstract
Description
さらに、in vivo系におけるHCV増殖抑制効果の検討したところ、当該siRNAはin vivo系においても有意なHCV増殖抑制効果を示すことが明らかとなった。
〔1〕配列番号:1~20のいずれかに示すヌクレオチド配列を有するオリゴリボヌクレオチド。
〔2〕〔1〕に記載のオリゴリボヌクレオチドと相補的な配列を有するHCVのRNA領域、あるいは該オリゴリボヌクレオチドとストリンジェントな条件下でハイブリダイズするHCVのRNA領域とストリンジェントな条件下でハイブリダイズするオリゴリボヌクレオチド。
〔3〕配列番号:24~29のいずれかに示すヌクレオチド配列において連続する19~23塩基からなるヌクレオチド配列で示されるオリゴリボヌクレオチド。
〔4〕〔3〕に記載のオリゴリボヌクレオチドと相補的な配列を有するHCVのRNA領域、あるいは該オリゴリボヌクレオチドとストリンジェントな条件下でハイブリダイズするHCVのRNA領域とストリンジェントな条件下でハイブリダイズするオリゴリボヌクレオチド。
〔5〕〔1〕~〔4〕のいずれかに記載のオリゴリボヌクレオチドを発現するベクター。
〔6〕〔1〕~〔4〕のいずれかに記載のオリゴリボヌクレオチド若しくはペプチド核酸、または〔5〕に記載のベクターを有効成分とするC型肝炎治療剤。
〔7〕〔1〕~〔4〕のいずれかに記載のオリゴリボヌクレオチドまたはペプチド核酸をHCVのRNAに結合させて、HCVの複製能を阻害する方法。
〔8〕標的遺伝子に対する効率的なRNAi活性を有するsiRNAの設計法であって、以下の工程を含む設計方法;
i)標的遺伝子又はその断片に対応するRNAをdicerで切断する工程
ii)当該RNAの上記切断部位を特定する工程
iii)当該RNAの、上記切断部位を含み且つ連続する18~23塩基からなる配列を選択する工程
iv)iii)で選択した塩基配列を有するsiRNAを設計する工程。
〔9〕前記標的遺伝子が宿主細胞遺伝子ある〔8〕記載の設計方法。
〔10〕前記標的遺伝子が動物細胞遺伝子である〔8〕記載の設計方法。
〔11〕前記標的遺伝子がウイルス遺伝子である〔8〕記載の設計方法。
〔12〕前記ウイルス遺伝子がRNAウイルス遺伝子である〔11〕記載の設計方法。
〔13〕前記標的遺伝子又はその断片に対応するRNAが、高次構造を有し且つ株間で80~90%以上保存されている配列を含むRNAである〔11〕又は〔12〕記載の設計方法。
〔14〕前記標的遺伝子又はその断片に対応するRNAが、高次構造を有し且つ株間で80~90%以上保存されている配列が、インターナル・リボソーム・エントリー・サイト(IRES領域)を含む配列である〔13〕記載の設計方法。
〔15〕前記RNAの配列が20~400塩基である〔8〕の設計方法。
〔16〕前記ウイルスが、HCV、HIV、インフルエンザウイルス、HBV、デングウイルス、又は麻疹ウイルス、ノロウイルス、SARSウイルス、風疹ウイルス、ポリオウイルス、RSウイルス、マーブルグウイルス、エボラウイルス、クリミア・コンゴ出血熱ウイルス、黄熱病ウイルス、デング熱ウイルス、G型肝炎ウイルス、狂犬病ウイルス、又はヒトTリンパ好性ウイルスである〔11〕又は〔12〕記載の設計方法。
本発明のオリゴRNAまたはペプチド核酸は当業者に公知の方法で作製することが可能である。
本発明の設計方法において、Dicerで切断する際の標的RNAの長さは特に制限されないが、好ましくは20~400塩基の長さである。
本発明の方法でsiRNAを設計する場合、対応する標的遺伝子の切断部位が設計したsiRNAの配列の中心付近に存在することが好ましく、具体的には、切断部位を中心に5'側に5~12塩基又は3'側に5~12塩基存在することが好ましく、5'側に8~12塩基又は3'側に8~12塩基存在することがさらに好ましい。
なお、本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。
〔実施例1〕 Diced-siRNAのHCVレプリコンRNA切断部位の解析
Diced-siRNAのトランスフェクション
<Lipofectamine2000を用いたトランスフェクション法>
実験開始前日に、HCVレプリコン保持細胞div. bla n3を6ウェルプレート(BECTON DICKINSON, cat.#353046)に250000個/2ml/ウェルで撒いた。この時5%非働化済みFCS(invitrogen, cat.#26140-079)を含むDMEM+GlutaMAX-I(invitrogen, cat.#10569-044)を使用した。
実験開始当日、終濃度が30 nMになるようにDiced siRNA(D5-50, D5-197)(図3)をLipofectamine 2000 Reagent(invitrogen, cat.#11668-019)を用いてdiv. bla n3にトランスフェクションした。以下に、その方法(製品のマニュアルに従った方法)を示す。
トランスフェクション用のDiced-siRNAは、0.23% のNaHCO3を含むOPTI-MEM I(invitrogen, cat.#22600-050)で300 nMに希釈した。一方、Lipofectamine2000 は、0.23% NaHCO3/OPTI-MEM Iで3%に希釈し、ゆるやかに混合し、5分間室温で放置した。300 nM Diced-siRNA溶液と、3% Lipofectamine 2000溶液を等量で混合し、20分間室温で放置した。前日に調整したdiv. bla n3 に、siRNA-Lipofectamine 混合溶液を500 μL/wellで6ウェルプレートに加え、なじませた。37℃、5% CO2インキュベーターにて培養し、トランスフェクション開始後6時間後にサンプルを回収した。
実験開始当日、終濃度が30 nMになるようにsiRNA (siE-R5)(図3)をLipofectamine RNAiMAX Reagent(invitrogen, cat.#13778-015)を用いてdiv. bla n3にトランスフェクションした。以下に、その方法(製品のマニュアルに従った方法)を示す。
トランスフェクション用のsiRNA (siE-R5)は、0.23% NaHCO3/OPTI-MEM Iで180 nMに希釈した。希釈したsiRNAを6ウェルプレートに500 μL/wellで分注し、Lipofectamine RNAiMAX Reagentを2.5 μL/wellで加えた。緩やかに混合し、20分間室温で放置した。div. bla n3細胞をsiRNA-Lipofectamine 混合溶液が入った6ウェルプレートに380000個/2.5ml/wellで撒いた。このとき10%非働化済みFCSを含むDMEM+GlutaMAX-Iを使用した。37℃、5% CO2インキュベーターにて培養し、トランスフェクション開始後6時間後にサンプルを回収した。
トランスフェクション開始後6時間後に、培地を吸引廃棄し、1.4%の2-Mercaptoethanol(nacalai tesque, cat.#21438-82)を含む5 M GTC溶液(5 M Guanidine thiocyanate (Fluka, cat.#50980), 37.5 mM Sodium Citrate(pH 7.0)(WAKO, cat.#191-01785), 0.75% Sarkosyl ; N-Lauroylsarcosine Sodium Salt (nacalai tesque, cat.#201-17)を1300 μL/wellで加え、ピペッティングで完全に溶解した。
400 μLの5M GTC細胞溶解液に対し、27 μLの3M Sodium Acetate(pH 5.2)(Wako, cat.#198-01055)を加えて混合し、さらに400 μLのTE飽和フェノール(Wako, cat.#160-12725)を加えて1分間緩やかに混和した。その後氷上にて15分静置した。次にクロロホルム/イソアミルアルコール(49:1)(Chloroform, Wako, cat.#038-02606, Isoamyl Alcohol, Wako, cat.#017-03676)溶液を90 μL加え、1分間緩やかに混和し、その後氷上にて15分間静置した。15000 rpm、4℃にて、20分間遠心し、上層を新しいチューブに移し、等量の2-プロパノール(Wako, 166-04836)を加え、混和した。-20℃で2時間処理し、15000 rpm、4℃にて、10分間遠心し、RNAを沈殿させた。RNAのペレットを-20℃で冷やした80% エタノール(Wako, cat.#057-00451)で洗浄し、 15000 rpm、4℃にて、5分間遠心した。この洗浄作業を4回繰り返した。ペレットを数秒風乾させ、11 μLのRNA/DW(10 mM DTT(Fluka, cat.#43815), 200U/ml Ribonuclease Inhibitor (TaKaRa, cat.#2310A))で溶解し、RNA濃度を定量した。
GeneRacer Kit (invitrogen, cat.#L1502-01)の一部を使用して行った(製品マニュアルの「RNA Oligoの付加」以降のプロトコールに従って行った)。
1~5 μgのRNAサンプルを総量 7 μLに調整し、凍結乾燥されたGeneRacer RNA Oligoのtubeへ加え、数回ピペッティングしてRNA Oligoと混ぜた。65℃で5分間処理し、氷上にて急冷した。ライゲーション用の試薬(10 x Ligase Buffer, 10 mM ATP, RNase OUT, T4 RNA Ligase)を各1 μLずつ加え、37℃で1時間処理した。Nuclease-Free Water(Ambion, cat.#9932)を90 μL加えた後、等量のフェノール/クロロホルム溶液で混和後遠心し、上清を新しいチューブへ移した。2 μLの10mg/ml mussel glycogenと、10 μLの3 M Na-Acetate (pH5.2)を加えて混合し、220 μLのEtOHを加えて混合した。-80℃で15分間冷やし、15000 rpm、4℃にて、20分間遠心した。ペレットを70% EtOHで洗浄し、軽く風乾させた。9 μLのNuclease-Free Waterに溶解した。
GeneRacer Kit付属のSuperScript III RT Moduleを使用して行った。
RNA Oliogoを付加したRNAに100 μMのGene Specific Reverse Primer(R6-876-R20)(配列番号:21、表1参照)を1 μL加え、10 mM dNTP Mixture solution(GE Healthcare, cat.# 28-4065-51)を1 μL加えた。70℃で3分間処理し、氷上にて急冷した。2 μLの10 x RT Buffer、4 μLの25 mM MgCl2、2 μLの0.1M DTT、1 μLのRNase OUTを加え、ピペッティングで混合した。25℃で2分間処理し、SuperScript III RTを1 μL加え混合した。その後25℃ 10分間、50℃ 30分間、55℃ 30分間、85℃ 5分間処理し、氷上に静置した。RNase H を1 μL加え、37℃で20分間処理した。本反応液のうち2 μLをテンプレートにして1st PCRを行った。
Phusion DNA Polymerase(Finnzymes, cat.#F-530L)を使用した。
テンプレートcDNA 2 μLに対して、ddH2Oを28.2 μL, 5 x Phusion HF Bufferを10 μL, 10 mM dNTPsを1 μL, 10 μM GeneRacer 5’ Primerを5 μL, 10 μM R6 610-R24 reverse primer (配列番号:22、表1参照)を3.3 μL, Phusion DNA Polymeraseを0.5 μL加え、計50 μLでPCR反応を行った。PCRのプログラムは98℃ 2分の後、98℃ 10秒→72℃ 30秒のサイクルを20サイクル繰り返し、72℃ 5分処理後、4℃に急冷した。この1st PCR産物をテンプレートにして、2nd PCRを行った。
AmpliTaq Gold with GeneAmp(Applied Biosystems, cat.#N888-0249)を使用した。
1st PCR DNAサンプル5 μLに対して、ddH2Oを24.2 μL, 10 x PCR Buffer IIを5 μL, 25 mM MgCl2を6 μL, 10 mM dNTPsを1 μL, 10 μM GeneRacer 5’ Primerを5 μL, 10 μM R6 536-R20 reverse primer (配列番号:23、表1参照)を3.3 μL, Taq Gold DNA Polymeraseを0.5 μL加え、計50 μLでPCR反応を行った。95℃ 5分の後、95℃ 30秒、55℃ 30秒、72℃ 1分のサイクルを20サイクル繰り返し、72℃ 7分処理後、4℃に急冷した。3 μLを電気泳動で確認し、残りすべてをエタノール沈殿し、増幅したDNA断片の切り出しを行った。
5’ Race System (invitrogen, cat.#18374-058)の一部を使用して行った(製品のマニュアルに従った方法で行った)。
0.5 mlシリコナイズドチューブ(アシスト、cat.#72.699Z)に2.5 μMのGene Specific Reverse Primer (R6-876-R20;配列番号:21、表1参照)を1 μLと、HCV RNAサンプルを1~5 μg加え、Nuclease-Free Water(Ambion, cat.#9932)で総量15.5 μLにした。70℃で10分間処理し、氷上にて急冷した。2.5 μLの10 x PCR Buffer、2.5 μLの25 mM MgCl2、1 μLの10 mM dNTP Mixture solution(GE Healthcare, cat.# 28-4065-51)、2.5 μLの0.1 M DTTを加え、ピペッティングで混合した。42℃で1分間処理し、SuperScript II RTを1 μL加え混合した。42℃で50分間反応させた。70℃で15分間処理し、反応を停止させ、37℃に置いた。RNase mixを1 μL加え、37℃で30分間処理した。
室温にしたBinding solution (6 M sodium iodide)120 μLをcDNAに加え、S.N.A.P.カラムに全量移し、遠心(13000 x g, 20秒)した。冷やしたWash Bufferを加え、同様に遠心し、4回洗浄を行った。次に冷やした70% エタノールを加え、同様に2回洗浄した。最後のエタノールを除いてから1分間遠心した。65℃に暖めた50 μLのNuclease-Free Waterをカラムに加え、遠心(13000 x g, 20秒)し、cDNAを溶出した。
溶出したcDNA, 10 μLに対して、Nuclease-Free Waterを6.5 μL, 5x tailing Bufferを5 μL, 2mM dCTP (GE Healthcare, cat.# 28-4065-51)を2.5 μLを加え、穏やかに混合した。94℃で2分間処理し、氷中で急冷した。1 μL のTdTを加え、穏やかに混合し、37℃で10分間処理した。10分後すぐに65℃で10分間処理し、TdTを不活化した。軽く遠心して氷上に置いた。
TaKaRa Ex Taq(TaKaRa, cat.#RR001A)を使用した。
テンプレートcDNA 5 μLに対して、Nuclease-Free Waterを34.5 μL, 10 x Ex Taq Bufferを5 μL, 10 mM dNTPsを1 μL, 10 μM 5’ RACE Abridged Anchor Primer(AAP)を2 μL, 10 μM R6 610-R24 reverse primer(配列番号:22、表1参照)を2 μL, TaKaRa Ex Taq Polymeraseを0.5 μL加え、計50 μLでPCR反応を行った。94℃ 2分の後、94℃ 30秒→55℃ 30秒→72℃ 1分間のサイクルを35サイクル繰り返し、72℃ 5分処理後、4℃に急冷した。この1st PCR産物をテンプレートにして、2nd PCRを行った。
1st PCR産物 1 μLに対して、ddH2Oを40.5 μL, 10 x Ex Taq Bufferを5 μL, 10 mM dNTPsを1 μL, 10 μM Abridged Universal Amplification Primer(AUAP)を1 μL, 10μM R6 536-R20 reverse primer(配列番号:23、表1参照)を1 μL, TaKaRa Ex Taq Polymeraseを0.5 μL加え、計50 μLで2nd PCR反応を行った。反応後、3 μLを電気泳動で確認し、残りすべてをエタノール沈殿し、増幅したDNA断片の切り出しを行った。
2nd PCRで増幅したPCR産物の断片を切り出し、精製を行った。
2nd PCRの結果から、siE-R5, siD5-50は200bp-300bpの間に、siD5-197は150bp-400bpの間にバンドが確認されたので、その領域のバンドをゲルから切り出し、マックスイールドNP (ATTO, Model ; AE-6580)を用いて、50V、1時間泳動し、DNAを溶出した。溶出したDNAサンプルをTE飽和フェノール処理とクロロホルム処理を行い、エタノール沈殿を行った。沈殿したDNAサンプルをライゲーションに用いた。
pGEM-T Easy Vector System I(Promega, cat.#A1360)使用した。
沈殿したDNAサンプルのペレットを7 μLの2mM Tris (Trizma base ; SIGMA, cat.#T1503-1KG) /0.4mM EDTA (2NA ; Dojindo, cat.#345-01865)(T2E0.4)に溶解し、1 μLを5% ポリアクリルアミドゲルで泳動して、同時に泳動したDNA分子量マーカー(100 bp DNA Ladder ; New England Biolabs, cat.#N3231L)からおおよそのDNA濃度を決定した。pGET-T Easyのクローニングベクター50 ngに対し、4.1 ng相当量のDNAサンプルを混合し、T4 DNA Ligaseを加えて4℃にて一晩放置し、ライゲーション反応させた。
<ケミカルトランスフォーム法>(製品のマニュアルに従った方法で行った)
ライゲーション反応後のサンプル4 μLを、氷中で溶解したワンショットTOP10コンピテントセル(invitrogen, cat.# C4040-10)(50 μL/tube)に加え、緩やかに混ぜ、氷上にて30分間置いた。42℃で30秒処理し、氷水で急冷した。250 μLのHi-Competence Broth(ニッポンジーン, cat.# 319-01343)を加え、37℃で1時間振とう培養した。その後、37℃で暖めておいたLBプレート(A)に150 μLの培養液を撒いた。37℃でインキュベートし、10時間後にコロニーを確認した。250~300のコロニーが確認された。70個のコロニーを各々10 mlのRich LB培地(B)に植菌し、37℃で13時間振とう培養した。
LBプレート(A);1% Bacto-tryptone(BD, cat.#211705)、0.5% Bacto-yeast extract(BD, cat.#212750)、1% NaCl(Wako, cat.#191-01665)、0.002N NaOH(Wako, cat.#197-02125)、1.5% Bacto Agar(BD, cat.#214010)
Rich LB(B);2.5% Bacto-tryptone(BD, cat.#211705)、0.75% Bacto-yeast extract(BD, cat.#212750)、0.6% NaCl(Wako, cat.#191-01665)、0.005N NaOH(Wako, cat.#197-02125)
ライゲーション反応後のサンプルに、グリコーゲンを10 μg加えてエタノール沈殿した。一晩-20℃で処理し、翌日遠心してDNAを沈殿させた。ペレットを70% エタノールでリンスし、風乾後6 μLの2mM Tris/0.4mM EDTA(T2E0.4)に溶解した。2 μLのライゲーションサンプルを50 μLのJM109とともにキュベットに入れ、2.5 kV, 200 OHMS, 25 μFDの条件でエレクトロポレーションした。450 μLのHi-Competence Brothで回収し、1時間、37℃で振とう培養した。その後、37℃で暖めておいたLBプレートに数μLから100 μLの培養液を撒いた。37℃でインキュベートし、10時間後にコロニーを確認した。70個のコロニーを各々10 mlのRich LB 培地に植菌し、37℃で13時間振とう培養した。
振とう培養したRich LB培地10 ml中1 mlの菌液を1.5 mlチューブにとり、15 krpm, 4℃、1分間遠心した。上清を捨て、ボルテックス後、300 μLのTENS溶液(10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 N NaOH, 0.5% SDS (Sodium Dodecyl Sulfate ; Wako, 191-07145))を加え、緩やかに混ぜた。次に150 μLの3 M NaOAc (Sodium Acetate Trihydrate ; Wako, 198-01055)を加え、緩やかに混ぜ、15 krpm, 4℃、10分間遠心した。遠心後の上清を1 mlの99.5%エタノールに加え、よく混合し、15 krpm, 4℃、10分間遠心した。上清を捨て、沈殿したDNAのペレットを、70%エタノールで2回リンスした。風乾し、0.1 μg/μL RNase A (BOEHRINGER MANNHEIM #1119915) を含むT2E0.4を15 μL加えて、ペレットを溶解した。2 μLをEcoRI(TaKaRa, cat.#1040A)で制限酵素処理し、電気泳動してインサートのチェックを行った。
インサートが認められたサンプルのみ、90 μLのTEを加え、TE飽和フェノール/クロロホルム処理とクロロホルム処理を行い、エタノール沈殿を行った(-80 ℃、15分間)。15krmp, 4℃、10分間遠心し、ペレットを70%エタノールでリンスした。ペレットを風乾し、20 μL のT2E0.4で溶解後、12 μLの20% PEG/2.5M NaCl(Polyethylene Glycol #6000 ; nacalai tesque, cat.#28254-85)を加えて、十分に混合した。氷上に1時間ほど置き、15krmp, 4℃、5分間遠心した。ペレットを70%エタノールで2回リンスし、風乾後、60 μLのT2E0.4に溶解した。DNAを定量し、100-200 ng/μLに希釈した。
BigDye Terminator v3.1 Cycle Seqencing Kit(Applied Biosystems, cat.#4337456) と、3130xl Genetic Analyzerを用いた。
100-200 ng/μLのプラスミドDNAサンプル1 μLに対し、1 μM R6 536-R20 reverse primer を1 μL、5 x BigDye Terminator v1.1/v3.1 Sequencing Buffer(Applied Biosystems, cat.#4336697)を1.5 μL、BigDye Terminator v3.1 Cycle Seqencing RR100(Applied Biosystems, cat.#4337456)を1 μL、ddH2Oを5.5 μL加えて混合し、PCR Systemを用いて反応させた。反応条件は、95℃ 1分の後、95℃ 30秒→58℃ 20秒→60℃ 3分間のサイクルを30サイクル繰り返した後、4℃に急冷した。
MultiScreen-HV(Millipore, cat.#MAHVN4550)のウェルにSephadex G-50 Superfine (GE Healthcare, cat.#17-0041-01)を詰め、ddH2Oを300 μL/wellで加えて3時間ほど膨潤し、セファデックスカラムを作製した。プレート用の遠心機で余分な水分を除き、シークエンス用のサンプルを10 μL/wellでカラムに加え、2500rpm, 室温, 5分間遠心し、シークエンス用の96プレートに回収した。プレート用エバポレーターで45℃、20分間処理し、水分を蒸発させた。各ウェルにHi-Di Formamide(Applied Biosystems, cat.#4311320)を15 μL/wellで加え、95℃、5分間処理し、氷水で急冷した。
3130xl Genetic Analyzerにシークエンスサンプルのプレートをセットし、シークエンス解析を行った。
解析された配列情報の、HCV相同配列とRNA Oligo配列またはCの連続配列の境界が、切断されたHCV配列の5’末端を示しており、すなわち、Diced siRNAによるHCV RNAの切断部位と考えられた(図3)。同じHCV RNA切断部位をもつクローン数を数え、クローン数が多いHCV RNA切断部位を中心にsiRNAをデザインした。デザイン方法は、HCV RNA切断部位を中心に5’末端側に9塩基、3’末端側に12塩基の計21塩基をsiRNAのセンス鎖としてデザインした。siRNAのアンチセンス鎖は、HCV RNA切断部位を中心に3’末端側に11塩基、5’末端側に10塩基の計21塩基となる。デザインしたsiRNAの配列を表2に示す。siRNAの合成はインビトロジェンのカスタムsiRNA合成および、DhamaconのカスタムsiRNA合成に依頼した。
<siRNAのリバーストランスフェクション>
各siRNAサンプルを0.23%のNaHCO3を含むopti-MEMで、0.108 μMになるように希釈し、3倍希釈で10段階の希釈系列を作製した。各希釈系列のサンプルを、ルシフェラーゼアッセイ用の細胞培養用マルチプレート96FII(白)(住友ベークライト, cat.#MS-8096W)と細胞毒性測定用の96ウェルプレート(BD, cat.#353072)にそれぞれn=3で10 μL/wellで96ウェルプレートに加えた。Lipofectamine RNAiMAX Reagent (invitrogen, cat.#13778-150)を、0.23%のNaHCO3を含むOPTI-MEM Iで1%に希釈し、siRNAを分注したウェルに10 μL/wellで96ウェルプレートに加えて十分に混和後、室温で20分間インキュベートして、siRNA-Lipofectamene複合体を作製した。Lipofectamine RNAiMAXのみを加えたウェルをコントロールとした。本実施例では、ルシフェラーゼ遺伝子を含むHCVレプリコン細胞として、R6 FLR41-14細胞と、FLR3-1細胞、JFH luc 3-13細胞を用いてsiRNAアッセイを行った。各々の細胞の細胞数を数え、10% 非働化済FCSを含むDMEM+GlutaMax-Iにけん濁し、R6 FLR41-14細胞とFLR3-1細胞の場合は5200cells/100 μL/wellで、JFH luc 3-13細胞の場合は6000cells/100 μL/wellで96ウェルプレートに撒いた。siRNAの終濃度は、0.5、1.4、4.1、12.3、37.0、111.1、333.3、1000、3000、9000 pMとなった。37℃、5% CO2にてインキュベートし、72時間後にルシフェラーゼアッセイおよびWST-8による細胞毒性試験を行った。
ルシフェラーゼ活性測定には、Bright-Glo Luciferase Assay System (Promega, cat.#E2620)を使用した。
ルシフェラーゼアッセイ用96ウェルプレートの全ウェルの培地を廃棄し、5% 非働化済FCSを含むDMEM+GlutaMax-Iを75 μL/wellで加え、培地交換した。Bright-Glo Luciferase Assay Systemを75 μL/wellで加えて、Mithras LB 940(Berthold)で1分間振とうした後、ルシフェラーゼ活性を測定した。
細胞毒性測定には、Cell Counting Kit-8(Dojindo, cat.#343-07623)を使用した。
Cell Counting Kit-8溶液を、5%非働化済FCSを含むDMEM+GlutaMax-Iで7%に希釈し、アッセイ用の溶液を作製した。細胞毒性試験用の96ウェルプレートの全ウェルの培地を廃棄し、上述のアッセイ用の溶液を100 μL/wellで加え、37℃、5% CO2にて1時間インキュベートした。マイクロプレートリーダー(Bio-Rad model 550)を用いて、波長450 nm(参照波長655 nm)を測定した。
その結果、si197-#1, si197-#6が最も強力なHCVレプリコン複製阻害活性を示し、細胞毒性については軽微であることが明らかになった(図4a,b,c)。
<siRNAのリバーストランスフェクション>
各siRNAサンプルを0.23%のNaHCO3を含むopti-MEMで、18 nMになるように希釈し、500 μL/wellで6ウェルプレートに加えた。Lipofectamine RNAiMAXを、siRNAを分注したウェルに2.5 μL/wellで6ウェルプレートに加えて十分に混和し、室温で20分間インキュベートした。R6 FLR41-14細胞の細胞数を数え、10% 非働化済FCSを含むDMEM+GlutaMax-Iにけん濁し、155000 cells/2.5 ml/wellで6ウェルプレートに撒いた(siRNAの終濃度は3 nM)。37℃、5% CO2にてインキュベートし、72時間後に培地を捨て、PBS(-)で1回洗浄し、PBS(-)を吸引除去した後、60 μL/well 6ウェルプレートのRIPA(1% SDS, 0.5% Nonidet P-40 (nacalai tesque, cat.#23640-94), 150 mM NaCl, 10 mM Tris-HCl(pH 7.5), 1x Complete (Roche, cat.#11697 498001))でサンプリングした。
上述のサンプルをソニケーターで処理した後、RC DC Protein Assay Reagents(Bio-Rad, cat.#500-0120)を用いて蛋白濃度を定量した。得られたタンパク質10 μgを10% アクリルアミドゲルでトリス-グリシンSDS緩衝液を用いて電気泳動した。分子量サイズマーカーはプレシジョンPlusブルースタンダード(Bio-Rad, cat.#161-0373)とBroad range Marker(Bio-Rad, cat.#161-0317)を用いた。電気泳動したタンパク質をTrans-BLOT SD SEMI-DRY TRANSFER CELL(Bio-Rad)を用いて1 mA/cm2で80分間Immobilon-P (Millipore, cat.#IPVH00010)に転写し、5%スキムミルク(雪印)で1時間ブロッキングした。その後、一次抗体にNS3抗体(1 μg/ml, R212 Rabbit抗体)またはNS5A抗体(Rabbit抗体)を用いて4℃で一晩反応させ、翌日0.1% Tween/TBS溶液で5分間、3回洗浄し、二次抗体に5%スキムミルクで2000倍に希釈したECL Anti-Rabbit IgG, HRP-Linked F(ab’)2 Fragment(GE Healthcare, cat.#NA9340)を用いて室温で一時間反応させた。その後0.1% Tween/TBS溶液で10分間、3回洗浄し、ECL Western Blotting Detection Reagents(GE Healthcare, cat.#RPN2106)で検出した。β-アクチンの検出は、一次抗体はMonoclonal Anti-β-Actin antibody produced in mouse(SIGMA, cat.#A5441)を5000倍希釈で使用し、二次抗体はECL Anti-Mouse IgG, HRP-Linked Whole Ab(GE Healthcare, cat.#NA931)を用いて検出した。その結果、合成したsiRNA si50とsiRNA si197によるHCVタンパク質合成阻害が確認された。
<HCV発現マウスの作成>
C型肝炎ウイルス発現マウスであるMxCre/CN2-29マウス(関口敏、飛田良美、小原道法:C型肝炎ウイルスの持続感染機構と病原性 Medical Science Digest. 35(6):14-17 2009)にポリIC(GE社、アメリカ)を300μg / 匹で48時間おきに3回投与後、180日間飼育した。
siRNAのリポソーム導入はInvivofectamin(Invitrogen社)を用いた。すなわち、100μgのsiRNAに100μLのInvivofectaminを加え、緩やかに混和した。Invivofectamin とsiRNA混液を室温で30分間振とうした(オービタル振とう機使用)。14倍量の5%ブトウ糖(1400μL)を加え混和した。アミコンウルトラ-15(ミリポア Cat. UFC900308)にて5,000×G、4℃、室温で遠心した。濃縮部分を回収し、ブトウ糖で2 mLとした。マウスあたり0.2 mLのリポソーム投与液にsiRNAを混ぜ、1日目で1回静脈投与を行い、2日目で肝臓を回収後、HCVコア定量キット(オーソ・クリニカル・ダイアグノスティックス株式会社)にて肝臓中のHCV量を定量した。
コントロールsiRNAを投与した群(A-1からA-3)はHCVコア量が平均211pg/mgであったが、si197-#1投与群(B-1からB-3)はHCVコアが検出されなかった(図8)。図8には各個体のHCVコア量の測定結果を示した。以上のことから、siSB-197は極めて短時間でHCV発現マウスに対して抗ウイルス作用を示すことが明らかとなった。
Claims (16)
- 配列番号:1~20のいずれかに示すヌクレオチド配列を有するオリゴリボヌクレオチド。
- 請求項1に記載のオリゴリボヌクレオチドと相補的な配列を有するHCVのRNA領域、あるいは該オリゴリボヌクレオチドとストリンジェントな条件下でハイブリダイズするHCVのRNA領域とストリンジェントな条件下でハイブリダイズするオリゴリボヌクレオチド。
- 配列番号:24~29のいずれかに示すヌクレオチド配列において連続する19~23塩基からなるヌクレオチド配列で示されるオリゴリボヌクレオチド。
- 請求項3に記載のオリゴリボヌクレオチドと相補的な配列を有するHCVのRNA領域、あるいは該オリゴリボヌクレオチドとストリンジェントな条件下でハイブリダイズするHCVのRNA領域とストリンジェントな条件下でハイブリダイズするオリゴリボヌクレオチド。
- 請求項1~4のいずれかに記載のオリゴリボヌクレオチドを発現するベクター。
- 請求項1~4のいずれかに記載のオリゴリボヌクレオチド若しくはペプチド核酸、または請求項5に記載のベクターを有効成分とするC型肝炎治療剤。
- 請求項1~4のいずれかに記載のオリゴリボヌクレオチドまたはペプチド核酸をHCVのRNAに結合させて、HCVの複製能を阻害する方法。
- 標的遺伝子に対する効率的なRNAi活性を有するsiRNAの設計法であって、以下の工程を含む設計方法;
i)標的遺伝子又はその断片に対応するRNAをdicerで切断する工程
ii)当該RNAの上記切断部位を特定する工程
iii)当該RNAの、上記切断部位を含み且つ連続する18~23塩基からなる配列を選択する工程
iv)iii)で選択した塩基配列を有するsiRNAを設計する工程。 - 前記標的遺伝子が宿主細胞遺伝子ある請求項8記載の設計方法。
- 前記標的遺伝子が動物細胞遺伝子である請求項8記載の設計方法。
- 前記標的遺伝子がウイルス遺伝子である請求項8記載の設計方法。
- 前記ウイルス遺伝子がRNAウイルス遺伝子である請求項11記載の設計方法。
- 前記標的遺伝子又はその断片に対応するRNAが、高次構造を有し且つ株間で80~90%以上保存されている配列を含むRNAである請求項11又は12記載の設計方法。
- 前記標的遺伝子又はその断片に対応するRNAが、高次構造を有し且つ株間で80~90%以上保存されている配列が、インターナル・リボソーム・エントリー・サイト(IRES領域)を含む配列である請求項13記載の設計方法。
- 前記RNAの配列が20~400塩基である請求項8の設計方法。
- 前記ウイルスが、HCV、HIV、インフルエンザウイルス、HBV、デングウイルス、又は麻疹ウイルス、ノロウイルス、SARSウイルス、風疹ウイルス、ポリオウイルス、RSウイルス、マーブルグウイルス、エボラウイルス、クリミア・コンゴ出血熱ウイルス、黄熱病ウイルス、デング熱ウイルス、G型肝炎ウイルス、狂犬病ウイルス、又はヒトTリンパ好性ウイルスである請求項11又は12記載の設計方法。
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EP09829126A EP2368989A4 (en) | 2008-11-26 | 2009-11-26 | OLOGORIBONUCLEOTIDE OR PEPTIDE NUCLEIC ACID CAPABLE OF INHIBITING HEPATITIS C VIRUS ACTIVITY |
JP2010540503A JPWO2010061881A1 (ja) | 2008-11-26 | 2009-11-26 | C型肝炎ウイルスの働きを阻害するオリゴリボヌクレオチドまたはペプチド核酸 |
US13/130,673 US8957199B2 (en) | 2008-11-26 | 2009-11-26 | Oligoribonucleotide or peptide nucleic acid capable of inhibiting activity of hepatitis C virus |
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US8957199B2 (en) | 2015-02-17 |
EP2610343A2 (en) | 2013-07-03 |
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