WO2002006297A1 - Novel antisense oligonucleotide derivatives against wilms's tumor gene - Google Patents

Novel antisense oligonucleotide derivatives against wilms's tumor gene Download PDF

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WO2002006297A1
WO2002006297A1 PCT/JP2001/006049 JP0106049W WO0206297A1 WO 2002006297 A1 WO2002006297 A1 WO 2002006297A1 JP 0106049 W JP0106049 W JP 0106049W WO 0206297 A1 WO0206297 A1 WO 0206297A1
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oligonucleotide derivative
antisense
oligonucleotide
antisense oligonucleotide
present
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PCT/JP2001/006049
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French (fr)
Japanese (ja)
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Takeshi Imanishi
Haruo Sugiyama
Yoshiyuki Ohsugi
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Chugai Seiyaku Kabushiki Kaisha
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Priority to AU2001271042A priority Critical patent/AU2001271042A1/en
Publication of WO2002006297A1 publication Critical patent/WO2002006297A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA

Definitions

  • the present invention relates to a novel antisense to the Wilms oncogene (WT 1). More specifically, the present invention relates to a WT 1 comprising an oligonucleotide derivative containing at least one nucleotide analog unit having a sugar moiety modified. A new antisense to the Wilms oncogene (WT 1).
  • Antisense oligonucleotides are one of the most promising fields in recent years, as they specifically regulate the expression of unwanted genes.
  • the antisense method is based on the concept of controlling the flow of so-called central dharma using antisense oligonucleotides, namely DNA-RNA-protein.
  • nucleic acid derivatives and analogs have been synthesized and studied. For example, phosphorothioate in which an oxygen atom on a phosphorus atom is substituted with an iodo atom, methylphosphonate in which a methyl group is substituted, or more recently one in which a phosphorus atom is also substituted with a carbon atom, and even a sugar moiety Structures that have converted the structure of nucleosides or modified nucleic acid bases have also been synthesized.
  • Japanese Patent Application Laid-Open No. 10-304889 discloses a nucleotide analog unit in which the sugar moiety of an oligonucleotide constituting antisense is modified.
  • This nucleotide analog unit has a structure represented by the following formula 1 in which a sugar moiety conformation is immobilized to an N-type.
  • an oligonucleotide derivative incorporating one or more nucleotide analog units having a sugar moiety having the above structure is synthesized, and the basic physical properties of the oligonucleotide derivative are measured extracellularly. That is, the duplex forming ability is studied by measuring the melting temperature of the oligonucleotide derivative and the annealing treatment of the sense strand composed of natural DNA or RNA. The resistance of the oligonucleotide derivative to the nuclease enzyme was measured in vitro.
  • this publication describes whether the oligonucleotide derivative exhibits nuclease resistance and acts stably as an antisense even in cells, as in the case of the extracellular experimental results. However, it does not disclose whether or not it can form a double-stranded or triple-stranded form by binding to a naturally-occurring gene, particularly in a cell, to actually inhibit the expression of a specific gene.
  • Wilms tumor is a pediatric kidney tumor caused by inactivation of both Wilms tumor gene (WT1) alleles located on chromosome 11p13 (Cal KM et al., Cell 60: 509, 1990).
  • WT1 Wilms tumor gene
  • Non-coding upstream sequence of WT1 CE Camphell et al., Oncogene 9: 583-595, 1994
  • DA Haber et al., Proc. Natl. Acad. Sc. USA, 88: 9618-9622 (1991) have already been reported and are expected to be involved in the growth and differentiation of tumors and the like (DA Haber et al., Supra).
  • WT1 is strongly expressed in some tumor cells such as human leukocyte-derived K562 cells and human gastric cancer-derived Az521 cells, and when its expression is suppressed, cells become apoptotic. It has been reported to cause cis (Kentaro Senba, Cell Engineering, 14, 5 4 6- 5 5 3 (1995).
  • Japanese Patent Application Laid-Open No. 9-104629 discloses an antisense oligonucleotide derivative against WT1.
  • This oligonucleotide derivative includes not only a natural oligonucleotide that has not been modified but also a modified oligonucleotide derivative.
  • the modified oligonucleotide derivative is obtained by modifying the phosphodiester portion of a nucleotide, for example, a modified lower alkylphosphonate such as a methylphosphonate type or an ethylphosphonate type, a modified phospholipid thioate or a modified phosphothioate. Modified oral amidates are mentioned.
  • an oligonucleotide derivative containing a nucleotide analog unit having a modified sugar moiety is not disclosed.
  • the antisense oligonucleotide derivative disclosed in this publication has various properties required for antisense, that is, whether or not it has nuclease resistance in cells as well as in the results of extracellular experiments. In addition, it does not mention whether it can bind to a naturally-occurring gene, particularly in vivo, to form a double-stranded or triple-stranded chain, and actually inhibit the expression of a specific gene.
  • the inventors of the present invention designed a nucleic acid analog having a sugar moiety conformation immobilized on a nucleic acid, which is considered to be useful in the antisense method, and synthesized a nucleotide analog unit having a unit structure thereof. It was confirmed that the oligonucleotide derivative prepared using this was extremely useful as a WT1 antisense molecule. The details of the present invention will be described below.
  • FIG. 1 is a diagram showing a nucleotide sequence of an oligonucleotide used as a sample in Experimental Example 1 and a band pattern obtained by subjecting a sample treated with nuclease (snake venom phosphodiesterase) to polyacrylamide gel electrophoresis.
  • Figure 2 shows the nucleotide sequence of the oligonucleotide used as a sample in Experimental Example 1 and the band pattern obtained by subjecting a sample treated with nuclease (fetal bovine serum: FBS) to polyacrylamide gel electrophoresis. It is.
  • nuclease fetal bovine serum
  • FIG. 3 is a graph showing the antisense effect of the natural oligonucleotide.
  • FIG. 4 is a graph showing the concentration dependence of the antisense effect of the oligonucleotide derivative of the present invention.
  • FIG. 5 is a graph showing the antisense effect of comparing the oligonucleotide derivative of the present invention with a phosphorothioate-type oligonucleotide derivative.
  • FIG. 6 is a graph showing the concentration-dependent antisense effect of the oligonucleotide derivative of the present invention and the scrambled sequence oligonucleotide.
  • FIG. 7 is a graph showing the antisense effect of the oligonucleotide derivative of the present invention with two different scrambled sequence oligonucleotides.
  • the oligonucleotide or polynucleotide derivative of the present invention has the following general formula
  • B is a pyrimidine nucleobase or a purine nucleobase or an analog thereof
  • B has one or more nucleotide analog units having a structure represented by the following formula:
  • the oligonucleotide or polynucleotide derivative of the present invention has the general formula
  • BB 2 is the same or different, is a pyrimidine nucleobase or purine nucleobase or an analog thereof, R is hydrogen, a hydroxyl group, a halogen, or an alkoxy group, and WW 2 is the same or different, hydrogen, Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aralkyl group, aryl group, acyl group, silyl group or natural nucleotide via phosphate residue or phosphate diester bond, synthetic nucleotide or these nucleotides O Rigo nucleotide if Ku is a polynucleotide, n 1 or n 2 are the same or different, 0-5 is a integer of 0 (where, n 1 or n 2 do not become zero simultaneously.
  • N 3 is an integer from 1 to 50, provided that n 1 and Z or n 2 are B 1 and B in the case of 2 or more may not be the same, R is also an oligonucleotide or polynucleotide derived material represented by may not be the same.
  • the number of bases in the antisense oligonucleotide derivative of the present invention is not particularly limited, but is usually 5 to 50, preferably 9 to 30.
  • the pyrimidine nucleobase or purine nucleobase or an analog thereof is thymine, peracyl, cytosine, adenine, guanine and an analog thereof.
  • These analogs include purine nucleic acid salt analogs and pyrimidine nucleobase analogs.
  • the purine nucleobase analog can be preferably selected from the following compounds.
  • Guanosine diphosphate 8-oxo-adenosine, 8-oxo-guanosine, 8-fluoro-adenosine, 8-fluoro-guanosine, 8-methoxy- Adenosine, 8-methoxy-guanosine, 8-azaadenosine, 8-azaguanosine, azacytin, fludarabine phosphate, 6-MP, 6-TG, azasammlungne, aloprinol, acyclovir, ganciclovia, deoxyphormycin, arabinosilagenin ( ara-A), guanosine diphosphate tofucoose, guanosine diphosphate- 12-fluorofurose, guanosine diphosphate toe j3 L-2-aminofucose, guanosine diphosphate-D-arabinose and 2-aminoadesine And so on.
  • the pyridine nucleobase analog can be preferably selected from the following compounds. That is,
  • 5-fluoroperacil 5-chlorouracil, 5-bromouracil, dihydrouracil, 5-methylcytosine, 5-propierthymine, 5-propynylperacyl, 5-propiercytosine, 5-fluorocytosine, floxyperidine, peridine, thymine , 3'-azidodeoxythymidine, 2-fluorodeoxycytidine, 3_fluoro--3, -deoxythymidine, 3, -dideoxycytidine- 2, -ene, 3, -doxy-3, dedeo Xitymidine-2, -ene, and cytosine arabinose.
  • nucleotide analog unit which can be used as an antisense in the present invention
  • oligonucleotide derivative containing the nucleotide analog unit are described in detail in the above-mentioned Japanese Patent Application Laid-Open No. 10-304889. I have. This description shall be included in this document.
  • the antisense oligonucleotide derivative to WT1 of the present invention may be any oligo or polynucleotide that can bind to WT1 and inhibit or suppress the expression of its gene, It suffices that one or more of the nucleotides is replaced with the nucleotide analog unit of the present invention.
  • antisense oligonucleotide derivatives include those for the transcriptional capping site of WT1, those for the translation initiation region, those for exons, those for introns, and the like. More specifically, the following bases Contains an array.
  • Antisense sequence of transcription capping site AGGGTCGAAT GCGGTGGG (SEQ ID NO: 1)
  • Antisense sequence of transcription capping region (AS 2) TCAAATAAGA GGGGCCGG (SEQ ID NO: 2)
  • Antisense sequence of translation initiation region (AS 3) GTCGGAGCCC ATTTGCTG
  • the vicinity of the translation initiation region of WT1 is a region where an antisense effect is observed, and is expected as a target for cancer treatment by antisense.
  • the present inventors have synthesized the oligonucleotide derivative of the present invention having an antisense sequence in this region, and administered it to Az521 cells that strongly express WT1, using the decrease in cell number due to apoptosis as an index. The antisense effect was examined and an excellent effect was confirmed.
  • the antisense of the present invention can be used as an antitumor drug by introducing into tumor cells expressing WT1, thereby inhibiting or suppressing the expression to induce the cells to undergo apoptosis.
  • oligonucleotide derivative of the present invention are represented by the following formula:
  • 5′-GUCGGAGCCCAUUUGCUG-3 ′ (SEQ ID NO: 5) A sequence in which one or two or more bases of the antisense sequence to the translation initiation region of the WT1 translation initiation region are replaced with the nucleotide analog unit of the present invention and a sequence thereof.
  • Antisense oligonucleotide derivatives containing the same, and oligonucleotide derivatives that hybridize under stringent conditions to DNA or RNA, which is a sequence complementary to the above antisense sequence, can be mentioned. Hybridization is a well-known technique (Sambrook, l et al., Molecular Cloning 2nd ed., Cold spring Harbor Lab. Press, 1989, etc.).
  • Stringent end conditions in the present invention may be appropriately selected by those skilled in the art. Is possible, for example, under low stringent conditions.
  • the low stringent conditions are, for example, in washing after hybridization, at 42 ° C, 0.1 XSSC, 0.1% SDS, preferably 50 ° (: 0.1 XS SC, 0.1% SDS More preferred stringent conditions include high stringency conditions High stringency end conditions are 65 ° C, 6 XSSC, 0.1% SDS.
  • the oligonucleotide derivative of the present invention exhibits superior nuclease resistance as compared to a natural oligo nucleotide having the same base sequence. Therefore, it is stable even when administered intracellularly, and has the advantage of maintaining its excellent antisense effect.
  • the antisense oligonucleotide derivative of the present invention requires the use of a nucleotide analog unit in which only the sugar moiety is modified, but further modifies the other moiety, for example, the phosphodiester moiety as in the case of phosphorothioate. Nucleotide analog units may be used. Further, an oligonucleotide derivative antisense can also be prepared by combining the nucleotide analog unit having a modified sugar moiety of the present invention with a nucleotide analog unit which is another known antisense structural unit. The antisense oligonucleotide derivative of the present invention can be used as an external preparation such as a liniment or a poultice by mixing with a suitable base material which is inactive against the derivative.
  • excipients can be prepared according to a conventional method.
  • the antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is adapted to the patient so that it can reach the affected area as a result of intravenous administration or the like.
  • an antisense-encapsulated material that enhances durability and membrane permeability can be used.
  • ribosome, poly-L-lysine, lipid, cholesterol, lipofectin, or an analog thereof can be mentioned.
  • the dose of the antisense oligonucleotide derivative of the present invention depends on the patient's condition, A suitable amount can be used after appropriately adjusting according to age, sex, body weight, and the like.
  • various administration methods such as oral administration, intramuscular administration, intraperitoneal administration, intradermal administration, subcutaneous administration, intravenous administration, intravenous administration, rectal administration, etc.
  • a suitable method can be used as appropriate from the administration method.
  • Example 1 Nuclease resistance of oligonucleotide derivatives containing nucleotide analog units of the present invention
  • Oligonucleotide derivatives containing five nucleotide analog units of the present invention were prepared and used as samples for the nuclease resistance test. On the other hand, a natural oligonucleotide having the same sequence was prepared and used as a control.
  • the nuclease of the oligonucleotide derivative of the present invention is obtained by using a snake venom phosphodiesterase as a nuclease, treating the oligonucleotide labeled with radioisotope with 32 P with nuclease, and separating the oligonucleotide by polyacrylamide gel electrophoresis. Monozyme resistance was evaluated.
  • Figure 1 shows the sequence of the oligonucleotide prepared as a sample and the results of electrophoresis after nuclease treatment.
  • the ratio of the undegraded oligonucleotide was less than 30% for the natural oligonucleotide.
  • the undegraded oligonucleotide completely disappeared.
  • the oligonucleotide derivative of the present invention about 90% of undegraded oligonucleotide was confirmed even 8 hours after the start of the nuclease treatment. From these results, it was found that the oligonucleotide derivative of the present invention has at least 100 times or more the nuclease resistance performance as compared with the natural oligonucleotide.
  • the oligonucleotide derivatives of the present invention showed extremely strong resistance to degradation by nuclease.
  • the following oligonucleotide derivative to WT1 synthesized by the method described in the example of JP-A-10-304889 was used as an antisense strand, and was subjected to annealing treatment with a sense strand consisting of natural DNA or RNA.
  • Tm value melting temperature
  • a sample solution (5001) with final concentrations of 100 mM NaC, 10 mM sodium phosphate buffer (pH 7.2), 4 M for antisense strand and 4 M for sense strand was bathed in boiling water. And slowly cooled to room temperature over 10 hours.
  • a nitrogen stream was passed through the cell chamber of the spectrophotometer (Bec kman DU650) to prevent dew condensation, the sample solution was gradually cooled to 5 ° C, and kept at 5 ° C for another 20 minutes before measuring. Started.
  • the sample temperature was increased by 0.2 ° C per minute to 90 ° C, and ultraviolet absorption at 260 nm was measured at 0.1 ° C intervals.
  • Tm value Melting temperature (ATm / mod.) /.
  • Antisense molecules 1, 2, 3, and 4 correspond to SEQ ID NOs: 3, 6, 7, and 1, respectively.
  • the oligomer (Test 2 (referred to as 5bc_AS) and Test 3) in which five nucleotide analog units (underlined) of the present invention were inserted into the natural DNA chain was found to be complementary to the complementary DNA oligomer.
  • Hypride forming ability was increased 7-8 degrees over the native chain (Test 1) as assessed by Tm values.
  • the Tm value of the oligomer of the present invention was found to be 5 to 9 degrees higher than that of the natural chain (D-origo).
  • D-origo no analogs whose Tm value is so markedly higher than that of the natural chain have been known.
  • the oligonucleotide derivative antisense of the present invention showed an excellent double strand-forming ability to both DNA and RNA.
  • Example 3 Antisense effect of oligonucleotide derivative on WT1
  • the antisense effect of oligonucleotide derivative of the present invention on WT1 was confirmed using cultured cells.
  • oligonucleotide derivative having an antisense sequence near the translation initiation region of WT1 was prepared in the same manner as in Example 2.
  • the oligonucleotide derivative of the present invention having the antisense sequence was administered to Az521 cells, and the antisense effect was evaluated using the decrease in the number of cells as an index.
  • experiments were performed under the same conditions for natural (D-origo) and phosphorothioate-type oligonucleotides (S-oligo).
  • oligonucleotide solution in the absence of serum (1.25 / 1) was added to 21 (final concentration 25 fig / m 1), and cultured for 2 hours. Then, 10 il of FBS (final concentration 10%) was added, followed by further culturing. Oligonucleotide solution 11 (final concentration + 12.5 g / ml) was added on the second, third, and fourth days, respectively, and the cells were further cultured for 2 days. (In experiments where the oligonucleotide concentration was varied, an appropriate volume of the oligonucleotide solution was used.) On day 6, the number of living cells was counted using a microscope.
  • the antisense effect of the oligonucleotide derivative of the present invention was examined using a system in which the number of cells was not reduced by D-oligo in (1) above. At this time, the oligonucleotide derivative was also administered to PC 14 cells that did not express WT1, and changes in the number of cells were compared. Fig. 4 shows the results. In the case of Az521 cells, the number of cells was not reduced when the D-oligo sequence was administered, but the number of cells was decreased when the oligonucleotide derivative of the present invention was administered. .
  • the oligonucleotide derivative of the present invention exhibited an antisense effect, but the S-oligo and D-oligo did not show an antisense effect. From these results, it was confirmed that the oligonucleotide derivative of the present invention had a higher antisense effect than S-oligo widely known as antisense. This result shows a good correlation with the value obtained in the in vitro experiment (Tm value).
  • the oligonucleotide derivative antisense of the present invention was administered to Az521 cells in the same manner as in Example 3, and the antisense effect was evaluated using the decrease in the number of cells as an index. For comparison, an experiment was performed under the same conditions using an oligonucleotide derivative having a scrambled sequence represented by the following formula.
  • FIG. 5 shows the growth inhibitory effect of Az521 cells when the antisense oligonucleotide derivative (5bc-AS) of the present invention was administered at different concentrations. At the same time, the Sc-B sequence was tested under the same conditions.
  • the antisense oligonucleotide derivative of the present invention exhibited a remarkable cell growth inhibitory effect as compared with the two scrambled sequence oligonucleotides.
  • An antisense oligonucleotide derivative having the following base sequence in which peracyl (U) of the above antisense oligonucleotide derivatives (first and second) is substituted with thymine (T) is expected to have the same antisense effect as the above antisense.

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Abstract

Antisenses against WT1 which comprise oligonucleotide derivatives containing 1 or more sugar-modified nucleotide analog units represented by the following general formula (2) wherein B represents a pyrimidine nucleic acid base, a purine nucleic acid base or an analog thereof. These antisenses, which are antisenses against Wilms's tumor (WT1) gene, are expected as antisense oligonucleotide derivatives capable of binding to WT1 at a high affinity in cells to thereby efficiently regulate or inhibit the expression of this gene and being highly resistant to nucleases.

Description

明 細 書 ウィルムス腫瘍遺伝子に対する新規なアンチセンスオリゴヌクレオチド誘導体 [技術分野]  Description New antisense oligonucleotide derivative against Wilms tumor gene [Technical field]
本発明は、 ウィルムス腫瘍遺伝子 （WT 1 ) に対する新規なアンチセンスに関 し、 更に詳細には、 本発明は糖部を修飾したヌクレオチド類縁体ユニットを 1つ 以上含有するオリゴヌクレオチド誘導体からなる WT 1に対する新規なアンチセ ンスに関する。  The present invention relates to a novel antisense to the Wilms oncogene (WT 1). More specifically, the present invention relates to a WT 1 comprising an oligonucleotide derivative containing at least one nucleotide analog unit having a sugar moiety modified. A new antisense to
[背景技術]  [Background technology]
1 9 7 8年にアンチセンス分子がインフルエンザウイルスの感染を阻害したと の報告が初めて成された。 以後、 ガン遺伝子発現や A I D S感染を阻害したとの 報告もなされている。 アンチセンスオリゴヌクレオチドが望ましくない遺伝子の 発現を特異的に制御することから、 近年、 医薬品として最も期待されている分野 の一つである。  In 1978, the first report was made that antisense molecules inhibited influenza virus infection. Since then, it has been reported that it inhibited oncogene expression and AIDS infection. Antisense oligonucleotides are one of the most promising fields in recent years, as they specifically regulate the expression of unwanted genes.
アンチセンス法とは、 D N A—R NA—タンパク質という、 いわゆるセントラ ルドダマの一連の流れをアンチセンスオリゴヌクレオチドを用いて制御しょうと いう概念に基づいている。  The antisense method is based on the concept of controlling the flow of so-called central dharma using antisense oligonucleotides, namely DNA-RNA-protein.
しかしながら、 天然型オリゴヌクレオチドをアンチセンス分子としてこの方法 に適用した場合、 生体内の各種ヌクレアーゼにより分解を受けたり、 細胞膜透過 性が高くないなどの問題が生じた。 そのため、 様々な核酸誘導体や類縁体が数多 く合成され、 研究が重ねられてきた。 例えば、 リン原子上の酸素原子をィォゥ原 子に置換したホスホロチォエート、 メチル基に置換したメチルホスホネート、 ま た最近になっては、 リン原子も炭素原子で置換したもの、 さらには糖部の構造を 変換したもの、 核酸塩基を修飾したものなども合成されている。 しかし、 いずれ の場合も、 細胞内での安定性、 合成の容易さ、 配列の結合特異性 （特定の遺伝子 発現のみを選択的に制御する） などの点で十分に満足のいく誘導体や類縁体が得 られていないのが現状である。 特開平 1 0— 3 0 4 8 8 9号公報には、 アンチセンスを構成するオリゴヌクレ ォチドの糖部分を修飾したヌクレオチド類縁体ュニットが開示されている。 この ヌクレオチド類縁体ュニットは、 糖部コンホメーシヨンを N型に固定化した下記 の式 1で表される構造を有するものである。 However, when a natural oligonucleotide was applied to this method as an antisense molecule, there were problems such as degradation by various nucleases in the living body and poor permeability to cell membranes. As a result, many nucleic acid derivatives and analogs have been synthesized and studied. For example, phosphorothioate in which an oxygen atom on a phosphorus atom is substituted with an iodo atom, methylphosphonate in which a methyl group is substituted, or more recently one in which a phosphorus atom is also substituted with a carbon atom, and even a sugar moiety Structures that have converted the structure of nucleosides or modified nucleic acid bases have also been synthesized. However, in each case, derivatives and analogs that are sufficiently satisfactory in terms of stability in cells, ease of synthesis, and sequence specificity (selective control of specific gene expression only), etc. Is currently not available. Japanese Patent Application Laid-Open No. 10-304889 discloses a nucleotide analog unit in which the sugar moiety of an oligonucleotide constituting antisense is modified. This nucleotide analog unit has a structure represented by the following formula 1 in which a sugar moiety conformation is immobilized to an N-type.
Figure imgf000004_0001
Figure imgf000004_0001
1  1
この公報では、 上記構造の糖部を有するヌクレオチド類縁体ユニットを 1以上 組み込んだオリゴヌクレオチド誘導体を合成し、 そのオリゴヌクレオチド誘導体 の基本的物性を細胞外で測定している。 すなわち、 当該オリゴヌクレオチド誘導 体と天然 D N Aあるいは R N Aからなるセンス鎖とをァニーリング処理したもの の融解温度を測定して、 二重鎖形成能を検討している。 また、 このオリゴヌクレ ォチド誘導体のヌクレアーゼ酵素に対する耐性をィンビトロで測定している。 しかしながら、 この公報には当該オリゴヌクレオチド誘導体が、 細胞内におい ても、 細胞外の実験結果と同ようにヌクレアーゼ耐性を示してアンチセンスとし て安定に作用するのか否か、 また、 当該オリゴヌクレオチド誘導体が、 天然に存 在する遺伝子と特に細胞内 結合して二重鎖、 三重鎖を形成し、 実際に特定遺伝 子の発現を阻止できるのか否かについては開示されていない。  In this publication, an oligonucleotide derivative incorporating one or more nucleotide analog units having a sugar moiety having the above structure is synthesized, and the basic physical properties of the oligonucleotide derivative are measured extracellularly. That is, the duplex forming ability is studied by measuring the melting temperature of the oligonucleotide derivative and the annealing treatment of the sense strand composed of natural DNA or RNA. The resistance of the oligonucleotide derivative to the nuclease enzyme was measured in vitro. However, this publication describes whether the oligonucleotide derivative exhibits nuclease resistance and acts stably as an antisense even in cells, as in the case of the extracellular experimental results. However, it does not disclose whether or not it can form a double-stranded or triple-stranded form by binding to a naturally-occurring gene, particularly in a cell, to actually inhibit the expression of a specific gene.
一方、 ウィルムス（Wi lms) 腫瘍は、 染色体 1 1 p 1 3に位置するウィルムス腫 瘍遺伝子 （WT 1 ) の両対立遺伝子の不活性化により生ずる小児腎腫瘍である (Cal l KM et al. , Ce l l 60 : 509, 1990)。 WT 1の非コード上流配列 (C. E. Camphe l l ら、 Oncogene 9： 583- 595, 1994)及びイントロンを含むコード領域 (D. A. Haberら、 Proc. Nat l. Acad. Sc i. USA, 88： 9618-9622, (1991) )はすでに報告されており、 腫 瘍等の増殖及び分化に関与することが予想される（D. A. Haberら、 前掲）。  On the other hand, Wilms tumor is a pediatric kidney tumor caused by inactivation of both Wilms tumor gene (WT1) alleles located on chromosome 11p13 (Cal KM et al., Cell 60: 509, 1990). Non-coding upstream sequence of WT1 (CE Camphell et al., Oncogene 9: 583-595, 1994) and coding region containing introns (DA Haber et al., Proc. Natl. Acad. Sc. USA, 88: 9618-9622). , (1991)) have already been reported and are expected to be involved in the growth and differentiation of tumors and the like (DA Haber et al., Supra).
WT 1はヒト白血球由来の K 5 6 2細胞やヒト胃ガン由来の A z 5 2 1細胞な ど一部の腫瘍細胞では強く発現されており、 その発現が抑制されると細胞がアポ ト一シスを起すことが報告されている （仙波憲太郎、 細胞工学、 1 4、 5 4 6 - 5 5 3 ( 1 9 9 5 ) )。 WT1 is strongly expressed in some tumor cells such as human leukocyte-derived K562 cells and human gastric cancer-derived Az521 cells, and when its expression is suppressed, cells become apoptotic. It has been reported to cause cis (Kentaro Senba, Cell Engineering, 14, 5 4 6- 5 5 3 (1995).
特開平 9— 1 0 4 6 2 9号公報には、 この WT 1に対するアンチセンスオリゴ ヌクレオチド誘導体が開示されている。 このオリゴヌクレオチド誘導体は、 修飾 されていない天然型オリゴヌクレオチドだけでなく、 修飾されたオリゴヌクレオ チド誘導体が含まれている。 この修飾オリゴヌクレオチド誘導体としては、 ヌク レオチド体のリン酸ジエステル部分を修飾したものであり、 例えば、 メチルホス ホネート型又はェチルホスホネート型のような低級アルキルホスホネート修飾体、 その他ホスホ口チォエート修飾体あるいはホスホ口アミデート修飾体等が挙げら れている。 しかしながら、 糖部分を修飾したヌクレオチド類縁体ユニットを含む オリゴヌクレオチド誘導体は開示されていない。  Japanese Patent Application Laid-Open No. 9-104629 discloses an antisense oligonucleotide derivative against WT1. This oligonucleotide derivative includes not only a natural oligonucleotide that has not been modified but also a modified oligonucleotide derivative. The modified oligonucleotide derivative is obtained by modifying the phosphodiester portion of a nucleotide, for example, a modified lower alkylphosphonate such as a methylphosphonate type or an ethylphosphonate type, a modified phospholipid thioate or a modified phosphothioate. Modified oral amidates are mentioned. However, an oligonucleotide derivative containing a nucleotide analog unit having a modified sugar moiety is not disclosed.
しかしながら、 この公報に開示されているアンチセンスオリゴヌクレオチド誘 導体は、 アンチセンスとして要求される種々の特性、 すなわち、 細胞内において も細胞外の実験結果と同ようにヌクレアーゼ耐性を有するのか否か、 また、 天然 に存在する遺伝子と、 特にインビポで結合して二重鎖、 三重鎖を形成し、 実際に 特定遺伝子の発現を阻止できるのか否かについては言及していない。  However, the antisense oligonucleotide derivative disclosed in this publication has various properties required for antisense, that is, whether or not it has nuclease resistance in cells as well as in the results of extracellular experiments. In addition, it does not mention whether it can bind to a naturally-occurring gene, particularly in vivo, to form a double-stranded or triple-stranded chain, and actually inhibit the expression of a specific gene.
細胞内でヌクレアーゼによる分解を受けにくく、 高い親和力で標的の WT 1に 結合してその遺伝子の発現を効率良く制御、 阻止できるアンチセンスオリゴヌク レオチド誘導体の創製が望まれている。  There is a need for an antisense oligonucleotide derivative that is less susceptible to degradation by nucleases in cells, and that can bind to the target WT1 with high affinity and efficiently control and block gene expression.
[発明の開示]  [Disclosure of the Invention]
本発明の発明者らは、 アンチセンス法において有用と考えられる、 核酸におけ る糖部のコンホメーシヨンの固定化を施したした核酸類縁体を設計し、 その単位 構造となるヌクレオチド類縁体ュニットの合成を行い、 これを用いて調製したォ リゴヌクレオチド誘導体が WT 1アンチセンス分子として極めて有用であること を確認した。 以下に本発明の詳細を説明する。  The inventors of the present invention designed a nucleic acid analog having a sugar moiety conformation immobilized on a nucleic acid, which is considered to be useful in the antisense method, and synthesized a nucleotide analog unit having a unit structure thereof. It was confirmed that the oligonucleotide derivative prepared using this was extremely useful as a WT1 antisense molecule. The details of the present invention will be described below.
[図面の簡単な説明]  [Brief description of drawings]
図 1は、 実験例 1に試料として使用したオリゴヌクレオチドのヌクレオチド配 列、 及びヌクレア一ゼ （蛇毒ホスホジエステラーゼ） 処理した試料をポリアクリ ルアミドゲル電気泳動にかけて得られたバンドのパターンを示す図である。 図 2は、 実験例 1に試料として使用したオリゴヌクレオチドのヌクレオチド配 列、 及びヌクレア一ゼ (牛胎児血清： F B S ) 処理した試料をポリアクリルアミ ドゲル電気泳動にかけて得られたバンドのパターンを示す図である。 FIG. 1 is a diagram showing a nucleotide sequence of an oligonucleotide used as a sample in Experimental Example 1 and a band pattern obtained by subjecting a sample treated with nuclease (snake venom phosphodiesterase) to polyacrylamide gel electrophoresis. Figure 2 shows the nucleotide sequence of the oligonucleotide used as a sample in Experimental Example 1 and the band pattern obtained by subjecting a sample treated with nuclease (fetal bovine serum: FBS) to polyacrylamide gel electrophoresis. It is.
図 3は、 天然型オリゴヌクレオチドによるアンチセンス効果を示すグラフであ る。  FIG. 3 is a graph showing the antisense effect of the natural oligonucleotide.
図 4は、 本発明のオリゴヌクレオチド誘導体のアンチセンス効果の濃度依存性 を示すグラフである。  FIG. 4 is a graph showing the concentration dependence of the antisense effect of the oligonucleotide derivative of the present invention.
図 5は、 本発明のオリゴヌクレオチド誘導体とホスホロチォエート型オリゴヌ クレオチド誘導体とを比較したアンチセンス効果を示すグラフである。  FIG. 5 is a graph showing the antisense effect of comparing the oligonucleotide derivative of the present invention with a phosphorothioate-type oligonucleotide derivative.
図 6は、 本発明のオリゴヌクレオチド誘導体とスクランブル配列オリゴヌクレ ォチドとの濃度依存アンチセンス効果を示すグラフである。  FIG. 6 is a graph showing the concentration-dependent antisense effect of the oligonucleotide derivative of the present invention and the scrambled sequence oligonucleotide.
図 7は、 本発明のォリゴヌクレオチド誘導体と異なる 2種のスクランブル配列 オリゴヌクレオチドとのアンチセンス効果を示すグラフである。 本発明のオリゴヌクレオチド又はポリヌクレオチド誘導体は下記の一般式  FIG. 7 is a graph showing the antisense effect of the oligonucleotide derivative of the present invention with two different scrambled sequence oligonucleotides. The oligonucleotide or polynucleotide derivative of the present invention has the following general formula
Figure imgf000006_0001
Figure imgf000006_0001
[式中、 Bはピリミジン核酸塩基もしくはプリン核酸塩基又はそれらの類縁体で ある] で表わされる構造を有するヌクレオチド類縁体ユニットを 1又は 2以上有 する。  [Wherein B is a pyrimidine nucleobase or a purine nucleobase or an analog thereof] has one or more nucleotide analog units having a structure represented by the following formula:
本発明のオリゴヌクレオチド又はポリヌクレオチド誘導体は一般式 The oligonucleotide or polynucleotide derivative of the present invention has the general formula
Figure imgf000007_0001
Figure imgf000007_0001
[式中、 B B 2は同一または異なり、 ピリミジン核酸塩基もしくはプリン核酸 塩基又はそれらの類縁体であり、 Rは水素、 水酸基、 ハロゲン、 またはアルコキ シ基であり、 W W2は同一または異なり、 水素、 アルキル基、 アルケニル基、 アルキニル基、 シクロアルキル基、 ァラルキル基、 ァリール基、 ァシル基、 シリ ル基またはリン酸残基もしくはリン酸ジエステル結合を介した天然型ヌクレオチ ド、 合成ヌクレオチドまたはこれらヌクレオチドを含むォリゴヌクレオチドもし くはポリヌクレオチドであり、 n 1または n 2は同一または異なり、 0〜5 0の整 数である （ただし、 n 1または n 2が同時にゼロになることはない。 また、 n 2の全 てが同時にゼロになることはない。）、 n3は 1〜5 0の整数である、 ただし、 n 1 および Zまたは n 2が 2以上の場合には B 1と Bは同一でなくてもよく、 Rも同一 でなくてもよい] で表されるオリゴヌクレオチドもしくはポリヌクレオチド誘導 体である。 Wherein BB 2 is the same or different, is a pyrimidine nucleobase or purine nucleobase or an analog thereof, R is hydrogen, a hydroxyl group, a halogen, or an alkoxy group, and WW 2 is the same or different, hydrogen, Alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aralkyl group, aryl group, acyl group, silyl group or natural nucleotide via phosphate residue or phosphate diester bond, synthetic nucleotide or these nucleotides O Rigo nucleotide if Ku is a polynucleotide, n 1 or n 2 are the same or different, 0-5 is a integer of 0 (where, n 1 or n 2 do not become zero simultaneously. also, n 2 cannot be zero at the same time.), N 3 is an integer from 1 to 50, provided that n 1 and Z or n 2 are B 1 and B in the case of 2 or more may not be the same, R is also an oligonucleotide or polynucleotide derived material represented by may not be the same.
本発明のアンチセンスオリゴヌクレオチド誘導体の塩基の数は特に制限はない が、 通常 5〜5 0、 好ましくは 9〜3 0個である。  The number of bases in the antisense oligonucleotide derivative of the present invention is not particularly limited, but is usually 5 to 50, preferably 9 to 30.
本発明における、 ピリミジン核酸塩基もしくはプリン核酸塩基またはその類縁 体とは、 チミン、 ゥラシル、 シトシン、 アデニン、 グァニン及びそれらの類縁体 である。 これらの類縁体としてはプリン核酸塩 ¾類縁体及びピリミジン核酸塩基 類縁体を挙ることができる。  In the present invention, the pyrimidine nucleobase or purine nucleobase or an analog thereof is thymine, peracyl, cytosine, adenine, guanine and an analog thereof. These analogs include purine nucleic acid salt analogs and pyrimidine nucleobase analogs.
プリン核酸塩基類縁体としては、 好ましくは下記の化合物から選択することが できる。  The purine nucleobase analog can be preferably selected from the following compounds.
即ち、 グアノシンジフォスフェート、 8—ォキソ一アデノシン、 8—ォキソ一グ ァノシン、 8—フロロ一アデノシン、 8—フロロ一グアノシン、 8—メトキシー アデノシン、 8—メトキシ—グアノシン、 8 _ァザーアデノシン、 8—ァザーグ ァノシン、 ァザシチヂン、 フルダラビンフォスフェート、 6— M P、 6—T G、 ァザチプリン、 ァロプリノール、 ァシクロビア、 ガンシクロビア、 デォキシホル ミシン、 ァラビノシラジェニン （ara- A )、 グアノシンジフォスフエ一トフコー ス、 グアノシンジフォスフェート一 2 _フロロフコース、 グアノシンジフォスフ ェ一トー j3 L— 2—アミノフコース、 グアノシンジフォスフェート一 D—ァラビ ノース及び 2—アミノアデシン等である。 Guanosine diphosphate, 8-oxo-adenosine, 8-oxo-guanosine, 8-fluoro-adenosine, 8-fluoro-guanosine, 8-methoxy- Adenosine, 8-methoxy-guanosine, 8-azaadenosine, 8-azaguanosine, azacytin, fludarabine phosphate, 6-MP, 6-TG, azatipurine, aloprinol, acyclovir, ganciclovia, deoxyphormycin, arabinosilagenin ( ara-A), guanosine diphosphate tofucoose, guanosine diphosphate- 12-fluorofurose, guanosine diphosphate toe j3 L-2-aminofucose, guanosine diphosphate-D-arabinose and 2-aminoadesine And so on.
一方、 ピリジン核酸塩基類縁体としては、 好ましくは下記の化合物から選択す ることができる。 即ち、  On the other hand, the pyridine nucleobase analog can be preferably selected from the following compounds. That is,
5—フロロゥラシル、 5 _クロロウラシル、 5—ブロモウラシル、 ジヒドロウ ラシル、 5—メチルシトシン、 5—プロピエルチミン、 5—プロピニルゥラシル、 5—プロピエルシトシン、 5—フロロシトシン、 フロキシゥリジン、 ゥリジン、 チミン、 3 ' —アジドーデォキシチミジン、 2—フロロデォキシシチジン、 3 _ フロロ— 3， —デォキシチミジン、 3， —ジデォキシシチジン— 2， —ェン、 3， —デォキシー 3， ーデォキシチミジン— 2， —ェン、 及びシトシンァラビノース 等である。  5-fluoroperacil, 5-chlorouracil, 5-bromouracil, dihydrouracil, 5-methylcytosine, 5-propierthymine, 5-propynylperacyl, 5-propiercytosine, 5-fluorocytosine, floxyperidine, peridine, thymine , 3'-azidodeoxythymidine, 2-fluorodeoxycytidine, 3_fluoro--3, -deoxythymidine, 3, -dideoxycytidine- 2, -ene, 3, -doxy-3, dedeo Xitymidine-2, -ene, and cytosine arabinose.
本発明でアンチセンスとして使用できるヌクレオチド類縁体ュニット、 及びそ のヌクレオチド類縁体ュニットを含有するオリゴヌクレオチド誘導体の合成は、 前記特開平 1 0— 3 0 4 8 8 9号公報に詳細に記載されている。 この説明は本明 細書に含まれるものとする。  The synthesis of a nucleotide analog unit which can be used as an antisense in the present invention, and the synthesis of an oligonucleotide derivative containing the nucleotide analog unit are described in detail in the above-mentioned Japanese Patent Application Laid-Open No. 10-304889. I have. This description shall be included in this document.
本発明の WT 1に対するアンチセンスオリゴヌクレオチド誘導体としては、 W T 1に結合して、 その遺伝子の発現を阻止、 又は抑制できるものであればどのよ うなオリゴ又はポリヌクレオチドであってもよく、 構成単位であるヌクレオチド の 1つ又は 2以上が本発明のヌクレオチド類縁体ュニットで置換されていればよ い。  The antisense oligonucleotide derivative to WT1 of the present invention may be any oligo or polynucleotide that can bind to WT1 and inhibit or suppress the expression of its gene, It suffices that one or more of the nucleotides is replaced with the nucleotide analog unit of the present invention.
アンチセンスオリゴヌクレオチド誘導体の例としては、 WT 1の転写キヤッピ ング部位に対するもの、 翻訳開始領域に対するもの、 ェクソンに対するもの又は イントロンに対するものなどが挙げられる。 更に具体的には、 下記のような塩基 配列が含まれる。 Examples of antisense oligonucleotide derivatives include those for the transcriptional capping site of WT1, those for the translation initiation region, those for exons, those for introns, and the like. More specifically, the following bases Contains an array.
転写キヤッピング部位のアンチセンス配列 （A S 1 ) AGGGTCGAAT GCGGTGGG (配列番号 1 )  Antisense sequence of transcription capping site (A S 1) AGGGTCGAAT GCGGTGGG (SEQ ID NO: 1)
転写キヤッピング領域のアンチセンス配列 （A S 2 ) TCAAATAAGA GGGGCCGG (配列番号 2 )  Antisense sequence of transcription capping region (AS 2) TCAAATAAGA GGGGCCGG (SEQ ID NO: 2)
翻訳開始領域のアンチセンス配列 （AS 3) GTCGGAGCCC ATTTGCTG Antisense sequence of translation initiation region (AS 3) GTCGGAGCCC ATTTGCTG
(配列番号 3 ) (SEQ ID NO: 3)
ェクソン 6のアンチセンス配列 （AS 4) CGTTGTGTGG TTATCGCT Exon 6 antisense sequence (AS 4) CGTTGTGTGG TTATCGCT
(配列番号 4) (SEQ ID NO: 4)
WT 1の翻訳開始領域近傍は、 アンチセンス効果の見られる領域であることが 報告されており、 アンチセンスによるガン治療の標的として期待されている。 本 発明者等はこの領域のアンチセンス配列を有する本発明のオリゴヌクレオチド誘 導体を合成し、 WT 1を強く発現する Az 52 1細胞に投与し、 アポト一シスに よる細胞数の減少を指標にアンチセンス効果を検討して、優れた効果を確認した。 このように、 本発明のアンチセンスは、 WT 1を発現する腫瘍細胞に揷入するこ とにより、 その発現を阻止、 抑制して細胞をアポトーシスに導くことにより抗腫 瘍薬剤として使用できる。  It has been reported that the vicinity of the translation initiation region of WT1 is a region where an antisense effect is observed, and is expected as a target for cancer treatment by antisense. The present inventors have synthesized the oligonucleotide derivative of the present invention having an antisense sequence in this region, and administered it to Az521 cells that strongly express WT1, using the decrease in cell number due to apoptosis as an index. The antisense effect was examined and an excellent effect was confirmed. As described above, the antisense of the present invention can be used as an antitumor drug by introducing into tumor cells expressing WT1, thereby inhibiting or suppressing the expression to induce the cells to undergo apoptosis.
本発明のオリゴヌクレオチド誘導体の具体例としては、 下記の式で表される Specific examples of the oligonucleotide derivative of the present invention are represented by the following formula:
5 ' 一 GTCGGAGCCC ATTTGCTG— 3 ' (配列 3 ) 5 'One GTCGGAGCCC ATTTGCTG— 3' (Sequence 3)
5 ' -GUCGGAGCCCAUUUGCUG- 3 ' (配列 5 ) で表される WT 1翻訳開始領域に対するアンチセンス配列の 1又は配列 2以上の 塩基が本発明のヌクレオチド類縁体ュニットで置換されている配列及びその配列 を含むァンチセンスオリゴヌクレオチド誘導体、 及び上記ァンチセンス配列に対 して相補的な配列である DN A又は RN Aとストリンジェントな条件下でハイブ リダィズするオリゴヌクレオチド誘導体を挙ることができる。 ハイブリダィゼー シヨンは既に良く知られた技術である （Sambrook, l et al. , Molecular Cloning 2nd ed. , Cold spring Harbor Lab. Press, 1989 等）。  5′-GUCGGAGCCCAUUUGCUG-3 ′ (SEQ ID NO: 5) A sequence in which one or two or more bases of the antisense sequence to the translation initiation region of the WT1 translation initiation region are replaced with the nucleotide analog unit of the present invention and a sequence thereof. Antisense oligonucleotide derivatives containing the same, and oligonucleotide derivatives that hybridize under stringent conditions to DNA or RNA, which is a sequence complementary to the above antisense sequence, can be mentioned. Hybridization is a well-known technique (Sambrook, l et al., Molecular Cloning 2nd ed., Cold spring Harbor Lab. Press, 1989, etc.).
本発明でいうストリンジエンドな条件は、 当業者であれば、 適宜選択すること が可能であるが、 例えば、 低ストリンジエンドな条件である。 低ストリ トな条件とは、 例えば、 ハイブリダィゼ一シヨン後の洗浄において、 42°C、 0. 1 XS SC、 0. 1 %SD Sの条件であり、 好ましくは 50° (：、 0. 1 XS SC、 0. 1 %SDSである。 さらに好ましいストリンジェントな条件としては高スト リンジェントな条件がある。 高ストリンジエンドの条件としては、 65°C、 6 X S S C、 0. 1 %SDSである。 Stringent end conditions in the present invention may be appropriately selected by those skilled in the art. Is possible, for example, under low stringent conditions. The low stringent conditions are, for example, in washing after hybridization, at 42 ° C, 0.1 XSSC, 0.1% SDS, preferably 50 ° (: 0.1 XS SC, 0.1% SDS More preferred stringent conditions include high stringency conditions High stringency end conditions are 65 ° C, 6 XSSC, 0.1% SDS.
また、 本発明のオリゴヌクレオチド誘導体は、 同一の塩基配列を有する天然型 ォリゴヌクレオチドに比較して優れたヌクレアーゼ耐性を示すことも確認されて いる。 したがって、 細胞内に投与された場合にも安定であり、 その優れたアンチ センス効果が持続するという利点がある。  It has also been confirmed that the oligonucleotide derivative of the present invention exhibits superior nuclease resistance as compared to a natural oligo nucleotide having the same base sequence. Therefore, it is stable even when administered intracellularly, and has the advantage of maintaining its excellent antisense effect.
本発明のアンチセンスオリゴヌクレオチド誘導体は、 糖部のみを修飾したヌク レオチド類縁体ユニットの使用が必須であるが、 さらに他の部分、 例えば、 ホス ホロチォェ一トのようにリン酸ジエステル部を修飾したヌクレオチド類縁体ュニ ットを使用しても良い。 また、 本発明の糖部を修飾したヌクレオチド類縁体ュニ ッ卜と他の公知のアンチセンス構成単位であるヌクレオチド類縁体ュニッ卜とを 組み合わせてオリゴヌクレオチド誘導体ァンチセンスを作成することもできる。 本発明のアンチセンスオリゴヌクレオチド誘導体は、 それらに対して不活性な 適当な基剤と混和して塗布剤、 パップ剤などの外用剤とすることができる。  The antisense oligonucleotide derivative of the present invention requires the use of a nucleotide analog unit in which only the sugar moiety is modified, but further modifies the other moiety, for example, the phosphodiester moiety as in the case of phosphorothioate. Nucleotide analog units may be used. Further, an oligonucleotide derivative antisense can also be prepared by combining the nucleotide analog unit having a modified sugar moiety of the present invention with a nucleotide analog unit which is another known antisense structural unit. The antisense oligonucleotide derivative of the present invention can be used as an external preparation such as a liniment or a poultice by mixing with a suitable base material which is inactive against the derivative.
また、 必要に応じて、 賦形剤、 等張化剤、 溶解補助剤、 安定化剤、 防腐剤、 無痛 化剤等を加えて錠剤、 散剤、 顆粒剤、 カプセル剤、 リボソームカプセル剤、 注射 剤、 液剤、 点鼻剤など、 さらに凍結乾燥剤とすることができる。 これらは常法に 従つて調製することができる。 If necessary, excipients, isotonic agents, solubilizing agents, stabilizers, preservatives, soothing agents, etc., and tablets, powders, granules, capsules, ribosome capsules, injections Lyophilized agents such as liquids, nasal drops and the like. These can be prepared according to a conventional method.
本発明のアンチセンスオリゴヌクレオチド誘導体は患者の患部に直接適用する か、 または血管内に投与するなどして結果的に患部に到達し得るように患者に適 応させる。 さらに持続性、 膜透過性を高めるアンチセンス封入素材を用いること もできる。 例えば、 リボゾーム、 ポリ一 L—リジン、 リピッド、 コレステロール、 リポフエクチン又はこれらの類縁体が挙げられる。  The antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is adapted to the patient so that it can reach the affected area as a result of intravenous administration or the like. Furthermore, an antisense-encapsulated material that enhances durability and membrane permeability can be used. For example, ribosome, poly-L-lysine, lipid, cholesterol, lipofectin, or an analog thereof can be mentioned.
本発明のアンチセンスオリゴヌクレオチド誘導体の投与量は、 患者の状態、 年 齢、 性別、体重などに応じて適宜調整し好ましい量を用いることができる。 また、 その投与方法は、 患者の状態、 薬剤形態などに応じ、 経口投与、 筋肉内投与、 腹 腔内投与、 皮内投与、 皮下投与、 静脈内投与、 脈内投与、 直腸投与などの種々の 投与方法から適宜好ましい方法を用いることができる。 The dose of the antisense oligonucleotide derivative of the present invention depends on the patient's condition, A suitable amount can be used after appropriately adjusting according to age, sex, body weight, and the like. Depending on the condition of the patient, the form of the drug, and the like, various administration methods such as oral administration, intramuscular administration, intraperitoneal administration, intradermal administration, subcutaneous administration, intravenous administration, intravenous administration, rectal administration, etc. A suitable method can be used as appropriate from the administration method.
発明の WT 1に対するアンチセンスオリゴヌクレオチド誘導体は、 アンチセン ス D N Aとして優れた種々の特性を示すことを以下の実施例によって詳しく説明 する。 .  The following examples illustrate in detail that the antisense oligonucleotide derivative against WT1 of the present invention exhibits various excellent properties as antisense DNA. .
実施例 1 ： 本発明のヌクレオチド類縁体ユニットを含むオリゴヌクレオチド 誘導体のヌクレアーゼ耐性  Example 1: Nuclease resistance of oligonucleotide derivatives containing nucleotide analog units of the present invention
本発明のヌクレオチド類縁体ュニットを 5個含むオリゴヌクレオチド誘導体を 作成して、 ヌク.レアーゼ耐性試験の試料とした。 一方、 同じ配列を有する天然型 ォリゴヌクレオチドを作成して対照とした。  Oligonucleotide derivatives containing five nucleotide analog units of the present invention were prepared and used as samples for the nuclease resistance test. On the other hand, a natural oligonucleotide having the same sequence was prepared and used as a control.
( 1 ) ヌクレア一ゼとしては蛇毒ホスホジエステラーゼを用い、 32 Pにて放射性 同位体ラベルしたオリゴヌクレオチドをヌクレア一ゼ処理し、 ポリアクリルアミ ドゲル電気泳動により分離する方法によって、 本発明オリゴヌクレオチド誘導体 のヌクレア一ゼ耐性を評価した。 図 1に試料として作成したォリゴヌクレオチド の配列とヌクレアーゼ処理後の電気泳動の結果を示す。 (1) The nuclease of the oligonucleotide derivative of the present invention is obtained by using a snake venom phosphodiesterase as a nuclease, treating the oligonucleotide labeled with radioisotope with 32 P with nuclease, and separating the oligonucleotide by polyacrylamide gel electrophoresis. Monozyme resistance was evaluated. Figure 1 shows the sequence of the oligonucleotide prepared as a sample and the results of electrophoresis after nuclease treatment.
蛇毒ホスホジエステラーゼは 3 ' ェキソヌクレアーゼであるので、 ァ一32 P— A T Pを用いて、 T 4キナーゼによってオリゴヌクレオチドの 5 ' 末端をリン酸 化し、 ラベル化した。 ラベル化した各配列のオリゴヌクレオチドをそれぞれ、 0 .'Because in E exonuclease, using § one 32 P- ATP, 5 oligonucleotides by T 4 kinase' snake venom phosphodiesterase 3 turned into phosphoric acid ends were labeled. Oligonucleotides of each labeled sequence were assigned 0.
0 8、 0. 5、 1、 2、 4、 8時間ヌクレアーゼ処理したもの及びヌクレアーゼ未 処理 （0時間） のものをポリアクリルアミドゲル電気泳動にかけて分離した。 この分解反応は一次反応としては進行しないので、 単純に半減期を求めること は出来ない。そこで、各時間での未分解のオリゴヌクレオチドのバンドを定量し、 これがヌクレアーゼ未処理 （0分） のバンドの半分になった時間を指標とし、 こ れを比較した。 Those treated with nuclease for 08, 0.5, 1, 2, 4, and 8 hours and those not treated with nuclease (0 hour) were separated by polyacrylamide gel electrophoresis. Since this decomposition reaction does not proceed as a primary reaction, it is not possible to simply determine the half-life. Therefore, the band of the undegraded oligonucleotide at each time was quantified, and the time at which this band was half of the band untreated with nuclease (0 min) was used as an index, and this was compared.
その結果、 天然型オリゴヌクレオチドではヌクレアーゼ処理開始から 0 . 0 8 時間 （約 5分） 後には、 未分解のオリゴヌクレオチドの割合が 3 0 %以下であつ た。 さらに、 0. 5時間後には未分解のオリゴヌクレオチドは完全に消失した。 これに対して、 本発明のオリゴヌクレオチド誘導体の場合では、 ヌクレアーゼ処 理開始から 8時間後においても未分解のオリゴヌクレオチドが 90 %程度確認で きた。 このことから、 本発明のオリゴヌクレオチド誘導体は天然型オリゴヌクレ ォチドに比べて少なくとも 1 0 0倍以上のヌクレアーゼ耐性能を有していること が分かった。 As a result, after 0.08 hours (about 5 minutes) from the start of the nuclease treatment, the ratio of the undegraded oligonucleotide was less than 30% for the natural oligonucleotide. Was. Further, after 0.5 hours, the undegraded oligonucleotide completely disappeared. On the other hand, in the case of the oligonucleotide derivative of the present invention, about 90% of undegraded oligonucleotide was confirmed even 8 hours after the start of the nuclease treatment. From these results, it was found that the oligonucleotide derivative of the present invention has at least 100 times or more the nuclease resistance performance as compared with the natural oligonucleotide.
本発明のオリゴヌクレオチド誘導体は、 ヌクレア一ゼによる分解に対して極め て強い抵抗性を示した。  The oligonucleotide derivatives of the present invention showed extremely strong resistance to degradation by nuclease.
(2) 上記 （1) と同一のオリゴヌクレオチドを使用し、 ヌクレア一ゼとしては 牛胎児血清 （FBS) を用いた。 10%FBSを含有するダルべコ変性イーグル 培地に 0. 02mMの試料オリゴヌクレオチドを添加し、 37°Cで 0. 5、 1、 2、 4、 8、 24時間、 加水分解を行った。 加水分解物はポリアクリルアミドゲル電 気泳動にかけて分離した。 図 2に試料として作成したオリゴヌクレオチドの配列 とヌクレアーゼ処理後の電気泳動の結果を示す。  (2) The same oligonucleotide as in (1) above was used, and fetal bovine serum (FBS) was used as nuclease. 0.02 mM sample oligonucleotide was added to Dulbecco's modified Eagle's medium containing 10% FBS, and the mixture was hydrolyzed at 37 ° C for 0.5, 1, 2, 4, 8, 24 hours. The hydrolyzate was separated by polyacrylamide gel electrophoresis. Figure 2 shows the sequence of the oligonucleotide prepared as a sample and the results of electrophoresis after nuclease treatment.
実施例 2 ：融解温度 （Tm) の測定 Example 2: Measurement of melting temperature (Tm)
特開平 10— 304889号公報の実施例記載の方法により合成した WT 1に 対する下記のオリゴヌクレオチド誘導体をアンチセンス鎖とし、 天然の DNAあ るいは RNAからなるセンス鎖とァニ一リング処理したものの融解温度（Tm値） を測定することにより、 本発明のオリゴヌクレオチド誘導体の相補 D N Aおよび RNAに対するハイブリッド形成能を調べた。  The following oligonucleotide derivative to WT1 synthesized by the method described in the example of JP-A-10-304889 was used as an antisense strand, and was subjected to annealing treatment with a sense strand consisting of natural DNA or RNA. By measuring the melting temperature (Tm value), the ability of the oligonucleotide derivative of the present invention to form a hybrid with complementary DNA and RNA was examined.
終濃度をそれぞれ、 N a C 1 100 mM、 リン酸ナトリゥム緩衝液（ p H 7. 2) 10mM、 アンチセンス鎖 4 M、 センス鎖 4 Mとしたサンプル溶液 （5 00 1) を沸騰水中に浴し、 10時間をかけてゆっくり室温まで冷却した。 分 光光度計 （B e c kman DU650) のセル室内に結露防止のために窒素気 流を通し、 サンプル溶液を 5 °Cまで徐々に冷却し、 さらに 20分間 5°Cに保った 後、 測定を開始した。 サンプル温度は 90°Cまで毎分 0. 2 °Cずつ上昇させ、 0. 1°C間隔で 260 nmにおける紫外線吸収を測定した。  A sample solution (5001) with final concentrations of 100 mM NaC, 10 mM sodium phosphate buffer (pH 7.2), 4 M for antisense strand and 4 M for sense strand was bathed in boiling water. And slowly cooled to room temperature over 10 hours. A nitrogen stream was passed through the cell chamber of the spectrophotometer (Bec kman DU650) to prevent dew condensation, the sample solution was gradually cooled to 5 ° C, and kept at 5 ° C for another 20 minutes before measuring. Started. The sample temperature was increased by 0.2 ° C per minute to 90 ° C, and ultraviolet absorption at 260 nm was measured at 0.1 ° C intervals.
結果は、 下記の表に示した。 ヌクレオチド配列のうち、 下線を引いたものが本 発明のヌクレオチド類縁体ュニットである。 表 1. ァンチセンスォリゴヌクレオチド誘導体の相補 DNA及び MA The results are shown in the table below. The underlined nucleotide sequence in the book 1 is a nucleotide analog unit of the invention. Table 1. Complementary DNA and MA of antisense oligonucleotide derivatives
に対する融解温度 （Tm値） 試験番号 アンチセンス分子 Tm値 （ATm/mod. )/ 。C  Melting temperature (Tm value) for test No. Antisense molecule Tm value (ATm / mod.) /. C
相補 DNAとの Tm 相補 RNAとの Tm  Tm with complementary DNA Tm with complementary RNA
1 5' -GTCGGAGCCCATTTGCTG-3' (D-ol igo) 67 67 1 5 '-GTCGGAGCCCATTTGCTG-3' (D-ol igo) 67 67
2 5' -GUCGGAGCCCAUUUGCUG-3' (5 be -AS) 74 (+ 1. 4) 82 (+ 3. 0)2 5 '-GUCGGAGCCCAUUUGCUG-3' (5 be -AS) 74 (+1.4) 82 (+3.0)
3 5' -GTCGGAGCCCATTTGCTG-3' 75 (+ 1. 6) 86 (+ 3. 8)3 5 '-GTCGGAGCCCATTTGCTG-3' 75 (+1.6) 86 (+3.8)
4 5' -GTCGGAGCCCATTTGCTG-3' (S-oligo) 59 60 4 5 '-GTCGGAGCCCATTTGCTG-3' (S-oligo) 59 60
(アンチセンス分子 1、 2、 3、 4は、 それぞれ、 配列番号 3、 6、 7、 1に対 応する。）  (Antisense molecules 1, 2, 3, and 4 correspond to SEQ ID NOs: 3, 6, 7, and 1, respectively.)
表から明らかなように、天然 DN A鎖中に本発明のヌクレオチド類縁体ュニッ ト （下線） を 5個揷入したオリゴマー （試験 2 (5bc_ASという）、 及び試験 3) は、 相補 DNAオリゴマーとのハイプリド形成能が、 Tm値で評価して天然鎖（試験 1 ) よりも 7— 8度上昇した。、一方、相補 RN Aに対するハイブリツド形成能を評価 したところ、 本発明のオリゴマーでは天然鎖のもの （D- origo)よりも 5— 9度の Tm値の上昇が認められた。 このように、 天然鎖よりも Tm値がこのように著し く上昇する類縁体は従来知られていない。  As is clear from the table, the oligomer (Test 2 (referred to as 5bc_AS) and Test 3) in which five nucleotide analog units (underlined) of the present invention were inserted into the natural DNA chain was found to be complementary to the complementary DNA oligomer. Hypride forming ability was increased 7-8 degrees over the native chain (Test 1) as assessed by Tm values. On the other hand, when the ability to form a hybrid to complementary RNA was evaluated, the Tm value of the oligomer of the present invention was found to be 5 to 9 degrees higher than that of the natural chain (D-origo). Thus, no analogs whose Tm value is so markedly higher than that of the natural chain have been known.
比較のために、 天然鎖と同じ塩基配列を有するホスホロチォェ一ト型オリゴヌ クレオチド誘導体 （S - oligo) (試験 4) と比較した。 このホスホロチォェ一ト型 のオリゴヌクレオチド誘導体は天然型のものと比較しても T m値が著しく低下し ており、 本発明のオリゴヌクレオチド誘導体と比較するとその差は更に著しかつ た。  For comparison, comparison was made with a phosphorothioate-type oligonucleotide derivative (S-oligo) (Test 4) having the same base sequence as the natural chain. The Tm value of this phosphorothioate-type oligonucleotide derivative was significantly lower than that of the natural-type oligonucleotide derivative, and the difference was even more remarkable when compared with the oligonucleotide derivative of the present invention.
本発明のオリゴヌクレオチド誘導体ァンチセンスは、 D N A及び R N Aの何れ に対しても優れた二重鎖形成能を示した。 実施例 3 ：オリゴヌクレオチド誘導体の WT 1に対するアンチセンス効果 この実施例では、 培養細胞を用いて本発明のオリゴヌクレオチド誘導体の WT 1に対するアンチセンス効果を確認した。 The oligonucleotide derivative antisense of the present invention showed an excellent double strand-forming ability to both DNA and RNA. Example 3: Antisense effect of oligonucleotide derivative on WT1 In this example, the antisense effect of oligonucleotide derivative of the present invention on WT1 was confirmed using cultured cells.
WT 1の翻訳開始領域近傍のアンチセンス配列を有するオリゴヌクレオチド誘 導体を実施例 2と同様に作成した。 このアンチセンス配列を有する本発明のオリ ゴヌクレオチド誘導体を、 Az 521細胞に投与し、 細胞数の減少を指標にアン チセンス効果を評価した。 比較のために、 天然型 （D- origo)、 ホスホロチォエー ト型オリゴヌクレオチド （S- oligo)についても同じ条件で実験を行った。  An oligonucleotide derivative having an antisense sequence near the translation initiation region of WT1 was prepared in the same manner as in Example 2. The oligonucleotide derivative of the present invention having the antisense sequence was administered to Az521 cells, and the antisense effect was evaluated using the decrease in the number of cells as an index. For comparison, experiments were performed under the same conditions for natural (D-origo) and phosphorothioate-type oligonucleotides (S-oligo).
1日目に各ゥエル（10 O 1) に Az 521細胞もしくは P C 14細胞（2. 5 X 1 03c e 1 1， DMEM培地） を蒔き、 血清非存在下でオリゴヌクレオチド 溶液 （1. 25 / 1) を2 1 (終濃度 25 fi g/m 1 ) を加え、 2時間 培養後、 FBS 10 i l (終濃度 10%) を加え、 さらに培養した。 2日目、 3 日目、 4日目にそれぞれオリゴヌクレオチド溶液 1 1 (終濃度 + 12. 5 g /m l) を加え、 さらに 2日間培養した。 （オリゴヌクレオチドの濃度を変えた実 験ではそれにあった容量のオリゴヌクレオチド溶液を用いた。） 6日目に生きてい る細胞数を顕微鏡でカウントした。 Each Ueru to 1 day (10 O 1) to the Az 521 cells or PC 14 cells (2. 5 X 1 0 3 ce 1 1, DMEM medium) were plated, oligonucleotide solution in the absence of serum (1.25 / 1) was added to 21 (final concentration 25 fig / m 1), and cultured for 2 hours. Then, 10 il of FBS (final concentration 10%) was added, followed by further culturing. Oligonucleotide solution 11 (final concentration + 12.5 g / ml) was added on the second, third, and fourth days, respectively, and the cells were further cultured for 2 days. (In experiments where the oligonucleotide concentration was varied, an appropriate volume of the oligonucleotide solution was used.) On day 6, the number of living cells was counted using a microscope.
(1) 天然型オリゴヌクレオチド （D-oligo) によるアンチセンス効果の確認 まず、 種々の濃度での天然型オリゴヌクレオチド （D- oligo) によるアンチセン ス効果を調べた。 対照としてセンス配列 （Sと表示） を用いた。  (1) Confirmation of antisense effect by natural oligonucleotide (D-oligo) First, antisense effect by natural oligonucleotide (D-oligo) at various concentrations was examined. The sense sequence (labeled S) was used as a control.
結果を図 3に示す。  The results are shown in Figure 3.
1日目のオリゴヌクレオチド濃度を 50， 100, 200 g/m lとした系 ではアンチセンス配列 （Aと表示） で細胞数の減少が見られたものの、 1日目の オリゴヌクレオチドの濃度を 25 1とすると細胞数の減少は見られなかった。 ( 2 ) 本発明のオリゴヌクレオチド誘導体のアンチセンス効果の確認  In the system in which the oligonucleotide concentration on day 1 was 50, 100, or 200 g / ml, the number of cells was reduced in the antisense sequence (denoted as A), but the oligonucleotide concentration on day 1 was 25 1 No decrease in cell number was observed. (2) Confirmation of antisense effect of oligonucleotide derivative of the present invention
上記 （1) の D-oligoで細胞数の減少が見られなかった系を用いて、 本発明の オリゴヌクレオチド誘導体のアンチセンス効果を検討した。 この時、 WT 1を発 現しない P C 14細胞にもオリゴヌクレオチド誘導体を投与し、 その細胞数の変 化を比較した。 結果を図 4に示す。 Az 52 1細胞の場合では D-oligoの各配列を投与した時では、細胞数の減少 は見られなかったが、 本発明のオリゴヌクレオチド誘導体を投与した時は、 細胞 数の減少が見られた。 The antisense effect of the oligonucleotide derivative of the present invention was examined using a system in which the number of cells was not reduced by D-oligo in (1) above. At this time, the oligonucleotide derivative was also administered to PC 14 cells that did not express WT1, and changes in the number of cells were compared. Fig. 4 shows the results. In the case of Az521 cells, the number of cells was not reduced when the D-oligo sequence was administered, but the number of cells was decreased when the oligonucleotide derivative of the present invention was administered. .
一方、 PC 14細胞の時には、 何れの場合も細胞数の減少が見られなかった。 このことから、 Az 52 1細胞の細胞数の減少はオリゴヌクレオチド誘導体の毒 性に由来するものでなく、 WT 1の発現の抑制によるアポトーシス死の結果であ ることが認められた。 また、 本発明のオリゴヌクレオチド誘導体は、 天然型オリ ゴヌクレオチドに比較して、 高いアンチセンス効果を示すことが認められた。  On the other hand, in the case of PC 14 cells, no decrease in cell number was observed in any case. From this, it was confirmed that the decrease in the number of Az521 cells was not due to the toxicity of the oligonucleotide derivative, but was due to apoptotic death due to suppression of WT1 expression. In addition, it was confirmed that the oligonucleotide derivative of the present invention exhibited a higher antisense effect than the natural oligonucleotide.
(3) 本発明のオリゴヌクレオチド誘導体と S- oligo との比較  (3) Comparison between oligonucleotide derivative of the present invention and S-oligo
さらに、 ホスホロチォェ一ト型オリゴヌクレオチド誘導体（S_oligo) を用いて 上記 （2) と同様の条件で実験を行った。 なお、 初日の投与濃度は、 2 5 2 g/ m lであった。 結果を図 5に示す。  Further, an experiment was performed using the phosphorothioate-type oligonucleotide derivative (S_oligo) under the same conditions as in (2) above. The concentration on the first day was 252 g / ml. Fig. 5 shows the results.
結果から明らかなように、 本発明のォリゴヌクレオチド誘導体についてはアン チセンス効果が見られたが、 S-oligo、 D- oligoの場合にはアンチセンス効果は見 られなかった。 この結果から、 本発明のオリゴヌクレオチド誘導体は、 アンチセ ンスとして広く知られている S- oligo と比較しても高いアンチセンス効果が確 認された。 この結果はインビトロでの実験で得られた （Tm値） と良い相関を示 している。  As is clear from the results, the oligonucleotide derivative of the present invention exhibited an antisense effect, but the S-oligo and D-oligo did not show an antisense effect. From these results, it was confirmed that the oligonucleotide derivative of the present invention had a higher antisense effect than S-oligo widely known as antisense. This result shows a good correlation with the value obtained in the in vitro experiment (Tm value).
実施例 4 ： 本発明のオリゴヌクレオチド誘導体の WT 1に対する Example 4: Oligonucleotide derivatives of the invention against WT1
アンチセンス効果  Antisense effect
本発明のオリゴヌクレオチド誘導体ァンチセンスを実施例 3と同じ方法で A z 52 1細胞に投与し、 細胞数の減少を指標にアンチセンス効果を評価した。 比較 のために、 下記の式で表されるスクランブル配列のオリゴヌクレオチド誘導体を 用いて同じ条件で実験を行った。  The oligonucleotide derivative antisense of the present invention was administered to Az521 cells in the same manner as in Example 3, and the antisense effect was evaluated using the decrease in the number of cells as an index. For comparison, an experiment was performed under the same conditions using an oligonucleotide derivative having a scrambled sequence represented by the following formula.
5' -GUCGGAGCCCAUUUGCUG-3' 本発明アンチセンス（5bc- AS) (配列番号 6 ) 5' -UCCGUCGGUAGUCCGUAG-3' 比較スクランブル配列 （Sc) (配列番号 8 ) 5' -GCUAGCGCUUGCGAGUUC-3' 比較スクランブル配列 （Sc-B) (配列番号 1 0 ) 【化 6】
Figure imgf000016_0001
5'-GUCGGAGCCCAUUUGCUG-3 'Antisense of the present invention (5bc-AS) (SEQ ID NO: 6) 5'-UCCGUCGGUAGUCCGUAG-3' Comparative scramble sequence (Sc) (SEQ ID NO: 8) 5'-GCUAGCGCUUGCGAGUUC-3 'Comparative scramble sequence ( Sc-B) (SEQ ID NO: 10)
Figure imgf000016_0001
( 1 ) 本発明のアンチセンスオリゴヌクレオチド誘導体（5bc-AS)を濃度を変え て投与した場合の、 Az 521細胞の増殖抑制効果を図 5に示した。 同時に S c 一 B配列についても同じ条件で試験した。 (1) FIG. 5 shows the growth inhibitory effect of Az521 cells when the antisense oligonucleotide derivative (5bc-AS) of the present invention was administered at different concentrations. At the same time, the Sc-B sequence was tested under the same conditions.
投与は、 96ゥエルプレートを用い、 各ゥエル当り 5 X 103個の Az 52 1 細胞を蒔き、 各オリゴヌクレオチドを、 図 6に示した異なる濃度 （初期濃度を表 示している） でゥエルに添加し、 更に 24時間毎に初期濃度の半分の濃度で 3回 添加した。 最初のオリゴヌクレオチド添加時から 4日後に生存細胞数をカウント した。 Administration, using 96 © El plates, plated each Ueru per 5 X 10 3 cells of Az 52 1 cells, each oligonucleotide in Ueru at different concentrations indicated in FIG. 6 (the initial concentration are shown Table) It was added three times every 24 hours at half the initial concentration. The number of surviving cells was counted 4 days after the first oligonucleotide addition.
図 6から明らかなように、 5bc-AS では、 濃度依存的に細胞増殖を抑制するこ とが判った。 また、 スクランブル配列である比較オリゴヌクレオチド （S c—B) の細胞増殖抑制作用は、 いずれの濃度においても 5bc- AS よりも著しく低いこと が確認された。  As is evident from FIG. 6, it was found that 5bc-AS suppressed cell growth in a concentration-dependent manner. In addition, it was confirmed that the cell growth inhibitory effect of the comparative oligonucleotide (Sc-B), which is a scrambled sequence, was significantly lower than that of 5bc-AS at any concentration.
(2) 同様の試験を 50 i g/m l濃度 （初期濃度） で行った。 比較例として異 なる 2種のスクランブル配列のオリゴヌクレオチド （S c) 及び （S c—B) を 用いて細胞増殖抑制効果を試験した。 結果を図 7に示す。  (2) The same test was performed at a concentration of 50 ig / ml (initial concentration). As a comparative example, cell proliferation inhibitory effects were tested using oligonucleotides (Sc) and (Sc-B) having two different scrambled sequences. Fig. 7 shows the results.
図 7から明らかなように、 2種のスクランブル配列のオリゴヌクレオチドに比 較した、 本発明のアンチセンスオリゴヌクレオチド誘導体は顕著な細胞増殖抑制 効果を示した。  As is evident from FIG. 7, the antisense oligonucleotide derivative of the present invention exhibited a remarkable cell growth inhibitory effect as compared with the two scrambled sequence oligonucleotides.
(3) 上記実施例 3と同様な方法で、 オリゴヌクレオチド類縁体ユニットの置換 数が異なる下記塩基配列を有する本発明のアンチセンスオリゴヌクレオチド誘導 体を合成し、 上記 5 bc-AS と同様に細胞増殖抑制作用を試験したところ、 上記 5 be - ASとほぼ匹敵する作用が確認された。 (3) In the same manner as in Example 3 above, the antisense oligonucleotide derivative of the present invention having the following base sequence in which the number of substitutions of the oligonucleotide analog unit is different The body was synthesized and tested for cell growth inhibitory activity in the same manner as in 5bc-AS. As a result, an effect almost equivalent to that in 5be-AS was confirmed.
5' -GUCGGAGCCCAUUUGCUG-3' (配列番号 10)  5'-GUCGGAGCCCAUUUGCUG-3 '(SEQ ID NO: 10)
5 ' -GUCGGAGCCCAUUUGCUG-3' (配列番号 11 )  5'-GUCGGAGCCCAUUUGCUG-3 '(SEQ ID NO: 11)
上記アンチセンスオリゴヌクレオチド誘導体（1番目と 2番目）のゥラシル（U) をチミン （T) に置換した下記塩基配列のアンチセンスオリゴヌクレオチド誘導 体も上記アンチセンスと同程度のアンチセンス効果が予測される。  An antisense oligonucleotide derivative having the following base sequence in which peracyl (U) of the above antisense oligonucleotide derivatives (first and second) is substituted with thymine (T) is expected to have the same antisense effect as the above antisense. You.
5' -GTCGGAGCCCATTTGCTG-3' (配列番号 7)  5'-GTCGGAGCCCATTTGCTG-3 '(SEQ ID NO: 7)
5' -GTCGGAGCCCATTTGCTG-3' (配列番号 12)  5'-GTCGGAGCCCATTTGCTG-3 '(SEQ ID NO: 12)
5' -GTCGGAGCCCATTTGCTG-3' (配列番号 13) 5'-GTCGGAGCCCATTTGCTG-3 '(SEQ ID NO: 13)

Claims

請 求 の' 範 囲 The scope of the claims
1. 糖部を修飾したヌクレオチド類縁体ユニットを 1つ以上含有する、 ウィル ムス腫瘍遺伝子 （WT 1) に対するアンチセンスオリゴヌクレオチド誘導体。 1. An antisense oligonucleotide derivative against the Wilms oncogene (WT 1), which contains one or more nucleotide analog units with modified sugar moieties.
2. 糖部を修飾したヌクレオチド類縁体ュニットが下記の一般式  2. Nucleotide analogue unit with modified sugar moiety has the following general formula
Figure imgf000018_0001
Figure imgf000018_0001
[式中、 Bはピリミジン核酸塩基もしくはプリン核酸塩基又はそれらの類縁体で ある] で表される構造を有する請求項 1記載のアンチセンスオリゴヌクレオチド 誘導体。  2. The antisense oligonucleotide derivative according to claim 1, having a structure represented by the formula: wherein B is a pyrimidine nucleobase or purine nucleobase or an analog thereof.
3. 糖部を修飾したヌクレオチド類縁体ュニッ卜が下記の一般式  3. The sugar unit-modified nucleotide analog unit has the following general formula:
Figure imgf000018_0002
Figure imgf000018_0002
で表されることを特徴とする請求項 2記載のアンチセンスオリゴヌクレオチド誘 導体。 3. The antisense oligonucleotide derivative according to claim 2, which is represented by:
4. WT 1の翻訳開始領域を含む領域に対する請求項 1、 2又は 3記載のアン チセンスオリゴヌクレオチド誘導体。  4. The antisense oligonucleotide derivative according to claim 1, 2 or 3, corresponding to a region containing the translation initiation region of WT1.
5. 次の塩基配列： 5. The following nucleotide sequence:
5 ' —GTCGGAGCCCATTTGCTG - 3 '  5 '—GTCGGAGCCCATTTGCTG-3'
5 ' -GUCGGAGCCCAUUUGCUG-3 ' を含む請求項 1〜 4の何れかの項に記載のアンチセンスオリゴヌクレオチド誘導 体。 5 '-GUCGGAGCCCAUUUGCUG-3' The antisense oligonucleotide derivative according to any one of claims 1 to 4, comprising:
6. 下線部分の塩基の 1つ以上を請求項 2記載のヌクレオチド類縁体ュニット で置換したオリゴヌクレオチド誘導体を含む請求項 1〜 5のいずれかの項に記載 のアンチセンスオリゴヌクレオチド誘導体。  6. The antisense oligonucleotide derivative according to any one of claims 1 to 5, comprising an oligonucleotide derivative in which one or more underlined bases have been substituted with the nucleotide analog unit according to claim 2.
5  Five
5 一 GTCGGAGCCCATTTGCTG— 3 '  5 One GTCGGAGCCCATTTGCTG— 3 '
5 -GTCGGAGCCCATTTGCTG- 3 '  5 -GTCGGAGCCCATTTGCTG- 3 '
5 -GUCGGAGCCCAUUUGCUG- 3 '  5 -GUCGGAGCCCAUUUGCUG- 3 '
5 -GUCGGAGCCCAUUUGCUG- 3 '  5 -GUCGGAGCCCAUUUGCUG- 3 '
5 -GUCGGAGCCCAUUUGCUG- 3 '  5 -GUCGGAGCCCAUUUGCUG- 3 '
7. 下線の塩基部分が請求項 2記載のヌクレオチド類縁体ュニットで置換した ォリゴヌクレオチド誘導体を含む請求項 6記載のアンチセンスォリゴヌクレオチ ド誘導体。  7. The antisense oligonucleotide derivative according to claim 6, wherein the underlined base moiety contains an oligonucleotide derivative substituted with the nucleotide analog unit according to claim 2.
5  Five
5 -GTCGGAGCCCATTTGCTG- 3 '  5 -GTCGGAGCCCATTTGCTG- 3 '
5 -GTCGGAGCCCATTTGCTG- 3 '  5 -GTCGGAGCCCATTTGCTG- 3 '
5 -GUCGGAGC C C AUUUGCUG- 3 '  5 -GUCGGAGC C C AUUUGCUG- 3 '
5 -GUCGGAGCCCAUUUGCUG- 3 '  5 -GUCGGAGCCCAUUUGCUG- 3 '
5 -GUCGGAGC CC AUUUGCUG- 3 '  5 -GUCGGAGC CC AUUUGCUG- 3 '
8. 請求項 5記載の塩基配列と相補的な配列の DNA又は RNAとストリンジ ェントな条件下でハイプリダイズするオリゴヌクレオチド誘導体を含む請求項 1 〜 4記載のァンチセンスォリゴヌクレオチド誘導体。  8. The antisense oligonucleotide derivative according to any one of claims 1 to 4, further comprising an oligonucleotide derivative that hybridizes with DNA or RNA having a sequence complementary to the base sequence according to claim 5 under stringent conditions.
9 請求項 1〜8の何れかの項に記載の WT 1に対するアンチセンスオリゴヌ クレオチド誘導体を有効成分として含有する抗腫瘍剤。  9. An antitumor agent comprising the antisense oligonucleotide derivative for WT1 according to any one of claims 1 to 8 as an active ingredient.
PCT/JP2001/006049 2000-07-19 2001-07-12 Novel antisense oligonucleotide derivatives against wilms's tumor gene WO2002006297A1 (en)

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Publication number Priority date Publication date Assignee Title
US11234995B2 (en) 2016-01-07 2022-02-01 Osaka University α-synuclein expression inhibitor

Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0841068A1 (en) * 1995-06-01 1998-05-13 Kishimoto, Tadamitsu Leukemic cell growth inhibitor containing antisense oligonucleotide derivative against wilms' tumor gene (wt1)
EP1004319A1 (en) * 1997-07-16 2000-05-31 Haruo Sugiyama Remedies for solid tumor containing wilms' tumor gene (wt1) expression inhibitors
EP1013661A1 (en) * 1997-03-07 2000-06-28 Takeshi Imanishi Novel bicyclonucleoside and oligonucleotide analogues

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0841068A1 (en) * 1995-06-01 1998-05-13 Kishimoto, Tadamitsu Leukemic cell growth inhibitor containing antisense oligonucleotide derivative against wilms' tumor gene (wt1)
EP1013661A1 (en) * 1997-03-07 2000-06-28 Takeshi Imanishi Novel bicyclonucleoside and oligonucleotide analogues
EP1004319A1 (en) * 1997-07-16 2000-05-31 Haruo Sugiyama Remedies for solid tumor containing wilms' tumor gene (wt1) expression inhibitors

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11234995B2 (en) 2016-01-07 2022-02-01 Osaka University α-synuclein expression inhibitor

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