JPH04235919A - Anticancer drug - Google Patents
Anticancer drugInfo
- Publication number
- JPH04235919A JPH04235919A JP3003528A JP352891A JPH04235919A JP H04235919 A JPH04235919 A JP H04235919A JP 3003528 A JP3003528 A JP 3003528A JP 352891 A JP352891 A JP 352891A JP H04235919 A JPH04235919 A JP H04235919A
- Authority
- JP
- Japan
- Prior art keywords
- ribozyme
- nucleotide
- ras
- activated
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000002246 antineoplastic agent Substances 0.000 title claims abstract description 7
- 229940041181 antineoplastic drug Drugs 0.000 title abstract 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical group O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims abstract description 5
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical class N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims abstract description 5
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical class NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims abstract description 5
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000004480 active ingredient Substances 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 4
- 108090000994 Catalytic RNA Proteins 0.000 abstract description 30
- 102000053642 Catalytic RNA Human genes 0.000 abstract description 27
- 108091092562 ribozyme Proteins 0.000 abstract description 27
- 108020004999 messenger RNA Proteins 0.000 abstract description 14
- 108700042226 ras Genes Proteins 0.000 abstract description 6
- 108700020796 Oncogene Proteins 0.000 abstract description 4
- 101150040459 RAS gene Proteins 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 239000002773 nucleotide Substances 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 14
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 8
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 108091027075 5S-rRNA precursor Proteins 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 239000012888 bovine serum Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 3
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000012869 ethanol precipitation Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- HWCKGOZZJDHMNC-UHFFFAOYSA-M tetraethylammonium bromide Chemical compound [Br-].CC[N+](CC)(CC)CC HWCKGOZZJDHMNC-UHFFFAOYSA-M 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241001131785 Escherichia coli HB101 Species 0.000 description 2
- 102100029974 GTPase HRas Human genes 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 108090001102 Hammerhead ribozyme Proteins 0.000 description 2
- 101000584633 Homo sapiens GTPase HRas Proteins 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 229940124276 oligodeoxyribonucleotide Drugs 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 150000008300 phosphoramidites Chemical class 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 101710086015 RNA ligase Proteins 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- -1 dimethoxytrityl group Chemical group 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002681 effect on RNA Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- AFQIYTIJXGTIEY-UHFFFAOYSA-N hydrogen carbonate;triethylazanium Chemical compound OC(O)=O.CCN(CC)CC AFQIYTIJXGTIEY-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960003646 lysine Drugs 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
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- 230000007017 scission Effects 0.000 description 1
- ZNJHFNUEQDVFCJ-UHFFFAOYSA-M sodium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;hydroxide Chemical compound [OH-].[Na+].OCCN1CCN(CCS(O)(=O)=O)CC1 ZNJHFNUEQDVFCJ-UHFFFAOYSA-M 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
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- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Landscapes
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は、癌遺伝子より転写され
るｍＲＮＡに対して特異的な切断活性を有するリボザイ
ムを有効成分とする抗腫瘍剤に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antitumor agent containing as an active ingredient a ribozyme that has specific cleaving activity against mRNA transcribed from cancer genes.
【０００２】0002
【従来の技術】ＲＮＡは、遺伝情報伝達物質としての役
割を担っているばかりでなく、それ自身が触媒的に作用
してＲＮＡの切断や連結を行なう機能を有する（以下、
このような機能を有するＲＮＡを「リボザイム」という
。）ことが知られている。そして、リボザイムは、切断
部位近傍の塩基配列や反応の性質からさらに、■ハンマ
−ヘッド型ＲＮＡ、■ヘアピン型ＲＮＡ、■クロ−バ−
リ−フ状ＲＮＡ、■その他の自己切断反応ＲＮＡの４種
類に分類できる（蛋白質 核酸 酵素、ｖｏｌ．３
５、 Ｎｏ．１３、 ２１９１−２２００、 （１９９
０））。[Prior Art] RNA not only plays a role as a genetic information transmitter, but also has the function of catalytically acting to cleave and link RNA (hereinafter referred to as
RNA having such a function is called a "ribozyme." )It is known. Based on the base sequence near the cleavage site and the nature of the reaction, ribozymes can be further classified into: (1) hammerhead RNA, (2) hairpin RNA, and (2) cloverhead RNA.
It can be classified into four types: leaf-like RNA, and other self-cleavage reaction RNA (Protein, Nucleic Acids, Enzymes, vol. 3).
5. No. 13, 2191-2200, (199
0)).
【０００３】本発明者らは、ハンマ−ヘッド型リボザイ
ムが、標的ＲＮＡを配列特異的に切断するエンドヌクレ
ア−ゼとして利用できることを見出し、この内容につい
てはすでに報告しており、以下に詳細を記載する（Ｎｕ
ｃｌｅｉｃ Ａｃｉｄ Ｒｅｓ．， ｖｏｌ．１７，
７０５９−７０７１，（１９８９） ）。ｃ−Ｈａ−ｒ
ａｓ遺伝子は、分子量２１，０００の蛋白質（ｐ２１
）をコ−ドしている。ヒトの癌から検出された活性化ｒ
ａｓ 遺伝子は、塩基の点突然変異によりｐ２１ のア
ミノ酸にいくつかの変異が生じている。リボザイムがこ
のうちの１塩基の違いを認識できるか否かを調べた。リ
ボザイムは、ｐ２１ の１２番目のＧｌｙ（ＧＧＵ）が
Ｖａｌ（ＧＵＵ）に変異した活性化ｒａｓ 遺伝子ｍＲ
ＮＡを切断し、正常ｍＲＮＡは切断しないように設計し
た。そして、試験管内の実験においては、このリボザイ
ムが、活性化ｒａｓ 遺伝子ｍＲＮＡ分子のみを切断し
、正常ｍＲＮＡ分子は切断しないことを確認したのであ
る。[0003] The present inventors have discovered that a hammerhead ribozyme can be used as an endonuclease that cleaves target RNA in a sequence-specific manner, and this content has already been reported and will be described in detail below. (Nu
cleic acid Res. , vol. 17,
7059-7071, (1989)). c-Har-r
The as gene is a protein with a molecular weight of 21,000 (p21
) is coded. Activated r detected from human cancer
The as gene has several mutations in the p21 amino acids due to point mutations in the bases. We investigated whether ribozymes could recognize differences in one of these bases. Ribozyme is an activated ras gene mR in which Gly (GGU) at position 12 of p21 is mutated to Val (GUU).
It was designed to cleave NA but not normal mRNA. In vitro experiments confirmed that this ribozyme only cleaves activated ras gene mRNA molecules, but not normal mRNA molecules.
【０００４】0004
【発明が解決しようとする課題】本発明者らは、リボザ
イムの有する触媒活性が１塩基の差異をも認識する機能
を利用して、腫瘍遺伝子より転写されるｍＲＮＡを特異
的に切断するリボザイムが有効な抗腫瘍効果を示すこと
を見出し、本発明を完成した。[Problems to be Solved by the Invention] The present inventors have developed a ribozyme that specifically cleaves mRNA transcribed from tumor genes by utilizing the ability of ribozyme's catalytic activity to recognize even a single base difference. The present invention was completed based on the discovery that it exhibits an effective antitumor effect.
【０００５】[0005]
【課題を解決するための手段】本発明は、一般式[Means for Solving the Problems] The present invention provides the general formula
【００
０６】00
06]
【化３】[Chemical formula 3]
【０００７】または[0007] or
【０００８】[0008]
【化４】[C4]
【０００９】「式中、Ａはアデニンヌクレオチド、Ｕは
ウラシルヌクレオチド、Ｃはシトシンヌクレオチド、Ｇ
はグアニンヌクレオチドを表わし、３’および５’はそ
れぞれ３’末端および５’末端を表わす。」で表わされ
る化合物を有効成分とする抗腫瘍剤に関する。"In the formula, A is an adenine nucleotide, U is a uracil nucleotide, C is a cytosine nucleotide, G
represents a guanine nucleotide, and 3' and 5' represent the 3' and 5' ends, respectively. The present invention relates to an antitumor agent containing a compound represented by the following as an active ingredient.
【００１０】本発明において、「腫瘍」とは、活性化し
た癌遺伝子によってもたらされる疾患を意味するが、と
くに好適には、活性化ｃ−Ｈａ−ｒａｓ遺伝子によって
もたらされる疾患を意味するものとする。[0010] In the present invention, the term "tumor" refers to a disease caused by an activated oncogene, and particularly preferably refers to a disease caused by an activated c-Ha-ras gene. .
【００１１】本発明の化合物（Ｉ ）および（ＩＩ）は
、公知の方法（Ｎｕｃｌｅｉｃ Ａｃｉｄ Ｒｅｓ．，
ｖｏｌ．１７， ７０５９−７０７１）で製造するこ
とができる。Compounds (I) and (II) of the present invention can be prepared by a known method (Nucleic Acid Res.,
vol. 17, 7059-7071).
【００１２】本発明において、リボザイムの抗腫瘍効果
の確認は、以下の方法により行なうことができる。[0012] In the present invention, the antitumor effect of ribozyme can be confirmed by the following method.
【００１３】すなわち、ＮＩＨ−３Ｔ３ 細胞を、目的
とするリボザイムを発現するプラスミドにより形質転換
した細胞を調製し、さらにこの細胞に活性化ｃ−Ｈａ−
ｒａｓ遺伝子を形質導入して、リボザイムを発現しない
プラスミドにより形質転換した細胞とのフォ−カス形成
率を比較する方法である。Specifically, NIH-3T3 cells are transformed with a plasmid expressing the ribozyme of interest, and then activated c-Ha-
This method involves transducing the ras gene and comparing the focus formation rate with cells transformed with a plasmid that does not express ribozyme.
【００１４】本発明により得られるリボザイムは、様々
な態様で投与することにより極めて有効な抗腫瘍効果を
示す。[0014] The ribozyme obtained according to the present invention exhibits extremely effective antitumor effects when administered in various ways.
【００１５】本発明のリボザイムを投与するための方法
は、経口投与または非経口投与（例えば、静注、筋注、
皮下注または腹腔内注）であり、投与される組成物には
、治療上有効量の生成物が、薬理上許容される希釈剤、
担体および／又はアジュバントとともに通常含有される
。[0015] The method for administering the ribozyme of the present invention includes oral administration or parenteral administration (eg, intravenous injection, intramuscular injection,
(subcutaneous or intraperitoneal injection) and the administered composition includes a therapeutically effective amount of the product in a pharmaceutically acceptable diluent,
It is usually included together with a carrier and/or an adjuvant.
【００１６】ヒト血清アルブミンのような標準的希釈剤
が本発明の医薬的組成物として考えられ、生理的食塩水
のような標準的担体が同様に考えられうる。Standard diluents such as human serum albumin are contemplated for the pharmaceutical compositions of the present invention, as are standard carriers such as physiological saline.
【００１７】投与形態としては、皮下注射、静脈内注射
、筋肉注射、座剤などの非経口投与法、または、錠剤、
カプセル剤、散剤、顆粒剤などによる経口投与法が挙げ
られる。[0017] As for the administration form, subcutaneous injection, intravenous injection, intramuscular injection, parenteral administration methods such as suppositories, tablets, etc.
Oral administration methods include capsules, powders, granules, and the like.
【００１８】なお、リボザイム分子をリポソ−ムに封入
して、経口または非経口により投与することも可能であ
る。[0018] It is also possible to encapsulate the ribozyme molecule in a liposome and administer it orally or parenterally.
【００１９】投与量は、投与経路または投与回数などに
よって異なるが、例えば、成人に対しては、通常は、一
日１ 〜１，０００ μｇ／ｋｇ体重を一日に１回また
は数回に分けて投与するのが好ましい。The dosage varies depending on the route of administration and the number of administrations, but for adults, it is usually 1 to 1,000 μg/kg body weight per day or divided into several doses per day. It is preferable to administer the drug.
【００２０】以下、参考例、実施例を挙げて本発明をよ
り具体的に説明するが、これらは本発明の範囲を制限す
るものではない。The present invention will be explained in more detail below with reference to reference examples and examples, but these are not intended to limit the scope of the present invention.
【００２１】なお、以下の、参考例、実施例においては
、アデニンヌクレオチドをＡ、グアニンヌクレオチドを
Ｇ、シトシンヌクレオチドをＣ、ウラシルヌクレオチド
をＵと記載する。In the following Reference Examples and Examples, adenine nucleotides are indicated as A, guanine nucleotides as G, cytosine nucleotides as C, and uracil nucleotides as U.
【００２２】［参考例］オリゴデオキシリボヌクレオチ
ドは、アプライド−バイオシステムズ社製３８０Ａ
ＤＮＡシンセサイザ−を用いホスホロアミダイト法で合
成した。オリゴリボヌクレオチドは、５’水酸基の保護
基にジメトキシトリチル基、２’−水酸基の保護基にテ
トラヒドロピラニル基を用いたホスホロアミダイト法（
Ｎｕｃｌｅｉｃ Ａｃｉｄ Ｒｅｓ．， ｖｏｌ．１７
７０５９−７０７１， （１９８９） ）で合成した
。[Reference Example] The oligodeoxyribonucleotide is 380A manufactured by Applied Biosystems.
It was synthesized by the phosphoramidite method using a DNA synthesizer. Oligoribonucleotides can be prepared using the phosphoramidite method (using a dimethoxytrityl group as the protecting group for the 5'-hydroxyl group and a tetrahydropyranyl group as the protecting group for the 2'-hydroxyl group).
Nucleic Acid Res. , vol. 17
7059-7071, (1989)).
【００２３】ｐＲＳＶ−ｒｖ１２ 、ｐＲＳＶ−ｒ１６
１ の構築は、文献（Ｃａｎｃｅｒ Ｒｅｓ．， ｖｏ
ｌ．８０， ２００−２０３，（１９８９））に記載さ
れた方法に従った。[0023] pRSV-rv12, pRSV-r16
The construction of 1 is described in the literature (Cancer Res., vo
l. 80, 200-203, (1989)).
【００２４】参考例１ リボザイムＣＵＡＣＡＣＣＣ
ＵＧＡＵＧＡＡＧＧＧＵＧＡＵＡＣＣＣＵＧＡＡＡＣＡ
ＧＣＧＣｐの調製オリゴリボヌクレオチド（ＵＡＣＣＣ
ＵＧＡＡＡＣＡＧＣＧＣＧ， ５ ｎｍｏｌ ）にポリ
ヌクレオチドキナ−ゼ（１０単位、寶酒造（株）製）、
０．６７ ｍＭ ＡＴＰ 、５０ ｍＭ Ｔｒｉｓ−Ｈ
Ｃｌ （ｐＨ ７．６）、１０ ｍＭ ＭｇＣｌ２ 、
１０ ｍＭ メルカプトエタノ−ルを加え、３７
℃で１時間加温した。フェノ−ルクロロホルムにより処
理し、セファデックスＧ−２５（ファルマシア社製：φ
１．８ ｘ ３７ ｃｍ 、溶媒は ０．１ Ｍ ト
リエチルアンモニウムバイカ−ボネ−ト（以下「ＴＥＡ
Ｂ」という。）で脱塩して、ＡＴＰ 、ＡＤＰ を除い
た。Reference Example 1 Ribozyme CUACACCC
UGAUGAAGGGGUGAUACCCUGAAACA
Preparation of GCGCp oligoribonucleotide (UACCC
UGAAACAGCGCG, 5 nmol), polynucleotide kinase (10 units, manufactured by Takara Shuzo Co., Ltd.),
0.67mM ATP, 50mM Tris-H
Cl (pH 7.6), 10 mM MgCl2,
Add 10 mM mercaptoethanol and
It was heated at ℃ for 1 hour. Treated with phenol chloroform, Sephadex G-25 (manufactured by Pharmacia: φ
1.8 x 37 cm, the solvent was 0.1 M triethylammonium bicarbonate (hereinafter referred to as "TEA").
B”. ) to remove ATP and ADP.
【００２５】得られた ５’−リン酸化オリゴリボヌク
レオチド（ｐＵＡＣＣＣＵＧＡＡＡＣＡＧＣＧＣＧ）を
滅菌水（２０ μｌ ）に溶解し、０．１ Ｍ Ｎａ
ＩＯ４（２．５ μｌ ）を加え、暗所で０ ℃、７０
分放置し、０．１ Ｍ エチレングリコ−ル（２５
μｌ ）を加え、０ ℃、３０分反応させた。これに
２ Ｍ リジン−ＨＣｌ（ｐＨ ８．２、 ５０ μ
ｌ ）を加え、４５℃、 ２ 時間加温し、セファデッ
クスＧ−２５（ φ１．８ ｘ ３７ ｃｍ 、溶媒は
０．１ Ｍ ＴＥＡＢ）で脱塩した。これを陰イオ
ン交換ＨＰＬＣ（ＴＳＫ ｇｅｌ ＤＥＡＥ−２ＳＷ、
φ０．４６ ｘ ２５ ｃｍ：東ソ−（株）製）で精製
することによって５’，３’−ジリン酸を有するオリゴ
リボヌクレオチド（ｐＵＡＣＣＣＵＧＡＡＡＣＡＧＣＧ
Ｃｐ ）を得た。The obtained 5'-phosphorylated oligoribonucleotide (pUACCCUGAAACAGCGCG) was dissolved in sterile water (20 μl), and 0.1 M Na
Add IO4 (2.5 μl) and incubate at 0 °C in the dark for 70 min.
Leave for 0.1 M ethylene glycol (25
μl) was added and reacted at 0°C for 30 minutes. To this was added 2 M lysine-HCl (pH 8.2, 50 μl
1) was added, heated at 45°C for 2 hours, and desalted with Sephadex G-25 (φ1.8 x 37 cm, solvent: 0.1 M TEAB). This was subjected to anion exchange HPLC (TSK gel DEAE-2SW,
φ0.46 x 25 cm: manufactured by Tosoh Corporation) to obtain an oligoribonucleotide having 5',3'-diphosphoric acid (pUACCCUGAAACAGCG).
Cp) was obtained.
【００２６】別途調整したオリゴリボヌクレオチド（Ｃ
ＵＡＣＡＣＣＣＵＧＡＵＧＡＡＧＧＧＵＧＡ， ２．５
ｎｍｏｌ ）および上記のオリゴリボヌクレオチド（
ｐＵＡＣＣＣＵＧＡＡＡＣＡＧＣＧＣｐ， ２．５ ｎ
ｍｏｌ）を ５０ｍＭ ＨＥＰＥＳ−ＮａＯＨ （ｐＨ
７．５），２０ ｍＭ ＭｇＣｌ２， ３．３ ｍＭ
ＤＴＴ， ０．００１％ ＢＳＡ， １０％（ｖ／ｖ
）ＤＭＳＯ， １．３８ 単位／μｌ ＲＮＡ リ
ガ−ゼ（寶酒造（株）製）、１００ μＭ ＡＴＰ の
溶液５０ μｌ 中、６℃で１３．５時間加温した。
フェノ−ルクロロホルム処理をし、陰イオン交換ＨＰＬ
Ｃで目的の鎖長のリボザイムＣＵＡＣＡＣＣＣＵＧＡＵ
ＧＡＡＧＧＧＵＧＡＵＡＣＣＣＵＧＡＡＡＣＡＧＣＧＣ
ｐを得た。[0026] Separately prepared oligoribonucleotide (C
UACACCCUGAUGAAGGGUGA, 2.5
nmol) and the above oligoribonucleotides (
pUACCCUGAAACAGCGCp, 2.5 n
mol) to 50mM HEPES-NaOH (pH
7.5), 20mM MgCl2, 3.3mM
DTT, 0.001% BSA, 10% (v/v
) DMSO, 1.38 units/μl RNA ligase (manufactured by Takara Shuzo Co., Ltd.), and 50 μl of a solution of 100 μM ATP was heated at 6° C. for 13.5 hours. Phenol chloroform treatment and anion exchange HPL
Ribozyme CUACACCCUGAU of desired chain length in C
GAAGGGUGAUACCCUGAAACAGCGC
I got p.
【００２７】参考例２ 活性化ｃ−Ｈａ−ｒａｓ
ＤＮＡテンプレ−トの調製
１２番目のアミノ酸をコ−ドするコドンに点突然変異を
起こした（Ｇｌｙ１２（ＧＧＵ）→Ｖａｌ１２（ＧＵＵ
））活性化ｃ−Ｈａ−ｒａｓＤＮＡを調製した。Reference Example 2 Activated c-Ha-ras
Preparation of DNA template A point mutation was made in the codon encoding the 12th amino acid (Gly12 (GGU) → Val12 (GUU
)) Activated c-Ha-ras DNA was prepared.
【００２８】７本の合成オリゴデオキシリボヌクレオチ
ド（１ ｎｍｏｌ）をそれぞれＴ４ポリヌクレオチドキ
ナ−ゼ（１ μｌ，１０単位）、１ ｍＭ ＡＴＰ（１
５ μｌ）、緩衝液（４ μｌ，２５０ ｍＭ Ｔｒ
ｉｓ−ＨＣｌ （ｐＨ ７．６）， ５０ ｍＭ Ｍｇ
Ｃｌ２，５０ ｍＭ メルカプトエタノ−ル）を加え
、３７℃で１ 時間加温した。Seven synthetic oligodeoxyribonucleotides (1 nmol) were each mixed with T4 polynucleotide kinase (1 μl, 10 units) and 1 mM ATP (1 nmol).
5 μl), buffer (4 μl, 250 mM Tr
is-HCl (pH 7.6), 50 mM Mg
Cl2, 50 mM mercaptoethanol) was added, and the mixture was heated at 37°C for 1 hour.
【００２９】これらを合わせ、緩衝液（４０ μｌ、
３３０ ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ ７．６）
， ３３ ｍＭ ＭｇＣｌ２）、２．５ ｍＭ ＡＴＰ
（４８ μｌ ）を加えて７５℃で５ 分間加熱し
室温まで徐冷した。０．２ Ｍ ２−メルカプトエタノ
−ル（１０ μｌ ）、Ｔ４ ＤＮＡ リガ−ゼ（
２ μｌ，７００単位、寶酒造（株）製）を加え、１５
℃で１５時間反応させた。エタノ−ル沈殿後、１０％ポ
リアクリルアミドゲル電気泳動で分取し、約３ μｇ
の活性化ｃ−Ｈａ−ｒａｓＤＮＡテンプレ−トを得た（
図１）。[0029] Combine these and add buffer (40 μl,
330 mM Tris-HCl (pH 7.6)
, 33mM MgCl2), 2.5mM ATP
(48 μl) was added, heated at 75°C for 5 minutes, and slowly cooled to room temperature. 0.2 M 2-mercaptoethanol (10 μl), T4 DNA ligase (
Add 2 μl, 700 units, manufactured by Takara Shuzo Co., Ltd., and add 15
The reaction was carried out at ℃ for 15 hours. After ethanol precipitation, approximately 3 μg was fractionated by 10% polyacrylamide gel electrophoresis.
An activated c-Ha-ras DNA template was obtained (
Figure 1).
【００３０】なお、正常ｃ−Ｈａ−ｒａｓＤＮＡテンプ
レ−トも同様の方法で調製した。[0030] A normal c-Ha-ras DNA template was also prepared in the same manner.
【００３１】参考例３ Ｔ７ ポリメラ−ゼによる
活性化ｃ−Ｈａ−ｒａｓ ｍＲＮＡの調製
転写反応は、公知の方法（実験医学、第８巻、（１３）
、１６８５−１６８９頁、（１９９０））により、次の
条件で行なった。得られた活性化ｃ−Ｈａ−ｒａｓＤＮ
Ａ テンプレ−ト（３０ ｐｍｏｌ ）、緩衝液（４０
ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ ８．１）， ２０
ｍＭ ＭｇＣｌ２， ５ ｍＭＤＴＴ， １ ｍＭス
ペルミジン， ０．０１％（ｖ／ｖ） Ｔｒｉｔｏｎ
Ｘ−１００， ０．０５ μｇ／μｌ 牛血清アルブミ
ン、４ｍＭ ４ＮＴＰｓ（ＡＴＰ，ＧＴＰ，ＣＴＰ，
およびＵＴＰ：以下「４ＮＴＰｓ」 という。）、８％
（Ｗ／Ｖ） ポリエチレングリコ−ル）に溶解し、Ｔ７
ＲＮＡ ポリメラ−ゼ（８４０ 単位、ファルマシ
ア社製）を加え、３７℃で２ 時間加温することにより
転写反応を行なった（図１）。フェノ−ルクロロホルム
処理をして、セファデックスＧ−５０（φ１．８ ｘ
３７ｃｍ， 溶媒は０．１ Ｍ ＴＥＡＢ）で脱塩し
、未反応の ４ＮＴＰｓを除いた後、８ Ｍ 尿素を含
む１０％ ポリアクリルアミドゲル電気泳動で精製した
。Reference Example 3 Preparation of activated c-Ha-ras mRNA using T7 polymerase The transcription reaction was performed using a known method (Jikken Igaku, Vol. 8, (13)
, pp. 1685-1689, (1990)) under the following conditions. The obtained activated c-Ha-rasDN
A template (30 pmol), buffer solution (40 pmol)
mM Tris-HCl (pH 8.1), 20
mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.01% (v/v) Triton
X-100, 0.05 μg/μl Bovine serum albumin, 4mM 4NTPs (ATP, GTP, CTP,
and UTP: hereinafter referred to as "4NTPs". ), 8%
(W/V) dissolved in polyethylene glycol), T7
A transcription reaction was performed by adding RNA polymerase (840 units, manufactured by Pharmacia) and heating at 37°C for 2 hours (Fig. 1). After treatment with phenol chloroform, Sephadex G-50 (φ1.8 x
After desalting with 0.1 M TEAB (solvent: 0.1 M TEAB) to remove unreacted 4NTPs, it was purified by electrophoresis on a 10% polyacrylamide gel containing 8 M urea.
【００３２】なお、正常ｃ−Ｈａ−ｒａｓ ｍＲＮＡ
も同様の方法で調製した。[0032] Normal c-Ha-ras mRNA
was also prepared in a similar manner.
【００３３】参考例４ 活性化ｃ−Ｈａ−ｒａｓ
ｍＲＮＡの５’末端標識
活性化ｃ−Ｈａ−ｒａｓＲＮＡ（２０ ｐｍｏｌ ）を
０．１ Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ ８．０ ）に溶
解し、大腸菌由来アルカリ性フォスファタ−ゼ（０．０
２単位、寶酒造（株）製）を加え、３７℃１ 時間加温
した後、フェノ−ルクロロホルム処理を２回行ない、［
γ−３２Ｐ］ＡＴＰ （５ μＣｉ）、５０ ｍＭ Ｔ
ｒｉｓ−ＨＣｌ（ｐＨ ７．６）、１０ ｍＭ ＭｇＣ
ｌ２ 、１０ ｍＭ ２−メルカプトエタノ−ル、Ｔ４
− ポリヌクレオチド・キナ−ゼ（１ 単位、 寶酒造
（株）製で５’末端をリン酸化し、ＮＥＮＳＯＲＢ ２
０（デュポン社製）脱塩、除蛋白を行なった。Reference Example 4 Activated c-Ha-ras
5'-end labeled activated c-Ha-rasRNA (20 pmol) of mRNA was dissolved in 0.1 M Tris-HCl (pH 8.0), and E. coli alkaline phosphatase (0.0
2 units of Takara Shuzo Co., Ltd.) were added, heated at 37°C for 1 hour, and then treated with phenol chloroform twice.
γ-32P]ATP (5 μCi), 50 mM T
ris-HCl (pH 7.6), 10 mM MgC
l2, 10mM 2-mercaptoethanol, T4
- Polynucleotide kinase (1 unit, manufactured by Takara Shuzo Co., Ltd., phosphorylates the 5' end, and NENSORB 2
0 (manufactured by DuPont) and was subjected to desalting and protein removal.
【００３４】なお、正常ｃ−Ｈａ−ｒａｓ ｍＲＮＡ
も同様の方法で標識した。[0034] Note that normal c-Ha-ras mRNA
were also labeled in the same manner.
【００３５】参考例５ ｉｎ ｖｉｔｒｏ での切
断反応参考例４で得られた活性化ｃ−Ｈａ−ｒａｓ
ｍＲＮＡ（５ ｐｍｏｌ）に、参考例１で得られたリボ
ザイム（７．５ ｐｍｏｌ）を加え、４０ ｍＭ Ｔｒ
ｉｓ−ＨＣｌ（ｐＨ ７．５）、２０ ｍＭ ＮａＣｌ
、２５ ｍＭ ＭｇＣｌ２ になる溶液を加え、３７℃
で 加温し、５０ ｍＭ ＥＤＴＡ を加え反応を停
止した。５５℃、３３分急冷したものを ８ Ｍ 尿
素を含む １０％ ポリアクリルアミドゲル電気泳動
で分析した。切断率はゲルを切り出し、液体シンチレ−
ションカウンタ−で測定することにより求めた。Reference Example 5 In vitro cleavage reaction Activated c-Ha-ras obtained in Reference Example 4
The ribozyme (7.5 pmol) obtained in Reference Example 1 was added to mRNA (5 pmol), and 40 mM Tr
is-HCl (pH 7.5), 20 mM NaCl
, add a solution of 25 mM MgCl2 and heat to 37 °C.
The reaction was stopped by adding 50 mM EDTA. The mixture was rapidly cooled at 55° C. for 33 minutes and analyzed by electrophoresis on a 10% polyacrylamide gel containing 8 M urea. Cutting rate is determined by cutting out the gel and cutting out the liquid scintillator.
It was determined by measuring with a tion counter.
【００３６】対照として、正常ｃ−Ｈａ−ｒａｓ ｍ
ＲＮＡについても同様の実験を行なった。As a control, normal c-Ha-ras m
Similar experiments were conducted with RNA.
【００３７】この結果、参考例１で得られたリボザイム
は、正常ｃ−Ｈａ−ｒａｓｍ ＲＮＡは切断せず、活
性化ｃ−Ｈａ−ｒａｓ ｍＲＮＡのみを切断すること
が確認された。As a result, it was confirmed that the ribozyme obtained in Reference Example 1 did not cleave normal c-Ha-rasm RNA, but only cleaved activated c-Ha-ras mRNA.
【００３８】[0038]
【実施例】実施例１ プラスミドｐＫＣＳの構築ｐＲ
ＳＶｎｅｏ （５ μｌ， １．４ ｐｍｏｌ ）をＨ
ｉｎｄ ＩＩＩ（１２ 単位） で３７℃、 一夜加温
して消化し、エタノ−ル沈殿により消化物を得た。これ
に滅菌水（１０ μｌ ）、緩衝液（４ μｌ， ３
３０ ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ ７．６）， ３
３ ｍＭ ＭｇＣｌ２， ２．５ ｍＭ ＡＴＰ ）、
０．２Ｍ ２−メルカプトエタノ−ル（１ μｌ）、
４０ ｍｅｒ ＣｌａＩ−ＳａｌＩ リンカ−（５ μ
ｌ， ７ ｐｍｏｌ：図２） 、Ｔ４リガ−ゼ（１０単
位） を加え、１６℃、２時間加温した。エタノ−ル沈
殿後、ｐＫＣＳを得た。これを大腸菌ＨＢ１０１ にト
ランスフェクトし、文献公知の方法（Ａｎａｌ． Ｂｉ
ｏｃｅｍ．， ｖｏｌ．１１３ ３４−４２， （１９
８９））に従い、２５０ ｍｌ の培地から約４５０
μｇのｐＫＣＳを単離した（図３）。[Example] Example 1 Construction of plasmid pKCS pR
SVneo (5 μl, 1.4 pmol) was added to H
The mixture was digested with indIII (12 units) at 37° C. overnight, and the digested product was obtained by precipitation with ethanol. To this, sterile water (10 μl) and buffer solution (4 μl, 3
30 mM Tris-HCl (pH 7.6), 3
3mM MgCl2, 2.5mM ATP),
0.2M 2-mercaptoethanol (1 μl),
40 mer ClaI-SalI linker (5μ
1,7 pmol (Fig. 2) and T4 ligase (10 units) were added, and the mixture was heated at 16°C for 2 hours. After ethanol precipitation, pKCS was obtained. This was transfected into Escherichia coli HB101, and a method known in the literature (Anal.
ocem. , vol. 113 34-42, (19
89)) from 250 ml of medium.
μg of pKCS was isolated (Figure 3).
【００３９】実施例２ プラスミドｐＲＺ１の調製ｐ
ＫＣＳ（７．５ μｌ， ２．５ ｐｍｏｌ）をＣｌ
ａ Ｉ （１５単位）で３７℃で２ 時間加温し消化後
、Ｓａｌ Ｉ （３０ 単位）で３７℃で一夜加温して
消化した。０．１ Ｍ Ｔｒｉｓ−ＨＣｌ （ｐＨ
８．０，４８ μｌ ）に溶解し、大腸菌由来アルカ
リ性フォスファタ−ゼ（０．７ 単位）を加え、３７℃
で２時間加温した。フェノ−ルクロロフォルム処理後、
エタノ−ル沈殿により消化物を得た。別途調製した、リ
ボザイム（ＣＵＡＣＡＣＣＣＵＧＡＵＧＡＡＧＧＧＵＧ
ＡＵＡＣＣＣＵＧＡＡＡＣＡＧＣＧＣｐ）遺伝子を含む
２本鎖オリゴリボヌクレオチドも同様にＣｌａ Ｉ 、
Ｓａｌ Ｉ で消化した（図４）。このｐＫＣＳＣｌａ
Ｉ − Ｓａｌ Ｉ 消化物に上記のリボザイム遺伝
子を加え、Ｔ４ ＤＮＡリガ−ゼで連結し、ｐＲＺ１
を得た。これを大腸菌ＨＢ１０１ にトランスフェクト
し、文献公知の方法（Ａｎａｌ． Ｂｉｏｃｅｍ．，
ｖｏｌ．１１３ ３４−４２， （１９８９））に従い
、２５０ ｍｌ の培地から約４７０ μｇ のｐＲ
Ｚ１を単離した（図３）。Example 2 Preparation of plasmid pRZ1 p
KCS (7.5 μl, 2.5 pmol) was dissolved in Cl
After digestion with a I (15 units) at 37°C for 2 hours, the mixture was digested with Sal I (30 units) at 37°C overnight. 0.1 M Tris-HCl (pH
8.0.48 μl), added E. coli alkaline phosphatase (0.7 units), and incubated at 37°C.
The mixture was heated for 2 hours. After treatment with phenol chloroform,
A digestate was obtained by ethanol precipitation. Separately prepared ribozyme (CUACACCCUGAUGAAGGGUG
Similarly, the double-stranded oligoribonucleotide containing the AUACCCUGAAACAGCGCp) gene is Cla I,
Digested with Sal I (Figure 4). This pKCSCla
The above ribozyme gene was added to the I-Sal I digest and ligated with T4 DNA ligase to create pRZ1.
I got it. This was transfected into Escherichia coli HB101, and a method known in the literature (Anal. Biocem.,
vol. 113 34-42, (1989)), approximately 470 μg of pR was obtained from 250 ml of medium.
Z1 was isolated (Figure 3).
【００４０】実施例３ プラスミドｐＲＺ２の調製上
記と同様の方法により、リボザイム（ＵＧＣＣＵＡＣＡ
ＣＣＣＵＧＡＵＧＡＧＵＣＧＵＧＡＵＡＣＧＡＣＧＡＡ
ＡＣＡＧＣＧＣＣＡｐ ）遺伝子を組み込んだプラスミ
ドｐＲＺ２を調製した。Example 3 Preparation of plasmid pRZ2 Ribozyme (UGCCUACA
CCCUGAUGAGUCGUGAUACGACGAA
A plasmid pRZ2 incorporating the ACAGCGCCAp) gene was prepared.
【００４１】実施例４ プラスミドｐＲＺ３の調製上
記と同様の方法により、リボザイム（ＣＵＡＣＡＣＣＣ
ＵＧＡＵＧＡＧＧＧＧＧＣＵＵＵＵＣＣＣＣＣＧＡＡＡ
ＣＡＧＣＧＣｐ）を組み込んだプラスミドｐＲＺ３を調
製した。Example 4 Preparation of plasmid pRZ3 Ribozyme (CUACACCC
UGAUGAGGGGGGCUUUUCCCCCGAAA
A plasmid pRZ3 incorporating CAGCGCp) was prepared.
【００４２】実施例５ ｐＲＺ１、ｐＲＺ２またはｐ
ＲＺ３でトランスフォ−ムさせたＮＩＨ３Ｔ３細胞の調
製ｐＲＺ１（１ μｇ ）、ＮＩＨ３Ｔ３細胞ＤＮＡ（
３０ μｇ ）に２ Ｍ ＣａＣｌ２ （６２．５
μｌ ）を加え、滅菌水で全量 ５００μｌ とした
。これをＨＢＳ 溶液（１１．９２ ｇ ＨＥＰＥＳ，
１６．３６ ｇ ＮａＣｌ， ０．２３ ｇ ＮａＨ
２ＰＯ４ （ｐＨ ７．１） ／１０００ ｍｌ）５０
０ μｌ 中に滴下し、３０分放置し、リン酸カルシウ
ム沈殿を形成させた。
ＮＩＨ３Ｔ３細胞を（８ ｘ １０５ 細胞）培養した
シャ−レにこれを加え、３７℃で５ 時間培養した（リ
ン酸カルシウム法）。そして、１０％ ウシ血清を含む
ＤＭＥＭ培地（ダルベッコ変法イ−グル最少培地）を除
き、１７％ グリセリン（２ ｍｌ）を加え１ 分４５
秒放置後、ＤＭＥＭ培地（１０ ｍｌ ）を加えた。Ｄ
ＭＥＭ培地（１０％ ウシ血清を含む。）に交換し、３
７℃で１５時間培養した。トリプシン処理をし、細胞を
シャ−レよりはがし３枚のシャ−レに広げ、ＤＭＥＭ培
地（５％ウシ血清を含む。）で培養した。４ 日後、Ｇ
ＥＮＥＴＩＣＩＮ（３００ ｍｇ／５００ ｍｌ，ギブ
コ社製）を加えたＤＭＥＭ培地（５％ ウシ血清を含
む。）に交換して７日間培養し、ｐＲＺ１／ＮＩＨ３Ｔ
３ 細胞を得た。Example 5 pRZ1, pRZ2 or p
Preparation of NIH3T3 cells transformed with RZ3 pRZ1 (1 μg), NIH3T3 cell DNA (
30 μg) to 2 M CaCl2 (62.5
μl) was added, and the total volume was made up to 500 μl with sterile water. This was mixed with HBS solution (11.92 g HEPES,
16.36 g NaCl, 0.23 g NaH
2PO4 (pH 7.1) /1000ml)50
0 μl and left for 30 minutes to form a calcium phosphate precipitate. This was added to a petri dish in which NIH3T3 cells (8 x 105 cells) had been cultured, and cultured at 37°C for 5 hours (calcium phosphate method). Then, the DMEM medium containing 10% bovine serum (Dulbecco's modified Eagle's minimal medium) was removed, and 17% glycerin (2 ml) was added for 1 minute.
After standing for a second, DMEM medium (10 ml) was added. D
Replace with MEM medium (containing 10% bovine serum) and
The cells were cultured at 7°C for 15 hours. After trypsin treatment, the cells were peeled from the petri dish, spread on three petri dishes, and cultured in DMEM medium (containing 5% bovine serum). 4 days later, G.
The medium was replaced with DMEM medium (containing 5% bovine serum) supplemented with ENETICIN (300 mg/500 ml, manufactured by Gibco) and cultured for 7 days.
3 cells were obtained.
【００４３】ｐＫＣＳ、 ｐＲＺ２、 ｐＲＺ３でトラ
ンスフォ−ムさせたｐＫＣＳ／ＮＩＨ３Ｔ３ 細胞、ｐ
ＲＺ２／ＮＩＨ３Ｔ３細胞、ｐＲＺ３／ＮＩＨ３Ｔ３
細胞も同様にして得た。[0043] pKCS/NIH3T3 cells transformed with pKCS, pRZ2, and pRZ3, p
RZ2/NIH3T3 cells, pRZ3/NIH3T3
Cells were also obtained in the same manner.
【００４４】実施例６ 活性化ｃ−Ｈａ−ｒａｓ遺伝
子（ｐＲＳＶ−ｒｖ１２ ）によるトランスフォ−メ−
ションｐＲＳＶ−ｒｖ１２（２０ ｎｇ）、ＮＩＨ３Ｔ
３細胞ＤＮＡ （３０ μｇ ）を用いて上記と同様の
リン酸カルシウム法で、ｐＫＣＳ／ＮＩＨ３Ｔ３ 細胞
、ｐＲＺ２／ＮＩＨ３Ｔ３ 細胞、ｐＲＺ３／ＮＩＨ３
Ｔ３ 細胞をトランスフォ−ムした。その後、ＤＭＥＭ
培地（５％ ウシ血清を含む。）で約２週間培養した
。Example 6 Transformation by activated c-Ha-ras gene (pRSV-rv12)
pRSV-rv12 (20 ng), NIH3T
pKCS/NIH3T3 cells, pRZ2/NIH3T3 cells, pRZ3/NIH3 cells were isolated using the same calcium phosphate method as above using 3 cell DNA (30 μg).
T3 cells were transformed. Then DMEM
The cells were cultured in a medium (containing 5% bovine serum) for about 2 weeks.
【００４５】なお、ｐＲＳＶ−ｒｖ１２ は、正常ｃ−
Ｈａ−ｒａｓの１２番目のアミノ酸をコ−ドするコドン
に点突然変異を起こした（Ｇｌｙ １２（ＧＧＵ） →
Ｖａｌ １２（ＧＵＵ） ）活性化ｃ−Ｈａ−ｒａｓＤ
ＮＡを組み込んだものである。[0045] pRSV-rv12 is normal c-
A point mutation was caused in the codon encoding the 12th amino acid of Ha-ras (Gly 12 (GGU) →
Val 12 (GUU)) activated c-Ha-rasD
It incorporates NA.
【００４６】対照として、正常ｃ−Ｈａ−ｒａｓの１２
番目のアミノ酸をコ−ドするコドンに変化がないが、６
１番目のアミノ酸をコ−ドするコドンに点突然変異を起
こした（Ｇｌｎ６１→Ｌｅｕ ６１）活性化ｃ−Ｈａ−
ｒａｓ ＤＮＡを組み込んだｐＲＳＶ−ｒｌ６１ を
用いて、上記の各細胞をトランスフォ−ムし、培養をし
た。As a control, normal c-Ha-ras 12
There is no change in the codon encoding the 6th amino acid, but
Activated c-Ha- with a point mutation in the codon encoding the first amino acid (Gln61→Leu61)
Each of the above cells was transformed using pRSV-rl61 incorporating ras DNA and cultured.
【００４７】培養後、細胞をギムザ染色し、生じたフォ
−カスの数を数えた。After culturing, the cells were stained with Giemsa, and the number of foci produced was counted.
【００４８】結果を図５に示す。The results are shown in FIG.
【００４９】（Ａ）のグラフがｐＲＳＶ−ｒｖ１２ に
よりｐＫＣＳ／ＮＩＨ３Ｔ３ 細胞、ｐＲＺ１／ＮＩＨ
３Ｔ３ 細胞、ｐＲＺ２／ＮＩＨ３Ｔ３ 細胞、ｐＲＺ
３／ＮＩＨ３Ｔ３ 細胞をそれぞれトランスフェクトし
たときのフォ−カスの数である。The graph in (A) shows that pKCS/NIH3T3 cells, pRZ1/NIH
3T3 cells, pRZ2/NIH3T3 cells, pRZ
This is the number of foci when transfecting 3/NIH3T3 cells.
【００５０】（Ｂ）のグラフがｐＲＳＶ−ｒｌ６１ に
よりｐＫＣＳ／ＮＩＨ３Ｔ３ 細胞、ｐＲＺ１／ＮＩＨ
３Ｔ３ 細胞、ｐＲＺ２／ＮＩＨ３Ｔ３ 細胞、ｐＲＺ
３／ＮＩＨ３Ｔ３ 細胞をそれぞれトランスフェクトし
たときのフォ−カスの数である。The graph in (B) shows that pKCS/NIH3T3 cells, pRZ1/NIH
3T3 cells, pRZ2/NIH3T3 cells, pRZ
This is the number of foci when transfecting 3/NIH3T3 cells.
【００５１】ｐＲＺ１／ＮＩＨ３Ｔ３ 細胞、ｐＲＺ２
／ＮＩＨ３Ｔ３ 細胞、においては、ｐＲＳＶ−ｒｖ１
２ によるトランスフェクションにおいて、約５０％
のフォ−カス形成阻害が見られたが、ｐＲＺ３／ＮＩＨ
３Ｔ３ 細胞では阻害が見られなかった。pRZ1/NIH3T3 cells, pRZ2
/NIH3T3 cells, pRSV-rv1
In transfection with 2, approximately 50%
Although inhibition of focus formation was observed in pRZ3/NIH
No inhibition was seen in 3T3 cells.
【００５２】ｐＲＳＶ−ｒｌ６１ でトランスフェクト
した場合、ｐＫＣＳ／ＮＩＨ３Ｔ３ 細胞と比較して、
フォ−カス形成阻害を示さなかったところから、該プラ
スミドにより転写される活性化ｃ−Ｈａ−ｒａｓｍ
ＲＮＡには、リボザイムが何の効果も示さないことが確
認された。したがって、本発明のリボザイムは、正常ｃ
−Ｈａ−ｒａｓの１２番目のアミノ酸をコ−ドするコド
ンに点突然変異を起こした（Ｇｌｙ １２（ＧＧＵ）
→Ｖａｌ １２（ＧＵＵ） ）活性化ｃ−Ｈａ−ｒａｓ
ｍＲＮＡを特異的に切断することが結論付けられた
。When transfected with pRSV-rl61, compared to pKCS/NIH3T3 cells,
The activated c-Ha-rasm transcribed by the plasmid showed no inhibition of focus formation.
It was confirmed that ribozyme had no effect on RNA. Therefore, the ribozymes of the present invention are suitable for normal c.
-A point mutation was caused in the codon encoding the 12th amino acid of Ha-ras (Gly 12 (GGU)
→Val 12 (GUU) ) activated c-Ha-ras
It was concluded that it specifically cleaves mRNA.
【００５３】[0053]
【発明の効果】本発明の、癌遺伝子より転写されるｍＲ
ＮＡに対して特異的な切断活性を有するリボザイムは、
有効な抗腫瘍効果を示すものであり、このものを有効成
分とする抗腫瘍剤としての使用を期待しうるものである
。Effect of the invention: mR transcribed from oncogenes of the present invention
Ribozymes with specific cleaving activity for NA are
It exhibits an effective antitumor effect, and can be expected to be used as an antitumor agent containing it as an active ingredient.
図１は、活性化ｃ−Ｈａ−ｒａｓＤＮＡテンプレ−トお
よびそれより転写された活性化ｃ−Ｈａ−ｒａｓ ｍ
ＲＮＡを示し、図２は、Ｃｌａ Ｉ −Ｓａｌ Ｉ
リンカ−のヌクレオチド配列を示し、図３は、プラスミ
ドｐＲＺ１の構築方法を示し、図４は、リボザイム遺伝
子を含むオリゴリボヌクレオチドを示し、図５は、リボ
ザイム遺伝子を運搬するプラスミドによりトランスフェ
クトされた細胞における癌遺伝子によるフォ−カス形成
の阻害率を示す。Figure 1 shows activated c-Ha-ras DNA template and activated c-Ha-ras m transcribed from it.
Figure 2 shows Cla I-Sal I
The nucleotide sequence of the linker is shown, FIG. 3 shows the method of construction of plasmid pRZ1, FIG. 4 shows the oligoribonucleotide containing the ribozyme gene, and FIG. 5 shows the cells transfected with the plasmid carrying the ribozyme gene. Figure 2 shows the inhibition rate of focus formation by oncogenes.
Claims (2)
レオチド、Ｃはシトシンヌクレオチド、Ｇはグアニンヌ
クレオチドを表わし、３’および５’はそれぞれ３’末
端および５’末端を表わす。」で表わされる化合物を有
効成分とする抗腫瘍剤。Claim 1: General formula: [Formula 1] In the formula, A represents an adenine nucleotide, U represents a uracil nucleotide, C represents a cytosine nucleotide, and G represents a guanine nucleotide, and 3' and 5' represent the 3' end and 5', respectively. An antitumor agent whose active ingredient is a compound represented by the following formula:
レオチド、Ｃはシトシンヌクレオチド、Ｇはグアニンヌ
クレオチドを表わし、３’および５’はそれぞれ３’末
端および５’末端を表わす。」で表わされる化合物を有
効成分とする抗腫瘍剤。[Claim 2] General formula: [Formula 2] In the formula, A represents an adenine nucleotide, U represents a uracil nucleotide, C represents a cytosine nucleotide, and G represents a guanine nucleotide, and 3' and 5' represent the 3' end and 5', respectively. An antitumor agent whose active ingredient is a compound represented by the following formula:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3003528A JPH04235919A (en) | 1991-01-17 | 1991-01-17 | Anticancer drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3003528A JPH04235919A (en) | 1991-01-17 | 1991-01-17 | Anticancer drug |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04235919A true JPH04235919A (en) | 1992-08-25 |
Family
ID=11559887
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3003528A Pending JPH04235919A (en) | 1991-01-17 | 1991-01-17 | Anticancer drug |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04235919A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6090546A (en) * | 1994-11-11 | 2000-07-18 | Medinnova Sf | Method for the detection of Ras oncogenes, in particular the K-ras oncogene |
-
1991
- 1991-01-17 JP JP3003528A patent/JPH04235919A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6090546A (en) * | 1994-11-11 | 2000-07-18 | Medinnova Sf | Method for the detection of Ras oncogenes, in particular the K-ras oncogene |
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