JPH06296492A - Anti-hepatitis c virus oligonucleotide - Google Patents

Anti-hepatitis c virus oligonucleotide

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Publication number
JPH06296492A
JPH06296492A JP5087195A JP8719593A JPH06296492A JP H06296492 A JPH06296492 A JP H06296492A JP 5087195 A JP5087195 A JP 5087195A JP 8719593 A JP8719593 A JP 8719593A JP H06296492 A JPH06296492 A JP H06296492A
Authority
JP
Japan
Prior art keywords
hepatitis
sequence
antisense
oligonucleotide
hcv
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.)
Withdrawn
Application number
JP5087195A
Other languages
Japanese (ja)
Inventor
Kazuya Hoshiko
和哉 星子
Chikahide Nozaki
周英 野崎
Tsukasa Nishihara
司 西原
Hiroshi Nakatake
博 中武
Fukusaburo Hamada
福三郎 濱田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AISUISU PHARMACEUT Inc
Chemo Sero Therapeutic Research Institute Kaketsuken
Ionis Pharmaceuticals Inc
Original Assignee
AISUISU PHARMACEUT Inc
Chemo Sero Therapeutic Research Institute Kaketsuken
Isis Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AISUISU PHARMACEUT Inc, Chemo Sero Therapeutic Research Institute Kaketsuken, Isis Pharmaceuticals Inc filed Critical AISUISU PHARMACEUT Inc
Priority to JP5087195A priority Critical patent/JPH06296492A/en
Priority to AU49837/93A priority patent/AU680435B2/en
Priority to DK93919644T priority patent/DK0662157T3/en
Priority to US08/397,220 priority patent/US6284458B1/en
Priority to JP6507055A priority patent/JPH08506479A/en
Priority to CA002143678A priority patent/CA2143678A1/en
Priority to PCT/JP1993/001293 priority patent/WO1994005813A1/en
Priority to NZ255578A priority patent/NZ255578A/en
Priority to NZ286209A priority patent/NZ286209A/en
Priority to DE69330372T priority patent/DE69330372T2/en
Priority to AT93919644T priority patent/ATE202383T1/en
Priority to EP93919644A priority patent/EP0662157B1/en
Publication of JPH06296492A publication Critical patent/JPH06296492A/en
Priority to US10/457,304 priority patent/US20040033978A1/en
Priority to JP2004268347A priority patent/JP3803354B2/en
Withdrawn legal-status Critical Current

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Abstract

PURPOSE: To obtain a therapeutic agent hepatitis C, having an antisense nucleic acid sequence against hepatic C virus(HCV).
CONSTITUTION: A compound having extremely high inhibiting activity against HCV replication characterized by the possession of an antisense nucleic acid targeting a specific nucleic acid sequence in a 5' untranslated region of a HCV gene is prepared to give a therapeutic agent effective for hepatitis C.
COPYRIGHT: (C)1994,JPO

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】本発明は、抗Ｃ型肝炎ウイルスオ
リゴヌクレオチドに関する。さらに詳細には、本発明
は、Ｃ型肝炎ウイルスの核酸に特異的にハイブリダイズ
するアンチセンスオリゴヌクレオチドを有するＣ型肝炎
治療に有効なアンチセンス核酸治療剤に関する。FIELD OF THE INVENTION The present invention relates to anti-hepatitis C virus oligonucleotides. More specifically, the present invention relates to an antisense nucleic acid therapeutic agent effective for treating hepatitis C, which has an antisense oligonucleotide that specifically hybridizes to a nucleic acid of hepatitis C virus.

【０００２】[0002]

【発明の背景及び従来技術】ウイルス性肝炎にはＡ型肝
炎(伝染性肝炎)とＢ型肝炎(血清肝炎)の２種類があるこ
とは古くから知られていた。これは主として感染経路の
相違に基づいたもので、Ａ型肝炎は経口感染で流行を起
こし、Ｂ型肝炎は主として血液を介して伝播されるもの
であることが確認された。これらの２つの肝炎の原因ウ
イルスは既に分離同定され、Ａ型肝炎ウイルスはピコル
ナウイルスに属する直径27nmのＲＮＡウイルスであり(F
inestone, S.M. et al., Science 182 p.1026(1973))、
一方、Ｂ型肝炎ウイルスはヘパドナウイルスに属する直
径42nmのエンベロープを有するＤＮＡウイルスであるこ
とが突き止められた(Dane, O.S., et al., Lancet,I p.
695(1970))。現在ではこれらの肝炎ウイルスの免疫血清
学的診断方法が確立され、その予防対応策もほぼ確立さ
れている。BACKGROUND OF THE INVENTION and Prior Art It has long been known that there are two types of viral hepatitis, hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis). This was mainly based on the difference in the infection route, and it was confirmed that hepatitis A causes an epidemic by oral infection and hepatitis B is mainly transmitted via blood. These two hepatitis-causing viruses have already been isolated and identified, and hepatitis A virus is an RNA virus with a diameter of 27 nm belonging to picornavirus (F
inestone, SM et al., Science 182 p.1026 (1973)),
On the other hand, hepatitis B virus was found to be a DNA virus belonging to hepadnavirus and having an envelope of 42 nm in diameter (Dane, OS, et al., Lancet, I p.
695 (1970)). At present, immunological serological diagnostic methods for these hepatitis viruses have been established, and their preventive measures have also been established.

【０００３】これらの２つの肝炎ウイルスの確定診断方
法が確立されるに従い、このいずれにも属さない非Ａ非
Ｂ型肝炎の存在が明らかになってきた(Prince,A.M.,et
al.,Lancet I p.241(1974))。輸血後肝炎は、Ｂ型肝炎
ウイルス表面抗原(HBsAg)のスクリーニングの導入によ
り大幅に減少したがゼロにはならず、しかも、発生した
肝炎患者からは、Ａ型、Ｂ型肝炎の感染の証拠は得られ
なかった。このことから、この肝炎は一般に非Ａ非Ｂ型
肝炎と呼ばれた。この種の肝炎は、わが国では散発性肝
炎の約50％、輸血後肝炎の約90％以上にのぼり、さらに
慢性肝炎、肝硬変、肝癌の50％以上が非Ａ非Ｂ型肝炎に
起因すると推定されており大きな社会問題となってい
る。With the establishment of definitive diagnostic methods for these two hepatitis viruses, the existence of non-A non-B hepatitis that does not belong to either of these has become clear (Prince, AM, et.
al., Lancet I p. 241 (1974)). Post-transfusion hepatitis was significantly reduced by the introduction of hepatitis B virus surface antigen (HBsAg) screening, but it did not become zero, and there was no evidence of hepatitis A or B infection from hepatitis patients who developed it. I couldn't get it. For this reason, this hepatitis was generally called non-A non-B hepatitis. It is estimated that this kind of hepatitis accounts for about 50% of sporadic hepatitis and over 90% of post-transfusion hepatitis in Japan, and more than 50% of chronic hepatitis, liver cirrhosis and liver cancer are caused by non-A non-B hepatitis. Has become a major social problem.

【０００４】これとは別に、インド、ミャンマー、アフ
ガニスタン、または北アフリカなどで経口感染で流行す
る第２のウイルス性非Ａ非Ｂ型肝炎が存在することが明
らかになった(Khuroo,M.S. Am.J.Med., 68 p.818(198
0))。これは、一般には水系、または流行性非Ａ非Ｂ型
肝炎と呼ばれている。わが国では、この肝炎の流行は観
察されていないが、渡航者による流行地からの肝炎の輸
入は若干観られるようである［福原ら、第２５回日本肝
臓学会総会講演要旨集(1989)］。Apart from this, it has been revealed that there is a second viral non-A non-B hepatitis which is prevalent by oral infection in India, Myanmar, Afghanistan, North Africa and the like (Khuroo, MS Am. J. Med., 68 p.818 (198
0)). It is commonly referred to as waterborne or epidemic non-A non-B hepatitis. In Japan, no outbreak of this hepatitis has been observed, but it seems that there is some import of hepatitis from endemic areas by travelers [Fukuhara et al., 25th Annual Meeting of the Japanese Society of Hepatology (1989)].

【０００５】本発明は、上記で言う前者の主に血液を介
して感染する血液型非Ａ非Ｂ型肝炎ウイルスに関するも
のであり、本明細書中ではこのウイルスをＣ型肝炎ウイ
ルスと呼ぶ。The present invention relates to the above-mentioned blood group non-A non-B hepatitis virus which is mainly transmitted via blood, and this virus is referred to as hepatitis C virus in the present specification.

【０００６】Ｃ型肝炎は、患者血清中のウイルス濃度が
102〜103と低いこと、同じ接種材料で再感染を起こした
チンパンジーが存在するなど抗体の存在が疑わしいこ
と、感染実験モデルがチンパンジー、マーモセットに限
られることなどの問題点のため、その研究に困難を来た
している。Hepatitis C has a virus concentration in the serum of patients.
Due to problems such as low as 10 2 to 10 3 , suspected presence of antibody such as chimpanzee reinfected with the same inoculum, and limited experimental model of infection to chimpanzee and marmoset, etc. Is coming to hardship.

【０００７】このため、Ｃ型肝炎については未だウイル
ス本体の分離同定はなされておらず、また、この種類の
肝炎の治療法、及び予防法は確立されてはいない。従
来、この肝炎の診断は除外診断によるしかなかった。す
なわち、患者の血清について診断方法が確立されている
Ａ型、Ｂ型肝炎の検査を行ない、これらの肝炎であるこ
とを否定し、さらに全身感染の一部の症状として肝炎様
症状を示すヘルペス、サイトメガロ、エプスタインバー
ウイルス感染の可能性を否定し、また薬物性やアルコー
ル性肝炎、自己免疫性肝炎を否定してＣ型肝炎として診
断されていた。Therefore, for hepatitis C, the virus body has not yet been isolated and identified, and the therapeutic and preventive methods for this type of hepatitis have not been established. Conventionally, the diagnosis of this hepatitis has been based only on exclusion diagnosis. That is, a test for hepatitis A and B for which the diagnostic method has been established is performed on the serum of a patient, and these hepatitis are denied, and further, herpes which shows hepatitis-like symptoms as a part of systemic infection, He was diagnosed as hepatitis C, denying the possibility of infection with cytomegalo and Epstein-Barr virus, and denying drug-induced or alcoholic hepatitis and autoimmune hepatitis.

【０００８】この肝炎の病因ウイルスが感染性を有する
ことは、1978年アメリカの研究グループにより、チンパ
ンジーを用いた感染実験で証明された(Tabor,E.,et a
l., Lancet I p.463(1978))。しかし、世界中の多くの
努力にもかかわらず、10年以上経た現在も、病因ウイル
スの実態は判明していない。感染チンパンジーの血液や
肝組織を材料として、寒天ゲル内沈降反応、免疫電気向
流法、ラジオイムノアッセイ、蛍光抗体法、電顕法など
のＡ型及びＢ型肝炎の研究で用いられた殆どすべてのア
プローチにより、ウイルスや関連する抗原抗体系の探索
が行なわれてきたが、いまだ確実といわれるものを得る
には至っていない。[0008] The infectivity of this pathogenic virus for hepatitis was proved by an American research group in 1978 in an infection experiment using chimpanzees (Tabor, E., et a.
L., Lancet I p.463 (1978)). However, despite the efforts of many people around the world, the actual condition of the causative virus is still unknown even more than 10 years ago. Almost all approaches used in hepatitis A and B studies, such as agar gel precipitation, immunoelectric countercurrent method, radioimmunoassay, fluorescent antibody method, electron microscopy method, using blood or liver tissue of infected chimpanzee Have sought to search for viruses and related antigen-antibody systems, but have yet to obtain what is said to be reliable.

【０００９】最近になって、Ｃ型肝炎ウイルス（ＨＣ
Ｖ）のcDNAを捕えたという報告が米国のカイロン社（欧
州特許公開 EP 388232A）、及び自治医科大学の岡本ら
［VOROLOGY 188, P321-341 (1992)］によって発表さ
れ、このウイルス遺伝子の全容が明らかにされつつあ
る。このような状況の中、本発明者らも単一種HCVゲノ
ムを確立するために、単一の日本人血漿を出発材料とし
てHCV-cDNAのクローニングを行った。その結果、HCV−R
NAの全長アミノ酸をコードすると考えられるcDNAをクロ
ーニングすることに成功した（特願平3-203884）。Recently, hepatitis C virus (HC
V) was reported to have captured the cDNA of Chiron (European Patent Publication EP 388232A) and Jichi Medical University Okamoto et al. [VOROLOGY 188, P321-341 (1992)], and the entire viral gene was published. It is being revealed. Under these circumstances, the present inventors have also cloned HCV-cDNA using a single Japanese plasma as a starting material in order to establish a single-species HCV genome. As a result, HCV-R
We have succeeded in cloning a cDNA that is thought to encode the full-length amino acid of NA (Japanese Patent Application No. 3-203884).

【００１０】このようにHCVの遺伝子が捕えられたこと
により、治療薬開発の方法の新しい可能性の道が開かれ
ることになった。例えば、HCVの機能蛋白を活性を持っ
た形で遺伝子組換え技術を用いて発現させ、この機能を
阻害するような物質のスクリーニングを行うことにより
抗HCV化合物を調製することも可能になろう。また、別
の例としては遺伝子配列情報をもとに、近年注目されて
いる、遺伝子レベルでの制御をかける、アンチセンス核
酸によるウイルス増殖阻止という可能性も考えられる。[0010] The capture of the HCV gene in this way has opened the way for new possibilities for therapeutic drug development. For example, it may be possible to prepare an anti-HCV compound by expressing a functional protein of HCV in an active form by gene recombination technology and screening a substance that inhibits this function. Further, as another example, there is a possibility that anti-sense nucleic acid may be used to prevent viral growth, which is attracting attention in recent years based on gene sequence information, and is applied at the gene level.

【００１１】このアンチセンス核酸とは、ウイルスが増
殖する際に合成するメッセンジャーRNA (mRNA)の特定な
領域と相補的な配列を持つ核酸のことで、これをmRNAが
翻訳されている場に与えることによって、mRNAそのもの
またはその成熟に至る中間体の適当な領域とハイブリダ
イズさせることにより、リボソームの結合、転移の阻害
あるいはRNA分解酵素による分解などの効果を与え、当
核mRNAの発現を阻止することを可能とするものである。
(渡辺信元ら、実験医学713 p138 (1990))The antisense nucleic acid is a nucleic acid having a sequence complementary to a specific region of messenger RNA (mRNA) synthesized when the virus grows, and gives this to the site where the mRNA is translated. By hybridizing with an appropriate region of the mRNA itself or an intermediate that leads to its maturation, it exerts effects such as ribosome binding, inhibition of translocation, or degradation by RNA degrading enzyme, and the expression of the nuclear mRNA is blocked. It makes it possible.
(Watanabe Nobumoto et al., Experimental Medicine 713 p138 (1990))

【００１２】これらアンチセンス配列を持つオリゴヌク
レオチドは、核酸分解に対する抵抗性は増大しているが
培養細胞の原形質膜に侵入して相補的なDNA配列またはR
NA配列と特異的にハイブリダイズする能力は保持される
ような形で修飾され、(TS’O, P.O.P. et al. in
Development of Target-Oriented Antisencer Drug
s, eds. Cheng, Y.C., et al. (Raven Press,
NewYork) p189 (1983) ; Miller, P.S. et al.
Biochimie, 67, p769 (1985))ウイルス阻止に対して
インビトロで活性であることが明らかにされているが、
ウイルス阻止活性を示すためには実際に治療薬として用
いるには不適当な程の高い濃度が要求された。今後、ア
ンチセンス技術を治療薬の分野に応用していくためには
ウイルス阻止活性を示すために要求される濃度をより低
くできるような方法を求めていかなければならない。そ
のための手段として、生体内に投与した核酸が分解され
るのを防ぐ、あるいは膜透過性を高めるべく、核酸の効
果的な修飾法が開発されている。Oligonucleotides having these antisense sequences have increased resistance to nucleolytic degradation, but enter the plasma membrane of cultured cells and enter a complementary DNA sequence or R
The ability to specifically hybridize with NA sequences was modified in a manner that preserves it (TS'O, POP et al. In
Development of Target-Oriented Antisencer Drug
s, eds. Cheng, YC, et al. (Raven Press,
NewYork) p189 (1983); Miller, PS et al.
Biochimie, 67, p769 (1985)) has been shown to be active in vitro against viral inhibition,
In order to exhibit the virus-blocking activity, a concentration that is unsuitable for practical use as a therapeutic drug was required. In the future, in order to apply the antisense technology to the field of therapeutic drugs, it is necessary to seek a method capable of lowering the concentration required for exhibiting virus blocking activity. As a means for that purpose, an effective modification method of a nucleic acid has been developed in order to prevent the nucleic acid administered in the living body from being decomposed or to enhance the membrane permeability.

【００１３】しかしながら、このような核酸の効果的な
修飾法の前に、対象となるウイルスのどの遺伝子領域の
アンチセンス核酸をいかに調製するかということを決定
しなければならない。アンチセンス核酸を細胞に対して
働かせようとした場合、膜透過性とハイブリッド形成能
の兼合いから15-30merの長さ、好ましくは20mer程度が
適当とされている。従って、対象となるウイルスの遺伝
子の配列から20mer程度のターゲット領域（アンチセン
ス核酸をハイブリッドさせる標的配列）を選び出す必要
があるが、どこを選んでも同じというわけではない。な
ぜならば、mRNAの配列上の任意の連続する20merの果た
す機能は場所によって異なるので、その領域に相補的な
アンチセンス核酸がmRNAの全体の機能に与える影響も当
然異なってくる。したがって、ウイルスの増殖機能によ
り効果的な阻害を与えるようなウイルス遺伝子上のター
ゲット領域を見いだすことが重要な意味を持つことにな
る。However, prior to such effective modification of nucleic acids, it must be decided how and how to prepare the antisense nucleic acid of which gene region of the virus of interest. When trying to make an antisense nucleic acid act on a cell, a length of 15-30 mer, preferably about 20 mer is considered to be suitable in view of the combination of membrane permeability and hybridizing ability. Therefore, it is necessary to select a target region of about 20 mer (target sequence for hybridizing antisense nucleic acid) from the gene sequence of the virus of interest, but it is not the same no matter which one is selected. Because the function of any continuous 20-mer on the mRNA sequence differs depending on the location, the influence of the antisense nucleic acid complementary to the region on the overall function of mRNA naturally also differs. Therefore, it is important to find a target region on the viral gene that gives more effective inhibition to the growth function of the virus.

【００１４】[0014]

【本発明が解決しようとする課題】このように、Ｃ型肝
炎ウイルスに対するアンチセンスを調製する際には、ア
ンチセンス核酸のターゲット領域となる、HCV-RNA中の2
0mer程度の有効な核酸配列部位を見いだすことが極めて
重要な要件となる。しかしながら、Ｃ型肝炎に関しては
アンチセンスのターゲット領域となるような特に有効な
部位に関する報告はこれまでのところ一切なされていな
い。As described above, when preparing an antisense against hepatitis C virus, 2 of HCV-RNA, which is a target region of an antisense nucleic acid, is prepared.
Finding an effective nucleic acid sequence site of about 0mer is a very important requirement. However, regarding hepatitis C, no report has been made so far regarding a particularly effective site as a target region of antisense.

【００１５】[0015]

【課題を解決するための手段】ウイルス遺伝子上のアン
チセンス核酸のターゲット部位を探索するという目的の
ためには通常はインビトロのウイルス増殖系を使用して
いるがHCVの場合現時点ではこのような系は存在しな
い。そこで本発明らは、ウサギ網状赤血球抽出液と人工
的に合成したHCV-mRNAの5'非翻訳領域からコアのN端側
までの部分を用いた無細胞翻訳系を用いることによっ
て、アンチセンス治療薬のターゲットとなるHCV-RNA中
の核酸配列について特に有効な領域を見いだすことに成
功した。[Means for Solving the Problems] An in vitro virus propagation system is usually used for the purpose of searching for a target site of an antisense nucleic acid on a viral gene, but in the case of HCV, such a system is currently used. Does not exist. Therefore, the present inventors have used a cell-free translation system that uses a portion from the 5'untranslated region of HCV-mRNA artificially synthesized with rabbit reticulocyte extract to the N-terminal side of the core, thereby performing antisense treatment. We succeeded in finding a particularly effective region in the nucleic acid sequence in HCV-RNA, which is a drug target.

【００１６】発明者らが構築した上記無細胞翻訳系はHC
Vの生活環にとって重要な過程である翻訳開始の段階を
試験管内で模擬していると考えられる。翻訳開始の段階
においてはmRNAとリボゾームの結合、開始コドンの位置
での複合体の形成といった一連の反応が行われ、HCV-mR
NAの5'非翻訳領域とコア領域のN端側がこの過程に関与
していると考えられる。しかし、この領域は約300塩基
の長さがあり、全体をアンチセンスによってカバーする
ことは極めて困難である。本発明者らは、この領域のう
ちどの部分の塩基配列をアンチセンス核酸のターゲット
とすることが、他の部分に較べて強く翻訳開始が阻害さ
れるかについて鋭意研究を進めた結果、他の部分に較べ
て極めて強く翻訳開始を阻害する部分を見いだした。The above cell-free translation system constructed by the inventors is HC
It is considered that the stage of translation initiation, which is an important process for V's life cycle, is simulated in vitro. At the translation initiation stage, HCV-mR undergoes a series of reactions such as binding of mRNA to ribosome and formation of complex at the position of initiation codon.
The 5'untranslated region of NA and the N-terminal side of the core region are considered to be involved in this process. However, this region has a length of about 300 bases, and it is extremely difficult to cover the entire region with antisense. The inventors of the present invention have conducted extensive studies on which part of the region, which is the base sequence of the antisense nucleic acid, strongly inhibits translation initiation compared to other parts, and as a result, other We found a part that strongly inhibited translation initiation compared to the part.

【００１７】すなわち、本発明に従えば、Ｃ型肝炎ウイ
ルスゲノムの塩基配列中の5'非翻訳領域に存在する下記
の塩基配列（Ａ）もしくはこの核酸配列に極めて相同性
の高い塩基配列（塩基配列（Ａ）と比較し、１ないし２
個の塩基のみが異なる塩基配列）とハイブリッドするよ
うなアンチセンス塩基配列を有することを特徴とするア
ンチセンス化合物を調製することにより、Ｃ型肝炎治療
にきわめて効果的なアンチセンス核酸治療剤を調製する
ことが可能となる。 （Ａ）GCCUCCAGGACCCCThat is, according to the present invention, the following base sequence (A) present in the 5'untranslated region in the base sequence of the hepatitis C virus genome or a base sequence (base having a very high homology to this nucleic acid sequence: 1 to 2 compared to sequence (A)
A highly effective antisense nucleic acid therapeutic agent for the treatment of hepatitis C is prepared by preparing an antisense compound characterized by having an antisense base sequence that hybridizes with a base sequence having only one base) It becomes possible to do. (A) GCCUCCAGGACCCC

【００１８】そのようなアンチセンス化合物とは、少な
くとも１４merの塩基を有するオリゴヌクレオチドまた
はその化合物であり、好ましくは１４〜２６merのオリ
ゴヌクレオチドを有する化合物をいう。また、さらに好
ましくは、上記の14個の塩基からなる塩基配列もしくは
この配列に極めて相同性の高い塩基配列に相補的な塩基
配列を有し、さらに、HCVゲノム由来の下記の塩基配列
（Ｂ）もしくはHCVゲノムの、この配列からなる領域に
相当する塩基配列中の連続した約２０mer程度の塩基配
列に相補的な塩基配列からなる化合物を言う。 （Ｂ）CGUGCAGCCUCCAGGACCCCCCCUCCSuch an antisense compound is an oligonucleotide having a base of at least 14 mer or a compound thereof, preferably a compound having an oligonucleotide of 14 to 26 mer. Further, more preferably, it has a base sequence complementary to the above-mentioned base sequence consisting of 14 bases or a base sequence highly homologous to this sequence, and further has the following base sequence (B) derived from HCV genome: Alternatively, it refers to a compound having a nucleotide sequence complementary to a continuous nucleotide sequence of about 20 mer in the nucleotide sequence corresponding to the region consisting of this sequence in the HCV genome. (B) CGUGCAGCCUCCAGGACCCCCCCUCC

【００１９】上記の本発明の最も好ましい化合物の具体
的な例としては、下記の(A1)〜(A4)の核酸配列からなる
２０merのアンチセンス化合物が挙げられる。 (A1) GGGGTCCTGGAGGCTGCACG (A2) GGGGGGTCCTGGAGGCTGCA (A3) AGGGGGGGTCCTGGAGGCTG (A4) GGAGGGGGGGTCCTGGAGGC 上記(A1)〜(A4)においては、アンチセンス核酸としてＤ
ＮＡを用いた例を示しているが、ＤＮＡの代わりにＲＮ
Ａを用いた場合でも同様のアンチセンス化合物を調製す
ることができる。そのような場合には、上記T（チミ
ン）をU（ウラシル）に代えることにより調製される。Specific examples of the above-mentioned most preferred compounds of the present invention include 20-mer antisense compounds comprising the following nucleic acid sequences (A1) to (A4). (A1) GGGGTCCTGGAGGCTGCACG (A2) GGGGGGTCCTGGAGGCTGCA (A3) AGGGGGGGTCCTGGAGGCTG (A4) GGAGGGGGGGTCCTGGAGGC In the above (A1) to (A4), D was used as the antisense nucleic acid.
An example using NA is shown, but RN is used instead of DNA.
Similar antisense compounds can be prepared even when A is used. In such a case, it is prepared by replacing the above T (thymine) with U (uracil).

【００２０】HCVの5'非翻訳領域の塩基配列はウイルス
株間での相同性が他の領域と比較して高いが、それでも
株間で塩基の変異率の高い箇所が5'非翻訳領域全体で数
十箇所報告されている。上記配列A中にもそのような株
間での変異率の高い塩基が一箇所あり、塩基番号119
（配列A中では5'側より10番目）のアデニンがそれであ
る。また、上記配列B中では塩基番号107（配列B中では
5'側より４番目）のグアニン、塩基番号104(配列B中で
は5'側より１番目）のシトシンが変異率が高い。The nucleotide sequence of the 5'untranslated region of HCV has a high homology among virus strains as compared with other regions, but the number of sites with high mutation rate of nucleotides among the strains is high in the entire 5'untranslated region. Ten places have been reported. In the above sequence A, there is one base with a high mutation rate among such strains.
That is adenine (10th from the 5'side in sequence A). Also, in the above sequence B, the base number 107 (in the sequence B,
The mutation rate of guanine at the 4th position from the 5'side and cytosine at base number 104 (1st position from the 5'side in Sequence B) is high.

【００２１】15-20mer程度の鎖長のオリゴヌクレオチド
に対しその相補鎖をハイブリダイズさせる際に両鎖間に
一箇所塩基対のミスマッチがあると全体の結合能が著し
く落ちることが知られているが、この事実に拠れば今回
の実験に用いたHCV株の塩基配列に基づいて作製したア
ンチセンス化合物は上記の位置の塩基に変異を有する他
のHCV株に対しては著しく活性が低下する可能性が想定
される。そこで我々は上記の119の位置のアデニンに対
応するアンチセンスDNA配列中のチミンをイノシンに置
換したアンチセンスDNAを作製した。イノシンとは、オ
リゴヌクレオチドプローブを調製する際に変異の可能性
のある位置に本来の塩基対を形成する塩基の替わりに入
れるというような使い方をされているプリン類似化合物
で、配列中にイノシンが入っているオリゴヌクレオチド
はそれと相補的なオリゴヌクレオチドでイノシンの位置
に対応する塩基がＡ、Ｇ、Ｃ、Ｔ（Ｕ）のどれであって
もその部分の塩基対がミスマッチである場合に比較して
はるかに高いハイブリッド形成能を持つという性質を持
つ。この塩基番号119の位置をイノシンに置換したアン
チセンスDNAを上記の系にて評価した結果、完全に相補
的な場合とほぼ同等の活性を示した。このことから、上
記の位置の塩基をイノシンに置換したアンチセンス治療
薬はHCVの複数の株に対して有効であることが示され
た。It is known that when hybridizing a complementary strand of an oligonucleotide having a chain length of about 15 to 20 mer, if there is a single base pair mismatch between both strands, the overall binding ability is significantly reduced. However, based on this fact, the antisense compound prepared based on the nucleotide sequence of the HCV strain used in this experiment may have significantly reduced activity against other HCV strains having a mutation at the base at the above position. Sex is assumed. Therefore, we prepared an antisense DNA in which thymine in the antisense DNA sequence corresponding to the adenine at position 119 was replaced with inosine. Inosine is a purine-like compound that is used to prepare an oligonucleotide probe in place of a base that forms an original base pair at a position where mutation may occur. The oligonucleotide contained in the oligonucleotide is complementary to that in the case where the base pair corresponding to the position of inosine is A, G, C or T (U) and the base pair of that portion is mismatched. And has a much higher hybridizing ability. The antisense DNA in which the position of the base number 119 was substituted with inosine was evaluated in the above system, and as a result, almost the same activity as in the case of being completely complementary was shown. From this, it was shown that the antisense therapeutic agent in which the base at the above position was substituted with inosine was effective against multiple strains of HCV.

【００２２】また、このようなアンチセンス核酸を有す
ることを特徴とする本発明のＣ型肝炎治療剤を調製する
際には、核酸を公知の方法で一部化学修飾することによ
り、生体内に投与した際に安定な医薬品として有用な核
酸改変体を調製することができる。このようなアンチセ
ンス核酸の安定性を高める修飾としては、ホスホジエス
テル結合を形成するリン酸基をメチル基に置換する方法
［C.Smithら、Proc. Natl. Acad. Sci. USA 83, p2787
(1986)］あるいは、イオウに置換する方法［松倉ら、Pr
oc. Natl. Acad. Sci. USA 84, p7706 (1987)］、また
3'末端にアクリジン［J.Toulmeら、Proc. Natl. Acad.
Sci. USA 83, p1227 (1986)］やポリジン［M.Lemait
ら、Proc. Natl. Acad. Sci. USA 84, p1648 (1987)］
を共有結合させる方法などがその一例として挙げられ
る。また最近では、骨格部分の核酸構造を他の化合物に
置き換える試みもなされている。このような修飾は、標
的臓器への移行性、ｍＲＮＡとの結合能またはウイルス
に作用後の代謝性など、治療薬として使用する際に要求
される種々の事項に対応すべく必要に応じて行われる。
特にホスホジエステル結合を形成するリン酸基の酸素原
子をイオウ原子に置換する方法は、本発明の治療剤調製
の上で有用な好ましい手法と考えられる。Further, when preparing the therapeutic agent for hepatitis C of the present invention, which comprises such an antisense nucleic acid, the nucleic acid is partially chemically modified by a known method to give it in vivo. A nucleic acid variant useful as a drug that is stable when administered can be prepared. As a modification for improving the stability of such an antisense nucleic acid, a method of substituting a methyl group for a phosphate group forming a phosphodiester bond [C. Smith et al., Proc. Natl. Acad. Sci. USA 83, p2787].
(1986)] or a method of substituting with sulfur [Matsukura et al., Pr
oc. Natl. Acad. Sci. USA 84, p7706 (1987)],
Acridine at the 3'end [J. Toulme et al., Proc. Natl. Acad.
Sci. USA 83, p1227 (1986)] and polyzine [M.Lemait
Proc. Natl. Acad. Sci. USA 84, p1648 (1987)]
An example thereof is a method of covalently bonding. Recently, attempts have been made to replace the nucleic acid structure of the skeleton with another compound. Such modification is carried out as necessary to cope with various matters required for use as a therapeutic drug, such as transferability to a target organ, ability to bind to mRNA, and metabolism after action on virus. Be seen.
In particular, the method of substituting the sulfur atom for the oxygen atom of the phosphate group forming a phosphodiester bond is considered to be a preferable method useful for preparing the therapeutic agent of the present invention.

【００２３】本発明においては、ウサギ網状赤血球抽出
液を用いた無細胞翻訳系において、HCV-RNAよりHCVコア
蛋白を発現させ、この反応系に本発明のアンチセンスＤ
ＮＡ等を加えてＨＣＶ由来の蛋白発現に阻害がかかるか
どうかをみた結果、上記に示した GCCUCCAGGACCCCに対
するアンチセンス核酸を有するアンチセンス化合物が他
の領域に対するアンチセンス核酸に比較し、極めて高い
翻訳阻害活性を示すことが確認された。以下、実施例に
沿って本発明を詳細に説明する。In the present invention, an HCV core protein is expressed from HCV-RNA in a cell-free translation system using a rabbit reticulocyte extract, and the antisense D of the present invention is added to this reaction system.
As a result of checking whether HCV-derived protein expression was inhibited by adding NA or the like, the antisense compound having the antisense nucleic acid against GCCUCCAGGACCCC shown above showed extremely high translation inhibition as compared with the antisense nucleic acid against other regions. It was confirmed to show activity. Hereinafter, the present invention will be described in detail with reference to Examples.

【００２４】[0024]

【実施例】1. In vitro translationに用いるためのＣ型肝炎ウイ
ルス（HCV）RNAの調製 HCV遺伝子配列の塩基番号1-686と相同な配列を持つRNA
を、In vitro translation に用いるため3'末端に終
止コドン（TGA）を付加させた形で以下の方法により調
製した。[Example] 1. Hepatitis C virus for use in in vitro translation
Preparation of ruth (HCV) RNA RNA having a sequence homologous to nucleotide numbers 1-686 of HCV gene sequence
Was prepared by the following method with a stop codon (TGA) added to the 3'end for use in in vitro translation.

【００２５】(1) Polymerase Chain Reaction（PCR）
用鋳型のHCV-cDNAの調製 本発明者らが一人の日本人Ｃ型肝炎患者血清よりクロー
ニングしたHCV全長アミノ酸をコードしていると考えら
れるcDNAの塩基配列をもとに、既知の方法を用いて341
塩基のHCV遺伝子5'非翻訳領域全長と本研究に必要な145
塩基のコア領域5'端側が連続する686塩基を含むcDNAを
クローニングして、下記(3)で行ったPCRの鋳型とした。[1] Polymerase Chain Reaction (PCR)
Preparation of HCV-cDNA as a template for use by the present inventors Based on the nucleotide sequence of cDNA which is considered to encode the full-length amino acid of HCV cloned from the serum of one Japanese hepatitis C patient, the known method was used. 341
HCV gene 5'untranslated region of the base and the 145 necessary for this study
A cDNA containing 686 bases in which the 5'end side of the base region was continuous was cloned and used as a template for PCR performed in the following (3).

【００２６】(2) PCR用プライマーの調製 5'末端より、EcoRI切断部位を含む7塩基、T7プロモータ
ーとして機能する20塩基、HCV配列の1-14までの14塩
基、の順に並んだ41塩基をセンスのプライマーとして、
5'末端より、EcoRI切断部位を含む9塩基、終止コドン
（TGA）の相補鎖である3塩基、HCV配列の672-686の部分
の相補配列である15塩基の順に並んだ27塩基をアンチセ
ンスのプライマーとして、それぞれMilliGen / Biosear
ch 社製 Cyclone Plus DNA Synthesizer を用いて
固相化ホスホアミダイト法により合成した。得られたセ
ンス及びアンチセンスのプライマーをそれぞれ Sy-NC-
1及びSy-CR-1と命名した。図１にその配列を示す。(2) Preparation of PCR primer From the 5'end, 41 bases arranged in the order of 7 bases containing an EcoRI cleavage site, 20 bases functioning as a T7 promoter, and 14 bases up to 1-14 of the HCV sequence were prepared. As a sense primer,
From the 5'end, antisense 27 bases including 9 bases including the EcoRI cleavage site, 3 bases complementary to the stop codon (TGA), and 15 bases complementary to the 672-686 portion of the HCV sequence in order. As a primer of each, MilliGen / Biosear
It was synthesized by the solid-phased phosphoamidite method using Cyclone Plus DNA Synthesizer manufactured by ch. The obtained sense and antisense primers were respectively labeled with Sy-NC-
1 and Sy-CR-1. The sequence is shown in FIG.

【００２７】(3) PCRによるRNA合成鋳型DNAの調製 上記(1)に記されたcDNAを鋳型とし、上記(2)に記された
Sy-NC-1及びSy-CR-1のプライマーにてPCR（20サイク
ル）を行った。PCRは変性：94℃1分、アニール：55℃2
分、ポリメラーゼ反応：72℃2分の反応組成にて行っ
た。得られたDNA断片をEcoRIにて処理し、pUC19のEcoRI
サイトに挿入して大腸菌JM109株を常法によりトランス
フォームした。コロニーとして得られた複数のクローン
より調製した組換えプラスミドの挿入部位をジデオキシ
法にてシークエンスを行って、すべてのクローン由来の
プラスミドにて挿入された686塩基のHCV由来の配列が鋳
型としたcDNAの対応する領域と一致することを確認し
た。それらのうちのひとつのクローンよりプラスミドを
pUIA1と名付けた。(3) Preparation of RNA synthesis template DNA by PCR Using the cDNA described in (1) above as a template, described in (2) above
PCR (20 cycles) was performed using Sy-NC-1 and Sy-CR-1 primers. PCR denaturation: 94 ℃ for 1 minute, annealing: 55 ℃ 2
Min, polymerase reaction: Reaction was performed at 72 ° C. for 2 minutes. The obtained DNA fragment was treated with EcoRI to obtain EcoRI of pUC19.
Escherichia coli JM109 strain was inserted into the site and transformed by a conventional method. Sequencing the insertion site of the recombinant plasmid prepared from multiple clones obtained as colonies by the dideoxy method, cDNA with HCV-derived sequences of 686 bases inserted in the plasmids derived from all clones as a template It was confirmed that the area and the corresponding area of A plasmid from one of those clones
It was named pUIA1.

【００２８】(4) HCV遺伝子配列の一部を持つRNAの調製 pUIA1を大量調製して挿入されたフラグメントをEcoRIに
て切り出し、これを鋳型として、MEGAscript in vitr
o Transcription Kit(Ambion社)を用いてRNA合成を行
い、5'側よりHCV配列の1-686の部分、終止コドン（UG
A）、EcoRI切断部位を含む９塩基、の順に並んだ698塩
基のRNA断片を得た。この断片をR-IA-1と命名した。R-I
A-Iの配列の内、HCV由来の686塩基の配列を図２に示
す。(4) Preparation of RNA Having a Part of HCV Gene Sequence pUIA1 was prepared in large quantities and the inserted fragment was cut out with EcoRI. Using this as a template, MEGAscript in vitr
o Transcription Kit (Ambion) was used to synthesize RNA, and from the 5'side, the 1-686 portion of the HCV sequence, the stop codon (UG
An RNA fragment of 698 bases arranged in the order of A) and 9 bases containing an EcoRI cleavage site was obtained. This fragment was named R-IA-1. RI
Among the AI sequences, the HCV-derived 686-base sequence is shown in FIG.

【００２９】2. 無細胞翻訳系によるHCVコア蛋白の合成 R-IA-1より、ウサギ網状赤血球抽出液を用いてHCVコア
蛋白を以下の方法により無細胞的に翻訳し、発現をELIS
Aにより確認した。 2. Synthesis of HCV core protein by cell-free translation system HCV core protein was cell-free translated from R-IA-1 by the following method using a rabbit reticulocyte extract, and expression was expressed by ELIS.
Confirmed by A.

【００３０】(1) HCVコア蛋白を定量するためのELISA系
の構築 HCVのコア領域を従来法にて大腸菌で直接発現させた。
得られた発現蛋白をマウスに免疫し、従来法にてIgM型
であるRJC4-1、IgGであるRJC4-2の二種類のモノクロー
ナル抗体を得た。RJC4-1を10mMPBSに希釈しマキソープ
イモビュランプレートの各ウェルに50μg/mlの濃度で50
μl加え、4℃に一夜放置して固相化した後、ウェルより
抗体溶液を吸引除去した。次に、1%牛血清アルブミンの
溶解したPBSを各ウェル150μl加えて4℃に一夜放置して
ブロッキングを行った後、洗浄操作を行った。次に被験
物質であるウサギ網状血球抽出液にて合成した上記コア
蛋白を1%牛血清アルブミンの溶解したPBSにて適当な濃
度に希釈して50μl加え、室温で2時間反応させた後、洗
浄操作を行った。次に、西洋ワサビペルオキシダーゼを
結合させたRJC4-2抗体を50μl加え、37℃で1時間反応さ
せた後、洗浄操作を行った。次に、3,3',5,5'-テトラメ
チルベンジジン溶液を50μl加え、室温にて、暗所で15
分反応させた後、1N硫酸で反応を止め、直ちに450nmの
吸光度を測定した。その結果、上記ELISAの系にてHCVコ
ア蛋白の定量が可能であることが示された。(1) Construction of ELISA system for quantifying HCV core protein The core region of HCV was directly expressed in E. coli by a conventional method.
Mice were immunized with the obtained expressed proteins, and two types of monoclonal antibodies, IgM type RJC4-1 and IgG RJC4-2, were obtained by a conventional method. RJC4-1 was diluted in 10 mM PBS and 50 μg / ml was added to each well of a Maxorp Immobilin plate at a concentration of 50 μg / ml.
After adding μl, the mixture was allowed to stand at 4 ° C. overnight for immobilization, and then the antibody solution was removed by suction from the well. Next, 150 μl of PBS in which 1% bovine serum albumin was dissolved was added to each well, left at 4 ° C. overnight for blocking, and then washed. Next, the above core protein synthesized with rabbit reticulocyte extract, which is a test substance, was diluted to an appropriate concentration with PBS containing 1% bovine serum albumin, added 50 μl, and reacted at room temperature for 2 hours, then washed. The operation was performed. Next, 50 μl of RJC4-2 antibody conjugated with horseradish peroxidase was added, reacted at 37 ° C. for 1 hour, and then washed. Next, add 50 μl of 3,3 ', 5,5'-tetramethylbenzidine solution, and in the dark at room temperature for 15 minutes.
After reacting for minutes, the reaction was stopped with 1N sulfuric acid, and the absorbance at 450 nm was immediately measured. As a result, it was shown that HCV core protein can be quantified by the above ELISA system.

【００３１】(2) ウサギ網状赤血球抽出液によるHCVコ
ア蛋白の発現 20pmolのR-IA-1が10μlのTEに溶解している溶液、なら
びに全くRNAの溶解していない10μlのTE溶液に最終濃度
10μMの メチオニン溶液を2μl加え、さらにこの12μl
の混合溶液に20μlのウサギ網状赤血球抽出液（In vit
ro translationkit : STARATAGENE社）を加えて30℃で
2時間インキュベイトした。この反応液を段階希釈し、
上記ELISAにてコア蛋白量を定量したところ、陽性コン
トロールではHCVコア蛋白が合成されていることを確認
したが、陰性コントロールではHCVコア蛋白は見出され
なかった。(2) Expression of HCV core protein by rabbit reticulocyte extract 20 pmol of R-IA-1 dissolved in 10 μl of TE and 10 μl of TE in which RNA was not dissolved at a final concentration
Add 2 μl of 10 μM methionine solution, and further add this 12 μl
20 μl of rabbit reticulocyte extract (In vit
ro translationkit: STARATAGENE) at 30 ℃
I incubated for 2 hours. This reaction solution is serially diluted,
When the amount of core protein was quantified by the above ELISA, it was confirmed that the HCV core protein was synthesized in the positive control, but the HCV core protein was not found in the negative control.

【００３２】3. アンチセンス化合物のターゲット領域
の探索 HCV-RNAの5'非翻訳領域のうち、アンチセンス化合物が
結合することにより蛋白合成に他の領域に較べて強く阻
害がかかると考えられるような領域を以下に述べる方法
にて探索した。 3. Target area of antisense compounds
In the 5'untranslated region of HCV-RNA, a region that is thought to strongly inhibit protein synthesis by binding with an antisense compound as compared with other regions was searched by the method described below. .

【００３３】(1) 合成アンチセンスDNAの調製 アンチセンスDNAは、MilliGen / Biosearch 社製 Cyclo
ne Plus DNA Synthesizer を用いて固相化ホスホア
ミダイト法により合成した。得られたDNA溶液中のRNA分
解酵素を失活させるためにフェノール処理を行い、エタ
ノール沈殿法により沈殿させた後、10mM Tris-HCl(pH
8.0) , 1mM EDTA溶液（TE）に適当な濃度に溶解させ使
用した。今回合成したアンチセンスDNAは長さがそれぞ
れ20merで、対応するRNA領域の5'端の塩基の図２での塩
基番号の前にCAS-という文字を入れ、そのアンチセンス
DNAに対する命名とした。(1) Preparation of Synthetic Antisense DNA Antisense DNA was prepared by MilliGen / Biosearch Cyclo.
It was synthesized by a solid-phased phosphoamidite method using ne Plus DNA Synthesizer. Phenol treatment was performed to inactivate the RNA degrading enzyme in the obtained DNA solution, and after precipitation by the ethanol precipitation method, 10 mM Tris-HCl (pH
8.0), dissolved in 1 mM EDTA solution (TE) to an appropriate concentration and used. The antisense DNAs synthesized this time each have a length of 20 mer, and the characters CAS- are inserted before the base number in Fig. 2 of the base at the 5'end of the corresponding RNA region.
It was named as DNA.

【００３４】(2) 阻害度の評価法 20pmolのR-IA-1及び100pmolの評価対象となるアンチセ
ンスDNAを最終容積10μlのTE中で混合して室温で10分放
置する。この溶液に2μlの10mM メチオニンを加え、さ
らにこの12μlの混合溶液に20μlのウサギ網状赤血球抽
出液（In vitrotranslation kit : STARATAGENE社）
を加えて30℃で2時間インキュベイトした。反応終了
後、溶液中にて合成されたコア蛋白量を上記ELISAにて
定量し、アンチセンスDNAが最初のTE中に存在しなかっ
た場合のコア蛋白合成量に対する比を求め、この比を1
より減じて得られた数値を%で表示して阻害度とする。(2) Method for evaluating degree of inhibition 20 pmol of R-IA-1 and 100 pmol of antisense DNA to be evaluated are mixed in a final volume of 10 μl of TE and left at room temperature for 10 minutes. 2 μl of 10 mM methionine was added to this solution, and 20 μl of rabbit reticulocyte extract (In vitro translation kit: STARATAGENE) was further added to this 12 μl mixed solution.
Was added and incubated at 30 ° C. for 2 hours. After the reaction was completed, the amount of core protein synthesized in the solution was quantified by the above-mentioned ELISA, and the ratio to the amount of core protein synthesized when antisense DNA was not present in the initial TE was calculated.
The numerical value obtained by further subtracting is displayed as% to be the degree of inhibition.

【００３５】(3) 阻害領域の大まかな探索 HCV-RNAの塩基番号1-20までの配列と相補的な20merの合
成DNAであるCAS-1を合成し、次に対応領域を3'側に9塩
基ずらした塩基番号10-29までと相補的なCAS-10を合成
した。以下同様にして対応領域を3'側に10塩基ずつずら
して相補的な20merのアンチセンス合成DNAをCAS-320ま
で合計33種類合成しそれぞれの阻害効果を評価したとこ
ろ、CAS-110が70％以上の阻害度を示し、他と比較して
非常に高かった。(3) Rough Search for Inhibitory Region CAS-1 which is a 20-mer synthetic DNA complementary to the sequence of HCV-RNA up to nucleotide numbers 1-20 was synthesized, and then the corresponding region was placed on the 3 ′ side. CAS-10 was synthesized, which was complementary to base numbers 10-29, which were shifted by 9 bases. In the same manner, 33 corresponding complementary 20-mer antisense synthetic DNAs were synthesized up to CAS-320 by shifting the corresponding region to the 3'side by 10 bases each, and the inhibitory effect of each was evaluated. The above-mentioned degree of inhibition was shown, and it was much higher than the others.

【００３６】(4) 塩基番号110-129付近の詳しい解析 阻害効果の高かったCAS-110と相補的な塩基番号110-129
付近について詳しく解析するため、CAS-100とCAS-110の
対応領域の間を2塩基ずづに区切ってCAS-102、CAS-10
4、CAS-106、CAS-108の4種類の20merのアンチセンスDNA
を合成し、さらにCAS-110とCAS-120の対応領域の間につ
いても同様にしてCAS-112、CAS-114、CAS-116、CAS-118
の4種類を合成した。新しく合成したアンチセンスDNAに
ついて上記の方法にて阻害効果の評価を行った結果、対
応領域が連続するCAS-104、CAS-106、CAS-108の3種類の
アンチセンスDNAが70％以上の高い阻害度を示した。前
の結果と合わせると、CAS-104、CAS-106、CAS-108、CAS
-110の対応領域が連続する4種類の20merのアンチセンス
DNAが70％の高い阻害度を示したこととなり、塩基番号1
04から129の26塩基の範囲の連続する20塩基と相補的な
アンチセンスDNAは、5'NCRの他の領域と相補的なアンチ
センスDNAと比較してHCV-RNAに対し、強い翻訳阻害効果
を持つことが示された。(4) Detailed analysis around base numbers 110-129 Base numbers 110-129 complementary to CAS-110, which had a high inhibitory effect
In order to analyze the neighborhood in detail, the corresponding regions of CAS-100 and CAS-110 are divided into two bases, CAS-102 and CAS-10.
4, 4 types of 20-mer antisense DNA, CAS-106, CAS-108
, And similarly between the corresponding areas of CAS-110 and CAS-120, CAS-112, CAS-114, CAS-116, CAS-118
4 types were synthesized. The inhibitory effect of the newly synthesized antisense DNA was evaluated by the above method. As a result, three types of antisense DNA, CAS-104, CAS-106, and CAS-108, in which the corresponding regions are continuous, have a high level of 70% or more. The degree of inhibition was shown. Combined with previous results, CAS-104, CAS-106, CAS-108, CAS
-4 types of 20mer antisense with 110 corresponding regions in a row
DNA showed a high degree of inhibition of 70%, and base number 1
Antisense DNA complementary to consecutive 20 bases in the range of 26 bases from 04 to 129 has a strong translational inhibitory effect on HCV-RNA as compared to antisense DNA complementary to other regions of 5'NCR. Was shown to have.

【００３７】(5) 塩基番号119をイノシンに置換したア
ンチセンスDNAの評価 塩基番号119のアデニンはHCV株間で変異率の高いことが
知られているので、この位置をイノシンに置換すること
で複数のウイルス株に効果を持つように出来ないかどう
かを以下に述べるような方法で調べた。CAS-110の配列
中、塩基番号119のアデニンに対応するチミンをイノシ
ンに置換したCAS-110-I-119と、対照として上記チミン
をグアニンに置換して人為的にミスマッチを導入したCA
S-110-G-119を合成し、この二つのアンチセンスDNAの阻
害度を評価したところ、CAS-110-I-119はCAS-110と同程
度の70%以上の高い阻害度を示したが、CAS-110-G-119で
は著しく減少した。この結果より上記チミンをイノシン
に置換することにより、今回実験に用いた配列をもつウ
イルス株と比較して119番のアデニンが他の塩基に置換
されている他のウイルス株に対しても効果を期待しうる
ことが示された。今回合成した43種類それぞれのアンチ
センスDNAの配列を図３に記す。(5) Evaluation of antisense DNA in which base number 119 is replaced with inosine Since adenine with base number 119 is known to have a high mutation rate among HCV strains, it can be replaced by inosine at this position. The following method was used to examine whether or not the virus strain could not be effective. In the sequence of CAS-110, CAS-110-I-119 in which thymine corresponding to adenine at base number 119 was replaced with inosine, and as a control, CA was introduced with artificial mismatch by replacing thymine with guanine.
When S-110-G-119 was synthesized and the inhibition of these two antisense DNAs was evaluated, CAS-110-I-119 showed a high inhibition of 70% or more, which was similar to CAS-110. However, it was markedly reduced in CAS-110-G-119. From this result, by substituting the above thymine with inosine, compared with the virus strain having the sequence used in this experiment, the effect on other virus strains in which the 119th adenine is replaced with other bases is also effective. It was shown to be promising. The sequences of each of the 43 types of antisense DNA synthesized this time are shown in Fig. 3.

【００３８】[0038]

【配列表】[Sequence list]

配列番号：１ 配列の長さ：１４ 配列の型：核酸 トポロジー：直鎖状 配列の種類：ゲノムＤＮＡ 起源、生物名：Ｃ型肝ウイルス 配列 GCCUCCAGGACCCC SEQ ID NO: 1 Sequence length: 14 Sequence type: Nucleic acid Topology: Linear Sequence type: Genomic DNA Origin, Organ name: C-type liver virus Sequence GCCUCCAGGACCCC

【００３９】配列番号：２ 配列の長さ：２６ 配列の型：核酸 トポロジー：直鎖状 配列の種類：ゲノムＤＮＡ 起源、生物名：Ｃ型肝炎ウイルス 配列 CGUGCAGCCUCCAGGACCCCCCCUCCSEQ ID NO: 2 Sequence length: 26 Sequence type: Nucleic acid Topology: Linear Sequence type: Genomic DNA Origin, Organ name: Hepatitis C virus Sequence CGUGCAGCCUCCAGGACCCCCCCUCC

【００４０】配列番号：３ 配列の長さ：２０ 配列の型：核酸 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ アンチセンス：Ｙｅｓ 配列 ＧＧＧＧＴＣＣＴＧＧＡＧＧＣＴＧＣＡＣＧSEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence GGGGTCCTGGAGGCTGCACCG

【００４１】配列番号：４ 配列の長さ：２０ 配列の型：核酸 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ アンチセンス：Ｙｅｓ 配列 ＧＧＧＧＧＧＴＣＣＴＧＧＡＧＧＣＴＧＣＡSEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence GGGGGGTCCTGGAGGGCTGCA

【００４２】配列番号：５ 配列の長さ：２０ 配列の型：核酸 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ アンチセンス：Ｙｅｓ 配列 AGGGGGGGTCCTGGAGGCTGSEQ ID NO: 5 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence AGGGGGGGTCCTGGAGGCTG

【００４３】配列番号：６ 配列の長さ：２０ 配列の型：核酸 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ アンチセンス：Ｙｅｓ 配列 GGAGGGGGGGTCCTGGAGGCSEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence GGAGGGGGGGTCCTGGAGGC

【図面の簡単な説明】[Brief description of drawings]

【図１】 HCV-RNAの塩基配列中の配列番号1-686に対応
するHCV-cDNAをクローニングし、あわせて5'側にT7プロ
モーター、3'側に終止コドン（TGA）を付加させるため
のPCRプライマー（センスとアンチセンス）の塩基配列
を示す。[FIG. 1] A clone of HCV-cDNA corresponding to SEQ ID NO: 1-686 in the nucleotide sequence of HCV-RNA for adding a T7 promoter on the 5'side and a stop codon (TGA) on the 3'side in total The nucleotide sequences of PCR primers (sense and antisense) are shown.

【図２】 HCV-RNAの塩基配列中の配列番号1-686に対応
する塩基配列を表す。FIG. 2 shows a nucleotide sequence corresponding to SEQ ID NOs: 1-686 in the nucleotide sequence of HCV-RNA.

【図３】 実施例中で評価したアンチセンスオリゴヌク
レオチドの名前と配列を表す。FIG. 3 shows the names and sequences of antisense oligonucleotides evaluated in the examples.

フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｇ０１Ｎ 33/576 Ｚ 8310−2Ｊ (72)発明者 星子 和哉 熊本県菊池郡合志町幾久富字建山1909−47 ベルテック武蔵野台Ａ−102 (72)発明者 野崎 周英 熊本県熊本市武蔵ケ丘１−444 (72)発明者 西原 司 熊本県熊本市龍田町上立田1749−６ (72)発明者 中武 博 熊本県菊池郡菊陽町津久礼2333−15 松本 ハイツ202号 (72)発明者 濱田 福三郎 熊本県菊池郡西合志町須屋2679−２Continuation of front page (51) Int.Cl. 5 Identification number Reference number within the agency FI Technical indication location G01N 33/576 Z 8310-2J (72) Inventor Kazuya Hoshiko Ikukyu, Koshimachi, Kikuchi-gun, Kumamoto 1909 47 Veltech Musashinodai A-102 (72) Inventor Shuei Nozaki 1-444 Musashigaoka, Kumamoto City, Kumamoto Prefecture (72) Inventor Tsukasa Nishihara 1749-6 Kamitsutada, Tatsuta-cho, Kumamoto City, Kumamoto Prefecture (72) Hiroshi Nakatake, Kumamoto 2333-15 Tsukure, Kikuyo-cho, Kikuchi-gun, Matsumoto Matsumoto Heights No. 202 (72) Inventor Fukusaburo Hamada 2679-2 Suya, Nishigoshi-cho, Kikuchi-gun, Kumamoto

Claims (8)

【特許請求の範囲】[Claims] 【請求項１】 Ｃ型肝炎ウイルスゲノムの塩基配列のう
ち、5'非翻訳領域に存在する下記の塩基配列（Ａ）もし
くはこの塩基配列に相同性の高い塩基配列とハイブリッ
ドしうることを特徴とするオリゴヌクレオチド。 （Ａ）GCCUCCAGGACCCC1. A base sequence of the hepatitis C virus genome, which is capable of hybridizing with the following base sequence (A) present in the 5'untranslated region or a base sequence highly homologous to this base sequence: An oligonucleotide that does. (A) GCCUCCAGGACCCC 【請求項２】 該オリゴヌクレオチドが、少なくとも１
４個の塩基を有する請求項１のオリゴヌクレオチド。2. The oligonucleotide comprises at least 1
The oligonucleotide of claim 1 having 4 bases. 【請求項３】 該オリゴヌクレオチドが、上記の塩基配
列（Ａ）に対するアンチセンス塩基配列を少なくとも含
む請求項１のオリゴヌクレオチド。3. The oligonucleotide according to claim 1, wherein the oligonucleotide contains at least an antisense base sequence for the base sequence (A). 【請求項４】 該オリゴヌクレオチドが、上記の塩基配
列（Ａ）に対するアンチセンス塩基配列を有する２０個
の塩基を有する核酸化合物である請求項３のオリゴヌク
レオチド。4. The oligonucleotide according to claim 3, which is a nucleic acid compound having 20 bases having an antisense base sequence for the base sequence (A). 【請求項５】 該オリゴヌクレオチドが、少なくとも上
記配列（Ａ）に対するアンチセンス塩基配列を有し、さ
らに下記の塩基配列（Ｂ）中の連続した１４〜２６個の
核酸に相補的なアンチセンス塩基配列を有する請求項３
のオリゴヌクレオチド。 （Ｂ） CGUGCAGCCUCCAGGACCCCCCCUCC （下線部は上記
配列（Ａ））5. The antisense base which has at least an antisense base sequence for the above sequence (A) and is complementary to 14 to 26 consecutive nucleic acids in the following base sequence (B). 4. Having an array
Oligonucleotide. (B) CGUGCA GCCUCCAGGACCCC CCCUCC (The underlined part is the above sequence (A)) 【請求項６】 該オリゴヌクレオチドが、化学修飾され
た化合物である請求項１のオリゴヌクレオチド。6. The oligonucleotide according to claim 1, wherein the oligonucleotide is a chemically modified compound. 【請求項７】 該オリゴヌクレオチドが、フォスフォロ
チオエート化された化合物である請求項６のオリゴヌク
レオチド。7. The oligonucleotide of claim 6, wherein the oligonucleotide is a phosphorothioated compound. 【請求項８】 請求項１〜７のいずれかに記載のオリゴ
ヌクレオチドを有効成分とするＣ型肝炎治療剤。8. A hepatitis C therapeutic agent comprising the oligonucleotide according to any one of claims 1 to 7 as an active ingredient.
JP5087195A 1992-09-10 1993-04-14 Anti-hepatitis c virus oligonucleotide Withdrawn JPH06296492A (en)

Priority Applications (14)

Application Number Priority Date Filing Date Title
JP5087195A JPH06296492A (en) 1993-04-14 1993-04-14 Anti-hepatitis c virus oligonucleotide
NZ255578A NZ255578A (en) 1992-09-10 1993-09-10 Antisense oligonucleotides complementary and hybridizable to a portion of hepatitis c virus and their use in treating hepatitis c virus associated diseases
NZ286209A NZ286209A (en) 1992-09-10 1993-09-10 Use of HCV RNA anti-sense nucleotide sequences for treating Hepatitis C virus related disease
US08/397,220 US6284458B1 (en) 1992-09-10 1993-09-10 Compositions and methods for treatment of hepatitis C virus-associated diseases
JP6507055A JPH08506479A (en) 1992-09-10 1993-09-10 Compositions and methods for the treatment of hepatitis C virus-related disorders
CA002143678A CA2143678A1 (en) 1992-09-10 1993-09-10 Compositions and methods for treatment of hepatitis c virus-associated diseases
PCT/JP1993/001293 WO1994005813A1 (en) 1992-09-10 1993-09-10 Compositions and methods for treatment of hepatitis c virus-associated diseases
AU49837/93A AU680435B2 (en) 1992-09-10 1993-09-10 Compositions and methods for treatment of hepatitis C virus-associated diseases
DK93919644T DK0662157T3 (en) 1992-09-10 1993-09-10 Preparations and Methods for the Treatment of Hepatitis C Virus-Associated Diseases
DE69330372T DE69330372T2 (en) 1992-09-10 1993-09-10 COMPOSITIONS AND METHODS FOR TREATING DISEASES ASSOCIATED WITH HEPATITIS C VIRUSES
AT93919644T ATE202383T1 (en) 1992-09-10 1993-09-10 COMPOSITIONS AND METHODS FOR THE TREATMENT OF HEPATITIS C VIRUS-ASSOCIATED DISEASES
EP93919644A EP0662157B1 (en) 1992-09-10 1993-09-10 Compositions and methods for treatment of hepatitis c virus-associated diseases
US10/457,304 US20040033978A1 (en) 1992-09-10 2003-06-09 Compositions and methods for treatment of Hepatitis C virus-associated diseases
JP2004268347A JP3803354B2 (en) 1992-09-10 2004-09-15 Compositions and methods for treatment of hepatitis C virus related diseases

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008283975A (en) * 2002-02-20 2008-11-27 Sirna Therapeutics Inc RNA INTERFERENCE MEDIATED INHIBITION OF HEPATITIS C VIRUS (HCV) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008283975A (en) * 2002-02-20 2008-11-27 Sirna Therapeutics Inc RNA INTERFERENCE MEDIATED INHIBITION OF HEPATITIS C VIRUS (HCV) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

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