JP4705370B2 - より新規形態の干渉rna分子 - Google Patents
より新規形態の干渉rna分子 Download PDFInfo
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- JP4705370B2 JP4705370B2 JP2004526885A JP2004526885A JP4705370B2 JP 4705370 B2 JP4705370 B2 JP 4705370B2 JP 2004526885 A JP2004526885 A JP 2004526885A JP 2004526885 A JP2004526885 A JP 2004526885A JP 4705370 B2 JP4705370 B2 JP 4705370B2
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Description
このとき
上記第1鎖および/または第2鎖は、2’−位置が修飾されている修飾ヌクレオチドの複数の群を含むものであり、また該鎖内に於いて各修飾ヌクレオチド群は片側部または両側部でヌクレオチドのフランキング群(flanking group)によりフランキングされており、ヌクレオチドのフランキング群を形成するフランキングヌクレオチドは未修飾ヌクレオチドか、または該修飾ヌクレオチドの修飾とは異なる修飾を有するヌクレオチドである、リボ核酸によって解決される。
本実施例では、2本鎖RNAi分子の活性に及ぼすNH2末端保護基の影響を調べた。合成siRNAはBiospring(Frankfurt、Germany)より購入した。リボ−オリゴヌクレオチドを、RNaseを含まないTEに最終濃度50μMになるように懸濁した。2分子型siRNAの場合は、等量(100μM)を組み合わせて最終濃度を50μMとした。分子間二本鎖を形成する場合には、siRNAを50℃で2分間、アニーリング緩衝液(25mM NaCl;5mM MgCl2)の中でインキュベーションしてから室温まで冷却した。トランスフェクションは、オリゴフェクタミン(Oligofectamine)、リポフェクタミン(Lipofectamine(Life Technologies))、NC388(Ribozyme Pharmaceuticals、Inc.,CO)またはFuGene 6(Roche)といった各種カチオン性脂質を製造元指示書に従って使用し、96ウエルまたは10−cmプレートの中で(30〜50%集密度)行った。RNAi分子のトランスフェクションは、前もって無血清培地に5倍濃度で調製しておいたアニーリング済みRNAiと脂質との複合物を、完全培地中の細胞に加える形で実施した。トランスフェクション前、96ウエル形式でのトランスフェクションを行う15〜18時間前に、ウエル当たり2500個のHeLa細胞をプレーティングした。
96ウエル内でトランスフェクションした細胞のRNAを、トランスフェクション24時間後にInvisorb RNA HTS96キット(InVitek GmbH、Berlin)を用いて単離、精製した。PTEN mRNA発現の阻害は、300nMのPTEN5’プライマーCACCGCCAAATTTAACTGCAGA、300nMのPTEN3’プライマーAAGGGTTTGATAAGTTCTAGCTGTおよび100nMのPTEN TaqmanプローブFam−TGCACAGTATCCTTTTGAAGACCATAACCCA−Tamraを、40nMのβ−アクチン5’プライマーGTTTGAGACCTTCAACACCCCA、40nMのβ−アクチン3’プライマーGACCAGAGGCATACAGGGACAおよび100nMのβ−アクチンTaqmanプローブVic−CCATGTACGTAGCCATCCAGGCTGTG−Tamraと組み合わせて用いるリアルタイムRT−PCR(Taqman)分析により検出した。Aktプライマーおよびプローブは、Sternberger et al.(Sternberger、a.a.O)で決定されたものであり、製造元指示書(Applied Biosystem;Amplicon Setを使用)に従い使用した。前記プライマーおよびプローブはPrimer Express(Applied Biosystem)を用いて設計してもよい。反応は50μlで実施され、ABI PRISM 7700シーケンス検出装置(Applied Biosystems)を製造元の指示書に従い用いて、以下の条件で行った:48℃30分間、95℃10分間の後に95℃15秒および60℃1分間を40サイクル。
実験手法は、干渉RNAi分子を標的とするPTEN mRNAの設計が異なる外は実施例1の説明と同じであった。結果は図4Aに用量反応曲線の形で示し、図4Bに図4Aに示したデータを得るのに用いた干渉RNAi分子の具体的な配列および修飾を示した。表記法は、例えばRNAi18はアンチセンス鎖としての鎖18Aと、センス鎖としての鎖18Bとから成ることを示すものである。
実験方法は、干渉RNA分子がAkt1のmRNAを標的とする外は、実施例1に概要示したものと同様であった。RNAi分子の特異性を示すための陰性コントロールは、ここでもp110 mRNAであった。実験結果を図5Aに示し、図5Bには用いた干渉RNAi分子の詳細を示した。図7A、左パネルに示した別のsiRNA構築物についても同様の実験を実施したが、この場合不一致を矢印で示し、デオキシリボヌクレオチドは大文字で表した。表示量のsiRNA分子をトランスフェクションしたHeLa細胞でのAkt1 mRNA発現の阻害を図7Aの右パネルに示した。
実験構成は実施例1に記載のものと同等であるが、この場合RNAiはAKt1特異的である。さらにPTEN特異的干渉RNA分子を設計し、陰性コントロールに用いた。結果は図6Aおよび図6Bに示した。図7Bに示す更に別のsiRNA分子を用いて、基本的には同一な実験を行い、その結果をそれぞれ図7B(右パネル)および図7Cに示した。
オリゴヌクレオチドをヒト血清中で15分間および2時間インキュベーションし、未処理のコントロールと共に10%ポリアクリルアミドゲルにかけた。結果は図8Aに示した。使用した各種RNAi分子を図8Bに示し、詳しく説明した。
実験構成は実施例1に関連して記載したものと同等であるが、この場合の標的核酸配列はPTEN mRNAである。HeLa細胞濃度は2,000細胞/ウエルであった。PTENのRNAは、各種修飾RNAi分子トランスフェクション後にTaqmanアッセイにて分析した。用いた各種干渉RNA分子は図9Bに示し、実験結果は図9Aに示した。
RNAノックダウンを再度示すために、図10Aに示すようなPTEN mRNAを標的としたRNAi二本鎖分子をトランスフェクションしたHeLa細胞にリアルタイムRT−PCR分析を行った。実験手法は基本的には実施例1に記載の手順に同じである。図10Aに示す、研究対象のRNAi分子の構造およびその用量反応は図10Cに示した。太字で示したヌクレオチドは2’−O−メチル修飾を有するヌクレオチドである。
本試験に関する実験方法は、実際的には実施例1に記載の方法と同一であった。ここでもPTEN RNAは、各種用量のRNAi分子をトランスフェクションした密度2000細胞/ウエルのHeLa細胞にリアルタイムRT−PCRを行い分析した。RNAi分子を血清中で2時間インキュベーションし、10%ポリアクリルアミドゲルで分析した。本試験の結果を図11A〜11Cに示すが、図11Aは図11Cに示した各種RNAi分子の用量反応を表しており、図11Bは図11Cに示したRNAi分子の幾つかを用いた安定性試験の結果を示している。図11Cに太字で書かれたヌクレオチドは修飾を有するヌクレオチドであり、本例での修飾はリボース部分の2’−O−メチル修飾である。
この種の試験の実施に関する実験構成は、実施例1および実施例8にそれぞれ記載した試験に関係し使用した構成と同一であり、今回も標的核酸はPTEN mRNAであった。HeLa細胞に図12Bに記載の各種RNAi分子をトランスフェクションし、PTEN RNAを対象に用量依存的に行ったリアルタイムRT−PCRを用いてRNAのノックダウンを実証した(図12A)。37℃、血清中、15分および2時間後の各種RNAi分子の安定性を図12Cに、各種RNAi分子の標的タンパク質としてのp110およびPTENに関するウエスタンブロットを図12Dに示すが、図12Cおよび図12Dの基礎となる両実験で試験したRNAi分子は同じものである。
実験方法は実施例1で概要説明した方法と同様であった。
実験方法は、トランスフェクションを5時間後にトランスフェクション培地を新規培地と交換することで停止した以外は実施例10に概要を示した方法と同様であった。プロトコールを若干変更し、各RNAi分子の濃度が、実施例1に関し記載した1μgRNAi/mlカチオン脂質の保存液を用いて40nMになるようにした。トランスフェクション後5時間目に、培地を取り去り、新鮮なEMEMを加えた。72時間後に細胞を分割し、半分を溶解し、残り半分は新たにプレーティングしてから24時間後(トランスフェクション後96時間目)に溶解した。3種類のRNAi分子(80AB、94A1/B2、94A2/B1)を用いたウエスタンブロットの結果を図14に示す。陽性コントロールとして、未処理細胞を用いた。図14は72時間および96時間後それぞれのPTENの発現を示している。該各種RNAi分子の構造特性を考慮すると、図14からは、非修飾RNAi分子(例えば80AB)およびRNAi分子94A1B2に対し、92A2B1のような交互型分子では、細胞分割、再プレーティング後96時間においてもタンパク質ノックダウンが持続することが分かる。
mRNAおよびタンパク質発現阻害に及ぼす各種ループ構造の影響を試験した。これら実験については、標的としてAkt1およびAkt2を選択した。実験方法は実施例12に記載の方法と同様であった。
Claims (44)
- 2本鎖構造を含み、前記2本鎖構造が第1鎖および第2鎖を含み、前記第1鎖が連続するヌクレオチドの第1ストレッチを含み、前記第1ストレッチは標的核酸と少なくとも部分的に相補的であり、そして前記第2鎖が連続するヌクレオチドの第2ストレッチを含み、前記第2ストレッチは標的核酸と少なくとも部分的に同一である、標的遺伝子またはその誘導物の発現を阻害するためのリボ核酸であって、
前記第1ストレッチおよび前記第2ストレッチが2’−位置に修飾を有する修飾ヌクレオチドの複数の群からなるパターンを含み、ストレッチ内の修飾ヌクレオチドの各群はヌクレオチドのフランキング群により片側または両側をフランキングされており、ヌクレオチドのフランキング群を形成しているフランキングヌクレオチドが非修飾ヌクレオチドまたは前記修飾ヌクレオチドの修飾とは異なる修飾を有するヌクレオチドであり、第1ストレッチのパターンが第2ストレッチのパターンに整合することを特徴とする、リボ核酸。 - 修飾ヌクレオチドの群および/またはフランキングヌクレオチドの群が、1ヌクレオチド〜10ヌクレオチドを含む群より選択される数のヌクレオチドを含む、請求項1に記載のリボ核酸。
- 第1ストレッチの修飾ヌクレオチドのパターンが第2ストレッチの修飾ヌクレオチドのパターンと同一である、請求項1〜2のいずれか1項に記載のリボ核酸。
- 修飾がアミノ、フルオロ、メトキシ、アルコキシおよびアルキルを含む群より選択される、請求項1〜3のいずれか1項に記載のリボ核酸。
- 2本鎖構造が平滑端である、請求項1〜4のいずれか1項に記載のリボ核酸。
- 2本鎖構造が両側部において平滑端である、請求項1〜5のいずれか1項に記載のリボ核酸。
- 2本鎖構造が、第1鎖の5’−末端および第2鎖の3’−末端により画定される2本鎖構造上の側部において平滑端である、請求項1〜6のいずれか1項に記載のリボ核酸。
- 2本鎖構造が、第1鎖の3’−末端および第2鎖の5’−末端により画定される2本鎖構造上の側部において平滑端である、請求項1〜7のいずれか1項に記載のリボ核酸。
- 2本鎖の少なくとも一方が5’−末端に少なくとも1ヌクレオチドの突出を有している、請求項1〜4のいずれか1項に記載のリボ核酸。
- 突出が、リボヌクレオチドおよびデオキシリボヌクレオチドを含む群より選択される少なくとも1つのヌクレオチドから成る、請求項9に記載のリボ核酸。
- ヌクレオチドが修飾を有し、前記修飾が逆方向脱塩基であるヌクレオチド及び2’−位置にNH2−修飾を有するヌクレオチドを含む群より選択される、請求項10に記載のリボ核酸。
- 5’−末端の突出が第2鎖上にある、請求項9〜11のいずれか1項に記載のリボ核酸。
- 第1鎖も突出を含むことを特徴とする、請求項12に記載のリボ核酸。
- 第1鎖が突出を、5’−末端に含むことを特徴とする、請求項13に記載のリボ核酸。
- 5’−末端の突出が第1鎖上にある、請求項9〜14のいずれか1項に記載のリボ核酸。
- 第2鎖も突出を含むことを特徴とする、請求項15に記載のリボ核酸。
- 第2鎖が突出を、5’−末端に含むことを特徴とする、請求項16に記載のリボ核酸。
- 鎖の少なくとも1つが、3’−末端に少なくとも1ヌクレオチドの突出を有する、請求項1〜4のいずれか1項に記載のリボ核酸。
- 3’−末端のヌクレオチドがリボヌクレオチドおよびデオキシリボヌクレオチドを含む群より選択される、請求項18に記載のリボ核酸。
- 第1鎖の3’−末端が突出を含んでいる、請求項18〜19のいずれか1項に記載のリボ核酸。
- 2本鎖構造の長さが19〜21塩基長を有する、請求項1〜20のいずれか1項に記載のリボ核酸。
- 2本鎖構造の長さが19塩基長を有する、請求項1〜20のいずれか1項に記載のリボ核酸。
- 第1鎖の長さ、および前記第2鎖の長さが、互いに独立に19〜23塩基、19〜21塩基の範囲、および19塩基を含む群から選択される、請求項1〜22のいずれか1項に記載のリボ核酸。
- 第1鎖と標的核酸間の相補性が完全である、請求項1〜23のいずれか1項に記載のリボ核酸。
- 第1鎖と標的核酸間に形成される二本鎖が少なくとも19ヌクレオチドを含み、前記2本鎖構造を形成している前記第1鎖と標的核酸との間に1個の不一致または2個の不一致がある、請求項1〜24のいずれか1項に記載のリボ核酸。
- 修飾ヌクレオチドの各群が単一ヌクレオチドから成り、そして/またはヌクレオチドの各フランキング群が単一ヌクレオチドから成る、請求項1〜25に記載のリボ核酸。
- 第1鎖上でヌクレオチドのフランキング群を形成しているヌクレオチドが修飾ヌクレオチド群を形成しているヌクレオチドに対し3’方向に配置される非修飾ヌクレオチドであり、そして第2鎖上で修飾ヌクレオチド群を形成しているヌクレオチドがヌクレオチドのフランキング群を形成しているヌクレオチドに対し5’方向に配置される修飾ヌクレオチドである、請求項26に記載のリボ核酸。
- 第1鎖が8〜11の修飾ヌクレオチド群を含み、そして第2鎖が8〜11の修飾ヌクレオチド群を含む、請求項26〜27のいずれか1項に記載のリボ核酸。
- 標的遺伝子が構造遺伝子、ハウスキーピング遺伝子、転写因子、運動因子、細胞周期因子、細胞周期インヒビター、酵素、増殖因子、サイトカインおよび腫瘍サプレッサーを含む群から選択される、請求項1〜28のいずれか1項に記載のリボ核酸。
- 請求項1〜29のいずれか1項に記載の第1鎖および第2鎖がループ構造で連結している、リボ核酸。
- ループ構造が非核酸ポリマーを含んでいる、請求項30に記載のリボ核酸。
- 非核酸ポリマーがポリエチレングリコールであることを特徴とする、請求項31に記載のリボ核酸。
- ループ構造が核酸を含んでいる、請求項30〜32のいずれか1項に記載のリボ核酸。
- 第1鎖の5’−末端が第2鎖の3’−末端に連結していることを特徴とする、請求項30〜33のいずれか1項に記載のリボ核酸。
- 第1鎖の3’−末端が第2鎖の5’−末端に連結していることを特徴とする、請求項30〜33のいずれか1項に記載のリボ核酸。
- 医薬製造のための、請求項1〜35のいずれか1項に記載のリボ核酸の使用。
- 医薬が神経膠芽腫、前立腺癌、乳癌、肺癌、肝臓癌、結腸癌、膵臓癌および白血病、糖尿病、肥満、心臓血管病、ならびに代謝性疾患を含む群から選択される疾患または状態の処置を目的とするものである、請求項36に記載の使用。
- 請求項1〜35のいずれか1項に記載のリボ核酸を含有する細胞。
- 請求項1〜35のいずれか1項に記載のリボ核酸を含有するノックダウン細胞。
- 請求項1〜35のいずれか1項に記載のリボ核酸を含有する、ヒトを除く生物。
- 請求項1〜35のいずれか1項に記載のリボ核酸を含有する、ヒトを除くノックダウン生物。
- 請求項1〜35のいずれか1項に記載のリボ核酸を含有する組成物。
- 請求項1〜35のいずれか1項に記載のリボ核酸および医薬的に許容可能な担体を含有する医薬組成物。
- 生体外において請求項1〜35のいずれか1項に記載のリボ核酸を標的遺伝子の発現を阻害するのに十分な量で細胞内に導入することを含み、前記標的遺伝子が請求項1〜35のいずれか1項に記載のリボ核酸の標的遺伝子である、生体外において細胞内での標的遺伝子またはその誘導物の発現を阻害する方法。
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US20070265220A1 (en) * | 2004-03-15 | 2007-11-15 | City Of Hope | Methods and compositions for the specific inhibition of gene expression by double-stranded RNA |
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KR20120029002A (ko) | 2012-03-23 |
TR201816291T4 (tr) | 2018-11-21 |
HUE032289T2 (en) | 2017-09-28 |
ES2627779T3 (es) | 2017-07-31 |
ES2665274T3 (es) | 2018-04-25 |
PT3222724T (pt) | 2018-12-17 |
ES2627779T5 (es) | 2021-05-04 |
ES2695050T3 (es) | 2018-12-28 |
HUE038749T2 (hu) | 2018-11-28 |
JP2006500014A (ja) | 2006-01-05 |
PT2258847T (pt) | 2017-06-22 |
SI1527176T1 (sl) | 2007-06-30 |
PT1857547T (pt) | 2018-02-08 |
TR201708347T4 (tr) | 2019-02-21 |
ES2665274T5 (es) | 2021-06-30 |
HUE039866T2 (hu) | 2019-02-28 |
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