JP2014509852A - マイクロRNA−155(miR−155)により誘導される変異誘発活性は炎症および癌を結び付ける - Google Patents
マイクロRNA−155(miR−155)により誘導される変異誘発活性は炎症および癌を結び付ける Download PDFInfo
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Abstract
【選択図】 なし
Description
関連出願への相互参照
[0001] この出願は2011年3月7日に出願された米国仮出願第61/449,854号の利益を主張し、その開示全体を明確に本明細書に援用する。
[0002] 本発明は、国立衛生研究所により与えられた助成金番号CA123541の下での政府援助によりなされた。政府は本発明において一定の権利を有する。
[0003] 本出願はEFS-webを介して提出された配列表を含み、それをそのまま本明細書に援用する。2012年3月6日に作成されたASCIIコピーは604_52873_SEQ_LIST_11137.txtと名付けられており、大きさは34,668バイトである。
[0004] この発明は、概して分子生物学の分野に関する。本発明の特定の観点には、癌および白血病と関係する障害の診断法、療法、および予後徴候における適用が含まれる。
a)miR-155を評価すべき薬剤と接触させ;
b)miR-155の1個以上の標的遺伝子を評価すべき薬剤と接触させ;または
c)それらの組み合わせを接触させる;ここで、その薬剤がmiR-155の発現を阻害する、その標的遺伝子の発現を増進する、またはそれらの組み合わせを行う場合、その薬剤はその炎症と関連する癌の増殖を阻害するために用いることができる。
[0068] 前記の一般的な記述および以下の詳細な記述は両方とも典型的かつ説明的なものでしかなく、本教示の範囲を限定することは意図していないことは理解されるべきである。この出願において、別途明確に記載しない限り、単数形の使用には複数形が含まれる。本開示の様々な態様の再考を容易にするため、以下の特定の用語の説明を提供する。
[00102] miR-155をコードする核酸分子は、本発明の様々な態様において用いられる。成熟したmiR-155、pre-miR-155に関するmiR-155配列を一部の態様において用いることができる。他の態様において、成熟したmiR-155およびpre-miR-155をコードするcDNAを用いることができる。pri-miR-155をコードする核酸分子も一部の態様において用いることができる。miRNA配列は、約6ヌクレオチドから約99ヌクレオチドまで、またはより多くのヌクレオチドを含んでいてよい。一部の態様において、miRNA配列は、pre-miRNA-155の最初の約6ヌクレオチド〜最初の約22ヌクレオチドを含む。miR-155のコード配列またはその相補物の少なくとも6ヌクレオチド(すなわちハイブリダイズ可能な部分)を有する単離または精製されたポリヌクレオチドが、一部の態様において用いられる。他の態様において、miR-155ポリヌクレオチドは好ましくは少なくとも(連続した)22ヌクレオチド、または完全長のmiR-155のコード配列を含む。
[00112] miR-155または抗miR-155コード配列を含有する発現ベクターを、miR-155または抗miR-155を標的細胞に送達するために用いることができる。特定の態様において、発現ベクターは、標的細胞におけるそのコード配列の発現を方向付ける調節エレメントと場合により結合した、miR-155配列および/または抗miR-155配列を含有することができる。個々のベクターおよび/またはそれにコード配列が操作可能であるように連結される発現制御配列の選択は、一般に(当業者により理解されているように)望まれる個々の機能的特性、例えば形質転換される宿主細胞に依存することは理解されるべきである。
[00117] 特定の態様において、miR-155または抗miR-155オリゴヌクレオチドが標的細胞に送達される。他の態様において、miR-155または抗miR-155をコードする発現ベクターが標的細胞に送達され、そこでmiR-155または抗miR-155が発現される。オリゴヌクレオチドおよび発現ベクターの標的細胞への送達のために様々な方法を用いることができることは理解されるべきである。
[00126] 本明細書において、miR-155の過剰発現および炎症促進環境の、6-チオグアニン(6-GT)への耐性に基づいて検出することができるヒポキサンチンホスホリボシルトランスフェラーゼ(HPRT)の自然突然変異の発生頻度への作用を記述する。miR-155の過剰発現および炎症促進環境は両方ともHPRT変異の発生頻度を増大させ、細胞周期の進行を遮断するキナーゼであるWEE1を下方制御した。HPRTの突然変異の増大した発生頻度は、ミスマッチ修復機構における欠陥にはわずかにしか帰することができなかった。この結果は、miR-155は変異を抑制する異なる遺伝子を同時に標的にして、WW1のような細胞周期制御因子の標的化によりDNA保護機構の効率を低下させることにより突然変異率を高めることを示している。
[00135] 結果
[00136] MiR-155の過剰発現は結果として高められた突然変異率をもたらす。
[00161] この実施例において、miR-155の、およびmiR-155と関連する炎症促進環境の突然変異誘発活性を分析した。炎症性刺激が結果としてmiR-155の上方制御をもたらした細胞は、HPRTアッセイにより推定した際に、突然変異率における2〜3倍の増大を示した。
[00171] 細胞培養、形質移入、および処理。
[00177] WEE1レポーターコンストラクトは、HEK-293細胞のゲノムDNAからPCRにより増幅したヒトWEE1の3’UTRを、pGL3-Controlベクター(Promega)のXbaI部位においてルシフェラーゼ遺伝子の下流に挿入することにより調製された。成熟したmiR-155およびmiR-155前駆体(pre-miR-155)を、以下のプライマーの再アニーリングにより調製された二本鎖DNAのNotIおよびEcoRIによる消化後に、pRetroX-tight-Purベクター中にクローニングした。
V155MatForward:
5’-ATAGCGGCCGCTTAATGCTAATCGTGATAGGGGTGAATTCGCG-3’[SEQ ID NO: 1]および
V155-MatReverse:
5’-CGCGAATTCACCCCTATCACGATTAGCATTAAGCGGCCGCTAT-3’[SEQ ID NO: 2];
pre-miR-155:
V155PreForward:
5’ATAGCGGCCGCCTGTTAATGCTAATCGTGATAGGGGTTTTTGCCTCCAACTGACTCCTACATATTAGCATTAACAGGAATTCGCG-3’[SEQ ID NO: 3]
および
V155PreReverse:
5’CGCGAATTCCTGTTAATGCTAATATGTAGGAGTCAGTTGGAGGCAAAAACCCCTATCACGATTAGCATTAACAGGCGGCCGCTAT-3’[SEQ ID NO: 4]。
Claims (32)
- マイクロRNA-155(miR-155)オリゴヌクレオチドを標的細胞に投与することを含む、標的細胞においてWEE1キナーゼの発現レベルを調節するための方法。
- miR-155オリゴヌクレオチドを対象中の標的細胞に投与し;そして該標的細胞の変異を測定することを含む、対象中の標的細胞の変異を調節する方法であって、該標的細胞が癌細胞または前癌細胞である、前記方法。
- 内因性のmiR-155のレベルを低減することを含む、細胞の自然突然変異率を低減することを必要とする対象において、細胞の自然突然変異率を低減する方法。
- 内因性のmiR-155のレベルを低減することを含む、炎症関連癌細胞の自然突然変異率を低減することを必要とする対象において、炎症関連癌細胞の自然突然変異率を低減する方法。
- 癌細胞または癌細胞集団において細胞増殖を遅くする、または阻害する方法であって、該細胞または細胞集団を成熟したマイクロRNA-155と少なくとも90%同一である配列に相補的であるmiR-155オリゴヌクレオチドを含むmiR-155アンチセンス化合物と接触させ、それにより該細胞または細胞集団の変異を遅くする、または阻害することを含む、前記方法。
- miR-155癌を有する、または有することが疑われる対象を同定し;そして標的細胞にmiR-155オリゴヌクレオチドを投与することを含む、miR-155と関係する癌を処置または予防する方法。
- miR-155と関係する癌を有する、または有することが疑われる対象を同定し、そして該対象に成熟したマイクロRNA-155と少なくとも90%同一である配列に相補的であるmiR-155オリゴヌクレオチドを含むmiR-155アンチセンス化合物を投与することを含む、miR-155と関係する癌を処置または予防する方法。
- 標的細胞において1個以上の遺伝子の発現を調節する方法であって、該標的細胞をmiR-155オリゴヌクレオチドと接触させることを含み、該遺伝子が以下:APC、腺腫性結腸ポリープ症;FADD、デスドメインを介してFas(TNFRSF6)と会合する;FOXO3、フォークヘッドボックスO3;KGF、ケラチノサイト成長因子;HIVEP2、HIV I型エンハンサー結合タンパク質2;MYO10、ミオシンX;RHOA、Rasホモログ遺伝子ファミリー、メンバーA;RIP1、受容体相互作用タンパク質キナーゼ1;SHIP1、イノシトールポリリン酸-5-ホスファターゼ;SMAD1/5、SMADファミリーメンバー1/5;SOCS1、サイトカインシグナル伝達の抑制因子1;TP53INP、腫瘍タンパク質53に誘導される核タンパク質1から選択される、前記方法。
- miR-155オリゴヌクレオチドがアンチセンスmiR-155オリゴヌクレオチドを含む、請求項1〜8のいずれか1項に記載の方法。
- miR-155オリゴヌクレオチドがmiR-155アンチセンス化合物を含む、請求項1〜9のいずれか1項に記載の方法。
- miR-155オリゴヌクレオチドがmiR-155アンタゴニスト化合物を含む、請求項1〜10のいずれか1項に記載の方法。
- miR-155オリゴヌクレオチドが成熟したmiR-155オリゴヌクレオチド、pre-miR-155オリゴヌクレオチド、およびmiR-155シード配列からなる群から選択される、請求項1〜11のいずれか1項に記載の方法。
- miR-155アンチセンス化合物が12〜30個の連結されたヌクレオシドからなる修飾されたオリゴヌクレオチドを含み、該修飾されたオリゴヌクレオチドの核酸塩基配列が成熟したmiR-155、pre-miR-155、miR-155シード配列と少なくとも80%同一である配列に相補的であり、または成熟したmiR-155、pre-miR-155、もしくはmiR-155の配列に完全に相補的な配列である、請求項1〜12のいずれか1項に記載の方法。
- miR-155オリゴヌクレオチドの投与が:アンチセンスmiR-155発現ベクターを標的細胞に投与し;そしてアンチセンスmiR-155を該標的細胞中で発現させる工程;を含む、請求項1〜13のいずれか1項に記載の方法。
- miR-155オリゴヌクレオチドの投与が:miR-155発現ベクターを標的細胞に投与する工程;そしてmiR-155を該標的細胞中で発現させる工程;を含む、請求項1〜14のいずれか1項に記載の方法。
- miRNA-155発現ベクターが操作可能であるようにプロモーターに連結されたmiRNA-155をコードする核酸配列を含む、請求項1〜15に記載の方法。
- 標的細胞が癌細胞である、請求項1〜16のいずれか1項に記載の方法。
- 標的細胞が乳癌または前癌細胞である、請求項1〜17のいずれか1項に記載の方法。
- 標的細胞が結腸癌または前癌細胞である、請求項1〜18のいずれか1項に記載の方法。
- 標的細胞が胃癌または前癌細胞である、請求項1〜19のいずれか1項に記載の方法。
- 標的細胞が肺癌または前癌細胞である、請求項1〜20のいずれか1項に記載の方法。
- 調節が1個以上の遺伝子の発現を低下させることを含む、請求項1〜21のいずれか1項に記載の方法。
- 修飾されたオリゴヌクレオチドが成熟したmiR-155の核酸塩基配列に対して2個より多くないミスマッチを有する、請求項1〜22のいずれか1項に記載の方法。
- 調節が1個以上の遺伝子の発現を低下させることを含む、請求項1〜23に記載の方法。
- 細胞をアンチセンスmiR-155阻害RNA(155-I)と接触させることを含む、請求項1〜24のいずれか1項に記載の方法。
- 細胞をWEE1レベルを増大させるのに十分な量のアンチセンスmiR-155阻害RNA(155-I)と接触させる、請求項1〜25のいずれか1項に記載の方法。
- 炎症関連癌細胞の自然突然変異率を低減することを必要とする対象において、炎症関連癌細胞の自然突然変異率を低減する方法であって、該細胞をアンチセンスmiR-155阻害RNA(155-I)と接触させることを含む、前記方法。
- 細胞をWEE1レベルを増大させるのに十分な量のアンチセンスmiR-155阻害RNA(155-I)と接触させる、請求項27に記載の方法。
- miR-155の上方制御を調節することを必要とする対象において、miR-155の上方制御を調節することを含む、炎症と関連する癌の発病を予防する方法。
- 対象がヒトである、請求項1〜29のいずれか1項に記載の方法。
- アンチセンスmiR-155を含む、それを必要とする対象において細胞の自然突然変異率を低減するために有用な組成物。
- 以下の工程:
a)miR-155を評価すべき薬剤と接触させ;
b)miR-155の1個以上の標的遺伝子を評価すべき薬剤と接触させ;または
c)それらの組み合わせを接触させる;
を含む、炎症と関連する癌を阻害するために用いることができる薬剤を同定する方法であって、該薬剤がmiR-155の発現を阻害する、その標的遺伝子の発現を増進する、またはそれらの組み合わせを行う場合、該薬剤は該炎症と関連する癌の増殖を阻害するために用いることができる、前記方法。
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AU2012225506B2 (en) | 2016-11-17 |
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WO2012122239A1 (en) | 2012-09-13 |
US20160289683A1 (en) | 2016-10-06 |
CN103561750A (zh) | 2014-02-05 |
EP2683387A1 (en) | 2014-01-15 |
US20130065938A1 (en) | 2013-03-14 |
EP2683387A4 (en) | 2014-09-03 |
AU2012225506A1 (en) | 2013-09-19 |
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