JP5296328B2 - 1本鎖環状rnaおよびその製造方法 - Google Patents
1本鎖環状rnaおよびその製造方法 Download PDFInfo
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- JP5296328B2 JP5296328B2 JP2007125045A JP2007125045A JP5296328B2 JP 5296328 B2 JP5296328 B2 JP 5296328B2 JP 2007125045 A JP2007125045 A JP 2007125045A JP 2007125045 A JP2007125045 A JP 2007125045A JP 5296328 B2 JP5296328 B2 JP 5296328B2
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- C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance
Description
(1)センス鎖配列と、該センス鎖配列に相補的なアンチセンス鎖配列と、該センス鎖と該アンチセンス鎖の間に両鎖を結合する同じか又は異なる2つのループ配列とを含み、該センス鎖と該アンチセンス鎖が対合してステムを形成することを特徴とする、持続的又は徐放的RNA干渉作用を有する1本鎖環状RNA。
(2)対照細胞における標的RNAの発現を1とした場合に、細胞に導入後24時間の標的RNAの発現が0.4以下を示す、(1)に記載の1本鎖環状RNA。
(3)ヒト血清中で8時間経過したときに70%以上が保持されている、(1)または(2)に記載の1本鎖環状RNA。
(4)相補鎖を形成しない塩基配列を5’末端および3’末端に有する、センス鎖およびアンチセンス鎖をそれぞれ合成し、センス鎖が有する相補鎖を形成しない塩基配列の5’末端の塩基とアンチセンス鎖が有する相補鎖を形成しない塩基配列の3’末端の塩基、およびセンス鎖が有する相補鎖を形成しない塩基配列の3’末端の塩基とアンチセンス鎖が有する相補鎖を形成しない塩基配列の5’末端の塩基を、リガーゼで同時に連結することを含み、
その際、センス鎖が5’末端に有する相補鎖を形成しない塩基配列とアンチセンス鎖が3’末端に有する相補鎖を形成しない塩基配列が互いに結合してループを形成し、センス鎖が3’末端に有する相補鎖を形成しない塩基配列とアンチセンス鎖が5’末端に有する相補鎖を形成しない塩基配列が互いに結合してループを形成し、ならびにセンス鎖とアンチセンス鎖が対合してステムを形成する、(1)〜(3)のいずれかに記載の1本鎖環状RNAの製造方法。
(5)センス鎖が有する相補鎖を形成しない塩基配列とアンチセンス鎖が有する相補鎖を形成しない塩基配列の5’末端をリン酸化することを更に含む、(4)に記載の製造方法。
(6)前記ループの配列が互いに同じかまたは異なる、(4)または(5)に記載の製造方法。
(7)前記ループの長さが2〜20塩基である、(4)〜(6)のいずれかに記載の製造方法。
(8)前記ステムの長さが19〜31塩基である、(4)〜(7)のいずれかに記載の製造方法。
(9)(1)〜(3)のいずれかに記載の1本鎖環状RNAをヒト由来の細胞に導入し、標的RNAを不能にしてタンパク質への翻訳を持続的に阻害することを含む、該タンパク質をコードする遺伝子の発現をin vitroで抑制する方法。
(10)(1)〜(3)のいずれかに記載の1本鎖環状RNAを非ヒト動物、植物又はそれの細胞に導入し、標的RNAを不能にしてタンパク質への翻訳を持続的に阻害することを含む、該タンパク質をコードする遺伝子の発現を抑制する方法。
(11)(1)〜(3)のいずれかに記載の1本鎖環状RNAを有効成分として含む、医薬組成物。
RNA干渉とは、RNA interference(RNAi)ともいい、標的RNAと相補的な配列を持つ小RNA分子が標的RNAに結合し、それを分解するか又は翻訳を抑制する現象である。
本発明によれば、本発明の1本鎖環状RNAを細胞に導入し、標的RNAを不能にしてタンパク質への翻訳を持続的に阻害することができ、前記細胞は、ヒト細胞または非ヒト細胞を用いることができる。
1本鎖環状RNAの医薬組成物への配合量は、組成物の種類や目的等によって調整することができ、組成物の全量に対して、例えば1重量%、3重量%、5重量%、10重量%、20重量%、30重量%、40重量%、50重量%、60重量%、70重量%、80重量%、90重量%、100重量%であるが、これらに限定されない。
ダンベル型RNAの原料となる5’リン酸化RNAは、全てホスホロアミダイト法に基づきDNA合成機(GeneWorld H8−SE)により合成した。RNAアミダイトはTBDMS保護体(Proligo)、5’リン酸化はChemical Phosphorylation Reagent(Glen Research)を用いた。脱保護は定法に従い、PAGE精製した。合成したRNA配列を、配列番号1(センス鎖28mer)、配列番号2(アンチセンス鎖28mer)、配列番号3(56mer)および図2に示す。なお、図2のRNA配列のうち、下線の配列がダンベル型RNAのループ部を形成する配列である。
レーン1:28塩基のセンス鎖および28塩基のアンチセンス鎖の混合物(マーカー)
レーン2:57塩基のRNA(マーカー)
レーン3:28塩基のセンス鎖と28塩基のアンチセンス鎖から形成したダンベル型RNA
レーン4:56塩基(1本鎖)から形成したダンベル型RNA
レーン5:RNAリガーゼで処理した28塩基のセンス鎖(参考)
レーン6:RNAリガーゼで処理した28塩基のアンチセンス鎖(参考)
上記DNA合成機で、配列番号4〜13に示すRNAを合成した。配列番号4および5は18塩基対を形成する2本鎖RNA(siRNA−1)、配列番号6および7はステムの長さ19塩基のダンベル型RNA(Db−19)、配列番号8および9はステムの長さ23塩基のダンベル型RNA(Db-23)、配列番号10及び11はステムの長さ27塩基のダンベル型RNA(Db-27)、配列番号12および13はステムの長さ31塩基のダンベル型RNA(Db-31)を形成する(図3)。
上記ダンベル型RNA(Db−19、Db−23、Db−27、Db−31)のRNA干渉効果を蛍ルシフェラーゼレポーター遺伝子pGL3の発現阻害実験により評価した。
GeneSilencer 1μl
DMEM培地 25μl
siRNA希釈液 2.5μl
DMEM培地 15μl
RNA(5pmol/μl) 0.5μl
pGL3−Control(1μg/μl) 0.2μl
pRL−TK(0.1μg/μl) 0.2μl
44.4μl
Db−23とsiRNA−1のRNA干渉効果の持続性について比較した。
NIH 3T3細胞を、10%ウシ仔牛血清(FCS、Invitro/Gibco)を含むダルベッコ改変イーグル培地(DMEM、Gibco)で、5%CO2加湿チャンバーで培養した。約70%コンフルエントの状態のトランスフェクションの40時間前に、細胞を96穴プレートに、100μlずつ、1.6×104細胞/ウェルになるように分注した。レポータープラスミドとRNAのコトランスフェクションをGeneSilencer(Gene Therapy Systems)を用いてNIH 3T3細胞の製造者による説明に従って行った。実施例3と比べてトランスフェクション試薬で10倍に希釈した16ng/mlのpGL3−Control(Promega)、1.6ng/mlのpRL−TK(Promega)および25nMのRNAを、ウェルに100μl適用した。4時間のインキュベーション後、20%FCSを含むDMEMを100μl添加した。更に3日以上インキュベーションを行い、培地を随時交換した。ルシフェラーゼの発現を、Wallac ARVO SX 1420 Multilabel Counter(Perkin−Elmer)に添付の指示書に従い、Dual−Luciferase Reporter Assay System(Promega)で、24時間、72時間、120時間後にモニターした(図6)。RNAの入っていないものをコントロールとした。
Db-23とsiRNAのヒト血清中での安定性を比較した。
アニーリングバッファー(100mM酢酸カリウム、30mM HEPES−KOH(pH7.4)、2mM酢酸マグネシウム)にアニーリング前のdsRNA(siRNA−1、20μM)またはダンベル型RNA(Db−23、20μM)を加えたその4μlと、PBS32μl、正常ヒト血清(Chemicon International)4μlを混合して37℃でインキュベーションした。アリコート5μlを0.5、1、1.5、3、8および20時間後に採取し、15%nativePAGEで、SYBR Green Iで染色、MolecularImager FX(BioRad)を使用して分析した。
Claims (9)
- センス鎖配列と、該センス鎖配列に相補的なアンチセンス鎖配列と、該センス鎖と該アンチセンス鎖の間に両鎖を結合する同じか又は異なる、ループ長6〜9塩基の2つのループ配列とを含み、該センス鎖と該アンチセンス鎖が対合してステム長19〜31塩基対のステムを形成することを特徴とする、持続的又は徐放的RNA干渉作用を有する1本鎖環状RNA。
- 対照細胞における標的RNAの発現を1とした場合に、細胞に導入後24時間の標的RNAの発現が0.4以下を示す、請求項1に記載の1本鎖環状RNA。
- ヒト血清中で8時間経過したときに70%以上が保持されている、請求項1または2に記載の1本鎖環状RNA。
- 相補鎖を形成しない塩基配列を5’末端および3’末端に有する、センス鎖およびアンチセンス鎖をそれぞれ合成し、センス鎖が有する相補鎖を形成しない塩基配列の5’末端の塩基とアンチセンス鎖が有する相補鎖を形成しない塩基配列の3’末端の塩基、およびセンス鎖が有する相補鎖を形成しない塩基配列の3’末端の塩基とアンチセンス鎖が有する相補鎖を形成しない塩基配列の5’末端の塩基を、リガーゼで同時に連結することを含み、
その際、センス鎖が5’末端に有する相補鎖を形成しない塩基配列とアンチセンス鎖が3’末端に有する相補鎖を形成しない塩基配列が互いに結合してループを形成し、センス鎖が3’末端に有する相補鎖を形成しない塩基配列とアンチセンス鎖が5’末端に有する相補鎖を形成しない塩基配列が互いに結合してループを形成し、ならびにセンス鎖とアンチセンス鎖が対合してステムを形成する、請求項1〜3のいずれか1項に記載の、ループ長6〜9塩基及びステム長19〜31塩基対からなる1本鎖環状RNAの製造方法。 - センス鎖が有する相補鎖を形成しない塩基配列とアンチセンス鎖が有する相補鎖を形成しない塩基配列の5’末端をリン酸化することを更に含む、請求項4に記載の製造方法。
- 前記ループの配列が互いに同じかまたは異なる、請求項4または5に記載の製造方法。
- 請求項1〜3のいずれか1項に記載の1本鎖環状RNAをヒト由来の細胞に導入し、標的RNAを不能にしてタンパク質への翻訳を持続的に阻害することを含む、該タンパク質をコードする遺伝子の発現をin vitroで抑制する方法。
- 請求項1〜3のいずれか1項に記載の1本鎖環状RNAを非ヒト動物、植物又はそれらの細胞に導入し、標的RNAを不能にしてタンパク質への翻訳を持続的に阻害することを含む、該タンパク質をコードする遺伝子の発現を抑制する方法。
- 請求項1〜3のいずれか1項に記載の1本鎖環状RNAを有効成分として含む、医薬組成物。
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