JP2007031443A - 特定遺伝子の発現を抑制するための方法および薬剤 - Google Patents
特定遺伝子の発現を抑制するための方法および薬剤 Download PDFInfo
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Abstract
【解決手段】本発明によれば、その二本鎖RNAの1の鎖は、少なくとも一部が標的遺伝子に相補的である。標的遺伝子は下記の群から選択することができる:ガン遺伝子、サイトカイン遺伝子、Id蛋白質遺伝子、発生遺伝子、プリオン遺伝子。標的遺伝子は、病原性生物(特に、プラスモディウム)において発現し得る。標的遺伝子はまた、好ましくはヒトに対して病原性であるウイルスまたはウイロイドの一部であり得る。
【選択図】なし
Description
転写抑制を、ヒトHeLa細胞からの核抽出物を用いたインビトロ転写システムにおける配列相同的なdsRNAによって検出した。この実験に対するDNAテンプレートは、BamHIで線状化されたプラスミドpCMV1200であった。
図1に示されるプラスミドを、dsRNAの酵素的合成において使用するために構築した。この目的のために、Promega(Madison、米国)によるHelaScribe(登録商標)核抽出物インビトロ転写キットの「陽性コントロールDNA」をDNAテンプレートとして用いたポリメラーゼ連鎖反(PCR)を最初に行った。使用したプライマーの1つには、配列番号1に示されているように、EcoRI切断部位およびT7RNAポリメラーゼプロモーターの配列が含まれた。もう一方のプライマーには、配列番号2に示されているように、BamHI切断部位およびSP6RNAポリメラーゼプロモーターの配列が含まれた。さらに、この2つのプライマーは、3’末端に、DNAテンプレートと同一である領域、またはDNAテンプレートに対して相補的である領域を有した。PCRを、Qiagen(Hilden、ドイツ)による「Taq PCR Coreキット」で製造者の説明書に従って行った。1.5mMのMgCl2、200μMの各dNTP、0.5μMの各プライマー、2.5UのTaqDNAポリメラーゼ、およびテンプレートとしての約100ngの「陽性コントロールDNA」を、100μlの容量のPCR緩衝液において用いた。94℃で5分間加熱することによってテンプレートDNAを最初に変性した後、増幅を、それぞれの場合において、94℃で60秒間の変性、プライマーの計算された融解温度よりも5℃低い温度で60秒間のアニーリング、および72℃で1.5分間〜2分間の重合の30サイクルで行った。72℃で5分間の最後の重合を行った後、反応液の5μlをアガロースゲル電気泳動により分析した。このように増幅されたDNAフラグメントの長さは400塩基対であった。340塩基対は「陽性コントロールDNA」に対応した。PCR産物を精製し、EcoRIおよびBamHIで加水分解し、そして再び精製した後、EcoRIおよびBamHIで同様に加水分解されたpUC18ベクターとの連結において用いた。その後、大腸菌XL1−blueを形質転換した。得られたプラスミド(pCMV5)は、5’末端にはT7プロモーターが隣接し、3’末端にはSP6プロモーターが隣接するDNAフラグメントを有する。プラスミドをBamHIで線状化することによって、プラスミドは、長さが340ヌクレオチドであり、配列番号3に示される一本鎖RNAのラン・オフ転写のためにT7RNAポリメラーゼとともにインビトロで用いることができる。プラスミドをEcoRIで線状化すれば、プラスミドは、SP6RNAポリメラーゼを用いたラン・オフ転写のために用いることができ、これにより相補鎖が得られる。本明細書中上記に概略された方法により、長さが23ヌクレオチドのRNAもまた合成された。この目的に対して、配列番号4に示されるDNAを、EcoRIおよびBamHIの切断部位を介してpUC18ベクターに連結した。
pCMV5プラスミドDNAをEcoRIまたはBamHIで線状化した。これを、SP6RNAポリメラーゼおよびT7RNAポリメラーゼをそれぞれ用いた相補的なRNA一本鎖のインビトロ転写のためのDNAテンプレートとして使用した。Promega(Madison、米国)による「Riboprobeインビトロ転写」システムをこの目的のために用いた。製造者の説明書に従い、2μgの線状化プラスミドDNAを100μlの転写緩衝液において40UのT7RNAポリメラーゼまたはSP6RNAポリメラーゼと37℃で5時間〜6時間インキュベーションした。続いて、DNAテンプレートを、2.5μlのRNase非含有DNase RQ1を加え、37℃で30分間インキュベーションすることによって分解した。転写反応液をH2Oで300μlにしてフェノール抽出により精製した。RNAを、150μlの7M酢酸アンモニウム[sic]および1125μlのエタノールを加えることによって沈殿させて、ハイブリダイゼーションに使用されるまで−65℃で保存した。
ハイブリダイゼーションのために、エタノール中に保存され、沈殿させた500μlの一本鎖RNAを遠心分離した。得られたペレットを乾燥し、80%ホルアミド、400mMのNaClおよび1mMのEDTAの存在下、30μlのPIPES緩衝液(pH6.4)に溶解した。それぞれの場合において、15μlの相補的な一本鎖を一緒にして、85℃で10分間加熱した。続いて、反応液を50℃で一晩インキュベーションして、室温に冷却した。
Promega(Madison、米国)によるHeLaScribe(登録商標)核抽出物インビトロ転写キットを使用して、プラスミドpCMV1200に存在し、かつその「陽性コントロールDNA」に対して相同的である上記DNAフラグメントの転写効率を、配列相同性を有するdsRNA(dsRNA−CMV5)の存在下で測定した。また、配列相同性を有さず、黄色蛍光蛋白質(YFP)遺伝子に対応するdsRNA(dsRNA−YFP)の影響も調べた。このdsRNAは、配列相同性を有するdsRNAと同様に作製された。このdsRNAの鎖の配列は配列番号5に見出され得る。プラスミドpCMV1200をラン・オフ転写のテンプレートとして使用した。このプラスミドは、真核生物RNAポリメラーゼIIによって認識される「極初期」サイトメガロウイルスプロモーターと、転写可能なDNAフラグメントとを有する。転写を、転写に必要な蛋白質をすべて含有するHeLa核抽出物によって行った。[・−32P]rGTPを転写反応液に加えることによって、放射能標識された転写物が得られた。使用した[・−32P]rGTPは、比活性が400Ci/mmol(10mCi/ml)であった。転写緩衝液において、3mMのMgCl2、それぞれが400μMのrATP、rCTP、rUTP、16μMのrGTP、0.4μMの[・−32P]rGTP、ならびに実験に依存して、1fmolの線状化プラスミドDNA、および様々な量のdsRNAを反応毎に用いた。各バッチを8.5μlの容量にH2Oで作製した。反応液を注意深く混合した。転写を開始させるために、4UのHeLa核抽出物を4μlの容量で加え、30℃で60分間インキュベーションした。反応を、30℃に暖められた87.5μlの停止混合液を加えることによって停止させた。蛋白質を除くために、反応液を、TE緩衝液(pH5.0)を飽和させた100μlのフェノール/クロロホルム/イソアミルアルコール(25:24:1 v/v/v)で処理した。反応液を1分間激しく混合した。相分離のために、反応液を12000rpmで約1分間遠心分離して、上部相を新しい反応容器に移した。それぞれの反応液を250μlのエタノールで処理した。反応液を十分に混合し、ドライアイス/メタノールにおいて少なくとも15分間インキュベーションした。RNAを沈殿させるために、反応液を40℃において12000rpmで20分間遠心分離した。上清を捨てた。ペレットを真空下で15分間乾燥して、10μlのH2Oに再懸濁した。それぞれの反応液を10μlの変性ローディング緩衝液で処理した。遊離GTPを、7M尿素を含む8%ゲルでの変性ポリアクリルアミドゲル電気泳動によって、形成された転写物から分離した。HeLa核抽出物を用いて転写させたときに形成されたRNA転写物を、変性ローディング緩衝液中で90℃で10分間加熱して、10μl部分を、新しく洗浄したポケットに直ちに加えた。電気泳動を40mAで行った。転写時に形成された放射活性なssRNAの量を、電気泳動後、Instant Imagerの助けによって分析した。
レーン1:テンプレートDNAなし、dsRNAなし;
レーン1:50ngのテンプレートDNA、dsRNAなし;
レーン3:50ngのテンプレートDNA、0.5μgのdsRNA YFP;
レーン4:50ngのテンプレートDNA、1.5μgのdsRNA YFP;
レーン5:50ngのテンプレートDNA、3μgのdsRNA YFP;
レーン6:50ngのテンプレートDNA、5μgのdsRNA YFP;
レーン7:テンプレートDNAなし、1.5μgのdsRNA YFP;
レーン8:50ngのテンプレートDNA、dsRNAなし;
レーン9:50ngのテンプレートDNA、0.5μgのdsRNA CMV5:
レーン10:50ngのテンプレートDNA、1.5μgのdsRNA CMV5:
レーン11:50ngのテンプレートDNA、3μgのdsRNA CMV5:
レーン12:50ngのテンプレートDNA、5μgのdsRNA CMV5:
これらのインビボ実験に使用される試験システムはマウスの繊維芽細胞株NIH3T3(ATCC CRL−1658)であった。YFP遺伝子をマイクロインジェクションによって核内に導入した。YFPの発現を、配列相同性を有する同時にコトランスフェクションされたdsRNAの影響のもとで調べた。このdsRNA YFPは、315bpの長さにわたってYFP遺伝子の5’領域との相同性を示す。dsRNA YFPの鎖のヌクレオチド配列は配列番号5に示される。蛍光顕微鏡による評価を、形成されたYFPの緑黄色蛍光に関して、インジェクションの3時間後に行った。
プラスミドを、T7およびSP6のインビトロ転写によってYFP dsRNAを産生するためのテンプレートとして作用するように、使用例1に記載されるのと同じ原理に従って構築した。配列番号6に示されるプライマーEco_T7_YFPおよび配列番号7に示されるプライマーBam_SP6_YFPを使用して、所望する遺伝子フラグメントをPCRにより増幅し、上記記載と同様に使用して、dsRNAを調製した。得られたdsRNA YFPは、配列非特異的なコントロールとして使用例1で使用されたdsRNAと同一である。
細胞を、7.5%CO2雰囲気のもと、37℃で、培養皿において、4.5g/lのグルコース、10%のウシ胎児血清を補充したDMEMでインキュベーションし、コンフルエンスに達する前に継代した。細胞をトリプシン/EDTAによって剥がした。マイクロインジェクション用に調製するために、細胞をペトリ皿に移し、微小コロニーが形成されるまでさらにインキュベーションした。
マイクロインジェクションのために、培養皿をインキュベーターから約10分間取り出した。約50個の核を、Carl Zeiss(Gottingen、ドイツ)から得られるAISマイクロインジェクションシステムを使用して、印を付けられた領域内に反応あたり1個づつ注入した。続いて、細胞をさらに3時間インキュベーションした。マイクロインジェクションのために、先端の直径が0.5μm未満であるHilgenberg GmbH(Malsfeld、ドイツ)から得られるホウケイ酸塩ガラスキャピラリーを準備した。マイクロインジェクションを、ナリシゲ科学機械(東京、日本)から得られるマイクロマニピュレーターを使用して行った。注入時間は0.8秒であり、圧力は約100hPaであった。トランスフェクションを、ベクターpcDNA3内にYFP遺伝子とともに約800bpのBamHI/EcoRIフラグメントを含有するプラスミドpCDNA−YFPを使用して行った。核に注入されたサンプルには、0.01μg/μlのpCDNA−YFPと、デキストラン−7000に結合させたテキサスレッドとが、14mMのNaCl、3mMのKCl、10mMのKPO4[sic](pH7.5)に含まれた。約100plのRNAを、1μMの濃度で、あるいはL−dsRNAの場合には375μMの濃度でさらに加えた。
Claims (50)
- 哺乳類細胞中の所定の標的遺伝子の発現を阻害することができるオリゴヌクレオチドを含む、ウイルス感染によって媒介されるか又は誘導される病気又は疾患の治療用組成物であって、当該オリゴヌクレオチドは、15から49の塩基対を有する二本鎖構造を含むオリゴリボヌクレオチド(dsRNA)を含み、当該dsRNAの1の鎖は、少なくとも一部が当該標的遺伝子に相補的な、49以下の連続的ヌクレオチド対を有する領域Iを有しており、二本鎖構造内部の相補的領域IIが、二つの分離したRNA一本鎖により形成されるものであり、当該標的遺伝子がウイルス遺伝子である、治療用組成物。
- 前記のdsRNAが、ミセル構造、好ましくはリポソームにより封入されているものである請求項1に記載の治療用組成物。
- 前記のdsRNAが、天然のウイルスキャプシド、又は化学的若しくは酵素学的に産生された人工のキャプシド、又はこれらに由来する構造によって封入されているものである請求項1又は2に記載の治療用組成物。
- 前記のdsRNAの末端が修飾されて、細胞内での分解又は一本鎖への分離が生じないようになっているものである請求項1乃至3の何れ一項に記載の治療用組成物。
- ヌクレオチド対により形成される、前記の二本鎖構造の結合が、少なくとも1の、好ましくは2の更なる化学結合により増大しているものである請求項1乃至4の何れか一項に記載の治療用組成物。
- 前記の化学結合が、共有若しくはイオン結合、水素結合、疎水性相互作用、好ましくはファンデルワールス若しくはスタッキング相互作用、又は金属-イオン配位により形成されるものである請求項5に記載の治療用組成物。
- 前記の化学結合が、前記の二本鎖構造の少なくとも一方の、好ましくは両方の末端において生じているものである請求項5又は6に記載の治療用組成物。
- 前記の化学結合が、1以上の化合物群、好ましくは:ポリ(オキシホスフィニコオキシ-1,3-プロパンジオール)鎖及び/又はポリエチレングリコール鎖により形成されているものである請求項5乃至7の何れか一項に記載の治療用組成物。
- 前記の化学結合が、プリンの代わりに、前記の二本鎖構造において使用されるプリン類縁体によって形成されているものである請求項5乃至7の何れか一項に記載の治療用組成物。
- 前記の化学結合が、前記の二本鎖構造に導入されたアザベンゼン単位により形成されているものである請求項5乃至7の何れか一項に記載の治療用組成物。
- 前記の化学結合が、ヌクレオチドの代わりに、前記の二本鎖構造において使用される分枝ヌクレオチド類縁体により形成されるものである請求項5乃至7の何れか一項に記載の治療用組成物。
- 下記の基:メチレンブルー;二官能性基、好ましくはビス(2-クロロエチル)アミン;N-アセチル-N’-(p-グリオキシルベンゾイル)-シスタミン;4-チオウラシル; ソラレンの少なくとも1を使用して前記の化学結合が形成されているものである請求項5乃至7の何れか一項に記載の治療用組成物。
- 前記の化学結合が、前記の二本鎖構造の末端にあるチオホスホリル基により形成されているものである請求項5乃至7の何れか一項に記載の治療用組成物。
- 前記の化学結合が、前記の二本鎖構造の末端にある三重らせん結合であるものである請求項5乃至7の何れか一項に記載の治療用組成物。
- 前記の二本鎖構造内の前記のdsRNAのヌクレオチドの2’-ヒドロキシル基の少なくとも1つが、化学基、好ましくは2’-アミノ基又は2’-メチル基により置換されているものである請求項1乃至14の何れか一項に記載の治療用組成物。
- 前記の二本鎖構造の少なくとも1の鎖中の少なくとも1のヌクレオチドが、好ましくは2’-O、4’-Cメチレン架橋により、化学的に修飾された糖環を有する、ロックされたヌクレオチドであるものである請求項1乃至15の何れか一項に記載の治療用組成物。
- 前記のdsRNAが、少なくとも1のウイルス外殻蛋白質であって、ウイルス起源のもの、それに由来するもの、又は合成されたものに、結合しているか、会合しているか、又は囲まれているものである請求項1乃至16の何れか一項に記載の治療用組成物。
- 前記の外殻蛋白質が、ポリオーマウイルス由来であるものである請求項17に記載の治療用組成物。
- 前記の外殻蛋白質が、ポリオーマウイルスのウイルス蛋白質1(VP1)及び/又はウイルス蛋白質2(VP2)を含むものである請求項17又は18に記載の治療用組成物。
- キャプシド又はキャプシドタイプの構造が、外殻蛋白質より形成される場合に、1の側面が当該キャプシド又はキャプシドタイプの構造の内部に面しているものである請求項17乃至19の何れか一項に記載の治療用組成物。
- 前記のdsRNAの1の鎖が、前記の標的遺伝子の第一次RNA転写産物又はプロセッシングされたRNA転写産物に相補的であるものである請求項1乃至20の何れか一項に記載の治療用組成物。
- 前記の哺乳類細胞が、ヒトの細胞であるものである請求項1乃至21の何れか一項に記載の治療用組成物。
- 互いに異なる少なくとも2つの請求項1から22の何れか一項に記載のdsRNAを、前記の治療用組成物内に含ませ、それぞれのdsRNAの1の鎖の少なくとも一部が、それぞれの場合に、少なくとも2つの異なる標的遺伝子の一つに相補的であるものである請求項1乃至22の何れか一項に記載の治療用組成物。
- 前記のdsRNAが、15から21の塩基対からなる請求項1乃至23の何れか一項に記載の治療用組成物。
- 前記の二本鎖構造が、一本鎖の化学結合によって安定化されているものである請求項1乃至24の何れか一項に記載の治療用組成物。
- 哺乳類細胞中の所定の標的遺伝子の発現を阻害することができるオリゴヌクレオチドを含む、望ましくない細胞増殖によって媒介されるか又は誘導される病気又は疾患の治療用組成物であって、当該オリゴヌクレオチドは、15から49の塩基対を有する二本鎖構造を含むオリゴリボヌクレオチド(dsRNA)を含み、当該dsRNAの1の鎖は、少なくとも一部が当該標的遺伝子に相補的な、49以下の連続的ヌクレオチド対を有する領域Iを有しており、二本鎖構造内部の相補的領域IIが、二つの分離したRNA一本鎖により形成されるものであり、当該標的遺伝子がガン遺伝子、Id−蛋白質遺伝子、 サイトカイン遺伝子、又は発生遺伝子である、治療用組成物。
- 前記のdsRNAが、ミセル構造、好ましくはリポソームにより封入されているものである請求項26に記載の治療用組成物。
- 前記のdsRNAが、天然のウイルスキャプシド、又は化学的若しくは酵素学的に産生された人工のキャプシド、又はこれらに由来する構造によって封入されているものである請求項26又は27に記載の治療用組成物。
- 前記のdsRNAの末端が修飾されて、細胞内での分解又は一本鎖への分離が生じないようになっているものである請求項26乃至28の何れ一項に記載の治療用組成物。
- ヌクレオチド対により形成される、前記の二本鎖構造の結合が、少なくとも1の、好ましくは2の更なる化学結合により増大しているものである請求項26乃至29の何れか一項に記載の治療用組成物。
- 前記の化学結合が、共有若しくはイオン結合、水素結合、疎水性相互作用、好ましくはファンデルワールス若しくはスタッキング相互作用、又は金属-イオン配位により形成されるものである請求項30に記載の治療用組成物。
- 前記の化学結合が、前記の二本鎖構造の少なくとも一方の、好ましくは両方の末端において生じているものである請求項30又は31に記載の治療用組成物。
- 前記の化学結合が、1以上の化合物群、好ましくは:ポリ(オキシホスフィニコオキシ-1,3-プロパンジオール)鎖及び/又はポリエチレングリコール鎖により形成されているものである請求項30乃至32の何れか一項に記載の治療用組成物。
- 前記の化学結合が、プリンの代わりに、前記の二本鎖構造において使用されるプリン類縁体によって形成されているものである請求項30乃至32の何れか一項に記載の治療用組成物。
- 前記の化学結合が、前記の二本鎖構造に導入されたアザベンゼン単位により形成されているものである請求項30乃至32の何れか一項に記載の治療用組成物。
- 前記の化学結合が、ヌクレオチドの代わりに、前記の二本鎖構造において使用される分枝ヌクレオチド類縁体により形成されるものである請求項30乃至32の何れか一項に記載の治療用組成物。
- 下記の基:メチレンブルー;二官能性基、好ましくはビス(2-クロロエチル)アミン;N-アセチル-N’-(p-グリオキシルベンゾイル)-シスタミン;4-チオウラシル; ソラレンの少なくとも1を使用して前記の化学結合が形成されているものである請求項30乃至32の何れか一項に記載の治療用組成物。
- 前記の化学結合が、前記の二本鎖構造の末端にあるチオホスホリル基により形成されているものである請求項30乃至32の何れか一項に記載の治療用組成物。
- 前記の化学結合が、前記の二本鎖構造の末端にある三重らせん結合であるものである請求項30乃至32の何れか一項に記載の治療用組成物。
- 前記の二本鎖構造内の前記のdsRNAのヌクレオチドの2’-ヒドロキシル基の少なくとも1つが、化学基、好ましくは2’-アミノ基又は2’-メチル基により置換されているものである請求項26乃至39の何れか一項に記載の治療用組成物。
- 前記の二本鎖構造の少なくとも1の鎖中の少なくとも1のヌクレオチドが、好ましくは2’-O、4’-Cメチレン架橋により、化学的に修飾された糖環を有する、ロックされたヌクレオチドであるものである請求項26乃至40の何れか一項に記載の治療用組成物。
- 前記のdsRNAが、少なくとも1のウイルス外殻蛋白質であって、ウイルス起源のもの、それに由来するもの、又は合成されたものに、結合しているか、会合しているか、又は囲まれているものである請求項26乃至41の何れか一項に記載の治療用組成物。
- 前記の外殻蛋白質が、ポリオーマウイルス由来であるものである請求項42に記載の治療用組成物。
- 前記の外殻蛋白質が、ポリオーマウイルスのウイルス蛋白質1(VP1)及び/又はウイルス蛋白質2(VP2)を含むものである請求項42又は43に記載の治療用組成物。
- キャプシド又はキャプシドタイプの構造が、外殻蛋白質より形成される場合に、1の側面が当該キャプシド又はキャプシドタイプの構造の内部に面しているものである請求項42乃至44の何れか一項に記載の治療用組成物。
- 前記のdsRNAの1の鎖が、前記の標的遺伝子の第一次RNA転写産物又はプロセッシングされたRNA転写産物に相補的であるものである請求項26乃至45の何れか一項に記載の治療用組成物。
- 前記の哺乳類細胞が、ヒトの細胞であるものである請求項26乃至46の何れか一項に記載の治療用組成物。
- 互いに異なる少なくとも2つの請求項26から47の何れか一項に記載のdsRNAを、前記の治療用組成物内に含ませ、それぞれのdsRNAの1の鎖の少なくとも一部が、それぞれの場合に、少なくとも2つの異なる標的遺伝子の一つに相補的であるものである請求項26乃至47の何れか一項に記載の治療用組成物。
- 前記のdsRNAが、15から21の塩基対からなる請求項26乃至48の何れか一項に記載の治療用組成物。
- 前記の二本鎖構造が、一本鎖の化学結合によって安定化されているものである請求項26乃至49の何れか一項に記載の治療用組成物。
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