JP2010536380A - 転写後遺伝子サイレンシングのための方法及び組成物 - Google Patents
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Abstract
Description
適用外。
配列番号6及び配列番号7に与えられるshRNA配列を含む核酸分子がHsp25発現を低下させる能力を調べた。マウスの乳癌4T1細胞は、変異原処理することなく410.4腫瘍から選択した6−チオグアニン耐性細胞系である。2mMのL−グルタミンを含むDulbecco’s modified Eagle medium(Invitrogen、Carlsbad、Calif.、USA)中に細胞を維持し、5%CO2雰囲気の加湿したインキュベーターにおいて37℃で、1.5g/lの炭酸水素ナトリウム、4.5g/lのグルコース、10mMのHEPES、1.0mMのピルビン酸ナトリウム、及び10%のウシ胎仔血清を含むように調製した。細胞を指数関数的増殖率で増殖させ、培養物がおよそ80%集密のとき、0.1%のトリプシン−EDTAを用いて収穫した。新鮮な細胞を使用する前に、細胞を5〜8回だけ継代培養した。
例1に記載のベクターを形質移入した腫瘍細胞の増殖を調べた。4T1の細胞増殖を合計4日間、血球計測器を用いて測定し、増殖結果を図3に示し、グラフを次のようにラベルする。対照shRNAは4T1/対照shRNA1に対応し、AS1は4T1/HSP25shRNA1に対応、DS1は4T1/HSP25shRNA2に対応する。結果は、対照shRNA、AS1又はDS1を発現する細胞が類似の増殖曲線を表すことを実際に示す。
腫瘍細胞の転移能を低下させる例1に記載のAS1及びDS1分子の能力を、細胞遊走アッセイを用いて調べた。製品の説明書に従って、Matrigel invasion chamber(BD Biocoat Cellware、San Jose、Calif.、USA)及び例1に記載の4T1腫瘍細胞を使用して細胞遊走を測定した。簡潔に述べると、条件培地を化学遊走物質として下部の容器に置いた。単一細胞の懸濁液を上部の容器に置いた。22時間後、フィルターを通過しない細胞を洗い落とし、膜を0.5%クリスタルバイオレットで染色し、顕微鏡のスライド上に乗せ、視覚化して写真を撮った。15カ所の異なる部分を10×の倍率で光学顕微鏡を用いて視覚化した。図7は各コンストラクトに関する浸潤細胞数のプロットであり、浸潤は化学遊走物質の方へ遊走する腫瘍細胞の数を指し、グラフは次のようにラベルする。対照shRNAは4T1/対照shRNA1に対応し、AS1は4T1/HSP25shRNA1に対応し、DS1は4T1/HSP25shRNA2に対応する。結果は、AS1又はDS1コンストラクトのいずれかを形質移入した腫瘍細胞が、対照shRNAを形質移入した腫瘍細胞より狭い範囲を遊走したことを実際に示す。
簡潔に述べると、DOTAP及びコレステロール(モル比1:1)からなるリポソームを、薄膜水和の後、膜押出しにより、N4 PLUS Coulter粒径散乱測定器を用いて測定したとき、80〜100nmの粒子サイズを得ることにより調製した。リポソームナノ粒子には、DOTAP/コレステロール、プロタミンスルホン酸、並びに配列番号4〜6及び対照配列中に提示されているタイプのHsp標的指向化siRNAオリゴヌクレオチドを含ませた。体重1kg当たり1mgのsiRNA製剤を調製するために、200μlのリポソームナノ粒子は、13.5μlのsiRNA、10μl(20μg)のプロタミンスルホン酸、40μlのDOTAP及びコレステロール(モル比1:1)、15μlのトランスフェリン(300μg)、リボヌクレアーゼを含まない121.5μlの水を含む。DOTAP、コレステロールは、Avanti Polar Lipids,Inc.から商業的に入手でき、ヒトトランスフェリンは、鉄飽和の熱失活した形態でBD Biosciencesから商業的に入手でき、サケから単離したプロタミンスルホン酸GradeXは、Sigma−Aldrichから商業的に入手することができる。ナノ粒子複合体を、プロタミンスルホン酸と、リボヌクレアーゼを含まない水と、siRNAとを混合し、10分間室温で放置した後、DOTAP/コレステロールリポソーム、トランスフェリン複合体を加えることにより調製されよう。リポソームナノ粒子を、動物に注射する前に10分間室温でインキュベートした。
以下は、本明細書に開示されている組成物を利用するsiRNA遺伝子治療に関する机上のプロトコールである。簡潔に述べると、DOTAP及びコレステロール(モル比1:1)からなるリポソームは、薄膜水和の後、膜押出しにより、80〜100nmの粒子サイズを得ることによって調製されよう。粒子サイズは、N4 PLUS Coulter散乱測定器を用いて測定されよう。リポソームナノ粒子には、DOTAP/コレステロール、プロタミンスルホン酸、及び配列番号4〜6中に提示されているタイプのHsp標的指向化siRNAオリゴヌクレオチドを含むことになろう。体重1kg当たり1mgのsiRNA製剤を調製するために、200μlのリポソームナノ粒子は、13.5μlのsiRNA、10μl(20μg)のプロタミンスルホン酸、40μlのDOTAP及びコレステロール(モル比1:1)、15μlのトランスフェリン(300μg)、リボヌクレアーゼを含まない121.5μlの水を含む。DOTAP、コレステロールは、Avanti Polar Lipids,Inc.から商業的に入手でき、ヒトトランスフェリンは、鉄飽和の熱失活した形態でBD Biosciencesから商業的に入手でき、サケから単離したプロタミンスルホン酸GradeXは、Sigma−Aldrichから商業的に入手することができる。ナノ粒子複合体を、プロタミンスルホン酸と、リボヌクレアーゼを含まない水と、siRNAとを混合することにより調製すると、DOTAP/コレステロールリポソーム、トランスフェリン複合体を加える前に、10分間室温で維持することができよう。リポソームナノ粒子は、動物に注射する前に10分間室温でインキュベートされよう。
Claims (28)
- 標的遺伝子の発現を阻害する、単離した二本鎖リボ核酸(dsRNA)分子であって、前記dsRNAが2本のヌクレオチド鎖と、第1鎖の3’末端を第2鎖の5’末端に結合させる結合部分とを含み、第1鎖が19〜28個の連続ヌクレオチドの長さを示し、標的遺伝子中の配列と実質的に同一であり、第2鎖が第1鎖に対して実質的に相補的である、上記単離したdsRNA。
- 結合部分がポリヌクレオチドリンカーを含む、請求項1に記載のdsRNA。
- ポリヌクレオチドリンカーが5〜12塩基対の長さである、請求項2に記載のdsRNA。
- 標的遺伝子が熱ショックタンパク質をコードする、請求項1に記載のdsRNA。
- 標的遺伝子が配列番号3を含む、請求項1に記載のdsRNA。
- 第1鎖が配列番号4、5又は6を含む、請求項1に記載のdsRNA。
- 第1鎖、第2鎖又は両方がさらにマーカータンパク質を含む、請求項1に記載のdsRNA。
- マーカータンパク質が蛍光タンパク質である、請求項7に記載のdsRNA。
- 請求項1に記載のdsRNAを含む細胞の形質導入のためのベクターファミリー。
- ベクターがレトロウイルスベクターである、請求項9に記載のベクターファミリー。
- ベクターがレンチウイルスベクターである、請求項10に記載のベクターファミリー。
- プロモーター、リボソーム結合部位、エンハンサー配列、応答エレメント、誘導性エレメント、選択可能マーカー、調節エレメント、又はそれらの組合せをさらに含む、請求項9に記載のベクターファミリー。
- プロモーターが、マウスUG RNAプロモーター、合成ヒトH1RNAプロモーター、SV40プロモーター、CMVプロモーター、RSVプロモーター、RNAポリメラーゼIIプロモーター、RNAポリメラーゼIIIプロモーター、それらの誘導体、又はそれらの組合せを含む、請求項12に記載のベクターファミリー。
- 請求項1に記載のdsRNAを含む細胞系。
- 細胞系がパッケージング細胞系である、請求項14に記載の細胞系。
- 請求項1に記載のdsRNAを含む、ヒト以外の動物。
- 請求項1に記載のdsRNAを含む、治療量の組成物の投与を含む、増殖性障害に罹っている生物を処置する方法。
- 増殖性障害が腫瘍増殖により証明される、請求項17に記載の方法。
- 腫瘍増殖が約10%〜約95%まで阻害される、請求項18に記載の方法。
- 腫瘍の転移能が約10%〜約95%まで低下する、請求項18に記載の方法。
- 請求項1に記載のdsRNAと賦形剤とを含む、医薬品組成物。
- デリバリーシステムと腫瘍標的指向性部分とをさらに含む、請求項21に記載の医薬品組成物。
- デリバリーシステムがリポソームを含む、請求項22に記載の医薬品組成物。
- 腫瘍標的指向性部分が、抗体、トランスフェリン又はそれらの組合せを含む、請求項22に記載の医薬品組成物。
- 配列番号3と実質的に同一であるヌクレオチドの第1鎖と、第1鎖に対して実質的に相補的である第2鎖とを含む、単離した二本鎖リボ核酸分子。
- 配列番号3中に提示される配列を含む核酸分子によりコードされているタンパク質の発現を阻害する単離した二本鎖リボ核酸であって、dsRNAの第1鎖が配列番号3と実質的に同一であり、第2鎖が第1鎖に対して実質的に相補的である、上記単離した二本鎖リボ核酸。
- 請求項26に記載のdsRNAを含む細胞の形質導入のための、ベクターファミリー。
- 請求項26に記載のdsRNAと賦形剤とを含む、腫瘍増殖及び/又は転移能を低下させるための医薬品組成物。
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JPN6013009102; THE JOURNAL OF BIOLOGICAL CHEMISTRY vol.282 no.23, 200706, pp.17297-17305 * |
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