JP2013500960A - ポリヌクレオチド剤を含む細胞標的化ナノ粒子およびその使用 - Google Patents
ポリヌクレオチド剤を含む細胞標的化ナノ粒子およびその使用 Download PDFInfo
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Abstract
【選択図】なし
Description
(a)ポリヌクレオチドを、カチオン性分子を含む組成物と接触させるステップであって、カチオン性分子は静電的相互作用によりポリヌクレオチドを凝縮して複合体を生成し、カチオン性分子はリポソームに含まれないステップと、
(b)約4.5以下のpHで、複合体を標的化部分に共有結合させて、標的細胞にポリヌクレオチド剤を送達するための粒子を生成するステップと
を含む、標的細胞にポリヌクレオチドを送達するための粒子を生成する方法が提供される。
(a)ポリヌクレオチド剤を、カチオン性分子を含む組成物と接触させるステップであって、カチオン性分子は静電的相互作用によりポリヌクレオチド剤を凝縮して複合体を生成し、カチオン性分子はリポソームに含まれないステップと、
(b)約4.5未満のpHで、複合体を標的化部分に共有結合させて、標的細胞にポリヌクレオチド剤を送達するためのナノ粒子を生成するステップと
を含む、標的細胞にポリヌクレオチドを送達するためのナノ粒子を生成する方法が提供される。
siRNAおよびカチオン性分子の核、ならびに標的化部分(例えば、HA)を含む殻を含む粒子であって、
粒子の集団の粒子の各々は、約−40mVのゼータ電位を含み、粒子の集団の粒子の各々は、溶液中の場合、直径が約100〜300nmである。
miRNAおよびカチオン性分子の核、ならびに標的化部分(例えば、HA)を含む殻を含む粒子であって、
粒子の集団の粒子の各々は、溶液中の場合、直径が約30〜50nmである。
実施例
実施例1
DOTAPをベースとする粒子
材料および方法
siRNAおよびmiRNA模倣体(アンタゴmir)の調製:
siRNAをカプセル化したH−mer(ヒアルロン酸被覆ナノ粒子)の調製−方法1
siRNAをカプセル化したH−mer(ヒアルロン酸被覆ナノ粒子)の調製−方法2
ヒトサイクリンD1(CCND1)
順方向:TGCTCCTGGTGAACAAGCTCAAGT(配列番号1)
逆方向:TGATCTGTTTGTTCTCCTCCGCCT(配列番号2)
GAPDH
順方向:GACCCCTTCATTGACCTCAAC(配列番号3)
逆方向:CTTCTCCATGGTGGTGAAGA(配列番号4)
STAT1
順方向:GTGCATCATGGGCTTCATCAGCAA(配列番号5)
逆方向:TAGGGTTCAACCGCATGGAAGTCA(配列番号6)
結果
siRNAを封入したヒアルロン酸被覆ナノ粒子(H−mer)の調製および構造評価
最良の製剤をスクリーニングするためのCD44発現細胞への形質移入
H−merは、T−IC中のリファレンス遺伝子を選択的にノックダウンすることができる。
いくつかの発がん遺伝子に対するsiRNAの混合物を封入したH−merは、T−ICを選択的に根絶する。
siRNAを封入したH−merは、望まない免疫応答を誘導しない。
miRNAを封入したH−mer
アンタゴmirは、Dharmaconから購入した。アンタゴmir分子の数は、リボグリーン(Invitrogen)を使用して測定した。
実施例2
DLPE:DLPGをベースとする粒子
材料および方法
結果
siRNAを封入したヒアルマーの調製および構造評価
実施例3
本発明の実施形態の粒子は、AML初代細胞およびヒト卵巣腺癌細胞を標的化することができる。
材料および方法
結果
Claims (49)
- (a)ポリヌクレオチドを、カチオン性分子を含む組成物と接触させるステップであって、前記カチオン性分子は静電的相互作用により前記ポリヌクレオチドを凝縮して複合体を生成し、前記カチオン性分子はリポソームに含まれないステップと、
(b)約4.5以下のpHで、前記複合体を標的化部分に共有結合させて、標的細胞にポリヌクレオチド剤を送達するための粒子を生成するステップと
を含む、標的細胞にポリヌクレオチドを送達するための粒子を生成する方法。 - 前記ポリヌクレオチドがDNAを含む、請求項1に記載の方法。
- 前記ポリヌクレオチドがRNAを含む、請求項1に記載の方法。
- 前記ポリヌクレオチドが一本鎖である、請求項1に記載の方法。
- 前記ポリヌクレオチドが二本鎖である、請求項1に記載の方法。
- 前記ポリヌクレオチドがRNAサイレンシング剤を含む、請求項1に記載の方法。
- 前記RNAサイレンシング剤が、siRNA、miRNA、アンチセンスオリゴヌクレオチドおよびリボザイムからなる群から選択される、請求項6に記載の方法。
- 前記RNAサイレンシング剤が、siRNAまたはmiRNAを含む、請求項6に記載の方法。
- 前記カチオン性分子が、カチオン性ポリペプチド、カチオン性脂質、カチオン性界面活性剤および合成ポリマーからなる群から選択される、請求項1に記載の方法。
- 前記カチオン性脂質が、1,2−ジラウロイル−sn−グリセロ−3−ホスホエタノールアミン(DLPE)および1,2−ジラウロイル−sn−グリセロ−3−グリセロール(DLPG)、ジオレオイル−1,2−ジアシル−3−トリメチルアンモニウム−プロパン(DOTAP、18:1;14:0;16:0;18:0)およびN−[1−(2,3−ジオレイルオキシ)プロピル]−N,N,N−トリメチルアンモニウムクロリド(DOTMA);ジメチルジオクタデシルアンモニウム(DDAB);1,2−ジラウロイル−sn−グリセロ−3−エチルホスホコリン(エチルPC、12:0;14:0;16:0;18:0;18:1;16:0〜18:1);1,2−ジ−(9Z−オクタデセノイル)−3−ジメチルアンモニウム−プロパンおよび3β−[N−(N’,N’−ジメチルアミノエタン)−カルバモイル]コレステロール塩酸塩(DC−コレステロール)からなる群から選択される、請求項9に記載の方法。
- 前記組成物が、中性脂質をさらに含む、請求項1に記載の方法。
- 前記中性脂質が、ジオレイルホスファチジルエタノールアミン(DOPE)を含む、請求項11に記載の方法。
- 前記組成物が、アニオン性リン脂質をさらに含む、請求項1に記載の方法。
- 前記アニオン性リン脂質が、ホスファチジルセリン、ホスファチジン酸、ホスファチジルコリンおよびホスファチジルグリセロールからなる群から選択される、請求項13に記載の方法。
- 前記組成物が、コレステロールをさらに含む、請求項1に記載の方法。
- 前記合成ポリマーが、ポリエチレンイミン(PEI)またはポリ−L−リジンを含む、請求項9に記載の方法。
- 前記標的化部分が、ポリペプチド標的化部分を含む、請求項1に記載の方法。
- 前記標的化部分が、抗体、抗体断片、アプタマーおよび受容体リガンドからなる群から選択される、請求項17に記載の方法。
- 前記標的化部分が、グリコサミノグリカンを含む、請求項1に記載の方法。
- 前記グリコサミノグリカンが、ヒアルロン酸(HA)、ケラタン硫酸、コンドロイチン硫酸、ヘパリン硫酸、ヘパラン硫酸、デルマチン硫酸、これらの塩および混合物からなる群から選択される、請求項19に記載の方法。
- 前記グリコサミノグリカンが、HAを含む、請求項20に記載の方法。
- ステップ(b)の前に前記グリコサミノグリカンを活性化するステップをさらに含む、請求項19に記載の方法。
- 前記活性化が、酸性緩衝液中で前記グリコサミノグリカンをインキュベートすることにより行われる、請求項22に記載の方法。
- 前記組成物が、水素化ホスファチジルコリン(HSPC)、コレステロールおよびジオレオイルトリメチルアンモニウムプロパン(DOTAP)を含む、請求項1に記載の方法。
- 前記組成物が、1,2−ジラウロイル−sn−グリセロ−3−ホスホエタノールアミン(DLPE)および1,2−ジラウロイル−sn−グリセロ−3−グリセロール(DLPG)を含む、請求項1に記載の方法。
- 前記標的化部分がHAを含み、前記ポリヌクレオチド剤がsiRNAまたはmiRNAを含む、請求項1に記載の方法。
- 前記複合体が、ステップ(b)の前に押し出し成形されない、請求項1に記載の方法。
- 請求項1から27に記載の方法のいずれかに従って生成される粒子。
- シリコン表面上で乾燥させると直径が約100nmである、請求項28に記載の粒子。
- シリコン表面上で乾燥させると直径が約30nmである、請求項28に記載の粒子。
- 溶液中で直径が約30〜300nmである、請求項28に記載の粒子。
- 形が丸い、請求項28に記載の粒子。
- 約−40mVのゼータ電位を含む、請求項28に記載の粒子。
- 帯電している、請求項28に記載の粒子。
- 中性である、請求項28に記載の粒子。
- ナノ粒子である、請求項28に記載の粒子。
- 前記標的化部分が、HAを含む、請求項28に記載の粒子。
- 抗体、抗体断片、受容体リガンドおよびアプタマーからなる群から選択される少なくとも1つの追加の標的化部分をさらに含む、請求項37に記載の粒子。
- 約6000個を超えるsiRNAまたはmiRNA分子を含む、請求項28に記載の粒子。
- 請求項28から39のいずれかに記載の複数の粒子を含む組成物。
- 実質的に均一である、請求項40に記載の組成物。
- siRNAおよびカチオン性分子の核、ならびに標的化部分を含む殻を含む粒子の実質的に均一な集団を含む組成物であって、前記粒子の集団の粒子の各々が、約−40mVのゼータ電位を含み、前記粒子の集団の粒子の各々が、溶液中の場合、直径が約100〜300nmである組成物。
- miRNAおよびカチオン性分子の核、ならびに標的化部分を含む殻を含む粒子の実質的に均一な集団を含む組成物であって、前記粒子の集団の粒子の各々が、溶液中の場合、直径が約30〜50nmである組成物。
- 請求項7に記載の方法に従って生成される粒子を、CD44を発現している標的細胞と接触させ、それにより標的細胞内の目的の遺伝子を下方制御するステップを含む、標的細胞内の目的の遺伝子を下方制御する方法。
- 被験体に治療上有効量の請求項7に記載の方法に従って生成される粒子を投与し、それによりがんを治療するステップを含む、それを必要とする被験体のがんを治療する方法。
- 前記投与が生体内で行われる、請求項45に記載の方法。
- 前記投与が生体外で行われる、請求項45に記載の方法。
- 前記RNAサイレンシング剤が、発がん遺伝子の発現を下方制御するよう選択される、請求項45に記載の方法。
- 前記RNAサイレンシング剤が、生存率と関連する遺伝子の発現を下方制御するよう選択される、請求項45に記載の方法。
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EP2459724B1 (en) | 2019-09-25 |
CA2767976A1 (en) | 2011-02-03 |
AU2010277166A1 (en) | 2012-02-23 |
GB0913442D0 (en) | 2009-09-16 |
KR20120059491A (ko) | 2012-06-08 |
EP2459724A2 (en) | 2012-06-06 |
WO2011013130A3 (en) | 2011-03-24 |
US10179113B2 (en) | 2019-01-15 |
US20170143641A1 (en) | 2017-05-25 |
US9574210B2 (en) | 2017-02-21 |
CN104491872A (zh) | 2015-04-08 |
JP2017014229A (ja) | 2017-01-19 |
WO2011013130A2 (en) | 2011-02-03 |
CN102482685B (zh) | 2015-01-07 |
AU2010277166B2 (en) | 2016-08-04 |
AU2016250495A1 (en) | 2016-11-17 |
KR101755049B1 (ko) | 2017-07-06 |
US20120129916A1 (en) | 2012-05-24 |
CN102482685A (zh) | 2012-05-30 |
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