JP6346265B2 - Dnaインターカレーティング剤の細胞送達 - Google Patents
Dnaインターカレーティング剤の細胞送達 Download PDFInfo
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Description
本発明の複数の模範的実施形態を記載する前に、本発明が以下の説明に示された構築物または工程ステップの詳細に限定されないことが理解されなければならない。本発明は、他の実施形態で可能であり、様々な方法で実践または実行することが可能である。
実施例1:横紋筋肉腫細胞におけるG8 mAbコンジュゲートデンドリマーの内在化
細胞
二層DNAデンドリマーの調製:DNAデンドリマーは、過去に開示された通り製造した(例えば、全体として参照により本明細書に組み入れられる、特許明細書第5,175,270号、同第5,484,904号、同第5,487,973、同第6,110,687号および同第6,274,723号参照)。簡潔に述べると、各DNA鎖の中心部分に相補的に配置された配列の一領域を共有する2つのDNA鎖から生成されたDNAモノマーから、DNAデンドリマーを構築した。2つの鎖をアニーリングしてモノマーを形成するが、得られた構造は、4つの一本鎖「アーム」に隣接する中央の二本鎖「ウエスト」を有すると説明することができる。このウエスト+アーム構造は、塩基性の3DNA(登録商標)モノマーを含む。5つのモノマータイプそれぞれの端部にある一本鎖アームは、精密で特殊な方法により互いに相互作用するように設計されている。相補性モノマーのアームの間の塩基対合が、モノマー層の連続付加を通してデンドリマーの定方向の構築を可能にする。デンドリマーの各層の構築は、DNA鎖が互いに共有結合する架橋工程を含み、それにより別の方法ではデンドリマー構造の変形を引き起こす変性条件に耐える完全に共有結合の分子を形成する。加えて、相補性キャプチャーオリゴとして働く38塩基のオリゴヌクレオチドが、以下に示す通り、簡単なT4 DNAリガーゼ依存性ライゲーション反応により、利用可能なデンドリマーアームの5’末端にライゲートされる。
1.1×TE緩衝液 5.4μL(2680ng)中の二層DNAデンドリマー(500ng/μL)
2.a(−)LIG−BR7架橋オリゴ(14マー)(50ng/μL) 2.7μL(134ng)
3.10× リガーゼ緩衝液 10.2μL
4.ヌクレアーゼ不含水 81.7μL
5.Cap03キャプチャーオリゴ(38マー)(50ng/μL) 4.0μL(200ng)
6.T4DNAリガーゼ(1U/μL) 10.0μL(10単位)
ヒト胎児型および胞巣型横紋筋肉腫細胞(ATCCの細胞株136および2061)を、培地8mlが入った100nm組織培養皿中のガラス製カバースリップ(12カバースリップ/プレート)上で24時間培養した。136細胞株は、10%ウシ胎仔血清を含むDMEMで培養した。2061細胞株は、10%FBSを含むRPMIで培養した。カバースリップを以下の培地1mlを含有する35mm組織培養皿に移した(2枚カバースリップ/皿):
1.2μM希釈標準溶液に1:500希釈されたLysoSensor Green DND 189原液(1mM)(Molecular Probe/Invitrogen)を含む培地
2.2μM LysoSensor+1:75希釈されたG8:4n デンドリマー−Cy3を含む培地
二層DNAデンドリマーの調製:二層DNAデンドリマーを、実施例1に記載された通り付着されたキャプチャー配列を用いて調製した。キャプチャー配列を、デンドリマーアームの10〜15%にライゲートさせた。
1.添加なし
2. G8Ab/Cap03オリゴ/CPL −Dox DFに1:8希釈された
3. 二層G8 Ab/Cap03オリゴ/Cplオリゴ − DFで1:8に希釈
4. 二層Cap03 − DF中に1:8に高度に希釈
二層DNAデンドリマーの調製:二層DNAデンドリマーを、デンドリマーアームの10〜15%へのキャプチャー配列の付着をはじめとし、実施例1に記載された通り調製する。
Claims (16)
- 全体的または部分的に二本鎖の合成DNA担体と、前記DNA担体に連結された標的化剤と、前記DNA担体の二本鎖部分にインターカレートされた活性剤とを含み、前記標的化剤が、G8抗原に結合する抗体またはペプチドである、
細胞または組織への活性剤の送達のための組成物。 - 前記二本鎖DNAが、5塩基対を超える長さである、請求項1に記載の組成物。
- 前記活性剤が、水素結合を通して前記二本鎖DNAにインターカレートされている、請求項1または2に記載の組成物。
- 前記合成DNA担体がDNAデンドリマーである、請求項1〜3のいずれか1項に記載の組成物。
- 前記DNAデンドリマーが、3〜216のハイブリダイズされたDNAの一本鎖を含む、請求項4に記載の組成物。
- 前記活性剤が、化学療法薬、抗感染剤、抗マラリア剤、抗ウイルス剤および抗真菌剤からなる群から選択される、請求項1〜5のいずれか1項に記載の組成物。
- 前記活性剤が、ベルベリン、アクリジン、ダウノマイシン、ドキソルビシン、ダウノルビシン、ダクチノマイシン、シスプラチン、カルボプラチンまたはサリドマイドである、請求項6に記載の組成物。
- 請求項1〜7のいずれか1項に記載の組成物と、医薬的に許容し得る賦形剤とを含む医薬組成物。
- 細胞または組織に活性剤を送達するためのDNA担体を作製する方法であって、一本鎖DNAオリゴヌクレオチドを構築して全体的または部分的に二本鎖のDNAを形成させること、標的化剤を前記全体的または部分的に二本鎖のDNAに連結させること、および前記全体的または部分的に二本鎖のDNAを活性剤と接触させて、前記活性剤を前記DNAの二本鎖部分にインターカレートさせることを含み、前記標的化剤が、G8抗原に結合する抗体またはペプチドである、方法。
- 前記全体的または部分的に二本鎖のDNAを前記活性剤と接触させると同時に、前記一本鎖DNAオリゴヌクレオチドが構築される、請求項9に記載の方法。
- 前記全体的または部分的に二本鎖のDNAを、構築の後に前記活性剤と接触させる、請求項9に記載の方法。
- 前記活性剤と接触させる前に、前記標的化剤が前記全体的または部分的に二本鎖のDNAに連結している、請求項11に記載の方法。
- 細胞または組織を、請求項8に記載の医薬組成物と接触させることによって活性剤を前記細胞または組織に送達する方法において使用するための請求項8に記載の医薬組成物。
- 請求項8に記載の医薬組成物を前記患者に投与することによって患者における疾患または状態を処置する方法において使用するための請求項8に記載の医薬組成物。
- 細胞または組織への活性剤の送達のための、請求項8に記載の医薬組成物。
- 患者における疾患または状態を処置するための、請求項8に記載の医薬組成物。
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US201361803863P | 2013-03-21 | 2013-03-21 | |
US61/803,863 | 2013-03-21 | ||
PCT/US2014/031192 WO2014153394A1 (en) | 2013-03-21 | 2014-03-19 | Cellular delivery of dna intercalating agents |
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WO2017127033A1 (en) * | 2016-01-22 | 2017-07-27 | Yeditepe Universitesi | A preparation method for a dna origami based carrier system |
US20210162064A1 (en) * | 2016-02-19 | 2021-06-03 | Genisphere Llc | Nucleic Acid Carriers And Therapeutic Methods Of Use |
US10982210B2 (en) | 2018-03-02 | 2021-04-20 | Sixfold Bioscience Ltd. | Compositions for delivery of cargo to cells |
US20220175958A1 (en) * | 2019-03-25 | 2022-06-09 | University Of Baltimore, Maryland | Doxorubicin Containing Nanoplexes and Uses Thereof |
EP4022058A2 (en) | 2019-08-30 | 2022-07-06 | Sixfold Bioscience Ltd. | Compositions for transfer of cargo to cells |
CN115997016A (zh) | 2020-04-27 | 2023-04-21 | 西克斯福德生物科学有限公司 | 包含具有模块化官能团的核酸纳米颗粒的组合物 |
CN113975399A (zh) * | 2021-11-09 | 2022-01-28 | 启东启晨生物科技有限公司 | 一种dna作为药物载体的抗体偶联物抗肿瘤药物 |
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US20070225213A1 (en) * | 2006-03-23 | 2007-09-27 | Kosak Matthew K | Nucleic acid carriers for delivery of therapeutic agents |
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US9707299B2 (en) * | 2007-05-23 | 2017-07-18 | The Trustees Of The University Of Pennsylvania | Targeted carriers for intracellular drug delivery |
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CA2816121C (en) * | 2010-11-01 | 2017-06-13 | Syracuse University | System and method for delivery of dna-binding chemotherapy drugs using nanoparticles |
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EP2976109A1 (en) | 2016-01-27 |
US20170312299A1 (en) | 2017-11-02 |
EP2976109B1 (en) | 2018-07-25 |
JP2016523811A (ja) | 2016-08-12 |
WO2014153394A1 (en) | 2014-09-25 |
US20200148763A1 (en) | 2020-05-14 |
CN105188764A (zh) | 2015-12-23 |
CN105188764B (zh) | 2020-03-20 |
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