JP2016530303A - 生細胞内への分子の送達のための組成物および方法 - Google Patents
生細胞内への分子の送達のための組成物および方法 Download PDFInfo
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- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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Abstract
Description
本出願は、2013年9月10日に出願された仮出願第61/876,006号の利益を請求し、その全体が参照によって本明細書に明確に組み込まれる。
本出願に関連する配列表は、紙の写しの代わりにテキストフォーマットで提供され、本明細書により参照によって本明細書に組み込まれる。配列表を含むテキストファイルの名称は、52683_Seq_ST25.txtである。テキストファイル2KBであり、2014年9月9日に作成され、本明細書の提出と共に、EFS−Webにより提出された。
本発明は、米国国立衛生研究所によって授与されたNIH助成金番号GM087227および助成金番号GM087981のもと、米国政府による支援によってなされた。米国政府は、本発明における特定の権利を有する。
Xは、連結部分であり、
Yは、疎水性部分に共有結合したアミノ酸残基であり、
Zは、細胞透過性ペプチド(CPP)部分であり、CPP部分は、正味の正電荷を有し、かつ50%またはそれ超の残基がグアニジニウム基を有しているアミノ酸配列を有し、
mおよびnは、独立して0または1である)
を有する化合物を提供する。
X−Y−Z
(式中、
Xは、システイン(C)であり、
Yは、疎水性部分に共有結合したアミノ酸残基であり、
Zは、細胞透過性ペプチド(CPP)部分であり、CPP部分は、正味の正電荷を有し、かつ50%またはそれ超の残基がグアニジニウム基を有しているアミノ酸配列を有し、
XおよびYと、YおよびZとは、アミド結合によって連結されている)
を有する組成物を提供する。
一部の実施形態では、CPPは、アミノ酸残基の50%またはそれ超が側鎖にグアニジニウム基を有しているアミノ酸配列を有する。例えば、アミノ酸配列は、そのアミノ酸の50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、もしくは全て、またはその中の任意の導出可能な範囲が、側鎖にグアニジニウム基を有するものを、有することができる。グアニジニウム基は、式(II):
−NH−C(=NH2 +)−NH2 式(II)
によって表される。
結果
TATのジスルフィド結合されたダイマーは、モノマー対応物よりも効率的に細胞のサイトゾルを透過する
dTATは、通常はトランスフェクションに耐性のある細胞のサイトゾルを透過する
dTATは、エンドサイトーシスとその後に続くエンドソーム脱出により細胞を透過する
dTAT媒介型エンドソーム漏出は効率的である
dTAT媒介型サイトゾル送達は、細胞生存率、細胞増殖、およびdTATにより使用されるエンドサイトーシス経路に影響を及ぼさない
dTATは、細胞内で強力な転写応答を誘発しない
dTATは、タンパク質を、トランス状態で生細胞のサイトゾルおよび核に送達する
dTATにおける各TATモノマー間の構造連結を、細胞透過活性に影響を及ぼすことなく変化させることができる
考察
ペプチド設計、合成、および精製
全てのペプチドを、標準的なFmocプロトコールを使用したSPPSによって、rink amide MBHA resin(Novabiochem、San Diego、CA)において、組織内で合成した。Fmoc−Lys(Mtt)−OH、Fmoc−Lys(Boc)−OH、Fmoc−Gly−OH、Fmoc−Arg(Pbf)−OH、Fmoc−Gln(Trt)−OH、およびFmoc−Cys(Trt)−OH(Novabiochem)を使用してペプチドを組み立てた。反応を、撹拌が得られるように乾燥N2の流れを使用して、室温でSPPS容器内で実施した。Fmoc脱保護は、ピペリジンをジメチルホルムアミド(DMF)(Fisher、Waltham、MA)(20%、10mL)に加えたものを、Fmoc−ペプチド樹脂(0.30mmol)に添加することによって行った。脱保護反応は、1×5分および1×15分にわたり実施し、これらの反応の間には洗浄ステップを設けた。アミノ酸結合反応は、Fmoc−アミノ酸(1.2mmol)、HBTU(Novabiochem)(0.44g、1.1mmol)、およびジイソプロピルエチルアミン(DIEA)(Sigma、St.Louis、MO)(0.51mL、3.0mmol)をDMFに加えた混合物と共に4時間実施した。反応の終了後、樹脂をDMFおよびジクロロメタン(DCM)(Fisher、Waltham、MA)で洗浄した。DEAC−K9の場合、DEAC、HBTU、およびDIEA(ペプチドに対して4、3.9、および10当量)をDMFに加えた混合物を使用して、9番目のFmoc−Lys(Boc)−OHを結合させた後に、DEACフルオロフォア(AnaSpec、Fremont、CA)をペプチドのN末端に結合させた。反応は、撹拌が得られるように乾燥N2の流れを使用して一晩実施した。CK(ε−NH−TMR)TAT(TAT)の場合、CK(ε−NH−Mtt)TAT上のLysのε−アミノ基にあるMtt保護基を、2%トリフルオロ酢酸(TFA)(Fisher)および2%トリイソプロピルシラン(TIS)(Sigma)をDCMに加えたもので切断し、樹脂をDCMおよびDMFで洗浄した。TMR、HBTU、およびDIEA(ペプチドに対して4、3.9、および10当量)をDMFに加えた混合物を樹脂に添加し、反応を、撹拌が得られるように乾燥N2を使用して一晩実施した。Fmoc脱保護およびペプチド組立ての後、樹脂をDCMで洗浄し、真空乾燥した。次いで樹脂を、2.5%H2O、2.5%TIS、および2.5%エタンジチオール(EDT)(Sigma)を含有するTFAで、室温で3時間処置して、全体的な脱保護と樹脂からの切断とを実現した。粗製ペプチド生成物を沈殿させ、冷無水Et2O(Fisher)で洗浄した。沈殿物を水中に再懸濁し、凍結乾燥した。次いで得られた生成物を、0.1%水性TFA/アセトニトリルに再懸濁した。ペプチドを、逆相HPLCにより分析し精製した。HPLC分析は、Hewlett−Packard 1200シリーズ機器および分析Vydac C18カラム(5μm、4×150mm)で行った。流量は1mL/分であり、検出は214nmおよび550nmで行った。半分取HPLCを、Vydac C18 10×250mmカラムで行った。流量は4mL/分であり、検出は214nmおよび550nmで行った。全ての実験操作は、0.1%水性TFA(溶媒A)、および90%アセトニトリル、9.9%の水、および0.1%TFA(溶媒B)の線形勾配を使用した。ペプチドの正しい同一性を、Shimadzu/Kratos機器(AXIMA−CFR、島津製作所、京都)で行ったMALDI−TOFにより確認した。TAT、予測質量:2039.16、観察された質量:2040.66。DEAC−K9、予測質量:1412.97、観察された質量:1415.59。
アセトアミド化C(S−CH2CONH2)K(ε−NH−TMR)TATの合成
CK(TMR)TAT(TAT)のダイマー化によるdTATの発生
TAT−Cre、TAT−mCherry、HoxB4、およびTAT−HoxB4のクローニング、過発現、および精製
生細胞内のペプチドの送達
dTATとの同時インキュベーションによる、生細胞内での小分子、ペプチド、およびタンパク質の送達
細胞内でのペプチド取込みの定量的決定
ルシフェラーゼレポーターを使用した、TATおよびdTATによるTAT−HoxB4 & HoxB4送達の定量分析
細胞生存率アッセイ
dTATおよびカーゴの相互作用の決定
排他的性質または特権が主張されている本発明の実施形態は、下記の通り定義される。
Claims (54)
- 式:
Xは、連結部分であり、
Yは、疎水性部分に共有結合したアミノ酸残基であり、
Zは、細胞透過性ペプチド(CPP)部分であり、該CPP部分は、正味の正電荷を有し、かつ50%またはそれ超の残基がグアニジニウム基を有しているアミノ酸配列を有し、
mおよびnは、独立して0または1である)
を有する化合物。 - 前記CPP部分が、3から30の間のアミノ酸を含む、請求項1に記載の化合物。
- 前記CPP部分が、配列番号1に示される配列に対して少なくとも85%の同一性を有するアミノ酸配列を含む、請求項2に記載の化合物。
- 前記CPP部分が、配列番号1に示されるアミノ酸配列を含む、請求項3に記載の化合物。
- mおよびnの少なくとも1つが1である、請求項1に記載の化合物。
- 前記疎水性部分が、C6〜C30直鎖、分岐状、または環式基を含む、請求項5に記載の化合物。
- 前記基が、N、O、およびSから選択される1つまたは複数のヘテロ原子を含む、請求項6に記載の化合物。
- 前記環式基が、単環、二環、または三環式である、請求項6に記載の化合物。
- 前記疎水性部分がローダミン基を含む、請求項6に記載の化合物。
- 前記ローダミン基がテトラメチルローダミン(TMR)である、請求項9に記載の化合物。
- 前記アミノ酸残基がリシン(K)である、請求項5に記載の化合物。
- mおよびnが0であり、Z1および/またはZ2が、疎水性部分に連結されたアミノ酸残基を含むCPPである、請求項1に記載の化合物。
- 前記アミノ酸残基が、CPPアミノ酸配列中の最もN末端側にあるリシン(K)残基である、請求項12に記載の化合物。
- Xがシステイン(C)残基である、請求項1に記載の化合物。
- X1およびX2が、ジスルフィド結合により連結されたシステイン(C)残基である、請求項14に記載の化合物。
- X、Y、および/またはZが、アミド結合により連結される、請求項1に記載の化合物。
- Z1のCPPおよびZ2のCPPが、互いに少なくとも85%の同一性を有するアミノ酸配列を有する、請求項1に記載の化合物。
- X−Y−Zの組合せが、30個以下のアミノ酸残基を含む、請求項1に記載の化合物。
- エンドソーム溶解を容易にすることが可能である、請求項1に記載の化合物。
- 式:
X−Y−Z
(式中、
Xは、システイン(C)であり、
Yは、疎水性部分に共有結合したアミノ酸残基であり、
Zは、細胞透過性ペプチド(CPP)部分であり、前記CPP部分は、正味の正電荷を有し、かつ50%またはそれ超の残基がグアニジニウム基を有しているアミノ酸配列を有し、
XおよびYと、YおよびZとは、アミド結合によって連結されている)
を有する組成物。 - 前記CPP部分が、配列番号1に示される配列に対して少なくとも85%の同一性を有するアミノ酸配列を含む、請求項20に記載の組成物。
- 前記CPP部分が、配列番号1に示される配列を持つアミノ酸配列を含む、請求項21に記載の化合物。
- Yがリシン(K)である、請求項20に記載の化合物。
- 前記疎水性部分がローダミン基を含む、請求項20に記載の化合物。
- 前記ローダミン基がテトラメチルローダミン(TMR)である、請求項24に記載の化合物。
- N末端システイン(C)残基間でのジスルフィド結合の形成によって、ホモダイマーを形成することが可能である、請求項20に記載の化合物。
- 細胞内のエンドソーム浸透性を高める方法であって、細胞を請求項1または請求項20に記載の化合物と接触させることを含む方法。
- 前記細胞を、前記化合物のエンドサイトーシスを可能にするのに十分な条件下で接触させる、請求項27に記載の方法。
- 前記細胞を少なくとも1μMの濃度の前記化合物と接触させる、請求項27に記載の方法。
- 前記細胞を少なくとも5μMの濃度の前記化合物と接触させる、請求項29に記載の方法。
- 前記細胞が、アルブミンを欠いている培養物中にある、請求項27に記載の方法。
- 前記細胞を細胞不浸透性分子と接触させることをさらに含む、請求項27に記載の方法。
- 前記細胞が、生きている対象から得られ、ex vivoで前記化合物と接触させる、請求項27に記載の方法。
- 前記細胞が、生きている対象内にあり、in vivoで接触させる、請求項27に記載の方法。
- 前記細胞の前記エンドソーム浸透性を高めるのに有効な量の前記化合物を前記対象に投与することによって、前記細胞をin vivoで接触させる、請求項34に記載の方法。
- 前記対象が哺乳動物である、請求項33または請求項34に記載の方法。
- 細胞不浸透性分子を細胞のサイトゾルに送達する方法であって、細胞を請求項1または請求項20に記載の化合物および細胞不浸透性分子と、エンドサイトーシスを可能にするのに十分な条件下で接触させることを含む方法。
- 前記細胞を少なくとも1μMの濃度の化合物と接触させる、請求項37に記載の方法。
- 前記細胞不浸透性分子が、ペプチド、ポリペプチドおよび核酸を含むポリマー、または小分子医薬品である、請求項37に記載の方法。
- 前記ポリペプチドが転写因子である、請求項39に記載の方法。
- 前記細胞不浸透性分子が、前記化合物に共有結合的に連結していない、請求項37に記載の方法。
- 前記細胞不浸透性分子が正味の正電荷を有する、請求項37に記載の方法。
- 前記細胞が、生きている対象から得られ、ex vivoで前記化合物および細胞不浸透性分子と接触させられる、請求項37に記載の方法。
- 前記細胞が、生きている対象内にあり、in vivoで前記化合物および細胞不浸透性分子と接触させられる、請求項37に記載の方法。
- 細胞不浸透性分子を前記細胞の前記サイトゾルに送達するのに有効な量の前記化合物を前記対象に投与することによって、前記細胞をin vivoで接触させる、請求項44に記載の方法。
- 前記対象が哺乳動物である、請求項43または請求項44に記載の方法。
- ex vivoで細胞における多能性を誘導する方法であって、対象から得られた細胞を請求項1または請求項20に記載の化合物および転写因子と、エンドサイトーシスを可能にするのに十分な条件下で接触させることと、前記細胞を培養して前記細胞の多能性を得ることとを含む方法。
- 前記細胞が、前記対象中の体細胞組織から得られる、請求項47に記載の方法。
- 前記対象が哺乳動物である、請求項47に記載の方法。
- 前記転写因子が、Oct4、Sox2、Klf4、およびc−Mycの1つである、請求項47に記載の方法。
- 多能性細胞またはその子孫細胞を、前記対象に引き続き投与することをさらに含む、請求項50に記載の方法。
- 幹細胞をex vivoで拡張する方法であって、幹細胞を請求項1または請求項20に記載の化合物および転写因子と、エンドサイトーシスを可能にするのに十分な条件下で接触させることと、前記細胞を培養して前記細胞の拡張を得ることとを含む方法。
- 前記幹細胞が造血幹細胞である、請求項52に記載の方法。
- 前記転写因子が、HoxB4、HoxA4/10、Gata2、Gf1、AML1、JunB、NF−Y、Bmi1Ezh2、Dmnt3a、Cbx7、p18、p21、p27、p57、PTEN、Myc、Fbxw7などからなる群から選択される、請求項52に記載の方法。
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US20110300111A1 (en) * | 2008-11-20 | 2011-12-08 | Cedars-Sinai Medical Center | Generation of induced pluripotent stem cells without the use of viral vectors |
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US20140056811A1 (en) * | 2010-12-27 | 2014-02-27 | Compugen Ltd. | New cell-penetrating peptides and uses thereof |
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AIDS RESEARCH AND THERAPY, vol. Vol.3:12, JPN6018031773, 2006, pages 1 - 15 * |
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 21, no. 8, JPN6018031774, 2011, pages 802 - 807 * |
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JPWO2018174158A1 (ja) * | 2017-03-22 | 2020-03-05 | 国立大学法人京都大学 | 細胞質送達ペプチド |
JP7068711B2 (ja) | 2017-03-22 | 2022-05-17 | 国立大学法人京都大学 | 細胞質送達ペプチド |
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US20150099690A1 (en) | 2015-04-09 |
EP3043811A4 (en) | 2017-09-20 |
WO2015038662A1 (en) | 2015-03-19 |
CA2923664A1 (en) | 2015-03-19 |
ES2794599T3 (es) | 2020-11-18 |
AU2014318839B2 (en) | 2019-12-05 |
CA2923664C (en) | 2023-05-02 |
US9662404B2 (en) | 2017-05-30 |
JP6659546B2 (ja) | 2020-03-04 |
EP3043811A1 (en) | 2016-07-20 |
EP3043811B1 (en) | 2020-03-25 |
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