JP2023523262A - 新規な細胞透過性ペプチド及びその用途 - Google Patents
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Abstract
Description
X1-X2-...Xn-1-Xn
前記一般式Iにおいて、
n≧16であり、
X2は、His(H)及びX6は、Leu(L)であり、
前記X2及びX6を除いたアミノ酸は、Gly(G)、His(H)、Glu(E)、Arg(R)、Lys(K)、Ser(S)、Asp(D)、Trp(W)、Val(V)、Thr(T)、Ala(A)、Asn(N)及びTyr(Y)からなる群から選択されるいずれか1つである。
X1-X2-...Xn-1-Xn
前記一般式Iにおいて、
n≧16であり、
X2は、His(H)及びX6は、Leu(L)であり、
前記X2及びX6を除いたアミノ酸は、Gly(G)、His(H)、Glu(E)、Arg(R)、Lys(K)、Ser(S)、Asp(D)、Trp(W)、Val(V)、Thr(T)、Ala(A)、Asn(N)及びTyr(Y)からなる群から選択されるいずれか1つである。
1-1.細胞透過性ペプチド候補群の設計
本発明者らは、先行研究で従来の多くの細胞透過性ペプチドに対する分析結果に基づいて多様な配列及び長さを有する細胞透過性ペプチドを設計及び合成し、その細胞透過性を検証した。その結果、最も高い細胞透過性を示した配列番号1のアミノ酸配列からなる細胞透過性ペプチドを発掘し、それをtranspep-1と名付けた。
本発明者らは、前記実施例1-1に記載の各ペプチドを合成するために固相ペプチド合成(Solid phase peptide synthesis, SPPS)方法を用いた。前記方法は、レジンのN末端に、N末端がF-mocで保護されているアミノ酸のC末端を1つずつ結合する有機合成法である。全ての反応溶媒は、N,N-ジメチルホルムアミド (N,N-dimethylformamide, DMF)を利用し、アミノ酸のカップリングは、2M濃度のアミノ酸溶液を0.5MのN,N’-ジイソプロピルカルボジイミド(N,N′-Diisopropylcarbodiimide, DIC)1ml、1Mのエチルシアノ(ヒドロキシイミノ)アセテート(Ethyl cyano(hydroxyimino)acetate, Oxyma)0.5mlと混合してマイクロウエーブ合成機(Microwave synthesizer)で反応させて進めた。また、各アミノ酸配列ごとに反応時間、温度またはマイクロウエーブの電圧を異ならせてアミノ酸を製造した。この際、次のアミノ酸を合成するためには、以前アミノ酸のF-mocを除去せねばならないので、このために、80%DMFと20%ピペリジン(piperidine)溶液を使用して、F-moc保護基を、80℃で2分間2回デプロテクティング(deprotecting)させた。全てのカップリング過程及びデプロテクティング過程の間には、DMFと塩化メチレン(dichloromethane、DCM)を用いて互いに3回ずつ洗浄する過程を遂行した。
本発明者らは、前記実施例1で合成した、総12個ペプチド候補群に対してin vitroで細胞透過性分析を実施した。具体的に、ヒト血液脳関門細胞であるhCMEC/D3細胞を96ウェルプレートに18,000cells/wellで分注し、細胞がプレート面積の80~90%に到逹するまで、上皮細胞成長培地である EGM(endothelial cell growth medium)で37℃、CO2条件で一晩培養した。次いで、前記実施例1-2で製造したFITCが連結された12個候補群ペプチドと陰性対照群であるFITC単独を4μMで前記培地に希釈し、各ウェル当たり100μlずつ処理する量を製造した。次いで、細胞を培養したプレートで培養液を吸入(suction)して除去し、前記ペプチドを希釈し、予め製造した溶液を処理した後、2時間37℃、CO2条件で培養した。次いで、各ウェルでペプチドを処理した溶液をいずれもsoakingして除去し、EGM100μlを添加して5~6回tappingした後、吸入して除去する過程を2回繰り返した。以後、Hoechst 33342をEGMに1:5000に希釈し、各ウェルに100μlずつ添加し、30分間37℃、CO2条件で培養した。培養後、Cytation 5装備でDAPIとGFP蛍光をイメージリーディング(image reading)した後、映像処理してDAPIで細胞核を区画した。次いで、細胞核周辺20μMのFITC値を測定し、DAPIで割ってMean-FITC値を求め、FITC実験群を陰性対照群として補正して血液脳関門細胞に対する透過度を計算した。
本発明者らは、前記実施例1で説明したように、前記transpep-1細胞透過性ペプチドにおいて2番目(AA2)及び6番目(AA6)アミノ酸残基が細胞透過性に重要な影響を与えると予想したところ、それを検証するために、下記のような実験を進めた。
本発明者らは、下記in vivo細胞透過性分析において本発明による細胞透過性ペプチドの体内組織への伝達をイメージングし、そのcargo輸送体としての機能を検証するために、蛍光を帯びるタンパク質であるGFP(Green Fluorescence Protein)を、本発明の細胞透過性ペプチドに結合させて細胞透過性ペプチド-GFPを製造しようとした。GFPは、代表的な蛍光タンパク質であって、27kDaの大きさを有し、395nmと475nmで励起ピーク(emission peak)を有し、509nmで放出ピーク(emission peak)を有する。
本発明者らは、本発明による細胞透過性ペプチドに対してin vivoで細胞透過性を検証しようとした。
本発明者らは、本発明による12個細胞透過性ペプチドに対して、当該技術分野で公知されている細胞透過性ペプチドと細胞透過程度を比較しようとした。このために、下記のようにそれぞれフローサイトメトリー法による細胞透過能及びin vivo 血液脳関門(BBB)透過度分析実験を実施し、前記公知された細胞透過性ペプチドとしては、angiopep-2を用いた。angiopep-2ペプチドは、19個のアミノ酸配列からなるものであって、BBBで発現される受容体である低密度脂質タンパク質受容体関連のタンパク質-1(low-density lipoprotein receptor-related protein-1; LRP-1)に結合して細胞内に流入されると知られている。
まず、前記実施例1-2で製造したtranspep-1-FITC及びangiopep-2-FITCに対してフローサイトメトリー法(Flow cytometry)を通じて単一細胞内部の蛍光物質の流入量を確認し、それを定量化した。具体的に、hCMEC/D3細胞株を2% BCS(bovine calf serum)が含有されたEGM培養液を用いて15,000cells/wellで分注し、24時間後、前記2個のペプチドをそれぞれ10μM濃度で細胞に処理した後、、37℃、CO2条件で2時間培養した。以後、1X PBSで細胞を洗浄し、1.1% TE(Trypsinase-EDTA)を入れ、3分間培養して細胞をプレート底から離した後、TEの3倍程度のEGM細胞培養液を入れ、100rpmで5分間遠心分離した。次いで、上澄液は、吸入して除去し、細胞を250μl 1xPBSで再懸濁させた後、BD Falcon 12 x 75 mm Tubewith Cell Strainer Capに移して各ペプチドによる蛍光物質の流入量をフローサイトメータで分析した。
次いで、本発明によるtranspep-1とangiopep-2の in vivo BBB透過度を比較するために、下記のような方法で実験を進めた。
Claims (11)
- 下記一般式Iで表示されるアミノ酸からなる、細胞透過性ペプチド:
[一般式I]
X1-X2-...Xn-1-Xn
前記一般式Iにおいて、
n≧16であり、
X2は、His(H)及びX6は、Leu(L)であり、
前記X2及びX6を除いたアミノ酸は、Gly(G)、His(H)、Glu(E)、Arg(R)、Lys(K)、Ser(S)、Asp(D)、Trp(W)、Val(V)、Thr(T)、Ala(A)、Asn(N)及びTyr(Y)からなる群から選択されるいずれか1つである。 - 前記一般式Iにおいて、nは、16であることを特徴とする、請求項1に記載の細胞透過性ペプチド。
- 前記細胞透過性ペプチドは、配列番号1ないし配列番号12からなる群から選択されるいずれか1つのアミノ酸配列からなることを特徴とする、請求項1に記載の細胞透過性ペプチド。
- 前記細胞は、血液脳関門内皮細胞(brain endothelial cell)、癌細胞、血液細胞(blood cell)、リンパ球(lymphocyte)、兔疫細胞、幹細胞、多能性幹細胞(induced pluripotent stem cell; iPSC)、神経幹細胞(neural stem cell; NSC)、T細胞、B細胞、ナチュラルキラー細胞(natural Killer cell; NK cell)、大食細胞(macrophage)、ミクログリア細胞(microglia)、神経細胞(neuron)、星状細胞(astrocyte)及び筋肉細胞(muscle cell)からなる群から選択されることを特徴とする、請求項1に記載の細胞透過性ペプチド。
- 請求項1~4のうち、いずれか1項に記載の細胞透過性ペプチド及び生物学的活性物質を含む、複合体。
- 前記生物学的活性物質は、化合物(chemical compound)、タンパク質、糖タンパク質、ペプチド、抗体(antibody)、酵素(enzyme)、核酸分解酵素(nuclease)、ホルモン、サイトカイン(cytokine)、転写因子(transcription factor)、毒素、核酸、炭水化物、脂質、糖脂質、天然物(natural product)、半合成物質(semi-synthetic drug)、薬物(drug)、マイクロ粒子、ナノ粒子、リポソーム、ウイルス、量子点(quantum dots)及び蛍光色素(fluorochrome)からなる群から選択される1つ以上であることを特徴とする請求項5に記載の複合体。
- 前記核酸分解酵素は、CAS9(CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, Cfp1(CxxC-finger protein-1), ZEN(Zinc-finger nucleases)及びTALEN(Transcription activator-like effector nuclease)からなる群から選択されることを特徴とする請求項6に記載の複合体。
- 前記核酸は、DNA、RNA、ASO(Antisense oligonucleotide)、マイクロRNA(microRNA; miRNA)、小さい干渉RNA(small interfering RNA; siRNA)、アプタマー(aptamer)、LNA(locked nucleic acid)、PNA(peptide nucleic acid)及びモルフォリノ(morpholino)からなる群から選択されることを特徴とする請求項6に記載の複合体。
- 請求項5に記載の複合体を含む、物質伝達用組成物。
- 請求項9に記載の組成物を細胞に処理する段階を含む、物質伝達方法。
- 前記細胞は、血液脳関門内皮細胞(brain endothelial cell)、癌細胞、血液細胞(blood cell)、リンパ球(lymphocyte)、兔疫細胞、幹細胞、多能性幹細胞(induced pluripotent stem cell; iPSC)、神経幹細胞(neural stem cell; NSC)、T細胞、B細胞、ナチュラルキラー細胞(natural Killer cell; NK cell)、大食細胞(macrophage)、ミクログリア細胞(microglia)、神経細胞(neuron)、星状細胞(astrocyte)及び筋肉細胞(muscle cell)からなる群から選択されることを特徴とする請求項10に記載の物質伝達方法。
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