WO2017135273A1 - Peptide having cell membrane permeability, construct, and method for transporting cargo molecule into cell - Google Patents

Peptide having cell membrane permeability, construct, and method for transporting cargo molecule into cell Download PDF

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WO2017135273A1
WO2017135273A1 PCT/JP2017/003520 JP2017003520W WO2017135273A1 WO 2017135273 A1 WO2017135273 A1 WO 2017135273A1 JP 2017003520 W JP2017003520 W JP 2017003520W WO 2017135273 A1 WO2017135273 A1 WO 2017135273A1
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peptide
cell
formula
construct
cell membrane
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庸介 出水
栗原　正明
隆史 三澤
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公益財団法人ヒューマンサイエンス振興財団
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00

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  • the present invention relates to a peptide having cell membrane permeability, and specifically to a peptide having cell membrane permeability that can efficiently transport plasmid DNA or the like into cells.
  • the present invention also relates to a construct comprising the peptide and a cargo molecule. Furthermore, the present invention relates to a method for transporting cargo molecules into cells.
  • CPPs cell-penetrating peptide
  • CPPs that are actually used include (1) those rich in basic amino acids such as arginine, (2) amphiphilic peptides having a basic part and a hydrophobic part, and (3) hydrophobic sequences. And (4) a hydrophobic peptide.
  • Peptide vectors are effective for introducing various substances, but may be unsuitable for introducing genes (plasmids).
  • the molecular weight of the plasmid is very large, the negatively charged plasmid and the positively charged peptide vector form insoluble aggregates, and the guanidino group and cell surface of the vector exhibit important functions for intracellular translocation. The reason for this is thought to be the reason that the interaction between the two is impaired.
  • Non-Patent Document 1 Non-Patent Document 2 and Non-Patent Document 3
  • CPPs that enable efficient intracellular introduction even if the target of introduction is a plasmid have been studied.
  • the importance of the cationic functional group and the fact that the peptide helical structure of oligoarginine contributes to the improvement of cell membrane permeability are described.
  • An object of the present invention is to provide a cell membrane-permeable peptide that enables more efficient intracellular introduction than the above-described technique.
  • the cell membrane permeable peptide according to the present invention has the following formula X F- (L-Arg-L-Arg-Xaa) m- (Gly) n -NH 2 Formula X [Where, m is an integer from 2 to 4, n is an integer from 0 to 3, F is a fluorescent label attached to the N-terminus of the peptide, with or without a linker, Xaa is any of the following formula A, formula B (n is 1 to 5), formula C (n is 1 to 5), or formula D (n is 1 to 5)
  • a cell membrane-permeable peptide that enables efficient intracellular introduction can be obtained.
  • the present inventors based on nonaarginine (R9) having cell membrane permeability, introduce a predetermined cationic proline derivative to form a random structure in a hydrophilic environment, while imitating the vicinity of the cell membrane.
  • R9 nonaarginine
  • the cell membrane permeable peptide according to the present invention has the following formula X F- (L-Arg-L-Arg-Xaa) m- (Gly) n -NH 2 Formula X [Where, m is an integer from 2 to 4, n is an integer from 0 to 3, F is a fluorescent label attached to the N-terminus of the peptide, with or without a linker, Xaa is any one of the following formula A, formula B (n is 1 to 5), formula C (n is 1 to 5), or formula D (n is 1 to 5).
  • the cationic proline derivative is a derivative represented by the above-described formula A, formula B, formula C or formula D, but is preferably a derivative represented by formula A.
  • F is preferably a fluorescent label via a linker, and examples of the linker include ⁇ -Ala. Although it does not specifically limit as a fluorescent label, Preferably it labels with a fluorescein compound. In the case of labeling with a fluorescein compound, for example, a compound represented by the following formula can be mentioned.
  • formula IV (5-FAM) or formula V (6-FAM).
  • n is preferably 3.
  • R is —NHCNHNH 2 .
  • the cell membrane-permeable peptide can also be represented by the following formula.
  • the method for synthesizing the cell membrane-permeable peptide according to the present embodiment is not particularly limited, but can be synthesized by, for example, the Fmoc solid phase method.
  • Fmoc is an abbreviation for Fluorenyl-Methoxy-Carbonyl and is a protecting group.
  • the construct according to this embodiment includes the cell membrane-permeable peptide according to this embodiment and a cargo molecule to be transported into the cell.
  • the cell membrane permeable peptide and the cargo molecule are bound covalently or non-covalently.
  • the cargo molecule is not particularly limited, and is, for example, a nucleic acid, a protein, a drug, or a nanoparticle.
  • the nucleic acid that is a cargo molecule may be a polynucleotide or an oligonucleotide, and may be a DNA or RNA molecule.
  • DNA plasmid DNA, cDNA, genomic DNA or synthetic DNA may be used.
  • DNA and RNA may be double-stranded or single-stranded. In the case of a single strand, it can be a coding strand or a non-coding strand.
  • Nucleic acids include DNA derivatives or RNA derivatives, which means nucleic acids with phosphorothioate linkages or nucleic acids that have been chemically modified at the phosphate, sugar, or base of the internucleotide to avoid degradation by enzymes. To do.
  • the nucleic acid includes viruses such as adenovirus and retrovirus.
  • viruses such as adenovirus and retrovirus.
  • the nucleic acid is a vector used for gene therapy such as plasmid DNA or virus, a form configured to express the genetic information encoded in the cell when introduced into the cell is preferable.
  • the method of transporting a cargo molecule according to this embodiment into a cell includes a step of binding a cargo molecule to be transported into a cell and the peptide according to this embodiment to obtain a construct, and introducing the construct into a cell. And a process.
  • any of oral, injection, eye drop, nasal drop, transpulmonary, and absorption through the skin may be used.
  • it is an injection.
  • systemic administration by intravenous injection or local administration by injecting the construct according to the present embodiment into an affected area is possible.
  • the construct according to this embodiment it is possible to examine the function in a cell into which a gene or protein is introduced.
  • it is possible to examine the function of a gene by introducing and expressing a plasmid DNA into which the gene whose function is to be examined is introduced into the cell, and to install siRNA nucleic acid that suppresses the expression of the gene whose function is to be examined. It is possible to examine the function of a gene by introducing it into the cell and suppressing the expression of the gene.
  • candidate substances that can treat various diseases such as cancer, genetic diseases, AIDS, and rheumatoid arthritis can be screened.
  • the properties of the cells can be changed.
  • siRNA capable of degrading a specific mRNA is administered to a cell as a cargo molecule
  • a cell with a reduced expression level of a functional protein encoded by the mRNA can be obtained.
  • an antagonist that suppresses the activity of a specific intracellular receptor is administered to a cell as a cargo molecule
  • the cell can reduce the activity of the receptor compared to a target cell to which no antagonist is administered.
  • Formula 1 is a peptide according to a comparative example, and Pro is as follows.
  • Formula 2 is a peptide according to another comparative example, and Pro NH2 is as follows.
  • Formula 3 is a peptide according to this example, and Pro Gu is as follows.
  • the synthesized peptide according to this example shown below has its structure in accordance with the environmental change from the physiological condition to the amphiphilic environment. It was shown to change from random to helical.
  • the CD spectrum observed at 190-260 nm reflects secondary structures such as a-helix, b-sheet, and random structure that are often found in the protein backbone.
  • the CD spectrum of a peptide forming an a-helix structure shows a minimum value near 205 to 208 nm and 220 to 225 nm and a maximum value near 192 nm.
  • the ratio R value ([q] 222 / [q] 208 ) between the minimum values of 208 nm and 222 nm is 0.6 to 1.2.
  • the b-sheet structure shows positive and negative maxima at 195 to 200 nm and 216 to 218 nm, respectively.
  • the CD spectrum of a peptide having a random structure is said to have a negative maximum at around 200 nm, but these characteristics are slightly different depending on the type of amino acid and the measurement solvent.
  • a PBS buffer (pH 7.4) in which 1% sodium dodecyl sulfate (SDS) is dissolved. It was adjusted.
  • SDS sodium dodecyl sulfate
  • the R value was about 0.6. This feature is very similar to the CD spectral pattern exhibited by peptides that form a-helical structures, suggesting that peptide 3 forms helical structures in amphiphilic environments (FIG. 2).
  • the R value was about 0.6. This feature is very similar to the CD spectral pattern exhibited by
  • peptide 1 is (FAM- ⁇ -Ala- (L-Arg-L-Arg-Pro) 3- (Gly) 3 -NH 2 ...
  • Formula 2 where peptide 3 is (FAM- ⁇ -Ala- (L-Arg -L-Arg-Pro Gu ) 3- (Gly) 3 -NH 2 ...
  • Formula 3 and peptide R9 is an oligoarginine.
  • FIGS. 3 (A) and 3 (B) show the results of cell membrane permeability of each peptide in adherent cells (relative permeability when R9 is 1) (peptide concentration: 1 ⁇ M, culture at 37 ° C. for 2 hours).
  • FIG. 3B shows the results of cell membrane permeability of each peptide in floating cells (relative permeability when R9 is 1) (peptide concentration: 1 ⁇ M, culture at 37 ° C. for 2 hours).
  • FIGS. 3 (A) shows the results of cell membrane permeability of each peptide in adherent cells (relative permeability when R9 is 1) (peptide concentration: 1 ⁇ M, culture at 37 ° C. for 2 hours).
  • peptide 3 according to this embodiment has high permeability at low concentrations for adherent cells (HeLa, A549, CHO-K1) and suspension cells (Jurkat). Indicated.
  • (4) Cargo molecular transport efficiency of peptides Luciferase assay using luciferase-encoded pDNA (Plasmid pCAcc + Luc, coding for firefly luciferase under the control of the CAG promoter, was provided by the RIKEN Gene Bank (Tsukuba, Japan)) Thus, the cargo molecule transport efficiency of the peptide was evaluated. The results are shown in FIG. FIG. 4 shows the delivery of plasmid DNA by each peptide (cultured at 37 ° C. for 24 hours). As shown in FIG. 4, the peptide according to this example achieved high pDNA transport efficiency in HeLa cells compared to oligoarginine.
  • It can be used for investigating gene functions in target cells, screening for cargo molecules that can treat diseases, and modifying target cells.

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Abstract

Provided is a cell-permeating peptide which enables efficient introduction into a cell. A cell-permeating peptide shown below can be produced by introducing a specific cationic proline derivative using nona-arginine having cell membrane permeability as a base.

Description

細胞膜透過性を有するペプチド、構築物、及び、カーゴ分子を細胞内に輸送する方法Peptide having cell membrane permeability, construct, and method for transporting cargo molecule into cell
　本発明は、細胞膜透過性を有するペプチドに関し、具体的には、プラスミドDNA等を効率的に細胞内に輸送できる細胞膜透過性を有するペプチドに関する。また、そのペプチドとカーゴ分子とからなる構築物に関する。更に、カーゴ分子を細胞内に輸送する方法に関する。 The present invention relates to a peptide having cell membrane permeability, and specifically to a peptide having cell membrane permeability that can efficiently transport plasmid DNA or the like into cells. The present invention also relates to a construct comprising the peptide and a cargo molecule. Furthermore, the present invention relates to a method for transporting cargo molecules into cells.
　細胞透過性を有するペプチド(以下、細胞膜透過性ペプチド(Cell-Penetrating Peptides;CPPs)と略することがある。)を用いて、細胞内にタンパク質等を導入する手法が注目されている。細胞内に導入したいタンパク質等にCPPsを細胞内導入ベクターとして化学的に結合させるか、又は、遺伝子工学的にCPPsと導入したいタンパク質等との融合タンパク質を調製し、細胞培養液に混合することで，効率よく細胞内に目的分子が導入される。 A technique for introducing a protein or the like into a cell by using a peptide having cell permeability (hereinafter sometimes abbreviated as “cell-penetrating peptide (CPPs)”) has attracted attention. Either chemically bind CPPs to the protein to be introduced into the cell as an intracellular introduction vector, or prepare a fusion protein between the CPPs and the protein to be introduced by genetic engineering and mix it with the cell culture medium. , The target molecule is efficiently introduced into the cell.
　CPPsとして実際に用いられている代表的なものとして、(1)アルギニン等の塩基性アミノ酸に富むもの、(2)塩基性部分と疎水性部分を有する両親媒性ペプチド、(3)疎水性配列に若干の塩基性配列を含むペプチド、(4)疎水性ペプチド等が挙げられる。 Typical examples of CPPs that are actually used include (1) those rich in basic amino acids such as arginine, (2) amphiphilic peptides having a basic part and a hydrophobic part, and (3) hydrophobic sequences. And (4) a hydrophobic peptide.
　ペプチドベクターは様々な物質の導入に有効であるが、遺伝子（プラスミド）の導入には不向きである場合がある。プラスミドの分子量が非常に大きいこと、負電荷を帯びたプラスミドと正電荷を帯びたペプチドベクターとが不溶性の凝集体を形成すること、細胞内移行に重要な働きを示すベクターのグアニジノ基と細胞表層の相互作用が損なわれたりすること、等がその理由と考えられている。 Peptide vectors are effective for introducing various substances, but may be unsuitable for introducing genes (plasmids). The molecular weight of the plasmid is very large, the negatively charged plasmid and the positively charged peptide vector form insoluble aggregates, and the guanidino group and cell surface of the vector exhibit important functions for intracellular translocation. The reason for this is thought to be the reason that the interaction between the two is impaired.
　そこで、非特許文献１、非特許文献２及び非特許文献３には、導入目的物がプラスミドであっても効率的な細胞内導入を可能とするCPPsが検討されており、CPPsの膜透過におけるカチオン性官能基の重要性や、オリゴアルギニンのペプチドヘリカル構造が細胞膜透過性の向上に寄与すること等が記載されている。 Therefore, in Non-Patent Document 1, Non-Patent Document 2 and Non-Patent Document 3, CPPs that enable efficient intracellular introduction even if the target of introduction is a plasmid have been studied. The importance of the cationic functional group and the fact that the peptide helical structure of oligoarginine contributes to the improvement of cell membrane permeability are described.
　本発明は上述の技術よりも更なる効率的な細胞内導入を可能とする細胞膜透過性ペプチドを提供することを目的とする。 An object of the present invention is to provide a cell membrane-permeable peptide that enables more efficient intracellular introduction than the above-described technique.
　本発明にかかる細胞膜透過性ペプチドは、下記の式Ｘ
F-(L-Arg-L-Arg-Xaa)m-(Gly)n-NH2・・・式Ｘ
〔式中、
ｍは２～４のいずれかの整数であり、
ｎは０～３のいずれかの整数であり、
Fは、リンカーを介して又は介さないで、ペプチドのＮ末端に結合した蛍光標識であり、
Xaaは、下記の式Ａ、式Ｂ（ｎは１～５）、式Ｃ（ｎは１～５）、又は、式Ｄ（ｎは１～５）の何れかである The cell membrane permeable peptide according to the present invention has the following formula X
F- (L-Arg-L-Arg-Xaa) m- (Gly) n -NH 2 Formula X
[Where,
m is an integer from 2 to 4,
n is an integer from 0 to 3,
F is a fluorescent label attached to the N-terminus of the peptide, with or without a linker,
Xaa is any of the following formula A, formula B (n is 1 to 5), formula C (n is 1 to 5), or formula D (n is 1 to 5)
Figure JPOXMLDOC01-appb-C000005
  
Figure JPOXMLDOC01-appb-C000005
  
Figure JPOXMLDOC01-appb-C000006
  
Figure JPOXMLDOC01-appb-C000006
  
Figure JPOXMLDOC01-appb-C000007
  
Figure JPOXMLDOC01-appb-C000007
  
Figure JPOXMLDOC01-appb-C000008
  
Figure JPOXMLDOC01-appb-C000008
  
〕で表されるペプチドである。 ] It is a peptide represented by this.
　本発明によれば、効率的な細胞内導入を可能とする細胞膜透過性ペプチドが得られる。 According to the present invention, a cell membrane-permeable peptide that enables efficient intracellular introduction can be obtained.
ペプチドの生理的条件下における二次構造を示すＣＤスペクトル測定の結果である。It is the result of the CD spectrum measurement which shows the secondary structure under the physiological conditions of a peptide. ペプチドの両親媒環境下における二次構造を示すＣＤスペクトル測定の結果である。It is the result of the CD spectrum measurement which shows the secondary structure in the amphiphilic environment of a peptide. ペプチドの細胞膜透過性を示すフローサイトメーターでの測定結果であり、そのうち（Ａ）は接着細胞における結果であり、（Ｂ）は浮遊細胞における結果である。It is the measurement result in the flow cytometer which shows the cell membrane permeability | transmittance of a peptide, (A) is a result in an adherent cell, (B) is a result in a floating cell. ペプチドによるプラスミドDNAのデリバリー結果を示す図である。It is a figure which shows the delivery result of plasmid DNA by a peptide.
　以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.
　本発明者らは、細胞膜透過性を有するノナアルギニン(R9)をベースとして、所定のカチオン性プロリン誘導体を導入することにより、親水性環境下でランダム構造を形成するのに対し、細胞膜付近を模した両親媒性環境下ではヘリカル構造へとその二次構造を動的に変化させる新規の膜透過性ペプチドの開発に成功した。この二次構造変化により、膜透過性の向上及びカーゴ分子の効率的なリリースが得られる。 The present inventors, based on nonaarginine (R9) having cell membrane permeability, introduce a predetermined cationic proline derivative to form a random structure in a hydrophilic environment, while imitating the vicinity of the cell membrane. In the amphiphilic environment, we have succeeded in developing a novel membrane-permeable peptide that dynamically changes its secondary structure into a helical structure. This secondary structure change results in improved membrane permeability and efficient release of cargo molecules.
　本発明にかかる細胞膜透過性ペプチドは、下記の式Ｘ
F-(L-Arg-L-Arg-Xaa)m-(Gly)n-NH2・・・式Ｘ
〔式中、
ｍは２～４のいずれかの整数であり、
ｎは０～３のいずれかの整数であり、
Ｆは、リンカーを介して又は介さないで、ペプチドのＮ末端に結合した蛍光標識であり、
Xaaは、下記の式Ａ、式Ｂ（ｎは１～５）、式Ｃ（ｎは１～５）、又は、式Ｄ（ｎは１～５）の何れかである。 The cell membrane permeable peptide according to the present invention has the following formula X
F- (L-Arg-L-Arg-Xaa) m- (Gly) n -NH 2 Formula X
[Where,
m is an integer from 2 to 4,
n is an integer from 0 to 3,
F is a fluorescent label attached to the N-terminus of the peptide, with or without a linker,
Xaa is any one of the following formula A, formula B (n is 1 to 5), formula C (n is 1 to 5), or formula D (n is 1 to 5).
Figure JPOXMLDOC01-appb-C000009
  
Figure JPOXMLDOC01-appb-C000009
  
Figure JPOXMLDOC01-appb-C000010
  
Figure JPOXMLDOC01-appb-C000010
  
Figure JPOXMLDOC01-appb-C000011
  
Figure JPOXMLDOC01-appb-C000011
  
Figure JPOXMLDOC01-appb-C000012
  
Figure JPOXMLDOC01-appb-C000012
  
〕で表される。 ] Is represented.
　カチオン性プロリン誘導体は、上述の式Ａ、式Ｂ、式Ｃ又は式Ｄで表される誘導体であるが、好ましくは式Ａで表される誘導体である。 The cationic proline derivative is a derivative represented by the above-described formula A, formula B, formula C or formula D, but is preferably a derivative represented by formula A.
　Ｆは、リンカーを介した蛍光標識である場合が好ましく、リンカーとしては例えばβ-Ala等がある。蛍光標識としては特に限定されるものではないが、好ましくはフルオレセイン化合物で標識する。フルオレセイン化合物で標識する場合、例えば下記の式で表される化合物が挙げられる。 F is preferably a fluorescent label via a linker, and examples of the linker include β-Ala. Although it does not specifically limit as a fluorescent label, Preferably it labels with a fluorescein compound. In the case of labeling with a fluorescein compound, for example, a compound represented by the following formula can be mentioned.
Figure JPOXMLDOC01-appb-C000013
  
Figure JPOXMLDOC01-appb-C000013
  
　好ましくは式IV(5-FAM)又は式V(6-FAM)である。 Preferred is formula IV (5-FAM) or formula V (6-FAM).
　また、ｍは３が好ましく、ｎは３が好ましい。 Further, m is preferably 3, and n is preferably 3.
　下記に本実施形態にかかる細胞膜透過性ペプチドの好適な場合の構造式を示す。 The structural formula of the preferred case of the cell membrane permeable peptide according to this embodiment is shown below.
Figure JPOXMLDOC01-appb-C000014
  
Figure JPOXMLDOC01-appb-C000014
  
　上記式においてＲは-ＮＨＣＮＨＮＨ２である。上記の細胞膜透過性ペプチドは下記の式で表すことも可能である。 In the above formula, R is —NHCNHNH 2 . The cell membrane-permeable peptide can also be represented by the following formula.
　　　FAM-β-Ala-(L-Arg-L-Arg-ProGu)3-(Gly)3-NH2・・・式３
　ここで、ProGuは下記である。 FAM-β-Ala- (L-Arg-L-Arg-Pro Gu ) 3- (Gly) 3 -NH 2 ... Formula 3
Here, Pro Gu is as follows.
Figure JPOXMLDOC01-appb-C000015
  
Figure JPOXMLDOC01-appb-C000015
  
　本実施形態にかかる細胞膜透過性ペプチドの合成方法は、特に限定されるものではないが、例えばFmoc固相法により合成することが可能である。FmocはFluorenyl-Methoxy-Carbonylの略であり、保護基である。 The method for synthesizing the cell membrane-permeable peptide according to the present embodiment is not particularly limited, but can be synthesized by, for example, the Fmoc solid phase method. Fmoc is an abbreviation for Fluorenyl-Methoxy-Carbonyl and is a protecting group.
　下記に式Ａで表されるカチオン性プロリン誘導体の合成例を示す。 Synthesis examples of cationic proline derivatives represented by the formula A are shown below.
Figure JPOXMLDOC01-appb-C000016
  
Figure JPOXMLDOC01-appb-C000016
  
　また、下記に式Ｃで表されるカチオン性プロリン誘導体の合成例を示す。 Further, synthesis examples of cationic proline derivatives represented by the formula C are shown below.
Figure JPOXMLDOC01-appb-C000017
  
Figure JPOXMLDOC01-appb-C000017
  
　本実施形態にかかる構築物は、本実施形態にかかる細胞膜透過性ペプチドと、細胞内に輸送すべきカーゴ分子とを含む。細胞膜透過性ペプチドとカーゴ分子とは共有結合的又は非共有結合的に結合する。 The construct according to this embodiment includes the cell membrane-permeable peptide according to this embodiment and a cargo molecule to be transported into the cell. The cell membrane permeable peptide and the cargo molecule are bound covalently or non-covalently.
　カーゴ分子は、特に限定されるものではないが、例えば、核酸、タンパク質、薬剤、又は、ナノ粒子の何れかである。 The cargo molecule is not particularly limited, and is, for example, a nucleic acid, a protein, a drug, or a nanoparticle.
　カーゴ分子である核酸は、ポリヌクレオチドでもオリゴヌクレオチドでもよく、DNAでもRNA分子でもよい。DNAの場合、プラスミドDNA、cDNA、ゲノミックDNA又は合成DNAでもよい。DNA及びRNAは２本鎖でも１本鎖でもよい。１本鎖の場合、コード鎖又は非コード鎖であり得る。核酸にはDNA誘導体又はRNA誘導体が含まれ、該誘導体とはホスホロチオエート結合を有する核酸又は酵素による分解を避ける為にインターヌクレオチドのリン酸部位、糖部分、塩基部分に化学修飾を施した核酸を意味する。また、核酸にはアデノウイルス、レトロウイルス等のウイルスも含まれる。核酸がプラスミドDNA又はウイルス等の遺伝子治療に用いられるベクターである場合、細胞内に導入されたときにコードした遺伝情報を細胞内で発現するように構成された形態が好ましい。 The nucleic acid that is a cargo molecule may be a polynucleotide or an oligonucleotide, and may be a DNA or RNA molecule. In the case of DNA, plasmid DNA, cDNA, genomic DNA or synthetic DNA may be used. DNA and RNA may be double-stranded or single-stranded. In the case of a single strand, it can be a coding strand or a non-coding strand. Nucleic acids include DNA derivatives or RNA derivatives, which means nucleic acids with phosphorothioate linkages or nucleic acids that have been chemically modified at the phosphate, sugar, or base of the internucleotide to avoid degradation by enzymes. To do. The nucleic acid includes viruses such as adenovirus and retrovirus. When the nucleic acid is a vector used for gene therapy such as plasmid DNA or virus, a form configured to express the genetic information encoded in the cell when introduced into the cell is preferable.
　本実施形態にかかるカーゴ分子を細胞内に輸送する方法は、細胞内に輸送すべきカーゴ分子と、本実施形態にかかるペプチドとを結合させて構築物を得る工程と、その構築物を細胞に導入する工程と、を有する。 The method of transporting a cargo molecule according to this embodiment into a cell includes a step of binding a cargo molecule to be transported into a cell and the peptide according to this embodiment to obtain a construct, and introducing the construct into a cell. And a process.
　本実施形態にかかる構築物を生体（ヒトを含む動物、特に、ヒトを含む哺乳類）に投与する方法としては、経口、注射、点眼、点鼻、経肺、皮膚を介した吸収のいずれでも良く、好ましくは注射である。例えば、本実施形態にかかる構築物を静脈注射による全身投与又は患部に注射することによる局所投与が可能である。 As a method for administering the construct according to the present embodiment to a living body (animals including humans, in particular mammals including humans), any of oral, injection, eye drop, nasal drop, transpulmonary, and absorption through the skin may be used. Preferably, it is an injection. For example, systemic administration by intravenous injection or local administration by injecting the construct according to the present embodiment into an affected area is possible.
　本実施形態にかかる構築物によれば、遺伝子又はタンパク質の導入される細胞内での機能を調べることが可能である。例えば、細胞内に機能を調べたい遺伝子を組み込んだプラスミドDNAを導入して発現させることによりその遺伝子の機能を調べることが可能であり、また機能を調べたい遺伝子の発現を抑制するsiRNA核酸を細胞内に導入して遺伝子の発現を抑制することによってその遺伝子の機能を調べることが可能である。 According to the construct according to this embodiment, it is possible to examine the function in a cell into which a gene or protein is introduced. For example, it is possible to examine the function of a gene by introducing and expressing a plasmid DNA into which the gene whose function is to be examined is introduced into the cell, and to install siRNA nucleic acid that suppresses the expression of the gene whose function is to be examined. It is possible to examine the function of a gene by introducing it into the cell and suppressing the expression of the gene.
　また本実施形態にかかる構築物によれば、癌、遺伝子疾患、AIDS、慢性関節リウマチ等の様々な疾患を処置し得る候補物質をスクリーニングすることができる。 Further, according to the construct according to this embodiment, candidate substances that can treat various diseases such as cancer, genetic diseases, AIDS, and rheumatoid arthritis can be screened.
　また本実施形態にかかる構築物によれば、細胞の性質を変えることができる。例えば、特定のmRNAを分解できるsiRNAをカーゴ分子として細胞に投与すれば、そのmRNAがコードする機能性タンパク質の発現量を低下させた細胞が得られる。また例えば特定の細胞内受容体の活性を抑えるアンタゴニストをカーゴ分子として細胞に投与すれば、その細胞はアンタゴニストが投与されていない標的細胞と比較して受容体の活性を低くすることができる。 Moreover, according to the construct according to this embodiment, the properties of the cells can be changed. For example, when siRNA capable of degrading a specific mRNA is administered to a cell as a cargo molecule, a cell with a reduced expression level of a functional protein encoded by the mRNA can be obtained. Further, for example, when an antagonist that suppresses the activity of a specific intracellular receptor is administered to a cell as a cargo molecule, the cell can reduce the activity of the receptor compared to a target cell to which no antagonist is administered.
（１）ペプチド合成
　細胞内に導入するプロリン誘導体は全て、有機化学的に合成した。ペプチドはマイクロウェーブを用いたFmoc固相法により簡便に行い、下記３つのペプチドを合成した。 (1) Peptide synthesis All proline derivatives introduced into cells were synthesized organically. Peptides were easily prepared by the Fmoc solid phase method using microwaves, and the following three peptides were synthesized.
　　　FAM-β-Ala-(L-Arg-L-Arg-Pro)3-(Gly)3-NH2・・・式１
　式１は比較例にかかるペプチドであり、Proは下記である。 FAM-β-Ala- (L-Arg-L-Arg-Pro) 3- (Gly) 3 -NH 2 ... Formula 1
Formula 1 is a peptide according to a comparative example, and Pro is as follows.
Figure JPOXMLDOC01-appb-C000018
  
Figure JPOXMLDOC01-appb-C000018
  
　　　FAM-β-Ala-(L-Arg-L-Arg-ProNH2)3-(Gly)3-NH2・・・式２
　式２は別の比較例にかかるペプチドであり、ProNH2は下記である。 FAM-β-Ala- (L- Arg-L-Arg-Pro NH2) 3 - (Gly) 3 -NH 2 ··· type 2
Formula 2 is a peptide according to another comparative example, and Pro NH2 is as follows.
Figure JPOXMLDOC01-appb-C000019
  
Figure JPOXMLDOC01-appb-C000019
  
　　　FAM-β-Ala-(L-Arg-L-Arg-ProGu)3-(Gly)3-NH2・・・式３
　式３は本実施例にかかるペプチドであり、ProGuは下記である。 FAM-β-Ala- (L-Arg-L-Arg-Pro Gu ) 3- (Gly) 3 -NH 2 ... Formula 3
Formula 3 is a peptide according to this example, and Pro Gu is as follows.
Figure JPOXMLDOC01-appb-C000020
  
Figure JPOXMLDOC01-appb-C000020
  
（２）ペプチドの二次構造解析
　得られた粗ペプチドは逆相HPLCにより精製し、MALDI-MSによって同定した。ペプチドの溶液状態における二次構造は20 mM PBS buffer solution (pH = 7.4)及び1% SDS in PBS buffer solution (pH = 7.4)を用い、CDスペクトル測定によって解析を行なった。 (2) Secondary structure analysis of peptide The obtained crude peptide was purified by reverse phase HPLC and identified by MALDI-MS. The secondary structure of the peptide in the solution state was analyzed by CD spectrum measurement using 20 mM PBS buffer solution (pH = 7.4) and 1% SDS in PBS buffer solution (pH = 7.4).
　溶液状態における二次構造解析の結果、合成した下記に示す本実施例にかかるペプチド（式３で示される）は、生理的条件下から両親媒環境下への環境変化に応じて、その構造をランダムからヘリカルへと変化させることが示された。 As a result of secondary structure analysis in the solution state, the synthesized peptide according to this example shown below (shown by Formula 3) has its structure in accordance with the environmental change from the physiological condition to the amphiphilic environment. It was shown to change from random to helical.
　即ち、実際に細胞膜を透過する際、ペプチドはpH＝7.2～7.4の培地内に分散した状態から細胞膜付近の両親媒環境へと近づき、直接透過ないしエンドサイトーシスを介して細胞内へと移行した後にサイトゾルへと拡散する。したがって、細胞から十分に離れた生理的親水性環境下における二次構造と細胞膜付近及びエンドソーム内を模した両親媒環境下における二次構造の解析をCD（円偏光二色性：Circular Dichroism）スペクトル測定（190～260 nm）により行った。タンパク質のアミド結合は240 nm以下の遠紫外波長領域にいくつかの電子遷移を有しているが、これらはアミド結合の状態によって異なることが知られている。そのため、190～260 nmで観察されるCDスペクトルはタンパク質主鎖内によく見られるa-ヘリックスやb-シート、ランダム構造などの二次構造を反映している。例えばa-ヘリックス構造を形成するペプチドのCDスペクトルは、205～208nm及び220～225 nm付近に極小値と192 nm付近に極大値を示す。また、208 nmと222 nmの極小値の比R値（[q]222/[q]208）が0.6～1.2を示す。一方で、b-シート構造では195～200 nmと216～218 nmにそれぞれ正と負の極大を示す。また、ランダム構造を有するペプチドのCDスペクトルは200 nm付近に負の極大を持つとされているが、こうした特徴は構成しているアミノ酸の種類や測定溶媒によって若干異なる。親水性環境下においては20 mM PBS buffer (pH=7.4)を、両親媒環境下においては1%のドデシル硫酸ナトリウム（SDS）を溶解したPBS buffer（pH=7.4）を用いて100μMのペプチド溶液を調整した。親水性環境下における二次構造解析の結果、全てのペプチドが同様の特徴（243～247 nm及び200 nm付近に負の極大、215～220 nm付近に正の極大）を持つスペクトルを示し、本結果からこれらのペプチドが生理的環境下において主にランダム構造を形成していることが示唆された（図１）。図１は、2mM PBS buffer solution(pH＝7.4)中のＣＤスペクトル（ペプチド濃度:0.1mM）である。一方で、両親媒環境下においては、親水性条件下で見られていた243～247nm付近の負の極大や215～220 nm付近の正の極大が消失した反面、206 nm及び228 nm付近に負の極大が現れR値は約0.6を示した。本特徴はa-ヘリカル構造を形成するペプチドが示すCDスペクトルパターンに非常に良く似ており、ペプチド３が両親媒環境下でヘリカル構造を形成することを示唆している（図２）。図２は、1%SDS in PBS buffer solution(pH＝7.4)中のＣＤスペクトル（ペプチド濃度:0.1mM）である。ここで図１及び図２において、ペプチド１は（FAM-β-Ala-(L-Arg-L-Arg-Pro)3-(Gly)3-NH2・・・式１であり、ペプチド２は（FAM-β-Ala-(L-Arg-L-Arg-ProNH2)3-(Gly)3-NH2・・・式２であり、ペプチド３は（FAM-β-Ala-(L-Arg-L-Arg-ProGu)3-(Gly)3-NH2・・・式３であり、ペプチドR9はオリゴアルギニンである。
（３）ペプチドの細胞膜透過性
　ペプチドの細胞膜透過性に関しては、フローサイトメーターを用い、細胞内の蛍光強度から測定した。結果を図３（Ａ）及び（Ｂ）に示す。図３（Ａ）は接着細胞における各ペプチドの細胞膜透過性の結果（R9を１とした時の相対的透過性）である（ペプチド濃度:1μM、37℃、２時間培養）。図３（Ｂ）は浮遊細胞における各ペプチドの細胞膜透過性の結果（R9を１とした時の相対的透過性）である（ペプチド濃度:1μM、37℃、２時間培養）である。図３（Ａ）及び（Ｂ）に示されるように、本実施にかかるペプチド３は、接着細胞（HeLa, A549, CHO-K1）及び浮遊細胞（Jurkat）に対し、低濃度において高い透過性を示した。
（４）ペプチドのカーゴ分子輸送効率
　ルシフェラーゼをコードしたpDNA（Plasmid pCAcc+Luc, coding for firefly luciferase under the control of the CAG promoter, was provided by the RIKEN Gene Bank (Tsukuba, Japan)）を利用したルシフェラーゼアッセイにより、ペプチドのカーゴ分子輸送効率を評価した。結果を図４に示す。図４は、各ペプチドによるプラスミドDNAのデリバリー（37℃、24時間培養）を示す。図４に示されるように、本実施例にかかるペプチドは、HeLa細胞において、オリゴアルギニンと比較して高いpDNA輸送効率を達成した。 That is, when actually permeating the cell membrane, the peptide approached the amphipathic environment near the cell membrane from the state dispersed in the medium of pH = 7.2 to 7.4, and transferred into the cell through direct permeation or endocytosis. Later diffuses into the cytosol. Therefore, the CD (Circular Dichroism) spectrum is used to analyze the secondary structure in a physiologically hydrophilic environment sufficiently away from the cell and the secondary structure in an amphiphilic environment that mimics the vicinity of the cell membrane and the endosome. Measurement was performed (190 to 260 nm). Protein amide bonds have several electronic transitions in the far-ultraviolet wavelength region below 240 nm, and these are known to vary depending on the state of the amide bond. Therefore, the CD spectrum observed at 190-260 nm reflects secondary structures such as a-helix, b-sheet, and random structure that are often found in the protein backbone. For example, the CD spectrum of a peptide forming an a-helix structure shows a minimum value near 205 to 208 nm and 220 to 225 nm and a maximum value near 192 nm. The ratio R value ([q] 222 / [q] 208 ) between the minimum values of 208 nm and 222 nm is 0.6 to 1.2. On the other hand, the b-sheet structure shows positive and negative maxima at 195 to 200 nm and 216 to 218 nm, respectively. In addition, the CD spectrum of a peptide having a random structure is said to have a negative maximum at around 200 nm, but these characteristics are slightly different depending on the type of amino acid and the measurement solvent. In a hydrophilic environment, use a 20 mM PBS buffer (pH = 7.4). In an amphiphilic environment, use a PBS buffer (pH = 7.4) in which 1% sodium dodecyl sulfate (SDS) is dissolved. It was adjusted. As a result of secondary structure analysis in a hydrophilic environment, all peptides showed spectra with the same characteristics (negative maximum near 243 to 247 nm and 200 nm, positive maximum near 215 to 220 nm). The results suggested that these peptides mainly formed random structures in a physiological environment (FIG. 1). FIG. 1 is a CD spectrum (peptide concentration: 0.1 mM) in 2 mM PBS buffer solution (pH = 7.4). On the other hand, in the amphiphilic environment, the negative maximum near 243 to 247 nm and the positive maximum near 215 to 220 nm disappeared under hydrophilic conditions, but negative at 206 nm and 228 nm. The R value was about 0.6. This feature is very similar to the CD spectral pattern exhibited by peptides that form a-helical structures, suggesting that peptide 3 forms helical structures in amphiphilic environments (FIG. 2). FIG. 2 is a CD spectrum (peptide concentration: 0.1 mM) in 1% SDS in PBS buffer solution (pH = 7.4). Here, in FIG. 1 and FIG. 2, peptide 1 is (FAM-β-Ala- (L-Arg-L-Arg-Pro) 3- (Gly) 3 -NH 2 ... (FAM-β-Ala- (L-Arg-L-Arg-Pro NH2 ) 3- (Gly) 3 -NH 2 ... Formula 2 where peptide 3 is (FAM-β-Ala- (L-Arg -L-Arg-Pro Gu ) 3- (Gly) 3 -NH 2 ... Formula 3 and peptide R9 is an oligoarginine.
(3) Cell membrane permeability of peptide The cell membrane permeability of the peptide was measured from the intracellular fluorescence intensity using a flow cytometer. The results are shown in FIGS. 3 (A) and 3 (B). FIG. 3A shows the results of cell membrane permeability of each peptide in adherent cells (relative permeability when R9 is 1) (peptide concentration: 1 μM, culture at 37 ° C. for 2 hours). FIG. 3B shows the results of cell membrane permeability of each peptide in floating cells (relative permeability when R9 is 1) (peptide concentration: 1 μM, culture at 37 ° C. for 2 hours). As shown in FIGS. 3 (A) and 3 (B), peptide 3 according to this embodiment has high permeability at low concentrations for adherent cells (HeLa, A549, CHO-K1) and suspension cells (Jurkat). Indicated.
(4) Cargo molecular transport efficiency of peptides Luciferase assay using luciferase-encoded pDNA (Plasmid pCAcc + Luc, coding for firefly luciferase under the control of the CAG promoter, was provided by the RIKEN Gene Bank (Tsukuba, Japan)) Thus, the cargo molecule transport efficiency of the peptide was evaluated. The results are shown in FIG. FIG. 4 shows the delivery of plasmid DNA by each peptide (cultured at 37 ° C. for 24 hours). As shown in FIG. 4, the peptide according to this example achieved high pDNA transport efficiency in HeLa cells compared to oligoarginine.
　標的細胞における遺伝子機能の調査、疾患を処置できるカーゴ分子のスクリーニング、標的細胞の改変等に利用できる。 It can be used for investigating gene functions in target cells, screening for cargo molecules that can treat diseases, and modifying target cells.

Claims (5)

  1. 　細胞膜透過性を有する下記の式Ｘ
    F-(L-Arg-L-Arg-Xaa)m-(Gly)n-NH2・・・式Ｘ
    〔式中、
    ｍは２～４のいずれかの整数であり、
    ｎは０～３のいずれかの整数であり、
    Ｆは、リンカーを介して又は介さないで、ペプチドのＮ末端に結合した蛍光標識であり、
    Xaaは、下記の式Ａ、式Ｂ（ｎは１～５）、式Ｃ（ｎは１～５）、又は、式Ｄ（ｎは１～５）の何れかである
    Figure JPOXMLDOC01-appb-C000001
      
    Figure JPOXMLDOC01-appb-C000002
      
    Figure JPOXMLDOC01-appb-C000003
      
    Figure JPOXMLDOC01-appb-C000004
      
    〕で表されるペプチド。     Formula X below having cell membrane permeability
    F- (L-Arg-L-Arg-Xaa) m- (Gly) n -NH 2 Formula X
    [Where,
    m is an integer from 2 to 4,
    n is an integer from 0 to 3,
    F is a fluorescent label attached to the N-terminus of the peptide, with or without a linker,
    Xaa is any of the following formula A, formula B (n is 1 to 5), formula C (n is 1 to 5), or formula D (n is 1 to 5)
    Figure JPOXMLDOC01-appb-C000001
    Figure JPOXMLDOC01-appb-C000002
    Figure JPOXMLDOC01-appb-C000003
    Figure JPOXMLDOC01-appb-C000004
    ] The peptide represented by this.
  2. 　Ｆはフルオレセイン化合物である請求項１記載のペプチド。 2. The peptide according to claim 1, wherein F is a fluorescein compound.
  3. 　請求項１又は２に記載のペプチドと、細胞内に輸送すべきカーゴ分子とを含む構築物。 A construct comprising the peptide according to claim 1 or 2 and a cargo molecule to be transported into a cell.
  4. 　前記カーゴ分子は、核酸、タンパク質、薬剤、又は、ナノ粒子の何れかである請求項３記載の構築物。 The construct according to claim 3, wherein the cargo molecule is any one of a nucleic acid, a protein, a drug, and a nanoparticle.
  5. 　細胞内に輸送すべきカーゴ分子と、請求項１又は２に記載のペプチドとを結合させて構築物を得る工程と、
    　前記構築物を細胞に導入する工程と、を有する、
    カーゴ分子を細胞内に輸送する方法。     A step of binding a cargo molecule to be transported into a cell and the peptide according to claim 1 or 2 to obtain a construct;
    Introducing the construct into cells.
    A method of transporting cargo molecules into cells.
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