WO2014104227A1 - Peptides for transferring target molecules into cells - Google Patents

Peptides for transferring target molecules into cells Download PDF

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Publication number
WO2014104227A1
WO2014104227A1 PCT/JP2013/084963 JP2013084963W WO2014104227A1 WO 2014104227 A1 WO2014104227 A1 WO 2014104227A1 JP 2013084963 W JP2013084963 W JP 2013084963W WO 2014104227 A1 WO2014104227 A1 WO 2014104227A1
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Prior art keywords
peptide
target molecule
introduction
cell
seq
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PCT/JP2013/084963
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French (fr)
Japanese (ja)
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鈴木　康弘
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国立大学法人東北大学
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Priority to JP2014554564A priority Critical patent/JP6373762B2/en
Publication of WO2014104227A1 publication Critical patent/WO2014104227A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • the present invention relates to a target molecule intracellular introduction peptide used for introducing a desired molecule into a cell, a target molecule complex obtained by binding the target molecule intracellular introduction peptide and a target molecule, and the target molecule cell.
  • the present invention relates to a vector comprising a polynucleotide encoding a peptide for internal transfer.
  • Non-patent Documents 1, 2 When HIV-1 -derived trans-activator of transcription protein (TAT protein) is added to the culture solution, it has been transferred to the cell through the cell membrane (Non-patent Documents 1, 2). Later, it was revealed that the protein derived from flies and the structural protein of HSV-1 also have the property of passing through the cell membrane. The amino acid sequence necessary for passage through the membrane has been determined, and the domain consisting of 10 to 20 amino acids has come to be called Protein-transduction domain (PTD) / Cell-penetrating peptide (CPP).
  • PTD Protein-transduction domain
  • CPP Cell-penetrating peptide
  • PTD intracellular introduction efficiency is greatly influenced by the properties of the target protein itself and the polymer compound to be introduced, and in most cases, sufficient introduction efficiency cannot be obtained even in vitro, and cell introduction efficiency There is a problem that is unstable.
  • the problem to be solved by the present invention is to provide a target molecule cell-introducing peptide capable of introducing a target polymer compound and nanocarrier into cells with higher efficiency than conventional peptides.
  • the present inventor has found that the pH-dependent membrane fusion peptide portion of the peptide for introduction into a target molecule into which a pH-dependent membrane fusion peptide and a protein introduction domain (PTD) are fused is described. It has been found that the above-mentioned problems can be solved by setting the number of constituent amino acids to 21 amino acids or longer, which is longer than those conventionally used (20 amino acids at the longest). The present inventors further studied the amino acid sequence of the peptide, the number of peptides for introduction into cells to be bound to one target molecule, and the like based on the above-mentioned novel findings, and completed the present invention.
  • PTD protein introduction domain
  • Item 1 A target molecule intracellular introduction peptide having a pH-dependent membrane fusion peptide and a protein introduction domain (PTD), wherein the membrane fusion peptide has 21 or more constituent amino acids.
  • PTD protein introduction domain
  • the membrane fusion peptide has the sequence X 1 X 2 X 3 [Wherein X 1 , X 2 and X 3 are the same or different and represent W, H, G, F, Y, C, L, or T] Item 2.
  • Item 3 The target molecule intracellular introduction peptide according to Item 1, which is any of the following: (1) The amino acid sequence of the membrane fusion peptide is GLFGAIAGFIENGWEGMIDGWYGF (SEQ ID NO: 1), GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 2), GFFGAIAGFLEGGWEGMIAGWHGY (SEQ ID NO: 3), GFFGAIAGFLEGGWEGMIAGWHG (SEQ ID NO: 4), LAGVIMAGVAIGIGTIGTAGCTG 6), GTFTWTLSDSSGVENPGGYCLT (SEQ ID NO: 7), AFFSWSLTDSSGKDTPGGYCL (SEQ ID NO: 8), AFFSWSLTDSSGKDMPGGYCL (SEQ ID NO: 9), AFFSWSLSDPKGNDMPGGYCL (SEQ ID NO: 10) or GIFSWTITDAVGNDMPGGYCL (SEQ ID NO: 11) peptide for introduction into the target molecule cell
  • Item 4. The peptide for intracellular introduction of a target molecule according to any one of Items 1 to 3, which is any of the following: (1) The peptide for introduction into a target molecule, wherein the amino acid sequence of the PTD is YGRKKRRQRRR (SEQ ID NO: 12), RRRRRRRRRRR (SEQ ID NO: 13), or RQIKIWFQNRRMKWKK (SEQ ID NO: 14) (2) The amino acid sequence of the PTD is YGRKKRRQRRR (SEQ ID NO: 12), RRRRRRRRRRR (SEQ ID NO: 13) or RQIKIWFQNRRMKWKK (SEQ ID NO: 14) is an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and permeability through the cell membrane A peptide for introduction into the target molecule into the cell.
  • Item 5. A target molecule complex obtained by binding the target molecule intracellular introduction peptide according to any one of items 1 to 4 and a target molecule.
  • Item 6. The complex according to Item 5, wherein two or more target molecule intracellular introduction peptides are bound to one target molecule.
  • Item 7. A vector comprising a polynucleotide encoding the target molecule intracellular introduction peptide according to any one of items 1 to 4.
  • Item 8. A pharmaceutical composition comprising the complex according to item 6 or 7 as an active ingredient.
  • Item 9 A method for introducing a target molecule into a cell, comprising the step of adding the complex according to Item 6 or 7 to the cell.
  • Item 10 A target molecule intracellular introduction agent comprising the target molecule intracellular introduction peptide according to any one of Items 1 to 4.
  • Item 11 Use of the peptide for intracellular introduction of a target molecule according to any one of Items 1 to 4 for producing an agent for intracellularly introducing a target molecule.
  • a peptide for introducing a target molecule cell that can introduce a target substance into cells with higher efficiency than conventional peptides for introducing a target molecule cell.
  • FIG. 1 shows a schematic diagram of cell membrane permeation by the complex of the present invention.
  • the target molecule intracellular introduction peptide is simply indicated as PTD.
  • A Shows the number of quantum dots taken up per cell 45 minutes after adding 30 pM of a peptide loaded with a target molecule into the cell at various valences on the quantum dot in the present invention.
  • B Quantum taken up per cell 45 minutes after adding 30 pM of the known bivalent PTD and the target molecule intracellular introduction peptide (20 valence) of the present invention loaded on the quantum dot onto the cell Indicates the number of dots.
  • the amino acid sequence of the peptide of the present invention includes both the N-terminal on the left side and the C-terminal on the left side.
  • the sequence “GLFGAIAGFIENGWEGMIDGWYGF” represents N-GLFGAIAGFIENGWEGMIDGWYGF-C or N-FGYWGDIMGEWGNEIFGAIAGFLG-C
  • the sequence “YGRKKRRQRRR” represents N-YGRKKRRQRRR-C or N-RRRQRRKKRGY-C
  • the sequence “GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR” represents N-GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR-C or N-YGRKKRRQRRRFGYWGDIMGEWGNEIFGAIAGFLG-C.
  • the present invention relates to a peptide for introduction into a target molecule cell having a pH-dependent membrane fusion peptide and a protein introduction domain (PTD), and the membrane fusion peptide has 21 or more constituent amino acids.
  • PTD protein introduction domain
  • the protein transduction domain is a peptide having a function of causing local stimulation after adsorption to a cell membrane surface receptor and causing the target molecule intracellular transduction peptide to be taken into the cell by endocytosis and an equivalent function
  • the amino acid sequence is not particularly limited, and examples thereof include YGRKKRRQRRR, RRRRRRRRR, RQIKIWFQNRRMKWKK, QWTLNSAGYLLGKINLKALAALAKKIL, KLALKLALKALKAALKLA, MVTVLFRRLRIRRACGPPRVRV, and the like.
  • PTD includes YGRKKRRQRRR, RRRRRRRRRRR, RQIKIWFQNRRMKWKK, QWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 15), KLALKLALKALKAALKLA (SEQ ID NO: 16), or the number of amino acids represented by MVTVLFRRLRIRRACGPPRVRV (SEQ ID NO: 17).
  • amino acid sequence having an amino acid deleted, substituted or added for example, YGRKKRRQRRRPPQ (SEQ ID NO: 18) in which PPQ is added to the end of YGRKKRRQRRR), and GRKKRRQRRRPPQ (SEQ ID NO: 19 in which Y is deleted from the opposite end) ), RRRRRRRRR (SEQ ID NO: 20) in which RR is deleted from RRRRRRRRR, etc.) are also included.
  • the range of “one or more” is not particularly limited as long as the peptide for introducing into a target molecule, which is a combination of the PTD and a pH-dependent membrane fusion peptide, has cell membrane permeability.
  • the number is 15, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 4, particularly preferably 1 to 3, and still more preferably 1 or 2.
  • Techniques for deleting, substituting and / or adding one or more amino acids in a specific amino acid sequence are known.
  • the peptide thus deleted, substituted and / or added is composed of an amino acid sequence having 50% or more identity to the amino acid sequence before deletion, substitution and addition, and PTD.
  • the peptide having a cell membrane permeability as a peptide for introduction into a target molecule into which a pH-dependent membrane fusion peptide is combined.
  • the identity of amino acids is usually 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more, and still more preferably 98%. That's it.
  • cell membrane permeability refers to the property of not only being taken into endosomes by endocytosis but also having a small hole in the endosomal membrane to reach the cytoplasm.
  • the pH-dependent membrane fusion peptide refers to a peptide that is inserted into the endosome and then inserted into the endosome membrane, and whose secondary structure changes with pH. More specifically, a peptide whose steric structure changes under neutral conditions and an acidic region such as pH 5 or lower (for example, pH 4 or lower or pH 3 or lower) is shown.
  • pH-dependent membrane fusion peptides include the sequences X 1 X 2 X 3 [wherein X 1 , X 2 and X 3 are the same or different, and W, H, G, F, Y, A, C] , L, or T (preferably W, H, G, F, or Y)] The thing which has is mentioned.
  • the position of the sequence X 1 X 2 X 3 is not particularly limited, but the terminal on the side that binds to the PTD is preferable.
  • Examples of the array X 1 X 2 X 3 include YGF, WYG, HGY, WHG, ALY, CLT, and YCL.
  • pH-dependent membrane fusion peptides examples include all amino acid sequences of membrane fusion peptides derived from various viruses (eg, influenza A, influenza B, influenza C, etc.) and having pH dependency. Although a part can be used, it is important that the pH-dependent membrane fusion peptide has 21 or more amino acids, preferably 23 or more.
  • the upper limit of the constituent amino acids is not particularly limited, but is preferably 26 or less, and more preferably 24 or less.
  • the amino acid sequence of such pH dependent membrane fusion peptide but not particularly limited as long as it has a number of the constituent amino acids, for example, GLFGAIAGFIENGWEGMIDGWYGF, GLFGAIAGFIENGWEGMIDGWYG, GFFGAIAGFLEGGWEGMIAGWHGY, GFFGAIAGFLEGGWEGMIAGWHG, LAGVIMAGVAIGIATAAQITAGVALY, GTFTWTLSDSEGKDTPGGYCLT, GTFTWTLSDSSGVENPGGYCLT, AFFSWSLTDSSGKDTPGGYCL, AFFSWSLTDSSGKDMPGGYCL, AFFSWSLSDPKGNDMPGGYCL, GIFSWTITDAVGNDMPGGYCL, etc.
  • the pH dependent membrane fusion peptide the GLFGAIAGFIENGWEGMIDGWYGF, GLFGAIAGFIENGWEGMIDGWYG, GFFGAIAGFLEGGWEGMIAGWHGY, GFFGAIAGFLEGGWEGMIAGWHG, AGVIMAGVAIGIATAAQITAGVALY, GTFTWTLSDSEGKDTPGGYCLT, GTFTWTLSDSSGVENPGGYCLT, AFFSWSLTDSSGKDTPGGYCL, AFFSWSLTDSSGKDMPGGYCL, 1 or several amino acids in the amino acid sequence shown in AFFSWSLSDPKGNDMPGGYCL or GIFSWTITDAVGNDMPGGYCL In which amino acid sequences are deleted, substituted or added.
  • the range of “one or more” is not particularly limited as long as the peptide for introduction into a target molecule cell in which PTD is combined with the pH-dependent membrane fusion peptide has cell membrane permeability. Is, for example, 1 to 15, preferably 1 to 10, more preferably 1 to 7, and still more preferably 1 to 5. Techniques for deleting, substituting and / or adding one or more amino acids in a specific amino acid sequence are known.
  • the peptide thus deleted, substituted and / or added consists of an amino acid sequence having 50% or more identity to the amino acid sequence before deletion, substitution and addition, and Examples are peptides in which a peptide for introduction into a target molecule into which a PTD is combined with a pH-dependent membrane fusion peptide has cell membrane permeability.
  • the identity of amino acids is usually 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more, and still more preferably 98%. That's it.
  • GLFX 4 AIAX 5 FIEX 6 GWEGX 7 IX 8 GWYG SEQ ID NO: 21
  • X 4 represents D, E, G or N.
  • X 5 represents D, E, G or N.
  • X 6 represents D, E, G or N.
  • X 7 represents M or L.
  • X 8 represents D, E, G or N.
  • the combination of (X 4 , X 5 , X 6 , X 7 , X 8 ) (G, G, N, M, D) is excluded.
  • the like the like.
  • a sequence in which four of the sequence GLFGAIAGFIENGWEGMIDGWYG are substituted for example, a peptide consisting of GLF E AIA E FIE G GWEG L I E GWYG (underlined is a substituted portion, SEQ ID NO: 22) and the like can be mentioned.
  • the pH-dependent membrane fusion peptide and the PTD may be bonded directly or via a linker.
  • a linker what is used in the said field
  • the peptide for introduction into a target molecule of the present invention has a domain for binding a target substance, specifically, for example, a DNA binding domain (zinc finger domain, HMG-box, etc.), a double-stranded RNA binding domain ( Histone, RDE-4 protein, double-stranded RNA binding domain derived from protamine, etc., various labeled tag protein recognition compounds (anti-Myc binding protein (for Myc-tag)), Halotag ligand (Halotag protein binding molecule), nickel molecule (His-tag protein binding molecule) etc.) may be present at the end of the PTD side among the end of the fusion peptide and the end of the PTD.
  • a DNA binding domain zinc finger domain, HMG-box, etc.
  • Histone, RDE-4 protein double-stranded RNA binding domain derived from protamine, etc.
  • various labeled tag protein recognition compounds anti-Myc binding protein (for Myc-tag)
  • Halotag ligand Halotag protein
  • examples of the peptide for introduction into a target molecule cell of the present invention include the following: N-GLFGAIAGFIENGWEGMIDGWYGF YGRKKRRQRRR- C (Peptide 1, SEQ ID NO: 23) N-GLFGAIAGFIENGWEGMIDGWYGF RRRQRRKKRGY- C (Peptide 2, SEQ ID NO: 24) N- YGRKKRRQRRR FGYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 3, SEQ ID NO: 25) N- RRRQRRKKRGY FGYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 4, SEQ ID NO: 26) N-GFFGAIAGFLEGGWEGMIAGWHGY YGRKKRRQRRR- C (peptide 5, SEQ ID NO: 27) N-GFFGAIAGFLEGGWEGMIAGWHGY RRRQRRKKRGY- C (Peptide 6, SEQ ID NO: 28) N- YGRKKRRQRRQRR
  • the peptide for introduction into a target molecule cell of the present invention can be prepared by a conventionally known genetic engineering method, chemical synthesis method or the like.
  • a desired peptide may be obtained after inserting a polynucleotide encoding the aforementioned peptide for introduction into a target molecule into a vector and culturing a transformant in which the vector is incorporated.
  • the peptide for introduction into a target molecule of the present invention may be obtained by isolating and purifying from a microorganism transformed to have the ability to produce the peptide.
  • the target molecule intracellular introduction peptide of the present invention may be synthesized by a conventionally known chemical synthesis method in accordance with the information of the amino acid sequence of the target molecule intracellular introduction peptide or the nucleotide sequence encoding the peptide.
  • the chemical synthesis method includes a peptide synthesis method using a liquid phase method or a solid phase method.
  • the peptide for introducing a target molecule into the cell of the present invention is produced by the above method by binding a pH-dependent membrane fusion peptide and PTD (or further binding a domain for binding a target substance).
  • the pH-dependent membrane fusion peptide and PTD may be synthesized by the above method and then bound to each other.
  • the present invention provides a target molecule complex formed by binding the above-described target molecule intracellular introduction peptide and a target molecule.
  • the binding mode of the target molecule and the peptide for introduction into the target molecule into the cell is not particularly limited.
  • the target molecule may be bound through the above-described binding domain, may be cross-linked by —SS—bonding, etc.
  • the avidin system may be used, may be electrostatically bonded, or may be chemically modified and bonded.
  • the number of target molecule intracellular introduction peptides to be bound to one target molecule is not particularly limited, but 2 or more (eg, 3 or more, more preferably 8 or more) target molecule intracellular introduction peptides are target molecules. It is preferable from the viewpoint of increasing the efficiency of cell surface receptor binding and activating the cell surface receptor uptake mechanism.
  • the upper limit of the number of the target molecule-introducing peptide to be bound to one target molecule is not particularly limited, but is preferably 30 or less, and more preferably 20 or less.
  • the target substance is not particularly limited and may be either a polymer or a low molecule.
  • polymer compounds include nucleic acid molecules such as DNA and RNA (siRNA, shRNA, etc.); peptides such as oligopeptides and proteins (antibodies, antibody fragments, enzymes, cytokines, chemokines, receptor polypeptides, etc.), sugars, etc. Examples include chains.
  • small molecules include antibiotics, anticancer agents, anti-inflammatory agents, liposomes, micelles, dendrimers, nanotubes, nanocarriers such as amino acid nanoparticles, quantum dots, fluorescent dyes, intracellular molecular visualization reagents, nanomagnetic materials, Examples thereof include compounds that can visualize target cell dynamics in vivo, such as nanogold.
  • a complex of the present invention containing the transcription factor as a target molecule and administer this to a subject to treat the cancer.
  • an antigen when introduced into an antigen-presenting cell using the method of the present invention, the antigen is presented to MHC-class I and cytotoxic T cells are activated. Therefore, application to vaccines against many viral infections and cancers that require the induction of cytotoxic T cells is conceivable.
  • a substance for example, antibody, antibody fragment (scFv (Single-chain variable fragment), etc.) that specifically binds to a specific antigen is bound to the peptide and complex for introduction into the target molecule of the present invention.
  • a target molecule can be specifically introduced into a specific cell in a sample or subject containing various cells.
  • the complex of the present invention can be applied to a conditional knockout animal. Specifically, for example, in a flox mouse having a gene locus in which a target gene region is sandwiched between Cre recombinase target sequences loxP, the target molecule intracellular introduction peptide of the present invention and Cre recombinase or a gene encoding the same are added. A method of administering the bound complex is exemplified.
  • Cre recombinase or a gene encoding it can be introduced into somatic cells of flox mice, and the target gene can be deleted by the recombinase. Therefore, this embodiment is useful because a target gene region can be deleted at a desired timing.
  • deletion of a target gene region can be caused at a desired timing and at a desired site.
  • the complex of the present invention can also be used to produce iPS cells.
  • the OCT3 / 4 / SOX2 / NANOG / LIN28 4 gene expression vector or 4 protein or the like is used as a target molecule, and the complex of the present invention is added to cells in vitro, or By directly administering to a subject animal, a gene as a target molecule is introduced into the cell, and an iPS cell can be established.
  • a reaction substrate that reacts to changes in the environment outside the cell inside the cell, for example, a decrease in pH after incorporation into the endosome changes its structure in response to pH reactivity, and the target substance is introduced from the introduced peptide.
  • the method includes separation from the target substance in the cell using, for example, pH-reactive para-nitrophenyl (pNP) -PEG-PE, SS bond between cysteine and cysteine, and the like.
  • the present invention also provides a vector comprising a polynucleotide encoding a peptide for introduction into a target molecule into a cell.
  • a vector comprising a polynucleotide encoding a peptide for introduction into a target molecule into a cell.
  • any vector such as a plasmid vector, an adenovirus vector, or a retrovirus vector can be used.
  • a method for cloning a nucleotide sequence containing a polynucleotide encoding a peptide for introduction into a target molecule into a cell a method known per se can be used.
  • expression control signals transcription initiation and translation initiation signals
  • Etc. can be designed so that the gene can be self-expressed in microbial cells depending on the host microorganism.
  • the vector of the present invention includes not only those encoding the target molecule intracellular introduction peptide but also those encoding a complex in which the target molecule intracellular introduction peptide and the target substance are bound.
  • the target molecule can be produced by covalently binding the target molecule-introducing peptide and the target substance by gene expression.
  • compositions comprising the complex as an active ingredient.
  • the complex as the active ingredient can be used alone, but depending on the route of administration, it should be formulated into a suitable dosage form using a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be administered to a subject in various administration routes, specifically oral or parenteral administration (eg, intravenous injection, intramuscular injection, nasal mucosal administration, oral mucosal administration, transdermal Administration, subcutaneous administration, intradermal administration, rectal administration, etc.).
  • parenteral administration such as intravenous injection, intramuscular injection, or intranasal mucosal administration is used.
  • the subject include mammals (human or non-human mammal), and examples of the non-human mammal include mice, rats, rabbits, dogs, monkeys, and the like.
  • Preferred examples of the dosage form include parenteral preparations such as injections (including drops), ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions and the like.
  • parenteral preparations such as injections (including drops), ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions and the like.
  • oral preparations include tablets, powders, fine granules, granules, coated tablets, capsules, syrups, and lozenges.
  • Carriers that can be used to formulate these preparations include, for example, excipients, binders, disintegrants, lubricants, colorants, and flavoring agents that are commonly used in the pharmaceutical field, and, if necessary, Stabilizer, emulsifier, absorption promoter, surfactant, pH adjuster, preservative, antioxidant, extender, wetting agent, surface active agent, dispersant, buffer, preservative, solubilizer, Examples include soothing agents.
  • the present invention provides a method for introducing a target molecule into a cell, comprising the step of adding the complex to a cell.
  • the complex may be added to cells either in vitro or in vivo.
  • PTD binds to heparan sulfate proteoglycan etc. on the membrane surface near the cell membrane surface to cause local stimulation, and the target molecule intracellular introduction peptide is taken into the cell by endocytosis (FIG. 1). ). Then, a pH-dependent membrane fusion peptide is inserted into the endosome membrane. Thereafter, the pH in the endosome rapidly changes to acidic with time. Along with the change in pH, the three-dimensional structure of the pH-dependent membrane fusion peptide is changed while it is inserted into the endosomal membrane, and is bent to form small pores in the endosomal membrane. As a result, the peptide for introduction into the target molecule into the cell is released into the cytoplasm through the endosome membrane.
  • Target molecule intracellular introduction agent comprising the aforementioned target molecule intracellular introduction peptide.
  • the target molecule intracellular introduction peptide itself may be used as the target molecule intracellular introduction agent, or the target molecule intracellular introduction peptide is mixed with the above-described carrier or the like to prepare a formulation. May be.
  • Example 1 A peptide consisting of the following amino acid sequence was synthesized by Fmoc (N- (9-fluorenyl) methoxycarbonyl) solid phase synthesis method by requesting Toray Industries, Inc .: N-GLFGAIAGFIENGWEGMIDGWYGF YGRKKRRQRRR -C Peptide 1 The synthesized peptide was purified to 95% or more by reverse phase HPLC with an acetonitrile / H 2 O / trifluoroacetic acid gradient (molecular weight 4149.7).
  • Examples 2 to 6 A peptide was synthesized in the same manner as in Example 1 except that a peptide consisting of the following amino acid sequence was synthesized: N- YGRKKRRQRRR FGYWGDIMGEWGNEIFGAIAGFLG-C (peptide 3, molecular weight 4149.7) N- RRRQRRKKRGY YGHWGAIMGEWGGELFGAIAGFFG-C (peptide 8, molecular weight 4072.6) N- YGRKKRRQRRR GYWGDIMGEWGNEIFGAIAGFLG-C (peptide 11, molecular weight 4002.6) N- RRRRRRRRRRRR GYWGEILGEWGGEIFEAIAEFLG-C (peptide 26, molecular weight 4261.9) N- YGRKKRRQRRR GYWGEILGEWGGEIFEAIAEFLG-C (peptide 29, molecular weight 4085.6) In the above sequence, the underlined portion indicates PTD, and the other portion is a pH-dependent membrane
  • Example 1 Further, in the above Fmoc solid phase synthesis method, N N was biotinylated in the same manner as in Example 1 except that the PTD side was biotinylated using a biotinylated amino acid for the extension of the last amino acid (N-terminal G in peptide 1). A peptide biotinylated at the end was synthesized. The obtained compound is defined as Compound 1 (molecular weight 4394.1).
  • Example 2'-6 ' In the Fmoc solid phase synthesis method, the PTD side was biotinylated by using a biotinylated amino acid for the extension of the last amino acid (N-terminal G in peptide 1) (compounds 3, 8, 11, 26).
  • the molecular weight of each compound is as follows: Compound 3, molecular weight 4394.1 Compound 8, molecular weight 4316.9 Compound 11, molecular weight 4246.9 Compound 26, molecular weight 4329.9
  • a peptide having the following amino acid sequence and biotinylated at the N-terminus was also synthesized in the same manner (Compound 33).
  • N-GYWGEILGEWGGEIFEAIAEFLGRRRRRRRRR-C (peptide 33, molecular weight 4506.2) Comparative Examples 1 and 2
  • a biotinylated peptide was synthesized in the same manner as Example 1 ′ except that a peptide consisting of the following amino acid sequence was synthesized (referred to as compounds 31 to 32, respectively): N-RRRQRRKKRGYGDIMGEWGNEIFGAIAGFLG-C (Peptide 31) N-YGRKKRRQRRR-C (peptide 32)
  • Examples 31 and 32 and Comparative Examples 3 and 4 Reaction of above-mentioned compounds 11 and 26 with various molar ratios up to 1,2,4,8,12,16,20 to 1 ⁇ mol of commercially available streptavidin fluorescent quantum dots (QD) (QD655, invitrogen; diameter 30 nm) Then, various numbers of peptides for introduction into the target molecule were polymerized on QD (this is expressed as valence, and
  • complexes 11-1 to 11-20 and 26-1 to 26-20 are referred to as complexes 11-1 to 11-20 and 26-1 to 26-20, respectively.
  • HeLa cells were used as living cells, and the HeLa cells were cultured in a cell culture medium X-VIVO buffer.
  • Complexes 11-1 to 11-20, 26-1 to 26-20, and 50 pM were added to the cells at the same time as endosome staining reagent 0.25% DiO. After 1 hour, the cells were washed with the culture medium, and then 2 hours. After culture, reference 1.Duan H, Nie S. J Am Chem Soc. (2007) Vol. 129,3333-8.
  • composites 31 and 32 were prepared in the same manner as in Example 31 except that compounds 31 and 32 synthesized in 1 and 2 were used as comparative examples, and the residual ratio of QD particles to endosomes was measured.
  • complex 32 derived from HIV-1 Tat
  • H-PTD complex 31
  • a control (20, which has the highest efficiency in the same study)
  • the peptide for introducing a target molecule into the cell of the present invention it is possible not only to introduce the target molecule in the form of being encapsulated in the endosome but also to introduce a small pore in the endosome into the cytoplasm. Therefore, it is very useful for studying the function of the target molecule in the cytoplasm.
  • the action point of many drugs since the action point of many drugs is considered to be in the cytoplasm, it is very important from the viewpoint of application to a drug delivery system.
  • Examples of the use of the peptide for intracellular introduction of a target molecule of the present invention include introduction of an anti-apoptotic peptide from a coronary artery against a myocardial ischemic damage caused by acute ischemic heart disease and directly introducing it into the cell. For use in protecting the myocardium.
  • Target molecule Peptide SEQ ID No. 48 into introduction into cells
  • Target molecule Peptide sequence number 49 for introduction into cell Peptide sequence number 50 for introduction into target molecule cell Peptide sequence number 51 for introduction into target molecule cell Peptide sequence number 52 for introduction into target molecule cell Peptide for introduction into target molecule cell Plastid

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Abstract

The purpose of the present is to provide peptides for transferring target molecules into cells, said peptides enabling target polymer compounds and nanocarriers to be transferred into cells with greater efficiency than peptides of the prior art. The peptides for transferring target molecules into cells include pH-dependent membrane fusion peptides and protein transduction domains (PTDs). The number of amino acids constituting the membrane fusion peptides is not less than 21.

Description

ターゲット分子細胞内導入用ペプチドPeptides for intracellular introduction of target molecules
　［関連出願の相互参照］
　本出願は、２０１２年１２月２８日に出願された、日本国特許出願第2012-287128号明細書（その開示全体が参照により本明細書中に援用される）に基づく優先権を主張する。 [Cross-reference of related applications]
This application claims priority based on Japanese Patent Application No. 2012-287128 filed on Dec. 28, 2012, the entire disclosure of which is incorporated herein by reference.
　本発明は、所望の分子を細胞内に導入するために用いるターゲット分子細胞内導入用ペプチド、当該ターゲット分子細胞内導入用ペプチドとターゲット分子とを結合させてなるターゲット分子複合体及び当該ターゲット分子細胞内導入用ペプチドをコードするポリヌクレオチドを含むベクターに関する。 The present invention relates to a target molecule intracellular introduction peptide used for introducing a desired molecule into a cell, a target molecule complex obtained by binding the target molecule intracellular introduction peptide and a target molecule, and the target molecule cell. The present invention relates to a vector comprising a polynucleotide encoding a peptide for internal transfer.
　高分子化合物、ＤＮＡ、ＲＮＡ、タンパク質等の分子の生体内での機能解析、またドラックデリバリーシステムの構築のため、目的の物質を細胞内に導入することは非常に重要である。 In order to analyze the function of molecules such as polymer compounds, DNA, RNA, and proteins in vivo and to construct a drug delivery system, it is very important to introduce a target substance into cells.
　従来、HIV-1 由来Trans-activator of transcription protein (TAT蛋白質)を培養液中に添加すると細胞膜を通過し細胞内に移行することがしられている（非特許文献１, 2）。その後ハエ由来の蛋白質やHSV-1の構造タンパク質なども細胞膜を通過する性質を有することが明らかとなった。膜通過のために必要なアミノ酸配列が決定され，その10から20個のアミノ酸からなるドメインはProtein Transduction domain（PTD）/Cell penetrating peptide (CPP) と呼ばれるようになった。 Conventionally, when HIV-1 -derived trans-activator of transcription protein (TAT protein) is added to the culture solution, it has been transferred to the cell through the cell membrane (Non-patent Documents 1, 2). Later, it was revealed that the protein derived from flies and the structural protein of HSV-1 also have the property of passing through the cell membrane. The amino acid sequence necessary for passage through the membrane has been determined, and the domain consisting of 10 to 20 amino acids has come to be called Protein-transduction domain (PTD) / Cell-penetrating peptide (CPP).
　しかし、PTDの細胞内導入効率が導入しようとする目的の蛋白質自体の性質や高分子化合物の性質に大きく影響を受け、ほとんどの場合、in vitroでも十分な導入効率が得られず、細胞導入効率が不安定であるという問題がある。 However, PTD intracellular introduction efficiency is greatly influenced by the properties of the target protein itself and the polymer compound to be introduced, and in most cases, sufficient introduction efficiency cannot be obtained even in vitro, and cell introduction efficiency There is a problem that is unstable.
　本発明が解決しようとする課題は、従来のペプチドよりも高い効率で目的とする高分子化合物及びナノキャリアを細胞内に導入することができるターゲット分子細胞導入用ペプチドを提供することである。 The problem to be solved by the present invention is to provide a target molecule cell-introducing peptide capable of introducing a target polymer compound and nanocarrier into cells with higher efficiency than conventional peptides.
　本発明者は、上記の状況の下、鋭意研究した結果、ｐＨ依存性膜融合ペプチドと蛋白質導入ドメイン（PTD）を融合させたターゲット分子細胞内導入用ペプチドにおいて、ｐＨ依存性膜融合ペプチド部分の構成アミノ酸数を従来用いられていたもの（最長で２０アミノ酸）よりも長い２１アミノ酸以上とすることにより、上記課題を解決できることを見出した。本発明者らはさらに、上記新規の知見に基づき、ペプチドのアミノ酸配列、ターゲット分子１分子に対し結合させる細胞内導入用ペプチドの数等を検討し、本発明を完成させた。 As a result of diligent research under the above circumstances, the present inventor has found that the pH-dependent membrane fusion peptide portion of the peptide for introduction into a target molecule into which a pH-dependent membrane fusion peptide and a protein introduction domain (PTD) are fused is described. It has been found that the above-mentioned problems can be solved by setting the number of constituent amino acids to 21 amino acids or longer, which is longer than those conventionally used (20 amino acids at the longest). The present inventors further studied the amino acid sequence of the peptide, the number of peptides for introduction into cells to be bound to one target molecule, and the like based on the above-mentioned novel findings, and completed the present invention.
　従って、本発明は、以下の項を提供する：
　項１．ｐＨ依存性膜融合ペプチド及び蛋白質導入ドメイン（PTD）を有するターゲット分子細胞内導入用ペプチドであって、該膜融合ペプチドの構成アミノ酸数が２１以上である、ターゲット分子細胞内導入用ペプチド。 Accordingly, the present invention provides the following sections:
Item 1. A target molecule intracellular introduction peptide having a pH-dependent membrane fusion peptide and a protein introduction domain (PTD), wherein the membrane fusion peptide has 21 or more constituent amino acids.
　項２．前記膜融合ペプチドが配列Ｘ１Ｘ２Ｘ３
［式中、Ｘ１、Ｘ２及びＸ３は同一又は異なって、Ｗ、Ｈ、Ｇ、Ｆ、Ｙ、Ｃ、Ｌ、又はＴを示す］
を有する、項１に記載のターゲット分子細胞内導入用ペプチド。 Item 2. The membrane fusion peptide has the sequence X 1 X 2 X 3
[Wherein X 1 , X 2 and X 3 are the same or different and represent W, H, G, F, Y, C, L, or T]
Item 2. The peptide for intracellular introduction of a target molecule according to Item 1, which has
　項３．下記のいずれかである項１に記載のターゲット分子細胞内導入用ペプチド：
（１）前記膜融合ペプチドのアミノ酸配列がGLFGAIAGFIENGWEGMIDGWYGF（配列番号１）、GLFGAIAGFIENGWEGMIDGWYG（配列番号２）、GFFGAIAGFLEGGWEGMIAGWHGY（配列番号３）、GFFGAIAGFLEGGWEGMIAGWHG（配列番号４）、LAGVIMAGVAIGIATAAQITAGVALY（配列番号５）、GTFTWTLSDSEGKDTPGGYCLT（配列番号６）、GTFTWTLSDSSGVENPGGYCLT（配列番号７）、AFFSWSLTDSSGKDTPGGYCL（配列番号８）、AFFSWSLTDSSGKDMPGGYCL（配列番号９）、AFFSWSLSDPKGNDMPGGYCL（配列番号１０）もしくはGIFSWTITDAVGNDMPGGYCL （配列番号１１）であるターゲット分子細胞内導入用ペプチド
（２）前記膜融合ペプチドのアミノ酸配列がGLFGAIAGFIENGWEGMIDGWYGF（配列番号１）、GLFGAIAGFIENGWEGMIDGWYG（配列番号２）、GFFGAIAGFLEGGWEGMIAGWHGY（配列番号３）、GFFGAIAGFLEGGWEGMIAGWHG（配列番号４）、LAGVIMAGVAIGIATAAQITAGVALY（配列番号５）、GTFTWTLSDSEGKDTPGGYCLT（配列番号６）、GTFTWTLSDSSGVENPGGYCLT（配列番号７）、AFFSWSLTDSSGKDTPGGYCL（配列番号８）、AFFSWSLTDSSGKDMPGGYCL（配列番号９）、AFFSWSLSDPKGNDMPGGYCL（配列番号１０）もしくはGIFSWTITDAVGNDMPGGYCL（配列番号１１）で示されるアミノ酸配列において１個又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列であり、かつ細胞膜透過性を有するターゲット分子細胞内導入用ペプチド。 Item 3. Item 1. The target molecule intracellular introduction peptide according to Item 1, which is any of the following:
(1) The amino acid sequence of the membrane fusion peptide is GLFGAIAGFIENGWEGMIDGWYGF (SEQ ID NO: 1), GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 2), GFFGAIAGFLEGGWEGMIAGWHGY (SEQ ID NO: 3), GFFGAIAGFLEGGWEGMIAGWHG (SEQ ID NO: 4), LAGVIMAGVAIGIGTIGTAGCTG 6), GTFTWTLSDSSGVENPGGYCLT (SEQ ID NO: 7), AFFSWSLTDSSGKDTPGGYCL (SEQ ID NO: 8), AFFSWSLTDSSGKDMPGGYCL (SEQ ID NO: 9), AFFSWSLSDPKGNDMPGGYCL (SEQ ID NO: 10) or GIFSWTITDAVGNDMPGGYCL (SEQ ID NO: 11) peptide for introduction into the target molecule cell (SEQ ID NO: 11) The amino acid sequence of the membrane fusion peptide is GLFGAIAGFIENGWEGMIDGWYGF (SEQ ID NO: 1), GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 2), GFFGAIAGFLEGGWEGMIAGWHGY (SEQ ID NO: 3), GFFGAIAGFLEGGWEGMIAGDHGFTGT (SEQ ID NO: 4), LAGVIMAGVAIGIGTGTFTTP One or several amino acids are deleted in the amino acid sequence represented by SSGVENPGGYCLT (SEQ ID NO: 7), AFFSWSLTDSSGKDTPGGYCL (SEQ ID NO: 8), AFFSWSLTDSSGKDMPGGYCL (SEQ ID NO: 9), AFFSWSLSDPKGNDMPGGYCL (SEQ ID NO: 10) or GIFSWTITDAVGNDMPGGYCL (SEQ ID NO: 11) A peptide for introduction into a target molecule, which is a substituted or added amino acid sequence and has cell membrane permeability.
　項４．　下記のいずれかである項１～３のいずれか１項に記載のターゲット分子細胞内導入用ペプチド：
（１）前記PTDのアミノ酸配列が、YGRKKRRQRRR（配列番号１２）、RRRRRRRRRRR（配列番号１３）、もしくはRQIKIWFQNRRMKWKK（配列番号１４）であるターゲット分子細胞内導入用ペプチド
（２）前記PTDのアミノ酸配列がYGRKKRRQRRR（配列番号１２）、RRRRRRRRRRR（配列番号１３）もしくはRQIKIWFQNRRMKWKK（配列番号１４）で示されるアミノ酸配列において１個又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列であり、かつ細胞膜透過性を有する該ターゲット分子細胞内導入用ペプチド。 Item 4. Item 4. The peptide for intracellular introduction of a target molecule according to any one of Items 1 to 3, which is any of the following:
(1) The peptide for introduction into a target molecule, wherein the amino acid sequence of the PTD is YGRKKRRQRRR (SEQ ID NO: 12), RRRRRRRRRRR (SEQ ID NO: 13), or RQIKIWFQNRRMKWKK (SEQ ID NO: 14) (2) The amino acid sequence of the PTD is YGRKKRRQRRR (SEQ ID NO: 12), RRRRRRRRRRR (SEQ ID NO: 13) or RQIKIWFQNRRMKWKK (SEQ ID NO: 14) is an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and permeability through the cell membrane A peptide for introduction into the target molecule into the cell.
　項５．上記項１～４のいずれか１項に記載のターゲット分子細胞内導入用ペプチドとターゲット分子とを結合させてなるターゲット分子複合体。 Item 5. 5. A target molecule complex obtained by binding the target molecule intracellular introduction peptide according to any one of items 1 to 4 and a target molecule.
　項６．ターゲット分子１個に対し、ターゲット分子細胞内導入用ペプチドが２個以上結合されている、項５に記載の複合体。 Item 6. Item 6. The complex according to Item 5, wherein two or more target molecule intracellular introduction peptides are bound to one target molecule.
　項７．前記項１～４のいずれか１項に記載のターゲット分子細胞内導入用ペプチドをコードするポリヌクレオチドを含むベクター。 Item 7. 5. A vector comprising a polynucleotide encoding the target molecule intracellular introduction peptide according to any one of items 1 to 4.
　項８．前記項６又は７に記載の複合体を有効成分として含む医薬組成物。 Item 8. 8. A pharmaceutical composition comprising the complex according to item 6 or 7 as an active ingredient.
　項９．細胞に前記項６又は７に記載の複合体を添加する工程を含む、ターゲット分子を細胞内に導入する方法。 Item 9. Item 8. A method for introducing a target molecule into a cell, comprising the step of adding the complex according to Item 6 or 7 to the cell.
　項１０．項１～４のいずれか１項に記載のターゲット分子細胞内導入用ペプチドを含む、ターゲット分子の細胞内導入剤。 Item 10. Item 5. A target molecule intracellular introduction agent comprising the target molecule intracellular introduction peptide according to any one of Items 1 to 4.
　項１１．ターゲット分子の細胞内導入剤を製造するための、項１～４のいずれか１項に記載のターゲット分子細胞内導入用ペプチドの使用。 Item 11. Item 5. Use of the peptide for intracellular introduction of a target molecule according to any one of Items 1 to 4 for producing an agent for intracellularly introducing a target molecule.
　本発明によれば、従来のターゲット分子細胞導入用ペプチドよりも高い効率で目的とする物質を細胞内に導入することができるターゲット分子細胞導入用ペプチドを提供することができる。 According to the present invention, it is possible to provide a peptide for introducing a target molecule cell that can introduce a target substance into cells with higher efficiency than conventional peptides for introducing a target molecule cell.
本発明の複合体による細胞膜透過の概略図を示す。尚、図１においては、ターゲット分子細胞内導入用ペプチドを単にPTDと示す。1 shows a schematic diagram of cell membrane permeation by the complex of the present invention. In FIG. 1, the target molecule intracellular introduction peptide is simply indicated as PTD. （A）本発明におけるターゲット分子細胞内導入用ペプチドを様々な価数で量子ドット上に負荷したものを細胞上に30pM添加して４５分後に一細胞あたりに取り込まれた量子ドットの数を示す。（B）公知の2価PTD と本発明におけるターゲット分子細胞内導入用ペプチド(20価)の量子ドット上に負荷したものを細胞上に30pM添加して４５分後に一細胞あたりに取り込まれた量子ドットの数を示す。(A) Shows the number of quantum dots taken up per cell 45 minutes after adding 30 pM of a peptide loaded with a target molecule into the cell at various valences on the quantum dot in the present invention. . (B) Quantum taken up per cell 45 minutes after adding 30 pM of the known bivalent PTD and the target molecule intracellular introduction peptide (20 valence) of the present invention loaded on the quantum dot onto the cell Indicates the number of dots. 本発明におけるターゲット分子細胞内導入用ペプチドおよび公知のPTDを量子ドットでラベルし、蛍光色素でエンドソームを標識し双方を同時に観察することで、エンドソームから離脱した量子ドットの割合を測定することで、エンドソームから離脱する割合を測定した実験の代表的な一分子顕微鏡像（左）と、定量化した結果（右）を示す。By labeling the target molecule intracellular introduction peptide in the present invention and a known PTD with quantum dots, labeling the endosome with a fluorescent dye and observing both simultaneously, by measuring the proportion of quantum dots detached from the endosome, A representative single-molecule microscope image (left) of the experiment in which the ratio of detachment from the endosome was measured and the quantified result (right) are shown. Tag蛋白質を、ミトコンドリア上の蛋白質MFN2に融合して発現する発現ベクターを細胞に導入して４８時間後（Ａ）、細胞培養液に合成したターゲット分子細胞内導入用ペプチドをs-s結合で付着させ、さらにTag-ligandを付加した量子ドットを15pM濃度で添加すると（Ｂ）、量子ドットは細胞に取り込まれ、細胞内でＴａｇタンパク質とリガンドとの結合が起きる。その後、量子ドットはミトコンドリアに局在し、ミトコンドリア上で運動するのが確認された。48 hours after introducing an expression vector expressing the Tag protein fused with the protein MFN2 on the mitochondria into the cell (A), the synthesized peptide for target molecule introduction into the cell culture medium is attached by ss bond, Further, when a quantum dot to which Tag-ligand is added is added at a concentration of 15 pM (B), the quantum dot is taken up by the cell, and the binding between the Tag protein and the ligand occurs in the cell. After that, it was confirmed that the quantum dots were localized in the mitochondria and moved on the mitochondria.
　本明細書において、特段の規定がない場合、本発明のペプチドが有するアミノ酸配列には、左側がＮ末端のものと左側がＣ末端のものの両方が包含される。例えば、配列「GLFGAIAGFIENGWEGMIDGWYGF」は、N-GLFGAIAGFIENGWEGMIDGWYGF-C又はN-FGYWGDIMGEWGNEIFGAIAGFLG-Cを示し、
配列「YGRKKRRQRRR」は、N-YGRKKRRQRRR-C又はN-RRRQRRKKRGY-Cを示し、
配列「GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR」は、N-GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR-C又はN-YGRKKRRQRRRFGYWGDIMGEWGNEIFGAIAGFLG-Cを示す。 In the present specification, unless otherwise specified, the amino acid sequence of the peptide of the present invention includes both the N-terminal on the left side and the C-terminal on the left side. For example, the sequence “GLFGAIAGFIENGWEGMIDGWYGF” represents N-GLFGAIAGFIENGWEGMIDGWYGF-C or N-FGYWGDIMGEWGNEIFGAIAGFLG-C,
The sequence "YGRKKRRQRRR" represents N-YGRKKRRQRRR-C or N-RRRQRRKKRGY-C,
The sequence “GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR” represents N-GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR-C or N-YGRKKRRQRRRFGYWGDIMGEWGNEIFGAIAGFLG-C.
　ターゲット分子細胞内導入用ペプチド
　本発明は、ｐＨ依存性膜融合ペプチド及び蛋白質導入ドメイン（PTD）を有するターゲット分子細胞内導入用ペプチドであって、該膜融合ペプチドの構成アミノ酸数が２１以上である、ターゲット分子細胞内導入用ペプチドを提供する。 The present invention relates to a peptide for introduction into a target molecule cell having a pH-dependent membrane fusion peptide and a protein introduction domain (PTD), and the membrane fusion peptide has 21 or more constituent amino acids. Provided are peptides for intracellular introduction of target molecules.
　本発明において、蛋白質導入ドメイン（PTD）とは、細胞膜表面受容体に吸着後局所刺激を引き起こし、エンドサイトーシスによりターゲット分子細胞内導入用ペプチドを細胞内に取り込ませる機能を有するペプチドおよび同等の機能を有するアミノ酸配列を示す。 In the present invention, the protein transduction domain (PTD) is a peptide having a function of causing local stimulation after adsorption to a cell membrane surface receptor and causing the target molecule intracellular transduction peptide to be taken into the cell by endocytosis and an equivalent function The amino acid sequence having
　PTD自体としては、公知の配列を適宜用いることができる。アミノ酸配列は特に限定されないが、例えば、YGRKKRRQRRR、RRRRRRRRRRR、RQIKIWFQNRRMKWKK、QWTLNSAGYLLGKINLKALAALAKKIL、KLALKLALKALKAALKLA、MVTVLFRRLRIRRACGPPRVRV等が挙げられる。また、本発明においては、PTDには、YGRKKRRQRRR、RRRRRRRRRRR、RQIKIWFQNRRMKWKK、QWTLNSAGYLLGKINLKALAALAKKIL（配列番号１５）、KLALKLALKALKAALKLA（配列番号１６）、またはMVTVLFRRLRIRRACGPPRVRV（配列番号１７）で示されるアミノ酸配列において１個又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなるもの（例えば、YGRKKRRQRRR の末端にPPQを付加したYGRKKRRQRRRPPQ（配列番号１８）、さらに反対側の末端からYを欠失させたGRKKRRQRRRPPQ（配列番号１９）、RRRRRRRRRRRからRRを欠失させたRRRRRRRRR（配列番号２０）等）も含まれる。 As the PTD itself, a known sequence can be appropriately used. The amino acid sequence is not particularly limited, and examples thereof include YGRKKRRQRRR, RRRRRRRRRRR, RQIKIWFQNRRMKWKK, QWTLNSAGYLLGKINLKALAALAKKIL, KLALKLALKALKAALKLA, MVTVLFRRLRIRRACGPPRVRV, and the like. In the present invention, PTD includes YGRKKRRQRRR, RRRRRRRRRRR, RQIKIWFQNRRMKWKK, QWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 15), KLALKLALKALKAALKLA (SEQ ID NO: 16), or the number of amino acids represented by MVTVLFRRLRIRRACGPPRVRV (SEQ ID NO: 17). An amino acid sequence having an amino acid deleted, substituted or added (for example, YGRKKRRQRRRPPQ (SEQ ID NO: 18) in which PPQ is added to the end of YGRKKRRQRRR), and GRKKRRQRRRPPQ (SEQ ID NO: 19 in which Y is deleted from the opposite end) ), RRRRRRRRR (SEQ ID NO: 20) in which RR is deleted from RRRRRRRRRRR, etc.) are also included.
　上記PTDにおいて、「１または複数」の範囲は、当該PTDにｐＨ依存性膜融合ペプチドを組み合わせたターゲット分子細胞内導入用ペプチドが細胞膜透過性を有することを限度として特に制限されないが、例えば１～１５個、好ましくは１～１０個、より好ましくは１～５個、さらに好ましくは１～４個、特に好ましくは１～３個、さらに特に好ましくは１又は２個が挙げられる。特定のアミノ酸配列において、１または複数のアミノ酸を欠失、置換及び／または付加させる技術は公知である。なお、このように欠失、置換及び／または付加されたペプチドとしては、欠失、置換及び付加をする前のアミノ酸配列に対して５０％以上の同一性を有するアミノ酸配列からなり、且つ、PTDにｐＨ依存性膜融合ペプチドを組み合わせたターゲット分子細胞内導入用ペプチドが細胞膜透過性を有するペプチドが例示される。また、当該ぺプチドとして、アミノ酸の同一性は通常７０％以上、好ましくは８０％以上、より好ましくは９０％以上、さらに好ましくは９５％以上、特に好ましくは９７％以上、さらに特に好ましくは９８％以上である。 In the above PTD, the range of “one or more” is not particularly limited as long as the peptide for introducing into a target molecule, which is a combination of the PTD and a pH-dependent membrane fusion peptide, has cell membrane permeability. The number is 15, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 4, particularly preferably 1 to 3, and still more preferably 1 or 2. Techniques for deleting, substituting and / or adding one or more amino acids in a specific amino acid sequence are known. The peptide thus deleted, substituted and / or added is composed of an amino acid sequence having 50% or more identity to the amino acid sequence before deletion, substitution and addition, and PTD. Examples of the peptide having a cell membrane permeability as a peptide for introduction into a target molecule into which a pH-dependent membrane fusion peptide is combined. In addition, as the peptide, the identity of amino acids is usually 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more, and still more preferably 98%. That's it.
　本発明において、細胞膜透過性とは、単にエンドサイトーシスによりエンドソームに取り込まれるだけでなく、エンドソーム膜に小孔を開けて、細胞質内に到達する性質を示す。 In the present invention, cell membrane permeability refers to the property of not only being taken into endosomes by endocytosis but also having a small hole in the endosomal membrane to reach the cytoplasm.
　本発明において、ｐＨ依存性膜融合ペプチドとは、エンドソームに取り込まれた後、エンドソーム膜に挿入されるペプチドであって、ｐＨによりその二次構造が変化するものを示す。より具体的には、ｐＨが中性条件とｐＨ５以下（例えば、ｐＨ４以下又はｐＨ３以下）等の酸性領域とで立体構造が変化するペプチドを示す。 In the present invention, the pH-dependent membrane fusion peptide refers to a peptide that is inserted into the endosome and then inserted into the endosome membrane, and whose secondary structure changes with pH. More specifically, a peptide whose steric structure changes under neutral conditions and an acidic region such as pH 5 or lower (for example, pH 4 or lower or pH 3 or lower) is shown.
　かかるｐＨ依存性膜融合ペプチドとしては、例えば、配列Ｘ１Ｘ２Ｘ３［式中、Ｘ１、Ｘ２及びＸ３は同一又は異なって、Ｗ、Ｈ、Ｇ、Ｆ、Ｙ、Ａ、Ｃ、Ｌ、又はＴ（好ましくはＷ、Ｈ、Ｇ、Ｆ又はＹ）を示す］
を有するものが挙げられる。配列Ｘ１Ｘ２Ｘ３の位置としては特に限定されないが、PTDと結合する側の末端が好ましい。配列Ｘ１Ｘ２Ｘ３としては、例えば、ＹＧＦ、ＷＹＧ、ＨＧＹ、ＷＨＧ、ＡＬＹ、ＣＬＴ、ＹＣＬ等が挙げられる。 Examples of such pH-dependent membrane fusion peptides include the sequences X 1 X 2 X 3 [wherein X 1 , X 2 and X 3 are the same or different, and W, H, G, F, Y, A, C] , L, or T (preferably W, H, G, F, or Y)]
The thing which has is mentioned. The position of the sequence X 1 X 2 X 3 is not particularly limited, but the terminal on the side that binds to the PTD is preferable. Examples of the array X 1 X 2 X 3 include YGF, WYG, HGY, WHG, ALY, CLT, and YCL.
　かかるｐＨ依存性膜融合ペプチドとしては、例えば、各種ウイルス（例えば、インフルエンザＡ型、インフルエンザＢ型、インフルエンザＣ型等）に由来する膜融合ペプチドであってｐＨ依存性を有するもののアミノ酸配列の全部又は一部を用いることができるが、当該ｐＨ依存性膜融合ペプチドは構成アミノ酸数が２１以上、好ましくは２３個以上であることが重要である。構成アミノ酸の上限は特に限定されないが、例えば、26以下が好ましく、24以下がより好ましい。 Examples of such pH-dependent membrane fusion peptides include all amino acid sequences of membrane fusion peptides derived from various viruses (eg, influenza A, influenza B, influenza C, etc.) and having pH dependency. Although a part can be used, it is important that the pH-dependent membrane fusion peptide has 21 or more amino acids, preferably 23 or more. The upper limit of the constituent amino acids is not particularly limited, but is preferably 26 or less, and more preferably 24 or less.
　このようなｐＨ依存性膜融合ペプチドのアミノ酸配列としては、上記構成アミノ酸数を有するものであれば特に限定されないが、例えば、GLFGAIAGFIENGWEGMIDGWYGF、GLFGAIAGFIENGWEGMIDGWYG、GFFGAIAGFLEGGWEGMIAGWHGY、GFFGAIAGFLEGGWEGMIAGWHG、LAGVIMAGVAIGIATAAQITAGVALY、GTFTWTLSDSEGKDTPGGYCLT、GTFTWTLSDSSGVENPGGYCLT、AFFSWSLTDSSGKDTPGGYCL、AFFSWSLTDSSGKDMPGGYCL、AFFSWSLSDPKGNDMPGGYCL、GIFSWTITDAVGNDMPGGYCL等を挙げることができる。また、本発明において、ｐＨ依存性膜融合ペプチドには、上記GLFGAIAGFIENGWEGMIDGWYGF、GLFGAIAGFIENGWEGMIDGWYG、GFFGAIAGFLEGGWEGMIAGWHGY、GFFGAIAGFLEGGWEGMIAGWHG、AGVIMAGVAIGIATAAQITAGVALY、GTFTWTLSDSEGKDTPGGYCLT、GTFTWTLSDSSGVENPGGYCLT、AFFSWSLTDSSGKDTPGGYCL、AFFSWSLTDSSGKDMPGGYCL、AFFSWSLSDPKGNDMPGGYCLもしくはGIFSWTITDAVGNDMPGGYCLで示されるアミノ酸配列において１個又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなるものも含まれる。 The amino acid sequence of such pH dependent membrane fusion peptide, but not particularly limited as long as it has a number of the constituent amino acids, for example, GLFGAIAGFIENGWEGMIDGWYGF, GLFGAIAGFIENGWEGMIDGWYG, GFFGAIAGFLEGGWEGMIAGWHGY, GFFGAIAGFLEGGWEGMIAGWHG, LAGVIMAGVAIGIATAAQITAGVALY, GTFTWTLSDSEGKDTPGGYCLT, GTFTWTLSDSSGVENPGGYCLT, AFFSWSLTDSSGKDTPGGYCL, AFFSWSLTDSSGKDMPGGYCL, AFFSWSLSDPKGNDMPGGYCL, GIFSWTITDAVGNDMPGGYCL, etc. can be mentioned. Further, in the present invention, the pH dependent membrane fusion peptide, the GLFGAIAGFIENGWEGMIDGWYGF, GLFGAIAGFIENGWEGMIDGWYG, GFFGAIAGFLEGGWEGMIAGWHGY, GFFGAIAGFLEGGWEGMIAGWHG, AGVIMAGVAIGIATAAQITAGVALY, GTFTWTLSDSEGKDTPGGYCLT, GTFTWTLSDSSGVENPGGYCLT, AFFSWSLTDSSGKDTPGGYCL, AFFSWSLTDSSGKDMPGGYCL, 1 or several amino acids in the amino acid sequence shown in AFFSWSLSDPKGNDMPGGYCL or GIFSWTITDAVGNDMPGGYCL In which amino acid sequences are deleted, substituted or added.
　上記ｐＨ依存性膜融合ペプチドにおいて、「１または複数」の範囲は、当該ｐＨ依存性膜融合ペプチドにPTDを組み合わせたターゲット分子細胞内導入用ペプチドが細胞膜透過性を有することを限度として特に制限されないが、例えば１～１５個、好ましくは１～１０個、より好ましくは１～７個、さらに好ましくは１～５個が挙げられる。特定のアミノ酸配列において、１または複数のアミノ酸を欠失、置換及び／または付加させる技術は公知である。 In the pH-dependent membrane fusion peptide, the range of “one or more” is not particularly limited as long as the peptide for introduction into a target molecule cell in which PTD is combined with the pH-dependent membrane fusion peptide has cell membrane permeability. Is, for example, 1 to 15, preferably 1 to 10, more preferably 1 to 7, and still more preferably 1 to 5. Techniques for deleting, substituting and / or adding one or more amino acids in a specific amino acid sequence are known.
　なお、このように欠失、置換及び／または付加されたペプチドとして、欠失、置換及びおよび付加をする前のアミノ酸配列に対して５０％以上の同一性を有するアミノ酸配列からなり、且つ、当該ｐＨ依存性膜融合ペプチドにPTDを組み合わせたターゲット分子細胞内導入用ペプチドが細胞膜透過性を有するようなペプチドが例示される。また、当該ぺプチドとして、アミノ酸の同一性は通常７０％以上、好ましくは８０％以上、より好ましくは９０％以上、さらに好ましくは９５％以上、特に好ましくは９７％以上、さらに特に好ましくは９８％以上である。 The peptide thus deleted, substituted and / or added consists of an amino acid sequence having 50% or more identity to the amino acid sequence before deletion, substitution and addition, and Examples are peptides in which a peptide for introduction into a target molecule into which a PTD is combined with a pH-dependent membrane fusion peptide has cell membrane permeability. In addition, as the peptide, the identity of amino acids is usually 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more, and still more preferably 98%. That's it.
　このようなペプチドとしては、例えば、GLFX4AIAX5FIEX6GWEGX7IX8GWYG（配列番号２１）
［式中、Ｘ４はＤ、Ｅ、Ｇ又Ｎを示す。Ｘ５はＤ、Ｅ、Ｇ又Ｎを示す。Ｘ６はＤ、Ｅ、Ｇ又Ｎを示す。Ｘ７はＭ又はＬを示す。Ｘ８はＤ、Ｅ、Ｇ又Ｎを示す。但し、（Ｘ４、Ｘ５、Ｘ６、Ｘ７、Ｘ８）＝（Ｇ、Ｇ、Ｎ、Ｍ、Ｄ）の組み合わせを除く。］
からなるペプチド等を挙げることができる。より具体的には、例えば、配列GLFGAIAGFIENGWEGMIDGWYG のうち４個を置換した配列、例えば、GLFEAIAEFIEGGWEGLIEGWYG（下線は置換部分、配列番号２２）からなるペプチド等が挙げられる。 As such a peptide, for example, GLFX 4 AIAX 5 FIEX 6 GWEGX 7 IX 8 GWYG (SEQ ID NO: 21)
[Wherein X 4 represents D, E, G or N. X 5 represents D, E, G or N. X 6 represents D, E, G or N. X 7 represents M or L. X 8 represents D, E, G or N. However, the combination of (X 4 , X 5 , X 6 , X 7 , X 8 ) = (G, G, N, M, D) is excluded. ]
And the like. More specifically, for example, a sequence in which four of the sequence GLFGAIAGFIENGWEGMIDGWYG are substituted, for example, a peptide consisting of GLF E AIA E FIE G GWEG L I E GWYG (underlined is a substituted portion, SEQ ID NO: 22) and the like can be mentioned.
　本発明のターゲット分子細胞内導入用ペプチドにおいて、ｐＨ依存性膜融合ペプチドとPTDとは直接結合しても、リンカーを介して結合してもよい。リンカーとしては、当該分野において用いられているものを広く使用することができる。 In the target molecule intracellular introduction peptide of the present invention, the pH-dependent membrane fusion peptide and the PTD may be bonded directly or via a linker. As a linker, what is used in the said field | area can be used widely.
　本発明のターゲット分子細胞内導入用ペプチドには、目的物質を結合させるためのドメイン、具体的には、例えば、ＤＮＡ結合ドメイン（ジンクフィンガードメイン、HMG-box等）、二本鎖RNA結合ドメイン（ヒストン、RDE-4タンパク質、プロタミン等由来の二本鎖RNA結合ドメイン）、各種標識タグ蛋白質認識化合物（抗Myc結合蛋白質（Myc-tagに対して）、Halotagリガンド（Halotag蛋白質結合分子）、nickel分子 (His-tag蛋白質結合分子)等）等を融合ペプチド側の末端及びPTD側の末端のうち、PTD側の末端に有していてもよい。 The peptide for introduction into a target molecule of the present invention has a domain for binding a target substance, specifically, for example, a DNA binding domain (zinc finger domain, HMG-box, etc.), a double-stranded RNA binding domain ( Histone, RDE-4 protein, double-stranded RNA binding domain derived from protamine, etc., various labeled tag protein recognition compounds (anti-Myc binding protein (for Myc-tag)), Halotag ligand (Halotag protein binding molecule), nickel molecule (His-tag protein binding molecule) etc.) may be present at the end of the PTD side among the end of the fusion peptide and the end of the PTD.
　より具体的には、本発明のターゲット分子細胞内導入用ペプチドとしては、例えば、以下のものが挙げられる：
N-GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR-C　（ペプチド１、配列番号２３）
N-GLFGAIAGFIENGWEGMIDGWYGFRRRQRRKKRGY-C　（ペプチド２、配列番号２４）
N-YGRKKRRQRRRFGYWGDIMGEWGNEIFGAIAGFLG-C　（ペプチド３、配列番号２５）
N-RRRQRRKKRGYFGYWGDIMGEWGNEIFGAIAGFLG-C　（ペプチド４、配列番号２６）
N-GFFGAIAGFLEGGWEGMIAGWHGYYGRKKRRQRRR-C　（ペプチド５、配列番号２７）
N-GFFGAIAGFLEGGWEGMIAGWHGYRRRQRRKKRGY-C　（ペプチド６、配列番号２８）
N-YGRKKRRQRRRYGHWGAIMGEWGGELFGAIAGFFG-C　（ペプチド７、配列番号２９）
N-RRRQRRKKRGYYGHWGAIMGEWGGELFGAIAGFFG-C　（ペプチド８、配列番号３０）
N-GLFGAIAGFIENGWEGMIDGWYGYGRKKRRQRRR-C　（ペプチド９、配列番号３１）
N-GLFGAIAGFIENGWEGMIDGWYGRRRQRRKKRGY-C　（ペプチド１０、配列番号３２）
N-YGRKKRRQRRRGYWGDIMGEWGNEIFGAIAGFLG-C　（ペプチド１１、配列番号３３）
N-RRRQRRKKRGYGYWGDIMGEWGNEIFGAIAGFLG-C　（ペプチド１２、配列番号３４）
N-GFFGAIAGFLEGGWEGMIAGWHGYGRKKRRQRRR-C　（ペプチド１３、配列番号３５）
N-GFFGAIAGFLEGGWEGMIAGWHGRRRQRRKKRGY-C　（ペプチド１４、配列番号３６）
N-YGRKKRRQRRRGHWGAIMGEWGGELFGAIAGFFG-C　（ペプチド１５、配列番号３７）
N-RRRQRRKKRGYGHWGAIMGEWGGELFGAIAGFFG-C　（ペプチド１６、配列番号３８）
N-GLFGAIAGFIENGWEGMIDGWYGFRRRRRRRRRRR-C　（ペプチド１７、配列番号３９）
N-RRRRRRRRRRRFGYWGDIMGEWGNEIFGAIAGFLG-C　（ペプチド１８、配列番号４０）
N-GFFGAIAGFLEGGWEGMIAGWHGYRRRRRRRRRRR-C　（ペプチド１９、配列番号４１）
N-RRRRRRRRRRRYGHWGAIMGEWGGELFGAIAGFFG-C　（ペプチド２０、配列番号４２）
N-GLFGAIAGFIENGWEGMIDGWYGRRRRRRRRRRR-C　（ペプチド２１、配列番号４３）
N-RRRRRRRRRRRGYWGDIMGEWGNEIFGAIAGFLG-C　（ペプチド２２、配列番号４４）
N-GFFGAIAGFLEGGWEGMIAGWHGRRRRRRRRRRR-C　（ペプチド２３、配列番号４５）
N-RRRRRRRRRRRGHWGAIMGEWGGELFGAIAGFFG-C　（ペプチド２４、配列番号４６）
N-GLFEAIAEFIEGGWEGLIEGWYGRRRRRRRRRRR-C　（ペプチド２５、配列番号４７）
N-RRRRRRRRRRRGYWGEILGEWGGEIFEAIAEFLG-C　（ペプチド２６、配列番号４８）
N-GLFEAIAEFIEGGWEGLIEGWYGYGRKKRRQRRR-C　（ペプチド２７、配列番号４９）
N-GLFEAIAEFIEGGWEGLIEGWYGRRRQRRKKRGY-C　（ペプチド２８、配列番号５０）
N-YGRKKRRQRRRGYWGEILGEWGGEIFEAIAEFLG-C　（ペプチド２９、配列番号５１）
N-RRRQRRKKRGYGYWGEILGEWGGEIFEAIAEFLG-C　（ペプチド３０、配列番号５２）。
上記配列において、下線部はPTDを示し、それ以外の部分は、ｐＨ依存性膜融合ペプチドである。 More specifically, examples of the peptide for introduction into a target molecule cell of the present invention include the following:
N-GLFGAIAGFIENGWEGMIDGWYGF YGRKKRRQRRR- C (Peptide 1, SEQ ID NO: 23)
N-GLFGAIAGFIENGWEGMIDGWYGF RRRQRRKKRGY- C (Peptide 2, SEQ ID NO: 24)
N- YGRKKRRQRRR FGYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 3, SEQ ID NO: 25)
N- RRRQRRKKRGY FGYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 4, SEQ ID NO: 26)
N-GFFGAIAGFLEGGWEGMIAGWHGY YGRKKRRQRRR- C (peptide 5, SEQ ID NO: 27)
N-GFFGAIAGFLEGGWEGMIAGWHGY RRRQRRKKRGY- C (Peptide 6, SEQ ID NO: 28)
N- YGRKKRRQRRR YGHWGAIMGEWGGELFGAIAGFFG-C (Peptide 7, SEQ ID NO: 29)
N- RRRQRRKKRGY YGHWGAIMGEWGGELFGAIAGFFG-C (Peptide 8, SEQ ID NO: 30)
N-GLFGAIAGFIENGWEGMIDGWYG YGRKKRRQRRR- C (Peptide 9, SEQ ID NO: 31)
N-GLFGAIAGFIENGWEGMIDGWYG RRRQRRKKRGY- C (Peptide 10, SEQ ID NO: 32)
N- YGRKKRRQRRR GYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 11, SEQ ID NO: 33)
N- RRRQRRKKRGY GYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 12, SEQ ID NO: 34)
N-GFFGAIAGFLEGGWEGMIAGWHG YGRKKRRQRRR -C (Peptide 13, SEQ ID NO: 35)
N-GFFGAIAGFLEGGWEGMIAGWHG RRRQRRKKRGY- C (peptide 14, SEQ ID NO: 36)
N- YGRKKRRQRRR GHWGAIMGEWGGELFGAIAGFFG-C (peptide 15, SEQ ID NO: 37)
N- RRRQRRKKRGY GHWGAIMGEWGGELFGAIAGFFG-C (Peptide 16, SEQ ID NO: 38)
N-GLFGAIAGFIENGWEGMIDGWYGF RRRRRRRRRRR -C (peptide 17, SEQ ID NO: 39)
N- RRRRRRRRRRRR FGYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 18, SEQ ID NO: 40)
N-GFFGAIAGFLEGGWEGMIAGWHGY RRRRRRRRRRR -C (peptide 19, SEQ ID NO: 41)
N- RRRRRRRRRRRR YGHWGAIMGEWGGELFGAIAGFFG-C (Peptide 20, SEQ ID NO: 42)
N-GLFGAIAGFIENGWEGMIDGWYG RRRRRRRRRRR- C (Peptide 21, SEQ ID NO: 43)
N-RRRRRRRRRRRGYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 22, SEQ ID NO: 44)
N-GFFGAIAGFLEGGWEGMIAGWHG RRRRRRRRRRR -C (peptide 23, SEQ ID NO: 45)
N- RRRRRRRRRRRR GHWGAIMGEWGGELFGAIAGFFG-C (Peptide 24, SEQ ID NO: 46)
N-GLFEAIAEFIEGGWEGLIEGWYG RRRRRRRRRRR- C (peptide 25, SEQ ID NO: 47)
N- RRRRRRRRRRRR GYWGEILGEWGGEIFEAIAEFLG-C (Peptide 26, SEQ ID NO: 48)
N-GLFEAIAEFIEGGWEGLIEGWYG YGRKKRRQRRR -C (peptide 27, SEQ ID NO: 49)
N-GLFEAIAEFIEGGWEGLIEGWYG RRRQRRKKRGY- C (peptide 28, SEQ ID NO: 50)
N- YGRKKRRQRRR GYWGEILGEWGGEIFEAIAEFLG-C (peptide 29, SEQ ID NO: 51)
N- RRRQRRKKRGY GYWGEILGEWGGEIFEAIAEFLG-C (peptide 30, SEQ ID NO: 52).
In the above sequence, the underlined portion indicates PTD, and the other portion is a pH-dependent membrane fusion peptide.
　本発明のターゲット分子細胞内導入用ペプチドは、従来公知の遺伝子工学的手法や化学合成法などによって作製できる。例えば、前述のターゲット分子細胞内導入用ペプチドをコードするポリヌクレオチドをベクター等に挿入し、次いで、当該ベクターが組み込まれた形質転換体を培養したのち、所望のペプチドを取得すればよい。また、本発明のターゲット分子細胞内導入用ペプチドは、当該ペプチドの産生能を有するよう形質転換した微生物から単離・精製することによって取得してもよい。また、ターゲット分子細胞内導入用ペプチドのアミノ酸配列またはこれをコードするヌクレオチド配列の情報に従って、本発明のターゲット分子細胞内導入用ペプチドを従来公知の化学合成法により合成して取得してもよい。なお、化学合成法には、液相法や固相法によるペプチド合成法が包含される。 The peptide for introduction into a target molecule cell of the present invention can be prepared by a conventionally known genetic engineering method, chemical synthesis method or the like. For example, a desired peptide may be obtained after inserting a polynucleotide encoding the aforementioned peptide for introduction into a target molecule into a vector and culturing a transformant in which the vector is incorporated. In addition, the peptide for introduction into a target molecule of the present invention may be obtained by isolating and purifying from a microorganism transformed to have the ability to produce the peptide. Moreover, the target molecule intracellular introduction peptide of the present invention may be synthesized by a conventionally known chemical synthesis method in accordance with the information of the amino acid sequence of the target molecule intracellular introduction peptide or the nucleotide sequence encoding the peptide. The chemical synthesis method includes a peptide synthesis method using a liquid phase method or a solid phase method.
　その際、本発明のターゲット分子細胞内導入用ペプチドは、ｐＨ依存性膜融合ペプチドとPTDとが結合したもの（またはさらに目的物質を結合させるためのドメインが結合したもの）を上記の方法により製造しても、ｐＨ依存性膜融合ペプチド及びPTD（またはPTDに目的物質を結合させるためのドメインが結合したもの）をそれぞれ上記の方法により合成した後にこれらを結合させて製造してもよい。 At that time, the peptide for introducing a target molecule into the cell of the present invention is produced by the above method by binding a pH-dependent membrane fusion peptide and PTD (or further binding a domain for binding a target substance). Alternatively, the pH-dependent membrane fusion peptide and PTD (or the PTD bound to the domain for binding the target substance) may be synthesized by the above method and then bound to each other.
　複合体
　本発明は、前述ターゲット分子細胞内導入用ペプチドとターゲット分子とを結合させてなるターゲット分子複合体を提供する。 Complex The present invention provides a target molecule complex formed by binding the above-described target molecule intracellular introduction peptide and a target molecule.
　ターゲット分子とターゲット分子細胞内導入用ペプチドとの結合様式は特に限定されず、例えば、前述の結合ドメインを介して結合してもよく、－Ｓ－Ｓ－結合等により架橋されてもよく、ビオチン、アビジンシステムを利用してもよく、静電的に結合してもよく、化学的に修飾して結合させても良い。 The binding mode of the target molecule and the peptide for introduction into the target molecule into the cell is not particularly limited. For example, the target molecule may be bound through the above-described binding domain, may be cross-linked by —SS—bonding, etc. The avidin system may be used, may be electrostatically bonded, or may be chemically modified and bonded.
　ターゲット１分子に対しターゲット分子細胞内導入用ペプチドを結合させる数も特に限定されないが、２個以上（例えば、３個以上、より好ましくは８個以上）のターゲット分子細胞内導入用ペプチドがターゲット分子に結合することが、細胞表面受容体結合の効率上昇び細胞表面受容体の取り込み機序の活性化の観点から好ましい。ターゲット１分子に対しターゲット分子細胞内導入用ペプチドを結合させる数の上限も特に限定されないが、例えば、30個以下が好ましく、20個以下がより好ましい。 The number of target molecule intracellular introduction peptides to be bound to one target molecule is not particularly limited, but 2 or more (eg, 3 or more, more preferably 8 or more) target molecule intracellular introduction peptides are target molecules. It is preferable from the viewpoint of increasing the efficiency of cell surface receptor binding and activating the cell surface receptor uptake mechanism. The upper limit of the number of the target molecule-introducing peptide to be bound to one target molecule is not particularly limited, but is preferably 30 or less, and more preferably 20 or less.
　目的物質としては、特に限定されず、高分子及び低分子のいずれであってもよい。例えば、高分子化合物としては、ＤＮＡ、ＲＮＡ（siRNA、shRNA等）等の核酸分子；オリゴペプチド、タンパク質（抗体、抗体フラグメント、酵素、サイトカイン、ケモカイン、レセプターポリペプチド、等）等のペプチド等、糖鎖等が挙げられる。低分子としては、例えば、抗生物質、抗癌剤、抗炎症剤、リポソーム、ミセル、デンドリマー、ナノチューブ、アミノ酸ナノ粒子等のナノキャリア、量子ドット等、蛍光色素等、細胞内分子可視化試薬、ナノ磁性体、ナノゴールド等、生体内での標的細胞動態を可視化できる化合物等が挙げられる。 The target substance is not particularly limited and may be either a polymer or a low molecule. For example, polymer compounds include nucleic acid molecules such as DNA and RNA (siRNA, shRNA, etc.); peptides such as oligopeptides and proteins (antibodies, antibody fragments, enzymes, cytokines, chemokines, receptor polypeptides, etc.), sugars, etc. Examples include chains. Examples of small molecules include antibiotics, anticancer agents, anti-inflammatory agents, liposomes, micelles, dendrimers, nanotubes, nanocarriers such as amino acid nanoparticles, quantum dots, fluorescent dyes, intracellular molecular visualization reagents, nanomagnetic materials, Examples thereof include compounds that can visualize target cell dynamics in vivo, such as nanogold.
　例えば、転写因子の異常により発生する癌については、当該転写因子をターゲット分子として含む本発明の複合体を調製し、これを被験体に投与して癌の治療をすることも可能である。 For example, for cancers caused by abnormal transcription factors, it is also possible to prepare a complex of the present invention containing the transcription factor as a target molecule and administer this to a subject to treat the cancer.
　また、本発明の方法を用いて抗原提示細胞に抗原を導入すると抗原がMHC-クラスIに提示されて細胞障害性T細胞が活性される。そこで、細胞障害性T細胞を誘導する必要がある多くのウイルス感染症および癌に対するワクチンへの応用が考えられる。 In addition, when an antigen is introduced into an antigen-presenting cell using the method of the present invention, the antigen is presented to MHC-class I and cytotoxic T cells are activated. Therefore, application to vaccines against many viral infections and cancers that require the induction of cytotoxic T cells is conceivable.
　本発明のターゲット分子細胞内導入用ペプチド及び複合体には、さらに特定の抗原に対し特異的に結合する物質（例えば、抗体、抗体フラグメント（scFv(Single-chain variable fragment)等））を結合させてもよい。当該実施形態によれば、種々の細胞を含む試料又は被験体において、特定の細胞に特異的にターゲット分子を導入することができる。 Further, a substance (for example, antibody, antibody fragment (scFv (Single-chain variable fragment), etc.)) that specifically binds to a specific antigen is bound to the peptide and complex for introduction into the target molecule of the present invention. May be. According to this embodiment, a target molecule can be specifically introduced into a specific cell in a sample or subject containing various cells.
　本発明の複合体は、コンディショナルノックアウト動物への応用も可能である。具体的には、例えば、標的となる遺伝子領域をCreリコンビナーゼ標的配列loxPで挟んだ遺伝子座を有するfloxマウスに、本発明のターゲット分子細胞内導入用ペプチドとCreリコンビナーゼ又はこれをコードする遺伝子とを結合した複合体を投与する方法があげられる。当該実施形態においては、Creリコンビナーゼ又はこれをコードする遺伝子がfloxマウスの体細胞に導入され、リコンビナーゼにより標的遺伝子を欠失させることができる。そのため、当該実施形態においては、所望のタイミングで標的となる遺伝子領域を欠失させることができるため有用である。また、当該複合体に特定の抗原等に結合する物質を結合させておくことにより、さらに、標的となる遺伝子領域の欠失を所望のタイミングでかつ所望の部位で生じさせることができる。 The complex of the present invention can be applied to a conditional knockout animal. Specifically, for example, in a flox mouse having a gene locus in which a target gene region is sandwiched between Cre recombinase target sequences loxP, the target molecule intracellular introduction peptide of the present invention and Cre recombinase or a gene encoding the same are added. A method of administering the bound complex is exemplified. In this embodiment, Cre recombinase or a gene encoding it can be introduced into somatic cells of flox mice, and the target gene can be deleted by the recombinase. Therefore, this embodiment is useful because a target gene region can be deleted at a desired timing. In addition, by binding a substance that binds to a specific antigen or the like to the complex, deletion of a target gene region can be caused at a desired timing and at a desired site.
　別の実施形態において、本発明の複合体はｉＰＳ細胞の作製にも使用することができる。具体的には例えば、OCT3/4・SOX2・NANOG・LIN28の４遺伝子発現ベクター、もしくは４蛋白質等をターゲット分子として本発明の複合体を作製し、これをin vitroで細胞に添加するか、または被験体となる動物に直接投与することにより、ターゲット分子である遺伝子が細胞に導入され、iPS細胞を樹立することができる。 In another embodiment, the complex of the present invention can also be used to produce iPS cells. Specifically, for example, the OCT3 / 4 / SOX2 / NANOG / LIN28 4 gene expression vector or 4 protein or the like is used as a target molecule, and the complex of the present invention is added to cells in vitro, or By directly administering to a subject animal, a gene as a target molecule is introduced into the cell, and an iPS cell can be established.
　また、細胞内での細胞外との環境変化に反応する反応基質を活用する例えば、エンドソームに取り込まれた後のpHの低下をpH反応性に応答して構造が変化し目的物質が導入ペプチドから離れるようにする、例えばpH反応性のpara-nitrophenyl (pNP)-PEG-PE、システイン-システイン間のSS結合等を用いて細胞内で目的物質より乖離する方法を含む。 In addition, utilizing a reaction substrate that reacts to changes in the environment outside the cell inside the cell, for example, a decrease in pH after incorporation into the endosome changes its structure in response to pH reactivity, and the target substance is introduced from the introduced peptide. The method includes separation from the target substance in the cell using, for example, pH-reactive para-nitrophenyl (pNP) -PEG-PE, SS bond between cysteine and cysteine, and the like.
　ベクター
　本発明は、ターゲット分子細胞内導入用ペプチドをコードするポリヌクレオチドを含むベクターも提供する。本発明においては、プラスミドベクター、アデノウイルスベクター、レトロウイルスベクター等いずれのベクターであっても利用できる。 Vector The present invention also provides a vector comprising a polynucleotide encoding a peptide for introduction into a target molecule into a cell. In the present invention, any vector such as a plasmid vector, an adenovirus vector, or a retrovirus vector can be used.
　当該ベクターにターゲット分子細胞内導入用ペプチドをコードするポリヌクレオチドを含む塩基配列をクローニングする方法としては、自体公知の方法を用いて行うことができ、例えば、発現制御シグナル（転写開始及び翻訳開始シグナル）等をコード領域の上流に配置する等して、宿主微生物に応じて該遺伝子が微生物菌体中で自発現可能となるよう設計することができる。 As a method for cloning a nucleotide sequence containing a polynucleotide encoding a peptide for introduction into a target molecule into a cell, a method known per se can be used. For example, expression control signals (transcription initiation and translation initiation signals) can be used. Etc.) can be designed so that the gene can be self-expressed in microbial cells depending on the host microorganism.
　本発明のベクターには、上記ターゲット分子細胞内導入用ペプチドをコードするものだけでなく、ターゲット分子細胞内導入用ペプチドと目的物質とが結合した複合体をコードするものも含む。当該実施形態においては、遺伝子発現により、ターゲット分子細胞内導入用ペプチドと目的物質とを共有結合したかたちで産生することができる。 The vector of the present invention includes not only those encoding the target molecule intracellular introduction peptide but also those encoding a complex in which the target molecule intracellular introduction peptide and the target substance are bound. In this embodiment, the target molecule can be produced by covalently binding the target molecule-introducing peptide and the target substance by gene expression.
　医薬組成物
　本発明は、前記複合体を有効成分として含む医薬組成物を提供する。本発明の医薬組成物において、有効成分である前記複合体は単独で用いることも可能であるが、投与経路に応じ、薬学的に許容し得る担体を用いて適当な剤形に製剤化することができる。例えば、本発明の医薬組成物は、被験体に対し、種々の投与経路、具体的には、経口又は非経口投与（例えば静脈注射、筋肉注射、経鼻粘膜投与、経口腔粘膜投与、経皮投与、皮下投与、皮内投与、直腸投与等）で投与することができる。好ましくは静脈注射、筋肉注射、経鼻粘膜投与等どの非経口的な局所投与である。被験体としては、哺乳動物（ヒト又は非ヒト哺乳動物）等が挙げられ、非ヒト哺乳動物としては、例えば、マウス、ラット、ウサギ、イヌ、サル等が挙げられる。 Pharmaceutical composition The present invention provides a pharmaceutical composition comprising the complex as an active ingredient. In the pharmaceutical composition of the present invention, the complex as the active ingredient can be used alone, but depending on the route of administration, it should be formulated into a suitable dosage form using a pharmaceutically acceptable carrier. Can do. For example, the pharmaceutical composition of the present invention can be administered to a subject in various administration routes, specifically oral or parenteral administration (eg, intravenous injection, intramuscular injection, nasal mucosal administration, oral mucosal administration, transdermal Administration, subcutaneous administration, intradermal administration, rectal administration, etc.). Preferably, parenteral administration such as intravenous injection, intramuscular injection, or intranasal mucosal administration is used. Examples of the subject include mammals (human or non-human mammal), and examples of the non-human mammal include mice, rats, rabbits, dogs, monkeys, and the like.
　好ましい剤形としては、非経口剤では、例えば、注射剤（点滴剤を含む）、軟膏剤、点眼剤、眼軟膏剤、点鼻剤、点耳剤、パップ剤、ローション剤等が好ましく挙げられる。経口剤では、例えば、錠剤、散剤、細粒剤、顆粒剤、被覆錠剤、カプセル剤、シロップ剤、及びトローチ剤等が好ましく挙げられる。 Preferred examples of the dosage form include parenteral preparations such as injections (including drops), ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions and the like. . Preferred examples of oral preparations include tablets, powders, fine granules, granules, coated tablets, capsules, syrups, and lozenges.
　これら製剤の製剤化に用い得る担体としては、例えば、医薬分野において通常使用される、賦形剤、結合剤、崩壊剤、滑沢剤、着色剤、及び矯味矯臭剤、また必要に応じて、安定化剤、乳化剤、吸収促進剤、界面活性剤、ｐＨ調整剤、防腐剤、抗酸化剤、増量剤、湿潤化剤、表面活性化剤、分散剤、緩衝剤、保存剤、溶解補助剤、無痛化剤等が挙げられる。 Carriers that can be used to formulate these preparations include, for example, excipients, binders, disintegrants, lubricants, colorants, and flavoring agents that are commonly used in the pharmaceutical field, and, if necessary, Stabilizer, emulsifier, absorption promoter, surfactant, pH adjuster, preservative, antioxidant, extender, wetting agent, surface active agent, dispersant, buffer, preservative, solubilizer, Examples include soothing agents.
　細胞内にターゲット分子を導入する方法
　本発明は、細胞に前記複合体を添加する工程を含む、ターゲット分子を細胞内に導入する方法を提供する。当該方法は、前記複合体をin vitro及びin vivoのいずれで細胞に添加してもよい。 Method for Introducing Target Molecule into Cell The present invention provides a method for introducing a target molecule into a cell, comprising the step of adding the complex to a cell. In this method, the complex may be added to cells either in vitro or in vivo.
　添加された複合体は、細胞膜表面付近でPTDが膜表面のへパラン硫酸プロテオグリカン等に結合して局所刺激を引き起こし、エンドサイトーシスによりターゲット分子細胞内導入用ペプチドを細胞内に取り込まれる（図１）。そして、ｐＨ依存性膜融合ペプチドがエンドソーム膜に挿入される。その後、時間と共にエンドソーム内のｐＨは早急に酸性に変化する。ｐＨの変化に伴い、ｐＨ依存性膜融合ペプチドはエンドソーム膜に挿入された状態でその立体構造が変化して湾曲し、エンドソーム膜に小孔を生じさせる。その結果、ターゲット分子細胞内導入用ペプチドは、エンドソーム膜を介して細胞質内に放出される。 In the added complex, PTD binds to heparan sulfate proteoglycan etc. on the membrane surface near the cell membrane surface to cause local stimulation, and the target molecule intracellular introduction peptide is taken into the cell by endocytosis (FIG. 1). ). Then, a pH-dependent membrane fusion peptide is inserted into the endosome membrane. Thereafter, the pH in the endosome rapidly changes to acidic with time. Along with the change in pH, the three-dimensional structure of the pH-dependent membrane fusion peptide is changed while it is inserted into the endosomal membrane, and is bent to form small pores in the endosomal membrane. As a result, the peptide for introduction into the target molecule into the cell is released into the cytoplasm through the endosome membrane.
　ターゲット分子の細胞内導入剤
　本発明は、前述したターゲット分子細胞内導入用ペプチドを含むターゲット分子の細胞内導入剤を提供する。本発明においては、ターゲット分子細胞内導入用ペプチド自体をターゲット分子の細胞内導入剤として用いてもよく、またターゲット分子細胞内導入用ペプチドを、前述した担体等と混合し製剤化したものを用いてもよい。 Target molecule intracellular introduction agent The present invention provides a target molecule intracellular introduction agent comprising the aforementioned target molecule intracellular introduction peptide. In the present invention, the target molecule intracellular introduction peptide itself may be used as the target molecule intracellular introduction agent, or the target molecule intracellular introduction peptide is mixed with the above-described carrier or the like to prepare a formulation. May be.
　以下、実施例及び比較例を示し、本発明を具体的に説明するが、本発明がこれらに限定されないことは明らかである。 Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated concretely, it is clear that this invention is not limited to these.
　実施例１
　東レ株式会社に依頼し、Fmoc (N-(9-フルオレニル)メトキシカルボニル)固相合成法で下記アミノ酸配列からなるペプチドを合成した：
　N-GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR-C　ペプチド１
合成したペプチドはアセトニトリル/H2O/トリフルオロ酢酸グラディエントによる逆相HPLCで９５％以上に精製した（分子量4149.7）。 Example 1
A peptide consisting of the following amino acid sequence was synthesized by Fmoc (N- (9-fluorenyl) methoxycarbonyl) solid phase synthesis method by requesting Toray Industries, Inc .:
N-GLFGAIAGFIENGWEGMIDGWYGF YGRKKRRQRRR -C Peptide 1
The synthesized peptide was purified to 95% or more by reverse phase HPLC with an acetonitrile / H 2 O / trifluoroacetic acid gradient (molecular weight 4149.7).
　実施例２～６
　下記のアミノ酸配列からなるペプチドを合成する以外、実施例１と同様にして、ペプチドを合成した：
N-YGRKKRRQRRRFGYWGDIMGEWGNEIFGAIAGFLG-C（ペプチド３、分子量4149.7）
N-RRRQRRKKRGYYGHWGAIMGEWGGELFGAIAGFFG-C（ペプチド８、分子量4072.6）
N-YGRKKRRQRRRGYWGDIMGEWGNEIFGAIAGFLG-C（ペプチド１１、分子量4002.6）
N-RRRRRRRRRRRGYWGEILGEWGGEIFEAIAEFLG-C（ペプチド２６、分子量4261.9）
N-YGRKKRRQRRRGYWGEILGEWGGEIFEAIAEFLG-C（ペプチド２９、分子量4085.6）
上記配列において、下線部はPTDを示し、それ以外の部分は、ｐＨ依存性膜融合ペプチドである。 Examples 2 to 6
A peptide was synthesized in the same manner as in Example 1 except that a peptide consisting of the following amino acid sequence was synthesized:
N- YGRKKRRQRRR FGYWGDIMGEWGNEIFGAIAGFLG-C (peptide 3, molecular weight 4149.7)
N- RRRQRRKKRGY YGHWGAIMGEWGGELFGAIAGFFG-C (peptide 8, molecular weight 4072.6)
N- YGRKKRRQRRR GYWGDIMGEWGNEIFGAIAGFLG-C (peptide 11, molecular weight 4002.6)
N- RRRRRRRRRRRR GYWGEILGEWGGEIFEAIAEFLG-C (peptide 26, molecular weight 4261.9)
N- YGRKKRRQRRR GYWGEILGEWGGEIFEAIAEFLG-C (peptide 29, molecular weight 4085.6)
In the above sequence, the underlined portion indicates PTD, and the other portion is a pH-dependent membrane fusion peptide.
　実施例１’
　また、上記Fmoc固相合成法において最後のアミノ酸(ペプチド１においてはＮ末端のＧ)の伸長に、ビオチン化アミノ酸を用いてPTD側をビオチン化したこと以外、実施例１と同様にして、Ｎ末端をビオチン化したペプチドを合成した。得られた化合物を化合物１とする（分子量4394.1）。 Example 1 '
Further, in the above Fmoc solid phase synthesis method, N N was biotinylated in the same manner as in Example 1 except that the PTD side was biotinylated using a biotinylated amino acid for the extension of the last amino acid (N-terminal G in peptide 1). A peptide biotinylated at the end was synthesized. The obtained compound is defined as Compound 1 (molecular weight 4394.1).
　実施例２’～６’
　また、上記Fmoc固相合成法において最後のアミノ酸(ペプチド１においてはＮ末端のＧ)の伸長に、ビオチン化アミノ酸を用いることによりPTD側をビオチン化した（化合物３、８、１１、２６）。各化合物の分子量は以下の通り：
化合物３、分子量4394.1
化合物８、分子量4316.9
化合物１１、分子量4246.9
化合物２６、分子量4329.9
また、下記アミノ酸配列を有しＮ末端をビオチン化したペプチドも同様にして合成した（化合物３３）
N-GYWGEILGEWGGEIFEAIAEFLGRRRRRRRRRRR-C（ペプチド33、分子量4506.2）
　比較例１及び２
　下記のアミノ酸配列としてからなるペプチドを合成する以外、実施例１’と同様にして、ビオチン化ペプチドを合成した（それぞれ、化合物３１～３２とする）：
　N-RRRQRRKKRGYGDIMGEWGNEIFGAIAGFLG-C（ペプチド３１）
　N-YGRKKRRQRRR-C（ペプチド３２）
　実施例３１及び３２ならびに比較例３及び４
　市販のストレプトアビジン蛍光性量子ドット（QD）（QD655, invitrogen; 直径30nm）1 μｍｏｌに前述の化合物11及び26を1,2,4,8,12,16,20までの様々なモル比で反応させ、様々な数のターゲット分子細胞内導入用ペプチドをQD上に重合させた（これを価数と表現し、x価の場合、１個のQD上にx個のペプチドが結合していることになる）（価数１～２０の複合体をそれぞれ複合体11－1～11－20及び26－1～26－20とする）。実験では生細胞としてHeLa細胞を用い、HeLa細胞は細胞培養液X-VIVO bufferで培養した。複合体11－1～11－20及び26－1～26－20、50 pMをendosome染色試薬0.25%のDiOと同時に細胞に添加して1時間後に細胞を培養液で洗浄して、さらに2時間培養後に、文献1.Duan H, Nie S. J Am Chem Soc. (2007) Vol. 129,3333-8.と2.Imamura J, Suzuki Y, Gonda K, Roy CN, Gatanaga H, Ohuchi N, Higuchi H. J Biol Chem. (2011) Vol.286, 10581-92.に記載の方法に従って一分子顕微鏡で観察を行い何%のQD粒子がエンドソームに残存するか定量的に観察した。この結果、力価が2,4,8、16、20価と価数が上昇するに従い、QD粒子の取り込みの向上が観察された（図２A）。 Example 2'-6 '
In the Fmoc solid phase synthesis method, the PTD side was biotinylated by using a biotinylated amino acid for the extension of the last amino acid (N-terminal G in peptide 1) (compounds 3, 8, 11, 26). The molecular weight of each compound is as follows:
Compound 3, molecular weight 4394.1
Compound 8, molecular weight 4316.9
Compound 11, molecular weight 4246.9
Compound 26, molecular weight 4329.9
A peptide having the following amino acid sequence and biotinylated at the N-terminus was also synthesized in the same manner (Compound 33).
N-GYWGEILGEWGGEIFEAIAEFLGRRRRRRRRRRR-C (peptide 33, molecular weight 4506.2)
Comparative Examples 1 and 2
A biotinylated peptide was synthesized in the same manner as Example 1 ′ except that a peptide consisting of the following amino acid sequence was synthesized (referred to as compounds 31 to 32, respectively):
N-RRRQRRKKRGYGDIMGEWGNEIFGAIAGFLG-C (Peptide 31)
N-YGRKKRRQRRR-C (peptide 32)
Examples 31 and 32 and Comparative Examples 3 and 4
Reaction of above-mentioned compounds 11 and 26 with various molar ratios up to 1,2,4,8,12,16,20 to 1 μmol of commercially available streptavidin fluorescent quantum dots (QD) (QD655, invitrogen; diameter 30 nm) Then, various numbers of peptides for introduction into the target molecule were polymerized on QD (this is expressed as valence, and in the case of x valence, x peptides must be bound on one QD. (The complexes having a valence of 1 to 20 are referred to as complexes 11-1 to 11-20 and 26-1 to 26-20, respectively). In the experiment, HeLa cells were used as living cells, and the HeLa cells were cultured in a cell culture medium X-VIVO buffer. Complexes 11-1 to 11-20, 26-1 to 26-20, and 50 pM were added to the cells at the same time as endosome staining reagent 0.25% DiO. After 1 hour, the cells were washed with the culture medium, and then 2 hours. After culture, reference 1.Duan H, Nie S. J Am Chem Soc. (2007) Vol. 129,3333-8. And 2.Imamura J, Suzuki Y, Gonda K, Roy CN, Gatanaga H, Ohuchi N, Higuchi According to the method described in H. J Biol Chem. (2011) Vol.286, 10581-92., Observation was carried out with a single molecule microscope to quantitatively observe the percentage of QD particles remaining in the endosome. As a result, an improvement in QD particle uptake was observed as the titer increased to 2, 4, 8, 16, 20 and the valence (FIG. 2A).
　次に、比較例として１及び２で合成した化合物３１及び３２を用いる以外、実施例３１と同様にして、複合体３１及び３２を調製し、QD粒子のエンドソームへの残存率を測定した。この結果、コントロールとして複合体３２（HIV-1 Tat由来）（我々の検討で最も効率が高い8価）と、現時点で最も強力な細胞導入能を有するとされる複合体３１（HA-PTD）（同様の検討で最も効率が高い20価）を用いて観察しても、それぞれ平均で0.2%と2%前後しか細胞内に導入されないことが明らかになった(図3)。他方新たに合成した複合体ではエンドソームからの遊出が著名に改善し、うち２つの複合体（複合体11－20及び26－20）では、平均値でQDの60～70％がエンドソームから遊出し（図3）、高いものではQDの70-90%近くがエンドソームから遊出することが明らかになった。 Next, composites 31 and 32 were prepared in the same manner as in Example 31 except that compounds 31 and 32 synthesized in 1 and 2 were used as comparative examples, and the residual ratio of QD particles to endosomes was measured. As a result, complex 32 (derived from HIV-1 Tat) (octavalent with the highest efficiency in our study) and complex 31 (HA-PTD), which is considered to have the most powerful cell introduction ability at present, as a control (20, which has the highest efficiency in the same study), it was revealed that only 0.2% and 2% on average were introduced into cells (Fig. 3). On the other hand, newly synthesized complexes have markedly improved migration from endosomes, with two complexes (complexes 11-20 and 26-20) averaging 60-70% of the QD migrating from endosomes on average. It was revealed that nearly 70-90% of QDs migrate from endosomes at high levels (Figure 3).
　ストレプトアビジンQDに化合物11及び26を同様に様々な濃度で重合させ、様々な力価（複合体11－1～11－20及び26－1～26－20）で作成した複合体11－1～11－20及び26－1～26－20を30pM、Hela細胞に添加して、45分後に細胞内に取り込まれた１個の細胞あたりのQD数で比較した場合、20価複合体11及び26は、2価の複合体３２（HIV-1 TatPTD-QD）と比較して、取り込まれる数でそれぞれ7.5倍および5倍改善した（図2B）。この結果、細胞内に実際に導入されるQD数としては20価複合体11及び26で2価の複合体３２と比較して、それぞれ2000倍および1740倍改善したことになった。以上のことより、QD導入能において、従来のPTDに対して1000倍以上の改善を行うことが可能であることが示された。 Compounds 11 and 26 were similarly polymerized at various concentrations to streptavidin QD, and complexes 11-1 to 11 were prepared with various titers (complexes 11-1 to 11-20 and 26-1 to 26-20). When 11-20 and 26-1 to 26-20 were added to 30 pM Hela cells and compared with the number of QDs per cell incorporated into the cells after 45 minutes, 20- valent complexes 11 and 26 Compared to the bivalent complex 32 (HIV-1 TatPTD-QD), the number incorporated was improved by 7.5 times and 5 times, respectively (Fig. 2B). As a result, the number of QDs actually introduced into the cells was improved by 2000 times and 1740 times in the 20- valent complex 11 and 26 compared to the divalent complex 32, respectively. From the above, it was shown that the QD introduction ability can be improved 1000 times or more compared to the conventional PTD.
　複合体11及び26をQD上に15分子をSS結合を用いて重合させて、さらにHaloTagリガンド分子(Halo-L)（この実験では確実にHaloTagを認識できるように8分子NH2を用いて結合させた）を表面に結合させたQD(QD 564)の合成を四国産総研に依頼し作成した。このQD粒子を用いてミトコンドリア外膜蛋白Mitofusin-2(MFN2)を標的分子に選んで可視化できるか検討した。 Complexes 11 and 26 were polymerized on QD with 15 molecules using SS bonds, and further coupled with HaloTag ligand molecule (Halo-L) (8 molecules NH2 to ensure recognition of HaloTag in this experiment). QD (QD 564) with the surface bonded to the surface was commissioned to Shikoku AIST. We investigated whether the mitochondrial outer membrane protein Mitofusin-2 (MFN2) can be selected and visualized using these QD particles.
　HaloTag付加MFN2発現ベクターを細胞に導入後48時間経過した時点で、細胞培養液に合成したQDを15pM濃度で添加し、2時間後、細胞内に取り込まれなかったQDを洗浄し、さらに30分間培養後、一分子顕微鏡を用いて細胞内QDを観察した(図４)。MFN2とQDが共局在するか明らかにする目的で、HaloTag付加MFN2は更にHaloTag TMR Ligandで可視化した。その結果、我々の作成したQDは2時間ほどで細胞に取り込まれて、HaloTag融合MFN2に結合し、高速度でミトコンドリア表面を往復運動することが示された(図４)。一方HaloTag付加MFN2発現ベクターを細胞に導入していない細胞ではQDのミトコンドリア上への集積は認められず細胞内にびまん性に集積していた。以上より、我々が開発した複合体11及び26とHalo-LをQDに結合させた系を活用すると簡便な操作で、生細胞内分子の一分子イメージングが可能であることが示された。 48 hours after the introduction of the HaloTag-added MFN2 expression vector into the cells, the synthesized QD was added to the cell culture solution at a concentration of 15 pM, and after 2 hours, the QD that had not been taken up into the cells was washed, and another 30 minutes After culture, intracellular QD was observed using a single molecule microscope (FIG. 4). In order to clarify whether MFN2 and QD colocalize, HaloTag-added MFN2 was further visualized with HaloTag TMR Ligand. As a result, it was shown that the QD we created was taken up by the cells in about 2 hours, bound to the HaloTag fusion MFN2, and reciprocated on the mitochondrial surface at high speed (FIG. 4). On the other hand, QD did not accumulate on the mitochondria in cells that had not been introduced with the HaloTag-added MFN2 expression vector. From the above, it was shown that single molecule imaging of molecules in living cells is possible with simple operation by utilizing the complex 11 and 26 and Halo-L coupled to QD that we developed.
　本発明のターゲット分子細胞内導入用ペプチドを用いることにより、ターゲット分子を単にエンドソーム内に封入されたかたちで導入するだけでなく、エンドソームに小孔を生じさせて細胞質にまで導入することができる。従って、ターゲット分子の細胞質における機能の研究に非常に有用である。また、多くの医薬の作用点は細胞質内にあると考えられるため、ドラッグデリバリーシステムへの応用の点からも非常に重要である。本発明のターゲット分子細胞内導入用ペプチドの用途としては、例えば、急性虚血性心疾患による心筋の虚血性ダメージに対して、アンチアポプトシスペプチドを冠動脈から導入し、細胞内に直接導入することで心筋を保護することに用いること等が挙げられる。 By using the peptide for introducing a target molecule into the cell of the present invention, it is possible not only to introduce the target molecule in the form of being encapsulated in the endosome but also to introduce a small pore in the endosome into the cytoplasm. Therefore, it is very useful for studying the function of the target molecule in the cytoplasm. In addition, since the action point of many drugs is considered to be in the cytoplasm, it is very important from the viewpoint of application to a drug delivery system. Examples of the use of the peptide for intracellular introduction of a target molecule of the present invention include introduction of an anti-apoptotic peptide from a coronary artery against a myocardial ischemic damage caused by acute ischemic heart disease and directly introducing it into the cell. For use in protecting the myocardium.
配列番号１　膜融合ペプチド
配列番号２　膜融合ペプチド
配列番号３　膜融合ペプチド
配列番号４　膜融合ペプチド
配列番号５　膜融合ペプチド
配列番号６　膜融合ペプチド
配列番号７　膜融合ペプチド
配列番号８　膜融合ペプチド
配列番号９　膜融合ペプチド
配列番号１０　膜融合ペプチド
配列番号１１　膜融合ペプチド
配列番号１２　蛋白質導入ドメイン
配列番号１３　蛋白質導入ドメイン
配列番号１４　蛋白質導入ドメイン
配列番号１５　蛋白質導入ドメイン
配列番号１６　蛋白質導入ドメイン
配列番号１７　蛋白質導入ドメイン
配列番号１８　蛋白質導入ドメイン
配列番号１９　蛋白質導入ドメイン
配列番号２０　蛋白質導入ドメイン
配列番号２１　膜融合ペプチド
配列番号２２　膜融合ペプチド
配列番号２３　ターゲット分子細胞内導入用ペプチド
配列番号２４　ターゲット分子細胞内導入用ペプチド
配列番号２５　ターゲット分子細胞内導入用ペプチド
配列番号２６　ターゲット分子細胞内導入用ペプチド
配列番号２７　ターゲット分子細胞内導入用ペプチド
配列番号２８　ターゲット分子細胞内導入用ペプチド
配列番号２９　ターゲット分子細胞内導入用ペプチド
配列番号３０　ターゲット分子細胞内導入用ペプチド
配列番号３１　ターゲット分子細胞内導入用ペプチド
配列番号３２　ターゲット分子細胞内導入用ペプチド
配列番号３３　ターゲット分子細胞内導入用ペプチド
配列番号３４　ターゲット分子細胞内導入用ペプチド
配列番号３５　ターゲット分子細胞内導入用ペプチド
配列番号３６　ターゲット分子細胞内導入用ペプチド
配列番号３７　ターゲット分子細胞内導入用ペプチド
配列番号３８　ターゲット分子細胞内導入用ペプチド
配列番号３９　ターゲット分子細胞内導入用ペプチド
配列番号４０　ターゲット分子細胞内導入用ペプチド
配列番号４１　ターゲット分子細胞内導入用ペプチド
配列番号４２　ターゲット分子細胞内導入用ペプチド
配列番号４３　ターゲット分子細胞内導入用ペプチド
配列番号４４　ターゲット分子細胞内導入用ペプチド
配列番号４５　ターゲット分子細胞内導入用ペプチド
配列番号４６　ターゲット分子細胞内導入用ペプチド
配列番号４７　ターゲット分子細胞内導入用ペプチド
配列番号４８　ターゲット分子細胞内導入用ペプチド
配列番号４９　ターゲット分子細胞内導入用ペプチド
配列番号５０　ターゲット分子細胞内導入用ペプチド
配列番号５１　ターゲット分子細胞内導入用ペプチド
配列番号５２　ターゲット分子細胞内導入用ペプチド SEQ ID NO: 1 Membrane fusion peptide SEQ ID NO: 2 Membrane fusion peptide SEQ ID NO: 3 Membrane fusion peptide SEQ ID NO: 4 Membrane fusion peptide SEQ ID NO: 5 Membrane fusion peptide SEQ ID NO: 6 Membrane fusion peptide SEQ ID NO: 7 Membrane fusion peptide SEQ ID NO: 8 Membrane fusion peptide SEQ ID NO: 9 Membrane fusion peptide SEQ ID NO: 10 Membrane fusion peptide SEQ ID NO: 11 Membrane fusion peptide SEQ ID NO: 12 Protein introduction domain SEQ ID NO: 13 Protein introduction domain SEQ ID NO: 14 Protein introduction domain SEQ ID NO: 15 Protein introduction domain SEQ ID NO: 16 Protein introduction domain SEQ ID NO: 17 Protein Introducing Domain SEQ ID NO: 18 Protein Introducing Domain SEQ ID NO: 19 Protein Introducing Domain SEQ ID NO: 20 Protein Introducing Domain SEQ ID NO: 21 Membrane Fusion Peptide SEQ ID NO: 22 Membrane Fusion Peptide SEQ ID NO: 23 Peptide SEQ ID NO: 25 for introduction into a get molecule cell Peptide No. 26 for introduction into a target molecule cell Peptide SEQ ID NO: 27 for introduction into a target molecule cell Peptide sequence number 28 for introduction into a target molecule cell Peptide sequence number 29 for introduction into a target molecule cell Peptide sequence number 30 for introduction into target molecule cell Peptide sequence number 31 for introduction into target molecule cell Peptide sequence number 32 for introduction into target molecule cell Peptide sequence number 33 for introduction into target molecule cell Peptide sequence number 34 for introduction into target molecule cell Peptide SEQ ID NO: 35 for target molecule intracellular introduction Peptide SEQ ID NO: 36 for target molecule intracellular introduction Peptide SEQ ID NO: 37 for target molecule intracellular introduction Peptide SEQ ID NO: 38 for target molecule intracellular introduction Peptide sequence number 39 for introduction into cells Peptide sequence number 40 for target molecule introduction into cells Peptide sequence number 41 for introduction into target molecule cells Peptide sequence number 42 for introduction into target molecule cells Peptide sequence number 43 for introduction into intracellular cells Target molecule Peptide SEQ ID NO: 44 for introduction into cells Peptide SEQ ID NO: 45 for introduction into target molecules Peptide SEQ ID NO: 46 for introduction into cells Target molecule Peptide No. 47 for introduction into cells Target molecule Peptide SEQ ID No. 48 into introduction into cells Target molecule Peptide sequence number 49 for introduction into cell Peptide sequence number 50 for introduction into target molecule cell Peptide sequence number 51 for introduction into target molecule cell Peptide sequence number 52 for introduction into target molecule cell Peptide for introduction into target molecule cell Plastid

Claims (11)

  1. 　ｐＨ依存性膜融合ペプチド及び蛋白質導入ドメイン（PTD）を有するターゲット分子細胞内導入用ペプチドであって、該膜融合ペプチドの構成アミノ酸数が２１以上である、ターゲット分子細胞内導入用ペプチド。 A target molecule intracellular introduction peptide having a pH-dependent membrane fusion peptide and a protein introduction domain (PTD), wherein the membrane fusion peptide has 21 or more constituent amino acids.
  2. 　前記膜融合ペプチドが配列Ｘ１Ｘ２Ｘ３
    ［式中、Ｘ１、Ｘ２及びＸ３は同一又は異なって、Ｗ、Ｈ、Ｇ、Ｆ、Ｙ、Ａ、Ｃ、Ｌ、又はＴを示す］
    を有する、請求項１に記載のターゲット分子細胞内導入用ペプチド。     The membrane fusion peptide has the sequence X 1 X 2 X 3
    [Wherein X 1 , X 2 and X 3 are the same or different and represent W, H, G, F, Y, A, C, L, or T]
    The peptide for intracellular introduction | transduction of the target molecule | numerator of Claim 1 which has these.
  3. 　下記のいずれかである請求項１に記載のターゲット分子細胞内導入用ペプチド：
    （１）前記膜融合ペプチドのアミノ酸配列がGLFGAIAGFIENGWEGMIDGWYGF、GLFGAIAGFIENGWEGMIDGWYG、GFFGAIAGFLEGGWEGMIAGWHGY、GFFGAIAGFLEGGWEGMIAGWHG、LAGVIMAGVAIGIATAAQITAGVALY、GTFTWTLSDSEGKDTPGGYCLT、GTFTWTLSDSSGVENPGGYCLT、AFFSWSLTDSSGKDTPGGYCL、AFFSWSLTDSSGKDMPGGYCL、AFFSWSLSDPKGNDMPGGYCLもしくはGIFSWTITDAVGNDMPGGYCL であるターゲット分子細胞内導入用ペプチド
    （２）前記膜融合ペプチドのアミノ酸配列がGLFGAIAGFIENGWEGMIDGWYGF、GLFGAIAGFIENGWEGMIDGWYG、GFFGAIAGFLEGGWEGMIAGWHGY、GFFGAIAGFLEGGWEGMIAGWHG、LAGVIMAGVAIGIATAAQITAGVALY、GTFTWTLSDSEGKDTPGGYCLT、GTFTWTLSDSSGVENPGGYCLT、AFFSWSLTDSSGKDTPGGYCL、AFFSWSLTDSSGKDMPGGYCL、AFFSWSLSDPKGNDMPGGYCLもしくはGIFSWTITDAVGNDMPGGYCLで示されるアミノ酸配列において１個又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列であり、かつ細胞膜透過性を有するターゲット分子細胞内導入用ペプチド。     The peptide for introducing a target molecule into cells according to claim 1, which is any of the following:
    (1) the membrane fusion amino acid sequence of the peptide GLFGAIAGFIENGWEGMIDGWYGF, GLFGAIAGFIENGWEGMIDGWYG, GFFGAIAGFLEGGWEGMIAGWHGY, GFFGAIAGFLEGGWEGMIAGWHG, LAGVIMAGVAIGIATAAQITAGVALY, GTFTWTLSDSEGKDTPGGYCLT, GTFTWTLSDSSGVENPGGYCLT, AFFSWSLTDSSGKDTPGGYCL, AFFSWSLTDSSGKDMPGGYCL, AFFSWSLSDPKGNDMPGGYCL or target molecules intracellular introduction peptide is GIFSWTITDAVGNDMPGGYCL (2) amino acids of the fusogenic peptide sequence GLFGAIAGFIENGWEGMIDGWYGF, GLFGAIAGFIENGWEGMIDGWYG, GFFGAIAGFLEGGWEGMIAGWHGY, GFFGAIAGFLEGGWEGMIAGWHG, LAGVIMAGVAIGIATAAQITAGVALY, GTFTWTLSDSEGKDTPGGYCLT, GTFTWTLSDSSGVENPGGYCLT, AFFSWSLTDSSGKDTPGGYCL, AFFSWSLTDSSGKDMPGGYCL, an amino acid sequence wherein one or several amino acids in the amino acid sequence shown are deleted, substituted or added in AFFSWSLSDPKGNDMPGGYCL or GIFSWTITDAVGNDMPGGYCL, And a peptide for introduction into a target molecule having cell membrane permeability.
  4. 　　下記のいずれかである請求項１～３のいずれか１項に記載のターゲット分子細胞内導入用ペプチド：
    （１）前記PTDのアミノ酸配列が、YGRKKRRQRRR、RRRRRRRRRRR、もしくはRQIKIWFQNRRMKWKK、であるターゲット分子細胞内導入用ペプチド
    （２）前記PTDのアミノ酸配列がYGRKKRRQRRR、RRRRRRRRRRRもしくはRQIKIWFQNRRMKWKKで示されるアミノ酸配列において１個又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列であり、かつ細胞膜透過性を有する該ターゲット分子細胞内導入用ペプチド。     The target molecule intracellular introduction peptide according to any one of claims 1 to 3, which is any of the following:
    (1) The peptide for introduction into a target molecule cell, wherein the amino acid sequence of the PTD is YGRKKRRQRRR, RRRRRRRRRRR, or RQIKIWFQNRRMKWKK (2) One or several amino acids in the amino acid sequence represented by YGRKKRRQRRR, RRRRRRRRRRR, or RQIKIWFQNRRMKWKK The peptide for introduction into a target molecule, which has an amino acid sequence in which one amino acid is deleted, substituted or added and has cell membrane permeability.
  5. 請求項１～４のいずれか１項に記載のターゲット分子細胞内導入用ペプチドとターゲット分子とを結合させてなるターゲット分子複合体。 A target molecule complex obtained by binding the target molecule intracellular introduction peptide according to any one of claims 1 to 4 and the target molecule.
  6. ターゲット分子１個に対し、ターゲット分子細胞内導入用ペプチドが２個以上結合されている、請求項５に記載の複合体。 6. The complex according to claim 5, wherein two or more peptides for introduction into the target molecule cell are bound to one target molecule.
  7. 請求項１～４のいずれか１項に記載のターゲット分子細胞内導入用ペプチドをコードするポリヌクレオチドを含むベクター。 A vector comprising a polynucleotide encoding the peptide for introduction into a target molecule according to any one of claims 1 to 4.
  8. 請求項６又は７に記載の複合体を有効成分として含む医薬組成物。 A pharmaceutical composition comprising the complex according to claim 6 or 7 as an active ingredient.
  9. 細胞に請求項６又は７に記載の複合体を添加する工程を含む、ターゲット分子を細胞内に導入する方法。 The method to introduce | transduce a target molecule into a cell including the process of adding the composite_body | complex of Claim 6 or 7 to a cell.
  10. 請求項１～４のいずれか１項に記載のターゲット分子細胞内導入用ペプチドを含む、ターゲット分子の細胞内導入剤。 A target molecule intracellular introduction agent comprising the target molecule intracellular introduction peptide according to any one of claims 1 to 4.
  11. ターゲット分子の細胞内導入剤を製造するための、請求項１～４のいずれか１項に記載のターゲット分子細胞内導入用ペプチドの使用。 Use of the peptide for introducing a target molecule into the cell according to any one of claims 1 to 4, for producing an agent for introducing the target molecule into the cell.
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