WO2008108505A1 - 新規核内移行ペプチド - Google Patents
新規核内移行ペプチド Download PDFInfo
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- WO2008108505A1 WO2008108505A1 PCT/JP2008/054563 JP2008054563W WO2008108505A1 WO 2008108505 A1 WO2008108505 A1 WO 2008108505A1 JP 2008054563 W JP2008054563 W JP 2008054563W WO 2008108505 A1 WO2008108505 A1 WO 2008108505A1
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- peptide
- amino acid
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Classifications
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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
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
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- A61K49/0013—Luminescence
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- A61K49/0056—Peptides, proteins, polyamino acids
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- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
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- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
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- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
- A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
- A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
- A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
- A61K49/1863—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being a polysaccharide or derivative thereof, e.g. chitosan, chitin, cellulose, pectin, starch
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- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
- A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
- A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
- A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
- A61K49/1869—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid coated or functionalised with a protein being an albumin, e.g. HSA, BSA, ovalbumin
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
- C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a peptide having translocation into a cell and nucleus, and a complex of the peptide and a functional molecule.
- PTD Protein Transduction Domain
- PTDs identified so far include HIV Tat-derived peptides (see E. Vives et al., J. Biological Chemistry, 272 (25), 16010, (1997)), Penetrat ⁇ n (D. Derossi et al., J. Biological Chemistry, 271 (30), 18188, (1996)), herpes simplex virus type 1 coat protein VP 22 (G. El Iot et al., Cell, 88, 223, (1997)).
- the tat peptide is the most well-known PTD.
- US Pat. No. 5,565,21 discloses that galactosidase or horseradish peroxidase was transported intracellularly using the tat peptide. Yes.
- C45 D 1 8 was identified, and C45D 1 8 and its enzyme protein complex are peptides that can move not only into the cytoplasm but also into the nucleus, and have a better transfer function than the tat peptide (See T. Taguchi et al., Biochem. Biophys. Res. Comm., 320, 18, (2004)). However, this document does not mention any amino acid sequence necessary for the expression of the translocation function.
- An object of the present invention is to provide a peptide capable of transporting a functional molecule not only into a cell but also into a nucleus more efficiently than a conventional PTD.
- the present inventors synthesized a peptide having a 20 amino acid residue represented by the amino acid sequence: RILQQLLFIHFRIGCRHSRI in the context of a negative target, and its translocation property.
- RILQQLLFIHFRIGCRHSRI the amino acid sequence represented by the amino acid sequence: RILQQLLFIHFRIGCRHSRI
- the transferability was much better than that of C45 D 1 8.
- a part of the amino acid sequence of C45D 18 was markedly both in the ability to move into the cell and into the nucleus compared to the conventional PTD.
- a peptide that is superior and less cytotoxic than C45D18 was identified and the present invention was completed.
- the present invention provides a peptide comprising the amino acid sequences RI and FI and RIGC and having 25 or less amino acid residues.
- the peptide of the present invention is expected to be put to practical use in fields that have been difficult to put into practical use because of its low migration function.
- the peptide of the present invention has lower cytotoxicity than C 45 D 18 which is a peptide, it is advantageous from the viewpoint of safety when applied to medical applications.
- the peptide of the present invention has a small number of amino acid residues, it is advantageous for cost reduction in addition to reduction of cytotoxicity when used industrially.
- FIG. 1 is a graph showing the results of the ability of various beptide CM DM complexes to translocate into HeLa cells and into the nucleus.
- FIG. 2 is a graph showing test results comparing the ability of the LR 17-CM DM complex to translocate into HeLa cells and into the nucleus depending on the particle diameter.
- Fig. 3 is a graph showing the results of various beptido CMDM complexes in the ability to translocate into neurons and into the nucleus.
- Fig. 4 is a graph showing the results of various peptide-CM DM complex migration test results for peripheral blood mononuclear cells.
- FIG. 5 is a graph showing the test results of the ability of the LR 15 DL-Qdot complex to translocate into He La cells and into the nucleus.
- FIG. 6 is a FACS analysis diagram showing the results of the EK293 T intracellular translocation test of the LR 15 DL-EGF P complex.
- FIG. 7 is a FACS analysis diagram showing the results of (His) 6 —LR 1 5 DL—EG FP chimeric protein translocation test in HEK 293 T cells.
- Figure 8 is a photomicrograph of the cells tested and compared for cytotoxicity of LR20, C45 D 18 and Vpr.
- Fig. 9 is a photograph of a petri dish after cell staining showing the result of examining the viability of cells after LR20-CMDM complex was taken up into cells and irradiated with a high frequency magnetic field.
- the peptide of the present invention (hereinafter sometimes abbreviated as the present peptide) and its production method, examples of functional molecules that can be combined with the peptide, methods for synthesizing complexes of the peptide and functional molecules, the complex
- the evaluation of the properties of the body, such as the ability to move into the cell and nucleus, and the target cells will be explained in turn.
- amino acid sequences may be indicated by the peptide numbers shown in Table 1 below for simplicity. Table 1. Amino acid sequence of each peptide
- the peptide of the present invention comprises an amino acid residue containing the amino acid sequences RI, FI, and RIGC as a minimum necessary part for exerting intracellular and nuclear translocation functions.
- the peptide is a peptide having a number of radicals of 25 or less, and the order of the amino acid sequences in the peptide molecule is preferably R and F in order from the N-terminus and then RIGC.
- the above amino acid sequence is the minimum sequence necessary for exhibiting the transfer function, and by adding another amino acid sequence before, after, or between the above amino acid sequences, a higher transfer ability can be obtained. It is also possible.
- a peptide comprising the amino acid sequences RI and FIHFRIGC in which HF is inserted between the amino acid sequences FI and RIGC and having 25 or less amino acid residues.
- a peptide represented by peptide number 4 RIFIHFRIGC
- the peptide represented by the amino acid sequence RI Xa QQ Xb Xc FIHFRIGC in which Xa QQ Xb Xc is further inserted between RI and FIHFRIGC (where Xa, Xb and Xc are the same or different, Each of which represents a standard amino acid, and “standard amino acid” means 20 kinds of amino acids that constitute proteins that function in mammals), in particular, the peptide represented by peptide number 3 (RILQQLLFIHFRI 6 C) Shows an even higher migration ability.
- a peptide in which a specific amino acid is added to such a minimum necessary sequence shows a migration amount that is about 10 to several tens of times higher than that of the conventional PTD tat peptide. Manufacturing method
- the peptide of the present invention can be produced by a peptide synthesis method known per se.
- peptide synthesis methods include solid phase synthesis (arrifield, J. Am. Chem. Soc., 85, 2149-2154, 1963). This peptide is currently automated using this principle. In addition, it can be produced relatively easily in a relatively short time using a widely used peptide synthesizer.
- this peptide can also be produced by a method known per se by genetic engineering as shown in the literature (Methods in Enzymolog, 154, 350, 367-382, 1987).
- the peptide of the present invention since the peptide of the present invention has excellent intracellular and nuclear translocation ability, it is used as a vector.
- the peptide and functional molecules or functional particles are bound to each other. It can be applied to producing a complex and transferring it into a cell or nucleus where the function of the molecule or particle is expressed.
- the molecules and particles that can be combined with this peptide can be selected from a very wide range.
- the functional molecule examples include bioactive substances such as nucleic acids (DNA, RNA, etc.), amino acids (proteins, peptides, etc.), lipids, saccharides, and other polymer compounds: functionalities such as fluorescent substances
- functional particles include magnetic particles, fluorescent particles, and ribosomes, which can be used alone or in combination of two or more. . In particular, nano-sized particles are preferred.
- functional molecules and functional particles may be collectively referred to as functional molecules.
- Nucleic acids DNA, RNA, etc.
- nucleic acids that can be used for binding to the peptide of the present invention general nucleic acids such as plasmid DNA, mRNA, siRNA and the like can be used without limitation.
- this peptide can be used in one application.
- a form capable of expressing the function in cells is preferably used.
- suitable DNA is one in which DNA is transcribed in the introduced cell and the bioactive substance produced expresses the desired function.
- nucleic acids examples include cypress force-in genes (eg, TNF-a, IL), cancer antigen peptide genes (eg, gp-100, 1 ⁇ 1 8-chome-1) And LDL receptor gene.
- cypress force-in genes eg, TNF-a, IL
- cancer antigen peptide genes eg, gp-100, 1 ⁇ 1 8-chome-1
- LDL receptor gene egp-100, 1 ⁇ 1 8-chome-1
- genes for the treatment of diabetes arteriosclerosis, Alzheimer's disease, etc.
- amino acids that can be used for binding to the peptide of the present invention for example, antibodies and enzymes that express some physiological activity in the cell by being transported into the target cell are preferable.
- proteins that can be used as antigens can be generally used because they induce cytotoxic T cells, and cell cycle regulatory proteins (for example, cyclins, cyclins).
- Dependent kinases antibodies (eg, HE R2 antibodies), enzymes (eg, ⁇ -galactosidase, chloramphenicol transferase), immortalized proteins (eg, SV40 large antigen, telomerase, etc.), anti-apoptotic proteins (eg, For example, mutant ⁇ 53, Be I x L, etc.) can also be used.
- the ⁇ (c) method of binding the peptide of the present invention to a functional molecule will be described in detail later, but in addition to this, the amino acid sequence of the peptide of the present invention is incorporated in advance using bacteria. Amino acids can also be synthesized biologically. Further, the bonding position may be either the N-terminal side or the C-terminal side.
- Examples of a method for obtaining amino acids in which the amino acid sequence of the peptide of the present invention is preliminarily incorporated include amino acids desired to be transported to plasmids generally used as expression vectors and amino acids of the peptide of the present invention.
- An example is a method obtained by inserting a DNA encoding a sequence and then expressing it in E. coli or the like. According to this method, when the desired amino acids are obtained, the ability to transport them into the cell can be imparted at the same time, which is advantageous in terms of cost in practical use.
- the peptide-introduced amino acids of the invention and this method are also encompassed by the present invention.
- the magnetic particles that can be used for binding to the peptide of the present invention generally have a wide variety of magnetic particles due to their form, physical properties, etc.
- the form preferably used for this peptide is excellent in biocompatibility and nano-sized. So-called magnetic nanoparticles having an overall diameter in the metric order (nanosize) range are preferred, but are not limited thereto.
- Such magnetic nanoparticles include, for example, Japanese Patent Publication No. 59-153 5 21, Japanese Patent No. 2 9 3 9 3 3 6, US Patent No. 4 4 5 2 7 7 3
- these particles are put to practical use as MRI contrast agents and for cell magnetic separation.
- carboxyalkyl polysaccharide monomagnetic metal oxides described in Japanese Patent No. 2 7 2 6 5 20, especially carboxymethyl dextran magnetite (hereinafter abbreviated as CMDM) are separated on the particle surface. It has a functional group that can be used for binding to a molecule, and can be used conveniently.
- the specific synthesis method is replaced with a detailed description with reference to the patent publication.
- the magnetic nanoparticles may have an overall diameter in the range of generally 1 to 50 nm, preferably 1 to 20 nm, more preferably 1 to 150 nm, depending on the application, for It can be appropriately selected according to the way.
- the total particle diameter is a value when measured with a laser single light scattering measurement apparatus.
- T 2 relaxation ability As an index of the magnetic properties of magnetic nanoparticles, for example, T 2 relaxation ability can be mentioned. This value should be as high as possible from the viewpoints of detection sensitivity in MR ⁇ and exothermicity in hyperthermia, generally 1 to 1 000 (mM 'sec), preferably 20 to 1 000 (mM ⁇ sec) — ', More preferably within the range of 50 to 1 000 (m M-sec) _'.
- the T 2 relaxation capacity is a value when measured with a pulse N MR device at 0.47 mm.
- ribosome that can be used for binding to the peptide of the present invention
- known liposomes can be used in general.
- DDS which is one of the uses of the peptide
- examples of such liposomes include temperature sensitive liposomes described in JP-A-2006-306784. The synthetic method and preferred physical properties are replaced with detailed descriptions by citation of the publication.
- the fluorescent particles that can be used for binding to the peptide of the present invention are not particularly limited in the wavelength and form of fluorescence, but in terms of size, of course, those that do not exceed the size of the cell, preferably nanometers. It is preferable to use one that is less than the order, especially one that has good biocompatibility. Examples of such particles include quantum dots. Among them, “Qdot®J, which was developed by Quantum Dot Inc. in the United States, is the most well-known example, but it is not limited to this. (c) Binding method of the peptide of the present invention and a functional molecule (Production method of complex)
- the binding of the peptide of the present invention and the functional molecule may be performed directly or indirectly through a single linker molecule. Furthermore, some functional molecules may be bound by simply mixing with the peptide of the present invention.
- the bonding can be performed by a method known per se. Specifically, for example, by using a functional group present at the terminal or inside of a biomolecule as a functional molecule, chemical bonding is performed. The method can be performed directly or indirectly through a linker molecule if direct binding is difficult. In this case, for example, amide bond, ester bond, thioester bond, ether bond, thioether bond, S—S bond, covalent bond such as ionic bond, electrostatic bond, etc.
- Non-covalent bonds such as intermolecular force bonds and hydrogen bonds, but are not limited to these.
- the linker molecule is not particularly limited as long as it is a molecule having a reactive group at both ends and a structure capable of connecting two molecules. Examples of the reactive group include, but are not limited to, maleimide group, N-succinimide ester group, epoxy group, and avidin group.
- this peptide to ribosomes or magnetic particles may be performed directly, but since there are relatively few reactive groups available on the particle surface that can be used for direct binding, Is done indirectly via some linker molecule.
- CMDM is used as the magnetic nanoparticles, a linker is introduced using the surface carboxyl group, and a peptide is further bound to the end of the linker. Is the best.
- the property that the complex of the peptide of the present invention should have is not particularly limited as long as it does not hinder the intracellular and nuclear translocation.
- the property that may hinder the migration includes, for example, size, and more specifically, the overall diameter of the complex.
- the mechanism of the intracellular and nuclear translocation of the peptide of the present invention is thought to be due to macropinocytosis, and there is no problem if the overall diameter is small, but if it is too large, it may not pass through the cell membrane or nuclear membrane. There is. Therefore, it is preferable that the complex has an overall diameter in the range of usually 5 to 500 nm, particularly 5 to 200 nm, more particularly 5 to 150 nm.
- present peptide nanoparticles composite suitable for the present invention produced as described above can have the following properties.
- the composite is preferably generally the same as that of generally used magnetic nanoparticles, although depending on the production conditions, usually 5 to 500 nm, preferably 5 to 20 O nm, depending on the application. Preferably it can have an overall diameter in the range of 5 to 150 nm.
- the T 2 relaxation capacity is preferably as high as possible, usually 1 to 1 000 (mM-sec)-', preferably 20 to 1000 (mM-sec)-', more preferably 50 to 1000 ( mM ⁇ sec) can be in the range of 1 .
- the content of the peptide in the peptide molecular functional complex described above can be changed depending on the use of the complex, the size of the functional molecule, etc., but the peptide functions sufficiently. And preferably as few as possible, usually in the range of 1-30, preferably 1-25, more preferably 1-20, per functional molecule.
- Intracellular and nuclear translocation Intracellular and nuclear translocation of the base Puchido magnetic nanoparticle complex was added to complex the culture fine alveolar, separately cytoplasmic and nuclear recovered after left for a certain time, T 2 relaxation time by NMR By measuring the content of the incorporated magnetic nanoparticles, the migration ability of the peptide of the present invention and the conventional PTD can be compared.
- the target cell to which the peptide functional molecule complex of the present invention can be transferred is not particularly limited as long as it does not have a cell wall, and may be of any type, dividing or non-dividing, It can be arbitrarily selected depending on the application.
- normal cells tumor cells, or immortalized cells from bacteria to insects and mammals.
- mammalian normal cells, tumor cells or immortalized cells eg, CHO, COS, etc.
- varicella normal cells, tumor cells or immortalized cells eg, He La, Huh— 7, 2 9 3, MCF-7, nervous system cells, blood cell suspension cells, etc.
- the functional molecule complex into which the peptide of the present invention has been introduced has a markedly improved ability to migrate into cells and even into the nucleus. It can be used for transporting into the nucleus and can be applied to various applications depending on the properties of the functional molecule. For example, if a gene is used as a functional molecule, it can be applied to gene transfer, and if a cytotoxic molecule is used, it can be applied as DDS.
- the peptide of the present invention is a cell of various functional molecules. If it is an application form that uses the function as a nuclear vector, it can be used widely without being limited to these. In that case, it is also possible to use the peptide of the present invention in combination with another intracellular migration molecule.
- the gene can be introduced into non-dividing cells by binding the peptide of the present invention to a positively charged polymer that functions as a gene transfer vector. Further, by binding the peptide of the present invention on the surface of a virus particle for gene expression, it becomes possible to transport the virus particle directly into the nucleus and to express a foreign gene.
- the magnetic particle composite Fever and can be applied to hyperthermia.
- the magnetic nanoparticle complex vibrates simultaneously with heating, it is possible to impart mechanistic cytotoxicity that damages DNA and nuclear membrane functions in the nucleus.
- CMDM and large-particle CMDM having the following properties 1) and 2) synthesized according to the method described in Japanese Patent No. 2726520 were used.
- Iron concentration 44.2 mg / m I (iron yield 83%), magnetic iron oxide particle size: 5.1 nm, total particle size: 40 nm, CMD iron weight ratio: 0.7, relaxation capacity: 32 (mM-sec) — ', T 2 relaxation ability: 1 21 (mM-sec)
- the peptide LR20 is programmed using the amino acid 20-residue sequence of the title peptide LR20.
- An amino acid reagent for peptide synthesis necessary for the production of the product was set and automatic synthesis was performed.
- the peptide is separated from the carrier resin using TFA, the resin is removed, ether is added and the mixture is centrifuged (3000 r pmx 5 min), and the precipitated peptide is lyophilized to obtain the peptide. It was.
- the obtained peptide was measured for molecular weight by mass spectrum (Applied Biosyst EMS, Voyager RP type), and HP LC (manufactured by Kanto Chemical Co., Migtysi IRP—18 GP column, The purity was measured from the peak area with a detection wavelength of 220 nm and an eluate of 0.1% T FAZ H 2 0), and it was confirmed that the obtained peptide was LR 20 peptide.
- CMDM 8m I with an overall diameter of 40 nm (iron concentration 20 mgm I), 1-ethyl 3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) 623 mg, N-hydroxysuccinic acid Imide (hereinafter referred to as N HS) 1 87 mg and N-ethylaminomaleimide 'trifluoroacetate 22 mg of 0.2 M sodium borate buffer solution 8 ml in this order are sequentially added, and the reaction is carried out at room temperature. After 20 hours, ultrafiltration (fractional molecular weight 50.000 Dalton) was performed to remove unnecessary reagents, and a linker-bound CMDM aqueous solution 28 ml was obtained.
- EDC 1-ethyl 3- (3-dimethylaminopropyl) carbodiimide hydrochloride
- N HS N-hydroxysuccinic acid Imide
- N HS N-ethylaminomaleimide 'trifluor
- Example 1 synthesis and analysis were performed in the same manner as in Example 1 except that the sequence programming and the necessary set of amino acid reagents were matched to the sequence of LR 17 to obtain the LR 17 peptide.
- Example 2 LR 1 7-CMDM aqueous sol was obtained in the same manner as in Example 2 except that the added peptide was LR 1 7 (peptide No. 2) 8.4 mg.
- Example 1 synthesis and analysis were performed in the same manner as in Example 1 except that the sequence programming and the necessary amino acid reagent set were matched to the sequence of LR 15 to obtain LR 15 peptide.
- Example 2J LR 1 5—CMDM aqueous sol was obtained in the same manner as in Example 2 except that the peptide to be added was LR 15 (peptide number 3) 7.2 mg.
- Example 1 synthesis and analysis were performed in the same manner as in Example 1 except that the sequence programming and the necessary amino acid reagent set were matched to the sequence of LR 15 DL, and LR 15 DL peptide was obtained. .
- Example 2 LR1 5 DL-CMDM aqueous sol was obtained in the same manner as in Example 2 except that the added peptide was LR 15 DL (Peptide No. 4) 2.5 mg (Complex No. 4) .
- Example 9 Synthesis of LR8DH F (peptide number 5)
- Example 1 synthesis and analysis were performed in the same manner as in Example 1 except that the sequence programming and the necessary set of amino acid reagents were matched to the sequence of LR 8 DH F, to obtain LR 8 DH F peptide.
- Example 2 the same procedure as in Example 2 was carried out except that the peptide to be added was LR 8 DH F (peptide number 5) 3.8 mg, to obtain an LR8 DH F-CMDM aqueous sol (complex number 5). .
- Example 1 synthesis and analysis were performed in the same manner as in Example 1 except that the sequence programming and the necessary set of amino acid reagents were matched to the sequence of LR 1 1 + GGC. LR 1 1 peptide (GGC addition product) Got.
- Example 1 synthesis and analysis were performed in the same manner as in Example 1 except that the sequence programming and the necessary set of amino acid reagents were matched to the sequence of LR 8 DH FR I, and LR 8 DH FR I peptide was obtained. .
- Example 2 except that the peptide to be added was LR8 DH FR ⁇ (peptide number 7) 4.5 mg, treatment was carried out in the same manner as in Example 2 to obtain an LR8 DH FR I—CM DM aqueous sol (complex Number 7).
- Example 1 synthesis and analysis were carried out in the same manner as in Example 1 except that the sequence programming and the necessary amino acid reagent set were matched to the sequence of LR 8 DR I HF, and LR8 DR I H F peptide was obtained.
- Example 2 except that the peptide to be added was LR8 DR IHF (peptide No. 8) 4.5 mg, the same treatment as in Example 2 was carried out to obtain LR8 DR IHF-CMDM aqueous sol (Complex No. 8) .
- Example 1 synthesis and analysis were carried out in the same manner as in Example 1 except that the sequence programming and the necessary amino acid reagent settings were matched to the C 45 D 18 sequence, and the C 45 D 18 peptide was obtained. Obtained.
- Example 2 except that the peptide to be added was C45 D 1 8 (Peptide No. 9) 1 2.5 mg, it was treated in the same manner as in Example 2 to obtain C45 D 1 8—CMDM aqueous sol (complex Number 9).
- Example 1 Synthesis of Penetratin—CM DM
- Example 2 Penetratin-CMDM aqueous sol was prepared in the same manner as in Example 2 except that the peptide to be added was 9.5 mg obtained by adding GGC to the C-terminus of Penetratin (peptide number 10). Obtained (complex number 10).
- Example 1 synthesis and analysis were performed in the same manner as in Example 1 except that the sequence programming and the necessary amino acid reagent settings were matched to the tat + GGC sequence to obtain the tat peptide (GGC addition product). It was.
- Example 2 the peptide to be added was obtained by adding GGC to the C-terminal end of the tat peptide (peptide number 11). 7. Treated in the same manner as in Example 2 except that Omg was used, and the tat peptide CMDM aqueous sol was prepared. Obtained (complex number 1 1).
- Example 1 Iron concentration: 7.3 mgZml, CMDZ iron weight ratio: 0.57, number of peptide bonds per magnetic nanoparticle: 1 0.1. Comparative Example 1 3: YM-3 (Peptide No. 1 2) Synthesis of GGC addition product In Example 1, synthesis and analysis were performed in the same manner as in Example 1, except that the sequence programming and the necessary amino acid reagent settings were matched to the YM-3 + GGC sequence.
- Example 2 the peptide to be added was YM-3 (Peptide No. 1 2) with G GC added to the C-terminus. An aqueous sol was obtained (complex number 1 2).
- Example 4 except that the CMDM used had a total diameter of 75 nm, it was treated in the same manner as in Example 4 to obtain LR 17 -large particle CMDM aqueous sol (complex number 13).
- P ET 15 b plasmid was used as an expression plasmid DNA expressing 6 histidines as tags.
- a DNA sequence encoding EG FP was inserted into this vector.
- a recombinant was prepared by inserting DNA encoding LR 15 DL (AGG ATC TTC ATC CAC TTC CGG ATC GGC TGC) into the N de I-BamHI site in this DNA.
- Each expression plasmid DNA was introduced into BL21 (manufactured by Novagen), which is an Escherichia coli for expressing a recombinant protein, and expressed.
- the obtained crude protein is purified by a affinity column (Model No.
- the supernatant is the cytoplasmic fraction
- the pellet is the nuclear fraction (add 0.5% Triton X-100 / PBS solution to the pellet and sonicate).
- the T 2 relaxation time of each fraction thus obtained was measured by NMR.
- the complex number “! To 5 consisting of peptides obtained by the present invention is several times to several tens of times higher than the complex numbers 9 to 12 consisting of existing peptides.
- the migration function is shown.
- Human peripheral blood mononuclear cells 2 X 1 0 6 or to the complex number 2 and 1 2 (F e Concentration: 0.7MgZinl adjustment in culture) was added.
- Human peripheral blood mononuclear cells were prepared from the subject's blood using Lymphoprep (AXIS-SHIELD). After 18 hours, the cells were collected, washed 3 times with PBS, suspended in 0.6 ml of PBS containing 1% Triton X-100, and sonicated. The T 2 relaxation time of the obtained sample was measured by NMR, and the magnetic substance concentration was calculated. The results are shown in Fig. 4.
- the final concentration of 6 nM of the LR 15 D LQ dot complex (complex number 1 4) obtained in Example 12 is used for 4 X 10 5 He La cells whose cell cycle has been stopped by thymidine. Were added as follows. After 16 hours, the cells were washed 3 times with PBS, detached with a scraper and collected. The cells were washed once with PBS, and disrupted and centrifuged (2000 r pmx 5 min) with a Dounce homogenizer. At this time, the supernatant is the cytoplasmic fraction, and the pellet is the nuclear fraction (add 0.5O6Triton X-100ZP BS solution to the pellets and sonicate). The fluorescence brightness of each of the fractions thus obtained was measured.
- the intracellular migration test was performed by the following method.
- LR15DL-EGFP (complex number 15) obtained in Example 13 was added to HEK2 93 T cells at a concentration of 10 gZmI and incubated at 37 ° C for 4 hours. The cells were treated with trypsin, and the amount of cells that emitted fluorescence upon incorporation of EG FP was measured by FACS (Fluorescence Activated Cell Sorting) analysis. The result is shown in FIG.
- Example 14 For the (His) 6 -LR 15DL-EGFP chimeric protein (complex number 16) synthesized in Example 14, the intracellular translocation test was performed by the following method.
- the peptide of the present invention was tested for damage to intracellular DNA by the following method.
- Vpr Vpr-derived protein
- C45D18 peptide number 9
- LR20 peptide number 1
- Test Example 8 High-frequency magnetic field treatment of cells incorporating the present peptide-one-magnetic nanoparticle complex
- LR20—CMDM (complex number 1) obtained in Example 1, the raw material CMDM and LR 20 peptide alone, to e L a cell, were added 800 U g Zm I as F e, to the cells after incubation for 12 hours, and radiation frequency 350 k H Z and magnetic field strength 21 mT 1 hour high-frequency magnetic field.
- Figure 9 shows the state of cell growth after 3 days. The stained part shows viable cells. From the results in Fig. 9, it can be seen that the 2 0- 01 ⁇ 1 0 1 ⁇ 1 complex tends to damage cells by magnetic field irradiation.
- This peptide can be used as a transformation vector by binding to nucleic acids intended for genetic modification.
- the peptide can also be used as a DDS for a target cell by binding to a drug or a drug inclusion compound.
- This peptide can also be used for molecular imaging, cell magnetic labeling, or high-frequency magnetic field processing to kill cancer cells by binding to magnetic particles that can generate heat by MR imaging and magnetic field irradiation. it can.
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US12/450,010 US8455616B2 (en) | 2007-03-07 | 2008-03-06 | Nuclear translocation peptide |
EP08738644.7A EP2130837B1 (en) | 2007-03-07 | 2008-03-06 | Novel nuclear translocation peptide |
JP2009502646A JP5403681B2 (ja) | 2007-03-07 | 2008-03-06 | 新規核内移行ペプチド |
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JP2007057387 | 2007-03-07 | ||
JP2007-057387 | 2007-03-07 |
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EP (1) | EP2130837B1 (ja) |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9347062B2 (en) | 2012-01-06 | 2016-05-24 | Arrowhead Research Corporation | Interfering RNA delivery system and uses thereof |
WO2018110471A1 (ja) | 2016-12-12 | 2018-06-21 | アステラス製薬株式会社 | 転写調節融合ポリペプチド |
WO2019117057A1 (ja) | 2017-12-11 | 2019-06-20 | アステラス製薬株式会社 | 細胞膜透過性ペプチド |
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US9856496B2 (en) | 2013-12-12 | 2018-01-02 | Life Technologies Corporation | Membrane-penetrating peptides to enhance transfection and compositions and methods for using same |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US565211A (en) | 1896-08-04 | Half to jesse b | ||
US4452773A (en) | 1982-04-05 | 1984-06-05 | Canadian Patents And Development Limited | Magnetic iron-dextran microspheres |
JP2726520B2 (ja) | 1989-10-20 | 1998-03-11 | 名糖産業株式会社 | 有機磁性複合体 |
JP2939336B2 (ja) | 1991-06-11 | 1999-08-25 | 名糖産業株式会社 | 水溶性カルボキシ多糖−磁性酸化鉄酸化複合体 |
JP2006306784A (ja) | 2005-04-28 | 2006-11-09 | Noguchi Inst | 高度にフッ素化されたアルコール誘導体と再利用が容易な使用法 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5670617A (en) | 1989-12-21 | 1997-09-23 | Biogen Inc | Nucleic acid conjugates of tat-derived transport polypeptides |
US6087486A (en) * | 1996-01-29 | 2000-07-11 | The Trustees Of The University Of Pennsylvania | Nucleotide sequences encoding vpr receptor protein |
US20030077826A1 (en) * | 2001-02-02 | 2003-04-24 | Lena Edelman | Chimeric molecules containing a module able to target specific cells and a module regulating the apoptogenic function of the permeability transition pore complex (PTPC) |
WO2005103654A2 (fr) * | 2004-04-09 | 2005-11-03 | Bioalliance Pharma | Methode d’identification de composes actifs sur la replication du virus hiv. |
-
2008
- 2008-03-06 WO PCT/JP2008/054563 patent/WO2008108505A1/ja active Application Filing
- 2008-03-06 EP EP08738644.7A patent/EP2130837B1/en not_active Not-in-force
- 2008-03-06 JP JP2009502646A patent/JP5403681B2/ja not_active Expired - Fee Related
- 2008-03-06 US US12/450,010 patent/US8455616B2/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US565211A (en) | 1896-08-04 | Half to jesse b | ||
US4452773A (en) | 1982-04-05 | 1984-06-05 | Canadian Patents And Development Limited | Magnetic iron-dextran microspheres |
JP2726520B2 (ja) | 1989-10-20 | 1998-03-11 | 名糖産業株式会社 | 有機磁性複合体 |
JP2939336B2 (ja) | 1991-06-11 | 1999-08-25 | 名糖産業株式会社 | 水溶性カルボキシ多糖−磁性酸化鉄酸化複合体 |
JP2006306784A (ja) | 2005-04-28 | 2006-11-09 | Noguchi Inst | 高度にフッ素化されたアルコール誘導体と再利用が容易な使用法 |
Non-Patent Citations (13)
Title |
---|
D. DEROSSI ET AL., J. BIOLOGICAL CHEMISTRY, vol. 271, no. 30, 1996, pages 18188 |
E. VIVES ET AL., J. BIOLOGICAL CHEMISTRY, vol. 272, no. 25, 1997, pages 16010 |
G. ELLIOT ET AL., CELL, vol. 88, 1997, pages 223 |
M. ZHAO ET AL., BICONJUGATE CHEM., vol. 13, 2002, pages 840 |
MARRIFIELD, J., AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2154 |
MASAKATSU HASEGAWA: "Jisei Nano Ryushi eno Peptide Fuka, Saitekika ni Kansuru Kenkyu", HYOTEKI PEPTIDE FUKAGATA KAN'ONSEI NANOMICELLE OYOBI KOSHUHA SHOTEN SHOSHA NI YORU KYOKUSHO DDS NO KAIHATSU' NI KANSURU KENKYU HEISEI 18 NENDO SOKATSU KENKYU NENDO SHURYO HOKOKUSHO, March 2007 (2007-03-01), pages 9 - 10 * |
MASAKATSU HASEGAWA: "Jisei Nano Ryushi eno Peptide Fuka, Saitekika ni Kansuru Kenkyu", PEPTIDE FUKAGATA KAN'ONSEI NANOMICELLE OYOBI KOSHUHA SHOTEN SHOSHA NI YORU KYOKUSHO DDS NO KAIHATSU NO TAME NO KIBAN KENKYU HEISEI 17 NENDO SOKATSU KENKYU HOKOKUSHO, 2006, pages 8 - 9 * |
METHODS IN ENZYMOLOGY, vol. 154, no. 350, 1987, pages 367 - 382 |
MIZOGUCHI, 1. ET AL.: "Improved gene expression in resting macrophages using an oligopeptide derived from Vpr of human immunodeficiency virus type-1", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 338, no. 3, 15 November 2005 (2005-11-15), pages 1499 - 1506, XP027218589 * |
See also references of EP2130837A4 |
T. TAGUCHI ET AL., BIOCHEM. BIOPHYS. RES. COMM., vol. 320, 2004, pages 18 |
TAGUCHI, T. ET AL.: "Nuclear trafficking of macromolecules by an oligopeptide derived from Vpr of human immunodeficiency virus type-1, Biochem", BIOPHYS. RES. COMMUN., vol. 320, no. 1, July 2004 (2004-07-01), pages 18 - 26, XP004519059 * |
Y. MUKAI ET AL., BIOL. PHARM. BULL., vol. 29, no. 8, 2006, pages 1570 |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9347062B2 (en) | 2012-01-06 | 2016-05-24 | Arrowhead Research Corporation | Interfering RNA delivery system and uses thereof |
WO2018110471A1 (ja) | 2016-12-12 | 2018-06-21 | アステラス製薬株式会社 | 転写調節融合ポリペプチド |
WO2019117057A1 (ja) | 2017-12-11 | 2019-06-20 | アステラス製薬株式会社 | 細胞膜透過性ペプチド |
JPWO2019117057A1 (ja) * | 2017-12-11 | 2021-01-21 | アステラス製薬株式会社 | 細胞膜透過性ペプチド |
EP3725884A4 (en) * | 2017-12-11 | 2021-10-13 | Astellas Pharma Inc. | PEPTIDE CAPABLE OF CROSSING THE CELLULAR MEMBRANE |
US11414456B2 (en) | 2017-12-11 | 2022-08-16 | Astellas Pharma Inc. | Cell penetrating peptide |
JP7250283B2 (ja) | 2017-12-11 | 2023-04-03 | アステラス製薬株式会社 | 細胞膜透過性ペプチド |
Also Published As
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US20100203611A1 (en) | 2010-08-12 |
EP2130837A1 (en) | 2009-12-09 |
EP2130837A4 (en) | 2010-08-04 |
JPWO2008108505A1 (ja) | 2010-06-17 |
US8455616B2 (en) | 2013-06-04 |
EP2130837B1 (en) | 2013-11-27 |
JP5403681B2 (ja) | 2014-01-29 |
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