Carrier peptide fragment and use thereof

Abstract

The technical problem of the present invention is to provide a novel vector peptide fragment having cell membrane permeability, and the disclosed vector peptide fragment contains the amino acid sequence: KFRAQRRW (SEQ ID NO: 1), said vector peptide fragment being introduced into at least the cytoplasm of a eukaryotic cell from the outside of the cell.

Description

Technical Field

[0001] The present invention relates to a method for introducing (transporting) an exogenous substance from the outside of a eukaryotic cell into the inside of the cell and a carrier peptide fragment used in the method. It should be noted that this application claims priority based on Japanese Patent Application No. 2022-166306 filed on October 17, 2022, and the entire content of this application is incorporated herein by reference as reference content in this specification. Background Art

[0002] Heretofore, attempts have been made to introduce exogenous substances such as polypeptides, particularly physiologically active substances, into cells (eukaryotic cells) of humans or mammals other than humans, and to change the properties of the cells (and tissues or organs composed of the cells) or improve and enhance the functions of the cells.

[0003] To date, the inventors of the present invention have created several carrier peptide fragments that can function as cell penetrating peptides (also abbreviated as "CPPs") that allow exogenous substances to cross the cell membrane from the outside of the cell into the cytoplasm. Some of them are disclosed in, for example, Patent Documents 1 to 5.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: Japanese Patent Publication No. 7041853

[0007] Patent Document 2: Japanese Patent Publication No. 7096990

[0008] Patent Document 3: International Publication No. 2011 / 013698

[0009] Patent Document 4: International Publication No. 2011 / 013699

[0010] Patent Document 5: International Publication No. 2011 / 013700 Summary of the Invention

[0011] Technical Problem to be Solved by the Invention

[0012] However, in constructs having a CPP and an exogenous substance, fluctuations in cell membrane permeability efficiency may occur depending on the combination of the amino acid sequence of the CPP and the type of the exogenous substance. In recent years, drug delivery techniques using CPPs have attracted much attention, and it is necessary to develop various CPPs to be able to select a CPP suitable for the exogenous substance (such as nucleic acid drugs, etc.) to be used.

[0013] The technology disclosed by the present invention was created in view of the above circumstances. The main object of the present invention is to provide a novel carrier peptide fragment with cell membrane permeability. Additionally, another object of the present invention is to provide a construct for introducing an exogenous substance having such a carrier peptide fragment. Furthermore, yet another object of the present invention is to provide a method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell.

[0014] Technical solutions for solving technical problems

[0015] The carrier peptide fragment disclosed by the present invention is a carrier peptide fragment for being introduced from the outside of a eukaryotic cell into at least the cytoplasm of the cell, and includes the amino acid sequence: KFRAQRRW (SEQ ID NO: 1).

[0016] The carrier peptide fragment with such a composition was discovered by the inventors of the present invention through in-depth research and is a synthetic peptide that can be artificially manufactured. This carrier peptide fragment has cell membrane permeability and can introduce exogenous substances from the outside of the cell into the inside of the cell (at least into the cytoplasm).

[0017] In addition, according to the present invention, in order to achieve the above object, a construct for introducing an exogenous substance (hereinafter also simply referred to as "construct") prepared for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell can be provided. The construct disclosed by the present invention has the carrier peptide fragment disclosed by the present invention and the above-mentioned target exogenous substance bound to the N-terminal side and / or C-terminal side of the carrier peptide fragment. Through such a construct, the target exogenous substance can be introduced from the outside of the cell into the inside of the cell (at least into the cytoplasm).

[0018] In one embodiment of the construct disclosed by the present invention, the above-mentioned exogenous substance may be at least one organic compound selected from polypeptides, nucleic acids, pigments, and pharmaceuticals.

[0019] Among them, "polypeptide" refers to a polymer having a structure in which multiple amino acids are bonded by peptide bonds. The number of peptide bonds (i.e., the number of amino acid residues) of the polypeptide is not limited. That is, polypeptides include compounds generally referred to as peptides having 10 or more and less than about 300 amino acid residues and compounds generally referred to as proteins (typically high molecular compounds composed of 300 or more amino acid residues). In this technical field, polypeptides and proteins are not strictly distinguished. In this specification, polymers (including oligomers) composed of multiple amino acid residues are collectively referred to as polypeptides.

[0020] In addition, "nucleic acid" refers to a polymer of nucleotides and includes DNA and RNA. The number of bases of the "nucleic acid" is not limited.

[0021] In one mode of the construct disclosed in the present invention, the above-mentioned exogenous substance can be arranged on the C-terminal side of the above-mentioned carrier peptide fragment.

[0022] In addition, according to the present invention, there is provided a method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell in an in vitro or in vivo environment. The method disclosed in the present invention includes: (1) a step of preparing the construct disclosed in the present invention; and (2) a step of supplying the above-mentioned construct to a sample containing the target eukaryotic cell. Thereby, the target exogenous substance can be efficiently introduced into the eukaryotic cell.

[0023] In one mode of the method disclosed in the present invention, the target eukaryotic cell into which the above-mentioned construct is introduced can be a cell of a human or a mammal other than a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a histogram obtained by analyzing the cells with a flow cytometer after culturing the culture solution of NSC-34 cells by adding the construct or FAM according to one embodiment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] Hereinafter, embodiments of the technology disclosed in the present invention will be described. Matters required for implementing the technology of the present invention other than those specifically mentioned in this specification (for example, chemical synthesis methods of peptides, cell culture techniques, general matters related to the preparation of constructs containing peptides, nucleic acids, etc. as components) can be grasped by those skilled in the art as designable matters based on the prior art in the fields of cell engineering, physiology, medicine, pharmacy, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, genetics, etc.

[0026] In addition, the technology disclosed in the present invention can be implemented based on the content described in this specification and the common general knowledge in the art. It should be noted that in this specification, there are cases where amino acids are represented by single-letter symbols in accordance with the nomenclature of amino acids shown in the IUPAC-IUB guidelines. It should also be noted that in this specification, unless otherwise stated, "amino acid residue" is a term that includes the N-terminal amino acid and the C-terminal amino acid of the peptide chain.

[0027] In addition, in this specification, "synthetic peptide" is not a substance that independently and stably exists in nature by itself, but refers to a peptide fragment that is manufactured by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and can stably exist in a specified composition. Among them, "peptide" is a term referring to an amino acid polymer having multiple peptide bonds and is not limited to the number of amino acid residues.

[0028] In addition, in this specification, unless otherwise specified, the amino acid residues constituting a peptide or protein can be either L-isomers (L-forms) or D-isomers (D-forms).

[0029] It should be noted that the amino acid sequences described in this specification always represent the N-terminal side on the left and the C-terminal side on the right.

[0030] The vector peptide fragment disclosed in the present invention is a synthetic peptide comprising the amino acid sequence shown in SEQ ID NO: 1: KFRAQRRW. Typically, the vector peptide fragment disclosed in the present invention is composed of the amino acid sequence shown in SEQ ID NO: 1, which has cell membrane permeability and can be introduced into the interior of eukaryotic cells from the outside of the cell. The amino acid sequence shown in SEQ ID NO: 1 is composed of 8 amino acid residues and can exhibit cell membrane permeability with a relatively short sequence of 10 or fewer amino acid residues.

[0031] In addition, as long as the cell membrane permeability is not impaired, the vector peptide fragment disclosed in the present invention can also be based on a modified sequence of the amino acid sequence shown in SEQ ID NO: 1. Herein, a "modified sequence" refers to an amino acid sequence (modified amino acid sequence) formed by substituting, deleting, and / or adding (inserting) one or more (usually two or three) amino acid residues. Therefore, the vector peptide fragment disclosed in the present invention can be composed of, for example, 6 to 10, or 7 to 9 amino acid residues.

[0032] As typical examples of the modified sequences in this specification, there can be mentioned, for example, sequences generated by so-called conservative amino acid replacement in which 1, 2, or 3 amino acid residues are conservatively substituted, and sequences in which 1, 2, or 3 amino acid residues are added (inserted) or deleted from a specified amino acid sequence. As typical examples of conservative amino acid replacement, there can be mentioned, for example, sequences in which a basic amino acid residue is replaced by another basic amino acid residue (e.g., the mutual replacement of a lysine residue and an arginine residue), etc.

[0033] The construct for introducing an exogenous substance disclosed in the present invention has the vector peptide fragment disclosed in the present invention and an exogenous substance bound to the N-terminal side and / or C-terminal side of the vector peptide fragment.

[0034] The construct disclosed in the present invention can be designed and constructed by directly or indirectly (through an appropriate linker) binding (linking) to the N-terminal side and / or C-terminal side of the above-mentioned vector peptide fragment.

[0035] The linker is not particularly limited and can be a peptide linker or a non-peptide linker. Preferably but not particularly limitedly, the amino acid sequence constituting the peptide linker is an amino acid sequence that does not cause steric hindrance and has flexibility. The peptide linker can be, for example, a linker composed of one or more amino acid residues selected from glycine, alanine, serine, etc. and consisting of 10 or fewer (more preferably 1 or more and 5 or fewer, such as 1, 2, 3, 4, or 5 amino acid residues) amino acid residues. In addition, as such a linker, β-alanine can also be used. As the non-peptide linker, for example, an alkyl linker, a PEG (polyethylene glycol) linker, an aminohexanoyl spacer, etc. can be used but not particularly limitedly.

[0036] It should be noted that the mode of binding only the linker to the carrier peptide fragment can also be included in the construct disclosed in the present invention.

[0037] The foreign substance can be, for example, an organic compound such as a polypeptide, a nucleic acid, a pigment, a medicament, etc.

[0038] When the foreign substance is a polypeptide, the peptide chain is designed to include the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the carrier peptide fragment, and the peptide chain is biosynthesized or chemically synthesized, whereby a construct for introducing the target foreign substance can be produced. In addition, by using various known scientific methods, various nucleic acids such as various DNAs or RNAs (including nucleic acid medicines, etc.), pigments (such as various fluorescent pigment compounds such as FAM or FITC), or organic compounds that function as medicaments (such as antitumor agents including nucleic acid antitumor agents such as 5-fluorouracil (5FU), antiviral agents such as azidothymidine (AZT), etc.) are directly or indirectly bound to the N-terminal side and / or C-terminal side of the above carrier peptide fragment, a construct can be prepared.

[0039] The function possessed by the foreign substance can be, for example, but not particularly limitedly, promoting stem cell induced differentiation (stem cell induced differentiation activity), inhibiting the proliferation of tumor cells (antitumor activity), inhibiting the proliferation of virus-infected cells (antiviral activity), etc.

[0040] In the construct disclosed in the present invention, the number of foreign substances bound to the carrier peptide fragment is not particularly limited. For example, one or more foreign substances can be bound to one carrier peptide fragment. For example, it can be, but not particularly limitedly, a polypeptide, a nucleic acid, a medicament, etc. are bound to the C-terminal side of one carrier peptide fragment, and a pigment is bound to the N-terminal side. By binding the pigment to the carrier peptide fragment, it will be easy to evaluate the efficiency of introducing the construct into eukaryotic cells and its localization in the cells, so it is preferred.

[0041] It should be noted that when the exogenous substance is a polypeptide, the polypeptide (amino acid sequence) used is not particularly limited. For example, a peptide with about 1 to 100 amino acid residues, a polypeptide with about 100 to 1000 amino acid residues, or a substance with a relatively large number of amino acid residues such as a protein can be used as the exogenous substance.

[0042] The total number of amino acid residues constituting the synthetic peptide prepared as the construct (that is, the sum of the amino acid residues constituting the vector peptide fragment and the exogenous substance) should be, for example, several to dozens or more (for example, 10 or more), preferably 1000 or less, more preferably 600 or less, further preferably 500 or less, and particularly preferably 300 or less (for example, 10 to 300). Such a length of polypeptide is easy to synthesize (biosynthesis, chemical synthesis) and easy to use.

[0043] As the exogenous substance, a mature polypeptide or polypeptide precursor (including pro-form (pro-), pre-pro-form (pre-pro-)) related to functions such as the development, differentiation, proliferation, carcinogenesis, homeostasis (constancy) in the body, and regulation of metabolism of various cells and tissues (organs), etc. is preferred. In addition, in order to introduce a polypeptide with unknown functions in the past into cells and clarify the functions of the polypeptide in cells (in biological tissues), the exogenous substance introduction method disclosed in the present invention can also be implemented.

[0044] For example, when the eukaryotic cell to be introduced with the exogenous substance is a stem cell of a human or other mammal, a mature polypeptide or polypeptide precursor with various physiological activities involved in the differentiation induction of the stem cell is preferably used. It should be noted that "stem cells" include adult stem cells, embryonic stem cells, and induced pluripotent stem cells (Induced pluripotent stem cells: iPS cells). In addition, when the eukaryotic cell to be introduced with the exogenous substance is a cancer cell (tumor cell), various polypeptides involved in the apoptosis induction of the cancer cell (tumor cell) are preferably used. Or, in this case, a polypeptide that can hinder the inhibition of the function of the immune surveillance mechanism by the cancer cell (tumor cell) is preferably used. In addition, when the eukaryotic cell to be introduced is a bacterially infected cell or a virus-infected cell, various polypeptides involved in the apoptosis induction of the infected cell, polypeptides that can inhibit the proliferation of bacteria or viruses in the infected cell, or polypeptides that can inhibit the spread of bacterial or viral infection from the infected cell are preferably used.

[0045] It should be noted that the polypeptide as the exogenous substance, like the vector peptide fragment, can also contain an altered amino acid sequence formed by substitution, deletion, and / or addition (insertion) of one or more amino acid residues, as long as its function is maintained.

[0046] In a construct in which an exogenous substance is bound to the C-terminal side of a carrier peptide fragment, it is preferable to acetylate the α-amino group of the amino acid residue on the N-terminal side of the carrier peptide fragment. Although the detailed mechanism has not been clarified, since the α-amino group of the amino acid on the N-terminal side of most proteins in eukaryotic cells is acetylated, in such a configuration, the stability of the construct in cells can be improved.

[0047] It is preferable that the amino acid residue on the C-terminal side of the construct is amidated. If the carboxyl group of an amino acid residue (typically the C-terminal amino acid residue of a peptide chain) is amidated, the structural stability (e.g., protease resistance) of the construct in the cytoplasm and nucleolus can be improved. In addition, by amidating the carboxyl group, the hydrophilicity of the construct is increased, and thus the solubility of the construct in an aqueous solvent can be improved. Examples of such an aqueous solvent include water, various buffers, physiological saline (e.g., PBS), cell culture media, and the like.

[0048] For example, in the case of a construct in which an exogenous substance is bound to the N-terminal side of a carrier peptide fragment, it is preferable to amidate the carboxyl group of the amino acid residue on the C-terminal side of the carrier peptide fragment. Additionally, for example, when the exogenous substance is a polypeptide and the polypeptide is bound to the C-terminal side of the carrier peptide fragment, it is preferable to amidate the carboxyl group of the C-terminal amino acid residue of the polypeptide.

[0049] Peptides with a short peptide chain (including polypeptides, carrier peptide fragments, and peptide linkers that make up the exogenous substance) in the construct can be easily produced by conventional chemical synthesis methods. For example, any of the currently known solid-phase synthesis methods or liquid-phase synthesis methods can be used. A solid-phase synthesis method suitable for using Boc (tert-butoxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as the protecting group for the amino group is applicable. That is, by using a solid-phase synthesis method with a commercially available peptide synthesis device, it is also possible to synthesize the above peptide chain with a desired amino acid sequence and modified parts (such as N-terminal acetylation, C-terminal amidation, etc.). It should be noted that it is also possible to synthesize only a part of the peptide chain by the above method. For example, only the carrier peptide fragment can be synthesized, or a peptide chain containing the carrier peptide fragment and the peptide linker part can be synthesized.

[0050] Alternatively, the peptide part can also be produced by biosynthesis based on genetic engineering methods. That is, a polynucleotide (typically DNA) encoding a nucleotide sequence (including the ATG start codon) of a desired amino acid sequence is synthesized. Then, according to the host cell, a recombinant vector having an expression gene construct is constructed, and the expression gene construct includes the synthesized polynucleotide (DNA) and various regulatory elements (including promoters, ribosome binding sites, terminators, enhancers, various cis-elements that control the expression level) for expressing the amino acid sequence in the host cell.

[0051] Using conventional technical methods, the recombinant vector is introduced into a specified host cell (such as yeast, insect cells, plant cells), and the host cell or the tissue or individual containing the cell is cultured under specified conditions. Thereby, the target peptide can be produced intracellularly. Then, the peptide moiety can be obtained by separating it from the host cell (from the culture medium in the case of secretion) and, if necessary, performing refolding, purification, etc.

[0052] It should be noted that the method for constructing the recombinant vector and the method for introducing the constructed recombinant vector into the host cell, etc., can directly adopt the methods that have been carried out in the art. Since these methods themselves do not particularly embody the features of the technology described in the present invention, detailed descriptions are omitted.

[0053] For example, in order to achieve efficient and large-scale production in host cells, a fusion protein expression system can be utilized. That is, a gene (DNA) encoding the amino acid sequence of the target polypeptide is chemically synthesized, and the synthetic gene is introduced into a suitable site of an appropriate fusion protein expression vector (such as the pET series provided by Novagen and the GST (glutathione S-transferase) fusion protein expression vectors such as the pGEX series provided by Amersham Bioscience). Then, the host cell (typically Escherichia coli) is transformed using this vector. The obtained transformant is cultured to prepare the target fusion protein. Then, the protein is extracted and purified. Next, the obtained purified fusion protein is cleaved with a specified enzyme (protease), and the free target peptide fragment (i.e., the designed artificial polypeptide) is recovered by methods such as affinity chromatography. By adopting such currently well-known fusion protein expression systems (for example, the GST / His system provided by Amersham Bioscience), the target construct (artificial polypeptide) can be manufactured.

[0054] Alternatively, a template DNA for a cell-free protein synthesis system (i.e., a synthetic gene fragment containing a nucleotide sequence encoding the amino acid sequence of the peptide portion of the construct) can be constructed, and using various compounds (ATP, RNA polymerase, amino acids, etc.) required for the synthesis of the peptide portion, the target polypeptide can be synthesized in vitro using a so-called cell-free protein synthesis system. Regarding the cell-free protein synthesis system, reference can be made, for example, to the papers by Shimizu et al. (Nature Biotechnology, Vol. 19, No. 7, pp. 751-755, 2001, Shimizu et al.: Shimizu et al., Nature Biotechnology, 19, 751-755 (2001)) and Madin et al. (Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 2, pp. 559-564, 2000, Madin et al.: Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559-564 (2000)). Based on the techniques described in these papers, at the time of filing this application, many companies were engaged in the contract production of polypeptides, and cell-free protein synthesis kits (e.g., available from Cellfree Science, Japan) were already on the market.

[0055] A single-stranded or double-stranded polynucleotide containing a nucleotide sequence encoding the peptide portion of the construct and / or a nucleotide sequence complementary to the sequence can be easily produced (synthesized) by currently known methods. That is, by selecting the codons corresponding to each amino acid residue constituting the designed amino acid sequence, the nucleotide sequence corresponding to the amino acid sequence can be easily determined and provided. Moreover, once the nucleotide sequence is determined, a polynucleotide (single-stranded) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like. Furthermore, the obtained single-stranded DNA can be used as a template, and the target double-stranded DNA can be obtained by various enzyme synthesis means (typically PCR). In addition, the polynucleotide can be in the form of DNA or RNA (such as mRNA). The DNA can be provided in double-stranded or single-stranded form. When provided in single-stranded form, it can be either the coding strand (sense strand) or the non-coding strand (antisense strand) complementary to the sequence.

[0056] As described above, the polynucleotide thus obtained can be used as a material for constructing a recombinant gene (expression cassette) for peptide production in various host cells or in a cell-free protein synthesis system.

[0057] The constructs disclosed in the present invention can be suitably used as an active ingredient of a composition for use based on the function of an exogenous substance. It should be noted that the constructs can also be in the form of salts, as long as the function of the exogenous substance is not lost. For example, acid addition salts obtained by addition reactions of commonly used inorganic acids or organic acids using conventional methods can be used. Therefore, the "constructs" described in this specification and the claims also include constructs in such salt forms.

[0058] The constructs can also be used as an active ingredient of a composition that can contain various pharmaceutically acceptable carriers corresponding to the usage form.

[0059] As the above carriers, preferably, for example, carriers commonly used as diluents, excipients, etc. in peptide pharmaceuticals. As such carriers, they can vary appropriately according to the usage and form of the constructs for introducing exogenous substances, and typically, water, physiological buffers, and various organic solvents can be cited. In addition, the carrier can also be an aqueous solution of an alcohol (such as ethanol) at an appropriate concentration, glycerin, a non-drying oil such as olive oil, or it can also be a liposome. In addition, as minor components that may be included in the pharmaceutical composition, various fillers, extenders, binders, wetting agents, surfactants, pigments, flavors, etc. can be cited.

[0060] The form of the composition is not particularly limited. For example, forms such as liquid preparations, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, ointments, etc. can be cited. In addition, for use in injections, etc., it can also be made into a freeze-dried product or granulated product for dissolving in physiological saline or an appropriate buffer (such as PBS) immediately before use to prepare a medicinal solution.

[0061] The process of preparing various forms of medicaments (compositions) using the constructs (main components) and various carriers (minor components) as materials can be carried out according to currently known methods. These methods of manufacturing medicaments themselves do not embody the technical features of the present invention, so detailed descriptions are omitted. As a source of information on formulation-related details, for example, "Comprehensive Medicinal Chemistry" edited by Corwin Hansch and published by Pergamon Press (1990) can be cited.

[0062] According to the present invention, a method for introducing an exogenous substance in an in vivo or in vitro environment using the constructs disclosed in the present invention is provided. In this method, it mainly includes the following steps (1) to (2):

[0063] (1) The step of preparing the constructs disclosed in the present invention; and

[0064] (2) A step of supplying the above-mentioned construct to a sample containing target eukaryotic cells. In addition, in the method disclosed in the present invention, after the step (2) above, as the step (3), it may further include a step of incubating the sample supplied with the above-mentioned construct to introduce the construct into the eukaryotic cells in the sample.

[0065] In vivo, the above-mentioned "eukaryotic cells" include, for example, various tissues, internal organs, organs, blood, lymph fluid, etc. In vitro, the above-mentioned "eukaryotic cells" include, for example, various cell masses, tissues, internal organs, organs, blood, lymph fluid, and cell lines removed from organisms. Examples of eukaryotic cells include cells derived from the animal kingdom such as mammals, birds, fish, amphibians, reptiles, insects, etc., cells derived from the fungal kingdom, cells derived from the plant kingdom, etc., and preferably cells of humans or mammals other than humans.

[0066] The composition containing the construct disclosed in the present invention can be used in vivo according to methods and doses corresponding to its form and purpose. For example, as a liquid agent, it can be administered only in the desired amount to the affected area (such as malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient (i.e., an organism) by intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal injection. Alternatively, solid-form preparations such as tablets, gel-like or aqueous gel-like preparations such as ointments can be directly applied to a specified tissue (i.e., an affected area such as a tissue or organ including, for example, tumor cells, virus-infected cells, inflammatory cells, etc.). Alternatively, solid-form preparations such as tablets can also be administered orally. In the case of oral administration, in order to inhibit digestion by digestive enzymes in the digestive tract, it is preferably administered using a capsule or a protective (coated) material.

[0067] Alternatively, for eukaryotic cells cultured in vitro (in vitro), an appropriate amount of the construct can be supplied to the culture medium of the target eukaryotic cells at least once. The amount supplied each time and the number of supply times vary depending on conditions such as the type of eukaryotic cells cultured, cell density (cell density at the start of culture), passage number, culture conditions, type of culture medium, etc., and thus there is no particular limitation. For example, it is preferably added multiple times, such as 1 time, 2 times, or more times, so that the concentration of the carrier peptide fragment in the culture medium is generally in the range of 0.05 μM or more and 100 μM or less, for example, in the range of 0.5 μM or more and 50 μM or less, or in the range of, for example, 1 μM or more and 30 μM or less. In addition, the incubation time after adding the construct also varies depending on the type of eukaryotic cells and various conditions, and thus there is no particular limitation. For example, it can be 0.5 hours or more, 1 hour or more, 4 hours or more, 8 hours or more, 20 hours or more. It should be noted that the incubation conditions also vary depending on the type of eukaryotic cells, and thus there is no particular limitation. For example, it can be incubated in an environment of 5% CO 2 and at a temperature of 37°C.

[0068] Note that, regarding the introduction method in vitro, an example is shown in the following test examples.

[0069] There is no particular limitation on the method for evaluating the introduction efficiency of the construct. For example, when a pigment (typically a fluorescent pigment compound) is conjugated to the construct, the introduction efficiency into eukaryotic cells can be evaluated by microscopic observation (e.g., fluorescence microscopy) or flow cytometry. In addition, the introduction efficiency of the construct can also be evaluated by immunochemical methods (e.g., Western Blotting, immunocytochemical staining, etc.) using an antibody that specifically recognizes the peptide portion of the construct.

[0070] As described above, as specific embodiments of the technology disclosed in the present invention, the following embodiments can be cited.

[0071] Item 1: A vector peptide fragment, wherein the vector peptide fragment is a vector peptide fragment that is introduced at least into the cytoplasm of a eukaryotic cell from the outside of the cell, and contains the following amino acid sequence: KFRAQRRW (SEQ ID NO: 1).

[0072] Item 2: A construct, which is a construct for introducing a target foreign substance at least into the cytoplasm of a eukaryotic cell from the outside of the cell, and has: the vector peptide fragment according to Item 1, and the target foreign substance conjugated to the N-terminal side and / or C-terminal side of the vector peptide fragment.

[0073] Item 3: The construct according to Item 2, wherein the foreign substance is at least one organic compound selected from polypeptides, nucleic acids, pigments, and pharmaceuticals.

[0074] Item 4: The construct according to Item 2 or Item 3, wherein the foreign substance is disposed on the C-terminal side of the vector peptide fragment.

[0075] Item 5: A method for introducing a target foreign substance at least into the cytoplasm of a eukaryotic cell from the outside of the cell in vitro, which includes:

[0076] (1) A step of preparing the construct according to any one of Items 2 to 4; and

[0077] (2) A step of supplying the construct to a sample containing target eukaryotic cells.

[0078] Item 6: The method according to Item 5, wherein the target eukaryotic cells into which the construct is introduced are cells of a human or a non-human mammal.

[0079] Several test examples related to the technology disclosed by the present invention are described below, but it is not intended to limit the technology disclosed by the present invention to the content shown in these test examples.

[0080] In Example 1, the construct shown in Table 1 was prepared as Sample 1. Specifically, a synthetic peptide formed by the amino acid sequence shown in SEQ ID NO: 1 with the α-amino group of the N-terminal lysine residue acetylated and a fluorescent dye FAM (C 21 H 12 O 7 : 5(6)-carboxyfluorescein, molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) bound to the C-terminal side of the synthetic peptide were prepared. This construct was diluted with dimethyl sulfoxide (DMSO) to prepare a sample solution 1 with a sample concentration of 2 mM.

[0081] NSC-34 cells (mouse motor neuron-like hybrid cell line) were used as eukaryotic cells to evaluate the cell membrane permeability of Sample 1. In Example 1, the prepared sample solution 1 was used, and in Example 2, a FAM solution was used.

[0082] The NSC-34 cells were cultured in a medium containing 10% FBS (fetal bovine serum) and DMEM (Dulbecco's Modified Eagle's Medium: manufactured by Fujifilm Wako Pure Chemical Corporation, product number 044-29765).

[0083] After washing the NSC-34 cells adhered to the culture plate with PBS, a 0.25% trypsin / EDTA solution was added and incubated at 37°C for 3 minutes. After this incubation, the above-mentioned DMEM containing 10% FBS was added to inactivate the trypsin, and then centrifuged at 150×g for 5 minutes to precipitate the cells. After removing the supernatant generated by centrifugation, the above-mentioned DMEM containing 10% FBS was added to the precipitate (cell mass) to prepare a cell suspension of approximately 1×10 5 cells / mL. 1 mL of this cell suspension was added to the wells of a commercially available 6-well plate (manufactured by AGC TECHNO GLASS Co., Ltd.) for cell seeding (approximately 1×10 5 cells / well). And, the above-mentioned 2 mM sample solution 1 was diluted with the above-mentioned DMEM containing 10% FBS to prepare a sample solution 1 with a Sample 1 concentration of 20 μM. Then, 1 mL of the above-mentioned 20 μM sample solution 1 was added to this well (that is, the concentration of Sample 1 in the culture solution in the well was 10 μM, and the DMSO concentration was 0.5%). Then, in 5% CO 2Under the conditions of, the cells were incubated at 37 °C for 20 hours.

[0084] After 20 hours of incubation, the culture supernatant was removed from the wells, and the cells in the wells were washed twice with 1 mL of PBS. Subsequently, 100 μL of 0.25% trypsin / EDTA solution was added to the wells, and incubation was carried out at 37 °C for 3 minutes. After this incubation, 900 μL of the above-mentioned DMEM containing 10% FBS was added to the wells to inactivate the trypsin, and then the cell suspension in the wells was transferred to a test tube to recover the cells. Thereafter, further rinsing (total rinsing) was performed to collect the cells remaining in the wells into the above-mentioned test tube. This test tube was centrifuged at 4 °C and 210×g for 5 minutes. After centrifugation, the supernatant was removed, and the precipitate (cell pellet) was suspended (washed) with 1 mL of PBS and centrifuged under the same conditions as above. Then the supernatant was removed to obtain the cells (cell pellet) cultured with the medium containing Sample 1.

[0085] For the cells (cell pellet) obtained above, the cell membrane permeability of Sample 1 was analyzed using a flow cytometer. As the flow cytometer, an On-Chip flow cytometer (manufactured by On-Chip Biotechnologies Co., LTD.) was used.

[0086] To perform this analysis, the above-obtained cell pellet was suspended with 50 μL of On-Chip T buffer to prepare an analysis cell suspension.

[0087] Using the above flow cytometer, gating based on forward scatter (FSC) and side scatter (SSC) was performed to gate the cell population to be analyzed, and the fluorescence intensity of the cell population within this gate was measured. It should be noted that the analysis was carried out with the number of cells in the cell population to be analyzed set to 10,000 or more. The measurement of the fluorescence intensity was performed using the fluorescence detector FL2 (optimal detection wavelength around 543 nm) of the above flow cytometer capable of detecting the FAM fluorescence wavelength. The measurement results were analyzed using the commercially available analysis software "FlowJo" (manufactured by TreeStar Inc.) to obtain the median (median fluorescent intensity, MFI) of the fluorescence intensity of the cell population to be measured.

[0088] In Example 2, except that a FAM solution diluted with DMSO was used instead of Sample Solution 1, the same procedures as in Example 1 were carried out. It should be noted that the concentration of this FAM solution was the same as the concentration of Sample Solution 1 (i.e., the FAM concentration in the culture medium in the well was 10 μM and the DMSO concentration was 0.5%). The results obtained in Examples 1 and 2 are shown in Table 1 and Figure 1 . Figure 1 is a histogram obtained by a flow cytometer.

[0089] [Table 1]

[0090] Composition of the construct (additive) MFI Example 1 Ac-KFRAQRRW-FAM 15.8 Example 2 FAM 7.58

[0091] As Figure 1 shown, compared with the histogram of Example 2, the histogram of Example 1 was shifted to the right. From this, it can be known that: the construct with the synthetic peptide composed of the amino acid sequence shown in SEQ ID NO: 1 added to FAM has higher cell membrane permeability than using FAM alone. That is, the synthetic peptide composed of the amino acid sequence shown in SEQ ID NO: 1 is a carrier peptide fragment that exhibits cell membrane permeability.

[0092] The specific examples of the technology disclosed in the present invention have been described in detail above, but these are only examples and do not limit the scope of the claims. The technology described in the scope of the claims includes the specific examples described above and various modified and changed schemes.

[0093] It should be noted that although detailed data are not shown, according to the research of the inventors of the present invention, it has been confirmed that: the construct having the above synthetic peptide can efficiently introduce not only a fluorescent pigment as an exogenous substance but also any one of polypeptides, nucleic acids, and pharmaceuticals from the outside of the cell into the cytoplasm.

[0094] Industrial Applicability

[0095] Using the technology disclosed in the present invention, a carrier peptide fragment capable of introducing a target exogenous substance from the outside of a eukaryotic cell (especially various animal cells represented by humans or mammals other than humans without cell walls) into the cytoplasm and a construct having the carrier peptide fragment are provided. Using this construct, a target exogenous substance can be effectively introduced into a target cell, and a cell, an organ, and other biological tissues into which the exogenous substance has been introduced can be obtained. In addition, by using the carrier peptide fragment disclosed in the present invention for drug delivery technology, therapeutic drugs for various diseases can be provided.

Claims

1. A carrier peptide fragment that is a carrier peptide fragment imported from outside of a eukaryotic cell into at least the cytoplasm of the cell, characterized in that: the carrier peptide fragment contains the following amino acid sequence: KFRAQRRW (SEQ ID NO: 1).

2. A construct that is a construct for importing a target foreign substance from outside of a eukaryotic cell into at least the cytoplasm of the cell, characterized in that it has: the carrier peptide fragment described in claim 1; and the target foreign substance bound to the N-terminal side and / or C-terminal side of the carrier peptide fragment.

3. The construct according to claim 2, characterized in that: the foreign substance is at least one organic compound selected from polypeptides, nucleic acids, pigments, and pharmaceuticals.

4. The construct according to claim 2, characterized in that: the foreign substance is arranged on the C-terminal side of the carrier peptide fragment.

5. A method for importing a target foreign substance from outside of a eukaryotic cell into at least the cytoplasm of the cell in vitro, characterized in that it includes: (1) a step of preparing the construct according to any one of claims 2 to 4; and (2) a step of supplying the construct to a sample containing target eukaryotic cells.

6. The method according to claim 5, characterized in that: the target eukaryotic cells into which the construct is imported are cells of a human or a non-human mammal.

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