Vector peptide fragments and uses thereof

Abstract

According to the present invention, it is possible to provide a method capable of introducing an exogenous substance of interest into at least the cytoplasm of a eukaryotic cell from outside the cell. The method comprises: (1) a step for preparing a construct for introducing an exogenous substance, said construct comprising a vector peptide fragment containing any one of the following amino acid sequences: KKRTLRKSNSRKKR (SEQ ID NO: 1), KKRTLRKSSNRKKR (SEQ ID NO: 2), KKRTLRKSNNRKKR (SEQ ID NO: 3), and KKRTLRKNSNRKKR (SEQ ID NO: 4), and the target exogenous substance bound to the N-terminal side and / or C-terminal side of the vector peptide fragment; and (2) a step for supplying the construct to a sample containing a target eukaryotic cell.

Description

Technical Field

[0001] The present invention relates to a method for introducing (transferring) an exogenous substance from the outside of a eukaryotic cell to 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. 2022-105722 filed on June 30, 2022, and the entire contents of the application are introduced as a reference in this specification. Background Art

[0002] Traditionally, exogenous substances such as polypeptides, especially physiologically active substances, have been introduced into cells (eukaryotic cells) of humans and other mammals to cause biotransformation of the cells (and tissues and organs composed of the cells, etc.) or to improve and enhance the functions of the cells.

[0003] For example, Patent Document 1 discloses a construct for introducing an exogenous substance that contains a carrier peptide fragment composed of an amino acid sequence called a nucleolar localization signal (hereinafter referred to as "NoLS") and an exogenous substance. Since such a construct has cell membrane permeability, the exogenous substance can be effectively introduced into the target cell.

[0004] Prior art literature Patent Literature Patent Document 1: International Publication No. 2011 / 013699 Summary of the invention

[0005] In addition, in recent years, from the perspective of medical treatment, it is necessary to develop a new carrier peptide fragment that can introduce a target exogenous substance into the cytoplasm. The cell membrane permeability of the carrier peptide fragment fluctuates in efficiency depending on the type of cell to which the carrier peptide fragment is to be introduced. Therefore, the development of a new carrier peptide fragment will expand the scope of medical treatment.

[0006] Therefore, the present invention is an invention created in response to this demand, and its purpose is to provide a carrier peptide fragment capable of introducing a target exogenous substance from the outside of a eukaryotic cell at least into the cytoplasm of the cell. In addition, another purpose of the present invention is to provide a construct for introducing an exogenous substance having the carrier peptide fragment. In addition, another purpose of the present invention is to provide a method for introducing a target exogenous substance from the outside of a eukaryotic cell at least into the cytoplasm of the cell.

[0007] The method of the present invention can introduce the 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, wherein: (1) preparing a construct for introducing an exogenous substance, wherein the construct comprises a carrier peptide fragment consisting of any one of the following amino acid sequences and the above-mentioned target exogenous substance bound to the N-terminal side and / or the C-terminal side of the above-mentioned carrier peptide fragment, KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (sequence number 4); as well as (2) A step of supplying the above construct to a sample containing target eukaryotic cells.

[0008] Here, "exogenous substances" refer to inorganic compounds and organic compounds that can be directly or indirectly bound to the N-terminal side or C-terminal side of the above-mentioned carrier peptide fragment through appropriate linkers, and have a molecular size and chemical properties that can be introduced into eukaryotic cells.

[0009] According to the method of the above composition, a target exogenous substance can be efficiently introduced into the cytoplasm from the outside of a eukaryotic cell (outside of the cell membrane) through the cell membrane.

[0010] Furthermore, in one embodiment of the method of the present invention, the exogenous substance may be at least one organic compound selected from the group consisting of polypeptides, nucleic acids, pigments and pharmaceutical agents.

[0011] Among them, "polypeptide" refers to a polymer having a structure in which multiple amino acids are bound by peptide bonds. Polypeptides are not limited by the number of peptide bonds (i.e., the number of amino acid residues). That is, polypeptides include compounds generally called peptides with an amino acid residue number of more than 10 and less than 300, and compounds generally called proteins (polymer compounds generally composed of more than 300 amino acid residues). In this technical field, there is no strict distinction between polypeptides and proteins. In this specification, polymers (including oligomers) formed by multiple amino acid residues are collectively referred to as polypeptides.

[0012] In addition, "nucleic acid" refers to a polymer of nucleotides, including DNA and RNA. "Nucleic acid" is not limited by the number of bases.

[0013] Furthermore, in one embodiment of the method of the present invention, the exogenous substance may be disposed on the C-terminal side of the carrier peptide fragment.

[0014] In one embodiment of the method of the present invention, the eukaryotic cells into which the construct for introducing an exogenous substance is introduced may be cells of humans or mammals other than humans.

[0015] In addition, according to the present invention, there is also provided an artificially manufactured exogenous substance introduction construct that can achieve the above-mentioned purpose and can introduce (transport) the target exogenous substance from the outside (i.e., the outside of the cell membrane) of a eukaryotic cell (especially various animal cells represented by humans or non-human mammals without cell walls) at least into the cytoplasm of the cell (preferably deeper into the cell nucleus).

[0016] That is, the construct of the present invention comprises a carrier peptide fragment consisting of any one of the following amino acid sequences and the above-mentioned target exogenous substance bound to the N-terminal side and / or the C-terminal side of the above-mentioned carrier peptide fragment, KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (serial number 4).

[0017] Since the construct has cell membrane permeability, the target exogenous substance can be efficiently introduced into the target eukaryotic cells.

[0018] In one embodiment of the construct of the present invention, the exogenous substance may be at least one organic compound selected from the group consisting of polypeptides, nucleic acids, pigments and pharmaceutical agents.

[0019] Furthermore, in one embodiment of the construct of the present invention, the exogenous substance may be disposed on the C-terminal side of the carrier peptide fragment.

[0020] In addition, according to the present invention, there is provided a carrier peptide fragment capable of introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell. The carrier peptide fragment of the present invention is composed of any one of the following amino acid sequences,

[0021] KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (serial number 4).

[0022] Since the above-mentioned carrier peptide fragment exhibits cell membrane permeability, it is possible to efficiently introduce exogenous substances into cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is a graph showing MFI values ​​obtained by adding the constructs (additives) shown in Examples 1 to 5 to the culture medium of NSC-34 cells and culturing the cells, and then analyzing the cells using a flow cytometer.

[0024] Figure 2 It is a graph showing the cytotoxicity test results of samples 1 to 3 and the reference example. DETAILED DESCRIPTION

[0025] Several preferred embodiments of the technology described in this specification are described below. Matters required for the implementation of the present invention other than those specifically mentioned in this specification (such as general matters such as chemical synthesis methods of peptides, cell culture techniques, preparation of constructs containing peptides and nucleic acids as components) can be understood as designable matters by those skilled in the art based on existing technologies 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 described in this specification can be implemented based on the content recorded in this specification and the technical common sense in this field. It should be noted that in the following description, amino acids are represented by single-letter symbols that conform to the amino acid nomenclature shown in the IUPAC-IUB Guide, depending on the circumstances. 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" refers to a peptide fragment that is not stable and independent in nature, but is manufactured by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and can be stably present in a given composition. Among them, "peptide" refers to a term for amino acid polymers having multiple peptide bonds, and is not limited to the number of amino acid residues.

[0028] In addition, the amino acid residues constituting the amino acid sequence shown in this specification may be L isomers or D isomers. That is, each amino acid residue constituting the carrier peptide fragment described in the present invention may be L isomers or D isomers. In other words, in the amino acid sequence constituting a peptide or protein, there may be L isomers of amino acid residues and D isomers of amino acid residues.

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

[0030] The carrier peptide fragment described in this specification has the following amino acid sequence: KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (serial number 4) Any sequence in . The carrier peptide fragment comprising the amino acid sequence shown in SEQ ID NOs. 1 to 4 can have cell membrane permeability. Therefore, such a carrier peptide fragment can impart cell membrane permeability to a construct having the carrier peptide fragment.

[0031] The "carrier peptide fragment" described in this specification is generally an amino acid sequence identical to the above-mentioned amino acid sequence, but as long as the cell membrane permeability is not impaired, it also includes a modified sequence of such an amino acid sequence. Among them, a "modified sequence" refers to an amino acid sequence (modified amino acid sequence) formed by replacing, deleting and / or adding (inserting) one or more (usually two or three) amino acid residues. Since such a slightly modified sequence can be easily applied by a person skilled in the art according to the information described in this specification, it is also included in the "carrier peptide fragment" as the technical idea described in this specification.

[0032] As typical examples of modified sequences in the present specification, there can be cited, for example, sequences produced by so-called conservative amino acid replacements of 1, 2 or 3 amino acid residues, and sequences in which 1, 2 or 3 amino acid residues are added (inserted) or deleted in a prescribed amino acid sequence, etc. As typical examples of similar replacements, there can be cited, for example, sequences in which basic amino acid residues are replaced by other basic amino acid residues (e.g., mutual replacement of lysine residues and arginine residues), sequences in which hydrophobic amino acid residues are replaced by other hydrophobic amino acid residues (e.g., mutual replacement of leucine residues, isoleucine residues and valine residues), etc.

[0033] The construct for introducing an exogenous substance of the present invention (also referred to as "construct" in this specification) comprises the carrier peptide fragment of the present invention and an exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

[0034] The construct of the present invention can be designed and constructed by directly or indirectly combining (linking) with the N-terminal side and / or C-terminal side of the above-mentioned carrier peptide fragment through a suitable linker.

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

[0036] Exogenous substances can be organic compounds such as polypeptides, nucleic acids, pigments, and drugs.

[0037] In the case where the exogenous substance is a polypeptide, a peptide chain is designed in such a way as to contain the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the carrier peptide fragment, and the peptide chain is subjected to biosynthesis or chemical synthesis, thereby preparing a construct for introducing the target exogenous substance. In addition, an organic compound that functions as various nucleic acids such as DNA or RNA, pigments (e.g., various fluorescent pigment compounds such as FAM and FITC), or drugs (e.g., antitumor agents including nucleic acid-based antitumor agents such as 5-fluorouracil (5FU), antiviral agents such as azidothymidine (AZT), etc.) can be directly or indirectly bound to the N-terminal side and / or C-terminal side of the above-mentioned carrier peptide fragment using various conventionally known scientific methods, thereby preparing a construct.

[0038] The functions of the exogenous substance may be, but are not particularly limited to, promotion of stem cell differentiation induction (stem cell differentiation inducing activity), inhibition of tumor cell proliferation (anti-tumor activity), inhibition of virus-infected cell proliferation (anti-viral activity), etc.

[0039] In the construct of the present invention, the number of exogenous substances bound to the carrier peptide fragment is not particularly limited. For example, one or more exogenous substances may be bound to one carrier peptide fragment. For example, it may be, but is not particularly limited to, binding a polypeptide, nucleic acid, medicament, etc. to the C-terminal side of a carrier peptide fragment, and binding a pigment to the N-terminal side. By binding the pigment to the carrier peptide fragment, it is easy to evaluate the efficiency of the construct introduced into eukaryotic cells and its localization in the cell, so it is preferred.

[0040] 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 substance with a relatively large number of amino acid residues such as a polypeptide having about 100 to 1000 amino acid residues or a protein can be used as the exogenous substance.

[0041] Generally, the total number of amino acid residues constituting the synthetic peptide prepared as a construct for introduction of exogenous substances is several or even tens (e.g., 10) or more, and a suitable value is 1000 or less, preferably 600 or less, more preferably 500 or less, and particularly preferably 300 or less (e.g., 10 to 300). Polypeptides of such length are easy to synthesize (biosynthesize, chemically synthesize) and easy to use.

[0042] As exogenous substances, mature polypeptides or polypeptide precursors (including pro- and pre-pro-) related to functions such as development, differentiation, proliferation, carcinogenesis, homeostasis (constancy), and metabolic regulation of various cells and tissues (organs), etc. are preferred. In addition, the exogenous substance introduction method of the present invention can also be implemented to introduce a polypeptide whose function is previously unknown into a cell and to clarify the function of the polypeptide in the cell (in a living tissue).

[0043] For example, when the eukaryotic cells to be introduced into the exogenous substance are stem cells of humans or other mammals, it is preferred to use mature polypeptides or polypeptide precursors having various physiological activities related to the differentiation induction of the stem cells. It should be noted that "stem cells" include somatic stem cells, embryonic stem cells, and artificially induced pluripotent stem cells (Induced pluripotent stem cells: iPS cells). In addition, when the eukaryotic cells to be introduced into the exogenous substance are cancer cells (tumor cells), it is preferred to use various polypeptides related to the apoptosis induction of the cancer cells (tumor cells). Alternatively, in this case, it is preferred to use polypeptides that can hinder the function of cancer cells (tumor cells) to inhibit the immune surveillance mechanism. In addition, when the eukaryotic cells to be introduced into the object are bacterially infected cells or virally infected cells, it is preferred to use various polypeptides related to the apoptosis induction of the infected cells, polypeptides that can inhibit the proliferation of bacteria or viruses in the infected cells, or polypeptides that can inhibit the spread of bacterial or viral infections from the infected cells.

[0044] It should be noted that the polypeptide and carrier peptide fragment as exogenous substances may also contain modified amino acid sequences formed by replacement, deletion and / or addition (insertion) of one or more amino acid residues, as long as their functions are maintained.

[0045] In the construct in which the exogenous substance is bound to the C-terminal side of the carrier peptide fragment, the α-amino group of the amino acid residue on the N-terminal side of the carrier peptide fragment is preferably acetylated. Although the detailed mechanism has not yet been ascertained, it is known that most proteins in eukaryotic cells are modified by acetylation of the α-amino group of the amino acid on the N-terminal side, and therefore, such a configuration can improve the intracellular stability of the construct.

[0046] The construct preferably has an amino acid residue at the C-terminal side amidated. If the carboxyl group of the amino acid residue (usually the C-terminal amino acid residue of the peptide chain) is amidated, the structural stability (e.g., protease resistance) of the construct in the cytoplasm and in the nucleosome can be improved. In addition, by amidating the carboxyl group, the hydrophilicity of the construct can be improved, thereby improving the solubility of the construct in an aqueous solvent. As such an aqueous solvent, for example, water, various buffer solutions, physiological saline (e.g., PBS), cell culture fluid, etc. can be cited.

[0047] For example, in the case of a construct in which an exogenous substance binds to the N-terminal side of a carrier peptide fragment, the carboxyl group of the amino acid residue at the C-terminal side of the carrier peptide fragment is preferably amidated. In addition, for example, in the case in which the exogenous substance is a polypeptide and the polypeptide binds to the C-terminal side of a carrier peptide fragment, the carboxyl group of the C-terminal amino acid residue of the polypeptide is preferably amidated.

[0048] Constructs with shorter peptide chains (including polypeptides, carrier peptide fragments, and peptide linkers constituted as exogenous substances) in the constructs can be easily manufactured according to conventional chemical synthesis methods. For example, any of conventional known solid phase synthesis methods or liquid phase synthesis methods can be used. It is suitable to use a solid phase synthesis method using Boc (tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the amino group. That is, by using a solid phase synthesis method using commercially available peptide synthesis equipment, it is also possible to synthesize the above-mentioned peptide chains having the desired amino acid sequence and modified (N-terminal acetylation, C-terminal amidation, etc.) parts. It should be noted that only a part of the peptide chain can also be synthesized 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.

[0049] Alternatively, the peptide portion can also be biosynthesized based on genetic engineering methods. That is, a polynucleotide (usually DNA) encoding a nucleotide sequence (including an ATG start codon) encoding the desired amino acid sequence is synthesized. Then, a recombinant vector having an expression gene construct including the synthesized polynucleotide (DNA) and various acting factors (including promoters, ribosome binding sites, terminators, enhancers, and various cis-acting factors that control the expression level) for expressing the amino acid sequence in the host cell is constructed according to the host cell.

[0050] The recombinant vector is introduced into a predetermined host cell (e.g., yeast, insect cell, plant cell) by conventional techniques, and the host cell or a tissue or individual containing the cell is cultured under specified conditions. Thus, the target peptide can be produced in the cell. Then, the target peptide portion can be obtained by isolating the peptide portion from the host cell (in the culture medium in the case of secretion), and refolding, refining, etc. as needed.

[0051] It should be noted that the method for constructing a recombinant vector and the method for introducing the constructed recombinant vector into a host cell can directly adopt the methods that have been used in the art in the past. Since these methods themselves are not technical contents that bring special characteristics to the technology described in this specification, detailed descriptions are omitted.

[0052] For example, in order to achieve efficient mass production in host cells, a fusion protein expression system can be used. 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 a suitable fusion protein expression vector (for example, the pET series provided by Novogene and the pGEX series provided by Amersham Biosciences, etc. GST (glutathione S-transferase) fusion protein expression vector). Then, the host cell (usually Escherichia coli) is biotransformed using the vector. The resulting biotransformant is cultured to prepare the desired fusion protein. The protein is then extracted and refined. The resulting refined fusion protein is then cleaved with a predetermined enzyme (protease), and the released target peptide fragment (i.e., the designed artificial polypeptide) is recovered by affinity chromatography or the like. By adopting this conventional known fusion protein expression system (for example, the GST / His system provided by Amersham Biosciences can be used), the target construct (artificial polypeptide) can be manufactured.

[0053] 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 various compounds required for the synthesis of the peptide portion (ATP, RNA polymerase, amino acids, etc.) can be used to synthesize the target polypeptide in vitro using a so-called cell-free protein synthesis system. Regarding the cell-free protein synthesis system, reference can be made to, for example, the paper by Shimizu et al., Nature Biotechnology, 19, 751-755 (2001) and the paper by Martin et al., Proc. Natl. Acad. Sci. USA, 97 (2), 559-564 (2000)). According to the technology recorded in these papers, at the time of filing this application, many companies were already engaged in commissioned production of polypeptides, and they were already on the market as a cell-free protein synthesis kit (e.g., available from Cellfree Science Co., Ltd., Japan).

[0054] A single-stranded or double-stranded polynucleotide including 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 conventionally known methods. That is, by selecting codons corresponding to each amino acid residue constituting the designed amino acid sequence, a 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 synthesis device or the like. The obtained single-stranded DNA can then be used as a template to obtain a target double-stranded DNA using various enzymatic synthesis methods (usually PCR). In addition, the polynucleotide may be in the form of DNA or in the form of RNA (mRNA, etc.). DNA may be provided in a double-stranded or single-stranded form. When provided in a single-stranded form, it may be either a coding strand (sense strand) or a non-coding strand (antisense strand) having a complementary sequence thereto.

[0055] 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 cell-free protein synthesis systems as described above.

[0056] The construct can be used as an active ingredient of a composition for use based on the function of the exogenous substance. It should be noted that the construct can also be in the form of a salt, as long as the function of the exogenous substance is not lost. For example, an acid salt obtained by an addition reaction of an inorganic acid or an organic acid, which is usually used in a conventional manner, can be used. Therefore, the "construct" recorded in this specification and claims also includes the form of such a salt.

[0057] The construct can also be used as an active ingredient of a composition which may contain various pharmaceutically acceptable carriers depending on the form of use.

[0058] As the above-mentioned carrier, it is preferred that the carrier is commonly used in peptide drugs as a diluent, excipient, etc. As such a carrier, although it may be appropriately different according to the purpose and form of the construct for the introduction of exogenous substances, water, physiological buffer and various organic solvents can be generally cited. In addition, the carrier can also be a non-drying oil such as an alcohol (ethanol, etc.) aqueous solution, glycerol, olive oil, etc. of an appropriate concentration, or it can also be a liposome. In addition, as a minor component that may be included in the pharmaceutical composition, various fillers, extenders, binders, wetting agents, surfactants, pigments, fragrances, etc. can be cited.

[0059] The form of the composition is not particularly limited. Examples thereof include liquids, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, ointments, and the like. In addition, for use in injections, a lyophilized product or granulated product that is dissolved in physiological saline or an appropriate buffer (e.g., PBS) or the like to prepare a drug solution immediately before use may also be used.

[0060] The process of preparing various forms of drugs (compositions) using constructs (main components) and various carriers (secondary components) as materials can be carried out according to conventional methods. These manufacturing methods themselves are not methods that have the characteristics of the technology described in this specification, so detailed descriptions are omitted. As a source of information on the details of the formulation, for example, Comprehensive Medicinal Chemistry (1990) edited by Corwin Hansch and published by Pergamon Press can be cited.

[0061] According to the present invention, a method for introducing exogenous substances into a living organism (in vivo) or in vitro (in vitro) using the construct of the present invention is provided. The method mainly comprises the following steps (1) to (2):

[0062] (1) preparing a construct for introducing an exogenous substance: the construct comprising a carrier peptide fragment consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 4 and a target exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment; and (2) A step of supplying the construct to a sample containing target eukaryotic cells. In addition, the method of the present invention may further include, after the step (2), a step (3) of incubating the sample to which the construct has been supplied, and introducing the construct into the eukaryotic cells in the sample.

[0063] The above-mentioned "eukaryotic cells" include, for example, various tissues, internal organs, organs, blood and lymph in the living body. The above-mentioned "eukaryotic cells" include, for example, various cell clusters, tissues, internal organs, organs, blood and lymph, and cell lines removed from the living body in vitro.

[0064] The construct of the present invention or the composition comprising the construct of the present invention can be used in a living organism according to the method and dosage corresponding to its form and purpose. For example, as a liquid preparation, the required amount can be administered to the affected part (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of the patient (i.e., living 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 part 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, it is preferred to use a capsule or protective (coating) material that can inhibit the decomposition of digestive enzymes in the digestive tract.

[0065] Alternatively, for eukaryotic cells cultured in vitro, an appropriate amount of the construct may be supplied to the target eukaryotic cell culture medium at least once. The amount supplied each time and the number of times supplied vary depending on the type of eukaryotic cells cultured, cell density (cell density at the start of culture), number of passages, culture conditions, type of culture medium, and other conditions, and are therefore not particularly limited. For example, it is preferred to perform one, two, or more additions so that the concentration of the carrier peptide fragment in the culture medium is approximately 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, for example, in the range of 1 μM or more and 20 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 is therefore not particularly limited. For example, it may be more than 0.5 hours, more than 1 hour, more than 4 hours, more than 8 hours, or more than 20 hours. It should be noted that the incubation conditions also vary depending on the type of eukaryotic cells, and are therefore not particularly limited, and may be incubated at a temperature of 37° C. in an environment of 5% CO2.

[0066] It should be noted that in the test examples described below, an example of an in vitro introduction method will be given.

[0067] The method for evaluating the introduction efficiency of the construct is not particularly limited. For example, when a pigment (usually a fluorescent pigment compound) is bound to the construct, the introduction efficiency into eukaryotic cells can be evaluated using microscopic observation (e.g., fluorescence microscopic observation), flow cytometry, etc. In addition, the introduction efficiency of the construct can also be evaluated by immunochemical methods (e.g., Western Blotting, immunocytological staining, etc.) using antibodies that specifically recognize the peptide portion of the construct.

[0068] As described above, as specific embodiments of the technology described in this specification, the following items can be cited.

[0069] Item 1: A method for introducing an exogenous substance, wherein the method introduces a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell in an in vitro environment, comprising: (1) preparing a construct for introducing an exogenous substance, the construct comprising a carrier peptide fragment consisting of any one of the following amino acid sequences and the above-mentioned target exogenous substance bound to the N-terminal side and / or the C-terminal side of the above-mentioned carrier peptide fragment, KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (sequence number 4); as well as (2) A step of supplying the above construct to a sample containing target eukaryotic cells.

[0070] Item 2: The method according to Item 1, wherein the exogenous substance is at least one organic compound selected from the group consisting of polypeptides, nucleic acids, pigments and drugs.

[0071] Item 3: The method according to Item 1 or Item 2, wherein the exogenous substance is disposed on the C-terminal side of the carrier peptide fragment.

[0072] Item 4: The method according to any one of Items 1 to 3, wherein the eukaryotic cell into which the construct is introduced is a cell of a human or a non-human mammal.

[0073] Item 5: A construct comprising a carrier peptide fragment consisting of any one of the following amino acid sequences and the above-mentioned target exogenous substance bound to the N-terminal side and / or the C-terminal side of the above-mentioned carrier peptide fragment, KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (serial number 4).

[0074] Item 6: The construct according to Item 5, wherein the exogenous substance is at least one organic compound selected from the group consisting of polypeptides, nucleic acids, pigments and drugs.

[0075] Item 7: The construct according to Item 5 or Item 6, wherein the exogenous substance is disposed on the C-terminal side of the carrier peptide fragment.

[0076] Item 8: A carrier peptide fragment, which is composed of any of the following amino acid sequences: KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (serial number 4).

[0077] Several relevant test examples of the technology described in this specification are described below, but it is not intended to limit the technology described in this specification to the contents shown in these test examples.

[0078] <Preparation of construct for introduction of exogenous substances> Constructs of synthetic peptides having the amino acid sequence composition shown in Table 1 were prepared. The construct having peptide 1 was designated as sample 1, the construct having peptide 2 was designated as sample 2, the construct having peptide 3 was designated as sample 3, and the construct having peptide 3 was designated as sample 3. All samples 1 to 3 were synthesized by commissioning Eurofins Genomics Co., Ltd.

[0079] It should be noted that in constructs 1 to 4, peptides in which the α-amino groups of the N-terminal amino acid residues of peptides 1 to 4 were all acetylated were prepared. In addition, peptides in which the C-terminal amino acid residues of peptides 1 to 4 were bound with the fluorescent dye FAM (C 21 H 12 O7: 5(6)-carboxyfluorescein, molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) peptide. Furthermore, samples 1 to 3 were diluted with dimethyl sulfoxide (DMSO) to prepare sample solutions 1 to 4 with a sample concentration of 2 mM.

[0080] [Table 1] [Test 1] <Evaluation of cell membrane permeability by flow cytometry> Using NSC-34 cells (mouse motor neuron-like hybrid cell line) as eukaryotic cells, the cell membrane permeability of constructs 1 to 4 was analyzed. In Examples 1 to 4, the above-prepared samples 1 to 4 were used, respectively, and in Example 5, a FAM solution was used.

[0081] (Example 1) NSC-34 cells were cultured in DMEM (Dulbecco's modified Eagle's medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Catalog No. 044-29765)) containing 10% FBS (fetal bovine serum) as a regular culture medium.

[0082] 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 the incubation, the DMEM containing 10% FBS was added to inactivate the trypsin, and then the cells were centrifuged at 150×g for 5 minutes to precipitate the cells. After removing the supernatant generated by centrifugation, the DMEM containing 10% FBS was added to the precipitate (cell pellet) to prepare approximately 2×10 5 1 mL of the cell suspension was added to the wells of a commercially available 6-well plate (manufactured by AJEXA TECHNO GLASS CO., LTD.), and cells (approximately 2×10 5 cells / well). Furthermore, the 2 mM sample solution 1 was diluted with the DMEM containing 10% FBS to prepare a sample solution 1 with a sample 1 concentration of 20 μM. Then, 1 mL of the 20 μM sample solution 1 was added to the well (i.e., the sample 1 concentration of the medium in the well was 10 μM and the DMSO concentration was 0.5%). The cells were then incubated at 37°C for 20 hours under 5% CO2 conditions.

[0083] After incubation for 20 hours, the culture supernatant was removed from the wells, and the cells in the wells were washed twice with 1 mL of PBS. Next, 200 μL of 0.25% trypsin / EDTA solution was added to the wells, and incubated at 37°C for 3 minutes. After the incubation, 400 μL of the above-mentioned DMEM containing 10% FBS was added to the wells to inactivate trypsin, and then the cell suspension in the wells was pipetted into a test tube to recover the cells. Thereafter, 600 μL of PBS was further added to the wells to wash the wells. Then, the PBS in the wells was pipetted into the above-mentioned test tubes to recover the cells remaining in the wells into the above-mentioned test tubes. The test tubes were 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. This operation was repeated twice, and then the supernatant was removed to obtain cells (cell pellets) cultured in the medium containing Sample 1.

[0084] The cell membrane permeability of the sample 1 of the cells (cell pellets) obtained above was analyzed using a flow cytometer. As the flow cytometer, an On-Chip flow cytometer (manufactured by On-Chip Biotechnologies Co., LTD.) was used.

[0085] For this analysis, the cell pellet obtained above was suspended in 100 μL of On-Chip T buffer to prepare a cell suspension for analysis.

[0086] The flow cytometer was used to perform selection based on forward scatter (FSC) and side scatter (SSC), set gates for the cell population to be analyzed, and measured the fluorescence intensity of the cell population within the gate. It should be noted that the analysis was performed with the number of cells in the cell population being at least 10,000. The fluorescence intensity was measured using the fluorescence detector FL2 of the flow cytometer that can detect the FAM fluorescence wavelength (optimal detection wavelength is around 543nm). The commercially available analysis software "FlowJo ® ” (manufactured by TreeStar) analyzed the measurement results and obtained the median fluorescent intensity (MFI) of the measured cell population.

[0087] (Example 2) The same procedure as in Example 1 was carried out except that the sample solution 1 was changed to the sample solution 2 prepared above.

[0088] (Example 3) The same procedure as in Example 1 was carried out except that the sample solution 1 was changed to the sample solution 3 prepared above.

[0089] (Example 4) The same procedure as in Example 1 was carried out except that the sample solution 1 was changed to the sample solution 4 prepared above.

[0090] (Example 5) The same procedure as in Example 1 was followed except that the sample solution 1 was changed to a FAM solution diluted with DMSO. The concentration of the FAM solution was the same as that of the sample 1 solution (i.e., the FAM concentration of the medium in the well was 10 μM and the DMSO concentration was 0.5%).

[0091] The results of Examples 1 to 5 are shown in Tables 2 and Figure 1 . Figure 1 Schematic diagram of the MFI value curve in each case.

[0092] [Table 2] As shown in Table 2 and Figure 1As shown, the MFI values ​​of Examples 1 to 4 were significantly higher than those of Example 5. That is, it was confirmed that by binding at least one of Peptides 1 to 4 to FAM as an exogenous substance, cell membrane permeability can be imparted to the construct, and more FAM can be introduced into the cell.

[0093] It should be noted that, although no detailed data are given, the inventors of the present invention have confirmed through research that a construct having any one of peptides 1 to 4 can efficiently introduce the exogenous substance from outside the cell into the cytoplasm not only when the exogenous substance is a fluorescent dye, but also when it is a polypeptide, nucleic acid and drug.

[0094] [Test 2] <Evaluation of cytotoxicity of carrier peptide fragments> The cytotoxicity of the above peptides 1 to 3 was evaluated using WS-1 cells (human skin fibroblast-like cell line, obtained from the American Type Culture Collection, ATCC No. CRL-1502) as eukaryotic cells. As a culture medium, DMEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 044-29765) containing 10% FBS (fetal bovine serum) was used. In Test 2, samples 1 to 3 were dissolved in DMSO to prepare 6 mM sample solutions 1 to 3. The 6 mM sample solutions 1 to 3 were diluted with the above culture medium to prepare 12.5 μM, 25 μM, 50 μM and 100 μM sample solutions 1 to 3. In addition, as a reference example, a construct having a carrier peptide fragment showing cell membrane permeability containing the amino acid sequence shown in SEQ ID NO: 5 was prepared. The reference example adopts the same composition as samples 1 to 3 except for the amino acid sequence constituting the carrier peptide fragment. Reference Example A solution of Reference Example containing 12.5 μM to 100 μM of the compound was prepared in the same manner as in Sample Solutions 1 to 3 (hereinafter also referred to as “Solution (Reference Example)”).

[0095] After washing the WS-1 cells adhered to the culture plate with PBS, a 0.25% trypsin / 0.53 mM EDTA solution was added and incubated at 37°C for 3 to 5 minutes. After the incubation, the above-mentioned medium was added to inactivate the trypsin, and the cells were centrifuged at 900 rpm for 5 minutes at room temperature to precipitate the cells. After removing the supernatant generated by centrifugation, the above-mentioned medium was added to the precipitate (cell pellet) to prepare approximately 1×10 5 100 μL of the cell suspension was added to the wells of a commercially available 96-well plate (manufactured by AJEXA TECHNO GLASS CO., LTD.) and cells were seeded (approximately 1×10 4cells / well), and incubated for 24 hours in an incubator at 5% CO2 and 37°C. After the incubation, the culture medium in the wells was removed, and 90 μL of the sample solutions 1 to 3 and the solution (reference example) prepared above at 12.5 μM, 25 μM, 50 μM, and 100 μM was added to each of the three wells (i.e., the test was performed in three consecutive wells). In addition, as a control, 90 μL of the above culture medium containing 1.67% DMSO was added to the three wells (the same DMSO concentration as in the wells to which the 100 μM sample solution was added). In addition, 90 μL of the above culture medium without DMSO was added to each of the three wells. Then, the incubation was further performed for 24 hours in an incubator at 5% CO2 and 37°C. After the incubation, the culture medium in each well was removed, and 100 μL of the above culture medium without DMSO was added to each well. It should be noted that, at this time, 100 μL of the above culture medium was also added to the wells without cells as a blank group. Furthermore, 10 μL of the cell proliferation / cytotoxicity kit: Cell Counting Kit-8 (CCK-8, manufactured by Dojin Chemical) was added to each well. Then, the cells were incubated in an incubator at 5% CO2 and 37°C for 1.5 hours, and the absorbance at 450 nm was measured using a microplate reader. Then, the arithmetic mean of the absorbance of three wells for each type and concentration of the solution added to the culture medium was calculated. The average value of the absorbance of the three wells in which the cells were cultured with the above-mentioned culture medium without DMSO was taken as 100%, and the cell viability was calculated. The results are shown in Figure 2 . Figure 2 The horizontal axis of the curve represents the sample concentration in the medium, and the vertical axis represents the cell viability. It should be noted that the culture example of the above medium containing 1.67% DMSO tested as a control was plotted at the position of sample concentration of 100 μM.

[0096] like Figure 2 As shown, in samples 1 to 3, even at a sample concentration of 100 μM, the cell viability did not decrease compared to the example in which 1.67% DMSO was added. In other words, it can be seen that samples 1 to 3 showed almost no cytotoxicity even at a high concentration of 100 μM.

[0097] The above detailed description of the specific examples of the technology described in the present invention is only for illustration and does not constitute a limitation on the scope of protection claimed. The technology described in the scope of protection claimed includes various modifications and changes of the specific examples described above.

[0098] According to the technology described in the present invention, a carrier peptide fragment artificially produced for introducing a target exogenous substance from the outside of a eukaryotic cell (especially a human or a variety of animal cells represented by mammals other than humans without a cell wall) into the cytoplasm and a construct having the carrier peptide fragment are provided. Using the construct, the target exogenous substance can be more safely and effectively introduced into the target cell, and living tissues such as cells into which the exogenous substance has been introduced and organs containing the cells containing the exogenous substance are obtained. In addition, since the cytotoxicity of the carrier peptide fragment is low, a construct with a higher concentration than before can be used, so the exogenous substance can be introduced at a high concentration. In addition, by using the construct, a therapeutic agent targeted at the disease can be provided.

Claims

1. A method for introducing an exogenous substance, characterized in that: The method introduces a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell in an in vitro environment, comprising: (1) Preparation of a construct for introducing an exogenous substance, which contains the following amino acid sequence KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (serial number 4) A carrier peptide fragment having any sequence in and the target exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment; and (2) A step of supplying the construct to a sample containing target eukaryotic cells.

2. The method according to claim 1, characterized in that The exogenous substance is at least one organic compound selected from polypeptides, nucleic acids, pigments and drugs.

3. The method according to claim 1 or 2, characterized in that The exogenous substance is arranged on the C-terminal side of the carrier peptide fragment.

4. The method according to claim 1 or 2, characterized in that: The eukaryotic cells into which the construct is to be introduced are cells of humans or mammals other than humans.

5. A construct, characterized in that Contains the following amino acid sequence KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (serial number 4) A carrier peptide fragment having any sequence in and the target exogenous substance bound to the N-terminal side and / or C-terminal side of the carrier peptide fragment.

6. The construct according to claim 5, characterized in that The exogenous substance is at least one organic compound selected from polypeptides, nucleic acids, pigments and drugs.

7. The construct according to claim 5 or 6, characterized in that The exogenous substance is arranged on the C-terminal side of the carrier peptide fragment.

8. A carrier peptide fragment, characterized in that: Contains the following amino acid sequence KKRTLRKSNSRKKR (serial number 1), KKRTLRKSSNRKKR (serial number 2), KKRTLRKSNNRKKR (sequence number 3) and KKRTLRKNSNRKKR (serial number 4) Any sequence in .

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