Synthetic peptides and constructs

Abstract

The present invention addresses the problem of providing a novel synthetic peptide having cell membrane permeability. [Solution] A synthetic peptide disclosed by the present invention comprises any one of the following amino acid sequences: (1) an amino acid sequence in which two or more minimum structural units are continuously bonded in series, said minimum structural units being PD (proline residue-aspartic acid residue) or PE (proline residue-glutamic acid residue); and (2) an amino acid sequence obtained by binding 1-3 glycine residues to the C-terminal side of the amino acid sequence of (1).

Description

Technical Field

[0001] The present invention relates to a synthetic peptide having cell membrane permeability and a construct having the synthetic peptide. It should be noted that this application claims priority based on Japanese Patent Application No. 2022-201099 filed on December 16, 2022, and the entire content of the application is incorporated herein by reference as part of this specification. Background Art

[0002] Cell-penetrating peptides (CPPs) are peptides that can pass through the cell membrane from the outside of the cell and move at least into the cytoplasm. In Japanese Patent No. 7041853, a carrier peptide fragment that functions as a CPP is disclosed, and a technique for introducing a target exogenous substance into the interior of a eukaryotic cell by using the carrier peptide fragment is disclosed.

[0003] Prior Art Documents

[0004] Patent Documents

[0005] Patent Document 1: Japanese Patent No. 7041853 Summary of the Invention

[0006] Technical Problem to be Solved by the Invention

[0007] Generally, basic amino acids (for example, arginine, lysine) are included in the amino acid sequence constituting CPP, and it is said that basic amino acids contribute to the efficiency of cell membrane permeability. However, it is known that the cytotoxicity tends to increase, for example, when the proportion of basic amino acids in CPP is high.

[0008] Therefore, the main object of the present invention is to provide a new synthetic peptide having cell membrane permeability that does not contain basic amino acids. Another object is to provide a construct having the synthetic peptide.

[0009] Technical Solution for Solving the Technical Problem

[0010] The synthetic peptide disclosed in the present invention is a CPP that can introduce a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell. The synthetic peptide is composed of any of the following amino acid sequences:

[0011] (1) An amino acid sequence in which two or more of the minimum structural units of PD (proline residue - aspartic acid residue) or PE (proline residue - glutamic acid residue) are serially and continuously bound; and

[0012] (2) An amino acid sequence in which 1 to 3 glycine residues are bound to the C-terminal side of the amino acid sequence in (1).

[0013] The synthetic peptide thus constituted does not contain arginine residues, lysine residues, and histidine residues, which are known as basic amino acids. As described above, it has been considered that existing amino acid residues can exhibit high cell membrane permeability by having basic amino acids. However, the synthetic peptide disclosed in the present invention can exhibit high cell membrane permeability even though it does not contain basic amino acids.

[0014] In one mode of the synthetic peptide disclosed in the present invention, the amino acid sequences of the above (1) and the above (2) have two or more and six or less of the above minimum structural units. That is, the synthetic peptide disclosed in the present invention can have an amino acid sequence in which the above minimum structural unit is repeated two or more times and six or less times.

[0015] The synthetic peptide disclosed in the present invention includes, for example, any of the following amino acid sequences:

[0016] PDPD (SEQ ID NO: 1);

[0017] PEPE (SEQ ID NO: 2);

[0018] PDPEG (SEQ ID NO: 7);

[0019] PEPDG (SEQ ID NO: 8);

[0020] PDPDPDPDPDPDG (SEQ ID NO: 9);

[0021] PEPEPEPEPEPEG (SEQ ID NO: 10); and

[0022] PDPEPDPEPDPEG (SEQ ID NO: 11).

[0023] Any of the above amino acid sequences can exhibit high cell membrane permeability.

[0024] In addition, according to the present invention, in order to achieve the above object, there is provided a construct for importing a target foreign substance (hereinafter, also simply referred to as "construct") prepared for importing the target foreign substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell. The construct disclosed in the present invention has the synthetic peptide disclosed in the present invention and the target foreign substance bound to the N-terminal side and / or C-terminal side of the synthetic peptide. Since the construct has a synthetic peptide that functions as a CPP, it can be efficiently imported into the eukaryotic cell and can efficiently import the target foreign substance into the cell.

[0025] In one mode of the construct disclosed in the present invention, the foreign substance may be at least one organic compound selected from polypeptides, nucleic acids, pigments, and pharmaceuticals.

[0026] Here, "polypeptide" refers to a polymer having a structure in which multiple amino acids are bonded through peptide bonds. The polypeptide is not limited by the number of peptide bonds (i.e., the number of amino acid residues). That is, polypeptides include: compounds generally referred to as peptides with 10 or more and less than about 300 amino acid residues, and compounds generally referred to as proteins (typically macromolecular compounds composed of 300 or more amino acid residues). In this field, polypeptides and proteins are not strictly distinguished. In this specification, polymers composed of multiple amino acid residues (including oligomers) are collectively referred to as polypeptides.

[0027] In addition, "nucleic acid" refers to a polymer of nucleotides, including DNA and RNA. "Nucleic acid" is not limited by the number of bases.

[0028] 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 synthetic peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a graph showing the MFI values obtained by analyzing the cells by flow cytometry after adding the constructs (additives) shown in Examples 1 to 6 and Reference Example 1 to the culture solution of NSC-34 cells and culturing them.

[0030] Figure 2 It is a graph showing the MFI values obtained by analyzing the cells by flow cytometry after adding the constructs (additives) shown in Examples 7 to 9 and Reference Example 2 to the culture solution of NSC-34 cells and culturing them. DETAILED DESCRIPTION OF THE INVENTION

[0031] 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 as matters that can be designed by those skilled in the art based on existing technologies in fields such as cell engineering, physiology, medicine, pharmacy, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, and genetics. In addition, the technology disclosed in the present invention can be implemented based on the content described in this specification and common general knowledge in the art. It should be noted that in the following description, amino acids are represented by single-letter symbols of the nomenclature for amino acids shown in the IUPAC-IUB guidelines as appropriate. In addition, in this specification, the term "amino acid residue" includes the N-terminal amino acid and C-terminal amino acid of the peptide chain unless otherwise specified.

[0032] In addition, in this specification, a "synthetic peptide" is not a substance in which the peptide chain itself stably exists independently in nature, but refers to a peptide fragment manufactured by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and capable of stably existing in a specified composition. Herein, "peptide" is a term referring to a polymer of amino acids having peptide bonds (including dimers, trimers, oligomers, etc.), and is not limited by the number of amino acid residues.

[0033] In addition, in this specification, the amino acid residues constituting a peptide or protein may be L-forms (L-isomers) or D-forms. Furthermore, in the amino acid sequences described in this specification, the left side is always the N-terminal side and the right side represents the C-terminal side.

[0034] In one mode of the synthetic peptide disclosed in the present invention, the synthetic peptide comprises an amino acid sequence shown in the following (1) or (2):

[0035] (1) An amino acid sequence formed by tandemly linking two or more of the smallest structural units of PD (proline residue - aspartic acid residue) or PE (proline residue - glutamic acid residue);

[0036] (2) An amino acid sequence having 1 to 3 glycine residues bound to the C-terminal side of the amino acid sequence of (1) above.

[0037] In addition, in the amino acid sequence constituting the above synthetic peptide, as the smallest structural unit, only two or more of either PD or PE can be tandemly and continuously bound. Alternatively, it can also be a mixture of both PD and PE, with two or more tandemly and continuously bound. For example, PD and PE can be tandemly and continuously bound in an alternating configuration. Since both aspartic acid and glutamic acid are acidic amino acids, the synthetic peptide disclosed in the present invention can also be said to have a repeating sequence formed by repeating proline residues - acidic amino acid residues. In addition, "tandemly and continuously bound" means that another smallest structural unit is bound to the N-terminal side and / or C-terminal side of one smallest structural unit through a peptide bond.

[0038] In the synthetic peptide disclosed in the present invention, for example, two or more and ten or fewer of the above smallest structural units can be tandemly and continuously bound. In addition, the smallest structural unit can be tandemly and continuously bound in numbers of two or more and six or fewer, three or more and six or fewer, four or more and six or fewer, or five or more and six or fewer. In a preferred mode, six smallest structural units are tandemly and continuously bound. An amino acid sequence in which six smallest structural units are tandemly and continuously bound can exhibit particularly excellent cell membrane permeability.

[0039] As shown in the above (2), the synthetic peptide disclosed in the present invention may have 1 to 3 glycine residues bound to the C-terminal side of the amino acid sequence formed by serially and continuously binding the minimum structural units, and the number of the glycine residues is preferably 1 to 2, and more preferably 1. Glycine is a neutral amino acid with the smallest side chain among amino acids. Therefore, when the amino acid sequence formed by serially and continuously binding the minimum structural units has cell membrane permeability, even if 1 to 3 glycine residues are bound to the C-terminal side of the amino acid sequence, the cell membrane permeability will not be significantly impaired, which can be understood according to the common general knowledge in the technical field of the present invention.

[0040] The number of amino acid residues of the synthetic peptide disclosed in the present invention includes at least 2 minimum structural units, and thus can be more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, or more than 12. In addition, the upper limit of the number of amino acid residues of the synthetic peptide is not particularly limited. For example, it can be 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, or 13 or less. When the number of amino acid residues of the synthetic peptide is too large, the volume of the synthetic peptide becomes too large, and the cell membrane permeability may decrease.

[0041] In the amino acid sequence constituting the synthetic peptide disclosed in the present invention, as specific examples having 2 minimum structural units, PDPD (SEQ ID NO: 1), PEPE (SEQ ID NO: 2), PDPE (SEQ ID NO: 3), PEPD (SEQ ID NO: 4), PDPDG (SEQ ID NO: 5), PEPEG (SEQ ID NO: 6), PDPEG (SEQ ID NO: 7), and PEPDG (SEQ ID NO: 8) can be cited.

[0042] In addition, in the amino acid sequence constituting the synthetic peptide disclosed in the present invention, as specific examples having 6 minimum structural units, for example, PDPDPDPDPDPDG (SEQ ID NO: 9), PEPEPEPEPEPEG (SEQ ID NO: 10), and PDPEPDPEPDPEG (SEQ ID NO: 11) etc. can be cited.

[0043] In addition, the synthetic peptide disclosed in the present invention may be an altered sequence of the amino acid sequence shown in the above (1) or (2) as long as it does not significantly impair the cell membrane permeability. Among them, the "altered sequence" refers to an amino acid sequence (altered amino acid sequence) formed by substituting, deleting, and / or adding (inserting) one or several (typically 2 or 3) amino acid residues.

[0044] As a typical example of the change sequence in this specification, for example, a sequence generated by so-called conservative amino acid replacement in which 1, 2, or 3 amino acid residues are conservatively substituted, or a sequence formed by adding (inserting) or deleting 1, 2, or 3 amino acid residues to a specified amino acid sequence can be cited. As a typical example of conservative substitution, for example, a sequence in which a proline residue, which is a nonpolar amino acid, is substituted with another nonpolar amino acid residue such as a glycine residue can be cited.

[0045] Since the synthetic peptide disclosed in the present invention described above can have cell membrane permeability, it can introduce a target foreign substance from the outside of a eukaryotic cell into at least the cytoplasm (and further into the nucleus) of the cell. Therefore, in the present invention, a construct for introducing a foreign substance having the synthetic peptide disclosed in the present invention is provided.

[0046] The construct disclosed in the present invention has the synthetic peptide disclosed in the present invention described above and a target foreign substance bound to the N-terminal side and / or C-terminal side of the synthetic peptide.

[0047] The construct disclosed in the present invention can be designed and constructed by directly binding or indirectly binding (linking) a desired foreign substance to the N-terminal side and / or C-terminal side of the above synthetic peptide through an appropriate linker.

[0048] The linker is not particularly limited and can be a peptidic linker or a non-peptidic linker. Although not particularly limited, the amino acid sequence constituting the peptidic linker is preferably an amino acid sequence that does not cause steric hindrance and is flexible. The peptidic linker is, for example, a linker composed 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 selected from one or more of glycine, alanine, serine, and the like. In addition, β-alanine can also be used as the linker. The non-peptidic linker is not particularly limited, and for example, an alkyl linker, a PEG (polyethylene glycol) linker, an aminohexanoyl spacer, or the like can be used.

[0049] The foreign substance can be, for example, an organic compound such as a polypeptide, a nucleic acid, a pigment, or a drug.

[0050] In the case where the foreign substance is a polypeptide, it is possible to design a peptide chain in a manner that includes the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the above synthetic peptide, and biosynthesize or chemically synthesize the peptide chain, thereby producing a construct for introducing the target foreign substance. Various known scientific methods can also be used to directly or indirectly bind various nucleic acids such as DNA or RNA, pigments (such as various fluorescent pigment compounds such as FAM or FITC), or organic compounds that function as pharmaceuticals (such as antitumor agents containing nucleic acid-based antitumor agents such as 5-fluorouracil (5FU), antiviral agents such as azidothymidine (AZT), etc.) to the N-terminal side and / or C-terminal side of the above carrier peptide fragment to prepare a construct. Although not particularly limited, the functions possessed by the foreign substance can be, for example, promoting the differentiation induction of stem cells (stem cell differentiation induction activity), inhibiting the proliferation of tumor cells (antitumor activity), inhibiting the proliferation of virus-infected cells (antiviral activity), etc.

[0051] Although not particularly limited, the functions possessed by the foreign substance can be, for example, promoting the differentiation induction of stem cells (stem cell differentiation induction activity), inhibiting the proliferation of tumor cells (antitumor activity), inhibiting the proliferation of virus-infected cells (antiviral activity), etc.

[0052] In the construct disclosed in the present invention, the number of foreign substances bound to the above synthetic peptide is not particularly limited. For example, for one synthetic peptide, one or more than one foreign substance can be bound. Although not particularly limited, for example, a polypeptide, nucleic acid, pharmaceutical, etc. can be bound to the C-terminal side of one synthetic peptide, and a pigment can be bound to the N-terminal side. By binding a pigment to the synthetic peptide, it is possible to easily evaluate the import efficiency of the construct into eukaryotic cells and the intracellular localization, and thus it is preferred.

[0053] In addition, in the case where the foreign substance is a polypeptide, the polypeptide (amino acid sequence) used is not particularly limited. For example, a polypeptide with a relatively large number of amino acid residues, such as a polypeptide or protein with about 100 to 1000 amino acid residues, can also be used as the foreign substance.

[0054] Typically, the total number of amino acid residues constituting the synthetic peptide produced as a construct for introducing a foreign substance is several to dozens or more (for example, 10 or more), preferably 1000 or less, more preferably 600 or less, still more preferably 500 or less, and particularly preferably 300 or less (for example, 10 to 300). A polypeptide of such a length is easy to synthesize (biosynthesis, chemical synthesis) and easy to use.

[0055] As an exogenous substance, it preferably involves the mature form or precursor of polypeptides (including pro-form and prepro-form) that function in the development, differentiation, proliferation, carcinogenesis, homeostasis (homeostasis), and metabolism regulation of various cells or tissues (organs). In addition, in order to introduce a polypeptide with unknown current function into cells and clarify its function in cells (within biological tissues), the exogenous substance introduction method disclosed in the present invention can also be implemented.

[0056] For example, when the eukaryotic cell as the object of exogenous substance introduction is a stem cell of a human or other mammal, it is preferable to use the mature form or its precursor of polypeptides with various physiological activities involved in the differentiation induction of the stem cell. Among them, "stem cell" includes adult stem cells, embryonic stem cells, and induced pluripotent stem cells (iPS cells). In addition, when the eukaryotic cell as the object of exogenous substance introduction is a cancer cell (tumor cell), it is preferable to use various polypeptides involved in the apoptosis induction of the cancer cell (tumor cell). Alternatively, in this case, it is preferable to use polypeptides that can hinder the function of the cancer cell (tumor cell) from inhibiting the immune surveillance mechanism. In addition, when the eukaryotic cell as the introduction object is a bacterially infected cell or a virus-infected cell, it is preferable to use 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. In addition, similar to synthetic peptides, as long as the polypeptide as an exogenous substance maintains its function, it may also include an altered amino acid sequence formed by substituting, deleting, and / or adding (inserting) one or several amino acid residues.

[0057] In a construct in which an exogenous substance is bound to the C-terminal side of a synthetic peptide, it is preferable that the α-amino group of the amino acid residue on the N-terminal side of the synthetic peptide is acetylated. The detailed mechanism is not yet clear, but since many proteins in eukaryotic cells are acetylated at the α-amino group of the N-terminal side amino acid, using such a structure can improve the stability of the construct in cells.

[0058] Preferably, the amino acid residues on the C-terminal side of the construct are amidated. When 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 tolerance) of such a 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 aqueous solvents include water, various buffers, physiological saline (e.g., PBS), cell culture media, etc. For example, in the case of a construct in which an exogenous substance is bound to the N-terminal side of a synthetic peptide, it is preferred that the carboxyl group of the amino acid residue on the C-terminal side of the synthetic peptide is amidated. In addition, for example, when the exogenous substance is a polypeptide and such a polypeptide is bound to the C-terminal side of the synthetic peptide, it is preferred that the carboxyl group of the C-terminal amino acid residue of the polypeptide is amidated.

[0059] Peptides with relatively short peptide chains (including polypeptides, synthetic peptides, and peptide linkers that make up exogenous substances) 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. Preferably, a solid-phase synthesis method using Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxy carbonyl) as the protecting group for the amino group is applied. That is, a peptide chain with a desired amino acid sequence and modified parts (such as N-terminal acetylation, C-terminal amidation, etc.) can be synthesized by the solid-phase synthesis method using a commercially available peptide synthesizer. In addition, only a part of the peptide chain can be synthesized using the above method. For example, only the synthetic peptide can be synthesized, or a peptide chain containing the synthetic peptide and the peptide linker part can be synthesized.

[0060] 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, a recombinant vector with an expression gene construct is constructed according to the host cell, and the expression gene construct is composed of the synthesized polynucleotide (DNA) and various regulatory elements (including promoters, ribosome binding sites, terminators, enhancers, and various cis-elements that control the expression level) for expressing the amino acid sequence in the host cell.

[0061] Using ordinary 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 part is separated from the host cell (in the case of secretion, from the culture medium), and refolding, purification, etc. are performed as needed to obtain the target peptide part.

[0062] In addition, for the construction method of the recombinant vector and the method of introducing the constructed recombinant vector into a host cell, etc., the methods that have been carried out in this field can be directly adopted. The method itself does not particularly reflect the characteristics of this technology, so detailed description is omitted.

[0063] For example, in order to efficiently and massively produce in a host cell, 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 synthesized gene is introduced into an appropriate site of a fusion protein expression vector (for example, GST (Glutathione S-transferase) fusion protein expression vectors such as the pET series provided by Novagen and the pGEX series provided by Amersham Bioscience). Then, the host cell (typically Escherichia coli) is transformed with 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 using such currently well-known fusion protein expression systems (for example, the GST / His system provided by Amersham Bioscience can be utilized), the target construct (artificial polypeptide) can be manufactured.

[0064] 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 (ATP, RNA polymerase, amino acids, etc.) required for the synthesis of the peptide portion are used, and the so-called cell-free protein synthesis system is adopted to synthesize the target polypeptide in vitro. Regarding the cell-free protein synthesis system, for example, refer to the papers of Shimizu et al. (Shimizu et al., Nature Biotechnology, 19, 751 - 755 (2001)) and Madin et al. (Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559 - 564 (2000)). Based on the technologies described in these papers, many enterprises have been engaged in the contract production of polypeptides at the time of filing this invention application, and cell-free protein synthesis kits (for example, available from CellFree Sciences in Japan) are also commercially available.

[0065] Single-stranded or double-stranded polynucleotides containing the nucleotide sequence of the peptide moiety of the coding construct and / or nucleotide sequences complementary thereto can be readily produced (synthesized) by currently well-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, the polynucleotide (single-stranded) corresponding to the desired nucleotide sequence can be readily obtained using a DNA synthesizer or the like. Further, using the obtained single-stranded DNA as a template, various enzymatic synthesis methods (typically PCR) can be employed to obtain the target double-stranded DNA. Additionally, 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. In the case of single-stranded provision, it can be the coding strand (sense strand) or the non-coding strand (antisense strand) of the sequence complementary thereto. 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 as described above.

[0066] 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. In addition, as long as the function of the exogenous substance is not lost, the construct can also be in the form of a salt. For example, acid addition salts obtained by addition reactions of commonly used inorganic acids or organic acids according to conventional methods can be used. Therefore, the "construct" described in this specification and the claims can include constructs in such salt forms.

[0067] The construct can be used as an active ingredient of a composition that can contain various pharmaceutically acceptable carriers depending on the usage mode. As the above-mentioned carriers, for example, carriers commonly used as diluents, excipients, etc. in peptide pharmaceuticals are preferably used. As such carriers, they can vary appropriately depending on the use or form of the construct for exogenous substance introduction, and typically include water, physiological buffers, and various organic solvents. Additionally, such carriers can be aqueous solutions of alcohols (such as ethanol) at appropriate concentrations, glycerol, non-drying oils such as olive oil, or can also be liposomes. In addition, as minor components that can be contained in pharmaceutical compositions, various fillers, extenders, binders, wetting agents, surfactants, pigments, flavors, etc. can be cited.

[0068] The form of the composition is not particularly limited. For example, forms such as liquids, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, ointments, etc. can be cited. Additionally, 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) etc. immediately before use to prepare a medicinal solution.

[0069] The process of preparing various forms of medicaments (compositions) using constructs (main components) and various carriers (sub-components) as materials can be carried out according to currently well-known methods. This preparation method itself does not constitute the features of the present technology, so detailed description is omitted. As a detailed information source regarding formulations, for example, Comprehensive Medicinal Chemistry, edited by Corwin Hansch, published by Pergamon Press (1990) can be cited.

[0070] In addition, using the constructs disclosed in the present invention, exogenous substances can be introduced in vivo or in vitro. Generally speaking, the introduction method can include: a step of preparing the constructs disclosed in the present invention (preparation step); and a step of supplying the constructs to a specimen containing target eukaryotic cells (supply step). In addition, after the above supply step, it may further include a step of incubating the specimen to which the constructs have been supplied and introducing the constructs into the eukaryotic cells in the specimen (introduction step).

[0071] The above-mentioned "eukaryotic cells" in vivo include, for example, various tissues, organs, organs, blood, and lymph. The above-mentioned "eukaryotic cells" in vitro include, for example, various cell masses, tissues, organs, organs, blood, and lymph removed from organisms, as well as cell lines.

[0072] The composition containing the constructs disclosed in the present invention can be used in vivo in a method and dosage corresponding to its form and purpose. For example, as a liquid agent, it can be administered by intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal injection to the affected area (such as malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient (i.e., an organism) only in the desired amount. Or, a solid-form composition such as a tablet or a gel-like or aqueous gel-like composition such as an ointment can be directly administered to a specified tissue (i.e., the affected area of a tissue or organ containing, for example, tumor cells, virus-infected cells, inflammatory cells, etc.). Or, a solid-form composition such as a tablet can be administered orally. In the case of oral administration, in order to inhibit digestion by digestive enzymes in the digestive tract, it is preferably encapsulated or a protective (coating) material is applied.

[0073] Alternatively, for eukaryotic cells cultured 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 can 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 are not particularly limited. For example, it is preferably added once, twice, or multiple times such that the concentration of the synthetic peptide in the culture medium is generally in the range of 0.05 μM or more and 100 μM or less, such as 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 30 μM or less. In addition, regarding the incubation time after adding the construct, it can also vary depending on the type of eukaryotic cells and various conditions, and thus is not particularly limited. 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. Furthermore, regarding the incubation conditions, they can also vary depending on the type of eukaryotic cells and thus are not particularly limited. For example, incubation can be carried out in a 5% CO2 atmosphere at 37°C. In addition, an example of the in vitro introduction method is illustrated in the test examples described later.

[0074] The method for evaluating the introduction efficiency of the construct is not particularly limited. For example, when a pigment (typically a fluorescent pigment compound) is conjugated to the construct, microscopy (such as fluorescence microscopy) or flow cytometry, etc., can be used to evaluate the introduction efficiency into eukaryotic cells. In addition, the introduction efficiency of the construct can also be evaluated by an immunochemical method (such as Western blotting or immunocytochemical staining, etc.) using an antibody that specifically recognizes the peptide portion of the above construct.

[0075] As described above, as specific embodiments of the technology disclosed in the present invention, the embodiments described in the following items can be cited.

[0076] Item 1: A synthetic peptide that can introduce a target foreign substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, comprising any of the following amino acid sequences, Item 1: A synthetic peptide that can introduce a target foreign substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, comprising any of the following amino acid sequences,

[0077] (1) An amino acid sequence in which PD (proline residue - aspartic acid residue) or PE (proline residue - glutamic acid residue) is used as the minimum structural unit and two or more of these minimum structural units are continuously conjugated in series; and

[0078] (2) An amino acid sequence in which 1 to 3 glycine residues are conjugated to the C-terminal side of the amino acid sequence in (1) above.

[0079] Item 2: The synthetic peptide according to Item 1, wherein the amino acid sequence of (1) above and the amino acid sequence of (2) above have 2 or more and 6 or less of the above-mentioned minimum structural units.

[0080] Item 3: A synthetic peptide that can introduce a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, including any of the following amino acid sequences:

[0081] PDPD (SEQ ID NO: 1);

[0082] PEPE (SEQ ID NO: 2);

[0083] PDPEG (SEQ ID NO: 7);

[0084] PEPDG (SEQ ID NO: 8);

[0085] PDPDPDPDPDPDG (SEQ ID NO: 9);

[0086] PEPEPEPEPEPEG (SEQ ID NO: 10); and

[0087] PDPEPDPEPDPEG (SEQ ID NO: 11).

[0088] Item 4: A construct for introducing an exogenous substance, which is a construct for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, having:

[0089] The synthetic peptide according to any one of Items 1 to 3; and

[0090] The target exogenous substance bound to the N-terminal side and / or C-terminal side of the above synthetic peptide.

[0091] Item 5: The construct according to Item 4, wherein the above exogenous substance is at least 1 organic compound selected from polypeptides, nucleic acids, pigments, and pharmaceuticals.

[0092] Item 6: The construct according to Item 4 or 5, wherein the above exogenous substance is arranged on the C-terminal side of the above synthetic peptide.

[0093] In addition, the above Item 3 represents a specific example having the matters defined in Item 1 or 2, so Item 3 can be subordinate to Item 1 or 2.

[0094] Hereinafter, several test examples regarding the technology disclosed in the present invention will be described, but there is no intention to limit the technology disclosed in the present invention to the content shown in such test examples.

[0095] [Test 1]

[0096] <Production of the construct>

[0097] Prepare constructs with synthetic peptides consisting of the amino acid sequences shown in Table 1. Use the constructs with peptide n (n is a natural number from 1 to 6) shown in Table 1 as Sample n, and obtain Samples 1 to 6 from Eurofins Genomics Co., Ltd. Among Samples 1 to 6, prepare peptides in which the α-amino groups of the amino acid residues on the N-terminal side of Peptides 1 to 6 are all acetylated. In addition, prepare peptides in which a fluorescent dye FAM (C 21 H 12 O7:5(6)-Carboxyfluorescein, molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) is bound to the amino acid residues on the C-terminal side of Peptides 1 to 6.

[0098] [Table 1]

[0099] Table 1

[0100] Peptide No. Amino acid sequence Sequence number 1 PDPD 1 2 PEPE 2 3 PDPEG 7 4 PEPDG 8 5 PD - 6 PE -

[0101] <Evaluation of cell membrane permeability using flow cytometry>

[0102] As eukaryotic cells, use NSC-34 cells (mouse motor neuron-like hybrid cell line) to analyze the cell membrane permeability of Peptides 1 to 6. Use DMEM

[0103] (Dulbecco's modified Eagle's medium (manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No. 044-26765)) containing 10% FBS (fetal bovine serum) as the culture medium for NSC-34 cells. In addition, dissolve Samples 1 to 6 in dimethyl sulfoxide (DMSO) respectively to prepare Sample Solutions 1 to 6 with a sample concentration of 4 mM. Further, dilute these sample solutions with the above culture medium to prepare Sample Solutions 1 to 6 with a concentration of 40 μM. In Examples 1 to 6, use Sample Solutions 1 to 6 prepared above respectively, and use FAM solution in Reference Example 1.

[0104] (Example 1)

[0105] Suspend NSC-34 cells in the above culture medium to prepare a cell suspension of 2×10 5 cells / mL. Add 1 mL of this cell suspension to the wells of a commercially available 6-well plate (manufactured by AGC TECHNO GLASS Co., Ltd.) so that the number of NSC-34 cells becomes 2×10 5Inoculate in the manner of [number of cells] / well. Then, add 1 mL of 40 μM sample solution 1 to the wells so that the sample concentration in the culture medium in the wells becomes 20 μM. Then, let the 6-well plate stand in the cell culture device and incubate at 37 °C for 20 hours under 5% CO₂ conditions.

[0106] After incubating for 20 hours, remove the culture supernatant from the wells and wash the cells in the wells twice with 1 mL of PBS. Next, add 100 μL of 0.25% trypsin / EDTA solution to the wells and incubate at 37 °C for 3 minutes. After this incubation, add 900 μL of the above-mentioned culture medium to the wells to inactivate the trypsin, then transfer the cell suspension in the wells to a test tube and recover the cells. Centrifuge the test tube at 4 °C and 210×g for 5 minutes. After centrifugation, remove the supernatant, suspend (wash) the precipitate (cell pellet) with 1 mL of PBS, and centrifuge under the same conditions as above. Repeat this operation twice, then remove the supernatant to obtain the cells (cell pellet) cultured with the culture medium containing sample 1.

[0107] For the cells (cell pellet) obtained above, analyze the cell membrane permeability of sample 1 using a flow cytometer. As the flow cytometer, use On-Chip Flowcytometer (manufactured by On-Chip Biotechnologies Co.,LTD.).

[0108] For this analysis, suspend the cell pellet obtained above with 100 μL of On-Chip Tbuffer to prepare a cell suspension for analysis.

[0109] Use the above-mentioned flow cytometer to perform gating based on forward scatter (FSC) and side scatter (SSC), set the gate for the cell population to be analyzed, and measure the fluorescence intensity for the cell population within this gate. Among them, perform the analysis in such a way that the number of cells in this cell population is at least 10,000 or more. In the measurement of fluorescence intensity, use the fluorescence detector FL2 (optimal detection wavelength around 543 nm) of the above-mentioned flow cytometer that can detect the fluorescence wavelength of FAM. For this measurement result, use the commercially available analysis software "FlowJo (registered trademark)" (manufactured by TreeStar Inc.) for analysis to obtain the value of the fluorescence intensity (mean fluorescence intensity: MFI) of the cell population to be measured.

[0110] (Examples 2 - 6)

[0111] Change the sample solution 1 to any one of the sample solutions 2 to 6 prepared above, and otherwise, carry out in the same manner as in Example 1. Among them, the samples (constructs) used in each example are shown in Table 2.

[0112] (Reference Example 1)

[0113] Except for using the fluorescent dye FAM instead of Sample 1, carry out in the same manner as in Example 1. Among them, the concentration of FAM in the culture medium containing FAM is used in the same manner as the concentration of Sample 1 in Example 1 (that is, the concentration of FAM in the culture medium in the well is 20 μM).

[0114] The results obtained in Examples 1 to 6 and Reference Example 1 are shown in Table 2 and Figure 1 . Figure 1 is a chart showing the MFI values in each example.

[0115] [Table 2]

[0116] Table 2

[0117] Composition of the construct (additive) MFI Example 1 Ac-PDPD-FAM 15 Example 2 Ac-PEPE-FAM 16.2 Example 3 Ac-PDPEG-FAM 18.9 Example 4 Ac-PEPDG-FAM 19.9 Example 5 Ac-PD-FAM 7.8 Example 6 Ac-PE-FAM 7.4 Reference Example 1 FAM 10.9

[0118] As shown in Table 2 and Figure 1 shown, the MFI values of Examples 1 to 4 are higher than those of Reference Example 1. That is, compared with the case of adding FAM alone (Reference Example 1), Samples 1 to 4 in which any one of Peptides 1 to 4 is bound to FAM are more introduced into cells. From this, it can be known that Peptides 1 to 4 have cell membrane permeability.

[0119] On the other hand, the MFI values of Examples 5 and 6 are lower than those of Reference Example 1. From this, it can be known that Peptides 5 and 6 have cell membrane permeability. In addition, in the constructs having Peptide 5 or 6, the size of the construct becomes larger, and it is considered that it is more difficult to be introduced into cells compared with the case of FAM alone.

[0120] From the above, it can be known that the amino acid sequences PD and PE do not have cell membrane permeability, but when they have a repetitive sequence of amino acids, they can exhibit cell membrane permeability.

[0121] [Experiment 2]

[0122] Prepare Peptides 7 - 9 shown in Table 3. Use the constructs with Peptide m (m is a natural number from 7 to 9) shown in Table 3 as Sample m, and obtain Samples 7 - 9 from Eurofins Genomics. Among them, in Samples 7 - 9, prepare peptides in which the α - amino groups of the amino acid residues on the N - terminal side of Peptides 7 - 9 are all acetylated. Then, perform the same operations as in Test 1 above to evaluate the cell membrane permeability of Peptides 7 - 9 (Examples 7 - 9). In Reference Example 2, add the fluorescent dye FAM alone in the same manner as in Reference Example 1 above. However, in Examples 7 - 9, make the sample concentration in the culture medium in the wells 25 μM, and make the FAM concentration in the culture medium in the wells in Reference Example 2 25 μM. Table 4 shows the samples (constructs) used in each example. Table 4 and Figure 2 show the MFI values of each example.

[0123] [Table 3]

[0124] Table 3

[0125] Peptide No. Amino acid sequence Sequence number 7 PDPDPDPDPDPDG 9 8 PEPEPEPEPEPEG 10 9 PDPEPDPEPDPEG 11

[0126] [Table 4]

[0127] Table 4

[0128] Composition of the construct (additive) MFI Example 7 Ac-PDPDPDPDPDPDG-FAM 27.5 Example 8 Ac-PEPEPEPEPEPEG-FAM 29.3 Example 9 Ac-PDPEPDPEPDPEG-FAM 25.1 Reference Example 2 FAM 13.1

[0129] As shown in Table 4 and Figure 2 shown, the MFI values of Examples 7 - 9 are significantly higher than those of Reference Example 2. From this, it can be known that Peptides 7 - 9 have excellent cell membrane permeability. In addition, the MFI values of Examples 7 - 9 are higher than those of Examples 1 - 4 in Test 1. Therefore, it is considered that by having an amino acid sequence with PD or PE repeated 6 times, particularly excellent cell membrane permeability can be exhibited.

[0130] In addition, although detailed data are not shown, according to the research of the inventors of the present invention, it was confirmed that constructs having the synthetic peptides (such as Peptides 1 - 4, 7 - 9) disclosed in the present invention can efficiently introduce the exogenous substance from the outside of the cell into the cytoplasm not only when the exogenous substance is a fluorescent dye (such as FAM), but also in any case where the exogenous substance is a polypeptide, nucleic acid, and drug.

[0131] Above, specific examples of the technology disclosed in the present invention have been described in detail, but these are only examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and changes to the specific examples illustrated above.

[0132] Industrial Applicability

[0133] Using the technology disclosed in the present invention, it is possible to provide a carrier peptide fragment capable of introducing a target exogenous substance from the outside into the cytoplasm of eukaryotic cells (especially various animal cells represented by human or mammalian cells other than humans that do not have a cell wall), and a construct having such a carrier peptide fragment. By using such a construct, it is possible to effectively introduce a target exogenous substance into a target cell, and obtain a biological tissue such as a cell and an organ into which the exogenous substance has been introduced. In addition, by using the carrier peptide fragment disclosed in the present invention in drug delivery technology, it is possible to provide therapeutic drugs for various diseases.

Claims

1. A synthetic peptide, which is a synthetic peptide capable of importing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, and is characterized in that: it comprises any of the following amino acid sequences, (1) an amino acid sequence formed by tandemly and continuously binding two or more of the smallest structural units with PD (proline residue - aspartic acid residue) or PE (proline residue - glutamic acid residue); and (2) an amino acid sequence in which 1 to 3 glycine residues are bound to the C-terminal side of the amino acid sequence of (1).

2. The synthetic peptide according to claim 1, characterized in that: the amino acid sequence of (1) and the amino acid sequence of (2) have two or more and six or less of the smallest structural units.

3. A synthetic peptide, which is a synthetic peptide capable of importing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, and is characterized in that: it comprises any of the following amino acid sequences: PDPD (SEQ ID NO: 1); PEPE (SEQ ID NO: 2); PDPEG (SEQ ID NO: 7); PEPDG (SEQ ID NO: 8); PDPDPDPDPDPDG (SEQ ID NO: 9); PEPEPEPEPEPEG (SEQ ID NO: 10); and PDPEPDPEPDPEG (SEQ ID NO: 11).

4. A construct for introducing an exogenous substance, which is a construct for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, characterized in that, It has: the synthetic peptide according to any one of claims 1 to 3; and the target exogenous substance bound to the N-terminal side and / or C-terminal side of the synthetic peptide.

5. The construct according to claim 4, characterized in that: the exogenous substance is at least one organic compound selected from polypeptides, nucleic acids, pigments and pharmaceuticals.

6. The construct according to claim 4, characterized in that: the exogenous substance is arranged on the C-terminal side of the synthetic peptide.

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