Synthetic peptides and constructs

Abstract

The present invention addresses the problem of providing a novel synthetic peptide having cell membrane permeability. A synthetic peptide according to 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 SA (serine residue-alanine acid residue) are consecutively bonded in series and at least two minimum structural units SA (serine residue-alanine residue) are used as the minimum structural units; 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. 2022-201100 filed on December 16, 2022, and the entire content of the application is incorporated herein by reference. 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] Problems to be Solved by the Invention

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

[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 Means for Solving the Problems

[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 SA (serine residue - alanine residue) is used as a minimum structural unit and two or more of the minimum structural units are consecutively bound in series; 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 known as basic amino acids. As described above, it has been thought that existing amino acid residues can exhibit high cell membrane permeability by having basic amino acids, but 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 2 or more and 4 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 2 or more times and 4 or less times.

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

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

[0017] SASASAG (SEQ ID NO: 2); and

[0018] SASASASAG (SEQ ID NO: 3).

[0019] Any of the above amino acid sequences can exhibit high cell membrane permeability.

[0020] In addition, according to the present invention, in order to achieve the above object, there is provided a construct for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell (hereinafter, also simply referred to as "construct"). The construct disclosed in the present invention has the synthetic peptide disclosed in the present invention and the above target exogenous 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 introduced into the interior of a eukaryotic cell and can efficiently introduce the target exogenous substance into the cell interior.

[0021] In one mode of the construct disclosed in the present invention, the above exogenous substance may be at least one organic compound selected from polypeptides, nucleic acids, pigments, and pharmaceuticals.

[0022] Here, "polypeptide" refers to a polymer having a structure in which a plurality of amino acids are bonded by 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 called peptides having 10 or more and less than about 300 amino acid residues, and compounds generally called proteins (typically, high molecular 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 a plurality of amino acid residues (including oligomers) are collectively referred to as polypeptides.

[0023] In addition, "nucleic acid" refers to a polymer of nucleotides, including DNA and RNA. "Nucleic acid" is not limited by the number of bases.

[0024] 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

[0025] Figure 1 It is a graph showing the MFI values obtained by analyzing the cells by flow cytometry after culturing the culture solution of NSC-34 cells with the constructs (additives) shown in Examples 1 to 3 and Reference Example 1 added. DETAILED DESCRIPTION OF THE INVENTION

[0026] 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 the prior art in the fields of cell engineering, physiology, medicine, pharmacy, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, genetics, etc. In addition, the technology disclosed in the present invention can be implemented based on the content described in this specification and the common general knowledge in the art. It should be noted that in the following description, amino acids are represented by single-letter symbols of the nomenclature for amino acids shown in the IUPAC-IUB guidelines as the case may be. In addition, in this specification, the term "amino acid residue" includes the N-terminal amino acid and the C-terminal amino acid of the peptide chain unless otherwise specified.

[0027] In addition, in this specification, "synthetic peptide" is not a substance that independently and stably exists in nature by itself, but refers to a peptide fragment manufactured by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and capable of stably existing in a specified composition. Among them, "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.

[0028] In addition, in this specification, the amino acid residues constituting the peptide or protein can be L-forms (L-isomers) or D-forms. In addition, the amino acid sequences described in this specification always have the N-terminal side on the left and the C-terminal side on the right.

[0029] In one mode of the synthetic peptide disclosed in the present invention, the synthetic peptide contains the amino acid sequence shown in the following (1) or (2):

[0030] (1) An amino acid sequence with SA (serine residue - alanine residue) as the smallest structural unit and two or more of these smallest structural units connected in series;

[0031] (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) above.

[0032] In addition, in the present invention, "connected in series continuously" 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.

[0033] In the synthetic peptide disclosed in the present invention, for example, two or more and ten or less of the above-mentioned smallest structural units can be connected in series continuously. In addition, the smallest structural units can be connected in series continuously in two or more and six or less, two or more and four or less, or three or more and four or less. In a preferred embodiment, four smallest structural units are connected in series continuously. The amino acid sequence in which four smallest structural units are connected in series continuously can exhibit particularly excellent cell membrane permeability.

[0034] As shown in (2) above, the synthetic peptide disclosed in the present invention can have 1 to 3 glycine residues bound to the C-terminal side of the amino acid sequence formed by connecting the smallest structural units in series. The number of these glycine residues is preferably 1 to 2, and more preferably 1. Glycine residue is a neutral amino acid with the smallest side chain among amino acids. Therefore, when the amino acid sequence formed by connecting the smallest structural units in series has cell membrane permeability, even if 1 to 3 glycine residues are bound to the C-terminal side of this amino acid sequence, the cell membrane permeability will not be significantly impaired, which can be understood based on the common general knowledge in this technical field.

[0035] The number of amino acid residues of the synthetic peptide disclosed in the present invention includes at least two smallest structural units, and thus can be four or more, five or more, six or more, seven or more, or eight or more. In addition, there is no particular limitation on the upper limit of the number of amino acid residues of the synthetic peptide. For example, it can be 21 or less, 19 or less, 17 or less, 15 or less, 13 or less, 12 or less, 11 or less, 10 or less, or 9 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.

[0036] As specific examples of the synthetic peptides disclosed in the present invention, for example, as the amino acid sequences shown in the above (2), SASAG (SEQ ID NO: 1), SASASAG (SEQ ID NO: 3), SASASASAG (SEQ ID NO: 4), etc. can be cited. Additionally, as specific examples of the amino acid sequences shown in the above (1), SASA (SEQ ID NO: 4), SASASA (SEQ ID NO: 5), SASASASA (SEQ ID NO: 6), etc. can be cited. The synthetic peptides containing the amino acid sequences shown in SEQ ID NOs: 1 to 6 can all exhibit cell membrane permeability.

[0037] In addition, the synthetic peptides disclosed in the present invention can be altered sequences of the amino acid sequences shown in the above (1) or (2) as long as the cell membrane permeability is not significantly impaired. Herein, an "altered sequence" refers to an amino acid sequence (altered amino acid sequence) formed by substitution, deletion, and / or addition (insertion) of one or several (typically two or three) amino acid residues.

[0038] As typical examples of the altered sequences in this specification, for example, sequences generated by so-called conservative amino acid replacement in which one, two, or three amino acid residues are conservatively substituted, or sequences formed by adding (inserting) or deleting one, two, or three amino acid residues to a specified amino acid sequence, etc. can be cited. As typical examples of conservative replacement, for example, a sequence in which an alanine residue, which is a non-polar amino acid, is replaced with another non-polar amino acid residue such as a glycine residue can be cited.

[0039] Since the synthetic peptides disclosed in the present invention described above can have cell membrane permeability, they can introduce a target exogenous 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 an exogenous substance having the synthetic peptide disclosed in the present invention is provided.

[0040] The construct disclosed in the present invention has the above-described synthetic peptide disclosed in the present invention and a target exogenous substance bound to the N-terminal side and / or C-terminal side of the synthetic peptide.

[0041] The construct disclosed in the present invention can be designed and constructed by directly or indirectly binding (linking) a desired exogenous substance to the N-terminal side and / or C-terminal side of the above synthetic peptide through an appropriate linker.

[0042] The linker is not particularly limited and can be a peptide linker or a non-peptide linker. Although not particularly limited, the amino acid sequence constituting the peptide linker is preferably an amino acid sequence that does not cause steric hindrance and is flexible. The peptide linker includes, 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 glycine, alanine, serine, etc. In addition, β-alanine can also be used as the linker. As the non-peptide linker, there is no particular limitation, and for example, an alkyl linker, a PEG (polyethylene glycol) linker, an aminohexanoyl spacer, etc. can be used.

[0043] The exogenous substance can be an organic compound such as a polypeptide, a nucleic acid, a pigment, a pharmaceutical agent, etc.

[0044] When the exogenous 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 preparing a construct for introducing the target exogenous substance. Various currently 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 pharmaceutical agents (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 exogenous 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.

[0045] In the construct disclosed in the present invention, the number of exogenous substances bound to the above synthetic peptide is not particularly limited. For example, for 1 synthetic peptide, 1 or more than 1 exogenous substances can be bound. Although not particularly limited, for example, a polypeptide, a nucleic acid, a pharmaceutical agent, etc. can be bound to the C-terminal side of 1 synthetic peptide, and a pigment can be bound to the N-terminal side. Binding a pigment to the synthetic peptide can easily evaluate the import efficiency of the construct into eukaryotic cells and the intracellular localization, and thus is preferred.

[0046] In addition, when the exogenous substance is a polypeptide, the polypeptide (amino acid sequence) used is not particularly limited. For example, a polypeptide or protein with a relatively large number of amino acid residues, such as a polypeptide with about 100 to 1000 amino acid residues, can also be used as the exogenous substance.

[0047] Typically, the total number of amino acid residues constituting the synthetic peptide prepared as a construct for introducing an exogenous substance is several to several tens 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.

[0048] As the exogenous substance, a mature form or a precursor (including a pro-form and a prepro-form) of a polypeptide preferably involved in functions such as the development, differentiation, proliferation, carcinogenesis, homeostasis (homeostasis), and metabolism regulation of various cells or tissues (organs) is used. In addition, in order to introduce a polypeptide with an unknown function into cells and clarify the function of the polypeptide in cells (in biological tissues), the method for introducing an exogenous substance disclosed in the present invention can also be implemented.

[0049] For example, when the eukaryotic cell as the object for introducing an exogenous substance is a stem cell of a human or other mammal, a mature form or a precursor of a polypeptide having various physiological activities involved in the differentiation induction of the stem cell is preferably used. 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 for introducing an exogenous substance is a cancer cell (tumor cell), various polypeptides involved in the apoptosis induction of the cancer cell (tumor cell) are preferably used. Alternatively, in this case, a polypeptide capable of inhibiting the function of the cancer cell (tumor cell) that suppresses the immune surveillance mechanism is preferably used. In addition, when the eukaryotic cell as the object for introduction is a bacterially infected cell or a virally infected cell, various polypeptides involved in the apoptosis induction of the infected cell, a polypeptide capable of inhibiting the proliferation of bacteria or viruses in the infected cell, or a polypeptide capable of inhibiting the spread of the infection of bacteria or viruses from the infected cell are preferably used. In addition, similar to the synthetic peptide, as long as the polypeptide as the 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.

[0050] In the construct in which an exogenous substance is bound to the C-terminal side of the synthetic peptide, it is preferred 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, such a structure can improve the stability of the construct in cells.

[0051] 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 an aqueous solvent 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, in the case where 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.

[0052] 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 well-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.

[0053] Alternatively, the peptide part can also be produced by biosynthesis based on genetic engineering methods. That is, a polynucleotide (typically DNA) encoding a nucleotide sequence (including the ATG start codon) of a desired amino acid sequence is synthesized. Then, according to the host cell, a recombinant vector with an expression gene construct is constructed. 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.

[0054] 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 if necessary, refolding, purification, etc. are carried out to obtain the target peptide part.

[0055] 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 conventionally used in this field can be directly adopted. Since these methods themselves do not particularly reflect the characteristics of this technology, detailed descriptions are omitted.

[0056] For example, in order to efficiently and abundantly 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 this synthetic 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 using this vector. The obtained transformant is cultured to prepare the target fusion protein. Next, the protein is extracted and purified. Then, 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 using 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 produced.

[0057] Alternatively, a template DNA for a cell-free protein synthesis system (that is, 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 target polypeptide is synthesized in vitro using a so-called cell-free protein synthesis system. Regarding the cell-free protein synthesis system, for example, refer to the papers by 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 companies were 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) were also commercially available.

[0058] A single-stranded or double-stranded polynucleotide comprising a nucleotide sequence of a peptide portion of an encoding construct and / or a nucleotide sequence complementary to the sequence can be easily produced (synthesized) by currently known methods. That is, by selecting codons corresponding to each amino acid residue constituting the designed amino acid sequence, the nucleotide sequence corresponding to the amino acid sequence can be easily determined and provided. Moreover, once the nucleotide sequence is determined, a polynucleotide (single-stranded) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like. Further, using the obtained single-stranded DNA as a template, various enzyme 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 a coding strand (sense strand) or a non-coding strand (antisense strand) of a 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 a cell-free protein synthesis system as described above.

[0059] The construct 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.

[0060] The construct can be used as an active ingredient of a composition that can contain various pharmaceutically acceptable carriers depending on the usage. As the above 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, 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. Further, as minor components that can be contained in pharmaceutical compositions, various fillers, extenders, binders, wetting agents, surfactants, pigments, flavors, etc. can be listed.

[0061] 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 listed. Additionally, for use in injections, etc., it can also be made into freeze-dried products or granulated products for dissolving in physiological saline or an appropriate buffer (such as PBS) etc. immediately before use to prepare a medicinal solution.

[0062] The process of preparing various forms of medicaments (compositions) using constructs (main components) and various carriers (sub-components) as materials can be based on currently well-known methods, and this preparation method itself does not constitute the features of the present technology. Therefore, detailed description is omitted. As a source of detailed information on formulations, for example, Comprehensive Medicinal Chemistry, edited by Corwin Hansch, published by Pergamon Press (1990) can be cited.

[0063] In addition, the synthetic peptides disclosed in the present invention do not contain basic amino acids and acidic amino acids, so constructs of foreign substances that were difficult to combine with conventional CPPs due to problems such as charge can be produced.

[0064] Furthermore, using the constructs disclosed in the present invention, foreign substances can be introduced in vivo or in vitro. Generally speaking, this 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 sample containing target eukaryotic cells (supply step). Additionally, after the above supply step, it can further include a step of incubating the sample to which the constructs have been supplied and introducing the constructs into the eukaryotic cells in the sample (introduction step).

[0065] The above-mentioned "eukaryotic cells" in vivo include, for example, various tissues, organs, organs, blood, and lymph fluid, etc. The above-mentioned "eukaryotic cells" in vitro include, for example, various cell masses, tissues, organs, organs, blood, and lymph fluid, and cell lines, etc. taken out from organisms.

[0066] 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 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 by intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal injection. 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, etc. containing 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, encapsulation or application of a protective (coating) material is preferred.

[0067] Alternatively, for eukaryotic cells cultured in vitro, an appropriate amount of the construct may 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 may 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, and type of culture medium, and thus are not particularly limited. For example, it is preferable to perform one, two, or more multiple additions 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, 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 30 μM or less. In addition, the incubation time after adding the construct may also vary depending on the type of eukaryotic cells and various conditions, and thus is not particularly limited. For example, it may be 0.5 hours or more, 1 hour or more, 4 hours or more, 8 hours or more, 20 hours or more. In addition, the incubation conditions may also vary depending on the type of eukaryotic cells and thus are not particularly limited. For example, incubation may be performed under 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.

[0068] 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 bound to the construct, microscopic observation (e.g., fluorescence microscopic observation) 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 (e.g., Western blotting or immunocytochemical staining, etc.) using an antibody that specifically recognizes the peptide portion of the construct.

[0069] As described above, as specific embodiments of the technology disclosed in the present invention, the embodiments described in the following items can be cited.

[0070] Item 1: 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, comprising any of the following amino acid sequences,

[0071] (1) An amino acid sequence in which SA (serine residue - alanine residue) is used as the minimum structural unit and two or more of these minimum structural units are continuously bound in series; and

[0072] (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).

[0073] Item 2: The synthetic peptide according to Item 1, wherein the amino acid sequence in (1) and the amino acid sequence in (2) have two or more and four or less of the above minimum structural units.

[0074] Item 3: A synthetic peptide that is capable of importing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, comprising any of the following amino acid sequences:

[0075] SASAG (SEQ ID NO: 1);

[0076] SASASAG (SEQ ID NO: 2); and

[0077] SASASASAG (SEQ ID NO: 3).

[0078] Item 4: A construct for importing an exogenous substance, which is a construct for importing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell, having:

[0079] The synthetic peptide according to any one of Items 1 to 3; and

[0080] The target exogenous substance bound to the N-terminal side and / or C-terminal side of the above synthetic peptide.

[0081] Item 5: The construct according to Item 4, wherein the above exogenous substance is at least one organic compound selected from polypeptides, nucleic acids, pigments, and pharmaceuticals.

[0082] 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.

[0083] In addition, the above Item 3 represents a specific example having the matters defined in Item 1 or 2, and thus Item 3 can be subordinate to Item 1 or 2.

[0084] Hereinafter, several test examples regarding the technology disclosed in the present invention will be described, but there is no intention of limiting the technology disclosed in the present invention to the content shown in such test examples.

[0085] [Test 1]

[0086] <Preparation of Construct>

[0087] Prepare a construct having a synthetic peptide composed of the amino acid sequence shown in Table 1. The construct having the peptide n (n is a natural number from 1 to 3) shown in Table 1 is used as Sample n, and Samples 1 to 3 are obtained from Eurofins Genomics Co., Ltd. Among Samples 1 to 3, peptides in which the α-amino groups of the amino acid residues on the N-terminal side of Peptides 1 to 3 are all acetylated are prepared. In addition, a fluorescent pigment FAM (C 21 H 12Peptide of O7: 5(6)-Carboxyfluorescein (molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm).

[0088] [Table 1]

[0089] Table 1

[0090] Peptide No. Amino acid sequence Sequence number 1 SASAG 1 2 SASASAG 2 3 SASASASAG 3

[0091] <Evaluation of cell membrane permeability using flow cytometry>

[0092] As eukaryotic cells, NSC-34 cells (mouse motor neuron-like hybrid cell line) were used to analyze the cell membrane permeability of Peptides 1-3. The culture medium for NSC-34 cells was DMEM (Dulbecco's modified Eagle's medium, manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No. 044-29765) containing 10% FBS (fetal bovine serum). In addition, Samples 1-3 were dissolved in dimethyl sulfoxide (DMSO) respectively to prepare Sample Solutions 1-3 with a sample concentration of 4 mM. Furthermore, these sample solutions were diluted with the above culture medium to prepare Sample Solutions 1-3 with a concentration of 40 μM respectively. In Examples 1-3, Sample Solutions 1-3 prepared above were used respectively, and FAM solution was used in Reference Example 1.

[0093] (Example 1)

[0094] 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.) and inoculate in such a way that the number of NSC-34 cells becomes 2×10 5 cells / well. Then, add 1 mL of 40 μM Sample Solution 1 to the well so that the sample concentration in the culture medium in the well becomes 20 μM. Then, leave this 6-well plate stationary in a cell culture device and incubate at 37 °C for 20 hours under 5% CO2 conditions.

[0095] 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. Then, 100 μL of 0.25% trypsin / EDTA solution was added to the wells and incubated at 37 °C for 3 minutes. After this incubation, 900 μL of the above-mentioned culture medium was added to the wells to inactivate the trypsin, and then the cell suspension in the wells was transferred to a test tube to recover the cells. The test tube was centrifuged at 4 °C and 210×g for 5 minutes. After centrifugation, the supernatant was removed, and the precipitate (cell pellet) was suspended (washed) with 1 mL of PBS and centrifuged under the same conditions as above. After repeating this operation twice, the supernatant was removed to obtain the cells (cell pellet) cultured with the culture medium containing Sample 1.

[0096] For the cells (cell pellet) obtained above, the cell membrane permeability of Sample 1 was analyzed using a flow cytometer. As the flow cytometer, On-Chip Flowcytometer (manufactured by On-Chip Biotechnologies Co.,LTD.) was used.

[0097] To perform this analysis, the cell pellet obtained above was suspended in 100 μL of On-Chip Tbuffer to prepare a cell suspension for analysis.

[0098] Using the above-mentioned flow cytometer, gating was performed based on forward scatter (FSC) and side scatter (SSC), and a gate for the cell population to be analyzed was set. For the cell population within this gate, the fluorescence intensity was measured. Among them, the analysis was performed in such a way that the number of cells in this cell population was at least 10,000 or more. In the measurement of the fluorescence intensity, the fluorescence detector FL2 (optimal detection wavelength around 543 nm) of the above-mentioned flow cytometer capable of detecting the fluorescence wavelength of FAM was used. For this measurement result, it was analyzed using a commercially available analysis software “FlowJo (registered trademark)” (manufactured by TreeStar Inc.) to obtain the value of the fluorescence intensity (mean fluorescence intensity: MFI) of the cell population to be measured.

[0099] (Examples 2 - 3)

[0100] Sample solution 1 was changed to any one of the above-prepared sample solutions 2 - 3, and the rest was carried out in the same manner as in Example 1. Among them, the samples (constructs) used in each example are shown in Table 2.

[0101] (Reference Example 1)

[0102] This was carried out in the same manner as in Example 1, except that the fluorescent dye FAM was used instead of Sample 1. Among them, the concentration of FAM in the culture medium containing FAM was 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 wells was 20 μM).

[0103] The results obtained in Examples 1 to 3 and Reference Example 1 are shown in Table 2 and Figure 1 . Figure 1 is a chart showing the MFI values in each example.

[0104] [Table 2]

[0105] Table 2

[0106] Composition of the construct (additive) MFI Example 1 Ac-SASAG-FAM 20.9 Example 2 Ac-SASASAG-FAM 19.2 Example 3 Ac-SASASASAG-FAM 22.8 Reference Example 1 FAM 10.9

[0107] As shown in Table 2 and Figure 1 shown, the MFI values of Examples 1 to 3 were higher than those of Reference Example 1. That is, compared with the case of adding FAM alone (Reference Example 1), Samples 1 to 3 in which any one of Peptides 1 to 3 was bound to FAM were more introduced into the cells. From this, it can be seen that Peptides 1 to 3 have cell membrane permeability.

[0108] As described above, specific examples of the technology disclosed in the present invention have been described in detail, but these are merely illustrative and do not limit the scope of the claims. The technology described in the scope of the claims includes various modifications and changes to the specific examples illustrated above.

[0109] Industrial Applicability

[0110] By 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 of a eukaryotic cell (particularly, various animal cells represented by humans or mammals other than humans without cell walls) into the cytoplasm, and a construct having the carrier peptide fragment. 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 or 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, characterized in that: it contains 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 of SA (serine residue - alanine 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 four or less of the smallest structural units.

3. A synthetic peptide, characterized in that, It contains any of the following amino acid sequences: SASAG (SEQ ID NO: 1); SASASAG (SEQ ID NO: 2); and SASASASAG (SEQ ID NO: 3).

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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