Carrier peptide fragment and use thereof

Abstract

Provided is a carrier peptide fragment having excellent cell membrane permeability. A vector peptide fragment according to the present invention is composed of the following amino acid sequence represented by SEQ ID NO: 1: KKRTLRKSNRKKR, the 12th lysine residue from the N-terminal side of the amino acid sequence being D body, and the remaining amino acid residues being L body.

Description

Technical Field

[0001] The present invention relates to a method for introducing (transporting) an exogenous substance from the outside of a eukaryotic cell to the inside of the cell and a carrier peptide fragment used in the method. In addition, the present invention claims priority based on Japanese patent application No. 2022-137043 filed on August 30, 2022, and the entire contents of the application are introduced as a reference in this specification. Background Art

[0002] Currently, the following operations are performed: introducing exogenous substances such as polypeptides, especially physiologically active substances, into cells (eukaryotic cells) of humans or mammals other than humans to transform the properties of the cells (and tissues or organs formed by the cells) or to improve and enhance the functions of the cells.

[0003] For example, Patent Document 1 discloses a construct having a carrier peptide fragment consisting of an amino acid sequence: KKRTLRKSNRKKR (SEQ ID NO. 2) and an exogenous substance. The carrier peptide fragment is a cell-penetrating peptide (hereinafter also referred to as "CPP") that exhibits excellent cell membrane permeability, and thus the construct having the carrier peptide fragment can be effectively introduced from the outside of a eukaryotic cell into the inside.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2022-047613 Summary of the invention

[0007] For example, from a medical point of view, the use of CPP in drug delivery systems and the like has been studied. Therefore, the development of CPP having excellent cell membrane permeability is desired.

[0008] The technology disclosed in the present invention is completed in view of the above situation. The main purpose of the present invention is to provide a carrier peptide fragment with excellent cell membrane permeability. In addition, another purpose of the present invention is to provide a construct for introducing an exogenous substance having such a carrier peptide fragment. Another purpose of the present invention is to provide a method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell.

[0009] The inventors of the present invention have conducted intensive research to create a carrier peptide fragment that exhibits better cell membrane permeability based on the amino acid sequence (SEQ ID NO. 2) disclosed in Patent Document 1. As a result, it was found that cell membrane permeability is improved by making one of the amino acid residues constituting the amino acid sequence shown in SEQ ID NO. 2 a D-type amino acid residue. Among them, it was further found that very excellent cell membrane permeability can be achieved by making only the 12th lysine residue (also called Lysine residue) from the N-terminal side of the amino acid sequence shown in SEQ ID NO. 2 a D-type (D-type isomer).

[0010] That is, one embodiment of the carrier peptide fragment disclosed in the present invention is a carrier peptide fragment introduced from the outside of a eukaryotic cell into at least the cytoplasm of the cell. The carrier peptide fragment is composed of the following amino acid sequence shown in sequence number 1: KKRTLRKSNRKKR, wherein the lysine residue at the 12th position from the N-terminal side of the above amino acid sequence is a D-body, and the remaining amino acid residues are L-bodies (L-type isomers). With such a structure, excellent cell membrane permeability can be achieved.

[0011] In addition, the present invention can provide a construct for introducing an exogenous substance having the carrier peptide fragment disclosed in the present invention (hereinafter, also referred to as a "construct" for short). One embodiment of the construct disclosed in the present invention is a construct for introducing an exogenous substance made to introduce a target exogenous substance from the outside of a eukaryotic cell at least into the cytoplasm of the cell, comprising the carrier peptide fragment disclosed in the present invention and an exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment. With such a configuration, a construct that exhibits excellent cell membrane permeability can be realized.

[0012] In one embodiment of the construct disclosed in the present invention, the exogenous substance may be at least one organic compound selected from the group consisting of a polypeptide, a nucleic acid, a pigment, and a drug.

[0013] Among them, "polypeptide" refers to a polymer having a structure in which multiple amino acids are bound by peptide bonds. The number of peptide bonds (i.e., the number of amino acid residues) of a polypeptide is not limited. That is, polypeptides include: compounds generally referred to as peptides having more than 10 and less than about 300 amino acid residues; and compounds generally referred to as proteins (polymer compounds typically composed of more than 300 amino acid residues). In this field, there is no strict distinction between polypeptides and proteins. In this specification, polymers (including oligomers) composed of multiple amino acid residues are collectively referred to as polypeptides.

[0014] In addition, "nucleic acid" refers to a polymer of nucleotides, including DNA and RNA. The number of bases in "nucleic acid" is not limited.

[0015] In one embodiment of the construct disclosed in the present invention, the exogenous substance may be placed on the C-terminal side of the carrier peptide fragment.

[0016] In addition, the present invention can provide a method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell in vitro or in vivo. The method disclosed in the present invention comprises: (1) a step of preparing a construct disclosed in the present invention; and (2) a step of supplying the construct to a sample containing the target eukaryotic cell. Thus, the target exogenous substance can be efficiently introduced into the interior of the eukaryotic cell.

[0017] In one embodiment of the method disclosed in the present invention, the target eukaryotic cell into which the construct is introduced may be a human or non-human mammalian cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the MFI values ​​obtained by analyzing HeLa cells using a flow cytometer after adding the constructs (supplements) shown in Examples 1 to 16 to the culture medium of the cells and culturing the cells. DETAILED DESCRIPTION

[0019] The following is an explanation of the embodiments of the technology disclosed in the present invention. Matters other than those specifically mentioned in this specification and required for implementation (e.g., general matters related to the preparation of constructs containing peptides or nucleic acids as components) can be mastered as design matters for 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.

[0020] In addition, the technology disclosed in the present invention can be implemented based on the content disclosed in this specification and the technical common sense in this field. In addition, in this specification, amino acids are sometimes represented by single-letter symbols according to the nomenclature of amino acids shown in the IUPAC-IUB guide. Wherein, in this specification, except for the case of special mention, "amino acid residue" is a term comprising the N-terminal amino acid and the C-terminal amino acid of a peptide chain.

[0021] In addition, in this specification, "synthetic peptide" is not a substance whose peptide chain exists independently and stably in nature, but refers to a peptide fragment produced by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and can be stably present in a prescribed composition. Among them, "peptide" refers to a term for an amino acid polymer having multiple peptide bonds and is not limited by the number of amino acid residues.

[0022] In the present specification, unless otherwise specified, the amino acid residues constituting a peptide or protein may be either L-form or D-form. However, the amino acid sequences shown in the sequence listing distinguish between L-form and D-form.

[0023] In the amino acid sequence described in the present specification, the left side usually represents the N-terminal side, and the right side represents the C-terminal side.

[0024] One embodiment of the carrier peptide fragment disclosed in the present invention is composed of the amino acid sequence KKRTLRKSNRKKR shown in SEQ ID NO: 1. In the amino acid sequence, the lysine residue at the 12th position from the N-terminal side is a D-amino acid (i.e., a D-lysine residue), and the remaining amino acid residues (amino acid residues at positions 1 to 11 and position 13 from the N-terminal side) are composed of L-amino acids.

[0025] As long as the cell membrane permeability is not impaired, the carrier peptide fragment disclosed in the present invention may also be a modified sequence of the amino acid sequence shown in SEQ ID NO: 1. Here, the "modified sequence" is an amino acid sequence (modified amino acid sequence) formed by replacing, deleting and / or adding (inserting) one or several (typically 2 or 3) amino acid residues.

[0026] As typical examples of the altered sequence in this specification, for example, sequences generated by conservatively replacing 1, 2 or 3 amino acid residues, sequences obtained by adding (inserting) or deleting 1, 2 or 3 amino acid residues to a specified amino acid sequence, etc. As typical examples of conservative substitutions, for example, sequences obtained by replacing basic amino acid residues with other basic amino acid residues (e.g., mutual substitution of lysine residues with arginine residues), sequences obtained by replacing hydrophobic amino acid residues with other hydrophobic amino acid residues (e.g., mutual substitution of leucine residues, isoleucine residues and valine residues), etc. can be cited.

[0027] The construct for introducing a foreign substance disclosed in the present invention comprises the carrier peptide fragment disclosed in the present invention and a foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

[0028] The construct disclosed in the present invention can be designed and constructed by directly binding (linking) a desired exogenous substance to the N-terminal side and / or C-terminal side of the above-mentioned carrier peptide fragment or indirectly binding (linking) via an appropriate linker.

[0029] 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 produce steric hindrance and has flexibility. The peptide linker can be, for example, a linker comprising one or more amino acid residues selected from glycine, alanine, and serine, and consisting of less than 10 (more preferably more than 1 and less than 5, such as 1, 2, 3, 4 or 5 amino acid residues) amino acid residues. In addition, as such a linker, β-alanine can also be used. As a non-peptide linker, it is not particularly limited, for example, an alkyl linker, a PEG (polyethylene glycol) linker, an aminocaproyl spacer, etc. can be used.

[0030] Exogenous substances may be, for example, organic compounds such as polypeptides, nucleic acids, pigments, and drugs.

[0031] In the case where the exogenous substance is a polypeptide, a peptide chain is designed in such a manner as to contain the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the carrier peptide fragment, and the peptide chain is biosynthesized or chemically synthesized, thereby making it possible to prepare a construct for introducing the target exogenous substance. It is also possible to prepare a construct by directly or indirectly binding various nucleic acids such as DNA or RNA, pigments (e.g., various fluorescent pigment compounds such as FAM or FITC), or organic compounds that function as drugs (e.g., 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-mentioned carrier peptide fragment using various scientific methods known at present.

[0032] Although not particularly limited, the functions of the exogenous substance may be, for example, promoting differentiation induction of stem cells (stem cell differentiation inducing activity), inhibiting the proliferation of tumor cells (anti-tumor activity), inhibiting the proliferation of virus-infected cells (anti-viral activity), etc.

[0033] In the construct disclosed in the present invention, the number of exogenous substances bound to the carrier peptide fragment is not particularly limited. For example, one carrier peptide fragment can be bound to one or more exogenous substances. There is no particular limitation, for example, the C-terminal side of one carrier peptide fragment can be bound to a polypeptide, a nucleic acid, a drug, etc., and the N-terminal side can be bound to a pigment. By binding the pigment to the carrier peptide fragment, the efficiency of the construct introduced into eukaryotic cells and the localization in the cell can be easily evaluated, so it is preferred.

[0034] When the exogenous substance is a polypeptide, the polypeptide (amino acid sequence) used is not particularly limited. For example, a substance having a relatively large number of amino acid residues such as a polypeptide having about 100 to 1000 amino acid residues or a protein can be used as the exogenous substance.

[0035] Typically, the total number of amino acid residues constituting the synthetic peptide prepared as a construct for the introduction of exogenous substances is several to tens or more (e.g., 10 or more), preferably 1000 or less, preferably 600 or less, more preferably 500 or less, and particularly preferably 300 or less (e.g., 10 to 300). Polypeptides of such length are easy to synthesize (biosynthesize, chemically synthesize) and easy to use.

[0036] As exogenous substances, mature forms or precursors (including pro-forms and prepro-forms) of polypeptides involved in the functions of development, differentiation, proliferation, carcinogenesis, homeostasis (homeostasis), regulation of metabolism, etc. of various cells or tissues (organs) are preferred. In addition, the exogenous substance introduction method disclosed in the present invention can also be implemented to introduce a polypeptide whose function is currently unknown into a cell and clarify the function of the polypeptide in the cell (in a living tissue).

[0037] For example, when the eukaryotic cell to be introduced into the exogenous substance is a stem cell of a human or other mammal, it is preferred to use a mature form or a precursor thereof of a polypeptide having various physiological activities that participate in the differentiation induction of the stem cell. Among them, "stem cells" include adult stem cells, embryonic stem cells, and induced pluripotent stem cells (Induced pluripotentstem cells: iPS cells). In addition, when the eukaryotic cell to be introduced into the exogenous substance is a cancer cell (tumor cell), it is preferred to use various polypeptides that participate in the apoptosis induction of the cancer cell (tumor cell). Alternatively, in this case, it is preferred to use a polypeptide that can hinder the function of cancer cells (tumor cells) to inhibit the immune surveillance mechanism. In addition, when the eukaryotic cell to be introduced into the object is a bacterially infected cell or a virally infected cell, it is preferred to use various polypeptides that participate 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.

[0038] In addition, similar to the carrier peptide fragment, the polypeptide as an exogenous substance may also contain an altered amino acid sequence formed by substitution, deletion and / or addition (insertion) of one or several amino acid residues as long as its function is maintained.

[0039] In the construct in which the exogenous substance is bound to the C-terminal side of the carrier peptide fragment, the α-amino group of the amino acid residue on the N-terminal side of the carrier peptide fragment is preferably acetylated. The detailed mechanism is not yet clear, but since many proteins in eukaryotic cells have acetylated α-amino groups of amino acids on the N-terminal side, the stability of the construct in the cell can be improved by using such a structure.

[0040] The amino acid residue on the C-terminal side of the construct is preferably amidated. If the carboxyl group of the amino acid residue (typically the C-terminal amino acid residue of the peptide chain) is amidated, the structural stability (e.g., protease tolerance) of such a construct in the cytoplasm and in the nucleolus can be improved. In addition, by amidating the carboxyl group, the hydrophilicity of the construct is improved, and therefore the solubility of the construct in an aqueous solvent can be improved. As such an aqueous solvent, for example, water, various buffer solutions, physiological saline (e.g., PBS), cell culture fluid, etc. can be cited.

[0041] For example, in the case of a construct in which an exogenous substance is bound to the N-terminal side of a carrier peptide fragment, the carboxyl group of the amino acid residue on the C-terminal side of the carrier peptide fragment is preferably amidated. In addition, for example, in the case in which the exogenous substance is a polypeptide and such a polypeptide is bound to the C-terminal side of a carrier peptide fragment, the carboxyl group of the C-terminal amino acid residue of the polypeptide is preferably amidated.

[0042] A relatively short peptide chain (including a polypeptide constituting an exogenous substance, a carrier peptide fragment and a peptide linker) in the construct can be easily manufactured according to a conventional chemical synthesis method. For example, any method of the currently known solid phase synthesis method or liquid phase synthesis method can be adopted. Preferably, a solid phase synthesis method using Boc (t-butyloxycarbonyl, tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxy carbonyl, 9-fluorenylmethoxycarbonyl) as a protecting group for the amino group is used. That is, the above-mentioned peptide chain having a desired amino acid sequence and a modified (N-terminal acetylation, C-terminal amidation, etc.) portion can be synthesized by a solid phase synthesis method using a commercially available peptide synthesizer. In addition, only a part of the peptide chain can also be synthesized using the above method, for example, only the carrier peptide fragment can be synthesized, or a peptide chain containing the carrier peptide fragment and the peptide linker portion can be synthesized.

[0043] Alternatively, the peptide portion can also be made by biosynthesis based on a genetic engineering method. That is, a polynucleotide (typically DNA) encoding a nucleotide sequence (including an ATG start codon) of a desired amino acid sequence is synthesized. Then, a recombinant vector having 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 for controlling expression levels) for expressing the amino acid sequence in the host cell.

[0044] The recombinant vector is introduced into a specified host cell (e.g., yeast, insect cell, plant cell) using a common technical method, and the host cell or a tissue or individual containing the cell is cultured under specified conditions. Thus, the target peptide can be produced in the cell. Then, the peptide portion is separated from the host cell (from the culture medium in the case of secretion), and refolded, purified, etc. as needed to obtain the target peptide portion.

[0045] In addition, the method for constructing a recombinant vector and the method for introducing the constructed recombinant vector into a host cell can directly adopt the methods that have been used in this field. This method itself does not particularly reflect the characteristics of the present technology, so detailed description is omitted.

[0046] For example, in order to produce efficiently and in large quantities in a host cell, a fusion protein expression system can be used. That is, a gene (DNA) encoding the amino acid sequence of the target polypeptide is chemically synthesized, and the synthetic gene is introduced into a suitable site of a suitable fusion protein expression vector (for example, a GST (Glutathione S-transferase, glutathione S-transferase) fusion protein expression vector 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 the vector. The resulting transformant is cultured to prepare the target fusion protein. Then, the protein is extracted and purified. Then, the resulting purified fusion protein is cut with a prescribed 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 a currently known fusion protein expression system (for example, the GST / His system provided by Amersham Bioscience can be used.), the target construct (artificial polypeptide) can be manufactured.

[0047] Alternatively, a template DNA for a cell-free protein synthesis system (i.e., a synthetic gene fragment containing a nucleotide sequence encoding the amino acid sequence of the peptide portion of the construct) can be constructed, and various compounds required for the synthesis of the peptide portion (ATP, RNA polymerase, amino acids, etc.) can be used to synthesize the target polypeptide in vitro using a so-called cell-free protein synthesis system. Regarding the cell-free protein synthesis system, for example, reference is made 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 companies have been engaged in commissioned production of polypeptides at the time of the present application, and kits for cell-free protein synthesis (for example, available from Cell Free Sciences in Japan) are also commercially available.

[0048] The nucleotide sequence of the peptide portion of the coding construct and / or the single-stranded or double-stranded polynucleotide of the nucleotide sequence complementary to the sequence can be easily manufactured (synthesized) by currently known methods. That is, by selecting the codons corresponding to each amino acid residue constituting the designed amino acid sequence, the nucleotide sequence corresponding to the amino acid sequence can be easily determined and provided. Moreover, once the nucleotide sequence is determined, the polynucleotide (single-stranded) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer, etc. Further, the obtained single-stranded DNA can be used as a template, and various enzyme synthesis methods (typically PCR) are adopted to obtain the target double-stranded DNA. In addition, the polynucleotide can be in the form of DNA, or in the form of RNA (mRNA, etc.). DNA can be provided in the form of double strands or single strands. In the case of providing in a single strand, it can be a coding strand (sense strand) or a non-coding strand (antisense strand) of a sequence complementary thereto.

[0049] The polynucleotide thus obtained can be used as a material for constructing a recombinant gene (expression cassette) for producing a peptide in various host cells or a cell-free protein synthesis system as described above.

[0050] The construct disclosed in the present invention can be suitably used as an active ingredient of a composition for purposes 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, an acid addition salt obtained by addition reaction of a commonly used inorganic acid or organic acid can be used according to a conventional method. Therefore, the "construct" recorded in this specification and the scope of the claim can include such a construct in the form of a salt.

[0051] The construct can be used as an active ingredient of a composition that can contain various pharmaceutically acceptable carriers depending on the mode of use.

[0052] As the above-mentioned carrier, for example, it is preferred that the carrier is usually used as a diluent, excipient, etc. in peptide pharmaceuticals. As such a carrier, it can be appropriately different according to the purpose or form of the construct for the introduction of exogenous substances, and typically water, physiological buffer, and various organic solvents can be listed. In addition, such a carrier can be a non-drying oil such as an alcohol (ethanol, etc.) aqueous solution, glycerol, olive oil, etc. of appropriate concentration, or it can also be a liposome. In addition, as the minor components that can be contained in the pharmaceutical composition, various fillers, extenders, binders, wetting agents, surfactants, pigments, spices, etc. can be listed.

[0053] The form of the composition is not particularly limited. For example, liquid, suspension, emulsion, aerosol, foam, granule, powder, tablet, capsule, ointment and the like can be cited. In addition, for injection, etc., a lyophilized product or granulated product can be prepared for dissolving in physiological saline or an appropriate buffer (e.g., PBS) or the like just before use to prepare a drug solution.

[0054] The process of preparing various forms of medicaments (compositions) using constructs (main components) and various carriers (auxiliary components) as materials can be based on currently known methods. The preparation method itself does not constitute the characteristics of this technology, so detailed description is omitted. As a source of detailed information about the formulation, for example, Comprehensive Medicinal Chemistry, edited by Corwin Hansch, published by Pergamon Press (1990) can be cited.

[0055] The present invention can provide a method for introducing exogenous substances in vivo or in vitro using the construct disclosed in the present invention. The method generally includes the following steps (1) to (2):

[0056] (1) a step of preparing the construct disclosed in the present invention; and

[0057] (2) A step of supplying the above construct to a sample containing target eukaryotic cells.

[0058] Furthermore, the method disclosed in the present invention may further include, after the above step (2), a step of incubating a sample to which the above construct has been supplied, and introducing the construct into eukaryotic cells in the sample as step (3).

[0059] The above-mentioned "eukaryotic cells" include, for example, various tissues, organs, blood, lymph, etc. in vivo. The above-mentioned "eukaryotic cells" include, for example, various cell clusters, tissues, organs, blood, lymph, cell lines, etc. extracted from organisms in vitro. As eukaryotic cells, for example, cells from the animal kingdom such as mammals, birds, fish, amphibians, reptiles, insects, cells from the fungus kingdom, cells from the plant kingdom, etc. can be listed, and preferably, they can be cells of humans or mammals other than humans.

[0060] The composition containing the construct 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, it can be administered to the affected part (e.g., malignant tumor tissue, virally infected tissue, inflammatory tissue, etc.) of the patient (i.e., organism) by intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal injection only in the desired amount. Alternatively, a composition in a solid form such as a tablet or a gel-like or aqueous jelly-like composition such as an ointment can be directly administered to a prescribed tissue (i.e., an affected part of a tissue or organ containing tumor cells, virally infected cells, inflammatory cells, etc.). Alternatively, a composition in a solid form such as a tablet can be administered orally. In the case of oral administration, in order to inhibit the decomposition of digestive enzymes in the digestive tract, it is preferably encapsulated or a protective (coating) material is applied.

[0061] Alternatively, for eukaryotic cells cultured in vitro, an appropriate amount of the construct can be supplied to the culture fluid of the target eukaryotic cells at least once. The amount supplied each time and the number of times supplied can vary depending on the type of eukaryotic cells cultured, cell density (cell density at the start of culture), number of passages, culture conditions, type of culture medium, and other conditions, and are therefore not particularly limited. For example, it is preferred to perform one, two, or more additions so that the concentration of the carrier peptide fragment in the culture fluid is approximately within the range of 0.05 μM to 100 μM, for example, within the range of 0.5 μM to 50 μM, and for example, within the range of 1 μM to 30 μM. In addition, the incubation time after adding the construct can also vary depending on the type of eukaryotic cells and various conditions, and is therefore not particularly limited. For example, it can be more than 0.5 hours, more than 1 hour, more than 4 hours, more than 8 hours, or more than 20 hours. In addition, the conditions for incubation can also vary depending on the type of eukaryotic cells, and are therefore not particularly limited, and can be incubated at 37° C. under a 5% CO2 atmosphere.

[0062] In addition, an example of an in vitro introduction method is described in the test examples described later.

[0063] The method that the importing efficiency of construct is evaluated is not particularly limited.For example, when this construct is combined with pigment (typically fluorescent pigment compound), microscopic observation (for example fluorescence microscope observation) or flow cytometer etc. can be used to evaluate the importing efficiency to eukaryotic cells. In addition, the importing efficiency of the above-mentioned construct can also be evaluated by immunochemical method (for example Western blotting or immune cell staining etc.) of the antibody of the peptide part of the above-mentioned construct using specific recognition.

[0064] As described above, as specific aspects of the technology disclosed in the present invention, aspects described in the following items can be cited.

[0065] Item 1: A carrier peptide fragment, which is composed of the following amino acid sequence shown in sequence number 1: KKRTLRKSNRKKR, wherein the lysine residue at the 12th position from the N-terminal side of the above amino acid sequence is a D-form, and the remaining amino acid residues are L-forms.

[0066] Item 2: A construct comprising the carrier peptide fragment described in Item 1 and a foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

[0067] Item 3: The construct according to Item 2, wherein the exogenous substance is at least one organic compound selected from the group consisting of a polypeptide, a nucleic acid, a pigment, and a drug.

[0068] Item 4: The construct according to Item 2 or 3, wherein the exogenous substance is disposed on the C-terminal side of the carrier peptide fragment.

[0069] Item 5: A method for introducing a target exogenous substance into at least the cytoplasm of a eukaryotic cell in vitro from the outside of the cell, comprising the steps of (1) preparing a construct described in any one of items 2 to 4 and (2) supplying the construct to a sample containing the target eukaryotic cells.

[0070] Item 6: The method according to Item 5, wherein the eukaryotic cell into which the construct is introduced is a human or a non-human mammalian cell.

[0071] Several test examples of the technology disclosed in the present invention are described below, but the technology disclosed in the present invention is not intended to be limited to the contents shown in such test examples.

[0072] <Preparation of construct for introduction of exogenous substances>

[0073] A construct having a synthetic peptide consisting of the amino acid sequence shown in Table 1 was prepared. A construct having peptide n (n is a natural number of 1 to 15) shown in Table 1 was used as sample n, and samples 1 to 15 were obtained from Eurofins Genomics Co., Ltd. Among them, in the amino acid sequence shown in Table 1, amino acid residues marked with capital letters represent L-forms, and amino acid residues marked with lowercase letters represent D-forms. In addition, in samples 1 to 15, peptides in which the α-amino groups of the amino acid residues on the N-terminal side of peptides 1 to 15 were all acetylated were prepared. In addition, a fluorescent dye FAM (C 21 H 12 O7: 5(6)-carboxyfluorescein (5(6)-Carboxyfluorescein, molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) peptide. Then, samples 1 to 15 were diluted with dimethyl sulfoxide (DMSO) to prepare sample solutions 1 to 15 with a sample concentration of 2 mM.

[0074] [Table 1]

[0075] Table 1

[0076] peptide No. sequence serial number 1 kKRTLRKSNRKKR 3 2 KkRTLRKSNRKKR 4 3 KKrTLRKSNRKKR 5 4 KKRtLRKSNRKKR 6 5 KKRTIRKSNRKKR 7 6 KKRTLrKSNRKKR 8 7 KKRTLRkSNRKKR 9 8 KKRTLRKsNRKKR 10 9 KKRTLRKSnRKKR 11 10 KKRTLRKSNrKKR 12 11 KKRTLRKSNRkKR 13 12 KKRTLRKSNRKkR 1 13 KKRTLRKSNRKKr 14 14 kkrtlrksnrkkr 15 15 KKRTLRKSNRKKR 2

[0077] <Evaluation of Cell Membrane Permeability Using Flow Cytometry>

[0078] HeLa cells (established cell line derived from human cervical cancer cells) were used as eukaryotic cells, and the cell membrane permeability of samples 1 to 15 was analyzed. In Examples 1 to 15, the sample solutions 1 to 15 prepared above were used, respectively, and in Example 16, a FAM solution was used.

[0079] (Example 1)

[0080] HeLa cells were cultured in DMEM (Dulbecco's modified Eagle's medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 044-29765)) containing 10% FBS (fetal bovine serum) as a culture medium.

[0081] After washing the HeLa cells attached to the culture plate with PBS, 0.25% trypsin / EDTA solution was added and incubated at 37°C for 3 minutes. After the incubation, the DMEM containing 10% FBS was added to inactivate the trypsin, and the cells were centrifuged at 150×g for 5 minutes to precipitate the cells. After removing the supernatant produced by centrifugation, the DMEM containing 10% FBS was added to the precipitate (cell pellet) to prepare about 2×10 5 1 mL of the cell suspension was added to the wells of a commercially available 6-well plate (manufactured by AGC TECHNO GLASS Co., Ltd.), and cells (approximately 2×10 5 cells / well). In addition, the above 2 mM sample solution 1 was diluted with the above DMEM containing 10% FBS to prepare a sample solution 1 with a concentration of 20 μM. Then, 1 mL of the above 20 μM sample solution 1 was added to the well (i.e., the concentration of sample 1 in the culture solution in the well was 10 μM, and the DMSO concentration was 0.5%). Thereafter, the cells were incubated at 37°C for 20 hours under 5% CO2 conditions.

[0082] After incubation for 20 hours, the culture supernatant was removed from the wells, and the cells in the wells were washed twice with 1 mL of PBS. Next, 200 μL of 0.25% trypsin / EDTA solution was added to the wells and incubated at 37°C for 3 minutes. After the incubation, 400 μL of the above-mentioned DMEM containing 10% FBS was added to the wells to inactivate trypsin, and the cell suspension in the wells was moved to the tube to recover the cells. After that, 600 μL of PBS was added to the wells to wash the wells. Then, the PBS in the wells was moved to the above-mentioned tubes, thereby recovering the cells remaining in the wells into the above-mentioned tubes. The tube was centrifuged at 4°C and 210×g for 5 minutes. After centrifugation, the supernatant was removed, and the precipitate (cell mass) 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 cells (cell mass) cultured with a culture medium containing sample 1.

[0083] The cells (cell clusters) obtained above were analyzed for cell membrane permeability of sample 1 using a flow cytometer. As the flow cytometer, On-Chip Flowcytometer (manufactured by On-Chip Biotechnologies Co., LTD.) was used.

[0084] In order to perform the analysis, the cell pellet obtained above was suspended in 100 μL of On-Chip Tbuffer to prepare a cell suspension for analysis.

[0085] The flow cytometer described above is used for gating based on forward scatter (FSC) and side scatter (SSC), and a gate is set for the cell group to be analyzed. For the cell group within the gate, the fluorescence intensity is measured. The analysis is performed in such a way that the number of cells in the cell group is at least 10,000. In the measurement of fluorescence intensity, a fluorescence detector FL2 (near the optimal detection wavelength of 543 nm) of the flow cytometer described above that can detect the fluorescence wavelength of FAM is used. The measurement results are analyzed using the commercially available analysis software "FlowJo (registered trademark)" (manufactured by TreeStar) to obtain the median fluorescence intensity (MFI) of the cell group to be measured.

[0086] (Examples 2~15)

[0087] The same procedures as in Example 1 were carried out except that the sample solution 1 was changed to any one of the sample solutions 2 to 15 prepared above. The samples (constructs) used in each example are shown in Table 2.

[0088] (Example 16)

[0089] The same procedure as in Example 1 was followed except that a FAM solution diluted with DMSO was used instead of the sample solution 1. The FAM solution was used at the same concentration as the sample solution 1 (i.e., the FAM concentration in the culture solution in the well was 10 μM and the DMSO concentration was 0.5%).

[0090] The results obtained for Examples 1 to 16 are shown in Tables 2 and Figure 1 . Figure 1 It is a graph which shows the MFI value of each example.

[0091] [Table 2]

[0092] Table 2

[0093] The structure of the structure MFI Example 1 Ac-kKRTLRKSNRKKR-FAM 13.9 Example 2 Ac-KkRTLRKSNRKKR-FAM 14.1 Example 3 Ac-KKrTLRKSNRKKR-FAM 13.9 Example 4 Ac-KKRtLRKSNRKKR-FAM 15.9 Example 5 Ac-KKRTIRKSNRKKR-FAM 16.5 Example 6 Ac-KKRTLrKSNRKKR-FAM 17.5 Example 7 Ac-KKRTLRkSNRKKR-FAM 16.9 Example 8 Ac-KKRTLRKsNRKKR-FAM 14.3 Example 9 Ac-KKRTLRKSnRKKR-FAM 15.6 Example 10 Ac-KKRTLRKSNrKKR-FAM 15.5 Example 11 Ac-KKRTLRKSNRkKR-FAM 17.4 Example 12 Ac-KKRTLRKSNRKkR-FAM 33.8 Example 13 Ac-KKRTLRKSNRKKr-FAM 15.2 Example 14 Ac-kkrtlrksnrkkr-FAM 16.1 Example 15 Ac-KKRTLRKSNRKKR-FAM 13.0 Example 16 FAM 10.6

[0094] As shown in Table 2 and Figure 1 As shown in Tables 2 and 3, the MFI values ​​in Examples 1 to 14 were higher than those in Example 15. Therefore, it can be seen that the cell membrane permeability is improved by at least one of the amino acid residues constituting the amino acid sequence shown in SEQ ID NO: 2 being a D-form amino acid residue. Figure 1 As shown, the MFI value was particularly high in Example 12. This shows that the cell membrane permeability was significantly improved by converting only the 12th lysine residue from the N-terminal side of the amino acid sequence: KKRTLRKSNRKKR to the D form.

[0095] The specific examples of the technology disclosed in the present invention have been described in detail above, but these are only examples and do not limit the scope of the claims. The technology described in the scope of the claims includes various modifications and changes to the specific examples described above.

[0096] By using the technology disclosed in the present invention, it is possible to provide a carrier peptide fragment artificially produced for the purpose of introducing a target exogenous substance from the outside of a eukaryotic cell (especially various animal cells represented by humans or mammals other than humans that do not have a cell wall) into the cytoplasm and a construct having the carrier peptide fragment. By using such a construct, the target exogenous substance can be effectively introduced into the target cell, and biological tissues such as cells into which the exogenous substance has been introduced and organs containing cells containing the exogenous substance can be obtained. In addition, by using a carrier peptide fragment or a construct, therapeutic drugs for various diseases (such as therapeutic drugs for ophthalmic treatment, nucleic acid medicines, etc.) can be provided. In addition, the peptide fragment or construct disclosed in the present invention can be used as an active ingredient or additive for external-use medicines (such as eye drops).

Claims

1. A carrier peptide fragment, characterized in that: It is composed of the following amino acid sequence shown in SEQ ID NO: 1: KKRTLRKSNRKKR, The 12th lysine residue from the N-terminal side of the amino acid sequence is a D-form, and the remaining amino acid residues are L-forms.

2. A construct, characterized in that have: The carrier peptide fragment of claim 1; and A foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

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

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

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

6. The method according to claim 5, characterized in that: The eukaryotic cell into which the construct is introduced is a human or a non-human mammal cell.

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