Carrier peptide fragment and use thereof

Abstract

Provided is a novel vector peptide fragment having cell membrane permeability. A vector peptide fragment according to the present invention comprises an amino acid sequence satisfying any of the following conditions: (i) the following amino acid sequence in which the amino acid residues at positions 8 and 9 from the N-terminal side of KKRTLRKSNRKKR (SEQ ID NO: 1) are each substituted with an aspartic acid residue or a glutamic acid residue; and (ii) one to six aspartic acid residues and / or glutamic acid residues inserted between the ninth and tenth amino acid residues from the N-terminal side of the amino acid sequence satisfying the condition (i).

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. 2022-117396 filed on July 22, 2022, and the entire contents of the application are introduced as a reference in this specification. Background Art

[0002] Currently, there is a process of introducing exogenous substances such as polypeptides, especially physiologically active substances, into cells (eukaryotic cells) of humans or non-human mammals 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 and a foreign substance. The carrier peptide fragment is a cell-penetrating peptide (hereinafter also referred to as "CPP") having excellent cell membrane permeability, and thus the construct having the carrier peptide fragment can be efficiently introduced from the outside to the inside of a eukaryotic cell.

[0004] Prior art literature

[0005] Patent Literature

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

[0007] However, in a construct having a CPP and an exogenous substance, the cell membrane permeation efficiency may vary depending on the combination of the amino acid sequence of the CPP and the type of the exogenous substance. In recent years, drug delivery technology using CPPs has attracted attention, and in order to be able to select CPPs suitable for various exogenous substances, it is desired to develop a variety of CPPs.

[0008] Therefore, the present invention is an invention completed in response to such a demand, and its purpose is to provide a novel carrier peptide fragment (CPP) having cell membrane permeability. In addition, another object of the present invention is to provide a construct for introducing an exogenous substance having such a carrier peptide fragment. In addition, another object of the present invention is to provide a method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell.

[0009] The carrier peptide fragment (CPP) 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 an amino acid sequence that satisfies any of the following conditions:

[0010] (i) the following amino acid sequence: KKRTLRKSNRKKR (SEQ ID NO: 1), wherein the 8th and 9th amino acid residues from the N-terminal side are substituted with an aspartic acid residue or a glutamic acid residue, respectively; and

[0011] (ii) One to six aspartic acid residues and / or glutamic acid residues are inserted between the 9th and 10th amino acid residues from the N-terminal side of the amino acid sequence satisfying the condition of (i) above.

[0012] In general, CPP contains basic amino acid residues (such as lysine residues, arginine residues) in a high proportion, and the cell membrane permeability of CPP tends to be improved by being strongly positively charged. Therefore, peptides with continuous acidic amino acid residues (aspartic acid residues, glutamic acid residues) tend to be difficult to exert cell membrane permeability. Therefore, the inventors of the present invention have conducted careful research and found that as long as the carrier peptide fragment of the above-mentioned structure created based on the amino acid sequence of sequence number 1 disclosed in patent document 1, sufficient cell membrane permeability can be achieved. By having continuous acidic amino acid residues in the carrier peptide fragment, exogenous substances that are poorly combined with CPPs with a high ratio of existing basic amino acid residues can also be easily introduced into the cell. In addition, the carrier peptide fragment disclosed in the present invention significantly inhibits cytotoxicity.

[0013] In one embodiment of the carrier peptide fragment disclosed in the present invention, it is an amino acid sequence that satisfies the condition (ii) above, and 2 or more and 4 or less aspartic acid residues and / or glutamic acid residues are inserted between the 9th and 10th amino acid residues from the N-terminal side of the amino acid sequence.

[0014] In one embodiment of the carrier peptide fragment disclosed in the present invention, it is composed of any of the following amino acid sequences:

[0015] KKRTLRKDERKKR (sequence number 2);

[0016] KKRTLRKEDRKKR (sequence number 3);

[0017] KKRTLRKEDEDRKKR (sequence number 4);

[0018] KKRTLRKEEDDRKKR (sequence number 5);

[0019] KKRTLRKDDEERKKR (sequence number 6);

[0020] KKRTLRKDEDERKKR (sequence number 7); and

[0021] KKRTLRKEDEDEDRKKR (serial number 8).

[0022] In addition, in order to achieve the above-mentioned purpose, the present invention provides a construct for introducing an exogenous substance (hereinafter also referred to as a "construct"), which is prepared for introducing a target exogenous substance from the outside of a eukaryotic cell at least into the cytoplasm of the cell. The construct disclosed in the present invention has a 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. Such a construct can be efficiently introduced into the target eukaryotic cell, so the target exogenous substance can be introduced into the cell.

[0023] 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.

[0024] 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.

[0025] In addition, "nucleic acid" refers to a polymer of nucleotides, including DNA and RNA. The number of bases in "nucleic acid" is not limited.

[0026] 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.

[0027] In addition, the present invention provides a method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell in order to achieve the above-mentioned object. The method disclosed in the present invention comprises: (1) preparing a construct having a carrier peptide fragment disclosed in the present invention and the target exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment; and (2) supplying the construct to a sample containing the target eukaryotic cell.

[0028] In one embodiment of the method 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.

[0029] In one embodiment of the method disclosed in the present invention, the exogenous substance may be placed on the C-terminal side of the carrier peptide fragment.

[0030] 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

[0031] Figure 1 The graph shows the MFI values ​​obtained by analyzing NSC-34 cells using flow cytometry after adding the constructs (supplements) shown in Examples 1 to 8 to the culture medium of the cells and culturing the cells.

[0032] Figure 2 It is a graph showing the results of the cytotoxicity test of samples 1, 2, and 7.

[0033] Figure 3 It is a graph showing the results of the cytotoxicity test of samples 3 to 6. DETAILED DESCRIPTION

[0034] 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 matters required for implementation (such as chemical synthesis of peptides, cell culture techniques, and general matters involved in 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 existing technologies in the fields of cell engineering, physiology, medicine, pharmacy, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, and genetics.

[0035] In addition, the technology disclosed in the present invention can be implemented based on the disclosed content in this specification and the technical common sense in this field. In addition, in the following description, according to the circumstances, the single-letter symbol about the nomenclature of amino acid shown in the IUPAC-IUB guide is used to represent amino acid. Wherein, in this specification, except for the case mentioned in particular, "amino acid residue" is a term comprising the N-terminal amino acid and the C-terminal amino acid of the peptide chain.

[0036] 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.

[0037] In the present specification, the amino acid residues constituting a peptide or a protein may be either L-form or D-form.

[0038] 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.

[0039] The carrier peptide fragment disclosed in the present invention is composed of an amino acid sequence that satisfies at least any of the following conditions (i) and (ii).

[0040] Condition (i): The serine residue at position 8 and the asparagine residue at position 9 from the N-terminal side of the amino acid sequence: KKRTLRKSNRKKR (SEQ ID NO: 1) are substituted with an aspartic acid residue or a glutamic acid residue, respectively.

[0041] Condition (ii): An aspartic acid residue and / or a glutamic acid residue is inserted between the 9th and 10th amino acid residues from the N-terminal side of the amino acid sequence satisfying the condition (i) above. In other words, an aspartic acid residue and / or a glutamic acid residue is inserted so that the aspartic acid residue or the glutamic acid residue introduced in place of the asparagine residue in condition (i) is further continuous.

[0042] Specific examples of amino acid sequences satisfying condition (ii) include:

[0043] KKRTLRKDERKKR (sequence number 2);

[0044] KKRTLRKEDRKKR (sequence number 3);

[0045] KKRTLRKDDRKKR (sequence number 9); and

[0046] KKRTLRKEERKKR (serial number 10).

[0047] In these sequences, although acidic amino acid residues are continuously present at positions 8 to 9 from the N-terminal side, they can exhibit high cell membrane permeability.

[0048] In the amino acid sequence satisfying condition (ii), the number of aspartic acid residues and / or glutamic acid residues inserted between the 9th and 10th amino acid residues from the N-terminal side of the amino acid sequence satisfying the above-mentioned condition (i) may be, for example, more than 1, or more than 2. That is, in the amino acid sequence constituting the carrier peptide fragment disclosed in the present invention, more than 3, or more than 4 acidic amino acid residues may be continuous. In addition, the upper limit of the number of the inserted aspartic acid residues and / or glutamic acid residues is, for example, less than 6, and may be less than 5, or less than 4. That is, in the amino acid sequence constituting the carrier peptide fragment disclosed in the present invention, the number of consecutive acidic amino acid residues is, for example, less than 8, and may be less than 7, or less than 6. When the number of inserted aspartic acid residues and / or glutamic acid residues is too large, the cell membrane permeability of the carrier peptide fragment may be impaired.

[0049] As the amino acid sequence satisfying condition (ii), for example, the following amino acid sequences can be cited:

[0050] KKRTLRKEDEDRKKR (sequence number 4);

[0051] KKRTLRKEEDDRKKR (sequence number 5);

[0052] KKRTLRKDDEERKKR (sequence number 6);

[0053] KKRTLRKDEDERKKR (sequence number 7); and

[0054] KKRTLRKEDEDEDRKKR (serial number 8) etc.

[0055] As long as the cell membrane permeability is not impaired, the carrier peptide fragment disclosed in the present invention may also be based on 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.

[0056] 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.

[0057] The cytotoxicity of the carrier peptide fragment disclosed in the present invention is suppressed to a very low level. Generally speaking, in CPPs containing many basic amino acid residues, due to strong positive charge, they can show high cell membrane permeability, on the other hand, the cytotoxicity tends to increase. In contrast, the carrier peptide fragment disclosed in the present invention contains multiple acidic amino acid residues, so it is presumed that by alleviating the positive charge caused by the basic amino acid residues, cytotoxicity can be suppressed. It should be noted that this mechanism is only presumed and does not limit the present technology in any way.

[0058] 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.

[0059] 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.

[0060] 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 a flexible amino acid sequence that does not produce steric hindrance. 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, for example 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.

[0061] Exogenous substances may be, for example, organic compounds such as polypeptides, nucleic acids, pigments, and drugs.

[0062] In the case where the exogenous substance is a polypeptide, a peptide chain is designed in a manner that contains 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 the introduction of the target exogenous substance. It is also possible to use various scientific methods currently known to make 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 drugs (such as antitumor agents containing nucleic acid-based antitumor agents such as 5-fluorouracil (5FU), antiviral agents such as azidothymidine (AZT), etc.) directly or indirectly bind to the N-terminal side and / or C-terminal side of the above-mentioned carrier peptide fragment to prepare a construct.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Typically, the total number of amino acid residues constituting a synthetic peptide prepared as a construct for introducing an exogenous substance is several to several tens (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 are easy to use.

[0067] 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).

[0068] For example, in the case where the eukaryotic cell to be introduced as the object of exogenous material 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 participates in the differentiation induction of the stem cell. Among them, "stem cells" include adult stem cells, embryonic stem cells, and artificial pluripotent stem cells (Induced pluripotentstem cells: iPS cells). In addition, in the case where the eukaryotic cell to be introduced as the object of exogenous material 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, in the case where the eukaryotic cell to be introduced 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, a polypeptide that can inhibit the proliferation of bacteria or viruses in the infected cell, or a polypeptide that can inhibit the spread of bacterial or viral infection from the infected cell.

[0069] 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.

[0070] 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 most proteins in eukaryotic cells are acetylated at the α-amino group of the amino acid on the N-terminal side, such a structure can improve the stability of the construct in the cell.

[0071] 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.

[0072] 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.

[0073] A relatively short peptide chain (including a polypeptide constituted as 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.

[0074] 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.

[0075] 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.

[0076] In addition, the method of constructing a recombinant vector and the method of introducing the constructed recombinant vector into a host cell can directly adopt the method conventionally performed in this field. This method itself does not particularly characterize the present technology, and thus a detailed description is omitted.

[0077] 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.

[0078] 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 already commissioned the production of polypeptides at the time of the present application, and kits for cell-free protein synthesis are also commercially available (for example, available from Cell Free Sciences, Japan).

[0079] 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 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.

[0080] 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.

[0081] 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 an addition reaction of a commonly used inorganic acid or organic acid can be used. Therefore, the "construct" recorded in this specification and the scope of the claim can include such a construct in the form of a salt.

[0082] 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.

[0083] As the above-mentioned carrier, for example, it is preferred that the carrier used in peptide pharmaceuticals as a diluent, excipient, etc. is usually used. 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, 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 secondary components that can be contained in the pharmaceutical composition, various fillers, extenders, binders, wetting agents, surfactants, pigments, spices, etc. can be listed.

[0084] 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 to prepare a medicinal solution just before use.

[0085] The process of preparing various forms of drugs (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 give characteristics to the present 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.

[0086] The present invention provides 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):

[0087] (1) a step of preparing a construct for introducing an exogenous substance comprising the carrier peptide fragment disclosed in the present invention and a target exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment; and

[0088] (2) A step of supplying the above construct to a sample containing target eukaryotic cells.

[0089] Furthermore, the method disclosed in the present invention further comprises, after the step (2), as the step (3), culturing a sample to which the construct has been supplied, and introducing the construct into eukaryotic cells in the sample.

[0090] 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, and cell lines removed from a living body in vitro.

[0091] 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.

[0092] 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 culture 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 culture conditions can also vary depending on the type of eukaryotic cells, and are therefore not particularly limited, and can be cultured at 37° C. under a 5% CO2 atmosphere.

[0093] In addition, regarding the in vitro introduction method, examples thereof will be described in the test examples described later.

[0094] 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.

[0095] As described above, as specific aspects of the technology disclosed in the present invention, aspects described in the following items can be cited.

[0096] Item 1: A carrier peptide fragment consisting of an amino acid sequence that satisfies any of the following conditions:

[0097] (i) the following amino acid sequence: KKRTLRKSNRKKR (SEQ ID NO: 1), wherein the 8th and 9th amino acid residues from the N-terminal side are substituted with an aspartic acid residue or a glutamic acid residue, respectively; and

[0098] (ii) One to six aspartic acid residues and / or glutamic acid residues are inserted between the 9th and 10th amino acid residues from the N-terminal side of the amino acid sequence satisfying the condition of (i) above.

[0099] Item 2: A carrier peptide fragment as described in Item 1, which is an amino acid sequence that satisfies the condition of (ii) above, and has 2 or more and 4 or less aspartic acid residues and / or glutamic acid residues inserted between the 9th and 10th amino acid residues from the N-terminal side of the amino acid sequence.

[0100] Item 3: The carrier peptide fragment as described in Item 1, which is composed of any sequence of the following amino acid sequences:

[0101] KKRTLRKDERKKR (sequence number 2);

[0102] KKRTLRKEDRKKR (sequence number 3);

[0103] KKRTLRKEDEDRKKR (sequence number 4);

[0104] KKRTLRKEEDDRKKR (sequence number 5);

[0105] KKRTLRKDDEERKKR (sequence number 6);

[0106] KKRTLRKDEDERKKR (sequence number 7); and

[0107] KKRTLRKEDEDEDRKKR (serial number 8).

[0108] Item 4: A construct comprising: the carrier peptide fragment according to any one of Items 1 to 3; and the target exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

[0109] Item 5: The construct according to Item 4, 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.

[0110] Item 6: The construct according to Item 4 or 5, wherein the exogenous substance is disposed on the C-terminal side of the carrier peptide fragment.

[0111] Item 7: 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, comprising:

[0112] (1) a step of preparing a construct comprising the carrier peptide fragment described in any one of items 1 to 3 and a foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment; and

[0113] (2) A step of supplying the above construct to a sample containing target eukaryotic cells.

[0114] Item 8: The method according to Item 7, 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.

[0115] Item 9: The method according to Item 7 or 8, wherein the exogenous substance is disposed on the C-terminal side of the carrier peptide fragment.

[0116] Item 10: The method according to any one of Items 7 to 9, wherein the eukaryotic cell into which the construct is introduced is a human or a non-human mammalian cell.

[0117] 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 the examples.

[0118] [Test 1]

[0119] <Preparation of construct for introduction of exogenous substances>

[0120] A construct having a synthetic peptide consisting of the amino acid sequence shown in Table 1 was prepared. A construct having a peptide n (n is a natural number of 1 to 7) shown in Table 1 was used as sample n, and samples 1 to 7 were obtained from Eurofinsgenomics Co., Ltd. Among them, in samples 1 to 7, peptides in which the α-amino groups of the amino acid residues on the N-terminal side of peptides 1 to 7 were all acetylated were prepared. In addition, a fluorescent dye FAM (C21 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 7 were diluted with dimethyl sulfoxide (DMSO) to prepare sample solutions 1 to 7 with a sample concentration of 6 mM.

[0121] [Table 1]

[0122] Table 1

[0123] PeptideNo. sequence Serial Number 1 KKRTLRKDERKKR 2 2 KKRTLRKEDRKKR 3 3 KKRTLRKEDEDRKKR 4 4 KKRTLRKEEDDRKKR 5 5 KKRTLRKDDEERKKR 6 6 KKRTLRKDEDERKKR 7 7 KKRTLRKEDEDEDRKKR 8

[0124] <Evaluation of Cell Membrane Permeability Using Flow Cytometry>

[0125] As eukaryotic cells, NSC-34 cells (mouse motor neuron-like hybrid cell line) were used to analyze the cell membrane permeability of samples 1 to 7. In Examples 1 to 7, the sample solutions 1 to 7 prepared above were used, respectively, and in Example 8, a FAM solution was used.

[0126] (Example 1)

[0127] NSC-34 cells were cultured in a common medium, 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).

[0128] After washing the NSC-34 cells attached to the culture plate with PBS, 0.25% trypsin / EDTA solution was added and cultured at 37°C for 3 minutes. After the culture, 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 5cells / well). In addition, the above 6 mM sample solution 1 was diluted with the above DMEM containing 10% FBS to prepare a sample solution 1 with a concentration of 50 μM. Then, 1 mL of the above 50 μM sample solution 1 was added to the well (i.e., the concentration of sample 1 in the culture solution in the well was 25 μM, and the DMSO concentration was 0.42%). Thereafter, the cells were cultured at 37°C for 20 hours under 5% CO2 conditions.

[0129] After 20 hours of culture, 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 cultured at 37°C for 3 minutes. After this culture, 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 transferred 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 transferred 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.

[0130] 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.

[0131] 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.

[0132] 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, and the fluorescence intensity is measured for the cell group within the gate. The analysis is performed in such a way that the number of cells in the cell group is at least 10,000. In the determination of the 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.

[0133] (Examples 2~7)

[0134] The same procedures as in Example 1 were carried out except that the sample solution 1 was changed to any of the sample solutions 2 to 7 prepared above. The samples (constructs) used in each example are shown in Table 2.

[0135] (Example 8)

[0136] The same procedure as in Example 1 was performed except that the sample solution 1 was a FAM solution diluted with DMSO. 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 25 μM and the DMSO concentration was 0.42%).

[0137] The results obtained for Examples 1 to 8 are shown in Tables 2 and Figure 1 . Figure 1 It is a graph showing the MFI value of each example.

[0138] [Table 2]

[0139] Table 2

[0140] Composition of constructs (additives) MFI Example 1 Ac-KKRTLRKDERKKR-FAM 16.3 Example 2 Ac-KKRTLRKEDRKKR-FAM 15.7 Example 3 Ac-KKRTLRKEDEDRKKR-FAM 13.8 Example 4 Ac-KKRTLRKEEDDRKKR-FAM 14.3 Example 5 Ac-KKRTLRKDDEERKKR-FAM 14.8 Example 6 Ac-KKRTLRKDEDERKKR-FAM 15.2 Example 7 Ac-KKRTLRKEDEDEDRKKR-FAM 14.3 Example 8 FAM 11

[0141] As shown in Table 2 and Figure 1As shown, the MFI values ​​of Examples 1 to 7 are significantly higher than those of Example 8. Therefore, it is known that the peptides (Examples 1 and 2) in which the amino acid residues at the 8th and 9th positions from the N-terminal side of the amino acid sequence shown in SEQ ID NO. 1 are substituted with aspartic acid residues or glutamic acid residues, respectively, or the peptides (Examples 3 to 7) in which the amino acid residues at the 8th and 9th positions from the N-terminal side of the amino acid sequence shown in SEQ ID NO. 1 are substituted with aspartic acid residues or glutamic acid residues, respectively, and one or more but less than four aspartic acid residues and / or glutamic acid residues are inserted between the amino acid residues at the 9th and 10th positions from the N-terminal side of the amino acid sequence have at least cell membrane permeability. It is also known that by using the peptides of Examples 1 to 7, exogenous substances can be introduced into cells.

[0142] In addition, although detailed data are not shown, the research of the inventors of the present invention has confirmed that the exogenous substance is not only a fluorescent dye (such as FAM), but also any substance among polypeptides, nucleic acids and drugs. The construct having peptides 1 to 7 can efficiently introduce the exogenous substance from the outside of the cell into the cytoplasm.

[0143] [Test 2]

[0144] <Evaluation of cytotoxicity of carrier peptide fragments>

[0145] As eukaryotic cells, WS-1 cells (human skin fibroblastoid cell line, obtained from American Type Culture Collection, ATCC No.CRL-1502) were used to evaluate the cytotoxicity of the above-mentioned peptides 1 to 7. As a culture medium, DMEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No.044-29765)) containing 10% FBS (fetal bovine serum) was used. In Experiment 2, samples 1 to 7 were dissolved in DMSO to prepare 6 mM sample solutions 1 to 3. The 6 mM sample solutions 1 to 7 were diluted with the above-mentioned culture medium to prepare 12.5 μM, 25 μM, 50 μM and 100 μM sample solutions 1 to 7. In addition, as a reference example, a construct having a carrier peptide fragment that exhibits cell membrane permeability and is composed of the amino acid sequence shown in SEQ ID NO: 11 was prepared. The composition of the construct of the reference example is the same as that of samples 1 to 7 except for the amino acid sequence constituting the carrier peptide fragment. Regarding the reference example, the same procedure as for the sample solutions 1 to 3 was performed to prepare a solution containing 12.5 μM to 100 μM of the reference example (hereinafter also referred to as “solution (reference example)”).

[0146] WS-1 cells attached to the culture plate were washed with PBS, and then a 0.25% trypsin / 0.53 mM EDTA solution was added and cultured at 37°C for 3 to 5 minutes. After the culture, the above-mentioned culture medium was added to inactivate the trypsin, and then the cells were centrifuged at 900 rpm for 5 minutes at room temperature to precipitate the cells. After removing the supernatant produced by centrifugation, the above-mentioned culture medium was added to the precipitate (cell mass) to prepare about 1×10 5 100 μL of the cell suspension was added to the wells of a commercially available 96-well plate (manufactured by AGC TECHNO GLASS Co., Ltd.), and cells (approximately 1×10 4 cells / well), and cultured for 24 hours in an incubator at 5% CO2 and 37°C. After the culture, the culture medium in the wells was removed, and 90 μL of the sample solutions 1 to 7 and solution (reference example) of 12.5 μM, 25 μM, 50 μM and 100 μM prepared above were added to each of the three wells (i.e., the test was performed in parallel 3 times). In addition, as a control, 90 μL of the above culture medium containing 1.67% DMSO (the same DMSO concentration as in the wells to which the 100 μM sample solution was added) was added to the three wells. In addition, 90 μL of the above culture medium without DMSO was added to each of the three wells. Thereafter, cultured for another 24 hours in an incubator at 5% CO2 and 37°C. After the culture, the culture medium in each well was removed, and 100 μL of the above culture medium without DMSO was added to each well. At this time, 100 μL of the above culture medium was also added to the wells without cells seeded as a blank. In addition, for each well, add 10 μL of the cell proliferation / cytotoxicity kit: Cell Counting Kit-8 (CCK-8, manufactured by Dojindo Laboratories) to each well. After that, culture was carried out in an incubator with 5% CO2 and 37°C for 1.5 hours, and the absorbance at 450 nm was measured using a microplate reader. Then, the arithmetic mean of the absorbance of the three wells was calculated based on the type and concentration of the solution added to the culture medium. The average value of the absorbance of the three wells in which cells were cultured in the above-mentioned culture medium without DMSO was set to 100%, and the cell viability was calculated. The results are shown in Figure 2 , 3 . Figure 2 , 3 The horizontal axis of the graph represents the sample concentration in the culture medium, and the vertical axis represents the cell viability. The example of culturing in the above culture medium containing 1.67% DMSO as a control is plotted at the position of the sample concentration of 100 μM.

[0147] like Figure 2 , 3As shown, in samples 1 to 7, even at a sample concentration of 100 μM, the cell viability did not decrease compared to the case where 1.67% DMSO was added. In other words, it was found that even at a high concentration of 100 μM, samples 1 to 7 showed little cytotoxicity.

[0148] 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.

[0149] 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 medicines (such as eye drops).

Claims

1. A carrier peptide fragment, characterized in that: It consists of an amino acid sequence that satisfies any of the following conditions: (i) the following amino acid sequence: KKRTLRKSNRKKR (SEQ ID NO: 1), wherein the 8th and 9th amino acid residues from the N-terminal side are substituted with an aspartic acid residue or a glutamic acid residue, respectively; and (ii) One to six aspartic acid residues and / or glutamic acid residues are inserted between the 9th and 10th amino acid residues from the N-terminal side of the amino acid sequence satisfying the condition of (i).

2. The carrier peptide fragment according to claim 1, characterized in that: This is an amino acid sequence that satisfies the condition (ii) above, wherein 2 or more and 4 or less aspartic acid residues and / or glutamic acid residues are inserted between the 9th and 10th amino acid residues from the N-terminal side of the amino acid sequence.

3. The carrier peptide fragment according to claim 1, characterized in that: It is composed of any of the following amino acid sequences: KKRTLRKDERKKR (sequence number 2); KKRTLRKEDRKKR (sequence number 3); KKRTLRKEDEDRKKR (sequence number 4); KKRTLRKEEDDRKKR (sequence number 5); KKRTLRKDDEERKKR (sequence number 6); KKRTLRKDEDERKKR (sequence number 7); and KKRTLRKEDEDEDRKKR (serial number 8).

4. A construct, characterized in that have: The carrier peptide fragment according to any one of claims 1 to 3; and A foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

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

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

7. 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, characterized in that: include: (1) a step of preparing a construct comprising the carrier peptide fragment according to any one of claims 1 to 3 and the target exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment; and (2) A step of supplying the construct to a sample containing target eukaryotic cells.

8. The method according to claim 7, characterized in that: The exogenous substance is at least one organic compound selected from the group consisting of polypeptides, nucleic acids, pigments and drugs.

9. The method according to claim 7, characterized in that: The exogenous substance is disposed on the C-terminal side of the carrier peptide fragment.

10. The method according to claim 7, characterized in that: The eukaryotic cell into which the construct is introduced is a human or a non-human mammal cell.

Images