Peptide fragment and use thereof

Abstract

A peptide fragment disclosed herein comprises any of the following amino acid sequences: YLIQFKKRTLRKSNRKKR (SEQ ID NO: 1), FQILYKKRTLRKSNRKKR (SEQ ID NO: 2), YQFLIKKRTLRKSNRKKR (SEQ ID NO: 3), and ILFQYKKRTLRKSNRKKR (SEQ ID NO: 4).

Description

Peptide fragments and their uses

[0001] The present disclosure relates to peptide fragments and uses thereof. This application claims priority based on Japanese Patent Application No. 2023-128578, filed on August 7, 2023, the entire contents of which are incorporated herein by reference.

[0002] Conventionally, foreign substances such as polypeptides and nucleic acids have been introduced into cells (eukaryotic cells) of humans and other mammals to transform the characteristics of the cells (and even tissues and organs made up of the cells) or to improve or enhance the functions of the cells.

[0003] The above-mentioned technology uses, for example, a cell-penetrating peptide (CPP) that passes through the cell membrane from the outside of the cell and introduces a foreign substance into the cytoplasm. Using CPPs allows various foreign substances to be efficiently introduced into cells, and thus is expected to be applied to pharmaceuticals, etc. For example, Japanese Patent Publication No. 7041853 and International Publication No. 2020 / 030928 disclose amino acid sequences that function as CPPs.

[0004] Japanese Patent No. 7041853 International Publication No. 2020 / 030928

[0005] The present inventors are currently investigating further improvement of the cell membrane permeability of the peptide fragment consisting of the amino acid sequence KKRTLRKSNRKKR (SEQ ID NO: 5) disclosed in Japanese Patent No. 7041853.

[0006] A primary object of the present disclosure is to provide a technique relating to peptide fragments that have excellent cell membrane permeability.

[0007] The present disclosure provides a peptide fragment (CPP) having cell membrane permeability. This peptide fragment consists of any of the following amino acid sequences: YLIQFKKRTLRKSNRKKR (SEQ ID NO: 1); FQILYKKRTLRKSNRKKR (SEQ ID NO: 2); YQFLIKKRTLRKSNRKKR (SEQ ID NO: 3); or ILFQYKKRTLRKSNRKKR (SEQ ID NO: 4). A peptide fragment consisting of any of the above amino acid sequences exhibits excellent cell membrane permeability.

[0008] The present disclosure also provides a construct for introducing a foreign substance (hereinafter simply referred to as a "construct") that can introduce a foreign substance of interest into a eukaryotic cell from the outside of the cell. The construct disclosed herein comprises a peptide fragment and a foreign substance bound to the N-terminus and / or C-terminus of the peptide fragment, wherein the peptide fragment consists of any of the following amino acid sequences: YLIQFKKRTLRKSNRKKR (SEQ ID NO: 1); FQILYKKRTLRKSNRKKR (SEQ ID NO: 2); YQFLIKKRTLRKSNRKKR (SEQ ID NO: 3); ILFQYKKRTLRKSNRKKR (SEQ ID NO: 4). Such a construct allows the foreign substance to be efficiently introduced into the eukaryotic cell (e.g., at least the cytoplasm) from the outside of the cell.

[0009] In some embodiments, the exogenous substance is at least one organic compound selected from the group consisting of polypeptides, nucleic acids, dyes, and drugs, thereby enabling the introduction of organic compounds with diverse functions into eukaryotic cells.

[0010] In some embodiments, the foreign substance is located at the C-terminus of the peptide fragment, which allows the construct to be efficiently introduced into eukaryotic cells.

[0011] The present disclosure also provides a method for introducing a foreign substance of interest into at least the cytoplasm of a eukaryotic cell from the outside of the cell in vitro or in vivo. The method disclosed herein comprises the steps of (1) preparing a construct disclosed herein and (2) providing the construct in a sample containing the eukaryotic cell of interest. This allows the foreign substance of interest to be introduced into the eukaryotic cell.

[0012] In some embodiments, the eukaryotic cells into which the construct is introduced are mammalian cells, thereby allowing the introduction of a desired foreign substance into the mammalian cells.

[0013] FIG. 1 is a graph showing the mean fluorescence intensity (MFI) for each example obtained by adding the constructs of Examples 1 to 6 or FAM to the culture medium of NSC-34 cells, culturing the cells, and then analyzing the cells using a flow cytometer.

[0014] Several embodiments of the technology disclosed herein are described below. Matters necessary for implementing the technology (e.g., general matters related to chemical synthesis of peptides, cell culture techniques, and preparation of constructs containing peptides or nucleic acids as components) other than those specifically mentioned herein can be understood as design matters of a person skilled in the art based on conventional techniques in the fields of cell engineering, physiology, medicine, pharmacology, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, genetics, etc. Furthermore, the technology disclosed herein can be implemented based on the content disclosed herein and the common general technical knowledge in the relevant fields. Note that, in this specification, amino acids may be represented by single-letter symbols in accordance with the nomenclature for amino acids set forth in the IUPAC-IUB guidelines. Note that, in this specification, the term "amino acid residue" encompasses the N-terminal amino acid and the C-terminal amino acid of a peptide chain, unless otherwise specified.

[0015] Furthermore, as used herein, the term "synthetic peptide" refers to a peptide (peptide fragment) whose peptide chain does not exist independently and stably in nature, but is produced by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and can exist stably in a given composition. Here, the term "peptide" refers to an amino acid polymer (including dimers, trimers, and oligomers) having one or more peptide bonds, and is not limited by the number of amino acid residues.

[0016] Furthermore, in this specification, amino acid residues constituting peptides or proteins may be in either the L- or D-form unless otherwise specified. That is, the amino acid residues constituting the amino acid sequences described in the claims are intended to include both the L- and D-forms. In addition, in the amino acid sequences described in this specification, the left side always represents the N-terminus and the right side represents the C-terminus.

[0017] In some embodiments, the peptide fragment disclosed herein comprises any of the following amino acid sequences: YLIQFKKRTLRKSNRKKR (SEQ ID NO: 1); FQILYKKRTLRKSNRKKR (SEQ ID NO: 2); YQFLIKKRTLRKSNRKKR (SEQ ID NO: 3); ILFQYKKRTLRKSNRKKR (SEQ ID NO: 4). The peptide fragment disclosed herein consists of, for example, any of the amino acid sequences set forth in SEQ ID NOs: 1 to 4. The peptide fragment disclosed herein is, for example, a synthetic peptide.

[0018] The peptide fragment disclosed herein has an amino acid sequence consisting of YLIQF (SEQ ID NO: 6), FQILY (SEQ ID NO: 7), YQFLI (SEQ ID NO: 8), or ILFQY (SEQ ID NO: 9) added to the N-terminus of the amino acid sequence KKRTLRKSNRKKR (SEQ ID NO: 5) disclosed in Patent Document 1. As will be described in detail in the test examples below, a peptide fragment consisting of any of the amino acid sequences shown in SEQ ID NOs: 1 to 4 exhibits superior cell membrane permeability to an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 5.

[0019] The number of amino acid residues in the peptide fragments disclosed herein is not limited as long as cell membrane permeability is not impaired. For example, the number of amino acid residues constituting the peptide fragment may be, for example, 20 or less, 19 or less, or 18 or less.

[0020] Furthermore, the "peptide fragment" disclosed herein typically has the same amino acid sequence as any of SEQ ID NOS: 1 to 4. In addition to the identical sequence, the "peptide fragment" also encompasses modified sequences in which one or several (e.g., two or three) amino acid residues have been substituted, deleted, and / or added (inserted) without impairing cell membrane permeability. That is, such minor modified sequences can be easily utilized based on the technical information disclosed herein and are encompassed by the "peptide fragment" as a technical concept disclosed herein. Typical examples of modified sequences herein include sequences resulting from conservative substitutions of one, two, or three amino acid residues (so-called conservative amino acid replacements). Typical examples of conservative substitutions include sequences in which a basic amino acid residue is substituted with another basic amino acid residue (e.g., mutual substitution of an arginine residue with a lysine residue).

[0021] The constructs disclosed herein comprise a peptide fragment disclosed herein and a foreign substance, which is bound to the N-terminus and / or C-terminus of the peptide fragment.

[0022] The construct is designed and constructed, for example, by directly or indirectly binding (linking) a foreign substance to the N-terminus and / or C-terminus of the peptide fragment. When the peptide fragment and the foreign substance are indirectly bound, for example, a linker is placed between the peptide fragment and the foreign substance. The linker is not particularly limited, and may be a peptidic linker or a non-peptidic linker. Alternatively, a peptidic linker and a non-peptidic linker may be used in combination. The method for directly or indirectly binding the peptide fragment and the foreign substance is not particularly limited, and can be carried out according to various conventionally known scientific techniques.

[0023] The peptidic linker may be composed of one or two or more amino acid residues. The number of amino acid residues constituting the peptidic linker is not particularly limited. The number of amino acid residues constituting the peptidic linker may be, for example, one or more or two or more. The number of amino acid residues constituting the peptidic linker may be, for example, 10 or less or 5 or less. In some embodiments, the amino acid sequence constituting the peptidic linker is an amino acid sequence that does not cause steric hindrance and is flexible. Such a peptidic linker may contain, for example, glycine, alanine, and / or serine. In some embodiments, the peptidic linker may contain β-alanine.

[0024] Non-peptidic linkers include, but are not limited to, alkyl linkers, PEG (polyethylene glycol) linkers, aminohexanoyl spacers, and the like. Furthermore, cross-linkers having homobifunctional or heterobifunctional groups may also be used as non-peptidic linkers. Typical functional groups possessed by cross-linkers include N-hydroxysuccinimide activated esters (NHS esters), maleimides, azides, and iodoacetamides. Among these, NHS esters can react efficiently with amines at neutral or higher pH levels to form bonds. Furthermore, maleimides can selectively react with SH groups to form bonds. For example, when using a cross-linker containing maleimide, the peptide fragment can be easily bonded to the cross-linker by adding a cysteine ​​residue as a linker to the N-terminus or C-terminus of the peptide fragment, or by preparing a peptide fragment having a cysteine ​​residue at the N-terminus or C-terminus.

[0025] Suitable cross-linking agents having homobifunctional groups include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS ３ ), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), etc. In particular, bis(sulfosuccinimidyl) suberate (BS ３ ) can be preferably used.

[0026] Preferred examples of suitable crosslinking agents having heterobifunctional groups include N-(6-maleimidocaproyloxy)succinimide (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl 4-[maleimidophenyl]butyrate (SMPB), succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-(γ-maleimidobutyloxy)succinimide ester (GMBS), m-maleimidopropionic acid-N-hydroxysuccinimide ester (MPS), and N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB). In particular, EMCS and MBS having NHS ester and maleimide reactive functional groups are preferred.

[0027] The foreign substance is not particularly limited as long as it has a molecular size and chemical properties that allow it to be introduced into eukaryotic cells, and may be, for example, an organic compound or an inorganic compound. Organic substances include, for example, amino acids, polypeptides, nucleic acids, dyes, drugs, etc. The foreign substance may contain one type alone or two or more types. When two or more types of foreign substances are contained, two or more types of foreign substances may be bound to the N-terminal or C-terminal side of the peptide fragment. Furthermore, one or two or more types of foreign substances may be bound to each of the N-terminal and C-terminal sides of the peptide fragment.

[0028] When the foreign substance is a polypeptide, the polypeptide (amino acid sequence) employed is not particularly limited. As used herein, "polypeptide" refers to a polymer (including dimers, trimers, and oligomers) having a structure in which multiple amino acids are linked by peptide bonds. It is not limited by the number of peptide bonds (i.e., the number of amino acid residues). Polypeptides with a relatively large number of amino acid residues, such as peptides with approximately 2 to 100 amino acid residues, polypeptides with approximately 100 to 300 amino acid residues, or proteins (polymeric compounds typically consisting of 300 or more amino acid residues), can also be employed as foreign substances. In this field, polypeptides and proteins are not strictly distinguished. While there is no particular upper limit on the number of amino acid residues constituting a polypeptide, from the viewpoint of ease of synthesis (biosynthesis or chemical synthesis), it is preferable that the number be, for example, 1,000 or less, 600 or less, 500 or less, 400 or less, or 300 or less.

[0029] The polypeptide may be a mature or precursor (including pro- and prepro-) polypeptide involved in functions such as the development, differentiation, proliferation, canceration, homeostasis, and metabolic regulation of various cells and tissues (organs). The polypeptide may also be a polypeptide whose function is unknown. The technology disclosed herein can be used to elucidate the function of a polypeptide whose function is unknown within a cell (in a living tissue).

[0030] For example, when the eukaryotic cells to be introduced with a foreign substance are mammalian stem cells (human or other mammalian cells), it is preferable to use mature forms or precursors of polypeptides with various physiological activities involved in the differentiation induction of the stem cells. Note that "stem cells" encompass somatic stem cells, embryonic stem cells, and induced pluripotent stem cells (iPS cells). Furthermore, when the eukaryotic cells to be introduced with a foreign substance are cancer cells (tumor cells), it is preferable to use various polypeptides involved in the induction of apoptosis of the cancer cells (tumor cells). Alternatively, in this case, it is preferable to use polypeptides that can inhibit the suppression of the immune surveillance mechanism of cancer cells (tumor cells). Furthermore, when the eukaryotic cells to be introduced with a foreign substance are bacterially or virally infected cells, it is preferable to use various polypeptides involved in the induction of apoptosis of the infected cells, polypeptides that can inhibit the proliferation of bacteria or viruses in the infected cells, or polypeptides that can inhibit the spread of bacterial or viral infection from the infected cells. As with peptide fragments, a polypeptide as a foreign substance may contain a modified amino acid sequence formed by substitution, deletion, and / or addition (insertion) of one or several amino acid residues, as long as it retains its function.

[0031] When the foreign substance is a polypeptide, for example, a peptide chain can be designed to contain the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the peptide fragment, and the peptide chain can be biosynthesized or chemically synthesized to produce a construct in which the polypeptide is directly linked to the peptide fragment.

[0032] The type of nucleic acid employed as the foreign substance is not particularly limited and may include, for example, so-called nucleic acid drugs. As used herein, "nucleic acid" refers to a polymer of nucleotides, encompassing DNA, RNA, and DNA / RNA hybrids (also referred to as DNA-RNA chimeras) containing both DNA and RNA. Furthermore, "nucleic acid" is not limited by the number of bases. Nucleic acids may be single-stranded (including hairpin-type single-stranded) or double-stranded. Furthermore, the nucleotides constituting the nucleic acid may contain modified groups in the phosphate moiety, sugar moiety, or base moiety. The type of nucleic acid drug is not particularly limited and may include, for example, siRNA, miRNA, antisense, aptamer, decoy, ribozyme, CpG oligo, etc.

[0033] The type of dye used as the foreign substance is not particularly limited. The dye may include various fluorescent dye compounds such as FAM and FITC. When the construct contains a dye, it becomes easier to evaluate the introduction of the construct. For example, the efficiency of introduction into eukaryotic cells can be evaluated using microscopic observation (e.g., fluorescence microscopic observation), flow cytometry, immunochemical techniques (e.g., Western blotting, immunocytostaining, etc.), etc.

[0034] The type of drug employed as the foreign substance is not particularly limited, and the drug may include various organic compounds, such as antitumor agents including nucleic acid-based antitumor agents such as 5-fluorouracil (5FU) and antiviral agents such as azidothymidine (AZT).

[0035] In the construct, when a foreign substance is bound to the C-terminus of a peptide fragment, it is preferable that the α-amino group of the N-terminal amino acid residue of the peptide fragment is acetylated. Although the detailed mechanism is unknown, the α-amino group of the N-terminal amino acid of many proteins in eukaryotic cells is acetylated, and such a configuration can improve the stability of the construct within the cell.

[0036] Furthermore, it is preferable that the C-terminal amino acid residue of the construct be amidated. Amidation of the carboxyl group of an amino acid residue (typically the C-terminal amino acid residue of a peptide chain) can improve the structural stability (e.g., protease resistance) of the construct in the cytoplasm or nucleus. Furthermore, amidation of the carboxyl group improves the hydrophilicity of the construct, thereby improving the solubility of the construct in aqueous solvents. Examples of such aqueous solvents include water, various buffer solutions, physiological saline (e.g., PBS), cell culture medium, etc. For example, in the case of a construct in which a foreign substance is bound to the N-terminus of a peptide fragment, it is preferable that the carboxyl group of the C-terminal amino acid residue of the peptide fragment be amidated. Furthermore, for example, when the foreign substance is a polypeptide and the polypeptide is bound to the C-terminus of the peptide fragment, it is preferable that the carboxyl group of the C-terminal amino acid residue of the polypeptide be amidated.

[0037] The peptide portion of the peptide fragment or construct (the polypeptide as the foreign substance, the peptide fragment, and the peptidic linker) can be easily produced, for example, according to a general chemical synthesis method. For example, either a conventionally known solid-phase synthesis method or a liquid-phase synthesis method may be employed. A solid-phase synthesis method using Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as the amino group protecting group is preferred. That is, the peptide portion having the desired amino acid sequence and modified portions (N-terminal acetylation, C-terminal amidation, etc.) can be synthesized by solid-phase synthesis using a commercially available peptide synthesizer. Note that only a portion of the peptide chain may be synthesized by the above method; for example, only the peptide fragment, or a peptide chain containing the peptide fragment and the peptidic linker portion may be synthesized.

[0038] Alternatively, peptide fragments or peptide portions of constructs can be produced biosynthetically using genetic engineering techniques. Specifically, a polynucleotide (typically DNA) encoding a desired amino acid sequence (including an ATG start codon) is synthesized. A recombinant vector carrying an expression gene construct consisting of the synthesized polynucleotide (DNA) and various regulatory elements (including promoters, ribosome binding sites, terminators, enhancers, and various cis-elements controlling expression levels) for expressing the amino acid sequence in host cells is then constructed for the host cell. This recombinant vector is then introduced into a desired host cell (e.g., yeast, insect cells, or plant cells) using standard techniques, and the host cell or a tissue or individual containing the cell is cultured under specified conditions. This allows the desired peptide to be produced intracellularly. The peptide portion is then isolated from the host cell (or from the culture medium if secreted) and, if necessary, refolded, purified, or otherwise processed to obtain the desired peptide portion. The method for constructing a recombinant vector and the method for introducing the constructed recombinant vector into a host cell may be any method conventionally used in the relevant field, and such methods themselves do not particularly characterize the present technology, so detailed explanations thereof will be omitted.

[0039] Furthermore, to produce the peptide portion of the construct, for example, a fusion protein expression system can be used to efficiently mass-produce the polypeptide in host cells. For example, a gene (DNA) encoding the amino acid sequence of the polypeptide of interest is chemically synthesized, and the synthetic gene is introduced into a suitable site of a suitable fusion protein expression vector (e.g., a GST (Glutathione S-transferase) fusion protein expression vector such as the pET series from Novagen and the pGEX series from Amersham Biosciences). Host cells (typically Escherichia coli) are then transformed with the vector. The resulting transformant is cultured to prepare the fusion protein of interest. The protein is then extracted and purified. The purified fusion protein is then cleaved with a specific enzyme (protease), and the released peptide fragment of interest (i.e., the designed artificial polypeptide) is recovered by affinity chromatography or other methods. The polypeptide of interest (artificial polypeptide) can be produced using such a conventional fusion protein expression system (e.g., the GST / His system from Amersham Biosciences). 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 necessary for synthesizing the peptide portion (ATP, RNA polymerase, amino acids, etc.) can be used to synthesize the desired peptide portion in vitro using a so-called cell-free protein synthesis system. Cell-free protein synthesis systems are described, for example, in the papers by Shimizu et al. (Shimizu et al., Nature Biotechnology, 19, 751-755 (2001)) and Madin et al. (Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559-564 (2000)). Based on the techniques described in these papers, many companies were already contracted to produce polypeptides at the time of filing the present application, and cell-free protein synthesis kits (available, for example, from CellFree Science Co., Ltd. in Japan) were commercially available.

[0040] Single-stranded or double-stranded polynucleotides containing a nucleotide sequence encoding the peptide portion of a construct and / or a nucleotide sequence complementary to said sequence can be easily produced (synthesized) by conventional methods. For example, by selecting codons corresponding to the amino acid residues constituting the peptide portion, the nucleotide sequence corresponding to the amino acid sequence can be easily determined and provided. Once the nucleotide sequence is determined, a polynucleotide (single-stranded) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like. Furthermore, the obtained single-stranded DNA can be used as a template to obtain the desired double-stranded DNA using various enzymatic synthesis methods (typically PCR). Furthermore, the polynucleotide may be in the form of DNA or RNA (e.g., mRNA). DNA may be provided as double-stranded or single-stranded. When provided as a single-stranded DNA, it may be the coding strand (sense strand) or the non-coding strand (antisense strand) of a complementary sequence. The polynucleotide thus obtained can be used as a material for constructing recombinant genes (expression cassettes) for peptide production in various host cells or cell-free protein synthesis systems, as described above. Nucleic acids as foreign substances can also be prepared in the same manner as in the above-mentioned production method.

[0041] The constructs disclosed herein can be suitably used as active ingredients in compositions for applications based on the function of the foreign substance. The constructs may be in the form of a salt, as long as the function of the foreign substance is not lost. For example, acid addition salts obtainable by addition reaction of commonly used inorganic or organic acids according to conventional methods can be used. Therefore, the "construct" described in this specification and claims can encompass such salt forms.

[0042] The construct can be used as an active ingredient in a composition that may contain various pharmaceutically acceptable carriers depending on the form of use. Preferred carriers include those commonly used in peptide drugs as diluents, excipients, etc. While such carriers may vary depending on the use and form of the construct for foreign substance introduction, typical examples include water, physiological buffer solutions, and various organic solvents. Furthermore, such carriers may be aqueous solutions of alcohol (e.g., ethanol) at an appropriate concentration, glycerol, non-drying oils such as olive oil, or liposomes. Secondary components that may be contained in pharmaceutical compositions include various fillers, extenders, binders, humectants, surfactants, dyes, fragrances, etc.

[0043] The form of the composition is not particularly limited. Examples include solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, and ointments. Furthermore, for use in injections, etc., the composition can be made into a lyophilized or granulated product that is dissolved in physiological saline or an appropriate buffer solution (e.g., PBS) immediately before use to prepare a medicinal solution. The process of preparing various forms of drugs (compositions) using a construct (main component) and various carriers (secondary components) can be performed according to conventionally known methods. Since the formulation method itself does not characterize the present technology, a detailed description thereof will be omitted. A detailed source of information regarding formulations is, for example, Comprehensive Medicinal Chemistry, edited by Corwin Hansch, published by Pergamon Press (1990).

[0044] In some embodiments, a composition comprises a peptide fragment disclosed herein and a foreign substance that is not bound to the N- or C-terminus of the peptide fragment. In such embodiments, the peptide fragment may form a complex with the foreign substance, for example, through electrical or molecular interaction. Such a complex is more easily introduced into eukaryotic cells, thereby enabling efficient introduction of the foreign substance into eukaryotic cells. In a preferred embodiment, the foreign substance contained in such a composition is a negatively charged substance (e.g., a nucleic acid). The peptide fragment disclosed herein has a high proportion of basic amino acids and is positively charged, so it easily forms a complex with a negatively charged foreign substance, etc. The ratio of the peptide fragment to the foreign substance contained in the composition is not particularly limited, but may be, for example, 5 to 100 times the molar ratio of the peptide fragment to the foreign substance, and preferably 40 to 60 times the molar ratio.

[0045] The present disclosure provides a method for introducing a foreign substance of interest from the outside of a eukaryotic cell into the inside of the cell in vivo or ex vivo (in vitro) using the construct disclosed herein. Here, "inside a eukaryotic cell" refers to the inside of a eukaryotic cell surrounded by a cell membrane, and includes, for example, the cytoplasm, cytosol, and intracellular organelles (e.g., nucleus, mitochondria, endoplasmic reticulum, microtubules, lysosomes, Golgi, etc.). In the method disclosed herein, for example, a construct can be introduced into at least the cytoplasm (and even intracellular organelles) of a eukaryotic cell.

[0046] The method disclosed herein broadly comprises the following steps (1) and (2): (1) preparing the construct disclosed herein, and (2) supplying the construct into a sample containing a target eukaryotic cell. The method disclosed herein may further comprise, after step (2), step (3) of incubating the sample to which the construct has been supplied, thereby introducing the construct into the eukaryotic cell in the sample.

[0047] The "eukaryotic cells" mentioned above include, for example, various tissues, organs, blood, and lymph in vivo. The "eukaryotic cells" mentioned above include, for example, various cell masses, tissues, organs, blood, and lymph, as well as cell lines, extracted from living organisms. Examples of eukaryotic cells include cells derived from the animal kingdom, such as mammals, birds, fish, amphibians, reptiles, and insects, cells derived from the fungi kingdom, and cells derived from the plant kingdom, but preferably they are cells of human or non-human mammals (mammalian cells).

[0048] The method disclosed herein involves, for example, preparing a composition containing the construct disclosed herein and delivering it to a sample containing eukaryotic cells. In vivo, such compositions can be used in a manner and dosage appropriate for their form and purpose. For example, as a liquid formulation, a desired amount can be administered to an affected area (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of a patient (i.e., living organism) by intravenous, intramuscular, subcutaneous, intradermal, or intraperitoneal injection. Alternatively, a solid form such as a tablet, or a gel or aqueous jelly such as an ointment, can be administered directly to a specific tissue (e.g., an affected area, such as a tissue or organ containing tumor cells, virus-infected cells, inflammatory cells, etc.). Alternatively, a solid form such as a tablet can be administered orally. For oral administration, encapsulation or application of a protective (coating) material is preferred to prevent degradation by digestive enzymes in the digestive tract.

[0049] In vitro, an appropriate amount of the construct or composition may be added to the culture medium of the eukaryotic cells of interest at least once. The amount and frequency of addition per addition are not particularly limited, as they may vary depending on the type of eukaryotic cells being cultured, cell density (cell density at the start of culture), number of passages, culture conditions, type of medium, and other conditions. For example, it is preferable to add the construct once, twice, or more times so that the peptide fragment concentration in the culture medium is approximately in the range of 0.05 μM to 100 μM, e.g., 0.5 μM to 50 μM, or e.g., 1 μM to 30 μM. Furthermore, the incubation time after addition of the construct is also not particularly limited, as it may vary depending on the type of eukaryotic cells and various conditions. For example, it may be 0.5 hours or more, 1 hour or more, 4 hours or more, 8 hours or more, or 20 hours or more. The incubation conditions may also vary depending on the type of eukaryotic cells, and are not particularly limited, but may be, for example, 5% CO ２ The mixture can be incubated at 37° C. in an ambient atmosphere. An example of an in vitro introduction method is shown in the test example below.

[0050] As described above, specific aspects of the technology disclosed herein include those described in the following items. Item 1: A peptide fragment consisting of any of the following amino acid sequences: YLIQFKKRTLRKSNRKKR (SEQ ID NO: 1); FQILYKKRTLRKSNRKKR (SEQ ID NO: 2); YQFLIKKRTLRKSNRKKR (SEQ ID NO: 3); ILFQYKKRTLRKSNRKKR (SEQ ID NO: 4). Item 2: A construct comprising a peptide fragment and a foreign substance bound to the N-terminus and / or C-terminus of the peptide fragment, wherein the peptide fragment consists of any of the following amino acid sequences: YLIQFKKRTLRKSNRKKR (SEQ ID NO: 1); FQILYKKRTLRKSNRKKR (SEQ ID NO: 2); YQFLIKKRTLRKSNRKKR (SEQ ID NO: 3); ILFQYKKRTLRKSNRKKR (SEQ ID NO: 4). Item 3: The construct according to Item 2, wherein the foreign substance is at least one organic compound selected from the group consisting of a polypeptide, a nucleic acid, a dye, and a drug. Item 4: The construct according to Item 2 or 3, wherein the foreign substance is bound to the C-terminus of the peptide fragment. Item 5: A method for introducing a foreign substance of interest into a eukaryotic cell in vitro from the outside of the cell, the method comprising the steps of: (1) preparing the construct according to any one of claims 2 to 4; and (2) supplying the construct into a sample containing the eukaryotic cell of interest. Item 6: The method according to Item 5, wherein the eukaryotic cell into which the construct is introduced is a mammalian cell.

[0051] Below, several test examples relating to the technology disclosed herein will be described, but it is not intended that the technology disclosed herein be limited to those shown in these test examples.

[0052] <Preparation of constructs> In Examples 1 to 5, the constructs shown in Table 1 were prepared. As shown in Table 1, in each example, a peptide fragment (synthetic peptide) consisting of any one of SEQ ID NOs: 1 to 5 and FAM (C ２１ H １２ O ７A construct consisting of 5(6)-carboxyfluorescein (molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm) was prepared. In each example, the amino group of the N-terminal amino acid residue of the peptide fragment was acetylated. Each construct was dissolved in dimethyl sulfoxide (DMSO) to prepare a sample solution with a 2 mM construct concentration. A 2 mM FAM solution was also prepared by dissolving the fluorescent dye FAM in DMSO. For the sake of explanation, the construct used in Example X will be referred to as Construct X, and the sample solution as Sample Solution X.

[0053] <Evaluation of Cell Membrane Permeability> NSC-34 cells (mouse motor neuron-like hybrid cell line) were used as eukaryotic cells to evaluate the cell membrane permeability of the constructs prepared above.

[0054] (Example 1) NSC-34 cells were cultured in a culture medium, DMEM (Dulbecco's modified Eagle's medium (Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 044-29765)) containing 10% FBS (fetal bovine serum). After washing the NSC-34 cells adhered to the culture plate with PBS, a 0.25% trypsin / EDTA solution was added and the cells were incubated at 37°C for 3 minutes. After this incubation, the above-mentioned 10% FBS-containing DMEM was added to inactivate the trypsin, and the cells were precipitated by centrifugation at 150 × g for 5 minutes. After removing the supernatant resulting from centrifugation, the above-mentioned 10% FBS-containing DMEM was added to the precipitate (cell pellet), and approximately 2 × 10 ５ A cell suspension of 100 cells / mL was prepared. 1 mL of the cell suspension was added to each well of a commercially available 6-well plate (AGC Technoglass Co., Ltd.), and the cells were seeded (approximately 2 × 10 ５The cells were incubated for 2-3 hours until they adhered to the bottom of the wells. The 2 mM sample solution 1 prepared above was then diluted with the 10% FBS-containing DMEM to prepare a sample solution 1 with a sample concentration of 20 μM. Then, 1 mL of the 20 μM sample solution 1 was added to the well (i.e., the concentration of construct 1 in the culture solution in the well was adjusted to 10 μM, and the DMSO concentration was adjusted to 0.5%). The cells were incubated in a 5% CO atmosphere. ２ The incubation was carried out under the conditions at 37°C for 20 hours.

[0055] After 20 hours of incubation, the culture supernatant was removed from the wells, and the cells in the wells were washed twice with 1 mL of PBS. Next, 100 μL of 0.25% trypsin / EDTA solution was added to the wells and incubated at 37°C for 3 minutes. After this incubation, 900 μL of the above-mentioned 10% FBS-containing DMEM was added to the wells to inactivate the trypsin, and the cell suspension in the wells was transferred to a tube to recover the cells. After further washing, the remaining cells in the wells were recovered into the tube. This tube was centrifuged at 4°C and 210 × g for 5 minutes. After centrifugation, the supernatant was removed, and the precipitate (cell pellet) was suspended (washed) in 1 mL of PBS and centrifuged under the same conditions as above. The supernatant was then removed, and cells (cell pellet) cultured in the construct-containing medium were obtained.

[0056] The obtained cells (cell pellet) were analyzed for cell membrane permeability using a flow cytometer. The flow cytometer used was an On-Chip Flowcytometer (manufactured by On-Chip Biotechnologies Co., Ltd.). For this analysis, the obtained cell pellet was suspended in 50 μL of On-Chip T buffer to prepare a cell suspension for analysis.

[0057] Using the above flow cytometer, gating based on forward scatter (FSC) and side scatter (SSC) was performed, a gate was set for the cell population to be analyzed, and the fluorescence intensity of the cell population within the gate was measured. The number of cells in the cell population to be analyzed was set to approximately 10,000, and the analysis was performed. To measure the fluorescence intensity, the fluorescence detector FL2 (optimal detection wavelength: around 543 nm) of the above flow cytometer, which can detect the fluorescence wavelength of FAM, was used. The measurement results were analyzed using commercially available analysis software "FlowJo" (manufactured by TreeStar), and the mean fluorescence intensity (MFI) of the cell population to be measured was obtained.

[0058] Examples 2 to 5 Examples 2 to 5 were carried out in the same manner as in Example 1, except that sample solution 1 was replaced with one of sample solutions 2 to 5.

[0059] Example 6 Example 6 was carried out in the same manner as Example 1, except that a FAM solution was used instead of sample solution 1. The concentration of the FAM solution was the same as that of 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%).

[0060] The MFI results obtained in Examples 1-6 are shown in Table 1 and FIG.

[0061]

[0062] As shown in Table 1 and Figure 1, Examples 1 to 4 had higher MFI values ​​than Example 5. This shows that the peptide fragments consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 4 exhibit superior cell membrane permeability to the peptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 5.

[0063] Although detailed data are not shown, the inventors' studies have confirmed that constructs containing a peptide fragment consisting of any of the amino acid sequences shown in SEQ ID NOs: 1 to 4 and a polypeptide, nucleic acid, or drug as a foreign substance can be efficiently introduced into eukaryotic cells from the outside of the cells.

[0064] While specific examples of the technology disclosed herein have been described in detail above, these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and alterations of the specific examples exemplified above.

[0065] The present disclosure provides peptide fragments and constructs containing the peptide fragments that can introduce a foreign substance of interest into a eukaryotic cell (particularly various animal cells, such as those of humans and other mammals, that do not have a cell wall) from the outside to the inside of the cell. This technology allows the foreign substance of interest to be effectively introduced into a target cell, thereby enabling the production of cells and biological tissues, such as organs, into which the foreign substance has been introduced. Furthermore, the peptide fragments or constructs disclosed herein can be used in drug delivery technology to provide therapeutic agents for various diseases (e.g., nucleic acid pharmaceuticals, therapeutic agents for ophthalmic treatment, etc.). Furthermore, the peptide fragments or constructs disclosed herein can be used as active ingredients or additives in topical medications (e.g., ointments, eye drops, etc.).

Claims

1. A peptide fragment consisting of any of the following amino acid sequences: YLIQFKKRTLRKSNRKKR (SEQ ID NO: 1); FQILYKKRTLRKSNRKKR (SEQ ID NO: 2); YQFLIKKRTLRKSNRKKR (SEQ ID NO: 3); ILFQYKKRTLRKSNRKKR (SEQ ID NO: 4).

2. A construct comprising a peptide fragment and a foreign substance bound to the N-terminus and / or C-terminus of the peptide fragment, wherein the peptide fragment has any one of the following amino acid sequences: YLIQFKKRTLRKSNRKKR (SEQ ID NO: 1); FQILYKKRTLRKSNRKKR (SEQ ID NO: 2); YQFLIKKRTLRKSNRKKR (SEQ ID NO: 3); ILFQYKKRTLRKSNRKKR (SEQ ID NO: 4).

3. The construct of claim 2, wherein the exogenous substance is at least one organic compound selected from the group consisting of polypeptides, nucleic acids, dyes and drugs.

4. The construct according to claim 2, wherein the foreign substance is bound to the C-terminus of the peptide fragment.

5. A method for introducing a desired foreign substance into a eukaryotic cell in vitro from the outside of the cell, comprising the steps of: (1) preparing a construct according to any one of claims 2 to 4; and (2) supplying the construct into a sample containing the desired eukaryotic cell.

6. The method of claim 5, wherein the eukaryotic cell into which the construct is introduced is a mammalian cell.

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