Peptide fragment and use thereof

Abstract

Provided is a novel peptide fragment that is cell membrane-permeable. The peptide fragment disclosed herein includes any amino acid sequence among KKRTLRKSARKKR (SEQ ID NO:1) , KKRTLRKSASARKKR (SEQ ID NO:2) , KKRTLRKSNSNRKKR (SEQ ID NO:3) , KKRTLRKSASNRKKR (SEQ ID NO:4) , KKRTLRKSNSARKKR (SEQ ID NO:5) , KKRTLRKSNSASARKKR (SEQ ID NO:6) , KKRTLRKSASASNRKKR (SEQ ID NO:7) , KKRTLRKSNSNSNRKKR (SEQ ID NO:8), and KKRTLRKSNSNSNSNRKKR (SEQ ID NO:9).

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. 2023-151637, filed on September 19, 2023, the entire contents of which are incorporated herein by reference.

[0002] Conventionally, foreign substances such as polypeptides 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 technique uses, for example, a peptide fragment (cell membrane penetrating peptide, CPP) that has cell membrane permeability and can pass through the cell membrane from the outside of the cell to introduce a foreign substance into the cytoplasm. Japanese Patent Application Publication No. 2022-047613 and International Publication No. 2011 / 013700 disclose an example of a construct for introducing a foreign substance, which comprises a cell membrane permeable peptide and a foreign substance bound to the peptide.

[0004] JP 2022-047613 A International Publication No. 2011 / 013700

[0005] Since cell membrane-permeable peptides can be used in technologies such as drug delivery systems, there is a demand for the development of a variety of cell membrane-permeable peptides.

[0006] Therefore, the present disclosure has been made in view of the above points, and aims to provide peptide fragments and techniques for utilizing peptide fragments.

[0007] The present disclosure provides a peptide fragment (synthetic peptide) having cell membrane permeability. One embodiment of the peptide fragment disclosed herein is a peptide fragment consisting of, from the N-terminus of the peptide, part A, part B, and part C, in which part A is the amino acid sequence shown in SEQ ID NO: 11 (KKRTLRK), part C is the amino acid sequence shown in SEQ ID NO: 12 (RKKR), and part B is located between part A and part C and is an amino acid sequence in which 1 unit, 2 units, 3 units, or 4 units selected from the group shown by SA or SN are arranged (excluding sequences in which part B is only SN).

[0008] The inventors conducted various studies on the construct for introducing foreign substances disclosed in the aforementioned Patent Document 1 in order to more efficiently introduce foreign substances from the outside of eukaryotic cells through the cell membrane into the cytoplasm. For example, they attempted to create a construct with higher cell membrane permeability by substituting various amino acid residues in the amino acid sequence (KKRTLRKSNRKKR) shown in SEQ ID NO: 10. As a result, they found that the amino acid sequence (KKRTLRKSNRKKR) can be divided into part A (SEQ ID NO: 11: KKRTLRK), part B, and part C (SEQ ID NO: 12: RKKR). Furthermore, by substituting the amino acid sequence of part B, which is located between part A and part C, they were able to complete a peptide fragment without impairing cell permeability.

[0009] The substitution of amino acid residues in the part B is not simply a conservative substitution (e.g., a sequence in which a basic amino acid residue is substituted with another basic amino acid residue). As a result of repeated trial and error, the inventors have found that the part B, which contains 1 to 4 SA or SN units, exhibits cell permeability without increasing cytotoxicity.

[0010] A peptide fragment having such a structure has cell membrane permeability, and therefore a construct containing the peptide fragment is introduced from the outside of a cell into the inside of the cell (at least into the cytoplasm).

[0011] In one embodiment of the peptide fragment disclosed herein, the part B is an amino acid sequence in which one, two, or three consecutive units of the amino acid sequence shown in SA are present. Such a peptide fragment can significantly improve cell membrane permeability.

[0012] The peptide fragment in one embodiment disclosed herein consists of any of the following amino acid sequences: KKRTLRKSARKKR (SEQ ID NO: 1); KKRTLRKSASARKKR (SEQ ID NO: 2); KKRTLRKSNSNRKKR (SEQ ID NO: 3); KKRTLRKSASNRKKR (SEQ ID NO: 4); KKRTLRKSNSARKKR (SEQ ID NO: 5); KKRTLRKSNSASARKKR (SEQ ID NO: 6); KKRTLRKSASASNRKKR (SEQ ID NO: 7); KKRTLRKSNSSNSNRKKR (SEQ ID NO: 8); and KKRTLRKSNSSNSSNSNRKKR (SEQ ID NO: 9). Such peptide fragments exhibit cell permeability without increasing cytotoxicity.

[0013] The present disclosure provides a construct for introducing an exogenous substance (hereinafter also referred to simply as a "construct") that can introduce a desired exogenous substance into a eukaryotic cell from the outside of the cell. One embodiment of the construct disclosed herein is a construct comprising a peptide fragment and an exogenous substance bound to the N-terminus and / or C-terminus of the peptide fragment, wherein the peptide fragment consists of, from the N-terminus, an A part, a B part, and a C part, wherein the A part has the amino acid sequence shown in SEQ ID NO: 11 (KKRTLRK), the C part has the amino acid sequence shown in SEQ ID NO: 12 (RKKR), and the B part is located between the A and C parts and is an amino acid sequence comprising one, two, three, or four units selected from the group SA or SN (excluding sequences in which the B part is only SN). Using such a construct, the exogenous substance can be introduced into the eukaryotic cell from the outside of the cell (e.g., at least into the cytoplasm).

[0014] In one embodiment of the construct disclosed herein, the part B is an amino acid sequence in which one, two, or three consecutive units of the amino acid sequence shown in the SA are present, thereby enabling the foreign substance to be introduced more reliably from the outside of a eukaryotic cell into the inside of the cell.

[0015] In one embodiment of the construct disclosed herein, the peptide fragment consists of any of the following amino acid sequences: KKRTLRKSARKKR (SEQ ID NO: 1); KKRTLRKSASARKKR (SEQ ID NO: 2); KKRTLRKSNSNRKKR (SEQ ID NO: 3); KKRTLRKSASNRKKR (SEQ ID NO: 4); KKRTLRKSNSARKKR (SEQ ID NO: 5); KKRTLRRKSNSASARKKR (SEQ ID NO: 6); KKRTLRKSASASNRKKR (SEQ ID NO: 7); KKRTLRRKSNSSNSNRKKR (SEQ ID NO: 8); and KKRTLRKSNSSNSSNSNRKKR (SEQ ID NO: 9). Such a construct introduces the foreign substance from the outside of a eukaryotic cell into the inside of the cell.

[0016] In one embodiment of the construct disclosed herein, the foreign 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 substances with diverse functions into eukaryotic cells.

[0017] In one embodiment of the construct disclosed herein, the foreign substance is located at the C-terminus of the peptide fragment, allowing the construct to be efficiently introduced into eukaryotic cells.

[0018] The present disclosure 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. One aspect of 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.

[0019] In one embodiment of the method disclosed herein, the eukaryotic cell into which the construct is introduced is a mammalian cell, thereby enabling the introduction of a desired foreign substance into the mammalian cell.

[0020] 1 is a graph showing the mean fluorescence intensity (MFI) for each example obtained by adding the constructs of Examples 1 to 3 and Comparative Examples 1 and 2 or FAM to a culture medium of NSC-34 cells, culturing the cells, and then analyzing the cells with a flow cytometer. 2 is a graph showing the mean fluorescence intensity (MFI) for each example obtained by adding the constructs of Examples 4 to 9 and Comparative Example 3 or FAM to a culture medium of NSC-34 cells, culturing the cells, and then analyzing the cells with a flow cytometer.

[0021] <Definitions> 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, biochemical genetic engineering, protein engineering, molecular biology, genetics, etc. Furthermore, the patented technology disclosed herein can be implemented based on the contents 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 acid residues 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.

[0022] 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.

[0023] In this specification, amino acid residues constituting peptides or proteins may be in either the L- or D-configuration unless otherwise specified. That is, the amino acid residues constituting the amino acid sequences described in the claims include both the L- and D-configurations. 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.

[0024] <Peptide Fragment> The peptide fragment disclosed herein is a peptide fragment consisting of part A, part B, and part C from the N-terminus of the peptide, in which part A has the amino acid sequence shown in SEQ ID NO: 11 (KKRTLRK), part C has the amino acid sequence shown in SEQ ID NO: 12 (RKKR), and part B is a synthetic peptide located between parts A and C and containing an amino acid sequence in which 1 unit, 2 units, 3 units, or 4 units from the group shown by SA or SN are arranged (however, sequences in which part B is only SN are excluded).

[0025] The peptide fragment disclosed herein can be divided from the N-terminus into parts A, B, and C. Part B is composed of an amino acid sequence in which any number of 1 to 4 SA units or SN units are arranged in any combination.

[0026] A preferred embodiment of the peptide fragment disclosed herein is a synthetic peptide comprising KKRTLRKSARKKR (SEQ ID NO: 1), KKRTLRKSASARKKR (SEQ ID NO: 2), KKRTLRKSNSNRKKR (SEQ ID NO: 3), KKRTLRKSASNRKKR (SEQ ID NO: 4), KKRTLRKSNSARKKR (SEQ ID NO: 5), KKRTLRRKSNSASARKKR (SEQ ID NO: 6), KKRTLRKSASASNRKKR (SEQ ID NO: 7), KKRTLRRKSNSSNSNRKKR (SEQ ID NO: 8), or KKRTLRKSNSSNSSNSNRKKR (SEQ ID NO: 9). In one aspect, the peptide fragment disclosed herein consists of any of the amino acid sequences set forth in SEQ ID NOs: 1 to 9. Peptide fragments of the amino acid sequences shown in SEQ ID NOs: 1 to 9 have cell membrane permeability and are efficiently introduced from the outside to the inside of the cell membrane of eukaryotic cells. Therefore, constructs containing the peptide fragments disclosed herein can be efficiently introduced from the outside to the inside of the cell membrane of eukaryotic cells.

[0027] The number of amino acid residues of the peptide fragments disclosed herein is not limited as long as cell membrane permeability is not impaired. The number of amino acid residues of the peptide fragments may be, for example, 19 or less, 17 or less, 15 or less, or 13 or less.

[0028] In the peptide fragments disclosed herein, the amino acid sequences set forth in Part A of SEQ ID NO: 11 (KKRTLRK) and Part C of SEQ ID NO: 12 (RKKR), excluding Part B, are typically identical. However, in addition to the identical sequences, modified sequences may also be included as long as cell membrane permeability is not impaired. Here, a "modified sequence" refers to an amino acid sequence (modified amino acid sequence) formed by the substitution, deletion, and / or addition (insertion) of one or several (e.g., two) amino acid residues. In other words, such minor modified sequences can be easily utilized based on the technical information disclosed herein and are encompassed by the peptide fragment as the technical concept disclosed herein. Typical examples of modified sequences herein include sequences resulting from conservative substitution of one or two amino acid residues (so-called conservative amino acid replacement), and sequences in which one, two, or three amino acid residues are added (inserted) or deleted from a given amino acid sequence. Typical examples of conservative substitutions include sequences in which a basic amino acid residue is substituted for another basic amino acid residue (eg, mutual substitution of a lysine residue with an arginine residue).

[0029] In the peptide fragments disclosed herein, the B part is composed of an amino acid sequence in which 1 to 4 SA units or SN units are arranged. For example, in the amino acid sequence shown in SEQ ID NO: 1, the B part consists of 1 SA unit. In the amino acid sequence shown in SEQ ID NO: 2, the B part consists of 2 SA units. In the amino acid sequence shown in SEQ ID NO: 3, the B part consists of 2 SN units. In the amino acid sequence shown in SEQ ID NO: 4, the B part consists of 1 SA unit and 1 SN unit from the N-terminus. In the amino acid sequence shown in SEQ ID NO: 5, the B part consists of 1 SN unit and 1 SA unit from the N-terminus. In the amino acid sequence shown in SEQ ID NO: 6, the B part consists of 1 SN unit and 2 SA units from the N-terminus. In the amino acid sequence shown in SEQ ID NO: 7, the B part consists of 2 SA units and 1 SN unit from the N-terminus. In the amino acid sequence shown in SEQ ID NO: 8, the B part consists of 3 SN units. In the amino acid sequence shown in SEQ ID NO: 9, the B part consists of 4 SN units.

[0030] 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.

[0031] <Linker> In some embodiments, a construct is designed and constructed by directly or indirectly binding (linking) a foreign substance to the N-terminal and / or C-terminal sides of a 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 does not need to be particularly limited. Furthermore, 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.

[0032] The peptidic linker may be constructed from 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.

[0033] 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.

[0034] Suitable cross-linking agents having homobifunctional groups include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS ３), dithiobis(succinimidyl propionate) (DSP), dithiobis(succinimidyl 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.

[0035] Preferred examples of 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.

[0036] <Foreign Substances> 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.

[0037] 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 that have 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.

[0038] 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).

[0039] 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 having various physiological activities involved in inducing the differentiation 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 inducing apoptosis of the 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 inducing apoptosis of the infected cells, polypeptides capable of suppressing cell or viral proliferation in the infected cells, or polypeptides capable of suppressing the spread of bacterial or viral infection from the infected cells. Note that, like peptide fragments, the polypeptides used as foreign substances may contain modified amino acid sequences formed by substitution, deletion, and / or addition (insertion) of one or several amino acid residues, so long as they retain their function.

[0040] 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.

[0041] The type of nucleic acid employed as a 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 a 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 includes, for example, siRNA, miRNA, antisense, aptamer, decoy, ribozyme, CpG oligo, etc.

[0042] 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.

[0043] 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).

[0044] <Construct> In the case where a foreign substance is bound to the C-terminus of a peptide fragment in a construct, it is preferable that the α-amino group of the amino acid residue on the N-terminus of the peptide fragment is acetylated. Although the detailed mechanism is unknown, the α-amino group of the amino acid on the N-terminus of many proteins in eukaryotic cells is acetylated, and therefore, such a configuration can improve the stability of the construct within the cell.

[0045] The construct preferably has an amidated amino acid residue at the C-terminus. Amidating 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, amidating 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 buffers, 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 amino acid residue at the C-terminus of the peptide fragment is amidated. 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 amino acid residue at the C-terminus of the peptide fragment is amidated. Furthermore, 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 is amidated.

[0046] 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-fluorenylmerhoxycarbonyl) as an amino-protecting group is preferred. That is, the peptide portion having the desired amino acid sequence and modifications (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 including the peptide fragment and the peptidic linker may be synthesized.

[0047] The peptide portion of a peptide fragment or construct may be produced by biosynthesis 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 containing 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 that control 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 specific host cell (e.g., yeast, insect cells, or plant cells) using standard techniques, and the host cells or tissues or solids containing the cells are cultured under specified conditions. This allows the intracellular production of the desired peptide. The peptide portion is then isolated from the host cells (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 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.

[0048] To produce the peptide portion of the construct, for example, a fusion protein expression system can be used to efficiently mass-produce it within a host cell. For example, a gene (DNA) encoding the amino acid sequence of the desired polypeptide is chemically synthesized, and the synthetic gene is introduced into a suitable site of an appropriate 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 desired fusion protein. The protein is then extracted and purified. The resulting purified fusion protein is then cleaved with a specific enzyme (protease), and the released peptide fragment of interest (i.e., the designed artificial peptide) is recovered by affinity chromatography or other methods. By using such a conventionally known fusion protein expression system (for example, the GST / His system provided by Masham Biosciences) the desired construct (artificial polypeptide) can be produced.

[0049] 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) is constructed, and various compounds necessary for synthesizing the peptide portion (ATP, RNA polymerase, amino acids, etc.) are used to employ a so-called cell-free protein synthesis system to synthesize the desired peptide portion in vitro. Regarding cell-free protein synthesis systems, for example, Shimizu et al.'s paper (Shimizu et al., Nature Biotechnology, 19, 751-755 (2001)) and Madin et al.'s paper (Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559-564 (2000)) are useful references. Based on the techniques described in these papers, at the time of filing this application, many companies were already undertaking contract production of polypeptides, and kits for cell-free protein synthesis (available, for example, from Cell-Free Sciences Inc. in Japan) were commercially available.

[0050] 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.

[0051] 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.

[0052] 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 medicines 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, physiologically acceptable 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.

[0053] <Composition> 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 product 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) as materials may be in accordance with conventionally known methods. Since such formulation methods do not characterize the present technology, detailed explanations are omitted. For example, a detailed source of information regarding formulations is "Comprehensive Medicinal Chemistry," edited by Corwin Hansch, published by Pergamon Press (1990).

[0054] 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 may be easily introduced into eukaryotic cells, thereby enabling efficient introduction of the foreign substance into eukaryotic organisms. In a preferred embodiment, the foreign substance contained in such a composition is a negatively charged substance (e.g., nucleic acid). Since the peptide fragment disclosed herein has a high proportion of basic amino acids and is positively charged, the ratio of the foreign substance to the peptide fragment may be limited. For example, the molar ratio of the peptide fragment to the foreign substance may be 5 to 100 times, preferably 40 to 60 times.

[0055] <Use of Construct> 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 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., mitochondria, endoplasmic reticulum, microtubules, lysosomes, Golgi, etc.). In the method disclosed herein, for example, the construct can be introduced into at least the cytoplasm (and even intracellular organelles) of a eukaryotic cell.

[0056] 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.

[0057] The "eukaryotic cells" in vivo include, for example, various tissues, organs, blood, and lymph. In vitro, the "eukaryotic cells" 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).

[0058] 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.

[0059] 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. 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 cells can be incubated at 37° C. in an ambient atmosphere. An example of an in vitro introduction method is also shown in the test example described below.

[0060] 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.

[0061] <Preparation of Constructs> The constructs shown in Table 1 of this example were prepared. In Example 1, a peptide fragment (synthetic peptide) consisting of the amino acid sequence shown in SEQ ID NO: 1, in which the amino group of the N-terminal lysine residue is acetylated, and a fluorescent dye, FAM (C ２１ H １２ O ７Construct 1 was prepared, which consisted of 5(6)-carboxyfluorescein (molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm). Construct 1 was diluted with dimethyl sulfoxide (DMSO) to prepare sample solution 1 with a concentration of 2 mM. Similarly to Example 1, in Examples 2 to 9 and Comparative Example 1, constructs 2 to 10 were prepared, each consisting of a synthetic peptide with the amino acid sequence shown in SEQ ID NOs: 2 to 10, in which the amino group of the N-terminal lysine residue was acetylated, and the fluorescent dye FAM attached to the C-terminus of the synthetic peptide. Constructs 2 to 10 were diluted with DMSO to prepare sample solutions 2 to 10 with a concentration of 2 mM. A 2 mM FAM solution was also prepared by dissolving the fluorescent dye FAM in DMSO.

[0062] <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.

[0063] 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 then centrifuged at 150 × g for 5 minutes to precipitate the cells. After removing the supernatant resulting from the 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 approximately 2 to 3 hours until the cells 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 5% CO ２ The incubation was carried out under the conditions at 37°C for 20 hours.

[0064] 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. 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 sample-containing medium were obtained.

[0065] 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.

[0066] 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 10,000 or more, and analysis was performed. Fluorescence intensity was measured using the fluorescence detector FL2 (optimal detection wavelength around 543 nm) of the above flow cytometer, which can detect the fluorescence wavelength of FAM. The measurement results were analyzed using commercially available analysis software "FlowJo" (manufactured by TreeStar), and the median fluorescence intensity (Mean Fluorescence Intensity: MFI) of the cell population to be measured was obtained (see Table 1).

[0067] Examples 2 to 8, Comparative Example 1 The same procedures as in Example 1 were carried out except that sample solutions 2 to 10 were used instead of sample solution 1.

[0068] Comparative Examples 2 and 3 were carried out in the same manner as in 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%).

[0069] The results obtained in Examples 1 to 9 and Comparative Examples 1 to 3 are shown in Tables 1 and 2 and Figures 1 and 2. Figures 1 and 2 show histograms measured using the flow cytometer. The vertical axis of Figures 1 and 2 represents the number of cells, and the horizontal axis represents the fluorescence intensity. Tables 1 and 2 show tests conducted independently on different days.

[0070]

[0071]

[0072] As shown in Tables 1 and 2 and Figures 1 and 2, Examples 1 to 9 showed MFIs equal to or higher than that of Comparative Example 1. Furthermore, Examples 1 to 9 had higher MFIs than Comparative Examples 2 and 3. This indicates that a greater amount of FAM was introduced into cells by the peptide fragments comprising the amino acid sequences shown in SEQ ID NOs: 1 to 9. This indicates that the peptide fragments comprising the amino acid sequences shown in SEQ ID NOs: 1 to 9 exhibit cell membrane permeability and are capable of introducing foreign substances from the outside to the inside of cells.

[0073] Although detailed data are not shown, the inventors' studies have shown that peptide fragments consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 9 have the same or lower cytotoxicity as the existing peptide fragment (SEQ ID NO: 10).

[0074] Although detailed data are not shown, the inventors' studies have confirmed that constructs containing peptide fragments consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 9 and foreign substances such as polypeptides, nucleic acids, or drugs can be efficiently introduced into eukaryotic cells from the outside of the cells.

[0075] 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.

[0076] In the technology disclosed herein, each component and each process mentioned herein may be omitted or combined as appropriate, unless a particular problem arises. This specification also includes the disclosures described in the following sections.

[0077] Item 1: A peptide fragment consisting of an A part, a B part, and a C part from the N-terminal side of the peptide, wherein the A part is the amino acid sequence shown in SEQ ID NO: 11 (KKRTLRK), the C part is the amino acid sequence shown in SEQ ID NO: 12 (RKKR), and the B part is located between the A part and the C part and is an amino acid sequence in which 1 unit, 2 units, 3 units, or 4 units from the group shown by SA or SN are arranged (however, a sequence in which the B part is only SN is excluded).

[0078] Item 2: The peptide fragment according to Item 1, wherein the part B is an amino acid sequence in which the amino acid sequence shown in SA is one unit, two units, or three units consecutively.

[0079] Item 3: The peptide fragment according to Item 1, wherein the peptide fragment consists of any of the following amino acid sequences: KKRTLRKSARKKR (SEQ ID NO: 1); KKRTLRKSASARKKR (SEQ ID NO: 2); KKRTLRKSASNRKKR (SEQ ID NO: 3); KKRTLRKSNSARKKR (SEQ ID NO: 4); KKRTLRKSNSASARKKR (SEQ ID NO: 5); KKRTLRKSASASNRKKR (SEQ ID NO: 6); KKRTLRKSNSNRKKR (SEQ ID NO: 3); KKRTLRKSNSSNSNRKKR (SEQ ID NO: 8); and KKRTLRKSNSSNSSNSNRKKR (SEQ ID NO: 9).

[0080] Item 4: A construct comprising a peptide fragment and a foreign substance bound to the N-terminal side and / or C-terminal side of the peptide fragment, wherein the peptide fragment consists of, from the N-terminal side, an A part, a B part, and a C part, wherein the A part is the amino acid sequence shown in SEQ ID NO: 11 (KKRTLRK), the C part is the amino acid sequence shown in SEQ ID NO: 12 (RKKR), and the B part is located between the A part and the C part and is an amino acid sequence in which 1 unit, 2 units, 3 units, or 4 units from the group shown by SA or SN are arranged (however, a sequence in which the B part is only SN is excluded).

[0081] Item 5: The construct according to Item 4, wherein the part B is an amino acid sequence in which the amino acid sequence shown in SA is one unit, two units, or three units consecutively.

[0082] Item 6: The construct according to Item 4, wherein the peptide fragment consists of any of the following amino acid sequences: KKRTLRKSARKKR (SEQ ID NO: 1); KKRTLRKSASARKKR (SEQ ID NO: 2); KKRTLRKSASNRKKR (SEQ ID NO: 3); KKRTLRKSNSARKKR (SEQ ID NO: 4); KKRTLRKSNSASARKKR (SEQ ID NO: 5); KKRTLRKSASASNRKKR (SEQ ID NO: 6); KKRTLRKSNSNRKKR (SEQ ID NO: 3); KKRTLRKSNSSNSNRKKR (SEQ ID NO: 8); and KKRTLRKSNSSNSSNSNRKKR (SEQ ID NO: 9).

[0083] Item 7: The construct according to Items 4 to 6, 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.

[0084] Item 8: The construct according to any one of Items 4 to 7, wherein the foreign substance is located on the C-terminal side of the peptide fragment.

[0085] Item 9: 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 a construct according to any one of Items 4 to 8; and (2) supplying the construct into a sample containing the eukaryotic cell of interest.

[0086] Item 10: The method according to Item 9, wherein the eukaryotic cell into which the construct is to be introduced is a mammalian cell.

[0087] The present disclosure provides peptide fragments and constructs comprising 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. By using such constructs, a foreign substance of interest can be effectively introduced into a target cell, and biological tissues, such as cells and organs, into which the foreign substance has been introduced can be obtained. Furthermore, the peptide fragments or constructs disclosed herein can be used in drug delivery technologies to provide therapeutic agents for various diseases (e.g., therapeutic agents for ophthalmology, etc.). Furthermore, the peptide fragments or constructs disclosed herein can be used as active ingredients or additives in topical medications (e.g., eye drops, etc.).

Claims

1. A peptide fragment consisting of A part, B part and C part from the N-terminus of a peptide, wherein the A part is an amino acid sequence shown in SEQ ID NO: 11 (KKRTLRK), the C part is an amino acid sequence shown in SEQ ID NO: 12 (RKKR), and the B part is located between the A part and the C part and is an amino acid sequence consisting of 1 unit, 2 units, 3 units or 4 units selected from the group shown by SA or SN (excluding a sequence in which the B part is only an SN 1 unit).

2. The peptide fragment according to claim 1, wherein the B part is an amino acid sequence in which the amino acid sequence shown in SA is one, two or three consecutive units.

3. The peptide fragment comprising parts A, B and C of claim 1, comprising any one of the following amino acid sequences: KKRTLRKSARKKR (SEQ ID NO: 1); KKRTLRKSASARKKR (SEQ ID NO: 2); KKRTLRKSNSNRKKR (SEQ ID NO: 3); KKRTLRKSASNRKKR (SEQ ID NO: 4); KKRTLRKSNSARKKR (SEQ ID NO: 5); KKRTLRKSNSASARKKR (SEQ ID NO: 6); KKRTLRKSASASNRKKR (SEQ ID NO: 7); KKRTLRKSNSNSNRKKR (SEQ ID NO: 8); and KKRTLRKSNSNSNSNRKKR (SEQ ID NO: 9).

4. 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 A part, B part and C part from the N-terminus, the A part being an amino acid sequence shown in SEQ ID NO: 11 (KKRTLRK), the C part being an amino acid sequence shown in SEQ ID NO: 12 (RKKR), and the B part being located between the A part and the C part and being an amino acid sequence consisting of 1 unit, 2 units, 3 units or 4 units selected from the group shown by SA or SN (excluding a sequence in which the B part is only SN).

5. The construct according to claim 4, wherein the B part is an amino acid sequence in which the amino acid sequence shown in SA is consecutively arranged in one, two or three units.

6. The construct of claim 4, wherein the peptide fragment consisting of parts A, B and C consists of any of the following amino acid sequences: KKRTLRKSARKKR (SEQ ID NO: 1); KKRTLRKSASARKKR (SEQ ID NO: 2); KKRTLRKSNSNRKKR (SEQ ID NO: 3); KKRTLRKSASNRKKR (SEQ ID NO: 4); KKRTLRKSNSARKKR (SEQ ID NO: 5); KKRTLRKSNSASARKKR (SEQ ID NO: 6); KKRTLRKSASASNRKKR (SEQ ID NO: 7); KKRTLRKSNSNSNRKKR (SEQ ID NO: 8); and KKRTLRKSNSNSNSNRKKR (SEQ ID NO: 9).

7. The construct of claim 4, wherein the exogenous substance is at least one organic compound selected from the group consisting of polypeptides, nucleic acids, dyes and drugs.

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

9. A method for introducing a desired foreign substance in vitro from the outside of a eukaryotic cell to the inside of the cell, comprising the steps of: (1) preparing a construct according to any one of claims 4 to 7; and (2) supplying the construct into a sample containing the desired eukaryotic cell.

10. The method of claim 9, wherein the eukaryotic cell into which the construct is introduced is a mammalian cell.

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