Modified peptide having gel degradation activity

Abstract

The present invention addresses the problem of providing a novel method for easily removing gel under physiological conditions without using protease.　Provided is a modified peptide having gel degradation activity and including at least one modified core peptide comprising an amino acid sequence represented by formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (in the formula, Xaa is independently Ile or Met; Yaa is independently Asp, Glu, Lys, or Arg; and Zaa is independently Ala or Gly). A nitrogen atom constituting any one or more peptide bonds constituting the modified core peptide has a substituent.

Description

Modified peptides with gel-degrading activity

[0001] The present invention relates to a modified peptide having gel-degrading activity, a composition for degrading a gel, a kit for preparing and degrading a gel, a method for degrading a gel, and a method for preparing a cell aggregate and / or tissue.

[0002] Organoids are three-dimensional structures formed by the self-organization of cells differentiated from stem cells such as ES cells and iPS cells, or cells derived from living tissue, that have structures and functions similar to those of in vivo organs. To date, organoids resembling various organs, including the brain, kidney, pancreas, and intestine, have been created. In particular, organoids composed of human-derived cells (human organoids) are being developed as organ models for testing the effects of therapeutic drugs in vitro, and as groundbreaking experimental materials for human biology research. Furthermore, they are expected to be used in the field of regenerative medicine as transplant tissues.

[0003] In general, organoids are produced by embedding and culturing tissue stem cells, ES cells, iPS cells, or other pluripotent stem cells in an artificial extracellular matrix (ECM) to support and maintain their three-dimensional structure.

[0004] The artificial extracellular matrix used in organoid production must have various properties, such as cell adhesiveness and mechanical strength to support the organoids three-dimensionally. Conventional organoid production methods mainly use Matrigel derived from mouse sarcoma cells or collagen extracted from animal tissues as gelling agents to create the artificial extracellular matrix.

[0005] However, animal-derived gelling agents are not necessarily uniform in molecular weight distribution or component composition, and there are quality differences between production lots. Furthermore, when introduced into the body, there is an unavoidable risk of allergies or unknown infectious diseases due to trace components or contamination in the extract.

[0006] Furthermore, when organoids are used as organ models or tissues for transplantation, the artificial extracellular matrix (ECM) must be removed after organoid formation. In the case of Matrigel or collagen, the gel is degraded by protease treatment, but protease treatment inevitably damages the surface of the organoids at the same time.

[0007] Therefore, there is a need for a new gelling agent that can be provided with high purity, and a new technique for easily removing the gel under physiological conditions without using proteases.

[0008] WO 2022 / 025209

[0009] An object of the present invention is to provide a new method for easily removing gel under physiological conditions without using proteases.

[0010] Chemically synthesized low-molecular-weight peptide gelators (synthetic low-molecular-weight peptide gelators) are being developed as potential peptide gelators that can form hydrogels and overcome the above-mentioned problems of animal-derived peptide gelators. Synthetic low-molecular-weight peptide gelators are peptides with specific amino acid sequences that can be synthesized with high purity by solution synthesis or solid-phase synthesis. Therefore, they have the advantage of stable quality for each production lot and extremely low contamination.

[0011] In previous research, the present inventors developed a jigsaw-shaped self-assembling peptide (JigSAP), an amphiphilic peptide consisting of alternating hydrophilic and hydrophobic amino acids. This amphiphilic peptide can gel at physiological temperature and pH and has a significantly higher elastic modulus than conventional peptide gelators (WO 2022 / 025209). However, no method for removing JigSAP gels under physiological conditions was previously known.

[0012] To solve the above problems, the present inventors newly created a modified JigSAP in which the hydrogen atom bonded to the nitrogen atom in the peptide bond constituting the peptide chain of JigSAP was replaced with a methyl group. The present inventors found that, unlike unmodified JigSAP, the modified JigSAP does not form a gel by itself, but when contacted with a gel composed of unmodified JigSAP, it exhibits the activity of rapidly degrading the gel under physiological conditions. The present invention is based on the above research results and provides the following:

[0013] (1) A modified peptide having gel-degrading activity, comprising at least one modified core peptide consisting of an amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (Formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), wherein a nitrogen atom constituting one or more peptide bonds constituting the modified core peptide has a substituent. (2) The modified peptide according to (1), wherein the substituent is an optionally substituted hydrocarbon group. (3) The modified peptide according to (2), wherein the hydrocarbon group is selected from the group consisting of a methyl group, an ethyl group, a propyl group, an isopropyl group, and a benzyl group. (4) The modified peptide according to any one of (1) to (3), wherein the nitrogen atom is contained in the seventh amino acid residue from the N-terminus in the amino acid sequence shown in Formula I. (5) The modified peptide according to (1), comprising an Arg residue or an Arg-Arg dipeptide sequence linked to the N-terminus and / or C-terminus of the modified core peptide. (6) The modified peptide according to (1), wherein the C-terminus is amidated and / or the N-terminus is carboxylated. (7) A composition for degrading a gel, comprising as an active ingredient the modified peptide according to any one of (1) to (6). (8) The composition for degrading a gel according to (7), for degrading a gel in vitro or in vivo. (9) A kit for producing and decomposing a gel, comprising: a self-assembling peptide for gel production; and a modified peptide according to any one of (1) to (6) for gel degradation, wherein the self-assembling peptide comprises at least one unmodified core peptide consisting of an amino acid sequence represented by the following formula II: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (Formula II) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), wherein the nitrogen atom constituting the peptide bond in the unmodified core peptide has no substituent. (10) The kit according to (9), wherein the gel is used for culturing organoids.(11) A method for decomposing a gel, comprising: a gel decomposition step of contacting a modified peptide having gel decomposition activity with a gel composed of a self-assembling peptide to decompose the gel, wherein the modified peptide comprises at least one modified core peptide consisting of an amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), wherein a nitrogen atom constituting one or more peptide bonds constituting the modified core peptide has a substituent, and the self-assembling peptide is represented by the following formula II: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula II) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), wherein in the unmodified core peptide sequence, the nitrogen atom constituting the peptide bond has no substituent. (12) The method of (11), further comprising: a mixing step of mixing the self-assembling peptide with water or an aqueous solution; and a gelation step of gelling the mixture obtained after the mixing step by maintaining the mixture at a temperature below the gelation temperature. (13) The method of (12), wherein in the mixing step, one or more anions selected from the group consisting of bicarbonate ions, carbonate ions, citrate ions, tartrate ions, and sulfate ions are further mixed. (14) The method according to any one of (12) to (14), wherein the gelation step is carried out in the presence of isolated cells, cell aggregates, or tissues to form a gel containing the cells, cell aggregates, or tissues therein, and the method further comprises a culture step of culturing the cells, cell aggregates, or tissues in the gel. (15) The gel degradation method according to any one of (11) to (14), wherein Xaa and Zaa in formula I have the same amino acid side chains as Xaa and Zaa at the corresponding positions, respectively, in formula II.(16) The gel degradation method described in (15), wherein all amino acid residues in formula I have the same amino acid side chains as the amino acid residues at the corresponding positions in formula II. (17) A method for producing a cell aggregate and / or tissue, comprising: a mixing step of mixing a self-assembling peptide with water or an aqueous solution; a gelation step of gelling the mixture obtained after the mixing step by maintaining the mixture at a temperature equal to or lower than the gelation temperature in the presence of isolated cells, cell aggregates, and / or tissues to produce a gel containing the cells, cell aggregates, and / or tissues therein; a culture step of culturing the cells, cell aggregates, and / or tissues in the gel; and a gel decomposition step of contacting the gel with a modified peptide having gel decomposition activity to decompose the gel, and removing the gel from the cell aggregates and / or tissues obtained after the culture step to isolate the cell aggregates and / or tissues, wherein the modified peptide is a peptide represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (Formula I) wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly, and a nitrogen atom constituting one or more peptide bonds constituting the modified core peptide has a substituent; and the self-assembling peptide comprises at least one unmodified core peptide having an amino acid sequence represented by the following formula II: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (Formula II) (where Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), and wherein a nitrogen atom constituting a peptide bond in the unmodified core peptide sequence does not have a substituent. This specification includes the disclosure of Japanese Patent Application No. 2023-192489, from which the present application claims priority.

[0014] According to the present invention, a new method for easily removing gel under physiological conditions without using proteases is provided.

[0015] Figure 1A is a schematic diagram showing the gelation and degradation of a self-assembling peptide. Figure 1A is a schematic diagram showing the gelation of a self-assembling peptide, the maintenance of the gel state, and the degradation of a gel based on the modified peptide of the present invention. Figure 1B is a schematic diagram showing the steps involved in isolating cultured tissues or organoids by embedding cell aggregates or tissue fragments in a gel and then removing the gel components. Figure 2A shows the structure and gelation ability of C-JigSAP, an unmodified JigSAP, and Me-C-JigSAP, a modified JigSAP. Figure 2A shows the chemical structure and gel-forming ability of C-JigSAP. Figure 2B shows the chemical structure of Me-C-JigSAP and the results of Me-C-JigSAP not gelling. Figure 2B shows the dissolution of a C-JigSAP peptide gel containing an Me-C-JigSAP dispersion and its subsequent drop into the cap of a microtube. Figure 4A shows the cell adhesiveness of the C-JigSAP peptide gel. Figure 4A shows cells adhered to a chamber slide. Figure 4B shows the number of cells adhered to the chamber slide. Error bars indicate standard error. Figure 6A shows the results of measuring the rheological properties of samples, including C-JigSAP peptide gel. Figure 6B shows three-dimensional retinal culture using C-JigSAP peptide gel. Figure 6A shows a schematic diagram of the preparation and culture of retinal encapsulation gels. Figure 6B shows the structure of a normal retina developed in vivo (left), a retina cultured in C-JigSAP peptide gel (center), and a retina cultured in Matrigel (right). Figure 6B shows the results of adding Me-C-JigSAP to C-JigSAP peptide gel or RADA16 peptide. Figure 7A shows the results of adding Me-C-JigSAP to RADA16 peptide gel. Figure 7B shows the results of adding Me-C-JigSAP to C-JigSAP peptide gel.

[0016] 1. Modified Peptides 1-1. Overview A first aspect of the present invention is a modified peptide having gel-degrading activity. The modified peptide of this aspect has a modified core peptide consisting of a specific amino acid sequence, in which one or more nitrogen atoms constituting the peptide bond have a substituent, and is capable of rapidly degrading gels composed of self-assembling peptides containing an unmodified core peptide under physiological conditions.

[0017] 1-2. Definition of Terms The following terms frequently used in this specification are defined below. As used herein, "self-assembly" refers to the spontaneous assembly of small molecules in a dispersion medium through intermolecular interactions or the like to form a three-dimensional structure. In this specification, a substance that gels through self-assembly is often referred to as a "gelator." As used herein, self-assembling peptides are a type of gelator.

[0018] As used herein, the term "self-assembling peptide" refers to a peptide that can solidify from a sol state dissolved in water or an aqueous solution to a gel state under specific temperature and pressure conditions. Examples of such peptides include collagen (including glue, gelatin, and jelly), (RADA)4 peptide, and JigSAP, which will be described in the Examples below.

[0019] In this specification, the term "gel" refers to a substance in which colloidal particles self-assemble in a dispersion medium, lose fluidity, solidify, and become solid.

[0020] In this specification, the term "gel state" refers to a state in which colloidal particles self-assemble in a dispersion medium, lose fluidity, and solidify. Generally, this refers to a state in which a sol is solidified by lowering its temperature. "Gellation" refers to a phase transition phenomenon from a sol state to a gel state.

[0021] As used herein, the term "sol" refers to a liquid state in which colloidal particles are dispersed in a dispersion medium and have fluidity. For example, a sol refers to a gel in which a colloid made of a gelling agent is fluidized in the dispersion medium by heating the gel.

[0022] The "sol state" refers to a liquid state in which colloidal particles are dispersed in a dispersion medium and have fluidity. For example, this refers to a state in which a gelling agent is dispersed in a dispersion medium such as water or an aqueous solution, or a state in which a gel is fluidized by heating. "Solation" is a phase transition phenomenon from a gel state to a sol state.

[0023] As used herein, the term "gelation temperature" refers to the temperature at which a gelling agent undergoes a phase transition from a sol state to a gel state. The term "solation temperature" refers to the temperature at which a gelling agent undergoes a phase transition from a gel state to a sol state.

[0024] As used herein, "disassembly" refers to dissociating at least some of the small molecules from a three-dimensional structure formed by the assembly of the small molecules. For example, "disassembly" of a gel formed by the assembly of self-assembling peptides means dissociating the self-assembling peptides that make up at least a part of the gel from the gel. Disassembly may be either partial or complete. Furthermore, it is sufficient that small molecules such as self-assembling peptides dissociate between molecules, regardless of whether intramolecular cleavage reactions occur.

[0025] As used herein, "gel-disassembling activity" refers to the activity of dissociating at least some small molecules from a gel formed by the assembly of small molecules such as self-assembling peptides. Substances with gel-degrading activity are often referred to herein as "gel-disintegrating agents."

[0026] As used herein, the term "peptide" refers to an amino acid polymer having one or more peptide bonds. The term "peptide" is not limited by the number of amino acid residues contained in the peptide. Therefore, "peptide" encompasses everything from oligopeptides containing a few amino acid residues, such as dipeptides and tripeptides, to polypeptides containing many amino acid residues. Therefore, it encompasses not only so-called proteins, but also fragments and peptides linked to other peptides by peptide bonds.

[0027] As used herein, the term "fusion peptide" refers to a self-assembling peptide to which a functional peptide is linked. The link between the self-assembling peptide and the functional peptide may be a covalent bond or a supramolecular interaction. The covalent bond is not limited, and examples include a peptide bond and a disulfide bond. Although not limited thereto, the functional peptide is preferably linked to the N-terminus and / or C-terminus of the self-assembling peptide, and the bond is preferably a covalent bond, with a preferred covalent bond being a peptide bond.

[0028] As used herein, the term "functional peptide" refers to a peptide that has a specific biological function in vivo or ex vivo, or intracellularly or extracellularly. As used herein, the term "specific biological function" is not limited to a function that can have any effect on biomolecules such as proteins and nucleic acids, cells, tissues, or individuals. The specific biological function may be natural or non-natural, and examples thereof include cell adhesion function, signal transduction function, binding function, linking function, labeling function, metabolic function, etc.

[0029] As used herein, "biocompatibility" refers to the property of being capable of being introduced into a living organism. In particular, it refers to the property of a material having no toxicity or side effects to the living organism, or having only very slight toxicity or side effects, and / or the property of not being recognized as a foreign body in the living organism and not being eliminated. As used herein, "a peptide having biocompatibility" refers, for example, to a peptide that is free from biological contamination and therefore has no or very little risk of causing allergies or unknown infectious diseases in the human body. Examples of biocompatible peptides include chemically synthesized peptides.

[0030] As used herein, the term "living body" refers to cells (including cultured cells), tissues, organs, or individuals. Examples include, but are not limited to, cells, tissues, or organs derived from humans or non-human individuals, or cells derived from humans or non-human individuals, such as cells or tissues obtained by differentiation from ES cells or iPS cells. Preferably, the organism is a human-derived cell, a tissue or organ composed of human-derived cells, or a human individual.

[0031] As used herein, "physiological conditions" refers to conditions such as temperature and pH that do not substantially impair the structure or activity of biomolecules, the structure or function of cells or tissues, or the activity or survival of individuals. More specifically, these conditions refer to conditions that may exist in a living organism or within a cell. As used herein, physiological conditions refer to conditions under which biomolecules, particularly proteins, do not denature or are unlikely to denature. As used herein, physiological pH is not limited to any pH at which biomolecules, such as proteins, do not denature or are unlikely to denature. For example, it is within the range of pH 4.0 to 10.0, pH 5.0 to 9.0, pH 6.0 to 8.0, or pH 6.5 to 7.5, e.g., pH 7.4. As used herein, physiological temperature is not limited to any temperature at which biomolecules, such as proteins, do not denature or are unlikely to denature. For example, it is within the range of 0 to 65°C, 4 to 60°C, 20 to 50°C, or 30 to 40°C, e.g., 37°C.

[0032] As used herein, the term "amino acid" encompasses both natural and unnatural amino acids. Unnatural amino acids are, for example, amino acids having any chemically modified group or substituent. As used herein, amino acids include any optical isomers, and may be either D- or L-isomers.

[0033] As used herein, the term "nitrogen atom constituting a peptide bond" refers to a nitrogen atom constituting a peptide bond (-CO-NH- bond) that connects adjacent amino acid residues in a peptide chain. The group consisting of a nitrogen atom and a hydrogen atom in a peptide bond is also called an imino group.

[0034] As used herein, the phrase "the nitrogen atom constituting the peptide bond is contained in the Xth amino acid residue from the N-terminus" in a peptide chain refers to the nitrogen atom constituting the peptide bond (-CO-NH- bond) connecting the X-1th amino acid residue and the Xth amino acid residue from the N-terminus of the peptide chain.

[0035] As used herein, the term "hydrophobic amino acid" refers to an amino acid having hydrophobicity or a highly hydrophobic amino acid. Examples include alanine (Ala / A), glycine (Gly / G), proline (Pro / P), valine (Val / V), leucine (Leu / L), isoleucine (Ile / I), methionine (Met / M), cysteine ​​(Cys / C), phenylalanine (Phe / F), tyrosine (Tyr / Y), and tryptophan (Trp / W). Among these amino acids, cysteine ​​has low hydrophobicity and is therefore sometimes classified as a hydrophilic amino acid.

[0036] As used herein, the term "hydrophilic amino acid" refers to an amino acid that has hydrophilicity or has high hydrophilicity, including, for example, aspartic acid (Asp / D), glutamic acid (Glu / E), lysine (Lys / K), histidine (His / H), and arginine (Arg / R).

[0037] As used herein, "plurality" refers to, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, 2 to 3, or 2.

[0038] As used herein, "amino acid identity" refers to the percentage (%) of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two peptides being compared are aligned by inserting appropriate gaps into one or both of them as needed to maximize the number of identical amino acid residues. Alignment of two amino acid sequences to calculate amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW.

[0039] As used herein, unless otherwise specified, "(amino acid) substitution" refers to a substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to alter the properties of peptides.

[0040] 1-3. Structure The structure of the modified peptide of this embodiment is described in detail below. The modified peptide of this embodiment comprises at least one modified core peptide or consists of the modified core peptide. The modified core peptide contained in the modified peptide of this embodiment consists of an amino acid sequence selected based on specific rules, and at least one nitrogen atom constituting a peptide bond in the amino acid sequence has a substituent.

[0041] As used herein, a "modified core peptide" refers to a 9-amino acid peptide that constitutes part or all of the modified peptide of the present invention, in which at least one nitrogen atom constituting a peptide bond has a substituent. In the present invention, the modified core peptide is a peptide formed by alternating peptide bonds between hydrophobic and hydrophilic amino acids, and the amino acid sequence is selected based on specific rules. The modified core peptide is characterized in that at least one nitrogen atom constituting a peptide bond in its amino acid sequence has a substituent, regardless of whether or not substitutions other than the nitrogen atom constituting the peptide bond (e.g., substitution of the amino acid side chain) exist. For example, the modified core peptide may have no substituents other than the nitrogen atom constituting at least one peptide bond in its amino acid sequence, except for an N-terminal acetyl group and / or a C-terminal NH2 amide, and the amino acid side chains in the modified core peptide may be natural side chains.

[0042] Specifically, the modified peptide of this embodiment comprises or consists of a modified core peptide consisting of the amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), and in the modified core peptide, at least one nitrogen atom constituting a peptide bond has a substituent.

[0043] Specific examples of modified core peptides consisting of the amino acid sequence shown in formula I above include IRARMDADI (sequence number 1), IRADMRADI (sequence number 2), IRADMDARI (sequence number 3), IDARMRADI (sequence number 4), IDARMDARI (sequence number 5), IDADMRARI (sequence number 6), IRGDIRGDI (sequence number 7), IRGDMRGDI (sequence number 8), IRADIRADM (sequence number 9), IDARMRADM (sequence number 10), MDARIDARI (sequence number 11), MDADMRARI (sequence number 12), IRGDMRADI (sequence number 13), IRADMRGDI (sequence number 14), IRGDIRGDI (sequence number 15), IRGDIRADI (sequence number 16), and IRADIRGDI (sequence number 17).

[0044] The modified peptide of this embodiment comprises or consists of at least one modified core peptide, for example, the modified peptide of this embodiment may comprise one or two modified core peptides.

[0045] When the modified peptide of this embodiment contains two or more modified core peptides, the two or more modified core peptides may be composed of the same amino acid sequence or may be composed of different amino acid sequences.

[0046] In one embodiment, the modified peptide of this aspect may not contain any additional amino acid residues on the N-terminal and / or C-terminal sides of the modified core peptide, in which case the modified peptide of this aspect consists solely of the modified core peptide.

[0047] In one embodiment, the modified peptide of this embodiment may comprise a peptide consisting of one amino acid residue or multiple amino acid residues (hereinafter referred to as the "N-terminal peptide" and the "C-terminal peptide") at the N-terminus and / or C-terminus of the modified core peptide. The amino acid residues at the N-terminus and / or C-terminus of the modified peptide of this embodiment may be hydrophilic amino acids. The N-terminal peptide and / or the C-terminal peptide of the modified peptide of this embodiment may each comprise one or more hydrophilic amino acids.

[0048] In one embodiment, Arg may be linked to the N-terminus and / or C-terminus of the modified peptide. That is, the modified peptide of this embodiment may have Arg only at its N-terminus, only at its C-terminus, or both at its N-terminus and C-terminus. Examples of such modified peptides include RIRARMDADIR (SEQ ID NO: 18), RIRADMRADIR (SEQ ID NO: 19), RIRADMDARIR (SEQ ID NO: 20), RIDARMRADIR (SEQ ID NO: 21), RIDARMDARIR (SEQ ID NO: 22), RIDADMRARIR (SEQ ID NO: 23), RIRGDIRGDIR (SEQ ID NO: 24), RIRGDMRGDIR (SEQ ID NO: 25), RIRADIRADMR (SEQ ID NO: 26), RIDARMRADMR (SEQ ID NO: 27), RMDARIDARIR (SEQ ID NO: 28), RMDADMRARIR (SEQ ID NO: 29), RIRGDMRADIR (SEQ ID NO: 30), RIRADMRGDIR (SEQ ID NO: 31), RIRGDIRGDIR (SEQ ID NO: 32), RIRGDIRADIR (SEQ ID NO: 33), and RIRADIRGDIR (SEQ ID NO: 34).

[0049] In a further embodiment, both of the two amino acids at the N-terminus and / or C-terminus of the modified peptide may be Arg. That is, the modified peptide of this embodiment may have only an Arg-Arg dipeptide at its N-terminus, only an Arg-Arg dipeptide at its C-terminus, or both an Arg-Arg dipeptide at its N-terminus and C-terminus.

[0050] The amino acids other than glycine constituting the modified peptide of this embodiment can be used regardless of their optical isomers. That is, either the D- or L-form may be used. For example, all of the amino acids other than glycine constituting the modified peptide may be D- or L-forms.

[0051] The total length of the amino acid sequence constituting the modified peptide is, but is not limited to, for example, 50 or less amino acids or 25 or less amino acids. Specific examples of the amino acid length include 20 or less amino acids, 15 or less amino acids, or 10 or less amino acids, such as 14, 13, 12, 11, 10, or 9 amino acids.

[0052] In one embodiment, the modified peptide of this aspect consists of a modified core peptide, in which case the total length of the amino acid sequence is 9 amino acids.

[0053] In another embodiment, the modified peptide of this aspect is an 11 amino acid peptide comprising a core peptide, both of whose N- and C-termini may be Arg.

[0054] In yet another embodiment, the modified peptide of this aspect is a 13 amino acid peptide comprising a core peptide, both of whose N-terminus and C-terminus are Arg-Arg dipeptides.

[0055] The amino group of the N-terminal amino acid residue and the carboxyl group of the C-terminal amino acid residue of the modified peptide of this embodiment may optionally have a modifying group added. For example, an acetyl group may be added to the N-terminus of the modified peptide of this embodiment. Furthermore, an NH2 amide may be added to the C-terminus of the modified peptide of this embodiment.

[0056] In the modified peptide of this embodiment, at least one nitrogen atom constituting a peptide bond in the modified core peptide has a substituent. As used herein, the "substituent" on a nitrogen atom constituting a peptide bond refers to any group other than a hydrogen atom. For example, it may be an optionally substituted hydrocarbon group having one or more carbon atoms.

[0057] In one embodiment, the hydrocarbon group is an optionally substituted alkyl group having one or more carbon atoms. As used herein, "alkyl" or "alkyl group" refers to a saturated hydrocarbon having one or more carbon atoms. Specific examples of the alkyl group include linear alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (also called cycloalkyl groups, alicyclic groups, or carbocyclic groups, e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), and branched alkyl groups (e.g., isopropyl, tert-butyl, sec-butyl, isobutyl, etc.).

[0058] The number of carbon atoms in the alkyl group is not limited as long as it is 1 or more. The number of carbon atoms in the alkyl group may be, for example, 1 or more, 2 or more, 3 or more, or 4 or more, and / or 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less. For example, it may be 1 to 10, 2 to 9, 3 to 8, or 4 to 7. More specifically, the number of carbon atoms in the alkyl group may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0059] Examples of substitution of hydrocarbon groups such as alkyl groups include substitution with hydroxyl, carboxyl, cyano, nitro, halogen (e.g., Cl, F, Br, or I), thiol, alkyl group, alkoxyl group, ester, thioether, thioester, nitro, or amine. The number of substitutions on the hydrocarbon group is not limited, and may be, for example, one or more substitutions.

[0060] In further embodiments, the hydrocarbon group may be an optionally substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or benzyl group. A substituted benzyl group may be, for example, a nitrobenzyl group.

[0061] In the modified peptide of this embodiment, the number of peptide bonds containing a nitrogen atom having a substituent in the modified core peptide is not particularly limited, and may be, for example, one or more, two or more, or three or more, and / or five or less, four or less, or three or less, preferably one or two.

[0062] In the modified peptide of this embodiment, the position of the nitrogen atom having a substituent in the modified core peptide is not particularly limited. For example, the nitrogen atom having a substituent may be a nitrogen atom constituting any peptide bond in the amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (Formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly). Specifically, the nitrogen atom having a substituent in the modified core peptide of the modified peptide of this embodiment may be any of the nitrogen atoms forming the peptide bond between the first and second amino acid residues from the N-terminus, the peptide bond between the second and third amino acid residues from the N-terminus, the peptide bond between the third and fourth amino acid residues from the N-terminus, the peptide bond between the fourth and fifth amino acid residues from the N-terminus, the peptide bond between the fifth and sixth amino acid residues from the N-terminus, the peptide bond between the sixth and seventh amino acid residues from the N-terminus, the peptide bond between the seventh and eighth amino acid residues from the N-terminus, or the peptide bond between the eighth and ninth amino acid residues from the N-terminus in the amino acid sequence shown in Formula I above, or any combination thereof. Preferably, the nitrogen atom forming the peptide bond between the sixth and seventh amino acid residues from the N-terminus, i.e., the nitrogen atom forming the peptide bond contained in the seventh amino acid residue from the N-terminus in the amino acid sequence shown in Formula I above, has a substituent.

[0063] In a further embodiment, the modified peptide of this embodiment may be linked to a functional peptide such as a labeling peptide or a peptide tag for purification. The labeling peptide may be, but is not limited to, a fluorescent protein such as GFP, a luminescent protein such as luciferin or aequorin, or an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Furthermore, the peptide tag is an artificially synthesized oligopeptide consisting of several to a dozen amino acids, and examples thereof include epitope tags such as FLAG tags, histidine tags, HA tags, and DAP tags, as well as His tags, GST tags, and Halo tags.

[0064] The modified peptide of this embodiment may also be optionally bound to one or more chemical modifying groups via a mechanism other than a peptide bond. The structure of the chemical modifying group is not particularly limited, but it is a moiety that confers a desired function to the modified peptide to which it is bound. Examples of the desired function include a labeling function, a linker function, a linking function, and a binding function. Examples of chemical modifying groups that impart a labeling function include chromophores and fluorescent groups (e.g., fluorescein). Examples of chemical modifying groups that impart a linker function include any polymer (e.g., alkylene). Examples of chemical modifying groups that impart a binding function include compounds such as biotin. Other examples of chemical modifying groups that can be bound to the self-assembling peptide or fusion peptide of this embodiment include lipids, sugars, aptamers, receptor ligands, and the like. Examples of lipids include cholesterol, fatty acids, and other lipids (e.g., vitamin E, vitamin A, vitamin D), fat-soluble vitamins such as vitamin K, intermediate metabolites such as acyl-CoA, glycolipids, glycerides, and derivatives thereof. Examples of sugars include glucose and sucrose.

[0065] 1-4. Effects The modified peptide of this embodiment can degrade gels composed of self-assembling peptides and the like. For example, by contacting the modified peptide of this embodiment with a gel composed of self-assembling peptides having a structure similar to that of the modified peptide of this embodiment, intermolecular interactions such as hydrogen bonds between the self-assembling peptides that make up the gel can be broken, thereby degrading the gel. In particular, efficient gel degradation is achieved when the Xaa and Zaa in the amino acid sequence of the modified peptide in Formula I above have the same amino acid side chains as the Xaa and Zaa at the corresponding positions in the amino acid sequence of the self-assembling peptide in Formula II below, or when the modified peptide has the same structure as the self-assembling peptide except for the substituents on the nitrogen atoms that form the peptide bonds.

[0066] 2. Gel Degradation Composition 2-1. Overview A second aspect of the present invention is a composition for gel degradation. The gel degradation composition of this aspect contains the modified peptide of the first aspect as an essential component, and also contains a carrier and other components. The gel degradation composition of this aspect can degrade a gel in vitro or in vivo by contacting it with a gel composed of a self-assembling peptide.

[0067] 2-2. Composition 2-2-1. Constituent Components The gel decomposing composition of the present invention is composed of an active ingredient and other ingredients. The components other than the active ingredient are not particularly limited, but examples include components that can promote the gel decomposition action of the gel decomposing composition and carriers. Each of the constituent components will be described in detail below.

[0068] (1) Active ingredient The gel decomposing composition of this embodiment contains, as an essential active ingredient, one or more modified peptides according to the first embodiment. The modified peptides according to the first embodiment as active ingredients may be one type or a combination of two or more different types.

[0069] The amount (content) of the modified peptide according to the first aspect to be incorporated into the composition for decomposing a gel is not particularly limited. It may be determined appropriately taking into consideration the conditions for gel degradation, such as in vitro or in vivo use. Specifically, the concentration of the modified peptide in the composition for decomposing a gel is not limited, but may be, for example, 0.4% by weight to 10% by weight, or 1.0% by weight to 10% by weight, or may be, for example, 1.0% by weight to 2.0% by weight.

[0070] When the gel-degrading composition of the present invention is administered into a living body, the target is, in principle, a living body to which a gel composed of self-assembling peptides has been transplanted as an artificial extracellular matrix or the like. In this case, the content of the modified peptide in the gel-degrading composition can be determined appropriately depending on the type and / or effective amount of the modified peptide contained in the gel-degrading composition, information about the subject, the dosage form of the gel-degrading composition, and the type of carrier or additive (described below). As used herein, the term "effective amount" refers to the amount of the modified peptide necessary to function as an active ingredient in the gel-degrading composition and which causes little or no harmful side effects in the living body to which it is applied. This effective amount may vary depending on various conditions, such as information about the subject, the route of administration, and the number of administrations. Here, the term "subject" refers to a living body to which the gel-degrading composition is applied. Examples of such living bodies include humans, livestock (e.g., cows, horses, sheep, goats, pigs, chickens, ostriches), racehorses, pets (e.g., dogs, cats, rabbits), and laboratory animals (e.g., mice, rats, guinea pigs, monkeys, marmosets). Preferably, the subject is a human. Furthermore, "subject information" refers to various individual information about the living body to which the gel-degrading composition is applied, including, for example, in the case of a subject, the subject's overall health condition, the progression and severity of any disease or injury, age, weight, sex, diet, drug sensitivity, the presence or absence of concomitant medications, and resistance to treatment. The final effective dose of the gel-degrading agent and the dosage calculated based on it are ultimately determined by the judgment of a physician, dentist, veterinarian, or the like, depending on the individual subject's information, etc.

[0071] (2) Carrier The gel-degrading composition of this embodiment may contain a pharmaceutically acceptable carrier, if necessary. As used herein, the term "pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. Examples include solvents, excipients, fillers, emulsifiers, flow control agents, lubricants, and human serum albumin.

[0072] The solvent may be, for example, water or another pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent, preferably water or another pharmaceutically acceptable aqueous solution. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other supplements, phosphate buffer, sodium acetate buffer, and any medium used in cell culture or tissue culture. Examples of supplements include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like. Commercially available media may be used, such as DMEM, Ham's F12, DMEM / F12, McCoy's 5A, Eagle's MEM, αMEM, MEM, RPMI 1640, Iscove's modified Dulbecco's medium, MCDB131, William's medium E, IPL41, and Fischer's medium.

[0073] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metallic salts, citric acid, tartaric acid, glycine, polyethylene glycol, Pluronic®, kaolin, silicic acid, or combinations thereof.

[0074] Examples of fillers include petrolatum, the aforementioned sugars and / or calcium phosphate.

[0075] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0076] Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

[0077] In addition to the above, if necessary, the composition may appropriately contain solubilizers, suspending agents, diluents, dispersing agents, surfactants, soothing agents, stabilizers, pH adjusters, absorption enhancers, bulking agents, moisturizing agents, humectants, wetting agents, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, antioxidants, fragrances, flavoring agents, sweeteners, buffers, isotonic agents, and the like that are commonly used in pharmaceuticals.

[0078] Such carriers are primarily used to facilitate the formation of dosage forms, maintain the dosage form and drug efficacy, and also to make the active ingredient, the gel decomposing agent, less susceptible to degradation by enzymes in the body, etc., and may be used appropriately as needed.

[0079] The pH of the gel-disintegrating composition of this embodiment is not limited, and may be, for example, a physiological pH, such as a pH in the range of 4.0 to 10.0, a pH in the range of 5.0 to 9.0, a pH in the range of 6.0 to 8.0, or a pH in the range of 6.5 to 7.5, e.g., pH 7.4.

[0080] 2-2-2. Dosage Form The dosage form of the gel-degrading composition of this embodiment is not particularly limited. For example, it may be a liquid or solid. In the case of a solid, its shape is not important. It may be a common solid dosage form such as a powder, a powder, a granule, or a tablet.

[0081] 2-2-3. Application Method The application method of the gel-degrading composition of this embodiment can be appropriately selected depending on whether it is to be used in vitro or in vivo. For in vitro use, for example, the gel can be degraded by adding the gel-degrading composition to a gel or a medium containing the gel. For in vivo use, there are no particular limitations, but parenteral administration is preferred, and local administration to the target site where the gel is implanted is more preferred. Examples of local administration include intramuscular administration, subcutaneous administration, tissue administration, and organ administration. For example, the target site may be incised by surgical operation and then locally administered. The dosage may be an amount effective for the active ingredient to have a beneficial effect, and is appropriately selected depending on subject information.

[0082] 2-3. Effects The gel-degrading composition of this embodiment can degrade gels in vitro or in vivo. For example, by administering the gel-degrading composition to a living body, it is possible to rapidly degrade a gel implanted in the living body and remove it from the implantation site.

[0083] 3. Self-assembling peptides and fusion peptides 3-1. Overview The third aspect of the present specification relates to self-assembling peptides and fusion peptides. The self-assembling peptide of this aspect comprises at least one unmodified core peptide consisting of an amino acid sequence selected based on specific rules. The fusion peptide of this aspect is a peptide formed by linking the self-assembling peptide of this aspect to a functional peptide. The self-assembling peptide and fusion peptide of this aspect can gel under physiological conditions, and the gel obtained by this gelation can be rapidly degraded by the modified peptide of the first aspect or the gel-degrading composition of the second aspect.

[0084] 3-2. Configuration 3-2-1. Self-assembling peptide The self-assembling peptide of this embodiment comprises at least one unmodified core peptide or consists of the unmodified core peptide. The unmodified core peptide contained in the self-assembling peptide of this embodiment consists of an amino acid sequence selected based on specific rules.

[0085] As used herein, an "unmodified core peptide" refers to a 9-amino acid peptide that constitutes part or all of the self-assembling peptide of this embodiment, in which the nitrogen atoms that constitute its peptide bonds are unsubstituted. In the present invention, an unmodified core peptide is a peptide formed by alternating peptide bonds between hydrophobic and hydrophilic amino acids, and its amino acid sequence is selected based on specific rules. Furthermore, an unmodified core peptide is characterized in that none of the nitrogen atoms that constitute peptide bonds in the peptide chain are substituted, regardless of whether or not substitutions other than the nitrogen atoms that constitute the peptide bonds (e.g., substitution of amino acid side chains) are present. For example, an unmodified core peptide may have no substituents except for an acetyl group at the N-terminus and / or an NH2 amide at the C-terminus. For example, the amino acid side chains in an unmodified core peptide may be natural side chains.

[0086] Specifically, the self-assembling peptide of this embodiment comprises at least one unmodified core peptide consisting of the amino acid sequence represented by the following formula II: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula II), wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly.

[0087] Specific examples of unmodified core peptides contained in the self-assembling peptides of this embodiment include peptides consisting of the amino acid sequences shown in SEQ ID NOS: 1-17.

[0088] The self-assembling peptide of this embodiment comprises or consists of one or more unmodified core peptides. For example, the self-assembling peptide of this embodiment may comprise one or two unmodified core peptides.

[0089] When the self-assembling peptide of this embodiment comprises two unmodified core peptides, the two unmodified core peptides may be composed of the same amino acid sequence or different amino acid sequences.

[0090] In one embodiment, the self-assembling peptide of this embodiment may not contain any additional amino acid residues on the N-terminal and / or C-terminal sides of the unmodified core peptide, in which case the self-assembling peptide of this embodiment consists solely of the unmodified core peptide.

[0091] In one embodiment, the self-assembling peptide of this embodiment may comprise a peptide consisting of one amino acid residue or multiple amino acid residues (hereinafter referred to as the "N-terminal peptide" and the "C-terminal peptide") at the N-terminus and / or C-terminus of the unmodified core peptide. The amino acid residues at the N-terminus and / or C-terminus of the self-assembling peptide of this embodiment may be hydrophilic amino acids. The N-terminal peptide and / or the C-terminal peptide in the self-assembling peptide of this embodiment may each comprise one or more hydrophilic amino acids.

[0092] In one embodiment, the N-terminus and / or C-terminus of the self-assembling peptide may be Arg. That is, the self-assembling peptide of this embodiment may have only the N-terminus as Arg, only the C-terminus as Arg, or both the N-terminus and C-terminus as Arg. Examples of such self-assembling peptides include peptides consisting of the amino acid sequences set forth in SEQ ID NOs: 18 to 34.

[0093] In a further embodiment, both of the two amino acids at the N-terminus and / or C-terminus of the self-assembling peptide may be Arg. That is, the self-assembling peptide of this embodiment may have only an Arg-Arg dipeptide at its N-terminus, only an Arg-Arg dipeptide at its C-terminus, or both an Arg-Arg dipeptide at its N-terminus and C-terminus.

[0094] The amino acids other than glycine that make up the self-assembling peptide of this embodiment can be used regardless of their optical isomers. That is, either the D- or L-form may be used. For example, all of the amino acids other than glycine that make up the self-assembling peptide may be D- or L-forms.

[0095] The total length of the amino acid sequence constituting the self-assembling peptide is, but is not limited to, for example, 25 amino acids or less. Specific examples of the amino acid length include 20 amino acids or less, 15 amino acids or less, or 10 amino acids or less, for example, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids.

[0096] In one embodiment, the self-assembling peptide of this embodiment consists of an unmodified core peptide, in which case the total length of the amino acid sequence is 9 amino acids.

[0097] In another embodiment, the self-assembling peptide of this aspect is an 11 amino acid peptide comprising an unmodified core peptide, both of whose N- and C-termini may be Arg.

[0098] In yet another embodiment, the self-assembling peptide of this aspect is a 13-amino acid peptide comprising an unmodified core peptide, both of whose N- and C-termini may be Arg-Arg dipeptides.

[0099] The amino group of the N-terminal amino acid residue and the carboxyl group of the C-terminal amino acid residue of the self-assembling peptide of this embodiment may optionally have a modifying group added. For example, an acetyl group may be added to the N-terminus of the self-assembling peptide of this embodiment. Furthermore, an NH2 amide may be added to the C-terminus of the self-assembling peptide of this embodiment.

[0100] 3-2-2. Fusion Peptide The fusion peptide of this embodiment is a peptide formed by linking a functional peptide to the self-assembling peptide of this embodiment. The self-assembling peptide and the functional peptide may be linked, for example, by a covalent bond or a supramolecular interaction. In one embodiment, the supramolecular interaction may be a hydrogen bond, a hydrophobic interaction, an electrostatic interaction, a coordinate bond, or the like.

[0101] The fusion peptide of this embodiment is not limited to, but may be, for example, a peptide obtained by linking a functional peptide to any position of the self-assembling peptide of this embodiment via a covalent bond such as a peptide bond or via a linker. The position at which the functional peptide is linked to the self-assembling peptide of this embodiment may be, for example, the N-terminus and / or C-terminus of the self-assembling peptide. When the self-assembling peptide of this embodiment is "linked to the N-terminus and / or C-terminus of the functional peptide by a peptide bond," this encompasses cases in which the self-assembling peptide of this embodiment is linked only to the N-terminus, only to the C-terminus, or to both the N-terminus and C-terminus of the functional peptide. In addition, the functional peptide may be linked to the side chain of an amino acid residue other than the N-terminus or C-terminus of the self-assembling peptide of this embodiment.

[0102] As described above, the functional peptide constituting the fusion peptide of this embodiment is a peptide that has a specific biological function in vivo or in a cell, such as, but not limited to, cell adhesion function, signal transduction function, binding function, linking function, labeling function, or metabolic function.

[0103] Furthermore, the functional peptide constituting the fusion peptide of this embodiment can be selected depending on the intended use, the purpose of the fusion peptide, such as cell culture, control of cell adhesion, proliferation, differentiation, etc., tissue or organ culture, formation, regeneration, or vascular induction.

[0104] Functional peptides of the present invention include, but are not limited to, cell adhesion molecules, extracellular matrix molecules, secreted proteins, binding proteins, enzymes, marker proteins, and artificial peptides, as well as peptide fragments thereof. "Cell adhesion molecules" as used herein refer to molecules involved in adhesion between cells or between cells and the extracellular matrix on the surface of cells. Examples include, but are not limited to, cadherins such as N-cadherin, integrins, and selectins. "Extracellular matrix molecules" refer to molecules that constitute the extracellular matrix. Examples include, but are not limited to, laminin, collagen, and fibronectin. "Secreted proteins" refer to proteins produced within cells and secreted outside the cells. Examples include, but are not limited to, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and cytokines. "Binding proteins" refer to proteins that specifically bind to specific molecules. Examples of functional peptides include, but are not limited to, antibodies or antibody fragments or antigens that mediate antigen-antibody binding, (strept)avidin, maltose-binding protein (MBP), receptors or ligands that mediate receptor-ligand interactions, DNA-binding proteins, and RNA-binding proteins. In particular, when the functional peptide is a DNA-binding protein or an RNA-binding protein, the nucleic acid molecule to which the functional peptide binds can bind to one or more other nucleic acid molecules by forming a multistranded structure. A "marker protein" is a protein that can be used as a label when detecting cells, proteins, etc. Typically, this refers to a polypeptide whose activity can be used to determine the expression or presence of a target protein. Examples include, but are not limited to, fluorescent proteins such as GFP, luminescent proteins such as luciferin or aequorin, and enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). An "artificial peptide," also known as a tag peptide, is an artificially synthesized oligopeptide consisting of several to a dozen amino acids. Examples include epitope tags such as FLAG tags, histidine tags, HA tags, and DAP tags, as well as His tags, GST tags, and Halo tags.

[0105] Furthermore, the self-assembling peptide or fusion peptide of this embodiment may optionally be bound with one or more chemical modifying groups via a mechanism other than a peptide bond. The structure of the chemical modifying group is not particularly limited, but it is a moiety that imparts a desired function to the self-assembling peptide or fusion peptide to which it is bound. Examples of the desired function include a labeling function, a linker function, a linking function, and a binding function. Examples of chemical modifying groups that impart a labeling function include chromophores and fluorescent groups (e.g., fluorescein). Examples of chemical modifying groups that impart a linker function include any polymer (e.g., alkylene). Examples of chemical modifying groups that impart a binding function include compounds such as biotin. Other examples of chemical modifying groups that can be bound to the self-assembling peptide or fusion peptide of this embodiment include lipids, sugars, aptamers, receptor ligands, and the like. Examples of lipids include cholesterol, fatty acids, and other lipids (e.g., vitamin E, vitamin A, vitamin D), fat-soluble vitamins such as vitamin K, intermediate metabolites such as acyl-CoA, glycolipids, glycerides, and derivatives thereof. Examples of sugars include glucose and sucrose.

[0106] 3-3. Effects The self-assembling peptide of this embodiment can gel under physiological conditions, and the gel obtained by this gelation can be rapidly degraded by the modified peptide of the first embodiment or the gel-degrading composition of the second embodiment. The self-assembling peptide of this embodiment can gel, for example, under conditions of 1 atmosphere, at a temperature within the range of 20 to 50°C or 30 to 40°C, and a pH within the range of 6.0 to 8.0 or 6.5 to 7.5. Therefore, a gel can be formed without losing the activity of biomolecules such as proteins embedded in the gel, or while at least partially retaining that activity.

[0107] The self-assembling peptides of this embodiment have a relatively short total amino acid sequence, allowing for low-cost chemical synthesis in large quantities. They can be synthesized with high purity by solution synthesis or solid-phase synthesis, which offers the advantages of consistent quality for each production lot and extremely low contamination. Furthermore, the self-assembling peptides and fusion peptides of this embodiment are biocompatible and can be introduced into living organisms.

[0108] 4. Kit for Preparing and Degrading Gels 4-1. Overview A fourth aspect of the present invention is a kit for preparing and degrading gels. The kit for preparing and degrading gels of this aspect comprises a self-assembling peptide for gel preparation and the modified peptide described in the first aspect. With the kit of this aspect, a gel can be prepared using the self-assembling peptide, and the gel can be degraded using a gel-degrading agent comprising the modified peptide.

[0109] 4-2. Configuration The kit for producing and decomposing a gel of this embodiment includes, as essential components, a self-assembling peptide for producing a gel and the modified peptide described in the first embodiment. The self-assembling peptide and modified peptide included in the kit of this embodiment may be of a single type or of multiple types. The self-assembling peptide included in the kit of this embodiment conforms to the configuration described in the third embodiment. Furthermore, the modified peptide included in the kit of this embodiment conforms to the configuration described in the first embodiment.

[0110] Furthermore, the gel production and degradation kit of this embodiment includes, as optional components, a fusion peptide formed by linking a functional peptide to a self-assembling peptide, a component or carrier that can promote the gelation of the self-assembling peptide, a component or carrier that can promote the gel degradation action of the self-assembling peptide, etc. Furthermore, the kit of this embodiment may include a buffer, a culture medium, and / or instructions for use in gel production and gel degradation.

[0111] The gel production and degradation kit of this embodiment can also be used for culturing, for example, cell aggregates or tissues, such as spheroids, organoids, and assembloids.

[0112] 5. Gel Decomposition Method 5-1. Overview A fifth aspect of the present invention is a gel decomposition method. The gel decomposition method of this aspect makes it possible to decompose a gel composed of self-assembling peptides.

[0113] 5-2. Method The gel decomposition method of this embodiment includes a gel decomposition step as an essential step, and includes a mixing step, a gelation step, and / or an incubation step as optional steps.

[0114] (Gel Decomposition Step) The gel decomposition step is a step in which a modified peptide having gel decomposition activity is brought into contact with a gel composed of a self-assembling peptide to decompose the gel. The modified peptide used in this step is similar to that described in the first embodiment, and its description will be omitted here. The self-assembling peptide used in this step is similar to that described in the third embodiment, and its description will be omitted here.

[0115] In one embodiment, Xaa and Zaa in the above formula I, which shows the amino acid sequence of the modified peptide used in this step, have the same amino acid side chains as Xaa and Zaa at the corresponding positions, respectively, in the above formula II, which shows the amino acid sequence of the self-assembling peptide.

[0116] In a further embodiment, all amino acid residues in Formula I, which shows the amino acid sequence of the modified peptide used in this step, have the same amino acid side chains as the amino acid residues at the corresponding positions in Formula II, which shows the amino acid sequence of the self-assembling peptide.

[0117] In this step, "contact" refers to direct physical contact of a target substance with a target substance. In the present invention, this refers to contact of the target modified peptide having gel-degrading activity with the target gel. The contacting method is not particularly limited. Examples include spraying, sprinkling, dropping, immersing, or applying a liquid containing the modified peptide, or a powder or granular modified peptide, to the gel, or a combination of these methods. The contacting may be repeated multiple times as necessary.

[0118] The time and temperature for contacting the modified peptide with the gel in this step vary depending on the type of self-assembling peptide and modified peptide used, and can be determined appropriately depending on the type. For example, the modified peptide may be maintained in contact with the gel for 10 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, or 1 hour or more at a temperature within a range of 4 to 80°C, 10 to 70°C, 15 to 60°C, 20 to 50°C, or 30 to 40°C, such as 37°C. Furthermore, the method for controlling the temperature used in this step is not particularly limited, and any known method can be used. For example, a method in which the self-assembling peptide and modified peptide are placed in a thermostatic bath or the like can be used.

[0119] The pH at which the modified peptide is contacted with the gel in this step is not limited, and may be, for example, within the range of pH 4.0 to 10.0, pH 5.0 to 9.0, pH 6.0 to 8.0, or pH 6.5 to 7.5, e.g., pH 7.4.

[0120] The concentration of the modified peptide used in this step is not limited, but may be, for example, 0.4% to 10% by weight, or 1.0% to 10% by weight, or may be, for example, 1.0% to 2.0% by weight. The amount of the modified peptide used in this step is not limited, but may be, for example, 1 μL or more, 5 μL or more, 10 μL or more, 100 μL or more, or 1 mL or more, and / or 100 mL or less, 50 mL or less, 10 mL or less, 5 mL or less, or 2 mL or less.

[0121] (Mixing Step) The mixing step is a step of mixing the self-assembling peptide with water or an aqueous solution. The mixing method is not particularly limited, and sufficient mixing may be achieved by stirring or the like. Furthermore, in this step, a component capable of promoting gelation in the gelation step described below may be further mixed. Examples of gelation-promoting components include, but are not limited to, components that have the effect of reducing protein solubility. Examples of anions that have this effect include bicarbonate ions, carbonate ions, citrate ions, tartrate ions, and sulfate ions. Examples of cations that have this effect include lithium ions, sodium ions, potassium ions, magnesium ions, and calcium ions. The concentrations of the above components are not particularly limited. For example, the concentration may be 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, or 40 mM or more. When bicarbonate ions or carbonate ions are used, taking into consideration that bicarbonate ions and carbonate ions are usually in equilibrium in an aqueous solution, the total concentration of bicarbonate ions and carbonate ions may be 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, or 40 mM or more, for example, 44 mM or more.

[0122] In one embodiment of this step, a fusion peptide formed by linking a functional peptide to a self-assembling peptide may be mixed together with the self-assembling peptide.

[0123] (Gelling Step) The gelling step is a step in which the mixture obtained after the mixing step is gelled by maintaining the mixture at a temperature equal to or lower than the gelling temperature.

[0124] The duration and temperature of this step vary depending on the type of self-assembling peptide used and may be determined appropriately depending on the type. For example, the self-assembling peptide may be maintained for at least 1 minute, at least 10 minutes, or at least 1 hour at a temperature ranging from 4 to 80°C, 10 to 70°C, 15 to 60°C, 20 to 50°C, or 30 to 40°C, such as 37°C. In particular, when a fusion peptide is mixed with the self-assembling peptide in the mixing step, temperature conditions are preferred that maintain the activity of the functional peptide constituting the fusion peptide or at least partially retain that activity. For example, a temperature range of 4 to 80°C, 10 to 70°C, 15 to 60°C, 20 to 50°C, or 30 to 40°C, such as 37°C, can be used. The temperature control method used in this step is not particularly limited, and examples include placing the self-assembling peptide in a thermostatic bath.

[0125] The pH conditions used for gelation in this step are not limited, and may be, for example, within the range of pH 4.0 to 10.0, pH 5.0 to 9.0, pH 6.0 to 8.0, or pH 6.5 to 7.5, e.g., pH 7.4.

[0126] The concentration of the self-assembling peptide used in this step is not limited, but may be, for example, 0.4% by weight to 10% by weight, or 1.0% by weight to 10% by weight, for example, 1.0% by weight to 2.0% by weight.

[0127] In one embodiment of this process, gelation is carried out in the presence of isolated cells, cell aggregates, or tissues. For example, by immersing cells, cell aggregates, or tissues in the mixture obtained after the mixing process and carrying out gelation, a gel containing cells, cell aggregates, or tissues can be produced. The cell aggregates or tissues can be, for example, spheroids, organoids, or assembloids. The spheroids, organoids, or assembloids can be used for transplantation.

[0128] (Culturing Step) The culturing step is a step of culturing cells, cell aggregates, or tissues in the gel after the gelling step.

[0129] This step can be carried out by cell or tissue culture methods known in the art, for example, known organoid culture methods.

[0130] The culture conditions in this step, such as culture temperature, CO2 concentration, culture period, and medium exchange frequency, are not limited. For example, static culture may be performed at 37°C under 5% CO2 for 30 minutes or more or 2 hours or more, for example, 3 hours to 2 weeks, 6 hours to 1 week, 9 hours to 3 days, or 12 hours to 24 hours.

[0131] In one embodiment of this process, culture can be performed by maintaining the gel in the medium. A known medium can be appropriately selected and used. For example, in the case of animal cells, any liquid medium for animal cell culture can be used as the basal medium, and other components (serum, serum replacement reagents, growth factors, etc.; N2 supplement, B27(R) supplement, insulin, bFGF, activin A, heparin, ROCK inhibitor, and / or GSK-3 inhibitor, etc.) can be appropriately added as needed. For example, DMEM medium, Ham's F12 medium, DMEM / F12 medium, McCoy's 5A medium, Eagle's MEM medium, αMEM medium, MEM medium, RPMI1640 medium, Iscove's modified Dulbecco's medium, MCDB131 medium, William's medium E, IPL41 medium, and Fischer's medium may also be used. The method of embedding cells, cell populations, or tissues such as organoids in a gel, placing the gel on a porous membrane, and culturing them on the surface of the culture solution is called the air-liquid interface culture method.

[0132] 6. Method for Producing Cell Aggregates and / or Tissues 6-1. Overview A sixth aspect of the present invention is a method for producing cell aggregates and / or tissues. According to the production method of this aspect, isolated cells, cell aggregates, and / or tissues can be cultured in a gel to obtain cell aggregates and / or tissues after culture.

[0133] 6-2. Method The production method of this embodiment includes, as essential steps, a mixing step, a gelation step, a culture step, and a gel degradation step. The configuration of each step is similar to that described in the fifth embodiment, and therefore further explanation is omitted here. In the gelation step of this embodiment, gelation is performed in the presence of isolated cells, cell aggregates, and / or tissues, thereby producing a gel containing cells, cell aggregates, and / or tissues. In addition, in the gel degradation step of this embodiment, the modified peptide is brought into contact with the gel to degrade the gel, and the gel is removed from the cell aggregates and / or tissues obtained after the culture step, thereby isolating the cell aggregates and / or tissues after culture.

[0134] The present invention will be specifically described below with reference to examples, but these examples are merely illustrative and the present invention is not limited to the scope described in the examples.

[0135] Example 1: Preparation of Me-C-JigSAP (Objective) To prepare a modified jigsaw-shaped self-assembling peptide (JigSAP) by substituting a methyl group for the hydrogen atom attached to the nitrogen atom in the peptide bond that constitutes the peptide chain of JigSAP, and to verify the gel-forming ability of the modified JigSAP and unmodified JigSAP that does not have this substitution.

[0136] (Methods and Results) (1) Synthesis of C-JigSAP The unmodified JigSAP used in the following examples was a peptide (hereinafter referred to as "C-JigSAP") consisting of the amino acid sequence shown in SEQ ID NO: 19 (Arg-Ile-Arg-Ala-Asp-Met-Arg-Ala-Asp-Ile-Arg), acetylated at the N-terminus, and amidated at the C-terminus. C-JigSAP has an unmodified core peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (Ile-Arg-Ala-Asp-Met-Arg-Ala-Asp-Ile), with Arg residues attached to both the N- and C-termini.

[0137] C-JigSAP was synthesized on a 0.10 mmol scale by Fmoc peptide solid-phase synthesis using polystyrene resin according to the method described in WO 2022 / 025209, and the synthesized peptide was cleaved from the resin and lyophilized.

[0138] (2) Synthesis of Me-C-JigSAP The modified JigSAP used in the following examples was a peptide (hereinafter referred to as "Me-C-JigSAP") consisting of the amino acid sequence shown in SEQ ID NO: 19 (Arg-Ile-Arg-Ala-Asp-Met-Arg-Ala-Asp-Ile-Arg), in which the hydrogen atom bound to the nitrogen atom of the eighth amino acid residue (Ala) from the N-terminus in this amino acid sequence (i.e., the nitrogen atom constituting the peptide bond between the seventh amino acid residue (Arg) and the eighth amino acid residue (Ala) from the N-terminus in the amino acid sequence shown in SEQ ID NO: 19) was substituted with a methyl group, and the N-terminus was acetylated and the C-terminus was amidated. The nitrogen atom into which a methyl group has been introduced is the nitrogen atom contained in the seventh amino acid residue (Ala) from the N-terminus in the modified core peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (Ile-Arg-Ala-Asp-Met-Arg-Ala-Asp-Ile) (i.e., the nitrogen atom constituting the peptide bond located between the sixth amino acid residue (Arg) and the seventh amino acid residue (Ala) from the N-terminus in the amino acid sequence shown in SEQ ID NO: 2).

[0139] Me-C-JigSAP was synthesized on a 0.10 mmol scale by Fmoc solid-phase peptide synthesis using polystyrene resin, as described in (1) above. The synthesized peptide was cleaved from the resin and lyophilized. However, in the peptide elongation reaction to introduce the eighth amino acid residue (Ala) from the N-terminus, Fmoc-MeAla-OH (Watanabe Chemical Industry Co., Ltd., M00432) was used instead of Fmoc-Ala-OH·HO (Watanabe Chemical Industry Co., Ltd.) used in (1) above, thereby introducing a methyl group to the nitrogen atom constituting the peptide bond at that position.

[0140] (3) Gel-forming ability: 2.5 mg of the lyophilized peptide powder and 250 μL of D-MEM (containing 1× penicillin, 4.0 mM HEPES, and 44 mM NaHCO3; pH 7.4) were added to a 1 mL microtube (a glass container with an inner diameter of 5 mm and a height of 5 cm; Maruem, Model No. 1). The mixture was sonicated in a water bath ultrasonicator (AS12GTU, 35 kHz, 60 W) to form a 1.0 wt% dispersion of C-JigSAP (1) and Me-C-JigSAP (2). The microtube was then placed vertically in a CO2 incubator (37°C, 5% CO2) for 48 hours. Penicillin (Life Technologies Corporation, model number 15140148) was used at 100 U / mL as an antibacterial agent to prevent mold growth, and a CO2 incubator was used to perform gelation under conditions similar to those in vivo.

[0141] To test whether the peptide dispersion had lost fluidity and gelled, the sample was sealed in a CO2 incubator (37°C, CO2 concentration 5%), removed from the incubator, and the microtube was quickly inverted and photographed. If some or all of the peptide dispersion remained on the bottom side of the microtube, it was judged that the peptide dispersion had lost fluidity and gelled. In contrast, if all of the peptide dispersion fell onto the cap side of the microtube, it was judged that the peptide sample had not gelled.

[0142] The results are shown in Figure 2. After the microtube was inverted, the unmodified JigSAP, C-JigSAP, remained entirely at the bottom of the microtube, lost its fluidity, and turned into a gel (Figure 2A).

[0143] On the other hand, in the case of the modified JigSAP, Me-C-JigSAP, almost all of the peptide dispersion fell onto the cap side of the microtube and did not gel (Fig. 2B).

[0144] Example 2: Verification of gel-decomposing activity of Me-C-JigSAP (Objective) The modified JigSAP, Me-C-JigSAP, synthesized in Example 1, was allowed to act on a gel formed by the unmodified JigSAP, C-JigSAP, to verify whether the gel would be decomposed.

[0145] (Method and Results) 400 μL of the dispersion containing 1.0 wt % Me-C-JigSAP in D-MEM (hereinafter referred to as "Me-C-JigSAP dispersion") that did not gel in Example 1 was added to 100 μL of the gel containing 1.0 wt % C-JigSAP in D-MEM (hereinafter referred to as "C-JigSAP peptide gel") formed on the bottom surface of the microtube prepared in Example 1. The microtube was placed upright with its bottom facing downwards in a CO2 incubator (37°C, CO2 concentration 5%) and gently shaken at 400 rpm for 10 minutes. The tube was then removed from the incubator, quickly turned upside down, and photographs of the microtube were taken and recorded.

[0146] The results are shown in Figure 3. The C-JigSAP peptide gel to which the Me-C-JigSAP dispersion solution was added dissolved, and almost all of the mixture of the two fell into the cap side of the microtube as a peptide dispersion solution (Figure 3).

[0147] This result indicates that Me-C-JigSAP does not form a gel by itself, but has the activity of decomposing a gel composed of C-JigSAP.

[0148] Example 3: Evaluation of cell adhesiveness (Objective) The cell adhesiveness of the C-JigSAP peptide gel was evaluated.

[0149] (Method) A dispersion containing 1.0 wt% C-JigSAP in D-MEM was prepared in the same manner as in Example 1. 100 μL of the dispersion was dropped onto a culture cover glass (Matsunami Glass Industry Co., Ltd., 13 mm diameter, non-coated type, C1100) and coated with C-JigSAP peptide using a spin coater (Mikasa Co., Ltd., MS-B100). 5Mouse embryonic 3T3 cells (NIH3T3 cells) were suspended in 1 mL of DMEM medium (containing 44 mM NaHCO3, Gibco, product number 11995-065) and seeded onto culture cover glasses coated with C-JigSAP peptide (SEQ ID NO: 19) or two other self-assembling peptides (SEQ ID NO: 21 and SEQ ID NO: 22). The cells were then incubated for 30 minutes in a CO2 incubator (37°C, 5% CO2). Nonadherent cells were removed by washing the culture cover glasses with PBS. The remaining adherent cells were fixed with 4% PFA and stained with DAPI. The number of adherent cells was determined using stereology, a rigorous quantitative method that eliminates sampling bias. Specifically, five 638.9 μm × 638.9 μm frames were created within the culture cover glass, and the number of cells within each frame was counted. The resulting cell counts were then converted to the number of cells per 1 mm × 1 mm area. In this example, fibronectin, which is known to have high cell adhesive properties, was used as a control.

[0150] (Results) DAPI staining of adherent cells and the number of adherent cells are shown in Figure 4. When the chamber slide surface was siliconized with Sigmacote (Sigma-Aldrich, SL2-25ML) (Figure 4, "Sigmacote"), and when the chamber slide surface was not treated to actively promote adhesion (Figure 4, "noncoat"), the number of cells adhering to the chamber slide was low. In contrast, the C-JigSAP peptide of the present invention and two other self-assembling peptides (SEQ ID NO: 21 and SEQ ID NO: 22) were shown to have cell adhesive properties equal to or greater than those of fibronectin.

[0151] Example 4: Rheological properties of C-JigSAP peptide gel (Objective) To analyze the rheological properties of C-JigSAP peptide gel.

[0152] (Method) To measure the rheological properties of the 1.0 wt% C-JigSAP peptide gel prepared in Example 1, a 250 μL sample was measured using a rheometer (Malvern Panalytical, Kinexus lab+). Specifically, the sample was sandwiched between plates with a 0.2 mm gap and measured at 20°C. The rheological property measurements yield the storage modulus G' and loss modulus G" of the sample. The storage modulus G' represents the component of energy generated by external force and strain that is stored within the object, while the loss modulus G" represents the component that diffuses to the outside. When the sample being measured is a gel, the storage modulus G' generally exceeds the loss modulus G". In this example, live brain (unfixed brain excised from an ICR mouse), 2.4 mg / mL collagen gel, and undiluted Matrigel were used as controls.

[0153] (Results) The results of measuring the rheological properties are shown in Figure 5. The C-JigSAP peptide gel was shown to have a significantly higher elastic modulus than collagen gel and Matrigel. That is, C-JigSAP can form a significantly harder gel than known peptide gelators. Furthermore, the C-JigSAP peptide gel exhibited an elastic modulus equal to or greater than that of living brain.

[0154] The above results demonstrate that C-JigSAP has a significantly superior elastic modulus compared to known peptide gelators and has excellent durability equivalent to or superior to that of biological tissue.

[0155] Example 5: Three-dimensional culture of retina using C-JigSAP peptide gel (Objective) To perform three-dimensional culture of developing retina using C-JigSAP peptide gel and compare the results with those using Matrigel.

[0156] (Method) Retinal tissue was harvested from ICR mice on embryonic day 14. The harvested retinal tissue included the retina, sclera-free, and the lens. A 1.0 wt% C-JigSAP dispersion was prepared in D-MEM as described in Example 1. 50 μL of the dispersion was dropped onto the retinal tissue placed on the lid of a PCR tube and then incubated in a CO2 incubator (37°C, 5% CO2) for 2 hours to produce a retinal inclusion gel (Figure 6A). The resulting retinal inclusion gel was cultured at the air-liquid interface for 3 days in explant medium (D-MEM / F12 medium containing 10% bovine serum, 20 mM hydroxyethylpiperazineethanesulfonic acid, and 2 μg / mL insulin).

[0157] A 1.0 wt% Me-C-JigSAP dispersion was prepared in D-MEM using the same method as in Example 1. The cultured retinal tissue-containing gel was immersed in the dispersion and gently inverted three times to disintegrate the gel within 5 minutes. The retinal tissue separated from the gel was thinly sectioned, and the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The 3D structure of the cultured retina was then reconstructed by imaging using a confocal microscope.

[0158] As a control, the same experiment as above was performed using Matrigel (BD Biosciences, product number 354234) instead of the C-JigSAP peptide gel. After three-dimensional culture, the Matrigel was removed by cutting it off with tweezers.

[0159] The results are shown in Figure 6B. Retinas cultured in C-JigSAP peptide gel exhibited a three-dimensional structure similar to that of normal retinas. In contrast, normal retinal structure was lost in retinas cultured in Matrigel. These results indicate that Matrigel is unable to maintain retinal three-dimensional structure due to its insufficient strength, whereas C-JigSAP peptide gel provides sufficient strength for the formation of retinal three-dimensional structure, thereby supporting the formation of organoids with normal three-dimensional structure.

[0160] Example 6: Verification of gel decomposition rate and decomposition specificity by Me-C-JigSAP (Objective) The gel decomposition rate and gel decomposition specificity were verified by applying Me-C-JigSAP to a C-JigSAP peptide gel and a peptide gel formed by (RADA)4 peptide (also referred to as "RADA16 peptide" in this specification) (hereinafter referred to as "RADA16 peptide gel").

[0161] (Method and Results) 100 μL of the Me-C-JigSAP dispersion prepared in Example 1 was added to 400 μL of the C-JigSAP peptide gel prepared in Example 1. After shaking at 400 rpm for 1 to 15 minutes in a CO2 incubator (37°C, CO2 concentration 5%), the microtube was removed from the incubator and placed upright with its bottom facing downwards, and a photograph of the microtube was taken and recorded.

[0162] As a control, a similar experiment was performed using a 1 wt% RADA16 peptide gel instead of the C-JigSAP peptide gel. The RADA16 peptide gel was prepared by preparing a 1.0 wt% (RADA)4 peptide (SEQ ID NO: 35) dispersion in D-MEM using the same method as in Example 1. The gel was then placed in a microtube in a CO2 incubator (37°C, 5% CO2) with the bottom facing upright for 2 days.

[0163] The results are shown in Figure 7. The C-JigSAP peptide gel (Figure 7B, dashed box) dissolved rapidly after the addition of Me-C-JigSAP dispersion, and almost all of the gel was dissolved within 7 minutes. In contrast, the RADA16 peptide gel (Figure 7A, dashed box) remained intact after the addition of Me-C-JigSAP dispersion. Thus, while Me-C-JigSAP rapidly degraded the C-JigSAP peptide gel, it did not degrade the peptide gel formed by the RADA16 peptide, which has a completely different amino acid sequence. This result demonstrates the specificity of the degradation activity of Me-C-JigSAP. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A modified peptide having gel decomposition activity, comprising at least one modified core peptide consisting of an amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), wherein any one or more nitrogen atoms constituting the peptide bonds constituting the modified core peptide have a substituent.

2. The modified peptide of claim 1, wherein the substituent is an optionally substituted hydrocarbon group.

3. The modified peptide of claim 2, wherein the hydrocarbon group is selected from the group consisting of a methyl group, an ethyl group, a propyl group, an isopropyl group, and a benzyl group.

4. The modified peptide of claim 1, wherein the nitrogen atom is contained in the seventh amino acid residue from the N-terminus in the amino acid sequence shown in formula I.

5. The modified peptide of claim 1, comprising an Arg residue or an Arg-Arg dipeptide sequence linked to the N-terminus and / or C-terminus of the modified core peptide.

6. The modified peptide of claim 1, wherein the C-terminus is amidated and / or the N-terminus is carboxylated.

7. A composition for decomposing a gel, comprising the modified peptide of claim 1 as an active ingredient.

8. The composition for decomposing a gel according to claim 7, for decomposing a gel in vitro or in vivo.

9. A kit for producing and decomposing a gel, comprising: a self-assembling peptide for producing a gel; and a modified peptide according to claim 1 for decomposing a gel, wherein the self-assembling peptide comprises at least one unmodified core peptide having an amino acid sequence represented by the following formula II: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula II), wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly, and wherein in the unmodified core peptide, a nitrogen atom constituting a peptide bond has no substituent.

10. The kit according to claim 9, wherein the gel is used for culturing organoids.

11. A method for decomposing a gel, comprising: a gel decomposition step of contacting a modified peptide having gel decomposition activity with a gel composed of a self-assembling peptide to decompose the gel, wherein the modified peptide comprises at least one modified core peptide consisting of an amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), wherein a nitrogen atom constituting one or more peptide bonds constituting the modified core peptide has a substituent, and the self-assembling peptide comprises at least one modified core peptide consisting of an amino acid sequence represented by the following formula II: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula II) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly), wherein in the unmodified core peptide sequence, a nitrogen atom constituting a peptide bond does not have a substituent.

12. The gel decomposition method according to claim 11, further comprising: a mixing step of mixing the self-assembling peptide with water or an aqueous solution; and a gelation step of gelling the mixture obtained after the mixing step by maintaining the mixture at a temperature equal to or lower than the gelation temperature.

13. The method according to claim 12, wherein in the mixing step, any one or more anions selected from the group consisting of bicarbonate ions, carbonate ions, citrate ions, tartrate ions, and sulfate ions are further mixed.

14. The method according to claim 12, wherein the gelation step is carried out in the presence of isolated cells, cell aggregates, or tissues to form a gel containing the cells, cell aggregates, or tissues therein, and the method further comprises a culture step of culturing the cells, cell aggregates, or tissues in the gel.

15. The method of claim 11, wherein Xaa and Zaa in formula I have the same amino acid side chains as Xaa and Zaa at the corresponding positions, respectively, in formula II.

16. The method of claim 15, wherein all amino acid residues in formula I have the same amino acid side chain as each amino acid residue at the corresponding position in formula II.

17. A method for producing a cell aggregate and / or tissue, comprising: a mixing step of mixing a self-assembling peptide with water or an aqueous solution; a gelation step of gelling the mixture obtained after the mixing step by maintaining a temperature equal to or lower than the gelation temperature in the presence of isolated cells, cell aggregates, and / or tissues to produce a gel containing the cells, cell aggregates, and / or tissues therein; a culture step of culturing the cells, cell aggregates, and / or tissues in the gel; and a gel decomposition step of decomposing the gel by contacting the gel with a modified peptide having gel decomposition activity, and isolating the cell aggregate and / or tissue by removing the gel from the cell aggregate and / or tissue obtained after the culture step, wherein the modified peptide is represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (Formula I) wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly; at least one nitrogen atom constituting a peptide bond constituting the modified core peptide has a substituent; and the self-assembling peptide comprises at least one unmodified core peptide having an amino acid sequence represented by the following formula II: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula II); wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly; and in the unmodified core peptide sequence, a nitrogen atom constituting a peptide bond does not have a substituent.