Kit for promoting translation into protein, and method for producing protein

Abstract

Provided is a new system that allows introduction of a protein-encoding nucleic acid into a cell and that can improve the efficiency of translating the introduced encoding nucleic acid to a protein.　A translation promotion kit according to the present invention is for introducing a nucleic acid and promoting translation of the nucleic acid that has been introduced, the kit being characterized by comprising: an introduction agent for introducing a protein-encoding nucleic acid and a translation promoter; and a translation promoter, and characterized in that the introduction agent is a plant-derived vesicle and the translation promoter is a membrane-permeable peptide.

Description

Protein translation promotion kit and protein production method

[0001] The present invention relates to a kit for promoting translation into a protein and a method for producing a protein.

[0002] In various fields of medicine and biochemistry, DDS (drug delivery system) platforms for delivering target substances to target cells have become important. For example, if a disease is caused by a deficiency or shortage of a specific protein, a DDS can be used to perform specific protein replacement therapy. That is, by introducing a coding nucleic acid encoding a target protein into cells using a DDS and expressing the target protein within the introduced cells, the target protein can be replenished. Furthermore, in the widely used genome editing, a DDS for introducing a coding nucleic acid for a nuclease into cells is an important tool.

[0003] In recent years, various DDSs have been reported. However, when considering their use in animals and plants in fields such as medicine and agriculture, safety for the target organisms is required, and further improvement in the translation efficiency of proteins based on the introduced coding nucleic acid is also required.

[0004] Therefore, an object of the present invention is to provide a new system that can introduce a protein-encoding nucleic acid into cells and can enhance the translation efficiency of the protein from the introduced encoding nucleic acid.

[0005] In order to achieve the above-mentioned object, the translation promotion kit of the present invention is a kit for introducing a nucleic acid and promoting the translation of the introduced nucleic acid, and is characterized in that it includes an introduction agent for introducing a protein-encoding nucleic acid and a translation promotion agent, and the translation promotion agent, wherein the introduction agent is a plant-derived vesicle, and the translation promotion agent is a membrane-permeable peptide.

[0006] The method for producing a protein of the present invention is characterized by comprising: a mixing step of using the translation promotion kit of the present invention and mixing an introduction agent of the translation promotion kit with a nucleic acid encoding a protein to prepare a mixed solution; and a culturing step of culturing cells in the presence of the mixed solution and the translation promotion agent of the translation promotion kit.

[0007] The method for producing a protein of the present invention is characterized by comprising: a mixing step of using the translation promotion kit of the present invention and mixing an introduction agent of the translation promotion kit with a nucleic acid encoding a protein to prepare a mixed solution; and an administration step of administering the mixed solution and the translation promotion agent of the translation promotion kit to a subject.

[0008] The translation promotion kit of the present invention can introduce a protein-encoding nucleic acid and a membrane-permeable peptide, which is the translation promoter, into cells using the plant-derived vesicle, which is the introduction agent. By introducing the membrane-permeable peptide into cells together with the protein-encoding nucleic acid, protein translation from the protein-encoding nucleic acid can be promoted. Therefore, the translation promotion kit of the present invention is useful for methods of introducing a protein-encoding nucleic acid into cells to synthesize a protein within the cells, such as protein replacement therapy and genome editing.

[0009] 1 is a graph showing the particle size distribution of vesicle sample A in Example 1. FIG. 2 is a graph showing the particle size distribution of vesicle sample B in Example 1. FIG. 3 is a graph showing the particle size distribution of vesicle sample C in Example 1. FIG. 4 is a micrograph of vesicle sample C in Example 1. FIG. 5 is a graph of relative values ​​showing the amount of Flue mRNA in cells in Example 2. FIG. 6 is a graph showing the hAPOA1 protein concentration in serum in Example 3. FIG. 7 is a graph showing the hAPOA1 protein concentration in liver in Example 3. FIG. 8 is a graph showing the hAPOA1 protein concentration in serum in Example 3. FIG. 9 is a graph showing the hAPOA1 protein concentration in liver in Example 3. FIG. 10 is a graph showing the relative intensity of luminescence due to the luciferase reaction of the culture in Example 4. FIG. 11 is a graph showing the relative intensity of luminescence due to the luciferase reaction of the culture in Example 4. FIG. 12 is a graph showing the relative luminescence amount corresponding to the amount of hAPOA1 protein in the culture in Example 5. FIG. 13 is a graph showing the particle size distribution of vesicle complexes containing hAPOA1 mRNA in Example 6. 1 is a graph showing the particle size distribution of vesicle complexes containing Fluc mRNA in Example 6. FIG. 2 is a graph showing the resistance of vesicle complexes in Example 6.

[0010] Unless otherwise specified, terms used in this specification can be used in the sense commonly used in the art.

[0011] The present invention includes, for example, the following embodiments. [1] A translation promotion kit for introducing a nucleic acid and promoting the translation of the introduced nucleic acid, comprising an introduction agent for introducing a protein-encoding nucleic acid and a translation promotion agent, and the translation promotion agent, wherein the introduction agent is a plant-derived vesicle, and the translation promotion agent is a membrane-permeable peptide. [2] The translation promotion kit according to [1], wherein the membrane-permeable peptide is a basic peptide. [3] The translation promotion kit according to [1] or [2], wherein the membrane-permeable peptide is an arginine-rich peptide. [4] The translation promotion kit according to any one of [1] to [3], wherein the membrane-permeable peptide contains 8 to 16 arginine residues. [5] The translation promotion kit according to any one of [1] to [4], wherein the membrane-permeable peptide has 80 to 100% arginine residues among all amino acid residues. [6] The translation promotion kit according to any one of [1] to [5], wherein the plant-derived vesicles are vesicles derived from the fruit of a plant. [7] The translation promotion kit according to any one of [1] to [6], wherein the plant-derived vesicles are vesicles derived from a plant of the family Canthaceae. [8] The translation promotion kit according to [7], wherein the plant of the family Canthaceae is a plant of the Acerola species. [9] The translation promotion kit according to any one of [1] to [8], further comprising a protein-encoding nucleic acid.

[10] The translation promotion kit according to [9], wherein the protein-encoding nucleic acid has DNA encoding the protein as the protein-coding sequence.

[11] The translation promotion kit according to [9] or

[10] , wherein the protein-encoding nucleic acid is an expression vector having the protein-encoding sequence.

[12] A method for producing a protein using the translation promotion kit according to any one of [1] to [8], comprising: a mixing step of mixing an introduction agent of the translation promotion kit with the protein-encoding nucleic acid to prepare a mixed solution; and a culturing step of culturing cells in the presence of the mixed solution and the translation promoter of the translation promotion kit.

[13] The method for producing a protein according to

[12] , wherein in the mixing step, an introduction agent and a translation promoter of the translation promotion kit are mixed with a nucleic acid encoding the protein to prepare the mixed solution; and in the culturing step, cells are cultured in the presence of the mixed solution.

[14] The method for producing a protein according to

[13] , further comprising a step of incubating the mixture, wherein cells are cultured in the presence of the incubated mixture.

[15] The method for producing a protein, comprising using the translation promotion kit according to any one of [1] to [8], a mixing step of mixing an introduction agent of the translation promotion kit with a nucleic acid encoding a protein to prepare a mixture, and an administration step of administering the mixture and the translation promoter of the translation promotion kit to a subject.

[16] The method for producing a protein according to

[15] , wherein the mixing step involves mixing an introduction agent and a translation promoter of the translation promotion kit with the nucleic acid encoding the protein to prepare the mixture, and the administration step involves administering the mixture to a subject.

[17] The method for producing a protein according to

[16] , further comprising a step of incubating the mixture, wherein the incubated mixture is administered to a subject.

[18] The method for producing a protein according to any one of

[15] to

[17] , wherein the subject is a human or a non-human animal.

[19] The method for producing a protein according to any one of

[15] to

[18] , wherein the administration of the mixture is oral administration.

[20] The method for producing a protein according to any one of

[15] to

[18] , wherein the administration is parenteral administration.

[21] The method for producing a protein according to any one of

[15] to

[20] , wherein the method is a method for supplementing protein in a living organism.

[22] The method for producing a protein according to any one of

[12] to

[21] , wherein the protein-encoding nucleic acid has DNA encoding the protein as the protein-coding sequence.

[24] The method for producing a protein according to any one of

[12] to

[22] , wherein the protein-encoding nucleic acid is an expression vector having the protein-coding sequence.

[0012] [1] Translation Promotion Kit As described above, the translation promotion kit of the present invention is a translation promotion kit for introducing a nucleic acid and promoting the translation of the introduced nucleic acid, and is characterized in that it includes an introduction agent for introducing a protein-encoding nucleic acid and a translation promotion agent, and the translation promotion agent, wherein the introduction agent is a plant-derived vesicle, and the translation promotion agent is a membrane-permeable peptide.

[0013] The present inventors have found that the plant-derived vesicles can carry a nucleic acid encoding a protein (hereinafter referred to as "encoding nucleic acid") as a delivery agent and can deliver the carried encoding nucleic acid into cells (Patent Application No. PCT / JP2023 / 35106). As a result of extensive research into the expression of a protein from the encoding nucleic acid delivered to cells, they have found that the expression of a protein from the encoding nucleic acid in cells can be promoted by delivering the membrane-permeable peptide using the plant-derived vesicles when delivering the encoding nucleic acid to cells.

[0014] (1-1) Delivery Agent As described above, the delivery agent in the present invention is the plant-derived vesicle. The plant-derived vesicle can carry (also referred to as retain) nucleic acids and peptides and can deliver the carried nucleic acids and peptides into cells. Therefore, according to the present invention, the plant-derived vesicle, which is the delivery agent, can carry the encoding nucleic acid and the membrane-permeable peptide and deliver them to cells. Therefore, in the present invention, the delivery agent is also referred to as, for example, a carrier, a delivery reagent, or a DDS reagent. The state in which the plant-derived vesicle carries the nucleic acid and / or peptide is hereinafter also referred to as a vesicle complex.

[0015] The type of plant from which the vesicles serving as the introduction agent are derived is not particularly limited, and examples thereof include plants of the Malpighiaceae family.

[0016] The type of plant of the Malpighiaceae family is not particularly limited, and examples thereof include the genus Malpighia, and specifically include acerola species (Malpighia sp.), and preferably acerola such as M. emarginata DC., M. glabra, and M. punicifolia.

[0017] The plant-derived vesicles can be prepared from, for example, the entire plant (individual) or any part of the plant. Examples of the part include fruit, fruit peel, pulp, pulp including seeds, fruit juice, seeds, leaves, etc. The raw material may be, for example, one type of the part, or may contain two or more types of the part. The raw material is preferably, for example, fruit, pulp, fruit juice, etc. Fruit juice can be prepared, for example, by squeezing fruit or pulp.

[0018] The method for preparing the plant-derived vesicles is not particularly limited, but as an example, a method for preparing the vesicles from the fruits of plants is shown below.

[0019] The fruit may be, for example, fully ripe, unripe, or a mixture thereof. The fruit may be, for example, any of room temperature fruit stored at room temperature, refrigerated fruit stored in a refrigerator, and frozen fruit stored in a freezer. The fruit juice may be, for example, any of room temperature fruit juice stored at room temperature, refrigerated fruit juice stored in a refrigerator, and frozen fruit juice stored in a freezer. The vesicles are preferably, for example, a vesicle fraction described below recovered from the juice of the fruit.

[0020] The plant-derived vesicles can be prepared, for example, by fractionation from the fruit. The preparation method is not particularly limited, and examples thereof include a method of recovering a liquid fraction containing vesicles from the fruit and fractionating the vesicles from the liquid fraction. The liquid fraction may be, for example, fruit juice squeezed from the fruit, crushed fruit, a suspension of the crushed fruit, or an extract from the crushed fruit. The fruit juice can be prepared by squeezing the fruit using, for example, a grinder or a squeezer. The suspension can be prepared, for example, by crushing the fruit and mixing the crushed fruit with a solvent. The extract can be prepared, for example, by mixing the crushed fruit with a solvent and recovering the liquid fraction. The solvent is not particularly limited, and for example, an aqueous solvent can be used. Examples of the aqueous solvent include water, a buffer solution, physiological saline, and buffered physiological saline. Examples of buffered physiological saline include phosphate-buffered saline (PBS).

[0021] The method for fractionating vesicles from the liquid fraction is not particularly limited, and examples thereof include ultrafiltration, ultracentrifugation, concentration gradient methods, and separation methods using a microfluidic system. For the preparation method, for example, a commercially available kit may be used, such as ExoEasy Maxi Kit (trade name, QIAGEN), ExoQuick (trade name, System Bioscience), or Total Exosome Isolation reagent (trade name, Invitrogen).

[0022] The introduction agent of the present invention can be, for example, a vesicle fraction containing a plurality of the plant-derived vesicles. The size of the vesicles is not particularly limited, and examples of particle sizes include 30 to 400 nm, 80 to 300 nm, 150 to 300 nm, 150 to 250 nm, 200 to 250 nm, 100 to 200 nm, and 80 to 200 nm. The vesicles are also called, for example, microvesicles or nanovesicles. The vesicle fraction, when expressed in terms of particle size distribution, has a particle size peak that is not particularly limited and is, for example, 30 to 400 nm, 80 to 300 nm, 150 to 300 nm, 150 to 250 nm, 200 to 250 nm, 100 to 200 nm, 80 to 200 nm, 200±100 nm, 200±50 nm, 200±30 nm, or 200±20 nm. Furthermore, in the particle size distribution, when all vesicles are taken as 100%, the proportion of vesicles at the peak (e.g., 200±50 nm, 200±20 nm) is not particularly limited and has a lower limit of, for example, 30% or more, 50% or more, or 80% or more, and an upper limit of, for example, 70% or less, 80% or less, 90% or less, or 100%. The vesicle fraction is, for example, a fraction extracted from the liquid fraction so as to have the above particle size and particle size distribution. When the vesicle fraction is used as the introducing agent, for example, a fraction extracted from the liquid fraction by an extraction method so as to have the above particle size and particle size distribution can be used. The vesicle fraction may, for example, contain other components derived from the liquid fraction (e.g., the fruit juice or the extract).

[0023] The method for measuring the particle size of vesicles is not particularly limited, and can be, for example, a light scattering method, a measurement method based on Brownian motion, an electrical resistance method, etc. Measurement methods based on Brownian motion include, for example, nanoparticle tracking analysis, and a commercially available nanoparticle analyzer (trade name: NanoSight, Malvern) can be used. Furthermore, the electrical resistance method can also be performed using, for example, a commercially available nanoparticle multi-analyzer (trade name: qNANO, trade name: Exoid). When using NanoSight, measurement conditions include, for example, a measurement time of 90 seconds and three repetitions.

[0024] The plant-derived vesicles may be, for example, either extracellular vesicles or intracellular vesicles, with extracellular vesicles being preferred. Examples of the vesicles include exosome-like vesicles. The exosome-like vesicles are, for example, vesicles of a size equivalent to that of extracellular vesicles (EVs) derived from human cells, more specifically, vesicles of a size equivalent to that of small EVs (also referred to as exosomes) derived from human cells. The exosome-like vesicles are, for example, vesicles obtained from the plant by the same isolation method as that for human cell-derived extracellular vesicles, preferably human cell-derived exosomes.

[0025] The extracellular vesicles can also be detected using, for example, plant extracellular vesicle markers. Examples of the markers include TET8, Heat Shock Proteins (HSP60, HSP70, HSP80, HSP90), Aquaporin family Patellines (Patellin 1, Patellin 2, Patellin 3), Syntaxins, Clathrin heavy chain family (Clathrin 1, Clathrin 2), and Sphingolipids. For information on the markers, see, for example, the following literature: Nemati et al., Plant-derived extracellular vesicles: a novel nanomedicine approach with advantages and challenges. Cell Communication and Signaling 2022

[0026] (1-2) Translation-Enhancing Agent As described above, the translation-enhancing agent of the present invention is a cell-penetrating peptide, hereinafter also referred to as a CPP. A CPP is generally a peptide that permeates a cell membrane.

[0027] In the present invention, the CPP used as the translation promoter is not particularly limited. The length of the peptide is not particularly limited, and the number of amino acid residues is, for example, 6 to 21, 6 to 16, 6 to 8, 9 to 16, or 17 to 21.

[0028] Examples of the CPP include basic peptides. Specific examples of the basic peptides include arginine-rich peptides. The arginine-rich peptides contain, for example, multiple arginine residues, and the number of arginine residues is, for example, 6 to 16, 6 to 8, 9 to 16, 9 to 12, 12 to 16, or 13 to 16. Furthermore, the arginine-rich peptides contain, for example, a certain number or more of arginine residues among all amino acid residues, and the proportion of arginine residues among all amino acid residues is, for example, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 70 to 80%, or 80 to 90%. Examples of arginine-rich peptides include polyarginine and FHV peptide derived from flock house virus (total length 15 amino acid residues, number of arginine residues 11, database Uniprot, accession number P12870).

[0029] The polyarginine is a peptide consisting of only consecutive arginine residues, and can be represented by, for example, nR (it can also be represented by Rn). n is a positive integer and indicates the number of arginine residues. n is, for example, 6 to 16, or 12 to 16. Specific examples of polyarginine include 9R (a peptide consisting of nine consecutive arginine residues), 12R, and 16R.

[0030] (1-3) Protein-Encoding Nucleic Acid As described above, the translation promotion kit of the present invention allows the encoding nucleic acid to be introduced into cells. Then, within the introduced cells, translation into a target protein is carried out based on the encoding nucleic acid. There are no particular limitations on the type of encoding nucleic acid to be introduced into cells using the translation promotion kit of the present invention. Hereinafter, the protein encoded by the encoding nucleic acid will also be referred to as a target protein.

[0031] The encoding nucleic acid may be a nucleic acid consisting of a nucleic acid sequence (coding sequence) that encodes a target protein, or may be a nucleic acid that includes the coding sequence. When the encoding nucleic acid is introduced into a cell using the translation enhancement kit of the present invention, the target protein is synthesized by the protein translation mechanism of the cell. Therefore, the encoding nucleic acid may include, for example, a sequence that serves as an open reading frame (e.g., an open reading frame fragment (ORF)) that enables the target protein to be synthesized by the cellular translation mechanism. In other words, the encoding nucleic acid may be, for example, capable of expressing the target protein based on the coding sequence in the cell into which it is introduced.

[0032] In the present invention, the term "protein" is used in a broad sense, and may refer to, for example, a protein in which multiple amino acid residues are linked by peptide bonds, with no particular limitation on its length. When the length (number of amino acid residues) is used as a standard, the term "protein" in the present invention also includes, for example, peptides such as so-called oligopeptides and polypeptides (e.g., 2 to 50 residues), as well as longer proteins in the narrower sense (e.g., 50 or more residues). The length of a protein in the broad sense is not particularly limited, and examples of such lengths include a lower limit of 2, 4, 6, 10, or 50 amino acid residues, an upper limit of 800, 500, 100, or 49 amino acid residues, and a range of 2 to 800 or 10 to 800 amino acid residues. The number of amino acid residues of the peptide can be, for example, 2 to 49, 4 to 49, 6 to 49, or 10 to 49, and the number of amino acid residues of the protein in the narrow sense can be, for example, 50 to 800, 50 to 500, 100 to 500, or 50 to 100.

[0033] Since the coding sequence encodes a target protein, its length can be, for example, the length calculated from the length of the protein (number of amino acid residues). Specific examples of the length of the coding sequence include a lower limit of the number of bases of 6, 12, 18, 30, or 150, an upper limit of the number of bases of 2400, 1500, 300, or 441, and a range of the number of bases of 6 to 2400 or 30 to 2400. Examples of the number of bases encoding the peptide include 6 to 441, 12 to 441, 18 to 441, or 30 to 441, and examples of the number of bases encoding the protein in the narrow sense include 150 to 2400, 150 to 1500, 300 to 1500, or 150 to 300. The length of the encoding nucleic acid is not particularly limited, and may be, for example, the same as the length of the coding sequence, or may be about 1 to 300 bases, 1 to 200 bases, or 1 to 100 bases longer than the length of the coding sequence.

[0034] The type of the target protein is not particularly limited and can be determined depending on the purpose, for example. Specific examples include functional proteins such as enzymes, antibodies, antigen-binding fragments, and transcription factors, as well as peptides thereof.

[0035] The constituent units of the coding sequence are, for example, nucleotides, and specific examples include ribonucleotides and deoxyribonucleotides. The coding sequence may be, for example, a ribonucleotide nucleic acid (ribonucleic acid, RNA), a deoxyribonucleotide nucleic acid (deoxyribonucleic acid, DNA), or a chimera of RNA and DNA. When the coding sequence is RNA, it may be, for example, pre-mRNA or mRNA, and preferably mRNA. When the coding sequence is DNA, it is preferably cDNA. The nucleotides may be, for example, unmodified or modified. The constituent units of the coding sequence may be, for example, natural nucleic acids, artificial nucleic acids, or both. Examples of the artificial nucleic acids include LNA (locked nucleic acid).

[0036] When the encoding nucleic acid is a nucleic acid containing the coding sequence, the constituent units of the region other than the coding sequence may be, for example, the same constituent units as those of the coding sequence.

[0037] Furthermore, when the encoding nucleic acid is a nucleic acid having the coding sequence, the encoding nucleic acid may be, for example, an expression vector capable of expressing the protein. Specifically, the encoding nucleic acid may be an expression vector into which the coding sequence is inserted so that the protein can be expressed. The coding sequence in the expression vector may be, for example, DNA, preferably cDNA, or may be, for example, RNA, preferably mRNA. The type of vector is not particularly limited, and examples thereof include a plasmid vector and a viral vector. Examples of the viral vector include an adenovirus vector and a Sendai virus vector.

[0038] (1-4) Form of Translation Promotion Kit In the translation promotion kit of the present invention, for example, the introduction agent and the translation promoter may be in an independent state (non-contact state) until use by a user, or the introduction agent and the translation promoter may be in contact with each other in advance. In the latter case, for example, the introduction agent may be in a state where the translation promoter is supported by the introduction agent.

[0039] The translation promotion kit of the present invention may further include, for example, the encoding nucleic acid. When the translation promotion kit of the present invention includes the encoding nucleic acid, for example, the introduction agent and / or the translation promoting agent and the encoding nucleic acid may be in a separate state (non-contact state) until use by the user, or the introduction agent and / or the translation promoting agent and the encoding nucleic acid may be in a contact state in advance. When the introduction agent and the encoding nucleic acid are in a contact state, for example, the introduction agent may carry the encoding nucleic acid.

[0040] (1-5) Method of Use The translation enhancement kit of the present invention can be used as a tool for introducing the encoding nucleic acid into cells to synthesize proteins. The application fields are not particularly limited, and it is useful as a research tool for, for example, pharmaceuticals, diagnostic agents, and pesticides, as well as agriculture, medicine, food science, life science, and the like.

[0041] The translation promotion kit of the present invention can be used, for example, in vivo and in vitro. In the present invention, the introduction agent is derived from a plant as described above, and no transfection reagent or the like is required, so that, for example, effects on the target cells or living organisms can be suppressed. The translation promotion kit of the present invention is also less susceptible to the effects of gastric acid, so that it is also effective for oral administration when used in vivo.

[0042] Genome editing, which is widely used in the fields of medicine, agriculture, etc., requires, for example, the function of nuclease in cells. Therefore, in genome editing, for example, by using the present invention, the encoding nucleic acid of nuclease can be introduced by the introduction agent, and the nuclease can be expressed. The nuclease is not particularly limited, and examples thereof include Cas proteins such as Cas9 proteins, ZFN proteins, TALEN proteins, etc.

[0043] In the field of medicine, the translation promotion kit of the present invention can be used as a replacement therapy for diseases caused by, for example, non-expression or insufficient expression of a necessary protein. For example, for low HDL cholesterol, a condition caused by dyslipidemia, replacement of apolipoprotein A1, a major component of HDL cholesterol, can be considered. In this case, for example, by administering mRNA encoding human apolipoprotein A1 (hAPOA1) to a patient with low HDL cholesterol using the translation promotion kit of the present invention, replacement therapy can be performed to promote the translation (synthesis) of apolipoprotein A1 in vivo.

[0044] The method for using the translation enhancement kit of the present invention will be described later in the method for producing the protein of the present invention.

[0045] [2] Protein Production Method According to the translation promotion kit of the present invention, the encoding nucleic acid and the membrane-permeable peptide can be introduced into cells using the introduction agent, for example, in vitro and in vivo. Then, in the cells, the target protein can be produced by translation of the encoding nucleic acid in the presence of the membrane-permeable peptide. The present invention is characterized by the use of the introduction agent and the translation promotion agent in the translation promotion kit, and other steps and conditions are not particularly limited.

[0046] The method for producing a protein of the present invention uses the translation promoting kit of the present invention. The introduction agent and the translation promoting agent in the translation promoting kit can be, for example, the translation promoting kit of the present invention.

[0047] The methods for producing the protein of the present invention will be explained below by giving examples of a first in vitro production method and a second in vivo production method.

[0048] (2-1) First manufacturing method: in vitro The first manufacturing method is characterized by comprising a mixing step of using the translation promotion kit of the present invention and mixing the introduction agent of the translation promotion kit with the encoding nucleic acid to prepare a mixed solution, and a culturing step of culturing cells in the presence of the mixed solution and the translation promotion agent of the translation promotion kit.

[0049] In the mixing step, the mixture may contain, for example, the introduction agent (the plant-derived vesicle) and the encoding nucleic acid, or may contain the introduction agent, the encoding nucleic acid, and the translation enhancer (the CPP), preferably the latter. In this case, the mixing step preferably involves, for example, mixing the introduction agent and translation enhancer of the translation enhancement kit with the encoding nucleic acid to prepare the mixture. The mixture is preferably prepared using, for example, a solvent, and the introduction agent, the encoding nucleic acid, and preferably the translation enhancer are mixed in the solvent.

[0050] The solvent is not particularly limited, and for example, an aqueous solvent can be used, specific examples of which include water, physiological saline, a buffer solution such as PBS, a liquid medium, etc. The liquid medium is not particularly limited, and can be appropriately determined depending on the type of cells into which the encoding nucleic acid is introduced.

[0051] In the mixed solution, the ratio of the plant-derived vesicles to the encoding nucleic acid is not particularly limited, and may be, for example, 5 × 10 plant-derived vesicles per 1 μmol of the encoding nucleic acid. ４ ~8 x 10 ４ particles, 2×10 ５ ~5 x 10 ５ particles, 2×10 ６ ~5 x 10 ６ In the mixed solution, the ratio of the CPP to the encoding nucleic acid is not particularly limited, and may be, for example, 0.1 nmol to 10 μmol, 0.1 nmol to 5 μmol, 0.1 nmol to 2.5 μmol, 0.05 μmol to 10 μmol, 1 μmol to 10 μmol, 0.05 μmol to 0.1 μmol, or 0.1 μmol to 1 μmol of the CPP relative to 1 μmol of the encoding nucleic acid.

[0052] The concentration of the plant-derived vesicles in the mixture is not particularly limited, and may be, for example, 5 × 10 ７ ~8 x 10 ７ particles / mL, 2×10 ８ ~5 x 10 ８ particles / mL, 2×10 ９ ~5 x 10 ９ The concentration of the encoding nucleic acid in the mixture is not particularly limited and is, for example, 5 to 500 μmol / mL, 5 to 100 μmol / mL, 5 to 50 μmol / mL, or 10 to 50 μmol / mL.

[0053] As described above, the plant-derived vesicles can carry nucleic acids and peptides. Therefore, by allowing the plant-derived vesicles to coexist with the encoding nucleic acid and the membrane-permeable peptide in the mixed solution, a vesicle complex containing the plant-derived vesicles, the encoding nucleic acid, and the membrane-permeable peptide can be formed. The form of the vesicle complex is not particularly limited, and may be, for example, a form in which the encoding nucleic acid and the membrane-permeable peptide are encapsulated inside the vesicles, or a form in which the encoding nucleic acid and the membrane-permeable peptide are retained on the outer wall (outer surface) of the vesicles.

[0054] The nucleic acid and peptide can be loaded into the plant-derived vesicles using common introduction methods such as electroporation, lipofection, etc. In the present invention, the plant-derived vesicles can retain the encoding nucleic acid and the membrane-permeable peptide without using these common introduction methods, simply by allowing the vesicles to coexist with the encoding nucleic acid and the membrane-permeable peptide.

[0055] The first production method preferably further comprises, for example, a step of incubating the mixture after the mixing step. By further incubating the mixture, the vesicle complex can be formed more efficiently.

[0056] The incubation conditions are not particularly limited. The incubation temperature is not particularly limited and may be, for example, in the room temperature range (e.g., 30±10°C) or a lower temperature range (e.g., above 0°C and below 20°C), preferably in the low temperature range (e.g., 4±5°C or 4±3°C), and more preferably under ice-cooled conditions (e.g., 1 to 6°C). In this way, for example, by incubating at a temperature in the low temperature range or lower, it is possible to prevent, for example, degradation of the encoding nucleic acid before the formation of the vesicle complex due to the presence of a nuclease such as RNase.

[0057] The incubation time is not particularly limited, and the lower limit is, for example, 5 minutes or more, 15 minutes or more. The upper limit of the incubation time is not particularly limited, and for example, incubation for about 30 minutes allows the formation of the vesicle complex to reach a plateau.

[0058] Next, cells are cultured in the presence of the mixed solution. Specifically, for example, cells are cultured in a medium to which the mixed solution has been added. When a liquid medium is used as the solvent in preparing the mixed solution, for example, the mixed solution prepared using the liquid medium may be used as is to culture cells.

[0059] In this embodiment, as described above, it is preferable that a mixed solution containing the introduction agent, the translation enhancer, and the encoding nucleic acid is prepared in the mixing step, and cells are cultured in a medium to which the mixed solution has been added. Note that, for example, if the mixed solution does not contain the translation enhancer of the translation enhancement kit, the mixed solution containing the introduction agent and the encoding nucleic acid and the translation enhancer can also be added to the medium to culture cells.

[0060] The concentration of the plant-derived vesicles in the medium is not particularly limited, and examples thereof include the following concentrations: ７ ~5 x 10 ９ particles / mL, 2×10 ８ ~5 x 10 ９ particles / mL, 2×10 ９ ~5 x 10 ９ particles / mL, 5×10 ７ ~5 x 10 ８ particles / mL, 2×10 ８ ~5 x 10 ８ particles / mL, 5×10 ７ ~8 x 10 ７ The concentrations of the encoding nucleic acid and the CPP in the medium are not particularly limited, and can be calculated, for example, from the concentration of the vesicles in the medium and the concentrations of each component in the mixed solution.

[0061] The type of the medium is not particularly limited and can be determined appropriately depending on the type of cells. Examples of the medium include liquid media such as DMEM / F12 and RPMI1640.

[0062] The culture conditions are not particularly limited and can be determined appropriately depending on the type of cells, and examples thereof include the following: Culture temperature: 35 to 37°C, 36 to 37°C Culture time: 18 to 48 hours

[0063] The mixture contains the plant-derived vesicles (vesicle complexes) carrying the encoding nucleic acid and the membrane-permeable peptide. Therefore, by culturing cells in the presence of the mixture, i.e., culturing the cells in a state where the vesicle complexes in the mixture are in contact with the cells, the encoding nucleic acid and the membrane-permeable peptide can be introduced into the cells via the plant-derived vesicles. The target protein encoded by the encoding nucleic acid can then be synthesized in the cells by the cellular translation machinery. Furthermore, according to the present invention, the membrane-permeable peptide can also be introduced into cells, thereby promoting translation into the target protein.

[0064] In the present invention, the promotion of translation into the target protein refers to, for example, increasing the expression level (E ０ ) compared to the expression level of the target protein (E １ ) is relatively high. １ / E ０ ) is, for example, greater than 1.0, and preferably, for example, 1.1 or more, 1.2 or more, 1.5 or more, or 2 or more.

[0065] In the first production method, the subject to be cultured in the culture step may be, for example, a cell, or a tissue or organ composed of cells. The cell may be, for example, a cell collected from a living organism, a cell line, a cultured cell, or a genetically engineered cell. The subject may be derived from, for example, a human or a non-human animal. Examples of the non-human animal include a mouse, rat, rabbit, dog, monkey, camel, and cow.

[0066] (2-2) Second Production Method: In Vivo The second production method is an in vivo protein production method, which includes a mixing step using the translation promotion kit of the present invention to mix the introduction agent of the translation promotion kit with the encoding nucleic acid to prepare a mixed solution, and an administration step of administering the mixed solution and the translation promotion agent of the translation promotion kit to a subject. The protein production method of the present invention can also be referred to as, for example, a method for supplementing a protein into a living body.

[0067] In the mixing step, the mixture may contain, for example, the introduction agent (the plant-derived vesicles) and the encoding nucleic acid, or may contain the introduction agent, the encoding nucleic acid, and the translation enhancer (the CPP), preferably the latter. In this case, the mixing step preferably involves, for example, mixing the introduction agent and translation enhancer of the translation enhancement kit with the encoding nucleic acid to prepare the mixture. The mixture is preferably prepared using, for example, a solvent, and the introduction agent, the encoding nucleic acid, and preferably the translation enhancer, are mixed in the solvent. The solvent is not particularly limited, and for example, a pharmaceutically acceptable solvent can be used. The solvent is, for example, an aqueous solvent, and specific examples include water, physiological saline, and buffer solutions such as PBS. The description of the mixing step in the first manufacturing method can be applied to the mixing step. The mixture may be, for example, incubated, and the description of the incubation step in the first manufacturing method can be applied to the mixing step.

[0068] Next, in the administering step, the mixture is administered to the subject. In this embodiment, as described above, in the mixing step, it is preferable to prepare a mixture containing the introduction agent, the translation enhancer, and the encoding nucleic acid, and administer the mixture. Note that, for example, if the mixture does not contain the translation enhancer of the translation enhancement kit, the mixture containing the introduction agent and the encoding nucleic acid and the translation enhancer can also be administered.

[0069] The administration method is not particularly limited and includes, for example, oral administration and parenteral administration, with oral administration being preferred. By using the plant-derived vesicles, the translation promotion kit of the present invention can introduce the encoding nucleic acid and CPP into living cells, for example, by oral administration as well as parenteral administration, thereby promoting intracellular protein expression. Examples of parenteral administration include intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, and topical administration.

[0070] The type of subject to be administered is not particularly limited, and examples thereof include humans and non-human animals. Examples of the non-human animals include mice, rats, rabbits, dogs, monkeys, camels, and cows.

[0071] The dosage of the mixture is not particularly limited and can be determined appropriately depending on, for example, the type of the target protein, the type, age, sex, and purpose of administration of the subject. The dosage of the mixture can be expressed, for example, based on the amount of the plant-derived vesicles contained in the mixture. The dosage of the plant-derived vesicles is, for example, 2 x 10 per kg of body weight for a human adult. ７ ~8 x 10 ９ particles, 2×10 ８ ~8 x 10 ９ particles, 5×10 ９ ~8 x 10 ９ particles, 2×10 ７ ~8 x 10 ８ particles, 2×10 ８ ~8 x 10 ８ particles, 2×10 ７ ~5 x 10 ７When the dosage form is oral administration or parenteral administration, for example, these dosages can be referred to.

[0072] In the present invention, the method of administration is preferably, for example, oral administration. In the case of oral administration, the following specific conditions can be mentioned.

[0073] The dose of the plant-derived vesicles is, for example, 2×10 per kg of body weight for an adult human. ８ ~8 x 10 １０ particles, 2×10 ９ ~8 x 10 １０ particles, 5×10 １０ ~8 x 10 １０ particles, 2×10 ８ ~8 x 10 ９ particles, 2×10 ８ ~5 x 10 ８ particles, 2×10 ９ ~8 x 10 ９ The ratio of the plant-derived vesicles to the encoding nucleic acid is not particularly limited, and may be, for example, 5 × 10 plant-derived vesicles per 1 μmol of the encoding nucleic acid. ５ ~5 x 10 ７ particles, 2×10 ６ ~5 x 10 ７ particles, 2×10 ７ ~5 x 10 ７ particles, 5×10 ５ ~5 x 10 ６ particles, 2×10 ６ ~5 x 10 ６ particles, 5×10 ５ ~8 x 10 ５ The ratio of the CPP to the encoding nucleic acid is not particularly limited, and may be, for example, 0.5 to 100 μmol, 1 to 100 μmol, 10 to 100 μmol, 0.5 to 10 μmol, 0.5 to 1 μmol, or 1 to 10 μmol of the CPP relative to 1 μmol of the encoding nucleic acid.

[0074] The concentration of the plant-derived vesicles in the mixture is not particularly limited, and may be, for example, 5 × 10 ８ ~5 x 10 １０ particles / mL, 2×10 ９ ~5 x 10 １０ particles / mL, 2×10 １０ ~5 x 10 １０ particles / mL, 5×10 ８ ~5 x 10 ９ particles / mL, 2×10 ９ ~5 x 10 ９ particles / mL, 5×10 ８ ~8 x 10 ８ The concentration of the encoding nucleic acid in the mixed solution is not particularly limited and is, for example, 50 to 5000 μmol / mL, 50 to 1000 μmol / mL, 50 to 500 μmol / mL, or 100 to 500 μmol / mL.

[0075] According to the present invention, which uses the introduction agent (the plant-derived vesicles), the encoding nucleic acid and the translation promoter (the CPP) can be efficiently delivered to the blood and organs such as the liver, for example, by oral administration, and the target protein can be efficiently expressed from the encoding nucleic acid in the blood and organs such as the liver.

[0076] (2-3) Modifications In the first and second production methods, a specific example has been given in which the encoding nucleic acid and the membrane-permeable peptide are brought into contact with the plant-derived vesicles to form a vesicle complex carrying the encoding nucleic acid and the membrane-permeable peptide. This is not limiting, and for example, the encoding nucleic acid and the membrane-permeable peptide may be carried by the plant-derived vesicles in separate steps.

[0077] That is, the mixing step may include, for example, a step of mixing the introduction agent (the plant-derived vesicles) with the encoding nucleic acid to prepare a mixed solution 1, and a step of mixing the introduction agent (the plant-derived vesicles) with the translation enhancer (the CPP) to prepare a mixed solution 2. The preparation of the mixed solution 1 and the preparation of the mixed solution 2 may be performed, for example, simultaneously in parallel or sequentially. For example, the solvent described above can be used to prepare the mixed solution.

[0078] In the incubation step, the incubation of the mixed solution 1 and the incubation of the mixed solution 2 may be carried out separately, or the mixed solution 1 and the mixed solution 2 may be further mixed, and the resulting mixed solution (mixed solution 3) may be incubated.

[0079] When the mixed solution 1 and the mixed solution 2 are separate (independent), in the culture step in the first production method, the mixed solution 1 and the mixed solution 2 may be added to the same medium, and cells may be cultured in the medium containing the mixed solution 1 and the mixed solution 2. When the mixed solution 1 and the mixed solution 2 are mixed, the mixed solution 3 may be added to the medium, and cells may be cultured.

[0080] When the mixed solution 1 and the mixed solution 2 are separate (independent), the mixed solution 1 and the mixed solution 2 may be administered to the subject simultaneously or sequentially in the administration step in the second production method. Also, when the mixed solution 1 and the mixed solution 2 are mixed, the mixed solution 3 may be administered to the subject.

[0081] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0082] Example 1 Acerola-derived vesicles were prepared by the following method.

[0083] Vesicle fractionation was performed using Brazilian acerola fruit juice using a commercially available vesicle isolation kit (exoEasy Maxi kit, Qiagen) according to the manufacturer's instructions. Specifically, juices squeezed from early-ripened acerola fruit, mid-ripened acerola fruit, and fully ripe acerola fruit were used. Five milliliters of each juice was loaded into the kit, and vesicle-containing elution fractions were isolated by elution with 400 μL of the kit's buffer (Buffer XE). The elution fractions were then ultracentrifuged (100,000 × g, 49,000 rpm, 70 minutes, 4°C) to recover vesicle pellets. The pellets were suspended in 50 μL of PBS(-) to obtain acerola-derived vesicle samples. The vesicle sample was then subjected to a nanoparticle analysis system (trade name NanoSight LM10, Malvern) to confirm the particle size distribution of the vesicles contained in the vesicle sample. The analysis conditions were a measurement time of 90 seconds and three repetitions. The vesicle sample was also observed by a cryo-electron microscope (TEM) and a cryo-electron microscopy (Cryo-EM). PBS(-) was used to measure the Ca ２＋ and Mg ２＋ In the following drawings, PBS refers to the PBS(-).

[0084] The particle size distribution results (average) of the vesicle samples collected from each of the early-ripening juices (n=3), mid-ripening juices (n=3), and fully ripe juices (n=3) are shown in Figure 1 and the table below. Figure 1A shows the results for Sample A, Figure 1B shows the results for Sample B, and Figure 1C shows the results for Sample C. In each figure in Figure 1, the solid black line indicates the average value, and the gray area indicates the standard error.

[0085]

[0086] As shown in these results, the vesicle concentration tended to increase with fruit ripeness, but no difference was observed in the vesicle size.

[0087] FIG. 2 shows cryo-EM and TEM photographs of acerola juice-derived vesicle sample C. The vesicles in the vesicle sample were confirmed to be spherical with a double membrane structure, as indicated by the arrows in FIG. 2. This shape was similar to so-called animal-derived exosomes. The shapes of the vesicles in the other vesicle samples were also similar to those of acerola juice-derived vesicle sample C in FIG. 2. Hereinafter, the acerola-derived vesicles in the vesicle samples are also referred to as AELN.

[0088] [Example 2] Using the vesicle sample C derived from ripe acerola prepared in Example 1, the introduction of the encoding nucleic acid (luciferase mRNA) in vitro and the promotion of expression of the target protein (luciferase) by CPP were confirmed.

[0089] (1) Evaluation of the efficiency of introduction of the encoding nucleic acid The encoding nucleic acid used was a 1800-base-long firefly luciferase mRNA (Fluc mRNA). The sequence of the luciferase mRNA is registered in the database (NCBI GenBank) under accession number QPB74360.

[0090] The concentration of the vesicle sample was 5×10 １０ The concentration of the vesicle sample was adjusted to 100 μL of particles / mL, and 50 μL of the vesicle sample, Fluc mRNA, and 50 μL of PBS(-) were mixed (total volume: 100 μL), and the mixture was incubated on ice for 30 minutes. The final concentrations of Fluc mRNA in the mixture were 1, 2, and 5 mg / mL. Since the vesicles in the vesicle sample carried Fluc mRNA and CPP, the mixture was hereinafter referred to as a vesicle complex sample (2 × 10 ８ particles / 100 μL).

[0091] DMEM / F12 (Gibco) medium containing 10% FBS was used, and the medium volume per well was 0.5 mL. Unless otherwise specified, the medium volume per well was similar (as in other examples). Human embryonic kidney cells (HEK293) were seeded (1 x 10 ４The wells were incubated (100 μL / well) at 37° C. After 24 hours of incubation, 100 μL of the vesicle complex sample after incubation was added to each well, and further incubation was carried out.

[0092] After 24 hours of incubation from the addition of the vesicle complex sample, the cultured cells in the wells were collected, RNA was extracted from the cultured cells, and real-time RT-PCR was performed on the obtained RNA samples to analyze the efficiency of Fluc mRNA transfection into the cells.

[0093] Control 1 was cultured and analyzed in the same manner, except that PBS(-) was added instead of the vesicle complex sample. Control 2 was cultured and analyzed in the same manner, except that Fluc mRNA was introduced using a commercially available transfection reagent (trade name: messengerMax Lipofectamin, ThermoFisher) instead of the vesicle sample. The transfection using the transfection reagent was performed according to the manufacturer's instructions, and the final concentration of Fluc mRNA in the medium was 1 μg / μL. Control 3 was cultured and analyzed in the same manner, except that only the vesicle sample was used without Fluc mRNA.

[0094] For normalization, actin β (ACTB) was also analyzed in the same manner. The expression level of Fluc mRNA was normalized by the expression level of ACTB mRNA. The expression level of Fluc mRNA in the normalized control 1 (PBS(-)) was set to 1, and the relative value of Fluc mRNA was calculated.

[0095] These results are shown in Figure 3. Figure 3 shows relative values ​​indicating the amount of Fluc mRNA in cells. In Figure 3, the units on the vertical axis represent the relative value of the expression level of Fluc mRNA, and the numbers in parentheses on the horizontal axis (0.1, 1, 2, and 5) represent the final concentration (µg / µL) of Fluc mRNA in the vesicle complex sample (100 µL). In Figure 3, AELN represents the vesicle sample (acerola-derived vesicles), and mRNA represents Fluc mRNA.

[0096] As shown in Figure 3, when only PBS(-) (Control 1) or only vesicles (Control 3) were added to cells, no Fluc mRNA was detected. On the other hand, when a commercially available transfection reagent (not shown) was used (Control 2), Fluc mRNA was detected. Furthermore, as an example, it was confirmed that Fluc mRNA could be introduced in a concentration-dependent manner by using the vesicle complex sample instead of the transfection reagent.

[0097] In addition, in Control 2 (transfection using the transfection reagent), the final concentration of Fluc mRNA in the medium was 1 μg / μL, as described above. When compared with an example (AELN / mRNA (1)) in which the same mRNA concentration was used, this example showed a 50-fold higher relative expression value than Control 2, confirming that mRNA can be transferred very efficiently using the vesicle sample.

[0098] (2) Evaluation of translation promotion by CPP In the above (1), the introduction of the encoding nucleic acid into cells by the vesicle complex sample was confirmed. Therefore, it was further confirmed that the expression of the target protein, luciferase, can be promoted by introducing the membrane-permeable peptide CPP into cells. Polyarginine 12R, which has 12 consecutive arginines, was used as the CPP.

[0099] In this example, 50 μL of the vesicle sample was mixed with 10 nmol of Fluc mRNA, 10 nmol of CPP, and 50 μL of PBS(-) (total volume: 100 μL), and the mixture was incubated on ice for 30 minutes. The vesicle samples used were vesicle sample B (hereinafter also referred to as AELN(B)) derived from medium-ripe acerola prepared in Example 1 and vesicle sample C (hereinafter also referred to as AELN(C)) derived from fully ripe acerola, respectively.

[0100] Then, in the same manner as in (1) above, HEK293 cells were cultured for 24 hours using the well plate, and the vesicle complex sample (the mixed solution) was added. The final concentration of Fluc mRNA in the medium based on the added vesicle complex sample was 1 μg / μL. D-luciferin, a luciferase substrate, was then added to each well of the well plate at a concentration of 1 μg / μL, and the fluorescence intensity in each well was measured. The measurement was performed using a commercially available measuring device (trade name GloMax (registered trademark) - Multi Detection System, Promega).

[0101] Comparative examples and controls were analyzed in the same manner as in the above Examples, except for the following: First, in a comparative example, a mixture containing the vesicle sample and Flue mRNA was prepared and incubated in the same manner, except that the CPP was not added, and the vesicle complex sample (the mixture) was added to the medium. In another comparative example, a mixture containing the Flue mRNA and the CPP was prepared and incubated in the same manner, except that the vesicle sample was not added, and the mixture was added to the medium. For the control, PBS(-) was added to the medium instead of the vesicle complex sample.

[0102] As a result, in the control where only PBS(-) was added to the medium and the comparative example where the vesicle sample was not added but a mixture of Fluc mRNA and the CPP was added to the medium, no luminescence intensity indicating protein expression was detected, and luciferase expression was not confirmed. On the other hand, for acerola-derived vesicle sample C, the luminescence intensity was significantly increased by the presence of CPP when the vesicle complex sample with CPP and mRNA was added (luminescence intensity 40,000) was used compared to the vesicle complex sample without CPP and with mRNA added (luminescence intensity 1,000). In other words, the use of the vesicle complex sample with CPP and mRNA added significantly increased luciferase expression. Similar results were also obtained for acerola-derived vesicle sample B. This confirms that the use of a vesicle complex sample with CPP added can promote the translation of target proteins from protein-encoding nucleic acids introduced into cells.

[0103] [Example 3] Using the vesicle sample C derived from ripe acerola prepared in Example 1, the in vivo transfer of a protein-encoding nucleic acid (apolipoprotein A1 mRNA) and the promotion of expression of the target protein (apolipoprotein A1) by CPP were confirmed.

[0104] The protein-encoding nucleic acid used was 900-base-long human apolipoprotein A1 mRNA (hAPOA1 mRNA). The sequence of hAPOA1 mRNA is registered in the database (NCBI GenBank) under accession number NM_000039.3.

[0105] (1) 12R Polyarginine 12R, which is a polyarginine having 12 consecutive arginines, was used as the CPP.

[0106] The concentration of the vesicle sample was 5×10 １０The concentration of the vesicle sample was adjusted to 100 μL, and 50 μL of the vesicle sample, 10 nmol of hAPOA1 mRNA, 10 nmol of CPP(12R), and 50 μL of PBS(-) were mixed (total volume: 100 μL), and the mixture was incubated on ice for 30 minutes. Since the vesicles of the vesicle sample contained Fluc mRNA and CPP, the mixture was hereinafter referred to as a vesicle complex sample (2 × 10 ８ particles / 100 μL).

[0107] C57BL / 6 mice were used, with n = 3 per group. The entire amount of the vesicle complex sample was orally administered to the mice using a stomach tube. 24 hours after oral administration, blood was collected and the livers were excised from the mice. Serum was collected from the whole blood and diluted 10,000-fold with PBS(-). The diluted samples were used as evaluation samples, and hAPOA1 protein was detected by ELISA. Meanwhile, livers were treated as follows to prepare evaluation samples, and hAPOA1 protein was detected by ELISA using these evaluation samples. Specifically, liver tissue was lysed in a cell lysis solution (RIPA buffer) to extract proteins, and APOA1 protein was detected using a detection kit (trade name: Human APOA1 ELISA kit, Proteintech) according to the protocol.

[0108] The comparative examples and controls were analyzed in the same manner as in the above examples, except for the following: First, in the comparative example, a mixture containing the vesicle complex sample and hAPOA1 mRNA was prepared and incubated in the same manner, except that the CPP was not added, and the vesicle complex sample (the mixture) was administered. As a control, PBS(-) was administered instead of the vesicle complex sample. It was confirmed by PCR that the evaluation sample of the example in which the vesicle complex sample (with CPP) was administered and the evaluation sample of the comparative example in which the vesicle complex sample (without CPP) was administered had increased hAPOA1 mRNA expression compared to the control.

[0109] These results are shown in Figure 4. Figure 4 is a graph showing hAPOA1 protein concentrations; Figure 4A shows the concentration in serum, and Figure 4B shows the concentration in liver. As shown in Figure 4A, when no CPP was added, the serum hAPOA1 concentration was similar to that observed when only PBS(-) was administered (control). In contrast, the serum hAPOA1 concentration was significantly increased by administering a vesicle complex sample containing CPP together with mRNA. This confirms that the use of vesicle complex samples allows for the introduction of protein-encoding nucleic acids and CPPs into cells by oral administration, and that the CPPs can also promote the translation of target proteins from protein-encoding nucleic acids in vivo. Furthermore, as shown in Figure 4B, the serum hAPOA1 concentration was significantly increased in the liver, as in the serum. This confirms that the use of vesicle complex samples allows for the introduction of protein-encoding nucleic acids and CPPs into cells by oral administration, and that the CPPs can also promote the translation of target proteins from protein-encoding nucleic acids in vivo.

[0110] (2) 16R Polyarginine 16R, which is a polyarginine having 16 consecutive arginines, was used as the CPP.

[0111] Oral administration to mice was performed in the same manner as in (1) above, except that 16R was used as the CPP, and hAPOA1 protein was detected by ELISA. Detection of hAPOA1 protein was performed in serum and liver. The control was the same as in (1) above, except that 10 nmol of hAPOA1 mRNA alone was administered instead of the vesicle complex sample.

[0112] These results are shown in Figure 5. Figure 5 is a graph showing hAPOA1 protein concentration, with Figure 5A representing the concentration in serum and Figure 5B representing the concentration in liver. In Figure 5, the vertical axis represents absorbance at 450 nm, which corresponds to the hAPOA1 protein concentration. As shown in Figures 5A and 5B, the hAPOA1 concentration in both serum and liver was significantly increased compared to the case where only mRNA was administered (control). Thus, it was found that even when the CPP was 16R, hAPOA1 mRNA could be delivered to the blood and liver and efficiently translated into the target protein, as in (1) above.

[0113] In patients with dyslipidemia and hypo-HDL cholesterol, supplementation with APOA1 protein can increase HDL cholesterol and lower LDL cholesterol in the blood. Furthermore, according to the present invention, hAPOA1 mRNA and CPP can be introduced into the body (specifically, the liver and blood) by oral administration, and the CPP can promote translation of mAPOA1 protein from hAPOA1 mRNA. Unlike injection, oral administration is a very convenient method of administration, and therefore the present invention can reduce the workload of patients and those assisting in treatment.

[0114] [Example 4] Using the vesicle sample C derived from ripe acerola prepared in Example 1, a protein-encoding nucleic acid (luciferase mRNA) and a CPP were introduced in vitro, and the promotion of expression of the target protein (luciferase) by the CPP was confirmed.

[0115] The CPPs used were polyarginine 12R (12 consecutive arginines), polyarginine 16R (16 consecutive arginines), and FHV. The protein-encoding nucleic acid used was the same firefly luciferase mRNA (Fluc mRNA) as in Example 1.

[0116] (1) 12R and FHV The concentration of the vesicle sample was 3 × 10 ５The concentration of the vesicle sample was adjusted to 100 μL per 1000 μL of vesicle sample, 20 μL of 10 nmol / L Fluc mRNA, a predetermined amount of CPP, and 30 μL of PBS(-) were mixed (total volume: 100 μL), and the mixture was incubated on ice for 30 minutes. The CPP concentrations in the mixture were adjusted to 0.2, 0.4, 0.6, and 1 μmol / L. Since the vesicles in the vesicle sample contained Fluc mRNA and CPP, the mixture is hereinafter referred to as the vesicle complex sample. The amount of CPP added to the mixture was adjusted so that when the vesicle complex sample was added to the medium, the final CPP concentration in the medium based on the vesicle complex sample was 1, 2, 3, or 5 μM. HEK293 cells were then cultured and the fluorescence intensity of the luciferase reaction was measured in the same manner as in Example 2(2).

[0117] As a control, PBS(-) was added to the medium instead of the vesicle complex sample, and fluorescence intensity was measured in the same manner. In a comparative example, instead of the vesicle sample, only Fluc mRNA was added to the medium, and fluorescence intensity was measured in the same manner. In another comparative example, a mixture containing the vesicle sample and Fluc mRNA was prepared and incubated in the same manner, except that the CPP was not added, and the vesicle complex sample (the mixture) was added to the medium.

[0118] These results are shown in Figure 6. Figure 6 is a graph showing the luminescence intensity of luminescence produced by the reaction of luciferase with the substrate luciferin. The relative intensity of luminescence correlates with the amount of luciferase expressed in the cells. In Figure 6, the vertical axis represents the luminescence intensity of the luciferase reaction (unit: photons / second), and the concentration in µM in the figure represents the final concentration of CPP in the medium based on the vesicle complex sample added. M can be converted to mol / L.

[0119] As shown in Figure 6, in the comparative example in which Fluc mRNA was used alone, no luminescence was detected, and luciferase expression could not be confirmed, just like the control. Furthermore, in the comparative example in which a vesicle complex sample without added CPP was used, slight luminescence was detected compared to the control, and luciferase expression was confirmed. In contrast, the use of a vesicle complex sample with added CPP significantly increased the luminescence intensity. Furthermore, luciferase expression increased depending on the CPP concentration. This confirms that the use of a vesicle complex sample with added CPP can promote the translation of target proteins from protein-encoding nucleic acids introduced into cells.

[0120] (2) 12R and 16R: 12R and 16R were used as the CPPs, respectively. HEK293 cells were cultured and the fluorescence intensity of the luciferase reaction was measured in the same manner as in (1). In preparing the endoplasmic reticulum sample, the CPP concentration in the mixture was 1 μmol / L. When the endoplasmic reticulum sample was added to the medium, the final CPP concentration in the medium based on the vesicle complex sample was 0.1 μM.

[0121] These results are shown in Figure 7. Figure 7 is a graph showing the relative light units (RLU) of luminescence produced by the reaction of luciferase with its substrate luciferin. The relative light units correlate with the amount of luciferase expressed in the cells.

[0122] In the above (1), it was confirmed that 12R and FHV were effective as CPPs in increasing protein expression, but as shown in Figure 7, it was found that protein expression could be further increased by using 16R as a CPP.

[0123] [Example 5] Using the vesicle sample C derived from ripe acerola prepared in Example 1, a protein-encoding nucleic acid (apolipoprotein A1 mRNA) and a CPP were introduced in vitro, and the promotion of expression of the target protein (apolipoprotein A1) by the CPP was confirmed.

[0124] The CPP used was polyarginine 16R, which is a sequence of 16 arginines. The encoding nucleic acid used was the same human apolipoprotein A1 mRNA (hAPOA1 mRNA) as in Example 3.

[0125] The concentration of the vesicle sample was 5×10 １０ The concentration of hAPOA1 mRNA was adjusted to 1000 μL per 1000 μL of the vesicle sample, 10 nmol of hAPOA1 mRNA, 10 nmol of CPP(16R), and 50 μL of PBS(-) were mixed (total volume: 100 μL), and the mixture was incubated on ice for 30 minutes. The final concentrations of hAPOA1 mRNA in the mixture were 0.1, 1, 2, and 5 μg / μL. Since the vesicles in the vesicle sample carried hAPOA1 mRNA and CPP, the mixture was hereinafter referred to as a vesicle complex sample (2×10 ８ particles / 100 μL).

[0126] Human embryonic kidney cells (HEK293) were seeded (1 × 10 cells) in a 96-well plate using DMEM / F12 (Gibco) medium containing 10% FBS. ４ The wells were incubated (100 μL / well) at 37° C. After 24 hours of incubation, 100 μL of the vesicle complex sample after incubation was added to each well, and further incubation was carried out.

[0127] After 24 hours of incubation following the addition of the vesicle complex sample, the cultured cells in the wells were harvested. The cultured cells were lysed in a cell lysis solution (RIPA buffer) to extract proteins, and APOA1 protein was detected using a detection kit (Human APOA1 ELISA kit, Proteintech) according to the protocol. The amount of APOA1 protein was measured as relative light units (RLU), which correspond to the amount of protein.

[0128] Control 1 (PBS(-)) was prepared by adding PBS(-) instead of the vesicle complex sample, and culture and analysis were performed in the same manner. Control 2 (AELN) was prepared by preparing a vesicle complex sample in which only hAPOA1 mRNA was loaded onto vesicles (AELN) without adding CPP(16R), and culture and analysis were performed in the same manner. Control 3 (mMax / mRNA) was prepared by introducing hAPOA1 mRNA using a commercially available transfection reagent (trade name messengerMax Lipofectamin, ThermoFisher) instead of the vesicle sample, and analysis was performed in the same manner. Transfection using the transfection reagent was performed according to the manufacturer's instructions, and the final concentration of hAPOA1 mRNA in the medium was 1 μg / μL.

[0129] These results are shown in Figure 8. Figure 8 shows relative values ​​indicating the amount of hAPOA1 mRNA in the cells. In Figure 8, the units on the horizontal axis are relative light units (RLU). In Figure 8, the numbers in parentheses on the vertical axis (0.1, 1, 2, and 5) indicate the final concentration (µg / µL) of hAPOA1 mRNA in the medium based on the vesicle complex sample added to the medium. Since the relative light unit corresponds to the amount of hAPOA1 protein, it can also be said to be the efficiency of translation into protein.

[0130] As shown in Figure 8, hAPOA1 protein was not detected in Control 1 (PBS(-)), but was detected in Control 2 (AELN / mRNA) without added CPP. When the vesicle complex sample carrying 16R and hAPOA1 mRNA was used, the amount of hAPOA1 protein was significantly increased compared to Control 2, and specifically, the increase in protein amount depended on the hAPOA1 mRNA concentration. This confirmed that the use of a vesicle complex sample with added CPP enabled the introduction of the encoding nucleic acid into cells and further promoted translation of the target protein from the introduced encoding nucleic acid.

[0131] Example 6 Using the vesicle sample C derived from the fully ripe acerola prepared in Example 1 and the encoding nucleic acid, the properties of the complex were confirmed.

[0132] (1) Confirmation of the Shape of the Complex The encoding nucleic acids used were the same Fluc mRNA as in Example 2 and the same hAPOA1 mRNA as in Example 3.

[0133] The concentration of the vesicle sample was 5×10 １０ The vesicle sample was adjusted to a concentration of 5 × 10 particles / mL, and 50 μL of the vesicle sample, 10 nmol of mRNA (hAPOA1 mRNA or Fluc mRNA), 10 nmol of CPP(12R), and 50 μL of PBS(-) were mixed (total volume: 100 μL). The mixture was incubated on ice for 30 minutes to prepare a vesicle complex sample. As a control, the vesicle sample was diluted to a concentration of 5 × 10 １０ The vesicle concentration was adjusted to 100 μL / mL, and 50 μL of the vesicle sample was mixed with 50 μL of PBS(-) (total volume: 100 μL), followed by incubation in the same manner. The particle concentration and particle size distribution of the mixed solution were measured using a nanoparticle analysis system (trade name: NanoSight, Malvern Instruments).

[0134] These results are shown in Figure 9. Figure 9 is a graph showing the particle size distribution of the mixed solution. The upper graph shows the results for the vesicle complex sample in which the mRNA was hAPOA1 mRNA, and the lower graph shows the results for the vesicle complex sample in which the mRNA was Fluc mRNA. Both graphs also show the results for the vesicle sample alone (control). In Figure 9, the vertical axis represents particle concentration (particles / mL) and the horizontal axis represents particle size (nm). As shown in Figure 9, regardless of the mRNA used, the vesicle complex sample complexed with mRNA and CPP had a larger distribution of particles with larger particle sizes than the vesicle sample alone (AELN alone). This result suggests that the vesicle sample formed aggregates with the mRNA and CPP.

[0135] (2) Confirmation of the resistance of the complex The concentration of the vesicle sample C was 5 × 10 １０The vesicle sample (50 μL), 10 nmol of Fluc mRNA, and 50 μL of PBS(−) were mixed (total volume: 100 μL), and the mixture was incubated on ice for 30 minutes. The mixture was hereinafter referred to as a vesicle complex sample (2 × 10 ８ The incubation mixture (the vesicle complex sample) was then subjected to ultracentrifugation to recover the precipitated fraction. Various damaging reagents that damage nucleic acids were added to the precipitated fraction, and the fraction was treated under predetermined conditions, after which RNA was extracted.

[0136] The reagents used were RNase, HCl, and NaOH. For the RNase system, RNase was added to the precipitate fraction to a concentration of 10 mg / mL, and the precipitate fraction was treated at 37°C for 1 hour, after which RNA was extracted. For the HCl system, 1 x 10 －２ HCl was added to the precipitate fraction to a concentration of 1×10 mol / L (pH 2), and the mixture was treated at 37° C. for 15 minutes, after which RNA was extracted. －２ NaOH was added to the mixture to a concentration of 1000 mol / L (pH 10), and the mixture was treated at 37°C for 15 minutes, after which RNA was extracted.

[0137] As a control, the encoding nucleic acid alone was incubated at 37°C for 1 hour, and then RNA was extracted. As a comparative example, the damaging reagent was added only to the encoding nucleic acid instead of the precipitate fraction, and the resulting mixture was treated under predetermined conditions, followed by RNA extraction in the same manner. The amount of the damaging reagent added to the encoding nucleic acid was the same as that added to the encoding nucleic acid contained in the precipitate fraction, and the treatment under the predetermined conditions was also the same.

[0138] The extracted RNA samples were subjected to a fully automated electrophoresis apparatus (TapeStation, Agilent) and RNA degradation (mRNA fragment size) was visualized according to the protocol. The results are shown in Figure 10. Figure 10 is a photograph of the electrophoresis of the extracted RNA.

[0139] As shown in Figure 10, compared to the control without the damaging agent, the comparative example (lane 1) with HCl added and no AELN added showed a fainter Fluc mRNA band (arrow), confirming a significant reduction in Fluc mRNA. In contrast, the example (lane 2) with HCl added and no AELN added showed suppressed reduction in Fluc mRNA. The comparative example (lane 3) with NaOH added and no AELN added showed a fainter band, confirming a significant reduction in Fluc mRNA. In contrast, the example (lane 4) with NaOH added and no AELN added showed suppressed reduction in Fluc mRNA. The comparative example (lane 5) with RNase added and no AELN added showed a fainter band, confirming a significant reduction in Fluc mRNA. In contrast, the example (lane 6) with RNase added and no AELN added showed suppressed reduction in Fluc mRNA.

[0140] Thus, it was found that, in the absence of AELN, the encoding nucleic acid would be degraded by the damaging reagent, but that the addition of AELN can inhibit degradation of the encoding nucleic acid by the damaging reagent. In a living body, for example, the digestive organs, the stomach and intestines, are acidic due to gastric acid, and RNase is present in the blood. Therefore, it is believed that nucleic acids are affected by acid when administered orally, and by RNase when administered intravenously. However, according to the present invention, by forming a complex with AELN, the nucleic acid can inhibit the effects of acid, alkali, and RNase. Therefore, according to the present invention, damage to the encoding nucleic acid can be inhibited by, for example, oral administration or parenteral administration such as intravenous administration, and a wide range of administration methods can be employed.

[0141] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-described embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0142] This application claims priority based on Japanese Patent Application No. 2024-45241, filed March 21, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0143] The translation promotion kit of the present invention can introduce a protein-encoding nucleic acid and a membrane-permeable peptide, which is the translation promoter, into cells using the plant-derived vesicle, which is the introduction agent. The introduction of the membrane-permeable peptide into cells together with the protein-encoding nucleic acid can promote protein translation from the protein-encoding nucleic acid. Therefore, the translation promotion kit of the present invention is useful for methods of introducing a protein-encoding nucleic acid into cells to synthesize a protein within the cells, such as protein replacement therapy and genome editing.

Claims

1. A translation promotion kit for introducing nucleic acid and promoting the translation of the introduced nucleic acid, comprising an introduction agent for introducing a protein-encoding nucleic acid and a translation promotion agent, and the translation promotion agent, wherein the introduction agent is a plant-derived vesicle, and the translation promotion agent is a membrane-permeable peptide.

2. The translation enhancing kit according to claim 1, wherein the membrane-permeable peptide is a basic peptide.

3. The translation enhancement kit according to claim 1 or 2, wherein the membrane-permeable peptide is an arginine-rich peptide.

4. A translation enhancement kit according to any one of claims 1 to 3, wherein the membrane-permeable peptide contains 8 to 16 arginine residues.

5. A translation enhancement kit according to any one of claims 1 to 4, wherein 80 to 100% of all amino acid residues in the membrane-permeable peptide are arginine residues.

6. A translation enhancement kit according to any one of claims 1 to 5, wherein the plant-derived vesicles are vesicles derived from the fruit of a plant.

7. A translation enhancement kit according to any one of claims 1 to 6, wherein the plant-derived vesicles are vesicles derived from a plant of the family Pyrolaceae.

8. The translation enhancing kit according to claim 7, wherein the plant of the Acerola family is an Acerola species plant.

9. A translation enhancement kit according to any one of claims 1 to 8, further comprising a nucleic acid encoding a protein.

10. The translation enhancement kit according to claim 9, wherein the protein-encoding nucleic acid has DNA encoding the protein as a protein-coding sequence.

11. The translation enhancement kit according to claim 9 or 10, wherein the protein-encoding nucleic acid is an expression vector having a protein-encoding sequence.

12. A method for producing a protein, comprising: a mixing step of using a translation promotion kit according to any one of claims 1 to 8, mixing an introduction agent of the translation promotion kit with a nucleic acid encoding a protein to prepare a mixed solution; and a culturing step of culturing cells in the presence of the mixed solution and the translation promotion agent of the translation promotion kit.

13. The method for producing a protein according to claim 12, wherein in the mixing step, the introduction agent and translation promoter of the translation promotion kit are mixed with the nucleic acid encoding the protein to prepare the mixed solution, and in the culturing step, cells are cultured in the presence of the mixed solution.

14. The method for producing a protein according to claim 13, further comprising the step of incubating the mixture, and culturing cells in the presence of the incubated mixture.

15. A method for producing a protein, comprising: a mixing step of using a translation promotion kit according to any one of claims 1 to 8, mixing an introduction agent of the translation promotion kit with a nucleic acid encoding a protein to prepare a mixed solution; and an administration step of administering the mixed solution and the translation promotion agent of the translation promotion kit to a subject.

16. The method for producing a protein according to claim 15, wherein in the mixing step, the introduction agent and translation promoter of the translation promotion kit are mixed with the nucleic acid encoding the protein to prepare the mixture, and in the administration step, the mixture is administered to a subject.

17. The method for producing a protein according to claim 16, further comprising the step of incubating the mixture, and administering the incubated mixture to a subject.

18. A method for producing a protein according to any one of claims 15 to 17, wherein the subject is a human or non-human animal.

19. A method for producing a protein described in any one of claims 15 to 18, wherein the mixed solution is administered orally.

20. A method for producing a protein according to any one of claims 15 to 18, wherein the administration is parenteral administration.

21. A method for producing a protein according to any one of claims 15 to 20, which is a method for supplementing proteins in a living body.

22. A method for producing a protein according to any one of claims 12 to 21, wherein the protein-encoding nucleic acid has a DNA encoding the protein as the protein-coding sequence.

23. A method for producing a protein according to any one of claims 12 to 22, wherein the protein-encoding nucleic acid is an expression vector having a coding sequence for the protein.

Images