Carrier for protein, and protein introduction method

Abstract

Provided is a novel carrier for introducing a protein such as a Cas protein into cells.　A carrier for a protein according to the present invention is characterized by containing vesicles of the fruit of the Malpighiaceae plant, wherein an object to be held is a protein or a nucleic acid encoding the protein. The Malpighiaceae plant is, for example, the Malpighia sp. plant. A carrier for a protein according to the present invention can be used as, for example, a genome editing tool.

Description

Protein carrier and protein introduction method

[0001] The present invention relates to a carrier for proteins and a method for introducing proteins.

[0002] In the field of biochemistry, great importance is attached to a DDS (drug delivery system) platform for delivering a target substance to a target cell.

[0003] For example, genome editing, which is widely used in various fields such as agriculture, livestock farming, fisheries, and medicine, involves contacting a Cas9 protein with nuclease activity with a guide RNA, which is an RNA molecule, with a target sequence to cleave the target sequence and thereby knock out a gene or knock in a foreign gene. Thus, genome editing requires the delivery of, for example, a protein with a relatively large molecular weight. Furthermore, when it comes to introducing it into animals and plants that are used as food, or into humans for treatment, for example, there are concerns about the effects of artificial DDS on living organisms.

[0004] Therefore, an object of the present invention is to provide a new carrier that can serve as a DDS platform for proteins.

[0005] To achieve the above object, the protein carrier of the present invention is characterized in that it contains vesicles from the fruit of a plant of the Malpighiaceae family, and the object to be held is a protein or a nucleic acid encoding the protein.

[0006] The method for producing a protein delivery reagent of the present invention is characterized by comprising a step of forming a complex between a vesicle of the protein carrier and the protein or the nucleic acid encoding the protein by causing the protein carrier containing the vesicle of the present invention and the protein to be delivered or the nucleic acid encoding the protein to coexist in a solvent.

[0007] The method for introducing a protein into a cell of the present invention is characterized by comprising a contact step of contacting a protein carrier containing the vesicle of the present invention with a cell, the protein to be delivered, or a complex of a nucleic acid encoding the protein.

[0008] The protein carrier of the present invention contains vesicles from the fruit of a plant of the Acerola family, such as acerola, and can retain proteins or nucleic acids encoding the proteins. Therefore, the protein carrier of the present invention can be used as a DDS tool for proteins such as nucleases used in genome editing, for example.

[0009] FIG. 1 is a graph showing the cell survival rate of cells into which anti-mTOR antibody was introduced using the protein carrier of the present invention in Example 3.

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

[0011] [1] A protein carrier comprising vesicles from the fruit of a plant of the Malpighiaceae family, wherein the retained target is a protein or a nucleic acid encoding the protein. [2] The protein carrier according to [1], wherein the plant of the Malpighiaceae family is an acerola species (Malpighia sp.). [3] The protein carrier according to [1] or [2], wherein the average particle size of the vesicles is 30 to 400 nm. [4] The protein carrier according to any one of [1] to [3], wherein the vesicles are extracellular vesicles. [5] The protein carrier according to any one of [1] to [4], wherein the encoding nucleic acid is an expression vector in which a coding sequence for the protein has been inserted into a vector. [6] The protein carrier according to any one of [1] to [5], wherein the protein is a sequence-specific nuclease. [7] The protein carrier according to [6], wherein the sequence-specific nuclease is a Cas protein. [8] The protein carrier according to [7], wherein the Cas protein is a Cas9 protein. [9] The protein carrier according to any one of [1] to [8], wherein the retention target further comprises a co-combinant component for the protein or the nucleic acid encoding the protein.

[10] The protein carrier according to [9], wherein the co-combinant component is a nucleic acid substance, and the nucleic acid substance is a guide RNA.

[11] The protein carrier according to any one of [1] to

[10] , further comprising the protein or the nucleic acid encoding the protein.

[12] A method for producing a protein delivery reagent, comprising a step of forming a complex between the vesicles of the protein carrier and the protein or the nucleic acid encoding the protein by coexisting the protein carrier containing the vesicles according to any one of [1] to

[10] with a protein to be delivered or the nucleic acid encoding the protein in a solvent.

[13] A method for producing a protein delivery reagent according to

[12] , comprising a step of mixing the protein carrier with the protein or the nucleic acid encoding the protein in the solvent and incubating.

[14] The method for producing a protein delivery reagent according to

[12] or

[13] , further comprising allowing a co-existence of a component for the protein or the nucleic acid encoding the protein with the protein carrier in the solvent.

[15] A method for introducing a protein into a cell, comprising a contacting step of contacting a cell with a complex of a protein carrier containing the vesicle according to any one of [1] to

[10] and a protein to be delivered or a nucleic acid encoding the protein.

[16] The method for introducing a protein according to

[15] , wherein the complex further comprises a component for the protein or the nucleic acid encoding the protein.

[17] The method for introducing a protein according to

[15] or

[16] , further comprising, prior to the contacting step, a complex formation step of forming a complex between the protein carrier and the protein or the nucleic acid encoding the protein.

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

[15] to

[17] , wherein the contacting step is a step of contacting the complex with a cell in vivo, ex vivo, or in vitro.

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

[15] to

[18] , wherein the contacting step is a step of administering the complex to a human or a non-human animal.

[20] A protein delivery reagent comprising the protein carrier according to any one of [1] to

[10] and a protein or a nucleic acid encoding the protein.

[21] The protein delivery reagent according to

[20] , wherein the encoding nucleic acid is an expression vector in which the coding sequence of the protein has been inserted into a vector.

[22] The protein delivery reagent according to

[20] or

[21] , wherein the protein is a sequence-specific nuclease.

[23] The protein delivery reagent according to

[22] , wherein the sequence-specific nuclease is a Cas protein.

[24] The protein delivery reagent according to

[23] , wherein the Cas protein is a Cas9 protein.

[25] The protein delivery reagent according to

[20] to

[24] , further comprising a co-administered component for the protein or the nucleic acid encoding the protein.

[26] The protein delivery reagent according to

[25] , wherein the concomitant component is a nucleic acid substance, and the nucleic acid substance is a guide RNA.

[0012] (1) Protein Carrier As described above, the protein carrier of the present invention is characterized by comprising vesicles from the fruit of a plant of the Malpighiaceae family. The present inventors discovered that vesicles from the fruit of a plant of the Malpighiaceae family hold a protein or a nucleic acid encoding the protein to form a complex, that the complex is taken up into a cell (hereinafter also referred to as a "target cell"), and that the protein or a protein expressed from the encoding nucleic acid functions within the cell, thereby establishing the present invention. The present invention is characterized by comprising the vesicles, and other configurations and conditions are not particularly limited.

[0013] As described above, the protein carrier of the present invention can retain the protein or the protein-encoding nucleic acid using the vesicles, and by retaining the protein or the protein-encoding nucleic acid, delivery of the protein or the protein-encoding nucleic acid can be achieved. Therefore, the protein carrier of the present invention is also referred to as, for example, a delivery reagent or a DDS reagent. In the present invention, the term "protein carrier" refers not only to retaining the protein itself, but also to retaining the protein-encoding nucleic acid. For example, when the encoding nucleic acid retained by the protein carrier is introduced into a cell by the protein carrier, the encoded protein can be synthesized by the cell's protein translation mechanism. Therefore, the encoding nucleic acid may contain, for example, a sequence (e.g., an open reading frame (ORF)) that enables protein synthesis by the cell's translation mechanism. When the coding sequence is retained, the protein carrier of the present invention can also be referred to as, for example, a coding nucleic acid carrier.

[0014] 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.

[0015] The vesicles can be obtained from the fruit of the plant of the family Canthariaceae. The fruit may be, for example, fully ripe, unripe, or a mixture thereof. The vesicles are preferably, for example, a vesicle fraction recovered from the juice of the fruit, as described below.

[0016] The vesicles can be prepared, for example, by extraction from the fruit of the plant belonging to the family Canthariaceae. The preparation method is not particularly limited, and can be, for example, obtained by crushing the fruit, preparing the crushed material or a suspension of the crushed material, and fractionating the vesicles. The fractionation method is not particularly limited, and examples thereof include ultrafiltration, ultracentrifugation, concentration gradient analysis, and separation methods using a microfluidic system. For example, commercially available kits such as ExoEasy Maxi Kit (product name, QIAGEN), ExoQuick (product name, System Bioscience), and Total Exosome Isolation Reagent (product name, Invitrogen) can be used for the preparation method. The vesicles can be obtained, for example, by subjecting the juice obtained by squeezing the fruit to various separation methods as described above. For example, a grinder, a press, or the like can be used to prepare the juice. The juice can be, for example, juice from ripe fruit, juice from unripe fruit, or juice from ripe or unripe frozen fruit.

[0017] The protein carrier of the present invention can be, for example, a vesicle fraction containing a plurality of the 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 juice of the fruit so as to have the above particle size and particle size distribution. When the vesicle fraction is used as a protein carrier of the present invention, for example, a fraction extracted from the fruit (preferably the juice) 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 fruit juice.

[0018] The method for measuring the particle size of vesicles is not particularly limited, and methods such as nanoparticle tracking analysis can be employed, for example, using commercially available devices (such as NanoSight and qNANO). The 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 by the same isolation method as that for human cell-derived extracellular vesicles, preferably human cell-derived exosomes.

[0019] A "protein" is a compound in which amino acids are linked by peptide bonds. The length (number of amino acid residues) of a "protein" in the present invention is not particularly limited, and in terms of length, it also includes peptides such as so-called oligopeptides and polypeptides (e.g., less than 50 residues) and longer proteins (e.g., 50 residues or more). The protein in the present invention may be, for example, an unmodified protein or a modified protein. The size of the protein held by the protein carrier of the present invention is not particularly limited, and the lower limit is, for example, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, or 80 kDa, and the upper limit is, for example, 150 kDa, 140 kDa, 130 kDa, 120 kDa, 110 kDa, 100 kDa, 90 kDa, 80 kDa, 70 kDa, 60 kDa, or 50 kDa, and the range is, for example, 10 to 150 kDa, 20 to 150 kDa, 10 to 110 kDa, 20 to 110 kDa, 80 to 110 kDa, 50 to 80 KDa, or 20 to 50 KDa.

[0020] The target to be retained by the protein carrier of the present invention may be, for example, a protein or a nucleic acid encoding the protein. The encoding nucleic acid is, for example, a ribonucleic acid, such as RNA or DNA. The RNA is, for example, pre-mRNA or mRNA, preferably mRNA. Furthermore, the protein carrier of the present invention may retain, for example, the protein, the encoding nucleic acid, or both the protein and the encoding nucleic acid, or may retain at least one of the protein and the encoding nucleic acid, as well as other components. The other components can be appropriately selected depending on, for example, the type of target to be retained, the purpose of introducing the target to cells, and the like. When the other components are selected depending on the type and / or purpose of the target to be retained, the other components are hereinafter also referred to as "combined components."

[0021] The type of protein to be retained is not particularly limited and can be appropriately selected depending on the purpose. Examples of the type of protein include functional proteins such as enzymes, antibodies, antigen-binding fragments, and transcription factors. The size of the protein is not particularly limited and includes the above-mentioned examples. The protein carrier of the present invention can be used, for example, in genetic engineering techniques such as genome editing, which will be described later. Therefore, the protein is, for example, an enzyme, and a specific example is a nuclease such as a sequence-specific nuclease.

[0022] Examples of the nuclease include Cas proteins such as Cas9 protein, Zinc Finger Nuclease (ZFN) proteins, and Transcription Activator-Like Effector Nuclease (TALEN) proteins.

[0023] The encoding nucleic acid to be protected may be a nucleic acid consisting of a coding sequence for the protein, or may be a nucleic acid including a coding sequence for the protein. That is, the encoding nucleic acid may be, for example, capable of expressing the protein of interest based on the coding sequence in a cell into which it is introduced. The coding sequence in the encoding nucleic acid may be, for example, RNA such as mRNA, or DNA such as cDNA. The length of the coding sequence is not particularly limited, and examples thereof include the length calculated from the size or length of the protein, and the size of the protein is, for example, as described above. Specific examples of the length of the coding sequence include a lower limit of 480 bases or 1200 bases and an upper limit of 2640 bases, and the range is, for example, 480 to 2640 bases, 1200 to 2640 bases, 480 to 1200 bases, 1200 to 1920 bases, or 1920 to 2640 bases. 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.

[0024] Examples of the encoding nucleic acid include a DNA molecule, an RNA molecule, a chimeric molecule of DNA and RNA, etc. The DNA molecule, the RNA molecule, and the chimeric molecule may be composed of, for example, a natural nucleic acid, an artificial nucleic acid, or a combination of the natural nucleic acid and the artificial nucleic acid.

[0025] The encoding nucleic acid may be, for example, an expression vector capable of expressing the protein. Specifically, it may be an expression vector into which a coding sequence for the protein is inserted so that the protein can be expressed. The coding sequence in the expression vector is, for example, DNA, preferably cDNA. For example, an antisense strand (template strand) for the amino acid sequence of the protein is inserted as DNA (e.g., cDNA), and specifically, double-stranded DNA consisting of the antisense strand and the sense strand is inserted into the expression vector. The type of the vector encoding the amino acids of the protein is not particularly limited, and examples thereof include a plasmid vector, a viral vector, etc., and examples of the viral vector include an adenovirus vector, a Sendai virus vector, etc.

[0026] Furthermore, as described above, the protein carrier of the present invention may hold, for example, at least one of the protein and the encoding nucleic acid, and further other components (the concomitant components).

[0027] The combined component is, for example, a nucleic acid substance (hereinafter also referred to as combined nucleic acid component). Examples of the combined nucleic acid component include nucleic acid molecules such as DNA molecules, RNA molecules, and chimeric molecules of DNA and RNA. The combined nucleic acid component may be, for example, composed of natural nucleic acids, artificial nucleic acids, or a combination of the natural nucleic acids and the artificial nucleic acids. The combined nucleic acid component may be, for example, an expression vector capable of expressing the nucleic acid molecule. When the nucleic acid molecule is a DNA molecule, for example, an antisense strand (template strand) of DNA relative to the DNA molecule is inserted into the expression vector, specifically, a double-stranded DNA consisting of the antisense strand and the sense strand is inserted. The type of the expression vector is not particularly limited, and examples include a plasmid vector, a viral vector, etc. Examples of the viral vector include a lentiviral vector, an adenoviral vector, etc.

[0028] When the protein carrier of the present invention is used for genome editing, the combined nucleic acid component may be, for example, a guide RNA (gRNA), specifically a single-stranded guide RNA (sgRNA). The combined nucleic acid component may be, for example, an expression vector capable of expressing the guide RNA (gRNA expression vector). Specifically, the gRNA expression vector may be, for example, an expression vector into which the guide RNA is inserted, or an expression vector into which the antisense strand (template strand) of DNA relative to the guide RNA, or a double-stranded DNA consisting of the antisense strand and the sense strand, is inserted. The guide RNA expression vector may be, for example, a commercially available viral gRNA expression vector. The guide RNA will be described later.

[0029] The protein carrier of the present invention may, for example, hold an expression vector for the protein and an expression vector for the concomitant nucleic acid component, which are independent of each other, or may hold an expression vector for the protein that co-expresses the protein and the nucleic acid molecule.

[0030] The protein carrier of the present invention may contain the protein or the encoding nucleic acid, or may not contain the protein or the encoding nucleic acid, for example, before use by a user. When the protein carrier of the present invention contains the protein or the encoding nucleic acid, it is also referred to as a protein delivery reagent, as described below. When the protein carrier of the present invention contains the protein or the encoding nucleic acid, it is preferable that the vesicle and the protein or the encoding nucleic acid form a complex (hereinafter also referred to as a "vesicle complex"). The form of the vesicle complex is not particularly limited, and may be, for example, a form in which the protein or the encoding nucleic acid is encapsulated inside the vesicle, or a form in which the protein or the encoding nucleic acid is retained on the outer wall (external surface) of the vesicle; specifically, for example, the latter.

[0031] The method for forming the vesicle complex is not particularly limited. Examples of methods for forming a vesicle complex carrying the protein and a vesicle complex carrying the encoding nucleic acid are given below. However, the present invention is not limited to these examples.

[0032] First, the vesicle complex holding the protein can be formed, for example, by causing the protein carrier of the present invention (i.e., the vesicle) and the protein to coexist in a solvent.

[0033] Specifically, for example, the vesicles and the protein can be coexisted in a solvent and incubated to form a complex between the vesicles and the protein. The incubation temperature is not particularly limited and may be, for example, room temperature (e.g., 30±10°C) or a lower temperature range (e.g., above 0°C and below 20°C), preferably a low temperature range (e.g., 4±5°C, 4±3°C), and more preferably under ice-cooled conditions (e.g., 1 to 6°C).

[0034] In the coexistence, the ratio of the vesicles to the protein is not particularly limited, and may be, for example, 5 × 10 vesicles per 1 μmol of the protein. 4 ~8 x 10 4 particles, 2×10 5~5 x 10 5 particles, 2×10 6 ~5 x 10 6 When the vesicles are used for genome editing to retain a Cas protein such as a Cas9 protein, the ratio of vesicles to 1 μmol of Cas protein is preferably within the above range, for example. The same applies to nucleases and other proteins other than Cas proteins.

[0035] The incubation time is not particularly limited. The lower limit is not particularly limited and is, for example, 5 minutes or more, 15 minutes or more, and the upper limit is not particularly limited and, for example, a plateau of complex formation can be reached by incubation for about 30 minutes. The solvent is not particularly limited and, for example, an aqueous solvent can be used, specific examples of which include water, physiological saline, and buffer solutions such as PBS.

[0036] For the coexistence of the vesicles and the protein, for example, general introduction methods such as electroporation, lipofection, etc. may also be used. Note that, according to the protein carrier of the present invention, the protein can be retained in the vesicles simply by allowing the vesicles and the protein to coexist without using these general introduction methods.

[0037] Thus, with the protein carrier of the present invention, for example, a complex with the vesicles can be formed simply by coexisting the target protein to be delivered to the target cell with the vesicles in the solvent. Therefore, the DDS target (the protein) can be held in the vesicles very easily. Furthermore, with the protein carrier of the present invention, for example, the held protein can be introduced into the target cell via the vesicles. Specifically, with the protein carrier of the present invention, for example, the vesicles holding the protein can be fused with the target cell, and the held protein can be released into the target cell, thereby allowing the protein to be introduced into the target cell from outside the cell.

[0038] When the co-conjugated component is retained in the vesicles together with the protein, the vesicles, the protein, and the co-conjugated component may be incubated together in the solvent. The incubation conditions are not particularly limited and are the same as those described above. When the co-conjugated component is the co-conjugated nucleic acid component, the incubation temperature is preferably within the low temperature range or lower. This can sufficiently prevent, for example, degradation of the co-conjugated nucleic acid component before the formation of the vesicle complex due to the presence of nucleases such as RNase.

[0039] Next, a vesicle complex carrying a nucleic acid encoding the protein can be similarly formed, for example, by allowing the protein carrier of the present invention (i.e., the vesicle) and the encoding nucleic acid to coexist in a solvent. Unless otherwise specified, the examples of the method for forming a vesicle complex carrying the protein can be used.

[0040] Specifically, for example, the vesicles and the encoding nucleic acid can be coexisted in a solvent and incubated to form a complex between the vesicles and the encoding nucleic acid. The incubation temperature is not particularly limited and may be, for example, room temperature (e.g., 30±10°C) or a lower temperature range (e.g., above 0°C and below 20°C), preferably a 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). When the encoding nucleic acid is to be maintained in this manner, it is preferable to incubate at a temperature within the low temperature range or lower. This can prevent, for example, degradation of the encoding nucleic acid before the formation of the vesicle complex due to the presence of nucleases such as RNase.

[0041] In the coexistence, the ratio of the vesicles to the encoding nucleic acid is not particularly limited, and may be, for example, 5 × 10 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 6The vesicles are preferably composed of particles. When the vesicles are used for genome editing and contain a Cas9-encoding nucleic acid encoding a Cas9 protein, the ratio of vesicles to 1 μmol of the Cas9 coding sequence is preferably within the above range. The coding sequence is, for example, Cas9 mRNA or Cas9 DNA encoding the Cas9 protein, preferably Cas9 mRNA. The coding nucleic acid may also be, for example, an expression vector into which the Cas9 mRNA or Cas9 DNA has been inserted. The same applies to, for example, coding nucleic acids for nucleases other than Cas proteins, and coding nucleic acids for other proteins.

[0042] The incubation time is not particularly limited. The lower limit is not particularly limited and is, for example, 5 minutes or more, 15 minutes or more, and the upper limit is not particularly limited and, for example, a plateau of complex formation can be reached by incubation for about 30 minutes. The solvent is not particularly limited and, for example, an aqueous solvent can be used, specific examples of which include water, physiological saline, and buffer solutions such as PBS.

[0043] For the coexistence of the vesicles and the encoding nucleic acid, for example, general introduction methods such as electroporation and lipofection may be used. Note that, according to the protein carrier of the present invention, the encoding nucleic acid can be retained in the vesicles simply by allowing the vesicles and the encoding nucleic acid to coexist without using these general introduction methods.

[0044] Thus, with the protein carrier of the present invention, for example, a complex can be formed between the vesicles and a nucleic acid encoding a protein of interest to be delivered to a target cell simply by coexisting the nucleic acid with the vesicles in the solvent. This makes it extremely easy to retain the DDS target (the encoding nucleic acid) in the vesicles. Furthermore, with the protein carrier of the present invention, for example, the retained encoding nucleic acid can be introduced into the target cell via the vesicles. Specifically, with the protein carrier of the present invention, for example, the vesicles retaining the encoding nucleic acid can be fused with the target cell, and the retained encoding nucleic acid can be released into the target cell, thereby allowing the encoding nucleic acid to be introduced into the target cell from outside the target cell. Then, when the encoding nucleic acid is introduced into the target cell via the vesicles, for example, the protein encoded by the encoding nucleic acid is expressed in the target cell, resulting in the target cell being introduced into the target cell via the vesicles.

[0045] In addition, when the encoding nucleic acid and the concomitant component are retained in the vesicles, for example, the vesicles, the encoding nucleic acid, and the concomitant component may be allowed to coexist in the solvent and incubated under conditions that are not particularly limited and are the same as those exemplified above.

[0046] The delivery of the protein or the encoding nucleic acid by the protein carrier of the present invention may be, for example, in vitro, ex vivo, or in vivo.

[0047] The target to which the protein or the encoding nucleic acid is delivered by the protein carrier of the present invention may be, for example, a cell, or a tissue, organ, or living organism composed of cells. The type of target is not particularly limited, and examples thereof include humans and non-human animals. Examples of non-human animals include mice, rats, rabbits, dogs, monkeys, camels, and cows.

[0048] The protein carrier of the present invention is useful, for example, as a pharmaceutical, diagnostic agent, agricultural chemical, and research tool in the fields of agriculture, medicine, food science, life science, and the like.

[0049] (2) Protein Delivery Reagent and Manufacturing Method Thereof The protein delivery reagent of the present invention is characterized by comprising the protein carrier of the present invention and the protein or a nucleic acid encoding the protein. The protein delivery reagent of the present invention is characterized by comprising vesicles from the fruit of a plant of the Malpighiaceae family as a carrier for the protein or the encoding nucleic acid, and other configurations and conditions are not particularly limited.

[0050] In the protein delivery reagent of the present invention, the vesicles retain the protein or the encoding nucleic acid. That is, the protein delivery reagent of the present invention includes, for example, a complex of the vesicle and the protein or the encoding nucleic acid (the vesicle complex). The protein delivery reagent of the present invention can be applied to the protein carrier of the present invention.

[0051] The method for producing a protein delivery reagent of the present invention is characterized by comprising the step of forming a complex between the vesicles of the protein carrier of the present invention and the protein or the encoding nucleic acid to be delivered by coexisting the protein carrier of the present invention (a carrier comprising vesicles of the fruit of a plant of the family Cantharinaceae) and the protein or the encoding nucleic acid in a solvent. The production method of the present invention is characterized by using the vesicles as the carrier, and other configurations and conditions are not particularly limited.

[0052] The method for producing the protein delivery reagent of the present invention can be based on the explanation of the method for forming the vesicle complex in the protein carrier of the present invention.

[0053] (3) Protein Introduction Method The protein introduction method of the present invention is characterized by, as described above, a contact step in which a complex (the vesicle complex) of the protein carrier of the present invention and the protein to be delivered or the nucleic acid encoding the protein is contacted with a cell. The protein introduction method of the present invention is characterized by using the vesicles from the fruit of a plant of the Malpighiaceae family, i.e., the protein carrier of the present invention, as the delivery carrier; other configurations and conditions are not particularly limited. Furthermore, in the protein introduction method of the present invention, for example, the descriptions of (1) and (2) above can be used for the protein carrier, the protein delivery reagent in which the protein or the encoding nucleic acid is carried on the protein carrier, and the production method thereof, unless otherwise specified.

[0054] In the protein introduction method of the present invention, the vesicle complex may be a complex that further contains the concomitant component in addition to the protein or the encoding nucleic acid, for example.

[0055] The protein introduction method of the present invention may, for example, use the vesicle complex (the protein delivery reagent of the present invention) prepared in advance, or may further include a step of forming the vesicle complex. That is, the protein introduction method of the present invention may further include, for example, a step of forming a vesicle complex between the protein carrier and the protein or a nucleic acid encoding the protein, prior to the contacting step.

[0056] Furthermore, in the protein introduction method of the present invention, for example, in the formation step, the vesicle complex may be formed as a complex of the protein carrier, the protein or a nucleic acid encoding the protein, and the concomitant component.

[0057] In the protein introduction method of the present invention, the contacting step is, for example, a step of contacting the vesicle complex with cells in vivo, ex vivo, or in vitro.

[0058] The subject to which the vesicle complex is administered is not particularly limited, and examples thereof include cells, tissues, organs, living organisms, etc., and the type thereof is also not particularly limited. When the subject to which the vesicle complex is administered is a living organism, the contacting step is, for example, a step of administering the vesicle complex to a human or a non-human animal. Examples of the non-human animal include mice, rats, rabbits, dogs, monkeys, camels, and cows.

[0059] When the vesicle complex is administered in vivo in the contacting step, the administration method is not particularly limited and includes, for example, oral administration and parenteral administration. The parenteral administration includes, for example, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, and topical administration.

[0060] According to the present invention, by using the protein carrier of the present invention containing the vesicles, the DDS target held by the vesicle complex can be introduced into the target cells simply by contacting the vesicle complex with the target cells, without using common introduction methods such as electroporation, transfection reagents, or microinjection. Furthermore, as described above, the protein carrier of the present invention is derived from plants, and no transfection reagents or the like are required, so that, for example, effects on the target cells or living organisms can be suppressed. The protein carrier of the present invention is also effective for oral administration, for example, because it is less susceptible to the effects of gastric acid.

[0061] [Embodiment 1] The protein carrier of the present invention is capable of delivering proteins to target cells, and can therefore be used, for example, as a tool for genome editing as described above.

[0062] When the protein carrier of the present invention is used for genome editing, it is preferable that the protein carrier of the present invention retains, for example, a nuclease that can be used for genome editing. Examples of the nuclease include a Cas protein such as a Cas9 protein, a ZFN protein, and a TALEN protein.

[0063] Below are examples of using the protein carrier of the present invention to retain the Cas9 protein, which is a Cas protein, and to use it for genome editing, or to retain a Cas9 expression vector in which the coding sequence has been inserted into a vector (e.g., a plasmid vector) and to use it for genome editing. Note that the present invention is not limited to these examples, and for example, Cas9 can be replaced with the Cas family or other nucleases.

[0064] (1) Cas9 Protein A genome editing system that utilizes the Cas9 protein is the so-called CRISPER / Cas9 system. In this system, a complex RNP (ribonucleptide protein) (Cas9 / gRNA) is used to cleave a target sequence in the genome within a cell. The RNP complex is a complex of the Cas9 protein and a guide RNA (gRNA). The gRNA specifically recognizes the target sequence of a target gene in the genome, i.e., is a sequence designed to specifically bind to the target sequence. A single-stranded gRNA (hereinafter also referred to as sgRNA) is typically used to form the RNP. When the RNP complex is introduced into a target cell, the gRNA of the RNP complex specifically recognizes the target sequence, and the Cas9 protein of the RNP complex cleaves the target sequence. This cleavage makes it possible, for example, to knock out a target gene in the genome. Furthermore, knock-in can be achieved by inserting a foreign gene using the cellular repair mechanism associated with the cleavage. In the latter case, for example, donor DNA is allowed to coexist as the foreign gene.

[0065] (1-1) Cas9 protein + gRNA When the protein carrier of the present invention is used in the CRISPER / Cas9 system, for example, the vesicles, Cas9 protein, and gRNA may be allowed to coexist in the solvent and incubated. The incubation conditions are not particularly limited, and the examples described above can be used. This allows the CAS9 protein and the gRNA to be retained in the vesicles, forming the vesicle complex. Then, by adding this vesicle complex to a target cell of interest, the vesicle complex fuses with the target cell, and the RNP complex of the CAS9 protein and gRNA retained in the vesicle is introduced into the target cell.

[0066] For example, the Cas9 protein and the gRNA can be contacted with each other in advance to form the RNP, and then the RNP can be coexisted with the vesicle in the solvent; or the vesicle, Cas9 protein, and gRNA can be added to the solvent without contacting each other in advance, and then coexisted. Alternatively, the Cas9 protein can be contacted with the vesicle in the solvent, and then the gRNA can be contacted. The Cas9 protein and the gRNA can, for example, be contacted with each other to form the RNP. In this way, the vesicle (vesicle complex) holding the RNP can be formed.

[0067] The method for introducing the vesicle complex carrying the RNP into the target cell is not particularly limited, and may be, for example, by allowing the vesicle complex to coexist with the target cell. For example, by allowing the vesicle complex to coexist with the target cell, the vesicles of the vesicle complex fuse with the cell membrane of the target cell, and the RNP is released from the vesicle complex into the cell. The RNP released into the cell is transported to the nucleus of the target cell, where it acts on the target sequence and is then degraded within the cell.

[0068] (1-2) Cas9 protein + oligonucleotides for gRNA The vesicles may contain, for example, an oligonucleotide for the gRNA (e.g., an antisense strand of DNA) instead of the gRNA. In the case of the oligonucleotide, the oligonucleotide may be held in the vesicle together with the Cas9 protein by contacting the vesicle with the vesicle, thereby forming a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex fuses with the target cell, and the Cas9 protein and the oligonucleotide are introduced into the target cell. Then, in the cytoplasm of the target cell, the gRNA is expressed from the oligonucleotide, forming a complex RNP of the Cas9 protein and the gRNA.

[0069] (1-3) Cas9 Protein + gRNA Expression Vector The vesicles may, for example, retain the gRNA expression vector instead of the gRNA. In the case of the gRNA expression vector, the Cas9 protein and the gRNA expression vector may be brought into contact with the vesicles, thereby retaining both in the vesicles and forming a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex fuses with the target cell, and the Cas9 protein and the gRNA expression vector are introduced into the target cell. Then, the gRNA is expressed in the cytoplasm of the target cell, forming a complex RNP between the Cas9 protein and the gRNA. The RNP is transferred to the nucleus of the target cell, functions on the target sequence, and is then degraded within the cell.

[0070] (2) Cas9 Protein Coding Sequence When a coding sequence (e.g., Cas9 mRNA) is used as the encoding nucleic acid for the Cas9 protein, the vesicles, the Cas9 mRNA, and the gRNA can be coexisted and incubated in the solvent, for example. The incubation conditions are not particularly limited, and the examples described above can be used. This allows the Cas9 mRNA and the gRNA to be retained in the vesicles, forming a vesicle complex. Then, by adding this vesicle complex to the target cell of interest, the vesicle complex fuses with the target cell, and the Cas9 mRNA and the gRNA retained in the vesicle are introduced into the target cell. The Cas9 mRNA introduced into the target cell expresses the Cas9 protein in the cytoplasm of the target cell and forms a complex RNP with the co-introduced gRNA. The RNP is then transported to the nucleus of the target cell, where it acts on the target sequence and is then degraded within the cell.

[0071] The gRNA may be exemplified by the example in (1) above. That is, for example, the gRNA may be retained in the vesicle as is as shown in (1-1) above, or an oligonucleotide for the gRNA may be retained in the vesicle as shown in (1-2) above, or the gRNA expression vector may be retained in the vesicle as shown in (1-3) above.

[0072] In the case of the gRNA, for example, by contacting the vesicle with the Cas9 mRNA, both may be retained in the vesicle to form a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex fuses with the target cell, and the Cas9 mRNA and the gRNA are introduced into the target cell. Then, in the cytoplasm of the target cell, the Cas9 protein is expressed, forming a complex RNP of the Cas9 protein and the gRNA. The RNP is transported to the nucleus of the target cell, functions on the target sequence, and then degraded within the cell.

[0073] In addition, in the case of the oligonucleotide, the oligonucleotide and the Cas9 mRNA may be brought into contact with the vesicle, so that both are retained in the vesicle and form a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex fuses with the target cell, and the Cas9 mRNA and the oligonucleotide are introduced into the target cell. Then, in the cytoplasm of the target cell, the Cas9 protein and the gRNA are expressed, forming a complex RNP. The RNP is transported to the nucleus of the target cell, functions on the target sequence, and then degraded within the cell.

[0074] In addition, in the case of the gRNA expression vector, the gRNA expression vector and the Cas9 mRNA may be brought into contact with the vesicle, so that both are retained in the vesicle and form a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex fuses with the target cell, and the Cas9 mRNA and the gRNA expression vector are introduced into the target cell. Then, in the cytoplasm of the target cell, the Cas9 protein and gRNA are expressed, forming a complex RNP. The RNP is transported to the nucleus of the target cell, functions against the target sequence, and then degraded within the cell.

[0075] (3) Cas9 Expression Vector When using a Cas9 expression vector, for example, the vesicles, the Cas9 expression vector, and the gRNA may be coexisted in the solvent and incubated. The incubation conditions are not particularly limited, and the examples described above can be used. This allows the Cas9 expression vector and the gRNA to be retained in the vesicles, forming a vesicle complex. Then, by adding this vesicle complex to the target cell of interest, the vesicle complex fuses with the target cell, and the Cas9 expression vector and the gRNA retained in the vesicle are introduced into the target cell. The Cas9 expression vector introduced into the target cell expresses the Cas9 protein in the cytoplasm of the target cell, forming a complex RNP with the co-introduced gRNA. The RNP is then transported to the nucleus of the target cell, functions on the target sequence, and is then degraded intracellularly.

[0076] The gRNA may be exemplified by the example in (1) above. That is, for example, the gRNA may be retained in the vesicle as is as shown in (1-1) above, or an oligonucleotide for the gRNA may be retained in the vesicle as shown in (1-2) above, or the gRNA expression vector may be retained in the vesicle as shown in (1-3) above.

[0077] In the case of the gRNA, for example, by contacting the vesicle with the Cas9 expression vector, both can be retained in the vesicle to form a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex fuses with the target cell, and the Cas9 expression vector and the gRNA are introduced into the target cell. Then, in the cytoplasm of the target cell, the Cas9 protein is expressed, forming a complex RNP of the Cas9 protein and the gRNA. The RNP is transported to the nucleus of the target cell, functions on the target sequence, and then degraded within the cell.

[0078] In addition, in the case of the oligonucleotide, the oligonucleotide and the Cas9 expression vector may be brought into contact with the vesicle, so that both are retained in the vesicle and form a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex fuses with the target cell, and the Cas9 expression vector and the oligonucleotide are introduced into the target cell. Then, in the cytoplasm of the target cell, the Cas9 protein and gRNA are expressed, forming a complex RNP. The RNP is transported to the nucleus of the target cell, functions on the target sequence, and is then degraded within the cell.

[0079] In addition, in the case of the gRNA expression vector, the gRNA expression vector and the Cas9 expression vector may be brought into contact with the vesicle, so that both are retained in the vesicle and form a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex fuses with the target cell, and the Cas9 expression vector and the gRNA expression vector are introduced into the target cell. Then, in the cytoplasm of the target cell, the Cas9 protein and gRNA are expressed, forming a complex RNP. The RNP is transported to the nucleus of the target cell, and the Cas9 protein of the RNP is degraded intracellularly.

[0080] [Embodiment 2] As shown in the above-mentioned embodiment 1, the protein carrier of the present invention can easily introduce proteins such as Cas proteins or their encoding nucleic acids into target cells. Therefore, the present invention can be used to treat various diseases using genome editing, etc. The type of disease, the type of target cell, and the type of target gene are not particularly limited, and examples of use for the following diseases can be given. Note that these are examples and do not limit the present invention. (1) Treatment target: transthyretin amyloidosis Target organ: liver (2) Hypercholesterolemia Target gene: PCSK9

[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: The function of acerola-derived vesicles as protein carriers was confirmed by genome editing. Kits, reagents, and equipment were used according to their instruction manuals. Unless otherwise specified, the same conditions were used for each treatment in the following examples.

[0083] (Example 1-1) The genome editing was carried out in vitro.

[0084] (1) Vesicle Preparation Ten milliliters of juice from ripe Okinawan acerola fruit was concentrated tenfold (1 mL) using an ultrafiltration filter with a nominal molecular weight limit (NMWL) of 50,000 (Amicon™ Ultra-15 Centrifugal Filter Units - 50,000 NMWL, Millipore). One milliliter of the resulting concentrate was ultracentrifuged (4°C, 4 hours, 145,000 x g) to recover a precipitated fraction containing vesicles. The precipitated fraction was suspended in 120 μL of PBS to obtain Vesicle Sample A. Vesicle Sample A obtained from 10 mL of juice was subjected to a nanoparticle analysis system (NanoSight, Malvern) to confirm the particle size distribution of the vesicles contained in the vesicle fraction. The results are shown below. Particle mass: 1.9 x 10 ９ particles / μL Particle size: Mean 248nm Mode 138nm SD 128nm

[0085] Vesicle Sample B containing vesicles was prepared using a commercially available kit (Qiagen exoEasy kit, Qiagen) with 10 mL of the same fruit juice used to prepare Sample A. Specifically, the procedure was as follows: 10 mL of the juice was filtered using a 0.45 μm pore size membrane filter (Durapore™ PVDF Membrane Filter, Millipore). 10 mL of the resulting filtrate was applied to the kit, and a vesicle-containing elution fraction was isolated by elution with 400 μL of the kit's Buffer (Buffer XE). The elution fraction was then ultracentrifuged (100,000 × g, 49,000 rpm, 70 minutes, 4°C) to recover a vesicle pellet. This pellet was suspended in 50 μL of PBS to prepare Vesicle Sample B. Furthermore, the vesicle sample B obtained from 10 mL of fruit juice was subjected to a nanoparticle analysis system (trade name NanoSight, Malvern) to confirm the particle size distribution of the vesicles contained in the vesicle fraction. The results are shown below. Particle amount: 2.2 x 10 ８particles / μL Particle size: Mean 208nm Mode 155nm SD 108nm

[0086] (2) RNP formation. Synthetic gRNA against human alpha-galactosidase (GLA) and Cas9 recombinant protein (trade name Alt-R Sp Cas9 Nuclease V3, IDT) were used as co-components. The synthetic gRNA was prepared using commercially available reagents (trade name Alt-R CRISPR-Cas9 crRNA, tracrRNA, IDT) according to their instructions. Specifically, the GLA-recognizing crRNA and the CAS9 protein-binding tracrRNA were mixed, heated (95°C), and annealed at room temperature for 15 minutes to prepare the synthetic gRNA. The sequence of the Cas9 protein is registered in the GenBank database under accession number CDJ55032. 15 μL of 1 μmol / L of the gRNA and 15 μL of 1 μmol / L of the Cas9 protein were mixed, and the mixture was reacted at room temperature for 15 minutes to generate RNP. The mixture after the reaction was used as an RNP sample.

[0087] (3) Formation of Vesicle Complexes 20 μL of the vesicle sample (A or B) and 30 μL of the RNP sample were mixed in a microtube and allowed to react on ice for 30 minutes. The entire mixture after the reaction was used as an RNP-vesicle complex sample. RNP-vesicle complex sample A was obtained using vesicle sample A, and RNP-vesicle complex sample B was obtained using vesicle sample B.

[0088] (4) Transfection into cells Human embryonic kidney cells HEK293 were seeded (1 × 10 cells) in a 96-well plate using DMEM / F12 (Gibco) medium containing 10% FBS. ４Cells / well) were cultured at 37°C. After 24 hours of culture, 50 μL of RNP-vesicle complex sample (A or B) was added to each well, and further culture was continued. After 48 hours of culture from the addition of the RNP-vesicle complex sample, genomic DNA was extracted from the cultured cells in the well. Then, for the obtained DNA extract sample, a region (500 bp) containing the GLA PAM sequence and the gRNA target sequence was amplified by PCR, and the sequence was analyzed by direct sequencing using the Sanger method.

[0089] For the negative control (NC), the vesicle sample was not used, and 30 μL of the RNP sample was added to the cells instead of the RNA-vesicle complex sample. The cells were cultured in the same manner, and the sequence was analyzed.

[0090] For the positive control (PC), a commercially available transfection reagent (trade name Lipofectamin RNAiMAX, Thermo Fisher Scientific) was used instead of the vesicle sample to transfect the RNP into the cells. Specifically, 30 μL of the RNP sample and 1.2 μL of the transfection reagent were mixed in a microtube and reacted at room temperature for 20 minutes. After the reaction, the entire mixture was added to the cells. The cells were cultured in the same manner, and the sequence was analyzed.

[0091] The waveform data from the sequence analysis was analyzed using the TIDE (Tracking of Indels by Decomposition) web tool (http: / / shinyapps.datacurators.nl / tide / ), and the INDEL rate (INDEL insertion rate) in the sequence of the amplified region was calculated. The results are shown in the table below.

[0092]

[0093] As shown in Table 1, in the NC control using neither the vesicle sample nor lipofectamine, no INDEL (insertion and / or deletion of bases) was observed downstream of the PAM sequence in the GLA gene when cells were contacted with the RNP (0%). In the PC control using lipofectamine, the RNP was introduced into cells by lipofectamine, and double-strand breaks in the genomic DNA at the PAM sequence in the GLA gene and insertion of sequences during genomic DNA repair following the double-strand break were confirmed. In contrast, in the examples using acerola-derived vesicle samples, double-strand breaks and insertions were confirmed, similar to the PC control, regardless of the vesicle sample used. These results demonstrate that the vesicle sample can form RNP-vesicle complexes holding the RNP simply by contacting the RNP. Furthermore, it was confirmed that the RNP could be introduced into cells simply by contacting the RNP-vesicle complexes with cells, and that the introduced RNP functioned intracellularly.

[0094] (Example 1-2) The genome editing was carried out in vivo.

[0095] The vesicle sample used was vesicle sample A from Example 1-1. The vesicle sample was adjusted to an appropriate concentration using PBS. Then, in the same manner as in Example 1-1, a Cas9 / gRNA complex (RNP) was generated between the Cas recombinant protein (trade name Alt-R Sp Cas9 Nuclease V3, IDT) and the synthetic gRNA for human alpha-galactosidase (GLA).

[0096] C57BL / 6 mice were used (n=3). １１0.05 mL of 100 nmol / L RNP was mixed with 0.05 mL of 100 nmol / L RNP and incubated on ice for 30 minutes to complex the two. 100 μL of the reaction mixture was used as the RNP-vesicle complex sample. PBS was added to 100 μL of the RNP-vesicle complex sample to make a total of 200 μL, and the mixture was orally administered to mice using a stomach tube. 24 hours after oral administration, the small intestine and liver were collected from the mice, and DNA was extracted from each tissue using a DNeasy Blood & Tissue kit (Qiagen). For the DNA extraction samples from each tissue, the region containing the GLA PAM sequence and the gRNA target sequence was amplified by PCR in the same manner as in Example 1-1, and the sequence was analyzed by direct sequencing using the Sanger method.

[0097] As a negative control, the vesicle sample was not used, and instead of the RNA-vesicle complex sample, 0.05 mL of 100 nmol / L RNP was added to PBS to prepare a total volume of 200 μL, which was then administered to mice. The mice were cultured in the same manner, and the sequence was analyzed.

[0098] Then, in the same manner as in Example 1-1, the INDEL rate in the sequence of the amplified region was calculated using the TIDE web tool. The results are shown in the table below.

[0099]

[0100] As shown in Table 2, in the NC control without the vesicle sample or Lipofectamine, administration of the RNP alone failed to detect INDEL (insertion and / or deletion of bases) downstream of the PAM sequence in the GLA gene in either the small intestine or liver (0.1%, 0%). In contrast, in the example using the acerola-derived vesicle sample, double-strand breaks and insertions were detected in both the small intestine and liver. This demonstrates that the vesicle sample can form an RNP-vesicle complex that retains the RNP simply by contacting it with the RNP. Furthermore, administration of the RNP-vesicle complex alone can introduce the RNP into cells in vivo, confirming that the introduced RNP functions within the cells.

[0101] Thus, the protein carrier of the present invention allows proteins and co-components (nucleic acid substances) to be retained in the vesicles simply by contacting them, without the need for transfection reagents such as lipofectamine. Furthermore, the protein carrier allows the retained proteins and co-components to be introduced into cells, for example, in vivo and in vitro. Therefore, the protein carrier can be a useful delivery tool for genome editing, as exemplified in this example, and other applications involving the introduction of proteins into cells.

[0102] [Example 2] In this example, instead of the Cas9 protein used in Example 1, Cas9 mRNA encoding the ORF of the Cas9 protein (GenBank database, Accession No. CDJ55032) was used, and the function of the protein vesicles was confirmed by genome editing.

[0103] The vesicle sample used was vesicle sample B from Example 1. Instead of the Cas9 protein, 500 ng of 4104-base Cas9 mRNA and 15 μL of the same 1 μmol / L gRNA as in Example 1 were mixed with 20 μL of the vesicle sample and allowed to react on ice for 30 minutes. The total volume of the mixture after the reaction was used as a vesicle complex sample. Except for using the vesicle complex sample, the addition to cells (HEK293), incubation, extraction of genomic DNA, sequence analysis, and calculation of the INDEL rate were performed in the same manner as in Example 1. The results are shown in the table below.

[0104]

[0105] As shown in Table 3, in the NC without the vesicle sample or lipofectamine, INDEL (insertion and / or deletion of bases) was not substantially confirmed (0.2%) downstream of the PAM sequence in the GLA gene. In the PC using lipofectamine, double-strand breaks in genomic DNA at the PAM sequence of the GLA gene and insertion of sequences during genomic DNA repair following the double-strand breaks were confirmed. In contrast, in the example using acerola-derived vesicle sample, double-strand breaks and insertions were confirmed, similar to the PC. From this, it can be seen that the vesicle sample can form a vesicle complex holding the mRNA and the gRNA simply by contacting the RNP with the vesicle sample. Furthermore, the mRNA and the gRNA can be introduced into the cell simply by contacting the cell with the vesicle complex, and the introduced mRNA is translated into the Cas9 protein in the cell. Furthermore, it was confirmed that the Cas9 protein and the gRNA form RNP, and that the RNP functions in the cell.

[0106] Thus, the protein carrier of the present invention allows the protein-encoding nucleic acid and the co-component (nucleic acid substance) to be retained in the vesicles and then introduced into cells simply by contacting them, without using a transfection reagent such as lipofectamine. Therefore, it can be said that the protein carrier is a useful delivery tool for genome editing, as exemplified in this example, and other applications for introducing proteins into cells.

[0107] [Example 3] It was confirmed that the acerola-derived vesicles can also be used as protein carriers for proteins other than the Cas9 protein.

[0108] The vesicle sample used was the vesicle sample B of Example 1-1. The vesicle sample (2 × 10 ７ 0.01 mL of the antibody-vesicle complex (0.01 mL of vesicle complex (0.01 μg / mL)) was mixed with 0.005 mL of anti-mTOR antibody (mouse IgG, Cell Signaling Technology, 2 μg) directed against human mTOR, and the mixture was incubated on ice for 30 minutes to allow the two to complex. The mixture after the reaction was used as an antibody-vesicle complex sample.

[0109] SiHa cells (a cell line derived from human cervical cancer) were seeded in a 6-well plate using DMEM / F12 medium containing 10% FCS (fetal calf serum) and cultured at 37°C until they reached 50% confluence. 10 μL of the antibody-vesicle complex sample (a final concentration of 1 × 10 acerola vesicle particles) was added to each well. ５ After culturing for 24 hours, the number of cells in each well was measured using a measuring device (trade name: Cell Titer-Glo, Promega).

[0110] In addition, as control 1, the antibody-vesicle complex sample was not added, and as control 2, only the vesicle sample was added (2 × 10 ７ Control 1 was cultured in a system containing only anti-mTOR antibody (2 μg), and Control 2 was cultured in a system containing only anti-mTOR antibody (2 μg), and the number of cells was measured in the same manner. The number of cells in Control 1 was set as a relative value of 1, and the relative value of the cells was calculated as the cell viability.

[0111] The results are shown in Figure 1. Figure 1 is a graph showing cell viability, with the vertical axis representing cell viability. As shown in Figure 1, Controls 1, 2, and 3 showed approximately the same viability. In contrast, in the examples to which the antibody-vesicle complex samples were added, a decrease in cell number, i.e., a decrease in viability, was observed compared to the respective controls. The anti-mTOR antibody is known to reduce cell viability by neutralizing the function of mTOR, which is involved in the PI3K / Akt / mTOR signaling pathway. Therefore, the above results confirm that the acerola vesicles delivered the anti-mTOR antibody to cells and activated mTOR within the cells.

[0112] Thus, it has become clear that the protein carrier of the present invention can hold and introduce into cells various proteins, including antibodies as in this example, in addition to the Cas9 protein as in Examples 1 and 2.

[0113] 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.

[0114] This application claims priority based on Japanese Patent Application No. 2022-156126, filed September 29, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0115] The protein carrier of the present invention contains vesicles derived from the fruit of a plant belonging to the Acerola family, such as acerola, and can retain proteins or nucleic acids encoding the proteins. Therefore, the protein carrier of the present invention can be used as a DDS tool for proteins such as nucleases used in genome editing, for example.

Claims

1. It contains vesicles from the fruit of plants in the Malpighiaceae family. A protein carrier characterized in that the object to be held is a protein or the nucleic acid encoding the protein.

2. The protein carrier according to claim 1, wherein the Malpighiaceae plant is an acerola plant.

3. The protein carrier according to claim 1 or 2, wherein the average particle size of the vesicles is 30 to 400 nm.

4. The protein carrier according to claim 1 or 2, wherein the vesicle is an extracellular vesicle.

5. The protein carrier according to claim 1 or 2, wherein the coding nucleic acid is an expression vector in which the coding sequence of the protein is inserted into the vector.

6. The protein carrier according to claim 1 or 2, wherein the protein is a sequence-specific nuclease.

7. The protein carrier according to claim 6, wherein the sequence-specific nuclease is a Cas protein.

8. The protein carrier according to claim 7, wherein the Cas protein is a Cas9 protein.

9. The protein carrier according to claim 1 or 2, wherein the retaining object further comprises a co-component for the protein or the nucleic acid encoding the protein.

10. The protein carrier according to claim 9, wherein the combined component is a nucleic acid substance, and the nucleic acid substance is a guide RNA.

11. Furthermore, the protein carrier according to claim 1 or 2, comprising the protein or the nucleic acid encoding the protein.

12. A method for producing a protein delivery reagent, characterized by comprising the step of forming a complex between the vesicles of the protein carrier and the protein or the nucleic acid encoding the protein by coexisting the protein carrier containing the vesicles described in claim 1 or 2 with the protein to be delivered or the nucleic acid encoding the protein in a solvent.

13. A method for producing a protein delivery reagent according to claim 12, comprising the step of mixing the protein carrier and the protein or the nucleic acid encoding the protein in the solvent and incubating them.

14. Furthermore, the method for producing a protein delivery reagent according to claim 12, wherein the protein or the nucleic acid encoding the protein is coexisted with the protein carrier in the solvent.

15. The method includes a contact step of bringing a complex of a protein carrier containing vesicles as described in claim 1 or 2 and a protein for delivery or a nucleic acid encoding the protein into contact with a cell. The contact step is a step of bringing the complex into contact with cells ex vivo or in vitro, or a step of administering the complex to a non-human animal in vivo. A method for introducing proteins into cells, characterized by the features described above.

16. The method for introducing a protein according to claim 15, wherein the complex further comprises a compound component for the protein or the nucleic acid coding for the protein.

17. Prior to the aforementioned contact process, A method for introducing a protein according to claim 15, comprising a complex formation step of forming a complex between the protein carrier and the protein or the nucleic acid encoding the protein.