Protein carrier and protein introduction method

Abstract

Provided is a novel carrier for introducing a protein such as a Cas protein into a cell. The carrier for a protein according to the present invention is characterized by comprising vesicles of a Malpigaceae plant fruit, and the carrier is a protein or a nucleic acid encoding the protein to be held. The present invention relates to a method for producing a plant comprising the following steps: (1) selecting a plant of Malpighiaceae, for example, a Malpighiaceae plant of the Malpighiaceae plant of the Malpighiaceae plant of the Malpighiaceae plant; the carrier for proteins according to the present invention can be used, for example, as a tool for genome editing.

Description

Technical Field

[0001] The present invention relates to a protein carrier and a protein introduction method. Background Art

[0002] In the field of biochemistry, DDS (drug delivery system) platforms for delivering target substances to target cells are highly valued.

[0003] For example, in the genome editing widely used in various fields such as agriculture, livestock, aquatic products, and medical treatment, the Cas9 protein with nuclease activity and the guide RNA as an RNA molecule are contacted with the target sequence, the above-mentioned target sequence is cut off, and the gene is knocked out and the foreign gene is knocked in. In this way, in the above-mentioned genome editing, for example, a protein with a relatively large molecular weight needs to be delivered. In addition, if it is introduced into animals and plants as food materials or introduced into humans for treatment, it is worried that, for example, artificial DDS will affect the organism. Summary of the invention

[0004] Technical problem to be solved by the invention Therefore, the object of the present invention is to provide a new carrier as a DDS platform for proteins.

[0005] Technical solutions for solving technical problems To achieve the above object, the protein carrier of the present invention is characterized in that it comprises a vesicle of a fruit of a plant of the family Malpighiaceae, and the object to be retained 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 in that it includes the following steps: by allowing the above-mentioned protein carrier containing vesicles of the present invention and the protein as the delivery target or the encoding nucleic acid of the above-mentioned protein to coexist in a solvent, a complex of the vesicle of the above-mentioned protein carrier and the above-mentioned protein or the encoding nucleic acid of the above-mentioned protein is formed.

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

[0008] Effects of the Invention The protein carrier of the present invention comprises a vesicle of a fruit of a Malpighiaceae plant such as acerola, and the protein or the nucleic acid encoding the protein can be retained by the vesicle. Therefore, the protein carrier of the present invention can be used as a DDS tool for proteins such as nucleases used in genome editing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0010] Unless otherwise specified, the terms used in this specification shall be used with the meanings generally used in the relevant technical field.

[0011] [1] A protein carrier comprising a vesicle of a fruit of a plant of the family Malpighiaceae, The object of maintenance is a protein or a nucleic acid encoding the protein.

[0012] [2] The protein carrier according to [1], wherein the plant of the Malpighiaceae family is a Malpighia sp. plant.

[0013] [3] The protein carrier according to [1] or [2], wherein the average particle size of the vesicles is 30 to 400 nm.

[0014] [4] The protein carrier according to any one of [1] to [3], wherein the vesicle is an extracellular vesicle.

[0015] [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 is inserted into a vector.

[0016] [6] The protein carrier according to any one of [1] to [5], wherein the protein is a sequence-specific nuclease.

[0017] [7] The protein carrier according to [6], wherein the sequence-specific nuclease is a Cas protein.

[0018] [8] The protein carrier according to [7], wherein the Cas protein is a Cas9 protein.

[0019] [9] The protein carrier according to any one of [1] to [8], wherein the retention object further comprises a component for use with the protein or a nucleic acid encoding the protein.

[0020]

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

[0021]

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

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

[0022]

[12] A method for producing a protein delivery reagent, comprising the following steps: forming a complex of the vesicle of the protein carrier and the protein or the nucleic acid encoding the protein by allowing the protein carrier containing a vesicle as described in any one of [1] to

[10] and a protein as a delivery target or a nucleic acid encoding the protein to coexist in a solvent.

[0023]

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

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

[0024]

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

[12] or

[13] , wherein a component for the protein or a nucleic acid encoding the protein is further contained in the solvent together with the protein carrier.

[0025]

[15] A method for introducing a protein into a cell, comprising a contact step of bringing a complex of a protein carrier containing a vesicle as described in any one of [1] to

[10] and a protein as a delivery target or a nucleic acid encoding the protein into contact with the cell.

[0026]

[16] The protein introduction method according to

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

[0027]

[17] The protein introduction method according to

[15] or

[16] , further comprising, before the contacting step: The complex formation step is to form a complex between the protein carrier and the protein or the nucleic acid encoding the protein.

[0028]

[18] The protein introduction method according to any one of

[15] to

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

[0029]

[19] The protein introduction method according to any one of

[15] to

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

[0030]

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

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

[0031]

[21] The protein delivery agent according to

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

[0032]

[22] The protein delivery reagent according to

[20] or

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

[0033]

[23] The protein delivery reagent according to

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

[0034]

[24] The protein delivery reagent according to

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

[0035]

[25] The protein delivery reagent according to any one of

[20] to

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

[0036]

[26] The protein delivery reagent according to

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

[0037] (1) Protein carriers As described above, the protein carrier of the present invention is characterized in that it comprises a vesicle of the fruit of a Malpighiaceae plant. The present inventors have found that the vesicle of the fruit of a Malpighiaceae plant retains a protein or a nucleic acid encoding the protein to form a complex, and the complex is taken into a cell (hereinafter also referred to as a "target cell"), and the protein expressed by the protein or the nucleic acid encoding the protein functions in the cell, thereby establishing the present invention. The present invention is characterized by comprising the vesicle, and other configurations and conditions are not particularly limited.

[0038] As described above, the protein carrier of the present invention can maintain the above-mentioned protein or the coding nucleic acid of the above-mentioned protein through the above-mentioned vesicle, thereby enabling the above-mentioned protein or the coding nucleic acid of the above-mentioned protein to be delivered. Therefore, the protein carrier of the present invention is also referred to as a delivery reagent, a DDS reagent, for example. In the present invention, "protein carrier" means, for example, in addition to the meaning of maintaining the protein itself, maintaining the coding nucleic acid of the protein. When the above-mentioned coding nucleic acid maintained by the above-mentioned protein carrier is introduced into a cell, for example, through the translation mechanism to the protein in the above-mentioned cell, the encoded above-mentioned protein can be synthesized. Therefore, the above-mentioned coding nucleic acid, for example, only needs to contain a sequence (such as ORF) as a reading frame that can synthesize a protein through the translation mechanism of the cell. When the above-mentioned coding sequence is maintained, the protein carrier of the present invention, for example, can also be referred to as a coding nucleic acid carrier.

[0039] The species of the Malpighiaceae plant is not particularly limited, and examples thereof include the genus Malpighia, and specifically, examples thereof include Malpighia sp., and preferably Malpighia sp. such as M. emarginata DC., M. glabra, and M. punicifolia.

[0040] The vesicles can be obtained from the fruit of the Malpighiaceae plant. The fruit can be, for example, ripe or unripe, or a mixture thereof. The vesicles are preferably, for example, the vesicle fraction recovered from the fruit juice, described below.

[0041] The vesicles can be prepared, for example, by extracting from the fruit of the Malpighiaceae plant. The preparation method is not particularly limited, for example, the vesicles can be obtained by crushing the fruit, preparing the crushed product or a suspension of the crushed product, and fractionating the vesicles. The classification method is not particularly limited, for example, ultrafiltration, ultracentrifugation, concentration gradient method, separation method using microfluidic system, etc. can be cited. In the preparation method, for example, commercially available kits can be used, such as ExoEasy Maxi Kit (trade name, QIAGEN), ExoQuick (trade name, System Bioscience), Total Exosome Isolation reagent (Total Exosome Isolation reagent) (trade name, Invitrogen), etc. can be used. The vesicles, for example, can be provided to the above-mentioned various separation methods by squeezing the fruit. The preparation of the juice can use, for example, a pulverizing device, a squeezing device, etc. The juice can be, for example, the juice of a ripe fruit, the juice of an unripe fruit, or the juice of a ripe or unripe frozen fruit.

[0042] The protein carrier of the present invention can use, for example, a vesicle fraction containing a plurality of the above-mentioned vesicles. The size of the above-mentioned vesicle is not particularly limited, and its particle diameter can be exemplified by, for example, 30 to 400 nm, 80 to 300 nm, 150 to 300 nm, 150 to 250 nm, 200 to 250, 100 to 200 nm, and 80 to 200 nm. The above-mentioned vesicles are also called, for example, microvesicles or nanovesicles. When the above-mentioned vesicle fraction is represented by a particle size distribution, the peak of the particle size 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, 100 to 200 nm, 80 to 200 nm, 200 ± 100 nm, 200 ± 50 nm, 200 ± 30 nm, and 200 ± 20 nm. In addition, in the above-mentioned particle size distribution, when all vesicles are set to 100%, the proportion of vesicles in the above-mentioned peak (for example, 200±50nm, 200±20nm) is not particularly limited, and its lower limit is, for example, 30% or more, 50% or more, 80% or more, and its upper limit is, for example, 70% or less, 80% or less, 90% or less, 100%. The above-mentioned vesicle fraction is, for example, a fraction extracted from the fruit juice of the above-mentioned fruit in a manner to achieve the above-mentioned particle size and the above-mentioned particle size distribution. As a protein carrier of the present invention, when the above-mentioned vesicle fraction is used, for example, a fraction extracted from the above-mentioned fruit (preferably fruit juice) by an extraction method to achieve the above-mentioned particle size and the above-mentioned particle size distribution can be used. The above-mentioned vesicle fraction can be, for example, in a state containing other components derived from the above-mentioned fruit juice.

[0043] The method for measuring the vesicle particle size is not particularly limited, for example, a method such as nanoparticle tracking analysis can be used, for example, a commercially available device (device name NanoSight, qNANO) can be used. The above-mentioned vesicles can be, for example, any of extracellular vesicles and intracellular vesicles, preferably extracellular vesicles. The above-mentioned vesicles can be, for example, exosome-like vesicles. The above-mentioned exosome-like vesicles are, for example, vesicles of the same size as extracellular vesicles (EVs) derived from human cells, more specifically, vesicles of the same size as small EVs (also called exosomes) derived from human cells. The above-mentioned exosome-like vesicles are, for example, extracellular vesicles derived from human cells, preferably vesicles obtained by the same separation method as exosomes derived from human cells.

[0044] "Protein" is a compound obtained by peptide bonding of amino acids. The length (number of amino acid residues) of "protein" in the present invention is not particularly limited. When the length is used as a reference, it also includes peptides such as oligopeptides and polypeptides (for example, less than 50 residues) and proteins longer than them (for example, more than 50 residues). 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, and 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, and 50 kDa, and the range is, for example, 10-150 kDa, 20-150 kDa, 10-110 kDa, 20-110 kDa, 80-110 KDa, 50-80 KDa, 20-50 KDa, etc.

[0045] The object of retention held by the protein carrier of the present invention may be, for example, a protein or a nucleic acid encoding the above-mentioned protein. The encoding nucleic acid is, for example, a ribonucleic acid, and RNA, DNA, etc. may be mentioned. RNA is, for example, premRNA, mRNA, preferably mRNA. In addition, the protein carrier of the present invention may, for example, hold the above-mentioned protein, or the above-mentioned encoding nucleic acid, or both the above-mentioned protein and the above-mentioned encoding nucleic acid, or may hold other components together with at least one of the above-mentioned protein and the above-mentioned encoding nucleic acid. The above-mentioned other components may be appropriately selected, for example, according to the type of the above-mentioned object of retention, the purpose of introducing the above-mentioned object of retention into cells, etc. In the case where the above-mentioned other components are components selected according to the type and / or purpose of the above-mentioned object of retention, the above-mentioned other components will also be referred to as "combination components" hereinafter.

[0046] The type of the above-mentioned protein as the above-mentioned object of maintenance is not particularly limited and can be appropriately selected according to the purpose. The type of the above-mentioned protein can be exemplified by functional proteins such as enzymes, antibodies, antigen-binding fragments, transcription factors, etc. The size of the above-mentioned protein is not particularly limited, and the above-mentioned examples can be cited. The protein carrier of the present invention can be used, for example, in genetic engineering technologies such as genome editing described later. Therefore, as the above-mentioned protein, for example, an enzyme, as a specific example, nucleases such as sequence-specific nucleases can be cited.

[0047] Examples of the nuclease include Cas proteins such as Cas9 protein, ZFN (Zinc Finger Nuclease) protein, TALEN (Transcription Activator-Like Effector Nuclease) protein, and the like.

[0048] The above-mentioned coding nucleic acid as the above-mentioned retention object can be a nucleic acid composed of the coding sequence of the above-mentioned protein, or a nucleic acid containing the coding sequence of the above-mentioned protein. That is, the above-mentioned coding nucleic acid, for example, can express the above-mentioned protein as the target based on the above-mentioned coding sequence in the introduced cell. The above-mentioned coding sequence in the above-mentioned coding nucleic acid can be, for example, RNA such as mRNA, or DNA such as cDNA. The length of the above-mentioned coding sequence is not particularly limited, and for example, the length converted from the size or length of the above-mentioned protein can be exemplified, and the size of the above-mentioned protein is, for example, as described above. Regarding the length of the above-mentioned coding sequence, as a specific example, the lower limit is, for example, 480 bases, 1200 bases, the upper limit is, for example, 2640 bases, etc., and its range is, for example, 480 to 2640 bases, 1200 to 2640 bases, 480 to 1200 bases, 1200 to 1920 bases, 1920 to 2640 bases. The length of the encoding nucleic acid is not particularly limited, and may be, for example, the same as the encoding sequence, or may be about 1 to 300 bases, 1 to 200 bases, or 1 to 100 bases longer than the length of the encoding sequence.

[0049] Examples of the coding nucleic acid include DNA molecules, RNA molecules, chimeric molecules of DNA and RNA, etc. The DNA molecules, RNA molecules and chimeric molecules may be composed of natural nucleic acids, artificial nucleic acids, or both natural nucleic acids and artificial nucleic acids.

[0050] The above-mentioned encoding nucleic acid may be, for example, an expression vector capable of expressing the above-mentioned protein, specifically, an expression vector in which the coding sequence of the above-mentioned protein is inserted into a vector in a manner capable of expressing the above-mentioned protein. The above-mentioned coding sequence in the above-mentioned expression vector is, for example, DNA, preferably cDNA. The above-mentioned expression vector, for example, is DNA (for example, cDNA), into which an antisense strand (template strand) for the amino acid sequence of the above-mentioned protein is inserted, and as a specific example, a double-stranded DNA of the above-mentioned antisense strand and sense strand is inserted. The type of the above-mentioned vector encoding the amino acids of the above-mentioned protein is not particularly limited, and examples thereof include plasmid vectors, viral vectors, etc., and examples of the above-mentioned viral vectors include adenovirus vectors, Sendai virus vectors, etc.

[0051] Furthermore, as described above, the protein carrier of the present invention may hold, for example, at least one of the above-mentioned protein and the above-mentioned encoding nucleic acid, and other components (the above-mentioned combined components).

[0052] The above-mentioned combined components are, for example, nucleic acid substances (hereinafter, also referred to as combined nucleic acid components). Examples of the above-mentioned combined nucleic acid components include nucleic acid molecules such as DNA molecules, RNA molecules, and chimeric molecules of DNA and RNA. The above-mentioned combined nucleic acid components may be composed of, for example, natural nucleic acids, artificial nucleic acids, or the above-mentioned natural nucleic acids and the above-mentioned artificial nucleic acids. The above-mentioned combined nucleic acid components may be, for example, expression vectors capable of expressing the above-mentioned nucleic acid molecules. In the case where the above-mentioned nucleic acid molecule is a DNA molecule, the above-mentioned expression vector, for example, inserts an antisense strand (template strand) of the DNA for the above-mentioned DNA molecule, and as a specific example, inserts a double-stranded DNA of the above-mentioned antisense strand and sense strand. The type of the above-mentioned expression vector is not particularly limited, and examples thereof include plasmid vectors, viral vectors, and the like. Examples of the above-mentioned viral vectors include lentiviral vectors, adenoviral vectors, and the like.

[0053] When the protein carrier of the present invention is used for the above-mentioned genome editing, the above-mentioned combined nucleic acid component can include, for example, guide RNA (gRNA), etc. As a specific example, single-stranded guide RNA (sgRNA) etc. can be mentioned. The above-mentioned combined nucleic acid component can be, for example, an expression vector (gRNA expression vector) capable of expressing the above-mentioned guide RNA. Specifically, the above-mentioned gRNA expression vector can be, for example, an expression vector in which the above-mentioned guide RNA is inserted into the vector, or an expression vector in which the antisense strand (template strand) of the DNA for the above-mentioned guide RNA, or the double-stranded DNA of the above-mentioned antisense strand and sense strand is inserted into the vector. The expression vector of the above-mentioned guide RNA can, for example, utilize a commercially available viral gRNA expression vector, etc. The above-mentioned guide RNA will be described later.

[0054] The protein vector of the present invention may contain, for example, an independent expression vector for the protein and an expression vector for the combined nucleic acid component, or an expression vector for the protein that co-expresses the protein and the nucleic acid molecule.

[0055] The protein carrier of the present invention may contain the above-mentioned protein or the above-mentioned encoding nucleic acid, or may not contain the above-mentioned protein or the above-mentioned encoding nucleic acid, for example, at the stage before use by the user. In the case where the above-mentioned protein or the above-mentioned encoding nucleic acid is contained, the protein carrier of the present invention is also referred to as a protein delivery agent as described later. In the case where the protein carrier of the present invention contains the above-mentioned protein or the above-mentioned encoding nucleic acid, for example, it is preferred that the above-mentioned vesicle forms a complex with the above-mentioned protein or the above-mentioned encoding nucleic acid (hereinafter also referred to as "vesicle complex"). The form of the above-mentioned vesicle complex is not particularly limited, for example, it may be a form in which the above-mentioned protein or the above-mentioned encoding nucleic acid is enclosed in the interior of the above-mentioned vesicle, or it may be a form in which the above-mentioned protein or the above-mentioned encoding nucleic acid is retained on the outer wall (outer surface) of the above-mentioned vesicle. Specifically, for example, it is the latter.

[0056] The method for forming the vesicle complex is not particularly limited. The following examples illustrate the method for forming a vesicle complex that retains the protein and the method for forming a vesicle complex that retains the encoding nucleic acid. It should be noted that the present invention is not limited to these examples.

[0057] First, the vesicle complex retaining the above-mentioned protein can be formed by, for example, allowing the protein carrier of the present invention (ie, the above-mentioned vesicle) and the above-mentioned protein to coexist in a solvent.

[0058] Specifically, for example, by making the vesicle and the protein coexist in a solvent and incubating them, a complex of the vesicle and the protein can be formed. The incubation temperature is not particularly limited, and can be, for example, room temperature (e.g., 30±10°C), or a temperature range below this range (e.g., higher than 0°C and lower than 20°C), preferably a low temperature range (e.g., 4±5°C, 4±3°C), and more preferably ice-cold conditions (e.g., 1 to 6°C).

[0059] In the above coexistence, the ratio of the above vesicles to the above protein is not particularly limited, for example, 1 m mol, vesicles 5×10 4 ~8×10 4 particles, 2×10 5 ~5×10 5 particles, 2×10 6 ~5×10 6In order to be used for the above-mentioned genome editing, when the above-mentioned vesicles hold Cas proteins such as Cas9 protein, the vesicles are 1 m The ratio of mol is preferably within the above range, for example. The same is true for nucleases and other proteins other than Cas proteins.

[0060] The incubation time is not particularly limited. The lower limit is not particularly limited, for example, it is 5 minutes or more, 15 minutes or more, and the upper limit is not particularly limited, for example, the plateau phase of complex formation can be reached by incubation for about 30 minutes. The above-mentioned solvent is not particularly limited, for example, an aqueous solvent can be used, as a specific example, water, physiological saline, PBS and other buffer solutions can be cited.

[0061] In the coexistence of the vesicle and the protein, for example, electroporation, lipofection, etc., which are common introduction methods, can be further utilized. It should be noted that, according to the protein carrier of the present invention, for example, even if the vesicle and the protein are merely coexisted without utilizing these common introduction methods, the protein can be retained in the vesicle.

[0062] Thus, according to the protein carrier of the present invention, for example, a complex with the vesicle can be formed simply by allowing the target protein to be delivered to the target cell to coexist with the vesicle in the solvent. Therefore, the object of DDS (the protein) can be held in the vesicle very simply. Further, according to the protein carrier of the present invention, for example, the held protein can be introduced into the target cell via the vesicle. Specifically, according to the protein carrier of the present invention, for example, the vesicle holding the protein can be fused with the target cell, and the held protein can be released into the target cell, so that the protein can be introduced from the extracellular part of the target cell into the cell.

[0063] It should be noted that, when the above-mentioned combined component is also maintained in the above-mentioned vesicle together with the above-mentioned protein, for example, the above-mentioned vesicle, the above-mentioned protein and the above-mentioned combined component are coexisted in the above-mentioned solvent and incubated. The conditions are not particularly limited and are the same as the above-mentioned examples. When the above-mentioned combined component is the above-mentioned combined nucleic acid component, regarding incubation, for example, the above-mentioned low temperature range or a temperature below it is preferably used. Thus, for example, the decomposition of the above-mentioned combined nucleic acid component before the formation of the above-mentioned vesicle complex caused by the mixing of nucleases such as RNase can be fully prevented.

[0064] Next, the vesicle complex holding the above-mentioned protein encoding nucleic acid can also be formed by, for example, allowing the protein carrier of the present invention (i.e., the above-mentioned vesicle) and the above-mentioned encoding nucleic acid to coexist in a solvent. Unless otherwise specified, the above-mentioned examples of the method for forming the vesicle complex holding the protein can be cited.

[0065] Specifically, for example, by making the above-mentioned vesicles and the above-mentioned encoding nucleic acid coexist in a solvent and incubate, a complex of the above-mentioned vesicles and the above-mentioned encoding nucleic acid can be formed. The incubation temperature is not particularly limited, for example, it can be a room temperature range (for example, 30±10°C), or a temperature range below this range (for example, higher than 0°C and lower than 20°C), preferably a low temperature range (for example, 4±5°C, 4±3°C), and more preferably under ice-cold conditions (for example, 1-6°C). In this way, while maintaining the above-mentioned encoding nucleic acid, it is preferably incubated at a temperature in the above-mentioned low temperature range or below. Thus, for example, the decomposition of the above-mentioned encoding nucleic acid before the formation of the above-mentioned vesicle complex caused by the mixing of nucleases such as RNase can be prevented.

[0066] In the above coexistence, the ratio of the above vesicles to the above encoding nucleic acid is not particularly limited, for example, 1 m mol, vesicles 5×10 4 ~8×10 4 particles, 2×10 5 ~5×10 5 particles, 2×10 6 ~5×10 6 In order to be used for the above-mentioned genome editing, when the Cas9 encoding nucleic acid encoding the Cas9 protein is retained in the above-mentioned vesicle, the vesicle is 1 m The ratio of mol is preferably in the above range. The above coding sequence is, for example, Cas9 mRNA or Cas9 DNA encoding Cas9 protein, preferably Cas9 mRNA. In addition, the above coding nucleic acid can be, for example, an expression vector inserted with the above Cas9 mRNA or Cas9 DNA. The coding nucleic acid of nucleases other than Cas protein and the coding nucleic acid of other proteins are also the same.

[0067] The incubation time is not particularly limited. The lower limit is not particularly limited, for example, it is 5 minutes or more, 15 minutes or more, and the upper limit is not particularly limited, for example, the plateau phase of complex formation can be reached by incubation for about 30 minutes. The above-mentioned solvent is not particularly limited, for example, an aqueous solvent can be used, as a specific example, water, physiological saline, PBS and other buffer solutions can be cited.

[0068] In the coexistence of the vesicle and the encoding nucleic acid, for example, electroporation, lipofection, etc., which are common introduction methods, can be further utilized. It should be noted that, according to the protein carrier of the present invention, for example, even if the vesicle and the encoding nucleic acid are only coexisted without utilizing these common introduction methods, the encoding nucleic acid can be retained in the vesicle.

[0069] Thus, according to the protein carrier of the present invention, for example, a complex with the vesicle can be formed simply by making the coding nucleic acid of the target protein to be delivered to the target cell coexist with the vesicle in the solvent. Therefore, the object of DDS (the coding nucleic acid) can be held in the vesicle extremely simply. Further, according to the protein carrier of the present invention, for example, the held coding nucleic acid can be introduced into the target cell via the vesicle. Specifically, according to the protein carrier of the present invention, for example, the vesicle holding the coding nucleic acid can be fused with the target cell, and the held coding nucleic acid can be released into the target cell, so that the coding nucleic acid can be introduced from the extracellular part of the target cell into the cell. Furthermore, if the coding nucleic acid is introduced into the target cell via the vesicle, for example, the protein encoded by the coding nucleic acid is expressed in the target cell, and as a result, the target protein is introduced into the target cell via the vesicle.

[0070] It should be noted that, when the above-mentioned combined component is also maintained in the above-mentioned vesicle together with the above-mentioned encoding nucleic acid, for example, the above-mentioned vesicle, the above-mentioned encoding nucleic acid and the above-mentioned combined component are coexisted in the above-mentioned solvent and incubated. The conditions are not particularly limited and are the same as those exemplified above.

[0071] The protein or the encoding nucleic acid described above can be delivered using the protein carrier of the present invention, for example, in vitro, ex vivo, or in vivo.

[0072] The object to which the above-mentioned protein or the above-mentioned encoding nucleic acid is delivered using the protein carrier of the present invention may be, for example, a cell, or a tissue, organ, or organism composed of cells. The type of the above-mentioned object is not particularly limited, and examples thereof include humans and non-human animals. The above-mentioned non-human animals include mice, rats, rabbits, dogs, monkeys, camels, cattle, etc.

[0073] The protein carrier of the present invention is useful as, for example, pharmaceuticals, diagnostic reagents, agricultural chemicals, and research tools in agriculture, medicine, food science, life science, and the like.

[0074] (2) Protein delivery reagent and method for manufacturing the same The protein delivery agent of the present invention is characterized by comprising the above-mentioned protein carrier of the present invention and the above-mentioned protein or the above-mentioned protein encoding nucleic acid. The protein delivery agent of the present invention is characterized by comprising, as a carrier for the above-mentioned protein or the above-mentioned encoding nucleic acid, a vesicle of the fruit of the above-mentioned Malpighiaceae plant, and other configurations and conditions are not particularly limited.

[0075] In the protein delivery reagent of the present invention, the vesicle holds the protein or the encoding nucleic acid. That is, the protein delivery reagent of the present invention, for example, comprises 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 refer to the description of the protein carrier of the present invention.

[0076] In addition, the method for producing a protein delivery agent of the present invention is characterized in that it includes the following step: forming a complex of the vesicle of the protein delivery agent and the protein or the encoding nucleic acid by making the above-mentioned protein carrier of the present invention (the carrier comprising the vesicle of the fruit of the Malpighiaceae plant) and the above-mentioned protein or the encoding nucleic acid of the above-mentioned protein as a delivery target coexist in a solvent. The production method of the present invention is characterized in that the above-mentioned vesicle is used as a carrier, and other configurations and conditions are not particularly limited.

[0077] The method for producing the protein delivery agent of the present invention can refer to the description of the method for forming the above-mentioned vesicle complex of the protein carrier of the present invention.

[0078] (3) Protein introduction method As described above, the protein introduction method of the present invention for introducing a protein into a cell is characterized by comprising a contact step of bringing a complex of the above-mentioned protein carrier of the present invention and a protein as a delivery target or a nucleic acid encoding the above-mentioned protein (the above-mentioned vesicle complex) into contact with a cell. The protein introduction method of the present invention is characterized by using a vesicle of the fruit of the above-mentioned Malpighiaceae plant, that is, the protein carrier of the present invention, as a delivery carrier, and other configurations and conditions are not particularly limited. In addition, in the protein introduction method of the present invention, for example, the above-mentioned protein carrier, the above-mentioned protein delivery reagent that holds the above-mentioned protein or the above-mentioned encoding nucleic acid in the above-mentioned protein carrier, and its production method, etc., can be cited as described in the above-mentioned (1) and (2) unless otherwise specified.

[0079] In the protein introduction method of the present invention, the vesicle complex may be, for example, a complex containing the above-mentioned combined component in addition to the above-mentioned protein or the above-mentioned encoding nucleic acid.

[0080] The protein delivery method of the present invention may, for example, use the previously prepared vesicle complex (the protein delivery reagent of the present invention), or may further include a step of forming the vesicle complex. That is, the protein delivery method of the present invention may, for example, further include a step of forming a vesicle complex of the protein carrier and the protein or the nucleic acid encoding the protein before the contacting step.

[0081] In the protein introduction method of the present invention, for example, a complex of the protein carrier, the protein or the nucleic acid encoding the protein, and the combined component can be formed as the vesicle complex in the forming step.

[0082] In the protein introduction method of the present invention, the contact step is, for example, a step of bringing the vesicle complex into contact with cells in vivo, in vitro or in vitro.

[0083] The subject to which the vesicle complex is administered is not particularly limited, and examples thereof include cells, tissues, organs, organisms, etc., and the type thereof is also not particularly limited. When the subject to be administered is an organism, the contacting step is, for example, a step of administering the vesicle complex to a human or non-human animal, and examples of the non-human animal include mice, rats, rabbits, dogs, monkeys, camels, cattle, etc.

[0084] In the contacting step, when the vesicle complex is administered in vivo, the administration method is not particularly limited, and examples thereof include oral administration and parenteral administration. Examples of parenteral administration include intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, and topical administration.

[0085] According to the present invention, since the above-mentioned protein carrier of the present invention comprising the above-mentioned vesicle is used, even without using a common introduction method such as electroporation, transfection reagent, microinjection, etc., the DDS object held by the above-mentioned vesicle complex can be introduced into the above-mentioned target cell simply by bringing the above-mentioned vesicle complex into contact with the above-mentioned target cell. In addition, as described above, the protein carrier of the present invention is derived from plants, and a transfection reagent or the like is not required, so, for example, the influence on the cell, organism, etc. as the introduction object can also be suppressed. The protein carrier according to the present invention is, for example, not easily affected by gastric acid, and is therefore effective when administered orally.

[0086] [Implementation Method 1] The protein carrier of the present invention can deliver proteins to target cells, for example, and therefore, as described above, can be used as a tool for genome editing, for example.

[0087] When the protein carrier of the present invention is used for genome editing, it is preferred that, for example, a nuclease that can be used for genome editing is retained in the protein carrier of the present invention. Examples of the nuclease include Cas proteins such as Cas9 protein, ZFN proteins, TALEN proteins, and the like.

[0088] The following are examples of using a Cas9 protein as a Cas protein in a protein carrier of the present invention for genome editing, or using a Cas9 expression vector that holds its coding sequence inserted into a vector (e.g., a plasmid vector) for genome editing. It should be noted that the present invention is not limited to these examples, and for example, Cas9 can be replaced by Cas family or other nucleases.

[0089] (1) Cas9 protein As a system for genome editing using Cas9 protein, the so-called CRISPER / Cas9 system can be cited. In this system, a complex RNP (ribonucleptide protein) (Cas9 / gRNA) is used to cut the target sequence on the genome in the cell. The above-mentioned complex RNP is a complex of Cas9 protein and guide RNA (gRNA). gRNA is a sequence designed to specifically recognize the target sequence of the target gene in the genome, that is, a sequence that specifically binds to the above-mentioned target sequence. Regarding gRNA, the formation of the above-mentioned RNP usually uses a single-stranded gRNA (hereinafter also referred to as sgRNA). After the above-mentioned complex RNP is introduced into the target cell, the gRNA of the above-mentioned complex RNP specifically recognizes the above-mentioned target sequence, and the Cas9 protein of the above-mentioned complex RNP cuts off the target sequence. Through this cutting, for example, the target gene in the genome can be knocked out. In addition, knock-in can also be performed by inserting a foreign gene using the repair mechanism of the cell accompanied by the cutting. In the latter case, for example, the donor DNA is made to coexist as the above-mentioned foreign gene in advance.

[0090] (1-1) Cas9 protein + gRNA In the case of using the protein carrier of the present invention in the above-mentioned CRISPER / Cas9 system, for example, the above-mentioned vesicle, Cas9 protein and gRNA are coexisted in the above-mentioned solvent and incubated. The incubation conditions are not particularly limited, and the above-mentioned examples can be cited. Thus, the CAS9 protein and the above-mentioned gRNA can be maintained in the above-mentioned vesicle to form the above-mentioned vesicle complex. Then, by adding the vesicle complex to the target cell as the target, the above-mentioned vesicle complex is fused with the above-mentioned target cell, and the complex RNP of the CAS9 protein and gRNA maintained in the above-mentioned vesicle is introduced into the above-mentioned target cell.

[0091] Regarding the Cas9 protein and the gRNA, for example, they can be contacted with each other in advance to form the RNP, and then the RNP and the vesicle can be coexisted in the solvent, or the vesicle, Cas9 protein and gRNA can be added to the solvent separately without contacting each other in advance to make them coexist. In addition, the Cas9 protein can be contacted with the vesicle in the solvent, and then the gRNA can be further contacted. The Cas9 protein and the gRNA can form the RNP, for example, by contacting each other. In this way, the vesicle (the vesicle complex) holding the RNP can be formed.

[0092] There is no particular limitation on the method of introducing the vesicle complex holding the RNP into the target cell, for example, the vesicle complex can be coexisted with the target cell. According to the vesicle complex, for example, by coexisting with the target cell, the vesicle of the vesicle complex fuses 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 transferred to the nucleus of the target cell, functions on the target sequence, and is then decomposed in the cell.

[0093] (1-2) Cas9 protein + oligonucleotide targeting gRNA At the vesicle, for example, an oligonucleotide (e.g., an antisense strand of DNA) directed against the gRNA can be retained in the vesicle instead of the gRNA. In the case of the oligonucleotide, the oligonucleotide can be brought into contact with the vesicle together with the Cas9 protein, thereby being retained in the vesicle to form a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex is fused 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 by the oligonucleotide to form a complex RNP of the Cas9 protein and the gRNA.

[0094] (1-3) Cas9 protein + gRNA expression vector For example, the gRNA expression vector can be replaced by the gRNA and maintained in the vesicle. In the case of the gRNA expression vector, the gRNA expression vector and the Cas9 protein can be contacted with the vesicle to maintain the two in the vesicle to form a vesicle complex. The vesicle complex is added to the target cell, the vesicle complex is fused 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 to form a complex RNP of the Cas9 protein and the gRNA. The RNP is transferred to the nucleus of the target cell, and decomposes in the cell after exerting its function on the target sequence.

[0095] (2) Coding sequence of Cas9 protein When a coding sequence (e.g., Cas9 mRNA) is used as the coding nucleic acid for the Cas9 protein, for example, the vesicle, the Cas9 mRNA, and the gRNA are coexisted in the solvent and incubated. The incubation conditions are not particularly limited, and the above-mentioned examples can be cited. Thus, the Cas9 mRNA and the gRNA can be maintained in the vesicle to form the vesicle complex. Then, by adding the vesicle complex to the target cell as the target, the vesicle complex is fused with the target cell, and the Cas9 mRNA and the gRNA maintained 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, forming a complex RNP with the co-introduced gRNA. Then, the RNP is transferred to the nucleus of the target cell, and decomposes in the cell after exerting its function on the target sequence.

[0096] The gRNA can be referred to the examples in (1). That is, for example, the gRNA can be directly held in the vesicle as shown in (1-1), an oligonucleotide targeting the gRNA can be held in the vesicle as shown in (1-2), or the gRNA expression vector can be held in the vesicle as shown in (1-3).

[0097] In the case of the above-mentioned gRNA, for example, the above-mentioned Cas9 mRNA can be brought into contact with the above-mentioned vesicle together with the above-mentioned vesicle, so that the two are maintained in the above-mentioned vesicle to form a vesicle complex. By adding the above-mentioned vesicle complex to the above-mentioned target cell, the above-mentioned vesicle complex is fused with the above-mentioned target cell, and the above-mentioned Cas9 mRNA and the above-mentioned gRNA are introduced into the above-mentioned target cell. Then, the Cas9 protein is expressed in the cytoplasm of the above-mentioned target cell to form a complex RNP of the Cas9 protein and the above-mentioned gRNA. The above-mentioned RNP is transferred to the nucleus of the above-mentioned target cell, and after exerting its function on the above-mentioned target sequence, it is decomposed in the cell.

[0098] In addition, in the case of the above-mentioned oligonucleotide, the above-mentioned oligonucleotide and the above-mentioned Cas9 mRNA can also be brought into contact with the above-mentioned vesicle together, so that both are maintained in the above-mentioned vesicle to form a vesicle complex. By adding the above-mentioned vesicle complex to the above-mentioned target cell, the above-mentioned vesicle complex is fused with the above-mentioned target cell, and the above-mentioned Cas9 mRNA and the above-mentioned oligonucleotide are introduced into the above-mentioned target cell. Then, the Cas9 protein and the above-mentioned gRNA are expressed in the cytoplasm of the above-mentioned target cell to form a complex RNP. The above-mentioned RNP is transferred to the nucleus of the above-mentioned target cell, and after exerting its function on the above-mentioned target sequence, it is decomposed in the cell.

[0099] In addition, in the case of the above-mentioned gRNA expression vector, the above-mentioned gRNA expression vector and the above-mentioned Cas9mRNA can be contacted with the above-mentioned vesicle together, so that both are maintained in the above-mentioned vesicle to form a vesicle complex. By adding the above-mentioned vesicle complex to the above-mentioned target cell, the above-mentioned vesicle complex is fused with the above-mentioned target cell, and the above-mentioned Cas9 mRNA and the above-mentioned gRNA expression vector are introduced into the above-mentioned target cell. Then, the Cas9 protein and gRNA are expressed in the cytoplasm of the above-mentioned target cell to form a complex RNP. The above-mentioned RNP is transferred to the nucleus of the above-mentioned target cell, and after exerting its function on the above-mentioned target sequence, it is decomposed in the cell.

[0100] (3) Cas9 expression vector When using a Cas9 expression vector, for example, the vesicle, the Cas9 expression vector and the gRNA are coexisted in the solvent and incubated. The incubation conditions are not particularly limited, and the examples described above can be cited. Thus, the Cas9 expression vector and the gRNA can be maintained in the vesicle to form the vesicle complex. Then, by adding the vesicle complex to the target cell as the target, the vesicle complex is fused with the target cell, and the Cas9 expression vector and the gRNA maintained 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. Then, the RNP is transferred to the nucleus of the target cell, and decomposes in the cell after exerting its function on the target sequence.

[0101] The gRNA can be referred to the examples in (1). That is, for example, the gRNA can be directly held in the vesicle as shown in (1-1), an oligonucleotide targeting the gRNA can be held in the vesicle as shown in (1-2), or the gRNA expression vector can be held in the vesicle as shown in (1-3).

[0102] In the case of the above-mentioned gRNA, for example, the above-mentioned Cas9 expression vector can be contacted with the above-mentioned vesicle together, so that the two are maintained in the above-mentioned vesicle to form a vesicle complex. By adding the above-mentioned vesicle complex to the above-mentioned target cell, the above-mentioned vesicle complex is fused with the above-mentioned target cell, and the above-mentioned Cas9 expression vector and the above-mentioned gRNA are introduced into the above-mentioned target cell. Then, the Cas9 protein is expressed in the cytoplasm of the above-mentioned target cell to form a complex RNP of the Cas9 protein and the above-mentioned gRNA. The above-mentioned RNP is transferred to the nucleus of the above-mentioned target cell, and after exerting its function on the above-mentioned target sequence, it is decomposed in the cell.

[0103] In addition, in the case of the above-mentioned oligonucleotide, the above-mentioned oligonucleotide and the above-mentioned Cas9 expression vector can be contacted with the above-mentioned vesicle together, so that both are maintained in the above-mentioned vesicle to form a vesicle complex. By adding the above-mentioned vesicle complex to the above-mentioned target cell, the above-mentioned vesicle complex is fused with the above-mentioned target cell, and the above-mentioned Cas9 expression vector and the above-mentioned oligonucleotide are introduced into the above-mentioned target cell. Then, the Cas9 protein and gRNA are expressed in the cytoplasm of the above-mentioned target cell to form a complex RNP. The above-mentioned RNP is transferred to the nucleus of the above-mentioned target cell, and after exerting its function on the above-mentioned target sequence, it is decomposed in the cell.

[0104] In addition, in the case of the gRNA expression vector, the gRNA expression vector and the Cas9 expression vector can be contacted with the vesicle together, so that both are maintained in the vesicle to form a vesicle complex. By adding the vesicle complex to the target cell, the vesicle complex is fused with the target cell, and the Cas9 expression vector and the gRNA expression vector are introduced into the target cell. Then, the Cas9 protein and gRNA are expressed in the cytoplasm of the target cell to form a complex RNP. The RNP is transferred to the nucleus of the target cell, and the Cas9 protein of the RNP is decomposed in the cell.

[0105] [Implementation Method 2] As shown in the above embodiment 1, according to the protein carrier of the present invention, proteins such as Cas proteins or their encoding nucleic acids can be easily introduced into target cells. Therefore, the present invention can be used for the treatment of various diseases using genome editing, etc. There is no particular limitation on the type of disease, target cell, and target gene, and for example, the following diseases can be exemplified. It should be noted that these are illustrative and do not limit the present invention.

[0106] (1) Treatment target: Transthyretin amyloidosis Target organ: Liver (2) Hypercholesterolemia Target gene: PCSK9 Hereinafter, the present invention will be described in detail by way of Examples and the like, but the present invention is not limited to these Examples.

[0107] Example [Example 1] The function of acerola-derived vesicles as a protein carrier was confirmed by genome editing. The kits, reagents, and devices were used according to their instructions. Unless otherwise specified, the conditions of each treatment in the following examples are the same.

[0108] (Example 1-1) The above-described genome editing is performed in vitro.

[0109] (1) Preparation of vesicles Using an ultrafiltration filter with a nominal molecular weight cutoff (NMWL) of 50,000 (trade name: Amicon (trademark) Ultra-15 Centrifugal Filter Units - 50,000 NMWL, Millipore), 10 mL of juice from ripe acerola fruit grown in Okinawa was concentrated to 10 times (1 mL). 1 mL of the obtained concentrate was subjected to ultracentrifugation (4°C, 4 hours, 145,000 × g) to recover the precipitate fraction containing vesicles. The precipitate fraction was suspended in 120 m The vesicle sample A was prepared by adding 10 mL of PBS to the juice. The vesicle sample A obtained from 10 mL of the 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.

[0110] Particle quantity: 1.9×10 9 particles / m L Particle size: Average (Mean) 248nm Mode 138nm SD 128nm Using 10 mL of the same juice as used in the preparation of sample A, a commercially available kit (trade name Qiagen exoEasy kit, Qiagen) was used to prepare a vesicle sample B containing vesicles. Specifically, the treatment was performed as follows. m 10 mL of the juice was filtered through a membrane filter (trade name: Durapore (trademark) PVDF Membrane Filter, Millipore). 10 mL of the obtained filtrate was supplied to the kit and 400% Buffer (Buffer XE) of the kit was used. m The eluted fraction containing vesicles was separated (dissociated) by elution of 1 L. Then, the eluted fraction was subjected to ultracentrifugation (100,000 × g, 49,000 rpm, 70 minutes, 4°C) to recover the vesicle particles. m L of PBS was added to prepare vesicle sample B. In addition, the vesicle sample B obtained from 10 mL of 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.

[0111] Particle quantity: 2.2×10 8 particles / m L Particle size: Average (Mean) 208nm Mode 155nm SD 108nm (2) RNP formation As a combined component, a synthetic gRNA targeting human α-galactosidase (GLA) was used, and as the Cas9 protein, a Cas9 recombinant protein (trade name Alt-R Sp Cas9 Nuclease V3, IDT) was used. The preparation of the above-mentioned synthetic gRNA uses a commercially available reagent (trade name Alt-R CRISPR-Cas9 crRNA, tracrRNA, IDT) according to its instructions. Specifically, the crRNA that recognizes the above-mentioned GLA and the tracRNA that binds to the above-mentioned CAS9 protein are mixed, heated (95°C), and then annealed at room temperature for 15 minutes to prepare the above-mentioned synthetic gRNA. The sequence of the above-mentioned Cas9 protein is registered in the database (Gen Bank) with accession number CDJ55032. 1 m mol / L of the above gRNA 15 m L and 1 m 15 mol / L of the above Cas9 protein m The mixture was reacted at room temperature for 15 minutes to generate RNP. The reaction mixture was used as an RNP sample.

[0112] (3) Formation of vesicle complex The above vesicle samples (A or B) were m L and the above RNP samples 30 m L were mixed in a microtube and reacted on ice for 30 minutes. The total amount of the reaction mixture was used as the RNP-vesicle complex sample. The RNP-vesicle complex sample A was used as the RNP-vesicle complex sample A, and the RNP-vesicle complex sample B was used as the RNP-vesicle complex sample B.

[0113] (4) Introduction into cells Human embryonic kidney HEK293 cells were seeded in 96-well plates (1×10 4 cells / well) and cultured at 37°C. After 24 hours of culture, 50 mL RNP-vesicle complex sample (A or B), further cultured. After adding the above RNP-vesicle complex sample and culturing for 48 hours, genomic DNA was extracted from the cultured cells in the above wells. Then, for the obtained DNA extract sample, the region (500 bp) containing the PAM sequence of the above GLA and the target sequence of the above gRNA was amplified by PCR, and direct sequencing based on the Sanger method was performed to analyze the sequence.

[0114] Negative control (NC) did not use the above vesicle samples, but added 30 m The culture was carried out in the same manner, and the sequence was analyzed, except that the above-mentioned RNP sample was used instead of the above-mentioned RNA-vesicle complex sample.

[0115] As a positive control (PC), a commercially available transfection reagent (trade name: Lipofectamin RNAiMAX, ThermoFischer) was used instead of the vesicle sample to introduce RNP into the cells. m L and the above transfection reagent 1.2 m L were mixed in a microtube, reacted at room temperature for 20 minutes, and the whole amount of the mixed solution after the reaction was added to the above cells. Culture and sequence analysis were performed in the same manner.

[0116] Then, the waveform data of the sequence analysis was analyzed using the TIDE (Tracking of Indels by Decomposition) web tool (http: / / shinyapps.datacurators.nl / tide / ) to calculate the INDEL rate (INDEL insertion rate) in the sequence of the amplified region. The results are shown in the following table.

[0117] [Table 1] As shown in Table 1, for NCs not using any of the vesicle samples and liposomes, no INDEL (insertion and / or deletion of bases) (0%) could be confirmed downstream of the PAM sequence in the GLA gene by simply contacting the RNP with cells. In PCs using liposomes, the RNP was introduced into cells using liposomes, and double-stranded cleavage of genomic DNA in the PAM sequence of the GLA gene and insertion of the sequence during genomic DNA repair following the double-stranded cleavage could be confirmed. In contrast, in the example using a vesicle sample derived from acerola, double-stranded cleavage and insertion could be confirmed in the same manner as in PCs when any vesicle sample was used. Thus, it can be confirmed that, according to the vesicle sample, an RNP-vesicle complex holding the RNP can be formed simply by contacting the RNP, and further, the RNP can be introduced into the cell simply by contacting the RNP-vesicle complex with the cell, and the introduced RNP functions in the cell.

[0118] (Example 1-2) The above genome editing is performed in vivo.

[0119] The vesicle sample used was the vesicle sample A of Example 1-1. The concentration of the vesicle sample was appropriately adjusted using PBS. Then, the same operation as in Example 1-1 was performed to generate a Cas9 / gRNA complex (RNP) of the Cas recombinant protein (trade name Alt-RSp Cas9 NucleaseV3, IDT) and a synthetic gRNA for the human α-galactosidase (GLA).

[0120] C57BL / 6 mice were used (n=3). The above vesicle sample A (1×10 11 0.05 mL of 100 nmol / L RNPs were mixed and incubated on ice for 30 minutes to allow the two to form a complex. m L was used as the RNP-vesicle complex sample. m Add PBS to adjust the total volume to 200 m L, and the mice were orally administered using a gastric tube. 24 hours after oral administration, the small intestine and liver were recovered from the mice, and DNA was extracted from each tissue using a DNeasyBlood&Tissur kit (Qiagen). For the DNA extraction samples of each tissue, the region containing the PAM sequence of the above GLA and the target sequence of the above gRNA was amplified by PCR in the same manner as in Example 1-1, and direct sequencing based on the Sanger method was performed to analyze the sequence.

[0121] The negative control was prepared by adding PBS to 0.05 mL of 100 nmol / L RNP instead of the above vesicle sample and a total of 200 m The cells were cultured and sequenced in the same manner except that the sample of L was administered to mice.

[0122] Then, in the same manner as in Example 1-1, the INDEL ratio in the sequence of the amplified region was calculated using the TIDE web tool. The results are shown in the following table.

[0123] [Table 2] As shown in Table 2, for NCs without using any of the above-mentioned vesicle samples and liposomes, only by administering the above-mentioned RNP, no INDEL (insertion and / or deletion of bases) could be confirmed downstream of the PAM sequence in the GLA gene in either the small intestine or the liver (0.1%, 0%). In contrast, in the example using the vesicle sample derived from acerola cherry, double-strand cleavage and insertion were confirmed in both the small intestine and the liver. It can be confirmed that, according to the above-mentioned vesicle sample, only by contacting with the above-mentioned RNP, an RNP-vesicle complex that retains the above-mentioned RNP can be formed, and further, only by administering the above-mentioned RNP-vesicle complex, the above-mentioned RNP can be introduced into cells in the body, and the introduced RNP functions in the cells.

[0124] It can be seen that according to the protein carrier of the present invention, the protein and the combined component (nucleic acid substance) can be retained in the above-mentioned vesicles only by contacting them without using transfection reagents such as liposomes. In addition, according to the above-mentioned protein carrier, for example, the retained protein and the combined component can be introduced into cells, whether in vivo or in vitro. Therefore, in addition to the genome editing and protein introduction into cells exemplified in this embodiment, it can also be said to be a useful delivery tool.

[0125] [Example 2] In this example, Cas9 mRNA (database GenBank, accession number CDJ55032) encoding the ORF of the Cas9 protein was used instead of the Cas9 protein in the above Example 1, and the function of the vesicle for the above protein was confirmed by genome editing.

[0126] The vesicle sample used was the vesicle sample B of Example 1. Then, instead of the above-mentioned Cas9 protein, 500 ng of Cas9 mRNA with 4104 bases and the same 1 m mol / L of the above gRNA 15 m L and the above vesicle sample 20 m L was mixed and reacted on ice for 30 minutes. The total amount of the mixed solution after the reaction was used as a vesicle complex sample. In addition to using the above vesicle complex sample, the addition to cells (HEK293), culture, extraction of genomic DNA, analysis of sequences, and calculation of INDEL rates were performed in the same manner as in Example 1 above. The results are shown in the following table.

[0127] [Table 3] As shown in Table 3, NC without using any of the above-mentioned vesicle samples and liposomes could not substantially confirm INDEL (insertion and / or deletion of bases) downstream of the PAM sequence in the GLA gene (0.2%). PC using liposomes can confirm double-stranded scission of genomic DNA in the PAM sequence of the GLA gene, and insertion of the sequence during genomic DNA repair immediately after the double-stranded scission. In contrast, in the example using the vesicle sample derived from acerola, double-stranded scission and insertion can be confirmed in the same way as PC. It can be confirmed that according to the above-mentioned vesicle sample, a vesicle complex that retains the above-mentioned mRNA and the above-mentioned gRNA can be formed only by contacting with the above-mentioned RNP, and further, the above-mentioned mRNA and the above-mentioned gRNA can be introduced into the above-mentioned cell only by contacting the cell with the above-mentioned vesicle complex, and the introduced mRNA is translated into Cas9 protein in the cell. Further, Cas9 protein and the above-mentioned gRNA form RNP, and RNP functions in the cell.

[0128] Thus, according to the protein carrier of the present invention, the protein encoding nucleic acid and the conjugate component (nucleic acid substance) can be held in the above-mentioned vesicles and then introduced into cells simply by contacting them without using transfection reagents such as liposomes. Therefore, it can be said that it is a useful delivery tool in other uses besides genome editing and protein introduction into cells as exemplified in this embodiment.

[0129] [Example 3] It was confirmed that the acerola-derived vesicles can be used as protein carriers for proteins other than the Cas9 protein.

[0130] The vesicle sample used was the vesicle sample B of Example 1-1. The vesicle sample (2×10 7 particles / mL) 0.01mL and anti-mTOR antibody (mouse IgG, Cell Signaling Technology, 2 mg) 0.005 mL, and incubated on ice for 30 minutes to allow the above two to complex. The mixed solution after the reaction was used as the antibody-vesicle complex sample.

[0131] 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 were 50% confluent. The antibody-vesicle complex sample was added to each well for 10 min. m L (as the particle number of Acerola vesicles, the final concentration is 1×10 5 After 24 hours of culture, the number of cells in each well was measured using a measuring device (trade name Cell Titer-Glo, Promega).

[0132] In addition, for control 1, no antibody-vesicle complex sample was added, and for control 2, only the vesicle sample (2×10 7 particles / mL, 0.01mL), and as control 3, only anti-mTOR antibody was added (2 m The system g) was also cultured in the same manner and the number of cells was measured. Then, the number of cells in control 1 was taken as relative value 1, and the relative value of cells was calculated as the cell survival rate.

[0133] The results are shown in Figure 1 . Figure 1 is a graph showing cell survival rate, where the vertical axis represents the cell survival rate. Figure 1 As shown, control 1, control 2, and control 3 showed approximately the same survival rate. In contrast, in the example to which the antibody-vesicle complex sample was added, a decrease in the number of cells, i.e., a decrease in the survival rate, was observed compared to the above-mentioned controls. The above-mentioned anti-mTOR antibody is known to reduce cell survival by counteracting the function of mTOR involved in the PI3K / Akt / mTOR signaling pathway. Therefore, the above-mentioned results confirmed that the above-mentioned anti-mTOR antibody was delivered to the cells through the above-mentioned Acerola vesicles, and mTOR exerted its function in the cells.

[0134] This shows that the protein carrier according to the present invention is not limited to the Cas9 protein as in Examples 1 and 2 above, and can hold and introduce various proteins represented by antibodies as in this example into cells.

[0135] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above embodiments. The configuration and details of the present invention can be modified in various ways that can be understood by those skilled in the art within the scope of the present invention.

[0136] This application claims priority based on Japanese patent application No. 2022-156126 filed on September 29, 2022, and all the disclosed contents are incorporated into this application.

[0137] Industrial Applicability The protein carrier according to the present invention comprises a vesicle of a Malpighiaceae fruit such as acerola, and the protein or the nucleic acid encoding the protein can be retained by the vesicle. Therefore, the protein carrier according to the present invention can be used as a DDS tool for proteins such as nucleases used in genome editing, etc.

Claims

1. A protein carrier, characterized in that: Vesicles containing fruits of Malpighiaceae, The object held by the protein carrier is a protein or a nucleic acid encoding the protein.

2. The protein carrier according to claim 1, wherein The Malpighiaceae plant is a plant of the species Acerola.

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 any one of claims 1 to 3, wherein The vesicles are extracellular vesicles.

5. The protein carrier according to any one of claims 1 to 4, wherein The encoding nucleic acid is an expression vector in which the coding sequence of the protein is inserted.

6. The protein carrier according to any one of claims 1 to 5, 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 Cas9 protein.

9. The protein carrier according to any one of claims 1 to 8, wherein The retention object further includes a component for use with the protein or a 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. The protein carrier according to any one of claims 1 to 10, wherein The protein carrier further comprises the protein or a nucleic acid encoding the protein.

12. A method for producing a protein delivery reagent, characterized in that: The method comprises the step of forming a complex of the protein carrier vesicle and the protein or the protein encoding nucleic acid by allowing the protein carrier containing a vesicle according to any one of claims 1 to 10 and a protein as a delivery target or a nucleic acid encoding the protein to coexist in a solvent.

13. The method for producing a protein delivery reagent according to claim 12, wherein: The production method comprises the following steps: mixing the carrier for the protein and the protein or the nucleic acid encoding the protein in the solvent and incubating the mixture.

14. The method for producing a protein delivery reagent according to claim 12 or 13, wherein: Furthermore, a component for the protein or a nucleic acid encoding the protein and a carrier for the protein are allowed to coexist in the solvent.

15. A method for introducing a protein into a cell, characterized in that: The method comprises a contacting step of bringing a complex of the protein carrier comprising a vesicle according to any one of claims 1 to 10 and a protein as a delivery target or a nucleic acid encoding the protein into contact with a cell.

16. The protein introduction method according to claim 15, wherein The complex is a complex further comprising a combined component for the protein or a nucleic acid encoding the protein.

17. The protein introduction method according to claim 15 or 16, wherein: Before the contacting step, the method further comprises: The step of forming a complex comprises forming a complex between the protein carrier and the protein or a nucleic acid encoding the protein.

18. The protein introduction method according to any one of claims 15 to 17, wherein The contacting step is a step of bringing the complex into contact with cells in vivo, ex vivo or in vitro.

19. The protein introduction method according to any one of claims 15 to 18, wherein The contacting step is a step of administering the complex to a human or non-human animal.

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