Drug delivery systems using solutions

Abstract

The present invention provides a method for introducing nucleic acids, proteins, or complexes thereof into cells with excellent efficiency, in particular, a method using a transduction solution. The method for transducing a molecule of interest into a cell of the present invention comprises the step of contacting the cell with the molecule of interest and a transduction solution, wherein the transduction solution comprises at least one of the following (A1) to (A5) and (B) a salt: (A1) a compound represented by the following general formula (I) or a salt thereof, but excluding a specified compound; (A2) a compound represented by the following general formula (II) or a salt thereof; (A3) a nucleic acid base or a salt thereof; (A4) a compound represented by the general formula (III) or a salt thereof, but excluding malic acid; (A5) at least one selected from the group consisting of creatinine, hydroxyproline, 1,3-butanediol, trientine, D-cellobiose, 1,3-dimethylurea, pantothenic acid, and trimethyldione, or a salt thereof. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a solution for delivering a molecule of interest (nucleic acid, protein, etc.) into a cell for transduction. Further, the present invention relates to a method for transducing a molecule of interest into a cell using such a solution and a method for producing a cell transduced with a molecule of interest. Background Art

[0002] In order to efficiently transfect nucleic acids, proteins, or complexes thereof into cells, various intracellular delivery systems are being studied in the art.

[0003] Patent document 1 describes a transduction buffer comprising (i) a transduction compound, (ii) an activator / enhancer of a salt or sodium-hydrogen transporter, and preferably (iii) an osmotic pressure protectant. As transduction compounds, betaine (e.g., non-surfactant type sulfobetaine), GABA (γ-aminobutyric acid), etc. are described. In addition, as an example, it is described that gRNA and Cas9 protein were transduced into KBM7 cells and the cutting of the WDR85 gene was observed.

[0004] Non-patent document 1 describes that sgRNA-Cas9 protein was transduced using specific compounds described in patent document 1 (GABA and non-detergent sulfobetaine 201 (NDSB-201), etc.), and that cleavage of the WDR85 gene was observed.

[0005] Patent Document 2 describes a transduction buffer comprising (i) a transduction compound, (ii) 250-2500 mM of one or more salts (Na, Rb, Li, K or Cs), (iii) an osmotic pressure inducer (osmotic pressure: 500-5000 mOsmol / kg) and (iv) an osmotic pressure protectant. In addition, as an example, it is described that gRNA and Cas9 protein were transduced into KBM7 cells and the cleavage of the target gene was observed.

[0006] Non-patent document 2 states that by adding 1-4 SV40 NLSs to the N-terminus of the Cas9 protein, the cleavage of the target gene caused by transduction of the Cas9 / gRNA complex was observed in vitro (neural progenitor cells) and in vivo (injection into mouse brain).

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication No. 2015 / 028969

[0010] Patent Document 2: International Publication No. 2017 / 093326

[0011] Non-patent literature

[0012] Non-patent document 1: D'Astolfo et al., Cell, 161, 674-690, April 23, 2015

[0013] Non-patent document 2: Brett T Staahl et al., Nature Biotechnology, 35, 431-434, 2017 Summary of the invention

[0014] Problems to be solved by the invention

[0015] The object of the present invention is to provide a method for efficiently introducing nucleic acids, proteins, or complexes thereof into cells, in particular, a method using a transduction solution. In another aspect, the present invention is to provide a method for producing cells transduced using such a method.

[0016] Methods used to solve problems

[0017] The present inventors conducted intensive studies to solve the above problems and found that nucleic acids, proteins or complexes thereof can be introduced into cells with high efficiency by using solutions of specific compounds or molecules, thereby solving the above problems and completing the present invention.

[0018] That is, the present invention relates to at least the following inventions. [1]

[0020] A method for transducing a molecule of interest into a cell, comprising the steps of contacting the cell with the molecule of interest and a transduction solution,

[0021] The transduction solution comprises at least one of the following (A1) to (A5) and (B) a salt:

[0022] (A1) a compound represented by the following general formula (I) or a salt thereof, excluding 20 kinds of L-amino acids and S-methyl-L-methionine which are constituent units of proteins in living organisms,

[0023]

[0024] [where n is 0 or 1,

[0025] R 1 and R 2 Each independently represents a hydrogen atom or COR 3 ,

[0026] R 0It represents the side chain of the amino acid constituting the structural unit of the protein of the organism, excluding C 1-6 Alkyl, the side chain may have a C 1-6 alkyl,

[0027] Among them, R 0 When it is a hydrogen atom, R 1 Indicates COR 3 , R 2 represents a hydrogen atom,

[0028] R 3 represents a C group which may have a substituent (excluding a thiol group and a disulfide group) 1-6 Alkyl (excluding methyl) or NR 4 R 5 ,

[0029] R 4 and R 5 Each independently represents a hydrogen atom or a C 1-4 alkyl.];

[0030] (A2) a compound represented by the following general formula (II) or a salt thereof,

[0031]

[0032] [wherein, X represents an oxygen atom or NR 11 ,

[0033] R 11 represents a hydrogen atom, or a C optionally substituted 1-4 Alkyl or COR 12 ,

[0034] R 12 Represents C 1-6 alkyl,

[0035] Y represents a hydrogen atom or OR 10 ,

[0036] R 6 , R 7 , R 8 and R 10 each independently represents a hydrogen atom or a phosphonic acid group,

[0037] R 9 represents a hydrogen atom, a hydroxyl group or a phosphate group,

[0038] R 10 When it is a hydrogen atom, OR 6 , OR 7 , OR 8 , R 9 At least one of them is a phosphate group. ],

[0039] The compounds represented by the general formula (II) also include compounds having a chain structure that is in equilibrium with the general formula (II) in aqueous solution;

[0040] (A3) a nucleic acid base, a nucleoside or a nucleotide, or a salt thereof;

[0041] (A4) a compound represented by the general formula (III) or a salt thereof, excluding malic acid,

[0042] Rx-CRyRzCOOH III

[0043] [In the formula, Rx represents the 1-6 A straight-chain C substituted with at least one of the group consisting of an alkyl group, an optionally substituted hydroxyl group, an oxo group, and a carboxyl group 2-4 alkyl,

[0044] Ry and Rz each independently represent a hydrogen atom or a hydroxyl group (at least one of which is a hydroxyl group), or together represent an oxo group. ];

[0045] (A5) at least one selected from the group consisting of creatinine, hydroxyproline, 1,3-butanediol, trientine, D-cellobiose, 1,3-dimethylurea, pantothenolactone and trimethyldione, or a salt thereof. [2]

[0047] The method according to item 1, wherein

[0048] (A1) is a compound selected from argininosuccinic acid, 1-methyl-L-histidine, pantothenic acid, L-carnosine, N-carbamoyl-L-aspartic acid, or a salt thereof;

[0049] (A2) is a compound selected from the group consisting of miglitol, 2-deoxy-D-glucose-6-phosphate, α-D-glucose-1-phosphate, and 1-deoxynojirimycin, or a salt thereof;

[0050] (A3) is a compound selected from cytosine, cytidine, deoxyuridine, deoxycytidine, and 5'-inosinic acid, or a salt thereof;

[0051] (A4) is a compound selected from 6-phospho-D-gluconic acid, 3-methyl-2-oxobutanoic acid, and 2-hydroxyglutaric acid, or a salt thereof;

[0052] (A5) is a compound selected from the group consisting of creatinine, hydroxyproline, 1,3-butanediol, 1,3-dimethylurea, D-cellobiose, 1,3-dimethylurea, pantothenate, and trimethyldione, or a salt thereof. [3]

[0054] The method according to item 1, wherein at least one of (A1) to (A5) is a compound selected from 1-methyl-L-histidine, N-carbamoyl-L-aspartic acid, deoxycytidine, 2-deoxy-D-glucose-6-phosphate, and 6-phospho-D-gluconic acid, or a salt thereof. [4]

[0056] The method according to item 1, wherein the salt (B) is at least one selected from the group consisting of sodium chloride, magnesium chloride and potassium chloride. [5]

[0058] The method according to item 1, wherein the above-mentioned molecule of interest comprises protein and / or nucleic acid. [6]

[0060] The method according to item 1, wherein the above-mentioned molecule of interest comprises Cas protein and / or gRNA. [7]

[0062] The method according to item 1, wherein the cell exists in a living body. [8]

[0064] The method according to item 1, wherein the cells are muscle cells. [9]

[0066] A method for producing a cell transduced with a molecule of interest, comprising the steps of contacting the cell with the molecule of interest and a transduction solution,

[0067] The transduction solution comprises at least one of the following (A1) to (A5) and (B) a salt:

[0068] (A1) a compound represented by the following general formula (I) or a salt thereof, excluding 20 kinds of L-amino acids and S-methyl-L-methionine which are constituent units of proteins in living organisms,

[0069]

[0070] [where n is 0 or 1,

[0071] R 1 and R 2 Each independently represents a hydrogen atom or COR 3 ,

[0072] R 0 It represents the side chain of the amino acid constituting the structural unit of the protein of the organism, excluding C 1-6 Alkyl, the side chain may have a C 1-6 alkyl,

[0073] Among them, R 0 When it is a hydrogen atom, R 1 Indicates COR 3 , R 2 represents a hydrogen atom,

[0074] R 3 represents a C group which may have a substituent (excluding a thiol group and a disulfide group) 1-6 Alkyl (excluding methyl) or NR 4 R 5 ,

[0075] R 4 and R 5 Each independently represents a hydrogen atom or a C 1-4 alkyl.];

[0076] (A2) a compound represented by the following general formula (II) or a salt thereof,

[0077]

[0078] [wherein, X represents an oxygen atom or NR 11 ,

[0079] R 11 represents a hydrogen atom, or a C optionally substituted 1-4 Alkyl or COR 12 ,

[0080] R 12 Represents C 1-6 alkyl,

[0081] Y represents a hydrogen atom or OR 10 ,

[0082] R 6 , R 7 , R 8 and R 10 each independently represents a hydrogen atom or a phosphonic acid group,

[0083] R 9 represents a hydrogen atom, a hydroxyl group or a phosphate group,

[0084] R 10 When it is a hydrogen atom, OR 6 , OR 7 , OR 8 , R 9 At least one of them is a phosphate group. ],

[0085] The compound represented by the general formula (II) also includes a compound having a chain structure that is in equilibrium with the general formula (II) in aqueous solution;

[0086] (A3) a nucleic acid base, a nucleoside or a nucleotide, or a salt thereof;

[0087] (A4) a compound represented by the general formula (III) or a salt thereof, excluding malic acid,

[0088] Rx-CRyRzCOOH III

[0089] [wherein, Rx represents a 1-6 A straight-chain C substituted with at least one of the group consisting of an alkyl group, an optionally substituted hydroxyl group, an oxo group, and a carboxyl group 2-4 alkyl,

[0090] Ry and Rz each independently represent a hydrogen atom or a hydroxyl group (at least one of which is a hydroxyl group), or together represent an oxo group. ];

[0091] (A5) at least one selected from the group consisting of creatinine, hydroxyproline, 1,3-butanediol, trientine, D-cellobiose, 1,3-dimethylurea, pantothenolactone and trimethyldione, or a salt thereof.

[10]

[0093] The method according to item 9, wherein

[0094] (A1) is a compound selected from argininosuccinic acid, 1-methyl-L-histidine, pantothenic acid, L-carnosine, N-carbamoyl-L-aspartic acid, or a salt thereof;

[0095] (A2) is a compound selected from the group consisting of miglitol, 2-deoxy-D-glucose-6-phosphate, α-D-glucose-1-phosphate, and 1-deoxynojirimycin, or a salt thereof;

[0096] (A3) is a compound selected from cytosine, cytidine, deoxyuridine, deoxycytidine, and 5'-inosinic acid, or a salt thereof;

[0097] (A4) is a compound selected from 6-phospho-D-gluconic acid, 3-methyl-2-oxobutanoic acid, and 2-hydroxyglutaric acid, or a salt thereof;

[0098] (A5) is a compound selected from the group consisting of creatinine, hydroxyproline, 1,3-butanediol, 1,3-dimethylurea, D-cellobiose, 1,3-dimethylurea, pantothenate, and trimethyldione, or a salt thereof.

[11]

[0100] The method according to item 9, wherein at least one of (A1) to (A5) is a compound selected from 1-methyl-L-histidine, N-carbamoyl-L-aspartic acid, deoxycytidine, 2-deoxy-D-glucose-6-phosphate, and 6-phospho-D-gluconic acid, or a salt thereof.

[12]

[0102] The method according to item 9, wherein the salt (B) is at least one selected from the group consisting of sodium chloride, magnesium chloride and potassium chloride.

[13]

[0104] The method according to item 9, wherein the above-mentioned molecules of interest include proteins and / or nucleic acids.

[14]

[0106] The method according to item 9, wherein the above-mentioned molecule of interest comprises Cas protein and / or gRNA.

[15]

[0108] The method according to item 9, wherein the above-mentioned cell exists in a living body.

[16]

[0110] The method according to item 9, wherein the cells are muscle cells.

[17]

[0112] A solution for transduction, comprising at least one of the following (A1) to (A5) and (B) a salt:

[0113] (A1) a compound represented by the following general formula (I) or a salt thereof, excluding 20 kinds of L-amino acids and S-methyl-L-methionine which are constituent units of proteins in living organisms,

[0114]

[0115] [where n is 0 or 1,

[0116] R 1 and R 2 Each independently represents a hydrogen atom or COR 3 ,

[0117] R 0 It represents the side chain of the amino acid constituting the structural unit of the protein of the organism, excluding C 1-6 Alkyl, the side chain may have a C 1-6 alkyl,

[0118] Among them, R 0 When it is a hydrogen atom, R 1 Indicates COR 3 , R 2 represents a hydrogen atom,

[0119] R 3 represents a C group which may have a substituent (excluding a thiol group and a disulfide group) 1-6 Alkyl (excluding methyl) or NR 4 R5 ,

[0120] R 4 and R 5 Each independently represents a hydrogen atom or a C 1-4 alkyl.];

[0121] (A2) a compound represented by the following general formula (II) or a salt thereof,

[0122]

[0123] [wherein, X represents an oxygen atom or NR 11 ,

[0124] R 11 represents a hydrogen atom, or a C optionally substituted 1-4 Alkyl or COR 12 ,

[0125] R 12 Represents C 1-6 alkyl,

[0126] Y represents a hydrogen atom or OR 10 ,

[0127] R 6 , R 7 , R 8 and R 10 each independently represents a hydrogen atom or a phosphonic acid group,

[0128] R 9 represents a hydrogen atom, a hydroxyl group or a phosphate group,

[0129] R 10 When it is a hydrogen atom, OR 6 , OR 7 , OR 8 , R 9 At least one of them is a phosphate group. ],

[0130] The compounds represented by the general formula (II) also include compounds having a chain structure that is in equilibrium with the general formula (II) in aqueous solution;

[0131] (A3) nucleic acid bases, nucleosides or nucleotides, or salts thereof,

[0132] (A4) a compound represented by the general formula (III) or a salt thereof, excluding malic acid,

[0133] Rx-CRyRzCOOH III

[0134] [In the formula, Rx represents the 1-6A straight-chain C substituted with at least one of the group consisting of an alkyl group, an optionally substituted hydroxyl group, an oxo group, and a carboxyl group 2-4 alkyl,

[0135] Ry and Rz each independently represent a hydrogen atom or a hydroxyl group (at least one of which is a hydroxyl group), or together represent an oxo group. ];

[0136] (A5) at least one selected from the group consisting of creatinine, hydroxyproline, 1,3-butanediol, trientine, D-cellobiose, 1,3-dimethylurea, pantothenolactone and trimethyldione, or a salt thereof.

[18]

[0138] The solution for transduction according to item 17, wherein

[0139] (A1) is a compound selected from argininosuccinic acid, 1-methyl-L-histidine, pantothenic acid, L-carnosine, N-carbamoyl-L-aspartic acid, or a salt thereof;

[0140] (A2) is a compound selected from the group consisting of miglitol, 2-deoxy-D-glucose-6-phosphate, α-D-glucose-1-phosphate, and 1-deoxynojirimycin, or a salt thereof;

[0141] (A3) is a compound selected from cytosine, cytidine, deoxyuridine, deoxycytidine, and 5'-inosinic acid, or a salt thereof;

[0142] (A4) is a compound selected from 6-phospho-D-gluconic acid, 3-methyl-2-oxobutanoic acid, and 2-hydroxyglutaric acid, or a salt thereof;

[0143] (A5) is a compound selected from the group consisting of creatinine, hydroxyproline, 1,3-butanediol, 1,3-dimethylurea, D-cellobiose, 1,3-dimethylurea, pantothenate, and trimethyldione, or a salt thereof.

[19]

[0145] The solution for transduction according to item 17, wherein at least one of (A1) to (A5) is a compound selected from 1-methyl-L-histidine, N-carbamoyl-L-aspartic acid, deoxycytidine, 2-deoxy-D-glucose-6-phosphate, and 6-phospho-D-gluconic acid, or a salt thereof.

[20]

[0147] The solution for transduction according to Item 17, wherein the salt (B) is at least one selected from the group consisting of sodium chloride, magnesium chloride, and potassium chloride. [twenty one]

[0149] A pharmaceutical composition comprising the solution for transduction according to item 17 and a molecule of interest. [twenty two]

[0151] The pharmaceutical composition according to item 21, wherein the above-mentioned molecule of interest comprises a protein and / or a nucleic acid. [twenty three]

[0153] A pharmaceutical composition according to item 21, wherein the above-mentioned molecule of interest comprises Cas protein and / or gRNA.

[0154] It should be noted that in the present specification, the “compound represented by the general formula (I)”, the “compound represented by the general formula (II)”, and the “compound represented by the general formula (III)” are sometimes referred to as “compound (I)”, “compound (II)”, and “compound (III)”, respectively. The compounds described in the above-mentioned items (A1) to (A5) and their salts are sometimes referred to as “compound (A1)” to “compound (A5)”, respectively. At least one of the above-mentioned (A1) to (A5) and the salt (B) are sometimes referred to as “components (A1) to (A5) and (B)”.

[0155] Effects of the Invention

[0156] By using the transduction method of the present invention, nucleic acids, proteins, or their complexes can be introduced into cells with high efficiency. For example, by using the transduction method of the present invention to introduce the complex of gRNA and Cas9 protein used in the CRISPR system into cells with high efficiency, high DNA cleavage activity based on the CRISPR system can be achieved. By using such a transduction method of the present invention, transduced cells can be efficiently manufactured. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] Figure 1 A reporter cassette for establishing EGFP reporter cells is shown.

[0158] Figure 2 The results of image analysis of EGFP reporter cells, HOECHST staining, and scanning under visible light are shown.

[0159] Figure 3 The expression portion of Cas9 protein is shown.

[0160] Figure 4-1 :Figure 4( Figure 4-1 and Figure 4-2 ) shows the results of measuring the EGFP positivity and the number of HOECHST cells when using various concentrations of compounds (A1) to (A5) (see Example 1).

[0161] Figure 4-2 :Same as above.

[0162] Figure 5The results of measuring the EGFP-positive rate and the number of HOECHST cells when each of the compounds (A1) to (A5) was used at a specific concentration are shown (see Example 1).

[0163] Figure 6 The results of measuring the EGFP-positive rate and the number of HOECHST cells when each of the compounds (A1) to (A5) was used at a specific concentration are shown (see Example 2).

[0164] Figure 7 The results of measuring the EGFP positive rate and the number of HOECHST cells when using the salt (B) at various concentrations are shown (see Example 3).

[0165] Figure 8 The results of measuring the EGFP positive rate when the concentration of sodium chloride as the salt (B) was changed for the compounds (A1) to (A5) are shown (see Example 4).

[0166] Fig. 9 The results of measuring the mutation introduction efficiency (indel (%)) when a dosing solution (dosing solution 1) containing 2'-deoxycytidine as the compound (A3), various concentrations of sodium chloride, and a complex of mRosa26 gRNA and Cas9 protein was topically administered to the gastrocnemius muscle of the right lower limb of mice are shown (see Example 5).

[0167] Fig.10 The results of measuring the mutation introduction efficiency (indel (%)) when a dosing solution (dosing solution 2) containing various compounds (A1) to (A5), sodium chloride, and a complex of mRosa26gRNA and Cas9 protein was topically administered to the gastrocnemius muscle of the right lower limb of mice are shown (refer to Example 6).

[0168] Fig.11 The results of measuring the skipping efficiency (%) when a dosing solution (dosing solution 3) containing various concentrations of 6-phospho-D-gluconic acid or N-carbamoyl-L-aspartic acid, sodium chloride, and a complex of mRosa26gRNA and Cas9 protein was locally administered to the tibialis anterior muscle of mice are shown (refer to Example 7).

[0169] Fig.12 The fluorescence microscope photographs are shown when evaluating the intracellular uptake of fluorescently labeled dextran using a transduction solution containing 6-phospho-D-gluconic acid or N-carbamoyl-L-aspartic acid (or GABA as a control) and sodium chloride (see Example 8). The bright areas in the fluorescence microscope photographs represent fluorescently labeled dextran taken into cells. DETAILED DESCRIPTION

[0170] In this specification, "transduction" (transfection) means introducing any substance into the interior of a cell. The interior of a cell includes at least the cytoplasm and the nucleus.

[0171] "Culture" or "culturing" means maintaining, proliferating and / or differentiating cells outside a tissue or in vitro, for example, in a culture dish, a culture vessel, a culture flask or a culture tank.

[0172] "Include (comprise(s) or comprising)~" means: Although it indicates that the elements connected to the phrase are included, it is not limited to this. Therefore, it indicates that the elements connected to the phrase are included, but it does not indicate that any other elements are excluded.

[0173] "Consist(s) of or consisting of" means that all the elements connected to the phrase are included and are limited to these. Therefore, the phrase "consist(s) of" means that the listed elements are required or necessary, and other elements do not exist substantially. "Essentially consisting of" means that any element connected to the phrase is included, and other elements that do not affect the activity or function specified in the present disclosure are limited to the element. Therefore, the phrase "essentially consisting of" means that the listed elements are required or necessary, and other elements are arbitrarily selected, and sometimes they are present and sometimes they are absent depending on whether they affect the activity or function of the listed elements.

[0174] In this specification, the clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system is referred to as a "CRISPR system".

[0175] The following is a detailed description of the definition of each substituent used in the present specification. Unless otherwise specified, each substituent has the following definition.

[0176] In this manual, “C 1-4 Examples of the "alkyl group" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

[0177] In this manual, “C 1-6The “alkyl group” includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl.

[0178] In this specification, the term “straight-chain C 2-4 The "alkyl group" includes, for example, ethyl, propyl and butyl.

[0179] In this specification, C 1-6 Alkyl or C 1-4 The "substituent" that the alkyl group may have includes, for example, C 1-6 In this specification, unless otherwise specified, C 1-6 Alkyl or C 1-4 The alkyl group may have one substituent or a plurality of substituents which are independent of each other at any substitutable position.

[0180] The transduction solution of the present invention is described below.

[0181] The transduction solution of the present invention contains at least one of the following (A1) to (A5) and (B) a salt.

[0182] The compound (A1) is a compound represented by the following general formula (I) or a salt thereof, but does not include 20 L-amino acids and S-methyl-L-methionine, which are constituent units of proteins in organisms. The compound (A1) may be a hydrate. The compound (A1) may be used alone or in combination of two or more.

[0183]

[0184] In formula (I),

[0185] n is 0 or 1,

[0186] R 1 and R 2 Each independently represents a hydrogen atom or COR 3 ,

[0187] R 0 It represents the side chain of the amino acid constituting the structural unit of the protein of the organism, excluding C 1-6 Alkyl, the side chain may have a C 1-6 alkyl,

[0188] Among them, R 0 When it is a hydrogen atom, R 1 Indicates COR 3 , R2 represents a hydrogen atom,

[0189] R 3 represents a C group which may have a substituent (excluding a thiol group and a disulfide group) 1-6 Alkyl (excluding methyl) or NR 4 R 5 ,

[0190] R 4 and R 5 Each independently represents a hydrogen atom or a C 1-4 alkyl.

[0191] It should be noted that R 0 The "side chains of amino acids constituting the structural units of proteins in living organisms" involved in the definition of 0 The carbon atom bonded to R 1 The nitrogen atoms to which the amine is bonded are integrated to form a ring (pyrrolidine ring).

[0192] In formula (I), n and R 1 , R 2 , R 3 , R 0 , R 4 and R 5 Preferred examples of each are described below.

[0193] n is preferably 0 or 1.

[0194] R 1 Preferably, a hydrogen atom or COR 3 .

[0195] R 2 Preferred is a hydrogen atom.

[0196] R 3 Preferably, C 1-6 Alkyl (excluding methyl) or NR 4 R 5 .

[0197] R 0 Preferably, it is a hydrogen atom, or a C 1-6 Alkyl, constituting the side chain of arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, cysteine ​​or methionine.

[0198] R 4 Preferably, a hydrogen atom or a C 1-4 alkyl.

[0199] R 5 Preferably, a hydrogen atom or a C 1-4 alkyl.

[0200] Preferred specific examples of compound (I) are as follows: The following compounds may be salts and / or hydrates.

[0201] Compound (Ii): n is 0 or 1, R 1 is a hydrogen atom or COR 3 , R 2 is a hydrogen atom, R 3 is a C group which may have a substituent (excluding a thiol group and a disulfide group) 1-6 Alkyl (excluding methyl), R 0 is a hydrogen atom, or a C 1-6 Alkyl compounds forming the side chains of arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, cysteine ​​or methionine.

[0202] Compound (I-ii): n is 0 or 1, R 1 COR 3 , R 2 is a hydrogen atom, R 3 is a C group which may have a substituent (excluding a thiol group and a disulfide group) 1-6 Alkyl (excluding methyl), R 0 is a hydrogen atom, or a C 1-6 Alkyl compounds forming the side chains of arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, cysteine ​​or methionine.

[0203] Compound (I-iii): n is 0 or 1, R 1 COR 3 , R 2 is a hydrogen atom, R 3 NR 4 R 5 , R 0 is a hydrogen atom, or a C 1-6 Alkyl, constituting the side chain of arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, cysteine ​​or methionine, R 4 is a hydrogen atom, R 5 A compound containing hydrogen atoms.

[0204] More preferred specific examples of compound (I) are as follows: The following compounds may be salts and / or hydrates.

[0205] Compound (Ia): n is 0, R 1 is a hydrogen atom, R 2 is a hydrogen atom, R 0 is C which may or may not have a substituent 1-6 Compounds in which an alkyl group constitutes the side chain of arginine, for example, argininosuccinic acid.

[0206] Compound (Ib): n is 0, R 1 is a hydrogen atom, R 2 is a hydrogen atom, R 0 To have C 1-6 A compound in which an alkyl group constitutes a side chain of histidine, for example, 1-methyl-L-histidine.

[0207] Compound (Ic): n is 1, R 1 COR 3 , R 2 is a hydrogen atom, R 3 To have C 1-6 Alkyl or hydroxy propyl, R 0 Compounds containing hydrogen atoms, such as pantothenic acid.

[0208] Compound (Id): n is 0, R 1 COR 3 , R 2 is a hydrogen atom, R 3 is an ethyl group which may have an amino group, R 0 To have C 1-6 Compounds in which an alkyl group forms the side chain of histidine, such as L-carnosine.

[0209] Compound (Ie): n is 0, R 1 COR 3 , R 2 is a hydrogen atom, R 3 NR 4 R 5 , R 0 To have C 1-6 The alkyl group constitutes the side chain of aspartic acid, R 4 is a hydrogen atom, R 5 A compound containing a hydrogen atom, such as N-carbamoyl-L-aspartic acid (also sometimes referred to as N-carbamoyl-L-aspartic acid).

[0210] In one aspect of the present invention, (A1) is particularly preferably a compound selected from argininosuccinic acid, 1-methyl-L-histidine, pantothenic acid, L-carnosine, N-carbamoyl-L-aspartic acid or a salt thereof (which may be a hydrate), especially a compound selected from argininosuccinic acid, 1-methyl-L-histidine, L-carnosine, N-carbamoyl-L-aspartic acid or a salt thereof (which may be a hydrate).

[0211] In another aspect of the present invention, (A1) is particularly preferably a compound selected from 1-methyl-L-histidine, N-carbamoyl-L-aspartic acid, or a salt thereof (which may be a hydrate).

[0212] The compound (A2) is a compound represented by the following general formula (II) or a salt thereof. The compound (A2) may be a hydrate. The compound (A2) may be used alone or in combination of two or more.

[0213]

[0214] In formula (II),

[0215] X represents an oxygen atom or NR 11 ,

[0216] R 11 represents a hydrogen atom, or a C optionally substituted 1-4 Alkyl or COR 12 ,

[0217] R 12 Represents C 1-6 alkyl,

[0218] Y represents a hydrogen atom or OR 10 ,

[0219] R 6 , R 7 , R 8 and R 10 Each independently represents a hydrogen atom or a phosphonic acid group (-P(=O)(OH)2),

[0220] R 9 represents a hydrogen atom, a hydroxyl group or a phosphate group (-OP(=O)(OH)2),

[0221] R 10 When it is a hydrogen atom, OR 6 , OR 7 , OR 8 , R 9 At least one of them is a phosphate group.

[0222] The compound represented by the general formula (II) also includes a compound having a chain structure that is in equilibrium with the general formula (II) in an aqueous solution.

[0223] X, Y, and R in formula (II) 11 , R 6 , R 7 , R 8 , R 9 and R 10 Preferred examples of each are described below.

[0224] X is preferably an oxygen atom or NR 11 .

[0225] Y is preferably a hydrogen atom or OR 10 .

[0226] R 11 It is preferably a hydrogen atom or a C 1-4 alkyl.

[0227] R 6 Preferred is a hydrogen atom or a phosphonic acid group.

[0228] R 7 Preferred is a hydrogen atom or a phosphonic acid group.

[0229] R 8 Preferred is a hydrogen atom or a phosphonic acid group.

[0230] R 9 Preferred are hydrogen atoms, hydroxyl groups or phosphoric acid groups.

[0231] R 10 Preferred is a hydrogen atom or a phosphonic acid group.

[0232] Preferred specific examples of compound (II) are as follows: The following compounds may be salts and / or hydrates.

[0233] Compound (II-i): X is NR 11 , Y is a hydrogen atom, R 6 is a hydrogen atom or a phosphonic acid group, R 7 is a hydrogen atom or a phosphonic acid group, R 8 is a hydrogen atom or a phosphonic acid group, R 9 is a hydroxyl group or a phosphate group, R 10 is a hydrogen atom or a phosphonic acid group, R 11 is a hydrogen atom or a C 1-4 Alkyl compounds.

[0234] Compound (II-ii): X is an oxygen atom, Y is OR 10 , R 6 is a hydrogen atom or a phosphonic acid group, R 7is a hydrogen atom or a phosphonic acid group, R 8 is a hydrogen atom or a phosphonic acid group, R 9 is a hydrogen atom, a hydroxyl group or a phosphate group, R 10 A compound containing a hydrogen atom or a phosphonic acid group.

[0235] More preferred specific examples of compound (II) are as follows: The following compounds may be salts and / or hydrates.

[0236] Compound (II-a): X is NR 11 , Y is a hydrogen atom, R 6 is a hydrogen atom, R 7 is a hydrogen atom, R 8 is a hydrogen atom, R 9 is hydroxyl group, R 11 A compound which is a 2-hydroxyethyl group, for example, miglitol.

[0237] Compound (II-b): X is an oxygen atom, Y is OR 10 , R 6 is a phosphate group, R 7 is a hydrogen atom, R 8 is a hydrogen atom, R 9 is a hydrogen atom, R 10 A compound containing a hydrogen atom, such as 2-deoxy-D-glucose-6-phosphate.

[0238] Compound (II-c): X is an oxygen atom, Y is OR 10 , R 6 is a hydrogen atom, R 7 is a hydrogen atom, R 8 is a hydrogen atom, R 9 is hydroxyl group, R 10 A compound containing a phosphonic acid group, for example, α-D-glucose-1-phosphate.

[0239] Compound (II-d): X is NR 11 , Y is a hydrogen atom, R 6 is a hydrogen atom, R 7 is a hydrogen atom, R 8 is a hydrogen atom, R 9 is hydroxyl group, R 11 A compound in which the moiety is a hydrogen atom, for example, 1-deoxynojirimycin.

[0240] In one aspect of the present invention, (A2) is particularly preferably a compound selected from miglitol, 2-deoxy-D-glucose-6-phosphate, α-D-glucose-1-phosphate, 1-deoxynojirimycin, or a salt thereof (which may be a hydrate).

[0241] In another aspect of the present invention, (A2) is particularly preferably 2-deoxy-D-glucose-6-phosphate or a salt thereof (which may be a hydrate).

[0242] The compound (A3) is a nucleic acid base, a nucleoside or a nucleotide, or a salt thereof. The compound (A3) may be a hydrate. The compound (A3) may be used alone or in combination of two or more.

[0243] It should be noted that, in one embodiment, the target molecule may be a "nucleic acid" which is a polynucleotide or an oligonucleotide, whereas the "nucleotide" of the compound (A3) is a mononucleotide or a dinucleotide, and the two can be distinguished from each other in this respect.

[0244] Examples of the nucleic acid base include adenine, guanine, cytosine, thymine and uracil, as well as modified bases thereof, such as methylated cytosine, hydroxymethylated cytosine, methylated guanine, hypoxanthine (deaminated adenine, 6-hydroxyproline), pseudouracil, dihydrouracil, thiouracil, methylaminoselenouracil, taurinomethyluracil, rigisine, agmatine cytosine, archaeosine, quercetin, wyosine, and other modified nucleic acid bases contained in genomic DNA, mRNA, tRNA, rRNA, etc.

[0245] Nucleosides are compounds with ribose or other sugars bonded to the base, including ribonucleosides, deoxyribonucleosides, etc. As ribonucleosides, adenosine, guanosine, cytidine, uridine, 5-methyluridine, and other compounds with ribose bonded to the above-mentioned modified nucleic acid bases, etc. can be listed. As deoxyribonucleotides, deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, deoxyuridine, and other compounds with deoxyribose bonded to the above-mentioned modified nucleic acid bases, etc. can be listed. Nucleosides also include inosine (a compound with ribose bonded to hypoxanthine), riboflavin (vitamin B2, a compound with ribose (ribitol) bonded to dimethylisoalloxazine that is reduced and in a chain), etc.

[0246] Nucleotides are compounds in which a phosphate group is bonded to a sugar residue such as a ribonucleoside or a deoxyribonucleoside. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), and other compounds in which one or more phosphate groups are bonded to a ribonucleoside; deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine Triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), thymidine monophosphate (dTMP), thymidine diphosphate (dDMP), thymidine triphosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), and other compounds having one or more phosphate groups bonded to deoxyribonucleosides. Nucleotides also include inosinic acid (inosine monophosphate), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), etc. In addition, compounds having cyclic phosphate groups bonded to nucleosides such as cyclic AMP (cAMP) and cyclic GMP (cGMP) are also included in nucleotides.

[0247] Preferred specific examples of the compound (A3) are as follows: The following compounds may be salts and / or hydrates.

[0248] Nucleobases: adenine, guanine, cytosine, thymine, uracil and hypoxanthine.

[0249] Nucleosides: adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, deoxyuridine, inosine.

[0250] Nucleotides: adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, uridine monophosphate, deoxyadenosine monophosphate, deoxyadenosine diphosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate, deoxycytidine diphosphate, thymidine monophosphate, deoxyuridine monophosphate, inosinic acid.

[0251] More preferred specific examples of the compound (A3) are as follows: The following compounds may be salts and / or hydrates.

[0252] Nucleobase: cytosine.

[0253] Nucleosides: cytidine, deoxyuridine, deoxycytidine.

[0254] Nucleotide: 5'-inosinic acid.

[0255] In one aspect of the present invention, (A3) is particularly preferably a compound selected from cytosine, cytidine, deoxyuridine, deoxycytidine, 5'-inosinic acid or a salt thereof (which may be a hydrate), especially a compound selected from cytidine, deoxycytidine, 5'-inosinic acid or a salt thereof (which may be a hydrate).

[0256] In another aspect of the present invention, (A3) is particularly preferably deoxycytidine or a salt thereof (which may be a hydrate).

[0257] The compound (A4) is a compound represented by the general formula (III) or a salt thereof, excluding malic acid. The compound (A4) may be a hydrate. The compound (A4) may be used alone or in combination of two or more.

[0258] Rx-CRyRzCOOH III

[0259] In formula (III),

[0260] Rx means selected by C 1-6 A straight-chain C substituted with at least one of the group consisting of an alkyl group, an optionally substituted hydroxyl group, an oxo group, and a carboxyl group 2-4 alkyl,

[0261] Ry and Rz each independently represent a hydrogen atom or a hydroxyl group (at least one of which is a hydroxyl group), or together represent an oxo group.

[0262] Examples of the hydroxyl group which may be substituted in Rx include a hydroxyl group substituted with a phosphonic acid group.

[0263] Preferred examples of each of Rx, Ry and Rz in formula (III) are as follows.

[0264] Rx is preferably selected from C 1-6 A straight-chain C substituted with at least one of the group consisting of an alkyl group, a hydroxyl group, a phosphate group, an oxo group, and a carboxyl group 2-4 alkyl.

[0265] Ry is preferably a hydroxyl group, or forms an oxo group with Rz.

[0266] Rz is preferably a hydrogen atom, or forms an oxo group with Ry.

[0267] Preferred specific examples of compound (III) are as follows.

[0268] Compound (III-i): Rx is a compound which can be selected from C 1-6 A straight-chain C substituted with at least one of the group consisting of an alkyl group, a hydroxyl group, a phosphate group, an oxo group, and a carboxyl group 2-4A compound in which Ry is an alkyl group, Rz is a hydroxyl group, and Rz is a hydrogen atom.

[0269] Compound (III-ii): Rx is a compound which can be selected from C 1-6 A straight-chain C 2-4 A compound in which an alkyl group, Ry and Rz form an oxo group.

[0270] More preferred specific examples of compound (III) are shown below.

[0271] Compound (III-a): a compound in which Rx is a butyl group substituted with a hydroxyl group and a phosphate group, Ry is a hydroxyl group, and Rz is a hydrogen atom, for example, 6-phospho-D-gluconic acid.

[0272] Compound (III-b): Rx is C 1-6 The compound in which an ethyl group substituted with an alkyl group, Ry and Rz form an oxo group, for example, 3-methyl-2-oxobutanoic acid.

[0273] Compound (III-c): a compound wherein Rx is an ethyl group substituted with a carboxyl group, Ry is a hydroxyl group, and Rz is a hydrogen atom, for example, 2-hydroxyglutaric acid.

[0274] In one aspect of the present invention, (A4) is particularly preferably a compound selected from 6-phospho-D-gluconic acid, 3-methyl-2-oxobutanoic acid, 2-hydroxyglutaric acid, or a salt thereof (which may be a hydrate).

[0275] In another aspect of the present invention, (A4) is particularly preferably 6-phospho-D-gluconic acid or a salt thereof (which may be a hydrate).

[0276] The compound (A5) is at least one selected from the group consisting of creatinine, hydroxyproline, 1,3-butanediol, trientine, D-cellobiose, 1,3-dimethylurea, pantothenolactone and trimethyldione, or a salt thereof. The compound (A5) may be a hydrate.

[0277] In one aspect of the present invention, (A5) is particularly preferably a compound selected from creatinine, hydroxyproline, 1,3-butanediol, 1,3-dimethylurea, pantothenic acid lactone, trimethyldione, or a salt thereof (which may be a hydrate).

[0278] The salts included in the definitions of (A1) to (A5) are preferably pharmacologically acceptable salts, and examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids.

[0279] Preferred examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, and ammonium salts. Sodium salts, potassium salts, calcium salts, and magnesium salts are preferred, and sodium salts, potassium salts, and magnesium salts are more preferred.

[0280] Preferred examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, and N,N-dibenzhydrylethylenediamine.

[0281] Preferred examples of the salt with an inorganic acid include salts with hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid. Preferred are salts with hydrochloric acid and salts with phosphoric acid.

[0282] Preferred examples of the salt with an organic acid include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0283] Preferred examples of the salt with a basic amino acid include salts with arginine, lysine, and ornithine.

[0284] Preferred examples of the salt with an acidic amino acid include salts with aspartic acid and glutamic acid.

[0285] (B) Salt is different from the salts included in the definitions of (A1) to (A5), and is a compound contained in the transduction solution of the present invention. In the present invention, salts corresponding to (A1) to (A5) are not included in (B) salt.

[0286] As (B) salts, alkali metal salts and alkaline earth metal salts such as lithium salts, sodium salts, magnesium salts, potassium salts, calcium salts, rubidium salts, and cesium salts can be cited; inorganic acid salts such as carbonates, sulfonates, and sulfates; organic acid salts such as aspartate; sulfides; halides such as chlorides, bromides, iodides, and fluorides. Among (B) salts, alkali metal salts and alkaline earth metal salts, organic acid salts, and chlorides are preferred, and salts formed by alkali metals or alkaline earth metals (cations) and organic acids or halogen atoms (anions), such as sodium chloride, magnesium chloride, potassium chloride, and magnesium aspartate, are more preferred. (B) Salts are preferably a combination of two or more of these. As a combination of two or more (B) salts, there is no particular limitation, and there are three combinations of sodium chloride, magnesium chloride, and potassium chloride. When a combination of sodium chloride, magnesium chloride and potassium chloride is used, the molar ratios thereof are preferably 0.75 to 0.95 for sodium chloride, 0.05 to 0.15 for magnesium chloride and 0.025 to 0.075 for potassium chloride.

[0287] The types (combinations) and concentrations of the compounds used as components (A1) to (A5) and (B) contained in the transduction solution of the present invention are not particularly limited and can be appropriately adjusted, for example, depending on the type of target cells, from the viewpoint of the efficiency of introducing the molecule of interest into cells.

[0288] The concentration of the compounds (A1) to (A5) in the transduction solution is usually 0.1 mM to 1000 mM, but the optimal concentration varies depending on the transduction substance. In the case of 2'-deoxycytidine, it is preferably 12.5 mM or more, and more preferably 100 mM to 250 mM. In the case of calcium pantothenate, it is preferably 160 mM to 250 mM. In the case of argininosuccinic acid disodium salt hydrate, it is preferably 30 mM or more, and more preferably 100 mM to 250 mM. In the case of sodium 3-methyl 2-oxobutyrate, it is preferably 40 mM or more, and more preferably 100 mM to 200 mM. In the case of 1,3-butanediol, it is preferably 0.6 mM or more, and more preferably 0.6 mM to 3 mM. In the case of 1,3-dimethylurea, it is preferably 30 mM or more, and more preferably 60 mM to 500 mM. In the case of 2'-deoxyuridine, it is preferably 10 mM or more, and more preferably 100 mM or more. In the case of 6-phospho-D-gluconic acid, it is preferably 60 mM or more, and more preferably 100 mM to 500 mM. In the case of creatinine, it is preferably 5 mM or more, and more preferably 100 mM to 500 mM. In the case of cytosine, it is preferably 20 mM or more, and more preferably 25 mM to 250 mM. In the case of D(+)-cellobiose, it is preferably 3 mM or more, and more preferably 200 mM to 500 mM. In the case of 2-deoxy-D-glucose-6-phosphate, it is preferably 30 mM or more, and more preferably 125 mM to 500 mM. α-D-glucose-1-phosphate is preferably 60 mM or more, and more preferably 125 mM to 500 mM. In the case of N-carbamoyl-L-aspartic acid, it is preferably 60 mM or more, and more preferably 125 mM to 500 mM. In the case of L-carnosine, it is preferably 1 mM or more, and more preferably 60 mM to 500 mM. In the case of miglitol, it is preferably 1 mM or more, and more preferably 60 mM to 250 mM. In the case of inosine-5'-phosphate disodium salt hydrate, it is preferably 100 mM or more, and more preferably 250 mM to 500 mM. In the case of 2-hydroxyglutaric acid, it is preferably 1 mM or more, and more preferably 60 mM to 500 mM. In the case of 1-methyl-L-histidine, it is preferably 10 mM or more, and more preferably 100 mM to 250 mM. In the case of cytidine, it is preferably 100 mM or more, and more preferably 125 mM to 500 mM. In the case of 6-phospho-D-gluconic acid, it is preferably 30 mM or more, and more preferably 100 mM to 250 mM.

[0289] The concentration of the salt (B) in the transduction solution is usually 200 mM or more, preferably 200 to 2000 mM, and more preferably 300 to 900 mM, when used alone or in combination of two or more salts.

[0290] The compounds (A1) to (A5) and the salt (B) can be produced by known methods (synthesis, isolation, purification, etc.), and commercially available products can also be used.

[0291] The transduction solution of the present invention can be prepared by dissolving at least one compound among (A1) to (A5) and the salt (B) in an appropriate solvent.

[0292] The solvent for dissolving components (A1) to (A5) and (B) is not particularly limited as long as it can dissolve these components in a specified amount, and a buffer is preferred. Examples of the buffer include acidic buffers such as acetate buffer, citrate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, and phosphate buffer; and neutral buffers such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris (hydroxymethyl) aminomethane (Tris) buffer, phosphate buffer, and phosphate buffered saline (PBS).

[0293] The osmotic pressure of the transduction solution of the present invention is not particularly limited, but is usually 300 mOsmol / kg or more, preferably 300 to 2000 mOsmol / kg, and more preferably 700 to 1700 mOsmol / kg.

[0294] It should be noted that, in the present invention, even without the transmembrane carriers selected from viral plasmids, nanoparticles, liposomes, cationic lipids, vesicles or other lipid vesicles (including micelles), cell membrane permeable peptides, etc. used in the known transduction (transfection) methods, the effects of the present invention can be exerted, and the molecules of interest can be introduced into cells with high efficiency.

[0295] The transduction method of the present invention is described below.

[0296] The transduction method of the present invention includes at least a step of contacting a cell with a molecule of interest and a transduction solution (referred to as a "contact step" in this specification), and may further include other steps related to the introduction of the molecule of interest into the cell as needed. In addition, in the transduction method of the present invention, the prescribed transduction solution described in this specification is used to introduce the molecule of interest into the cell, and other technical matters can basically follow the method of introducing the molecule of interest into the cell using a conventional solution.

[0297] It should be noted that the effects of the present invention can be exerted even without performing special treatments such as electroporation used in known transduction (transfection) methods, and the molecule of interest can be introduced into cells with high efficiency.

[0298] ·Focus on the subject

[0299] In the present invention, the molecules of interest introduced into cells using the transduction solution are not particularly limited as long as they can exert the desired purpose in the cells. Examples of molecules of interest include nucleic acids, proteins, and complexes thereof, and polymer compounds (e.g., dextran), and examples of preferred molecules of interest include nucleic acids, proteins, and complexes thereof.

[0300] "Nucleic acid" refers to any molecule as long as it is a molecule obtained by polymerizing nucleotides and molecules having the same function as the nucleotides, and examples thereof include RNA as a polymer of ribonucleotides, DNA as a polymer of deoxyribonucleotides, a polymer formed by mixing ribonucleotides and deoxyribonucleotides, and a nucleotide polymer containing nucleotide analogs. Furthermore, it can be a nucleotide polymer containing a nucleic acid derivative. In addition, nucleic acid can be a single-stranded nucleic acid or a double-stranded nucleic acid. In addition, double-stranded nucleic acid also includes a double-stranded nucleic acid in which one chain is hybridized with the other chain under stringent conditions.

[0301] As nucleotide analogs, any molecule can be used as long as it is a molecule modified to form ribonucleotides, deoxyribonucleotides, RNA or DNA in order to improve nuclease resistance or stabilize it compared to RNA or DNA, to improve affinity with complementary strand nucleic acid, or to improve cell permeability, or to visualize it. As nucleotide analogs, it can be a naturally occurring molecule or a non-natural molecule, and examples thereof include sugar-modified nucleotide analogs, phosphodiester bond-modified nucleotide analogs, etc.

[0302] The sugar-modified nucleotide analogs may be any substance as long as they are substances in which any chemical structure substance is added or substituted to a part or all of the chemical structure of the sugar of the nucleotide. Specific examples thereof include nucleotide analogs substituted with 2'-O-methyl ribose, nucleotide analogs substituted with 2'-O-propyl ribose, nucleotide analogs substituted with 2'-methoxyethoxy ribose, nucleotide analogs substituted with 2'-O-methoxyethyl ribose, nucleotide analogs substituted with 2'-O-[2-(guanidinium)ethyl] ribose, nucleotide analogs substituted with 2'-fluororibose, nucleic acid analogs in which the sugar part is substituted with a morpholino ring (morpholino nucleic acid), bridged artificial nucleic acids (BNA) having two ring structures by introducing a cross-linked structure into the sugar part, and more specifically, locked artificial nucleic acids (BNA) in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via a methylene group. Acid) (LNA) and ethylene bridged nucleic acid (Ethylenebridged nucleic acid) (ENA) [Nucleic Acid Research, 32, e175 (2004)], amide bridged nucleic acid (Amido-Bridged Nucleic Acids) (AmNA) in which the carbon atom at the 2' position and the carbon atom at the 4' position are bridged via an amide bond, and peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxygen peptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)] and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like are also mentioned.

[0303] As phosphodiester bond modified nucleotide analogs, any substance may be used as long as it is a substance obtained by adding or replacing any chemical substance to a part or all of the chemical structure of the phosphodiester bond of the nucleotide. Specific examples thereof include nucleotide analogs replaced with phosphorothioate bonds, nucleotide analogs replaced with N3'-P5'-phosphonamidate bonds, etc. [Cell Engineering, 16, 1463-1473 (1997)] [RNAi method and antisense method, Kodansha (2005)].

[0304] As a nucleic acid derivative, any molecule can be used as long as other chemical substances are added to the nucleic acid in order to improve nuclease resistance compared with the nucleic acid, for stabilization, for improving affinity with complementary chain nucleic acid, for improving cell permeability, or for visualization. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0305] Specific examples of nucleic acids include mRNA, siRNA, shRNA, miRNA, miRNA mimic, antisense nucleic acid, ribozyme, decoy nucleic acid, aptamer, plasmid DNA, cosmid DNA, BAC DNA, and guide RNA (gRNA) in the CRISPR system. These nucleic acids may be artificially modified analogs or derivatives.

[0306] As specific examples of proteins, enzymes, transcription factors, cytokines, tissue growth factors, antibodies, and therapeutic proteins can be cited. As enzymes, for example, restriction enzymes, endonucleases, Cre recombinases, flip enzymes, etc., enzymes for targeting nucleic acids (especially genomic DNA in target cells) and changing their base sequences can be cited. As endonucleases, for example, TAL effector nucleases (TALEN), zinc finger nucleases (ZFN), large-range nucleases (homing nucleases), guide molecules (RNA) inducible endonucleases (e.g., Cas9, Cas12a), and other endonucleases for genome editing systems can be cited.

[0307] As a preferred specific example of the molecule of interest in the present invention, there can be cited so-called genome editing substances used to induce genetic modification of the target locus in the cell, in particular substances used to induce genetic modification through the CRISPR system. Specifically, in a preferred embodiment of the present invention, the nucleic acid is a guide RNA (gRNA) in the CRISPR system, and the protein is an RNA-induced nuclease in the CRISPR system. In a preferred embodiment of the present invention, the molecule of interest is a complex of gRNA in the CRISPR system and an RNA-induced nuclease such as Cas9.

[0308] The basic information about the CRISPR system is well known, and various applications are also well known. Those skilled in the art can design, select, and manufacture nucleic acids, proteins, and other elements of a suitable CRISPR system according to the purpose, and use them to perform genetic modification using the CRISPR system.

[0309] "Guide RNA" can be in the form of one RNA connected by crRNA and tracrRNA, i.e., chimeric RNA (sometimes also referred to as single guide RNA, sgRNA, etc.), or can be in the form of unconnected RNAs (a combination of two RNAs or a combination of more RNAs). In addition, the guide RNA can be in the form of targeting one base sequence (one sgRNA, or one set of crRNA and tracrRNA), or can be in the form of targeting two or more base sequences (two or more sgRNAs, or two or more sets of crRNA and tracrRNA).

[0310] crRNA hybridizes with the base sequence in the genome or locus in the cell that serves as the target of genetic modification (sometimes referred to as "target sequence" in this specification), and contains a nucleic acid sequence of about 17 to 20 bases (sometimes referred to as "target recognition sequence" in this specification). The target sequence is adjacent to a short sequence (PAM (protospacer adjacent motif)) recognized by the CRISPR system. The sequence and length conditions of PAM vary depending on the type of nuclease used. PAM is typically a 2 to 5 base pair sequence adjacent to the target sequence.

[0311] The target sequence is not particularly limited as long as it satisfies the above-mentioned PAM conditions and can be appropriately selected according to the purpose. The target sequence is typically related to a genetic disease and is a sequence contained in a locus that can be a target of gene therapy, or a sequence contained in a locus related to infection by pathogens such as bacteria or viruses.

[0312] In a preferred embodiment of the present invention, the target sequence is a base sequence in a gene locus containing a dystrophin gene. More specifically, the target sequence is a base sequence that can repair a frameshift mutation or a nonsense mutation in a dystrophin gene by modifying its base sequence (deletion or insertion), thereby preventing or treating dystrophin abnormalities (e.g., muscular dystrophy (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy), dystrophin genotype dilated cardiomyopathy). Alternatively, the target sequence is a base sequence on a dystrophin gene that can produce a repaired dystrophin by repairing a frameshift mutation or a nonsense mutation.

[0313] Dystrophinosis refers to various diseases caused by dystrophin gene mutations and caused by functional loss or abnormal dystrophin, including Duchenne muscular dystrophy, Becker muscular dystrophy, and dystrophin gene-related dilated cardiomyopathy. Skeletal muscle disorders are usually the main symptom, but there are cases where skeletal muscle symptoms are not observed. Sometimes it is accompanied by hypercreatinine, myoglobinuria, dilated cardiomyopathy, cognitive dysfunction, etc.

[0314] Muscular dystrophy is defined as "a genetic disease characterized by skeletal muscle degeneration and necrosis as the main pathological changes, and clinically observed progressive muscle strength loss". Known muscular dystrophy includes Duchenne muscular dystrophy, Beck muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, congenital muscular dystrophy, Miyoshi muscular dystrophy, distal muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic muscular dystrophy.

[0315] Duchenne muscular dystrophy is the disease with the largest number of patients among pediatric muscular dystrophy, with an incidence rate of 4 to 5 per 100,000 people. The main symptom is progressive muscle atrophy, and the cause is the dysfunction of the dystrophin gene on the X chromosome due to mutation. More than half of the patients with Duchenne muscular dystrophy have single or multiple exon deletions. Due to the mutation of the dystrophin gene, the protein reading frame shift occurs, and the presence of a stop codon in the middle makes it impossible to synthesize dystrophin, which causes a series of symptoms.

[0316] The dystrophin gene is a huge gene with more than 2.2 million bases and is present on the X chromosome. There are various isoforms depending on the transcription start site, and Dp71 is expressed throughout the body, Dp116 is expressed in peripheral nerve cells, Dp140 is expressed in the brain and kidney, Dp260 is expressed in the retina, Dp417p is expressed in Purkinje nerve cells, Dp427b is expressed in the brain, and Dp427m is expressed in skeletal muscle. Among them, the dystrophin produced by the Dp427m isoform is a protein mainly expressed in muscle cells, binds to the cytoskeletal actin through the actin binding domain present on the N-terminal side, and binds to the dystrophin glycan complex through the homocysteine ​​domain present on the C-terminal side, and together with actin, it constitutes the cytoskeleton. The Dp427m isoform dystrophin gene consists of 79 exons.

[0317] In the case of patients with Duchenne muscular dystrophy, functional dystrophin is hardly expressed (the amount of protein is less than 3% of that of healthy persons when detected by Western blotting) due to deletion or duplication mutation in any exon of the dystrophin gene, or due to point mutation (nonsense mutation) or insertion / deletion mutation (frameshift mutation) of bases in the exon. On the other hand, in the case of patients with Becker muscular dystrophy, which has milder symptoms than Duchenne muscular dystrophy, even if there is deletion of an exon or point mutation of a base, a dystrophin whose amino acid sequence is shorter than that of normal dystrophin or whose amino acids are partially substituted is expressed without generating a stop codon in the middle.

[0318] As a mutation of the dystrophin gene, in Duchenne muscular dystrophy and Becker muscular dystrophy, the deletion of a single or multiple exons accounts for more than half. As a particularly common site for deletion, it is known that there is between exon 44 and exon 55. According to the site of the deleted exon of the dystrophin gene, for example, referring to published papers (for example, van Deutekom JC, van Ommen GJ., Nat Rev Genet. 2003), it can be confirmed which exon is the object of exon skipping to express the appropriate repair dystrophin. In addition, as a method for expressing repair dystrophin using genome editing, in addition to exon skipping, it can also be implemented by introducing a small deletion or insertion into the dystrophin gene to adjust the reading frame, or inserting the deleted exon by homologous recombination.

[0319] When the dystrophin gene as described above is abnormal, the abnormality can be repaired by any of the following operations: (i) by not integrating one or two or more exons into mRNA (skipping), and connecting the exons before and after it in a way that does not cause frame shift; (ii) by inserting one or two or more bases or deleting them to correct the frame shift; (iii) knocking in the deleted exon; etc. In the case of (i) or (ii) above, a dystrophin whose amino acid sequence is shorter or longer than that of normal dystrophin, or whose amino acids are partially replaced, will be produced. In addition, by (ii) or (iii) above, normal dystrophin can also be produced. By correcting the dystrophin gene in this way, Mongolia can prevent or treat diseases such as muscular dystrophy.

[0320] The nucleotide sequence of the human dystrophin gene can be obtained, for example, from the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / gene / 1756).

[0321] Repair dystrophin refers to dystrophin whose expression is restored as a result of genome editing. In particular, for a dystrophin gene having a frameshift mutation or a nonsense mutation, it refers to a dystrophin whose expression is restored by using genome editing and whose N-terminal actin binding domain and C-terminal homocysteine ​​domain are retained. When a frameshift mutation occurs due to exon duplication, when one of the duplicated exons is skipped by genome editing, expression of a repair dystrophin having 100% homology to the healthy type is sometimes restored. As a repair dystrophin, it particularly refers to human dystrophin translated from mRNA in which exon 43 and exon 46 are linked while exon 45 in the human dystrophin gene in which exon 44 is deleted is skipped. In addition, the repair dystrophin protein also includes, for example, human dystrophin protein produced by skipping each of the specified exons in the human dystrophin gene of the missing exons 12-44, 18-44, 46-47, 46-48, 46-49, 46-51, 46-53 or 46-55, but is not limited thereto. Whether the repair dystrophin protein is produced can be confirmed, for example, by detecting the mRNA encoding the repair dystrophin protein in the cell using PCR. Alternatively, it can be confirmed based on the molecular weight of the dystrophin protein by Western blotting using an antibody that recognizes the dystrophin protein.

[0322] In another preferred embodiment of the present invention, the target sequence is a base sequence in a gene locus containing a myostatin gene. More specifically, the target sequence is a base sequence that can induce a frameshift mutation or a nonsense mutation in the myostatin gene by modifying its base sequence (deletion or insertion), thereby deleting or weakening the function of myostatin and thereby inducing muscle hypertrophy.

[0323] In one embodiment of the present invention, at least a portion of the guide RNA is preferably a nucleotide analog as described in this specification. As nucleotide analogs, sugar-modified nucleotides and phosphodiester-modified nucleotides are preferred, and more specifically, 2'-O-methyl ribose and phosphodiester bonds are preferably substituted with thiophosphate bonds. In the guide RNA, it is preferred that at least one base at each of the 3' and 5' ends of the sequence is a nucleotide analog, and more preferably at least two bases or three bases at each of the 3' and 5' ends of the sequence are nucleotide analogs.

[0324] When the guide RNA is a chimeric RNA of crRNA and tracrRNA, it is preferred that at least one base at each of the 3' and 5' ends of its sequence is a nucleotide analog. When the guide RNA is in the form of one RNA (a combination of two RNAs) or more RNAs each not connected to crRNA and tracrRNA, it is preferred that at least one base at each of the 3' and 5' ends of each RNA sequence is a nucleotide analog (for example, preferably the 3' and 5' ends of crRNA and the 3' and 5' ends of tracrRNA are nucleotide analogs).

[0325] The "RNA-guided endonuclease" comprises at least one nuclease domain and at least one domain that interacts with the gRNA, and is a protein that is induced to the target site by forming a complex with the gRNA.

[0326] The RNA-guided endonuclease may be from a CRISPR system. The CRISPR system may be set to a type I, type III, type IV in class 1, or a type II, type V, and type VI system in class 2. Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a (or Cpf1), Cas12b (or C2c1), Cas12c, Cas13a1 (or C2c2), Cas13a2, Cas13b, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966.

[0327] In one embodiment, the RNA-guided endonuclease is derived from a class II CRISPR system. In a specific embodiment, the RNA-guided endonuclease is derived from a Cas9 protein.The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Nocardia dasonvillei, Streptomyces spp., Streptomyces viridochromogenes, Streptomyces roseus, Alicyclobacillus acidoterrestris, Bacillus pseudomycoides, Bacillus selenate-reducing, Exiguobacterium sibiricum, Francisella novicida, Lactobacillus bulgaricus, Lactobacillus salivarius, Bacillus stearothermophilus, Microscilla marina, Burkholderia, Naphthomonas naphthalenevorus, species of the genus Monocystis, Crocosphaera watsonii, Cyanocotyle, Microcystis aeruginosa. aeruginosa), Synechococcus, Arabinacetic Halobacter, Ammonifex degensii, Caldicellulosiruptor becscii, Campylobacter jejuni, Campylobacter coli, Neisseria meningitides, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Fingoldiella, Natranaerobius thermophilusm, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Achromobacter vinifera, Marinobacter, Nitrosococcus halophilus, Nitrosococccus varro watsoni), Pseudoalteromonas marine, Ktedonobacter racemifer, Methanohalbiumevestigatum, Anabaena variabilis, Nodularia spp., Arthrospira maxima, Arthrospira platensis, Arthrospira spp., Microcoleus chthonoplastes, Oscillatoria spp., Spatholobus mobilis, Thermocoelenteroides africanus) or deep-sea unicellular cyanobacteria.

[0328] In one embodiment, the RNA-guided endonuclease is a CRISPR-Cas12a / Cpf1 system from type 2 V. In a specific embodiment, the RNA-guided endonuclease is from a Cpf1 protein. The Cpf1 protein can be from Acidaminococcus, Lachnospiraceae, Chlamydomonas reinhardtii, or Francisella novicida.

[0329] The CRISPR / Cas protein can be a wild-type CRISPR / Cas protein, a modified CRISPR / Cas protein, or a fragment of a wild-type or modified CRISPR / Cas protein. The CRISPR / Cas protein can be modified to increase nucleic acid binding affinity and / or specificity, to alter enzyme activity, or to alter other properties of the protein.

[0330] RNA-guided nucleases can be Cas nucleases or Cas nickases. Here, Cas nucleases or Cas nickases refer to essential protein components in the CRISPR / Cas system, and refer to endonucleases or nickases that are active when forming a complex with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Nickases refer to DNA cleavage enzymes that cut a nick only in one DNA strand. Typically, the Cas9 protein includes at least two nuclease (ie, DNase) domains. For example, the Cas9 protein may include a RuvC-like nuclease domain and an HNH-like nuclease domain. RuvC and HNH domains collaborate to cut single strands in order to cut double strands in DNA (Jinek et al., Science, 337: 816-821). In one embodiment, the protein from Cas9 can be modified in a manner that includes only one functional nuclease domain (either one of the RuvC-like or HNH-like nuclease domains). For example, the protein from Cas9 can be modified in a way that a deletion or mutation occurs so that one of the nuclease domains is no longer functional (i.e., does not have nuclease activity). In a way that one of the nuclease domains is inactive, the protein from Cas9 can be introduced into a nick in a double-stranded nucleic acid, but the double-stranded DNA cannot be cut off. For example, the conversion of aspartic acid to alanine (D10A) in the RuvC-like domain is to convert the protein from Cas9 into a nickase. Similarly, the conversion of histidine to alanine (H840A or H839A) in the HNH domain is to convert the protein from Cas9 into a nickase. Each nuclease domain can be modified using well-known methods such as site-directed mutagenesis, PCR-mediated mutagenesis, and full gene synthesis and other methods known in the art.

[0331] RNA-guided nucleases can use Cas nucleases or Cas nickases, especially from Streptococcus sp. or Staphylococcus sp., Francisella novicida, Campylobacter jejuni. Among them, as a source, Streptococcus pyogenes is preferred in Streptococcus, and Staphylococcus aureus is preferred in Staphylococcus. Cas9 nucleases or Cas9 nickases from Streptococcus pyogenes recognize NGG or NAG trinucleotides as PAM sequences.

[0332] As an RNA-guided nuclease, Cas9 is preferred. In a preferred embodiment of the present invention, Cas9 is Cas9 (SpCas9) from Streptococcus pyogenes (S. pyogenes). As Cas9, Cas9 from various bacteria or archaea is known. In the present invention, in addition to SpCas9, Cas9 having the desired nuclease activity, such as Cas9 (SaCas9) from Staphylococcus aureus (S. aureus), can also be used.

[0333] The amount of the target molecule used in the transduction method of the present invention can be changed according to the conditions and purpose of transduction, and can be appropriately adjusted by a person skilled in the art. For example, when the target molecule is gRNA, the amount used is relative to 1×10 4 The amount used per cell is usually 10 ng to 510 ng, preferably 16 ng to 255 ng. When the molecule of interest is Cas9 protein, the amount used is usually 60 ng to 3200 ng, preferably 100 ng to 1600 ng.

[0334] ·cell

[0335] The target cells into which the molecule of interest is introduced using the solution for transduction of the present invention are not particularly limited and can be appropriately selected depending on the intended purpose. As target cells, for example, mesenchymal stem cells, neural stem cells, skin stem cells, spleen cells, neural cells, glial cells, pancreatic B cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells (e.g., skeletal muscle cells, cardiomyocytes, myoblasts, muscle satellite cells, smooth muscle cells), adipocytes, blood cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, leukocytes, neutrophils, basophils, eosinophils, monocytes, megakaryocytes, hematopoietic stem cells), synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes, interstitial cells, oocytes and sperm cells, and precursor cells that can be differentiated and induced into these cells, stem cells (e.g., including induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells)), primordial germ cells, oocytes and fertilized eggs. These cells can be cancerous cells (cancer cells).

[0336] When the transduction method of the present invention is performed in vitro (ex vivo) or in a culture environment (in vitro), the tissue or organ from which the target cell comes, or the tissue or organ in which the target cell exists when the transduction method of the present invention is performed in vivo (in vivo) is not particularly limited as long as it is a tissue or organ containing the target cell. As the tissue or organ containing the target cell, for example, the brain, various parts of the brain (for example, olfactory bulb, amygdala, cerebral basal nucleus, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, corpus callosum, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, digestive tract (for example, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, placenta, uterus, bone, joint and muscle (for example, skeletal muscle, smooth muscle, myocardium) etc. can be listed. In a preferred embodiment of the present invention, the tissue or organ containing the target cells is muscle, brain and various parts of the brain. In a more preferred embodiment of the present invention, the tissue or organ containing the target cells is muscle. These tissues or organs can be cancerous tissues or organs (cancer tissues, etc.).

[0337] The target cells may be cells derived from humans or cells derived from mammals other than humans (non-human mammals). Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, and primates other than humans (non-human primates, e.g., cynomolgus monkeys, rhesus monkeys, and chimpanzees).

[0338] In a preferred embodiment of the present invention, the target cell is a muscle cell (e.g., cardiomyocyte, skeletal muscle cell, muscle satellite cell), neural stem cell, nerve cell, glial cell, fibroblast, mesenchymal stem cell, blood cell or iPS cell. In a more preferred embodiment of the present invention, the target cell is a muscle cell, especially a skeletal muscle cell or a muscle satellite cell. As muscle cells, for example, muscle cells collected from humans (patients or healthy people) or other mammals (e.g., non-human primates (e.g., cynomolgus monkeys, rhesus monkeys, chimpanzees), cattle, pigs, mice, rats and other disease model animals), muscle cells present in vivo (e.g., in tissues in vivo of humans, etc.), muscle cell lines, and muscle cells obtained by differentiation induction of stem cells (e.g., iPS cells, ES cells) can be cited.

[0339] · Step of contacting cells with the molecule of interest and the transduction solution

[0340] In the transduction method of the present invention, the method for contacting cells with the molecule of interest and the transduction solution is not particularly limited, and various methods can be selected as long as the molecule of interest can be introduced into cells with a desired efficiency.

[0341] When the transduction method of the present invention is performed in vitro or in a culture environment (in vitro), the molecule of interest and the transduction solution (in the form of a solution prepared by mixing them in advance, if necessary) can be added to a culture medium and the target cells can be cultured in the culture medium, thereby allowing the molecule of interest and the transduction solution to contact the cells.

[0342] As a culture medium, a known culture medium suitable for culturing a cell group containing the target cell can be used. For example, BME culture medium, BGJb culture medium, CMRL 1066 culture medium, Glasgow MEM culture medium, modified MEM (IMEM) culture medium, modified MDM (IMDM) culture medium, Medium 199 culture medium, Eagle MEM culture medium, αMEM culture medium, DMEM culture medium (high glucose, low glucose), DMEM / F12 culture medium, HAM culture medium, RPMI 1640 culture medium, Fischer's culture medium, StemFit AK02N, primate ES cell culture medium and mixed culture medium thereof can be used. The type and amount of the culture medium can be appropriately set by those skilled in the art according to the cells and culture conditions.

[0343] Essential or non-essential amino acids, GlutaMAX (product name), vitamins, antibiotics (e.g., penicillin, streptomycin or a mixture thereof), antimicrobial agents (e.g., amphotericin B), antioxidants, pyruvic acid, buffers, inorganic salts and other additives may be added to the culture medium as needed. The type and amount of additives used may be appropriately set by those skilled in the art according to the cells and culture conditions.

[0344] When the cells are contacted with the molecule of interest and the transduction solution in a culture environment (in vitro), the time (period) of this step is not particularly limited, and can be set to a time that can achieve the desired introduction efficiency, usually 10 minutes to 180 minutes, preferably 15 minutes to 60 minutes. Various culture conditions other than the period (time), such as temperature, atmosphere (carbon dioxide concentration), etc., can also be appropriately adjusted by those skilled in the art.

[0345] When the transduction method of the present invention is performed in vivo, the molecule of interest is mixed with a transduction solution, and an injection such as intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection is prepared and administered to the organism, thereby allowing the molecule of interest and the transduction solution to contact the target cells. The transduction solution used in such an embodiment may contain, in addition to components (A1) to (A5) and (B), pharmaceutically acceptable additives when prepared as an injection, such as physiological saline, buffered physiological saline, water for injection, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonic agents, fillers, lubricants, thickeners, and the like.

[0346] In a preferred embodiment of the present invention, a transduction method is used to modify the dystrophin gene in muscle cells by the CRISPR system, in other words, to obtain modified cells by modifying the dystrophin gene by the CRISPR system, or to produce repair dystrophin by modifying the dystrophin gene.

[0347] The cell production method of the present invention is described below.

[0348] The cell manufacturing method of the present invention includes at least a step of contacting cells with a molecule of interest and a transduction solution (contact step), and may further include other steps related to manufacturing cells transduced with the molecule of interest, as required. In addition, in the cell manufacturing method of the present invention, the transduction solution described in this specification is used to introduce the molecule of interest into the cell, and other technical matters can basically follow the method of manufacturing cells introduced with the molecule of interest using a conventional solution.

[0349] The contacting step included in the cell production method of the present invention is the same as the contacting step described in the present specification in terms of its relationship with the transduction method of the present invention.

[0350] As other steps related to the production of cells transduced with the molecule of interest, the cell production method of the present invention may further include, as needed, for example: (a) a step of selecting cells introduced with the molecule of interest, which is performed after the contacting step; and (b) a step of inducing the differentiation of the target cells from pluripotent stem cells such as iPS cells or other stem cells, which is performed before the contacting step.

[0351] The selection of cells into which the molecule of interest has been introduced in the above step (a) can be performed using known methods. For example, when the molecule of interest is a substance used in the CRISPR system (representatively, a complex of gRNA and Cas9 protein), cells into which the molecule of interest has been introduced and the desired genetic modification has been performed can be selected by PCR and sequencing of base sequences, or by confirming the expression of proteins using electrophoresis. Alternatively, a drug-resistant gene, a gene encoding a fluorescent protein, other positive selection marker genes or negative selection marker genes are introduced into cells together with the molecule of interest, and treatments corresponding to each gene are performed (for example, a drug corresponding to the drug-resistant gene is added to the culture medium; or, light of an excitation wavelength corresponding to the fluorescent protein is irradiated, and the fluorescence emitted by the irradiation is detected using a fluorescence microscope, a cell sorter, etc.; or, an antibody that recognizes the protein generated by the gene is added, the bound antibody is fluorescently labeled, and the emitted fluorescence is detected using a fluorescence microscope, a cell sorter, etc.), thereby selecting cells into which the molecule of interest has been introduced.

[0352] In the above-mentioned step (b), the differentiation induction of the target cell by pluripotent stem cells such as iPS cells or other stem cells can be carried out using a known method. For example, as a method for inducing differentiation of the target cell by pluripotent stem cells, the method reported by Laflamme MA et al. (Laflamme MA & Murry CE, Nature 2011, Review) can be cited. It should be noted that, when the target cell is obtained by such differentiation induction, the cell group comprising the target cell can be an embryoid.

[0353] The pharmaceutical composition of the present invention is described below. It should be noted that those skilled in the art can further transform the present invention involving "pharmaceutical composition" into, for example, an invention involving "treatment method" (for example, a method comprising the step of administering an effective amount of a transduction solution and a molecule of interest to a human or other animal) based on the following description and other descriptions in the specification and common technical knowledge.

[0354] The pharmaceutical composition of the present invention comprises the solution for transduction of the present invention and a molecule of interest. The details, preferred embodiments, etc. of the "solution for transduction" and the "molecule of interest" are as described elsewhere in the present specification.

[0355] The use of the pharmaceutical composition of the present invention is determined by the molecules of interest contained therein. Pharmaceutical compositions with various uses can be prepared by selecting molecules of interest.

[0356] In a preferred embodiment of the present invention, the pharmaceutical composition is a preventive / therapeutic drug for dystrophinosis or a repair-type dystrophin production drug. In this embodiment, the molecule of interest may be an element constituting a CRISPR system for targeting a base sequence of a locus containing a dystrophin gene and modifying the base sequence, such as a complex of a gRNA and a Cas protein targeting a predetermined base sequence.

[0357] The dosage form of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected according to the intended use. For example, in relation to the transduction method of the present invention, the pharmaceutical composition of the present invention can be prepared as an injection such as intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection as described above.

[0358] The pharmaceutical composition of the present invention may further contain a transduction solution and pharmaceutically acceptable additives other than the molecule of interest, depending on the dosage form. The amount or concentration of the active ingredient (molecule of interest) in the pharmaceutical composition of the present invention may be appropriately adjusted in consideration of the dosage form, administration route, dosage per administration, number of administrations within a certain period, etc., so that an effective amount of the active ingredient for the desired preventive effect or therapeutic effect can be delivered to the target cells.

[0359] Example

[0360] In the following examples, the compounds shown in the following table were used.

[0361] [Table 1-1]

[0362]

[0363] [Table 1-2]

[0364]

[0365] [Table 1-3]

[0366]

[0367] [Table 1-4]

[0368]

[0369] [Table 1-5]

[0370]

[0371] FUJIFILM Wako Pure Chemical Corporation, TCI-JP; Tokyo Chemical Industry Co., Ltd.,

[0372] TRC; Toronto research chemicals, OY; Oriental Yeast Co., Ltd.,

[0373] CC; Cayman Chemical Company

[0374] Material

[0375] BL21(DE3) competent Escherichia coli (NEB, C2527)

[0376] Ni-NTA Superflow Cartridges (GE, 30765)

[0377] HiLoad 26 / 60Superdex 200pg(GE, 17-1071-01)

[0378] Resource S (GE, 17-1180-01)

[0379] TEV protease (Sigma, T4455)

[0380] Complete, EDTA free (Roche, 1873580)

[0381] SEM Nuclease, recombinant, solution (WAKO, 196-16181)

[0382] DTT (WAKO, 049-08972)

[0383] Imidazole (WAKO, 095-00015)

[0384] 1M magnesium chloride aqueous solution (WAKO, 310-90361)

[0385] Magnesium aspartate (MP Biomedicals Inc., 150496)

[0386] 10×TBS (Takarabio, T9141)

[0387] 1M HEPES buffer (Nacalai Tesque Inc., 17557-94)

[0388] 0.5M EDTA solution (Invitrogen, 15575-038)

[0389] LB medium (Lennox), (Sigma, L7275-500TAB)

[0390] 2×YT medium, (Sigma, Y2627-1kg)

[0391] IPTG (WAKO, 096-05143)

[0392] Ampicillin sodium (WAKO, 014-23302)

[0393] Potassium chloride (WAKO, 161-03541)

[0394] Pierce 660nm protein assay (Thermo scientific, 22660)

[0395] D-MEM (high glucose) with L-glutamine and phenol red (WAKO, 044-29765)

[0396] 0.25w / v% trypsin-1mmol / lEDTA·4Na solution containing phenol red (WAKO, 201-16945)

[0397] D-PBS(-), (WAKO, 043-29791)

[0398] 5M Sodium Chloride, (Invitrogen, AM9759)

[0399] UltraPure DNase / RNase-Free distilled water (Invitrogen, 10977015)

[0400] USDA-approved fetal bovine serum (Gibco, 10437028)

[0401] CellCarrier Ultra PDL-coated 96-well plates (PerkinElmer, 6055500)

[0402] HOECHST (registered trademark) 33342 (ThermoFisher, H3570)

[0403] C2C12 (ATCC (registered trademark), CRL-1772 (trademark))

[0404] Millex-GV 0.22μm PVDF 4mm EtO Ster sterilized (Merck, SLGV004SL)

[0405] 3-(1-Pyridyl)propanesulfonic acid (NDSB-201) (TCI-JP, S0813)

[0406] Glycerin (WAKO, 075-00616)

[0407] Glycine (WAKO, 070-05281)

[0408] L-Glutamine aqueous solution, ×100 (WAKO, 073-05391)

[0409] NEAA, ×100 (Life technologies, 11140-035)

[0410] N2 additive, ×100 (Gibco, 17502-048)

[0411] B27 additive (Gibco, 12587-010)

[0412] EGF (WAKO, 059-07873)

[0413] bFGF (WAKO, 064-05381)

[0414] Opti-MEM (trademark) I reduced serum medium (Life technologies, 31985-062)

[0415] 10× PBS-phosphate buffered saline (10×) pH 7.4, RNase-free (Invitrogen)

[0416] Amicon (R) Ultra 2mL Centrifugal Filter 10kDa (Merck)

[0417] Dextran, Alexa Fluor TM 488; 10000MW, anionic, fixable (Thermo Scientific)

[0418] 4% paraformaldehyde in phosphate buffer (WAKO)

[0419] Contains NucBlue TM ProLong stain TM Glass antifade mounting medium (Thermo Scientific)

[0420] Base sequences of crRNA and gRNA

[0421] MmDmdEx51 crRNA (GeneDesign, Inc): Serial number 1

[0422]

[0423] (mN: 2'-O-methyl RNA modification, *: phosphorothioate modification)

[0424] tracrRNA (GeneDesign, Inc): Serial number 2

[0425]

[0426] (mN: 2'-O-methyl RNA modification, *: phosphorothioate modification)

[0427] MmDmd Ex51 gRNA#1(mod): Sequence number 3

[0428]

[0429] (mN: 2'-O-methyl RNA modification, *: phosphorothioate modification, GeneDesign, Inc)

[0430] mRosa26 gRNA (mod): Sequence No. 4

[0431]

[0432] (mN: 2'-O-methyl RNA modification, *: phosphorothioate modification, GeneDesign, Inc, or Sumitomokagaku, Inc.)

[0433] hEx45#1 gRNA (mod): Sequence number 5

[0434]

[0435] (mN: 2'-O-methyl RNA modification, *: phosphorothioate modification, GeneDesign, Inc)

[0436] hEx45#23 gRNA (mod): sequence number 6

[0437]

[0438] (mN: 2'-O-methyl RNA modification, *: phosphorothioate modification, GeneDesign, Inc)

[0439] <Supplies>

[0440] FACS Asia(Trademark)(BD)

[0441] NEPA21 Transfection System (NEPA GENE)

[0442] AKTAprime(GE)

[0443] Opera Phenix (trademark) High Content Screening System (PerkinElmer)

[0444] Harmony(Trademark)(PerkinElmer)

[0445] <Compound>

[0446] The suppliers and product numbers of the compounds and the concentrations of the compound stock solutions are summarized in Table 1 .

[0447] <Explanation of terms used>

[0448] EGFP-SSA assay: abbreviation for EGFP single-strand annealing assay. Figure 1 The reporter box contains the MmDMD-Ex51 sequence inserted in the middle of the EGFP sequence. Usually, the EGFP ORF is cut off when the MmDMD-Ex51 sequence is present, so the EGFP protein is not translated. However, if the target sequence of MmDMD-Ex51 is cut off by the Cas9 protein / gRNA complex, single-stranded annealing occurs upstream (EG site) and downstream (FP site) of the EGFP ORF with the help of homologous regions, and the full-length EGFP ORF appears, producing EGFP protein. The EGFP-SSA analysis using this principle can predict the intracellular delivery amount of the Cas9 protein / gRNA complex by investigating the proportion of EGFP-producing cells.

[0449] <Preparation of reagents, reaction solutions and dosing solutions>

[0450] Lysis buffer

[0451] Add 20 mL of 10×TBS, 0.002 mL of 500 U / μL SEM nuclease, 0.2 mL of 0.5 M EDTA solution, 0.2 mL of 1 M magnesium chloride solution, 30.85 mg of DTT, and 272.308 mg of imidazole to a final concentration of 1×TBS, 20 mM imidazole, 0.005 U / μL SEM nuclease, 1 mM DTT, 0.5 mM EDTA2Na, and 1 mM magnesium chloride, dissolve in milliQ water, and adjust to 200 mL. Add 6 tablets of Complete EDTA free and stir, then filter and sterilize using a filter with a pore size of 0.22 μm.

[0452] GF Buffer

[0453] 20 mL of 1 M HEPES solution, 11.182 g of potassium chloride, and 154.25 mg of DTT were added to a final concentration of 20 mM HEPES, 150 mM potassium chloride, and 1 mM DTT (pH 7.5), and dissolved in milliQ water. After adjusting to pH 7.5, the volume was fixed to 1000 mL and sterilized by filtration using a filter with a pore size of 0.22 μm.

[0454] C2C12 (mouse striated muscle cell) cell culture medium

[0455] D-MEM (high glucose) medium containing 10% FBS (vol / vol) with L-glutamine and phenol red.

[0456] Stock compound solutions

[0457] The suppliers of the compounds and the final concentrations of the stock compounds are shown in Table 1. Each compound was weighed and dissolved in UltraPure DNase / RNase-Free distilled water to achieve the final concentration shown in Table 1, and then sterilized by filtration using a 0.22 μm filter to prepare a stock compound solution.

[0458] Reaction solution 1

[0459] 0.4 μg of Cas9 protein, 34.18 ng of tracrRNA, 29.48 ng of MmDmd Ex51crRNA, 0.05 μL of 1M HEPES buffer, and 5.5 μL of 5M sodium chloride aqueous solution were mixed to a final concentration of 8 ng / μL Cas9 protein, 0.68 ng / μL tracr RNA, 0.59 ng / μL MmDmd Ex51crRNA, 1 mM HEPES, 550 mM sodium chloride, and 250 mM compound, and the volume was adjusted to 37.5 μL with UltraPure DNase / RNase-Free distilled water. 12.5 μL of 1000 mM compound solution was added to this solution and incubated at room temperature for 15 minutes. It should be noted that when the final concentration of the compound was 0.1mM, 0.15mM, 0.34mM, 0.69mM, 1mM, 1.38mM, 5mM, 6.38mM, 10mM, 20mM, 40mM, 60mM, 80mM, 100mM, 125mM, 200mM, and 500mM, the stock compound solution was diluted with UltraPure DNase / RNase-Free distilled water to a concentration of 0.4mM, 0.6mM, 1.36mM, 2.76mM, 4mM, 5.52mM, 20mM, 25.5mM, 40mM, 80mM, 160mM, 240mM, 320mM, 400mM, 500mM, 800mM, and 2000mM, respectively, and then added.

[0460] Reaction solution 2

[0461] 0.4 μg of Cas9 protein, 34.18 ng of tracrRNA, 29.48 ng of MmDmd Ex51crRNA, 2.5 μL of 1M HEPES buffer, and 4.85 μL of 5M sodium chloride aqueous solution were mixed to a final concentration of 8 ng / μL Cas9 protein, 0.68 ng / μL tracr RNA, 0.59 ng / μL MmDmdEx51crRNA, 50 mM HEPES, 485 mM sodium chloride, and 250 mM compound, and the volume was adjusted to 37.5 μL with UltraPure DNase / RNase-Free distilled water. 12.5 μL of 1000 mM compound solution was added to this solution, and incubated at room temperature for 15 minutes. It should be noted that when the final concentration of the compound was 0.69mM, 25mM, 100mM, 125mM, 160mM, 200mM, and 500mM, the stock compound solution was diluted with UltraPureDNase / RNase-Free distilled water to a concentration of 2.76mM, 100mM, 400mM, 500mM, 640mM, 800mM, and 2000mM, respectively, and then added.

[0462] iTOP-1250 Buffer

[0463] iTOP-1250 buffer was prepared according to Patent Document 2. For the Opti-MEM-based solution, each reagent was added in a manner such that the final concentration reached 200mM GABA, 50mM NDSB-201, 15mM glycine, 30mM glycerol, 425mM sodium chloride, 0.75× glutamine, 0.75× Non-Essentialamino Acids, 0.75× N-2 additive, 0.75× B-27 additive, 100ng / μL FGF2, 100ng / μL EGF, 8ng / μL Cas9 protein, 0.68ng / μL tracrRNA, and 0.59ng / μL MmDmd Ex51crRNA, and incubated at room temperature for 15 minutes. For preparation, refer to Non-Patent Document 1. iTOP-1250 buffer contains 60% Opti-MEM.

[0464] Reaction solution 3-1

[0465] 2.0 μg of Cas9 protein, 170.9 ng of tracrRNA, 147.4 ng of MmDmd Ex51crRNA, 12.5 μL of 1M HEPES buffer, 18.35 μL of 5M sodium chloride aqueous solution, and 62.5 μL of 1M GABA aqueous solution were mixed to a final concentration of 8 ng / μL Cas9 protein, 0.68 ng / μL tracr RNA, 0.59 ng / μL MmDmdEx51crRNA, 50 mM HEPES, 250 mM GABA, and 367 mM sodium chloride, and the liquid volume was adjusted to 250 μL with UltraPure DNase / RNase-Free distilled water. Incubate at room temperature for 15 minutes. It should be noted that in the case of 485 mM sodium chloride, the amount of 5M sodium chloride aqueous solution added was changed to 24.25 μL, and similarly, in the case of 550 mM sodium chloride, it was changed to 27.5 μL.

[0466] Reaction solution 3-2

[0467] 2.0 μg of Cas9 protein, 170.9 ng of tracrRNA, 147.4 ng of MmDmd Ex51crRNA, 12.5 μL of 1M HEPES buffer, 91.75 μL of 1M potassium chloride aqueous solution, and 62.5 μL of 1M GABA aqueous solution were mixed to a final concentration of 8 ng / μL Cas9 protein, 0.68 ng / μL tracr RNA, 0.59 ng / μL MmDmdEx51crRNA, 50 mM HEPES, 250 mM GABA, and 367 mM potassium chloride, and the liquid volume was adjusted to 250 μL with UltraPure DNase / RNase-Free distilled water. Incubate at room temperature for 15 minutes. It should be noted that the amount of 1M potassium chloride aqueous solution added was changed to 121.25 μL in the case of 485 mM potassium chloride, and similarly, it was changed to 137.5 μL in the case of 550 mM potassium chloride.

[0468] Reaction solution 3-3

[0469] 2.0 μg of Cas9 protein, 170.9 ng of tracrRNA, 147.4 ng of MmDmd Ex51crRNA, 12.5 μL of 1M HEPES buffer, 91.75 μL of 1M magnesium chloride aqueous solution, and 62.5 μL of 1M GABA aqueous solution were mixed to a final concentration of 8 ng / μL Cas9 protein, 0.68 ng / μL tracr RNA, 0.59 ng / μL MmDmdEx51crRNA, 50 mM HEPES, 250 mM GABA, and 367 mM magnesium chloride, and the volume was adjusted to 250 μL with UltraPure DNase / RNase-Free distilled water. Incubate at room temperature for 15 minutes. In the case of 323 mM magnesium chloride, the amount of 1 M magnesium chloride aqueous solution added was changed to 80.75 μL, and similarly, in the case of 485 mM magnesium chloride, it was changed to 121.25 μL, and in the case of 550 mM magnesium chloride, it was changed to 137.5 μL.

[0470] Reaction solution 3-4

[0471] 2.0 μg of Cas9 protein, 170.9 ng of tracrRNA, 147.4 ng of MmDmdEx51 crRNA, 12.5 μL of 1M HEPES buffer, 121.25 μL of 1M magnesium aspartate aqueous solution, and 62.5 μL of 1M GABA aqueous solution were mixed to a final concentration of 8 ng / μL Cas9 protein, 0.68 ng / μL tracrRNA, 0.59 ng / μL MmDmdEx51 crRNA, 50 mM HEPES, 250 mM GABA, and 485 mM magnesium aspartate, and the liquid volume was adjusted to 250 μL with UltraPureDNase / RNase-Free distilled water. Incubate at room temperature for 15 minutes. It should be noted that in the case of 550 mM magnesium aspartate, the amount of 1M magnesium aspartate aqueous solution was changed to 137.5 μL.

[0472] Reaction solution 3-5

[0473] 2.0 μg of Cas9 protein, 170.9 ng of tracrRNA, 147.4 ng of MmDmd Ex51 crRNA, 12.5 μL of 1M HEPES buffer, 19.8 μL of 5M sodium chloride solution, 11.3 μL of 1M magnesium chloride solution, 5.4 μL of 1M potassium chloride solution, and 62.5 μL of 1M GABA solution were mixed to a final concentration of 8 ng / μL Cas9 protein, 0.68 ng / μL tracr RNA, 0.59 ng / μL MmDmdEx51 crRNA, 50 mM HEPES, 250 mM GABA, 395.8 mM sodium chloride, 45.2 mM magnesium chloride, and 21.4 mM potassium chloride, and the volume was adjusted to 250 μL with UltraPure DNase / RNase-Free distilled water. Incubate at room temperature for 15 minutes.

[0474] Reaction solution 3-6

[0475] 2.0 μg of Cas9 protein, 170.9 ng of tracrRNA, 147.4 ng of MmDmd Ex51crRNA, 12.5 μL of 1M HEPES buffer, 22.4 μL of 5M sodium chloride solution, 12.8 μL of 1M magnesium chloride solution, 6.1 μL of 1M potassium chloride solution, and 62.5 μL of 1M GABA solution were mixed to a final concentration of 8 ng / μL Cas9 protein, 0.68 ng / μL tracrRNA, 0.59 ng / μL MmDmdEx51crRNA, 50 mM HEPES, 250 mM GABA, 447.4 mM sodium chloride, 51.1 mM magnesium chloride, and 24.2 mM potassium chloride, and the volume was adjusted to 250 μL with UltraPure DNase / RNase-Free distilled water. Incubate at room temperature for 15 minutes.

[0476] Reaction solution 4

[0477] 800ng of Cas9 protein, 200ng of MmDmd Ex51 gRNA, 0.05μL of 1M HEPES buffer, and 5.5μL of 5M sodium chloride aqueous solution were mixed in such a way that the final concentration reached 16ng / μL Cas9 protein, 4ng / μL MmDmd Ex51gRNA, 1mM HEPES, 550mM sodium chloride, and 250mM compound, and the liquid volume was adjusted to 25μL with UltraPure DNase / RNase-Free distilled water. For this solution, a prescribed amount of compound stock solution was added in such a way that the final concentration reached 250mM, and incubated at room temperature for 15 minutes. It should be noted that when the final concentration of sodium chloride was 150mM and 350mM, 1.5μL and 3.5μL of 5M sodium chloride aqueous solution were added, respectively.

[0478] Reaction solution 5

[0479] 2.5 μL of 100 mg / mL FITC-Dextran, 0.25 μL of 1M HEPES buffer, and 27.5 μL of 5M sodium chloride aqueous solution were mixed so that the final concentration was 1 mg / mL fluorescein isothiocyanate-dextran, average molar weight 70,000 (FITC-Dextran), 1 mM HEPES, 550 mM sodium chloride, and 250 mM compound, and the liquid volume was adjusted to 250 μL with UltraPure DNase / RNase-Free distilled water. To this solution, a predetermined amount of compound stock solution was added so that the final concentration was achieved to prepare a reaction solution.

[0480] Administration of solution 1

[0481] Mix 20μg of Cas9 protein, 5μg of mRosa26 gRNA, 5μL of 10×PBS, and 6.25μL of 2.0M2'-deoxycytidine aqueous solution so that the final concentration reaches 400ng / μL Cas9 protein, 100ng / μL mRosa26 gRNA, 533mM sodium chloride, and 250mM 2'-deoxycytidine aqueous solution. After adjusting the concentration to 533mM with 5M sodium chloride aqueous solution, adjust the liquid volume to 50μL with UltraPureDNase / RNase-Free distilled water and incubate at room temperature for 15 minutes. It should be noted that when the final concentration is 163mM, 233mM, 333mM, and 433mM, the amount of 5M sodium chloride added is appropriately adjusted. In addition, the Cas9 protein used is produced by Amicon (R) Ultra 2mL centrifugal filters were buffered with 250 mM sodium chloride.

[0482] Administration of solution 2

[0483] 40μg of Cas9 protein, 10μg of mRosa26gRNA, 5μL of 10×PBS, and 25μL of 500M compound stock solution were mixed in such a way that the final concentration reached 800ng / μL Cas9 protein, 200ng / μL mRosa26 gRNA, 533mM sodium chloride, and 250mM compound. After adjusting the concentration to 533mM with 5M sodium chloride aqueous solution, the liquid volume was adjusted to 50μL with UltraPure DNase / RNase-Free distilled water, and incubated at room temperature for 15 minutes. It should be noted that when the final concentration is 25mM compound, 2.5μL of 500mM compound stock solution was added. The Cas9 protein used was prepared by Amicon (R) Ultra 2mL centrifugal filters were buffered with 500mM sodium chloride.

[0484] Administration of solution 3

[0485] 120μg of Cas9 protein, 15μg of hDMDEx45#1gRNA, 15μg of hDMDEx45#23gRNA, and 25μL of 10×PBS were mixed to a final concentration of 2400ng / μL Cas9 protein, 300ng / μL hDMD Ex45#1gRNA, 300ng / μL hDMD Ex45#23gRNA, 158mM sodium chloride, and 25mM or 250mM compound. After adding 500mM compound stock solution to a final concentration of 25mM or 250mM, the liquid volume was adjusted to 250μL with UltraPureDNase / RNase-Free distilled water and incubated at room temperature for 15 minutes. The Cas9 protein used was prepared by Amicon (R) Ultra 2mL centrifugal filters were buffered with 250 mM sodium chloride.

[0486] <Method>

[0487] Establishment of EGFP-SSA reporter cells

[0488] To evaluate the uptake of Cas9 into cells, an EGFP-SSA reporter cell line was established. The plasmid containing the EF1α-EGxxFP-MmDMD-Ex51 reporter cassette was transfected into C2C12 cells by electroporation (NEPA21 transfection system). Figure 1 ). Electroporation with NEPA21 was performed using 1×10 6The cells were perforated at 200 V and 5 ms. Puromycin was then added to the culture medium at a concentration of 1 mg / mL to screen for cells containing the EF1α-EGxxFP-MmDMD-Ex51 reporter cassette, and then EGFP-negative cells were sorted using a cell sorter (FACS Aria, BD) to clone ( Figure 2 ).

[0489] Expression and purification of Cas9 protein

[0490] A protein expression plasmid with the gene of interest was constructed with reference to pMJ806 in the following paper (Jinek, et al. 2012). Specifically, a vector expressing a 6×His tag sequence, an MBP sequence, a TEV protease cleavage sequence, and two SV40-derived nuclear transfer (NLS) sequences in the Cas9 gene of Streptococcus pyogenes was constructed ( Figure 3). The vector was transformed into Escherichia coli system BL21 (DE3) competent cells to overexpress the protein. The transformation was carried out according to the manufacturer's protocol. The culture and purification procedures refer to (Jinek, et al. 2012). The transformant was cultured with shaking at 37°C overnight in LB medium supplemented with 0.1 mg / mL ampicillin sodium, and the culture solution was added to 2×YT medium and cultured with shaking at 37°C until OD 0.6. IPTG was added, and after standing on ice for 30 minutes, it was further cultured with shaking at 16°C for 18 hours, centrifuged, and the obtained bacteria were lysed with lysis buffer. The lysate was purified by centrifugation and loaded into Ni-NTA Superflow Cartridges, and the Cas9 protein with the His tag was eluted by imidazole gradient according to the manufacturer's protocol (Ni-NTA Superflow Cartridge manual). In order to remove the imidazole in the elution fraction, gel filtration chromatography using HiLoad 26 / 60Superdex200pg was performed to merge the protein molecular weight fractions. According to the manufacturer's instructions, the protein fraction was digested with TEV to cut and separate the 6×His-MBP tag and the Cas9 protein. The TEV digested protein solution was loaded onto the Ni-NTA column, and the 6×His-MBP tag was removed by adsorption according to the manufacturer's regulations, and the fraction containing the Cas9 protein was recovered from the penetration liquid. The recovered fraction was loaded onto the Resource S column, and the Cas9 protein was eluted by a calcium chloride gradient. In order to remove the calcium chloride in the elution buffer, gel filtration chromatography was performed using a HiLoad 26 / 60Superdex 200pg column equilibrated with GF buffer to recover the Cas9 protein fraction. For the elution fraction, protein determination was performed by Pierce 660nm protein analysis, SDS gel electrophoresis and Coomassie staining to determine the concentration and purity of the protein. Filter sterilization was performed after purity determination.

[0491] References to Examples

[0492] Jinek, Martin, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, JenniferA Doudna, and Emmanuelle Charpentier. "A Programmable Dual-RNA-Guided DNAEndonuclease in Adaptive Bacterial Immunity." Science, 2012: 816-821

[0493] EGFP-SSA analysis

[0494] EGFP reporter cells were cultured at 1×10 4 / well was inoculated in the corresponding culture medium of a 96-well plate and cultured at 5% CO2 and 37°C for 24 hours. After washing with 100 μL / well PBS, reaction solution 1 or reaction solution 2 was added at 50 μL / well and incubated at 5% CO2 and 37°C for 30 minutes. Then, it was replaced with 100 μL / well of C2C12 cell culture medium and cultured at 5% CO2 and 37°C for 48 hours. After replacing with C2C12 cell culture medium with 1 / 2000 amount of HOECHST (registered trademark) 33342, it was cultured at 5% CO2 and 37°C for 1 hour, and then scanned with Opera Phenix (trademark) using EGFP / HOECHST / visible light. The obtained image was analyzed with the attached Harmony (trademark) to count the nuclei stained with HOECHST and the EGFP-positive cells. The count of EGFP-positive cells was determined by whether EGFP could be detected in the cell area stained with HOECHST. The EGFP positive rate in each well was calculated by the following formula.

[0495] EGFP positive rate (%) = (EGFP positive cell number / HOECHST detection number) × 100

[0496] Generation of human DMD exon 45 knock-in-mouse DMD exon 44 knock-out mice

[0497] A knock-in vector containing 10 μg of human DMD exon 45 and 1.5 kb of sequences at the 5' side and 0.6 kb at the 3' side, a neomycin resistance gene expression unit sandwiched by an FRT sequence, and 1.5 kb of sequences derived from mouse Dmd intron 44 and intron 45 was used together with 2.5 μg of pCAG-Cas9 expression vector and two 2.5 μg of pU6-sgRNA expression vectors (target sequence; SEQ ID NOs: 7 and 8) to 5 × 10 5ES cells from C57BL / 6J mice were electroporated, and homologous recombinant cell lines were screened by PCR and sequencing. After removing the neomycin resistance unit by Flpe (flipase) treatment, the ES cell line was microinjected into the 4-ploid blastocysts of ICR mice to obtain chimeric mice. Mice with heterozygous knock-in of female human DMD exon 45 were obtained by in vitro fertilization of chimeric mice with female C57BL / 6J mice. Next, 100 ng / μL of Cas9 mRNA (TriLink BioTechnologies), two sgRNAs for mouse Dmd exon 44 knockout (target sequences; sequence numbers 9 and 10, FASMAC Co., Ltd.), and 50 ng / μL of ssODN (sequence number 11, Eurofins Genomics KK) were microinjected into fertilized eggs of male C57BL / 6J mice and female mice with heterozygous knock-in of human DMD exon 45. The resulting male offspring were subjected to genetic determination by PCR and sequencing to screen for human DMD exon 45 knock-in-mouse Dmd exon 44 knock-out mice.

[0498] Sequence Number 7

[0499]

[0500] Sequence Number 8

[0501]

[0502] Sequence Number 9

[0503]

[0504] Sequence number 10

[0505]

[0506] Sequence Number 11

[0507]

[0508] Evaluation of DNA mutation introduction efficiency using mRosa26 gRNA in C57BL / 6J mice

[0509] Dosage solution 1 or 2, 50 μL of PBS were administered to the right lower limb gastrocnemius muscle of 9-week-old male C57BL / 6J mice (CLEA Japan, Inc). Dosage solution 1 was administered to the same site once a day for a total of 3 times. Dosage solution 2 was administered only once. Four days after the last administration, the mice were killed by cervical dislocation under 3.5% isoflurane anesthesia, and the gastrocnemius muscle tissue of the right lower limb was removed and quickly frozen with dry ice. From the frozen muscle tissue, genomic DNA was extracted and purified using QIAamp Fast DNA Tissue Kit (Qiagen), and PCR was performed using PrimeSTAR GXL DNA polymerase (TAKARA) (forward primer; sequence number 12, reverse primer; sequence number 13). The PCR product was purified with QIAquick PCR purification kit (QIAGEN), treated with T7 endonuclease I (NEB), and analyzed with Agilent 4200 TapeStation (Agilent). The mutation introduction efficiency was calculated using the obtained numerical value according to the following calculation formula (Mathematical Formula 1).

[0510] Sequence Number 12

[0511]

[0512] Sequence Number 13

[0513]

[0514] [Mathematical formula 1]

[0515]

[0516] a: Peak area of ​​the uncut band

[0517] b, c: Peak areas of the bands cut into predicted molecular weights

[0518] Evaluation of exon skipping efficiency in skeletal muscle of human DMD exon 45 knock-in-mouse DMD exon 44 knock-out mice

[0519] The drug solution 3 was administered once in 50 μL to the tibialis anterior muscle of 4-week-old male human DMD exon 45 knock-in-mouse Dmd exon 44 knock-out mice. The tibialis anterior muscle tissue was removed 7 days after administration and quickly frozen with dry ice. After adding QIAzol Lysis Reagent (QIAGEN) to the frozen muscle tissue and breaking the tissue, chloroform (WAKO) was added, mixed and centrifuged, and the water tank containing RNA was separated and recovered, and the total RNA was reverse transcribed using High Capacity RNA-to-cDNA kit (Thermo Fisherscientific.Inc). Then, in the quantitative determination of the product after the jump, FastStart Universal Probe Master (Roche) and foreign substances of sequence numbers 14 and 15 and probes of sequence 16 were used. In addition, in the quantitative determination of the product that was not jumped, FastStart Universal Probe Master (Roche) and foreign substances of sequence numbers 17 and 18 and probes of sequence 19 were used. The PCR products were determined using ViiA7 TM The real-time PCR system was used. In any amplification, the standard product of a specified concentration was amplified simultaneously to calculate the absolute amount. The attached software was used for calculation. The obtained values ​​were used to calculate the exon skipping efficiency (exon skipping efficiency) by the following calculation formula (Mathematical Formula 2).

[0520] Sequence Number 14

[0521]

[0522] (mEx43-46skipTaqF 114: Thermo Fisher Scientific)

[0523] Sequence Number 15

[0524]

[0525] (mEx43-46skipTaqR191: Thermo Fisher Scientific)

[0526] Sequence Number 16

[0527]

[0528] (FAM label, mEx43-46skipTaqP144: Thermo Fisher Scientific)

[0529] Sequence Number 17

[0530]

[0531] (hEx45TaqF252: Thermo Fisher Scientific)

[0532] Sequence Number 18

[0533]

[0534] (hEx45 TaqR316: Thermo Fisher Scientific)

[0535] Sequence Number 19

[0536]

[0537] (FAM label, hEx45TaqP278: Thermo Fisher Scientific)

[0538] [Mathematical formula 2]

[0539] Exon skipping efficiency (%) = 100 × a / (a+b)

[0540] a: the absolute amount of the product that has undergone the jump

[0541] b: Absolute amount of product without jump

[0542] Evaluation of intracellular uptake using dextran

[0543] C2C12 cells were cultured at 1×10 4 / well was inoculated into 8-chamber slides, and 5250 μL of the reaction solution was added the next day, and incubated for 30 minutes at 5% CO2 and 37°C. After washing twice with culture medium, the cells were incubated in culture medium for 30 minutes or 90 minutes. Furthermore, the samples were fixed by standing in 4% paraformaldehyde·phosphate buffer at room temperature for 15 minutes and then stained with NucBlue. TM ProLong stain TM The samples were embedded in glass antifade mounting medium and examined under a confocal laser microscope.

[0544] Statistical processing

[0545] exist Fig.10 and Fig.11In order to investigate the statistical significance of the difference between GABA and each compound condition, Dunnett type multiple comparison test was performed using DNA mutation introduction efficiency Indel (%) and exon skipping efficiency. When the P value was less than 0.05, it was judged to be statistically significant. Statistical processing used EXSUSVer 8.0.

[0546] [Example 1]

[0547] A transduction solution containing various compounds (A1) to (A5) and a salt (B) was prepared by the following steps. After the complex of gRNA and Cas9 protein was further dissolved in the solution, it was contacted with mouse myoblasts (C2C12 cells) in vitro to evaluate the target gene cutting activity and cell survival rate.

[0548] Evaluation of transduction compound activity and relative comparison with GABA

[0549] The activity of each compound was evaluated by EGFP-SSA assay using Cas9 protein / gRNA complex. EGFP reporter cells were cultured at 1×10 4 / well was inoculated in a 96-well plate, and 150 μL of the reaction solution was added the next day, and incubated for 30 minutes at 5% CO2 and 37°C. Subsequent operations were performed according to the EGFP-SSA analysis. The stock compound solution was diluted with UltraPure DNase / RNase-Free distilled water in a final concentration range of 0.1mM to 500mM to prepare the compound. For each compound, a dilution series of the compound was evaluated in the same plate. In each plate, 250mM GABA and compound-free (H2O as a solvent) conditions were prepared in 3 wells each. The EGFP positivity rate of each compound concentration was calculated from the number of HOECHST detections and the number of EGFP-positive cells in each well. The results are shown in Figure 4.

[0550] Table 2 records Figure 4-1 and Figure 4-2 The EGFP positive rate and HOECHST cell count at the concentration indicated by the arrow are recorded, and the EGFP positive rate and HOECHST cell count of the positive control group set on the same plate as the compound are also recorded. The relative activity of each compound is calculated by the following formula (1) and (2) to obtain Figure 5 .

[0551] EGFP positive rate (GABA relative ratio) = EGFP positive rate at the recorded concentration of the compound (%) / EGFP positive rate of the positive control on the same plate (%) ... (1)

[0552] HOECHST detection number (GABA relative ratio) = HOECHST detection number at the recorded concentration of the compound / HOECHST detection number of the positive control on the same plate ... (2)

[0553] [Table 2]

[0554]

[0555] The compounds with an EGFP positivity (transduction efficiency) that was 1.2 times higher than that of GABA were 4-hydroxyproline, N-carbamoyl-L-aspartic acid, 1-methyl-L-histidine, 6-phospho-D-gluconic acid, 1,3-butanediol, 2-hydroxyglutaric acid, 1,3-dimethylurea, creatinine, miglitol, α-D-glucose-1-phosphate, argininosuccinic acid disodium salt hydrate, L-carnosine, cytidine, sodium 3-methyl-2-oxobutyrate, and inosine-5'-phosphate disodium salt hydrate.

[0556] [Example 2] Comparison of the activity of transduction compounds and iTOP-1250

[0557] The intracellular delivery efficiency of the Cas9 protein / gRNA complex of each compound was evaluated by EGFP-SSA analysis. EGFP reporter cells were cultured at 1×10 4 / well were inoculated in a 96-well plate, and 250μL of the reaction solution was added the next day, and incubated for 30 minutes at 5% CO2 and 37°C. Prepare wells to which 50μL of iTOP-1250 was added and incubated in the same manner. Subsequent operations were performed according to the EGFP-SSA analysis method. The compounds were prepared as follows: the stock compound solution was diluted with UltraPureDNase / RNase-Free distilled water in a manner to achieve the final concentration in Table 2. In each plate, wells with 250mM GABA and no compound (only H2O as a solvent) were prepared. For the obtained HOECHST detection number and EGFP-positive cell rate, the relative ratio of each compound to GABA was calculated using formulas (1) and (2). The assay was performed in 3 wells in each plate. Figure 6The mean value is plotted in the middle, and the standard deviation is shown in the form of error bars. Cells treated with reaction solution 2 are recorded by compound name. The EGFP positivity ratio of the positive control to the negative control is 2 or more, so the test is determined to be established. Untreated is a well that was only washed with PBS without adding Cas9 / gRNA. The compounds with an EGFP positivity rate that is more than 1.2 times higher than that of iTOP-1250 are N-carbamoyl-L-aspartic acid, sodium 3-methyl-2-oxobutyrate, 2-hydroxyglutaric acid, argininosuccinic acid disodium salt hydrate, 2'-deoxycytidine, inosine-5'-phosphate disodium salt hydrate, 1-deoxynojirimycin, 2-deoxy-D-glucose-6-phosphate, L-carnosine, α-D-glucose 1-phosphate, creatinine, (R)-pantothenolide, trimethyldione, and 6-phospho-D-gluconic acid.

[0558] [Example 3]

[0559] (B) Evaluation of the intracellular delivery efficiency of the Cas9 protein / gRNA complex in salt by EGFP-SSA analysis. EGFP reporter cells were cultured at 1×10 4 / well was inoculated in a 96-well plate, and 50 μL of reaction solution 3-1, 3-2, 3-3, 3-4, 3-5, or 3-6 was added the next day, and incubated for 30 minutes at 37°C with 5% CO2. Prepare wells to which 50 μL of iTOP-1250 was added and incubated. The subsequent operations were performed according to the EGFP-SSA analysis method. The obtained HOECHST detection number and EGFP positive cell rate are shown in Figure 7 . The projects with sodium chloride used reaction solution 3-1, the projects with potassium chloride used reaction solution 3-2, the projects with magnesium chloride used reaction solution 3-3, the projects with magnesium aspartate used reaction solution 3-4, the projects with low concentration of mixed solution used reaction solution 3-5, the projects with high concentration used reaction solution 3-6, and the projects with iTOP-1250 used iTOP-1250 buffer. The experiment was performed with 3 wells in each well on the same plate, and the average value was plotted, with the standard deviation shown as the error bar. In addition, the inorganic salt ions (Na + ,Mg 2+ , K + , C1-) The electrolyte concentration is expressed in milliequivalents (mEq / L). For all salts, the EGFP positive rate increases with the increase of electrolyte concentration. The highest activity is the condition of mixing the three salts of sodium chloride, potassium chloride and magnesium chloride, which is also more active than iTOP-1250. In addition, when sodium chloride and magnesium chloride are compared at the same electrolyte concentration, they have basically the same activity.

[0560] [Example 4] Evaluation of intracellular delivery of Cas9 / gRNA complex at various sodium chloride concentrations

[0561] The intracellular delivery efficiency of the Cas9 protein / gRNA complex of each compound at each sodium chloride concentration in (B) was evaluated by EGFP-SSA analysis. 4 / well of a 96-well plate, add 3-750 μL of the reaction solution the next day, and incubate for 30 minutes at 5% CO2 and 37°C. The subsequent operations are performed according to the EGFP-SSA analysis method. (A) Compounds are added at a final concentration of 250 mM. Prepare wells with 250 mM GABA and no compound (only H2O as a solvent) in the plate. Perform the assay in 3 wells in the same plate. Figure 8 The average value of EGFP positivity is plotted in the figure, and the standard deviation is shown in the form of error bars. When the sodium chloride concentration is 150mM to 550mM, the EGFP positivity of no compound is less than 1%. GABA shows an EGFP positivity of 1.4% at a sodium chloride concentration of 550mM, and a positivity of less than 1% at 150mM and 350mM. In comparison, the compounds with an EGFP positivity exceeding 1% at a sodium chloride concentration of 150mM are N-carbamoyl-L-aspartic acid and 6-phospho-D-gluconic acid, especially 6-phospho-D-gluconic acid, with an EGFP positivity higher than 2.2%, confirming a higher activity than GABA. The compounds that showed an activity of 1.4% higher than the EGFP positive rate of GABA at a sodium chloride concentration of 350 mM were argininosuccinic acid disodium salt hydrate, N-carbamoyl-L-aspartic acid, α-D-glucose 1-phosphate, 6-phospho-D-gluconic acid, 2-deoxy-D-glucose-6-phosphate, and inosine-5'-phosphate disodium salt hydrate. Regarding the EGFP positive rate of the compounds at a sodium chloride concentration of 550 mM, all compounds except 4-hydroxyproline showed activity higher than that of GABA.

[0562] [Example 5] Evaluation of DNA mutation introduction efficiency under salt concentration using C57BC / 6J mice (B)

[0563] A transduction solution containing 2'-deoxycytidine as a compound (A3) and sodium chloride as a salt (B) is prepared, and the complex of mRosa26gRNA and Cas9 protein is further dissolved in the solution, and it is used as the dosing solution of Example 5 (dosing solution 1, prepared as described above). Dosing solution 1 was topically administered to the gastrocnemius muscle of the right lower limb of mice to evaluate the target gene cutting activity. Dosing solution 1 was prepared in a manner such that the sodium chloride concentration was 163mM, 233mM, 333mM, 433mM or 533mM. The dosing solution was administered in a manner of 50μL each time for 3 consecutive days, and the gastrocnemius muscle tissue of the right lower limb was extracted on the 4th day from the last administration, and T7E1 analysis was performed. The results are shown in Fig. 9 Genome editing was confirmed in normal mice at all salt concentrations.

[0564] [Example 6] Evaluation of DNA mutation introduction efficiency at various compound concentrations (A) using C57BC / 6J mice

[0565] A transduction solution containing various compounds (A1) to (A5) and sodium chloride as the salt (B) is prepared, and the complex of mRosa26 gRNA and Cas9 protein is further dissolved in the solution, and it is used as the dosing solution of Example 6 (dosing solution 2, prepared as described above). Dosing solution 2 is topically administered to the gastrocnemius muscle of the right lower limb of mice to evaluate the target gene cutting activity. Dosing solution 2 contains 2'-deoxycytidine, 6-phospho-D-gluconic acid, N-carbamoyl-L-aspartic acid or 2-deoxy-D-glucose-6-phosphate as compounds, and is prepared in a manner such that the final concentration in the dosing solution is 25mM or 250mM. Dosing solution 2 is administered once at 50μL, and the gastrocnemius muscle tissue of the right lower limb is extracted on the 4th day for T7E1 analysis. The results are shown in Fig.10 All compounds showed genome editing efficiencies equal to or higher than those of 250 mM GABA, a control compound, in normal mice. In particular, N-carbamoyl-L-aspartate and 2-deoxy-D-glucose-6-phosphate showed statistically significant results compared to GABA.

[0566] [Example 7] Exon skipping effect using hDMD Ex45#1 gRNA and hDMD Ex45#23 gRNA in human DMD exon 45 knock-in-mouse DMD exon 44 knock-out mice

[0567] A transduction solution containing various compounds (A1) and (A4) and sodium chloride as (B) salt is prepared, and the complex of hEx45#1gRNA, hEx45#23gRNA and Cas9 protein is further dissolved in the solution, and it is used as the dosing solution of Example 7 (dosing solution 3, prepared as described above). Dosing solution 3 is topically administered to the tibialis anterior muscle of mice to evaluate the skipping efficiency of human DMD exon 45 sequence. Dosing solution 3 contains 6-phospho-D-gluconic acid or N-carbamoyl-L-aspartic acid as a compound or contains GABA as a control compound, and is prepared in a manner such that the final concentration in the dosing solution is 25mM or 250mM. Dosing solution 3 is administered once at 50μL, and the tibialis anterior muscle tissue is extracted on the 7th day to measure the exon skipping efficiency. The results are shown in Fig.11 All compounds showed higher exon skipping activity of the target gene (dystrophin gene) in hEx45KI-mdx44 mice than 250 mM GABA. In particular, 25 mM N-carbamoyl-L-aspartic acid obtained statistically significant results compared with 250 mM GABA.

[0568] [Example 8] Intracellular uptake test using dextran

[0569] The intracellular uptake of C2C12 cells was evaluated using fluorescently labeled dextran (FITC-Dextran). The sodium chloride concentration was 550 mM, and the final concentrations of the compounds were 250 mM GABA (control), 125 mM N-carbamoyl-L-aspartic acid, and 250 mM 2-deoxy-D-glucose-6-phosphate. The reaction solution 5 to which each compound was added was added to the cells, and after 30 minutes, the cells were switched to the culture medium and incubated for 30 minutes or 90 minutes. The results are shown in Fig.12 By using the compound specified in the present invention in combination with a salt, the intracellular permeation of fluorescently labeled dextran is promoted. It is confirmed that the present invention can efficiently deliver not only proteins (Cas9) and nucleic acids (gRNA), but also high molecular weight compounds such as dextran.

[0570] Industrial Applicability

[0571] The transduction solution of the present invention and the transduction method of the present invention using the same can efficiently introduce molecules of interest such as nucleic acids and proteins into various cells. For example, by bringing the complex of the gRNA and Cas protein used in the CRISPR system into contact with the cell together with the transduction solution of the present invention, it can be efficiently introduced into the cell, and genome editing using the CRISPR system can be performed. In addition, by the transduction method of the present invention, cells transduced with molecules of interest such as nucleic acids and proteins can be efficiently manufactured. Sequence Listing <110> National University Corporation Kyoto University Takeda Pharmaceutical Company Limited <120> Drug delivery systems using solutions <130> PT38-9043 <150> JP 2018-224965 <151> 2018-11-30 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 42 <212> RNA <213> Artificial sequence <220> <223> MmDmdEx51 crRNA <220> <221> modified_base <222> (1)..(1) <223> 2'-O-methylcytidine (cm) <220> <221> modified_base <222> (2)..(2) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (3)..(3) <223> 2'-O-methylcytidine (cm) <220> <221> modified_base <222> (39)..(39) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (40)..(40) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (41)..(41) <223> 2'-O-methyluridine (um) <400> 1 cacuagagua acagucugac guuuuagagc uaugcuguuu ug 42 <210> 2 <211> 70 <212> RNA <213> Artificial sequence <220> <223> tracrRNA <220> <221> modified_base <222> (1)..(1) <223> 2'-O-methylcytidine (cm) <220> <221> modified_base <222> (2)..(2) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (3)..(3) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (4)..(4) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (67)..(67) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (68)..(68) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (69)..(69) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (70)..(70) <223> 2'-O-methylcytidine (cm) <400> 2 caaaacagca uagcaaguua aaauaaggcu aguccguuau caacuugaaa aaguggcacc 60 gagucggugc 70 <210> 3 <211> 101 <212> RNA <213> Artificial sequence <220> <223> MmDmd Ex51 gRNA#1 <220> <221> modified_base <222> (1)..(1) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (2)..(2) <223> 2'-O-methylcytidine (cm) <220> <221> modified_base <222> (3)..(3) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (98)..(98) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (99)..(99) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (100)..(100) <223> 2'-O-methyladenosine <400> 3 ucacuagagu aacagucuga cguuuuagag cuagaaauag caaguuaaaa uaaggcuagu 60 ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu u 101 <210> 4 <211> 94 <212> RNA <213> Artificial sequence <220> <223> mRosa26 gRNA <220> <221> modified_base <222> (1)..(1) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (2)..(2) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (3)..(3) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (91)..(91) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (92)..(92) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (93)..(93) <223> 2'-O-methylguanosine (gm) <400> 4 gaugggcggg agucuucugu uuuagagcua gaaauagcaa guuaaaauaa ggcuaguccg 60 uuaucaacuu gaaaaagugg caccgagucg gugc 94 <210> 5 <211> 96 <212> RNA <213> Artificial Sequence <220> <223> hEx45#1 gRNA <220> <221> modified_base <222> (1)..(1) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (2)..(2) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (3)..(3) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (93)..(93) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (94)..(94) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (95)..(95) <223> 2'-O-methylguanosine (gm) <400> 5 ugguaucuua caggaacucc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugc 96 <210> 6 <211> 96 <212> RNA <213> Artificial sequence <220> <223> hEx45#23 gRNA <220> <221> modified_base <222> (1)..(1) <223> 2'-O-methyladenosine <220> <221> modified_base <222> (2)..(2) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (3)..(3) <223> 2'-O-methylcytidine (cm) <220> <221> modified_base <222> (93)..(93) <223> 2'-O-methylguanosine (gm) <220> <221> modified_base <222> (94)..(94) <223> 2'-O-methyluridine (um) <220> <221> modified_base <222> (95)..(95) <223> 2'-O-methylguanosine (gm) <400> 6 agcugucaga cagaaaaaag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugc 96 <210> 7 <211> 20 <212> DNA <213> Mouse (Mus musculus) <400> 7 atgaatgtgc ctacatatgg 20 <210> 8 <211> 20 <212> DNA <213> Mouse (Mus musculus) <400> 8 catagcatgc atttggcttc 20 <210> 9 <211> 20 <212> DNA <213> Mouse (Mus musculus) <400> 9 gaatgaggta gtgttgtagg 20 <210> 10 <211> 20 <212> DNA <213> Mouse (Mus musculus) <400> 10 gcaggaaatc atcttatagc 20 <210> 11 <211> 120 <212> DNA <213> Artificial sequence <220> <223> ssODN <400> 11 gagcaagctg ggttagaaca aaggtctgtc agagtcagca tgggaatgag gtagtgttgt 60 agcaggaaat agtgtggttt aggtctctcc ccgccctctg tgtatgtgtg tgtgtgtgtt 120 <210> 12 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Forward primer <400> 12 ctccgaggcg gatcacaagc aataataacc tgtag 35 <210> 13 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 13 tgcaagcacg tttccgactt gagttgcctc aagag 35 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> mEx43-46skipTaqF114 <400> 14 cgtggcacag atggatttcc 20 <210> 15 <211> 25 <212> DNA <213> Artificial sequence <220> <223> mEx43-46skipTaqR191 <400> 15 ttcttttgtt cttcaatccc ttgtc 25 <210> 16 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> FAM labeling, mEx43-46skipTaqP144 <400> 16 acttcataga atgtacaagg aa 22 <210> 17 <211> 25 <212> DNA <213> Artificial sequence <220> <223> hEx45 TaqF252 <400> 17 tcctcaaaaa cagatgccagtattc 25 <210> 18 <211> 18 <212> DNA <213> Artificial sequence <220> <223> hEx45 TaqR316 <400> 18 tcctgccacc gcagattc 18 <210> 19 <211> 19 <212> DNA <213> Artificial sequence <220> <223> FAM labeling, hEx45 TaqP278 <400> 19 acaggaaaaa ttgggaagc 19

Claims

1. A method for transducing a molecule of interest comprising a Cas protein and / or a gRNA into a mammalian cell in vitro or in a culture environment, comprising contacting the cell with the molecule of interest and a transduction solution, The transduction solution comprises the following (A1) and (B) salts: The (A1) is a compound selected from argininosuccinic acid, 1-methyl-L-histidine, L-carnosine and N-carbamoyl-L-aspartic acid or a salt thereof, The (B) salt is a salt selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride and magnesium aspartate, The concentration of the (B) salt in the transduction solution is 300 mM to 900 mM.

2. The method according to claim 1, wherein: (A1) is a compound selected from the group consisting of 1-methyl-L-histidine and N-carbamoyl-L-aspartic acid.

3. The method according to claim 1, wherein: The salt (B) is at least one selected from the group consisting of sodium chloride, magnesium chloride and potassium chloride.

4. The method according to claim 1, wherein: The cells are muscle cells.

5. A method for producing a mammalian cell transduced with a molecule of interest comprising a Cas protein and / or a gRNA in vitro or in a culture environment, comprising the step of contacting the cell with the molecule of interest and a transduction solution, The transduction solution comprises the following (A1) and (B) salts: The (A1) is a compound selected from argininosuccinic acid, 1-methyl-L-histidine, L-carnosine and N-carbamoyl-L-aspartic acid or a salt thereof, The (B) salt is a salt selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride and magnesium aspartate, The concentration of the (B) salt in the transduction solution is 300 mM to 900 mM.

6. The method according to claim 5, wherein: (A1) is a compound selected from the group consisting of 1-methyl-L-histidine and N-carbamoyl-L-aspartic acid.

7. The method according to claim 5, wherein: The salt (B) is at least one selected from the group consisting of sodium chloride, magnesium chloride and potassium chloride.

8. The method according to claim 5, wherein: The cells are muscle cells.

9. A solution for transduction, comprising the following (A1) and (B) salts: The (A1) is a compound selected from argininosuccinic acid, 1-methyl-L-histidine, L-carnosine and N-carbamoyl-L-aspartic acid or a salt thereof, The (B) salt is a salt selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride and magnesium aspartate, The concentration of the (B) salt in the transduction solution is 300 mM to 900 mM.

10. The transduction solution according to claim 9, wherein (A1) is a compound selected from the group consisting of 1-methyl-L-histidine and N-carbamoyl-L-aspartic acid.

11. The transduction solution according to claim 9, wherein The salt (B) is at least one selected from the group consisting of sodium chloride, magnesium chloride and potassium chloride.

12. A pharmaceutical composition comprising the transduction solution according to claim 9 and a molecule of interest comprising a Cas protein and / or a gRNA.

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