WO2017213092A1 - Peptide for transfection promotion - Google Patents

Peptide for transfection promotion Download PDF

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Publication number
WO2017213092A1
WO2017213092A1 PCT/JP2017/020837 JP2017020837W WO2017213092A1 WO 2017213092 A1 WO2017213092 A1 WO 2017213092A1 JP 2017020837 W JP2017020837 W JP 2017020837W WO 2017213092 A1 WO2017213092 A1 WO 2017213092A1
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transfection
cells
peptide
present
promoting
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PCT/JP2017/020837
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French (fr)
Japanese (ja)
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亮 請川
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和光純薬工業株式会社
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Publication of WO2017213092A1 publication Critical patent/WO2017213092A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a transfection promoting peptide, a transfection kit containing the peptide, and a method for transfecting a cell with a nucleic acid.
  • transfection in which nucleic acids such as plasmid DNA, siRNA, and mRNA are introduced into cells are widely used in the field of gene recombination as a general experimental technique.
  • a lipofection method, an electroporation method, a calcium phosphate method and the like are known, but the lipofection method having a high introduction efficiency without requiring a dedicated device is most widely used.
  • the lipofection method forms a lipid-nucleic acid complex by an electrical interaction between a cationically charged lipid (transfection agent) and the negative charge of the nucleic acid, and causes endocytosis such as cell phagocytosis and cell membrane fusion.
  • nucleic acid is introduced into cells by introducing the complex into cells using tosis.
  • the nucleic acid introduction efficiency may be lowered depending on the type of nucleic acid or cell. Therefore, various transfection promoting peptides (hereinafter sometimes abbreviated as enhancer peptides) have been developed for the purpose of improving nucleic acid introduction efficiency in the lipofection method (Patent Documents 1 and 2).
  • NLS nuclear translocation signal
  • Sp spermine
  • the present invention has been made in view of the situation as described above, and provides a highly versatile transfection promoting peptide that promotes transfection with a nucleic acid introduction efficiency equal to or higher than that of conventional transfection promoting peptides. Is an issue.
  • a peptide comprising a specific sequence not having NLS depends on the type of nucleic acid to be introduced.
  • the transfection-promoting peptide of the present invention with a transduction-promoting efficiency equal to or higher than that of a conventional transfection-promoting peptide having NLS, which has the function of transporting the introduced nucleic acid into the nucleus even though it has no NLS It was surprising that can promote the transfection of nucleic acids such as DNA into cells.
  • the conventional transfection-promoting peptide having a nuclear translocation signal may not promote (cannot promote) transfection depending on the type of cell into which the nucleic acid is introduced. It was found that transfection can be promoted regardless of the type of cells introduced.
  • the present inventors have discovered that the transfection agent has cytotoxicity and decreases the survival rate of the transfected cells.
  • transfection using the transfection-promoting peptide of the present invention can suppress the decrease in cell viability, that is, the transfection-promoting peptide of the present invention can suppress the cytotoxicity of the transfection agent.
  • the headline and the present invention were completed.
  • the present invention has the following configuration.
  • a peptide for promoting transfection of cells represented by the following general formula [1] or [2].
  • (X 1 ) n [2] (In the formula, each of n X 1 s independently represents arginine or lysine, X 2 represents glycine or alanine, X 3 represents tyrosine or alanine, and n represents an integer of 8 to 17)
  • ⁇ 2> The peptide according to ⁇ 1>, wherein the peptide is represented by the following general formula [1 ′], [1 ′′], [2 ′] or [2 ′′].
  • a kit for transfection of cells comprising a transfection agent comprising the peptide according to any one of ⁇ 1> to ⁇ 10> and a cationic lipid.
  • ⁇ 13> A method of transfecting a cell with a nucleic acid, comprising the step of contacting the cell and the nucleic acid in vitro in the presence of the peptide according to any one of ⁇ 1> to ⁇ 10> and a transfection agent (the present invention) Transfection method).
  • the transfection agent contains a cationic lipid.
  • the transfection agent comprises N- [2- (dimethylamino) ethyl] -4,5-bis (undecylthio) pentanamide.
  • the nucleic acid is RNA.
  • transfection can be promoted regardless of the type of nucleic acid to be introduced. Furthermore, according to the present invention, transfection can be promoted with a nucleic acid introduction efficiency equal to or higher than that when a conventional transfection-promoting peptide is used regardless of the type of cells to be introduced. That is, the present invention relates to a highly versatile transfection method and a transfection promoting peptide. Further, according to the transfection method of the present invention, transfection can be performed with high cell viability by suppressing the cytotoxicity of the transfection agent.
  • Example 2 is a fluorescence microscopic image of COS7 cells into which YFP-H2B gene was introduced, obtained in Example 1, Comparative Example 1-1, and Comparative Example 1-2.
  • the horizontal axis represents the type of transfection-promoting peptide.
  • SFA Plus + ESFA4 used ESFA4, a conventional transfection-promoting peptide.
  • SFA Plus + C2 represents the case where C2 which is the transfection promoting peptide of the present invention was used (Example 1).
  • YFP on the vertical axis represents the expression level of the introduced YFP-H2B gene
  • DIC differential interference microscope image
  • 2 is a fluorescence microscopic image of HuH-7 cells into which YFP-H2B gene was introduced, obtained in Example 2, Comparative Example 2-1 and Comparative Example 2-2.
  • the horizontal axis indicates the type of transfection-promoting peptide.
  • SFA Plus + C2 represents the case where C2 which is the transfection promoting peptide of the present invention was used (Example 2).
  • YFP on the vertical axis represents the expression level of the introduced YFP-H2B gene
  • DIC differential interference microscope image
  • 2 is a fluorescence microscopic image of HCT-116 # 2Luc cells into which YFP-H2B gene was introduced, obtained in Example 3, Comparative Example 3-1 and Comparative Example 3-2.
  • the horizontal axis represents the type of transfection-promoting peptide.
  • SFA Plus + ESFA4 used ESFA4, a conventional transfection-promoting peptide.
  • SFA Plus + C2 represents the case where C2 which is the transfection promoting peptide of the present invention was used (Example 3).
  • YFP on the vertical axis represents the expression level of the introduced YFP-H2B gene, and DIC (differential interference microscope image) represents the state of the cells. It is the fluorescence-microscope image of the Hela cell which introduce
  • the horizontal axis represents the type of transfection-promoting peptide
  • SFA Plus does not use the transfection-promoting peptide
  • SFA Plus + C2 uses the transfection-promoting peptide C2 of the present invention.
  • Example 17 is shown respectively.
  • GFP on the vertical axis represents the expression level of the introduced EGFP mRNA
  • DIC differential interference microscope image
  • the horizontal axis represents the type of transfection-promoting peptide
  • SFA Plus does not use the transfection-promoting peptide
  • SFA Plus + C2 uses the transfection-promoting peptide C2 of the present invention.
  • Example 17 6 is a graph showing the luminescence intensity (expression intensity) of firefly luciferase in CHO-K1 cells into which a firefly luciferase gene was introduced, obtained in Examples 18 to 24 and Comparative Examples 18 to 28.
  • the abscissa represents the type of transfection-promoting peptide, none transfected when no transfection was performed (control), None enhancer when no transfection-promoting peptide was used (Comparative Example 18), and C2
  • C2 which is the transfection-promoting peptide of the invention is used (Example 18)
  • C5 is the case where C5 which is the transfection-promoting peptide of the present invention is used (Example 19)
  • C2_AACAA is the transfection-promoting peptide of the present invention.
  • K11 is the transfection promoting peptide of the present invention
  • R9 is the transfection promoting peptide of the present invention R9.
  • ESFA4 is a peptide using R16 (Comparative Example 19), C4 is a peptide using C4 (Comparative Example 20), C2_C14A is a peptide using C2_C14A (Comparative Example 21), C2_A6 Is the peptide C2_A6 (Comparative Example 22), R3 is the peptide R3 (Comparative Example 23), R6 is the peptide R6 (Comparative Example 24), R19 is the peptide When R19 is used (Comparative Example 25), R22 is a peptide using R22 (Comparative Example 26), 30 If using the R30 is a peptide (Comparative Example 27), H11 represents a case of using the H11 is a peptide (Comparative Example 28).
  • the vertical axis represents the emission intensity.
  • 3 is a graph showing the survival rate (%) of CHO-K1 cells into which plasmid DNA was introduced, obtained in Examples 25 to 31 and Comparative Example 29.
  • the abscissa represents the type of transfection-promoting peptide, none transfected when no transfection was performed (control), None enhancer when no transfection-promoting peptide was used (Comparative Example 29), and C2
  • C2 which is the transfection facilitating peptide of the invention is used (Example 25)
  • C5 which is the transfection facilitating peptide of the present invention Example 26
  • C2_AACAA is the transfection facilitating peptide of the present invention
  • K11 is the transfection promoting peptide of the present invention
  • K11 Example 28
  • R9 is the transfection promoting peptide of the present invention R9.
  • R16 used the transfection promoting peptide of the present invention
  • R16 used the transfection promoting peptide of the present invention
  • RK12 used the transfection promoting peptide of the present invention
  • the vertical axis represents the emission intensity
  • transfection-promoting peptide of the present invention (hereinafter sometimes abbreviated as the enhancer peptide of the present invention) is a peptide represented by the following general formula [1] or [2].
  • the peptide for promoting transfection of the present invention is a polypeptide having a core sequence represented by X 2 -X 2 -Cys-X 2 -X 3 -X 2 and an additional sequence represented by (X 1 ) n . Additional sequence (X 1 ) n is added (bonded) to either the N-terminal side or the C-terminal side of the core sequence X 2 -X 2 -Cys-X 2 -X 3 -X 2 . That is, the transfection promoting peptide of the present invention is a polypeptide represented by the following general formula [1] or the following general formula [2].
  • X 2 in the core sequence shown in X 2 -X 2 -Cys-X 2 -X 3 -X 2 in the above general formula [1] and the formula [2] represents the glycine or alanine
  • in the core sequence four X 2 may be the same or different but are preferably identical.
  • X 3 is tyrosine or alanine. Therefore, the core sequence represented by X 2 -X 2 -Cys-X 2 -X 3 -X 2 in the general formula [1] and the general formula [2] can be represented as follows.
  • X 2 -X 2 -Cys-X 2 -X 3 -X 2 include, for example, the sequences 1 to 4 shown in the following table. Among them, the sequences 1 and 4 are preferable, and the sequence 1 is particularly preferable.
  • the additional sequence represented by (X 1 ) n is (Arg) n , (Lys) n , (Arg-Lys) m , (Lys-Arg) m , [(Arg) p (Lys) q ], [(Lys) q (Arg) p ] and the like (wherein n represents an integer of 8 to 17, m represents an integer of 4 to 8, and p represents 1 to 16). An integer, q is an integer from 1 to 16, and p + q represents an integer from 8 to 17.
  • (Arg) n , (Lys) n , (Arg-Lys) m and (Lys-Arg) m are preferred, (Arg) n , (Lys) n and (Arg-Lys) m are more preferred, (Arg) n and (Lys) n are particularly preferred.
  • n is preferably an integer of 9 to 16.
  • m is particularly preferably 6.
  • p + q is preferably an integer of 9 to 16, more preferably p is an integer of 8 to 10, and q is an integer of 1 to 6.
  • Preferred specific examples of the peptide for promoting transfection of the present invention represented by the above general formula [1] or the above general formula [2] include, for example, the following general formula [1 ′], the following general formula [1 ′′], the following Examples include polypeptides represented by the general formula [2 ′] and the following general formula [2 ′′], more preferably those represented by the following general formula [1 ′] and the following general formula [2 ′]. Those represented by [1 ′] are particularly preferred.
  • the following general formula [1 ′] includes, for example, the following polypeptides [1′-a] to [1′-l], [1′-a] to [1′-h], [1 ′ -J] and [1'-l] are preferred, [1'-c] to [1'-h] and [1'-l] are more preferred, and [1'-e] to [1'-h] And [1′-l] are particularly preferred.
  • Examples of the general formula [2 ′] include the following [2′-a] to [2′-h] polypeptides, [2′-a] to [2′-e], [2 ′ -G] and [2′-h] are preferable, [2′-c] to [2′-e] are more preferable, and [2′-e] is particularly preferable.
  • Examples of the general formula [1 ′′] include the following polypeptides [1 ′′ -a] to [1 ′′ -f], including [1 ′′ -a] and [1 ′′ -c]. ] And [1 ′′ -d] are preferred, [1 ′′ -c] and [1 ′′ -d] are more preferred, and [1 ′′ -d] is particularly preferred.
  • Examples of the general formula [2 ′′] include the following polypeptides [2 ′′ -a] to [2 ′′ -e], [2 ′′ -a] and [2 ′′ -c]. ] Is preferable, and [2 ′′ -c] is more preferable.
  • transfection-promoting peptide of the present invention among the above specific examples, for example, general formula [1 ′], general formula [1 ′′], general formula [2 ′] and general formula [2 ′′] are preferable,
  • the general formula [1 ′] and the general formula [2 ′] are more preferable, and [1′-a] to [1′-h], [1′-j], [1′-l], [2′-a] ] To [2'-e], [2'-g] and [2'-h] are more preferred, and [1'-c] to [1'-h], [1'-l] and [2 ' -C] to [2'-e] are even more preferred, [1'-e] to [1'-h], [1'-l] and [2'-e] are particularly preferred, and [1'- f] is most preferred.
  • the transfection-promoting peptide of the present invention may be synthesized according to a peptide synthesis method known per se. Examples of such a synthesis method include the method described in Japanese Patent No. 3569966.
  • the transfection method of the present invention is a transfection agent used in a known lipofection method, except that the transfection promoting peptide of the present invention coexists when a nucleic acid is transfected into cells by the lipofection method.
  • the various reagents such as those described above may be used according to a known operation.
  • a cell and a nucleic acid may be contacted in vitro in the presence of the transfection promoting peptide of the present invention and the transfection agent of the present invention.
  • the transfection agent according to the present invention may be any transfection agent usually used in this field, and includes those containing cationic lipids, and those containing cationic lipids and neutral lipids are preferred.
  • the cationic lipid may be a monovalent cationic lipid or a polyvalent cationic lipid.
  • DOTMA N- [1- (2,3-dioleoyloxy) -propyl] -N , N, N-trimethylammonium chloride
  • DOTAP 1,2-bis (oleoyloxy) -3- (trimethylammonium) propane
  • DMRIE 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy
  • Monovalent cationic lipids such as ethylammonium bromide), DDAB (dimethyldioctadecylammonium bromide), lipospermine, DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl -1-Propanaminium triflu (Roacetic acid), DOSPER (1,3-dioleoyloxy-2- (6 carboxyspermyl) -propylamide), di- and tetra-alky
  • the neutral lipid examples include DOPE (1,2-dioleoyl-Sn-glycero-3-phosphoethanolamine, dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine), cholesterol and the like. DOPE is preferred.
  • Examples of those containing cationic lipids and neutral lipids include Lipofectamine (trademark) 2000 (manufactured by Thermo Fisher Scientific), Lipofectamine (trademark) 3000 (manufactured by Thermo Fisher Scientific), ScreenFect TM A plus ( Wako Pure Chemical Industries, Ltd.), ScreenFect TM A (Wako Pure Chemical Industries, Ltd.), ScreenFect TM mRNA (Wako Pure Chemical Industries, Ltd.), etc., and ScreenFect TM A plus (Wako Pure Chemicals).
  • Lipofectamine trademark 2000 (manufactured by Thermo Fisher Scientific) 2000 (manufactured by Thermo Fisher Scientific), Lipofectamine (trademark) 3000 (manufactured by Thermo Fisher Scientific), ScreenFect TM A plus ( Wako Pure Chemical Industries, Ltd.), ScreenFect TM A (Wako Pure Chemical Industries, Ltd.), ScreenFect TM
  • the transfection agent according to the present invention includes N- [2- (dimethylamino) ethyl] -4,5-bis (undecylthio) pentanamide and DOPE (1,2-dioleoyl-Sn-glycero-3-phosphoethanolamine, among others.
  • ScreenFect series (ScreenFect TM A plus (manufactured by Wako Pure Chemical Industries, Ltd.), ScreenFect TM A (manufactured by Wako Pure Chemical Industries, Ltd.), ScreenFect TM mRNA (Wako Pure Chemical Industries, Ltd.) Etc.) are more preferable.
  • the cells according to the present invention usually include eukaryotic cells, preferably higher eukaryotic cells, and are HeLa cells, 293T cells, HEK293 cells, CHO-K1 cells, A549 cells, COS7 cells, MCF7 cells, NIH3T3.
  • hematopoietic cells such as 7, glial cells such as microglia (Hortega cells), insect cells such as Sf9 cells, Sf21 cells, SF + cells, High-Five cells, BmN4 cells, and particularly preferred are adherent cells and suspension cells.
  • the cell according to the present invention may be a primary (primary culture) cell or a passaged cell.
  • nucleic acid according to the present invention examples include genomic DNA, single-stranded DNA, double-stranded DNA, DNA such as plasmid DNA, RNA such as mRNA, siRNA, isRNA (immunostimulatory RNA), tRNA, shRNA, and mRNA. Is preferred.
  • the nucleic acid according to the present invention may be derived from any organism or artificially designed.
  • the number of bases of the nucleic acid according to the present invention is usually 10 bases to 30000 bases, preferably 50 bases to 20000 bases, more preferably 100 bases to 10,000 bases.
  • the base constituting the nucleic acid according to the present invention may be either a natural base, an artificial base or a natural base, and is preferably a natural base.
  • nucleic acids according to the present invention include nucleic acids that can encode and express therapeutic proteins and useful proteins in cells, nucleic acids that inhibit undesired expression of nucleic acids in cells, and those that inhibit undesired protein activity.
  • nucleic acids that activate the desired protein function nucleic acids that catalyze reactions (ribozymes), and nucleic acids that function in diagnostic assays (eg, diagnostic nucleic acids) are included.
  • the amount of the nucleic acid according to the present invention is not particularly limited.
  • the amount of the nucleic acid used when contacting the cell and the nucleic acid in the presence of the transfection promoting peptide and the transfection agent of the present invention in other words, the present invention.
  • the amount used in the solution containing the transfection-promoting peptide, the transfection agent according to the present invention, cells and nucleic acids is usually 0.0001 ⁇ g to 10,000 mg, preferably 0.001 ⁇ g to 5000 mg.
  • Per well area of 1 cm 2 is usually 0.005 ⁇ g to 500 ⁇ g, preferably 0.0075 ⁇ g to 250 ⁇ g, more preferably 0.01 ⁇ g to 75 ⁇ g, particularly preferably 0.1 ⁇ g to 10 ⁇ g.
  • 0.005 ⁇ g to 50 per mL [mu] g preferably 0.0075 ⁇ g ⁇ 250 ⁇ g, more preferably 0.01 [mu] g ⁇ 75 [mu] g, particularly preferably 0.1 [mu] g ⁇ 10 [mu] g.
  • the amount of the transfection promoting peptide of the present invention is not particularly limited.
  • the amount used in the solution containing the transfection promoting peptide of the present invention, the transfection agent according to the present invention, cells and nucleic acids is The amount is usually 4 ⁇ g to 20 ⁇ g, preferably 5 ⁇ g to 15 ⁇ g, more preferably 8 ⁇ g to 12 ⁇ g, particularly preferably 9 ⁇ g to 11 ⁇ g per 1 ⁇ g of nucleic acid according to the invention.
  • the use amount of the transfection agent of the present invention is not particularly limited.
  • the use amount in a solution containing the transfection-promoting peptide of the present invention, the transfection agent according to the present invention, cells and nucleic acids is usually 0. .04 ⁇ g ⁇ 240000 ⁇ g, preferably 0.06 ⁇ g ⁇ 120000 ⁇ g, for example when using the plate, the area 1 cm 2 per well, usually 0.05 [mu] g ⁇ 10 [mu] g, preferably 0.1 [mu] g ⁇ 7.5 [mu] g, more preferably 0.
  • a flask when used, it is usually 0.05 ⁇ g to 24000 ⁇ g, preferably 0.1 ⁇ g to 12000 ⁇ g, more preferably 0.25 ⁇ g to 2400 ⁇ g per 1 mL of the medium.
  • the transfection method of the present invention is not particularly limited as long as cells and nucleic acids are brought into contact in vitro in the presence of the transfection promoting peptide of the present invention and the transfection agent of the present invention.
  • a method for example, (Step 1) a nucleic acid, a peptide for promoting transfection of the present invention, and a transfection agent according to the present invention are mixed in a solution to prepare a transfection solution (Step 2).
  • Examples include a method of mixing the transfection solution with a medium containing cells and bringing the cells into contact with a nucleic acid to effect transfection.
  • Step 1 includes, for example, (Step 1-a) a method of mixing a solution containing the nucleic acid, the transfection-promoting peptide of the present invention, and a solution containing the transfection agent according to the present invention (Step 1-b). ) A method of mixing a solution containing the nucleic acid and the transfection agent according to the present invention with a solution containing the transfection promoting peptide of the present invention, (step 1-c) a solution containing the nucleic acid, A method of mixing a transfection promoting peptide and a solution containing the transfection agent according to the present invention, (step 1-d) a solution containing a nucleic acid, a solution containing the transfection promoting peptide of the present invention, Examples include a method of mixing a solution containing the transfection agent according to the present invention.
  • Solution containing the nucleic acid of step 1, the transfection promoting peptide of the present invention, and / or the transfection agent according to the present invention (solution containing the nucleic acid and the transfection promoting peptide of the present invention, the transfection according to the present invention)
  • the solution to be contained and the solution (solvent) containing the nucleic acid include purified water and a buffer having a buffering action at pH 5 to 7 (for example, TE buffer, phosphate buffer, Tris buffer, Good buffer, Glycine buffer, borate buffer, sodium bicarbonate buffer Sodium acetate buffer, etc.) can be mentioned, preferably a buffer solution having a buffering action in the pH 5 ⁇ 7, TE buffer
  • the concentration of the buffering agent in these buffers is appropriately selected from the range of usually 1 mM to 500 mM, preferably 5 to 300 mM.
  • the amount of the nucleic acid, the transfection-promoting peptide of the present invention, the transfection agent according to the present invention, etc. in an amount that does not adversely affect the aggregation, for example, sugars, salts such as NaCl, surface activity, etc. Agents, preservatives, proteins and the like may be included.
  • the nucleic acid, the transfection promoting peptide of the present invention and the transfection agent of the present invention are mixed in a solution, and then usually 15 ° C. to 30 ° C., preferably 18 ° C. to 28 ° C., more preferably 20 ° C.
  • the reaction is usually carried out at ⁇ 26 ° C. for 1 minute to 60 minutes, preferably 3 minutes to 40 minutes, more preferably 5 minutes to 20 minutes.
  • the solution containing the nucleic acid of Step 1 the transfection promoting peptide of the present invention and / or the transfection agent according to the present invention (the solution containing the nucleic acid and the transfection promoting peptide of the present invention, the transfection promoting peptide of the present invention).
  • a solution containing a transfection agent, a solution containing a nucleic acid and a transfection agent according to the present invention, a solution containing a transfection promoting peptide of the present invention, a transfection promoting peptide of the present invention and a transfection agent according to the present invention The amount of the nucleic acid to be contained in the solution containing the nucleic acid, the solution (solvent) containing the nucleic acid, the amount of the transfection-promoting peptide of the present invention, and the amount of the transfection agent of the present invention Accelerating peptides and tolans
  • the amount used when the cell and the nucleic acid are contacted in the presence of the transfection agent in other words, the amount used in the solution containing the peptide for promoting transfection of the present invention, the transfection agent, the cell and the nucleic acid is within the above-mentioned range. It may be appropriately selected so as to be contained in a solution containing the nucleic acid, the peptide for
  • step 1 may be performed, for example, by the following method (step 1-a above).
  • a buffer solution having a buffering action at pH 5 to 7 for example, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer solution, Sodium acetate buffer, etc.
  • a buffer solution having a buffering action at pH 5 to 7 for example, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer solution, Sodium acetate buffer, etc.
  • a buffer solution having a buffering action at pH 5 to 7 (eg, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer) Solution, sodium acetate buffer, etc.) 40 ⁇ L, and usually 0.1 ⁇ g-20 ⁇ g, preferably 0.2 ⁇ g-15 ⁇ g, more preferably 0.5 ⁇ g-6 ⁇ g of the transfection agent according to the present invention is added.
  • the solutions in the above two sterilized tubes are mixed, and if necessary, usually 15 to 30 ° C, preferably 18 to 28 ° C, more preferably 20 to 26 ° C, usually 1 to 60 minutes.
  • the reaction is preferably carried out for 3 to 40 minutes, more preferably 5 to 20 minutes to obtain 80 ⁇ L of the transfection solution.
  • a buffer solution having a buffering action at pH 5 to 7 for example, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer solution, Sodium acetate buffer, etc.
  • a buffer solution having a buffering action at pH 5 to 7 for example, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer solution, Sodium acetate buffer, etc.
  • 100 ⁇ L usually 0.005 ⁇ g to 500 ⁇ g, preferably 0.0075 ⁇ g to 250 ⁇ g, more preferably 0.01 ⁇ g to 75 ⁇ g, particularly preferably 0.1 ⁇ g to 10 ⁇ g, and usually 0.02 ⁇ g to 10000 ⁇ g, preferably 0.0375 ⁇ g to 3750 ⁇ g, more preferably 0.08 ⁇ g to 900 ⁇ g of the transfection promoting peptide of the present invention is added.
  • a buffer solution having a buffering action at pH 5 to 7 eg, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer) Solution, sodium acetate buffer, etc.
  • TE buffer solution phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer
  • sodium acetate buffer etc.
  • the solutions in the above two sterilized tubes are mixed, and usually 15 ° C to 30 ° C, preferably 18 ° C to 28 ° C, more preferably 20 ° C to 26 ° C, usually 1 minute to 60 minutes, preferably The reaction is carried out for 3 to 40 minutes, more preferably 5 to 20 minutes to obtain 200 ⁇ L of the transfection solution.
  • Step 2 is performed by mixing the transfection solution obtained in Step 1 with a medium containing cells and bringing the cells into contact with nucleic acids to transfect the cells.
  • the number of cells according to the present invention is not particularly limited as long as it is the number of cells usually used for transfection in this field.
  • the transfection solution obtained in step 1 The number of cells when mixed with a medium containing cells, in other words, the number of cells when transfection is carried out by bringing cells into contact with nucleic acids is usually from 1 ⁇ 10 2 cells to 1 ⁇ 10 8 cells, preferably It is 1 ⁇ 10 3 cells to 2 ⁇ 10 8 cells, and is appropriately selected according to a container containing a medium such as a plate or a flask.
  • the number of cells according to the present invention when performing step 2 using a plate, is usually 0.1 ⁇ 10 5 cells to 5 ⁇ 10 5 cells, preferably 0.2 ⁇ 10 5 cells per 1 cm 2 of the well area. ⁇ 3 ⁇ 10 5 cells, more preferably 0.3 ⁇ 10 5 cells to 2 ⁇ 10 5 cells.
  • the number of cells according to the present invention when performing step 2 using a flask, is usually 0.1 ⁇ 10 5 cells to usually 30 ⁇ 10 5 cells, preferably 0.5 ⁇ 10 5 cells to 15 per 1 mL of the medium. ⁇ 10 5 cells, more preferably 1 ⁇ 10 5 cells to 10 ⁇ 10 5 cells.
  • the medium in step 2 is not particularly limited as long as it is usually used in this field, and an appropriate medium may be selected for each cell. As long as the medium in step 2 does not inhibit transfection, for example, serum such as FBS, albumin, transferrin, Knockout Serum Replacement (KSR), N2 supplement (invitrogen), B27 supplement (invitrogen), fatty acid, insulin, Serum substitutes such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, non-essential amino acids, vitamins, growth factors, cytokines, antibiotics, antioxidants, pyruvate, buffers In addition, inorganic salts and the like may be included, and the selection of these depends on the transfection agent. Further, the amount of the medium in Step 2 may be any medium amount that is usually used when transfection is performed in this field, and is appropriately selected depending on the container containing the medium such as a plate or a flask.
  • KSR Knockout Serum Replacement
  • the amount of the medium per 1 cm 2 of the well area is usually 100 ⁇ L to 1000 ⁇ L, preferably 150 ⁇ L to 800 ⁇ L, more preferably 200 ⁇ L to 600 ⁇ L.
  • the amount of the medium is usually 1 mL to 1000 L, preferably 20 mL to 500 L, more preferably 30 mL to 100 L.
  • the amount of the transfection solution obtained in step 1 in step 2 may be the amount of the transfection solution usually used for transfection in this field, and is usually 0.001 mL to 1000 mL, preferably 0. It is 0.01 mL to 500 mL, more preferably 0.05 mL to 100 mL, and is appropriately selected according to the container containing the medium such as a plate or a flask.
  • the amount of the transfection solution is usually 1 ⁇ L to 200 ⁇ L, preferably 5 ⁇ L to 180 ⁇ L, more preferably 10 ⁇ L to 150 ⁇ L per 1 cm 2 of the well area.
  • the amount of the transfection solution is usually 10 ⁇ L to 1000 ⁇ L, preferably 50 ⁇ L to 600 ⁇ L, more preferably 100 ⁇ L to 300 ⁇ L per 1 mL of the medium.
  • Transfection in step 2 is performed by introducing nucleic acid into the cell. Specifically, after the transfection solution obtained in step 1 is added to the cells in the medium, it is usually 25 ° C. to 39 ° C., preferably 35 ° C. to 38 ° C., usually 1 hour to 2 days, preferably 6 hours to One day culture method is mentioned.
  • step 2 may be performed by the following method, for example.
  • step 2 is performed using 1 well (2 cm 2 ) of 24 plates
  • step 2 When performing step 2 using a flask containing 1 mL of medium] Usually 0.1 ⁇ 10 5 cells to usually 30 ⁇ 10 5 cells, preferably 0.5 ⁇ 10 5 cells to 15 ⁇ 10 5 cells, more preferably 1 ⁇ 10 5 cells to 10 ⁇ 10 5 cells.
  • the transfection kit of the present invention is used for the transfection method of the present invention as described above, and comprises the transfection promoting peptide of the present invention and the transfection agent of the present invention, Preferred embodiments and specific examples of each component are as described above.
  • the transfection-promoting peptide of the present invention contained in the transfection kit of the present invention is a buffer solution having a buffering action in water or pH 5-7 (for example, TE buffer solution, phosphate, etc.) even in a lyophilized solid state.
  • Buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, sodium bicarbonate buffer, sodium acetate buffer, etc. In the liquid state, the concentration is usually 0.5 mg / mL to 30 mg / mL, preferably 2 mg / mL to 10 mg / mL.
  • kits include reagents usually used in this field, such as buffers, sensitizers, surfactants, preservatives (eg, sodium azide, salicylic acid, benzoic acid, etc.), stabilizers (eg, albumin, Globulins, water-soluble gelatins, surfactants, saccharides, etc.), activators, coexisting substance avoidance agents, and others used in this field that do not interfere with the stability of coexisting reagents You may have.
  • the concentration range or the like of these reagents may be appropriately selected from the concentration range or the like that is usually used for exhibiting the effects of each reagent.
  • kit of the present invention may contain instructions for the transfection method of the present invention.
  • the “instructions” are the instruction manuals, package inserts, pamphlets (leaflets), etc. of the kits in which the features, principles, operation procedures, judgment procedures, etc. of the method are substantially described in text or diagrams. Means.
  • Example 1 Verification of transfection promoting effect of enhancer peptide C2
  • COS7 cells were transfected with plasmid DNA having YFP-H2B gene under the experimental conditions described in Table 1 below, and transfection promotion of enhancer peptide C2 was promoted. The effect was confirmed.
  • COS7 cells were cultured using D-MEM 10% FBS medium (manufactured by Wako Pure Chemical Industries, Ltd.) until 1 ⁇ 10 5 cells or more were obtained to obtain cultured cells. .
  • ScreenFect A Plus reagent (hereinafter referred to as ScreenFect) attached to ScreenFect TM A Plus (manufactured by Wako Pure Chemical Industries, Ltd.). 1.5 ⁇ L (sometimes abbreviated as A Plus reagent) (ScreenFect A Plus reagent 3 ⁇ L per 1 ⁇ g of plasmid DNA) was added.
  • a cell suspension was prepared as follows. Remove the medium from the culture vessel, add trypsin EDTA solution [0.25 w / v% trypsin-1 mmol / l EDTA ⁇ 4Na solution (containing phenol red)] to the culture vessel to detach the cells from the culture vessel, Got. After adding the suspension to the centrifuge tube, centrifugation was performed (1000 rpm, 5 minutes) to sediment the cells. After removing the supernatant, an appropriate amount of medium was added to the cell pellet to obtain a cell suspension. The number of adherent cells in the cell suspension was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad), and the concentration of the cells in the cell suspension was calculated.
  • Example 2-16 Verification of transfection promoting effect of enhancer peptide C2 in various cells The same method as in Example 1 under the conditions shown in Table 2 below (cell, plasmid DNA amount, number of seeded cells, medium, enhancer peptide, enhancer peptide amount) Then, the plasmid DNA having the YFP-H2B gene was transfected into the cells, and the transfection promoting effect of the enhancer peptide C2 was confirmed by fluorescence microscope observation and fluorescence intensity comparison using a flow cytometer. In Example 7 using floating cells K562, “(2) Gene transfer (ii) Preparation of cell suspension” was performed as follows instead of the method of Example 1.
  • the cell culture solution obtained in (1) is transferred to a centrifuge tube, centrifuged (1000 rpm, 5 minutes), an appropriate amount of medium is added to the cell pellet from which the supernatant has been removed, and suspended.
  • the number of floating cells contained therein was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad).
  • the sorting sample in “(4) Comparison of fluorescence intensity using a flow cytometer” was obtained as follows instead of the method of Example 1. Specifically, (2) gene transfer (iii) a culture solution containing cells cultured overnight by transfection was added to a strainer tube to obtain a sorting sample.
  • Example 2 The results of microscopic observation of Example 2 are shown in SFA Plus + C2 of FIG. 2, and the results of microscopic observation of Example 3 are shown in SFA Plus + C2 of FIG.
  • the results of fluorescence intensity measurement using a flow cytometer are shown in Tables 5 and 6 below in comparison with Comparative Examples 2-2 to 16-2.
  • YFP represents the result of measuring the expression level of the introduced plasmid DNA by the fluorescence of YFP-H2B
  • DIC differential interference microscope image
  • Comparative Example 1-1 to Comparative Example 16-1 Verification of transfection promoting effect of conventional enhancer peptide ESFA4 in various cells As in Example 1 under the conditions described in Table 3 below (cells, plasmid DNA amount, seeded cell number, medium, enhancer peptide, enhancer peptide amount)
  • the plasmid DNA having the YFP-H2B gene was transfected into the cells by the method described above, and the transfection promoting effect of ESFA4, a conventional enhancer peptide, was confirmed by fluorescence fluorescence observation and comparison of fluorescence intensity using a flow cytometer.
  • Comparative Example 7-1 using the floating cell K562 “(2) Gene transfer (ii) Preparation of cell suspension” was performed as follows instead of the method of Example 1.
  • the cell culture solution obtained in (1) is transferred to a centrifuge tube, centrifuged (1000 rpm, 5 minutes), an appropriate amount of medium is added to the cell pellet from which the supernatant has been removed, and suspended.
  • the number of floating cells contained therein was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad).
  • the sorting sample in “(4) Fluorescence intensity comparison using a flow cytometer” was obtained as follows instead of the method of Example 1. . Specifically, (2) gene transfer (iii) a culture solution containing cells cultured overnight by transfection was added to a strainer tube to obtain a sorting sample.
  • Comparative Example 1-2 to Comparative Example 16-2 Transfection of various cells Under the conditions described in Table 4 below (cells, amount of plasmid DNA, number of seeded cells, medium, enhancer peptide), plasmid DNA having the YFP-H2B gene was transfected into the cells in the same manner as in Example 1. The fluorescence intensity was observed using a fluorescence microscope and a flow cytometer. In Comparative Example 7-2 using floating cells K562, “(2) Gene transfer (ii) Preparation of cell suspension” was performed as follows instead of the method of Example 1.
  • the cell culture solution obtained in (1) is transferred to a centrifuge tube, centrifuged (1000 rpm, 5 minutes), an appropriate amount of medium is added to the cell pellet from which the supernatant has been removed, and suspended.
  • the number of floating cells contained therein was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad).
  • the sorting sample in “(4) Comparison of fluorescence intensity using a flow cytometer” was obtained as follows instead of the method of Example 1. . Specifically, (2) gene transfer (iii) a culture solution containing cells cultured overnight by transfection was added to a strainer tube to obtain a sorting sample.
  • Ratio of “fluorescence intensity when enhancer peptide is used” to “fluorescence intensity when enhancer peptide is not used” is 2 times or more
  • Ratio of “fluorescence intensity when using enhancer” is 1.5 times or more
  • Ratio of “fluorescence intensity when using enhancer peptide” to “fluorescence intensity when no enhancer peptide is used” is 1.1 times or more
  • Ratio of “fluorescence intensity with enhancer peptide” to “fluorescence intensity with no enhancer peptide” is less than 1.1 times
  • Example 1-16 Comparative Example 1-1 to Comparative Example 16-1
  • Comparative Example 1-2 Comparative Example 16-2
  • ESFA4 was used for transfection of COS7 cells, HCT-116 # 2Luc cells, K562 cells, and L cells (Comparative Example 1-1, Comparative Example 3-1, Comparative Example 7-1, Comparative Example). 13-1) and the case where no enhancer peptide was used (Comparative Example 1-2, Comparative Example 3-2, Comparative Example 7-2, Comparative Example 13-2), there was no significant difference in fluorescence intensity. No transfection promoting effect was shown. On the other hand, when C2 was used (Example 1, Example 3, Example 7, and Example 13), strong fluorescence intensity was measured as compared with the case where no enhancer peptide was used. The effect was confirmed.
  • C2 also shows a transfection-promoting effect even for cells in which ESFA4 does not show a transfection-promoting effect
  • C2 is a versatile enhancer peptide regardless of the type of cells to be transfected. It turns out that there is.
  • a peptide having a nuclear translocation signal exhibits a transfection promoting effect by being introduced into the cytoplasm and carrying a plasmid gene into the nucleus.
  • C2 without a nuclear translocation signal was predicted to be inferior in transfection promoting effect to ESFA4 having a nuclear translocation signal, but surprisingly, C2 is equivalent to or better than ESFA4. It was found to show an effect.
  • Example 17 Verification of transfection promoting effect of enhancer peptide C2 in mRNA transfection
  • the same as in Example 1 under the conditions described in Table 7 below (cells, EGFP mRNA amount, number of seeded cells, medium, enhancer peptide, enhancer peptide amount)
  • mRNA was transfected instead of plasmid DNA, and the transfection promoting effect of enhancer peptide C2 was confirmed by fluorescence microscopy and comparison of EGFP fluorescence intensity using a flow cytometer.
  • EGFP mRNA # L-6301, manufactured by Trilink Biotechnologies
  • Example 17 The addition amount of the transfection agent (ScreenFect A Plus reagent) in Example 17 and Comparative Example 17 was 0.8 ⁇ L (ScreenFect A Plus reagent 4 ⁇ L per 1 ⁇ g of EGFP mRNA).
  • Example 18 Verification of the effect on the transfection promoting effect due to the difference in amino acid sequence of various peptides
  • CHO-K1 cells were transfected with plasmid DNA having a firefly luciferase gene under the experimental conditions described in Table 8 below. The influence on the transfection promotion effect by the difference in the amino acid sequence of various peptides was confirmed.
  • a cell suspension was prepared as follows. After removing the medium from the culture vessel, trypsin EDTA solution [0.25 w / v% trypsin-1 mmol / l EDTA ⁇ 4Na solution (containing phenol red)] is added to the culture vessel to detach the cells from the culture vessel, and A turbid liquid was obtained. After adding the suspension to the centrifuge tube, centrifugation was performed (1000 rpm, 5 minutes) to sediment the cells. After removing the supernatant, an appropriate amount of medium was added to the cell pellet to obtain a cell suspension. The number of cells in the cell suspension was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad), and the concentration of cells in the cell suspension was calculated.
  • Luciferase assay For the cells cultured overnight in (2) above, the expression level of the introduced plasmid was measured by the light emission intensity (expression intensity) of the firefly luciferase gene by the following method. First, the 96-well plate was removed from the CO 2 incubator and returned to room temperature. Next, 100 ⁇ L of ONE-Glo TM EX Luciferase Assay System (progema) was added to the well, and the mixture was allowed to react at room temperature for 5 minutes while stirring with a plate mixer. Thereafter, the emission intensity was measured using a plate reader (TECAN).
  • TECAN plate reader
  • Results The results obtained by the luciferase assay are shown in FIG. In FIG. 6, the horizontal axis represents the types of various peptides, and the vertical axis represents the luminescence intensity.
  • Examples 19-24 Verification of the effect on the transfection promoting effect due to the difference in amino acid sequence of various peptides Examples under the conditions shown in Table 9 below (cell, plasmid DNA amount, number of seeded cells, medium, enhancer peptide, enhancer peptide amount)
  • the plasmid DNA having the firefly luciferase gene was transfected by the same method as in No. 18, and the effect on the transfection promoting effect due to the difference in the amino acid sequence of various peptides was confirmed by luciferase assay.
  • Each peptide was synthesized by Toray Research Co., Ltd.
  • the horizontal axis represents the type of peptide
  • the vertical axis represents the luminescence intensity
  • Comparative Example 18-28 Verification of the effect on the transfection promoting effect due to differences in amino acid sequences of various peptides Examples under the conditions described in Table 10 below (cells, plasmid DNA amount, number of seeded cells, medium, enhancer peptide, enhancer peptide amount)
  • the plasmid DNA having the firefly luciferase gene was transfected by the same method as in No. 18, and the effect on the transfection promoting effect due to the difference in the amino acid sequence of various peptides was confirmed by luciferase assay.
  • Each peptide was synthesized by Toray Research Co., Ltd.
  • C2 (Example 18) in which the additional sequence is composed of polyarginine, K11 (Example 21) in which the additional sequence is composed of polylysine, and RK12 (Example 24) in which 12 arginine and lysine are alternately arranged are transfection promoting effects. showed that.
  • histidine H11 (Comparative Example 28) to which 11 histidines, which are cationic amino acids, were bound in the same manner as arginine and lysine did not show a transfection promoting effect. From these results, it was found that the additional sequence must be an amino acid sequence consisting of arginine and / or lysine among the cationic amino acids.
  • R9 consisting of 9 arginines as the additional sequence
  • K11 consisting of 11 lysines as the additional sequence
  • R16 consisting of 16 arginines as the additional sequence
  • an additional sequence such as R6 (Comparative Example 24) composed of 6 arginines, a peptide composed of 6 or less arginine or lysine
  • an R19 composed of 19 arginines.
  • Peptides consisting of 19 or more arginine or lysine showed no transfection promoting effect. From these results, it was found that the number of amino acids in the additional sequence consisting of arginine and / or lysine is 9 or more and 16 or less, and exhibits a transfection promoting effect.
  • C4 consisting only of polyarginine (Comparative Example 20) did not show a transfection promoting effect, and the core sequence was found to be an important sequence for the peptide transfection promoting effect.
  • C2_C14A Comparative Example 21
  • C2_A6 Comparative Example 22
  • the third Cys residue of the core sequence is substituted with Ala do not show transfection promoting effect
  • other than the third Cys residue of the core sequence C2_AACAAAA Example 20 in which is replaced with Ala showed a transfection promoting effect. From these results, it was revealed that the third Cys residue in the core sequence contributes particularly to the transfection promoting effect.
  • Example 25 Verification of cytotoxic effects of various enhancer peptide transfection agents
  • CHO-K1 cells were transfected under the experimental conditions described in Table 11 below, and transfection was performed by differences in the amino acid sequences of the various peptides. The effect on the promotion effect was confirmed.
  • the gene introduction operation (2) below was performed without using plasmid DNA.
  • CHO-K1 cells are cultured using Ham'sF-12 10% FBS medium (manufactured by Wako Pure Chemical Industries, Ltd.) until 1.5 ⁇ 10 5 or more cells are obtained. And cultured cells were obtained.
  • the number of cells in the cell suspension was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad), and the concentration of adherent cells in the cell suspension was calculated. The calculated number of cells was compared with the number of cells that had not been transfected, and the ratio of the number of living cells was calculated.
  • Examples 26-31 Comparative Example 29. Verification of cytotoxic effect of various enhancer peptides by transfection agents Under the conditions described in Table 12 below (cells, number of seeded cells, medium, enhancer peptide, amount of enhancer peptide), in the same manner as in Example 25, The inhibitory effect of the enhancer peptide on the cytotoxicity of the transfection agent was confirmed. The enhancer peptide that was found to have the effect of promoting transfection as a result of Examples 18-24 was used. All of these enhancer peptides were synthesized by Toray Research Co., Ltd.
  • the transfection-promoting peptide of the present invention, the transfection kit of the present invention, and the transfection method of the present invention can introduce a nucleic acid equivalent to or higher than a conventional transfection-promoting peptide, regardless of the type of cell or nucleic acid. Since transfection can be promoted with efficiency, it is useful in the field of transfection such as functional analysis of transgenes and protein expression production. In addition, according to the transfection method of the present invention, since transfection can be performed with high cell viability by suppressing the cytotoxicity of the transfection agent, transfection such as transgene function analysis and protein expression production can be performed. Useful in the field to do.

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Abstract

The present invention addresses the problem of providing a peptide for transfection promotion for promoting transfection with nucleic acid introduction efficiency equivalent to or greater than that of conventional peptides for transfection promotion and having high versatility. The present invention pertains to: (a) a peptide for transfection promotion in cells; (b) a kit for transfection of cells including a transfection agent that includes the peptide described in (a) and a cationic lipid; and (c) a method for transfecting nucleic acid into cells including a step for contacting cells and a nucleic acid in vitro in the presence of the peptide described in (a) and the transfection agent.

Description

トランスフェクション促進用ペプチドPeptide for promoting transfection
　本発明は、トランスフェクション促進用ペプチド、当該ペプチドを含むトランスフェクション用キット、及び細胞に核酸をトランスフェクションする方法に関する。 The present invention relates to a transfection promoting peptide, a transfection kit containing the peptide, and a method for transfecting a cell with a nucleic acid.
　細胞にプラスミドＤＮＡ、ｓｉＲＮＡ、ｍＲＮＡ等の核酸を導入する遺伝子導入実験（トランスフェクション）は、汎用的な実験手法として遺伝子組換えの分野において広く用いられている。具体的な方法としては、リポフェクション法、エレクトロポレーション法、リン酸カルシウム法等が知られているが、専用機器を必要とせずに高い導入効率をもつリポフェクション法が最も広く用いられている。 Gene transfer experiments (transfection) in which nucleic acids such as plasmid DNA, siRNA, and mRNA are introduced into cells are widely used in the field of gene recombination as a general experimental technique. As specific methods, a lipofection method, an electroporation method, a calcium phosphate method and the like are known, but the lipofection method having a high introduction efficiency without requiring a dedicated device is most widely used.
　リポフェクション法は、カチオニックな電荷を有する脂質（トランスフェクション薬剤）と核酸のもつマイナス荷電との電気的相互作用により脂質と核酸の複合体を形成し、細胞の持つ貪食作用や細胞膜融合等のエンドサイトーシスを利用して当該複合体を細胞内に導くことで、核酸を細胞内に導入する方法である。 The lipofection method forms a lipid-nucleic acid complex by an electrical interaction between a cationically charged lipid (transfection agent) and the negative charge of the nucleic acid, and causes endocytosis such as cell phagocytosis and cell membrane fusion. In this method, nucleic acid is introduced into cells by introducing the complex into cells using tosis.
　リポフェクション法は、核酸や細胞等の種類により、核酸導入効率が低くなる場合がある。その為、リポフェクション法における核酸導入効率の向上を目的として、各種トランスフェクション促進用ペプチド（以下、エンハンサーペプチドと略記する場合がある）が開発されてきた（特許文献１、２）。 In the lipofection method, the nucleic acid introduction efficiency may be lowered depending on the type of nucleic acid or cell. Therefore, various transfection promoting peptides (hereinafter sometimes abbreviated as enhancer peptides) have been developed for the purpose of improving nucleic acid introduction efficiency in the lipofection method (Patent Documents 1 and 2).
　トランスフェクション促進用ペプチドとしては、例えば、スペルミン（Ｓｐ）に核移行シグナル（ＮＬＳ）をタンデムに結合させたペプチドＳｐ－ＮＬＳＮＬＳ（特許文献１）等が知られている。一般に、ＮＬＳはタンパク質を核膜孔から核内へ移行させる働きがある配列として知られており、トランスフェクション促進用ペプチドにおいても、導入した核酸を核内に運ぶ働きが、核酸導入効率の向上に寄与していると考えられている。 As a peptide for promoting transfection, for example, peptide Sp-NLSNLS (Patent Document 1) in which a nuclear translocation signal (NLS) is bound in tandem to spermine (Sp) is known. In general, NLS is known as a sequence that has the function of transferring proteins from the nuclear membrane pore into the nucleus, and even in the transfection promoting peptide, the function of transporting the introduced nucleic acid into the nucleus improves nucleic acid introduction efficiency. It is thought to contribute.
　しかし、ｍＲＮＡを細胞にトランスフェクションする場合、ＮＬＳがｍＲＮＡを核内に運ぶため、Ｓｐ－ＮＬＳＮＬＳ等のＮＬＳを有するトランスフェクション促進用ペプチドを用いることはできなかった。その為、ｍＲＮＡ等導入する核酸の種類にかかわらずトランスフェクションを促進することができる、汎用性の高いトランスフェクション促進用ペプチドの開発が望まれている。 However, when transfection of mRNA into cells, NLS transports mRNA into the nucleus, and therefore, transfection-promoting peptides having NLS such as Sp-NLSNLS could not be used. Therefore, development of a highly versatile transfection-promoting peptide that can promote transfection regardless of the type of nucleic acid to be introduced, such as mRNA, is desired.
特許第４２６５６９９号Japanese Patent No. 4265699 ＷＯ２０１５／０８９４８７WO2015 / 088487
　本発明は、上記した如き状況に鑑みなされたもので、従来のトランスフェクション促進用ペプチドと同等又はそれ以上の核酸導入効率でトランスフェクションを促進し、且つ汎用性の高いトランスフェクション促進用ペプチドの提供を課題とする。 The present invention has been made in view of the situation as described above, and provides a highly versatile transfection promoting peptide that promotes transfection with a nucleic acid introduction efficiency equal to or higher than that of conventional transfection promoting peptides. Is an issue.
　本発明者らは、上記した如き状況に鑑み鋭意研究した結果、ＮＬＳを有さない特定の配列からなるペプチド（本発明のエンハンサーペプチドと略記する場合がある）が、導入する核酸の種類にかかわらずトランスフェクションを促進することを見出した。
　ＮＬＳを有していないにもかかわらず、導入した核酸を核内に運ぶ働きを有する従来のＮＬＳを有するトランスフェクション促進用ペプチドと同等又はそれ以上の導入効率で、本発明のトランスフェクション促進用ペプチドがＤＮＡ等の核酸の細胞へのトランスフェクションを促進し得ることは意外なことであった。 As a result of intensive studies in view of the above-described circumstances, the present inventors have found that a peptide comprising a specific sequence not having NLS (sometimes abbreviated as an enhancer peptide of the present invention) depends on the type of nucleic acid to be introduced. First, it was found to promote transfection.
The transfection-promoting peptide of the present invention with a transduction-promoting efficiency equal to or higher than that of a conventional transfection-promoting peptide having NLS, which has the function of transporting the introduced nucleic acid into the nucleus even though it has no NLS It was surprising that can promote the transfection of nucleic acids such as DNA into cells.
　さらに、核移行シグナルを有する従来のトランスフェクション促進用ペプチドは、核酸を導入する細胞の種類によりトランスフェクションを促進しない（促進できない）場合があるが、本発明のトランスフェクション促進用ペプチドは、核酸を導入する細胞の種類にかかわらずトランスフェクションを促進できることを見出した。 Furthermore, the conventional transfection-promoting peptide having a nuclear translocation signal may not promote (cannot promote) transfection depending on the type of cell into which the nucleic acid is introduced. It was found that transfection can be promoted regardless of the type of cells introduced.
　また、本発明者らは、トランスフェクション薬剤が細胞毒性を有しており、トランスフェクションを行った細胞の生存率を低下させていることを発見した。しかし、本発明のトランスフェクション促進用ペプチドを用いてトランスフェクションを行うと、細胞の生存率の低下を抑制できること、すなわち、本発明のトランスフェクション促進用ペプチドがトランスフェクション薬剤の細胞毒性を抑制できることも見出し、本発明を完成するに至った。 In addition, the present inventors have discovered that the transfection agent has cytotoxicity and decreases the survival rate of the transfected cells. However, transfection using the transfection-promoting peptide of the present invention can suppress the decrease in cell viability, that is, the transfection-promoting peptide of the present invention can suppress the cytotoxicity of the transfection agent. The headline and the present invention were completed.
　本発明は、以下の構成よりなる。
＜１＞下記一般式［１］又は［２］で示される、細胞のトランスフェクション促進用ペプチド。
（Ｘ１）ｎ－Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２　［１］
Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２－（Ｘ１）ｎ　［２］
（式中、ｎ個のＸ１はそれぞれ独立してアルギニン又はリシンを表し、Ｘ２はグリシン又はアラニン、Ｘ３はチロシン又はアラニンをそれぞれ表し、ｎは８～１７の整数を表す）
＜２＞前記ペプチドが、下記一般式［１’］、［１’’］、［２’］又は［２’’］で示されるものである、＜１＞に記載のペプチド。
（Ｘ１）ｎ－Ｇｌｙ－Ｇｌｙ－Ｃｙｓ－Ｇｌｙ－Ｔｙｒ－Ｇｌｙ　［１’］
（Ｘ１）ｎ－Ａｌａ－Ａｌａ－Ｃｙｓ－Ａｌａ－Ａｌａ－Ａｌａ　［１’’］
Ｇｌｙ－Ｇｌｙ－Ｃｙｓ－Ｇｌｙ－Ｔｙｒ－Ｇｌｙ－（Ｘ１）ｎ　［２’］
Ａｌａ－Ａｌａ－Ｃｙｓ－Ａｌａ－Ａｌａ－Ａｌａ－（Ｘ１）ｎ　［２’’］
（式中、Ｘ１、ｎは上記に同じ）
＜３＞前記ペプチドが、一般式［１’］又は［２’］で示されるものである、＜２＞に記載のペプチド。
＜４＞前記一般式［１’］又は一般式［２’］中の（Ｘ１）ｎが、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ、（Ａｒｇ－Ｌｙｓ）ｍ、及び（Ｌｙｓ－Ａｒｇ）ｍ（ｎは前記に同じ、ｍは４～８の整数を表す）から選ばれるものである、＜３＞に記載のペプチド。
＜５＞前記一般式［１’］又は一般式［２’］中の（Ｘ１）ｎが、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ又は（Ａｒｇ－Ｌｙｓ）ｍ（ｎ及びｍは前記に同じ）である、＜４＞に記載のペプチド。
＜６＞ｎが９～１６の整数、ｍが６である、＜４＞又は＜５＞に記載のペプチド。
＜７＞前記ペプチドが、一般式［１’’］又は［２’’］で示されるものである、＜２＞に記載のペプチド。
＜８＞前記一般式［１’’］又は一般式［２’’］中の（Ｘ１）ｎが、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ、（Ａｒｇ－Ｌｙｓ）ｍ、及び（Ｌｙｓ－Ａｒｇ）ｍ（ｎ、ｍは前記に同じ）から選ばれるものである、＜７＞に記載のペプチド。
＜９＞前記一般式［１’’］又は一般式［２’’］中の（Ｘ１）ｎが、（Ａｒｇ）ｎ（ｎは前記に同じ）である、＜８＞に記載のペプチド。
＜１０＞ｎが９～１６の整数、ｍが６である、＜８＞又は＜９＞に記載のペプチド。
＜１１＞＜１＞～＜１０＞のいずれかに記載のペプチド及びカチオン性脂質を含むトランスフェクション薬剤を含む、細胞のトランスフェクション用キット（本発明のトランスフェクション用キット）。
＜１２＞前記トランスフェクション薬剤が、Ｎ－［２－（ジメチルアミノ）エチル］－４，５－ビス（ウンデシルチオ）ペンタンアミドを含むものである、＜１１＞に記載の細胞のトランスフェクション用キット。
＜１３＞＜１＞～＜１０＞のいずれかに記載のペプチド及びトランスフェクション薬剤の存在下、インビトロで細胞と核酸とを接触させる工程を包含する、細胞に核酸をトランスフェクションする方法（本発明のトランスフェクション方法）。
＜１４＞前記トランスフェクション薬剤が、カチオン性脂質を含むものである、＜１３＞に記載の方法。
＜１５＞前記トランスフェクション薬剤が、Ｎ－［２－（ジメチルアミノ）エチル］－４，５－ビス（ウンデシルチオ）ペンタンアミドを含むものである、＜１３＞に記載の方法。
＜１６＞前記核酸がＲＮＡである、＜１３＞～＜１５＞に記載の方法。 The present invention has the following configuration.
<1> A peptide for promoting transfection of cells represented by the following general formula [1] or [2].
(X 1 ) n -X 2 -X 2 -Cys-X 2 -X 3 -X 2 [1]
X 2 -X 2 -Cys-X 2 -X 3 -X 2- (X 1 ) n [2]
(In the formula, each of n X 1 s independently represents arginine or lysine, X 2 represents glycine or alanine, X 3 represents tyrosine or alanine, and n represents an integer of 8 to 17)
<2> The peptide according to <1>, wherein the peptide is represented by the following general formula [1 ′], [1 ″], [2 ′] or [2 ″].
(X 1 ) n -Gly-Gly-Cys-Gly-Tyr-Gly [1 ′]
(X 1 ) n -Ala-Ala-Cys-Ala-Ala-Ala [1 ″]
Gly-Gly-Cys-Gly-Tyr-Gly- (X 1 ) n [2 ′]
Ala-Ala-Cys-Ala-Ala-Ala- (X 1 ) n [2 ″]
(Wherein X 1 and n are the same as above)
<3> The peptide according to <2>, wherein the peptide is represented by the general formula [1 ′] or [2 ′].
<4> (X 1 ) n in the general formula [1 ′] or the general formula [2 ′] is (Arg) n , (Lys) n , (Arg-Lys) m , and (Lys-Arg) m The peptide according to <3>, wherein n is the same as defined above, and m is an integer of 4 to 8.
<5> (X 1 ) n in general formula [1 ′] or general formula [2 ′] is (Arg) n , (Lys) n or (Arg-Lys) m (n and m are the same as above) The peptide according to <4>, wherein
<6> The peptide according to <4> or <5>, wherein n is an integer of 9 to 16, and m is 6.
<7> The peptide according to <2>, wherein the peptide is represented by the general formula [1 ″] or [2 ″].
<8> (X 1 ) n in the general formula [1 ″] or the general formula [2 ″] is (Arg) n , (Lys) n , (Arg-Lys) m , and (Lys-Arg) ) M (wherein n and m are the same as above), and the peptide according to <7>.
<9> The peptide according to <8>, wherein (X 1 ) n in the general formula [1 ″] or the general formula [2 ″] is (Arg) n (n is the same as described above).
<10> The peptide according to <8> or <9>, wherein n is an integer of 9 to 16, and m is 6.
<11> A kit for transfection of cells (transfection kit of the present invention) comprising a transfection agent comprising the peptide according to any one of <1> to <10> and a cationic lipid.
<12> The kit for transfection of cells according to <11>, wherein the transfection agent comprises N- [2- (dimethylamino) ethyl] -4,5-bis (undecylthio) pentanamide.
<13> A method of transfecting a cell with a nucleic acid, comprising the step of contacting the cell and the nucleic acid in vitro in the presence of the peptide according to any one of <1> to <10> and a transfection agent (the present invention) Transfection method).
<14> The method according to <13>, wherein the transfection agent contains a cationic lipid.
<15> The method according to <13>, wherein the transfection agent comprises N- [2- (dimethylamino) ethyl] -4,5-bis (undecylthio) pentanamide.
<16> The method according to <13> to <15>, wherein the nucleic acid is RNA.
　本発明によれば、導入する核酸の種類にかかわらず、トランスフェクションを促進することができる。さらに、本発明によれば、導入する細胞の種類にかかわらず、従来のトランスフェクション促進用ペプチドを用いた場合と同等又はそれ以上の核酸導入効率でトランスフェクションを促進することができる。すなわち、本発明は、非常に汎用性の高いトランスフェクション方法、及びトランスフェクション促進用ペプチドに関する。また、本発明のトランスフェクション法によれば、トランスフェクション薬剤の細胞毒性を抑制し、高い細胞生存率でトランスフェクションを行うこともできる。 According to the present invention, transfection can be promoted regardless of the type of nucleic acid to be introduced. Furthermore, according to the present invention, transfection can be promoted with a nucleic acid introduction efficiency equal to or higher than that when a conventional transfection-promoting peptide is used regardless of the type of cells to be introduced. That is, the present invention relates to a highly versatile transfection method and a transfection promoting peptide. Further, according to the transfection method of the present invention, transfection can be performed with high cell viability by suppressing the cytotoxicity of the transfection agent.
実施例１、比較例１－１及び比較例１－２で得られた、ＹＦＰ－Ｈ２Ｂ遺伝子を導入したＣＯＳ７細胞の蛍光顕微鏡像である。図中、横軸はトランスフェクション促進ペプチドの種類を表し、ＳＦＡ　Ｐｌｕｓはトランスフェクション促進ペプチドを用いなかった場合（比較例１－２）、ＳＦＡ　Ｐｌｕｓ＋ＥＳＦＡ４は従来のトランスフェクション促進ペプチドであるＥＳＦＡ４を用いた場合（比較例１－１）、ＳＦＡ　Ｐｌｕｓ＋Ｃ２は本発明のトランスフェクション促進ペプチドであるＣ２を用いた場合（実施例１）をそれぞれ表す。また、縦軸のＹＦＰは導入したＹＦＰ－Ｈ２Ｂ遺伝子の発現レベルを表し、ＤＩＣ（微分干渉顕微鏡像）は細胞の状態を表す。2 is a fluorescence microscopic image of COS7 cells into which YFP-H2B gene was introduced, obtained in Example 1, Comparative Example 1-1, and Comparative Example 1-2. In the figure, the horizontal axis represents the type of transfection-promoting peptide. When SFA Plus did not use a transfection-promoting peptide (Comparative Example 1-2), SFA Plus + ESFA4 used ESFA4, a conventional transfection-promoting peptide. In the case (Comparative Example 1-1), SFA Plus + C2 represents the case where C2 which is the transfection promoting peptide of the present invention was used (Example 1). YFP on the vertical axis represents the expression level of the introduced YFP-H2B gene, and DIC (differential interference microscope image) represents the state of the cells. 実施例２、比較例２－１及び比較例２－２で得られた、ＹＦＰ－Ｈ２Ｂ遺伝子を導入したＨｕＨ－７細胞の蛍光顕微鏡像である。図中、横軸はトランスフェクション促進ペプチドの種類を表し、ＳＦＡ　Ｐｌｕｓはトランスフェクション促進ペプチドを用いなかった場合（比較例２－２）、ＳＦＡ　Ｐｌｕｓ＋ＥＳＦＡ４は従来のトランスフェクション促進ペプチドであるＥＳＦＡ４を用いた場合（比較例２－１）、ＳＦＡ　Ｐｌｕｓ＋Ｃ２は本発明のトランスフェクション促進ペプチドであるＣ２を用いた場合（実施例２）をそれぞれ表す。また、縦軸のＹＦＰは導入したＹＦＰ－Ｈ２Ｂ遺伝子の発現レベルを表し、ＤＩＣ（微分干渉顕微鏡像）は細胞の状態を表す。2 is a fluorescence microscopic image of HuH-7 cells into which YFP-H2B gene was introduced, obtained in Example 2, Comparative Example 2-1 and Comparative Example 2-2. In the figure, the horizontal axis indicates the type of transfection-promoting peptide. When SFA Plus did not use a transfection-promoting peptide (Comparative Example 2-2), SFA Plus + ESFA4 used ESFA4, which is a conventional transfection-promoting peptide. In the case (Comparative Example 2-1), SFA Plus + C2 represents the case where C2 which is the transfection promoting peptide of the present invention was used (Example 2). YFP on the vertical axis represents the expression level of the introduced YFP-H2B gene, and DIC (differential interference microscope image) represents the state of the cells. 実施例３、比較例３－１及び比較例３－２で得られた、ＹＦＰ－Ｈ２Ｂ遺伝子を導入したＨＣＴ－１１６＃２Ｌｕｃ細胞の蛍光顕微鏡像である。図中、横軸はトランスフェクション促進ペプチドの種類を表し、ＳＦＡ　Ｐｌｕｓはトランスフェクション促進ペプチドを用いなかった場合（比較例３－２）、ＳＦＡ　Ｐｌｕｓ＋ＥＳＦＡ４は従来のトランスフェクション促進ペプチドであるＥＳＦＡ４を用いた場合（比較例３－１）、ＳＦＡ　Ｐｌｕｓ＋Ｃ２は本発明のトランスフェクション促進ペプチドであるＣ２を用いた場合（実施例３）をそれぞれ表す。また、縦軸のＹＦＰは導入したＹＦＰ－Ｈ２Ｂ遺伝子の発現レベルを表し、ＤＩＣ（微分干渉顕微鏡像）は細胞の状態を表す。2 is a fluorescence microscopic image of HCT-116 # 2Luc cells into which YFP-H2B gene was introduced, obtained in Example 3, Comparative Example 3-1 and Comparative Example 3-2. In the figure, the horizontal axis represents the type of transfection-promoting peptide. When SFA Plus did not use a transfection-promoting peptide (Comparative Example 3-2), SFA Plus + ESFA4 used ESFA4, a conventional transfection-promoting peptide. In the case (Comparative Example 3-1), SFA Plus + C2 represents the case where C2 which is the transfection promoting peptide of the present invention was used (Example 3). YFP on the vertical axis represents the expression level of the introduced YFP-H2B gene, and DIC (differential interference microscope image) represents the state of the cells. 実施例１７及び比較例１７で得られた、ＥＧＦＰ　ｍＲＮＡを導入したＨｅｌａ細胞の蛍光顕微鏡像である。図中、横軸はトランスフェクション促進ペプチドの種類を表し、ＳＦＡ　Ｐｌｕｓはトランスフェクション促進ペプチドを用いなかった場合（比較例１７）、ＳＦＡ　Ｐｌｕｓ＋Ｃ２は本発明のトランスフェクション促進ペプチドであるＣ２を用いた場合（実施例１７）をそれぞれ表す。また、縦軸のＧＦＰは導入したＥＧＦＰ　ｍＲＮＡの発現レベルを表し、ＤＩＣ（微分干渉顕微鏡像）は細胞の状態を表す。It is the fluorescence-microscope image of the Hela cell which introduce | transduced EGFP mRNA obtained in Example 17 and Comparative Example 17. FIG. In the figure, the horizontal axis represents the type of transfection-promoting peptide, SFA Plus does not use the transfection-promoting peptide (Comparative Example 17), and SFA Plus + C2 uses the transfection-promoting peptide C2 of the present invention. (Example 17) is shown respectively. GFP on the vertical axis represents the expression level of the introduced EGFP mRNA, and DIC (differential interference microscope image) represents the state of the cells. 実施例１７及び比較例１７で得られた、ＥＧＦＰ　ｍＲＮＡを導入したＨｅｌａ細胞におけるＥＧＦＰの相対蛍光強度を示すグラフである。図中、横軸はトランスフェクション促進ペプチドの種類を表し、ＳＦＡ　Ｐｌｕｓはトランスフェクション促進ペプチドを用いなかった場合（比較例１７）、ＳＦＡ　Ｐｌｕｓ＋Ｃ２は本発明のトランスフェクション促進ペプチドであるＣ２を用いた場合（実施例１７）である。It is a graph which shows the relative fluorescence intensity of EGFP in the Hela cell which introduce | transduced EGFP mRNA obtained in Example 17 and Comparative Example 17. In the figure, the horizontal axis represents the type of transfection-promoting peptide, SFA Plus does not use the transfection-promoting peptide (Comparative Example 17), and SFA Plus + C2 uses the transfection-promoting peptide C2 of the present invention. (Example 17). 実施例１８～２４及び比較例１８～２８で得られた、ホタルルシフェラーゼ遺伝子を導入したＣＨＯ－Ｋ１細胞におけるホタルルシフェラーゼの発光強度（発現強度）を示すグラフである。図中、横軸はトランスフェクション促進ペプチドの種類を表し、ｎｏｎｅ　ｔｒａｎｓｆｅｃｔｅｄはトランスフェクションを行わなかった場合（コントロール）、Ｎｏｎｅ　ｅｎｈａｎｅｒはトランスフェクション促進ペプチドを用いなかった場合（比較例１８）、Ｃ２は本発明のトランスフェクション促進ペプチドであるＣ２を用いた場合（実施例１８）、Ｃ５は本発明のトランスフェクション促進ペプチドであるＣ５を用いた場合（実施例１９）、Ｃ２＿ＡＡＣＡＡは本発明のトランスフェクション促進ペプチドであるＣ２＿ＡＡＣＡＡを用いた場合（実施例２０）、Ｋ１１は本発明のトランスフェクション促進ペプチドであるＫ１１を用いた場合（実施例２１）、Ｒ９は本発明のトランスフェクション促進ペプチドであるＲ９を用いた場合（実施例２２）、Ｒ１６は本発明のトランスフェクション促進ペプチドであるＲ１６を用いた場合（実施例２３）、ＲＫ１２は本発明のトランスフェクション促進ペプチドであるＲＫ１２を用いた場合（実施例２４）、ＥＳＦＡ４はペプチドであるＲ１６を用いた場合（比較例１９）、Ｃ４はペプチドであるＣ４を用いた場合（比較例２０）、Ｃ２＿Ｃ１４ＡはペプチドであるＣ２＿Ｃ１４Ａを用いた場合（比較例２１）、Ｃ２＿Ａ６はペプチドであるＣ２＿Ａ６を用いた場合（比較例２２）、Ｒ３はペプチドであるＲ３を用いた場合（比較例２３）、Ｒ６はペプチドであるＲ６を用いた場合（比較例２４）、Ｒ１９はペプチドであるＲ１９を用いた場合（比較例２５）、Ｒ２２はペプチドであるＲ２２を用いた場合（比較例２６）、Ｒ３０はペプチドであるＲ３０を用いた場合（比較例２７）、Ｈ１１はペプチドであるＨ１１を用いた場合（比較例２８）をそれぞれ表す。縦軸は発光強度を表す。6 is a graph showing the luminescence intensity (expression intensity) of firefly luciferase in CHO-K1 cells into which a firefly luciferase gene was introduced, obtained in Examples 18 to 24 and Comparative Examples 18 to 28. In the figure, the abscissa represents the type of transfection-promoting peptide, none transfected when no transfection was performed (control), None enhancer when no transfection-promoting peptide was used (Comparative Example 18), and C2 When C2 which is the transfection-promoting peptide of the invention is used (Example 18), C5 is the case where C5 which is the transfection-promoting peptide of the present invention is used (Example 19), and C2_AACAA is the transfection-promoting peptide of the present invention. When C2_AACAA is used (Example 20), K11 is the transfection promoting peptide of the present invention K11 (Example 21), and R9 is the transfection promoting peptide of the present invention R9. In the case (Example 22), R16 used the transfection promoting peptide of the present invention R16 (Example 23), RK12 used the transfection promoting peptide of the present invention RK12 (Example 24) ESFA4 is a peptide using R16 (Comparative Example 19), C4 is a peptide using C4 (Comparative Example 20), C2_C14A is a peptide using C2_C14A (Comparative Example 21), C2_A6 Is the peptide C2_A6 (Comparative Example 22), R3 is the peptide R3 (Comparative Example 23), R6 is the peptide R6 (Comparative Example 24), R19 is the peptide When R19 is used (Comparative Example 25), R22 is a peptide using R22 (Comparative Example 26), 30 If using the R30 is a peptide (Comparative Example 27), H11 represents a case of using the H11 is a peptide (Comparative Example 28), respectively. The vertical axis represents the emission intensity. 実施例２５～３１及び比較例２９で得られた、プラスミドＤＮＡを導入したＣＨＯ－Ｋ１細胞の生存率（％）を示すグラフである。図中、横軸はトランスフェクション促進ペプチドの種類を表し、ｎｏｎｅ　ｔｒａｎｓｆｅｃｔｅｄはトランスフェクションを行わなかった場合（コントロール）、Ｎｏｎｅ　ｅｎｈａｎｅｒはトランスフェクション促進ペプチドを用いなかった場合（比較例２９）、Ｃ２は本発明のトランスフェクション促進ペプチドであるＣ２を用いた場合（実施例２５）、Ｃ５は本発明のトランスフェクション促進ペプチドであるＣ５を用いた場合（実施例２６）、Ｃ２＿ＡＡＣＡＡは本発明のトランスフェクション促進ペプチドであるＣ２＿ＡＡＣＡＡを用いた場合（実施例２７）、Ｋ１１は本発明のトランスフェクション促進ペプチドであるＫ１１を用いた場合（実施例２８）、Ｒ９は本発明のトランスフェクション促進ペプチドであるＲ９を用いた場合（実施例２９）、Ｒ１６は本発明のトランスフェクション促進ペプチドであるＲ１６を用いた場合（実施例３０）、ＲＫ１２は本発明のトランスフェクション促進ペプチドであるＲＫ１２を用いた場合（実施例３１）をそれぞれ表す。縦軸は発光強度を表す。3 is a graph showing the survival rate (%) of CHO-K1 cells into which plasmid DNA was introduced, obtained in Examples 25 to 31 and Comparative Example 29. In the figure, the abscissa represents the type of transfection-promoting peptide, none transfected when no transfection was performed (control), None enhancer when no transfection-promoting peptide was used (Comparative Example 29), and C2 When C2 which is the transfection facilitating peptide of the invention is used (Example 25), when C5 which is the transfection facilitating peptide of the present invention is used (Example 26), C2_AACAA is the transfection facilitating peptide of the present invention When C2_AACAA is used (Example 27), K11 is the transfection promoting peptide of the present invention K11 (Example 28), and R9 is the transfection promoting peptide of the present invention R9. In the case (Example 29), R16 used the transfection promoting peptide of the present invention R16 (Example 30), RK12 used the transfection promoting peptide of the present invention RK12 (Example 31) Respectively. The vertical axis represents the emission intensity.
　本発明のトランスフェクション促進用ペプチド（以下、本発明のエンハンサーペプチドと略記する場合がある）は、下記一般式［１］又は［２］で示されるペプチドである。 The transfection-promoting peptide of the present invention (hereinafter sometimes abbreviated as the enhancer peptide of the present invention) is a peptide represented by the following general formula [1] or [2].
　本発明のトランスフェクション促進用ペプチドは、Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２で示されるコア配列と（Ｘ１）ｎで示される付加配列を有するポリペプチドである。
　付加配列（Ｘ１）ｎは、コア配列Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２のＮ末端側又はＣ末端側の何れか一方に付加されている（結合している）。即ち、本発明のトランスフェクション促進用ペプチドは、下記一般式［１］又は下記一般式［２］で示されるポリペプチドである。
（Ｘ１）ｎ－Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２　［１］
Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２－（Ｘ１）ｎ　［２］
（式中、Ｘ１、Ｘ２、Ｘ３、及びｎは上記に同じ）
The peptide for promoting transfection of the present invention is a polypeptide having a core sequence represented by X 2 -X 2 -Cys-X 2 -X 3 -X 2 and an additional sequence represented by (X 1 ) n .
Additional sequence (X 1 ) n is added (bonded) to either the N-terminal side or the C-terminal side of the core sequence X 2 -X 2 -Cys-X 2 -X 3 -X 2 . That is, the transfection promoting peptide of the present invention is a polypeptide represented by the following general formula [1] or the following general formula [2].
(X 1 ) n -X 2 -X 2 -Cys-X 2 -X 3 -X 2 [1]
X 2 -X 2 -Cys-X 2 -X 3 -X 2- (X 1 ) n [2]
(Wherein X 1 , X 2 , X 3 and n are the same as above)
　上記一般式［１］及び上記一般式［２］のＸ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２で示されるコア配列におけるＸ２は、グリシン又はアラニンを表し、コア配列中の４個のＸ２は同一でも異なっていてもよいが、同一であるのが好ましい。また、Ｘ３は、チロシン又はアラニンである。
　従って、上記一般式［１］及び一般式［２］のＸ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２で示されるコア配列は、以下のように示すことができる。
Figure JPOXMLDOC01-appb-I000001
 X 2 in the core sequence shown in X 2 -X 2 -Cys-X 2 -X 3 -X 2 in the above general formula [1] and the formula [2] represents the glycine or alanine, in the core sequence four X 2 may be the same or different but are preferably identical. X 3 is tyrosine or alanine.
Therefore, the core sequence represented by X 2 -X 2 -Cys-X 2 -X 3 -X 2 in the general formula [1] and the general formula [2] can be represented as follows.
Figure JPOXMLDOC01-appb-I000001
　Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２で示されるコア配列の好ましい具体例としては、例えば下表の１～４の配列が挙げられる。中でも１及び４の配列が好ましく、１の配列が特に好ましい。
Figure JPOXMLDOC01-appb-I000002
Preferable specific examples of the core sequence represented by X 2 -X 2 -Cys-X 2 -X 3 -X 2 include, for example, the sequences 1 to 4 shown in the following table. Among them, the sequences 1 and 4 are preferable, and the sequence 1 is particularly preferable.
Figure JPOXMLDOC01-appb-I000002
　上記一般式［１］及び上記一般式［２］の（Ｘ１）ｎで示される付加配列におけるＸ１は、アルギニン又はリシンを表し、ｎ個のＸ１は同一でも異なっていてもよい。即ち、ｎ個のＸ１は、ｎ個のアルギニンのみから構成されるポリペプチド、ｎ個のリシンのみから構成されるポリペプチド、又は、アルギニンとリシンの２種類のペプチドから構成され当該ペプチドの総和がｎ個のポリペプチドを意味する。 X 1 in the general formula [1] and the formula [2] of (X 1) additional sequence represented by n represents arginine or lysine, n-number of X 1 may be the same or different. That is, n X 1 is a polypeptide composed only of n arginines, a polypeptide composed only of n lysines, or a total of the peptides composed of two kinds of peptides, arginine and lysine. Means n polypeptides.
　（Ｘ１）ｎで示される付加配列は、より具体的には、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ、（Ａｒｇ－Ｌｙｓ）ｍ、（Ｌｙｓ－Ａｒｇ）ｍ、［（Ａｒｇ）ｐ（Ｌｙｓ）ｑ］、［（Ｌｙｓ）ｑ（Ａｒｇ）ｐ］等が挙げられる（式中、ｎは８～１７の整数を表し、ｍは４～８の整数をそれぞれ表す。また、ｐは１～１６の整数であり、ｑは１～１６の整数であり、且つｐ＋ｑは８～１７の整数を表す）。これらの中でも、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ、（Ａｒｇ－Ｌｙｓ）ｍ及び（Ｌｙｓ－Ａｒｇ）ｍが好ましく、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ及び（Ａｒｇ－Ｌｙｓ）ｍがより好ましく、（Ａｒｇ）ｎ及び（Ｌｙｓ）ｎが特に好ましい。 More specifically, the additional sequence represented by (X 1 ) n is (Arg) n , (Lys) n , (Arg-Lys) m , (Lys-Arg) m , [(Arg) p (Lys) q ], [(Lys) q (Arg) p ] and the like (wherein n represents an integer of 8 to 17, m represents an integer of 4 to 8, and p represents 1 to 16). An integer, q is an integer from 1 to 16, and p + q represents an integer from 8 to 17. Among these, (Arg) n , (Lys) n , (Arg-Lys) m and (Lys-Arg) m are preferred, (Arg) n , (Lys) n and (Arg-Lys) m are more preferred, (Arg) n and (Lys) n are particularly preferred.
　上記一般式［１］及び上記一般式［２］におけるｎとしては、９～１６の整数が好ましい。また、ｍは６が特に好ましい。また、ｐ＋ｑは９～１６の整数が好ましく、中でもｐが８～１０の整数、ｑが１～６の整数が好ましい。 In the general formula [1] and the general formula [2], n is preferably an integer of 9 to 16. Further, m is particularly preferably 6. Further, p + q is preferably an integer of 9 to 16, more preferably p is an integer of 8 to 10, and q is an integer of 1 to 6.
　上記一般式［１］又は上記一般式［２］で示される本発明のトランスフェクション促進用ペプチドの好ましい具体例としては、例えば下記一般式［１’］、下記一般式［１’’］、下記一般式［２’］及び下記一般式［２’’］で示されるポリペプチドが挙げられ、下記一般式［１’］及び下記一般式［２’］で示されるものがより好ましく、下記一般式［１’］で示されるものが特に好ましい。 Preferred specific examples of the peptide for promoting transfection of the present invention represented by the above general formula [1] or the above general formula [2] include, for example, the following general formula [1 ′], the following general formula [1 ″], the following Examples include polypeptides represented by the general formula [2 ′] and the following general formula [2 ″], more preferably those represented by the following general formula [1 ′] and the following general formula [2 ′]. Those represented by [1 ′] are particularly preferred.
（Ｘ１）ｎ－Ｇｌｙ－Ｇｌｙ－Ｃｙｓ－Ｇｌｙ－Ｔｙｒ－Ｇｌｙ　［１’］
（式中、Ｘ１、ｎは上記に同じ） (X 1 ) n -Gly-Gly-Cys-Gly-Tyr-Gly [1 ′]
(Wherein X 1 and n are the same as above)
（Ｘ１）ｎ－Ａｌａ－Ａｌａ－Ｃｙｓ－Ａｌａ－Ａｌａ－Ａｌａ　［１’’］
（式中、Ｘ１、ｎは上記に同じ） (X 1 ) n -Ala-Ala-Cys-Ala-Ala-Ala [1 ″]
(Wherein X 1 and n are the same as above)
Ｇｌｙ－Ｇｌｙ－Ｃｙｓ－Ｇｌｙ－Ｔｙｒ－Ｇｌｙ－（Ｘ１）ｎ　［２’］
（式中、Ｘ１、ｎは上記に同じ） Gly-Gly-Cys-Gly-Tyr-Gly- (X 1 ) n [2 ′]
(Wherein X 1 and n are the same as above)
Ａｌａ－Ａｌａ－Ｃｙｓ－Ａｌａ－Ａｌａ－Ａｌａ－（Ｘ１）ｎ　［２’’］
（式中、Ｘ１、ｎは上記に同じ） Ala-Ala-Cys-Ala-Ala-Ala- (X 1 ) n [2 ″]
(Wherein X 1 and n are the same as above)
　下記一般式［１’］としては、例えば、下記［１’－ａ］～［１’－ｌ］のポリペプチドが挙げられ、［１’－ａ］～［１’－ｈ］、［１’－ｊ］及び［１’－ｌ］が好ましく、［１’－ｃ］～［１’－ｈ］及び［１’－ｌ］がより好ましく、［１’－ｅ］～［１’－ｈ］及び［１’－ｌ］が特に好ましい。
Figure JPOXMLDOC01-appb-I000003
The following general formula [1 ′] includes, for example, the following polypeptides [1′-a] to [1′-l], [1′-a] to [1′-h], [1 ′ -J] and [1'-l] are preferred, [1'-c] to [1'-h] and [1'-l] are more preferred, and [1'-e] to [1'-h] And [1′-l] are particularly preferred.
Figure JPOXMLDOC01-appb-I000003
　上記一般式［２’］としては、例えば、下記［２’－ａ］～［２’－ｈ］のポリペプチドが挙げられ、［２’－ａ］～［２’－ｅ］、［２’－ｇ］及び［２’－ｈ］が好ましく、［２’－ｃ］～［２’－ｅ］がより好ましく、［２’－ｅ］が特に好ましい。
Figure JPOXMLDOC01-appb-I000004
Examples of the general formula [2 ′] include the following [2′-a] to [2′-h] polypeptides, [2′-a] to [2′-e], [2 ′ -G] and [2′-h] are preferable, [2′-c] to [2′-e] are more preferable, and [2′-e] is particularly preferable.
Figure JPOXMLDOC01-appb-I000004
　上記一般式［１’’］としては、例えば、下記［１’’－ａ］～［１’’－ｆ］のポリペプチドが挙げられ、［１’’－ａ］、［１’’－ｃ］及び［１’’－ｄ］が好ましく、［１’’－ｃ］及び［１’’－ｄ］がより好ましく、［１’’－ｄ］が特に好ましい。

Figure JPOXMLDOC01-appb-I000005
Examples of the general formula [1 ″] include the following polypeptides [1 ″ -a] to [1 ″ -f], including [1 ″ -a] and [1 ″ -c]. ] And [1 ″ -d] are preferred, [1 ″ -c] and [1 ″ -d] are more preferred, and [1 ″ -d] is particularly preferred.

Figure JPOXMLDOC01-appb-I000005
　上記一般式［２’’］としては、例えば、下記［２’’－ａ］～［２’’－ｅ］のポリペプチドが挙げられ、［２’’－ａ］及び［２’’－ｃ］が好ましく、［２’’－ｃ］がより好ましい。
Figure JPOXMLDOC01-appb-I000006
Examples of the general formula [2 ″] include the following polypeptides [2 ″ -a] to [2 ″ -e], [2 ″ -a] and [2 ″ -c]. ] Is preferable, and [2 ″ -c] is more preferable.
Figure JPOXMLDOC01-appb-I000006
　本発明のトランスフェクション促進用ペプチドとしては、上記具体例の中でも、例えば一般式［１’］、一般式［１’’］、一般式［２’］及び一般式［２’’］が好ましく、一般式［１’］及び一般式［２’］がより好ましく、［１’－ａ］～［１’－ｈ］、［１’－ｊ］、［１’－ｌ］、［２’－ａ］～［２’－ｅ］、［２’－ｇ］及び［２’－ｈ］が更に好ましく、［１’－ｃ］～［１’－ｈ］、［１’－ｌ］及び［２’－ｃ］～［２’－ｅ］が更により好ましく、［１’－ｅ］～［１’－ｈ］、［１’－ｌ］及び［２’－ｅ］が特に好ましく、［１’－ｆ］が最も好ましい。 As the transfection-promoting peptide of the present invention, among the above specific examples, for example, general formula [1 ′], general formula [1 ″], general formula [2 ′] and general formula [2 ″] are preferable, The general formula [1 ′] and the general formula [2 ′] are more preferable, and [1′-a] to [1′-h], [1′-j], [1′-l], [2′-a] ] To [2'-e], [2'-g] and [2'-h] are more preferred, and [1'-c] to [1'-h], [1'-l] and [2 ' -C] to [2'-e] are even more preferred, [1'-e] to [1'-h], [1'-l] and [2'-e] are particularly preferred, and [1'- f] is most preferred.
　本発明のトランスフェクション促進用ペプチドは、自体公知のペプチド合成法に準じて合成すればよく、このような合成法としては、例えば特許３５６９９６６号に記載の方法が挙げられる。 The transfection-promoting peptide of the present invention may be synthesized according to a peptide synthesis method known per se. Examples of such a synthesis method include the method described in Japanese Patent No. 3569966.
　本発明のトランスフェクション方法は、細胞に核酸をリポフェクション法によりトランスフェクションする際に、本発明のトランスフェクション促進用ペプチドを共存させて行う以外は、自体公知のリポフェクション法に於いて用いられるトランスフェクション薬剤等の各種試薬を用い、自体公知の操作に準じて行えばよい。 The transfection method of the present invention is a transfection agent used in a known lipofection method, except that the transfection promoting peptide of the present invention coexists when a nucleic acid is transfected into cells by the lipofection method. The various reagents such as those described above may be used according to a known operation.
　具体的には、例えば、本発明のトランスフェクション促進用ペプチド及び本発明に係るトランスフェクション薬剤の存在下、インビトロで細胞と核酸とを接触させればよい。 Specifically, for example, a cell and a nucleic acid may be contacted in vitro in the presence of the transfection promoting peptide of the present invention and the transfection agent of the present invention.
　本発明に係るトランスフェクション薬剤としては、通常この分野で用いられるトランスフェクション薬剤であれば何れでもよく、カチオン性脂質を含むものが挙げられ、カチオン性脂質と中性脂質を含むものが好ましい。
　上記カチオン性脂質としては、１価カチオン性脂質でも多価カチオン性脂質でもよく、その具体例としては、例えばＤＯＴＭＡ（Ｎ－［１－（２，３－ジオレオイルオキシ）－プロピル］－Ｎ，Ｎ，Ｎ－トリメチルアンモニウムクロリド）、ＤＯＴＡＰ（１，２－ビス（オレオイルオキシ）－３－３－（トリメチルアンモニウム）プロパン）、ＤＭＲＩＥ（１，２－ジミリスチルオキシプロピル－３－ジメチル－ヒドロキシエチルアンモニウムブロミド）、ＤＤＡＢ（ジメチルジオクタデシルアンモニウムブロミド）等の１価カチオン性脂質、リポスペルミン、ＤＯＳＰＡ（２，３－ジオレイルオキシ－Ｎ－［２（スペルミンカルボキサミド）エチル］－Ｎ，Ｎ－ジメチル－１－プロパンアミニウム（ｐｒｏｐａｎａｍｉｎｉｕｍ）トリフルオロ酢酸）、ＤＯＳＰＥＲ（１，３－ジオレオイルオキシ－２－（６カルボキシスペルミル）－プロピルアミド）、ジおよびテトラ－アルキル－テトラ－メチルスペルミン、ＴＭＴＰＳ（テトラメチルテトラパルミトイルスペルミン）、ＴＭＴＯＳ（テトラメチルテトラオレイルスペルミン）、ＴＭＴＬＳ（テトラメチルテトララウリルスペルミン）、ＴＭＴＭＳ（テトラメチルテトラミリスチルスペルミン）、ＴＭＤＯＳ（テトラメチルジオレイルスペルミン）、Ｎ－［２－（ジメチルアミノ）エチル］－４，５－ビス（ウンデシルチオ）ペンタンアミド等の多価カチオン性脂質が挙げられ、多価カチオン性脂質が好ましく、Ｎ－［２－（ジメチルアミノ）エチル］－４，５－ビス（ウンデシルチオ）ペンタンアミドが特に好ましい。
　上記中性脂質の具体例としては、ＤＯＰＥ（１，２－ジオレオイル－Ｓｎ－グリセロ－３－ホスホエタノールアミン、ジオレオイルホスファチジルエタノールアミン）、ＤＰｈＰＥ（ジフィタノイルホスファチジルエタノールアミン）、コレステロール等が挙げられ、ＤＯＰＥが好ましい。
　カチオン性脂質と中性脂質を含むものとしては、例えば、Ｌｉｐｏｆｅｃｔａｍｉｎｅ（商標）２０００（サーモフィッシャーサイエンティフィック社製）、Ｌｉｐｏｆｅｃｔａｍｉｎｅ（商標）３０００（サーモフィッシャーサイエンティフィック社製）、ＳｃｒｅｅｎＦｅｃｔＴＭ　Ａ　ｐｌｕｓ（和光純薬工業（株）製）、ＳｃｒｅｅｎＦｅｃｔＴＭ　Ａ（和光純薬工業（株）製）、ＳｃｒｅｅｎＦｅｃｔＴＭ　ｍＲＮＡ（和光純薬工業（株）製）等が挙げられ、ＳｃｒｅｅｎＦｅｃｔＴＭ　Ａ　ｐｌｕｓ（和光純薬工業（株）製）、ＳｃｒｅｅｎＦｅｃｔＴＭ　Ａ（和光純薬工業（株）製）、ＳｃｒｅｅｎＦｅｃｔＴＭ　ｍＲＮＡ（和光純薬工業（株）製）が好ましい。
　本発明に係るトランスフェクション薬剤は、中でもＮ－［２－（ジメチルアミノ）エチル］－４，５－ビス（ウンデシルチオ）ペンタンアミドとＤＯＰＥ（１，２－ジオレオイル－Ｓｎ－グリセロ－３－ホスホエタノールアミン、ジオレオイルホスファチジルエタノールアミン）を含むＳｃｒｅｅｎＦｅｃｔシリーズ（ＳｃｒｅｅｎＦｅｃｔＴＭ　Ａ　ｐｌｕｓ（和光純薬工業（株）製）、ＳｃｒｅｅｎＦｅｃｔＴＭ　Ａ（和光純薬工業（株）製）、ＳｃｒｅｅｎＦｅｃｔＴＭ　ｍＲＮＡ（和光純薬工業（株）製）等）がより好ましい。 The transfection agent according to the present invention may be any transfection agent usually used in this field, and includes those containing cationic lipids, and those containing cationic lipids and neutral lipids are preferred.
The cationic lipid may be a monovalent cationic lipid or a polyvalent cationic lipid. Specific examples thereof include, for example, DOTMA (N- [1- (2,3-dioleoyloxy) -propyl] -N , N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonium) propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy) Monovalent cationic lipids such as ethylammonium bromide), DDAB (dimethyldioctadecylammonium bromide), lipospermine, DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl -1-Propanaminium triflu (Roacetic acid), DOSPER (1,3-dioleoyloxy-2- (6 carboxyspermyl) -propylamide), di- and tetra-alkyl-tetra-methylspermine, TMTPS (tetramethyltetrapalmitoylspermine), TMTOS ( Tetramethyltetraoleylspermine), TMTLS (tetramethyltetralaurylspermine), TTMMS (tetramethyltetramyristylspermine), TMDOS (tetramethyldioleylspermine), N- [2- (dimethylamino) ethyl] -4,5- Examples include polyvalent cationic lipids such as bis (undecylthio) pentanamide, polyvalent cationic lipids are preferred, and N- [2- (dimethylamino) ethyl] -4,5-bis (undecylthio) pentanamide is particularly preferred. .
Specific examples of the neutral lipid include DOPE (1,2-dioleoyl-Sn-glycero-3-phosphoethanolamine, dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine), cholesterol and the like. DOPE is preferred.
Examples of those containing cationic lipids and neutral lipids include Lipofectamine (trademark) 2000 (manufactured by Thermo Fisher Scientific), Lipofectamine (trademark) 3000 (manufactured by Thermo Fisher Scientific), ScreenFect ™ A plus ( Wako Pure Chemical Industries, Ltd.), ScreenFect ™ A (Wako Pure Chemical Industries, Ltd.), ScreenFect ™ mRNA (Wako Pure Chemical Industries, Ltd.), etc., and ScreenFect ™ A plus (Wako Pure Chemicals). Industrial Co., Ltd.), ScreenFect ™ A (Wako Pure Chemical Industries, Ltd.), and ScreenFect ™ mRNA (Wako Pure Chemical Industries, Ltd.) are preferred.
The transfection agent according to the present invention includes N- [2- (dimethylamino) ethyl] -4,5-bis (undecylthio) pentanamide and DOPE (1,2-dioleoyl-Sn-glycero-3-phosphoethanolamine, among others. ScreenFect series (ScreenFect ™ A plus (manufactured by Wako Pure Chemical Industries, Ltd.), ScreenFect ™ A (manufactured by Wako Pure Chemical Industries, Ltd.), ScreenFect ™ mRNA (Wako Pure Chemical Industries, Ltd.) Etc.) are more preferable.
　本発明に係る細胞としては、通常、真核生物細胞が挙げられ、高等真核生物細胞が好ましく、ＨｅＬａ細胞、２９３Ｔ細胞、ＨＥＫ２９３細胞、ＣＨＯ－Ｋ１細胞、Ａ５４９細胞、ＣＯＳ７細胞、ＭＣＦ７細胞、ＮＩＨ３Ｔ３細胞、Ｌ細胞、ＨＵＶＥＣ細胞、ＨｕＨ－７細胞、ＨＣＴ－１１６＃２Ｌｕｃ細胞、ＨＴ－１０８０細胞、Ｕ２ＯＳ細胞等の接着細胞、Ｋ５６２細胞等の浮遊系細胞、幹細胞、マクロファージ、ＴＨＰ－１、ＲＡＷ２６４．７等の血球系細胞、ミクログリア（Ｈｏｒｔｅｇａ細胞）等のグリア細胞、Ｓｆ９細胞、Ｓｆ２１細胞、ＳＦ＋細胞、Ｈｉｇｈ－Ｆｉｖｅ細胞、ＢｍＮ４細胞等の昆虫細胞がより好ましく、接着細胞及び浮遊系細胞が特に好ましい。本発明に係る細胞は、プライマリー（初代培養）細胞であっても、継代した細胞であってもよい。 The cells according to the present invention usually include eukaryotic cells, preferably higher eukaryotic cells, and are HeLa cells, 293T cells, HEK293 cells, CHO-K1 cells, A549 cells, COS7 cells, MCF7 cells, NIH3T3. Cells, L cells, HUVEC cells, HuH-7 cells, HCT-116 # 2Luc cells, HT-1080 cells, adherent cells such as U2OS cells, suspension cells such as K562 cells, stem cells, macrophages, THP-1, RAW264. More preferred are hematopoietic cells such as 7, glial cells such as microglia (Hortega cells), insect cells such as Sf9 cells, Sf21 cells, SF + cells, High-Five cells, BmN4 cells, and particularly preferred are adherent cells and suspension cells. . The cell according to the present invention may be a primary (primary culture) cell or a passaged cell.
　本発明に係る核酸としては、ゲノムＤＮＡ、１本鎖ＤＮＡ、２本鎖ＤＮＡ、プラスミドＤＮＡ等のＤＮＡ、ｍＲＮＡ、ｓｉＲＮＡ、ｉｓＲＮＡ（免疫賦活性ＲＮＡ）、ｔＲＮＡ、ｓｈＲＮＡ等のＲＮＡが挙げられ、ｍＲＮＡが好ましい。本発明に係る核酸は、任意の生物に由来するもの、人工的にデザインしたもののいずれであってもよい。本発明に係る核酸の塩基数は、通常１０塩基～３００００塩基、好ましくは５０塩基～２００００塩基、より好ましくは１００塩基～１００００塩基である。本発明に係る核酸を構成する塩基は、天然の塩基であっても、人工塩基であってもいずれであってもよく、天然の塩基が好ましい。本発明に係る核酸には、細胞中において治療用タンパク質や有用なタンパク質をコードして発現し得る核酸、細胞中において核酸の所望ではない発現を阻害する核酸、所望ではないタンパク質活性を阻害するか又は所望のタンパク質機能を活性化する核酸、反応を触媒する核酸（リボザイム）、及び診断アッセイにおいて機能する核酸（例えば、診断用核酸）が含まれる。 Examples of the nucleic acid according to the present invention include genomic DNA, single-stranded DNA, double-stranded DNA, DNA such as plasmid DNA, RNA such as mRNA, siRNA, isRNA (immunostimulatory RNA), tRNA, shRNA, and mRNA. Is preferred. The nucleic acid according to the present invention may be derived from any organism or artificially designed. The number of bases of the nucleic acid according to the present invention is usually 10 bases to 30000 bases, preferably 50 bases to 20000 bases, more preferably 100 bases to 10,000 bases. The base constituting the nucleic acid according to the present invention may be either a natural base, an artificial base or a natural base, and is preferably a natural base. The nucleic acids according to the present invention include nucleic acids that can encode and express therapeutic proteins and useful proteins in cells, nucleic acids that inhibit undesired expression of nucleic acids in cells, and those that inhibit undesired protein activity. Alternatively, nucleic acids that activate the desired protein function, nucleic acids that catalyze reactions (ribozymes), and nucleic acids that function in diagnostic assays (eg, diagnostic nucleic acids) are included.
　本発明に係る核酸の使用量は、特に限定されないが、例えば、本発明のトランスフェクション促進用ペプチド及びトランスフェクション薬剤の存在下で細胞と核酸とを接触させる際の使用量、言い換えれば、本発明のトランスフェクション促進用ペプチド、本発明に係るトランスフェクション薬剤、細胞及び核酸を含む溶液中の使用量としては、通常０．０００１μｇ～１００００ｍｇ、好ましくは０．００１μｇ～５０００ｍｇであり、例えばプレートを用いる場合、ウェルの面積１ｃｍ２あたり、通常０．００５μｇ～５００μｇ、好ましくは０．００７５μｇ～２５０μｇ、より好ましくは０．０１μｇ～７５μｇ、特に好ましくは０．１μｇ～１０μｇであり、例えばフラスコで用いる場合、培地１ｍＬあたり、通常０．００５μｇ～５００μｇ、好ましくは０．００７５μｇ～２５０μｇ、より好ましくは０．０１μｇ～７５μｇ、特に好ましくは０．１μｇ～１０μｇである。 The amount of the nucleic acid according to the present invention is not particularly limited. For example, the amount of the nucleic acid used when contacting the cell and the nucleic acid in the presence of the transfection promoting peptide and the transfection agent of the present invention, in other words, the present invention. The amount used in the solution containing the transfection-promoting peptide, the transfection agent according to the present invention, cells and nucleic acids is usually 0.0001 μg to 10,000 mg, preferably 0.001 μg to 5000 mg. For example, when using a plate Per well area of 1 cm 2 is usually 0.005 μg to 500 μg, preferably 0.0075 μg to 250 μg, more preferably 0.01 μg to 75 μg, particularly preferably 0.1 μg to 10 μg. Usually 0.005 μg to 50 per mL [mu] g, preferably 0.0075μg ~ 250μg, more preferably 0.01 [mu] g ~ 75 [mu] g, particularly preferably 0.1 [mu] g ~ 10 [mu] g.
　本発明のトランスフェクション促進用ペプチドの使用量は特に限定されないが、例えば、本発明のトランスフェクション促進用ペプチド、本発明に係るトランスフェクション薬剤、細胞及び核酸を含む溶液中の使用量としては、本発明に係る核酸１μｇ当たり、通常４μｇ～２０μｇ、好ましくは５μｇ～１５μｇ、より好ましくは８μｇ～１２μｇ、特に好ましくは９μｇ～１１μｇである。 The amount of the transfection promoting peptide of the present invention is not particularly limited. For example, the amount used in the solution containing the transfection promoting peptide of the present invention, the transfection agent according to the present invention, cells and nucleic acids is The amount is usually 4 μg to 20 μg, preferably 5 μg to 15 μg, more preferably 8 μg to 12 μg, particularly preferably 9 μg to 11 μg per 1 μg of nucleic acid according to the invention.
　本発明のトランスフェクション薬剤の使用量は、特に限定されないが、例えば、本発明のトランスフェクション促進用ペプチド、本発明に係るトランスフェクション薬剤、細胞及び核酸を含む溶液中の使用量としては、通常０．０４μｇ～２４００００μｇ、好ましくは０．０６μｇ～１２００００μｇであり、例えばプレートを用いる場合、ウェルの面積１ｃｍ２あたり、通常０．０５μｇ～１０μｇ、好ましくは０．１μｇ～７．５μｇ、より好ましくは０．２５μｇ～３μｇであり、例えばフラスコを用いる場合、培地１ｍＬあたり、通常０．０５μｇ～２４０００μｇ、好ましくは０．１μｇ～１２０００μｇ、より好ましくは０．２５μｇ～２４００μｇである。 The use amount of the transfection agent of the present invention is not particularly limited. For example, the use amount in a solution containing the transfection-promoting peptide of the present invention, the transfection agent according to the present invention, cells and nucleic acids is usually 0. .04μg ~ 240000μg, preferably 0.06μg ~ 120000μg, for example when using the plate, the area 1 cm 2 per well, usually 0.05 [mu] g ~ 10 [mu] g, preferably 0.1 [mu] g ~ 7.5 [mu] g, more preferably 0. For example, when a flask is used, it is usually 0.05 μg to 24000 μg, preferably 0.1 μg to 12000 μg, more preferably 0.25 μg to 2400 μg per 1 mL of the medium.
　本発明のトランスフェクション方法は、本発明のトランスフェクション促進用ペプチド及び本発明に係るトランスフェクション薬剤の存在下、インビトロで細胞と核酸とを接触させればよく、特に限定されない。
　このような方法としては、例えば、（工程１）核酸と本発明のトランスフェクション促進用ペプチドと本発明に係るトランスフェクション薬剤とを溶液中で混合してトランスフェクション溶液を調製し、（工程２）当該トランスフェクション溶液を、細胞を含む培地と混合して、細胞と核酸とを接触させてトランスフェクションを行う方法等が挙げられる。 The transfection method of the present invention is not particularly limited as long as cells and nucleic acids are brought into contact in vitro in the presence of the transfection promoting peptide of the present invention and the transfection agent of the present invention.
As such a method, for example, (Step 1) a nucleic acid, a peptide for promoting transfection of the present invention, and a transfection agent according to the present invention are mixed in a solution to prepare a transfection solution (Step 2). Examples include a method of mixing the transfection solution with a medium containing cells and bringing the cells into contact with a nucleic acid to effect transfection.
　工程１としては、例えば（工程１－ａ）核酸と本発明のトランスフェクション促進用ペプチドを含有する溶液と、本発明に係るトランスフェクション薬剤を含有する溶液とを混合させる方法、（工程１－ｂ）核酸と本発明に係るトランスフェクション薬剤を含有する溶液と、本発明のトランスフェクション促進用ペプチドを含有する溶液とを混合させる方法、（工程１－ｃ）核酸を含有する溶液と、本発明のトランスフェクション促進用ペプチドと本発明に係るトランスフェクション薬剤を含有する溶液とを混合させる方法、（工程１－ｄ）核酸を含有する溶液と、本発明のトランスフェクション促進用ペプチドを含有する溶液と、本発明に係るトランスフェクション薬剤を含有する溶液とを混合させる方法等が挙げられる。 Step 1 includes, for example, (Step 1-a) a method of mixing a solution containing the nucleic acid, the transfection-promoting peptide of the present invention, and a solution containing the transfection agent according to the present invention (Step 1-b). ) A method of mixing a solution containing the nucleic acid and the transfection agent according to the present invention with a solution containing the transfection promoting peptide of the present invention, (step 1-c) a solution containing the nucleic acid, A method of mixing a transfection promoting peptide and a solution containing the transfection agent according to the present invention, (step 1-d) a solution containing a nucleic acid, a solution containing the transfection promoting peptide of the present invention, Examples include a method of mixing a solution containing the transfection agent according to the present invention.
　工程１の核酸、本発明のトランスフェクション促進用ペプチド、及び／又は本発明に係るトランスフェクション薬剤を含有させる溶液（核酸と本発明のトランスフェクション促進用ペプチドを含有させる溶液、本発明に係るトランスフェクション薬剤を含有させる溶液、核酸と本発明に係るトランスフェクション薬剤を含有させる溶液、本発明のトランスフェクション促進用ペプチドを含有させる溶液、本発明のトランスフェクション促進用ペプチドと本発明に係るトランスフェクション薬剤を含有させる溶液、核酸を含有させる溶液（溶媒）等）としては、例えば精製水、ｐＨ５～７に緩衝作用を有する緩衝液（例えばＴＥ緩衝液、リン酸緩衝液、トリス緩衝液、グッド緩衝液、グリシン緩衝液、ホウ酸緩衝液、炭酸水素ナトリウム緩衝液、酢酸ナトリウム緩衝液等）が挙げられ、ｐＨ５～７に緩衝作用を有する緩衝液が好ましく、ＴＥ緩衝液及び酢酸ナトリウム緩衝液が特に好ましい。また、これらの緩衝液中の緩衝剤濃度としては、通常１ｍＭ～５００ｍＭ、好ましくは５～３００ｍＭの範囲から適宜選択される。また、この溶液中には、核酸、本発明のトランスフェクション促進用ペプチド、本発明に係るトランスフェクション薬剤等に凝集等の悪影響を及ぼさない量であれば、例えば糖類、ＮａＣｌ等の塩類、界面活性剤、防腐剤、蛋白質等が含まれていても良い。 Solution containing the nucleic acid of step 1, the transfection promoting peptide of the present invention, and / or the transfection agent according to the present invention (solution containing the nucleic acid and the transfection promoting peptide of the present invention, the transfection according to the present invention) A solution containing a drug, a solution containing a nucleic acid and a transfection agent according to the present invention, a solution containing a transfection promoting peptide of the present invention, a transfection promoting peptide of the present invention and a transfection agent according to the present invention Examples of the solution to be contained and the solution (solvent) containing the nucleic acid include purified water and a buffer having a buffering action at pH 5 to 7 (for example, TE buffer, phosphate buffer, Tris buffer, Good buffer, Glycine buffer, borate buffer, sodium bicarbonate buffer Sodium acetate buffer, etc.) can be mentioned, preferably a buffer solution having a buffering action in the pH 5 ~ 7, TE buffer and sodium acetate buffer is particularly preferred. Further, the concentration of the buffering agent in these buffers is appropriately selected from the range of usually 1 mM to 500 mM, preferably 5 to 300 mM. In this solution, the amount of the nucleic acid, the transfection-promoting peptide of the present invention, the transfection agent according to the present invention, etc., in an amount that does not adversely affect the aggregation, for example, sugars, salts such as NaCl, surface activity, etc. Agents, preservatives, proteins and the like may be included.
　要すれば、核酸と本発明のトランスフェクション促進用ペプチドと本発明に係るトランスフェクション薬剤とを溶液中で混合後、通常１５℃～３０℃、好ましくは１８℃～２８℃、より好ましくは２０℃～２６℃で通常１分～６０分、好ましくは３分～４０分、より好ましくは５分～２０分反応させる。
　なお、工程１の核酸、本発明のトランスフェクション促進用ペプチド及び／又は本発明に係るトランスフェクション薬剤を含有させる溶液（核酸と本発明のトランスフェクション促進用ペプチドを含有させる溶液、本発明に係るトランスフェクション薬剤を含有させる溶液、核酸と本発明に係るトランスフェクション薬剤を含有させる溶液、本発明のトランスフェクション促進用ペプチドを含有させる溶液、本発明のトランスフェクション促進用ペプチドと本発明に係るトランスフェクション薬剤を含有させる溶液、核酸を含有させる溶液（溶媒）等）中に含有させる核酸の量、本発明のトランスフェクション促進用ペプチドの量及び本発明に係るトランスフェクション薬剤の量は、本発明のトランスフェクション促進用ペプチド及びトランスフェクション薬剤の存在下で細胞と核酸とを接触させる際の使用量、言い換えれば、本発明のトランスフェクション促進用ペプチド、トランスフェクション薬剤、細胞及び核酸を含む溶液中の使用量が前述した範囲となるように適宜選択して、核酸、本発明のトランスフェクション促進用ペプチド及び／又は本発明に係るトランスフェション薬剤を含有させる溶液中に含有させればよい。 In short, the nucleic acid, the transfection promoting peptide of the present invention and the transfection agent of the present invention are mixed in a solution, and then usually 15 ° C. to 30 ° C., preferably 18 ° C. to 28 ° C., more preferably 20 ° C. The reaction is usually carried out at ˜26 ° C. for 1 minute to 60 minutes, preferably 3 minutes to 40 minutes, more preferably 5 minutes to 20 minutes.
In addition, the solution containing the nucleic acid of Step 1, the transfection promoting peptide of the present invention and / or the transfection agent according to the present invention (the solution containing the nucleic acid and the transfection promoting peptide of the present invention, the transfection promoting peptide of the present invention). A solution containing a transfection agent, a solution containing a nucleic acid and a transfection agent according to the present invention, a solution containing a transfection promoting peptide of the present invention, a transfection promoting peptide of the present invention and a transfection agent according to the present invention The amount of the nucleic acid to be contained in the solution containing the nucleic acid, the solution (solvent) containing the nucleic acid, the amount of the transfection-promoting peptide of the present invention, and the amount of the transfection agent of the present invention Accelerating peptides and tolans The amount used when the cell and the nucleic acid are contacted in the presence of the transfection agent, in other words, the amount used in the solution containing the peptide for promoting transfection of the present invention, the transfection agent, the cell and the nucleic acid is within the above-mentioned range. It may be appropriately selected so as to be contained in a solution containing the nucleic acid, the peptide for promoting transfection of the present invention and / or the transfection agent according to the present invention.
　工程１は、具体的には例えば以下の方法（上記工程１－ａ）により行えばよい。
　［２４ウェルプレートの１ウェル（２ｃｍ２）を用いて本発明の方法（工程２）を行う場合］
　まず、滅菌チューブに、ｐＨ５～７に緩衝作用を有する緩衝液（例えばＴＥ緩衝液、リン酸緩衝液、トリス緩衝液、グッド緩衝液、グリシン緩衝液、ホウ酸緩衝液、炭酸水素ナトリウム緩衝液、酢酸ナトリウム緩衝液等）４０μＬ、通常、０．０１μｇ～１０００μｇ、好ましくは０．０１５μｇ～５００μｇ、より好ましくは０．０２μｇ～１５０μｇ、特に好ましくは０．２μｇ～２０μｇの本発明に係る核酸、及び、通常０．０４μｇ～２００００μｇ、好ましくは０．０７５μｇ～７５００μｇ、より好ましくは０．１６μｇ～１８００μｇ、特に好ましくは１μｇ～２００μｇの本発明のトランスフェクション促進用ペプチドを添加する。
　次に、異なる滅菌チューブに、ｐＨ５～７に緩衝作用を有する緩衝液（例えばＴＥ緩衝液、リン酸緩衝液、トリス緩衝液、グッド緩衝液、グリシン緩衝液、ホウ酸緩衝液、炭酸水素ナトリウム緩衝液、酢酸ナトリウム緩衝液等）４０μＬ、及び通常０．１μｇ～２０μｇ、好ましくは０．２μｇ～１５μｇ、より好ましくは０．５μｇ～６μｇの本発明に係るトランスフェクション薬剤を添加する。
　次いで、上記の２本の滅菌チューブ中の溶液を混合して、要すれば通常１５℃～３０℃、好ましくは１８℃～２８℃、より好ましくは２０℃～２６℃で通常１分～６０分、好ましくは３分～４０分、より好ましくは５分～２０分反応させ、トランスフェクション溶液８０μＬを得る。 Specifically, step 1 may be performed, for example, by the following method (step 1-a above).
[When performing the method of the present invention (Step 2) using 1 well (2 cm 2 ) of a 24-well plate]
First, in a sterile tube, a buffer solution having a buffering action at pH 5 to 7 (for example, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer solution, Sodium acetate buffer, etc.) 40 μL, usually 0.01 μg-1000 μg, preferably 0.015 μg-500 μg, more preferably 0.02 μg-150 μg, particularly preferably 0.2 μg-20 μg, and Usually, 0.04 μg to 20000 μg, preferably 0.075 μg to 7500 μg, more preferably 0.16 μg to 1800 μg, particularly preferably 1 μg to 200 μg of the transfection-promoting peptide of the present invention is added.
Next, in a different sterilized tube, a buffer solution having a buffering action at pH 5 to 7 (eg, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer) Solution, sodium acetate buffer, etc.) 40 μL, and usually 0.1 μg-20 μg, preferably 0.2 μg-15 μg, more preferably 0.5 μg-6 μg of the transfection agent according to the present invention is added.
Next, the solutions in the above two sterilized tubes are mixed, and if necessary, usually 15 to 30 ° C, preferably 18 to 28 ° C, more preferably 20 to 26 ° C, usually 1 to 60 minutes. The reaction is preferably carried out for 3 to 40 minutes, more preferably 5 to 20 minutes to obtain 80 μL of the transfection solution.
　［培地１ｍＬを含有するフラスコを用いて本発明の方法（工程２）を行う場合］
　まず、滅菌チューブに、ｐＨ５～７に緩衝作用を有する緩衝液（例えばＴＥ緩衝液、リン酸緩衝液、トリス緩衝液、グッド緩衝液、グリシン緩衝液、ホウ酸緩衝液、炭酸水素ナトリウム緩衝液、酢酸ナトリウム緩衝液等）１００μＬ、通常０．００５μｇ～５００μｇ、好ましくは０．００７５μｇ～２５０μｇ、より好ましくは０．０１μｇ～７５μｇ、特に好ましくは０．１μｇ～１０μｇの本発明に係る核酸、及び、通常０．０２μｇ～１００００μｇ、好ましくは０．０３７５μｇ～３７５０μｇ、より好ましくは０．０８μｇ～９００μｇの本発明のトランスフェクション促進用ペプチドを添加する。
　次に、異なる滅菌チューブに、ｐＨ５～７に緩衝作用を有する緩衝液（例えばＴＥ緩衝液、リン酸緩衝液、トリス緩衝液、グッド緩衝液、グリシン緩衝液、ホウ酸緩衝液、炭酸水素ナトリウム緩衝液、酢酸ナトリウム緩衝液等）１００μＬ、及び、通常０．０５μｇ～２４０００μｇ、好ましくは０．１μｇ～１２０００μｇ、より好ましくは０．２５μｇ～２４００μｇの本発明に係るトランスフェクション薬剤を添加する。
　次いで、上記の２本の滅菌チューブ中の溶液を混合して、通常１５℃～３０℃、好ましくは１８℃～２８℃、より好ましくは２０℃～２６℃で通常１分～６０分、好ましくは３分～４０分、より好ましくは５分～２０分反応させ、トランスフェクション溶液２００μＬを得る。 [When performing the method of the present invention (Step 2) using a flask containing 1 mL of medium]
First, in a sterile tube, a buffer solution having a buffering action at pH 5 to 7 (for example, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer solution, Sodium acetate buffer, etc.) 100 μL, usually 0.005 μg to 500 μg, preferably 0.0075 μg to 250 μg, more preferably 0.01 μg to 75 μg, particularly preferably 0.1 μg to 10 μg, and usually 0.02 μg to 10000 μg, preferably 0.0375 μg to 3750 μg, more preferably 0.08 μg to 900 μg of the transfection promoting peptide of the present invention is added.
Next, in a different sterilized tube, a buffer solution having a buffering action at pH 5 to 7 (eg, TE buffer solution, phosphate buffer solution, Tris buffer solution, Good buffer solution, glycine buffer solution, borate buffer solution, sodium bicarbonate buffer) Solution, sodium acetate buffer, etc.) 100 μL and usually 0.05 μg to 24000 μg, preferably 0.1 μg to 12000 μg, more preferably 0.25 μg to 2400 μg of the transfection agent according to the present invention are added.
Next, the solutions in the above two sterilized tubes are mixed, and usually 15 ° C to 30 ° C, preferably 18 ° C to 28 ° C, more preferably 20 ° C to 26 ° C, usually 1 minute to 60 minutes, preferably The reaction is carried out for 3 to 40 minutes, more preferably 5 to 20 minutes to obtain 200 μL of the transfection solution.
　工程２は、工程１により得られたトランスフェクション溶液を、細胞を含む培地と混合して、細胞と核酸とを接触させて細胞のトランスフェクションを行うことによりなされる。 Step 2 is performed by mixing the transfection solution obtained in Step 1 with a medium containing cells and bringing the cells into contact with nucleic acids to transfect the cells.
　本発明に係る細胞の数（細胞数）としては、通常この分野においてトランスフェクションを行う際に用いられる細胞数であればよく、特に限定されないが、例えば、工程１により得られたトランスフェクション溶液を、細胞を含む培地と混合する際の細胞数、言い換えれば、細胞と核酸とを接触させてトランスフェクションを行う際の細胞数としては、通常１×１０２細胞～１×１０８細胞、好ましくは１×１０３細胞～２×１０８細胞であり、プレートやフラスコ等の培地を含有させる容器に応じて適宜選択される。
　例えばプレートを用いて工程２を行う場合、ウェルの面積１ｃｍ２あたり、本発明に係る細胞数は、通常０．１×１０５細胞～５×１０５細胞、好ましくは０．２×１０５細胞～３×１０５細胞、より好ましくは０．３×１０５細胞～２×１０５細胞である。
　例えばフラスコを用いて工程２を行う場合、本発明に係る細胞数は、培地１ｍＬあたり、通常０．１×１０５細胞～通常３０×１０５細胞、好ましくは０．５×１０５細胞～１５×１０５細胞、より好ましくは１×１０５細胞～１０×１０５細胞である。 The number of cells according to the present invention (the number of cells) is not particularly limited as long as it is the number of cells usually used for transfection in this field. For example, the transfection solution obtained in step 1 The number of cells when mixed with a medium containing cells, in other words, the number of cells when transfection is carried out by bringing cells into contact with nucleic acids is usually from 1 × 10 2 cells to 1 × 10 8 cells, preferably It is 1 × 10 3 cells to 2 × 10 8 cells, and is appropriately selected according to a container containing a medium such as a plate or a flask.
For example, when performing step 2 using a plate, the number of cells according to the present invention is usually 0.1 × 10 5 cells to 5 × 10 5 cells, preferably 0.2 × 10 5 cells per 1 cm 2 of the well area. ˜3 × 10 5 cells, more preferably 0.3 × 10 5 cells to 2 × 10 5 cells.
For example, when performing step 2 using a flask, the number of cells according to the present invention is usually 0.1 × 10 5 cells to usually 30 × 10 5 cells, preferably 0.5 × 10 5 cells to 15 per 1 mL of the medium. × 10 5 cells, more preferably 1 × 10 5 cells to 10 × 10 5 cells.
　工程２における培地は、通常この分野で用いられるものであれば特に限定されず、細胞毎に適切な培地を選択すればよい。工程２における培地は、トランスフェクションを阻害しないものであれば、例えば、ＦＢＳ等の血清、アルブミン、トランスフェリン、Ｋｎｏｃｋｏｕｔ　Ｓｅｒｕｍ　Ｒｅｐｌａｃｅｍｅｎｔ（ＫＳＲ）、Ｎ２サプリメント（ｉｎｖｉｔｒｏｇｅｎ）、Ｂ２７サプリメント（ｉｎｖｉｔｒｏｇｅｎ）、脂肪酸、インスリン、コラーゲン前駆体、微量元素、２－メルカプトエタノール、３’－チオールグリセロール等の血清代替物、脂質、アミノ酸、非必須アミノ酸、ビタミン、増殖因子、サイトカイン、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類等を含んでいてもよく、これらの選択可否はトランスフェクション薬剤に依存する。
　また、工程２における培地の量は、通常この分野においてトランスフェクションを行う際に用いられる培地量であればよく、プレートやフラスコ等の培地を含有させる容器に応じて適宜選択される。 The medium in step 2 is not particularly limited as long as it is usually used in this field, and an appropriate medium may be selected for each cell. As long as the medium in step 2 does not inhibit transfection, for example, serum such as FBS, albumin, transferrin, Knockout Serum Replacement (KSR), N2 supplement (invitrogen), B27 supplement (invitrogen), fatty acid, insulin, Serum substitutes such as collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, non-essential amino acids, vitamins, growth factors, cytokines, antibiotics, antioxidants, pyruvate, buffers In addition, inorganic salts and the like may be included, and the selection of these depends on the transfection agent.
Further, the amount of the medium in Step 2 may be any medium amount that is usually used when transfection is performed in this field, and is appropriately selected depending on the container containing the medium such as a plate or a flask.
　例えばプレートを用いて工程２を行う場合、ウェルの面積１ｃｍ２あたり、培地の量は通常１００μＬ～１０００μＬ、好ましくは１５０μＬ～８００μＬ、より好ましくは２００μＬ～６００μＬである。例えばフラスコを用いて工程２を行う場合、培地の量は通常１ｍＬ～１０００Ｌ、好ましくは２０ｍＬ～５００Ｌ、より好ましくは３０ｍＬ～１００Ｌである。 For example, when performing step 2 using a plate, the amount of the medium per 1 cm 2 of the well area is usually 100 μL to 1000 μL, preferably 150 μL to 800 μL, more preferably 200 μL to 600 μL. For example, when performing step 2 using a flask, the amount of the medium is usually 1 mL to 1000 L, preferably 20 mL to 500 L, more preferably 30 mL to 100 L.
　工程２における、工程１により得られたトランスフェクション溶液の使用量は、通常この分野においてトランスフェクションを行う際に用いられるトランスフェクション溶液の量であればよく、通常０．００１ｍＬ～１０００ｍＬ、好ましくは０．０１ｍＬ～５００ｍＬ、より好ましくは０．０５ｍＬ～１００ｍＬであり、プレートやフラスコ等の培地を含有させる容器に応じて適宜選択される。
　例えばプレートを用いて工程２を行う場合、トランスフェクション溶液の量はウェルの面積１ｃｍ２あたり、通常１μＬ～２００μＬ、好ましくは５μＬ～１８０μＬ、より好ましくは１０μＬ～１５０μＬである。
　例えばフラスコを用いて工程２を行う場合、トランスフェクション溶液の量は、培地１ｍＬあたり、通常１０μＬ～１０００μＬ、好ましくは５０μＬ～６００μＬ、より好ましくは１００μＬ～３００μＬである。 The amount of the transfection solution obtained in step 1 in step 2 may be the amount of the transfection solution usually used for transfection in this field, and is usually 0.001 mL to 1000 mL, preferably 0. It is 0.01 mL to 500 mL, more preferably 0.05 mL to 100 mL, and is appropriately selected according to the container containing the medium such as a plate or a flask.
For example, when performing step 2 using a plate, the amount of the transfection solution is usually 1 μL to 200 μL, preferably 5 μL to 180 μL, more preferably 10 μL to 150 μL per 1 cm 2 of the well area.
For example, when performing Step 2 using a flask, the amount of the transfection solution is usually 10 μL to 1000 μL, preferably 50 μL to 600 μL, more preferably 100 μL to 300 μL per 1 mL of the medium.
　工程２におけるトランスフェクションは、核酸が細胞内に導入されることによりなされる。具体的には、工程１により得られたトランスフェクション溶液を培地中の細胞に添加後、通常２５℃～３９℃、好ましくは３５℃～３８℃で通常１時間～２日、好ましくは６時間～１日培養する方法が挙げられる。 Transfection in step 2 is performed by introducing nucleic acid into the cell. Specifically, after the transfection solution obtained in step 1 is added to the cells in the medium, it is usually 25 ° C. to 39 ° C., preferably 35 ° C. to 38 ° C., usually 1 hour to 2 days, preferably 6 hours to One day culture method is mentioned.
　工程２は、具体的には例えば以下の方法により行えばよい。
　［２４プレートの１ウェル（２ｃｍ２）を用いて工程２を行う場合］
　通常０．２×１０５細胞～１０×１０５細胞、好ましくは０．４×１０５細胞～６×１０５細胞、より好ましくは０．６×１０５細胞～４×１０５細胞を含有する、通常２００μＬ～２０００μＬ、好ましくは３００μＬ～１６００μＬ、より好ましくは４００μＬ～１２００μＬの培地に、通常２μＬ～４００μＬ、好ましくは１０μＬ～３６０μＬ、より好ましくは２０μＬ～３００μＬの工程１で調製したトランスフェクション溶液（通常０．０１μｇ～１０００μｇ、好ましくは０．０１５μｇ～５００μｇ、より好ましくは０．０２μｇ～１５０μｇ、特に好ましくは０．２μｇ～２０μｇの本発明に係る核酸、通常０．０４μｇ～２００００μｇ、好ましくは０．０７５μｇ～７５００μｇ、より好ましくは０．１６μｇ～１８００μｇ、特に好ましくは１μｇ～２００μｇの本発明のトランスフェクション促進用ペプチド、及び、通常０．１μｇ～２０μｇ、好ましくは０．２μｇ～１５μｇ、より好ましくは０．５μｇ～６μｇの本発明に係るトランスフェクション薬剤をｐＨ５～７に緩衝作用を有する緩衝液中に含有する溶液）を添加し、通常２５℃～３９℃、好ましくは３５℃～３８℃で通常１時間～２日、好ましくは６時間～１日培養することによりなされる。 Specifically, step 2 may be performed by the following method, for example.
[When step 2 is performed using 1 well (2 cm 2 ) of 24 plates]
Usually contains 0.2 × 10 5 cells to 10 × 10 5 cells, preferably 0.4 × 10 5 cells to 6 × 10 5 cells, more preferably 0.6 × 10 5 cells to 4 × 10 5 cells Usually, 200 μL to 2000 μL, preferably 300 μL to 1600 μL, more preferably 400 μL to 1200 μL medium, usually 2 μL to 400 μL, preferably 10 μL to 360 μL, more preferably 20 μL to 300 μL of the transfection solution prepared in Step 1 (usually 0.01 μg to 1000 μg, preferably 0.015 μg to 500 μg, more preferably 0.02 μg to 150 μg, particularly preferably 0.2 μg to 20 μg of the nucleic acid according to the present invention, usually 0.04 μg to 20000 μg, preferably 0.075 μg ~ 7500 μg, more preferably 0.16 μg to 1800 μg, particularly preferred 1 to 200 μg of the transfection-promoting peptide of the present invention, and usually 0.1 μg to 20 μg, preferably 0.2 μg to 15 μg, more preferably 0.5 μg to 6 μg of the transfection agent according to the present invention at pH 5 To 7 to a solution containing a buffer having a buffering action), and is usually cultured at 25 ° C. to 39 ° C., preferably 35 ° C. to 38 ° C. for 1 hour to 2 days, preferably 6 hours to 1 day. Is made by
　［培地１ｍＬを含有するフラスコを用いて工程２を行う場合］
　通常０．１×１０５細胞～通常３０×１０５細胞、好ましくは０．５×１０５細胞～１５×１０５細胞、より好ましくは１×１０５細胞～１０×１０５細胞を含有する、通常１０μＬ～１０００μＬ、好ましくは５０μＬ～６００μＬ、より好ましくは１００μＬ～３００μＬの工程１で調製したトランスフェクション溶液（通常０．００５μｇ～５００μｇ、好ましくは０．００７５μｇ～２５０μｇ、より好ましくは０．０１μｇ～７５μｇ、特に好ましくは０．１μｇ～１０μｇの本発明に係る核酸、通常０．０２μｇ～１００００μｇ、好ましくは０．０３７５μｇ～３７５０μｇ、より好ましくは０．０８μｇ～９００μｇの本発明のトランスフェクション促進用ペプチド、及び、通常０．０５μｇ～２４０００μｇ、好ましくは０．１μｇ～１２０００μｇ、より好ましくは０．２５μｇ～２４００μｇの本発明に係るトランスフェクション薬剤を、ｐＨ５～７に緩衝作用を有する緩衝液中に含有する溶液）を添加し、通常２５℃～３９℃、好ましくは３５℃～３８℃で通常１時間～２日、好ましくは６時間～１日することによりなされる。 [When performing step 2 using a flask containing 1 mL of medium]
Usually 0.1 × 10 5 cells to usually 30 × 10 5 cells, preferably 0.5 × 10 5 cells to 15 × 10 5 cells, more preferably 1 × 10 5 cells to 10 × 10 5 cells. Usually 10 μL to 1000 μL, preferably 50 μL to 600 μL, more preferably 100 μL to 300 μL of the transfection solution prepared in Step 1 (usually 0.005 μg to 500 μg, preferably 0.0075 μg to 250 μg, more preferably 0.01 μg to 75 μg Particularly preferably 0.1 μg to 10 μg of the nucleic acid according to the present invention, usually 0.02 μg to 10000 μg, preferably 0.0375 μg to 3750 μg, more preferably 0.08 μg to 900 μg of the transfection-promoting peptide of the present invention, and In general, 0.05 μg to 24000 μg, preferably 0.1 μg to 12 000 μg, more preferably 0.25 μg to 2400 μg of the transfection agent according to the present invention in a buffer solution having a buffering action at pH 5 to 7, and usually 25 ° C. to 39 ° C., preferably 35 It is usually carried out at 1 to 2 ° C. for 1 hour to 2 days, preferably 6 hours to 1 day.
　本発明のトランスフェクション用キットは、上記の如き本発明のトランスフェクション方法に用いられるもので、本発明のトランスフェクション促進用ペプチドと、本発明に係るトランスフェクション薬剤とを含んでなるものであり、夫々の構成要素の好ましい態様、具体例については上で述べたとおりである。 The transfection kit of the present invention is used for the transfection method of the present invention as described above, and comprises the transfection promoting peptide of the present invention and the transfection agent of the present invention, Preferred embodiments and specific examples of each component are as described above.
　本発明のトランスフェクション用キットに含まれる本発明のトランスフェクション促進用ペプチドは、凍結乾燥した固体状態であっても、水やｐＨ５～７に緩衝作用を有する緩衝液（例えばＴＥ緩衝液、リン酸緩衝液、トリス緩衝液、グッド緩衝液、グリシン緩衝液、ホウ酸緩衝液、炭酸水素ナトリウム緩衝液、酢酸ナトリウム緩衝液等）に含有させた液体状態であってもいずれでもよい。液体状態の場合、その濃度は通常０．５ｍｇ／ｍＬ～３０ｍｇ／ｍＬ、好ましくは２ｍｇ／ｍＬ～１０ｍｇ／ｍＬである。 The transfection-promoting peptide of the present invention contained in the transfection kit of the present invention is a buffer solution having a buffering action in water or pH 5-7 (for example, TE buffer solution, phosphate, etc.) even in a lyophilized solid state. Buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, sodium bicarbonate buffer, sodium acetate buffer, etc.) In the liquid state, the concentration is usually 0.5 mg / mL to 30 mg / mL, preferably 2 mg / mL to 10 mg / mL.
　また、これらキットには、通常この分野で用いられる試薬類、例えば緩衝剤、増感剤、界面活性剤、防腐剤（例えばアジ化ナトリウム、サリチル酸、安息香酸等）、安定化剤（例えばアルブミン、グロブリン、水溶性ゼラチン、界面活性剤、糖類等）、賦活剤、共存物質の影響回避剤、その他この分野で用いられているものであって、共存する試薬との安定性を阻害したりしないものを有していてもよい。またこれら試薬類等の濃度範囲等も、各々の試薬類が有する効果を発揮するために通常用いられる濃度範囲等を適宜選択して用いればよい。 These kits include reagents usually used in this field, such as buffers, sensitizers, surfactants, preservatives (eg, sodium azide, salicylic acid, benzoic acid, etc.), stabilizers (eg, albumin, Globulins, water-soluble gelatins, surfactants, saccharides, etc.), activators, coexisting substance avoidance agents, and others used in this field that do not interfere with the stability of coexisting reagents You may have. In addition, the concentration range or the like of these reagents may be appropriately selected from the concentration range or the like that is usually used for exhibiting the effects of each reagent.
　さらにまた、本発明のキットには、本発明のトランスフェクション方法の説明書等を含ませておいても良い。当該「説明書」とは、当該方法における特徴・原理・操作手順、判定手順等が文章又は図表等により実質的に記載されている当該キットの取扱説明書、添付文書、あるいはパンフレット（リーフレット）等を意味する。 Furthermore, the kit of the present invention may contain instructions for the transfection method of the present invention. The “instructions” are the instruction manuals, package inserts, pamphlets (leaflets), etc. of the kits in which the features, principles, operation procedures, judgment procedures, etc. of the method are substantially described in text or diagrams. Means.
　以下に実施例、参考例等により本発明を更に詳細に説明するが、本発明はこれら実施例等により何等限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited to these Examples and the like.
　以下の実施例及び比較例で用いた（エンハンサー）ペプチドの配列を以下に示す。
　Ｃ２：ＲＲＲＲＲＲＲＲＲＲＲＧＧＣＧＹＧ（配列番号２）
　ＥＳＦＡ４：ＲＲＲＲＲＲＲＲＲＲＲＧＧＣＧＹＧＰＫＫＫＲＫＶＧＧ（配列番号３）
　Ｃ５：ＧＧＣＧＹＧＲＲＲＲＲＲＲＲＲＲＲ（配列番号５）
　Ｃ２＿ＡＡＣＡＡ：ＲＲＲＲＲＲＲＲＲＲＲＡＡＣＡＡＡ（配列番号６）
　Ｋ１１：ＫＫＫＫＫＫＫＫＫＫＫＧＧＣＧＹＧ（配列番号７）
　Ｒ９：ＲＲＲＲＲＲＲＲＲＧＧＣＧＹＧ（配列番号８）
　Ｒ１６：ＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＧＧＣＧＹＧ（配列番号９）
　ＲＫ１２：ＲＫＲＫＲＫＲＫＲＫＲＫＧＧＣＧＹＧ（配列番号１０）
　Ｃ４：ＲＲＲＲＲＲＲＲＲＲＲ（配列番号１１）
　Ｃ２＿Ｃ１４Ａ：ＲＲＲＲＲＲＲＲＲＲＲＧＧＡＧＹＧ（配列番号１２）
　Ｃ２＿Ａ６：ＲＲＲＲＲＲＲＲＲＲＲＡＡＡＡＡＡ（配列番号１３）
　Ｒ３：ＲＲＲＧＧＣＧＹＧ（配列番号１４）
　Ｒ６：ＲＲＲＲＲＲＧＧＣＧＹＧ（配列番号１５）
　Ｒ１９：ＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＧＧＣＧＹＧ（配列番号１６）
　Ｒ２２：ＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＧＧＣＧＹＧ（配列番号１７）
　Ｒ３０：ＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＲＧＧＣＧＹＧ（配列番号１８）
　Ｈ１１：ＨＨＨＨＨＨＨＨＨＨＨＧＧＣＧＹＧ（配列番号１９） The sequences of (enhancer) peptides used in the following examples and comparative examples are shown below.
C2: RRRRRRRRRRRRRGCGYG (SEQ ID NO: 2)
ESFA4: RRRRRRRRRRRRGGCCGYGPKKRKVGG (SEQ ID NO: 3)
C5: GGCGYGRRRRRRRRRRR (SEQ ID NO: 5)
C2_AACAA: RRRRRRRRRRRAACAAAA (SEQ ID NO: 6)
K11: KKKKKKKKKKKKGGCGYG (SEQ ID NO: 7)
R9: RRRRRRRRRRGGCGYG (SEQ ID NO: 8)
R16: RRRRRRRRRRRRRRRRRRGGCGYG (SEQ ID NO: 9)
RK12: RKRKRKRKRKRKGGCGYG (SEQ ID NO: 10)
C4: RRRRRRRRRRRR (SEQ ID NO: 11)
C2_C14A: RRRRRRRRRRRRGGAGG (SEQ ID NO: 12)
C2_A6: RRRRRRRRRRRAAAAAA (SEQ ID NO: 13)
R3: RRRGGCGYG (SEQ ID NO: 14)
R6: RRRRRRGGGCG (SEQ ID NO: 15)
R19: RRRRRRRRRRRRRRRRRRRRRGGCGYG (SEQ ID NO: 16)
R22: RRRRRRRRRRRRRRRRRRRRRRRRRGGCGYG (SEQ ID NO: 17)
R30: RRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRGGCGYG (SEQ ID NO: 18)
H11: HHHHHHHHHHHGGGCGY (SEQ ID NO: 19)
実施例１．エンハンサーペプチドＣ２のトランスフェクション促進効果の検証
　以下の方法により、下記表１に記載の実験条件で、ＣＯＳ７細胞にＹＦＰ－Ｈ２Ｂ遺伝子を有するプラスミドＤＮＡをトランスフェクションし、エンハンサーペプチドであるＣ２のトランスフェクション促進効果について確認した。 Example 1. Verification of transfection promoting effect of enhancer peptide C2 By the following method, COS7 cells were transfected with plasmid DNA having YFP-H2B gene under the experimental conditions described in Table 1 below, and transfection promotion of enhancer peptide C2 was promoted. The effect was confirmed.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
（１）細胞の調製
　ＣＯＳ７細胞をＤ－ＭＥＭ　１０％ＦＢＳ培地（和光純薬工業（株）製）を用いて、１×１０５個以上の細胞が得られるまで培養し、培養細胞を得た。 (1) Preparation of cells COS7 cells were cultured using D-MEM 10% FBS medium (manufactured by Wako Pure Chemical Industries, Ltd.) until 1 × 10 5 cells or more were obtained to obtain cultured cells. .
（２）遺伝子導入
　（ｉ）トランスフェクション溶液の調製
　まず、配列番号１に記載のＹＦＰ－Ｈ２Ｂ遺伝子を有するプラスミドＤＮＡ（Ｉｎｃｅｌｌａ社製）を、０．５ｍｇ／ｍＬとなるようにＴＥ　ｂｕｆｆｅｒ（１０ｍＭ　Ｔｒｉｓ－ＨＣｌ，　１ｍＭ　ＥＤＴＡ　ｐＨ８．０）で懸濁し、プラスミドＤＮＡ溶液を調製した。
　次いで、Ｃ２（（株）東レリサーチにて合成）を、トランスフェクション薬剤であるＳｃｒｅｅｎＦｅｃｔＴＭＡ　Ｐｌｕｓ（和光純薬工業（株）製）に付属のＤｉｌｕｔｉｏｎ　ｂｕｆｆｅｒ（以下、Ｄｉｌｕｔｉｏｎ　ｂｕｆｆｅｒと略記する場合がある）に５ｍｇ／ｍＬとなるように溶解し、エンハンサーペプチド溶液を調製した。 (2) Gene Introduction (i) Preparation of Transfection Solution First, a plasmid DNA having a YFP-H2B gene described in SEQ ID NO: 1 (manufactured by Incella) was added to TE buffer (10 mM Tris) so as to be 0.5 mg / mL. -Suspended with HCl, 1 mM EDTA pH 8.0) to prepare a plasmid DNA solution.
Next, C2 (synthesized by Toray Research Co., Ltd.) may be abbreviated as “Dilution buffer” (hereinafter referred to as “Dilution buffer”) attached to ScreenFect ™ A Plus (manufactured by Wako Pure Chemical Industries, Ltd.), a transfection agent. ) To 5 mg / mL to prepare an enhancer peptide solution.
　そして、滅菌したチューブを２本用意し、一本のチューブにＤｉｌｕｔｉｏｎ　ｂｕｆｆｅｒを２３．５μＬ添加し、ＳｃｒｅｅｎＦｅｃｔＴＭＡ　Ｐｌｕｓ（和光純薬工業（株）製）に付属のＳｃｒｅｅｎＦｅｃｔ　Ａ　Ｐｌｕｓ　ｒｅａｇｅｎｔ（以下、ＳｃｒｅｅｎＦｅｃｔ　Ａ　Ｐｌｕｓ　ｒｅａｇｅｎｔと略記する場合がある）を１．５μＬ（プラスミドＤＮＡ量１μｇあたりＳｃｒｅｅｎＦｅｃｔ　Ａ　Ｐｌｕｓ　ｒｅａｇｅｎｔ３μＬ）添加した。 Then, two sterilized tubes are prepared, 23.5 μL of Dilution buffer is added to one tube, and ScreenFect A Plus reagent (hereinafter referred to as ScreenFect) attached to ScreenFect ™ A Plus (manufactured by Wako Pure Chemical Industries, Ltd.). 1.5 μL (sometimes abbreviated as A Plus reagent) (ScreenFect A Plus reagent 3 μL per 1 μg of plasmid DNA) was added.
　もう一本の滅菌済みチューブに、プラスミドＤＮＡ量が０．５μｇになるようプラスミドＤＮＡ溶液１μＬを添加し、更にエンハンサーペプチド溶液を１μＬ（プラスミドＤＮＡ量１μｇあたりエンハンサーペプチド溶液２μＬ）添加し、Ｄｉｌｕｔｉｏｎ　ｂｕｆｆｅｒを添加して、最終２５μＬになるように調製した。
　次いで、上記の２本のチューブを混合して室温で１５分反応させ、トランスフェクション溶液を得た。 To another sterilized tube, add 1 μL of plasmid DNA solution so that the amount of plasmid DNA is 0.5 μg, add 1 μL of enhancer peptide solution (2 μL of enhancer peptide solution per 1 μg of plasmid DNA), and add Dilution buffer. Added to a final volume of 25 μL.
Next, the above two tubes were mixed and reacted at room temperature for 15 minutes to obtain a transfection solution.
　（ｉｉ）細胞懸濁液の調製
　（１）で得られた培養細胞を用いて、以下の通り細胞懸濁液を調製した。
　培養容器から培地を除き、トリプシンＥＤＴＡ溶液［０．２５ｗ／ｖ％　トリプシン－１ｍｍｏｌ／ｌ　ＥＤＴＡ・４Ｎａ溶液（フェノールレッド含有）］を培養容器に添加して細胞を培養容器から剥離し、懸濁液を得た。遠沈管に当該懸濁液を添加後、遠心分離処理を行い（１０００ｒｐｍ，５分）、細胞を沈降させた。上清を除いた後、細胞ペレットに適当量培地を加え、細胞懸濁液を得た。当該細胞懸濁液中の接着細胞の細胞数をＴＣ２０全自動セルカウンター（Ｂｉｏ－Ｒａｄ社製）により測定し、細胞懸濁液中の細胞の濃度を算出した。 (Ii) Preparation of cell suspension Using the cultured cells obtained in (1), a cell suspension was prepared as follows.
Remove the medium from the culture vessel, add trypsin EDTA solution [0.25 w / v% trypsin-1 mmol / l EDTA · 4Na solution (containing phenol red)] to the culture vessel to detach the cells from the culture vessel, Got. After adding the suspension to the centrifuge tube, centrifugation was performed (1000 rpm, 5 minutes) to sediment the cells. After removing the supernatant, an appropriate amount of medium was added to the cell pellet to obtain a cell suspension. The number of adherent cells in the cell suspension was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad), and the concentration of the cells in the cell suspension was calculated.
　（ｉｉｉ）トランスフェクション
　まず、２４ウェルプレートの１つのウェルに以下のものをそれぞれ添加した。
　・Ｄ－ＭＥＭ培地（和光純薬工業（株）製）４５０μＬ
　・１×１０５個の細胞が存在する量の（ｉｉ）で調製した細胞懸濁液
　次いで、（ｉ）で調製したトランスフェクション溶液を５０μＬ滴下し、ゆっくりと攪拌混合後、３７℃のＣＯ２インキュベーターで一晩細胞を培養した。 (Iii) Transfection First, the following were respectively added to one well of a 24-well plate.
-D-MEM medium (Wako Pure Chemical Industries, Ltd.) 450 μL
Cell suspension prepared in (ii) in an amount of 1 × 10 5 cells Next, 50 μL of the transfection solution prepared in (i) was dropped, and after gently stirring and mixing, CO 2 at 37 ° C. Cells were cultured overnight in an incubator.
（３）蛍光顕微鏡観察
　（２）（ｉｉｉ）で一晩培養した細胞中のＹＦＰ－Ｈ２Ｂ（ＹＦＰ－ｈｕｍａｎ　Ｈｉｓｔｏｎｅ　Ｈ２Ｂ）の蛍光強度（発現強度）を、蛍光顕微鏡（Ａｘｉｏ　ｏｂｓｅｖｅｒ　Ｚ１、ＺＥＩＳＳ社製）により観察した。その結果を、図１のＳＦＡ　Ｐｌｕｓ＋Ｃ２に示す。図中、ＹＦＰは導入したプラスミドＤＮＡの発現レベルをＹＦＰ－Ｈ２Ｂの蛍光により測定した結果を表し、ＤＩＣ（微分干渉顕微鏡像）は細胞の状態を表す。 (3) Fluorescence microscope observation (2) The fluorescence intensity (expression intensity) of YFP-H2B (YFP-human Histone H2B) in the cells cultured overnight in (iii) was measured with a fluorescence microscope (Axio Obsever Z1, manufactured by ZEISS) Was observed. The result is shown as SFA Plus + C2 in FIG. In the figure, YFP represents the result of measuring the expression level of the introduced plasmid DNA by the fluorescence of YFP-H2B, and DIC (differential interference microscope image) represents the state of the cells.
（４）フローサイトメーターを用いた蛍光強度比較
　（３）で顕微鏡観察を行ったウェルから培地をアスピレーターで除いた。培地を除いたウェルにＰＢＳを適量添加し、再度アスピレーターでＰＢＳを除いて洗浄を行った。その後、トリプシンＥＤＴＡ溶液（０．２５ｗ／ｖ％　トリプシン－１ｍｍｏｌ／ｌ　ＥＤＴＡ・４Ｎａ溶液（フェノールレッド含有））１５０μＬを、洗浄後のウェルに添加した。トリプシンＥＤＴＡ溶液をアスピレーターで除き、ＣＯ２インキュベーターで５分間反応させた。反応後のウェルにｓｏｒｔｉｎｇ　ｂｕｆｆｅｒ（１ｘＤ－ＰＢＳ（－），　１ｍＭ　ＥＤＴＡ，　１％ＢＳＡ）を３００μＬ添加し、ピペティングにより細胞を分散させ、ストレーナーサンプルチューブに細胞を回収してｓｏｒｔｉｎｇサンプルを得た。
　フローサイトメーター（Ｇａｌｌｉｏｓ、Ｂｅｃｋｍａｎ　Ｃｏｕｌｔｅｒ社製）を用いて、ｓｏｒｔｉｎｇサンプル中のＹＦＰ－Ｈ２Ｂの蛍光強度（発現強度）を測定し、非トランスフェクション細胞の蛍光強度に対する相対蛍光強度を算出した。
　その結果とエンハンサーペプチドを用いずにトランスフェクションして測定した結果（後述の比較例１－２）との比較を下記表５に示す。 (4) Comparison of fluorescence intensity using a flow cytometer The medium was removed with an aspirator from the well observed with a microscope in (3). An appropriate amount of PBS was added to the well from which the medium had been removed, and the plate was again washed with an aspirator. Thereafter, 150 μL of trypsin EDTA solution (0.25 w / v% trypsin-1 mmol / l EDTA · 4Na solution (containing phenol red)) was added to the well after washing. The trypsin EDTA solution was removed with an aspirator and allowed to react for 5 minutes in a CO 2 incubator. 300 μL of sorting buffer (1 × D-PBS (−), 1 mM EDTA, 1% BSA) was added to the well after the reaction, the cells were dispersed by pipetting, and the cells were collected in a strainer sample tube to obtain a sorting sample.
Using a flow cytometer (Gallios, manufactured by Beckman Coulter), the fluorescence intensity (expression intensity) of YFP-H2B in the sorting sample was measured, and the relative fluorescence intensity relative to the fluorescence intensity of the non-transfected cells was calculated.
Table 5 below shows a comparison between the results and the results obtained by transfection without using the enhancer peptide (Comparative Example 1-2 described later).
実施例２－１６．各種細胞におけるエンハンサーペプチドＣ２のトランスフェクション促進効果の検証
　下記表２に記載の条件（細胞、プラスミドＤＮＡ量、播種細胞数、培地、エンハンサーペプチド、エンハンサーペプチドの量）で、実施例１と同様の方法によりＹＦＰ－Ｈ２Ｂ遺伝子を有するプラスミドＤＮＡを細胞にトランスフェクションし、蛍光顕微鏡観察及びフローサイトメーターを用いた蛍光強度比較により、エンハンサーペプチドＣ２のトランスフェクション促進効果を確認した。
　浮遊細胞Ｋ５６２を用いた実施例７においては、「（２）遺伝子導入（ｉｉ）細胞懸濁液の調製」を、実施例１の方法に代えて以下のように行った。即ち、（１）で得た細胞培養液を遠沈管に移し、遠心分離処理（１０００ｒｐｍ，５分）を行い、上清を除いた細胞ペレットに適当量培地を添加して懸濁し、懸濁液中に含まれる浮遊系細胞の細胞数をＴＣ２０全自動セルカウンター（Ｂｉｏ－Ｒａｄ社製）により測定した。
　また、浮遊細胞Ｋ５６２を用いた実施例７においては、「（４）フローサイトメーターを用いた蛍光強度比較」におけるｓｏｒｔｉｎｇサンプルを、実施例１の方法に代えて以下のようにして得た。即ち、（２）遺伝子導入（ｉｉｉ）トランスフェクションで一晩培養した細胞が含まれる培養液を、ストレーナーチューブに添加してｓｏｒｔｉｎｇサンプルとした。 Example 2-16. Verification of transfection promoting effect of enhancer peptide C2 in various cells The same method as in Example 1 under the conditions shown in Table 2 below (cell, plasmid DNA amount, number of seeded cells, medium, enhancer peptide, enhancer peptide amount) Then, the plasmid DNA having the YFP-H2B gene was transfected into the cells, and the transfection promoting effect of the enhancer peptide C2 was confirmed by fluorescence microscope observation and fluorescence intensity comparison using a flow cytometer.
In Example 7 using floating cells K562, “(2) Gene transfer (ii) Preparation of cell suspension” was performed as follows instead of the method of Example 1. That is, the cell culture solution obtained in (1) is transferred to a centrifuge tube, centrifuged (1000 rpm, 5 minutes), an appropriate amount of medium is added to the cell pellet from which the supernatant has been removed, and suspended. The number of floating cells contained therein was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad).
Further, in Example 7 using the floating cells K562, the sorting sample in “(4) Comparison of fluorescence intensity using a flow cytometer” was obtained as follows instead of the method of Example 1. Specifically, (2) gene transfer (iii) a culture solution containing cells cultured overnight by transfection was added to a strainer tube to obtain a sorting sample.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　実施例２の顕微鏡観察の結果を図２のＳＦＡ　Ｐｌｕｓ＋Ｃ２に、また、実施例３の顕微鏡観察の結果を図３のＳＦＡ　Ｐｌｕｓ＋Ｃ２にそれぞれ示す。
　また、フローサイトメーターを用いた蛍光強度測定の結果については、比較例２－２～１６－２との比較を、下記表５及び表６に示す。図中、ＹＦＰは導入したプラスミドＤＮＡの発現レベルをＹＦＰ－Ｈ２Ｂの蛍光により測定した結果を表し、ＤＩＣ（微分干渉顕微鏡像）は細胞の状態を表す。　 The results of microscopic observation of Example 2 are shown in SFA Plus + C2 of FIG. 2, and the results of microscopic observation of Example 3 are shown in SFA Plus + C2 of FIG.
The results of fluorescence intensity measurement using a flow cytometer are shown in Tables 5 and 6 below in comparison with Comparative Examples 2-2 to 16-2. In the figure, YFP represents the result of measuring the expression level of the introduced plasmid DNA by the fluorescence of YFP-H2B, and DIC (differential interference microscope image) represents the state of the cells.
比較例１－１～比較例１６－１．各種細胞における従来のエンハンサーペプチドＥＳＦＡ４のトランスフェクション促進効果の検証
　下記表３に記載の条件（細胞、プラスミドＤＮＡ量、播種細胞数、培地、エンハンサーペプチド、エンハンサーペプチドの量）で、実施例１と同様の方法によりＹＦＰ－Ｈ２Ｂ遺伝子を有するプラスミドＤＮＡを細胞にトランスフェクションし、蛍光顕微鏡観察及びフローサイトメーターを用いた蛍光強度比較により、従来のエンハンサーペプチドであるＥＳＦＡ４のトランスフェクション促進効果を確認した。
　浮遊細胞Ｋ５６２を用いた比較例７－１においては、「（２）遺伝子導入（ｉｉ）細胞懸濁液の調製」を、実施例１の方法に代えて以下のように行った。即ち、（１）で得た細胞培養液を遠沈管に移し、遠心分離処理（１０００ｒｐｍ，５分）を行い、上清を除いた細胞ペレットに適当量培地を添加して懸濁し、懸濁液中に含まれる浮遊系細胞の細胞数をＴＣ２０全自動セルカウンター（Ｂｉｏ－Ｒａｄ社製）により測定した。
　また、浮遊細胞Ｋ５６２を用いた比較例７－１においては、「（４）フローサイトメーターを用いた蛍光強度比較」におけるｓｏｒｔｉｎｇサンプルを、実施例１の方法に代えて以下のようにして得た。即ち、（２）遺伝子導入（ｉｉｉ）トランスフェクションで一晩培養した細胞が含まれる培養液を、ストレーナーチューブに添加してｓｏｒｔｉｎｇサンプルとした。 Comparative Example 1-1 to Comparative Example 16-1. Verification of transfection promoting effect of conventional enhancer peptide ESFA4 in various cells As in Example 1 under the conditions described in Table 3 below (cells, plasmid DNA amount, seeded cell number, medium, enhancer peptide, enhancer peptide amount) The plasmid DNA having the YFP-H2B gene was transfected into the cells by the method described above, and the transfection promoting effect of ESFA4, a conventional enhancer peptide, was confirmed by fluorescence fluorescence observation and comparison of fluorescence intensity using a flow cytometer.
In Comparative Example 7-1 using the floating cell K562, “(2) Gene transfer (ii) Preparation of cell suspension” was performed as follows instead of the method of Example 1. That is, the cell culture solution obtained in (1) is transferred to a centrifuge tube, centrifuged (1000 rpm, 5 minutes), an appropriate amount of medium is added to the cell pellet from which the supernatant has been removed, and suspended. The number of floating cells contained therein was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad).
Further, in Comparative Example 7-1 using the floating cell K562, the sorting sample in “(4) Fluorescence intensity comparison using a flow cytometer” was obtained as follows instead of the method of Example 1. . Specifically, (2) gene transfer (iii) a culture solution containing cells cultured overnight by transfection was added to a strainer tube to obtain a sorting sample.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　比較例１－１の顕微鏡観察の結果を図１のＳＦＡ　Ｐｌｕｓ＋ＥＳＦＡ４に、比較例２－１の顕微鏡観察の結果を図２のＳＦＡ　Ｐｌｕｓ＋ＥＳＦＡ４に、また、比較例３－１の顕微鏡観察の結果を図３のＳＦＡ　Ｐｌｕｓ＋ＥＳＦＡ４にそれぞれ示す。
　また、フローサイトメーターを用いた蛍光強度比較の結果については、比較例１－２～１６－２との比較を下記表５及び表６に示す。 The results of microscopic observation of Comparative Example 1-1 are shown in SFA Plus + ESFA4 of FIG. 1, the results of microscopic observation of Comparative Example 2-1 are shown in SFA Plus + ESFA4 of FIG. 2, and the results of microscopic observation of Comparative Example 3-1 are shown. 3 SFA Plus + ESFA4 respectively.
As for the results of fluorescence intensity comparison using a flow cytometer, the comparison with Comparative Examples 1-2 to 16-2 is shown in Tables 5 and 6 below.
比較例１－２～比較例１６－２．各種細胞に対するトランスフェクション
　下記表４に記載の条件（細胞、プラスミドＤＮＡ量、播種細胞数、培地、エンハンサーペプチド）で、実施例１と同様の方法によりＹＦＰ－Ｈ２Ｂ遺伝子を有するプラスミドＤＮＡを細胞にトランスフェクションし、蛍光顕微鏡観察及びフローサイトメーターを用いた蛍光強度比較を行った。
　なお、浮遊細胞Ｋ５６２を用いた比較例７－２においては、「（２）遺伝子導入（ｉｉ）細胞懸濁液の調製」を、実施例１の方法に代えて以下のように行った。即ち、（１）で得た細胞培養液を遠沈管に移し、遠心分離処理（１０００ｒｐｍ，５分）を行い、上清を除いた細胞ペレットに適当量培地を添加して懸濁し、懸濁液中に含まれる浮遊系細胞の細胞数をＴＣ２０全自動セルカウンター（Ｂｉｏ－Ｒａｄ社製）により測定した。
　また、　浮遊細胞Ｋ５６２を用いた比較例７－２においては、「（４）フローサイトメーターを用いた蛍光強度比較」におけるｓｏｒｔｉｎｇサンプルを、実施例１の方法に代えて以下のようにして得た。即ち、（２）遺伝子導入（ｉｉｉ）トランスフェクションで一晩培養した細胞が含まれる培養液を、ストレーナーチューブに添加してｓｏｒｔｉｎｇサンプルとした。 Comparative Example 1-2 to Comparative Example 16-2. Transfection of various cells Under the conditions described in Table 4 below (cells, amount of plasmid DNA, number of seeded cells, medium, enhancer peptide), plasmid DNA having the YFP-H2B gene was transfected into the cells in the same manner as in Example 1. The fluorescence intensity was observed using a fluorescence microscope and a flow cytometer.
In Comparative Example 7-2 using floating cells K562, “(2) Gene transfer (ii) Preparation of cell suspension” was performed as follows instead of the method of Example 1. That is, the cell culture solution obtained in (1) is transferred to a centrifuge tube, centrifuged (1000 rpm, 5 minutes), an appropriate amount of medium is added to the cell pellet from which the supernatant has been removed, and suspended. The number of floating cells contained therein was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad).
In Comparative Example 7-2 using the floating cell K562, the sorting sample in “(4) Comparison of fluorescence intensity using a flow cytometer” was obtained as follows instead of the method of Example 1. . Specifically, (2) gene transfer (iii) a culture solution containing cells cultured overnight by transfection was added to a strainer tube to obtain a sorting sample.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　比較例１－２の顕微鏡観察の結果を図１のＳＦＡ　Ｐｌｕｓに、比較例２－２の顕微鏡観察の結果を図２のＳＦＡ　Ｐｌｕｓに、比較例３－２の顕微鏡観察の結果を図３のＳＦＡ　Ｐｌｕｓに、それぞれ示す。
　また、フローサイトメーターを用いた蛍光強度の結果と実施例１～１６及び比較例１－１～比較例１６－１の結果との比較を下記表５及び表６に示す。
　表５及び表６中の記号は以下の通りである。
　　◎：「エンハンサーペプチドを用いなかった場合の蛍光強度」に対する「エンハンサーペプチドを用いた場合の蛍光強度」の比が２倍以上
　　○：「エンハンサーペプチドを用いなかった場合の蛍光強度」に対する「エンハンサーペプチドを用いた場合の蛍光強度」の比が１．５倍以上
　　△：「エンハンサーペプチドを用いなかった場合の蛍光強度」に対する「エンハンサーペプチドを用いた場合の蛍光強度」の比が１．１倍以上
　　×：「エンハンサーペプチドを用いなかった場合の蛍光強度」に対する「エンハンサーペプチドを用いた場合の蛍光強度」の比が１．１倍未満 The result of microscopic observation of Comparative Example 1-2 is shown in SFA Plus of FIG. 1, the result of microscopic observation of Comparative Example 2-2 is shown in SFA Plus of FIG. 2, and the result of microscopic observation of Comparative Example 3-2 is shown in FIG. Each is shown in SFA Plus.
Tables 5 and 6 below show a comparison between the results of fluorescence intensity using a flow cytometer and the results of Examples 1 to 16 and Comparative Examples 1-1 to 16-1.
The symbols in Table 5 and Table 6 are as follows.
◎: Ratio of “fluorescence intensity when enhancer peptide is used” to “fluorescence intensity when enhancer peptide is not used” is 2 times or more ○: “enhancer peptide when fluorescence intensity is obtained when enhancer peptide is not used” Ratio of “fluorescence intensity when using enhancer” is 1.5 times or more Δ: Ratio of “fluorescence intensity when using enhancer peptide” to “fluorescence intensity when no enhancer peptide is used” is 1.1 times or more ×: Ratio of “fluorescence intensity with enhancer peptide” to “fluorescence intensity with no enhancer peptide” is less than 1.1 times
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000012
 　実施例１－１６、比較例１－１～比較例１６－１、及び比較例１－２～比較例１６－２の結果及び考察を以下に纏めて示す。
Figure JPOXMLDOC01-appb-T000012
 The results and discussion of Example 1-16, Comparative Example 1-1 to Comparative Example 16-1, and Comparative Example 1-2 to Comparative Example 16-2 are summarized below.
《実施例１－３、比較例１－１～比較例３－１及び比較例１－２～比較例３－２の蛍光顕微鏡観察の結果・考察》
　ＣＯＳ７細胞に対するトランスフェクション（図１）及びＨＣＴ－１１６＃２Ｌｕｃ細胞に対するトランスフェクション（図３）では、従来のエンハンサーペプチドであるＥＳＦＡ４を用いた場合（比較例１－１、比較例３－１）とエンハンサーペプチドを用いなかった場合（比較例１－２、比較例３－２）とでＹＦＰ－Ｈ２Ｂの蛍光強度（発現強度）に有意な差はなく、ＥＳＦＡ４のトランスフェクション促進効果は観察されなかった。一方、Ｃ２を用いた場合（実施例１、実施例３）では、エンハンサーペプチドを用いなかった場合（比較例１－２、比較例３－２）に比べて強いＹＦＰ－Ｈ２Ｂの蛍光強度が観察されたことから、Ｃ２がトランスフェクション促進効果を有することが確認された。
　ＨｕＨ－７細胞（図２）に対するトランスフェクションでは、Ｃ２を用いた場合（実施例２）は、ＥＳＦＡ４を用いた場合（比較例２－１）に比べて強いＹＦＰ－Ｈ２Ｂの蛍光強度が観察されたことから、Ｃ２はＥＳＦＡ４よりもＨｕＨ－７細胞に対するトランスフェクションにおけるトランスフェクション促進効果が高いことが判った。 << Results and Discussion of Fluorescence Microscope Observation of Example 1-3, Comparative Example 1-1 to Comparative Example 3-1, and Comparative Example 1-2 to Comparative Example 3-2 >>
In transfection of COS7 cells (FIG. 1) and transfection of HCT-116 # 2Luc cells (FIG. 3), when the conventional enhancer peptide ESFA4 was used (Comparative Example 1-1, Comparative Example 3-1), There was no significant difference in the fluorescence intensity (expression intensity) of YFP-H2B when the enhancer peptide was not used (Comparative Example 1-2, Comparative Example 3-2), and the transfection promoting effect of ESFA4 was not observed. . On the other hand, when C2 was used (Example 1 and Example 3), stronger fluorescence intensity of YFP-H2B was observed than when the enhancer peptide was not used (Comparative Example 1-2 and Comparative Example 3-2). Thus, it was confirmed that C2 has a transfection promoting effect.
In transfection of HuH-7 cells (FIG. 2), the fluorescence intensity of YFP-H2B was stronger when C2 was used (Example 2) than when ESFA4 was used (Comparative Example 2-1). Thus, it was found that C2 has a higher transfection promoting effect in transfection against HuH-7 cells than ESFA4.
《実施例１－１６及び比較例１－１～比較例１６－１及び比較例１－２～１６－２のフローサイトメーターを用いた蛍光強度比較の結果・考察》
　上記表５及び表６から明らかなとおり、使用した１６種の細胞株全てにおいて、Ｃ２をエンハンサーペプチドとして用いた場合は、ＥＳＦＡ４をエンハンサーペプチドとして用いた場合と同等又はそれ以上の蛍光強度が測定された。この結果から、Ｃ２はＥＳＦＡ４と同等又はそれ以上のトランスフェクション促進効果を有することが判った。
　特に、ＣＯＳ７細胞、ＨＣＴ－１１６＃２Ｌｕｃ細胞、Ｋ５６２細胞、Ｌ細胞に対するトランスフェクションおいては、ＥＳＦＡ４を用いた場合（比較例１－１、比較例３－１、比較例７－１、比較例１３－１）とエンハンサーペプチドを用いなかった場合（比較例１－２、比較例３－２、比較例７－２、比較例１３－２）とで蛍光強度に有意な差はなく、ＥＳＦＡ４はトランスフェクション促進効果を示さなかった。他方、Ｃ２を用いた場合（実施例１、実施例３、実施例７、実施例１３）では、エンハンサーペプチドを用いなかった場合に比べて強い蛍光強度が測定されことから、Ｃ２のトランスフェクション促進効果が確認された。これらの結果から、ＥＳＦＡ４がトランスフェクション促進効果を示さない細胞に対しても、Ｃ２はトランスフェクション促進効果を示しており、Ｃ２はトランスフェクションを行う細胞の種類を問わない汎用性の広いエンハンサーペプチドであることが判った。
　一般に、核移行シグナルを有するペプチドは、細胞質に導入されてプラスミド遺伝子を核内に運ぶことでトランスフェクション促進効果を発揮することが知られている。この知見を考慮すると、核移行シグナルを持たないＣ２は、核移行シグナルを有するＥＳＦＡ４よりもトランスフェクション促進効果が劣ると予測されたが、意外にもＣ２はＥＳＦＡ４と同等又はそれ以上のトランスフェクション促進効果を示すことが判った。 << Results and Discussion of Fluorescence Intensity Comparison Using Flow Cytometers of Example 1-16, Comparative Example 1-1 to Comparative Example 16-1 and Comparative Examples 1-2 to 16-2 >>
As is clear from Table 5 and Table 6 above, in all 16 types of cell lines used, when C2 was used as an enhancer peptide, fluorescence intensity equal to or higher than that when ESFA4 was used as an enhancer peptide was measured. It was. From this result, it was found that C2 has a transfection promoting effect equivalent to or higher than that of ESFA4.
In particular, ESFA4 was used for transfection of COS7 cells, HCT-116 # 2Luc cells, K562 cells, and L cells (Comparative Example 1-1, Comparative Example 3-1, Comparative Example 7-1, Comparative Example). 13-1) and the case where no enhancer peptide was used (Comparative Example 1-2, Comparative Example 3-2, Comparative Example 7-2, Comparative Example 13-2), there was no significant difference in fluorescence intensity. No transfection promoting effect was shown. On the other hand, when C2 was used (Example 1, Example 3, Example 7, and Example 13), strong fluorescence intensity was measured as compared with the case where no enhancer peptide was used. The effect was confirmed. From these results, C2 also shows a transfection-promoting effect even for cells in which ESFA4 does not show a transfection-promoting effect, and C2 is a versatile enhancer peptide regardless of the type of cells to be transfected. It turns out that there is.
In general, it is known that a peptide having a nuclear translocation signal exhibits a transfection promoting effect by being introduced into the cytoplasm and carrying a plasmid gene into the nucleus. Considering this finding, C2 without a nuclear translocation signal was predicted to be inferior in transfection promoting effect to ESFA4 having a nuclear translocation signal, but surprisingly, C2 is equivalent to or better than ESFA4. It was found to show an effect.
実施例１７．比較例１７．ｍＲＮＡのトランスフェクションにおけるエンハンサーペプチドＣ２のトランスフェクション促進効果の検証
　下記表７に記載の条件（細胞、ＥＧＦＰ　ｍＲＮＡ量、播種細胞数、培地、エンハンサーペプチド、エンハンサーペプチドの量）で、実施例１と同様の方法により、プラスミドＤＮＡの代わりにｍＲＮＡをトランスフェクションし、蛍光顕微鏡観察及びフローサイトメーターを用いたＥＧＦＰの蛍光強度比較により、エンハンサーペプチドＣ２のトランスフェクション促進効果を確認した。
　導入するｍＲＮＡは、ＥＧＦＰ　ｍＲＮＡ（＃Ｌ－６３０１、Ｔｒｉｌｉｎｋ　Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓ社製）を使用した。
　実施例１７及び比較例１７におけるトランスフェクション薬剤（ＳｃｒｅｅｎＦｅｃｔ　Ａ　Ｐｌｕｓ　ｒｅａｇｅｎｔ）の添加量は、０．８μＬ（ＥＧＦＰ　ｍＲＮＡ量１μｇあたりＳｃｒｅｅｎＦｅｃｔ　Ａ　Ｐｌｕｓ　ｒｅａｇｅｎｔ４μＬ）とした。 Example 17. Comparative Example 17 Verification of transfection promoting effect of enhancer peptide C2 in mRNA transfection The same as in Example 1 under the conditions described in Table 7 below (cells, EGFP mRNA amount, number of seeded cells, medium, enhancer peptide, enhancer peptide amount) In this method, mRNA was transfected instead of plasmid DNA, and the transfection promoting effect of enhancer peptide C2 was confirmed by fluorescence microscopy and comparison of EGFP fluorescence intensity using a flow cytometer.
As the mRNA to be introduced, EGFP mRNA (# L-6301, manufactured by Trilink Biotechnologies) was used.
The addition amount of the transfection agent (ScreenFect A Plus reagent) in Example 17 and Comparative Example 17 was 0.8 μL (ScreenFect A Plus reagent 4 μL per 1 μg of EGFP mRNA).
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
（１）蛍光顕微鏡観察の結果及び考察
　蛍光顕微鏡観察で得られた結果について、図４に示す。図４中、ＳＦＡ　Ｐｌｕｓは比較例１７の結果、ＳＦＡ　Ｐｌｕｓ＋Ｃ２は実施例１７の結果である。図中、ＧＦＰは導入したＥＧＦＰ　ｍＲＮＡの発現レベルをＥＧＦＰの蛍光により測定した結果を表し、ＤＩＣ（微分干渉顕微鏡像）は細胞の状態を表す。
　Ｃ２を用いた場合（実施例１７）、エンハンサーペプチドを用いなかった場合（比較例１７）に比べて強い蛍光強度が観察されており、Ｃ２はｍＲＮＡのトランスフェクションにおいてもトランスフェクション促進効果を有することが判った。
　（２）フローサイトメーターを用いた蛍光強度比較の結果及び考察
　フローサイトメーターを用いた蛍光強度比較について、図５に示す。図５中、ＳＦＡ　Ｐｌｕｓは比較例１７の結果、ＳＦＡ　Ｐｌｕｓ＋Ｃ２は実施例１７の結果である。図中、相対蛍光強度は、非トランスフェクション細胞の蛍光強度を１００とした場合のトランスフェクションした細胞における導入ｍＲＮＡの相対蛍光強度を示す。
　Ｃ２を用いた場合（実施例１７）、エンハンサーペプチドを用いなかった場合（比較例１７）に比べて強い蛍光強度が観察されており、Ｃ２はｍＲＮＡのトランスフェクションにおいてもトランスフェクション促進効果を有することが判った。
　以上のことから、Ｃ２はＤＮＡだけでなくＲＮＡのトランスフェクションにおいてもトランスフェクション促進効果を有することが明らかとなり、導入する核酸の種類を問わない汎用性の高いエンハンスペプチドであることが判った。 (1) Results of fluorescence microscope observation and discussion The results obtained by fluorescence microscope observation are shown in FIG. In FIG. 4, SFA Plus is the result of Comparative Example 17, and SFA Plus + C2 is the result of Example 17. In the figure, GFP represents the result of measuring the expression level of the introduced EGFP mRNA by fluorescence of EGFP, and DIC (differential interference microscope image) represents the state of the cells.
Strong fluorescence intensity was observed when C2 was used (Example 17) and when no enhancer peptide was used (Comparative Example 17), and C2 has an effect of promoting transfection also in the transfection of mRNA. I understood.
(2) Results of fluorescence intensity comparison using a flow cytometer and discussion Comparison of fluorescence intensity using a flow cytometer is shown in FIG. In FIG. 5, SFA Plus is the result of Comparative Example 17, and SFA Plus + C2 is the result of Example 17. In the figure, the relative fluorescence intensity indicates the relative fluorescence intensity of the introduced mRNA in the transfected cells when the fluorescence intensity of the non-transfected cells is 100.
Strong fluorescence intensity was observed when C2 was used (Example 17) and when no enhancer peptide was used (Comparative Example 17), and C2 has an effect of promoting transfection also in the transfection of mRNA. I understood.
From the above, it became clear that C2 has a transfection promoting effect not only in DNA but also in RNA transfection, and it was found that C2 is a highly versatile enhanced peptide regardless of the type of nucleic acid to be introduced.
実施例１８．各種ペプチドのアミノ酸配列の違いによるトランスフェクション促進効果への影響についての検証
　以下の方法により、下記表８に記載の実験条件で、ＣＨＯ－Ｋ１細胞にホタルルシフェラーゼ遺伝子を有するプラスミドＤＮＡをトランスフェクションし、各種ペプチドのアミノ酸配列の違いによるトランスフェクション促進効果への影響を確認した。 Example 18 Verification of the effect on the transfection promoting effect due to the difference in amino acid sequence of various peptides By the following method, CHO-K1 cells were transfected with plasmid DNA having a firefly luciferase gene under the experimental conditions described in Table 8 below. The influence on the transfection promotion effect by the difference in the amino acid sequence of various peptides was confirmed.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
（１）細胞の調製
　ＣＨＯ－Ｋ１細胞を、Ｈａｍ’ｓＦ－１２培地（和光純薬工業（株）製）を用いて、１×１０５個以上の細胞数が得られるまで培養し、培養細胞を得た。 (1) Preparation of cells CHO-K1 cells are cultured using Ham'sF-12 medium (manufactured by Wako Pure Chemical Industries, Ltd.) until a cell number of 1 × 10 5 or more is obtained. Got.
（２）遺伝子導入
　（ｉ）トランスフェクション溶液の調製
　まず、ホタルルシフェラーゼ遺伝子を有するｐＧＬ３　ｃｏｎｔｒｏｌ　ｖｅｃｔｏｒ（ｐｒｏｍｅｇａ社製、配列番号４）を、０．５ｍｇ／ｍＬとなるようにＴＥ　ｂｕｆｆｅｒ（１０ｍＭ　Ｔｒｉｓ－ＨＣｌ，　１ｍＭ　ＥＤＴＡ　ｐＨ８．０）で懸濁し、プラスミドＤＮＡ溶液を調製した。
　次いで、Ｃ２（（株）東レリサーチにて合成）を、トランスフェクション薬剤であるＳｃｒｅｅｎＦｅｃｔＴＭＡ　Ｐｌｕｓ（和光純薬工業（株）製）に付属のＤｉｌｕｔｉｏｎ　ｂｕｆｆｅｒ（以下、Ｄｉｌｕｔｉｏｎ　ｂｕｆｆｅｒと略記する場合がある）に５ｍｇ／ｍＬとなるように溶解し、エンハンサーペプチド溶液を調製した。
　そして、滅菌したチューブを２本用意し、一本のチューブにＤｉｌｕｔｉｏｎ　ｂｕｆｆｅｒを４．７μＬ添加し、ＳｃｒｅｅｎＦｅｃｔＴＭＡ　Ｐｌｕｓ（和光純薬工業（株）製）に付属のＳｃｒｅｅｎＦｅｃｔ　Ａ　Ｐｌｕｓ　ｒｅａｇｅｎｔを０．３μＬ（プラスミドＤＮＡ量１μｇあたりＳｃｒｅｅｎＦｅｃｔ　Ａ　Ｐｌｕｓ　ｒｅａｇｅｎｔ３μＬ）添加した。
　もう一本の滅菌済みチューブに、プラスミドＤＮＡ量が０．１μｇになるようプラスミドＤＮＡ溶液１μＬを添加し、更にエンハンサーペプチド溶液を０．２μＬ（プラスミドＤＮＡ量１μｇあたりエンハンサーペプチド溶液２μＬ）添加し、Ｄｉｌｕｔｉｏｎ　ｂｕｆｆｅｒを加えて全量５μＬになるように調製した。
　次いで、上記の２本のチューブを混合して、室温で１５分反応させトランスフェクション溶液を得た。 (2) Gene Introduction (i) Preparation of Transfection Solution First, pbuffer control vector (Promega, SEQ ID NO: 4) having a firefly luciferase gene was added to TE buffer (10 mM Tris-HCl) at 0.5 mg / mL. , 1 mM EDTA pH 8.0) to prepare a plasmid DNA solution.
Next, C2 (synthesized by Toray Research Co., Ltd.) may be abbreviated as “Dilution buffer” (hereinafter referred to as “Dilution buffer”) attached to ScreenFect ™ A Plus (manufactured by Wako Pure Chemical Industries, Ltd.), a transfection agent. ) To 5 mg / mL to prepare an enhancer peptide solution.
Then, prepare two sterilized tubes, add 4.7 μL of dilution buffer to one tube, and add 0.3 μL of ScreenFect A Plus reagent attached to ScreenFect ™ A Plus (manufactured by Wako Pure Chemical Industries, Ltd.). (ScreenFect A Plus reagent 3 μL per 1 μg of plasmid DNA) was added.
To another sterilized tube, add 1 μL of plasmid DNA solution so that the amount of plasmid DNA is 0.1 μg, and then add 0.2 μL of enhancer peptide solution (2 μL of enhancer peptide solution per 1 μg of plasmid DNA). Buffer was added to prepare a total volume of 5 μL.
Next, the above two tubes were mixed and reacted at room temperature for 15 minutes to obtain a transfection solution.
　（ｉｉ）細胞懸濁液の調製
　（１）で得られた培養細胞を用いて、以下の通り細胞懸濁液を調製した。
　培養容器から培地を除いたのち、トリプシンＥＤＴＡ溶液［０．２５ｗ／ｖ％　トリプシン－１ｍｍｏｌ／ｌ　ＥＤＴＡ・４Ｎａ溶液（フェノールレッド含有））を培養容器に添加して細胞を培養容器から剥離し、懸濁液を得た。遠沈管に当該懸濁液を添加後、遠心分離処理を行い（１０００ｒｐｍ，５分）、細胞を沈降させた。上清を除いた後、細胞ペレットに適当量培地を加えて細胞懸濁液を得た。当該細胞懸濁液中の細胞の細胞数をＴＣ２０全自動セルカウンター（Ｂｉｏ－Ｒａｄ社製）により測定し、細胞懸濁液中の細胞の濃度を算出した。 (Ii) Preparation of cell suspension Using the cultured cells obtained in (1), a cell suspension was prepared as follows.
After removing the medium from the culture vessel, trypsin EDTA solution [0.25 w / v% trypsin-1 mmol / l EDTA · 4Na solution (containing phenol red)] is added to the culture vessel to detach the cells from the culture vessel, and A turbid liquid was obtained. After adding the suspension to the centrifuge tube, centrifugation was performed (1000 rpm, 5 minutes) to sediment the cells. After removing the supernatant, an appropriate amount of medium was added to the cell pellet to obtain a cell suspension. The number of cells in the cell suspension was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad), and the concentration of cells in the cell suspension was calculated.
　（ｉｉｉ）トランスフェクション
　まず、９６ウェルプレートの１つのウェルに、以下のものをそれぞれ添加した。
　・Ｈａｍ’ｓＦ－１２培地（和光純薬工業（株）製）９０μＬ
　・１．５×１０５個の細胞が存在する量の（ｉｉ）で調製した細胞懸濁液
　次いで、（ｉ）で調製したトランスフェクション溶液を１０μＬ滴下し、ゆっくりと攪拌混合後、３７℃のＣＯ２インキュベーターで一晩細胞を培養した。 (Iii) Transfection First, the following were added to one well of a 96-well plate.
・ Ham'sF-12 medium (Wako Pure Chemical Industries, Ltd.) 90μL
-Cell suspension prepared in (ii) in an amount of 1.5 × 10 5 cells Next, 10 μL of the transfection solution prepared in (i) was dropped, and after gently stirring and mixing, Cells were cultured overnight in a CO 2 incubator.
（３）ルシフェラーゼアッセイ
　上記（２）で一晩培養した細胞について、導入したプラスミドの発現レベルをホタルルシフェラーゼ遺伝子の発光強度（発現強度）により、以下の方法で測定した。
　まず、ＣＯ２インキュベーターから９６ウェルプレートを取り出し、室温に戻した。次いで、ウェルにＯＮＥ－ＧｌｏＴＭ　ＥＸ　Ｌｕｃｉｆｅｒａｓｅ　Ａｓｓａｙ　Ｓｙｓｔｅｍ（ｐｒｏｇｅｍａ）を１００μＬ添加し、プレートミキサーで撹拌しながら５分間室温で反応させた。その後、プレートリーダー（ＴＥＣＡＮ）を用いて発光強度の測定を行った。 (3) Luciferase assay For the cells cultured overnight in (2) above, the expression level of the introduced plasmid was measured by the light emission intensity (expression intensity) of the firefly luciferase gene by the following method.
First, the 96-well plate was removed from the CO 2 incubator and returned to room temperature. Next, 100 μL of ONE-Glo ™ EX Luciferase Assay System (progema) was added to the well, and the mixture was allowed to react at room temperature for 5 minutes while stirring with a plate mixer. Thereafter, the emission intensity was measured using a plate reader (TECAN).
（４）結果
　ルシフェラーゼアッセイで得られた結果を、図６に示す。図６中、横軸は各種ペプチドの種類、縦軸は発光強度をそれぞれ表す。 (4) Results The results obtained by the luciferase assay are shown in FIG. In FIG. 6, the horizontal axis represents the types of various peptides, and the vertical axis represents the luminescence intensity.
実施例１９－２４．各種ペプチドのアミノ酸配列の違いによるトランスフェクション促進効果への影響についての検証
　下記表９に記載の条件（細胞、プラスミドＤＮＡ量、播種細胞数、培地、エンハンサーペプチド、エンハンサーペプチドの量）で、実施例１８と同様の方法により、ホタルルシフェラーゼ遺伝子を有するプラスミドＤＮＡをトランスフェクションし、ルシフェラーゼアッセイにより、各種ペプチドのアミノ酸配列の違いによるトランスフェクション促進効果への影響を確認した。各ペプチドは、全て（株）東レリサーチにて合成した。 Examples 19-24. Verification of the effect on the transfection promoting effect due to the difference in amino acid sequence of various peptides Examples under the conditions shown in Table 9 below (cell, plasmid DNA amount, number of seeded cells, medium, enhancer peptide, enhancer peptide amount) The plasmid DNA having the firefly luciferase gene was transfected by the same method as in No. 18, and the effect on the transfection promoting effect due to the difference in the amino acid sequence of various peptides was confirmed by luciferase assay. Each peptide was synthesized by Toray Research Co., Ltd.
Figure JPOXMLDOC01-appb-T000015
 　ルシフェラーゼアッセイで得られた結果を、図６に示す。図６中、横軸はペプチドの種類、縦軸は発光強度をそれぞれ表す。
Figure JPOXMLDOC01-appb-T000015
 The results obtained in the luciferase assay are shown in FIG. In FIG. 6, the horizontal axis represents the type of peptide, and the vertical axis represents the luminescence intensity.
比較例１８－２８．各種ペプチドのアミノ酸配列の違いによるトランスフェクション促進効果への影響についての検証
　下記表１０に記載の条件（細胞、プラスミドＤＮＡ量、播種細胞数、培地、エンハンサーペプチド、エンハンサーペプチドの量）で、実施例１８と同様の方法により、ホタルルシフェラーゼ遺伝子を有するプラスミドＤＮＡをトランスフェクションし、ルシフェラーゼアッセイにより、各種ペプチドのアミノ酸配列の違いによるトランスフェクション促進効果への影響を確認した。各ペプチドは、全て（株）東レリサーチにて合成した。 Comparative Example 18-28. Verification of the effect on the transfection promoting effect due to differences in amino acid sequences of various peptides Examples under the conditions described in Table 10 below (cells, plasmid DNA amount, number of seeded cells, medium, enhancer peptide, enhancer peptide amount) The plasmid DNA having the firefly luciferase gene was transfected by the same method as in No. 18, and the effect on the transfection promoting effect due to the difference in the amino acid sequence of various peptides was confirmed by luciferase assay. Each peptide was synthesized by Toray Research Co., Ltd.
Figure JPOXMLDOC01-appb-T000016
 　ルシフェラーゼアッセイで得られた結果について、図６に示す。図６中、横軸は各種ペプチドの種類、縦軸は発光強度をそれぞれ表す。
　実施例１８～２４及び比較例１８～２８の結果及び考察を以下に纏めて示す。
Figure JPOXMLDOC01-appb-T000016
 The results obtained in the luciferase assay are shown in FIG. In FIG. 6, the horizontal axis represents the types of various peptides, and the vertical axis represents the luminescence intensity.
The results and discussion of Examples 18 to 24 and Comparative Examples 18 to 28 are summarized below.
　《実施例１８～２４及び比較例１８～２８のルシフェラーゼアッセイの結果及び考察》
　ルシフェラーゼアッセイの結果、Ｃ４、Ｃ２＿Ｃ１４Ａ、Ｃ２＿Ａ６、Ｒ３、Ｒ６、Ｒ１９、Ｒ２２、Ｒ３０又はＨ１１をトランスフェクション時に用いた場合（比較例２０～２８）の発光強度は、ペプチドを用いなかった場合（比較例１８）の発光強度と同等かそれ以下であった。
　他方、Ｃ２、Ｃ５、Ｃ２＿ＡＡＣＡＡＡ、Ｋ１１、Ｒ９、Ｒ１６、又はＲＫ１２をトランスフェクション時に用いた場合（実施例１８～２４）の発光強度は、ペプチドを用いなかった場合（比較例１８）の発光強度に比べて２倍以上強かった。また、これらのペプチドは、従来のエンハンサーペプチドであるＥＳＦＡ４を用いた場合（比較例１９）よりも強い発光を示し、ＥＳＦＡ４よりも優れたトランスフェクション促進効果を示すことが判った。
　中でも、Ｃ２、Ｃ５、Ｃ２＿ＡＡＣＡＡＡ、Ｒ１６又はＲＫ１２をトランスフェクション時に用いた場合（実施例１８～２０、２３及び２４）の発光強度は、ペプチドを用いなかった場合（比較例１８）の発光強度に比べて５倍以上の強い発光を示しており、これらのペプチドはエンハンサーペプチドとして特に有用であることが判った。 << Results and Discussion of Luciferase Assay in Examples 18-24 and Comparative Examples 18-28 >>
As a result of the luciferase assay, the luminescence intensity when C4, C2_C14A, C2_A6, R3, R6, R19, R22, R30 or H11 was used at the time of transfection (Comparative Examples 20 to 28) was obtained when no peptide was used (Comparative Example). The emission intensity was equal to or less than 18).
On the other hand, when C2, C5, C2_AACAAA, K11, R9, R16, or RK12 was used at the time of transfection (Examples 18 to 24), the luminescence intensity was the same as that when no peptide was used (Comparative Example 18). It was more than twice as strong. Moreover, these peptides showed stronger luminescence than when the conventional enhancer peptide ESFA4 was used (Comparative Example 19), and it was found that these peptides exhibited a transfection promoting effect superior to ESFA4.
In particular, the luminescence intensity when C2, C5, C2_AACAAA, R16, or RK12 was used during transfection (Examples 18 to 20, 23, and 24) was compared with the luminescence intensity when no peptide was used (Comparative Example 18). Thus, it was found that these peptides are particularly useful as enhancer peptides.
　Ｃ２（実施例１８）及びＣ５（実施例１９）はいずれもトランスフェクション促進効果を示しており、エンハンサーペプチドにおけるポリアルギニン又はポリリシンからなる付加配列は、コア配列のＮ末端側、Ｃ末端側のいずれに結合していても、トランスフェクション促進効果を有することが判った。
　付加配列がポリアルギニンからなるＣ２（実施例１８）等や付加配列がポリリシンからなるＫ１１（実施例２１）、アルギニンとリシンを交互に１２個配置させたＲＫ１２（実施例２４）はトランスフェクション促進効果を示した。他方、アルギニンやリシンと同様にカチオニックなアミノ酸であるヒスチジンが１１個結合したヒスチジンＨ１１（比較例２８）はトランスフェクション促進効果を示さなかった。
　これらの結果から、付加配列はカチオニックなアミノ酸の中でも、アルギニン又は／及びリシンからなるアミノ酸配列であることが必要であると判った。 C2 (Example 18) and C5 (Example 19) both show transfection promoting effects, and the additional sequence consisting of polyarginine or polylysine in the enhancer peptide is either on the N-terminal side or on the C-terminal side of the core sequence. It was found that even if it is bound to, it has an effect of promoting transfection.
C2 (Example 18) in which the additional sequence is composed of polyarginine, K11 (Example 21) in which the additional sequence is composed of polylysine, and RK12 (Example 24) in which 12 arginine and lysine are alternately arranged are transfection promoting effects. showed that. On the other hand, histidine H11 (Comparative Example 28) to which 11 histidines, which are cationic amino acids, were bound in the same manner as arginine and lysine did not show a transfection promoting effect.
From these results, it was found that the additional sequence must be an amino acid sequence consisting of arginine and / or lysine among the cationic amino acids.
　さらに、付加配列が９個のアルギニンからなるＲ９（実施例２２）、付加配列として１１個のリシンからなるＫ１１（実施例２１）、付加配列が１６個のアルギニンからなるＲ１６（実施例２３）等もトランスフェクション促進効果を示した。一方、付加配列が６個のアルギニンからなるＲ６（比較例２４）等の付加配列が６個以下のアルギニン又はリシンからなるペプチドや、付加配列が１９個のアルギニンからなるＲ１９（比較例２５）等の付加配列が１９個以上のアルギニン又はリシンからなるペプチドは、トランスフェクション促進効果を示さなかった。これらの結果から、アルギニン又は／及びリシンからなる付加配列のアミノ酸数は、９個以上１６個以下でトランスフェクション促進効果を示すことが判った。 Furthermore, R9 (Example 22) consisting of 9 arginines as the additional sequence, K11 (Example 21) consisting of 11 lysines as the additional sequence, R16 (Example 23) consisting of 16 arginines as the additional sequence, etc. Also showed an effect of promoting transfection. On the other hand, an additional sequence such as R6 (Comparative Example 24) composed of 6 arginines, a peptide composed of 6 or less arginine or lysine, an R19 (Comparative Example 25) composed of 19 arginines. Peptides consisting of 19 or more arginine or lysine showed no transfection promoting effect. From these results, it was found that the number of amino acids in the additional sequence consisting of arginine and / or lysine is 9 or more and 16 or less, and exhibits a transfection promoting effect.
　なお、ポリアルギニンのみからなるＣ４（比較例２０）はトランスフェクション促進効果を示しておらず、コア配列がペプチドのトランスフェクション促進効果に重要な配列であることが明らかとなった。特に、コア配列の３番目のＣｙｓ残基をＡｌａに置換したＣ２＿Ｃ１４Ａ（比較例２１）、Ｃ２＿Ａ６（比較例２２）はトランスフェクション促進効果を示さず、一方、コア配列の３番目のＣｙｓ残基以外をＡｌａに置換したＣ２＿ＡＡＣＡＡＡＡ（実施例２０）がトランスフェクション促進効果を示した。これらの結果から、コア配列の３番目のＣｙｓ残基が特にトランスフェクション促進効果に寄与していることが明らかとなった。 Note that C4 consisting only of polyarginine (Comparative Example 20) did not show a transfection promoting effect, and the core sequence was found to be an important sequence for the peptide transfection promoting effect. In particular, C2_C14A (Comparative Example 21) and C2_A6 (Comparative Example 22) in which the third Cys residue of the core sequence is substituted with Ala do not show transfection promoting effect, while other than the third Cys residue of the core sequence C2_AACAAAA (Example 20) in which is replaced with Ala showed a transfection promoting effect. From these results, it was revealed that the third Cys residue in the core sequence contributes particularly to the transfection promoting effect.
実施例２５．各種エンハンサーペプチドのトランスフェクション薬剤の細胞毒性抑制効果についての検証
　以下の方法により、下記表１１に記載の実験条件の下、ＣＨＯ－Ｋ１細胞にトランスフェクションし、各種ペプチドのアミノ酸配列の違いによるトランスフェクション促進効果への影響を確認した。
　なお、本実施例においては、導入遺伝子の発現による細胞毒性の効果を排除するため、プラスミドＤＮＡは用いずに下記（２）の遺伝子導入の操作を行った。 Example 25. Verification of cytotoxic effects of various enhancer peptide transfection agents By the following method, CHO-K1 cells were transfected under the experimental conditions described in Table 11 below, and transfection was performed by differences in the amino acid sequences of the various peptides. The effect on the promotion effect was confirmed.
In this example, in order to eliminate the effect of cytotoxicity due to the expression of the transgene, the gene introduction operation (2) below was performed without using plasmid DNA.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
（１）細胞の調製
　ＣＨＯ－Ｋ１細胞を、Ｈａｍ’ｓＦ－１２　１０％ＦＢＳ培地（和光純薬工業（株）製）を用いて、１．５×１０５個以上の細胞が得られるまで培養し、培養細胞を得た。 (1) Preparation of cells CHO-K1 cells are cultured using Ham'sF-12 10% FBS medium (manufactured by Wako Pure Chemical Industries, Ltd.) until 1.5 × 10 5 or more cells are obtained. And cultured cells were obtained.
（２）遺伝子導入
　（ｉ）トランスフェクション溶液の調製
　まず、Ｃ２（（株）東レリサーチにて合成）を、トランスフェクション薬剤であるＳｃｒｅｅｎＦｅｃｔＴＭＡ　Ｐｌｕｓ（和光純薬工業（株）製）に付属のＤｉｌｕｔｉｏｎ　ｂｕｆｆｅｒ（以下、Ｄｉｌｕｔｉｏｎ　ｂｕｆｆｅｒと略記する場合がある）に５ｍｇ／ｍＬとなるように溶解し、エンハンサーペプチド溶液を調製した。
　次いで、滅菌したチューブを２本用意し、一本のチューブにＳｃｒｅｅｎＦｅｃｔＴＭＡ　Ｐｌｕｓ（和光純薬工業（株）製）に付属のＤｉｌｕｔｉｏｎ　ｂｕｆｆｅｒを２３．５μＬ添加し、ＳｃｒｅｅｎＦｅｃｔ　Ａ　Ｐｌｕｓ　ｒｅａｇｅｎｔを１．５μＬ添加した。
　もう一本の滅菌済みチューブに、エンハンサーペプチド溶液を１μＬ添加し、Ｄｉｌｕｔｉｏｎ　ｂｕｆｆｅｒを添加して最終２５μＬになるように調製した。
　次いで、上記の２本のチューブを混合して、室温で１５分反応させトランスフェクション溶液を得た。 (2) Gene transfer (i) Preparation of transfection solution First, C2 (synthesized by Toray Research Co., Ltd.) is attached to ScreenFect ™ A Plus (manufactured by Wako Pure Chemical Industries, Ltd.) which is a transfection agent. An enhancer peptide solution was prepared by dissolving in a dilution buffer (hereinafter sometimes abbreviated as “dilution buffer”) to 5 mg / mL.
Next, two sterilized tubes are prepared, 23.5 μL of the dilution buffer attached to ScreenFect ™ A Plus (manufactured by Wako Pure Chemical Industries, Ltd.) is added to one tube, and 1.5 μL of ScreenFect A Plus reagent is added. Added.
To another sterilized tube, 1 μL of the enhancer peptide solution was added, and a dilution buffer was added to prepare a final 25 μL.
Next, the above two tubes were mixed and reacted at room temperature for 15 minutes to obtain a transfection solution.
　（ｉｉ）細胞懸濁液の調製
　（１）で得られた培養細胞を用いて、細胞懸濁液を調製した。培養容器から培地を除いたのち、トリプシンＥＤＴＡ溶液（０．２５ｗ／ｖ％　トリプシン－１ｍｍｏｌ／ｌ　ＥＤＴＡ・４Ｎａ溶液（フェノールレッド含有））を培養容器に添加して細胞を培養容器から剥離し、懸濁液を得た。遠沈管に当該懸濁液を添加後、遠心分離処理を行い（１０００ｒｐｍ，　５分）、細胞を沈降させた。上清を除いた後、細胞ペレットに適当量培地を加え、細胞懸濁液を得た。当該細胞懸濁液中の細胞数をＴＣ２０全自動セルカウンター（Ｂｉｏ－Ｒａｄ社製）により測定し、細胞懸濁液中の浮遊系細胞の濃度を算出した。 (Ii) Preparation of cell suspension Using the cultured cells obtained in (1), a cell suspension was prepared. After removing the culture medium from the culture vessel, trypsin EDTA solution (0.25 w / v% trypsin-1 mmol / l EDTA · 4Na solution (containing phenol red)) is added to the culture vessel to detach the cells from the culture vessel. A turbid liquid was obtained. After adding the suspension to the centrifuge tube, centrifugation was performed (1000 rpm, 5 minutes) to sediment the cells. After removing the supernatant, an appropriate amount of medium was added to the cell pellet to obtain a cell suspension. The number of cells in the cell suspension was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad), and the concentration of floating cells in the cell suspension was calculated.
　（ｉｉｉ）トランスフェクション
　まず、２４ウェルプレートの１つのウェルに以下のものをそれぞれ添加した。
　・Ｈａｍ’ｓＦ－１２培地（和光純薬工業（株）製）４５０μＬ
　・１．５×１０５細胞が存在する量の（ｉｉ）で調製した細胞懸濁液
　次いで、ウェルに（ｉ）で調製したトランスフェクション溶液を５０μＬ滴下し、ゆっくりと攪拌混合後、３７℃のＣＯ２インキュベーターで細胞を一晩培養した。 (Iii) Transfection First, the following were respectively added to one well of a 24-well plate.
・ Ham'sF-12 medium (Wako Pure Chemical Industries, Ltd.) 450μL
・ Cell suspension prepared in (ii) in an amount of 1.5 × 10 5 cells Next, 50 μL of the transfection solution prepared in (i) was dropped into the well, and gently stirred and mixed. Cells were cultured overnight in a CO 2 incubator.
（３）細胞生存率測定
　上記（２）で一晩培養した細胞について、培養容器から培地を除いたのち、トリプシンＥＤＴＡ溶液［０．２５ｗ／ｖ％　トリプシン－１ｍｍｏｌ／ｌ　ＥＤＴＡ・４Ｎａ溶液（フェノールレッド含有））を培養容器に添加して細胞を培養容器から剥離し懸濁液を得た。遠沈管に当該懸濁液を添加後、遠心分離処理を行い（１０００ｒｐｍ，５分）、細胞を沈降させた。上清を除いた後、細胞ペレットに適当量培地を加え、細胞懸濁液を得た。当該細胞懸濁液中の細胞数をＴＣ２０全自動セルカウンター（Ｂｉｏ－Ｒａｄ社製）により測定し、細胞懸濁液中の接着細胞の濃度を算出した。
　算出された細胞数とトランスフェクションを行っていない細胞の細胞数とを比較し、生細胞数の割合を算出した。 (3) Cell viability measurement For the cells cultured overnight in (2) above, after removing the medium from the culture vessel, trypsin EDTA solution [0.25 w / v% trypsin-1 mmol / l EDTA · 4Na solution (phenol red) Containing)) was added to the culture vessel, and the cells were detached from the culture vessel to obtain a suspension. After adding the suspension to the centrifuge tube, centrifugation was performed (1000 rpm, 5 minutes) to sediment the cells. After removing the supernatant, an appropriate amount of medium was added to the cell pellet to obtain a cell suspension. The number of cells in the cell suspension was measured with a TC20 fully automatic cell counter (manufactured by Bio-Rad), and the concentration of adherent cells in the cell suspension was calculated.
The calculated number of cells was compared with the number of cells that had not been transfected, and the ratio of the number of living cells was calculated.
（４）結果
　細胞生存率測定の結果を図７に示す。本実施例の考察は、実施例２６～３１及び比較例２９と併せて、比較例２９に示す。図中、横軸はエンハンサーペプチドに関する条件、縦軸は生細胞の割合（％）を示す。 (4) Results The results of cell viability measurement are shown in FIG. The consideration of this example is shown in Comparative Example 29 together with Examples 26 to 31 and Comparative Example 29. In the figure, the horizontal axis represents the condition relating to the enhancer peptide, and the vertical axis represents the ratio (%) of viable cells.
実施例２６－３１．比較例２９．各種エンハンサーペプチドのトランスフェクション薬剤による細胞毒性抑制効果についての検証
　下記表１２に記載の条件（細胞、播種細胞数、培地、エンハンサーペプチド、エンハンサーペプチドの量）で、実施例２５と同様の方法により、トランスフェクション薬剤の細胞毒性に対するエンハンサーペプチドの抑制効果を確認した。
　エンハンサーペプチドは、実施例１８－２４の結果トランスフェクション促進効果を有することが明らかとなったものを用いた。これらのエンハンサーペプチドは、全て（株）東レリサーチで合成した。 Examples 26-31. Comparative Example 29. Verification of cytotoxic effect of various enhancer peptides by transfection agents Under the conditions described in Table 12 below (cells, number of seeded cells, medium, enhancer peptide, amount of enhancer peptide), in the same manner as in Example 25, The inhibitory effect of the enhancer peptide on the cytotoxicity of the transfection agent was confirmed.
The enhancer peptide that was found to have the effect of promoting transfection as a result of Examples 18-24 was used. All of these enhancer peptides were synthesized by Toray Research Co., Ltd.
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
（４）結果
　細胞生存率測定の結果を図７に示す。図中、横軸はエンハンサーペプチドに関する条件、縦軸は生細胞の割合（％）を示す。実施例２５～３１及び比較例２９の結果及び考察を以下に纏めて示す。 (4) Results The results of cell viability measurement are shown in FIG. In the figure, the horizontal axis represents the condition relating to the enhancer peptide, and the vertical axis represents the ratio (%) of viable cells. The results and considerations of Examples 25 to 31 and Comparative Example 29 are summarized below.
　《実施例２５～３１及び比較例２９の結果及び考察》
　エンハンサーペプチドを用いなかった場合（比較例２９）の細胞生存率は、約５０％であった。他方、本発明のエンハンサーペプチドを用いた場合（実施例２５～３１）はいずれも、細胞生存率は約７０％から約９０％の生存率であった。
　これらの結果から、本発明のエンハンサーペプチドはいずれもトランスフェクション薬剤の細胞毒性に対する抑制効果を有することが判った。とりわけ、Ｃ２を用いた場合の細胞生存率は約９０％であり、トランスフェクション薬剤の細胞毒性に対する抑制効果が特に高く、Ｃ２は細胞毒性抑制効果の点からも特に有用なエンハンサーペプチドであることが判った。 << Results and Discussion of Examples 25 to 31 and Comparative Example 29 >>
The cell viability when the enhancer peptide was not used (Comparative Example 29) was about 50%. On the other hand, when the enhancer peptide of the present invention was used (Examples 25 to 31), the cell viability was about 70% to about 90%.
From these results, it was found that all the enhancer peptides of the present invention have an inhibitory effect on the cytotoxicity of the transfection agent. In particular, the cell viability when C2 is used is about 90%, and the inhibitory effect on the cytotoxicity of the transfection agent is particularly high, and C2 is a particularly useful enhancer peptide from the viewpoint of the cytotoxic inhibitory effect. understood.
　本発明のトランスフェクション促進用ペプチド、本発明のトランスフェクション用キット、及び本発明のトランスフェクション方法は、細胞や核酸の種類にかかわらず、従来のトランスフェクション促進用ペプチドと同等又はそれ以上の核酸導入効率でトランスフェクションを促進することができることから、導入遺伝子の機能解析やタンパク質発現生産等のトランスフェクションを行う分野において有用である。
　また、本発明のトランスフェクション法によれば、トランスフェクション薬剤の細胞毒性を抑制し、高い細胞生存率でトランスフェクションを行うことができることから、導入遺伝子の機能解析やタンパク質発現生産等のトランスフェクションを行う分野において有用である。 The transfection-promoting peptide of the present invention, the transfection kit of the present invention, and the transfection method of the present invention can introduce a nucleic acid equivalent to or higher than a conventional transfection-promoting peptide, regardless of the type of cell or nucleic acid. Since transfection can be promoted with efficiency, it is useful in the field of transfection such as functional analysis of transgenes and protein expression production.
In addition, according to the transfection method of the present invention, since transfection can be performed with high cell viability by suppressing the cytotoxicity of the transfection agent, transfection such as transgene function analysis and protein expression production can be performed. Useful in the field to do.

Claims (16)

  1. 下記一般式［１］又は［２］で示される、細胞のトランスフェクション促進用ペプチド。
    （Ｘ１）ｎ－Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２　［１］
    Ｘ２－Ｘ２－Ｃｙｓ－Ｘ２－Ｘ３－Ｘ２－（Ｘ１）ｎ　［２］
    （式中、ｎ個のＸ１はそれぞれ独立してアルギニン又はリシンを表し、Ｘ２はグリシン又はアラニン、Ｘ３はチロシン又はアラニンをそれぞれ表し、ｎは８～１７の整数を表す）     A peptide for promoting transfection of cells represented by the following general formula [1] or [2].
    (X 1 ) n -X 2 -X 2 -Cys-X 2 -X 3 -X 2 [1]
    X 2 -X 2 -Cys-X 2 -X 3 -X 2- (X 1 ) n [2]
    (In the formula, each of n X 1 s independently represents arginine or lysine, X 2 represents glycine or alanine, X 3 represents tyrosine or alanine, and n represents an integer of 8 to 17)
  2. 前記ペプチドが、下記一般式［１’］、［１’’］、［２’］又は［２’’］で示されるものである、請求項１に記載のペプチド。
    （Ｘ１）ｎ－Ｇｌｙ－Ｇｌｙ－Ｃｙｓ－Ｇｌｙ－Ｔｙｒ－Ｇｌｙ　［１’］
    （Ｘ１）ｎ－Ａｌａ－Ａｌａ－Ｃｙｓ－Ａｌａ－Ａｌａ－Ａｌａ　［１’’］
    Ｇｌｙ－Ｇｌｙ－Ｃｙｓ－Ｇｌｙ－Ｔｙｒ－Ｇｌｙ－（Ｘ１）ｎ　［２’］
    Ａｌａ－Ａｌａ－Ｃｙｓ－Ａｌａ－Ａｌａ－Ａｌａ－（Ｘ１）ｎ　［２’’］
    （式中、Ｘ１、ｎは上記に同じ）     The peptide according to claim 1, wherein the peptide is represented by the following general formula [1 ′], [1 ″], [2 ′] or [2 ″].
    (X 1 ) n -Gly-Gly-Cys-Gly-Tyr-Gly [1 ′]
    (X 1 ) n -Ala-Ala-Cys-Ala-Ala-Ala [1 ″]
    Gly-Gly-Cys-Gly-Tyr-Gly- (X 1 ) n [2 ′]
    Ala-Ala-Cys-Ala-Ala-Ala- (X 1 ) n [2 ″]
    (Wherein X 1 and n are the same as above)
  3. 前記ペプチドが、一般式［１’］又は［２’］で示されるものである、請求項２に記載のペプチド。 The peptide according to claim 2, wherein the peptide is represented by the general formula [1 '] or [2'].
  4. 前記一般式［１’］又は一般式［２’］中の（Ｘ１）ｎが、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ、（Ａｒｇ－Ｌｙｓ）ｍ、及び（Ｌｙｓ－Ａｒｇ）ｍ（ｎは前記に同じ、ｍは４～８の整数を表す）から選ばれるものである、請求項３に記載のペプチド。 In the general formula [1 ′] or the general formula [2 ′], (X 1 ) n is (Arg) n , (Lys) n , (Arg-Lys) m , and (Lys-Arg) m (n is The peptide according to claim 3, wherein m is the same as above, and m represents an integer of 4 to 8.
  5. 前記一般式［１’］又は一般式［２’］中の（Ｘ１）ｎが、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ又は（Ａｒｇ－Ｌｙｓ）ｍ（ｎ及びｍは前記に同じ）である、請求項４に記載のペプチド。 (X 1 ) n in the general formula [1 ′] or the general formula [2 ′] is (Arg) n , (Lys) n or (Arg-Lys) m (n and m are the same as described above). The peptide according to claim 4.
  6. ｎが９～１６の整数、ｍが６である、請求項４又は５に記載のペプチド。 The peptide according to claim 4 or 5, wherein n is an integer of 9 to 16, and m is 6.
  7. 前記ペプチドが、一般式［１’’］又は［２’’］で示されるものである、請求項２に記載のペプチド。 The peptide according to claim 2, wherein the peptide is represented by the general formula [1 ″] or [2 ″].
  8. 前記一般式［１’’］又は一般式［２’’］中の（Ｘ１）ｎが、（Ａｒｇ）ｎ、（Ｌｙｓ）ｎ、（Ａｒｇ－Ｌｙｓ）ｍ、及び（Ｌｙｓ－Ａｒｇ）ｍ（ｎ、ｍは前記に同じ）から選ばれるものである、請求項７に記載のペプチド。 In the general formula [1 ″] or the general formula [2 ″], (X 1 ) n is (Arg) n , (Lys) n , (Arg-Lys) m , and (Lys-Arg) m ( The peptide according to claim 7, wherein n and m are selected from the same as above.
  9. 前記一般式［１’’］又は一般式［２’’］中の（Ｘ１）ｎが、（Ａｒｇ）ｎ（ｎは前記に同じ）である、請求項８に記載のペプチド。 The peptide according to claim 8, wherein (X 1 ) n in the general formula [1 ″] or the general formula [2 ″] is (Arg) n (n is the same as above).
  10. ｎが９～１６の整数、ｍが６である、請求項８又は９に記載のペプチド。 The peptide according to claim 8 or 9, wherein n is an integer of 9 to 16, and m is 6.
  11. 請求項１～１０のいずれかに記載のペプチド及びカチオン性脂質を含むトランスフェクション薬剤を含む、細胞のトランスフェクション用キット。 A cell transfection kit comprising a transfection agent comprising the peptide according to any one of claims 1 to 10 and a cationic lipid.
  12. 前記トランスフェクション薬剤が、Ｎ－［２－（ジメチルアミノ）エチル］－４，５－ビス（ウンデシルチオ）ペンタンアミドを含むものである、請求項１１に記載の細胞のトランスフェクション用キット。 The cell transfection kit according to claim 11, wherein the transfection agent comprises N- [2- (dimethylamino) ethyl] -4,5-bis (undecylthio) pentanamide.
  13. 請求項１～１０のいずれかに記載のペプチド及びトランスフェクション薬剤の存在下、インビトロで細胞と核酸とを接触させる工程を包含する、細胞に核酸をトランスフェクションする方法。 A method for transfecting a cell with a nucleic acid comprising the step of contacting the cell with the nucleic acid in vitro in the presence of the peptide according to any one of claims 1 to 10 and a transfection agent.
  14. 前記トランスフェクション薬剤が、カチオン性脂質を含むものである、請求項１３に記載の方法。 14. The method of claim 13, wherein the transfection agent comprises a cationic lipid.
  15. 前記トランスフェクション薬剤が、Ｎ－［２－（ジメチルアミノ）エチル］－４，５－ビス（ウンデシルチオ）ペンタンアミドを含むものである、請求項１３に記載の方法。 14. The method of claim 13, wherein the transfection agent comprises N- [2- (dimethylamino) ethyl] -4,5-bis (undecylthio) pentanamide.
  16. 前記核酸がＲＮＡである、請求項１３に記載の方法。 The method of claim 13, wherein the nucleic acid is RNA.
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US20130149374A1 (en) * 2010-05-14 2013-06-13 Korea Research Institute Of Bioscience And Biotechnology Asymmetric Liposomes for the Highly Efficient Encapsulation of Nucleic Acids and Hydrophilic Anionic Compounds, and Method for Preparing Same
JP2013539360A (en) * 2010-07-30 2013-10-24 キュアヴァック ゲーエムベーハー Nucleic acid complex formation with disulfide-bridged cationic components for transfection and immune stimulation
WO2015056216A2 (en) * 2013-10-18 2015-04-23 Universidade Do Minho Peptide composition and uses thereof

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JP2001517939A (en) * 1997-03-14 2001-10-09 ライフ テクノロジーズ，インコーポレイテッド Transfection enhanced by peptides
US20130149374A1 (en) * 2010-05-14 2013-06-13 Korea Research Institute Of Bioscience And Biotechnology Asymmetric Liposomes for the Highly Efficient Encapsulation of Nucleic Acids and Hydrophilic Anionic Compounds, and Method for Preparing Same
JP2013539360A (en) * 2010-07-30 2013-10-24 キュアヴァック ゲーエムベーハー Nucleic acid complex formation with disulfide-bridged cationic components for transfection and immune stimulation
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