JP2007166946A - Composition for suppressing expression of target gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for suppressing expression of a target gene, containing liposome enclosing DNA (such as antisence DNA) or RNA (such as siRNA), suppressing expression of the target gene. <P>SOLUTION: The composition for suppressing expression of the target gene comprises a liposome having on the surface a first peptide containing a plurality of successive arginine residues, and DNA or RNA for suppressing expression of the target gene, wherein the DNA is enclosed in the liposome as an agglomerated state by a protamine or its derivative, and the RNA is enclosed in the liposome as an agglomerated state by a second peptide containing a plurality of successive arginine residues, or by its derivative modified with a hydrophobic group. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a composition for suppressing the expression of a target gene, comprising a liposome encapsulating DNA (for example, antisense DNA) or RNA (for example, siRNA) capable of suppressing the expression of the target gene.

  In recent years, development of vectors for reliably delivering target substances such as drugs, nucleic acids, peptides, proteins, and sugars to target sites has been actively conducted. For example, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses have been developed as vectors for introducing a target gene into target cells. However, since viral vectors have problems such as difficulty in mass production, antigenicity, and toxicity, non-viral vectors (for example, liposome vectors) with few such problems are attracting attention, and target substances In order to improve the intracellular delivery efficiency, non-viral vectors into which various functional molecules have been introduced have been actively developed.

  For example, liposomes in which a hydrophilic polymer (for example, polyalkylene glycol such as polyethylene glycol) is introduced on the outer surface of the liposome membrane have been developed (Patent Document 1, Patent Document 2, Patent Document 3 and Patent Document 4). According to this liposome, the directivity of the liposome with respect to tumor cells can be improved by improving the blood retention of the liposome.

  In addition, a liposome in which a substance capable of binding to a receptor or an antigen present on the surface of the cell membrane (for example, trassferin, insulin, folic acid, hyaluronic acid, antibody or fragment thereof, sugar chain) is introduced on the outer surface of the liposome membrane Have been developed (Patent Document 3 and Patent Document 4). According to this liposome, the endocytosis efficiency of the liposome can be improved.

  In addition, liposomes in which stearylated octaarginine is introduced on the outer surface of the liposome membrane have been developed (Patent Document 5, Non-Patent Document 1). According to this liposome, the intracellular delivery efficiency of the target substance enclosed in the liposome can be improved.

  On the other hand, regarding gene expression suppression, it has been reported that a synthetic oligo DNA complementary to the mRNA of Rous sarcoma virus suppresses virus replication (Non-patent Documents 2 and 3). Such antisense DNA has the advantages of being able to target introns, being easy to synthesize, highly stable and easy to handle. However, in order to apply to gene therapy in vivo, there are problems such as suppression of degradation of antisense DNA by nuclease, extension of residence time in the body, delivery to target site, breakthrough of cell membrane, etc. 4). In order to solve these problems, it is necessary to establish an efficient packaging method for antisense DNA.

  Regarding a method for packaging antisense DNA, a method of encapsulating antisense DNA in a lipid membrane by hydrating the lipid membrane in the presence of the antisense DNA has been reported (Non-patent Document 5). However, this method has a problem that the pharmacokinetics (PK) and biodistribution (BD) of the liposome are greatly suppressed due to the positive charge on the surface of the liposome, and the delivery opportunity to the mononuclear phagocyte system and tissues other than the lung is considerably less. Has a point.

In addition, a method of encapsulating antisense DNA in cationic lipid by the freeze-thaw method (Non-Patent Document 6), a method of encapsulating antisense DNA in pH-sensitive cationic lipids called stabilized antisense-lipid particles (SALP) (non-patent document 6) Patent document 7) has been reported. These two methods are effective in causing negatively charged antisense DNA to electrostatically interact with cationic lipids, but both have the problems that preparation time is long and encapsulation efficiency is not high. Have.
JP-A-1-249717 JP-A-2-149512 JP-A-4-346918 JP 2004-10482 A International Publication WO2005 / 032593 Pamphlet Kogure, K. et al., Journal of Controlled Release, 2004, Vol. 98, p.317-323 Stephenson, ML et al., Proc. Natl. Acad. Sci. USA, 1978, Vol. 75, p.285-288 Zamecnik, PC, etc., Proc. Natl. Acad. Sci. USA, 1978, Vol. 75, p.280-284 Shi F. et al., J Control. Release, 2004, 97, p.189-209 Gokhale PC etc., Gene Ther., 1997, Vol. 4, p.1289-99 Shi N et al., Proc Natl Acad Sci USA, 2000, Vol. 97, p.7567-7572 Semple SC et al., Biochim Biophys Acta., 2001, 1510, p.152-166

  The present invention provides a composition for suppressing expression of a target gene, comprising a liposome encapsulating DNA (for example, antisense DNA) or RNA (for example, siRNA) capable of suppressing the expression of the target gene. With the goal.

In order to solve the above problems, the present invention provides the following compositions.
(1) A composition for suppressing the expression of a target gene, comprising a liposome having a first peptide containing a plurality of consecutive arginine residues on its surface and DNA or RNA capable of suppressing the expression of the target gene The DNA is enclosed in the liposome in an aggregated state with protamine or a derivative thereof, and the RNA is a second peptide or hydrophobic group containing a plurality of consecutive arginine residues. A composition encapsulated in the liposome in an aggregated state by a derivative thereof modified with 1.
(2) The composition according to (1), wherein the first peptide is a peptide consisting of 4 to 35 amino acid residues containing 4 to 20 consecutive arginine residues.
(3) The composition according to (1) or (2), wherein the first peptide consists of only an arginine residue.
(4) The composition according to any one of (1) to (3), wherein the amount of the first peptide is 2% (molar ratio) or more with respect to the total lipid constituting the lipid membrane of the liposome. .
(5) The composition according to any one of (1) to (4), wherein the second peptide is a peptide composed of 4 to 35 amino acid residues containing 4 to 20 consecutive arginine residues. object.
(6) The composition according to any one of (1) to (5), wherein the second peptide consists of only an arginine residue.
(7) The composition according to any one of (1) to (6), wherein the hydrophobic group that modifies the second peptide is a stearyl group.
(8) The composition according to any one of (1) to (7), wherein the ratio of the cationic lipid to the total lipid constituting the lipid membrane of the liposome is 0 to 40% (molar ratio).
(9) The first peptide is modified with a hydrophobic group or a hydrophobic compound, the hydrophobic group or the hydrophobic compound is inserted into the lipid membrane of the liposome, and the peptide is released from the lipid membrane of the liposome. The composition according to any one of (1) to (8), which is exposed.
(10) The composition according to (9), wherein the hydrophobic group that modifies the first peptide is a stearyl group.
(11) The composition according to any one of (1) to (10), wherein the DNA is antisense DNA and the RNA is siRNA.

  Liposomes contained in the composition of the present invention can be efficiently transferred into cells or nuclei via the first peptide present on the surface (see WO2005 / 032593), and aggregates enclosed in the liposomes. DNA (for example, antisense DNA) can be efficiently disaggregated from protamine or a derivative thereof in the cell or nucleus, and the aggregated RNA (for example, siRNA) encapsulated in the liposome is intracellular. Alternatively, it can be disaggregated from the second peptide or its derivative efficiently in the nucleus. Therefore, according to the composition of the present invention, the expression of the target gene can be effectively suppressed.

The present invention will be described in detail below.
Liposomes are closed vesicles having a lipid membrane (lipid bilayer membrane). The number of lipid membranes in the liposome is not particularly limited, and may be multilamellar liposomes (MLV), SUV (small unilamella vesicle), LUV (large unilamella vesicle), GUV (giant unilamella vesicle), etc. Single membrane liposomes may also be used. The size of the liposome is not particularly limited, but is preferably 100 to 500 nm in diameter, and more preferably 150 to 300 nm in diameter.

  Examples of the components of the lipid membrane of the liposome include lipids, membrane stabilizers, antioxidants, charged substances, membrane proteins, and the like.

  The lipid is an essential constituent of the lipid membrane, and the amount of lipid contained in the lipid membrane is usually 70% (molar ratio) or more, preferably 75% (molar ratio) or more of the total amount of the substance constituting the lipid membrane, More preferably, it is 80% (molar ratio) or more. The upper limit of the amount of lipid contained in the lipid membrane is 100% of the total amount of substances constituting the lipid membrane.

Examples of lipids include phospholipids, glycolipids, sterols, saturated or unsaturated fatty acids exemplified below.
[Phospholipids]
Phosphatidylcholine (eg, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.), phosphatidylglycerol (eg, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, Phosphatidylglycerol, distearoyl phosphatidylglycerol, etc.), phosphatidylethanolamine (eg dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidiethanol Phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen, egg yolk Lecithin, soybean lecithin, hydrogenated products thereof, etc.

[Glycolipid]
Glyceroglycolipid (eg, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride), glycosphingolipid (eg, galactosyl cerebroside, lactosyl cerebroside, ganglioside) and the like.

[Sterol]
Animal-derived sterols (eg, cholesterol, cholesterol succinic acid, cholestanol, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterol) (eg, stigmasterol, sitosterol, campesterol, brassicasterol) , Sterols derived from microorganisms (eg, timosterol, ergosterol) and the like.

[Saturated or unsaturated fatty acids]
C12-C20 saturated or unsaturated fatty acids such as palmitic acid, oleic acid, stearic acid, arachidonic acid, myristic acid, and the like.

  A membrane stabilizer is an optional component of a lipid membrane that can be included to physically or chemically stabilize the lipid membrane or to regulate the fluidity of the lipid membrane. The amount of the membrane stabilizer contained is usually 30% (molar ratio) or less, preferably 25% (molar ratio) or less, more preferably 20% (molar ratio) or less of the total amount of substances constituting the lipid membrane. . Note that the lower limit of the content of the film stabilizer is zero.

  Examples of the film stabilizer include sterol, glycerin or fatty acid ester thereof. Specific examples of sterols are the same as those described above, and examples of glycerin fatty acid esters include triolein and trioctanoin.

  An antioxidant is an optional component of the lipid membrane that can be included to prevent oxidation of the lipid membrane, and the amount of antioxidant contained in the lipid membrane is the total amount of substances that make up the lipid membrane. Is usually 30% (molar ratio) or less, preferably 25% (molar ratio) or less, more preferably 20% (molar ratio) or less. In addition, the lower limit of the content of the antioxidant is 0.

  Examples of the antioxidant include tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene and the like.

  A charged substance is an optional component of the lipid membrane that can be included to impart positive charge or negative charge to the lipid membrane, and the amount of charged substance contained in the lipid membrane is the total amount of the lipid membrane. The amount is usually 30% (molar ratio) or less, preferably 25% (molar ratio) or less, more preferably 20% (molar ratio) or less. The lower limit value of the charged substance content is zero.

  Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane, and the like. Examples of the charged substance to be provided include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.

  A membrane protein is an optional component of a lipid membrane that can be included to maintain the structure of the lipid membrane or to add functionality to the lipid membrane, and the amount of membrane protein contained in the lipid membrane Is usually 10% (molar ratio) or less, preferably 5% (molar ratio) or less, more preferably 2% (molar ratio) or less of the total amount of substances constituting the lipid membrane. Note that the lower limit of the content of membrane protein is zero.

  Examples of membrane proteins include membrane surface proteins and membrane integral proteins.

  A lipid derivative having a blood retention function, a temperature change sensitivity function, a pH sensitivity function, or the like may be used as the lipid constituting the lipid membrane. Thereby, 1 type, or 2 or more types of functions among the said functions can be provided to a liposome. By imparting a blood retention function to the liposome, the retention of the liposome in the blood can be improved, and the capture rate by reticuloendothelial tissues such as the liver and spleen can be reduced. By imparting a temperature change sensitive function and / or a pH sensitive function to the liposome, the releasability of the target substance enclosed in the liposome can be enhanced.

  Examples of the blood-retaining lipid derivative that can impart a blood-retaining function include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivative, glutamic acid derivative, polyglycerin phospholipid derivative, N- { Carbonyl-methoxypolyethyleneglycol-2000} -1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N- {carbonyl-methoxypolyethyleneglycol-5000} -1,2-dipalmitoyl-sn-glycero- 3-phosphoethanolamine, N- {carbonyl-methoxypolyethylene glycol-750} -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N- {carbonyl-methoxypolyethylene glycol-200 } -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N- {carbonyl-methoxypolyethylene glycol-5000} -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, etc. And polyethylene glycol derivatives.

  Examples of the temperature change sensitive lipid derivative capable of imparting a temperature change sensitive function include dipalmitoyl phosphatidylcholine and the like, and examples of the pH sensitive lipid derivative capable of imparting a pH sensitive function include, for example, dioleoylphosphatidyl. Examples include ethanolamine.

  The liposome has a first peptide containing a plurality of continuous arginine residues on the surface (outer surface). The liposome may have the first peptide in a portion other than the surface (for example, the inner surface of the lipid membrane).

  In the first peptide, the number of consecutive arginine residues is not particularly limited as long as it is plural, but is usually 4 to 20, preferably 6 to 12, and more preferably 7 to 10 . The total number of amino acid residues constituting the first peptide is not particularly limited, but is usually 4 to 35, preferably 6 to 30, and more preferably 7 to 23. The first peptide can contain any amino acid sequence at the C-terminal and / or N-terminal of a plurality of consecutive arginine residues, but preferably comprises only arginine residues.

  In the first peptide, the amino acid sequence added to the C-terminal or N-terminal of a plurality of consecutive arginine residues is preferably an amino acid sequence having rigidity (for example, polyproline). Unlike polyethylene glycol (PEG), which has a soft and irregular shape, polyproline is linear and retains a certain degree of rigidity. In addition, the amino acid residue contained in the amino acid sequence added to the C-terminal or N-terminal of a plurality of consecutive arginine residues is preferably an amino acid residue other than acidic amino acids. This is because an acidic amino acid residue having a negative charge may electrostatically interact with a positively charged arginine residue and attenuate the effect of the arginine residue.

  The amount of the first peptide present on the surface of the liposome is usually 0.1 to 30% (molar ratio), preferably 1 to 25% (molar ratio), more preferably based on the total lipid constituting the lipid membrane. 2 to 20% (molar ratio).

  Liposomes can migrate into the cell or nucleus via the first peptide present on the surface (see WO2005 / 032593). The amount of the first peptide present on the surface of the liposome is less than 2% (molar ratio), preferably less than 1.5% (molar ratio), more preferably 1% (molar ratio) with respect to the total lipid constituting the lipid membrane. If it is less than (molar ratio), the liposome can be transferred into the cell or nucleus mainly via endocytosis (see WO2005 / 032593). The lower limit of the amount of the first peptide at this time is usually 0.1% (molar ratio), preferably 0.5% (molar ratio), more preferably 0.8%, based on the total lipid constituting the lipid membrane. 7% (molar ratio). On the other hand, the amount of the first peptide present on the surface of the liposome is 2% (molar ratio) or more, preferably 3% (molar ratio) or more, more preferably 4% (total ratio) with respect to the total lipid constituting the lipid membrane. When the molar ratio is greater than or equal to, the liposome can migrate into the cell or nucleus mainly via macropinocytosis (see WO2005 / 032593). The upper limit of the amount of the first peptide at this time is usually 30% (molar ratio), preferably 25% (molar ratio), more preferably 20% (molar ratio) with respect to the total lipid constituting the lipid membrane. is there. In macropinocytosis, extracellular substances are taken into the cell as a fraction called macropinosome, and unlike macrosomes, macropinosomes do not fuse with lysosomes, so macropinosome inclusions avoid degradation by lysosomes. Can do. Therefore, when the liposome moves into the cell via macropinocytosis, the target substance encapsulated in the liposome can be efficiently delivered into the cell or nucleus.

  When the liposome internalization pathway depends on endocytosis, the lipid membrane must contain a cationic lipid as its main component. In the present invention, however, the liposome internalization pathway depends only on endocytosis. It is not necessary to include a cationic lipid in the lipid membrane. That is, in the present invention, the lipid membrane of the liposome may be composed of either a cationic lipid or a non-cationic lipid, or may be composed of both. However, the amount of cationic lipid contained in the lipid membrane can be reduced from the viewpoint of reducing cytotoxicity caused by cationic lipids and efficiently delivering the target substance encapsulated in liposomes via macropinocytosis. Is preferably as small as possible, and the ratio of the cationic lipid to the total lipid constituting the lipid membrane is preferably 0 to 40% (molar ratio), more preferably 0 to 20% (molar ratio). preferable.

  Examples of the cationic lipid include DODAC (dioctadecyldimethylammonium chloride), DOTMA (N- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium), DDAB (didodecylammonium bromide), DOTAP (1,2-dioleoyloxy- 3-trimethylammoniopropane), DC-Chol (3β-N- (N ', N',-dimethyl-aminoethane) -carbamol cholesterol), DMRIE (1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA (2,3 -dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate).

  “Non-cationic lipid” means a neutral lipid or an anionic lipid, and examples of the neutral lipid include diacylphosphatidylcholine, diacylphosphatidylethanolamine, cholesterol, ceramide, sphingomyelin, cephalin, and cerebroside. Examples of anionic lipids include cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-succinylphosphatidylethanolamine (N-succinyl PE), phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, phosphatidylethylene glycol, and cholesterol succinic acid. Can be mentioned.

  As a preferred embodiment of the liposome, the first peptide is modified with a hydrophobic group or a hydrophobic compound, the hydrophobic group or the hydrophobic compound is inserted into the lipid membrane, and the first peptide is exposed from the lipid membrane. Liposomes can be exemplified. In the present embodiment, “the peptide is exposed from the lipid membrane” includes the case where the peptide is exposed from either the outer surface or the inner surface of the lipid membrane or from both. It is.

  The hydrophobic group or the hydrophobic compound is not particularly limited as long as it can be inserted into the lipid membrane. The lipid membrane is composed of a hydrophilic portion and a hydrophobic portion, and the hydrophobic group or the hydrophobic compound is inserted into the lipid membrane in a state of being hydrophobically bonded to the hydrophobic portion of the lipid membrane. Examples of the hydrophobic group include a saturated or unsaturated fatty acid group such as a stearyl group, a cholesterol group, or a derivative thereof. Among these, a fatty acid group having 10 to 20 carbon atoms (for example, a palmitoyl group, an oleyl group). Group, stearyl group, arachidoyl group and the like). Examples of the hydrophobic compound include phospholipids, glycolipids or sterols exemplified above, long-chain aliphatic alcohols (for example, phosphatidylethanolamine, cholesterol, etc.), polyoxypropylene alkyls, glycerin fatty acid esters, and the like. Can be mentioned.

  Inside the liposome, DNA or RNA capable of suppressing the expression of the target gene is encapsulated. DNA is enclosed in an aggregated state with protamine or a derivative thereof, and RNA is aggregated with a second peptide containing a plurality of consecutive arginine residues or a derivative thereof modified with a hydrophobic group It is enclosed with.

  Examples of the protamine derivative include protamine sulfate, protamine phosphate, and protamine hydrochloride.

  In the second peptide, the number of consecutive arginine residues is not particularly limited as long as it is plural, but is usually 4 to 20, preferably 6 to 12, and more preferably 7 to 10 . The total number of amino acid residues constituting the first peptide is not particularly limited, but is usually 4 to 35, preferably 6 to 30, and more preferably 7 to 23. The second peptide can contain an arbitrary amino acid sequence at the C-terminal and / or N-terminal of a plurality of consecutive arginine residues, but preferably comprises only arginine residues.

  In the second peptide, the amino acid sequence added to the C-terminal or N-terminal of a plurality of consecutive arginine residues is preferably an amino acid sequence having rigidity (for example, polyproline). Unlike polyethylene glycol (PEG), which has a soft and irregular shape, polyproline is linear and retains a certain degree of rigidity. In addition, the amino acid residue contained in the amino acid sequence added to the C-terminal or N-terminal of a plurality of consecutive arginine residues is preferably an amino acid residue other than acidic amino acids. This is because an acidic amino acid residue having a negative charge may electrostatically interact with a positively charged arginine residue and attenuate the effect of the arginine residue.

  The hydrophobic group that modifies the second peptide is not particularly limited, and examples thereof include a saturated or unsaturated fatty acid group such as a stearyl group, a cholesterol group, or a derivative thereof. Several to 20 fatty acid groups (for example, palmitoyl group, oleyl group, stearyl group, arachidoyl group, etc.) are preferable.

  The type of target gene is not particularly limited, and can be appropriately determined according to the purpose of treatment. The DNA capable of suppressing the expression of the target gene is not particularly limited, and examples thereof include antisense DNA and decoy DNA. Antisense DNA is single-stranded DNA that can suppress transcription of target gene into mRNA, and its base length is not particularly limited, but is usually 12 to 30 bases, preferably 15 to 25 bases. More preferably, it is 18 to 20 bases. The decoy DNA is a double-stranded DNA that can bind to a transcriptional regulatory factor that regulates the transcription of the target gene and can suppress the transcription of the target gene, and its base length is not particularly limited. -30 bases, preferably 15-25 bases, more preferably 18-22 bases. The RNA capable of suppressing the expression of the target gene is not particularly limited, and examples thereof include microRNA. The microRNA is a double-stranded RNA that can suppress the expression of the target gene by RNA interference or the like, and examples of the microRNA include siRNA. The base length of the microRNA is not particularly limited, but is usually 15 to 33 bases, preferably 18 to 30 bases, and more preferably 20 to 27 bases.

  Since protamine or a derivative thereof has a positive charge and DNA has a negative charge, both form a complex by electrostatic interaction, whereby the DNA is aggregated by protamine or a derivative thereof. The Similarly, since the second peptide or derivative thereof has a positive charge and RNA has a negative charge, both form a complex by electrostatic interaction, whereby the RNA is second. Of the peptide or a derivative thereof. Preparing aggregates that are positively or negatively charged as a whole by adjusting the mixing ratio of DNA and protamine or a derivative thereof, or the mixing ratio of RNA and a second peptide or derivative thereof during aggregation. Can do.

  When aggregating DNA with protamine or a derivative thereof, the N / P ratio is usually 0.5 to 3.5, preferably 0.8 to 3.0, and more preferably 1.0 to 2.5. By agglomerating at such an N / P ratio, the particle size is usually 100 to 250 nm, preferably 100 to 200 nm, more preferably 100 to 150 nm, and the zeta potential is usually −50 to 50 mV, preferably −30. Aggregates can be prepared that are -30 mV, more preferably -20-25 mV. In addition, when RNA is aggregated with the second peptide or derivative thereof, the N / P ratio is usually 0.5 to 3.5, preferably 0.8 to 3.0, more preferably 1.0 to 2. 5. By agglomerating at such an N / P ratio, the particle size is usually 100 to 250 nm, preferably 100 to 200 nm, more preferably 100 to 150 nm, and the zeta potential is usually −50 to 50 mV, preferably −30. Aggregates can be prepared that are -30 mV, more preferably -20-25 mV.

  Liposomes are prepared by hydrating a lipid membrane in the presence of DNA aggregated with protamine or a derivative thereof, or RNA aggregated with a second peptide or derivative thereof, and then stirring or sonicating. be able to. Aggregated DNA or RNA has a positive charge or a negative charge as a whole. Therefore, the liposome in which DNA or RNA is encapsulated is efficiently obtained by electrostatic interaction with a lipid film having a negative charge or a positive charge. Can be made well.

Examples of liposome production by the hydration method are shown below.
A lipid membrane is obtained by dissolving a lipid, which is a component of a lipid membrane, and a first peptide modified with a hydrophobic group or a hydrophobic compound in an organic solvent, and then evaporating and removing the organic solvent. In this case, examples of the organic solvent include hydrocarbons such as pentane, hexane, heptane, and cyclohexane; halogenated hydrocarbons such as methylene chloride and chloroform; aromatic hydrocarbons such as benzene and toluene; methanol, ethanol, and the like Lower alcohols; esters such as methyl acetate and ethyl acetate; ketones such as acetone and the like. These may be used alone or in combination of two or more. Subsequently, the lipid membrane can be hydrated and stirred or sonicated to produce liposomes having the first peptide on the surface.

Another production example by the hydration method is shown below.
Lipids constituting the lipid membrane are dissolved in an organic solvent, and then the organic solvent is removed by evaporation to obtain a lipid membrane. The lipid membrane is hydrated and stirred or sonicated to produce liposomes. Subsequently, the said peptide can be introduce | transduced on the surface of a liposome by adding the 1st peptide modified with the hydrophobic group or the hydrophobic compound to the external solution of this liposome.

  By passing the liposome through a filter having a predetermined pore size, a liposome having a certain particle size distribution can be obtained. Further, according to a known method, conversion from multilamellar liposomes to single membrane liposomes and conversion from single membrane liposomes to multilamellar liposomes can be performed.

  A composition containing liposomes encapsulating DNA aggregated with protamine or a derivative thereof, or RNA aggregated with a second peptide or a derivative thereof can be used to suppress the expression of a target gene. .

  The biological species from which the cell having the target gene is derived is not particularly limited, and may be any animal, plant, microorganism, etc., but is preferably an animal, more preferably a mammal. Examples of mammals include humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Moreover, the kind of cell which should deliver a target substance is not specifically limited, For example, a somatic cell, a germ cell, a stem cell, or these cultured cells etc. are mentioned.

  Examples of the dosage form of the composition of the present invention include a liposome dispersion or a dried product thereof (for example, a lyophilized product, a spray-dried product, etc.). As the dispersion solvent, for example, a buffer solution such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer can be used. For example, additives such as sugars, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH regulators, hydration accelerators may be added to the dispersion.

  The composition of the present invention can be used both in vivo and in vitro. When the composition of the present invention is used in vivo, examples of the administration route include parenteral administration such as intravenous, intraperitoneal, subcutaneous, and nasal administration, and the dosage and frequency of administration are encapsulated in liposomes. It can be appropriately adjusted according to the type and amount of the target substance. Since the liposome contained in the composition of the present invention can exhibit intracellular and nuclear transferability in a wide temperature range of 0 to 40 ° C. (effective temperature range is 4 to 37 ° C.) ( WO2005 / 032593), temperature conditions can be set according to the purpose. In order to effectively exert intracellular and nuclear translocation at low temperatures (usually 4 to 10 ° C., preferably 4 to 6 ° C.), liposomes enter the cell or nucleus without endocytosis. It is necessary to migrate. In the present invention, the amount of the first peptide present on the surface of the liposome is 2% (molar ratio) or more, preferably 3% (molar ratio) or more, more preferably 4 with respect to the total lipid constituting the lipid membrane. % Or more (molar ratio) or more, the liposome can effectively exhibit intracellular and nuclear transfer properties at a low temperature (usually 4 to 10 ° C., preferably 4 to 6 ° C.) (WO2005 / 032593). The upper limit of the amount of the first peptide at this time is usually 30% (molar ratio), preferably 25% (molar ratio), more preferably 20% (molar ratio) with respect to the total lipid constituting the lipid membrane. is there.

[Example 1]
Multifunctional envelope nanostructure (MEND) has been developed as an efficient delivery system for plasmid DNA (Kogure, K. et al., Journal of Controlled Release, 2004, Vol. 98, p.317-323) ). Moreover, it has already succeeded in packaging antisense oligo DNA (antisense ODN) in MEND using stearyl octaargine (STR-R8) which is useful as a membrane-permeable peptide as a condensation reagent ( Yamada, Y. et al., Biol. Pharm. Bull. 2005, 28, p. 1939-1942). Therefore, in this study, an experiment was conducted for the purpose of optimizing the structure for improving the function of the antisense ODN-enclosed MEND.

1. Materials and Methods (1) Reagent 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine (DOPE) was purchased from AVANTI Polar Lipids. Cholesteryl hemisuccinate (CHEMS) and poly-L-lysine (PLL, molecular weight 27400) were purchased from SIGMA-Aldrich. Stearyl octaarginine (STR-R8) was synthesized according to a known method (S. Futaki et al., Bioconjug. Chem. 12 (2001) 1005-1011.). Protamine sulfate was purchased from Calbiochem. Antisense oligo DNA (antisense ODN) for luciferase gene (5'-aaccgcttccccgacttcc-3 '(SEQ ID NO: 1)) was purchased from SIGMA-Aldrich (sequences are Xu et al. (Xu Y, Zhang HY et al.) , Biochem Biophys Res Commun. 306 (2003) 712-717.)). NIH3T3 cells were obtained from the American Type Culture Collection.

(2) Preparation of Multifunctional Envelope Nanostructure (MEND) The MEND preparation process consists of the following three processes.
(A) DNA aggregation by polycation (PLL, STR-R8, protamine sulfate) DNA and polycation were dissolved in 10 mM HEPES buffer (pH 7.4), respectively, and DNA solution (0.1 mg / mL) and poly A cation solution (0.1 mg / mL) was mixed at room temperature under vortex to aggregate the DNA. DNA / PLL complex (DPC) suspension prepared with N / P (nitrogen / phosphate) ratio 2.4, DNA / STR-R8 complex (DPC) suspension prepared with N / P ratio 2.9 The DNA amount of the DNA / protamine sulfate complex (DPC) suspension prepared with an N / P ratio of 2.2 was 0.05 mg / mL. The DNA is a mixture of a luciferase gene and a full-length 7037 bp plasmid DNA having a CMV promoter upstream thereof (a luciferase gene incorporated into a pcDNA3.1 plasmid having a CMV promoter) and an antisense ODN for the luciferase gene.

(B) Hydration of lipid membrane After DNA aggregation, 0.25 mL of DPC suspension was added to the lipid membrane and incubated for 10 minutes to hydrate. The lipid membrane was formed by accommodating a 137.5 nmol (DOPE / CHEMS = 9: 2 (molar ratio)) chloroform solution in a glass test tube and removing the solvent. The final lipid concentration was 0.55 mM.

(C) Packaging of aggregated DNA by sonication In order to coat DPC with lipid, a glass test tube was sonicated in an ultrasonic bath (125 W, Branson Ultrasonics) for about 1 minute to form a lipid membrane surface. To bind the membrane permeable peptide octaarginine (R8), STR-R8 solution (5 mol% of lipid) was added to the suspension and the mixture was incubated at room temperature for 30 minutes. The hydrodynamic diameter was measured by the quasi-elastic light scattering method, and the zeta potential was analyzed by an electrophoretic light scattering spectrophotometer (ELS-8000, Otsuka electronics).

(3) Sucrose density gradient centrifugation MEND containing FITC labeled DNA (15% of total DNA) and rhodamine labeled DOPE (1 mol% of total lipid) on discontinuous sucrose density gradient (0-60%) And centrifuged at 20 ° C. and 160,000 g for 2 hours. Fractions were collected 1 mL each from the top, and the fluorescence intensity of FITC and rhodamine was measured. Of the fractions collected by discontinuous sucrose density gradient centrifugation, the fraction having a high DNA content was defined as a liposome-containing fraction. The liposome-containing fraction could be recovered from the boundary of 30-60% sucrose. Furthermore, FRET (fluorescence resonance energy transfer) between FITC and rhodamine was measured. Each fraction was dissolved in 1% SDS and used to measure fluorescence intensity. The encapsulation efficiency is calculated using the following formula:
Encapsulation efficiency = (ΣDN-containing fraction ODN amount / recovered total ODN amount) × 100
Calculated according to

(4) Cotransfection assay Sample containing 0.04 μg of DNA (plasmid DNA: ODN = 1: 748 (molar ratio)) or 0.04 μg of DNA treated with Lipofectamine 2000 (plasmid DNA: ODN = 1: 748 (molar ratio) )) Was suspended in 0.25 mL of DMEM (without serum and antibiotics), added to 4 × 10 ⁴ NIH3T3 cells, and incubated at 37 ° C. for 3 hours. Then 1 mL of DMEM containing 10% fetal calf serum was added and incubated for a further 5, 13, 21 and 45 hours. Thereafter, the cells were washed, lysed with a reporter lysis buffer (Promega), 100 μL of luciferase assay reagent (Promega) was added to 20 μL of the cell lysis solution, and luciferase activity was measured with a luminometer (Luminescencer-PSN). In addition, the amount of protein in the cell lysate was measured using a BCA protein assay kit (PIERCE).

2. Results and Discussion The luciferase expression plasmid DNA and the corresponding antisense ODN were aggregated together with STR-R8, encapsulated in MEND, and the lipid membrane was modified with STR-R8 to prepare antisense ODN-encapsulated MEND. This was cotransfected into NIH3T3 cells, and the inhibition rate of luciferase expression was examined. As a result, the luciferase expression suppression rate 8 hours after cotransfection was 30%, and the luciferase expression suppression effect was not very high. In addition, no effect of suppressing luciferase expression was observed from 16 hours after cotransfection.

  Using rhodamine-labeled DOPE and FITC-labeled antisense ODN, sucrose density gradient centrifugation of MEND was performed, and the difference in specific gravity was examined in detail. As a result, as shown in FIG. 1, the fractions # 7 (20-25%), # 9 (25-30%), and # 10 (30-40%) contain a large amount of antisense ODN, and the specific gravity is high. MEND with a light and rather heterogeneous specific gravity was made. In FIG. 1, ◯ represents the amount of DNA, and ● represents the amount of lipid. The non-uniformity of the MEND structure may lead to non-uniform functions. Therefore, in order to optimize the antisense ODN-encapsulated MEND, an experiment was conducted by changing the polycation that aggregates the antisense ODN to PLL or protamine sulfate. PLL is widely used as a condensation reagent, and protamine is a sperm-derived peptide known as a good condensation reagent with a large proportion of arginine (Brewer, LR et al., Science, 286, 120-123., Sorgi, FL). Et al., Gene Ther., 4, 961-968.).

  Antisense ODN and DOPE were labeled in the same manner as described above, and antisense ODN was aggregated with PLL or protamine sulfate, and sucrose density gradient centrifugation of MEND packaged with a lipid membrane was performed. As a result, in the case of PLL, as shown in FIG. 2, many antisense ODNs are contained in # 10 (30-40%) and # 11 (40-60%), and in the case of protamine, it is shown in FIG. Thus, # 10 (30-40%) contained a large amount of antisense ODN, and in both cases, the specific gravity was heavy, and MEND having a relatively uniform specific gravity was obtained. 2 and 3, ◯ represents the amount of DNA, and ● represents the amount of lipid. In addition, the presence of lipid and antisense ODN coexisted, and after solubilizing the lipid membrane with SDS, the fluorescence intensity of FITC increased several to several tens of times (data not shown). Since it was resolved, it was shown that antisense ODN was packaged with a lipid membrane.

  Table 1 shows the particle size, zeta potential, and antisense ODN encapsulation rate of each MEND. The particle size was approximately 200-350 nm, the zeta potentials all indicated-, and the antisense ODN encapsulation rates calculated from sucrose density gradient centrifugation fractions all showed high values of 90% or more.

  Antisense ODN-encapsulated MEND aggregated with PLL or protamine sulfate and the lipid membrane modified with STR-R8 was cotransfected into NIH3T3 cells in the same manner as described above. As a result, as shown in FIG. 4, in the case of PLL, no luciferase expression suppression effect was observed, but in the case of protamine, the luciferase expression suppression rate 8 hours after cotransfection was 90%, which is high. A luciferase expression inhibitory effect was observed, and the luciferase expression inhibitory rate after 48 hours was as high as 77%, maintaining a high luciferase expression inhibitory effect. Further, when co-transfection was performed in the same manner using Lipofectamine 2000 as a control, the luciferase expression inhibition rate after 8 hours after cotransfection was 55%, but the luciferase expression inhibition rate after 48 hours was 19%. It decreased considerably.

  Since such a difference was caused by the polycation, it was considered that this difference might be in an agglomerated state, and the particle diameter and zeta potential of the particles obtained by aggregating the antisense ODN with PLL or protamine, respectively, were expressed as N / The measurement was performed while changing the P ratio. As a result, as shown in FIGS. 5 and 6, in the case of the PLL, when the antisense ODN is added to the PLL, the N / P ratio aggregates in the vicinity of 1.8, and when the N / P ratio is higher than that, the particle size is increased. Particles having a zeta potential of + at 100 nm or less were formed, but particles having a particle diameter of 100-200 nm and a zeta potential of − were formed at an N / P ratio of less than 100 nm. In the case of protamine, no clear aggregation point was observed, and the particle size was within 100-200 nm. In FIGS. 5 and 6, ◯ represents protamine sulfate, and ● represents PLL.

  Regarding this result, PLL is composed only of lysine having a + charge, whereas protamine includes those having no net + charge such as neutral amino acids in addition to basic amino acids. It is thought that the interaction between particles is hindered to make aggregation difficult. There is also a difference in the particle size, and antisense ODN / protamine is better for the N / P ratio (PLL = 2.4, protamine = 2.2) used for aggregation in the cotransfection assay. It was larger than the antisense ODN / PLL. This seems to indicate that the antisense ODN / protamine is looser than the antisense ODN / PLL. Conversely, antisense ODN / protamine is considered to be easily decondensed. Regarding the zeta potential, the N / P ratio at which the potential becomes 0 is different. In the case of protamine, the N / P ratio is around 1.2, but in the case of PLL, the N / P ratio is around 1.8. It was. This indicates that the PLL has more + charge remaining inside the particles, and it is considered that the antisense ODN is less likely to decondense from the PLL. From the above, since antisense ODN / protamine is more easily decondensed than antisense ODN / PLL, it is considered that antisense ODN is freed and functions are easily exhibited.

[Example 2]
In the same manner as in Example 1, siRNA (5'-gcgcugcuggugccaaccctt-3 '(SEQ ID NO: 2) and 5'-ggguuggcaccagcagcgctt-3' (sequence) are used for luciferase expression plasmid and luciferase gene using PLL, protamine sulfate or STR-R8. Number 3)) was agglomerated and packaged into MEND. That is, polycation and siRNA were dissolved in 10 mM HEPES buffer (pH 7.4), and vortex condition was added to 0.2 mL of polycation solution (0.1 mg / mL), and siRNA solution (0.1 mg / mL). By adding 0.1 mL, siRNA was condensed to prepare positively charged particles. A chloroform solution containing 137.5 nmol of dioleoylphosphatidylethanolamine (DOPE) and cholesterol succinic acid (CHEMS) (DOPE: CHEMS = 9: 2 (molar ratio)) is fractionated into a test tube and sprayed with nitrogen gas. This was evaporated to dryness to form a lipid film having a negative charge. 0.25 mL of the aggregated siRNA suspension was added to the lipid membrane and allowed to stand at room temperature for 10 minutes or longer to hydrate the lipid membrane and bind the condensed siRNA particles to the lipid membrane. After hydration, the condensed siRNA was coated with a lipid membrane by sonication in an ultrasonic bath for 10-30 seconds. To the obtained lipid-coated condensed siRNA suspension, a STR-R8 (2 mg / mL) solution was added so as to be 5 mol% of the total lipid amount, and the mixture was allowed to stand at room temperature for 30 minutes. STR-R8 was distributed to the coated lipid membrane and the surface was modified with R8 peptide.

  FIG. 7 shows the results of subjecting each MEND to sucrose density gradient centrifugation in the same manner as in Example 1. As shown in FIG. 7, in the case of STR-R8, a large amount of siRNA was contained in # 10 (30-40%), and the specific gravity was heavy and a relatively uniform specific gravity MEND was produced. However, PLL and protamine sulfate were used. In this case, such a MEND could not be performed. In FIG. 7, ◯ represents the amount of DNA, and ● represents the amount of lipid.

  Each complex was applied to a 15% polyacrylamide gel at 200 ng or 400 ng (corresponding to RNA), electrophoresed at 50 mV for 45 minutes, and visualized by irradiating the ethidium bromide stained with UV. The results of subjecting the siRNA / polycation complex to polyacrylamide electrophoresis are shown in FIG. As shown in FIG. 8, in the case of PLL, siRNA was observed on the high molecular weight side, and it was found that aggregation was incomplete. In the case of protamine sulfate, siRNA was observed on the low molecular weight side and was found not to be aggregated. In the case of STR-R8, siRNA was confirmed at the starting point and found to be aggregated.

  HeLa cells were transfected with a luciferase expression plasmid pcDNA3.1-Luc and cultured in the presence of antibiotic G418, so that only plasmid-containing cells were selected and cultured, and a luciferase stable expression cell line was established by repeating the culture. In the same manner as in Example 1, each MEND was co-transfected into HeLa cells stably expressing luciferase (siRNA concentration was about 30 pmol / well). FIG. 9 shows the luciferase expression inhibition rate (RNAi effect) 24 hours after cotransfection. As shown in FIG. 9, in the case of STR-R8, the luciferase expression suppression rate was high (about 80%) as in Lipofectamine 2000, but in the case of PLL and protamine sulfate, it was significantly lower than those. It was the luciferase expression suppression rate. Moreover, as shown in FIG. 10, in the case of STR-R8, the time dependency and concentration dependency similar to Lipofectamine 2000 (LA2000) were shown. In FIG. 10, ● represents the result of MEND using STR-R8, and Δ represents the result of Lipofectamine 2000 (LA2000).

It is a figure showing the result of a sucrose density gradient centrifugation. It is a figure showing the result of a sucrose density gradient centrifugation. It is a figure showing the result of a sucrose density gradient centrifugation. It is a figure showing the luciferase expression suppression rate by various MEND. It is a figure showing the relationship between N / P ratio and particle size at the time of aggregating antisense oligo DNA with PLL or protamine. It is a figure showing the relationship between N / P ratio and zeta potential at the time of aggregating antisense oligo DNA with PLL or protamine. It is a figure showing the result of a sucrose density gradient centrifugation. It is a figure showing the result of a polyacrylamide electrophoresis. It is a figure showing the luciferase expression suppression rate by various MEND. It is a figure showing the time dependence and concentration dependence of a luciferase expression suppression rate.

Claims (11)

A composition for suppressing the expression of a target gene, comprising a liposome having a first peptide containing a plurality of continuous arginine residues on its surface and DNA or RNA capable of suppressing the expression of the target gene. ,
The DNA is encapsulated in the liposome in an aggregated state with protamine or a derivative thereof,
The composition in which the RNA is encapsulated in the liposome in a state of being aggregated with a second peptide containing a plurality of continuous arginine residues or a derivative thereof modified with a hydrophobic group.   The composition according to claim 1, wherein the first peptide is a peptide consisting of 4 to 35 amino acid residues containing 4 to 20 consecutive arginine residues.   The composition according to claim 1 or 2, wherein the first peptide consists only of arginine residues.   The composition according to any one of claims 1 to 3, wherein the amount of the first peptide is 2% (molar ratio) or more with respect to the total lipid constituting the lipid membrane of the liposome.   The composition according to any one of claims 1 to 4, wherein the second peptide is a peptide consisting of 4 to 35 amino acid residues containing 4 to 20 consecutive arginine residues.   The composition according to any one of claims 1 to 5, wherein the second peptide consists of only an arginine residue.   The composition according to any one of claims 1 to 6, wherein the hydrophobic group that modifies the second peptide is a stearyl group.   The composition according to any one of claims 1 to 7, wherein the ratio of the cationic lipid to the total lipid constituting the lipid membrane of the liposome is 0 to 40% (molar ratio).   The first peptide is modified with a hydrophobic group or a hydrophobic compound, the hydrophobic group or the hydrophobic compound is inserted into the lipid membrane of the liposome, and the peptide is exposed from the lipid membrane of the liposome. The composition according to any one of claims 1 to 8.   The composition according to claim 9, wherein the hydrophobic group that modifies the first peptide is a stearyl group. The composition according to any one of claims 1 to 10, wherein the DNA is antisense DNA and the RNA is siRNA.