JP2012147677A - Modified double-stranded rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a modified double-stranded RNA having inhibitory activity on the expression of a protein encoded by a target gene.SOLUTION: There is provided the modified double-stranded RNA having following characteristics (1)-(5): (1) the RNA has a sense strand which comprises a target sequence of 30-100 bases homologous to an mRNA of the target gene; (2) the RNA has an antisense strand which comprises a sequence complementary to the target sequence; (3) at least two contiguous nucleotides in the 22nd to 24th positions from the 5'-terminal of the sense strand and/or at least two contiguous nucleotides in the 22nd to 24th positions from the 3'-terminal of the antisense strand are nucleotides in each of which hydroxy at the position 2' in a ribose is substituted; (4) at least two contiguous nucleotides in the 22nd to 24th positions from the 3'-terminal of the sense strand and/or at least two contiguous nucleotides in the 22nd to 24th positions from the 5'-terminal of the antisense strand are nucleotides in each of which hydroxy at the position 2' in the ribose is substituted; and (5) the RNA has the inhibitory activity on the expression of the protein encoded by the target gene.

Description

  The present invention relates to a novel double-stranded modified RNA.

RNA interference refers to a phenomenon in which gene expression is suppressed in a sequence-specific manner by degradation or translation inhibition of mRNA having a base sequence complementary to a double-stranded RNA (short interfering RNA: siRNA) of about 19 to 23 bases ( For example, refer nonpatent literature 1 and 2.). The siRNA is generated by cleaving long double-stranded RNA present in the cell with Dicer having RNAase III-like activity.
Recently, a 25-base double-stranded modified RNA in which the hydroxyl group at the 2′-position of ribose of a 4-base nucleotide continuous from the 5′-end and the 3′-end of the sense strand has been replaced with a methoxy or fluorine atom has been produced by Dicer. It has been reported to cause RNA interference without being cleaved. However, no double-stranded RNA longer than 25 bases has been reported so far that causes RNA interference without being cleaved by Dicer.

Nature, 391, 1998, p. 806-811 Nature 411, 2001, p. 494-498

  The main object of the present invention is to provide a novel double-stranded modified RNA having an activity to suppress the expression of the protein encoded by the target gene, and a pharmaceutical comprising the complex of the double-stranded modified RNA and a carrier It is to provide a composition.

For example, the inventor found the inventions listed in the following 1 and 2 and completed the present invention.
1. Double-stranded modified RNA having the following characteristics (1) to (5) (hereinafter referred to as “the present RNA”):
(1) having a sense strand containing a 30-100 base target sequence homologous to the mRNA of the target gene;
(2) having an antisense strand comprising a sequence complementary to the target sequence;
(3) The 2 ′ position of ribose for at least two consecutive nucleotides 22-24 from the 5 ′ end of the sense strand and / or at least two consecutive nucleotides 22-24 from the 3 ′ end of the antisense strand A hydroxyl group of which is substituted,
(4) The 2 ′ position of ribose with respect to at least two consecutive nucleotides 22-24 from the 3 ′ end of the sense strand and / or at least two consecutive nucleotides 22-24 from the 5 ′ end of the antisense strand A hydroxyl group of which is substituted,
(5) It has the expression suppression activity of the protein which a target gene codes.
2. A pharmaceutical composition comprising a complex of the RNA of the present invention and a carrier (hereinafter referred to as “the composition of the present invention”).

FIG. 1 shows the cleavage activity by Dicer. In the figure, “−” represents a Dicer non-added group, and “+” represents a Dicer added group. FIG. 2 shows the cleavage activity by Dicer. In the figure, “−” represents a Dicer non-added group, and “+” represents a Dicer added group. FIG. 3 shows the cleavage activity by Dicer. In the figure, “−” represents a Dicer non-added group, and “+” represents a Dicer added group. FIG. 4 shows the cleavage activity by Dicer. In the figure, “−” represents a Dicer non-added group, and “+” represents a Dicer added group. FIG. 5 shows the expression-suppressing activity of Bcl-2 protein. The upper row shows the Bcl-2 protein, and the lower row shows the β-actin protein. FIG. 6 shows Bcl-2 protein expression inhibitory activity. The vertical axis represents the ratio of the expression level of Bcl-2 protein to the expression level of β-actin protein. FIG. 7 shows the expression suppression activity of SOD1 protein. The upper row shows SOD1 protein, and the lower row shows β-actin protein. FIG. 8 shows the expression suppression activity of SOD1 protein. The vertical axis represents the ratio of the expression level of SOD1 protein to the expression level of β-actin protein. FIG. 9 shows hnRNPH protein expression inhibitory activity. The upper row shows hnRNPH protein, and the lower row shows β-actin protein. FIG. 10 shows the expression suppression activity of hnRNPH protein. The vertical axis represents the ratio of the expression level of hnRNPH protein to the expression level of β-actin protein. FIG. 11 shows the expression suppression activity of luciferase protein. FIG. 12 shows the expression suppression activity of luciferase protein.

  Hereinafter, embodiments of the present invention will be described in detail.

I. RNA of the present invention
The RNA of the present invention is a double-stranded modified RNA having the following characteristics (1) to (5).
(1) having a sense strand containing a 30-100 base target sequence homologous to the mRNA of the target gene;
(2) having an antisense strand comprising a sequence complementary to the target sequence;
(3) The 2 ′ position of ribose for at least two consecutive nucleotides 22-24 from the 5 ′ end of the sense strand and / or at least two consecutive nucleotides 22-24 from the 3 ′ end of the antisense strand A hydroxyl group of which is substituted,
(4) The 2 ′ position of ribose with respect to at least two consecutive nucleotides 22-24 from the 3 ′ end of the sense strand and / or at least two consecutive nucleotides 22-24 from the 5 ′ end of the antisense strand A hydroxyl group of which is substituted,
(5) It has the expression suppression activity of the protein which a target gene codes.

In the RNA of the present invention, the 2′-position hydroxyl group of ribose of nucleotides 22 and 23 from the 5 ′ end of the sense strand and / or the antisense strand and / or the 23 ′ from the 3 ′ end of the sense strand and / or the antisense strand And the hydroxyl group at the 2 ′ position of ribose of the 24th nucleotide is preferably substituted.
Further, the hydroxyl group at the 2 ′ position of the ribose of the 24th nucleotide from the 5 ′ end of the sense strand and / or the antisense strand and / or the ribose of the 22nd nucleotide from the 3 ′ end of the sense strand and / or the antisense strand It is more preferable that the 2′-position hydroxyl group is substituted.

The “target gene” is not particularly limited and can be arbitrarily selected. For example, a gene that is considered to be the cause of a disease caused by abnormal expression of the protein encoded by the target gene, or a gene for which a part of the base sequence of the gene is known but the function of which is to be clarified it can. In addition, target genes include genes that act in a suppressive manner or genes that act in a stimulating manner against various diseases and the like.
Specifically, cancer-related genes, apoptosis-related genes, hematological malignant disease-related genes, metabolic disease-related genes, cardiovascular disease-related genes, neurological disease-related genes, urological disease-related genes, autoimmune disease-related genes, cytokines Examples include genes, development-related genes, growth-related genes, aging-related genes, immune-related genes, viral genes, viroid genes, prion genes, microorganism-derived genes, and protozoan-derived genes.

Examples of the “cancer-related gene” include cancer genes (for example, bcr / abl gene, c-fms gene, c-fos gene, c-myb gene, c-myc gene, K-ras gene, c-erbB-2). Gene) and tumor suppressor genes (for example, p53 gene, RB gene, BRCA1 / BRCA2 gene, WT1 gene, VHL gene, PTEN gene, bcl-2 gene, bcl-XI gene).
Examples of the “viral gene” include human papilloma virus gene, hepatitis B and C virus gene, and cytomegalovirus (CMV) gene.
“Viroid genes” include, for example, potato and potato viroid (PSTV) genes.
Examples of the “autoimmune disease-related gene” include tumor necrosis factor (TNF) -α gene.
Examples of the “cytokine gene” include an interferon gene and an interleukin gene.
Examples of “development-related genes” include homeogenes.
Examples of the “growth factor-related gene” include vascular endothelial growth factor (VEGF) gene, hepatocyte growth factor (HGF) gene, and fibroblast growth factor (FGF) gene.
Examples of the “prion gene” include a human prion gene and a bovine prion gene.
Examples of the “microbe-derived gene” include pathogenic E. coli such as O157.
Examples of the “protozoan-derived gene” include a malaria parasite-derived gene.

  The “target sequence of 30 to 100 bases homologous to the target gene mRNA” is suitably a sequence having 80% or more homology with a part of the target gene mRNA, and has 90% or more homology. Sequences are preferred, and sequences with 100% homology are more preferred. However, the homology is calculated by excluding the nucleotide in which the 2'-position hydroxyl group of ribose is substituted among the 22-24th nucleotides from the 5 'end and 3' end of the sense strand.

  The chain length of the target sequence is suitably in the range of 30 to 100 bases, preferably in the range of 30 to 80 bases, and more preferably in the range of 30 to 50 bases.

  The “sequence complementary to the target sequence” is suitably a sequence having 80% or more complementarity with the target sequence, preferably a sequence having 90% or more complementarity, and a sequence having 100% complementarity. More preferred. However, complementarity is calculated by excluding nucleotides in which the hydroxyl group at the 2'-position of ribose is substituted among the 22-24th nucleotides from the 5'-end and 3'-end of the antisense strand.

Examples of the “nucleotide in which the 2′-position hydroxyl group of ribose is substituted” include, for example, the 2′-position hydroxyl group of ribose is a hydrogen atom, alkyl, aryl, arylalkyl, alkoxyalkyl, aryloxyalkyl, alkoxy, aryloxy , Arylalkyloxy, alkoxyalkyloxy, aryloxyalkyloxy, thiol, alkylthio, amino, alkylamino, dialkylamino, aminoalkyloxy, alkylaminoalkyloxy, dialkylaminoalkyloxy, azide, cyano, nitro and halogen And nucleotides substituted with any group. More specifically, a hydrogen atom, a fluorine atom, methoxy, methoxyethoxy, aminopropyloxy, dimethylaminoethoxy, and dimethylaminoethoxyethoxy can be exemplified.
Among them, a hydrogen atom, a fluorine atom, and methoxy are preferable, and a hydrogen atom is particularly preferable.

  “Having the expression suppression activity of the protein encoded by the target gene” means that the suppression rate is 0% when the expression level of the protein encoded by the target gene is equal to that of the negative control in the cell into which the RNA of the present invention is introduced And the suppression rate is 50% or more, preferably 75% or more, and more preferably 90% or more.

  In the RNA of the present invention, the total length of the sense strand and the antisense strand is the same or different and is suitably in the range of 30 to 104 bases, preferably in the range of 30 to 84 bases, and in the range of 30 to 54 bases. More preferred.

  In the RNA of the present invention, a part of the ribonucleotide constituting the sense strand and / or the antisense strand may be substituted with a modified ribonucleotide, deoxyribonucleotide or modified deoxyribonucleotide.

  “Modified ribonucleotide” and “modified deoxyribonucleotide” refer to a nucleotide in which at least one of “sugar”, “nucleobase”, and “phosphate backbone” constituting the nucleotide is modified.

Examples of the modification of “sugar” include modification of the hydroxyl group and hydrogen atom at the 2 ′ position of ribose, modification of the hydrogen atom at the 2 ′ position of deoxyribose, modification with the oxygen atom at the 4 ′ position of sugar as a sulfur atom, Modification using a hydrogen atom at the 2 ′ position of ribose and a hydroxyl group as a carbonyl group, or a modification of bridging the hydroxyl group at the 2 ′ position of ribose and a carbon atom at the 4 ′ position with methylene [so-called Bridged Nucleic Acid (BNA) or Locked Nucleic Acid (LNA)].
Modification of the hydroxyl group at the 2 ′ position of ribose is the same as described above.
Examples of the modification of the hydrogen atom at the 2 ′ position of ribose or deoxyribose include, for example, alkyl, aryl, arylalkyl, alkoxyalkyl, aryloxyalkyl, alkoxy, aryloxy, arylalkyloxy, alkoxyalkyloxy, aryloxyalkyloxy, Examples of the modification include substitution with any group selected from thiol, alkylthio, amino, alkylamino, dialkylamino, aminoalkyloxy, alkylaminoalkyloxy, dialkylaminoalkyloxy, azide, cyano, nitro and halogen. .

Examples of the “nucleobase” include 2-aminopurine, 2-amino-1,9-dihydro-6H-purin-6-one (guanine), 6-amino-9H-purine (adenine), and 8-aminopurine. 2,6-diaminopurine, 2,6,8-triaminopurine, 6,8-diaminopurine, 6-amino-2-hydroxypurine, 2-amino-6-hydroxypurine, 8-amino-6-hydroxy Purine bases such as purine, 6-amino-8-hydroxypurine, 6-hydroxypurine, 2,6-dihydroxypurine or their tautomers: 5-methyl-1,2,3,4-tetrahydropyrimidine-2 , 4-dione (thymine), 4-aminopyrimidin-2 (1H) -one (cytosine), pyrimidine-2,4 (1H, 3H) -dione (uracil), 4,5-dia Nopyrimidin-2-one, 4,6-diaminopyrimidin-2-one, 5-aminopyrimidine-2,4-dione, 6-aminopyrimidine-2,4-dione, 5-aminopyrimidin-2-one, 6 -Pyrimidine bases such as aminopyrimidin-2-one, 2,4-dihydroxypyrimidine or their tautomers.
The modified form of nucleobase refers to a nucleobase modified with an appropriate substituent at any substitutable position in a purine base or pyrimidine base, for example. Examples of the substituent include 1 to 3 substituents selected from the group consisting of halogen, acyl, alkyl, arylalkyl, alkoxy, alkoxyalkyl, hydroxy, carboxy, cyano, and nitro.

    Examples of the modification of the phosphate backbone include modifications such as phosphorothioate, phosphorodithioate, alkylphosphonate, boranophosphate, or phosphoramidate.

Examples of “alkyl” include linear or branched alkyl having 1 to 6 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl. The alkyl may be substituted, and examples of the substituent include halogen, alkyl, alkoxy, cyano, and nitro, and 1 to 3 of these may be substituted.
Examples of “halogen” include fluorine, chlorine, bromine and iodine.
Examples of “alkoxy” include linear or branched alkoxy having 1 to 6 carbon atoms. Examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy and isohexyloxy. Among these, C1-C3 alkoxy is preferable.
Examples of “aryl” include aryl having 6 to 10 carbon atoms. Examples thereof include phenyl, α-naphthyl, and β-naphthyl. The aryl may be substituted, and examples of the substituent include halogen, alkyl, alkoxy, cyano, and nitro, and 1 to 3 of these may be substituted at any position. Of these, phenyl is preferred.
Examples of “acyl” include linear or branched alkanoyl having 1 to 6 carbon atoms and aroyl having 7 to 13 carbon atoms. Examples include formyl, acetyl, n-propionyl, isopropionyl, n-butyryl, isobutyryl, tert-butyryl, valeryl, hexanoyl, benzoyl, naphthoyl, and levulinyl.
“Arylalkyl”, “arylalkyloxy”, “alkoxyalkyl”, “alkoxyalkyloxy”, “alkylthio”, “alkylamino”, “dialkylamino”, “aminoalkyloxy”, “alkylaminoalkyloxy”, “ The “alkyl” part of “dialkylaminoalkyloxy”, “aryloxyalkyl” and “aryloxyalkyloxy” may be the same as the above “alkyl”.
Examples of the “alkoxy” part of “alkoxyalkyl” and “alkoxyalkyloxy” include the same as the above “alkoxy”.
Examples of the “aryl” part of “arylalkyl”, “aryloxyalkyl”, “arylalkyloxy”, “aryloxyalkyloxy” and “aryloxy” include the same as the above “aryl”.
Examples of the “halogen”, “alkyl” and “alkoxy” which are the substituents of “alkyl” and “aryl” are the same as those described above.

  The RNA of the present invention may have an overhang at the 3 'end and / or the 5' end of its sense strand and / or antisense strand.

  As the overhang, 1 to 4 nucleotides are suitable, and 2 nucleotides are preferred. The nucleotide may be ribonucleotide or deoxyribonucleotide. Further, it may be a modified ribonucleotide or a modified deoxyribonucleotide. Among those embodiments, deoxyribonucleotides are suitable, and deoxythymidine monophosphate is preferred. Modified ribonucleotides and modified deoxyribonucleotides are the same as described above.

  The RNA of the present invention may contain a base having one or more deletions, substitutions, insertions or additions in its sense strand and / or antisense strand.

  The RNA of the present invention can be produced by, for example, phosphoramidite method [for example, 2′-position hydroxyl group is converted to 2 ′-(2-cyanoethoxy) methyl (CEM), 2′-tert-butyldimethylsilyloxymethyl (TOM), Method using ribonucleotide phosphoramidite compound converted to 2′-bis (acetoxymethoxy) methylethyl (ACE) or 2′-tert-butyldimethylsilyl (TBDMS)] or solid phase synthesis method using H-phosphonate method Or by a liquid phase synthesis method by a triester method.

Modification of the phosphate backbone can be performed by methods known to those skilled in the art (for example, “Methods in Molecular Biology”, 20, 1993, Humana Press Inc.).
The phosphorothioate compound can be produced, for example, by using a sulfurizing agent such as sulfur or tetraethylthiodisulfide (TETD) instead of an oxidant by iodine-water in the oxidation step of solid phase synthesis by the phosphoramidite method, for example. it can.
The phosphorodithioate can be produced, for example, by using phosphorothioamidite instead of phosphoramidite and using a sulfurizing agent such as sulfur in the oxidation step.
The alkyl phosphonate body can be produced, for example, by using an alkyl phosphonamidite such as methylphosphonamidite instead of the phosphoramidite.
For example, in the solid-phase synthesis by the H-phosphonate method, the phosphoramidate body is produced by synthesizing the H-phosphonate body and then using a desired primary or secondary amine and an oxidizing agent such as iodine. Can do.
The boranophosphate body can be produced, for example, by using boron and a tertiary amine after synthesizing the H-phosphonate body in solid phase synthesis by the H-phosphonate method.

  The RNA of the present invention can be transfected into cells using a carrier described later. It can also be directly introduced into cells by the calcium phosphate method, electroporation method or microinjection method.

II. Composition of the present invention The composition of the present invention comprises a complex of the RNA of the present invention and a carrier.

  The carrier is not particularly limited as long as it is effective for transferring the RNA of the present invention into cells. Examples thereof include cationic carriers such as cationic liposomes, cationic polymers, and cationic dendrimers, and carriers using virus envelopes or nanoparticles.

As the “cationic liposome”, for example,
(1) A cationic liposome carrier (hereinafter referred to as “liposome A”) formed with 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol and phospholipid as essential components. ),
(2) 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol, 1,3-distearoylglycero-2-phosphatidyl-N- (methoxypolyethyleneglycol succinyl) ethanolamine and phosphorus A cationic liposome (hereinafter referred to as “liposome B”) containing lipid as an essential component;
(3) 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol, N- (methoxy polyethylene glycol succinyl) distearoyl phosphatidylethanolamine [SUNBRIGHT DSPE-020C; manufactured by NOF Corporation] And a cationic liposome (hereinafter, referred to as “liposome C”) containing phospholipid as an essential constituent component,
(4) Oligofectamine (manufactured by Invitrogen),
(5) Lipofectin (manufactured by Invitrogen),
(6) Lipofectamine (manufactured by Invitrogen),
(7) Cellfectin (manufactured by Invitrogen),
(8) Lipofectamine 2000 (manufactured by Invitrogen),
(9) DMRIE-C (manufactured by Invitrogen),
(10) GeneSilencer (manufactured by Gene Therapy Systems),
(11) TransMessenger (manufactured by QIAGEN),
(12) TransIT-TKO (manufactured by Miras) can be mentioned.
Among them, liposome A, liposome B, and liposome C are preferable.

  2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol can be produced by the method described in the literature (Example 20 of WO 94/19314).

  1,3-distearoylglycero-2-phosphatidyl-N- (methoxypolyethyleneglycol succinyl) ethanolamine is produced by the method described in the literature (Cancer Research vol. 68, no. 21 (2008) pp. 8843-8851). can do. The compound is preferably distributed within a molecular weight range of 2000 to 3800. The molecular weight can be measured by a mass spectrum using an electrospray ionization method.

  Phospholipids that can be used in liposomes A, B and C include, for example, phosphatidylcholine [eg, dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine], phosphatidyl List ethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, dipalmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg yolk lecithin, soybean lecithin or their hydrogenated phospholipids be able to.

Lipofectin is N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) as a neutral lipid 1: It is a cationic liposome containing 1 (mol / mol).
Lipofectamine is composed of 2,3-dioleoxyoroxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneammonium trifluoroacetate (DOSPA) and dioleoylphosphatidylethanolamine. It is a cationic liposome containing 3: 1 (w / w) of (DOPE).
Cellfectin is a cation containing N, N ¹ , N ² , N ³ -tetramethyl-tetrapalmitylspermine (TM-TPS) and dioleoylphosphatidylethanolamine (DOPE) at a ratio of 1: 1.5 (mol / mol). Liposomes.
DMRIE-C is a cationic liposome containing 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE) and cholesterol (Cholesterol) at 1: 1 (mol / mol).

  Examples of the “cationic polymer” include jetSI (Qbiogene), jetPEI (polyethyleneimine; Qbiogene), and atelocollagen.

  Examples of the “virus envelope” include GenomeONE (HVJ-E liposome; manufactured by Ishihara Sangyo Co., Ltd.).

  Examples of the “cationic dendrimer” include a cationic amino acid dendrimer (for example, dendritic poly (L-lysine) (for example, “Organic Biomolecular Chemistry”, 2003, Vol. 1, 1270-1273) or a polyamidoamine dendrimer. Can be mentioned.

  Examples of the “nanoparticles” include gold nanoparticles and silica nanoparticles, and the average particle diameter is suitably in the range of 1 nm to 5000 nm, for example.

  Examples of the “gold nanoparticles” include cationic gold nanoparticles (“Bioconjugate Chemistry”, 2002, 13, 3-6), cationic gold nanoparticles modified with polyethylene glycol (for example, “Journal of” Controlled Release ", 2006, 111, 382-389).

  The kind of RNA of the present invention contained in the composition of the present invention may be one kind, but may be 2 to 10 kinds.

The concentration of the RNA of the present invention contained in the composition of the present invention varies depending on the type of carrier and the like, but when used in vitro, for example, the range of 0.1 nM to 10 μM is appropriate, and 10 nM to 1 μM. Within the range of is preferable. Moreover, when using it in vivo, the inside of the range of 0.1-10 mg / mL is suitable, for example, and the inside of the range of 0.5-2 mg / mL is preferable.
The weight ratio of the RNA of the present invention to the carrier (carrier / RNA of the present invention) contained in the composition of the present invention varies depending on the nature of the RNA of the present invention, the type of the carrier, etc., but is suitably within the range of 0.01-100. In the range of 1-50, the range of 5-30 is more preferable.

  The composition of the present invention can optionally contain a pharmaceutically acceptable additive. Examples of such additives include emulsification aids (for example, fatty acids having 6 to 22 carbon atoms and pharmaceutically acceptable salts thereof, albumin, dextran), stabilizers (for example, cholesterol, phosphatidic acid), and isotonic agents. (For example, sodium chloride, glucose, maltose, lactose, sucrose, trehalose) and pH adjusters (for example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine) can be mentioned. One or more of these can be used. The content of the additive in the composition of the present invention is suitably 90% by weight or less, preferably 70% by weight or less, and more preferably 50% by weight or less.

  The composition of the present invention can be prepared by adding the RNA of the present invention to a carrier dispersion and stirring appropriately. Further, the additive can be added in an appropriate step before or after the RNA of the present invention is added. The solvent that can be used for adding the RNA of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, glucose solution, and maltose. Examples thereof include sugar liquids such as liquid. Further, conditions such as pH and temperature in such a case can be appropriately selected by those skilled in the art.

  The composition of the present invention can be, for example, a solution or a lyophilized preparation thereof. The lyophilized preparation can be prepared by lyophilizing the composition of the present invention having a liquid form according to a conventional method. For example, after appropriately sterilizing the composition of the present invention in the form of a liquid agent, a predetermined amount is dispensed into a vial, and pre-freezing is performed at about -40 to -20 ° C for about 2 hours. Primary drying can be performed under reduced pressure at about 0 to 10 ° C., followed by secondary drying at about 15 to 25 ° C. under reduced pressure and freeze-drying. In general, the inside of the vial is replaced with nitrogen gas and stoppered to obtain a lyophilized preparation of the composition of the present invention.

  In general, the above lyophilized preparation can be re-dissolved and used by adding any appropriate solution (re-dissolved solution). Examples of such redissolved solution include water for injection, physiological saline, and other general infusion solutions. The amount of the redissolved solution varies depending on the use and the like and is not particularly limited. However, an amount of 0.5 to 2 times the amount of the solution before lyophilization or 500 mL or less is appropriate.

III. Medicinal use of the RNA of the present invention and the composition of the present invention The RNA of the present invention or the composition of the present invention is used, for example, for the treatment and / or prevention of diseases caused by abnormal expression of the protein encoded by the target gene. be able to.
For example, used for the treatment and / or prevention of cancer, viral diseases, metabolic diseases, cardiovascular diseases, neurological diseases, urological diseases, hematological malignancies, or diseases where the promotion or suppression of apoptosis is desired Can do.

  The RNA or the composition of the present invention is administered to animals including humans by intravenous administration, intraarterial administration, oral administration, tissue administration (for example, intravesical administration, intrathoracic administration, intraperitoneal administration, intraocular administration, Administration into the brain), transdermal administration, transmucosal administration, pulmonary administration, or rectal administration. In particular, intravenous administration, transdermal administration, and transmucosal administration are desirable. Needless to say, these dosage forms are suitable for administration, for example, various injections, oral preparations, drops, inhalants, eye drops, ointments, lotions and suppositories.

The dosage of the RNA of the present invention or the composition of the present invention is determined, for example, for adults in consideration of the type of RNA of the present invention, dosage form, patient condition such as age and weight, administration route, nature and degree of disease. In general, it is generally within the range of 0.1 mg to 5 g / human, preferably within the range of 1 mg to 2 g per day. This value may vary depending on the type of target disease, dosage form, and target molecule. Therefore, in some cases, a lower dose may be sufficient, and conversely, a higher dose may be required. Moreover, it can be administered once to several times a day or at intervals of 1 day to several days.

  Hereinafter, the present invention will be described in detail with reference to production examples and test examples. However, it goes without saying that the present invention is not limited to the description of the examples.

Production Example 1 Preparation of Double-stranded RNA (1) Double-stranded RNA Sequence The double-stranded RNA used in Test Examples 1 to 3 is as follows. In the following sequences, “dA”, “dT”, “dG”, and “dC” represent nucleotides (DNA) in which the hydroxyl group at the 2′-position of ribose is replaced with a hydrogen atom. “A”, “u”, “g” and “c” represent nucleotides in which the hydroxyl group at the 2′-position of ribose is substituted with methoxy.
Bcl2-1:
Sense strand (SEQ ID NO: 1)
5'-GUG AUG AAG UAC AUC CAU UdTdT-3 '
Antisense strand (SEQ ID NO: 2)
5′-AAU GGA UGU ACU UCA UCA CdTdT-3 ′
Bcl2-2:
Sense strand (SEQ ID NO: 3)
5'-CGA UAA CCG GGA GAU AGU GAU
GAA GUA CAU CCA UUDT dT-3 '
Antisense strand (SEQ ID NO: 4)
5'-AAU GGA UGU ACU UCA UCA CUA
UCU CCC GGU UAU CGdT dT-3 '
Bcl2-3:
Sense strand (SEQ ID NO: 5)
5'-CGA UAA CCG GGA G dAdT AGU GAU
dGdA A GUA CAU CCA UUdT dT-3 '
Antisense strand (SEQ ID NO: 4)
5'-AAU GGA UGU ACU UCA UCA CUA
UCU CCC GGU UAU CGdT dT-3 '
Bcl2-4:
Sense strand (SEQ ID NO: 6)
5'-CGA UAA CCG GGA G au AGU GAU
ga A GUA CAU CCA UUdT dT-3 '
Antisense strand (SEQ ID NO: 4)
5'-AAU GGA UGU ACU UCA UCA CUA
UCU CCC GGU UAU CGdT dT-3 '
Bcl2-5:
Sense strand (SEQ ID NO: 7)
5'-GGG UAC GAU AAC CGG GAG AUA
GUG AUG AAG UAC AUC CAU UdTdT-3 '
Antisense strand (SEQ ID NO: 8)
5'-AAU GGA UGU ACU UCA UCA CUA
UCU CCC GGU UAU CGU ACC CdTdT-3 '
Bcl2-6:
Sense strand (SEQ ID NO: 9)
5'-GGG UAC GAU AAC CGG GAG dAdTA
dGdT G AUG AAG UAC AUC CAU UdTdT-3 '
Antisense strand (SEQ ID NO: 8)
5'-AAU GGA UGU ACU UCA UCA CUA
UCU CCC GGU UAU CGU ACC CdTdT-3 '
Bcl2-7:
Sense strand (SEQ ID NO: 10)
5'-GGG UAC GAU AAC CGG GAG au A
gu G AUG AAG UAC AUC CAU UdTdT-3 '
Antisense strand (SEQ ID NO: 8)
5'-AAU GGA UGU ACU UCA UCA CUA
UCU CCC GGU UAU CGU ACC CdTdT-3 '

SOD1-1:
Sense strand (SEQ ID NO: 11)
5′-GGU GGA AAU GAA GAA AGU AdTdT-3 ′
Antisense strand (SEQ ID NO: 12)
5′-UAC UUU CUU CAU UUC CAC CdTdT-3 ′
SOD1-2:
Sense strand (SEQ ID NO: 13)
5'-AGA UGA CUU GGG CAA AGG UGG AAA
UGA AGA AAG UAdT dT-3 '
Antisense strand (SEQ ID NO: 14)
5'-UAC UUU CUU CAU UUC CAC CUU UGC
CCA AGU CAU CUdT dT-3 '
SOD1-3:
Sense strand (SEQ ID NO: 15)
5'-AGA UGA CUU GGG C dAdA dA GG UGG
dAdAdA UGA AGA AAG UAdT dT-3 '
Antisense strand (SEQ ID NO: 14)
5'-UAC UUU CUU CAU UUC CAC CUU UGC
CCA AGU CAU CUdT dT-3 '
SOD1-4:
Sense strand (SEQ ID NO: 16)
5'-AAA GCA GAU GAC UUG GGC AAA GGU
GGA AAU GAA GAA AGU AdTdT-3 '
Antisense strand (SEQ ID NO: 17)
5'-UAC UUU CUU CAU UUC CAC CUU UGC
CCA AGU CAU CUG CUU UdTdT-3 '
SOD1-5:
Sense strand (SEQ ID NO: 18)
5'-AAA GCA GAU GAC UUG GGC dAdAdA
dGdGdT GGA AAU GAA GAA AGU AdTdT-3 ′
Antisense strand (SEQ ID NO: 17)
5'-UAC UUU CUU CAU UUC CAC CUU UGC
CCA AGU CAU CUG CUU UdTdT-3 '

hnRNPH-1
Sense strand (SEQ ID NO: 19)
5'-AAC UUG AAU CAG AAG AUG AdTdT-3 '
Antisense strand (SEQ ID NO: 20)
5'-UCA UCU UCU GAU UCA AGU UdTdT-3 '
hnRNPH-2
Sense strand (SEQ ID NO: 21)
5'-GGC GAG GCU UUU GUU GAA CUU GAA
UCA GAA GAU GAdT dT-3 '
Antisense strand (SEQ ID NO: 22)
5'-UCA UCU UCU GAU UCA AGU UCA ACA
AAA GCC UCG CCdT dT-3 '
hnRNPH-3
Sense strand (SEQ ID NO: 23)
5'-GGC GAG GCU UUU G dTdT dG AA CUU
dGdAdA UCA GAA GAU GAdT dT-3 ′
Antisense strand (SEQ ID NO: 22)
5'-UCA UCU UCU GAU UCA AGU UCA ACA
AAA GCC UCG CCdT dT-3 '
hnRNPH-4
Sense strand (SEQ ID NO: 24)
5'-CAA GUG GCG AGG CUU UUG UUG AAC
UUG AAU CAG AAG AUG AdTdT-3 '
Antisense strand (SEQ ID NO: 25)
5'-UCA UCU UCU GAU UCA AGU UCA ACA
AAA GCC UCG CCA CUU GdTdT-3 '
hnRNPH-5
Sense strand (SEQ ID NO: 26)
5'-CAA GUG GCG AGG CUU UUG dTdTdG
dAdAdC UUG AAU CAG AAG AUG AdTdT-3 '
Antisense strand (SEQ ID NO: 25)
5'-UCA UCU UCU GAU UCA AGU UCA ACA
AAA GCC UCG CCA CUU GdTdT-3 '

GL3-1:
Sense strand (SEQ ID NO: 27)
5'-CUU ACG CUG AGU ACU UCG AdTdT-3 '
Antisense strand (SEQ ID NO: 28)
5'-UCG AAG UAC UCA GCG UAA GdTdT-3 '
GL3-2:
Sense strand (SEQ ID NO: 29)
5'-GGU GGA CAU CAC UUA CGC UGA
GUA CUU CGA dTdT-3 '
Antisense strand (SEQ ID NO: 30)
5'-UCG AAG UAC UCA GCG UAA GUG
AUG UCC ACC dTdT-3 '
GL3-3:
Sense strand (SEQ ID NO: 31)
5'-GGU GGA CA dT dCdA C UUA CGC UGA
dGdT A CUU CGA dTdT-3 ′
Antisense strand (SEQ ID NO: 30)
5'-UCG AAG UAC UCA GCG UAA GUG
AUG UCC ACC dTdT-3 '
GL3-4:
Sense strand (SEQ ID NO: 32)
5'-AUC GAG GUG GAC AUC ACU UAC
GCU GAG UAC UUC GAdT dT-3 '
Antisense strand (SEQ ID NO: 33)
5'-UCG AAG UAC UCA GCG UAA GUG
AUG UCC ACC UCG AUDT dT-3 '
GL3-5:
Sense strand (SEQ ID NO: 34)
5'-AUC GAG GUG GAC A dTdC dA CU UAC
dGdC U GAG UAC UUC GAdT dT-3 '
Antisense strand (SEQ ID NO: 33)
5'-UCG AAG UAC UCA GCG UAA GUG
AUG UCC ACC UCG AUDT dT-3 '
GL3-6:
Sense strand (SEQ ID NO: 35)
5'-CAC AUA UCG AGG UGG ACA UCA
CUU ACG CUG AGU ACU UCG AdTdT-3 '
Antisense strand (SEQ ID NO: 36)
5'-UCG AAG UAC UCA GCG UAA GUG
AUG UCC ACC UCG AUA UGU GdTdT-3 '
GL3-7:
Sense strand (SEQ ID NO: 37)
5'-CAC AUA UCG AGG UGG ACA dTdCdA
dCdT U ACG CUG AGU ACU UCG AdTdT-3 '
Antisense strand (SEQ ID NO: 36)
5'-UCG AAG UAC UCA GCG UAA GUG
AUG UCC ACC UCG AUA UGU GdTdT-3 '

Bcl2-1 is described in WO2004 / 105774, SOD1-1 is described in literature (Biochemical and Biophysical Research Communications, 314, 2004, p.283-291), and hnRNPH-1 is described in literature (Nature Biotechnology, 23, 2004, p. 222-226), GL3-1 is a sequence described in the literature (Journal of Controlled Release, 131, 2008, p. 64-69), and all suppress the expression of the protein encoded by the target gene. To get.
(2) Synthesis of RNA strands constituting double-stranded RNA The synthesis of RNA strands constituting double-stranded RNA was commissioned to Japan Bioservice.
Each RNA strand was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Factory Co., Ltd., the same shall apply hereinafter) so as to have a concentration of 100 μM.

Production Example 2 Preparation of Pharmaceutical Composition (1) Preparation of Carrier (Liposome A) 2-O- (2-diethylaminoethyl) carbamoyl-1,3 produced by the method described in the literature (Example 20 of WO94 / 19314) -60 mg of O-dioleoylglycerol and 100 mg of egg yolk lecithin (manufactured by Kewpie) were dissolved in 2 mL of chloroform in a vial, and nitrogen gas was blown onto this to remove the chloroform, thereby forming a thin film on the wall of the vial. It was. This was further allowed to stand overnight under reduced pressure, 1000 mg of maltose (manufactured by Hayashibara), 4.0 mL of water for injection and 80 μL of 1N hydrochloric acid were added, and the thin film was dispersed with a vortex mixer. After leaving at 4 ° C. for 3 hours, ultrasonic treatment was performed for 10 minutes using a microprobe (Sonifier-250, manufactured by BRANSON). Thereafter, the volume was adjusted to 10 mL using water for injection to prepare a 16 mg / mL dispersion.
(2) Preparation of pharmaceutical composition The sense strand and antisense strand synthesized in Production Example 1 (2) are mixed in equimolar amounts and diluted with a 10% maltose solution (manufactured by Otsuka Pharmaceutical Factory Co., Ltd., the same shall apply hereinafter). Strand RNA was prepared. The carrier prepared in (1) above is appropriately diluted with a 10% maltose solution, and the same volume of double-stranded RNA is added dropwise little by little while stirring to form a complex of double-stranded RNA and carrier (pharmaceutical composition). ) Was formed.
The concentration of double-stranded RNA in the complex was 0.1 to 3 μM, and the ratio of double-stranded RNA and carrier in the complex was 1:16 (w / w). The prepared complex was appropriately diluted with 10% maltose.

Test Example 1 In Vitro Cleaving Activity by Dicer The sense strand and the antisense strand synthesized in Production Example 1 (2) were each 20 μM in annealing buffer (30 mM HEPES-KOH pH 7.4, 100 mM KCl, 2 mM MgCl ₂ ). It prepared so that it might become. Using PROGRAM TEMP CONTROL SYSTEM PC-800 (manufactured by ASTEC), it was heated to 95 ° C., held for 2 minutes, and then cooled to 30 ° C. over 60 minutes to prepare double-stranded RNA. The prepared double-stranded RNA was subjected to an in vitro cleavage reaction using Recombinant Dicer Enzyme Kit (manufactured by Genlantis). After the reaction, electrophoresis was carried out with TBE (× 0.5) on a 15% polyacrylamide gel so that the double-stranded RNA was 20 pmol / lane, stained with ethidium bromide, and ChemiDoc (manufactured by Bio-Rad Laboratories). Detected.
The results are shown in FIGS. As shown in FIGS. 1-4, it turned out that this invention RNA is hard to receive the cutting | disconnection by Dicer. On the other hand, natural double-stranded RNA was cleaved by Dicer.

Test Example 2 Bcl-2 protein, SOD1 protein or hnRNPH protein expression-inhibiting activity A431 cells (human epithelial cancer cell line) were seeded at a density of 1 × 10 ⁵ cells in a 6 cm petri dish. The cells were cultured overnight in 3 mL of DMEM medium containing 10% fetal bovine serum (manufactured by Sigma, the same shall apply hereinafter) under conditions of 37 ° C. and 5% CO ₂ . The medium was replaced with 2.7 mL of fresh medium, 0.3 mL of 30 nM pharmaceutical composition prepared in Production Example 2 was added, and the mixture was cultured for 3 days. The cells were washed twice with PBS (manufactured by Nissui, the same applies hereinafter), and then transferred to a 1.5 mL tube using a cell scraper. Centrifuge at 1000 × g for 2 minutes, remove the supernatant, suspend in 50-100 μL of buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40] and then on ice for 30 minutes. Left to stand. Centrifugation was performed at 15000 × g and 4 ° C. for 20 minutes, and the supernatant (cell extract) was recovered.
The protein concentration in the cell extract was measured using a BCA protein assay system (Pierce Biotechnology).
15 μg of protein in the cell extract was electrophoresed with e-PAGEEL (registered trademark) (manufactured by ATTO) and transferred to a polyvinylidene fluoride (PVDF) membrane (manufactured by Millipore). The membrane to which the protein was transferred was blocked by shaking for 30 minutes at room temperature in Blocking One (manufactured by Nacalai Tesque, the same applies hereinafter). The primary antibody was diluted with blocking one (5% blocking one) diluted 20-fold with PBS containing 0.1% Tween-20 (PBST), and shaken in the diluted solution for 1 hour at room temperature. After washing with PBST, the mixture was shaken in a secondary antibody diluted with 5% blocking one for 1 hour at room temperature. After washing with PBST, light is emitted using Western Lightening Chemiluminescence Reagent Plus (manufactured by PerkinElmer), and Bcl-2 protein, SOD1 protein, hnRNPH protein or β-actin protein is produced using ChemiDoc (manufactured by Bio-Rad Laboratories). The amount of expression of each protein was detected and quantified. In addition, the cell which added GL3-1 and the cell which added maltose instead of the pharmaceutical composition were made into the negative control.
In the detection of Bcl-2 protein, a mouse anti-human bcl-2 antibody (M0887, manufactured by DAKO CYTOMATION) was diluted 500 times as the primary antibody, and a peroxidase (HRP) -labeled anti-mouse IgG antibody was used as the secondary antibody. (P0260, DAKO CYTOMATION) diluted 2000 times was used.
In detection of the SOD1 protein, a primary antibody obtained by diluting a goat anti-human SOD1 antibody (# SOD-101, manufactured by Stressgen) 1000 times was used as a secondary antibody, and an HRP-labeled anti-rabbit IgG antibody (# 7074, CellSignaling). ) Were diluted 2000 times, respectively.
In the detection of hnRNPH protein, 200-fold diluted goat anti-human hnRNPH antibody (sc-10042, manufactured by Santa Cruz) was used as the primary antibody, and HRP-labeled anti-goat IgG antibody (P-0449, DAKO CYTOMATION Co., Ltd.) diluted 2000 times were used.
In detection of β-actin protein, a primary antibody obtained by diluting a goat anti-human β-actin antibody (sc-1615, manufactured by Santa Cruz) 2000-fold was used as a secondary antibody, and an HRP-labeled anti-goat IgG antibody (P0449) was used. , DAKO CYTOMATION) diluted 2000 times, or as a primary antibody, mouse anti-human β-actin antibody (sc-8432, manufactured by Santa Cruz) diluted 2000 times as a primary antibody. Used were HRP-labeled anti-mouse IgG antibodies (P-0260, manufactured by DAKO CYTOMATION) diluted 2000 times.
The results are shown in FIGS. As shown in FIGS. 5 to 10, the RNA of the present invention is a protein (Bcl-2 protein, SOD1 protein or hnRNPH protein) encoded by a target gene at the same level or stronger than the corresponding natural double-stranded RNA. Expression was suppressed.

Test Example 3 Luciferase Protein Expression Inhibitory Activity A549 cells (human lung cancer-derived cells) that stably express luciferase were seeded at 1000 cells / well in a 96-well plate, 37 in 100 μL of DMEM medium containing 10% fetal calf serum ° C., and cultured overnight in 5% CO _2. The next day, 11.11 μL of 100 nM pharmaceutical composition prepared in Production Example 2 was added. 72 hours after the addition of the pharmaceutical composition, the medium was replaced with 75 μL of the fresh medium, and 75 μL of a commercially available luciferase activity measurement kit (Steady-Glo Luciferase Assay System; manufactured by Promega) was added to cause luminescence dependent on luciferase. The amount of luminescence was measured with a microplate chemiluminescence detector (Top Count; manufactured by Packard). In addition, the cell which added maltose instead of the pharmaceutical composition was made into the negative control.
The results are shown in FIGS. As shown in FIGS. 11 and 12, the RNA of the present invention suppressed the expression of the luciferase protein to the same extent or more strongly than the corresponding natural double-stranded RNA even when the luciferase gene was targeted.

Claims (29)

Double-stranded modified RNA having the following characteristics (1) to (5):
(1) having a sense strand containing a 30-100 base target sequence homologous to the mRNA of the target gene;
(2) having an antisense strand comprising a sequence complementary to the target sequence;
(3) The 2 ′ position of ribose for at least two consecutive nucleotides 22-24 from the 5 ′ end of the sense strand and / or at least two consecutive nucleotides 22-24 from the 3 ′ end of the antisense strand A hydroxyl group of which is substituted,
(4) The 2 ′ position of ribose with respect to at least two consecutive nucleotides 22-24 from the 3 ′ end of the sense strand and / or at least two consecutive nucleotides 22-24 from the 5 ′ end of the antisense strand A hydroxyl group of which is substituted,
(5) It has the expression suppression activity of the protein which a target gene codes. Double-stranded modified RNA having the following characteristics (1) to (5):
(1) having a sense strand containing a 30-100 base target sequence homologous to the mRNA of the target gene;
(2) having an antisense strand comprising a sequence complementary to the target sequence;
(3) A nucleotide in which the hydroxyl group at the 2′-position of ribose is substituted for the 22nd and 23rd nucleotides from the 5 ′ end of the sense strand and / or the 23th and 24th nucleotides from the 3 ′ end of the antisense strand thing,
(4) The 23rd and 24th nucleotides from the 3 'end of the sense strand and / or the 22nd and 23rd nucleotides from the 5' end of the antisense strand are nucleotides in which the hydroxyl group at the 2 'position of ribose is substituted. thing,
(5) It has the expression suppression activity of the protein which a target gene codes.   2′-position hydroxyl group of ribose of the 24th nucleotide from the 5 ′ end of the sense strand and / or antisense strand and / or 2 of ribose of the 22nd nucleotide from the 3 ′ end of the sense strand and / or antisense strand The double-stranded modified RNA according to claim 2, which is a nucleotide further substituted at the 'position hydroxyl group.   The hydroxyl group at the 2 ′ position of ribose is a hydrogen atom, alkyl, aryl, arylalkyl, alkoxyalkyl, aryloxyalkyl, alkoxy, aryloxy, arylalkyloxy, alkoxyalkyloxy, aryloxyalkyloxy, thiol, alkylthio, amino, The alkylamino, dialkylamino, aminoalkyloxy, alkylaminoalkyloxy, dialkylaminoalkyloxy, azide, cyano, nitro and halogen are substituted with any group, characterized in that The described double-stranded modified RNA.   The double-stranded modified RNA according to claim 1, wherein the hydroxyl group at the 2'-position of ribose is substituted with any group selected from a hydrogen atom, a fluorine atom and methoxy.   The double-stranded modified RNA according to any one of claims 1 to 5, wherein a part of the ribonucleotide constituting the sense strand and / or the antisense strand is substituted with deoxyribonucleotide or modified deoxyribonucleotide.   The double strand according to any one of claims 1 to 5, wherein a nucleotide having 1 to 4 bases is added as a protruding portion to the 3 'end and / or the 5' end of the sense strand and / or the antisense strand. Modified RNA.   The double-stranded modified RNA according to claim 7, wherein the nucleotide added as the overhang is a deoxyribonucleotide.   The double-stranded modified RNA according to claim 7, wherein the nucleotide added as the overhang is 2-base deoxythymidine monophosphate.   The double-stranded modified RNA according to any one of claims 1 to 5, wherein a part of the ribose or phosphate backbone constituting the nucleotide of the sense strand and / or the antisense strand is modified.   Modification of the ribose or phosphate backbone is a hydrogen atom at the 2 'hydroxyl group of ribose, alkyl, aryl, arylalkyl, alkoxyalkyl, aryloxyalkyl, alkoxy, aryloxy, arylalkyloxy, alkoxyalkyloxy, aryloxyalkyl Modification of substitution of any group selected from oxy, thiol, alkylthio, amino, alkylamino, dialkylamino, aminoalkyloxy, alkylaminoalkyloxy, dialkylaminoalkyloxy, azide, cyano, nitro and halogen, ribose A modification in which the 4'-position is a thio form or a modification in which the phosphate backbone is a phosphorothioate form, a phosphorodithioate form, an alkylphosphonate form or a phosphoramidate form. Double-stranded modified RNA according to 0.   The double-stranded modified RNA according to any one of claims 1 to 5, comprising a base that has one or more deletions, substitutions, insertions or additions to the sense strand and / or the antisense strand.   A pharmaceutical composition comprising the complex of the double-stranded modified RNA according to any one of claims 1 to 12 and a carrier.   The pharmaceutical composition according to claim 13, wherein the carrier is a carrier utilizing a cationic carrier, a virus envelope or nanoparticles.   The pharmaceutical composition according to claim 14, wherein the cationic carrier is a cationic liposome.   Cationic liposome in which the cationic liposome has 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol and phospholipid as essential components: 2-O- (2-diethylaminoethyl) Cationic liposomes comprising carbamoyl-1,3-O-dioleoylglycerol, 1,3-distearoylglycero-2-phosphatidyl-N- (methoxypolyethyleneglycol succinyl) ethanolamine and phospholipid as essential components: Or a cationic liposome comprising 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol, N- (methoxypolyethyleneglycol succinyl) distearoyl phosphatidylethanolamine and phospholipid as essential components There, the pharmaceutical composition according to claim 15. The pharmaceutical composition according to claim 16, wherein the phospholipid is phosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, phosphatidylethanolamine, egg yolk lecithin, soybean lecithin or hydrogenated phospholipids thereof. object.   15. A pharmaceutical composition according to claim 14, wherein the cationic carrier is a cationic polymer.   15. A pharmaceutical composition according to claim 14, wherein the cationic carrier is a cationic dendrimer.   20. A pharmaceutical composition according to claim 19, wherein the cationic dendrimer is a polyamidoamine dendrimer.   15. The pharmaceutical composition according to claim 14, wherein the nanoparticles are gold nanoparticles.   The double-stranded modified RNA according to any one of claims 1 to 12, for the treatment and / or prevention of a disease caused by abnormal expression of a protein encoded by a target gene.   The double-stranded modified RNA according to any one of claims 1 to 12, for the treatment and / or prevention of cancer, viral diseases, metabolic diseases, cardiovascular diseases, neurological diseases or urological diseases.   The double-stranded modified RNA according to any one of claims 1 to 12, for treating and / or preventing a disease for which promotion or suppression of apoptosis is desired.   The double-stranded modified RNA according to any one of claims 1 to 12, for the treatment and / or prevention of a hematological malignancy.   The pharmaceutical composition according to any one of claims 13 to 21, for the treatment and / or prevention of a disease caused by abnormal expression of a protein encoded by a target gene.   The pharmaceutical composition according to any one of claims 13 to 21, for the treatment and / or prevention of cancer, viral diseases, metabolic diseases, cardiovascular diseases, neurological diseases or urological diseases.   The pharmaceutical composition according to any one of claims 13 to 21, for the treatment and / or prevention of a disease for which promotion or suppression of apoptosis is desired. The pharmaceutical composition according to any one of claims 13 to 21, for the treatment and / or prevention of a hematological malignancy.