JPWO2020090211A1 - Stabilization of double-stranded RNA by cationic artificial oligosaccharides - Google Patents

Abstract

(1) The step of introducing a modification into the skeleton of double-stranded RNA, (2) the double-stranded RNA obtained by step (1), and 2,6-diamino-2,6-dideoxy-β- (1). → 4) Provided is a method for regulating the stability of double-stranded RNA, which comprises a step of contacting with a cationic oligosaccharide such as -D-galactopyranose tetramer. In addition, a complex of double-stranded RNA with a modified skeleton and a cationic oligosaccharide such as 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer. A nucleic acid composition comprising , Nucleic acid composition is provided.

Description

The present invention relates to stabilization of double-stranded RNA with cationic artificial oligosaccharides.

In recent years, the development of nucleic acid drugs using double-stranded RNA such as siRNA or miRNA has become active. Since nucleic acid drugs can target any gene in the living body, they are strongly expected as a new-generation therapeutic drug that is effective for various diseases that were difficult to treat with conventional drugs. On the other hand, the fact that nucleic acids are easily degraded by nucleases in vivo is a major obstacle in the development of nucleic acid drugs.

Nucleic acid analogs into which various chemical modifications have been introduced are used in nucleic acid pharmaceuticals currently under development in order to prevent degradation by nucleases. Typical chemical modifications include modification (2'-O-methylation) in which the hydroxyl group at the 2'position of the ribose ring is replaced with a 2'-O-methyl group, and non-phosphate moiety that connects nucleic acids. There is phosphorothioate modification (thiophosphorylation) in which one of the crosslinked oxygen atoms is replaced with a sulfur atom. All of these chemical modifications are intended to confer nuclease resistance, but their effects are limited. Further, for example, when excessive chemical modification is introduced into siRNA or miRNA, there is a problem that the effect of suppressing the expression of a target gene is diminished. Therefore, it is desired to establish a method capable of imparting nuclease resistance while maintaining the original functional activity of nucleic acid drugs using double-stranded RNA.

As another measure for improving the nuclease resistance of nucleic acid drugs using double-stranded RNA, stabilization of the double-stranded structure by using a cationic compound as a carrier molecule has been attempted. Since RNase present in serum specifically cleaves single-strand RNA generated by dissociation of double strands, stabilization of the double-stranded structure results in RNase resistance of nucleic acid drugs using double-stranded RNA. Is expected to be improved. The present inventors have succeeded in synthesizing oligoamino sugars having a high affinity for double-stranded RNA (Patent Document 1), and found that they improve the RNase resistance of double-stranded RNA to some extent. Confirmed (Non-Patent Document 1). However, since it is necessary for the double chain to dissociate when the nucleic acid drug exhibits pharmaceutical activity, if the double chain structure is excessively stabilized, the pharmaceutical activity of the nucleic acid drug decreases. Becomes a problem.

International Publication No. 2010/104192

Hara, R. I. et al. , Org. Biomol. Chem. , (2017), Vol. 15, pp. 1710-1717

The present invention has been made to provide a method for solving various problems of the prior art and enhancing the stability without impairing the functional activity of the nucleic acid drug.

As a result of diligent research, the present inventors have dramatically reduced the serum half-life by combining a double-stranded RNA having a skeletal modification with a cationic oligosaccharide while maintaining the pharmaceutical activity of the double-stranded RNA. I found that it could be improved.

で表される２価の基（ここで、Ｒ^＋は、ＮＨ_３ ^＋または式（ＩＩ）：

（ここで、Ｒ^３、Ｒ^４およびＲ^５は、それぞれ独立して水素原子もしくはメチル基である）で表される基である）から選択される］で表されるカチオン性オリゴ糖とを接触させるステップとを含む方法を提供するものである。That is, according to one embodiment, the present invention is a method for regulating the stability of double-stranded RNA, in which (1) a step of introducing a modification into the skeleton of double-stranded RNA and (2) a step (2) ( Double-stranded RNA obtained by 1) and general formula (I):
R ^1- O- (X) _n- R ²
[During the ceremony,
R ¹ and R ² are independent hydrogen atoms or monovalent substituents, respectively.
n is an integer of 3 to 6 and
The n Xs may be the same or different, and independently of each of the following (a) to (i) :.

In expressed divalent group (wherein the, ^{R +} is, _NH ^{3 +} or formula (II):

(Here, R ³ , R ⁴ and R ⁵ are groups represented by independently hydrogen atoms or methyl groups)) are contacted with the cationic oligosaccharides represented by. It provides a method including a step to make it.

Further, according to one embodiment, the present invention is a complex of (1) double-stranded RNA in which a modification has been introduced into the skeleton and (2) a cationic oligosaccharide represented by the above general formula (I). A nucleic acid composition comprising , A nucleic acid composition is provided.

In the general formula (I), ^{it is preferable that R 1} is a hydrogen atom, R ² is a hydrogen atom or a substituted or unsubstituted alkyl group, and n is 3 or 4.

The cationic oligosaccharide represented by the general formula (I) is preferably 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer.

At least 10% of the entire backbone of the double-stranded RNA may be modified.

The modification may be selected from 2'-O-methylation and phosphorothioation, preferably phosphorothioation.

The modification is preferably introduced into the backbone of the 3'end region of at least one RNA strand constituting the double-stranded RNA.

The double-stranded RNA is preferably 12 to 50 base pair length.

The method according to the present invention is desired for double-stranded RNA while maintaining the pharmaceutical activity of double-stranded RNA by appropriately combining the type and number of skeletal modifications and the type of cationic oligosaccharide. Stability can be imparted. Therefore, the serum half-life of nucleic acid drugs using double-strand RNA can be freely controlled, and unnecessary and undesired actions can be reduced or avoided.

Further, since the nucleic acid composition according to the present invention has a dramatically improved serum half-life while maintaining the pharmaceutical activity, a sufficient effect can be obtained even at a small dose.

FIG. 1 shows the interaction of the cationic oligosaccharide 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer (ODAGal4) with the main groove of double-stranded RNA. It is a schematic diagram which shows a style. FIG. 2 is a diagram in which unmodified siRNA (HP2) complexed / unmodified with ODAGal4 treated with a serum solution was detected by electrophoresis. FIG. 3 is a graph quantifying the results of FIG. FIG. 4 is a graph showing the serum half-life of siRNA calculated from the results of FIG. FIG. 5 is a diagram in which unmodified siRNA (B2M2) complexed / unmodified with ODAGal4 treated with a serum solution was detected by electrophoresis. FIG. 6 is a graph quantifying the results of FIG. FIG. 7 is a graph showing the serum half-life of siRNA calculated from the results of FIG. FIG. 8 shows siRNAs (HP2, HP2-M1-HP2-M3 and HP2-S1-HP2-) with / without skeletal modification (2'-O-methylation or phosphorothioatetization) and / or complexation with ODAGal4. It is a graph which shows the serum half-life of S3). FIG. 9 is a graph showing the relative ratio of serum half-life calculated from the result of FIG. FIG. 10 shows siRNAs (B2M2, B2M2-M1-B2M2-M3 and B2M2-S1-B2M2-) with / without skeletal modification (2'-O-methylation or phosphorothioatetization) and / or complexation with ODAGal4. It is a graph which shows the serum half-life of S3). FIG. 11 is a graph showing the relative ratio of serum half-life calculated from the results of FIG. FIG. 12 shows siRNAs (HP2, HP2-M1-HP2-M3 and HP2-S1-HP2-) with / without skeletal modification (2'-O-methylation or phosphorothioatetization) and / or complexation with ODAGal4. It is a graph which shows the knockdown efficiency (relative ratio of hypoxanthine phosphoribosyl transferase 1 (HPRT1) gene expression level) of S3). FIG. 13 shows siRNAs (B2M2, B2M2-M1-B2M2-M3 and B2M2-S1-B2M2-) with / without skeletal modification (2'-O-methylation or phosphorothioatetization) and / or complexation with ODAGal4. It is a graph which shows the knockdown efficiency (relative ratio of β-2-microglobulin gene expression level) of S3). FIG. 14 (A) is a graph showing the serum half-life of siRNAs (HP2, HP2-S4 to HP2-S7) with / without skeletal modification (phosphorothioateization) and / or complexing with ODAGal4. FIG. 14 (B) is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 15 (A) is a graph showing the serum half-life of siRNAs (HP2, HP2-S8 to HP2-S11) with / without skeletal modification (phosphorothioateization) and / or complexing with ODAGal4. FIG. 15B is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 16 (A) is a graph showing the serum half-life of siRNAs (B2M2, B2M2-S4 to B2M2-S7) with / without skeletal modification (phosphorothioateization) and / or complexing with ODAGal4. FIG. 16B is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 17 (A) is a graph showing the serum half-life of siRNAs (B2M2, B2M2-S8 to B2M2-S11) with / without skeletal modification (phosphorothioateization) and / or complexing with ODAGal4. FIG. 17B is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 18 (A) is a graph showing the serum half-life of siRNAs (HP2, HP2-S7, HP2-S12 and HP2-S13) with / without skeletal modification (phosphorothioateization) and / or complexing with ODAGal4. Is. FIG. 18B is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 19 (A) is a siRNA with / without skeletal modification (phosphorothioateization) and / or complexation with ODAGal4, which has a 3'protruding DNA end (HP2, HP2-T and HP2-TS). ) Is a graph showing the serum half-life. FIG. 19B is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 20 (A) is a graph showing the serum half-life of siRNAs (HP2, HP2-S14-HP2-S16) with / without skeletal modification (phosphorothioateization) and / or complexing with ODAGal4. FIG. 20B is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 21 (A) shows siRNAs (HP2, HP2-M1, HP2-S1, HP2) with / without skeletal modification (2'-O-methylation and / or phosphorothioatetization) and / or complexation with ODAGal4. -It is a graph which shows the serum half-life of MS1, HP2-M4, HP2-S2 and HP2-MS2). FIG. 21 (B) is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 22 (A) shows siRNAs (HP2, HP2-F) with / without skeletal modifications (2'-F and / or phosphorothioated, LNA and / or phosphorothioated) and / or complexed with ODAGal4. , HP2-FS, HP2-L and HP2-LS) are graphs showing the serum half-life. FIG. 22B is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 23 (A) shows sera of short double-stranded RNA (12 base pairs) (12M and 12M-S) with / or blunt-ended skeletal modifications (phosphorothioatetization) and / or complexing with ODAGal4. It is a graph which shows the half-life. FIG. 23 (B) is a graph showing the relative ratio of serum half-life calculated from the result of (A). FIG. 24 (A) is a graph showing the serum half-life of siRNAs (HP2 and HP2-S3) with / without skeletal modification (phosphorothioateization) and / or complexing with various cationic oligosaccharides. FIG. 24 (B) is a graph showing the relative ratio of serum half-life calculated from the result of (A).

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the embodiments described in the present specification.

で表される２価の基（ここで、Ｒ^＋は、ＮＨ_３ ^＋または式（ＩＩ）：

（ここで、Ｒ^３、Ｒ^４およびＲ^５は、それぞれ独立して水素原子もしくはメチル基である）で表される基である）から選択される］で表されるカチオン性オリゴ糖とを接触させるステップとを含む方法である。According to the first embodiment, the present invention is a method for regulating the stability of double-stranded RNA, in which (1) a step of introducing a modification into the skeleton of double-stranded RNA and (2) a step (2) ( Double-stranded RNA obtained by 1) and general formula (I):
R ^1- O- (X) _n- R ²
[During the ceremony,
R ¹ and R ² are independent hydrogen atoms or monovalent substituents, respectively.
n is an integer of 3 to 6 and
The n Xs may be the same or different, and independently of each of the following (a) to (i) :.

In expressed divalent group (wherein the, ^{R +} is, _NH ^{3 +} or formula (II):

(Here, R ³ , R ⁴ and R ⁵ are groups represented by independently hydrogen atoms or methyl groups)) are contacted with the cationic oligosaccharides represented by. It is a method including a step to make it.

In this embodiment, "stability" of double-stranded RNA means the thermal stability of the double-stranded structure of RNA and / or resistance to nucleases such as blood RNase.

In the method of this embodiment, a modification is introduced into the skeleton of double-stranded RNA. The "double-strand RNA" in the present embodiment means a single-strand RNA consisting of an arbitrary sequence and a single-strand RNA having complementarity to the sequence hybridized with each other. The double-stranded RNA in the present embodiment may include one substantially composed of the double-stranded RNA. Here, "substantially composed" of double-stranded RNA means about 1 to 3 base pairs at the end of double-stranded RNA, for example, to the extent that it does not affect the overall structure of double-stranded RNA. Means that it contains double-stranded DNA or double-stranded DNA / RNA.

The length of the double-stranded RNA in the present embodiment is not particularly limited, but may be, for example, 10 to 200 base pairs, preferably 12 to 50 base pairs. Preferred double-stranded RNAs in this embodiment include, for example, siRNA, miRNA, and precursors thereof.

The double-stranded RNA in this embodiment does not have to form a complete double-strand throughout, and may be one or more (eg, 1), provided that substantially double strands are formed. One, two, three, four, five, or more) mismatches or bulges may be included. In other words, the double-stranded RNA in this embodiment does not have to have perfect or complete (ie 100%) complementarity and is at least 80% or more, 90% or more, 95% or more, or 100%. It suffices to have the complementarity of. Further, each of the RNA strands constituting the double strand may have the same length or may have different lengths. Therefore, the end of double-stranded RNA may be a blunt end or a sticky end (5'protruding or 3'protruding end). Alternatively, one of the ends of the double-stranded RNA may be linked to each other, in which case the double-stranded RNA can form a hairpin structure.

In this embodiment, the "skeleton" of double-stranded RNA means the binding moiety between nucleosides (in natural nucleic acids, the phosphodiester bond from 3'to 5') and / or the sugar moiety. Therefore, the method of this embodiment introduces modifications into the binding and / or sugar moieties between the nucleosides of the RNA strands that make up the double strand. Modifications at the binding moiety between nucleosides include, for example, phosphorothioation, boranophosphate, phosphorodithioate, boranophosphorothioate and the like. Modifications at the sugar moiety include modification at the 2'position (for example, 2'-O-methylation, 2'-O-methoxymethylation, 2'-F formation, etc.), and cross-linking between the 2'position and the 4'position (for example, 2'-O-methylation, 2'-F formation, etc.). For example, 2', 4'-BNA (also known as LNA (Locked Nucleic Acid)), amino-LNA, thio-LNA, α-L-oxy-LNA, ENA (2'-O, 4'-C-Ethylene-bridged). Nucleic Acid), AmNA (Amido-bridged Nucleic Acid), GuNA (Guandine-bridged Nucleic Acid), scpBNA (2'-O, 4'-C-spiroxyclocycleElectronvic Acid), 3'-amino-2', 4'-BNA, 5'-amino-2', 4'-BNA, PrNA (2'-O, 4'-C-Propyrene-bridged Nucleic Acid), 2' , 4'-BNANC (2'-O, 4'-C-aminomethylene-bridged Nucleic Acid), 2', 4'-BNACOC (2'-O, 4'-C-methylneoxymethylene-bridged Nucleic Acid), etc. Examples thereof include substitution of the sugar moiety with a morpholino ring. In the method of the present embodiment, only one of these modifications or a combination of two or more modifications can be used. Preferred modifications in the methods of this embodiment will vary depending on the use and function of the double-stranded RNA, for example, 2'-O-methylation and / or phosphorothioationation will be introduced into the double-stranded RNA such as siRNA and miRNA. It is particularly preferred that phosphorothioateization be introduced.

In the method of this embodiment, at least one modification can be introduced into the skeleton of either or both of the RNA strands constituting the double strand. The number of modifications contained in the backbone of the RNA strand may be appropriately determined according to the desired stability of the double strand RNA. The double-stranded RNA in this embodiment may contain, for example, 5, 10, 15, 20, 25, 30 or more skeletal modifications, depending on its length. In other words, the double-stranded RNA in this embodiment is, for example, about 10% or more of the entire skeleton of the double-stranded RNA (that is, the binding portion and / or the sugar portion between nucleosides contained in the entire double-stranded RNA). It may be 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% modified. Further, the site where the modification is introduced is not particularly limited, and for example, the modification may be introduced dispersedly over the entire length, or the modification may be introduced continuously only at the terminal portion, preferably a double chain. Modifications can be introduced into the 3'end region of at least one RNA strand that constitutes.

The introduction of modifications to the skeleton of RNA can be performed by a well-established and conventionally known method. Further, RNA having a skeletal modification may be synthesized by outsourcing to a nucleic acid synthesis contractor.

Next, the double-stranded RNA having a modification introduced into the skeleton is brought into contact with the cationic oligosaccharide represented by the general formula (I).

In the general formula (I), R ¹ and R ² are independently hydrogen atoms or monovalent substituents. The monovalent substituent is not limited to the following, and examples thereof include substituted or unsubstituted alkyl groups. The alkyl group may be, for example, a C _1-20 alkyl group, preferably a C _1-12 alkyl group, and even more preferably a C _1-6 alkyl group. Further, the alkyl group may include any of linear, branched and cyclic forms. Alkyl groups may have one or more hydrogen atoms substituted with substituents. Examples of the substituent in this case include a hydroxyl group, an alkoxy group, a halogen atom, an amino group, a monoalkylamino group, a dialkylamino group, a carboxyl group, an alkoxycarbonyl group, an oxo group and a sulfo group. The number of substituents and the position of substitution are not particularly limited, but the number of substituents is preferably 0 to 3. In the general formula (I), R ¹ is preferably a hydrogen atom and R ² is a hydrogen atom or a substituted or unsubstituted C _1-6 alkyl group.

In the general formula (I), the n Xs may be the same or different, and are independently selected from the divalent groups represented by the above (a) to (i). Here, in (a) ~ (i), R + is a guanidino group represented by _NH ^{3 +} or above Formula (II), in the general formula ^{(II), R 3, R} 4 and ^{R 5} Are independently hydrogen atoms or methyl groups. In the general formula (I), all n Xs may be the same divalent group. For example, in (a) ~ (i), R + is a _NH ^{3 +,} when the divalent group represented has n pieces bonded with (a) is 2,6-diamino-2,6 -Dideoxy-α- (1 → 4) -D-glucopyranose oligomer, and when n divalent groups represented by (b) are bonded, 3,6-diamino-3,6? Dideoxy- When α- (1 → 4) -D-glucopyranose oligomer is formed and n divalent groups represented by (d) are bonded, 2,6-diamino-2,6-dideoxy-α-(. 1 → 4) -D-mannopyranose oligomer, and when n divalent groups represented by (e) are bonded, 3,6-diamino-3,6-dideoxy-α- (1 →) 4) It becomes -D-mannopyranose oligomer. When n divalent groups represented by (g) are bonded, it becomes 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose oligomer, and ( When n divalent groups represented by h) are bonded, it becomes 3,6-diamino-3,6-dideoxy-β- (1 → 4) -D-galactopyranose oligomer. In the general formula (I), it is preferable that n Xs are all (a), n Xs are all (d), or n Xs are all (g), and n Xs are all (g). g) is particularly preferable. n is an integer of 3 to 6, preferably 3 or 4.

That is, the cationic oligosaccharides that can be used in the method of the present embodiment are 2,6-diamino-2,6-dideoxy-α- (1 → 4) -D-glucopyranose trimmer or tetramer, 3,6-. Diamino-3,6-dideoxy-α-(1 → 4) -D-glucopyranose trimmer or tetramer, 2,6-diamino-2,6-dideoxy-α- (1 → 4) -D-mannopyranose trimmer Or tetramer, 3,6-diamino-3,6-dideoxy-α- (1 → 4) -D-mannopyranose notrimer or tetramer, 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose trimmer or tetramer, 3,6-diamino-3,6-dideoxy-β- (1 → 4) -D-galactopyranose trimmer or tetramer, 2,6-diguanidino-2,6-dideoxy -Α- (1 → 4) -D-glucopyranose trimmer or tetramer, 3,6-diguanidino-3,6-dideoxy-α- (1 → 4) -D-glucopyranose trimmer or tetramer, 2,6-diguanidino -2,6-dideoxy-α- (1 → 4) -D-mannopyranose trimmer or tetramer, 3,6-diguanidino-3,6-dideoxy-α- (1 → 4) -D-mannopyranose trimmer Alternatively, tetramer, 2,6-diguanidino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose trimmer or tetramer, or 3,6-diguanidino-3,6-dideoxy-β- (1 → 4). ) -D-galactopyranose trimmer or tetramer is preferable, and in the method of this embodiment, only one of these or a combination of two or more cationic oligosaccharides can be used. The cationic oligosaccharide that can be used in the method of the present embodiment is particularly preferably 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer.

The cationic oligosaccharide that can be used in the method of the present embodiment is obtained by appropriately combining various conventionally known methods with the chemical synthesis method disclosed in International Publication No. 2010/104192 pamphlet and the chemical synthesis method equivalent thereto. It can be synthesized by.

The double-stranded RNA in which the skeleton has been modified and the cationic oligosaccharide represented by the general formula (I) are both water or a buffer solution having a low salt concentration (for example, phosphate buffered saline or Tris hydrochloric acid). It can be contacted by adding it to (such as a buffer) and incubating for a certain period of time. The concentration of double-stranded RNA can be appropriately selected, for example, in the range of 1 nM to 1 mM. The concentration of the cationic oligosaccharide varies depending on the desired stability of the double-stranded RNA, but can be appropriately selected, for example, in the range of 1 nM to 10 mM. The mixing ratio (molar ratio) of the double-stranded RNA and the cationic oligosaccharide is, for example, in the case of a double-strand RNA having a length of 21 base pairs, the double-strand RNA: cationic oligosaccharide = 1: 1 to 1:10. Can be. The incubation time may be, for example, 10 seconds to 24 hours, and the incubation temperature may be, for example, 0 to 40 ° C.

Alternatively, in an aqueous solution containing a cationic oligosaccharide represented by the general formula (I) or a buffer solution having a low salt concentration, the single-strand RNA having a modification introduced into the skeleton and the single-strand RNA having complement thereof are annealed. Thereby, the double-stranded RNA having the modification introduced into the skeleton may be brought into contact with the cationic oligosaccharide represented by the general formula (I). The annealing conditions may be, for example, holding the solution at 90 to 95 ° C. for 3 to 20 minutes and then cooling it to 0 to 30 ° C. at a rate of −0.5 to −1.5 ° C./min. The concentrations and mixing ratios of RNA and cationic oligosaccharides may be similar to those described above.

Double-stranded RNA generally has a three-dimensional structure called a type A helical structure, which has a narrow and deep main groove and a wide and shallow sub-groove. When the double-stranded RNA and the cationic oligosaccharide represented by the general formula (I) come into contact with each other, the cationic oligosaccharide interacts with the main groove of the spiral structure to stabilize the double-stranded structure. As an example, the interaction mode of the cationic oligosaccharide 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer with the main groove of double-stranded RNA is shown in FIG. Shown in. Here, RNase degrades double-stranded RNA by cleaving the single-strand RNA moiety that results from the disruption of the helical structure. Therefore, although we do not want to be bound by any particular theory, double-stranded RNA stabilized by cationic oligosaccharides indirectly acquires high RNase resistance as a result of the resistance to producing single-strand RNA moieties. It is thought that it can be done. In contrast, skeletal modifications such as 2'-O-methylation and phosphorothioatetization primarily confer RNase resistance directly on single-strand RNA. Therefore, it is considered that the stability of double-stranded RNA can be freely regulated by the combined use of a cationic oligosaccharide that stabilizes the double-stranded structure and a skeletal modification that enhances the RNase resistance of single-stranded RNA. .. Therefore, according to the method of the present embodiment, it is possible to obtain an RNA-based nucleic acid drug having a desired serum half-life, obtain a high pharmacological action, and reduce or avoid unnecessary and undesired actions. can.

Further, according to the method of the present embodiment, the type of cationic oligosaccharide that stabilizes the double-stranded structure and the type and number of skeletal modifications that enhance the RNase resistance of the single-stranded RNA are appropriately combined to obtain a pharmaceutical. It is possible to prepare nucleic acid compositions with extremely long serum half-life without compromising activity. According to the method of the present embodiment, it is possible to achieve a serum half-life of 5 times or more, preferably 10 times or more, particularly preferably 20 times or more, as compared with unmodified double-stranded RNA.

で表される２価の基（ここで、Ｒ^＋は、ＮＨ_３ ^＋または式（ＩＩ）：

（ここで、Ｒ^３、Ｒ^４およびＲ^５は、それぞれ独立して水素原子もしくはメチル基である）で表される基である）から選択される］で表されるカチオン性オリゴ糖と
の複合体を含んでなる核酸組成物であって、未修飾の二重鎖ＲＮＡと同等の薬学的活性を有し、かつ、未修飾の二重鎖ＲＮＡと比較して５倍以上長い血清半減期を有する、核酸組成物である。That is, according to the second embodiment, the present invention comprises (1) a double-stranded RNA in which a modification has been introduced into the skeleton, and (2) a general formula (I) :.
R ^1- O- (X) _n- R ²
[During the ceremony,
R ¹ and R ² are independent hydrogen atoms or monovalent substituents, respectively.
n is an integer of 3 to 6 and
The n Xs may be the same or different, and independently of each of the following (a) to (i) :.

In expressed divalent group (wherein the, ^{R +} is, _NH ^{3 +} or formula (II):

(Here, R ³ , R ⁴ and R ⁵ are groups represented by independently hydrogen atoms or methyl groups, respectively))] in combination with the cationic oligosaccharide. Nucleic acid composition comprising a body, having the same pharmaceutical activity as unmodified double-stranded RNA, and having a serum half-life that is 5 times or more longer than that of unmodified double-stranded RNA. It is a nucleic acid composition having.

The "double-stranded RNA", "skeleton", "modification" and "cationic oligosaccharide" in this embodiment are the same as those defined in the first embodiment.

The nucleic acid composition of the present embodiment is a complex of a double-stranded RNA having a modification introduced into the skeleton and a cationic oligosaccharide represented by the general formula (I) (hereinafter, "modified double-stranded nucleic acid-cation"). It is described as "sex oligosaccharide complex"). The modified double-stranded nucleic acid-cationic oligosaccharide complex in the present embodiment can be prepared according to the same procedure as the method of the first embodiment.

The nucleic acid composition of the present embodiment may be composed only of a modified double-stranded nucleic acid-cationic oligosaccharide complex, but generally, as a further optional component, a known pharmaceutically acceptable dilution. It may contain liquids, carriers, excipients and the like. In the nucleic acid composition of the present embodiment, the content of the modified double-stranded nucleic acid-cationic oligosaccharide complex is not particularly limited, but is usually about 0.001 to 100 parts by weight per 100 parts by weight of the nucleic acid composition. And it is sufficient.

The nucleic acid composition of the present embodiment can be formulated into various dosage forms, and the dosage forms include, for example, tablets, capsules, granules, powders, syrups, suspending agents, suppositories, ointments, and the like. Examples include creams, gels, patches, inhalants, and injections. Therefore, the nucleic acid composition of the present embodiment can be administered by various methods such as oral administration, intraperitoneal administration, intradermal administration, intravenous administration, intramuscular administration, and intracerebral administration. The nucleic acid composition of the present embodiment can be a solid preparation or a liquid preparation, and preferably a liquid preparation such as an injection or a rectal administration. In the case of solids, by conventional methods, appropriate additives such as starch, lactose, sucrose, mannitol, carboxymethyl cellulose, cornstarch, inorganic salts and, if desired, binders, disintegrants and abundance Agents and the like can be blended. When the solid agent is a tablet or a pill, it may be coated with a sugar coating such as sucrose, gelatin or hydroxypropyl cellulose or a film of a gastric-soluble or enteric-soluble substance, if desired. In the case of a liquid preparation, it is necessary to dissolve the modified double-chain nucleic acid-cationic oligosaccharide complex in a diluent such as distilled water for injection, physiological saline, aqueous glucose solution, propylene glycol, polyethylene glycol, etc. by a conventional method. It may be prepared by adding a bactericidal agent, a stabilizer, an isotonic agent, a pain-relieving agent, or the like.

The dose of the nucleic acid composition of the present embodiment may be appropriately set within a range in which the desired pharmaceutical activity can be obtained by converting it into the amount of double-stranded RNA or the active ingredient.

The nucleic acid composition of the present embodiment has pharmaceutical activity equivalent to that of unmodified double-stranded RNA, and is 5 times or more, preferably 10 times or more, particularly that of unmodified double-stranded RNA. It preferably has a serum half-life of 20 times or more. In order for the nucleic acid composition of the present embodiment to have a particularly long serum half-life, the 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer as a cationic oligosaccharide About 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more of the entire skeleton of double-stranded RNA. It is preferred that 90% or more, or 100%, is phosphorothioated. When modifying a part of the skeleton, it is preferable to introduce phosphorothioatetization into at least one 3'end region of the RNA strand constituting the double strand.

The nucleic acid composition of this embodiment has both intrinsic pharmaceutical activity and improved serum half-life. Therefore, it is useful because a desired effect (for example, in the case of siRNA, an effect of suppressing the expression of a target gene) can be obtained with a small dose.

Hereinafter, the present invention will be further described with reference to examples. It should be noted that these do not limit the present invention in any way.

<1. Materials and methods>
(1) Synthesis of siRNA siRNA (hereinafter referred to as “HP2”) and β-2- that target hypoxanthine phosphoribosyltransferase 1 (HPRT1) with / without 2'-O-methylation or phosphorothioatetization. SiRNA targeting microglobulin (hereinafter referred to as "B2M2") was prepared. The sense strand and antisense strand of siRNA were synthesized by commissioning Grinder Japan or Gene Design. The sense strand and antisense strand of siRNA were mixed in a buffer (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM HEPES-KOH, pH 7.4) and 95 by a thermal cycler (C1000, BIO-RAD). After heating at ° C. for 3 minutes, annealing was performed by cooling from 95 ° C. to 25 ° C. at a rate of −1.5 ° C./min to obtain double-stranded siRNA.

The sequence of siRNA is shown in Table 1. Uppercase letters indicate unmodified RNA, lowercase bold letters indicate 2'-O-methylation modification, and lowercase italics indicate phosphorothioateization modification (specifically, phosphorothioateization modification indicates between nucleosides in lowercase italicization). It is introduced in the joint portion. The same applies to Tables 3 and 4 below).

Table 1. sequence of siRNA

In addition, siRNA consisting of a sequence that does not act on either the HPRT1 gene or the β-2-microglobulin gene (sense strand: 5'-GUACCGCACGUCAUCGUAUC-3'(SEQ ID NO: 29) / antisense strand: 5'-UACGAUGACGUGCGUACGU- 3'(SEQ ID NO: 30)) was prepared by the same procedure as above (negative control).

The cationic oligosaccharide 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer (hereinafter referred to as “ODAGal4”) was used in the method of Hara et al. (Org. Biomol). Chem., (2017), Vol. 15, pp. 1710-1717).

(2) Analysis of serum half-life of siRNA By mixing siRNA (5 pmol) and ODAGal4 (20 pmol) in a buffer solution (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM HEPES-KOH, pH 7.4). A siRNA-ODAGal4 complex was formed. Then, mouse serum (Sigma-Aldrich) having a final concentration of 10% was added, and the mixture was incubated at 37 ° C. 0, 4, 6, 8, 12, 24, 36, 48, 72 or 96 hours after the addition of serum, an electrophoresis buffer (EXELDYE 6 × DNA Loading Dye, SMOBIO) was added to the reaction solution, and the reaction was carried out. Was stopped and subjected to 15% polyacrylamide gel electrophoresis (SuperSep DNA, Wako Pure Chemical Industries, Ltd.). The gel after migration was fluorescently stained with SYBR Green II (Takara Bio Inc.), and images were acquired with LAS-4000 (Fujifilm Inc.). The fluorescence intensity of siRNA was analyzed by ImageJ software (National Institute of Health, USA), and siRNA was quantified. The obtained measured values were analyzed by DeltaGraph software (RedLock software) or Excel software (Microsoft) to obtain an exponential approximation curve formula. From the functional formula of this curve, the time required for the residual amount of siRNA in the reaction solution to be half of that at the start of the reaction (decomposition half-life) was calculated.

(3) Analysis of siRNA melting temperature (Tm value) 100 μM siRNA sense strand or 100 μM siRNA sense strand or 100 μM siRNA sense strand or 100 μM siRNA sense strand or 100 μM siRNA sense strand or 5 μL of each of the antisense strand aqueous solutions was added and sufficiently mixed. After heating at 95 ° C. for 20 minutes using a thermal cycler, it was cooled to 20 ° C. at a rate of −0.5 ° C./min. 20 μL of 100 μM ODAGal4 aqueous solution or sterile water was added to prepare a total 200 μL aqueous solution. This was heated from 20 ° C. to 95 ° C. at a rate of 0.5 ° C./min using a UV-1650PC (Shimadzu Corporation), and the ultraviolet absorbances at 260 nm and 320 nm were measured at 0.2 ° C. intervals. The temperature was plotted on the horizontal axis, and the measured value obtained by subtracting the absorbance at 320 nm from the absorbance at 260 nm on the vertical axis was plotted to obtain a melting temperature curve. This curve was first-order differentiated, and the temperature indicating the peak value was calculated as the Tm value.

(4) Analysis of siRNA inhibitory effect on gene expression Liver cancer cell line Hep3B was sown in a 48-well plate (Gliner Japan) and cultured for 24 hours in DMEM medium supplemented with 10% fetal bovine serum (FCS). .. siRNA (2.5 pmol) and OGAGal4 (10 pmol) were mixed in buffer (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM HEPES-KOH, pH 7.4). Then, siRNA was introduced into cells by mixing with 0.75 μm of Lipofectamine RNAi max (Thermo Fisher Scientific), adding the obtained mixed solution to a well, and culturing for 4 hours. Then, the medium was replaced with fresh medium, and the culture was continued for another 2 days. Next, total RNA was extracted from the cells using a TRIzol reagent (Thermo Fisher Scientific), and a reverse transcription reaction was performed using PrimeScript RT Master Mix (Takara Bio Inc.). Using the obtained cDNA as a template, quantitative PCR was performed using TB Green Premix Ex Taq II (Takara Bio Inc.) and LightCycler 480 (Roche Inc.). The oligonucleotides used for quantitative PCR were designed by ProbeFinde Software (Roche).

<2. Analysis of serum half-life of unmodified siRNA-ODAGal4 complex>
Using the unmodified siRNAs HP2 and B2M2 prepared in (1) above, the serum half-life of the unmodified siRNA-ODAGal4 complex was analyzed by the procedure in (2) above. At the same time, HP2 and B2M2, which are not complexed with ODAGal4, were used instead of the HP2-ODAGal4 complex and the B2M2-ODAGal4 complex, but they were not complexed with ODAGal4 by the same procedure as in (2) above. The serum half-life of unmodified siRNA was analyzed (negative control).

The results for HP2 are shown in FIGS. 2-4, and the results for B2M2 are shown in FIGS. 5-7. Both HP2 and B2M2 unmodified siRNAs had a 2-fold increase in serum half-life by complexing with ODAGal4 (FIGS. 4 and 7). In addition to HP2 and B2M2, the same analysis was performed on unmodified siRNAs for 9 different genes. As a result, the serum half-life was extended in all of them, and all 11 types were averaged 2.05 times in serum. It was confirmed that the half-life was extended (data not shown). From this result, it was shown that the stabilizing effect of ODAGal4 is highly universal and does not depend on the sequence of siRNA.

<3. Analysis of serum half-life of skeletal-modified siRNA-ODAGal4 complex>
For various siRNAs containing / without 2'-O-methylation or phosphorothioatetization prepared in (1) above, the serum half-life of the siRNA-ODAGal4 complex was analyzed by the procedure in (2) above. At the same time, as a control, the serum half-life of siRNA not complexed with ODAGal4 was analyzed.

The results are shown in FIGS. 8-11. 8 and 10 show the serum half-life (time), and FIGS. 9 and 11 show the relative values when the serum half-life of the unmodified siRNA (HP2 or B2M2, respectively) not complexed with ODAGal4 is 1. SiRNA containing 2'-O-methylation in the skeleton has a serum half-life extended to about 1 to 5 times as the number of 2'-O-methylation increases when it is not complexed with ODAGal4. showed that. On the other hand, siRNA containing phosphorothioate formation in the skeleton had almost no change in serum half-life when it was not complexed with ODAGal4. In contrast, all complexes of siRNA with 2'-O-methylation or phosphorothioatetation in the skeleton and ODAGal4 had a significantly extended serum half-life. In particular, the complex of siRNA containing phosphorothioatetization and ODAGal4 has a serum half-life of about 10 to 20 times, and by combining phosphorothioateization and complexation with ODAGal4, the serum half-life of double-stranded RNA is halved. It was shown that the period can be extended synergistically and dramatically.

<4. Analysis of Tm value of siRNA-ODAGal4 complex>
In order to evaluate the skeletal modification and the stabilizing effect of siRNA on the double-stranded structure by ODAGal4, Tm values were measured for various siRNAs complexed with or not with ODAGal4 by the procedure of (3) above.

The results are shown in Table 2. The unmodified siRNAs HP2 and B2M2 increased their Tm values by 1.1 and 3.7 ° C., respectively, by complexing with ODAGal4. Further, the Tm value increased as the 2'-O-methylation was increased, and the Tm value was further increased by combining the 2'-O-methylation and the complexation with ODAGal4. On the other hand, it was shown that the Tm value of the phosphorothioated siRNA decreases when it is not complexed with ODAGal4, but the Tm value increases significantly when it is combined with the complexation with ODAGal4. In particular, siRNAs (HP2-S3 and B2M2-S3) in which both the sense and antisense strands were phosphorothioated increased their Tm values by 4.6 and 7.9 ° C, respectively. These results are also consistent with the above serum half-life analysis results, indicating that ODAGal4 has an excellent stabilizing effect on double-stranded RNA.

Table 2. Tm value of siRNA complexed with or not complexed with ODAGal4

<5. Analysis of inhibitory effect of siRNA-ODAGal4 complex on gene expression>
In order to evaluate the effect of skeletal modification and ODAGal4 on the activity of siRNA, gene expression was performed on various siRNAs prepared in (1) above, which were complexed with or not complexed with ODAGal4 by the procedure of (4) above. The inhibitory effect on the disease was analyzed.

The results are shown in FIGS. 12 and 13. In the figure, "None" indicates the result of performing the same procedure using a buffer solution containing no siRNA (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM HEPES-KOH, pH 7.4), and "Negative". "Control" shows the result of performing the same procedure using siRNA consisting of a sequence that does not act on either the HPRT1 gene or the β-2-microglobulin gene. Unmodified siRNAs (HP2 and B2M2) not complexed with ODAGal4 both suppressed gene expression levels to about 20%, but the introduction of 2'-O-methylation diminished the knockdown effect. The tendency to do was confirmed. In particular, siRNAs with high 2'-O-methylation introduced (HP2-M3 and B2M2-M3) showed only a weak knockdown effect. On the other hand, all phosphorothioated siRNAs suppressed gene expression levels to the same extent as unmodified siRNAs. Here, all of the siRNAs complexed with ODAGal4 had almost no change in the knockdown effect as compared with the siRNAs not complexed with ODAGal4. From these results, it was clarified that ODAGal4 does not affect the activity of siRNA and does not change the properties of double-stranded RNA.

From the above results, it was shown that a double-stranded RNA drug having both high activity and stability can be provided by combining skeletal modification and ODAGal4.

<6. Preparation of double-stranded RNA with different types of skeletal modification and introduction site>
By the same procedure as in (1) above, various double-stranded siRNAs having different types of skeletal modifications and introduction sites were prepared. The arrangement is shown in Table 3. Uppercase letters indicate unmodified RNA (where "T" indicates thymidine (DNA)). Lowercase letters indicate modified RNA: bold is 2'-O-methylated modification, italic is phosphorothioated modification, bold italic is 2'-O-methylated and phosphorothioated modification, and underlined bold is 2'-F modification is shown. The underlined uppercase bold letters indicate LNA.

Table 3. sequence of siRNA

For various siRNAs shown in Table 3, the serum half-life of the siRNA-ODAGal4 complex was analyzed by the procedure of (2) above. At the same time, as a control, the serum half-life of siRNA not complexed with ODAGal4 was analyzed (FIGS. 14-22). In FIGS. 14 to 22, (A) shows a relative value when the serum half-life (time) is set, and (B) shows a relative value when the serum half-life of unmodified siRNA not complexed with ODAGal4 is set to 1.

<7. Comparison of stability of siRNA-ODAGal4 complexes with different sites of introduction of skeletal modification>
The results for siRNA in which a phosphorothioated bond is introduced into a part of the sense strand or the antisense strand (between 6 bases) are shown in FIGS. 14 to 17. FIG. 14 shows the results for HP2 (HP2-S4 to HP2-S7) in which part of the sense strand was phosphorothioated, and FIG. 15 shows the results for HP2 (HP2-S8 to) in which part of the antisense strand was phosphorothioated. The results for HP2-S11) are shown, FIG. 16 shows the results for B2M2 (B2M2-S4 to B2M2-S7) in which part of the sense strand was phosphorothioated, and FIG. 17 shows the results for part of the antisense strand in phosphorothioate. The results for the modified B2M2 (B2M2-S8 to B2M2-S11) are shown. In each case, the closer the phosphorothioated region was to the 3'end, the higher the stabilizing effect of the complex with ODAGal4 was observed.

The results for siRNAs (HP2-S12 and HP2-S13) into which a phosphorothioated bond was introduced into a part of the sense strand (between 4 bases) are shown in FIG. The complex of HP2 (HP2-S12) with ODAGal4, which introduced a phosphorothioatetation bond between the 4 bases at the 3'end including the protruding region of the sense strand, showed a slightly improved serum half-life, while it showed a slightly improved serum half-life. The complex of HP2 (HP2-S13) and ODAGal4, in which a phosphorothioated bond was introduced between the 4 bases at the 3'end excluding the overhanging region, showed a significantly improved serum half-life. Similar results were obtained even when the protruding region was changed to DNA (FIG. 19, HP2-TS).

The results for siRNAs (HP2-S14 to HP2-S16) into which a phosphorothioated bond was introduced into the central region (between 11 bases) of the sense strand and / or the antisense strand are shown in FIG. In both cases, a high stabilizing effect was obtained by complexing with ODAGal4, but when phosphorothioation was introduced into only one strand of siRNA, it was stabilized by either the sense strand or the antisense strand. The degree of effect has changed.

From the above results, it was shown that in the siRNA-ODAGal4 complex, the desired stability can be imparted to the siRNA by changing the introduction site and the length of the skeletal modification to be introduced into the siRNA. Furthermore, it was shown that in the siRNA-ODAGal4 complex, a significantly high stabilizing effect can be obtained by introducing a phosphorothioated bond between only 4 bases in the double chain region at the 3'end of one strand of siRNA. rice field.

<8. Comparison of stability of siRNA-ODAGal4 complexes with different types of skeletal modification>
The serum half-life of the complex of HP2 with 2'-O-methylation, 2'-F, or LNA introduced, in place of or in combination with phosphorothioation, and ODAGal4, is described in (2) above. It was analyzed by the procedure of. The results are shown in FIGS. 21 and 22. When any type of skeletal modification was introduced, a stabilizing effect was obtained by complexing with ODAGal4, but in particular, an extremely high stabilizing effect was obtained when combined with phosphorothioate formation (FIG. 21, FIG. 21). HP2-MS1, HP2-MS2; FIG. 22, HP2-FS, HP2-LS).

<9. Stability of short double-stranded RNA-ODAGal4 complex>
A short double-stranded RNA (blunt end) consisting of 12 base pairs with or without phosphorothioation was prepared by the same procedure as in (1) above. The arrangement is shown in Table 4. Uppercase letters indicate unmodified RNA. Lowercase italics indicate post-transcriptional modified RNA.

Table 4. Sequence of short RNA

The serum half-life of the short double-stranded RNA was analyzed by the same procedure as in (2) above, except that the short double-stranded RNA was used instead of siRNA. The results are shown in FIG. (A) shows the serum half-life (time), and (B) shows the relative value when the serum half-life of the unmodified siRNA not complexed with ODAGal4 is 1. From this result, it was shown that even in the case of blunt-ended and short double-stranded RNA, a remarkably high stabilizing effect can be obtained by combining phosphorothioation and complexing with ODAGal4 (FIGS. 23 and 12M-S). ).

<10. Comparison of stability of phosphorothioated siRNA-various cationic oligosaccharide complexes>
The serum half-life of the complex of HP2-S3, which is a phosphorothioated siRNA, and various cationic oligosaccharides was analyzed by the same procedure as in (2) above. The results are shown in FIG. In FIG. 24, “ODAGlc4” indicates 2,6-diamino-2,6-dideoxy-α- (1 → 4) -D-glucopyranose tetramer; “ODAMan4” indicates 2,6-diamino-2, 6-Dideoxy-α- (1 → 4) -D-mannopyranose tetramer; "ODGGal3" is 2,6-diguanidino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose Indicates a trimmer. Further, (A) shows a relative value when the serum half-life (time) is set, and (B) shows a relative value when the serum half-life of unmodified siRNA not complexed with ODAGal4 is set to 1. From this result, it was confirmed that the double-stranded RNA can be remarkably stabilized by combining with the phosphorothioate formation in the complex with a cationic oligosaccharide other than ODAGal4.

Claims (11)

A method of regulating the stability of double-stranded RNA,
(1) Introducing at least one modification into the backbone of double-stranded RNA,
(2) The double-stranded RNA obtained in step (1) and the general formula (I):
R ^1- O- (X) _n- R ²
[During the ceremony,
R ¹ and R ² are independent hydrogen atoms or monovalent substituents, respectively.
n is an integer of 3 to 6 and
The n Xs may be the same or different, and independently of each of the following (a) to (i) :.

In expressed divalent group (wherein the, ^{R +} is, _NH ^{3 +} or formula (II):

(Here, R ³ , R ⁴ and R ⁵ are each independently represented by a hydrogen atom or a methyl group).]
A method comprising contacting with a cationic oligosaccharide represented by. In the general formula (I)
R ¹ is a hydrogen atom,
R ² is a hydrogen atom or a substituted or unsubstituted C _1-6 alkyl group.
n is 3 or 4,
The method according to claim 1. The cationic oligosaccharide represented by the general formula (I) is 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer, according to claim 1 or 2. The method described. The method of any one of claims 1-3, wherein the modification is selected from 2'-O-methylation and phosphorothioatetization. (1) Double-stranded RNA with modifications introduced into the skeleton,
(2) General formula (I):
R ^1- O- (X) _n- R ²
[During the ceremony,
R ¹ and R ² are independent hydrogen atoms or monovalent substituents, respectively.
n is an integer of 3 to 6 and
The n Xs may be the same or different, and independently of each of the following (a) to (i) :.

In expressed divalent group (wherein the, ^{R +} is, _NH ^{3 +} or formula (II):

(Here, R ³ , R ⁴ and R ⁵ are each independently represented by a hydrogen atom or a methyl group).]
A nucleic acid composition comprising a complex with a cationic oligosaccharide represented by, which has the same pharmaceutical activity as unmodified double-stranded RNA and is an unmodified double-stranded RNA. A nucleic acid composition having a serum half-life that is 5 times or more longer than that. In the general formula (I)
R ¹ is a hydrogen atom,
R ² is a hydrogen atom or a substituted or unsubstituted C _1-6 alkyl group.
n is 3 or 4,
The nucleic acid composition according to claim 5. The nucleic acid composition according to claim 5 or 6, wherein the cationic oligosaccharide is 2,6-diamino-2,6-dideoxy-β- (1 → 4) -D-galactopyranose tetramer. The nucleic acid composition according to any one of claims 5 to 7, wherein at least 10% of the entire skeleton of the double-stranded RNA is modified. The nucleic acid composition according to any one of claims 5 to 8, wherein the modification is selected from 2'-O-methylation and phosphorothioatetization. The nucleic acid composition according to any one of claims 5 to 9, wherein the modification is introduced into the skeleton of the 3'end region of at least one RNA strand constituting the double-strand RNA. The nucleic acid composition according to any one of claims 5 to 10, wherein the double-stranded RNA has a length of 12 to 50 base pairs.

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