Modified heterogeneous nucleic acid

Abstract

To provide double-stranded nucleic acid complexes with novel structures.SOLUTION: In one embodiment, the present invention relates to a nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand (1) being capable of hybridizing to at least a part of a target transcript, (2) having an antisense effect on the target transcript, and (3) being a gapmer comprising a central region, as well as 5' and 3' wing regions; the second nucleic acid strand comprising at least one sugar-unmodified central region (first exposed region) consisting of one or two to three consecutive sugar-unmodified ribonucleosides linked by internucleoside linkages that is complementary to a part of the first nucleic acid strand; and the first nucleic acid strand being annealed to the second nucleic acid strand.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] The present invention relates to a double-stranded nucleic acid complex, a composition containing the same, and the like. [Background technology]

[0002] In recent years, oligonucleotides have attracted attention in the ongoing development of pharmaceuticals known as nucleic acid drugs, and in particular, the development of nucleic acid drugs using antisense methods is being actively promoted due to their high selectivity for target genes and low toxicity. The antisense method involves using a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand and introducing a complementary oligonucleotide (antisense oligonucleotide: often referred to as "ASO (AntiSense Oligonucleotide)" in this specification) into cells to selectively modify or inhibit the expression of a protein encoded by a target gene or the activity of a miRNA.

[0003] As a nucleic acid utilizing the antisense method, the present inventors have developed a double-stranded nucleic acid complex (heteroduplex oligonucleotide (HDO)) in which an antisense oligonucleotide and its complementary strand are annealed (Patent Document 1, Non-Patent Documents 1 and 2).

[0004] The mechanism of action of the double-stranded nucleic acid complex is partially, but not exclusively, as follows: When introduced into a cell, the RNA region in the complementary strand that is complementary to a portion of the antisense oligonucleotide is cleaved by RNase H, releasing the antisense oligonucleotide, which can then act, for example, to modify the activity or function of a transcription product (see, for example, Patent Document 2). This is called the "RNase H-dependent pathway," and it is desirable for the nucleic acid portion to be unmodified in order for cleavage by RNase H to occur. On the other hand, if the nucleic acid portion is unmodified, it may be degraded by nucleases in vivo, preventing it from fully exerting its activity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 089283 [Patent Document 2] International Publication No. 2014 / 192310 [Non-patent literature]

[0006] [Non-Patent Document 1] Nishina K, et. al., "DNA / RNA heteroduplex oligonucleotide for highly efficient gene silencing", Nature Communication, 2015, 6:7969. [Non-patent document 2] Asami Y, et al., "Drug delivery system of therapeutic oligonucleotides", Drug Discoveries &Therapeutics. 2016; 10(5):256-262. Summary of the Invention [Problem to be solved by the invention]

[0007] There is insufficient knowledge about how nucleic acids should be modified to suppress degradation by nucleases in vivo while maintaining the activity of double-stranded nucleic acid complexes.

[0008] An objective of the present invention is to provide a double-stranded nucleic acid complex that can maintain activity and / or suppress degradation in vivo. [Means for solving the problem]

[0009] The present inventors have found that a complex of a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand is a gapmer and the second nucleic acid strand includes a sugar-unmodified central region (also referred to herein as a "first exposed region") consisting of one or two to three consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds that are complementary to a portion of the first nucleic acid strand, can maintain activity and / or be inhibited from being degraded in vivo. The inventors also discovered that the function of the above complex can be further improved by including a protective region in the second nucleic acid strand that contains modified or unmodified pyrimidine bases, and thus completed the present invention.

[0010] The present invention is based on the above findings and includes the following embodiments. [1] A nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid strand (1) capable of hybridizing to at least a portion of the target transcript; (2) have an antisense effect on the target transcript; and (3) A gapmer comprising a central region containing at least four consecutive deoxyribonucleosides, and 5' and 3' wing regions containing unnatural nucleosides, respectively, on the 5' and 3' ends of the central region, the second nucleic acid strand comprises at least one sugar-unmodified central region (first exposed region) that is complementary to a portion of the first nucleic acid strand and consists of one or two to three consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds; and The nucleic acid complex, wherein the first nucleic acid strand is annealed to the second nucleic acid strand. [2] The nucleic acid complex according to [1], wherein the first nucleic acid strand has a length of 13 to 20 bases. [3] the second nucleic acid strand linked by modified or unmodified internucleoside linkages, (1) the sugar-unmodified central region (first exposed region), and (2) (a) deoxyribonucleosides and / or (b) sugar-modified nucleosides The nucleic acid complex according to [1] or [2], comprising: [4] The nucleic acid complex according to [3], wherein the second nucleic acid strand contains a sugar-modified ribonucleoside. [5] The nucleic acid complex according to any one of [1] to [4], wherein the sugar-unmodified central region (first exposed region) consists of three consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds. [6] The nucleic acid complex according to any one of [1] to [5], wherein the second nucleic acid strand comprises only one sugar-unmodified central region (first exposed region). [7] The nucleic acid complex according to any one of [1] to [6], wherein the sugar-unmodified ribonucleoside in the sugar-unmodified central region (first exposed region) is a natural ribonucleoside. [8] The nucleic acid complex according to any one of [1] to [7], wherein the sugar-unmodified central region (first exposed region) contains at least one modified internucleoside bond. [9] The nucleic acid complex according to any one of [1] to [8], wherein the second nucleic acid strand comprises a sugar-unmodified terminal region containing at least one sugar-unmodified ribonucleoside at the 5' end and / or 3' end.

[10] The nucleic acid complex according to [9], wherein the sugar-unmodified ribonucleoside in the sugar-unmodified terminal region is a natural ribonucleoside.

[11] The nucleic acid complex according to any one of [1] to

[10] , wherein the sugar-unmodified central region (first exposed region) of the second nucleic acid strand contains at least one chiral-controlled phosphorothioate bond in the Rp or Sp configuration.

[12] The nucleic acid complex according to

[11] , wherein the second nucleic acid strand does not contain any sugar-unmodified region other than the sugar-unmodified terminal region and the sugar-unmodified central region (first exposed region).

[13] The nucleic acid complex according to any one of [1] to

[12] , wherein the base in the sugar-unmodified central region (first exposed region) is a purine base.

[14] The nucleic acid complex according to any one of [1] to

[13] , wherein the second nucleic acid strand is bound to a functional moiety having a function selected from a labeling function, a purification function, and a target delivery function.

[15] The nucleic acid complex according to any one of [1] to

[14] , wherein the sugar-unmodified central region (first exposed region) is located on the 3' side of the center of the second nucleic acid strand.

[0011] The present invention further includes the following embodiments. [1A] A nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid strand (1) capable of hybridizing to at least a portion of the target transcript; (2) have an antisense effect on the target transcript; and (3) A gapmer comprising a central region containing at least four consecutive deoxyribonucleosides, and 5' and 3' wing regions containing unnatural nucleosides, respectively, on the 5' and 3' ends of the central region, the second nucleic acid strand comprises at least one first exposed region and at least one protected region; the first exposed region is complementary to a portion of the first nucleic acid strand and consists of one or two to three consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds; The protective region is composed of one or more (a) deoxyribonucleosides, (b) sugar-modified nucleosides, and / or (c) nucleosides having a modified internucleoside bond at the 3' side, linked by an internucleoside bond; and The nucleic acid complex, wherein the first nucleic acid strand is annealed to the second nucleic acid strand. [2A] The nucleic acid complex according to [1A], wherein the first nucleic acid strand has a length of 13 to 22 bases. [3A] The nucleic acid complex according to [1A] or [2A], wherein the first exposed region consists of three consecutive unmodified sugar ribonucleosides linked by internucleoside bonds. [4A] The nucleic acid complex according to any one of [1A] to [3A], wherein the first exposed region contains a nucleoside containing a modified or unmodified purine base. [5A] The nucleic acid complex according to any one of [1A] to [4A], wherein the second nucleic acid strand contains only one first exposed region. [6A] The nucleic acid complex according to any one of [1A] to [4A], wherein the second nucleic acid strand comprises at least two first exposed regions. [7A] The nucleic acid complex according to any one of [1A] to [6A], wherein the second nucleic acid strand comprises at least two protected regions. [8A] The nucleic acid complex according to any one of [1A] to [7A], wherein the sugar-unmodified ribonucleoside in the first exposed region is a natural ribonucleoside. [9A] The nucleic acid complex according to any one of [1A] to [8A], further comprising a second exposed region consisting of four or more consecutive unsugar-modified ribonucleosides linked by internucleoside bonds, which are complementary to a portion of the first nucleic acid strand, and wherein the bases of the unsugar-modified ribonucleosides in the second exposed region include modified or unmodified purine bases. [10A] A nucleic acid complex according to any one of [1A] to [9A], wherein at least one of the protective regions comprises (a) a deoxyribonucleoside containing a modified or unmodified pyrimidine base, (b) a sugar-modified nucleoside, and / or (c) a nucleoside having a modified internucleoside bond on the 3' side. [11A] The nucleic acid complex according to [10A], wherein all of the protective regions contain (a) deoxyribonucleosides, (b) sugar-modified nucleosides, and / or (c) nucleosides having a modified internucleoside linkage at the 3' side, each of which contains a modified or unmodified pyrimidine base. [12A] A nucleic acid complex according to any one of [1A] to [11A], wherein at least one of the protective regions is composed of (a) a deoxyribonucleoside, (b) a sugar-modified nucleoside, and / or (c) a nucleoside having a modified internucleoside bond at the 3' side, each of which contains one or two or more modified or unmodified pyrimidine bases linked by an internucleoside bond. [13A-1] The nucleic acid complex according to any one of [1A] to [12A], wherein the sugar-modified nucleoside in the protective region is a 2'-sugar-modified nucleoside and / or a bridged nucleoside. [13A-2] The nucleic acid complex according to any one of [1A] to [12A] or [13A-1], wherein the sugar-modified nucleoside in the protective region is a 2'-sugar-modified nucleoside. [13A-3] The nucleoside having a modified internucleoside bond on the 3' side in the protective region is a phosphorothioate bond on the 3' side, 1 to 4 C 1~6 The nucleic acid complex according to any one of [1A] to [12A] or [13A-1] to [13A-2], wherein the nucleic acid complex is a nucleoside having an internucleoside bond containing a guanidine moiety substituted with an alkyl group and / or an internucleoside bond containing a cyclic guanidine moiety. [13A-4] A nucleic acid complex according to any one of [1A] to [12A] or [13A-1] to [13A-3], wherein the nucleoside having a modified internucleoside bond on the 3' side in the protective region is a nucleoside having a phosphorothioate bond on the 3' side. [13A] A nucleic acid complex according to any one of [1A] to [12A] or [13A-1] to [13A-4], wherein the sugar-modified nucleoside in the protective region is a 2'-O-methyl-modified nucleoside, and / or the nucleoside having a modified internucleoside bond on the 3' side is a nucleoside having a phosphorothioate bond on the 3' side. [14A] The nucleic acid complex according to any one of [1A] to [13A] or [13A-1] to [13A-4], wherein the first exposed region and / or the second exposed region contains at least one modified internucleoside bond. [15A] The nucleic acid complex according to any one of [1A] to [14A] or [13A-1] to [13A-4], wherein the second nucleic acid strand comprises a sugar-unmodified terminal region containing at least one sugar-unmodified ribonucleoside at the 5' end and / or 3' end. [16A] The nucleic acid complex according to [15A], wherein the sugar-unmodified ribonucleoside in the sugar-unmodified terminal region is a natural ribonucleoside. [17A] The nucleic acid complex according to any one of [1A] to [16A] or [13A-1] to [13A-4], wherein at least one protected region of the second nucleic acid strand is linked to the first exposed region and / or the second exposed region at the 5' end and 3' end by an internucleoside bond. [18A] The nucleic acid complex according to any one of [1A] to [17A] or [13A-1] to [13A-4], wherein the second nucleic acid strand does not contain a sugar-unmodified region other than the first exposed region and the second exposed region. [19A] The nucleic acid complex according to any one of [1A] to [17A] or [13A-1] to [13A-4], wherein the second nucleic acid strand comprises a sugar-modified terminal region containing at least one deoxyribonucleoside and / or sugar-modified nucleoside at the 5' end and / or 3' end. [20A] The nucleic acid complex according to any one of [1A] to [19A] or [13A-1] to [13A-4], wherein the second nucleic acid strand is composed of (1) at least one of the first exposed region and / or at least one of the second exposed region, (2) at least one of the protective region, and (3) the sugar-modified terminal region at the 5' end and the 3' end, all linked by an internucleoside bond. [21A] The first exposed region and / or the second exposed region of the second nucleic acid strand comprises at least one phosphorothioate bond chirally controlled in an Rp configuration or an Sp configuration, 1 to 4 C 1~6The nucleic acid complex according to any one of [1A] to [20A] or [13A-1] to [13A-4], which comprises an internucleoside bond containing a guanidine moiety substituted with an alkyl group and / or an internucleoside bond containing a cyclic guanidine moiety. [22A] The defense region of the second nucleic acid strand comprises at least one phosphorothioate bond chirally controlled in the Rp or Sp configuration, 1 to 4 C 1~6 The nucleic acid complex according to any one of [1A] to [21A] or [13A-1] to [13A-4], which comprises an internucleoside bond containing a guanidine moiety substituted with an alkyl group and / or an internucleoside bond containing a cyclic guanidine moiety. [23A] The nucleic acid complex according to [21A] or [22A], wherein the internucleoside bond containing a cyclic guanidine moiety is an internucleoside bond represented by a partial structure represented by the following formula (II): [ka] [24A] The above 1 to 4 C 1~6 The nucleic acid complex according to any one of [21A] to [23A], wherein the internucleoside bond containing a guanidine moiety substituted with an alkyl group is an internucleoside bond represented by a partial structure represented by the following formula (III): [ka] [25A] The nucleic acid complex according to any one of [17A] or [19A] to [24A], wherein the second nucleic acid strand does not contain a sugar-unmodified region other than the first exposed region, the second exposed region, and the sugar-unmodified terminal region. [26A] The nucleic acid complex according to any one of [17A] to [25A], wherein at least one of the first exposed region, the second exposed region and / or the protected region of the second nucleic acid strand contains one or more mismatched bases. [27A] The nucleic acid complex according to any one of [17A] to [25A], wherein the first exposed region, the second exposed region and / or the protected region of the second nucleic acid strand does not contain a mismatched base. [28A] The nucleic acid complex according to any one of [1A] to [27A] or [13A-1] to [13A-4], wherein the second nucleic acid strand is bound to a functional moiety having a function selected from a labeling function, a purification function, and a delivery function to a target. [29A] A pharmaceutical composition comprising, as an active ingredient, the nucleic acid complex according to any one of [1A] to [28A] or [13A-1] to [13A-4]. [30A] A nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid strand (1) capable of hybridizing to at least a portion of the target transcript; (2) have an antisense effect on the target transcript; and (3) A gapmer comprising a central region containing at least four consecutive deoxyribonucleosides, and 5' and 3' wing regions containing unnatural nucleosides, respectively, on the 5' and 3' ends of the central region, the second nucleic acid strand comprises at least one second exposed region and at least one protected region; the second exposed region is composed of four or more consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds that are complementary to a portion of the first nucleic acid strand; The protective region is composed of one or more (a) deoxyribonucleosides, (b) sugar-modified nucleosides, and / or (c) nucleosides having a modified internucleoside bond at the 3' side, linked by an internucleoside bond; and The nucleic acid complex, wherein the first nucleic acid strand is annealed to the second nucleic acid strand. [31A] The nucleic acid complex according to [30A], wherein the first nucleic acid strand has a length of 13 to 22 bases. [32A] The nucleic acid complex according to [30A] or [31A], wherein the second nucleic acid strand contains only one second exposed region. [33A] The nucleic acid complex according to any one of [30A] to [32A], wherein the second nucleic acid strand comprises at least two second exposed regions. [34A] The nucleic acid complex according to any one of [30A] to [33A], further comprising a first exposed region consisting of one or two to three consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds, which are complementary to a portion of the first nucleic acid strand. This specification includes the disclosure of Japanese Patent Application No. 2019-188042, from which this application claims priority. [Effects of the Invention]

[0012] The present invention provides a double-stranded nucleic acid complex having a novel structure. The nucleic acid complex of the present invention may have high activity and / or reduced degradation in vivo. [Brief explanation of the drawings]

[0013] [Figure 1] 1A and 1B are schematic diagrams showing a specific embodiment of the nucleic acid complex according to the present invention, in which the second nucleic acid strand contains a lipid. [Figure 2] 2A to 2C are schematic diagrams showing specific embodiments of the nucleic acid complex according to the present invention, in which the second nucleic acid strand contains a lipid and also contains a complementary region and an overhang region. [Figure 3] FIG. 3 shows an example of the general mechanism of antisense technology. [Figure 4] FIG. 4 shows the structures of various bridged nucleic acids. [Figure 5] FIG. 5 shows the structures of various natural and non-natural nucleotides. [Figure 6] Figure 6 is a schematic diagram of the structure of the nucleic acid used in Example 1. Chol represents cholesterol. [Figure 7]Figure 7 shows the results of electrophoresis of ASO alone, a double-stranded complex of ASO and Chol-cRNA (mMalat1) (Chol-HDO), and a double-stranded complex of ASO and Chol-cRNA (mMalat1) full OMe (Chol-HDO with full OMe cRNA) after cleavage with RNase H and / or RNase A. In the RNase H treatment group, "+" indicates the result when 5 μL of 10 μM nucleic acid was reacted with 10 U of Ribonuclease H, and "++" indicates the result when 5 μL of 20 μM nucleic acid was reacted with 10 U of Ribonuclease H. [Figure 8] FIG. 8 is a graph showing the concentration of the unresolved bands based on the electrophoresis results of FIG. 7, expressed as a relative intensity level (%) to that of the untreated bands. [Figure 9] Figure 9 is a graph showing the concentration of the undegraded band as a relative intensity level (%), similar to Figure 8, in a double-stranded nucleic acid agent containing three consecutive unsugar-modified ribonucleosides complementary to a portion of the gap region of the first nucleic acid strand, in which a cleavage test with RNase H and / or RNase A was performed by changing the positions of the three consecutive unsugar-modified ribonucleosides. [Figure 10] FIG. 10 shows the relative RNA expression levels when cells were treated with various double-stranded nucleic acid complexes, with the Malat1 / Actb (β-actin) expression level in PBS treatment set at 1. [Figure 11] 11 is a schematic diagram of the structure of the nucleic acid used in Example 3. Chol represents cholesterol. [Figure 12] 12 is a schematic diagram of the structure of the nucleic acid used in Example 3. Chol represents cholesterol. [Figure 13] Figure 13 shows the results of electrophoresis of a double-stranded nucleic acid agent containing one or two consecutive unsugar-modified ribonucleosides complementary to a portion of the gap region of the first nucleic acid strand, after changing the position of the unsugar-modified ribonucleoside and cleaving it with RNase A. [Figure 14]Figure 14 is a graph showing the concentration of the undegraded band, as in Figure 8, in terms of relative intensity level (%) after RNase A cleavage of a double-stranded nucleic acid molecule containing one or two consecutive RNAs complementary to a portion of the gap region of the first nucleic acid strand. [Figure 15] Figure 15 is a schematic diagram showing the structure of the nucleic acid used in Example 4. Chol represents cholesterol. Figure 15A shows the double-stranded nucleic acid complex Chol-HDO(Default) containing ASO(mDMPK) and Chol-cRNA(Default). Figure 15B shows the double-stranded nucleic acid complex Chol-HDO(CU OMe) containing ASO(mDMPK) and Chol-cRNA(CU OMe). [Figure 16] 16 shows the inhibitory effects of the double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CU OMe), as well as PBS (negative control), on Dmpk gene expression in the gastrocnemius (GC), triceps brachii (TB), tibialis anterior (TA), back, quadriceps femoris, and cardiac muscle. The relative RNA expression levels are shown when the Dmpk / Actb (β-actin) expression level is set to 1. [Figure 17] 17 shows the inhibitory effects of the double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CU OMe), as well as the negative control PBS, on Dmpk gene expression in the kidney and liver. The relative RNA expression levels are shown when the Dmpk / Actb (β-actin) expression level is set to 1. [Figure 18] Figure 18 is a schematic diagram showing the structure of the nucleic acid used in Example 5. Chol represents cholesterol. Figure 18A shows a double-stranded nucleic acid complex Chol-HDO(Default) containing ASO(hSOD1) and Chol-cRNA(Default). Figure 18B shows a double-stranded nucleic acid complex Chol-HDO(CU OMe) containing ASO(hSOD1) and Chol-cRNA(CU OMe). [Figure 19]19 shows the inhibitory effects of the double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CU OMe), as well as the negative control PBS, on the expression of the SOD1 gene in the back muscles, quadriceps muscles, heart muscles, and diaphragm. The relative RNA expression levels are shown when the expression level of SOD1 / Actb (β-actin) is set to 1. [Figure 20] Figure 20 is a schematic diagram showing the structure of the nucleic acid used in Example 6. Chol represents cholesterol. Figure 20A shows a double-stranded nucleic acid complex, Chol-HDO (Default), containing ASO (Malat1) and Chol-cRNA (Default). Figure 20B shows a double-stranded nucleic acid complex, Chol-HDO (CT DNA), containing ASO (Malat1) and Chol-cRNA (CT DNA). [Figure 21] 21 shows the inhibitory effect of the double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CU OMe), as well as the negative control PBS, on Malat1 gene expression in the cervical spinal cord. The relative RNA expression levels are shown when the expression level of Malat1 / Actb (β-actin) is set to 1. [Figure 22A] FIG. 22A shows the sequences, chemical modifications and structures of the oligonucleotides used in Example 7. [Figure 22B] FIG. 22B shows the sequences, chemical modifications and structures of the oligonucleotides used in Example 7. [Figure 23] Figure 23 shows the results of electrophoresis of a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing a modified ribonucleoside, which was mixed with mouse serum for the indicated times (0 hr, 24 hr). [Figure 24] FIG. 24 is a graph showing the concentration of the double-stranded band after 24 hours based on the electrophoresis results of FIG. 23, expressed as a relative intensity level (%) to the double-stranded band after 0 hours. [Figure 25]Figure 25 shows the results of electrophoresis of a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing a modified ribonucleoside, which was mixed with human serum for the indicated times (0 hr, 2 hr). [Figure 26] FIG. 26 is a graph showing the concentration of the double-stranded band after 2 hours based on the electrophoresis results of FIG. 25, expressed as a relative intensity level (%) to the double-stranded band after 0 hours. DETAILED DESCRIPTION OF THE INVENTION

[0014] <Nucleic acid complex> In one aspect, the present invention relates to a nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand annealed to the second nucleic acid strand. The first nucleic acid strand (1) is capable of hybridizing to at least a portion of a target transcript, (2) has an antisense effect on the target transcript, and (3) is a gapmer. The nucleic acid complex of the present invention and the nucleic acid strands that constitute it are described in detail below.

[0015] The first nucleic acid strand may be a nucleotide strand containing or consisting of an antisense oligonucleotide region against a target transcript. "Antisense oligonucleotide" or "antisense nucleic acid" refers to a single-stranded oligonucleotide containing a base sequence capable of hybridizing to (i.e., complementary to) at least a portion of a target transcript (primarily a transcript of a target gene) and exerting an antisense effect on the target transcript. In the present invention, the antisense oligonucleotide region in the first nucleic acid strand exerts an antisense effect on the target transcript. The target region of the target transcript can be at least 8 bases long, for example, 10 to 35 bases long, 12 to 25 bases long, 13 to 20 bases long, 14 to 19 bases long, or 15 to 18 bases long, or 13 to 22 bases long, 16 to 22 bases long, or 16 to 20 bases long.

[0016] The term "antisense effect" refers to the modulation of target transcript expression resulting from hybridization of a target transcript (sense RNA strand) with a strand (e.g., a DNA strand) that is complementary to a subsequence of the transcript and designed to induce an antisense effect. Modulation of target transcript expression can involve suppressing or reducing the expression of the target gene or the level of the target transcript, or, in certain instances, inhibiting translation or altering splicing function, such as exon skipping, or degrading the transcript (see Figure 3). For example, in the case of translation inhibition, when an RNA-containing oligonucleotide is introduced into a cell as an antisense oligonucleotide (ASO), the ASO binds to the target gene's transcript (mRNA), forming a partial duplex. This partial duplex acts as a cover to prevent translation by ribosomes, thereby inhibiting the expression of the protein encoded by the target gene at the translational level (Figure 3, dashed line, x). On the other hand, when a DNA-containing oligonucleotide is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase H, resulting in degradation of the target gene's mRNA, thereby inhibiting the expression of the protein encoded by the target gene at the expression level (Figure 3, dashed line). This is referred to as the "RNase H-dependent pathway." Furthermore, in certain instances, the antisense effect can be achieved by targeting an intron of a pre-mRNA. The antisense effect can also be achieved by targeting an miRNA, in which case the function of the miRNA is inhibited and the expression of the gene whose expression the miRNA normally controls can be increased. In one embodiment, the modulation of the expression of a target transcript can be a reduction in the amount of the target transcript.

[0017] The antisense oligonucleotide region in the first nucleic acid strand comprises a base sequence capable of hybridizing to at least a portion of a target transcript (e.g., any target region), which may include a 3' UTR, 5' UTR, exon, intron, coding region, translation initiation region, translation termination region, or other nucleic acid region.

[0018] The "target gene" whose expression is regulated (e.g., suppressed, altered, or modified) by the antisense effect is not particularly limited, but includes, for example, genes whose expression is increased in various diseases. The target transcript also includes mRNA transcribed from genomic DNA encoding the target gene, as well as mRNA that has not undergone base modification and unprocessed mRNA precursors. The "target transcript" may include not only mRNA but also non-coding RNA (ncRNA) such as miRNA. More generally, the "transcript" may be any RNA synthesized by a DNA-dependent RNA polymerase. In one embodiment, the "target transcript" may be, for example, a gene encoding scavenger receptor B1 (often referred to herein as "SR-B1"), myotonic dystrophy protein kinase (often referred to herein as "DMPK"), transthyretin (often referred to herein as "TTR"), apolipoprotein B (often referred to herein as "ApoB"), or metastasis associated lung adenocarcinoma transcript 1 (often referred to herein as "Malat1"), e.g., a non-coding RNA or mRNA thereof. SEQ ID NO: 1 shows the nucleotide sequence of mouse Malat1 non-coding RNA, and SEQ ID NO: 2 shows the nucleotide sequence of human Malat1 non-coding RNA. SEQ ID NO: 39 shows the nucleotide sequence of mouse SR-B1 mRNA, and SEQ ID NO: 40 shows the nucleotide sequence of human SR-B1 mRNA. The nucleotide sequence of mouse DMPK mRNA is shown in SEQ ID NO: 41, and the nucleotide sequence of human DMPK mRNA is shown in SEQ ID NO: 42. In SEQ ID NOs: 1 to 2 and 39 to 42, the nucleotide sequence of mRNA is replaced with the nucleotide sequence of DNA.The nucleotide sequence information of these genes and transcripts can be obtained from known databases such as the NCBI (National Center for Biotechnology Information) database (e.g., GenBank, Trace Archive, Sequence Read Archive, BioSample, BioProject), etc. The target region of the target transcript may include, for example, the nucleotide sequence of positions 1317 to 1332 of SEQ ID NO: 1 in the case of mouse Malat-1 non-coding RNA.

[0019] As used herein, the term "nucleic acid" or "nucleic acid molecule" may refer to a monomeric nucleotide or nucleoside, or to an oligonucleotide composed of multiple monomers. The term "nucleic acid chain" or "chain" is also used herein to refer to an oligonucleotide. Nucleic acid chains can be produced in full length or partial chains by chemical synthesis (e.g., using an automated synthesizer) or by enzymatic processes (e.g., but not limited to, polymerase, ligase, or restriction reactions).

[0020] The term "nucleobase" or "base" as used herein refers to a base component (heterocyclic moiety) that constitutes a nucleic acid, and the main known bases are adenine, guanine, cytosine, thymine, and uracil.

[0021] The term "complementary" used herein refers to the relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) through hydrogen bonds.In the present invention, the antisense oligonucleotide region in the first nucleic acid strand does not necessarily have to be completely complementary to at least a part of the target transcript (for example, the transcript of the target gene), but it is acceptable as long as the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (for example, 95%, 96%, 97%, 98%, or 99% or more) complementarity.The antisense oligonucleotide region in the first nucleic acid strand can hybridize to the target transcript when the base sequence is complementary (typically, when the base sequence is complementary to at least a part of the base sequence of the target transcript). Similarly, the complementary region in the second nucleic acid strand does not necessarily need to be completely complementary to at least a portion of the first nucleic acid strand; it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. The complementary region in the second nucleic acid strand can anneal to at least a portion of the first nucleic acid strand if the base sequence is complementary. The complementarity of the base sequences can be determined using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can anneal or hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design an antisense nucleic acid complementary to a target transcription product, for example, based on information about the base sequence of the target gene.

[0022] Hybridization conditions may be of various stringent conditions, such as low stringency conditions and high stringency conditions. Low stringency conditions may be conditions of relatively low temperature and high salt concentration, for example, 30°C, 2xSSC, 0.1% SDS. High stringency conditions may be conditions of relatively high temperature and low salt concentration, for example, 65°C, 0.1xSSC, 0.1% SDS. Hybridization stringency can be adjusted by changing conditions such as temperature and salt concentration. Here, 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0023] The antisense oligonucleotide region in the first nucleic acid strand may typically be, but is not limited to, at least 8 bases long, at least 9 bases long, at least 10 bases long, at least 11 bases long, at least 12 bases long, or at least 13 bases long. The antisense oligonucleotide region in the first nucleic acid strand may be 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. The antisense oligonucleotide region in the first nucleic acid strand may be, for example, 8 to 35 bases long, 9 to 30 bases long, 10 to 25 bases long, 10 to 20 bases long, 11 to 18 bases long, 12 to 16 bases long, or 13 to 22 bases long, 16 to 22 bases long, or 16 to 20 bases long.

[0024] The first nucleic acid strand is not particularly limited, and may be at least 9 bases long, at least 10 bases long, at least 11 bases long, at least 12 bases long, or at least 13 bases long. The first nucleic acid strand may be 50 bases or less, 45 bases or less, 40 bases or less, 35 bases or less, 30 bases or less, 28 bases or less, 26 bases or less, 24 bases or less, 22 bases or less, 20 bases or less, 18 bases or less, or 16 bases or less. The first nucleic acid strand may be, for example, 9 to 50 bases long, 10 to 40 bases long, 11 to 35 bases long, 12 to 30 bases long, 13 to 20 bases long, 13 to 22 bases long, 16 to 22 bases long, or 16 to 20 bases long.

[0025] The complementary region in the second nucleic acid strand may typically be, but is not limited to, at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, or at least 13 bases in length. The complementary region in the second nucleic acid strand may be 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less in length. In one embodiment, the complementary region in the second nucleic acid strand is 9 to 35 bases, 9 to 30 bases, 10 to 25 bases, 10 to 20 bases, 11 to 18 bases, or 12 to 16 bases in length. In one embodiment, the complementary region in the second nucleic acid strand is 13 to 22 bases, 16 to 22 bases, or 16 to 20 bases in length. In one embodiment, the complementary region in the second nucleic acid strand is 8 to 35 bases long, 8 to 30 bases long, 8 to 25 bases long, 8 to 20 bases long, 8 to 16 bases long, 8 to 12 bases long, or 8 to 10 bases long.

[0026] The second nucleic acid strand is not particularly limited, and may be at least 5 bases long, at least 6 bases long, at least 7 bases long, at least 8 bases long, at least 9 bases long, at least 10 bases long, at least 11 bases long, at least 12 bases long, or at least 13 bases long. The second nucleic acid strand may be 50 bases or less, 45 bases or less, 40 bases or less, 35 bases or less, 30 bases or less, 28 bases or less, 26 bases or less, 24 bases or less, 22 bases or less, 20 bases or less, 18 bases or less, 16 bases or less, 14 bases or less, 12 bases or less, 10 bases or less, or 9 bases or less in length. The second nucleic acid strand may be, for example, 9 to 50 bases long, 10 to 40 bases long, 11 to 35 bases long, 12 to 30 bases long, 13 to 20 bases long, 13 to 22 bases long, 16 to 22 bases long, 16 to 20 bases long, or 5 to 14 bases long, 6 to 12 bases long, 6 to 10 bases long, 7 to 10 bases long, 8 to 10 bases long, 8 to 9 bases long, or 7 to 9 bases long. The choice of length is generally determined by the balance between the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost, synthesis yield, etc.

[0027] The second nucleic acid strand comprises or consists of a complementary region that is complementary to at least a portion of the first nucleic acid strand.

[0028] In one embodiment, the complementary region in the second nucleic acid strand can be complementary to at least a portion of the antisense oligonucleotide region in the first nucleic acid strand. The complementary region in the second nucleic acid strand may be complementary to the entire antisense oligonucleotide region in the first nucleic acid strand. The complementary region in the second nucleic acid strand may be complementary to a portion of the first nucleic acid strand in addition to the antisense oligonucleotide region. An example of this embodiment is the heteroduplex oligonucleotide (HDO) disclosed in International Publication No. 2013 / 089283, Nishina K, et al., Nature Communication, 2015, 6:7969, and Asami Y, et al., Drug Discoveries & Therapeutics. 2016; 10(5):256-262 (FIGS. 1A and 1B).

[0029] In a further embodiment, the second nucleic acid strand may further include at least one overhang region located at one or both of the 5' and 3' ends of the complementary region. An example of this embodiment is described in International Publication No. 2018 / 062510. An "overhang region" refers to a region of nucleotides in the second nucleic acid strand that protrudes from the double-stranded structure adjacent to the complementary region, such that when the first and second nucleic acid strands anneal to form a double-stranded structure, the 5' end of the second nucleic acid strand extends beyond the 3' end of the first nucleic acid strand and / or the 3' end of the second nucleic acid strand extends beyond the 5' end of the first nucleic acid strand. The overhang region in the second nucleic acid strand may be located at the 5' end of the complementary region (FIG. 2A) or the 3' end (FIG. 2B). The overhang region in the second nucleic acid strand may be located at both the 5' and 3' ends of the complementary region (FIG. 2C).

[0030] In general, a "nucleoside" is a combination of a base and a sugar. The nucleobase (also known as base) portion of a nucleoside is usually a heterocyclic base moiety. A "nucleotide" further comprises a phosphate group covalently linked to the sugar portion of the nucleoside. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed by the covalent linkage of adjacent nucleosides to one another to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are generally considered to form the internucleoside linkages of the oligonucleotide.

[0031] A nucleic acid strand can contain natural and / or non-natural nucleotides. "Natural nucleotides" include deoxyribonucleotides found in DNA and ribonucleotides found in RNA. "Deoxyribonucleotides" and "ribonucleotides" are sometimes referred to as "DNA nucleotides" and "RNA nucleotides," respectively.

[0032] Similarly, "naturally occurring nucleosides" include the deoxyribonucleosides found in DNA and the ribonucleosides found in RNA. "Deoxyribonucleosides" and "ribonucleosides" are sometimes referred to as "DNA nucleosides" and "RNA nucleosides," respectively.

[0033] "Non-natural nucleotide" refers to any nucleotide other than a naturally occurring nucleotide, including modified nucleotides and nucleotide mimics. Similarly, "non-natural nucleoside" refers to any nucleoside other than a naturally occurring nucleoside, including modified nucleosides and nucleoside mimics. "Modified nucleotide" refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase. "Modified nucleoside" refers to a nucleoside having a modified sugar moiety and / or a modified nucleobase. Nucleic acid chains containing non-natural oligonucleotides are often preferred over natural forms due to desirable properties such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0034] "Modified internucleoside linkage" refers to an internucleoside linkage that has a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage). Modified internucleoside linkages include internucleoside linkages that contain a phosphorus atom and internucleoside linkages that do not contain a phosphorus atom. Representative phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages (e.g., methyl phosphotriester linkages and ethyl phosphotriester linkages described in U.S. Patent Registration No. 5,955,599), alkyl phosphonate linkages (e.g., methyl phosphonate linkages described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, and methoxypropyl phosphonate linkages described in WO 2015 / 168172), alkylthiophosphonate linkages, methylthiophosphonate linkages, boranophosphate linkages, and internucleoside linkages containing a cyclic guanidine moiety (e.g., a partial structure represented by the following formula (II): [ka] ), 1 to 4 C 1~6 an internucleoside linkage containing a guanidine moiety (e.g., a tetramethylguanidine (TMG) moiety) substituted with an alkyl group of the formula (III): [ka] Examples of suitable internucleoside linkages include, but are not limited to, the internucleoside linkages used in the self-neutralizing nucleic acids (ZONs) described in International Publication No. WO 2016 / 081600 and phosphoramidate linkages. A phosphorothioate linkage refers to an internucleoside linkage in which the non-bridging oxygen atom of a phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known. Modified internucleoside linkages are preferably those that are more nuclease-resistant than naturally occurring internucleoside linkages.

[0035] When an internucleoside bond has a chiral center, the internucleoside bond may be chiral controlled. "Chiral controlled" refers to a single diastereomer present with respect to the chiral center, e.g., chiral phosphorus. A chiral controlled internucleoside bond may be completely chirally pure or may have a high chiral purity, e.g., 90% de, 95% de, 98% de, 99% de, 99.5% de, 99.8% de, 99.9% de, or higher. As used herein, "chiral purity" refers to the proportion of one diastereomer in a mixture of diastereomers, expressed as diastereomeric excess (% de), and defined as (target diastereomer - other diastereomers) / (total diastereomers) × 100 (%).

[0036] For example, the internucleoside bond may be a phosphorothioate bond chirally controlled in the Rp or Sp configuration, or 1 to 4 C 1~6The internucleoside linkage may be an internucleoside linkage containing a guanidine moiety substituted with an alkyl group (e.g., a tetramethylguanidine (TMG) moiety; see, for example, Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811) (e.g., a partial structure represented by formula (III)), and / or an internucleoside linkage containing a cyclic guanidine moiety (e.g., a partial structure represented by formula (II)). Methods for preparing chiral internucleoside bonds are known. For example, chiral phosphorothioate bonds in the Rp or Sp configuration can be prepared by the methods described in Naoki Iwamoto et al., Angew. Chem. Int. Ed. Engl. 2009, 48(3), 496-9; Natsuhisa Oka et al., J. Am. Chem. Soc. 2003, 125, 8307-8317; Natsuhisa Oka et al., J. Am. Chem. Soc. 2008, 130, 16031-16037; Yohei Nukaga et al., J. Org. Chem. 2016, 81, 2753-2762; Yohei Nukaga et al., J. Org. Chem. 2012, 77, They can be synthesized according to the method described in [Publication ID No. 7913-7922]. Chiral phosphorothioate bonds in the Rp or Sp configuration are also known and are known to have the effects described, for example, in Naoki Iwamoto et al., Nat. Biotechnol., 2017, 35(9), 845-851 and Anastasia Khvorova et al., Nat. Biotechnol., 2017, 35(3), 238-248. For example, in one embodiment, phosphorothioate bonds in the Sp configuration are more stable than those in the Rp configuration, and / or ASOs in the Sp configuration are chiral to promote target RNA cleavage by RNase H1, resulting in a more sustained response in vivo. ASOs containing 1 to 4 C 1~6Methods for preparing internucleoside linkages containing a guanidine moiety (e.g., a TMG moiety) substituted with an alkyl group such as those described above are known, and the internucleoside linkages can be synthesized, for example, according to the method described in Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811.

[0037] As used herein, "nucleobase" or "base" encompasses both modified and unmodified nucleobases (bases) unless otherwise specified. Therefore, unless otherwise specified, a purine base may be either a modified or unmodified purine base. Furthermore, unless otherwise specified, a pyrimidine base may be either a modified or unmodified pyrimidine base.

[0038] "Modified nucleobase" or "modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. "Unmodified nucleobase" or "unmodified base" (natural nucleobase) refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Examples of modified nucleobases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, or N4-methylcytosine; N6-methyladenine or 8-bromoadenine; 2-thiothymine; and N2-methylguanine or 8-bromoguanine. The modified nucleobase is preferably 5-methylcytosine.

[0039] "Modified sugar" refers to a sugar having a substitution and / or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)), and "sugar modification" refers to a substitution and / or any change from a natural sugar moiety. A nucleic acid strand may optionally include one or more modified nucleosides, including modified sugars. "Sugar-modified nucleoside" refers to a nucleoside having a modified sugar moiety. Such sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to a nucleic acid strand. In certain embodiments, the nucleoside includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNAs), and the modification of ribosyl ring oxygen atoms to S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently H, C1-C 12 alkyl, or protecting groups), and combinations thereof.

[0040] Examples of sugar-modified nucleosides include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 5'-allyl (R or S), 4'-S, 2'-F (2'-fluoro), 2'-OCH (2'-OMe or 2'-O-methyl), and 2'-O(CH)OCH substituents. The 2'-position substituent can also be allyl, amino, azido, thio, -O-allyl, -O-C-C 10 alkyl, —OCF, —O(CH)SCH, —O(CH)—ON(R)(R), and O—CH—C(═O)—N(R)(R), where each R and R is independently H or a substituted or unsubstituted C—C 10 The term "2'-modified sugar" refers to a furanosyl sugar modified at the 2'-position. A nucleoside containing a 2'-modified sugar is also referred to as a "2'-sugar-modified nucleoside."

[0041] "Bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are commonly referred to as bridged nucleic acids (BNAs). Nucleosides containing a bicyclic sugar moiety are sometimes referred to as "bridged nucleosides" or "BNA nucleosides." Some examples of bridged nucleic acids are shown in Figure 4.

[0042] A bicyclic sugar may be a sugar in which the 2'- and 4'-carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. One subgroup of nucleic acids (BNAs) or BNA nucleosides containing bicyclic sugars is the 4'-(CH2) p -O-2',4'-(CH2) p -CH2-2',4'-(CH2) p -S-2',4'-(CH2) p -OCO-2',4'-(CH2) n -N(R3)-O-(CH2) m-2' [wherein p, m, and n represent an integer of 1 to 4, an integer of 0 to 2, and an integer of 1 to 3, respectively; and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a unit substituent (a fluorescent or chemiluminescent labeled molecule, a functional group having nucleic acid cleavage activity, an intracellular or intranuclear localization signal peptide, etc.)]. Furthermore, with respect to BNAs or BNA nucleosides according to certain embodiments, in the OR2 substituent on the 3' carbon atom and the OR1 substituent on the 5' carbon atom, R1 and R2 are typically hydrogen atoms, but may be the same or different from each other and may also be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R4)R5 (wherein R4 and R5 may be the same or different from each other and represent, respectively, a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms). Non-limiting examples of such BNAs include methyleneoxy (4'-CH2-O-2') BNA (also known as LNA (Locked Nucleic Acid®), 2',4'-BNA) (e.g., α-L-methyleneoxy (4'-CH2-O-2') BNA or β-D-methyleneoxy (4'-CH2-O-2') BNA), ethyleneoxy (4'-(CH2)2-O-2') BNA (also known as ENA), β-D-thio (4'-CH2-S-2') BNA, aminooxy (4'-CH2-ON(R3)-2') BNA, oxyamino (4'-CH2-N(R3)-O-2') BNA (2',4'-BNA NC Also known as; R=H is 2',4'-BNANC [NH], R=Me is 2',4'-BNA NC [N-Me]), 2',4'-BNA coc , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH3)-O-2')BNA (also known as cEt BNA), (4'-CH(CH2OCH3)-O-2')BNA (cMOE BNAs), amide BNAs, (4'-C(O)-N(R)-2')BNAs (R=H, Me) (also known as AmNAs; R=H is AmNA[NH], R=Me is AmNA[N-Me]), guanidine BNAs (also known as GuNAs (e.g., R=H is GuNA[NH], R=Me is GuNA[N-Me] in Figure 4), amine BNAs (also known as 2'-Amino-LNAs) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substitutions), 2'-O,4'-C-spirocyclopropylene bridged nucleic acids (also known as scpBNAs), and other BNAs known to those skilled in the art. Non-limiting examples of such BNA nucleosides include methyleneoxy(4'-CH2-O-2') BNA nucleosides (also known as LNA nucleosides, 2',4'-BNA nucleosides) (e.g., α-L-methyleneoxy(4'-CH2-O-2') BNA nucleosides, β-D-methyleneoxy(4'-CH2-O-2') BNA nucleosides), ethyleneoxy(4'-(CH2)2-O-2') BNA nucleosides (also known as ENA nucleosides), β-D-thio(4'-CH2-S-2') BNA nucleosides, aminooxy(4'-CH2-ON(R3)-2') BNA nucleosides, oxyamino(4'-CH2-N(R3)-O-2') BNA nucleosides (2',4'-BNA NC Also known as nucleosides; R=H is 2',4'-BNA NC [NH] nucleoside, R=Me is 2',4'-BNA NC [N-Me] nucleoside), 2',4'-BNA cocNucleosides, 3'-amino-2',4'-BNA nucleosides, 5'-methyl BNA nucleosides, (4'-CH(CH3)-O-2') BNA nucleosides (also known as cEt nucleosides), (4'-CH(CHOCH3)-O-2') BNA nucleosides (also known as cMOE nucleosides), amide BNA nucleosides, (4'-C(O)-N(R)-2') BNA nucleosides (R=H, Me) (also known as AmNA nucleosides; R=H is AmNA[NH]) nucleosides, where R=Me is an AmNA[N-Me] nucleoside), guanidine BNA nucleosides (also known as GuNA nucleosides (e.g., in Figure 4, where R=H is a GuNA[NH] nucleoside, and where R=Me is a GuNA[N-Me] nucleoside)), amine BNA nucleosides (also known as 2'-Amino-LNA nucleosides) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substituted nucleosides), 2'-O,4'-C-spirocyclopropylene bridged nucleosides (also known as scpBNA nucleosides), and other BNA nucleosides known to those of skill in the art.

[0043] As used herein, a "cationic nucleoside" is a modified nucleoside that exists in a cationic form relative to a neutral form (such as the neutral form of a ribonucleoside) at a certain pH (e.g., human physiological pH (about 7.4), the pH of a delivery site (e.g., organelle, cell, tissue, organ, organism, etc.)). A cationic nucleoside may contain one or more cationic modifying groups at any position of the nucleoside. In one embodiment, the cationic nucleoside is a 2'-Amino-LNA nucleoside (e.g., a 3-(Bis(3-aminopropyl)amino)propanoyl substituted nucleoside), an aminoalkyl modified nucleoside (e.g., a 2'-O-methyl and 4'-CH2CH2CH2NH2 substituted nucleoside), a GuNA nucleoside (e.g., in Figure 3, R = H is a GuNA[NH] nucleoside, and R = Me is a GuNA[N-Me] nucleoside), or the like.

[0044] Bicyclic nucleosides with a methyleneoxy (4'-CH2-O-2') bridge are sometimes referred to as LNA nucleosides.

[0045] Methods for preparing modified sugars are well known to those of skill in the art. In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) may be maintained for hybridization with an appropriate nucleic acid target.

[0046] "Nucleoside mimics" include structures used to replace sugars or sugars and bases, and, although not necessarily, linkages, at one or more positions in an oligomeric compound. "Oligomeric compound" refers to a polymer of linked monomeric subunits that can hybridize to at least a region of a nucleic acid molecule. Nucleoside mimics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimetics, e.g., nucleoside mimics having non-furanose sugar units. "Nucleotide mimics" include structures used to replace nucleosides and linkages at one or more positions in an oligomeric compound. Nucleotide mimics include, for example, peptide nucleic acids or morpholino nucleic acids (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Peptide nucleic acids (PNAs) are nucleotide mimics with a backbone in which N-(2-aminoethyl)glycine is linked via amide bonds in place of the sugar. An example of the structure of a morpholino nucleic acid is shown in Figure 5. The term "mimetic" refers to a group that replaces one or more of the sugar, nucleobase, and internucleoside linkage. Generally, a mimetic is used in place of the sugar or a combination of sugar and internucleoside linkage, while maintaining the nucleobase for hybridization to a selected target.

[0047] Generally, modifications can be made so that nucleotides in the same chain can be independently modified. The same nucleotide can also have a modified internucleoside linkage (e.g., a phosphorothioate linkage) and a modified sugar (e.g., a 2'-O-methyl modified sugar or a bicyclic sugar) to confer resistance to enzymatic cleavage. The same nucleotide can also have a modified nucleobase (e.g., a 5-methylcytosine) and a modified sugar (e.g., a 2'-O-methyl modified sugar or a bicyclic sugar).

[0048] The number, type, and position of non-natural nucleotides in a nucleic acid chain can affect the antisense effect provided by the nucleic acid complex. The choice of modification can vary depending on the sequence of the target gene, etc., but those skilled in the art can determine a suitable embodiment by referring to the descriptions in literature related to antisense methods (e.g., WO 2007 / 143315, WO 2008 / 043753, and WO 2008 / 049085). Furthermore, when the antisense effect of a modified nucleic acid complex is measured, if the measured value thus obtained is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of that of the nucleic acid complex before modification), the relevant modification can be evaluated.

[0049] The antisense effect can be measured, for example, by administering a test nucleic acid compound to a subject (e.g., a mouse) and measuring, for example, several days later (e.g., 2 to 7 days later), the expression level of a target gene or the level (quantity) of a target transcript (e.g., mRNA amount or RNA amount such as microRNA, cDNA amount, protein amount, etc.) whose expression is regulated by the antisense effect provided by the test nucleic acid compound.

[0050] For example, a decrease in the measured expression level of the target gene or level of the target transcript by at least 10%, at least 20%, at least 25%, at least 30%, or at least 40% compared to a negative control (e.g., vehicle administration) indicates that the test nucleic acid compound can produce an antisense effect (e.g., a reduction in the amount of the target transcript).

[0051] In this specification, the first nucleic acid strand is a gapmer. That is, the antisense oligonucleotide region in the first nucleic acid strand is a gapmer-type antisense oligonucleotide region (gapmer-type antisense oligonucleotide region). The term "gapmer type" refers to a nucleoside composition consisting of a central region (DNA gap region) containing at least four consecutive deoxyribonucleosides and regions (5' wing region and 3' wing region) containing unnatural nucleosides located on the 5' and 3' ends of the central region. A gapmer in which the unnatural nucleoside is a bridged nucleoside is specifically referred to as a "BNA / DNA gapmer." The length of the DNA gap region may be 13 to 22 bases, 16 to 22 bases, or 16 to 20 bases, or 4 to 20 bases, 5 to 18 bases, 6 to 16 bases, 7 to 14 bases, or 8 to 12 bases. The central region of a gapmer may comprise or consist of natural nucleosides, unmodified sugars (and modified or unmodified internucleoside linkages) in which the sugar moieties of the nucleic acid are not modified, for example, 13 to 22 bases, 16 to 22 bases, or 16 to 20 bases, or 4 to 20 bases, 5 to 18 bases, 6 to 16 bases, 7 to 14 bases, or 8 to 12 bases in length that are natural nucleosides or contain unmodified sugars. The central region may comprise or consist of natural nucleosides linked by modified internucleoside linkages such as phosphorothioate linkages.

[0052] The lengths of the 5' wing region and the 3' wing region may be, independently, typically 1 to 10 bases, 1 to 7 bases, 2 to 5 bases, or 2 to 3 bases. The 5' wing region and the 3' wing region may contain at least one unnatural nucleoside, and may further contain natural nucleosides. The gapmer-type antisense oligonucleotide region may have a BNA / DNA gapmer-type nucleoside composition, including a 5' wing region containing two or three bridged nucleosides, a 3' wing region containing two or three bridged nucleosides, and a DNA gap region therebetween. The bridged nucleosides may contain modified nucleobases (e.g., 5-methylcytosine). The gapmer may also be an "LNA / DNA gapmer" in which the bridged nucleosides are LNA nucleosides. The 5' and 3' wing regions can be non-natural nucleosides, e.g., 2'-O-methyl modified nucleosides, linked by modified internucleoside linkages, such as phosphorothioate linkages. In one embodiment, the nucleosides of the first nucleic acid strand comprise or consist of deoxyribonucleosides, e.g., 70% or more, 80% or more, 90% or more, or 95% or more of the nucleosides of the first nucleic acid strand are deoxyribonucleosides.

[0053] The internucleoside linkages in the first nucleic acid strand may be naturally occurring internucleoside linkages and / or modified internucleoside linkages.

[0054] At least one, at least two, or at least three internucleoside linkages from the 5'-end of the first nucleic acid strand may be modified internucleoside linkages. At least one, at least two, or at least three internucleoside linkages from the 3'-end of the first nucleic acid strand may be modified internucleoside linkages. For example, the two internucleoside linkages from the end of the nucleic acid strand refer to the internucleoside linkage closest to the end of the nucleic acid strand and the adjacent internucleoside linkage located in the opposite direction from the end of the nucleic acid strand. Modified internucleoside linkages in the terminal region of the nucleic acid strand are preferred because they can suppress or inhibit undesired degradation of the nucleic acid strand.

[0055] The modified internucleoside linkages may be at least 70%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or 100% of the internucleoside linkages in the antisense oligonucleotide region in the first nucleic acid strand, and may be phosphorothioate linkages.

[0056] The nucleosides in the first nucleic acid strand can be natural nucleosides (including deoxyribonucleosides, ribonucleosides, or both) and / or unnatural nucleosides.

[0057] The second nucleic acid strand comprises at least one, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, or at least 2 sugar-unmodified central regions (first exposed regions) that are complementary to a portion of the first nucleic acid strand (e.g., a portion of the central region of the first nucleic acid strand). Note that, in this specification, the sugar-unmodified central region is mainly referred to as the "first exposed region."

[0058] The first exposed region may be completely complementary to a portion of the first nucleic acid strand, or may have one or more mismatched bases. For example, the first exposed region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) to a portion of the first nucleic acid strand.

[0059] The first exposed region consists of one or two to three consecutive unsugar-modified ribonucleosides linked by internucleoside bonds. As used herein, "unsugar-modified ribonucleoside" refers to a ribonucleoside in which the sugar moiety of a natural ribonucleoside does not contain a sugar modification, such as a substitution at the 2' position and / or any other change. The first exposed region may contain at least one modified base and / or modified internucleoside bond.

[0060] In one embodiment, the sugar-unmodified ribonucleosides in the first exposed region comprise natural ribonucleosides, for example, all of the sugar-unmodified ribonucleosides are natural ribonucleosides, or some of them, for example, one or two of the sugar-unmodified ribonucleosides are natural ribonucleosides.

[0061] In one embodiment, the second nucleic acid strand comprises at least 1, e.g., 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, at least 2, e.g., 2 to 10, 2 to 5, 3 to 4, 2 to 3, or 2, or at least 3, e.g., 3 to 10, 3 to 5, 3 to 4, or 3, first exposed regions.

[0062] In one embodiment, the first exposed region consists of two or three consecutive unsugar-modified ribonucleosides linked by an internucleoside bond. In one embodiment, the first exposed region consists of three consecutive unsugar-modified ribonucleosides linked by an internucleoside bond. This embodiment may have the effect of maintaining a high level of RNase H cleavage activity compared to a first exposed region consisting of one or two consecutive unsugar-modified ribonucleosides. This embodiment may have the effect of maintaining or improving activity in the RNase H-dependent and / or -independent pathway compared to a central region consisting of unsugar-modified ribonucleosides.

[0063] In one embodiment, the first exposed region comprises nucleosides containing modified or unmodified purine bases. This embodiment may have the advantage of maintaining RNase H cleavage activity while reducing RNase A cleavage activity, compared to a first exposed region that does not contain nucleosides containing purine bases (e.g., nucleosides containing pyrimidine bases). This embodiment may have the advantage of maintaining or improving activity in RNase H-dependent and / or RNase H-independent pathways, compared to a first exposed region that does not contain nucleosides containing purine bases (e.g., nucleosides containing pyrimidine bases).

[0064] In one embodiment, the second nucleic acid strand comprises at least one protective region, e.g., 1-10, 1-5, 1-4, 1-3, 1-2, or 1, at least two, e.g., 2-10, 2-5, 3-4, 2-3, or 2, or at least three, e.g., 3-10, 3-5, 3-4, or 3 protective regions.

[0065] As used herein, the term "protective region" refers to a region that is resistant to cleavage by a nuclease. The nuclease is not limited thereto. Examples of nucleases include RNase A and RNase H. The protective region may be, for example, a region that is resistant to RNase A, a region that is resistant to RNase H, or a region that is resistant to both RNase A and RNase H. Furthermore, the resistance to cleavage by a nuclease does not need to be complete, but may be increased compared to a region that has the same base sequence and contains unmodified ribonucleosides.

[0066] A protective region consists of one or more (a) deoxyribonucleosides, (b) sugar-modified nucleosides, and / or (c) nucleosides with a modified internucleoside linkage at the 3' end, linked by an internucleoside linkage. The protective region may also contain at least one modified base and / or modified internucleoside linkage. The deoxyribonucleosides and / or sugar-modified nucleosides in the protective region are preferably deoxyribonucleosides, 2'-sugar-modified nucleosides (e.g., nucleosides containing a 2'-O-methyl group), bridged nucleosides (e.g., LNA nucleosides), and / or cationic nucleosides, more preferably deoxyribonucleosides, 2'-sugar-modified nucleosides (e.g., nucleosides containing a 2'-O-methyl group), and / or bridged nucleosides (e.g., LNA nucleosides), even more preferably deoxyribonucleosides and / or 2'-sugar-modified nucleosides (e.g., nucleosides containing a 2'-O-methyl group), and particularly preferably deoxyribonucleosides and / or nucleosides containing a 2'-O-methyl group. The nucleoside of the nucleoside having a modified internucleoside bond on the 3' side of the protective region is preferably a deoxyribonucleoside, a ribonucleoside, a 2'-sugar-modified nucleoside (e.g., a nucleoside containing a 2'-O-methyl group), a bridged nucleoside (e.g., an LNA nucleoside), and / or a cationic nucleoside, more preferably a deoxyribonucleoside, a ribonucleoside, a 2'-sugar-modified nucleoside (e.g., a nucleoside containing a 2'-O-methyl group), a bridged nucleoside (e.g., an LNA nucleoside), and / or a cationic nucleoside. The nucleoside having a modified internucleoside bond on the 3' side of the protective region is preferably a nucleoside containing a phosphorothioate bond, a phosphorothioate bond, or 1 to 4 C's on the 3' side. 1~6The nucleoside is preferably a nucleoside having an internucleoside bond (preferably a partial structure represented by formula (III)) containing a guanidine moiety (preferably a TMG moiety) substituted with an alkyl group such as 1 or 2, and / or an internucleoside bond (preferably a partial structure represented by formula (II)) containing a cyclic guanidine moiety, and more preferably a nucleoside having a phosphorothioate bond at the 3'-side. The modified internucleoside bond at the 3'-side may be chiral-controlled to have an Rp or Sp configuration. In this specification, a nucleoside having a modified internucleoside bond at the 3'-side means a nucleoside having a modified internucleoside bond at least at the 3'-side, and may also have a modified internucleoside bond at the 5'-side.

[0067] The protective region may be completely complementary to a portion of the first nucleic acid strand (e.g., a portion of the central region of the first nucleic acid strand), or may have one or more mismatched bases. For example, the protective region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) to a portion of the first nucleic acid strand.

[0068] In one embodiment, the protective region comprises (a) deoxyribonucleosides, (b) sugar-modified nucleosides, and / or (c) nucleosides having a modified internucleoside linkage at the 3'-side, each containing a pyrimidine base. Here, the pyrimidine base may be modified or unmodified. For example, at least one of the protective regions, or all of the protective regions, may comprise (a) deoxyribonucleosides, (b) sugar-modified nucleosides, and / or (c) nucleosides having a modified internucleoside linkage at the 3'-side, each containing a modified or unmodified pyrimidine base. This embodiment may have the effect of reducing the cleavage activity of RNase A against the second nucleic acid strand, compared to when a nucleoside containing a pyrimidine base is contained in a sugar-unmodified region (e.g., the first exposed region or the second exposed region). This embodiment can have the effect of maintaining or improving activity in the RNase H-dependent and / or RNase H-independent pathways compared to when a nucleoside containing a pyrimidine base is contained in the non-sugar-modified region (e.g., the first exposed region or the second exposed region).

[0069] In one embodiment, the sugar-modified nucleosides in the protective regions are sugar-modified ribonucleosides.

[0070] In one embodiment, the second nucleic acid strand comprises a first exposed region and a protected region. In a further embodiment, the second nucleic acid strand comprises alternating first exposed regions and protected regions. In a further embodiment, at least one of the protected regions, or all of the protected regions, comprises (a) deoxyribonucleosides containing modified or unmodified pyrimidine bases, (b) sugar-modified nucleosides, and / or (c) nucleosides with a modified 3' internucleoside linkage.

[0071] In one embodiment, the second nucleic acid strand comprises at least one, e.g., 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, or at least 2, second exposed regions complementary to a portion of the first nucleic acid strand.

[0072] The second exposed region is composed of four or more consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds, and the bases of the sugar-unmodified ribonucleosides in the second exposed region include purine bases. Here, the purine bases may be modified or unmodified. The number of sugar-unmodified ribonucleosides constituting the second exposed region may be, for example, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, or 4. The second exposed region may contain at least one modified base and / or modified internucleoside bond.

[0073] The second exposed region may be completely complementary to a portion of the first nucleic acid strand (e.g., a portion of the central region of the first nucleic acid strand), or may have one or more mismatched bases. For example, the second exposed region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) to a portion of the first nucleic acid strand.

[0074] In one embodiment, the sugar-unmodified ribonucleosides in the second exposed region comprise natural ribonucleosides, for example, all of the sugar-unmodified ribonucleosides are natural ribonucleosides, or some of them, for example, one or two of the sugar-unmodified ribonucleosides are natural ribonucleosides.

[0075] In one embodiment, the second nucleic acid strand comprises at least 1 second exposed region, e.g., 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, at least 2, e.g., 2 to 10, 2 to 5, 3 to 4, 2 to 3, or 2, or at least 3, e.g., 3 to 10, 3 to 5, 3 to 4, or 3, second exposed regions.

[0076] In one embodiment, the second nucleic acid strand includes a total of at least 1, for example, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, at least 2, for example, 2 to 10, 2 to 5, 3 to 4, 2 to 3, or 2, or at least 3, for example, 3 to 10, 3 to 5, 3 to 4, or 3, first exposed regions and / or second exposed regions.

[0077] In one embodiment, the second nucleic acid strand comprises a first exposed region, a second exposed region, and a protective region. In a further embodiment, the second nucleic acid strand comprises alternating first exposed regions or second exposed regions and protective regions. In a further embodiment, at least one of the protective regions, or all of the protective regions, comprises (a) deoxyribonucleosides containing modified or unmodified pyrimidine bases, (b) sugar-modified nucleosides, and / or (c) nucleosides with modified 3' internucleoside linkages.

[0078] In one embodiment, the second nucleic acid strand comprises a second exposed region and a protective region. In a further embodiment, the second nucleic acid strand comprises alternating second exposed regions and protective regions. In a further embodiment, at least one of the protective regions, or all of the protective regions, comprises (a) deoxyribonucleosides containing modified or unmodified pyrimidine bases, (b) sugar-modified nucleosides, and / or (c) nucleosides with a modified 3' internucleoside linkage.

[0079] In one embodiment, at least one protected region of the second nucleic acid strand is linked to the first exposed region and / or the second exposed region by an internucleoside bond at the 5'-end and 3'-end.

[0080] In one embodiment, the first exposed region and / or the second exposed region has at least one, e.g., one or two, modified internucleoside linkages, e.g., phosphorothioate linkages, e.g., chiral controlled phosphorothioate linkages in the Rp or Sp configuration, 1 to 4 C 1~6 The nucleoside bond includes an internucleoside bond containing a guanidine moiety (e.g., a TMG moiety) substituted with an alkyl group (e.g., a partial structure represented by formula (III)) and / or an internucleoside bond including a cyclic guanidine moiety (e.g., a partial structure represented by formula (II)).

[0081] In one embodiment, the second nucleic acid strand comprises or consists of (1) the first exposed region, and / or (2) the second exposed region, and (3) (a) deoxyribonucleosides and / or (b) sugar-modified nucleosides, e.g., sugar-modified ribonucleosides, linked by modified or unmodified internucleoside linkages.

[0082] In one embodiment, the second nucleic acid strand further comprises the following sugar-unmodified terminal region at the 5'-end and / or 3'-end. In one embodiment, the length of the sugar-unmodified terminal region at the 5'-end and / or 3'-end is, independently, typically 1 to 10 bases long, 1 to 7 bases long, 2 to 5 bases long, or 2 to 3 bases long. In one embodiment, the second nucleic acid strand comprises or consists of the first exposed region and / or the second exposed region and sugar-modified ribonucleosides (and optionally sugar-unmodified terminal regions) linked by modified or unmodified internucleoside bonds.

[0083] In one embodiment, the second nucleic acid strand comprises a sugar-unmodified terminal region at the 5'-end and / or 3'-end, which comprises at least one, e.g., 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, or at least two sugar-unmodified ribonucleosides. Because the toxicity of a nucleic acid complex tends to increase with an increase in sugar modifications, rendering the terminal region sugar-unmodified reduces the number of sugar-modified nucleic acids in the second nucleic acid strand, thereby potentially reducing toxicity. The sugar-unmodified terminal region may comprise modified bases and / or modified internucleoside linkages. In one embodiment, the sugar-unmodified terminal region is complementary to a portion of the first nucleic acid strand (e.g., the 5'-wing region, central region, or 3'-wing region of the first nucleic acid strand, or any portion thereof). The sugar-unmodified terminal region may be completely complementary to a portion of the first nucleic acid strand, or may have one or more mismatched bases. For example, the second exposed region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) with respect to a portion of the first nucleic acid strand. In one embodiment, the sugar-unmodified ribonucleosides in the sugar-unmodified terminal region include natural ribonucleosides, for example, all of the sugar-unmodified ribonucleosides are natural ribonucleosides, or a portion thereof, for example, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 sugar-unmodified ribonucleosides, are natural ribonucleosides. In one embodiment, the sugar-unmodified terminal region includes nucleosides including modified or unmodified purine bases and / or modified or unmodified pyrimidine bases. In one embodiment, the sugar-unmodified terminal region includes nucleosides including modified or unmodified purine bases.

[0084] In one embodiment, the second nucleic acid strand comprises (1) at least one first exposed region and / or at least one second exposed region, (2) at least one protective region, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 1 to 10 first exposed regions and / or 1 to 10 second exposed regions, (2) 1 to 10 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 1 to 5 first exposed regions and / or 1 to 5 second exposed regions, (2) 1 to 5 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 1 to 3 first exposed regions and / or 1 to 3 second exposed regions, (2) 1 to 3 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 2 to 10 first exposed regions and / or 2 to 10 second exposed regions, (2) 2 to 10 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 2 to 6 first exposed regions and / or 2 to 6 second exposed regions, (2) 2 to 6 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand consists of (1) 2 to 5 first exposed regions and / or second exposed regions, (2) 2 to 5 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand consists of (1) 2 to 4 first exposed regions and / or second exposed regions, (2) 2 to 4 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, which are linked by modified or unmodified internucleoside bonds.In one embodiment, the second nucleic acid strand comprises (1) 2 to 3 first exposed regions and / or second exposed regions, (2) 2 to 3 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 3 to 10 first exposed regions and / or second exposed regions, (2) 3 to 10 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 3 to 6 first exposed regions and / or second exposed regions, (2) 3 to 6 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand consists of (1) 3 to 5 first exposed regions and / or second exposed regions, (2) 3 to 5 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand consists of (1) 3 to 4 first exposed regions and / or second exposed regions, (2) 3 to 4 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all linked by modified or unmodified internucleoside bonds. In this embodiment, the second nucleic acid strand has (a) the first exposed regions or second exposed regions and (b) the protective regions arranged alternately.

[0085] In one embodiment, the second nucleic acid strand comprises a sugar-modified terminal region at the 5'-end and / or 3'-end comprising at least one deoxyribonucleoside, sugar-modified nucleoside, and / or nucleoside having a modified internucleoside linkage on the 3'-end. In one embodiment, the second nucleic acid strand comprises a sugar-modified terminal region at the 5'-end and / or 3'-end comprising at least one deoxyribonucleoside and / or sugar-modified nucleoside. In one embodiment, the sugar-modified terminal region is complementary to a portion of the first nucleic acid strand (e.g., the 5' wing region, central region, or 3' wing region of the first nucleic acid strand, or any portion thereof). The sugar-modified terminal region may be completely complementary to the portion of the first nucleic acid strand or may have one or more mismatched bases. For example, the second exposed region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) with the portion of the first nucleic acid strand. In one embodiment, the sugar-modified terminal region comprises deoxyribonucleosides containing pyrimidine bases, sugar-modified nucleosides, and / or nucleosides having a modified internucleoside linkage at the 3' side. Here, the pyrimidine base may be modified or unmodified. In one embodiment, the sugar-modified terminal region comprises deoxyribonucleosides containing purine bases, sugar-modified nucleosides, and / or nucleosides having a modified internucleoside linkage at the 3' side. In one embodiment, the sugar-modified terminal region comprises deoxyribonucleosides and / or sugar-modified nucleosides containing purine bases. Here, the purine base may be either modified or unmodified.

[0086] In one embodiment, the second nucleic acid strand comprises or consists of the first exposed region, the second exposed region, the protective region, and / or the sugar-modified terminal region, which are linked by modified or unmodified internucleoside bonds. In one embodiment, the length of the sugar-modified terminal region at the 5'-end and / or the 3'-end is, independently, typically 1 to 10 bases long, 1 to 7 bases long, 2 to 5 bases long, or 2 to 3 bases long.

[0087] In one embodiment, all of the nucleosides in the sugar-modified terminal region are deoxyribonucleosides, sugar-modified nucleosides, and / or nucleosides having a modified internucleoside bond on the 3' side (preferably, all of the nucleosides are deoxyribonucleosides and / or sugar-modified nucleosides), or some of them, for example, 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7, are deoxyribonucleosides, sugar-modified nucleosides, and / or nucleosides having a modified internucleoside bond on the 3' side (preferably, all of the nucleosides are deoxyribonucleosides and / or sugar-modified nucleosides). In one embodiment, the nucleosides in the sugar-modified terminal region contain deoxyribonucleosides, sugar-modified nucleosides, and / or nucleosides having a modified internucleoside bond on the 3' side at any ratio (preferably, all of the nucleosides are deoxyribonucleosides and / or sugar-modified nucleosides). For example, 10% or more, 30% or more, 50% or more, 70% or more, 80% or more, or 90% or more of the nucleosides are deoxyribonucleosides, sugar-modified nucleosides, and / or nucleosides having a modified internucleoside bond on the 3' side (preferably, all of the nucleosides are deoxyribonucleosides and / or sugar-modified nucleosides). The deoxyribonucleoside and / or sugar-modified nucleoside is preferably a deoxyribonucleoside, a 2'-sugar-modified nucleoside (e.g., a nucleoside containing a 2'-O-methyl group), a bridged nucleoside (e.g., an LNA nucleoside), and / or a cationic nucleoside, more preferably a deoxyribonucleoside, a 2'-sugar-modified nucleoside (e.g., a nucleoside containing a 2'-O-methyl group), and / or a bridged nucleoside (e.g., an LNA nucleoside''), even more preferably a deoxyribonucleoside, a nucleoside containing a 2'-O-methyl group, and / or an LNA nucleoside, even more preferably a deoxyribonucleoside and / or an LNA nucleoside, and particularly preferably an LNA nucleoside. The nucleoside having a modified internucleoside bond at the 3'-side is preferably a nucleoside having a phosphorothioate bond at the 3'-side.

[0088] In one embodiment, the second nucleic acid strand comprises (1) at least one first exposed region and / or at least one second exposed region, (2) at least one protective region, and (3) sugar-modified terminal regions at the 5'- and 3'-ends, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 1 to 10 first exposed regions and / or 1 to 10 second exposed regions, (2) 1 to 10 protective regions, and (3) sugar-modified terminal regions at the 5'- and 3'-ends, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 1 to 5 first exposed regions and / or 1 to 5 second exposed regions, (2) 1 to 5 protective regions, and (3) sugar-modified terminal regions at the 5'- and 3'-ends, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 1 to 4 first exposed regions and / or 1 to 4 second exposed regions, (2) 1 to 4 protective regions, and (3) 5'- and 3'-terminal sugar-modified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 1 to 3 first exposed regions and / or 1 to 3 second exposed regions, (2) 1 to 3 protective regions, and (3) 5'- and 3'-terminal sugar-modified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 2 to 5 first exposed regions and / or 1 to 5 second exposed regions, (2) 2 to 5 protective regions, and (3) 5'- and 3'-terminal sugar-modified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand consists of (1) 2 to 4 first exposed regions and / or 1 to 4 second exposed regions, (2) 2 to 4 protective regions, and (3) 5'- and 3'-terminal sugar-modified terminal regions, which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand consists of (1) 2 to 3 first exposed regions and / or 1 to 3 second exposed regions, (2) 2 to 3 protective regions, and (3) 5'- and 3'-terminal sugar-modified terminal regions, which are linked by modified or unmodified internucleoside bonds.In one embodiment, the second nucleic acid strand comprises (1) 3 to 5 first exposed regions and / or 1 to 5 second exposed regions, (2) 3 to 5 protective regions, and (3) 5'- and 3'-terminal sugar-modified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 3 to 4 first exposed regions and / or 1 to 4 second exposed regions, (2) 3 to 4 protective regions, and (3) 5'- and 3'-terminal sugar-modified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 2 to 10 first exposed regions and / or 2 to 10 second exposed regions, (2) 2 to 10 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 2 to 6 first exposed regions and / or second exposed regions, (2) 2 to 6 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 2 to 5 first exposed regions and / or second exposed regions, (2) 2 to 5 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand comprises (1) 2 to 4 first exposed regions and / or second exposed regions, (2) 2 to 4 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, all of which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand consists of (1) 2 to 3 first exposed regions and / or second exposed regions, (2) 2 to 3 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, which are linked by modified or unmodified internucleoside bonds. In one embodiment, the second nucleic acid strand consists of (1) 3 to 10 first exposed regions and / or second exposed regions, (2) 3 to 10 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions, which are linked by modified or unmodified internucleoside bonds.In one embodiment, the second nucleic acid strand is linked by modified or unmodified internucleoside bonds and consists of (1) 3 to 6 first exposed regions and / or second exposed regions, (2) 3 to 6 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions. In one embodiment, the second nucleic acid strand is linked by modified or unmodified internucleoside bonds and consists of (1) 3 to 5 first exposed regions and / or second exposed regions, (2) 3 to 5 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions. In one embodiment, the second nucleic acid strand is linked by modified or unmodified internucleoside bonds and consists of (1) 3 to 4 first exposed regions and / or second exposed regions, (2) 3 to 4 protective regions, and (3) 5'- and 3'-terminal sugar-unmodified terminal regions. In this embodiment, the second nucleic acid strand has (a) the first exposed region or the second exposed region, and (b) the protected region arranged alternately.

[0089] In one embodiment, the modified internucleoside linkage of the first nucleic acid strand and / or the second nucleic acid strand is such that at a certain pH (e.g., human physiological pH (about 7.4), the pH of the delivery site (e.g., organelle, cell, tissue, organ, organism, etc.)), the modified internucleoside linkage is in an anionic form (e.g., —OP(O)(O - )-O- (anionic form of the natural phosphate bond), -OP(O)(S -In one embodiment, the modified internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand comprise neutral internucleoside linkages. In one embodiment, the modified internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand comprise cationic internucleoside linkages. In one embodiment, the non-negatively charged internucleoside linkages (e.g., neutral internucleoside linkages), when in their neutral form, do not have moieties with a pKa less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, or less than 14. In one embodiment, the non-negatively charged internucleoside linkage is, for example, a methyl phosphonate linkage as described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, a methyl phosphotriester linkage as described in U.S. Patent Registration No. 5,955,599, an ethyl phosphotriester linkage, a methoxypropyl phosphonate linkage as described in WO 2015 / 168172, or an internucleoside linkage used in self-neutralizing nucleic acids (ZON) as described in WO 2016 / 081600. In one embodiment, the non-negatively charged internucleoside linkage comprises a triazole moiety or an alkyne moiety. In one embodiment, the non-negatively charged internucleoside linkage comprises a cyclic guanidine moiety and / or 1 to 4 C 1~6 In one embodiment, the modified internucleoside linkage containing a cyclic guanidine moiety has a moiety represented by formula (II): 1~6 The alkyl-substituted guanidine moiety has a moiety represented by formula (III): 1~6Neutral internucleoside linkages comprising a guanidine moiety substituted with an alkyl group of the formula (I) are chiral controlled. In one embodiment, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleoside linkage and at least one phosphorothioate internucleoside linkage. Without wishing to be bound by any particular theory, in at least some cases, the neutral internucleoside linkage can improve properties and / or activity compared to a comparable nucleic acid that does not comprise a neutral internucleoside linkage, such as improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape, and / or improved nuclear uptake.

[0090] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more modified internucleoside linkages. In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more modified internucleoside linkages.

[0091] In one embodiment, the first exposed region, the second exposed region, the protective region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid strand may each contain 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, or 1 to 22 modified internucleoside linkages. In one embodiment, the first exposed region, the second exposed region, the guard region, the non-sugar-modified terminal region and / or the sugar-modified terminal region of the second nucleic acid strand may each comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more modified internucleoside linkages.

[0092] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more chiral controlled internucleoside linkages. In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more chiral controlled internucleoside linkages.

[0093] In one embodiment, the first exposed region, the second exposed region, the protective region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid strand may each contain 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, or 1 to 22 chirality-controlled internucleoside linkages. In one embodiment, the first exposed region, the second exposed region, the guard region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid strand may each comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more chiral controlled internucleoside linkages.

[0094] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more non-negatively charged internucleoside linkages (preferably neutral internucleoside linkages). In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more non-negatively charged internucleoside linkages.

[0095] In one embodiment, the first exposed region, the second exposed region, the protective region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid strand may each contain 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, or 1 to 22 non-negatively charged internucleoside linkages (preferably neutral internucleoside linkages). In one embodiment, the first exposed region, the second exposed region, the guard region, the non-sugar-modified terminal region and / or the sugar-modified terminal region of the second nucleic acid strand may each comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more non-negatively charged internucleoside linkages.

[0096] In one embodiment, any of the above regions (first exposed region, protective region, second exposed region, sugar-unmodified terminal region, and / or sugar-modified terminal region) in the second nucleic acid strand contains at least one, for example, one or two modified internucleoside bonds, for example, a phosphorothioate bond, a phosphorothioate bond chiralized in the Rp or Sp configuration, 1 to 4 C 1~6 an internucleoside bond (e.g., a partial structure represented by formula (III)) containing a guanidine moiety (e.g., a TMG moiety) substituted with an alkyl group, 1 to 4 C chiral groups controlled in the Rp or Sp configuration 1~6 The internucleoside bond includes an internucleoside bond containing a guanidine moiety (e.g., a TMG moiety) substituted with an alkyl group (e.g., a partial structure represented by formula (III)), an internucleoside bond containing a cyclic guanidine moiety (e.g., a partial structure represented by formula (II)), and / or an internucleoside bond containing a cyclic guanidine moiety that is chiral controlled to the Rp or Sp configuration (e.g., a partial structure represented by formula (II)).

[0097] At least one, at least two, or at least three internucleoside linkages from the 5'-end of the second nucleic acid strand may be modified internucleoside linkages. At least one, at least two, or at least three internucleoside linkages from the 3'-end of the second nucleic acid strand may be modified internucleoside linkages, such as phosphorothioate linkages, 1 to 4 C 1~6 The modified internucleoside bond may be an internucleoside bond containing a guanidine moiety (e.g., a TMG moiety) substituted with an alkyl group (e.g., a partial structure represented by formula (III)) and / or an internucleoside bond containing a cyclic guanidine moiety (e.g., a partial structure represented by formula (II)). The modified internucleoside bond may be chiral controlled to have an Rp configuration or an Sp configuration.

[0098] The second nucleic acid strand may not contain a sugar-unmodified region other than the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region, if present). That is, the sugars of the nucleic acid in the region other than the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region, if present) are modified sugars, such as 2'-modified sugars.

[0099] In one embodiment, the bases in the region other than the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region, if present) comprise modified and / or unmodified pyrimidine bases. For example, all of the bases are modified and / or unmodified pyrimidine bases, or some of the bases, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 2, or 1, are modified and / or unmodified pyrimidine bases. The modified and / or unmodified pyrimidine bases are preferably cytosine, uracil, thymine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, and / or 2-thiothymine, more preferably cytosine, uracil, thymine, and / or 5-methylcytosine, and particularly preferably cytosine.

[0100] In one embodiment, the bases in the region other than the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region, if present) contain modified and / or unmodified pyrimidine bases at any ratio. For example, 10% or more, 30% or more, 50% or more, 70% or more, 80% or more, or 90% or more are modified and / or unmodified pyrimidine bases. Modified and / or unmodified pyrimidine bases are preferably cytosine, uracil, thymine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, and / or 2-thiothymine, more preferably cytosine, uracil, thymine, and / or 5-methylcytosine, and particularly preferably cytosine.

[0101] In one embodiment, the bases in the first exposed region include purine bases, for example, all of the bases are modified and / or unmodified purine bases, or some of the bases, for example, one or two bases, are modified and / or unmodified purine bases. This is because RNase A cleaves the phosphodiester bond on the 3' side of modified and / or unmodified pyrimidine bases, and if the bases in the first exposed region are modified and / or unmodified pyrimidine bases, this portion can be cleaved by RNase A. Therefore, by including modified and / or unmodified purine bases in the first exposed region, undesired degradation by RNase A can be reduced. For example, the bases in the first exposed region do not include any of the modified and / or unmodified pyrimidine bases C, UC, and UU, for example, any of the three sequences.

[0102] In one embodiment, the bases in the first exposed region include modified and / or unmodified purine bases. For example, all of the bases are modified and / or unmodified purine bases, or some of them, for example, 1 to 3, 1 to 2, or 1, are modified and / or unmodified purine bases. In one embodiment, the bases in the second exposed region include modified and / or unmodified purine bases. For example, all of the bases are modified and / or unmodified purine bases, or some of them, for example, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, are modified and / or unmodified purine bases. In one embodiment, any proportion of the bases in the first exposed region and / or the second exposed region are modified and / or unmodified purine bases. For example, 10% or more, 30% or more, 50% or more, 70% or more, 80% or more, or 90% or more are modified and / or unmodified pyrimidine bases. The modified and / or unmodified purine base is preferably adenine, guanine, N6-methyladenine, 8-bromoadenine, N2-methylguanine and / or 8-bromoguanine, more preferably adenine and / or guanine.

[0103] In one embodiment, the second nucleic acid strand comprises sugar-modified nucleosides and / or deoxyribonucleosides containing modified and / or unmodified pyrimidine bases. For example, all of the bases are modified and / or unmodified pyrimidine bases, or some of the bases, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 2, or 1, are modified and / or unmodified pyrimidine bases. In one embodiment, the second nucleic acid strand comprises sugar-modified nucleosides and / or deoxyribonucleosides containing modified and / or unmodified pyrimidine bases in any proportion. For example, 10% or more, 30% or more, 50% or more, 70% or more, 80% or more, or 90% or more of the second nucleic acid strand are pyrimidine bases. The pyrimidine base to be sugar-modified is preferably cytosine, uracil, thymine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, and / or 2-thio-thymine, more preferably cytosine, uracil, thymine, and / or 5-methylcytosine, and particularly preferably cytosine.

[0104] In one embodiment, the protective region of the second nucleic acid strand comprises modified and / or unmodified pyrimidine bases. For example, all of the bases are modified and / or unmodified pyrimidine bases, or a subset of the bases, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 2, or 1, are modified and / or unmodified pyrimidine bases. In one embodiment, the protective region of the second nucleic acid strand comprises modified and / or unmodified pyrimidine bases. For example, 10% or more, 30% or more, 50% or more, 70% or more, 80% or more, or 90% or more of the second nucleic acid strand are pyrimidine bases. The pyrimidine base to be sugar-modified is preferably cytosine, uracil, thymine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, and / or 2-thio-thymine, more preferably cytosine, uracil, thymine, and / or 5-methylcytosine, and particularly preferably cytosine.

[0105] The positions of the first exposed region and / or the second exposed region in the second nucleic acid strand are not limited. In one embodiment, at least one, for example, all, of the first exposed region and / or the second exposed region are located on the 3' side of the center of the second nucleic acid strand. Here, "the first exposed region and / or the second exposed region are located on the 3' side of the center of the second nucleic acid strand" includes the center, 5' end, or 3' end of the first exposed region and / or the second exposed region being located on the 3' side of the center of the second nucleic acid strand.

[0106] In one embodiment, the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region, if present) can be cleaved by RNase H. Whether or not a nucleic acid strand is cleaved by RNase H can be determined, for example, by reacting the nucleic acid strand with RNase H, performing electrophoresis, and measuring the concentration of the undegraded band, as described in detail in the Examples. It was surprising that a first exposed region consisting of one or two or three consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds can be cleaved by RNase H, given the conventional common knowledge that the cleavage activity of RNase H requires at least four consecutive natural ribonucleosides (see, for example, Figure 3 in FEBS J 2008; 276(6): 1494-505). In one embodiment, the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region, if present) can have activity in an RNase H-dependent and / or -independent pathway.

[0107] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may comprise, in whole or in part, cationic nucleosides, such as 2'-Amino-LNA nucleosides (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl-substituted nucleosides), aminoalkyl-modified nucleosides (e.g., 2'-O-methyl and 4'-CH2CH2CH2NH2-substituted nucleosides), and GuNA nucleosides (e.g., in Figure 4, R = H represents a GuNA[NH] nucleoside, and R = Me represents a GuNA[N-Me] nucleoside).

[0108] In one embodiment, the second nucleic acid strand may be bound to a functional moiety. The bond between the second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond via another substance, but in one embodiment, the second nucleic acid strand and the functional moiety are preferably directly bound to each other via a covalent bond, an ionic bond, a hydrogen bond, or the like, and a covalent bond is more preferred from the viewpoint of obtaining a more stable bond.

[0109] In one embodiment, the "functional moiety" is not particularly limited in structure, and it confers a desired function to the double-stranded nucleic acid complex to which it is bound. Desired functions include labeling, purification, and target delivery. Examples of moieties that impart labeling include compounds such as fluorescent proteins and luciferase. Examples of moieties that impart purification include compounds such as biotin, avidin, His-tag peptide, GST-tag peptide, and FLAG-tag peptide. Furthermore, in one embodiment, from the viewpoint of highly specific and efficient delivery of the first nucleic acid strand to a target site and highly effective suppression of target gene expression by the nucleic acid, it is preferable that a molecule having the activity of delivering the double-stranded nucleic acid complex of one embodiment to a target site be bound to the second nucleic acid strand as a functional moiety. Examples of moieties that impart target delivery function include lipids, antibodies, aptamers, and ligands for specific receptors. In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) is bound to a functional moiety.

[0110] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand is bound to a lipid, including, but not limited to, tocopherol, cholesterol, fatty acids, phospholipids, and their analogs; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstane, and their analogs; steroids and their analogs; ligands for LDLR, SRBI, or LRP1 / 2; FK-506, cyclosporine; and lipids described in PCT / JP2019 / 12077, PCT / JP2019 / 10392, and PCT / JP2020 / 035117.

[0111] As used herein, the term "analog" refers to a compound having a similar structure and properties, which has the same or a similar basic skeleton. Analogs include, for example, biosynthetic intermediates, metabolic products, compounds with substituents, etc. Whether a compound is an analog of another compound can be determined by one skilled in the art.

[0112] The tocopherol may be selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol. Tocopherol analogs include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, etc. Preferably, the tocopherol is α-tocopherol.

[0113] Cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols with a sterol skeleton, including, but not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0114] The lipid may be linked to the 5'-end, 3'-end, or both ends of the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand). Alternatively, the lipid may be linked to an internal nucleotide of the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand). The first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) contains two or more lipids, which may be linked to multiple positions on the second nucleic acid strand and / or may be linked as a group to a single position on the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand). One lipid may be linked to each of the 5'-end and 3'-end (preferably the 5'-end) of the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand).

[0115] The bond between the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) and the lipid may be a direct bond or an indirect bond mediated by another substance. However, in certain embodiments, the lipid is preferably directly bonded to the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) via a covalent bond, an ionic bond, a hydrogen bond, or the like, and a covalent bond is more preferred in view of obtaining a more stable bond.

[0116] The lipid may also be linked to the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) via a cleavable linker. A "cleavable linker" refers to a linker that is cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within a human body). In certain embodiments, the cleavable linker is selectively cleaved by an endogenous enzyme such as a nuclease. Examples of cleavable linkers include amides, esters, one or both phosphodiesters, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers.

[0117] The lipid may be linked to the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) via a non-cleavable linker. A "non-cleavable linker" refers to a linking group that is not cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within the human body). Examples of non-cleavable linkers include linkers consisting of phosphorothioate bonds and modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked via phosphorothioate bonds. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not limited, and may be 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.

[0118] A specific example of the linker is a linker represented by the following formula (I):

[0119] [ka] (In the formula, L 2 is a substituted or unsubstituted C1 to C 12(e.g., propylene, hexylene, dodecylene), a substituted or unsubstituted C3 to C8 cycloalkylene group (e.g., cyclohexylene), —(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-, —(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-, or CH(CH2-OH)-CH2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-; L 3 represents -NH- or a bond, and L 4 is a substituted or unsubstituted C1 to C 12 alkylene groups (e.g., ethylene, pentylene, heptylene, undecylene), substituted or unsubstituted C3-8 cycloalkylene groups (e.g., cyclohexylene), -(CH2)2-[O-(CH2)2] m - or a bond, where m represents an integer of 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond (wherein the substitution is preferably made by a halogen atom).

[0120] In one embodiment, the linker of formula (I) is L 2 is an unsubstituted C3 to C6 alkylene group (e.g., propylene, hexylene), —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, or —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, and L 3 is -NH-, and L 4 and L 5 is a bond.

[0121] The attachment position and type of attachment of the functional moiety on the first and / or second nucleic acid strand (preferably the second nucleic acid strand) are as described above for the attachment of the lipid to the first and / or second nucleic acid strand (preferably the second nucleic acid strand).

[0122] Those skilled in the art can prepare the first and second nucleic acid strands that constitute the nucleic acid complex by appropriately selecting known methods. For example, nucleic acids can be prepared by designing the respective base sequences of the nucleic acids based on information about the base sequence of the target transcript (or, in some cases, the base sequence of the target gene), synthesizing the nucleic acids using a commercially available automated nucleic acid synthesizer (such as a product from Applied Biosystems, Inc. or Beckman Coulter, Inc.), and then purifying the resulting oligonucleotides using a reverse-phase column or the like. The nucleic acids prepared in this manner can be mixed in an appropriate buffer solution and denatured at about 90°C to 98°C for several minutes (e.g., 5 minutes), and then annealing the nucleic acids at about 30°C to 70°C for about 1 to 8 hours, thereby producing a nucleic acid complex. The preparation of the annealed nucleic acid complex is not limited to these time and temperature protocols. Conditions suitable for promoting strand annealing are well known in the art. A nucleic acid complex having a functional moiety bound thereto may be prepared by carrying out the above-described synthesis, purification, and annealing using a nucleic acid species to which a functional moiety has already been bound, or the functional moiety may be subsequently bound to the nucleic acid. Numerous methods for linking a functional moiety to a nucleic acid are well known in the art. Alternatively, a nucleic acid chain can be ordered and obtained from a manufacturer (e.g., Gene Design, Inc.) by specifying the base sequence and the modification site or type.

[0123] <Composition> In one aspect, the present invention relates to a composition for suppressing the expression of a target gene or a target transcript by antisense effect, comprising the nucleic acid complex described above. The composition may be a pharmaceutical composition. The composition may be used to treat or prevent a disease associated with increased expression of the target gene in a subject, such as a neurological disease, a central nervous system disease, a metabolic disease, a tumor, or an infectious disease.

[0124] The subject may be an animal, including a human, but is not limited to any particular animal other than a human, and may include various livestock, poultry, pets, laboratory animals, and the like. The composition can be formulated by known pharmaceutical methods. For example, the composition can be used orally or parenterally in the form of capsules, tablets, pills, liquids, powders, granules, microgranules, film-coated formulations, pellets, troches, sublingual tablets, peptizers, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal formulations, lotions, inhalants, aerosols, eye drops, injections, and suppositories.

[0125] For the formulation of these preparations, a pharmaceutically acceptable carrier or solvent or a carrier or solvent acceptable for food and beverage products may be appropriately incorporated. Specific examples of such carriers or solvents include sterilized water, physiological saline, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavors, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonicity adjusters, soothing agents, bulking agents, disintegrating agents, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizing agents, and other additives.

[0126] The dosage of the composition can be appropriately selected based on the subject's age, weight, symptoms, and health condition, dosage form, etc. However, the dosage of the composition may be, for example, 0.0000001 mg / kg / day to 1,000,000 mg / kg / day, 0.00001 mg / kg / day to 10,000 mg / kg / day, or 0.001 mg / kg / day to 500 mg / kg / day of the nucleic acid complex. The composition may be administered in a single dose or multiple doses. In the case of multiple doses, the composition may be administered daily or at appropriate intervals (e.g., at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times. The single dose of the nucleic acid complex may be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.025 mg / kg or more, 0.1 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 3.0 mg / kg or more, 4.0 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more The dose can be 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, and can be appropriately selected from any amount within the range of, for example, 0.001 mg / kg to 500 mg / kg (for example, 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg).

[0127] The nucleic acid complex of the present invention may be administered at a dose of 0.01 to 10 mg / kg (e.g., about 6.25 mg / kg) twice a week for four doses. Alternatively, the nucleic acid complex may be administered at a dose of 0.05 to 30 mg / kg (e.g., about 25 mg / kg) once or twice a week for two to four doses, for example, twice a week for two doses. The adoption of such a dosing regimen (divided administration) can reduce toxicity and the burden on the subject compared to a single administration of a higher dose. In one embodiment, the nucleic acid complex of the present invention can be administered subcutaneously, which can reduce toxicity and the burden on the subject compared to intravenous administration.

[0128] In certain embodiments, the nucleic acid complex of the present invention has excellent properties as a pharmaceutical, such as excellent solubility in water, the second fluid of the Japanese Pharmacopoeia dissolution test, or the second fluid of the Japanese Pharmacopoeia disintegration test, excellent pharmacokinetics (e.g., blood drug half-life, brain transferability, metabolic stability, CYP inhibition), low toxicity (e.g., superior as a pharmaceutical in terms of acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interactions, carcinogenicity, phototoxicity, etc.), and few side effects (e.g., suppression of excessive sedation, avoidance of lamellar necrosis).

[0129] Herein, there is no particular limitation on the preferred administration form of the composition. For example, oral administration or parenteral administration may be used. Specific examples of parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, tracheal / bronchial administration, rectal administration, intrathecal administration, intraventricular administration, intranasal administration, and intramuscular administration, as well as administration by blood transfusion. Administration can also be by intramuscular injection, intravenous drip administration, or implanted continuous subcutaneous administration. Subcutaneous administration is preferred because it allows patients to self-inject. Furthermore, in the case of intravenous administration, the amount of nucleic acid complex contained in one dose of the composition, i.e., the single dose of the nucleic acid complex, can be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.025 mg / kg or more, 0.1 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more. For example, any amount within the range of 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg) can be appropriately selected.

[0130] In one aspect, there is provided a method for treating and / or preventing a disease, comprising administering to a subject the nucleic acid complex or composition described above. [Example]

[0131] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0132] [Example 1: Cleavage activity of a double-stranded nucleic acid complex containing a second nucleic acid strand containing consecutive three unmodified sugar ribonucleosides by RNase H / A] A double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing three consecutive unmodified sugar ribonucleosides bound to cholesterol was treated with RNase A or RNase H to examine its cleavage efficiency. The second nucleic acid strand and cholesterol are linked at the 5' end in a similar manner to chol#1-cRNA (DNA) (mMalat1) shown in WO 2018 / 056442.

[0133] (Preparation of nucleic acid agents) A 16-mer single-stranded LNA / DNA gapmer (ASO(mMalat1)1) (first nucleic acid strand) targeting the malat1 non-coding RNA was prepared. This LNA / DNA gapmer is a 16-base-long oligonucleotide containing three LNA nucleosides at the 5' end and three LNA nucleosides at the 3' end, with 10 DNA nucleosides between them. This LNA / DNA gapmer has a sequence complementary to positions 1317 to 1332 of mouse malat1 non-coding RNA (SEQ ID NO: 1).

[0134] We prepared a complementary RNA strand (Chol-cRNA(mMalat1)) (second nucleic acid strand) with a base sequence complementary to this ASO and covalently linked cholesterol at the 5' end. The second strand is a 16-base-long oligonucleotide containing three 2'-O-methyl-modified ribonucleosides at both ends, seven 2'-O-methyl-modified ribonucleosides between them, and three consecutive unmodified sugar ribonucleosides at different positions. As controls, we prepared a complementary strand (Chol-cRNA) containing three 2'-O-methyl-modified ribonucleosides at both ends and ten unmodified sugar ribonucleosides between them, as well as a complementary strand (Chol-cRNA full OMe) in which all 16 nucleic acid moieties were 2'-O-methyl-modified ribonucleosides.

[0135] The ASO was annealed with a second nucleic acid strand to prepare a double-stranded nucleic acid agent. Specifically, the two were dissolved in PBS, mixed in equimolar amounts, and the solution was heated to 98°C for 5 minutes, then cooled to 37°C and held for 1 hour. This annealed the nucleic acid strands to prepare the double-stranded nucleic acid agent.

[0136] The sequences, chemical modifications, and structures of the oligonucleotides used in Example 1 are shown in Table 1 and Figure 6. All oligonucleotides were manufactured by Gene Design Co., Ltd. (Osaka, Japan).

[0137] [Table 1]

[0138] (Cutting experiment) 5 μL of 10 μM double-stranded nucleic acid annealed as described above was prepared. The following enzymes were prepared: Ribonuclease H (RNase H) (60 U / μl : TAKARA BIO INC) RNase A (7000 U / ml (100 mg / ml): Qiagen) For RNase H, the following buffers were prepared (all final concentrations). 60 mM Tris-HCl (pH 7.8) 60 mM KCl 2.5 mM MgCl2 2 mM TCEP After preparing the buffer, 5 μL of 10 μM nucleic acid was reacted with 5 U or 10 U of Ribonuclease H.

[0139] Dulbecco's phosphate buffered saline (Ca, Mg-free) (Nacalai Tesque) was used for RNase A. 0.035 U of RNase A was reacted with 5 μL of 10 μM nucleic acid in the buffer.

[0140] Under the above reaction conditions, the enzyme was reacted in a PCR machine (LifeECO: thermal cycler) at 37°C for 20 minutes, after which the enzyme reaction was stopped with liquid nitrogen.

[0141] (Electrophoresis and Analysis) A 20% acrylamide gel (1x TBE) was prepared and prepared. The enzyme-reacted product was mixed with 6x Gel Loading Dye, Purple (New England Biolab) and electrophoresed on the gel at 150V for 2 hours. ASO alone was also run as a control. Next, a solution of GelRed (x10,000) solution (Biotium) was diluted to a concentration of 1 / 10,000 in 1x TBE. The gel was then permeated with this solution for 10 minutes. The gel was then photographed using a ChemiDoc Touch imaging system (BioRad). Band density was analyzed using Image Lab Version 5.2 build 14 (BioRad).

[0142] (result) Figure 7 shows the electrophoresis results of ASO alone, a double-stranded complex of ASO and Chol-cRNA (mMalat1) (Chol-HDO), and a double-stranded complex of ASO and Chol-cRNA (mMalat1) full OMe (Chol-HDO with full OMe cRNA) after cleavage with RNase H and / or RNase A. Figure 8 also shows a graph showing the concentration of the undegraded bands based on the electrophoresis results, expressed as a relative intensity level (%) compared to the untreated complex. As shown in Figures 7 and 8, when all 10 nucleotides in the second nucleic acid strand complementary to the gap region of the first nucleic acid strand were unsugar-modified ribonucleosides (Chol-HDO), the complementary strand was cleaved regardless of whether RNase H or A was used. In contrast, when all 10 nucleotides in the second nucleic acid strand complementary to the gap region of the first nucleic acid strand were 2'-O-methyl-modified ribonucleosides (Chol-HDO with full OMe cRNA), the complementary strand was not cleaved using either RNase H or A. These results suggest that the activity of Chol-HDO may be reduced due to degradation in vivo, whereas the activity of Chol-HDO with full OMe cRNA may be reduced due to its high resistance to degradation.

[0143] A similar test was performed on a double-stranded nucleic acid agent containing three consecutive unsugared ribonucleosides complementary to a portion of the gap region of the first nucleic acid strand, varying the position of the three consecutive unsugared ribonucleosides. Figure 9 shows the concentration of the undegraded bands as a relative intensity level (%) to the untreated band. Note that 3windows-1 to 8 represent the results for complexes of ASO with each of the Chol-cRNA 3windows-1 to 8 listed in Table 1. For complexes containing a second nucleic acid strand containing three consecutive unsugared ribonucleosides, RNase A resistance was strong when the second nucleic acid strand contained three consecutive unsugared ribonucleosides on the 3' side, where C / U ratios are low (3windows 4-8), whereas RNase A resistance was low when the second nucleic acid strand contained three consecutive unsugared ribonucleosides on the 5' side, where C / U ratios are high (3windows 1-3). These results suggest that RNase A resistance correlates with whether or not the triad of unmodified sugar ribonucleosides contains a C / U residue, which is consistent with the fact that RNase A cleaves the P–O bond 3' to the pyrimidine base C / U. In contrast, RNase H was more susceptible to cleavage when the second strand contained a triad of unmodified sugar ribonucleosides (3 windows 4-8) at the 3' end. These results suggest that it is possible to design nucleic acids that are less susceptible to RNase A degradation while maintaining activity via the RNase H-dependent pathway.

[0144] [Example 2: Gene suppression effect in cells] The double-stranded nucleic acid complex containing the first nucleic acid strand containing an antisense oligonucleotide and the cholesterol-conjugated complementary strand (second nucleic acid strand) was introduced into cells, and its gene silencing effect was examined.

[0145] (Preparation of nucleic acid agents) The same nucleic acid as in Example 1 was prepared and prepared.

[0146] (Cell experiments) Neuro2a cells were purchased from the American Type Culture Collection (ATC). Neuro2a cells were grown in 24-well plates to 60-80% confluency. After growth, the nucleic acids were transfected at 5, 10, or 20 nM using RNAimax (Thermo Fisher Scientific) at 1.5 μL per well according to the manufacturer's protocol. Cells were harvested 24 hours later. RNA was then extracted using ISOGEN (Nippon Gene) according to the manufacturer's protocol. cDNA was synthesized using PrimeScript RT Master Mix (TAKARA-Bio) according to the manufacturer's protocol. Quantitative RT-PCR was performed using a LightCycler 480 Probes Master (Roche Life Sciences) according to standard procedures. The primers and probes used in qRT-PCR are as follows:

[0147] [Table 2]

[0148] The Probes Master, cDNA, primers, and probes were mixed and PCR was carried out with one cycle of 95°C for 10 minutes, 45 cycles of 95°C for 10 seconds / 60°C for 30 seconds / 72°C for 1 second, and one cycle of 40°C for 30 seconds.

[0149] (result) Figure 10 shows the relative RNA expression levels when cells were treated with various double-stranded nucleic acid complexes, with the Malat1 / Actb (β-actin) expression level in PBS treatment set at 1. As above, Chol-HDO represents a double-stranded complex of ASO and Chol-cRNA (mMalat1), Chol-HDO with full OMe cRNA represents a double-stranded complex of ASO and Chol-cRNA (mMalat1) full OMe, and 3 windows 1-8 represent the results for the double-stranded complex of ASO with each of the Chol-cRNA 3 windows 1 to 8 listed in Table 1.

[0150] As a control, a second nucleic acid strand complementary to the gap region of the first nucleic acid strand, in which all ten nucleotides were unsugared ribonucleosides (Chol-HDO), showed a high antisense effect, whereas a second nucleic acid strand complementary to the gap region of the first nucleic acid strand, in which all ten nucleotides were 2'-O-methyl-modified ribonucleosides (Chol-HDO with full OMe cRNA), showed a low antisense effect. Furthermore, in a complex containing a second nucleic acid strand containing three consecutive unsugared ribonucleosides, the presence of three consecutive unsugared ribonucleosides on the 5' side of the second nucleic acid strand (3 windows 1-2) resulted in a lower gene silencing effect than the presence of three consecutive unsugared ribonucleosides on the 3' side of the second nucleic acid strand (3 windows 3-8). These results generally correspond to the results of RNase A / H cleavage activity in Example 1.

[0151] The antisense effect observed with Chol-HDO with full OMe cRNA was a cleavage-independent effect, and the difference in the antisense effects of 3windows 1-2 and 3windows 3-8 is thought to be due to activity dependent on RNase H cleavage.

[0152] [Example 3: Cleavage activity of a double-stranded nucleic acid complex containing a second nucleic acid strand containing one or two consecutive unmodified ribonucleosides by RNase H / A] A double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing one or two consecutive unmodified sugar ribonucleosides bound to cholesterol was treated with RNase A or RNase H, and the cleavage efficiency was examined.

[0153] (Preparation of nucleic acid agents) A complementary strand containing an ASO and one or two consecutive unsugar-modified ribonucleosides was prepared according to Example 1. These were dissolved in PBS and mixed in equimolar amounts. The solution was heated to 98°C for 5 minutes, then cooled to 37°C and held for 1 hour, thereby annealing the nucleic acid strands to prepare the double-stranded nucleic acid agent described above. As controls, a complementary strand (Chol-cRNA) containing three 2'-O-methyl-modified ribonucleosides at both ends and 10 unsugar-modified ribonucleosides between them, and a complementary strand (Chol-cRNAfull OMe) in which all 16 nucleic acid moieties were 2'-O-methyl-modified ribonucleosides were prepared.

[0154] The sequences of ASO, Chol-cRNA, and Chol-cRNA full OMe are as shown in Table 1, and the names and sequences of other second strands used in this example are shown below.

[0155] [Table 3]

[0156] Cleavage with RNase A or H and subsequent electrophoresis and analysis were carried out in accordance with Example 1.

[0157] (result) The electrophoresis results after RNase A digestion are shown in FIG. 13, and a graph showing the concentration of the undigested band based on the electrophoresis results as a relative intensity level (%) to Chol-HDO with full OMe cRNA is shown in FIG. 14.

[0158] 13 and 14, when all 10 nucleic acids in the second nucleic acid strand complementary to the gap region of the first nucleic acid strand were sugar-unmodified ribonucleosides (Chol-HDO), the complementary strand was cleaved by RNase A. In contrast, when all 10 nucleic acids in the second nucleic acid strand complementary to the gap region of the first nucleic acid strand were 2'-O-methyl-modified ribonucleosides (Chol-HDO with full OMe cRNA), the complementary strand was not cleaved by RNase A.

[0159] As shown in Figures 13 and 14, when a single unsugar-modified ribonucleoside was present, RNase A cleaved the second strand only when the unsugar-modified ribonucleoside was present at the C position (1 window-3), but not when the unsugar-modified ribonucleoside was present at the U position (1 window-1, 2, 6). On the other hand, when two consecutive unsugar-modified ribonucleosides were present, RNase A cleaved the second strand only when the unsugar-modified ribonucleoside was present at the CA (2 window-3), UC (2 window-2), or UU (2 window-1) positions. These results suggest that RNase A cleaves C or UC / UU consecutive sequences.

[0160] Example 4: In vivo inhibitory effect of DmpkmRNA expression by a double-stranded nucleic acid complex containing a second nucleic acid strand having a sugar-modified ribonucleoside at the C / U position We will verify the in vivo inhibitory effect of mRNA expression in tissues by a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide targeting the Dmpk gene and a complementary strand (second nucleic acid strand) bound to cholesterol and having a sugar-modified ribonucleoside at the C / U position.

[0161] (Preparation of nucleic acid agents) We prepared an antisense oligonucleotide ASO (ASO DMPK) targeting the Dmpk gene, a cholesterol-conjugated complementary strand Chol-cRNA (CU OMe) with 2'-O-methyl modifications at the C / U positions, and a control complementary strand Chol-cRNA (Default). The sequences of ASO (mDMPK), Chol-cRNA (Default), and Chol-cRNA (CU OMe) are shown below.

[0162] [Table 4]

[0163] The ASO (mDMPK) targets the mouse DMPK gene and is composed of a 16-mer single-stranded LNA / DNA gapmer with a base sequence complementary to positions 2682 to 2697 of its transcription product, DMPK mRNA (GenBank accession number NM_032418, sequence number 46). More specifically, this LNA / DNA gapmer is composed of three LNA nucleosides at each of the 5' and 3' ends, with ten DNA nucleosides between them (Figure 15).

[0164] The Chol-cRNA (Default) has a sequence complementary to the ASO (mDMPK), contains three 2'-O-methyl-modified ribonucleosides at both ends and ten unmodified sugar-modified ribonucleosides between them, and is bound to cholesterol at its 5' end (Figure 15A).

[0165] The Chol-cRNA (CU OMe) has 2'-O-methyl modifications at all C and U positions, except for the U position located at the 3' end, among the 10 sugar-unmodified ribonucleosides located in the center of the Chol-cRNA (Default) (Figure 15B).

[0166] As in Example 1, ASO(mDMPK) and Chol-cRNA(Default) or ASO(mDMPK) and Chol-cRNA(CU OMe) were dissolved in PBS and mixed in equimolar amounts. The solution was heated to 98°C for 5 minutes, then cooled to 37°C and maintained for 1 hour, thereby annealing the nucleic acid strands to prepare a double-stranded nucleic acid complex. The prepared double-stranded nucleic acid complex is referred to as "Chol-HDO(Default)" or "Chol-HDO(CU OMe)."

[0167] (in vivo experiment) The mice to be administered the double-stranded nucleic acid complex were male C57BL / 6 mice weighing 20 g, 4 to 5 weeks old. Four mice were used for each condition. The double-stranded nucleic acid complex was intravenously injected into each mouse via the tail vein at a dose of 12.5 mg / kg, and a negative control group of mice was also injected with PBS alone.

[0168] (Expression analysis) At 72 hours after administration, mice were perfused with PBS and then dissected to remove the heart, quadriceps, back, tibialis anterior (TA), gastrocnemius (GC), triceps brachii (TB), kidneys, and liver. mRNA was extracted from each tissue using a high-throughput automated nucleic acid extraction system, the MagNA Pure 96 (Roche Life Sciences). cDNA was synthesized according to the Transcriptor Universal cDNA Master (Roche Life Sciences) protocol. Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific, based on various gene numbers. The PCR conditions (temperature and time) were 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second, repeated 40 times. The resulting amplified products were quantified by quantitative RT-PCR, and the expression level of mRNA (DMPK) was calculated based on the results, divided by the expression level of mRNA (ACTB; internal control gene) to obtain relative expression levels. The mean and standard error of the relative expression levels were calculated.

[0169] (result) The results are shown in Figures 16 and 17. Figure 16 shows the inhibitory effect of the double-stranded nucleic acid complexes Chol-HDO(Default) and Chol-HDO(CU OMe) on the expression of the target mDMPK gene in the gastrocnemius (GC), triceps brachii (TB), tibialis anterior (TA), back, quadriceps femoris, and heart muscle. Figure 17 shows the inhibitory effect of the double-stranded nucleic acid complexes Chol-HDO(Default) and Chol-HDO(CU OMe) on the expression of the target mDMPK gene in the kidney and liver. Error bars indicate the standard error of each value.

[0170] Chol-HDO(CU OMe) showed stronger silencing effects than Chol-HDO(Default) in the gastrocnemius, triceps brachii, tibialis anterior, dorsi proper, quadriceps femoris, and cardiac muscle. These results suggest that the addition of sugar modifications to the ribonucleoside at the C / U position of the second nucleic acid strand confers resistance to RNase A cleavage, enabling more efficient silencing of target genes.

[0171] Furthermore, Chol-HDO(CU OMe) showed the same inhibitory effect on expression as Chol-HDO(Default) in the kidney and liver.

[0172] Example 5: In vivo inhibitory effect of a double-stranded nucleic acid complex containing a second nucleic acid strand having a sugar-modified ribonucleoside at the C / U position on human SOD1 mRNA expression We will verify the in vivo inhibitory effect of mRNA expression in tissues by a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide targeting the human SOD1 gene and a complementary strand (second nucleic acid strand) containing a cholesterol-bound sugar-modified ribonucleoside at the C / U position.

[0173] (Preparation of nucleic acid agents) We prepared an antisense oligonucleotide ASO (ASO1) targeting the SOD1 gene, a cholesterol-conjugated complementary strand Chol-cRNA (CU OMe) with 2'-O-methyl modifications at the C / U positions, and a control complementary strand Chol-cRNA (Default). The sequences of ASO (mDMPK), Chol-cRNA (Default), and Chol-cRNA (CU OMe) are shown below.

[0174] [Table 5]

[0175] The ASO (hSOD1) targets the human SOD1 gene and is composed of a 17-mer single-stranded LNA / DNA gapmer (SEQ ID NO: 67) with a base sequence complementary to positions 679 to 695 of its transcription product, hSOD1 mRNA (GenBank accession number NM_000454.4). More specifically, this LNA / DNA gapmer is composed of three LNA nucleosides at each of the 5' and 3' ends, with ten DNA nucleosides between them (Figure 18).

[0176] The Chol-cRNA (Default) has a sequence complementary to the ASO (hSOD1), contains three 2'-O-methyl-modified ribonucleosides at both ends and ten sugar-unmodified ribonucleosides between them, and is bound to cholesterol at its 5' end (Figure 18A).

[0177] The Chol-cRNA (CU OMe) has 2'-O-methyl modifications at all C and U positions among the 10 sugar-unmodified ribonucleosides located in the center of the Chol-cRNA (Default) (Figure 18B).

[0178] As in Example 1, ASO(hSOD1) and Chol-cRNA(Default), or ASO(hSOD1) and Chol-cRNA(CU OMe) were dissolved in PBS and mixed in equimolar amounts. The solution was heated to 98°C for 5 minutes, then cooled to 37°C and maintained for 1 hour, thereby annealing the nucleic acid strands to prepare a double-stranded nucleic acid complex. The prepared double-stranded nucleic acid complex is referred to as "Chol-HDO(Default)" or "Chol-HDO(CU OMe)."

[0179] (in vivo experiment) The mice administered with the double-stranded nucleic acid complex were male human SOD1 G93A transgenic mice (G93A Tg mice), 4 to 5 weeks old and weighing 20 g. Two to four mice were used for each condition. The double-stranded nucleic acid complex was subcutaneously injected into mice at a dose of 50 mg / kg in a single dose, and a negative control group of mice was also injected with PBS alone in a single dose.

[0180] (Expression analysis) At 72 hours after administration, mice were perfused with PBS and then dissected to remove the heart, quadriceps, diaphragm, and dorsal muscles. Subsequently, mRNA was extracted from each tissue using a high-throughput, fully automated nucleic acid extraction system, the MagNA Pure 96 (Roche Life Sciences), according to the protocol. cDNA was synthesized according to the Transcriptor Universal cDNA Master (Roche Life Sciences) protocol. Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific, based on various gene numbers. PCR conditions (temperature and time) consisted of 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second. The resulting amplified products were quantified by quantitative RT-PCR, and the relative expression levels were calculated by dividing the expression level of SOD1 mRNA by the expression level of ACTB mRNA (an internal control gene). The mean and standard error of the relative expression levels were calculated.

[0181] (result) The results are shown in Figure 19. Figure 19 shows the inhibitory effect of the double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-HDO (CU OMe) on the expression of the target hSOD1 gene in the back muscles, quadriceps, diaphragm, and heart. The error bars indicate the standard error of each value.

[0182] Chol-HDO(CU OMe) showed a stronger inhibitory effect than Chol-HDO(Default) in all of the dorsal proper, quadriceps, diaphragm, and cardiac muscle. These results suggest that the addition of sugar modifications to the ribonucleoside at the C / U position of the second nucleic acid strand confers resistance to RNase A cleavage, enabling more efficient target gene silencing.

[0183] Example 6: In vivo inhibitory effect of mouse Malat1 RNA expression by a double-stranded nucleic acid complex containing a second nucleic acid strand having a deoxyribonucleoside at the C / U position We will verify the in vivo inhibitory effect of mRNA expression in tissues by a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide targeting the mouse Malat1 gene and a complementary strand (second nucleic acid strand) containing a cholesterol-bound deoxyribonucleoside at the C / U position.

[0184] (Preparation of nucleic acid agents) We prepared an antisense oligonucleotide ASO (mMalat1) targeting the mouse Malat1 gene, a cholesterol-conjugated complementary strand Chol-cRNA (CT DNA) with DNA at the C / U position, and a control complementary strand Chol-cRNA (Default). The sequences of ASO (mMalat1), Chol-cRNA (Default), and Chol-cRNA (CT DNA) are shown below.

[0185] [Table 6]

[0186] The ASO (mMalat1) targets the mouse Malat1 gene and is composed of a 16-mer single-stranded LNA / DNA gapmer (SEQ ID NO: 49) with a base sequence complementary to positions 5032 to 5047 of its transcription product, Malat1 ncRNA (GenBank accession number NR_002847.3). More specifically, this LNA / DNA gapmer is composed of three LNA nucleosides at each of the 5' and 3' ends, with ten DNA nucleosides between them (Figure 20).

[0187] The Chol-cRNA (Default) has a sequence complementary to the ASO (mMalat1), contains three 2'-O-methyl-modified ribonucleosides at both ends and ten unmodified ribonucleosides between them, and is cholesterol-linked at its 5' end (Figure 20A).

[0188] The Chol-cRNA (CT DNA) has deoxyribonucleosides at all C and U positions among the 10 sugar-unmodified ribonucleosides located in the center of the Chol-cRNA (Default) (Figure 20B).

[0189] As in Example 1, ASO (mMalat1) and Chol-cRNA (Default), or ASO (mMalat1) and Chol-cRNA (CT DNA), were dissolved in PBS and mixed in equimolar amounts. The solution was heated to 98°C for 5 minutes, then cooled to 37°C and maintained for 1 hour, thereby annealing the nucleic acid strands to prepare a double-stranded nucleic acid complex. The prepared double-stranded nucleic acid complex is referred to as "Chol-HDO (Default)" or "Chol-HDO (CT DNA)."

[0190] (in vivo experiment) The mice administered with the double-stranded nucleic acid complex were male C57BL / 6 mice weighing 20 g and aged 4 to 5 weeks. Three mice were used for each condition. The double-stranded nucleic acid complex was intravenously injected into each mouse via the tail vein at a dose of 40 mg / kg. A negative control group of mice was also prepared, receiving a single injection of PBS alone.

[0191] (Expression analysis) At 72 hours after administration, mice were perfused with PBS and then dissected to remove the cervical spinal cord. Subsequently, mRNA was extracted from each tissue using a high-throughput, fully automated nucleic acid extraction system, the MagNA Pure 96 (Roche Life Sciences), according to the protocol. cDNA was synthesized according to the Transcriptor Universal cDNA Master (Roche Life Sciences) protocol. Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific, based on various gene numbers. PCR conditions (temperature and time) consisted of 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second. The resulting amplified products were quantified by quantitative RT-PCR, and the relative expression levels were calculated by dividing the expression level of mMalat1 RNA by the expression level of ACTB mRNA (an internal control gene). The mean and standard error of the relative expression levels were then calculated.

[0192] (result) The results are shown in Figure 21. Figure 21 shows the inhibitory effect of the double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-HDO (CT DNA) on the expression of the target mMalat1 gene in the cervical spinal cord. The error bars indicate the standard error of each.

[0193] Chol-HDO(CT DNA) also showed a stronger silencing effect than Chol-HDO(Default) in the cervical spinal cord. This result suggests that the substitution of deoxyribonucleosides for ribonucleosides at the C / U positions of the second nucleic acid strand confers resistance to RNase A cleavage, enabling more efficient silencing of target genes.

[0194] Example 7: Stability in mouse and human serum of a double-stranded nucleic acid complex containing a second nucleic acid strand containing a modified ribonucleoside A double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing a modified ribonucleoside was mixed with mouse or human serum, and the stability of the double strand in serum was evaluated.

[0195] (Preparation of nucleic acid agents) The same nucleic acid as in Example 1 was prepared and prepared. The sequences, chemical modifications, and structures of the oligonucleotides used in Example 1 are shown in Table 7 and Figures 22A and 22B. All oligonucleotides were manufactured by Gene Design Co., Ltd. (Osaka, Japan). [Table 7]

[0196] (Serum stability evaluation experiment) Ten μL of 10 μM double-stranded nucleic acid was prepared as described above. Serum was prepared from blood collected from Crl:CD1 (ICR) mice and human blood. 10 μL of 10 μM nucleic acid was mixed with 30 μL of mouse or human serum and incubated in a 37°C incubator. After incubation for 0, 2, or 24 hours, Reaction Stop Buffer (50 mM Tris-HCl, pH 7.5, 48.5 mM Borate, 20 mM EDTA, 10% SDS, final volume 60 μL) was added and the mixture was immersed in liquid nitrogen to terminate the reaction.

[0197] (extraction) The thawed sample was mixed with 60 μL of phenol:chloroform:isoamylalcohol (25:24:1) (Nacalai Tesque), vortexed, and incubated in a 37°C incubator for 20 minutes. It was then centrifuged at 35°C and 12,000 xg for 30 minutes. The supernatant was mixed again with 60 μL of phenol:chloroform:isoamylalcohol (25:24:1), and the same procedure was repeated three times. The final supernatant was collected and mixed with 7.5% sucrose solution to prepare the electrophoresis sample.

[0198] (Electrophoresis and Analysis) A 20% acrylamide gel (1x TBE) was prepared and prepared. The above electrophoresis samples were run on the gel at 100V for 80 minutes. ASO alone was run simultaneously as a control. Next, a solution was prepared by diluting GelRed (x10,000) aqueous solution (Biotium) to a concentration of 1 / 10,000 in 1x TBE. The gel was then permeated with this solution for 10 minutes. The gel was then photographed using a ChemiDocTouch imaging system (BioRad). Band density was analyzed using Image J software.

[0199] (result) Figure 23 shows the electrophoresis results of ASO (mDmpk) alone and a double-stranded complex of ASO (mDmpk) and cRNA (default, CU OMe, AG OMe, CU PS) after incubation with mouse serum. Figure 25 shows the electrophoresis results of ASO (mDmpk) alone and a double-stranded complex of ASO (mDmpk) and cRNA (default, CU OMe, CT DNA) after incubation with human serum. Figures 24 and 26 show the relative intensity (%) of the double-stranded band after 24 hours of incubation compared to the double-stranded band after 0 hours of incubation. As shown in Figures 23 and 24, nucleic acid strands in which C and U are 2'-O-methyl-modified ribonucleosides showed improved stability in mouse serum. In addition, changing the phosphodiester bond (PO) at the 3' end of ribonucleosides C and U in the second nucleic acid strand to a phosphorothioate bond (PS) also improved stability in mouse serum. Furthermore, as shown in Figures 25 and 26, when C and U or T in the second nucleic acid strand are 2'-O-methyl-modified ribonucleosides or DNA, stability in human serum was improved.

[0200] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A composition for inhibiting RNase A cleavage activity and / or RNase H-dependent and / or RNase H-independent pathways, comprising a nucleic acid complex including a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid strand (1) capable of hybridizing to at least a portion of the target transcript; (2) have an antisense effect on the target transcript; and (3) A gapmer comprising a central region containing at least four consecutive deoxyribonucleosides, and 5' and 3' wing regions containing unnatural nucleosides, respectively, on the 5' and 3' ends of the central region, the second nucleic acid strand comprises at least one first exposed region and at least one protected region; the first exposed region is complementary to a portion of the first nucleic acid strand and consists of one or two to three consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds; The protective region is composed of one or more (a) deoxyribonucleosides, (b) sugar-modified nucleosides, and / or (c) nucleosides having a modified internucleoside bond at the 3' side, which are linked by an internucleoside bond; and The composition wherein the first nucleic acid strand is annealed to the second nucleic acid strand.

2. 2. The composition of claim 1, wherein the first nucleic acid strand is 10 to 26 bases in length.

3. The composition of claim 1 or 2, wherein the first exposed region consists of three consecutive sugar-unmodified ribonucleosides linked by internucleoside bonds.

4. The composition of any one of claims 1 to 3, wherein the first exposed region comprises a nucleoside comprising a modified or unmodified purine base.

5. The composition of any one of claims 1 to 4, wherein the second nucleic acid strand comprises only one first exposed region.

6. The composition of any one of claims 1 to 4, wherein the second nucleic acid strand comprises at least two first exposed regions.

7. The composition according to any one of claims 1 to 6, wherein the sugar-unmodified ribonucleoside in the first exposed region is a natural ribonucleoside.

8. The composition according to any one of claims 1 to 7, further comprising a second exposed region consisting of four or more consecutive unsugar-modified ribonucleosides linked by internucleoside bonds that are complementary to a portion of the first nucleic acid strand, wherein the unsugar-modified ribonucleosides in the second exposed region comprise a modified or unmodified purine base.

9. 9. The composition of claim 1, wherein at least one of the protective regions comprises (a) deoxyribonucleosides containing modified or unmodified pyrimidine bases, (b) sugar-modified nucleosides, and / or (c) nucleosides having a modified internucleoside linkage at the 3' end.

10. 10. The composition of claim 9, wherein all of the protective regions comprise (a) deoxyribonucleosides containing modified or unmodified pyrimidine bases, (b) sugar-modified nucleosides, and / or (c) nucleosides having modified internucleoside linkages at the 3' side.

11. 11. The composition of claim 1, wherein at least one of the protective regions is comprised of (a) a deoxyribonucleoside containing one or two or more modified or unmodified pyrimidine bases linked by an internucleoside linkage, (b) a sugar-modified nucleoside, and / or (c) a nucleoside having a modified internucleoside linkage at its 3' end.

12. 12. The composition of any one of claims 1 to 11, wherein the sugar-modified nucleosides in the protective region are 2'-O-methyl-modified nucleosides and / or the nucleosides having a modified internucleoside linkage at the 3' side are nucleosides having a phosphorothioate linkage at the 3' side.

13. The composition of any one of claims 1 to 12, wherein the first exposed region and / or the second exposed region comprises at least one modified internucleoside linkage.

14. The composition according to any one of claims 1 to 13, wherein the second nucleic acid strand does not include a sugar-unmodified region other than the first exposed region and the second exposed region.

15. The first exposed region and / or the second exposed region of the second nucleic acid strand comprises at least one phosphorothioate bond chirally controlled in the Rp or Sp configuration, 1 to 4 C １～６ The composition according to any one of claims 1 to 14, comprising an internucleoside linkage comprising a guanidine moiety substituted with an alkyl group of the formula:

16. The defense region of the second nucleic acid strand comprises at least one phosphorothioate bond chirally controlled in the Rp or Sp configuration, 1 to 4 C １～６ The composition according to any one of claims 1 to 15, comprising an internucleoside linkage comprising a guanidine moiety substituted with an alkyl group of the formula:

17. The composition of claim 15 or 16, wherein the second nucleic acid strand does not include a sugar-unmodified region other than the first exposed region and the second exposed region.

18. The composition of any one of claims 1 to 17, wherein the second nucleic acid strand is linked to a functional moiety having a function selected from a labeling function, a purification function, and a target delivery function.

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