Heteronucleic acid containing scpbna or amna

Abstract

Provided is a double-stranded nucleic acid complex that exhibits a reduced toxicity without a loss of effectiveness.　Provided is a double-stranded nucleic acid complex, comprising a first nucleic acid strand and a second nucleic acid strand, wherein: the first nucleic acid strand can hybridize to at least a part of a target gene or a transcript thereof and has an antisense effect on the target gene or transcript thereof; the second nucleic acid strand contains a base sequence complementary to that of the first nucleic acid strand; and the first nucleic acid strand and / or the second nucleic acid strand contains at least one crosslinked unnatural nucleoside given by formula (I) or (II) (In the formulas, R represents a hydrogen atom or methyl group.).

Description

Heterogeneous nucleic acids containing scpBNA or AmNA

[0001] The present invention relates to a double-stranded nucleic acid complex containing scpBNA or AmNA, and a pharmaceutical composition containing the same.

[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 pursued due to their high selectivity for target genes and low toxicity. The antisense method involves selectively modifying or inhibiting the expression of proteins encoded by target genes or the activity of miRNAs by introducing complementary oligonucleotides (antisense oligonucleotides; often referred to herein as "ASOs (Antisense Oligonucleotides)") into cells, using a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand.

[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 is annealed to its complementary strand (Patent Document 1, Non-Patent Documents 1 and 2).

[0004] Double-stranded nucleic acid complexes have a strong antisense effect and are a groundbreaking technology that enables them to transcend the blood-brain barrier and control the central nervous system. However, high doses are required to treat neurological diseases such as Alzheimer's disease, and side effects such as severe liver damage can be a problem.

[0005] Therefore, there is a need for techniques that reduce the toxicity of double-stranded nucleic acid complexes without compromising their efficacy.

[0006] International Publication No. 2013 / 089283 International Publication No. 2014 / 192310

[0007] Nishina K, et. al., "DNA / RNA heteroduplex oligonucleotide for highly gene silencing", Nature Communication, 2015, 6:7969. Asami Y, et al., "Drug efficient delivery system of therapeutic oligonucleotides", Drug Discoveries & Therapeutics. 2016; 10(5):256-262.

[0008] The object is to provide a double-stranded nucleic acid complex with reduced toxicity without compromising efficacy.

[0009] The present inventors conducted extensive research to solve the above-mentioned problems and introduced 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid (scpBNA) or amide-bridged nucleic acid (AmNA) into double-stranded nucleic acid complexes. As a result, they found that the introduction of scpBNA or AmNA can dramatically reduce or eliminate the toxicity of double-stranded nucleic acid complexes. This toxicity-suppressing effect is a surprising effect that far exceeds previous expectations. Furthermore, it was found that the introduction of scpBNA or AmNA does not impair the efficacy of double-stranded nucleic acid complexes. The present invention is based on the above findings and provides the following:

[0010] (1) A double-stranded nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand can hybridize to at least a part of a target gene or a transcription product thereof and has an antisense effect on the target gene or the transcription product thereof, the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, and the first nucleic acid strand and / or the second nucleic acid strand is represented by the following formula (I) or formula (II): (wherein R represents a hydrogen atom or a methyl group). (2) The double-stranded nucleic acid complex according to (1), wherein the first nucleic acid strand is a gapmer. (3) The double-stranded nucleic acid complex according to (2), wherein the first nucleic acid strand comprises: [1] a central region comprising at least four consecutive deoxyribonucleosides, [2] a 5'-wing region disposed on the 5'-terminal side of the central region and comprising an unnatural nucleoside, and [3] a 3'-wing region disposed on the 3'-terminal side of the central region and comprising an unnatural nucleoside. (4) The double-stranded nucleic acid complex according to (3), wherein the 5'-wing region and / or the 3'-wing region comprises a bridged unnatural nucleoside represented by formula (I) or formula (II). (5) The double-stranded nucleic acid complex according to (3) or (4), wherein the second nucleic acid strand comprises at least four consecutive ribonucleosides complementary to at least four consecutive deoxyribonucleosides in the central region of the first nucleic acid strand. (6) The double-stranded nucleic acid complex according to (5), wherein the second nucleic acid strand further comprises at least two consecutive deoxyribonucleosides. (7) The double-stranded nucleic acid complex according to any one of (3) to (6), wherein the second nucleic acid strand comprises a bridged unnatural nucleoside represented by formula (I) or formula (II) in a region consisting of a base sequence complementary to the 5' wing region and / or 3' wing region of the first nucleic acid strand. (8) The double-stranded nucleic acid complex according to (1), wherein the first nucleic acid strand is a mixmer. (9) The double-stranded nucleic acid complex according to any one of (1) to (8), wherein the first nucleic acid strand and / or the second nucleic acid strand comprises at least one ribose 2'-position-modified nucleoside selected from the group consisting of 2'-O-methyl-modified nucleosides, 2'-O-methoxyethyl-modified nucleosides, and 2'-O-[2-(N-methylcarbamoyl)ethyl]-modified nucleosides. (10) The double-stranded nucleic acid complex according to any one of (1) to (9), wherein all or part of the internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand are modified internucleoside linkages. (11) The double-stranded nucleic acid complex according to (10), wherein the modified internucleoside linkages are phosphorothioate linkages.(12) The double-stranded nucleic acid complex according to any one of (1) to (11), wherein the second nucleic acid strand is bound to tocopherol, cholesterol, or an analog thereof. (13) The double-stranded nucleic acid complex according to any one of (1) to (11), which is not bound to a ligand. (14) The double-stranded nucleic acid complex according to any one of (1) to (13), wherein the first nucleic acid strand and the second nucleic acid strand are bound via a cleavable or non-cleavable linker. (15) A pharmaceutical composition comprising as an active ingredient the double-stranded nucleic acid complex according to any one of (1) to (14). (16) The pharmaceutical composition according to (15), for treating a central nervous system disease in a subject. (17) The pharmaceutical composition according to (15) or (16), which is administered intracerebroventricularly or intrathecally. (18) The pharmaceutical composition according to (17), wherein 0.1 mg to 200 mg of the double-stranded nucleic acid complex is administered. (19) The pharmaceutical composition according to (15) or (16), which is administered intravenously or subcutaneously. (20) The pharmaceutical composition according to (19), wherein the double-stranded nucleic acid complex is administered at a dose of 0.1 mg / kg to 100 mg / kg. (21) The pharmaceutical composition according to any one of (15) to (20), which reduces the induction of inflammation or gliosis, or the abnormal increase in cytokines or chemokines. This specification includes the disclosures of Japanese Patent Application No. 2021-087941, from which the present application claims priority.

[0011] According to the present invention, double-stranded nucleic acid complexes are provided that have reduced toxicity without compromising efficacy.

[0012] FIG. 1 shows the structures of various bridged nucleic acids. FIG. 2 shows the structures of various natural and non-natural nucleotides. FIG. 3 is a schematic diagram of the structures of the nucleic acids used in Example 1. FIG. 3A shows the structure of an ASO (LNA) containing three LNA nucleosides at the 5' and 3' ends and ten DNA nucleosides between them. FIG. 3B shows the structure of an HDO (LNA) containing an ASO (LNA) as the first nucleic acid strand and RNA having a sequence complementary to the first nucleic acid strand as the second nucleic acid strand. FIG. 3C shows the structure of an ASO (scpBNA) containing three scpBNA nucleosides at each of the 5' and 3' ends and ten DNA nucleosides between them. FIG. 3D shows the structure of an HDO (scpBNA) containing an ASO (scpBNA) as the first nucleic acid strand and RNA having a sequence complementary to the first nucleic acid strand as the second nucleic acid strand. Figure 4 shows the expression levels of Mapt mRNA in the hippocampus of mice administered various nucleic acid agents intracerebroventricularly. Error bars indicate standard error. Figure 5 shows the expression levels of TNFα mRNA and GFAP mRNA in the hippocampus of mice administered various nucleic acid agents intracerebroventricularly. Figure 5A shows the expression level of TNFα mRNA. Figure 5B shows the expression level of GFAP mRNA. Error bars indicate standard error. Figure 6 is a schematic diagram of the structure of the nucleic acids used in Example 2. Figure 6A shows the structure of Toc-HDO(LNA). Figure 6B shows the structure of Toc-HDO(scpBNA). Figure 6C shows the structure of Toc-HDO(AmNA). Toc stands for tocopherol. Figure 7 shows the expression levels of malat1 mRNA in mice administered a single intravenous dose of a double-stranded nucleic acid complex agent. Figure 7A shows the expression level of malat1 mRNA in the liver. Figure 7B shows the expression level of malat1 mRNA in the kidney. Error bars indicate standard error. Figure 8 shows the expression level of malat1 mRNA in mice that received a single intravenous administration of a double-stranded nucleic acid complex agent. Figure 8A shows the expression level of malat1 mRNA in the quadriceps muscle. Figure 8B shows the expression level of malat1 mRNA in the cardiac muscle. Error bars indicate standard error. Figure 9 shows the results of serum analysis and body weight measurement in mice that received a single intravenous administration of a double-stranded nucleic acid complex agent targeting malat1. Figure 9A shows the results of measuring serum AST and ALT. Figure 9B shows the results of body weight measurement.Error bars indicate standard error. Figure 10 is a schematic diagram of the structures of the nucleic acids used in Examples 3 and 4. Figure 10A shows the structure of Toc-HDO(LNA). Figure 10B shows the structure of Toc-HDO(scpBNA). Figure 10C shows the structure of Toc-HDO(AmNA). Toc stands for tocopherol. Figure 11 shows the expression levels of PTEN mRNA in mice administered a single intravenous dose of a double-stranded nucleic acid complex agent. Figure 11A shows the expression levels of PTEN mRNA in the liver. Figure 11B shows the expression levels of PTEN mRNA in the kidney. Error bars indicate standard error. Figure 12 shows the expression levels of PTEN mRNA in mice administered a single intravenous dose of a double-stranded nucleic acid complex agent. Figure 12A shows the expression levels of PTEN mRNA in the quadriceps muscle. Figure 12B shows the expression levels of PTEN mRNA in the cardiac muscle. Error bars indicate standard error. Figure 13 shows the results of serum analysis and body weight measurement in mice that received a single intravenous administration of a double-stranded nucleic acid complex agent targeting PTEN. Figure 13A shows the results of measuring serum AST and ALT. Figure 13B shows the change in body weight before and after administration of the double-stranded nucleic acid complex agent. Error bars indicate standard error. Figure 14 shows the expression level of SR-B1 mRNA in mice that received a single intravenous administration of the double-stranded nucleic acid complex agent. Figure 14A shows the expression level of SR-B1 mRNA in the liver. Figure 14B shows the expression level of SR-B1 mRNA in the kidney. Error bars indicate standard error. Figure 15 shows the expression level of SR-B1 mRNA in mice that received a single intravenous administration of the double-stranded nucleic acid complex agent. Figure 15A shows the expression level of SR-B1 mRNA in the quadriceps muscle. Figure 15B shows the expression level of SR-B1 mRNA in the cardiac muscle. Error bars indicate standard error. Figure 16 shows the results of serum analysis and body weight measurement in mice that received a single intravenous administration of a double-stranded nucleic acid complex agent targeting SR-B1. Figure 16A shows the results of measuring serum AST and ALT. Figure 16B shows the change in body weight before and after administration of the double-stranded nucleic acid complex agent. Error bars indicate standard error. Figure 17 is a schematic diagram of the structure of the nucleic acids used in Example 5. Figure 17A shows the structure of Chol-HDO(LNA). Figure 17B shows the structure of Chol-HDO(scpBNA). Chol represents cholesterol.Figure 18 shows the expression levels of SR-B1 mRNA in mice that received multiple intravenous administrations of double-stranded nucleic acid complexes. Figure 18A shows the expression levels of SR-B1 mRNA in the cortex, cerebellum, striatum, hippocampus, brainstem, cervical spinal cord, and lumbar spinal cord. Figure 18B shows the expression levels of SR-B1 mRNA in the liver, kidney, spleen, heart, quadriceps, back, diaphragm, adrenal gland, lung, colon, small intestine, and adipose tissue. Error bars indicate standard error. Figure 19 shows the results of measuring AST and ALT in the serum of mice that received multiple intravenous administrations of double-stranded nucleic acid complexes. For Chol-HDO (LNA), the administered mice died after a single administration, so the AST / ALT measurement results after a single administration are shown. Figure 20 shows the results of measuring TNFα in the blood of mice that received a single intravenous administration of the double-stranded nucleic acid complex agent. Figure 20A shows the results for Toc-HDO (PTEN) and Toc-HDO (Malat1). Figure 20B shows the results for Toc-HDO (SR-B1). Figure 21 shows the results of measuring IP-10 in the blood of mice that received a single intravenous administration of the double-stranded nucleic acid complex agent. Figure 21A shows the results for Toc-HDO (PTEN) and Toc-HDO (Malat1). Figure 21B shows the results for Toc-HDO (SR-B1). Figure 22 shows the results of measuring RANTES in the blood of mice that received a single intravenous administration of the double-stranded nucleic acid complex agent. Figure 22A shows the results for Toc-HDO (PTEN) and Toc-HDO (Malat1). FIG. 22B shows the results for Toc-HDO (SR-B1).

[0013] 1. Double-Stranded Nucleic Acid Complex 1-1. Overview A first aspect of the present invention is a double-stranded nucleic acid complex. The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand, and the first nucleic acid strand and / or the second nucleic acid strand comprises at least one scpBNA or AmNA. The double-stranded nucleic acid complex of the present invention has reduced toxicity, such as hepatotoxicity, and reduced induction of inflammation or gliosis, or abnormal increases in cytokines or chemokines.

[0014] 1-2. Definition of Terms As used herein, the term "transcription product" of a target gene refers to any RNA that is a direct target of the nucleic acid complex of the present invention and is synthesized by RNA polymerase. Specifically, it may include mRNA transcribed from the target gene (including mature mRNA, pre-mRNA, and mRNA without base modifications), non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA.

[0015] The "target gene" whose expression is regulated (e.g., suppressed, altered, or modified) by the antisense effect is not particularly limited, and examples thereof include genes whose expression is increased in various diseases. Examples of transcription products of the target gene include SR-B1 mRNA, which is a transcription product of the SR-B1 gene, Malat1 non-coding RNA, which is a transcription product of the Malat1 gene, and DMPK mRNA, which is a transcription product of the DMPK gene. Examples of "target transcripts" include genes 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"), and metastasis associated lung adenocarcinoma transcript 1 (often referred to herein as "Malat1"), including their non-coding RNAs or mRNAs. The nucleotide sequence of mouse Malat1 non-coding RNA is shown in SEQ ID NO: 19, and the nucleotide sequence of human Malat1 non-coding RNA is shown in SEQ ID NO: 20. The nucleotide sequence of mouse SR-B1 mRNA is shown in SEQ ID NO: 21, and the nucleotide sequence of human SR-B1 mRNA is shown in SEQ ID NO: 22. The nucleotide sequence of mouse DMPK mRNA is shown in SEQ ID NO: 23, and the nucleotide sequence of human DMPK mRNA is shown in SEQ ID NO: 24. Note that in all of SEQ ID NOs: 19 to 24, the mRNA nucleotide sequence is replaced with the DNA nucleotide sequence. Information on the nucleotide sequences of these genes and transcripts can be obtained from publicly known databases, such as the NCBI (National Center for Biotechnology Information) databases (e.g., GenBank, Trace Archive, Sequence Read Archive, BioSample, BioProject).

[0016] As used herein, the terms "antisense oligonucleotide (ASO)" and "antisense nucleic acid" refer to a single-stranded oligonucleotide that contains 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 exerts an antisense effect on the target transcript. In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand functions as the ASO, and its target region may include the 3' UTR, 5' UTR, exon, intron, coding region, translation initiation region, translation termination region, or any other nucleic acid region. The target region of the target transcript may be at least 8 bases long, e.g., 10-35 bases, 12-25 bases, 13-20 bases, 14-19 bases, or 15-18 bases, or 13-22 bases, 16-22 bases, or 16-20 bases long.

[0017] The term "antisense effect" refers to the effect of ASOs hybridizing to a target transcript (e.g., the RNA sense strand) to modulate the expression or editing of that target transcript. "Modulating the expression or editing of a target transcript" refers to suppression or reduction of the expression of a target gene or the expression level of the target transcript (herein, "expression level of a target transcript" is often referred to as "target transcript level"), translation inhibition, RNA editing, splicing function modification effects (e.g., splicing switch, exon inclusion, exon skipping, etc.), or transcript degradation. For example, in post-transcriptional inhibition of a target gene, when an RNA oligonucleotide is introduced into a cell as an ASO, the ASO forms a partial duplex with the mRNA, the transcript of the target gene. This partial duplex acts as a cover to prevent translation by ribosomes, thereby inhibiting the expression of the target protein encoded by the target gene at the translational level (steric blocking). On the other hand, when an oligonucleotide containing DNA 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 and inhibition of the expression of the protein encoded by the target gene. Furthermore, the antisense effect can also be achieved by targeting an intron in a pre-mRNA. Furthermore, the antisense effect can also be achieved by targeting an miRNA. In this case, inhibition of the function of the miRNA can increase the expression of the gene whose expression the miRNA normally controls. In one embodiment, the modulation of the expression of the target transcript can be a reduction in the amount of the target transcript.

[0018] 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.

[0019] 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).

[0020] 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.

[0021] As used herein, the term "translation product of a target gene" refers to any polypeptide or protein synthesized by translation of the target transcript or transcript of a target gene that is the direct target of the nucleic acid complex of the present invention.

[0022] As used herein, the term "nucleic acid" or "nucleic acid molecule" may refer to a monomeric nucleotide or nucleoside, or may refer to an oligonucleotide made up of multiple monomers.

[0023] As used herein, the term "nucleic acid strand" or simply "strand" refers to an oligonucleotide or polynucleotide. A nucleic acid strand can be produced as a full-length strand or a partial strand by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction reactions. A nucleic acid strand can include natural and / or non-natural nucleotides.

[0024] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, specific examples of which include ribose and deoxyribose. The base portion (nucleobase) of a nucleoside is typically a heterocyclic base moiety. Examples include, but are not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).

[0025] A "nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of a nucleoside. In the case of nucleotides containing a pentofuranosyl sugar, the phosphate group is typically linked to the 2', 3', or 5' hydroxyl group of the sugar.

[0026] An "oligonucleotide" refers to a linear oligomer formed by covalently linking several to several dozen hydroxyl groups in the sugar moieties and phosphate groups of adjacent nucleotides. A "polynucleotide" refers to a linear polymer formed by linking several dozens, preferably several hundred, of nucleotides, more numerous than an oligonucleotide, by such covalent bonds. Within an oligonucleotide or polynucleotide structure, the phosphate groups are generally considered to form internucleoside bonds.

[0027] As used herein, "natural nucleosides" refer to nucleosides that exist in nature. Examples include ribonucleosides consisting of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides consisting of deoxyribose and a base such as adenine, cytosine, guanine, or thymine. Ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to herein as "DNA nucleosides" and "RNA nucleosides," respectively.

[0028] As used herein, the term "natural nucleotide" refers to a naturally occurring nucleotide molecule in which a phosphate group is covalently bound to the sugar moiety of the natural nucleoside. Examples include ribonucleotides, which are known as building blocks of RNA and in which a phosphate group is bound to a ribonucleoside, and deoxyribonucleotides, which are known as building blocks of DNA and in which a phosphate group is bound to a deoxyribonucleoside.

[0029] As used herein, the term "non-natural nucleotide" refers to any nucleotide other than a natural nucleotide, including modified nucleotides and nucleotide mimetics. As used herein, "modified nucleotide" refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase. The term "nucleotide mimetics" includes structures used to replace nucleosides and linkages at one or more positions in an oligomeric compound. Examples of nucleotide mimetics include peptide nucleic acids and morpholino nucleic acids (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Peptide nucleic acids (PNAs) are nucleotide mimetics with a backbone containing N-(2-aminoethyl)glycine linked by amide bonds in place of sugars. Nucleic acid chains containing non-natural oligonucleotides often possess desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Therefore, they are preferred over natural nucleotides.

[0030] As used herein, the term "unnatural nucleoside" refers to any nucleoside other than a naturally occurring nucleoside. For example, this includes modified nucleosides and nucleoside mimetics. As used herein, the term "modified nucleoside" refers to a nucleoside having a modified sugar moiety and / or a modified nucleobase.

[0031] As used herein, the term "mimetic" refers to functional groups that replace sugars, nucleobases, and / or internucleoside linkages. Generally, mimetics are used in place of sugars or sugar-internucleoside linkage combinations, while maintaining the nucleobases for hybridization to a selected target. As used herein, "nucleoside mimetics" includes structures used to replace sugars, or sugars and bases, at one or more positions in an oligomeric compound, or to replace linkages between monomeric subunits that comprise the oligomeric compound. An "oligomeric compound" refers to a polymer of linked monomeric subunits that is at least capable of hybridizing to a region of a nucleic acid molecule. Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimetics, e.g., nucleoside mimetics having non-furanose sugar units.

[0032] "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, a 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.

[0033] 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), 2'-O-[2-(N-methylcarbamoyl)ethyl] (2'-O-MCE), and 2'-O-methoxyethyl (2'-O-MOE or 2-O(CH)OCH) substituents. The 2'-position substituent may also be an 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."

[0034] "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 also sometimes referred to as "bridged nucleosides," "bridged non-natural nucleosides," or "BNA nucleosides." Some examples of bridged nucleic acids are shown in Figure 1.

[0035] 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 Examples of BNAs include amide BNAs (amide-bridged nucleic acids) or (4'-C(O)-N(R)-2')BNAs (R=H, Me) (also known as AmNAs; in Figure 1, R=H is AmNA[NH] and R=Me is AmNA[N-Me]), guanidine BNAs (also known as GuNAs (e.g., in Figure 1, R=H is GuNA[NH] and R=Me is GuNA[N-Me])), 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 include 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, or (4'-C(O)-N(R)-2') BNA nucleosides (R = H, Me) (also known as AmNA nucleosides; in Figure 1, R = H represents AmNA[NH] nucleosides, and R = Me represents AmNA[N-M e]nucleosides), guanidine BNA nucleosides (also known as GuNA nucleosides (e.g., in Figure 1, R = H is a GuNA[NH] nucleoside, and 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.

[0036] 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'-CHCHCHNH-substituted nucleoside), a GuNA nucleoside (e.g., in Figure 3, R = H represents a GuNA[NH] nucleoside, and R = Me represents a GuNA[N-Me] nucleoside), etc. Bicyclic nucleosides having a methyleneoxy (4'-CH-O-2') bridge are sometimes referred to as LNA nucleosides.

[0037] As used herein, a "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 (III): ), 1 to 4 C １～６ and internucleoside linkages (e.g., a moiety represented by the following formula (IV): Examples of modified internucleoside linkages include, but are not limited to, the internucleoside linkages and phosphoramidate linkages used in the self-neutralizing nucleic acids (ZONs) described in International Publication No. WO 2016 / 081600. Phosphorothioate linkages refer to internucleoside linkages 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.

[0038] When an internucleoside bond has a chiral center, the internucleoside bond may be chiral controlled. "Chiral controlled" refers to a bond existing as a single diastereomer with respect to the chiral center, e.g., chiral phosphorus. A chiral controlled internucleoside bond may be completely chiral 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) x 100 (%).

[0039] 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 (IV)), and / or an internucleoside linkage containing a cyclic guanidine moiety (e.g., a partial structure represented by formula (III)). Methods for preparing chiral internucleoside bonds are known. For example, chiral phosphorothioate bonds having an 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. １～６Methods for preparing internucleoside linkages containing a guanidine moiety (e.g., a TMG moiety) substituted with an alkyl group are known, and the 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.

[0040] As used herein, the term "nucleobase" or "base" refers to a base component (heterocyclic moiety) constituting a nucleic acid, and the most commonly known are adenine, guanine, cytosine, thymine, and uracil. Unless otherwise specified, "nucleobase" or "base" herein encompasses both modified and unmodified nucleic acid bases (bases). 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.

[0041] "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.

[0042] The term "complementary" as used herein refers to a 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 need to be completely complementary to at least a portion of the target transcript (e.g., 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% (e.g., 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 portion 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 as long as the base sequence is at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementary. 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 to that of the first nucleic acid strand. 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 antisense nucleic acids complementary to target transcription products, for example, based on information about the base sequence of the target gene.

[0043] Hybridization conditions may be of various stringent conditions, such as low stringency and high stringency. 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.

[0044] As used herein, the term "subject" refers to a target to which the double-stranded nucleic acid complex or pharmaceutical composition of the present invention is applied. Subjects include individuals as well as organs, tissues, and cells. When the subject is an individual, it can be any animal, including humans. Examples of non-human subjects include various livestock, poultry, pets, and laboratory animals. The subject may be, but is not limited to, an individual in need of reducing the expression level of a target transcript or an individual in need of disease treatment or prevention.

[0045] The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand. The specific structure of each nucleic acid strand is shown below.

[0046] In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand can hybridize to at least a part of a target gene or its transcription product and has an antisense effect on the target gene or its transcription product, the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, and the first nucleic acid strand and / or the second nucleic acid strand are represented by the following formula (I) or formula (II): (wherein R represents a hydrogen atom or a methyl group).

[0047] The bridged unnatural nucleoside represented by the above formula (I) is a 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid, and is mainly referred to as "scpBNA" in this specification.

[0048] The bridged unnatural nucleoside represented by the above formula (II) is an amide BNA (amide-bridged nucleic acid), which can also be represented as (4'-C(O)-N(R)-2')BNA (R = H, Me), but is primarily represented as "AmNA" in this specification. In the above formula (II), R may be either a hydrogen atom or a methyl group. Unless otherwise specified herein, R may be either a hydrogen atom or a methyl group, but when distinguishing between the two, it can be represented as AmNA[NH] when R is a hydrogen atom, and AmNA[N-Me] when R is a methyl group.

[0049] The number of bridged unnatural nucleosides represented by Formula (I) or (II) contained in the first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex is at least one, and may be, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, or may be 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. For example, the number of bridged unnatural nucleosides represented by Formula (I) or Formula (II) may be 1 to 10, preferably 1 to 6. For example, it may be 1, 2, 3, 4, 5, or 6.

[0050] The first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex of the present invention may contain only the bridged unnatural nucleoside represented by formula (I) or formula (II) above, or may contain only the bridged unnatural nucleoside represented by formula (I), or may contain both the bridged unnatural nucleoside represented by formula (I) and formula (II).

[0051] Furthermore, the bridged unnatural nucleosides represented by the above formulas (I) and (II) may be contained only in the first nucleic acid strand, only in the second nucleic acid strand, or in both the first and second nucleic acid strands.

[0052] The base lengths of the first and second nucleic acid strands are not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. The base lengths of the first and second nucleic acid strands 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 first and second nucleic acid strands may be the same length or different lengths (e.g., one of them may be 1 to 3 bases shorter or longer). The double-stranded structure formed by the first and second nucleic acid strands may include a bulge. The length can be determined by balancing the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield.

[0053] The first nucleic acid strand, when hybridized to a target transcript, can contain at least 4, at least 5, at least 6, or at least 7 consecutive nucleosides recognized by RNase H. Typically, the region can be any region containing 4 to 20, 5 to 16, or 6 to 12 consecutive nucleosides. Nucleosides recognized by RNase H can be, for example, natural deoxyribonucleosides. Modified deoxyribonucleosides and suitable nucleosides containing other bases are well known in the art. It is also known that nucleosides containing a hydroxy group at the 2' position, such as ribonucleosides, are unsuitable for use as such nucleosides. The suitability of nucleosides for use in this region containing "at least 4 consecutive nucleosides" can be easily determined. In one embodiment, the first nucleic acid strand can contain at least 4 consecutive deoxyribonucleosides.

[0054] In one embodiment, the nucleosides of the first nucleic acid strand comprise or consist of deoxyribonucleosides, for example, 70% or more, 80% or more, 90% or more, or 95% or more of the nucleosides of the first nucleic acid strand are deoxyribonucleosides.

[0055] In one embodiment, the first nucleic acid strand may be a gapmer. As used herein, the term "gapmer" generally refers to a single-stranded nucleic acid consisting of a central region (DNA gap region) and wing regions (referred to as the 5' wing region and the 3' wing region, respectively) located directly at the 5' end and the 3' end of the central region. 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. In a gapmer, the central region contains at least four consecutive deoxyribonucleosides, and the wing regions contain at least one unnatural nucleoside. Although not limited thereto, unnatural nucleosides contained in the wing regions typically have stronger RNA-binding affinity and higher resistance to nucleases (such as nucleases) than natural nucleosides. When the non-natural nucleosides comprising the wing regions comprise or consist of bridged nucleosides, the gapmer is specifically referred to as a "BNA / DNA gapmer." The number of bridged nucleosides in the 5' and 3' wing regions is at least one, and may be, for example, two or three. The bridged nucleosides in the 5' and 3' wing regions may be contiguous or non-contiguous within the 5' and 3' wing regions. The bridged nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine). When the bridged nucleoside is an LNA nucleoside, the gapmer is specifically referred to as an "LNA / DNA gapmer." When the non-natural nucleosides comprising the 5' and 3' wing regions comprise or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer." When the unnatural nucleosides that make up the 5' wing region and the 3' wing region contain or consist of morpholino nucleic acid, the gapmer is specifically referred to as a "morpholino nucleic acid gapmer." The base lengths of the 5' wing region and the 3' wing region may each independently be at least 2 bases long, for example, 2 to 10 bases long, 2 to 7 bases long, or 3 to 5 bases long.In one embodiment, the 5' wing region and / or the 3' wing region may comprise at least one unnatural nucleoside and may further comprise natural nucleosides. The 5' wing region and the 3' wing region may comprise unnatural nucleosides linked by modified internucleoside linkages, such as phosphorothioate linkages, e.g., 2'-O-methyl modified nucleosides.

[0056] The first nucleic acid strand constituting the gapmer may be composed of, in order from the 5' end, a bridged nucleoside of 2 to 7 bases or 3 to 5 bases (e.g., 2 or 3 bases), a ribonucleoside or deoxyribonucleoside of 4 to 15 bases or 8 to 12 bases (e.g., 8 or 10 bases), and a bridged nucleoside of 2 to 7 bases or 3 to 5 bases (e.g., 2 or 3 bases).

[0057] In addition, a nucleic acid strand having a wing region only on either the 5'-end or the 3'-end is called a "hemigapmer" in the art, and in this specification, hemigapmers are also included in the term "gapmer."

[0058] The 5' wing region and / or the 3' wing region of the first nucleic acid strand may comprise a bridged unnatural nucleoside represented by Formula (I) or Formula (II) above. The number of bridged unnatural nucleosides represented by Formula (I) or Formula (II) contained in each of the 5' wing region and the 3' wing region may be at least one, for example, two or more, or three or more, or may be five or fewer, four or fewer, three or fewer, or two or fewer. For example, it may be one, two, three, or four. In one embodiment, the 5' wing region and / or the 3' wing region of the first nucleic acid strand may be composed of a bridged unnatural nucleoside represented by Formula (I) or Formula (II) above.

[0059] In one embodiment, the 5' wing region and / or the 3' wing region of the first nucleic acid strand comprises bridged unnatural nucleosides as shown in formula (I) or (II) above and LNA nucleosides.

[0060] In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand may be a mixmer. As used herein, a "mixmer" refers to a nucleic acid strand containing alternating natural and unnatural nucleosides of periodic or random segment lengths, but not containing four or more consecutive deoxyribonucleosides and ribonucleosides. A mixmer in which the unnatural nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "BNA / DNA mixmer." The bridged nucleoside may be a bridged unnatural nucleoside represented by formula (I) or (II) above. A mixmer in which the unnatural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "peptide nucleic acid / DNA mixmer." In a mixmer, a mixmer in which the unnatural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "morpholino nucleic acid / DNA mixmer." A mixmer is not limited to containing only two types of nucleosides. A mixmer can contain any number of types of nucleosides, regardless of whether they are natural or modified nucleosides or nucleoside mimics. For example, a mixmer may have one or two consecutive deoxyribonucleosides separated by a bridged nucleoside (e.g., an LNA nucleoside or a bridged unnatural nucleoside represented by formula (I) or (II) above). The bridged nucleoside may further contain a modified nucleobase (e.g., 5-methylcytosine).

[0061] All nucleosides in the second nucleic acid strand may be composed of ribonucleosides and / or modified nucleosides. For example, all nucleosides in the second nucleic acid strand may be composed of ribonucleosides. All nucleosides in the second nucleic acid strand may be composed of deoxyribonucleosides and / or modified nucleosides, or may not contain ribonucleosides.

[0062] In one embodiment, the second nucleic acid strand may contain at least four consecutive ribonucleosides complementary to the at least four consecutive nucleosides (e.g., deoxyribonucleosides) in the central region of the first nucleic acid strand, such that the second nucleic acid strand forms a partial DNA-RNA heteroduplex with the first nucleic acid strand and is recognized and cleaved by RNase H. The at least four consecutive ribonucleosides in the second nucleic acid strand are preferably linked by naturally occurring internucleoside linkages, i.e., phosphodiester bonds.

[0063] In a further embodiment, the second nucleic acid strand may further comprise at least two consecutive deoxyribonucleosides in addition to the at least four consecutive ribonucleosides. The at least two consecutive deoxyribonucleosides may be complementary to the first nucleic acid strand and may be contained in a region complementary to the central region of the first nucleic acid strand. The at least two consecutive deoxyribonucleosides may be located on either the 5' or 3' side of the at least four consecutive ribonucleosides, or may be located on both the 5' and 3' sides. The at least two consecutive deoxyribonucleosides may also be 2, 3, 4, 5, or six or more consecutive deoxyribonucleosides.

[0064] At least one, at least two (e.g., two), at least three, or at least four nucleosides from the end (5'-end, 3'-end, or both ends) of the second nucleic acid strand may be modified nucleosides. The modified nucleosides may contain a modified sugar and / or a modified nucleobase. The modified sugar may be a 2'-modified sugar (e.g., a sugar containing a 2'-O-methyl group). The modified nucleobase may also be 5-methylcytosine.

[0065] The second nucleic acid strand may be composed of, from the 5'-terminus, 2 to 7 or 3 to 5 (e.g., 2- or 3-base) modified nucleosides (e.g., modified nucleosides containing a 2'-modified sugar), 4 to 15 or 8 to 12 (e.g., 8- or 10-base) ribonucleosides or deoxyribonucleosides (optionally linked via a modified internucleoside bond), and 2 to 7 or 3 to 5 (e.g., 2- or 3-base) modified nucleosides (e.g., modified nucleosides containing a 2'-modified sugar). In this case, the first nucleic acid strand may be a gapmer.

[0066] In one embodiment, the second nucleic acid strand may contain a bridged unnatural nucleoside represented by formula (I) or (II) above in a region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand.

[0067] The number of bridged nucleosides other than the bridged unnatural nucleosides represented by formula (I) or (II) above contained in the first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex is not limited, and may be, for example, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 15, 0 to 12, 0 to 10, 0 to 8, or 0 to 6, and preferably 0 to 5, for example, 0, 1, 2, 3, 4, or 5.

[0068] Furthermore, the number of unnatural nucleosides other than the bridged unnatural nucleosides represented by formula (I) or (II) contained in the first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex is not limited, and may be, for example, 0 to 30, 0 to 20, 0 to 15, 0 to 12, 0 to 10, 0 to 8, or 0 to 6, and preferably 0 to 5, for example, 0, 1, 2, 3, 4, or 5.

[0069] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex may contain at least one ribose-2'-modified nucleoside in addition to the bridged unnatural nucleoside represented by formula (I) or (II). The ribose-2'-modified nucleoside may be at least one ribose-2'-modified nucleoside selected from the group consisting of 2'-O-methyl-modified nucleosides (2'-O-Me-modified nucleosides), 2'-O-methoxyethyl-modified nucleosides (2'-O-MOE-modified nucleosides), and 2'-O-[2-(N-methylcarbamoyl)ethyl]-modified nucleosides (2'-O-MCE-modified nucleosides).

[0070] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex contains at least one bridged unnatural nucleoside represented by formula (I) or formula (II) above, and at least one 2'-O-methyl modified nucleoside (2'-O-Me-modified nucleoside).

[0071] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex contains at least one bridged unnatural nucleoside represented by the above formula (I) or formula (II) and at least one 2'-O-methoxyethyl modified nucleoside (2'-O-MOE modified nucleoside).

[0072] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand constituting the double-stranded nucleic acid complex contains at least one bridged unnatural nucleoside represented by the above formula (I) or formula (II), and contains at least one 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleoside (2'-O-MCE-modified nucleoside).

[0073] In one embodiment, the 5' wing region of the first nucleic acid strand may comprise at least one bridged unnatural nucleoside represented by formula (I) or (II) above and at least one ribose-2'-modified nucleoside on the 3' side thereof, and the 3' wing region of the first nucleic acid strand may comprise at least one bridged unnatural nucleoside represented by formula (I) or (II) above and at least one ribose-2'-modified nucleoside on the 5' side thereof.

[0074] In one embodiment, the 5' wing region of the first nucleic acid strand may comprise at least one bridged unnatural nucleoside represented by formula (I) or (II) above and at least one ribose-2'-modified nucleoside on the 5' side thereof, and the 3' wing region of the first nucleic acid strand may comprise at least one bridged unnatural nucleoside represented by formula (I) or (II) above and at least one ribose-2'-modified nucleoside on the 5' side thereof.

[0075] In one embodiment, the 5' wing region of the first nucleic acid strand may comprise at least one bridged unnatural nucleoside represented by formula (I) or (II) above and at least one ribose-2'-modified nucleoside on the 3' side thereof, and the 3' wing region of the first nucleic acid strand may comprise at least one bridged unnatural nucleoside represented by formula (I) or (II) above and at least one ribose-2'-modified nucleoside on the 3' side thereof.

[0076] In a preferred embodiment, the 5' wing region of the first nucleic acid strand may comprise at least one bridged unnatural nucleoside represented by formula (I) or (II) above and at least one ribose-2'-modified nucleoside on the 5' side thereof, and the 3' wing region of the first nucleic acid strand may comprise at least one bridged unnatural nucleoside represented by formula (I) or (II) above and at least one ribose-2'-modified nucleoside on the 3' side thereof.

[0077] The first nucleic acid strand and / or the second nucleic acid strand may comprise, in whole or in part, nucleoside or nucleotide mimetics. The nucleotide mimetics may be peptide nucleic acids and / or morpholino nucleic acids.

[0078] The internucleoside linkages in the first and second nucleic acid strands may be naturally occurring internucleoside linkages and / or modified internucleoside linkages. While not limited thereto, it is preferred that at least one, at least two, or at least three internucleoside linkages from the termini (5'-terminus, 3'-terminus, or both) of the first and / or second nucleic acid strands are modified internucleoside linkages. Here, for example, the two internucleoside linkages from the terminus of the nucleic acid strand refer to the internucleoside linkage closest to the terminus of the nucleic acid strand and the adjacent internucleoside linkage located opposite the terminus. Modified internucleoside linkages in the terminal regions of the nucleic acid strand are preferred because they can suppress or inhibit undesired degradation of the nucleic acid strand.

[0079] In one embodiment, all or some of the internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand may be modified internucleoside linkages, and 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 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%, at least 93%, at least 95%, at least 98%, or 100% modified internucleoside linkages. In one embodiment, the modified internucleoside linkages may be phosphorothioate linkages.

[0080] 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.

[0081] 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 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.

[0082] In one embodiment, 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 １～６The 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 (IV)) and / or an internucleoside bond containing a cyclic guanidine moiety (e.g., a partial structure represented by formula (III)). The modified internucleoside bond may be chiral controlled to have an Rp or Sp configuration.

[0083] At least one (e.g., three) internucleoside linkages from the 3'-end of the second nucleic acid strand may be a modified internucleoside linkage such as a phosphorothioate linkage, which has high RNase resistance. Inclusion of a modified internucleoside linkage such as a phosphorothioate modification at the 3'-end of the second nucleic acid strand is preferred because it improves the gene silencing activity of the double-stranded nucleic acid complex.

[0084] 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 a 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 -The modified internucleoside linkages of the first and / or second nucleic acid strands comprise non-negatively charged (neutral or cationic, respectively) internucleoside linkages that exist in a neutral or cationic form compared to the non-negatively charged internucleoside linkages of the first and / or second nucleic acid strands (e.g., the anionic form of a phosphorothioate linkage). In one embodiment, the modified internucleoside linkages of the first and / or second nucleic acid strands comprise neutral 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 of 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 comprising a cyclic guanidine moiety has a moiety represented by formula (III): １～６ The alkyl-substituted guanidine moiety has a moiety represented by formula (IV): In one embodiment, the cyclic guanidine moiety and / or the 1-4 C 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 an equivalent 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.

[0085] In one embodiment, the second nucleic acid strand may be 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.

[0086] As used herein, "tocopherol" refers to a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) with a ring structure called chroman. Tocorol has strong antioxidant properties and therefore functions in vivo as an antioxidant to eliminate free radicals generated by metabolism and protect cells from damage.

[0087] Several different types of tocopherol are known, consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, based on the position of the methyl group bound to the chroman. The tocopherol referred to herein may be any tocopherol. Examples of tocopherol analogs include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. Preferably, the tocopherol is α-tocopherol.

[0088] As used herein, "cholesterol" refers to a type of sterol, also known as steroid alcohol, which is particularly abundant in animals. Cholesterol plays an important role in metabolic processes in vivo, and is also a major component of the cell membrane system in animal cells, along with phospholipids. Cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols having a sterol skeleton, and include, but are not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0089] 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 based on common general technical knowledge.

[0090] Cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols having a sterol skeleton, and include, but are not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0091] In one embodiment, the second nucleic acid strand may be bound to tocopherol, cholesterol, or an analog thereof. The second nucleic acid strand bound to cholesterol or an analog thereof may have a group represented by the following general formula (V): [In the formula, R c represents an alkylene group having 4 to 18 carbon atoms, preferably 5 to 16 carbon atoms, which may have a substituent (wherein the substituent is an alkyl group having 1 to 3 carbon atoms which may be substituted with a halogen atom or a hydroxy group, such as a hydroxymethyl group, and non-adjacent carbon atoms in the alkylene group may be substituted with an oxygen atom).

[0092] R c may be, but is not limited to, -(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-, -(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-CH2-CH(CH2OH)-, or -(CH2)6-.

[0093] The group represented by the above general formula (V) can be bound to the 5'-end or 3'-end of the second nucleic acid strand via a phosphate ester bond.

[0094] The lipid, such as cholesterol or its analog, may be bound to the 5'-end, 3'-end, or both ends of the second nucleic acid strand. Alternatively, the lipid, such as cholesterol or its analog, may be bound to an internal nucleotide of the second nucleic acid strand. While not limited thereto, cholesterol or its analog bound to the 5'-end of the second nucleic acid strand is particularly preferred.

[0095] When the second nucleic acid strand contains multiple cholesterols or their analogs, they may be the same or different. For example, one cholesterol may be bound to the 5'-end of the second nucleic acid strand and one other cholesterol analog may be bound to the 3'-end. Regarding the binding positions, cholesterol or its analogs may be bound to multiple positions on the second nucleic acid strand and / or may be bound as a group to one position. One cholesterol or its analog may be linked to each of the 5'-end and 3'-end of the second nucleic acid strand.

[0096] The bond between the second nucleic acid strand and the lipid may be a direct bond or an indirect bond mediated by another substance.

[0097] When the second nucleic acid strand and the lipid are directly bound to each other, the lipid may be bound to the second nucleic acid strand via, for example, a covalent bond, an ionic bond, a hydrogen bond, etc. In view of the fact that a more stable bond can be obtained, a covalent bond is preferred.

[0098] In one embodiment, the second nucleic acid strand is not bound to a ligand. As used herein, "not bound to a ligand" refers to the absence of a lipid such as tocopherol or cholesterol. In a further embodiment, the double-stranded nucleic acid complex of the present invention is not bound to a ligand, i.e., neither the first nor the second nucleic acid strand is bound to a ligand.

[0099] When the second nucleic acid strand and cholesterol or an analog thereof are indirectly bound to each other, they may be bound via a linking group (often referred to as a "linker" in this specification). The linker may be either a cleavable linker or an uncleavable linker.

[0100] "Cleavable linker" refers to a linker that can be cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within a human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases. Cleavable linkers include, but are not limited to, amides, esters, one or both phosphodiesters, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers. As an example, cholesterol or an analog thereof may be linked via a disulfide bond.

[0101] The term "non-cleavable linker" refers to a linker 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, but are not limited to, linkers consisting of phosphorothioate bonds, and modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by phosphorothioate bonds. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not particularly limited, but may typically be 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.

[0102] A specific example of the linker is a linker represented by the following formula (VI): (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)2-O—(CH2)3-, —(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 is an integer from 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond (wherein the substitution is preferably made by a halogen atom).

[0103] In one embodiment, the linker of formula (VI) 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.

[0104] Another specific example of the linker is a linker represented by the following general formula (VII): [In the formula, n represents 0 or 1.]

[0105] The first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) may further comprise at least one functional moiety bound to a polynucleotide constituting the nucleic acid strand. The term "functional moiety" refers to a moiety that confers a desired function to the double-stranded nucleic acid complex and / or the nucleic acid strand to which the functional moiety is bound. Examples of the desired function include labeling and purification. Examples of moieties that confer labeling include compounds such as fluorescent proteins and luciferase. Examples of moieties that confer purification include compounds such as biotin, avidin, His-tag peptides, GST-tag peptides, and FLAG-tag peptides. 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 preferred that the second nucleic acid strand be bound to a molecule that has the activity of delivering the double-stranded nucleic acid complex to a target site in certain embodiments. Examples of moieties that confer 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. The bond between the second nucleic acid strand and the functional moiety may be direct or indirect via another substance. In one embodiment, however, the second nucleic acid strand is preferably directly bound to the functional moiety 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.

[0106] In a further embodiment, the second nucleic acid strand may further comprise at least one overhang region located on one or both of the 5' and 3' ends of the complementary region, an example of which is described in WO 2018 / 062510.

[0107] The first and second nucleic acid strands may be linked via a linker. In this case, the first and second nucleic acid strands may be linked via the linker to form a single strand. However, even in this case, the functional region has the same structure as in the double-stranded nucleic acid complex, and therefore, this specification also encompasses such single-stranded nucleic acids as an embodiment of the double-stranded nucleic acid complex of the present invention. The linker may be any polymer. Examples include polynucleotides, polypeptides, and alkylenes. Specifically, it may be composed of natural nucleotides such as DNA and RNA, or unnatural nucleotides such as peptide nucleic acids and morpholino nucleic acids. When the linker is composed of a nucleic acid, the chain length of the linker may be at least one base, for example, 3 to 10 bases or 4 to 6 bases. A chain length of 4 bases is preferred. The linker may be located on either the 5' or 3' side of the first nucleic acid strand, but for example, in a configuration in which cholesterol or an analog thereof is bound to the 5' side of the second nucleic acid strand, the 5' end of the first nucleic acid strand and the 3' end of the second nucleic acid strand are linked via a linker. The linker may be either cleavable or non-cleavable.

[0108] In the double-stranded nucleic acid complex of the present invention, the antisense effect of the first nucleic acid strand on a target transcript can be measured by methods known in the art. For example, after introducing the double-stranded nucleic acid complex into cells, measurement can be performed using known techniques such as Northern blotting, quantitative PCR, or Western blotting. Whether target gene expression is suppressed by the double-stranded nucleic acid complex can be determined by measuring the expression level of the target gene or the level of the target transcript (e.g., mRNA level, RNA level such as microRNA, cDNA level, protein level, etc.). The criteria for this determination are not limited, but may include a determination that the double-stranded nucleic acid complex of the present invention has an antisense effect if the measured expression level of the target gene or the target transcript is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 40% compared to the measured value of a negative control (e.g., vehicle administration).

[0109] As described above, exemplary embodiments of the double-stranded nucleic acid complex of the present invention have been described, but the double-stranded nucleic acid complex of the present invention is not limited to the above exemplary embodiments.

[0110] 1-4. Method for Producing Double-Stranded Nucleic Acid Complexes Those skilled in the art can produce the double-stranded nucleic acid complexes of the present invention by appropriately selecting known methods. While not limited thereto, this typically begins with designing and producing each of the first and second nucleic acid strands that make up the double-stranded nucleic acid complex. For example, the first nucleic acid strand is designed based on the base sequence information of the target transcription product (e.g., the base sequence of the target gene), and the second nucleic acid strand is designed as its complementary strand. Next, based on the designed base sequence information, each nucleic acid strand can be synthesized using a commercially available automated nucleic acid synthesizer, for example, from GE Healthcare, Thermo Fisher Scientific, Beckman Coulter, or the like. The resulting oligonucleotides can then be purified using a reverse-phase column or the like.

[0111] Furthermore, in the case of a double-stranded nucleic acid complex to which a functional moiety is bound, the first nucleic acid strand may be prepared according to the above-described method. Meanwhile, the second nucleic acid strand to which a functional moiety is bound can be prepared by carrying out the above-described synthesis and purification using a nucleic acid species to which a functional moiety has already been bound. For example, the second nucleic acid strand may be prepared by carrying out the above-described synthesis and purification using a nucleic acid species to which cholesterol or its analog has already been bound. Alternatively, cholesterol or its analog may be bound to the second nucleic acid strand prepared by carrying out the above-described synthesis and purification using a known method. After preparing each nucleic acid strand, the first and second nucleic acid strands are annealed to produce a double-stranded nucleic acid complex to which the desired functional moiety is bound. Specifically, nucleic acids are mixed in an appropriate buffer solution and denatured at approximately 90°C to 98°C for several minutes (e.g., 5 minutes), and then the nucleic acids are annealed at approximately 30°C to 70°C for approximately 1 to 8 hours to produce one of the double-stranded nucleic acid complexes of the present invention. Methods for linking functional moieties to nucleic acids are well known in the art. Alternatively, nucleic acid chains can be ordered and obtained from various manufacturers (for example, Gene Design Inc.) by specifying the base sequence and the site and type of modification.

[0112] 1-5. Effects The double-stranded nucleic acid complex of the present invention can reduce or eliminate the toxicity of the double-stranded nucleic acid complex without impairing the efficacy of the double-stranded nucleic acid complex. That is, compared to conventional double-stranded nucleic acid complexes, liver toxicity and nephrotoxicity are reduced, and the induction of inflammation or gliosis, or abnormal increases in cytokines or chemokines, are reduced, without impairing the antisense effect on the target gene.

[0113] 2. Pharmaceutical Composition 2-1. Overview A second aspect of the present invention is a pharmaceutical composition. The pharmaceutical composition of the present invention contains the double-stranded nucleic acid complex of the first aspect as an active ingredient. The pharmaceutical composition of the present invention has low hepatotoxicity and / or low nephrotoxicity. Furthermore, the induction of inflammation or gliosis, or abnormal increases in cytokines or chemokines, is reduced. Each component that may be included in the pharmaceutical composition of the present invention will be specifically described below.

[0114] 2-2. Composition 2-2-1. Active Ingredient The pharmaceutical composition of the present invention contains at least the double-stranded nucleic acid complex described in the first aspect as an active ingredient. The pharmaceutical composition of the present invention may contain two or more types of double-stranded nucleic acid complex. The amount (content) of the double-stranded nucleic acid complex contained in the pharmaceutical composition varies depending on the type of double-stranded nucleic acid complex, the delivery site, the dosage form of the pharmaceutical composition, the dosage of the pharmaceutical composition, and the type of carrier (described below). Therefore, it can be determined appropriately taking into account each condition. Typically, the pharmaceutical composition is adjusted so that an effective amount of double-stranded nucleic acid complex is contained in a single dose. The "effective amount" refers to the amount of double-stranded nucleic acid complex necessary to exert its function as an active ingredient and that causes little or no harmful side effects in the living body to which it is applied. This effective amount may vary depending on various conditions, such as information about the subject, the route of administration, and the number of administrations. Ultimately, it is determined by the judgment of a physician, veterinarian, pharmacist, or other such person. "Subject information" refers to various individual information about the living body to which the pharmaceutical composition is applied. For example, if the subject is a human, the information includes age, weight, sex, diet, health condition, progression and severity of disease, drug sensitivity, and whether or not concomitant medication is being used.

[0115] 2-2-2. Carrier The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. Examples include solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavors, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonicity agents, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizers, and other additives.

[0116] The solvent may be, for example, water or any other pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0117] The above-mentioned carriers are used to avoid or inhibit the decomposition of the double-stranded nucleic acid complex, which is the active ingredient, by enzymes and the like in the body, as well as to facilitate formulation and administration methods and maintain the dosage form and medicinal efficacy, and may be used appropriately as needed.

[0118] The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a form that can deliver the double-stranded nucleic acid complex described in the first aspect, which is the active ingredient, to a target site without inactivating it through degradation or the like, and can exert the pharmacological effect of the active ingredient in vivo (antisense effect on the expression of a target gene).

[0119] The specific dosage form varies depending on the administration method and / or formulation conditions. The administration method can be roughly divided into parenteral administration and oral administration, and therefore, a dosage form suitable for each administration method may be used.

[0120] If the administration method is parenteral administration, the preferred dosage form is a liquid that can be administered directly to the target site or systemically via the circulatory system. Examples of liquids include injections. Injections can be formulated by appropriately combining the above-mentioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. Other forms include ointments, plasters, cataplasms, transdermal agents, lotions, inhalants, aerosols, eye drops, and suppositories.

[0121] If the administration method is oral administration, preferred dosage forms may be solid or liquid, such as tablets, capsules, drops, troches, pills, granules, powders, powders, oral solutions, emulsions, syrups, pellets, sublingual tablets, peptizers, buccal tablets, pastes, suspensions, elixirs, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, eye drops, injections, and suppositories. If the solid formulation is used, it may be made into a dosage form coated with a coating known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multi-layer tablets, as needed.

[0122] The specific shape and size of each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art. The pharmaceutical composition of the present invention may be formulated according to a conventional method in the art.

[0123] In certain embodiments, the double-stranded nucleic acid complex of the present invention has excellent properties as a pharmaceutical, such as excellent solubility in water, Japanese Pharmacopoeia Dissolution Test Fluid 2, or Japanese Pharmacopoeia Disintegration Test Fluid 2, excellent pharmacokinetics (e.g., drug half-life in blood, 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).

[0124] 2-3. Dosage Form and Dose As used herein, there are no particular limitations on the preferred administration form of the pharmaceutical composition. For example, oral administration or parenteral administration may be used. Specific examples of parenteral administration include intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, tracheal / bronchial administration, rectal administration, administration by transfusion, intraventricular administration, intrathecal administration, intranasal administration, and intramuscular administration.

[0125] When the pharmaceutical composition is administered or ingested, the dosage or intake may be, for example, such that the amount of the double-stranded nucleic acid complex contained therein is 0.00001 mg / kg / day to 10,000 mg / kg / day, or 0.001 mg / kg / day to 100 mg / kg / day. The pharmaceutical 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 double-stranded 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, 0.25 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 2.5 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, 75 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, 10 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, 10 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, 10 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, 1 ... The dose can be 0 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 (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).

[0126] The double-stranded nucleic acid complex of the present invention may be administered twice a week for four doses at a dose of 0.01 to 10 mg / kg (e.g., about 6.25 mg / kg). Alternatively, the double-stranded nucleic acid complex may be administered once or twice a week for two to four doses at a dose of 0.05 to 30 mg / kg (e.g., about 25 mg / kg), for example, twice a week for two doses. The use of such a dosing regimen (divided administration) can reduce toxicity (e.g., avoid platelet reduction) and the burden on the subject compared to a single administration of a higher dose.

[0127] The pharmaceutical composition exerts an additive inhibitory effect within cells even when administered repeatedly. Furthermore, when administering repeatedly, the efficacy can be improved by leaving a certain interval between administrations (e.g., half a day or more).

[0128] In one embodiment, the pharmaceutical composition of this aspect is administered intracerebroventricularly or intrathecally. When the pharmaceutical composition of this aspect is administered intracerebroventricularly or intrathecally, the amount administered to monkeys or humans may be 0.01 mg or more, 0.1 mg or more, or 1 mg or more, for example, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 75 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, or 500 mg or more, or 0.01 mg to 1000 mg, 0.1 mg to 200 mg, or 1 mg to 20 mg, and in the case of mice, 1 μg or more may be administered.

[0129] In one embodiment, the pharmaceutical composition of this aspect is administered intravenously or subcutaneously. When administered intravenously or subcutaneously, the pharmaceutical composition of this aspect may be administered at a dose of 0.01 mg / kg or more, 0.1 mg / kg or more, or 1 mg / kg or more, for example, 2 mg / kg or more, 3 mg / kg or more, 4 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, or at a dose of 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 100 mg / kg, or 1 mg / kg to 10 mg / kg.

[0130] 2-4. Target Diseases The diseases to which the pharmaceutical composition can be applied are not limited, and can include diseases associated with increased expression of target genes, such as neurological diseases, central nervous system diseases, metabolic diseases, tumors, and infectious diseases. In one embodiment, the pharmaceutical composition of this aspect can be used to treat a central nervous system disease in a subject. Central nervous system diseases to which the pharmaceutical composition of this aspect can be applied include, but are not limited to, brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, etc.

[0131] The pharmaceutical composition of the present invention has low hepatotoxicity and / or low nephrotoxicity. Furthermore, the pharmaceutical composition of the present invention reduces the induction of inflammation or gliosis, or the abnormal increase in cytokines or chemokines, when administered intracerebroventricularly, intrathecally, intravenously, subcutaneously, or the like.

[0132] The pharmaceutical composition of the present invention can treat or prevent diseases by suppressing or inhibiting the expression of a specific gene without substantially causing hepatotoxicity and / or nephrotoxicity, and can also treat or prevent diseases while reducing abnormal increases in cytokines or chemokines without substantially inducing inflammation or gliosis.

[0133] In particular, the treatment of neurological diseases such as Alzheimer's disease requires the administration of high doses of nucleic acid agents, which can cause serious liver damage as a side effect. However, the pharmaceutical composition of the present invention can significantly reduce such side effects.

[0134] There is also provided a method for treating and / or preventing a disease such as a central nervous system disease, which comprises administering the above-described double-stranded nucleic acid complex or pharmaceutical composition to a subject.

[0135] 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.

[0136] Example 1: Gene silencing and toxicity-reducing effects of intraventricular administration of HDO(scpBNA) (Objective) To verify the gene silencing and toxicity-reducing effects of intraventricular administration of a heteroduplex oligonucleotide (hereinafter referred to as "HDO(scpBNA)") comprising a first nucleic acid strand consisting of an scpBNA / DNA gapmer antisense nucleic acid (hereinafter referred to as "ASO(scpBNA)") and a second nucleic acid strand consisting of RNA having a base sequence complementary to the first nucleic acid strand, through in vivo experiments.

[0137] (Method) (1) Preparation of Nucleic Acid The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO(scpBNA) used in this example are shown in Table 1 and FIG. 3D.

[0138]

[0139] The first nucleic acid strand of HDO(scpBNA) is an scpBNA / DNA gapmer-type antisense nucleic acid, containing 5' and 3' wing regions composed of three scpBNA nucleosides at the 5' and 3' ends, respectively, with 10 DNA nucleosides between them. The base sequence of the first nucleic acid strand is complementary to a portion of mouse microtubule-associated protein tau (Mapt) mRNA. The second nucleic acid strand of HDO(scpBNA) is composed of RNA having a sequence complementary to the first nucleic acid strand. Note that neither the first nor second nucleic acid strand of HDO(scpBNA) is bound to a ligand.

[0140] The scpBNA used in the examples of the present specification has the following formula (I): It is an unnatural nucleoside represented by the formula:

[0141] As a control for comparison with HDO(scpBNA), a heteroduplex oligonucleotide (hereinafter referred to as "HDO(LNA)") containing an LNA / DNA gapmer antisense nucleic acid (hereinafter referred to as "ASO(LNA)") was used. The structures and base sequences of the first and second nucleic acid strands constituting the HDO(LNA) used in this example are shown in Table 2 and Figure 3B. Neither the first nucleic acid strand nor the second nucleic acid strand of HDO(LNA) is bound to a ligand.

[0142]

[0143] To prepare the double-stranded nucleic acid complex, the first and second nucleic acid strands were mixed in equimolar amounts, heated to 95°C for 5 minutes, and 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 annealed nucleic acid was stored at 4°C or on ice. All oligonucleotides were custom synthesized by Gene Design Co., Ltd. (Osaka, Japan).

[0144] In addition, in this example, the following experiments were also performed on single-stranded gapmer antisense nucleic acids, including ASOs (scpBNA) consisting only of the first nucleic acid strand shown in Table 1 and ASOs (LNA) consisting only of the first nucleic acid strand shown in Table 2.

[0145] (2) In vivo experiments. Seven-week-old female ICR mice were anesthetized with 2.5-4% isoflurane and placed in a stereotaxic apparatus. A 2-3 cm incision was made in the skin between the ears, and a 1 mm drill hole was created 1 mm left and 0.2 mm posterior to the bregma. A Hamilton syringe was filled with the nucleic acid agent. The needle was inserted approximately 3 mm into the hole, and the nucleic acid agent was administered intraventricularly into the left ventricle at a dose of 24 μmol / mouse at a rate of 2-3 μl / min (n=2). The skin was then sutured with nylon thread. Seven days after injection, the mice were perfused with PBS, and the left hippocampus was then removed by dissection.

[0146] (3) Expression Analysis RNA was extracted from the excised left hippocampus using the Isogen I kit (Gene Design Inc.). cDNA was synthesized using Transcriptor Universal cDNA Master, DNase (Roche Diagnostics) according to the protocol.

[0147] Next, quantitative RT-PCR was performed using the obtained cDNA as a template to evaluate the gene silencing effect and toxicity of various nucleic acid agents. Quantitative RT-PCR was performed using TaqMan (Roche Applied Science). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific (formerly Life Technologies Corp.) based on various gene numbers. The amplification conditions (temperature and time) were as follows: 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second (1 cycle), repeated 40 times.

[0148] To evaluate the gene silencing effect of nucleic acid agents, we measured the expression levels of Mapt mRNA and Actb mRNA (internal control genes) and calculated the ratio between them. To evaluate the toxicity of nucleic acid agents, we also measured the expression levels of Tumor Necrosis Factor-α (TNFα) mRNA as an indicator of inflammatory cytokines and of Glial fibrillary acidic protein (GFAP) mRNA as an indicator of astrocyte activation and gliosis.

[0149] (Results) Figure 4 shows the level of Mapt mRNA expression in the left hippocampus of mice administered various nucleic acid agents into the left ventricle. No significant difference was observed in the Mapt gene suppression effect between intraventricular administration of ASO(LNA), HDO(LNA), ASO(scpBNA), and HDO(scpBNA).

[0150] Figure 5 shows the expression levels of TNFα mRNA and GFAP mRNA in the left hippocampus of mice administered various nucleic acid agents into the left lateral ventricle. ASO (LNA) and HDO (LNA) significantly increased the expression levels of TNFα mRNA and GFAP mRNA compared to PBS administration. In contrast, ASO (scpBNA) and HDO (scpBNA) did not significantly increase the expression levels of TNFα mRNA and GFAP mRNA compared to PBS administration, and the induction of inflammatory cytokines and gliosis was reduced.

[0151] These results demonstrate that scpBNAs can reduce central nervous system side effects without weakening the gene-regulating effects of conventional LNAs, and this mitigation effect was observed not only in single-stranded ASOs but also in HDO structures.

[0152] Example 2: Gene suppression effect and hepatotoxicity reduction effect by single intravenous administration of mMalat1-targeting Toc-HDO (scpBNA) (Objective) To verify the target gene expression suppression effect and toxicity reduction effect by single intravenous administration of a double-stranded nucleic acid complex consisting of an scpBNA / DNA gapmer antisense nucleic acid and a tocopherol-bound complementary strand, and a double-stranded nucleic acid complex consisting of an AmNA / DNA gapmer antisense nucleic acid and a tocopherol-bound complementary strand, both of which target mMalat1.

[0153] (Method) (1) Preparation of Nucleic Acids The target gene was metastasis-associated lung adenocarcinoma transcript 1 (mMalat1). The base sequences of the first and second nucleic acid strands constituting the three types of double-stranded nucleic acid complexes used in this example are shown in Table 3 and Figure 6.

[0154]

[0155] The first nucleic acid strand targets the mMalat1 gene and is composed of a 13-mer gapmer with a base sequence complementary to a portion of the malat1 non-coding RNA, its transcription product. The first nucleic acid strand of Toc-HDO(LNA) comprises a 5' wing region consisting of three LNA nucleosides at the 5' end, a 3' wing region consisting of two LNA nucleosides at the 3' end, and eight DNA nucleosides between them. In Toc-HDO(scpBNA) and Toc-HDO(AmNA), some of the LNA nucleosides in the 5' and 3' wing regions are replaced with scpBNA or AmNA nucleosides.

[0156] On the other hand, the second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is composed of a tocopherol-bound complementary strand RNA having tocopherol bound to its 5' end.

[0157] The AmNA used in the examples of the present specification is represented by the following formula (II): (wherein R represents a methyl group).

[0158] The double-stranded nucleic acid complex was prepared in the same manner as in Example 1(1).

[0159] (2) In vivo experiments Mice administered with the double-stranded nucleic acid complex were male C57BL / 6 mice weighing 20 g, 6 to 7 weeks old. Experiments were performed with n=4 for each condition. A single dose of the double-stranded nucleic acid complex was intravenously injected into the mice via the tail vein at 50 mg / kg. Additionally, a negative control group of mice was administered with a single dose of PBS alone. Blood was collected 72 hours after administration, and the mice were perfused with PBS. The mice were then dissected, and the liver, kidneys, myocardium, and quadriceps muscles were removed.

[0160] (3) Expression Analysis. mRNA was extracted from each tissue using a high-throughput, fully automated nucleic acid extraction system, 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 qRT-PCR, and the expression levels of malat1 mRNA and GAPDH mRNA (internal control gene) were calculated. Relative expression levels were calculated from the ratio of the two, and the mean and standard error of the relative expression levels were calculated. Differences between groups were analyzed using t-tests.

[0161] (4) Analysis of Serum The serum obtained by blood collection was analyzed by contract with SRL Hachioji Lab, Inc.

[0162] (Results) The results of expression analysis are shown in Figures 7 and 8. Toc-HDO(scpBNA) and Toc-HDO(AmNA) exhibited gene silencing effects comparable to those of Toc-HDO(LNA) in the liver (Figure 7A), kidney (Figure 7B), quadriceps muscle (Figure 8A), and cardiac muscle (Figure 8B).

[0163] The results of serum analysis are shown in Figure 9A. In the Toc-HDO(LNA)-treated group, AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were significantly elevated, indicating impaired liver function. In contrast, in the Toc-HDO(scpBNA)- and Toc-HDO(AmNA)-treated groups, no significant increases in AST or ALT were detected, indicating reduced liver toxicity.

[0164] Furthermore, a decrease in body weight was observed in the Toc-HDO(LNA)-administered group, whereas no significant change in body weight was observed in the Toc-HDO(scpBNA)-administered group or the Toc-HDO(AmNA)-administered group (Figure 9B).

[0165] The above results demonstrate that by substituting a portion of LNA in the wing region of the first nucleic acid strand with scpBNA or AmNA, the toxicity of HDO can be reduced without weakening its gene suppression effect.

[0166] Example 3: Gene suppression effect and hepatotoxicity reduction effect by single intravenous administration of Toc-HDO (scpBNA) targeting PTEN (Objective) To verify the target gene expression suppression effect and toxicity reduction effect by single intravenous administration of a double-stranded nucleic acid complex consisting of an scpBNA / DNA gapmer antisense nucleic acid and a tocopherol-bound complementary strand, and a double-stranded nucleic acid complex consisting of an AmNA / DNA gapmer antisense nucleic acid and a tocopherol-bound complementary strand, both of which target PTEN.

[0167] (Method) The target gene was PTEN (Phosphatase and Tensin Homolog Deleted from Chromosome 10). The base sequences and chemical modifications of the first and second nucleic acid strands constituting the three types of double-stranded nucleic acid complexes used in this example are shown in Table 4 and Figure 10.

[0168]

[0169] The first nucleic acid strand targets the PTEN gene and is composed of a 14-mer gapmer having a base sequence complementary to a portion of the PTEN mRNA. The first nucleic acid strand of Toc-HDO(LNA) consists of a 5' wing region composed of two LNA nucleosides at the 5' end, a 3' wing region composed of two LNA nucleosides at the 3' end, and 10 DNA nucleosides between them. In Toc-HDO(scpBNA) and Toc-HDO(AmNA), all of the LNA nucleosides in the 5' and 3' wing regions are replaced with scpBNA or AmNA nucleosides.

[0170] On the other hand, the second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is composed of a tocopherol-bound complementary strand RNA having tocopherol bound to its 5' end.

[0171] The double-stranded nucleic acid complex was prepared in the same manner as in Example 1(1).

[0172] The in vivo experiment was carried out in the same manner as in Example 2, except that the double-stranded nucleic acid complex agent was intravenously injected into mice at 35 mg / kg. Expression analysis and serum analysis were carried out in the same manner as in Example 2.

[0173] (Results) The results of expression analysis are shown in Figures 11 and 12. Toc-HDO(scpBNA) and Toc-HDO(AmNA) exhibited gene silencing effects generally equivalent to or greater than those of Toc-HDO(LNA) in the liver (Figure 11A), kidney (Figure 11B), quadriceps muscle (Figure 12A), and cardiac muscle (Figure 12B).

[0174] The results of serum analysis are shown in Figure 13A. Significant increases in AST and ALT levels were observed in the Toc-HDO(LNA)-treated group, indicating impaired liver function. In contrast, no significant increases in AST and ALT levels were detected in the Toc-HDO(scpBNA)-treated group or the Toc-HDO(AmNA)-treated group, indicating reduced liver toxicity.

[0175] Furthermore, a significant decrease in body weight was observed in the Toc-HDO(LNA)-administered group, whereas no significant changes were observed in the Toc-HDO(scpBNA)-administered group or the Toc-HDO(AmNA)-administered group (Figure 13B).

[0176] These results demonstrate that by substituting all or part of the nucleosides constituting the wing region of the first nucleic acid strand with scpBNA or AmNA, the toxicity of HDO can be reduced without weakening its gene silencing effect.

[0177] Example 4: Gene suppression effect and hepatotoxicity reduction effect by single intravenous administration of Toc-HDO (scpBNA) targeting SR-B1 (Objective) To verify the target gene expression suppression effect and toxicity reduction effect by single intravenous administration of a double-stranded nucleic acid complex consisting of an scpBNA / DNA gapmer antisense nucleic acid and a tocopherol-bound complementary strand, and a double-stranded nucleic acid complex consisting of an AmNA / DNA gapmer antisense nucleic acid and a tocopherol-bound complementary strand, both of which target SR-B1.

[0178] (Method) The target gene was the scavenger receptor B1 (SR-B1) gene. The base sequences of the first and second nucleic acid strands constituting the three types of double-stranded nucleic acid complexes used in this example are shown in Table 5 and Figure 10.

[0179]

[0180] The first nucleic acid strand targets the SR-B1 gene and is composed of a 14-mer gapmer having a base sequence complementary to a portion of the SR-B1 mRNA. The first nucleic acid strand of Toc-HDO(LNA) consists of a 5' wing region composed of two LNA nucleosides at the 5' end, a 3' wing region composed of two LNA nucleosides at the 3' end, and 10 DNA nucleosides between them. In Toc-HDO(scpBNA) and Toc-HDO(AmNA), all of the LNA nucleosides in the 5' and 3' wing regions are replaced with scpBNA or AmNA nucleosides.

[0181] On the other hand, the second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is composed of a tocopherol-bound complementary strand RNA having tocopherol bound to its 5' end.

[0182] The double-stranded nucleic acid complex was prepared in the same manner as in Example 1(1).

[0183] The in vivo experiment, expression analysis, and serum analysis were performed in the same manner as in Example 2.

[0184] (Results) The results of expression analysis are shown in Figures 14 and 15. Toc-HDO(scpBNA) and Toc-HDO(AmNA) exhibited gene silencing effects comparable to those of Toc-HDO(LNA) in the liver (Figure 14A), kidney (Figure 14B), quadriceps muscle (Figure 15A), and cardiac muscle (Figure 15B).

[0185] The results of serum analysis are shown in Figure 16A. In the Toc-HDO(LNA)-treated group, AST and ALT levels were significantly elevated, indicating impaired liver function. In contrast, in the Toc-HDO(scpBNA)- and Toc-HDO(AmNA)-treated groups, AST and ALT levels were not elevated, and no hepatotoxicity was detected.

[0186] Furthermore, a decrease in body weight was observed in the Toc-HDO(LNA)-administered group, whereas no significant change in body weight was observed in the Toc-HDO(scpBNA)-administered group or the Toc-HDO(AmNA)-administered group (Figure 16B).

[0187] These results demonstrate that by substituting all or part of the nucleosides constituting the wing region of the first nucleic acid strand with scpBNA or AmNA, the toxicity of HDO can be reduced without weakening its gene silencing effect.

[0188] Example 5: Gene suppression effect and hepatotoxicity reduction effect by multiple intravenous administration of Chol-HDO (scpBNA) (Objective) To verify the target gene expression suppression effect and toxicity reduction effect of a double-stranded nucleic acid complex consisting of an scpBNA / DNA gapmer antisense nucleic acid targeting SR-B1 and a cholesterol-conjugated complementary strand by multiple intravenous administration.

[0189] (Method) (1) Preparation of nucleic acid The target gene was the SR-B1 gene. The base sequences of the first and second nucleic acid strands constituting the two types of double-stranded nucleic acid complexes used in this example are shown in Table 6 and Figure 17.

[0190]

[0191] The first nucleic acid strand targets the SR-B1 gene and is composed of a 14-mer gapmer having a base sequence complementary to a portion of the SR-B1 mRNA. The first nucleic acid strand of Chol-HDO(LNA) consists of a 5' wing region composed of two LNA nucleosides at the 5' end, a 3' wing region composed of two LNA nucleosides at the 3' end, and 10 DNA nucleosides between them. In Chol-HDO(scpBNA), all of the LNA nucleosides in the 5' and 3' wing regions are replaced with scpBNA nucleosides.

[0192] On the other hand, the second nucleic acid strand has a sequence complementary to the first nucleic acid strand and is composed of cholesterol-bound complementary RNA with cholesterol bound to its 5' end.

[0193] The double-stranded nucleic acid complex was prepared in the same manner as in Example 1(1).

[0194] (2) In vivo experiments Mice administered with the double-stranded nucleic acid complex were male C57BL / 6 mice weighing 20 g, aged 6 to 7 weeks. Experiments were performed with n=4 for each condition. The double-stranded nucleic acid complex was administered intravenously via the tail vein at 50 mg / kg multiple times (once a week, a total of four times). Additionally, a negative control group of mice was administered a single injection of PBS alone. Blood was collected 72 hours after administration, and the mice were perfused with PBS. The mice were then dissected, and various brain regions and whole bodies were removed.

[0195] (3) Expression Analysis. mRNA was extracted from each tissue using a high-throughput, fully automated nucleic acid extraction system, 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 qRT-PCR, and the expression levels of SR-B1 mRNA and Actin mRNA (internal control gene) were calculated based on the results. Relative expression levels were calculated from the ratio of the two. The mean and standard error of the relative expression levels were calculated. Differences between groups were analyzed by t-test.

[0196] (4) Analysis of Serum The serum obtained by blood collection was analyzed by contract with SRL Hachioji Lab, Inc.

[0197] (Results) The results of expression analysis are shown in Figure 18. Chol-HDO(scpBNA) demonstrated a strong gene silencing effect in the brain (Figure 18A) and in systemic organs (Figure 18B). The results of serum analysis are shown in Figure 19. In the Chol-HDO(LNA)-administered group, AST / ALT levels rose significantly three days after the first administration, and the mice died five to six days later. In contrast, the Chol-HDO(scpBNA)-administered group survived even after four administrations, with only a mild increase in AST / ALT levels.

[0198] These results demonstrate that the toxicity of HDO is reduced by placing scpBNA in the wing region of the first nucleic acid strand, even when administered multiple times.

[0199] Example 6: Effect of a single intravenous administration of Toc-HDO (scpBNA) on reducing the expression of inflammatory cytokines / chemokines in the blood (Objective) To examine the expression of inflammatory cytokines / chemokines in the blood when a double-stranded nucleic acid complex consisting of an scpBNA / DNA gapmer antisense nucleic acid and a tocopherol-bound complementary strand is administered intravenously.

[0200] (Methods) (1) Preparation of Nucleic Acids In this example, Toc-HDO(LNA), Toc-HDO(scpBNA), and Toc-HDO(AmNA) targeting mMalat1 listed in Table 3, Toc-HDO(LNA), Toc-HDO(scpBNA), and Toc-HDO(AmNA) targeting PTEN listed in Table 4, and Toc-HDO(LNA), Toc-HDO(scpBNA), and Toc-HDO(AmNA) targeting SR-B1 listed in Table 5 were used. Double-stranded nucleic acid complexes were prepared by the same method as in Example 1(1).

[0201] (2) In vivo Experiments In vivo experiments were performed in the same manner as in Example 2, except that the dose of mMalat1-targeting HDO was 50 mg / kg and the dose of PTEN-targeting HDO was 35 mg / kg. Specifically, a single dose of the double-stranded nucleic acid complex agent was intravenously administered via the tail vein. Blood samples were collected 72 hours after administration.

[0202] (3) Quantification of Inflammatory Cytokines / Chemokines in Blood Blood samples were analyzed using MILLIPLEX MAP Mouse Cytokine / Chemokine Magnetic Bead Panel - Immunology Multiplex (Millipore) according to the manufacturer's instructions. To assess nucleic acid-induced toxicity, the following proteins were measured: G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, VEGF, and Eotaxin / CCL11. As a result, TNFα, IP-10, and RANTES, which were elevated by Toc-HDO(LNA), were measured as indicators of inflammation.

[0203] (Results) The results of measuring the levels of inflammatory cytokines in the blood are shown in Figures 20 to 22. As shown in Figures 20 to 22, the Toc-HDO(scpBNA)-administered group and the Toc-HDO(AmNA)-administered group showed lower expression levels of TNFα (Figure 20), IP-10 (Figure 21), and RANTES (Figure 22) compared to the Toc-HDO(LNA)-administered group. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A double-stranded nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand can hybridize to at least a part of a target gene or its transcript, and has an antisense effect on the target gene or its transcript, the second nucleic acid strand contains a base sequence complementary to the first nucleic acid strand, the first nucleic acid strand (1) a central region containing at least 4 consecutive deoxyribonucleosides, (2) a 5' wing region containing a non-natural nucleoside, disposed on the 5'-terminal side of the central region, and (3) a 3' wing region containing a non-natural nucleoside, disposed on the 3'-terminal side of the central region, wherein the 5' wing region and / or the 3' wing region in the first nucleic acid strand has at least one crosslinked non-natural nucleoside and an LNA nucleoside represented by the following formula (I) or formula (II): 【Chemical 1】 (In the formula, R represents a hydrogen atom or a methyl group.) The double-stranded nucleic acid complex comprising.

2. The double-stranded nucleic acid complex according to claim 1, wherein the second nucleic acid strand contains at least 4 consecutive ribonucleosides complementary to at least 4 consecutive deoxyribonucleosides in the central region of the first nucleic acid strand.

3. The double-stranded nucleic acid complex according to claim 1, wherein the second nucleic acid strand further contains at least 2 consecutive deoxyribonucleosides.

4. The double-stranded nucleic acid complex according to claim 1, wherein in the second nucleic acid strand, a region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand contains a crosslinked non-natural nucleoside represented by the formula (I) or formula (II).

5. The double-stranded nucleic acid complex according to claim 1, wherein the first nucleic acid strand and / or the second nucleic acid strand contains at least one ribose 2'-position modified nucleoside selected from the group consisting of 2'-O-methyl modified nucleoside, 2'-O-methoxyethyl modified nucleoside, and 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleoside.

6. The double-stranded nucleic acid complex according to claim 1, wherein all or part of the internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand are modified internucleoside linkages.

7. The double-stranded nucleic acid complex according to claim 6, wherein the modified internucleoside linkage is a phosphorothioate linkage.

8. The double-stranded nucleic acid complex according to claim 1, wherein the second nucleic acid strand is bound to tocopherol or cholesterol or an analog thereof.

9. The double-stranded nucleic acid complex according to claim 1, which is not bound to a ligand.

10. The double-stranded nucleic acid complex according to claim 1, wherein the first nucleic acid strand and the second nucleic acid strand are bound via a cleavable or non-cleavable linker.

11. A pharmaceutical composition comprising the double-stranded nucleic acid complex according to claim 1 as an active ingredient.

12. The pharmaceutical composition according to claim 11, for treating a central nervous system disease of a subject.

13. The pharmaceutical composition according to claim 11, which is administered intracerebroventricularly or intrathecally.

14. The pharmaceutical composition according to claim 13, wherein the double-stranded nucleic acid complex is administered at 0.1 mg to 200 mg.

15. The pharmaceutical composition according to claim 11, which is administered intravenously or subcutaneously.

16. The pharmaceutical composition according to claim 15, wherein the double-stranded nucleic acid complex is administered at 0.1 mg / kg to 100 mg / kg.

17. The pharmaceutical composition according to claim 11, wherein the induction of inflammation or gliosis, or the abnormal increase in cytokines or chemokines is reduced.

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