Heteronucleotides 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

[Technical Field]

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

[0002] In recent years, oligonucleotides have attracted attention in the ongoing development of drugs 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 is a method that involves introducing a complementary oligonucleotide (antisense oligonucleotide: often referred to as "ASO (Antisense Oligonucleotide)" in this specification) into cells, using a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand, thereby selectively modifying or inhibiting the expression of proteins encoded by the target gene or the activity of miRNA.

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

[0004] Double-stranded nucleic acid complexes possess high antisense efficacy and represent a groundbreaking technology that enables control of the central nervous system across the blood-brain barrier. However, high doses are required for the treatment of neurological diseases such as Alzheimer's disease, and serious liver damage can be a problem as a side effect.

[0005] Therefore, there is a need for technologies that can reduce the toxicity of double-stranded nucleic acid complexes without compromising their effectiveness. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2013 / 089283 [Patent Document 2] International Publication No. 2014 / 192310 [Non-patent literature]

[0007] [Non-Patent Document 1] Nishina K, et. al., "DNA / RNA heteroduplex oligonucleotide for highly efficient gene silencing", Nature Communication, 2015, 6:7969. [Non-Patent Document 2] Asami Y, et al., "Drug delivery system of therapeutic oligonucleotides", Drug Discoveries & Therapeutics. 2016; 10(5):256-262. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The objective is to provide a double-stranded nucleic acid complex with reduced toxicity without compromising efficacy. [Means for solving the problem]

[0009] The inventors of the present invention conducted intensive research to solve the above problems, and introduced 2'-O,4'-C-spirocyclopropylene-crosslinked nucleic acid (scpBNA) or amide-crosslinked nucleic acid (AmNA) into a double-stranded nucleic acid complex. As a result, it was found that the introduction of scpBNA or AmNA can dramatically reduce or eliminate the toxicity of the double-stranded nucleic acid complex. This toxicity suppression effect is a surprising effect that greatly exceeds the conventional expectations. Furthermore, it was found that the introduction of scpBNA or AmNA does not impair the effectiveness of the double-stranded nucleic acid complex. 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 its transcript, 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, and the first nucleic acid strand and / or the second nucleic acid strand has at least one crosslinked unnatural nucleoside represented by the following formula (I) or formula (II):

Chemical formula

[0011] According to the present invention, a double-stranded nucleic acid complex is provided in which toxicity is reduced without impairing efficacy. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the structures of various cross-linked nucleic acids. [Figure 2] Figure 2 shows the structures of various natural and unnatural nucleotides. [Figure 3]Figure 3 is a schematic diagram of the nucleic acid structure used in Example 1. Figure 3A shows the structure of ASO(LNA) containing three LNA nucleosides at the 5' and 3' ends, with ten DNA nucleosides in between. Figure 3B shows the structure of HDO(LNA) containing 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. Figure 3C shows the structure of ASO(scpBNA) containing three scpBNA nucleosides at each of the 5' and 3' ends, with ten DNA nucleosides in between. Figure 3D shows the structure of HDO(scpBNA) containing 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] Figure 4 shows the levels of Mapt mRNA expression in the hippocampus of mice administered various nucleic acid agents intravenously. Error bars indicate the standard error. [Figure 5] Figure 5 shows the expression levels of TNFα mRNA and GFAP mRNA in the hippocampus of mice administered various nucleic acid agents intravenously. Figure 5A shows the expression level of TNFα mRNA. Figure 5B shows the expression level of GFAP mRNA. Error bars indicate the standard error. [Figure 6] Figure 6 is a schematic diagram of the nucleic acid structure 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] Figure 7 shows the expression levels of malat1 mRNA in mice that received a single intravenous dose of a double-stranded nucleic acid complex. Figure 7A shows the expression levels of malat1 mRNA in the liver. Figure 7B shows the expression levels of malat1 mRNA in the kidney. Error bars indicate the standard error. [Figure 8] Figure 8 shows the expression levels of malat1 mRNA in mice that received a single intravenous dose of a double-stranded nucleic acid complex. Figure 8A shows the expression levels of malat1 mRNA in the quadriceps femoris muscle. Figure 8B shows the expression levels of malat1 mRNA in the myocardium. Error bars indicate the standard error. [Figure 9]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 targeting malat1. Figure 9A shows the results of serum AST and ALT measurements. Figure 9B shows the body weight measurement results. Error bars indicate the standard error. [Figure 10] Figure 10 is a schematic diagram of the nucleic acid structures 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] Figure 11 shows the expression levels of PTEN mRNA in mice that received a single intravenous dose of a double-stranded nucleic acid complex. 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 the standard error. [Figure 12] Figure 12 shows the expression levels of PTEN mRNA in mice that received a single intravenous dose of a double-stranded nucleic acid complex. Figure 12A shows the expression levels of PTEN mRNA in the quadriceps femoris muscle. Figure 12B shows the expression levels of PTEN mRNA in the myocardium. Error bars indicate the standard error. [Figure 13] 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 targeting PTEN. Figure 13A shows the results of serum AST and ALT measurements. Figure 13B shows the change in body weight before and after administration of the double-stranded nucleic acid complex. Error bars indicate the standard error. [Figure 14] Figure 14 shows the expression levels of SR-B1 mRNA in mice that received a single intravenous dose of a double-stranded nucleic acid complex. Figure 14A shows the expression levels of SR-B1 mRNA in the liver. Figure 14B shows the expression levels of SR-B1 mRNA in the kidney. Error bars indicate the standard error. [Figure 15] Figure 15 shows the expression levels of SR-B1 mRNA in mice that received a single intravenous dose of a double-stranded nucleic acid complex. Figure 15A shows the expression levels of SR-B1 mRNA in the quadriceps femoris muscle. Figure 15B shows the expression levels of SR-B1 mRNA in the myocardium. Error bars indicate the standard error. [Figure 16] 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 targeting SR-B1. Figure 16A shows the results of serum AST and ALT measurements. Figure 16B shows the change in body weight before and after administration of the double-stranded nucleic acid complex. Error bars indicate the standard error. [Figure 17] Figure 17 is a schematic diagram of the nucleic acid structure 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] Figure 18 shows the expression levels of SR-B1 mRNA in mice that received multiple intravenous doses of double-stranded nucleic acid complex agents. 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, intrinsic back muscles, diaphragm, adrenal gland, lung, colon, small intestine, and adipose tissue. Error bars indicate the standard error. [Figure 19] 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 complex agents. For Chol-HDO (LNA), the mice that received the agent died after one administration, so the AST / ALT measurement results after one administration are shown. [Figure 20] Figure 20 shows the results of measuring TNFα in the blood of mice that received a single intravenous administration of a double-stranded nucleic acid complex. 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]Figure 21 shows the results of measuring IP-10 in the blood of mice that received a single intravenous administration of a double-stranded nucleic acid complex. 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] Figure 22 shows the results of measuring RANTES levels in the blood of mice that received a single intravenous administration of a double-stranded nucleic acid complex. Figure 22A shows the results for Toc-HDO(PTEN) and Toc-HDO(Malat1). Figure 22B shows the results for Toc-HDO(SR-B1). [Modes for carrying out the invention]

[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, the first nucleic acid strand and / or the second nucleic acid strand each containing at least one scpBNA or AmNA. The double-stranded nucleic acid complex of the present invention has reduced toxicity such as hepatotoxicity, and reduces the induction of inflammation or gliosis, or the abnormal increase of cytokines or chemokines.

[0014] 1-2. Definitions of Terms In this specification, the “transcript” of the 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, this may include mRNA transcribed from the target gene (including mature mRNA, mRNA precursors, unmodified mRNA, etc.), non-coding RNAs (ncRNAs) such as miRNAs, long non-coding RNAs (lncRNAs), and natural antisense RNAs.

[0015] The "target genes" whose expression is regulated (e.g., suppressed, altered, or modified) by the antisense effect are not particularly limited, but include, for example, genes whose expression is increased in various diseases. Examples of target gene transcripts include SR-B1 mRNA, the transcript of the SR-B1 gene; Malat1 non-coding RNA, the transcript of the Malat1 gene; and DMPK mRNA, the transcript of the DMPK gene. The "target transcript" may be, for example, the genes for scavenger receptor B1 (often referred to as "SR-B1" herein), myotonic dystrophy protein kinase (often referred to as "DMPK" herein), transthyretin (often referred to as "TTR" herein), apolipoprotein B (often referred to as "ApoB" herein), or metastasis-associated lung adenocarcinoma transcript 1 (often referred to as "Malat1" herein), such as their non-coding RNA or mRNA. Sequence ID 19 shows the nucleotide sequence of mouse Malat1 non-coding RNA, and Sequence ID 20 shows the nucleotide sequence of human Malat1 non-coding RNA. Sequence ID 21 shows the nucleotide sequence of mouse SR-B1 mRNA, and Sequence ID 22 shows the nucleotide sequence of human SR-B1 mRNA. Sequence ID 23 shows the base sequence of mouse DMPK mRNA, and Sequence ID 24 shows the base sequence of human DMPK mRNA. Note that Sequence IDs 19-24 all have the mRNA base sequences replaced with DNA base sequences. The base sequence information for these genes and transcripts can be obtained from publicly known databases such as the NCBI (National Center for Biotechnology Information) database (e.g., GenBank, Trace Archive, Sequence Read Archive, BioSample, BioProject).

[0016] In this specification, “antisense oligonucleotide (ASO)” or “antisense nucleic acid” refers to a single-stranded oligonucleotide that contains a nucleotide sequence capable of hybridizing (i.e., complementary) to at least a portion of a target transcript (primarily a transcript of a target gene) and can impart an antisense effect to the target transcript. In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand functions as an ASO, and its target region may include a 3'UTR, 5'UTR, exon, intron, coding region, translation start region, translation termination region, or any other nucleic acid region. The target region of the target transcript can be at least 8 nucleotides long, for example, 10-35 nucleotides, 12-25 nucleotides, 13-20 nucleotides, 14-19 nucleotides, or 15-18 nucleotides, or 13-22 nucleotides, 16-22 nucleotides, or 16-20 nucleotides.

[0017] "Antisense effect" refers to the effect of an ASO (Antisense Organisation) hybridizing to a target transcript (e.g., an RNA sense strand) to regulate the expression or editing of that target transcript. "Regulating the expression or editing of a target transcript" means suppression or reduction of the expression of the target gene or the expression level of the target transcript (in this specification, "expression level of the target transcript" is often referred to as "level of the target transcript"), inhibition of translation, RNA editing, splicing function modification effects (e.g., splicing switch, exon inclusion, exon skipping, etc.), or degradation of the transcript. 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 double helix by annealing with mRNA, which is the transcript of the target gene. This partial double helix 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 the degradation of the target gene's mRNA and the inhibition of the expression of the protein encoded by the target gene at the expression level. Furthermore, the antisense effect can also be induced by targeting introns in the mRNA precursor. Moreover, the antisense effect can also be induced by targeting miRNAs. In this case, inhibition of the miRNA's function can increase the expression of the gene that the miRNA normally regulates. In one embodiment, the regulation of target transcript expression may be achieved by reducing the amount of the target transcript.

[0018] The antisense effect can be measured, for example, by administering the test nucleic acid compound to a subject (e.g., a mouse) and, for example, several days later (e.g., 2 to 7 days later), by measuring the expression level of the target gene or the level (amount) of the 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, if the measured expression level of the target gene or the level of the target transcript is reduced 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), it can be shown that the test nucleic acid compound may produce an antisense effect (e.g., a decrease in the amount of the target transcript).

[0020] The number, type, and position of non-natural nucleotides in the nucleic acid chain can affect the antisense effect provided by the nucleic acid complex. The choice of modification may 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 literature related to antisense methods (e.g., WO 2007 / 143315, WO 2008 / 043753, and WO 2008 / 049085). Furthermore, when the antisense effect of the modified nucleic acid complex is measured, the relevant modification can be evaluated if the measured value obtained in this way is not significantly lower than the measured value of the nucleic acid complex before modification (for example, if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of the measured value of the nucleic acid complex before modification).

[0021] In this specification, "translation product of target gene" means any polypeptide or protein synthesized by translation of the target transcript or the target gene transcript that is the direct target of the nucleic acid complex of the present invention.

[0022] As used herein, the terms “nucleic acid” or “nucleic acid molecule” may refer to a monomeric nucleotide or nucleoside, or to an oligonucleotide composed of multiple monomers.

[0023] In this specification, “nucleic acid chain” or simply “chain” means oligonucleotide or polynucleotide. Nucleic acid chains can be prepared as full-length or partial chains by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerase, ligase, or restriction reactions. Nucleic acid chains may contain native and / or non-native nucleotides.

[0024] A "nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is not limited to but is usually composed of pentofuranosyl sugars, with specific examples including ribose and deoxyribose. The base portion (nucleic acid base) of a nucleoside is usually a heterocyclic base. While not limited to these, examples include adenine, cytosine, guanine, thymine, or uracil, or other modified nucleic acid bases (modified bases).

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

[0026] An "oligonucleotide" is a linear oligomer formed by the covalent linkage of several to tens of hydroxyl groups and phosphate groups of the sugar portion between adjacent nucleotides. A "polynucleotide" is a linear polymer formed by the covalent linkage of tens or more, preferably hundreds or more, nucleotides, which is more numerous than that of an oligonucleotide. Within the oligonucleotide or polynucleotide structure, the phosphate groups are generally considered to form internucleoside bonds.

[0027] In this specification, "natural nucleoside" refers to a nucleoside that exists in nature. Examples include ribonucleosides, which consist of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides, which consist of deoxyribose and a base such as adenine, cytosine, guanine, or thymine. In this specification, ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to as "DNA nucleosides" and "RNA nucleosides," respectively.

[0028] In this specification, "natural nucleotide" refers to a nucleotide that exists in nature, in which a phosphate group is covalently bonded to the sugar portion of the natural nucleoside. Examples include ribonucleotides, known as the building blocks of RNA, in which a phosphate group is bonded to a ribonucleoside, and deoxyribonucleotides, known as the building blocks of DNA, in which a phosphate group is bonded to a deoxyribonucleoside.

[0029] In this specification, "non-natural nucleotide" refers to any nucleotide other than natural nucleotides, and includes modified nucleotides and nucleotide mimetic. In this specification, "modified nucleotide" means a nucleotide having one or more of the following: a modified sugar moiety, a modified nucleoside bond, and a modified nucleic acid base. In this specification, "nucleotide mimetic" refers to a structure used to substitute nucleosides and bonds at one or more positions in an oligomeric compound. Examples of nucleotide mimetics include peptide nucleic acids or morpholino nucleic acids (morpholino linked by -N(H)-C(=O)-O- or other non-phosphodiester bonds). Peptide nucleic acid (PNA) is a nucleotide mimetic having a main chain in which N-(2-aminoethyl)glycine is linked by an amide bond instead of sugar. In this specification, nucleic acid chains containing non-natural oligonucleotides often have desirable properties such as enhanced cellular uptake, enhanced affinity to nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Therefore, they are preferred over natural nucleotides.

[0030] In this specification, "non-natural nucleoside" means any nucleoside other than a natural nucleoside. This includes, for example, modified nucleosides and nucleoside mimetic compounds. In this specification, "modified nucleoside" means a nucleoside having a modified sugar moiety and / or a modified nucleic acid base.

[0031] In this specification, “mimetic” refers to a functional group that substitutes sugars, nucleic acid bases, and / or nucleoside bonds. Generally, mimetics are used in place of sugars or combinations of sugar-nucleoside bonds, and nucleic acid bases are retained for hybridization to a selected target. “Nucreoside mimetic” as used herein includes structures used to substitute sugars at one or more positions in an oligomeric compound, or to substitute sugars and bases, or to substitute bonds between monomer subunits constituting an oligomeric compound. “Oligomer compound” means a polymer of linked monomer subunits that can hybridize to at least one region of a nucleic acid molecule. Examples of nucleoside mimetics include morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar mimetics, such as nucleoside mimetics having non-furanose sugar units.

[0032] "Modified sugar" refers to a sugar that has substitution and / or any alteration from the natural sugar moiety (i.e., the sugar moiety found in DNA(2'-H) or RNA(2'-OH)), and "sugar modification" refers to substitution and / or any alteration from the natural sugar moiety. A nucleic acid chain may optionally contain one or more modified nucleosides containing 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 any other beneficial biological properties to the nucleic acid chain. In certain embodiments, the nucleoside contains a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings, though not limited to them, include the addition of substituents (including 5' and 2' substituents), the formation of bicyclic nucleic acids (bridged nucleic acids, BNA) by crosslinking of non-geminal ring atoms, and the addition of S, N(R), or C(R1)(R2)(R, where R1 and R2 are independently H and C1-C) to the oxygen atom of the ribosyl ring. 12 Examples include substitution with alkyl groups or protecting groups, and combinations thereof.

[0033] Examples of sugar-modified nucleosides include, but are not limited to, nucleosides containing substituents such as 5'-vinyl, 5'-methyl(R or S), 5'-allyl(R or S), 4'-S, 2'-F (2'-fluoro group), 2'-OCH3 (2'-OMe group or 2'-O-methyl group), 2'-O-[2-(N-methylcarbamoyl)ethyl](2'-O-MCE group), and 2'-O-methoxyethyl (2'-O-MOE or 2-O(CH2)2OCH3) substituents. Substituents at the 2' position also include allyl, amino, azide, thio, -O-allyl, and -O-C1-C 10 The following can be selected: alkyl, -OCF3, -O(CH2)2SCH3, -O(CH2)2-ON(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn can independently be H or substituted or unsubstituted C1-C 10 It is alkyl. "2'-modified sugar" refers to a furanosyl sugar modified at the 2' position. Nucleosides containing 2'-modified sugars are sometimes called "2'-sugar-modified nucleosides."

[0034] "Bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are generally referred to as bridged nucleic acids (BNAs). Nucleosides containing a bicyclic sugar moiety may also be referred to as "bridged nucleosides", "bridged unnatural nucleosides", or "BNA nucleosides". A partial example of a bridged nucleic acid is illustrated in FIG. 1.

[0035] The bicyclic sugar may be a sugar in which the carbon atoms at the 2'-position and the 4'-position 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 a bicyclic sugar is 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 integers from 1 to 4, 0 to 2, and 1 to 3, respectively; and R3 represents a hydrogen atom, alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, sulfonyl group, and unit substituents (fluorescent or chemiluminescent labeled molecules, functional groups with nucleic acid cleavage activity, intracellular or nuclear localized signal peptides, etc.)] can be described as having a carbon atom at the 2' position and a carbon atom at the 4' position that are cross-linked by these substituents. Furthermore, with respect to BNA or BNA nucleosides according to a particular embodiment, 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 hydroxyl protecting 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 each represents 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 BNA include methyleneoxy(4'-CH2-O-2')BNA (LNA (Locked Nucleic Acid®, also known as 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, and 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'-methylBNA, (4'-CH(CH3)-O-2')BNA (also known as cEt BNA), (4'-CH(CH2OCH3)-O-2')BNA (cMOE Examples include BNA (also known as BNA), amide BNA (amide-crosslinked nucleic acid) or (4'-C(O)-N(R)-2')BNA (R=H,Me) (also known as AmNA; in Figure 1, R=H is AmNA[NH], R=Me is AmNA[N-Me]), guanidine BNA (also known as GuNA (e.g., in Figure 1, R=H is GuNA[NH], R=Me is GuNA[N-Me])), amine BNA (also known as 2'-Amino-LNA) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substituted), 2'-O,4'-C-spirocyclopropylene-crosslinked nucleic acid (also known as scpBNA), and other BNAs known to those skilled in the art. Non-exclusive examples of such BNA nucleosides include methyleneoxy(4'-CH2-O-2')BNA nucleosides (also known as LNA nucleosides or 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, and oxyamino(4'-CH2-N(R3)-O-2')BNA nucleosides (2',4'-BNA NC Also known as a nucleoside; R=H is 2',4'-BNA NC [NH] Nucleoside, R=Me is 2',4'-BNA NC [N-Me] nucleoside), 2',4'-BNA cocNucleosides, 3'-amino-2',4'-BNA nucleosides, 5'-methyl BNA nucleosides, (4'-CH(CH3)-O-2')BNA nucleosides (also known as cEt nucleosides), (4'-CH(CH2OCH3)-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 is AmNA[NH] nucleosides, R=Me is AmNA[N-M Examples include [e]nucleoside), guanidine BNA nucleoside (also known as GuNA nucleoside (e.g., R=H in Figure 1 is GuNA[NH]nucleoside, R=Me is GuNA[N-Me]nucleoside)), amine BNA nucleoside (also known as 2'-Amino-LNA nucleoside) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl-substituted nucleoside), 2'-O,4'-C-spirocyclopropylene-bridged nucleoside (also known as scpBNA nucleoside), and other BNA nucleosides known to those skilled in the art.

[0036] In this specification, "cationic nucleoside" refers to a modified nucleoside that exists in a cationic form at a certain pH (e.g., human physiological pH (approximately 7.4), the pH of a delivery site (e.g., organelles, cells, tissues, organs, organisms, etc.)) compared to its neutral form (e.g., the neutral form of a ribonucleoside). A cationic nucleoside may contain one or more cationic modifying groups at any position of the nucleoside. In one embodiment, cationic nucleosides include 2'-Amino-LNA nucleosides (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl-substituted nucleosides), aminoalkyl-modified nucleosides (e.g., 2'-O-methyl and 4'-CH2CH2CH2NH2-substituted nucleosides), GuNA nucleosides (e.g., in Figure 3, R=H is GuNA[NH] nucleoside, and R=Me is GuNA[N-Me] nucleoside). Bicyclic nucleosides having a methyleneoxy(4'-CH2-O-2') crosslink are sometimes referred to as LNA nucleosides.

[0037] In this specification, "modified nucleoside bond" refers to a nucleoside bond that has been substituted or modified from a naturally occurring nucleoside bond (i.e., a phosphodiester bond). Modified nucleoside bonds include nucleoside bonds containing a phosphorus atom and nucleoside bonds that do not contain a phosphorus atom. Typical phosphorus-containing nucleoside bonds include phosphodiester bonds, phosphorothioate bonds, phosphorodithioate bonds, phosphotryester bonds (methylphosphotryester bonds and ethylphosphotryester bonds described in U.S. Patent Registration No. 5,955,599), alkylphosphonate bonds (e.g., methylphosphonate bonds described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, and methoxypropylphosphonate bonds described in International Publication No. 2015 / 168172), alkylthiophosphonate bonds, methylthiophosphonate bonds, boranophosphate bonds, and nucleoside bonds containing a cyclic guanidine moiety (e.g., a substructure represented by the following formula (III): [ka] ), 1 to 4 C 1~6 Nucleoside bonds containing an alkyl group-substituted guanidine moiety (e.g., a tetramethylguanidine (TMG) moiety) (e.g., a substructure represented by formula (IV) below: [ka] Examples include, but are not limited to, the nucleoside-interconnected bonds and phosphoramidate bonds used in self-neutralizing nucleic acids (ZONs) described in International Publication No. 2016 / 081600. A phosphorothioate bond refers to a nucleoside-interconnected bond in which the non-bridged oxygen atom of a phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known. Modified nucleoside-interconnected bonds are preferably bonds that have higher nuclease resistance than naturally occurring nucleoside-interconnected bonds.

[0038] If the nucleoside bond has a chiral center, the nucleoside bond may be chiral-controlled. "Chiral-controlled" means that it exists as a single diastereomer with respect to the chiral center, for example, the chiral-bound phosphorus. A chiral-controlled nucleoside bond may be completely chiral pure, or it 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. In this specification, "chiral purity" refers to the proportion of one diastereomer in a mixture of diastereomers, expressed as diastereomer excess (%de), and defined as (diastereomer of interest - other diastereomers) / (total diastereomers) × 100 (%).

[0039] For example, the nucleoside bond is a phosphorothioate bond chiralized to an Rp or Sp configuration, with 1 to 4 carbon atoms. 1~6The internucleoside bond may include an alkyl-substituted guanidine moiety (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 substructure represented by formula (IV)), and / or an internucleoside bond including a cyclic guanidine moiety (e.g., a substructure represented by formula (III)). Methods for preparing chiralally controlled nucleoside bonds are known; for example, phosphorothioate bonds chiralally controlled to Rp or Sp configurations are 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. It can be synthesized according to the methods described in 7913-7922. Chiral-controlled phosphorothioate bonds in Rp or Sp configurations are also known and are known to exert effects such as those described, for example, 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, a chiral-controlled phosphorothioate bond in the Sp configuration is more stable than one in the Rp configuration, and / or a chiral-controlled ASO in the Sp configuration promotes target RNA cleavage by RNase H1, resulting in a more sustained response in vivo. 1-4 C 1~6Methods for preparing internucleoside bonds containing alkyl-substituted guanidine moieties (e.g., TMG moieties) are known and can be synthesized, for example, by following the method described in Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811.

[0040] As used herein, the terms "nucleic acid base" or "base" refer to the base components (heterocyclic portions) that constitute nucleic acids, primarily adenine, guanine, cytosine, thymine, and uracil. In this specification, "nucleic acid base" or "base" includes both modified and unmodified nucleic acid bases (bases) unless otherwise specified. Therefore, unless otherwise specified, purine bases may be either modified or unmodified. Similarly, unless otherwise specified, pyrimidine bases may be either modified or unmodified.

[0041] "Modified nucleic acid base" or "modified base" means any nucleic acid base other than adenine, cytosine, guanine, thymine, or uracil. "Unmodified nucleic acid base" or "unmodified base" (natural nucleic acid base) means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Examples of modified nucleic acid bases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, or N4-methylcytosine; N6-methyladenine or 8-bromoadenine; 2-thio-thymine; and N2-methylguanine or 8-bromoguanine. The modified nucleic acid base is preferably 5-methylcytosine.

[0042] As used herein, the term "complementary" means 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.) via hydrogen bonding. In the present invention, the antisense oligonucleotide region in the first nucleic acid chain does not necessarily have to be perfectly complementary to at least a portion of the target transcript (e.g., the transcript of the target gene), but is acceptable if the base sequence has complementarity of at least 70%, preferably at least 80%, and more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more). The antisense oligonucleotide region in the first nucleic acid chain can hybridize to the target transcript if the base sequence is complementary (typically, if 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 chain does not necessarily need to be perfectly complementary to at least a portion of the first nucleic acid chain; it is acceptable if the base sequence has at least 70%, preferably at least 80%, and more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. The complementary region in the second nucleic acid chain can be annealed if its base sequence is complementary to at least a portion of the first nucleic acid chain. Base sequence complementarity can be determined using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can anneal or hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design an antisense nucleic acid complementary to a target transcript, for example, based on the base sequence information of the target gene.

[0043] Hybridization conditions may vary, including low-stringent and high-stringent conditions. Low-stringent conditions may be relatively low temperature and high salt concentration conditions, for example, 30°C, 2×SSC, 0.1%SDS. High-stringent conditions may be relatively high temperature and low salt concentration conditions, for example, 65°C, 0.1×SSC, 0.1%SDS. The stringency of hybridization can be adjusted by changing conditions such as temperature and salt concentration. Here, 1×SSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0044] In this specification, "subject" refers to the object to which the double-stranded nucleic acid complex or pharmaceutical composition of the present invention is applied. The subject includes individuals, organs, tissues, and cells. When the subject is an individual, it can be any animal, including humans. Examples of non-human animals include various livestock, poultry, pets, and laboratory animals. The subject may also be an individual that needs to have the expression level of a target transcript reduced, or an individual that needs to be treated or prevented from having a disease.

[0045] 1-3. Structure The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand. The specific composition 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 portion 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, and the first nucleic acid strand and / or the second nucleic acid strand have the following formula (I) or formula (II): [ka] (In the formula, R represents a hydrogen atom or a methyl group.) It contains at least one cross-linked non-natural nucleoside represented by .

[0047] The cross-linked non-natural nucleoside represented by formula (I) above is a 2'-O,4'-C-spirocyclopropylene cross-linked nucleic acid, which is primarily referred to as "scpBNA" in this specification.

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

[0049] The number of cross-linked non-natural nucleosides represented by formula (I) or formula (II) in the first and / or second nucleic acid strands constituting the double-stranded nucleic acid complex is at least one, and may be, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, and may be 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less. For example, the number of cross-linked non-natural 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 second nucleic acid strand constituting the double-stranded nucleic acid complex of the present invention may contain only the cross-linked non-natural nucleoside represented by formula (I) and formula (II) above, or only the one represented by formula (II), or both the one represented by formula (I) and formula (II).

[0051] Furthermore, the cross-linked non-natural nucleosides represented by formulas (I) and (II) above may be contained only in the first nucleic acid chain, only in the second nucleic acid chain, or in both the first and second nucleic acid chains.

[0052] The base lengths of the first and second nucleic acid chains 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. Alternatively, the base lengths of the first and second nucleic acid chains 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 chains may be the same length or of different lengths (for example, one may be 1 to 3 bases shorter or longer than the other). The double-stranded structure formed by the first and second nucleic acid chains may include a bulge. The choice of length can be determined by balancing the strength of the antisense effect with the specificity of the nucleic acid chain to the target, among other factors such as cost and synthesis yield.

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

[0054] In one embodiment, the nucleoside of the first nucleic acid chain contains or consists of a deoxyribonucleoside, for example, 70% or more, 80% or more, 90% or more, or 95% or more of the nucleoside of the first nucleic acid chain is a deoxyribonucleoside.

[0055] In one embodiment, the first nucleic acid strand may be a gapmer. In this specification, "gapmer" generally refers to a single-stranded nucleic acid consisting of a central region (DNA gap region) and wing regions directly located at its 5' and 3' ends (referred to as the 5' wing region and the 3' wing region, respectively). The length of the DNA gap region may be 13-22 nucleotides, 16-22 nucleotides, 16-20 nucleotides, or 4-20 nucleotides, 5-18 nucleotides, 6-16 nucleotides, 7-14 nucleotides, or 8-12 nucleotides. In the gapmer, the central region contains at least four consecutive deoxyribonucleosides, and the wing region contains at least one non-natural nucleoside. Although not limited, non-natural nucleosides included in the wing region usually have a higher binding affinity to RNA and higher resistance to nucleases than natural nucleosides. If the non-natural nucleosides constituting the wing region include or consist of cross-linked nucleosides, the gapmer is specifically referred to as a "BNA / DNA gapmer." The number of cross-linked nucleosides included in the 5' wing region and the 3' wing region is at least one, and may be, for example, two or three. The cross-linked nucleosides included in the 5' wing region and the 3' wing region may be continuous or discontinuous within the 5' wing region and the 3' wing region. The cross-linked nucleosides may further include modified nucleic acid bases (e.g., 5-methylcytosine). If the cross-linked nucleosides are LNA nucleosides, the gapmer is specifically referred to as an "LNA / DNA gapmer." If the non-natural nucleosides constituting the 5' wing region and the 3' wing region include or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer." If the non-natural nucleosides constituting the 5' wing region and the 3' wing region contain or consist of morpholino nucleic acids, 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 be at least 2 base lengths, independently of each other, for example, 2 to 10 base lengths, 2 to 7 base lengths, or 3 to 5 base lengths.In one embodiment, the 5'-wing region and / or the 3'-wing region may contain at least one non-natural nucleoside, and may further contain a natural nucleoside. The 5'-wing region and the 3'-wing region may be non-natural nucleosides linked by modified nucleoside bonds such as phosphorothioate bonds, for example, 2'-O-methyl-modified nucleosides.

[0056] The first nucleic acid chain constituting the gapmer may consist, in order from the 5' end, of a cross-linked nucleoside of 2 to 7 nucleotides or 3 to 5 nucleotides (for example, 2 or 3 nucleotides), a ribonucleoside or deoxyribonucleoside of 4 to 15 nucleotides or 8 to 12 nucleotides (for example, 8 or 10 nucleotides), and a cross-linked nucleoside of 2 to 7 nucleotides or 3 to 5 nucleotides (for example, 2 or 3 nucleotides).

[0057] In this specification, nucleic acid chains having a wing region at either the 5' or 3' end are called "hemigapmers," but hemigapmers are also included in the definition of gapmers.

[0058] The 5' wing region and / or 3' wing region of the first nucleic acid chain may contain a cross-linked non-natural nucleoside represented by formula (I) or formula (II). The number of cross-linked non-natural nucleosides represented by formula (I) or formula (II) contained in each region of the 5' wing region or 3' wing region may be at least one, for example, two or more, or three or more, and may be five or less, four or less, three or less, or two or less. For example, it may be one, two, three, or four. In one embodiment, the 5' wing region and / or 3' wing region of the first nucleic acid chain may consist of a cross-linked non-natural nucleoside represented by formula (I) or formula (II).

[0059] In one embodiment, the 5' wing region and / or 3' wing region of the first nucleic acid chain comprises a cross-linked non-natural nucleoside represented by formula (I) or formula (II) and an LNA nucleoside.

[0060] In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand may be a mixmer. In this specification, "mixmer" means a nucleic acid strand that contains alternating natural and non-natural nucleosides with periodic or random segment lengths, and does not contain four or more consecutive deoxyribonucleosides and ribonucleosides. In a mixmer, a mixmer in which the non-natural nucleosides are cross-linked nucleosides and the natural nucleosides are deoxyribonucleosides is specifically referred to as a "BNA / DNA mixmer". The cross-linked nucleosides may be cross-linked non-natural nucleosides represented by formula (I) or formula (II) above. In a mixmer, a mixmer in which the non-natural nucleosides are peptide nucleic acids and the natural nucleosides are deoxyribonucleosides is specifically referred to as a "peptide nucleic acid / DNA mixmer". In a mixmer, a mixmer in which the non-natural 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 may contain any number of types of nucleosides, whether natural or modified nucleosides or nucleoside mimetic compounds. For example, it may have one or two consecutive deoxyribonucleosides separated by a cross-linked nucleoside (e.g., an LNA nucleoside or a cross-linked non-natural nucleoside represented by formula (I) or formula (II) above). The cross-linked nucleoside may further contain a modified nucleic acid base (e.g., 5-methylcytosine).

[0061] The second nucleic acid chain may consist entirely of ribonucleosides and / or modified nucleosides. For example, all nucleosides in the second nucleic acid chain may consist entirely of ribonucleosides. All nucleosides in the second nucleic acid chain may consist entirely of deoxyribonucleosides and / or modified nucleosides, and may not contain any 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. This is so that the second nucleic acid strand can form a partial DNA-RNA heteroduplex with the first nucleic acid strand and be recognized and cleaved by RNaseH. The at least four consecutive ribonucleosides in the second nucleic acid strand are preferably linked by naturally occurring nucleoside bonds, i.e., phosphodiester bonds.

[0063] In further embodiments, the second nucleic acid chain may further include at least two consecutive deoxyribonucleosides in addition to the at least four consecutive ribonucleosides described above. These at least two consecutive deoxyribonucleosides are complementary to the first nucleic acid chain and may be located in a region complementary to the central region of the first nucleic acid chain. These at least two consecutive deoxyribonucleosides may be located on either the 5' or 3' side of the at least four consecutive ribonucleosides, or on both the 5' and 3' sides. Furthermore, these at least two consecutive deoxyribonucleosides may be two, three, four, five, 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 ends of the second nucleic acid chain (5' end, 3' end, or both ends) may be modified nucleosides. Modified nucleosides may contain modified sugars and / or modified nucleic acid bases. Modified sugars may be 2'-modified sugars (e.g., sugars containing a 2'-O-methyl group). Modified nucleic acid bases may also be 5-methylcytosine.

[0065] The second nucleic acid chain may consist, in order from the 5' end, of a modified nucleoside of 2-7 nucleotides or 3-5 nucleotides (e.g., 2 or 3 nucleotides) (e.g., a modified nucleoside containing a 2'-modified sugar), a ribonucleoside or deoxyribonucleoside of 4-15 nucleotides or 8-12 nucleotides (e.g., 8 or 10 nucleotides) (optionally linked by intermodified nucleoside bonds), and a modified nucleoside of 2-7 nucleotides or 3-5 nucleotides (e.g., 2 or 3 nucleotides) (e.g., a modified nucleoside containing a 2'-modified sugar). In this case, the first nucleic acid chain may be a gapmer.

[0066] In one embodiment, the second nucleic acid chain may contain a cross-linked non-natural 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 chain.

[0067] The number of cross-linked nucleosides other than the cross-linked non-natural nucleosides represented by formula (I) or formula (II) above, which are included in the first and / or second nucleic acid strands constituting the double-stranded nucleic acid complex, is not limited, but 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 non-natural nucleosides other than the cross-linked non-natural nucleosides represented by formula (I) or formula (II) above, which are included in the first and / or second nucleic acid strands constituting the double-stranded nucleic acid complex, is not limited, but 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 and / or second nucleic acid strands constituting the double-stranded nucleic acid complex may include, in addition to the cross-linked non-natural nucleoside represented by formula (I) or formula (II), at least one ribose 2'-modified nucleoside. This 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 and / or second nucleic acid strands constituting the double-stranded nucleic acid complex contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one 2'-O-methyl modified nucleoside (2'-O-Me-modified nucleoside).

[0071] In one embodiment, the first and / or second nucleic acid strands constituting the double-stranded nucleic acid complex contain at least one cross-linked non-natural nucleoside represented by 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 and / or second nucleic acid strands constituting the double-stranded nucleic acid complex contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and 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 chain may contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one ribose 2'-modified nucleoside on its 3' side, and the 3' wing region of the first nucleic acid chain may contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one ribose 2'-modified nucleoside on its 5' side.

[0074] In one embodiment, the 5' wing region of the first nucleic acid chain may contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one ribose 2'-modified nucleoside on its 5' side, and the 3' wing region of the first nucleic acid chain may contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one ribose 2'-modified nucleoside on its 5' side.

[0075] In one embodiment, the 5' wing region of the first nucleic acid chain may contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one ribose 2'-modified nucleoside on its 3' side, and the 3' wing region of the first nucleic acid chain may contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one ribose 2'-modified nucleoside on its 3' side.

[0076] In a preferred embodiment, the 5' wing region of the first nucleic acid chain may contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one ribose 2'-modified nucleoside on its 5' side, and the 3' wing region of the first nucleic acid chain may contain at least one cross-linked non-natural nucleoside represented by formula (I) or formula (II) and at least one ribose 2'-modified nucleoside on its 3' side.

[0077] The first nucleic acid chain and / or the second nucleic acid chain may contain, in whole or in part, nucleoside mimetic or nucleotide mimetic. The nucleotide mimetic may be peptide nucleic acid and / or morpholino nucleic acid.

[0078] The nucleoside bonds in the first and second nucleic acid chains may be naturally occurring nucleoside bonds and / or modified nucleoside bonds. While not limited to these, it is preferable that at least one, at least two, or at least three nucleoside bonds from the ends (5' end, 3' end, or both ends) of the first and / or second nucleic acid chains are modified nucleoside bonds. Here, for example, the two nucleoside bonds from the end of the nucleic acid chain refer to the nucleoside bond closest to the end of the nucleic acid chain and the adjacent nucleoside bond located on the opposite side of the end. Modified nucleoside bonds in the terminal region of the nucleic acid chain are preferable because they can suppress or inhibit undesirable degradation of the nucleic acid chain.

[0079] In one embodiment, all or part of the nucleoside bonds of the first nucleic acid chain and / or the second nucleic acid chain may be modified nucleoside bonds. In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain 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 nucleoside bonds. In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain 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%, at least 93%, at least 95%, at least 98%, or 100% modified nucleoside bonds. In one embodiment, the modified nucleoside bonds may be phosphorothioate bonds.

[0080] In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain may each contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, thirty, thirty, thirty, thirty, thirty, forty, forty, fifty, fifty, or more chiral-controlled nucleoside bonds. In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain 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 nucleoside bonds.

[0081] In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain 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 unloaded nucleoside bonds (preferably neutral nucleoside bonds). In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain 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 unloaded nucleoside bonds.

[0082] In one embodiment, at least one, at least two, or at least three nucleoside bonds from the 5' end of the second nucleic acid chain may be modified nucleoside bonds. At least one, at least two, or at least three nucleoside bonds from the 3' end of the second nucleic acid chain may be modified nucleoside bonds, such as phosphorothioate bonds, or 1 to 4 C 1~6The modified nucleoside bond may include a guanidine moiety substituted with an alkyl group (e.g., the TMG moiety) (e.g., a substructure represented by formula (IV)) and / or a nucleoside bond including a cyclic guanidine moiety (e.g., a substructure represented by formula (III)). The modified nucleoside bond may be chiralized to an Rp configuration or an Sp configuration.

[0083] At least one (e.g., three) nucleoside bonds at the 3' end of the second nucleic acid strand may be modified nucleoside bonds, such as phosphorothioate bonds, which have high RNase resistance. It is preferable to include modified nucleoside bonds, such as phosphorothioate modifications, at the 3' end of the second nucleic acid strand because this improves the gene repression activity of the double-stranded nucleic acid complex.

[0084] In one embodiment, the modified nucleoside bonds of the first and / or second nucleic acid chains are such that, at a certain pH (e.g., human physiological pH (approximately 7.4), the pH of the delivery site (e.g., organelles, cells, tissues, organs, organisms, etc.)), the modified nucleoside bonds are in an anionic form (e.g., -OP(O)(O) - )-O-(anionic form of the natural phosphate bond), -OP(O)(S -Compared to )-O-(phosphorothioate bond anionic form), etc., it includes unloaded (neutral or cationic) nucleoside bonds that exist in a neutral or cationic form. In one embodiment, the modified nucleoside bonds of the first and / or second nucleic acid chain include neutral nucleoside bonds. In one embodiment, the modified nucleoside bonds of the first and / or second nucleic acid chain include cationic nucleoside bonds. In one embodiment, the unloaded nucleoside bonds (e.g., neutral nucleoside bonds) in their neutral form do not have portions with a pKa less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, or less than 14. In one embodiment, the unloaded electronucleoside bond is, for example, a methylphosphonate bond as described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, a methylphosphotriester bond as described in U.S. Patent Registration No. 5,955,599, an ethylphosphotriester bond, a methoxypropylphosphonate bond as described in International Publication No. 2015 / 168172, or a nucleoside bond used in self-neutralizing nucleic acids (ZONs) as described in International Publication No. 2016 / 081600. In one embodiment, the unloaded electronucleoside bond includes a triazole moiety or an alkyne moiety. In one embodiment, the unloaded electronucleoside bond includes a cyclic guanidine moiety and / or 1 to 4 C atoms. 1~6 It contains a guanidine moiety (preferably a TMG moiety) substituted with an alkyl group. In one embodiment, the modified nucleoside bond containing the cyclic guanidine moiety has a substructure represented by formula (III). In one embodiment, 1 to 4 C 1~6 The alkyl-substituted guanidine moiety has a substructure represented by formula (IV). In one embodiment, the cyclic guanidine moiety and / or 1 to 4 C 1~6The neutral nucleoside bond containing the alkyl group-substituted guanidine moiety is chiral-controlled. In one embodiment, the disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral nucleoside bond and at least one phosphorothioate nucleoside bond. While we do not wish to be bound by any particular theory, in at least some cases, the neutral nucleotide bond can improve properties and / or activity compared to equivalent nucleic acids that do not contain the neutral nucleotide bond, for example, by improving delivery, improving resistance to exonucleases and endonucleases, improving cellular uptake, improving endosomal escape, and / or improving nuclear uptake.

[0085] In one embodiment, the second nucleic acid chain may be bound to a lipid. Examples of lipids include, but are not limited to, tocopherol, cholesterol, fatty acids, phospholipids and their analogues; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstan and their analogues; steroids and their analogues; ligands for LDLR, SRBI, or LRP1 / 2; FK-506, and cyclosporine; and lipids described in PCT / JP2019 / 12077, PCT / JP2019 / 10392, and PCT / JP2020 / 035117.

[0086] In this specification, "tocopherol" is a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) having a cyclic structure called chroman. Tocorol has strong antioxidant properties, and therefore, in the body, it functions as an antioxidant to eliminate free radicals generated by metabolism and protect cells from damage.

[0087] Tocopherol is known to exist in several different forms, consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, based on the position of the methyl group bonded to chroman. In this specification, tocopherol may refer to any of these forms. Furthermore, analogs of tocopherol include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. Preferably, the tocopherol is α-tocopherol.

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

[0089] In this specification, "analog" refers to a compound having the same or similar basic skeleton and similar structure and properties. Analogs include, for example, biosynthetic intermediates, metabolites, and substituted compounds. Whether a compound is an analog of another compound can be determined by common technical knowledge of the art.

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

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

[0092] R c It may also be -(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 attached to the 5' or 3' end of the second nucleic acid chain via a phosphate ester bond.

[0094] Cholesterol or its analogous lipids may be bound to the 5' end, the 3' end, or either end of the second nucleic acid chain. Furthermore, cholesterol or its analogous lipids may be bound to nucleotides within the second nucleic acid chain. While not limited to these, cholesterol or its analogous lipids bound to the 5' end of the second nucleic acid chain are particularly preferred.

[0095] If the second nucleic acid chain contains multiple cholesterol or its analogs, they may be identical or different. For example, this applies when one cholesterol molecule is bound to the 5' end of the second nucleic acid chain and one other cholesterol analog is bound to the 3' end. Regarding binding sites, cholesterol or its analogs may be bound to multiple positions on the second nucleic acid chain, and / or as a group bound to a single position. Cholesterol or its analogs may be linked one at each of the 5' and 3' ends of the second nucleic acid chain.

[0096] The binding between the second nucleic acid chain and the lipid may be direct or indirect, mediated by another substance.

[0097] When the second nucleic acid chain and the lipid are directly bonded, the bond can be via a covalent bond, ionic bond, hydrogen bond, or the like. A covalent bond is preferred because it allows for a more stable bond.

[0098] In one embodiment, the second nucleic acid chain is not bound to a ligand. In this specification, "not bound to a ligand" means that lipids such as tocopherol or cholesterol are not bound to it. In a further embodiment, the double-stranded nucleic acid complex of the present invention is not bound to a ligand, that is, neither the first nor the second nucleic acid chain is bound to a ligand.

[0099] When the second nucleic acid chain and cholesterol or its analogs are indirectly bonded, they may be bonded via a linking group (often referred to herein as "linker"). The linker may be either a cleavable or uncleavable linker.

[0100] A "cleavable linker" refers to a linker that can be cleaved under physiological conditions, for example, within an intracellular or animal body (e.g., within a human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases. Examples of cleavable linkers, though not limited to them, include amides, esters, one or both of phosphodiesters, phosphate esters, carbamates, disulfide bonds, and natural DNA linkers. As an example, cholesterol or its analogues may be linked via disulfide bonds.

[0101] A "non-cleavable linker" refers to a linker that is not cleaved under physiological conditions, for example, within cells or within animals (e.g., within humans). Non-cleavable linkers are not limited to those consisting of phosphorothioate bonds and modified or unmodified deoxyribonucleosides linked by phosphorothioate bonds, or modified or unmodified ribonucleosides. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not particularly limited, but is usually 2 to 20 nucleotides, 3 to 10 nucleotides, or 4 to 6 nucleotides.

[0102] One specific example of a linker is the linker represented by the following equation (VI). [ka] (In the formula, L 2 C1~C (either replaced or not replaced) 12 The alkylene group (e.g., propylene, hexylene, dodecylene), substituted or unsubstituted C3-C8 cycloalkylene group (e.g., cyclohexylene), -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-, -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-, or CH(CH2-OH)-CH2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3- represents L 3 represents -NH- or a bond, L 4C1~C, whether substituted or not. 12 alkylene groups (e.g., ethylene, pentylene, heptylene, andesylene), substituted or unsubstituted C3-8 cycloalkylene groups (e.g., cyclohexylene), -(CH2)2-[O-(CH2)2] m - represents a combination, where m is an integer from 1 to 25, and L 5 represents -NH-(C=O)-, -(C=O)-, or a bond (where the substitution is preferably made by a halogen atom).

[0103] In one embodiment, the linker represented by formula (VI) is L 2 However, it is an unsubstituted C3-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)3-, L 3 However, it is -NH- and L 4 and L 5 However, it is a combination.

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

[0105] The first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain) may further include at least one functional moiety bound to a polynucleotide constituting the nucleic acid chain. The "functional moiety" refers to a moiety that imparts a desired function to the double-stranded nucleic acid complex and / or the nucleic acid chain to which the functional moiety is bound. Examples of desired functions include labeling function and purification function. Examples of moieties that impart labeling function include compounds such as fluorescent proteins and luciferases. Examples of moieties that impart purification function include compounds such as biotin, avidin, His-tagged peptides, GST-tagged peptides, and FLAG-tagged peptides. In one embodiment, from the viewpoint of delivering the first nucleic acid chain to the target site with high specificity and efficiency, and very effectively suppressing the expression of the target gene by the nucleic acid, it is preferable that a molecule having the activity to deliver the double-stranded nucleic acid complex in a certain embodiment to the target site is bound to the second nucleic acid chain as a functional moiety. Examples of moieties that impart target delivery function include lipids, antibodies, aptamers, ligands for specific receptors, etc. In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain) are bound to the functional moiety. The binding between the second nucleic acid chain and the functional moiety may be direct or indirect, mediated by other substances. In one embodiment, however, it is preferable that the second nucleic acid chain and the functional moiety are directly bound by a covalent bond, ionic bond, hydrogen bond, etc., and a covalent bond is more preferable from the viewpoint of obtaining a more stable bond.

[0106] In further embodiments, the second nucleic acid chain may further include at least one overhang region located at either or both of the 5' and 3' ends of the complementary region. An example of this embodiment is described in International Publication No. 2018 / 062510.

[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, since the functional region has the same configuration as the double-stranded nucleic acid complex in this case as well, this specification also includes such single-stranded nucleic acids as one embodiment of the double-stranded nucleic acid complex of the present invention. The linker can be any polymer. Examples include polynucleotides, polypeptides, alkylenes, etc. Specifically, it can be composed of natural nucleotides such as DNA and RNA, or unnatural nucleotides such as peptide nucleic acids or morpholino nucleic acids. When the linker is made of nucleic acid, the chain length of the linker can be at least 1 base, for example, 3 to 10 bases or 4 to 6 bases. Preferably, the chain length is 4 bases. The linker can be located on either the 5' or 3' end of the first nucleic acid strand. For example, in a configuration where cholesterol or its analogues are attached to the 5' end 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 will be linked via the 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 the target transcript can be measured by methods known in the art. For example, after introducing the double-stranded nucleic acid complex into cells, it can be measured using known techniques such as Northern blotting, quantitative PCR, or Western blotting. By measuring the expression level of the target gene or the level of the target transcript (e.g., mRNA level or RNA level such as microRNA, cDNA level, protein level, etc.), it can be determined whether or not the double-stranded nucleic acid complex suppresses the expression of the target gene. The criteria for this determination are not limited, but it may be determined that the double-stranded nucleic acid complex of the present invention has produced an antisense effect if the measured value of the 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 explained, but the double-stranded nucleic acid complex of the present invention is not limited to the exemplary embodiments described above.

[0110] 1-4. Method for producing double-stranded nucleic acid complexes The double-stranded nucleic acid complex of the present invention can be manufactured by those skilled in the art by appropriately selecting known methods. While not limited to these methods, the process typically begins with designing and manufacturing the first and second nucleic acid strands that constitute the double-stranded nucleic acid complex. For example, the first nucleic acid strand is designed based on the base sequence information of the target transcript (e.g., the base sequence of the target gene), and the second nucleic acid strand is designed as its complementary strand. Subsequently, each nucleic acid strand can be synthesized based on the designed base sequence information using commercially available automated nucleic acid synthesizers from companies such as GE Healthcare, Thermo Fisher Scientific, and Beckman Coulter. The resulting oligonucleotides can then be purified using reverse-phase columns or the like.

[0111] Furthermore, in the case of a double-stranded nucleic acid complex to which a functional portion is attached, the first nucleic acid chain may be manufactured according to the method described above. On the other hand, the second nucleic acid chain to which the functional portion is attached can be manufactured by performing the above synthesis and purification using a nucleic acid species to which the functional portion is already attached. For example, the second nucleic acid chain may be manufactured by performing the above synthesis and purification using a nucleic acid species to which cholesterol or its analogs are already attached. Alternatively, cholesterol or its analogs may be attached to the second nucleic acid chain manufactured by performing the above synthesis and purification using a known method. After manufacturing each nucleic acid chain, a double-stranded nucleic acid complex to which the desired functional portion is attached can be manufactured by performing annealing on the first and second nucleic acid chains. Specifically, one of the double-stranded nucleic acid complexes of the present invention can be manufactured by mixing nucleic acids in a suitable buffer solution, denaturing them at approximately 90°C to 98°C for several minutes (for example, 5 minutes), and then annealing the nucleic acids at approximately 30°C to 70°C for approximately 1 to 8 hours. Methods for attaching functional portions to nucleic acids are well known in the art. Furthermore, nucleic acid chains can also be ordered and obtained from various manufacturers (for example, Gene Design Co., Ltd.) by specifying the base sequence, modification sites, and types.

[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 its effectiveness. Specifically, compared to conventional double-stranded nucleic acid complexes, hepatotoxicity and nephrotoxicity are reduced without impairing the antisense effect on target genes, and the induction of inflammation or gliosis, or abnormal increases in cytokines or chemokines, are reduced.

[0113] 2. Pharmaceutical Compositions 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, it reduces the induction of inflammation or gliosis, or the abnormal increase of cytokines or chemokines. The following describes in detail each component that may be included in the pharmaceutical composition of the present invention.

[0114] 2-2. Composition 2-2-1. Active Ingredients The pharmaceutical composition of the present invention contains at least one double-stranded nucleic acid complex as described in the first embodiment as an active ingredient. The pharmaceutical composition of the present invention may contain two or more double-stranded nucleic acid complexes. The amount (content) of double-stranded nucleic acid complexes contained in a 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 later. Therefore, it should be determined appropriately considering each of these conditions. Usually, the pharmaceutical composition is adjusted so that an effective amount of double-stranded nucleic acid complex is contained in a single dose. "Effective amount" refers to the amount necessary for the double-stranded nucleic acid complex to exert its function as an active ingredient, and which does not cause little to no harmful side effects to the organism to which it is applied. This effective amount can change depending on various conditions such as the subject's information, the route of administration, and the number of administrations. Ultimately, it is determined by the judgment of a physician, veterinarian, or pharmacist. "Subject's information" refers to various individual information of the organism to which the pharmaceutical composition is applied. For example, if the subject is human, it includes age, weight, sex, diet, health status, disease progression and severity, drug sensitivity, and presence or absence of concomitant drugs.

[0115] 2-2-2. Carrier The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers. "pharmaceutically acceptable carriers" refers to additives commonly used in the pharmaceutical technology field. Examples include solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavorings, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonic agents, sedatives, bulking agents, disintegrants, buffering agents, 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 any pharmaceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, and sodium acetate buffers. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0117] The above-mentioned carrier is used to avoid or suppress the degradation of the active ingredient, the double-stranded nucleic acid complex, by enzymes in the body, as well as to facilitate formulation and administration methods, and to maintain the dosage form and efficacy. It should be used as appropriate as needed.

[0118] 2-2-3. Dosage Form The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it can deliver the double-stranded nucleic acid complex described in the first embodiment, which is the active ingredient, to the target site without inactivating it by degradation or the like, and exert the pharmacological effect of the active ingredient (antisense effect on the expression of the target gene) in the body.

[0119] The specific dosage form will vary depending on the method of administration and / or prescription conditions. Since the methods of administration can be broadly classified into parenteral administration and oral administration, the dosage form should be appropriate for each method of administration.

[0120] If the method of administration is parenteral, the preferred dosage form is a liquid preparation that can be administered directly to the target site or systemically via the circulatory system. An example of a liquid preparation is an injectable preparation. Injectable preparations can be formulated by mixing them with the aforementioned excipients, elixirs, emulsifiers, suspensions, surfactants, stabilizers, pH adjusters, etc., in a unit dose form generally accepted for pharmaceutical production. Other possible forms include ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, eye drops, and suppositories.

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

[0122] The specific shapes and sizes of each of the above dosage forms are not particularly limited, as long as they fall within the range of dosage forms known in the relevant art. The pharmaceutical composition of the present invention may be manufactured according to conventional methods in the relevant art.

[0123] In certain embodiments, the double-stranded nucleic acid complex of the present invention exhibits excellent solubility in water, Japanese Pharmacopoeia Dissolution Test Solution 2, or Japanese Pharmacopoeia Disintegration Test Solution 2, excellent pharmacokinetics (e.g., drug half-life in blood, brain penetration, 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 hypersedation, avoidance of laminar necrosis), thus possessing excellent properties as a pharmaceutical.

[0124] 2-3. Dosage Form and Dosage In this specification, there are no specific limitations on the preferred mode of administration of a pharmaceutical composition. For example, it may be administered orally or parenterally. Specific examples of parenteral administration include intramuscular, intravenous, intra-arterial, intraperitoneal, subcutaneous (including implantable continuous subcutaneous infusion), intradermal, tracheal / bronchial, rectal, transfusion, intraventricular, intrathecal, nasal, and intramuscular administration.

[0125] When a pharmaceutical composition is administered or ingested, the dosage or intake should be such that the contained double-stranded nucleic acid complex is 0.00001 mg / kg / day to 10000 mg / kg / day, or 0.001 mg / kg / day to 100 mg / kg / day. The pharmaceutical composition may be administered as a single dose or multiple doses. In the case of multiple doses, it may be administered daily or at appropriate time intervals (for example, 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 above double-stranded nucleic acid complex is, 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 The dosage 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. For example, any amount within the range of 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg) can be appropriately selected.

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

[0127] The inhibitory effects of pharmaceutical compositions are additive even with repeated administration. Furthermore, when administering repeatedly, allowing a certain interval between doses (for example, half a day or more) can improve efficacy.

[0128] In one embodiment, the pharmaceutical composition of this embodiment is administered intracerebroventricularly or intrathecally. When the pharmaceutical composition of this embodiment is administered intracerebroventricularly or intrathecally, in the case of monkeys or humans, the dose 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, the dose may be 1 μg or more.

[0129] In one embodiment, the pharmaceutical composition of this embodiment is administered intravenously or subcutaneously. When the pharmaceutical composition of this embodiment is administered intravenously or subcutaneously, it may be administered in doses 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 in doses 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. Applicable Diseases The diseases to which the pharmaceutical composition can be applied are not limited, but may 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 embodiment can be used to treat a central nervous system disease in a subject. Examples of central nervous system diseases to which the pharmaceutical composition of this embodiment can be applied are not particularly limited, but include brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, and the like.

[0131] 2-5. Effects The pharmaceutical composition of the present invention exhibits low hepatotoxicity and / or low nephrotoxicity. Furthermore, the pharmaceutical composition of the present invention reduces the induction of inflammation or gliosis, or abnormal increases in cytokines or chemokines, when administered by intraventricular, intrathecal, intravenous, or subcutaneous administration.

[0132] The pharmaceutical composition of the present invention can treat or prevent diseases by suppressing or inhibiting the expression of specific genes without substantially causing hepatotoxicity and / or nephrotoxicity. Furthermore, it can 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 poses a problem as a side effect of severe liver damage. However, the pharmaceutical composition of the present invention makes it possible to significantly reduce such side effects.

[0134] A method for treating and / or preventing diseases such as central nervous system disorders is also provided, comprising administering the above-mentioned double-stranded nucleic acid complex or pharmaceutical composition to a subject. [Examples]

[0135] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0136] <Example 1: Gene suppression and toxicity reduction effects of intracerebroventricular administration of HDO (scpBNA)> (the purpose) We will investigate the gene suppression and toxicity reduction effects of a heterodouble-stranded oligonucleotide (hereinafter referred to as "HDO(scpBNA)"), which includes a first nucleic acid strand composed of scpBNA / DNA gapmer type antisense nucleic acid (hereinafter referred to as "ASO(scpBNA)") and a second nucleic acid strand composed of RNA having a base sequence complementary to the first nucleic acid strand, by intracerebroventricular administration using in vivo experiments.

[0137] (method) (1) Preparation of nucleic acids Table 1 and Figure 3D show the base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO(scpBNA) used in this example.

[0138] [Table 1]

[0139] The first nucleic acid strand of HDO(scpBNA) is an scpBNA / DNA gapmer type antisense nucleic acid, containing a 5' wing region and a 3' wing region at the 5' and 3' ends, respectively, each composed of three scpBNA nucleosides, with ten DNA nucleosides in between. 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) consists of RNA with a sequence complementary to the first nucleic acid strand. Neither the first nor the second nucleic acid strand of HDO(scpBNA) is bound to a ligand.

[0140] The scpBNA used in the examples of this specification is given by the following formula (I): [ka] It is a non-natural nucleoside represented by [the formula shown].

[0141] As a comparative example to HDO(scpBNA), a heterodouble-stranded oligonucleotide (hereinafter referred to as "HDO(LNA)") containing LNA / DNA gapmer type antisense nucleic acid (hereinafter referred to as "ASO(LNA)") was used. The structures and base sequences of the first and second nucleic acid chains constituting the HDO(LNA) used in this example are shown in Table 2 and Figure 3B. Neither the first nor the second nucleic acid chain of HDO(LNA) is bound to a ligand.

[0142] [Table 2]

[0143] To prepare the double-stranded nucleic acid complex described above, the first and second nucleic acid strands were mixed in equimolar amounts, the solution was heated at 95°C for 5 minutes, and then cooled to 37°C and held for 1 hour to anneal the nucleic acid strands and prepare the double-stranded nucleic acid complex. The annealed nucleic acids were stored at 4°C or on ice. All oligonucleotides were synthesized by Gene Design Co., Ltd. (Osaka, Japan).

[0144] In addition, in this embodiment, the following experiments were also conducted on single-stranded gapmer-type antisense nucleic acids, specifically ASO(scpBNA) consisting only of the first nucleic acid strand as shown in Table 1, and ASO(LNA) consisting only of the first nucleic acid strand as shown in Table 2.

[0145] (2) in vivo experiment Seven-week-old female ICR mice were fixed to a stereotactic device under 2.5-4% isoflurane anesthesia. A 2-3 cm incision was then made between the ears, and a 1 mm diameter drill was used to perforate the area 1 mm to the left and 0.2 mm posterior to the bregma. A Hamilton syringe was filled with nucleic acid. A needle was inserted approximately 3 mm through the perforation site, and 24 μmol / mouse of nucleic acid was administered to the left ventricular cavity 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 mice were subsequently dissected to extract the left hippocampus.

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

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

[0148] To evaluate the gene suppression effect of nucleic acid agents, the expression levels of Mapt mRNA and Actb mRNA (internal standard genes) were measured, and their ratios were calculated. Furthermore, to assess the toxicity of nucleic acid agents, the expression level of Tumor Necrosis Factor-α (TNFα) mRNA was measured as an indicator of inflammatory cytokines, and the expression level of Glial fibrillary acidic protein (GFAP) mRNA was similarly measured as an indicator of astrocyte activation and gliosis.

[0149] (result) Figure 4 shows the level of Mapt mRNA expression in the left hippocampus of mice administered various nucleic acid agents intraventricularly. No significant difference in the suppression effect of the Mapt gene was observed with 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 intraventricularly. ASO(LNA) and HDO(LNA) showed a significant increase in TNFα mRNA and GFAP mRNA expression compared to PBS administration. In contrast, ASO(scpBNA) and HDO(scpBNA) did not show a significant increase in TNFα mRNA and GFAP mRNA expression compared to PBS administration, indicating a reduction in the induction of inflammatory cytokine gliosis.

[0151] These results demonstrate that scpBNA reduces central nervous system side effects without diminishing the gene regulatory effects of conventional LNA, and this mitigating effect was observed not only in single-stranded ASO but also in HDO structures.

[0152] <Example 2: Gene suppression and hepatotoxicity reduction effects of single intravenous administration of Toc-HDO (scpBNA) targeting mMalat1> (the purpose) We will investigate the inhibitory effect on target gene expression and toxicity reduction effects of single intravenous administration of double-stranded nucleic acid complexes consisting of scpBNA / DNA gapmer type antisense nucleic acid and a tocopherol-bound complementary strand, and double-stranded nucleic acid complex consisting of AmNA / DNA gapmer type antisense nucleic acid and a tocopherol-bound complementary strand, targeting mMalat1.

[0153] (method) (1) Preparation of nucleic acids The target gene was metastasis-associated lung adenocarcinoma transcript 1 (mMalat1). The nucleotide 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] [Table 3]

[0155] The first nucleic acid strand described above targets the mMalat1 gene and consists of a 13-mer gapmer having a base sequence complementary to a portion of its transcript, the malat1 non-coding RNA. The first nucleic acid strand of Toc-HDO(LNA) consists of a 5' wing region composed of three LNA nucleosides at the 5' end, a 3' wing region composed of two LNA nucleosides at the 3' end, and eight DNA nucleosides in between. 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 consists of a tocopherol-bound complementary RNA strand that has a sequence complementary to the first nucleic acid strand and has tocopherol bound to its 5' end.

[0157] The AmNA used in the examples of this specification is given by the following formula (II): [ka] (In the formula, R represents a methyl group.) It is a non-natural nucleoside represented by [the formula shown].

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

[0159] (2) in vivo experiment The mice administered with the double-stranded nucleic acid complex were male C57BL / 6 mice weighing 20g and 6-7 weeks old. Experiments were conducted with n=4 for each condition. A double-stranded nucleic acid complex was administered as a single dose to mice via the tail vein at a dose of 50 mg / kg. Furthermore, mice were created as a negative control group, receiving only PBS as a single dose. Blood samples were taken 72 hours after administration, and the mice were perfused with PBS. The mice were then dissected, and the liver, kidneys, myocardium, and quadriceps femoris muscle were removed.

[0160] (3) Expression analysis mRNA was extracted from each tissue according to the protocol using a high-throughput fully automated nucleic acid extraction system, MagNA Pure 96 (Roche Life Sciences). cDNA was synthesized according to the protocol of Transcriptor Universal cDNA Master (Roche Life Sciences). Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). The primers used in quantitative RT-PCR were products designed and manufactured by Thermo Fisher Scientific based on various gene counts. The PCR conditions (temperature and time) were 15 seconds at 95°C, 30 seconds at 60°C, and 1 second at 72°C, repeated for 40 cycles. The resulting amplification products were quantified by quantitative RT-PCR, and the expression levels of malat1 mRNA and GAPDH mRNA (internal standard gene) were calculated. Relative expression levels were obtained from the ratio of the two, and the mean and standard error of the relative expression levels were calculated. Differences between each group were also analyzed by t-test.

[0161] (4) Analysis of serum The serum obtained from blood samples was analyzed by SRL Hachioji Lab Co., Ltd.

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

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

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

[0165] These results demonstrate that by substituting a portion of LNA with scpBNA or AmNA in the wing region of the first nucleic acid chain, the toxicity of HDO can be reduced without diminishing its gene-suppressing effect.

[0166] <Example 3: Gene suppression and hepatotoxicity reduction effects of single intravenous administration of PTEN-targeting Toc-HDO (scpBNA)> (the purpose) We will investigate the inhibitory effect on target gene expression and toxicity reduction effects of single intravenous administration of double-stranded nucleic acid complexes consisting of scpBNA / DNA gapmer type antisense nucleic acid and a tocopherol-bound complementary strand, and double-stranded nucleic acid complex consisting of AmNA / DNA gapmer type antisense nucleic acid and a tocopherol-bound complementary strand, targeting 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] [Table 4]

[0169] The first nucleic acid strand described above targets the PTEN gene and consists 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 consists of a tocopherol-bound complementary RNA strand that has a sequence complementary to the first nucleic acid strand and has 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 performed in the same manner as in Example 2, except that the double-stranded nucleic acid complex was intravenously injected into mice at a dose of 35 mg / kg. Expression analysis and serum analysis were performed in the same manner as in Example 2.

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

[0174] The results of the serum analysis are shown in Figure 13A. In the Toc-HDO(LNA) administration group, AST and ALT levels were significantly elevated, indicating impaired liver function. On the other hand, no significant increases in AST and ALT were detected in the Toc-HDO(scpBNA) administration group or the Toc-HDO(AmNA) administration group, indicating reduced hepatotoxicity.

[0175] Furthermore, the Toc-HDO(LNA) administration group showed a significant decrease in body weight, while no significant changes were observed in the Toc-HDO(scpBNA) administration group or the Toc-HDO(AmNA) administration 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 chain with scpBNA or AmNA, the toxicity of HDO can be reduced without diminishing its gene-repressing effect.

[0177] <Example 4: Gene suppression and hepatotoxicity reduction effects of single intravenous administration of Toc-HDO (scpBNA) targeting SR-B1> (the purpose) We will investigate the inhibitory effect on target gene expression and toxicity reduction effects of single intravenous administration of double-stranded nucleic acid complexes consisting of scpBNA / DNA gapmer type antisense nucleic acid and a tocopherol-bound complementary strand, and double-stranded nucleic acid complex consisting of AmNA / DNA gapmer type antisense nucleic acid and a tocopherol-bound complementary strand, targeting 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] [Table 5]

[0180] The first nucleic acid strand described above targets the SR-B1 gene and consists 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 consists of a tocopherol-bound complementary RNA strand that has a sequence complementary to the first nucleic acid strand and has 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] In vivo experiments, expression analysis, and serum analysis were performed using the same methods as in Example 2.

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

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

[0186] Furthermore, weight loss was observed in the Toc-HDO(LNA) administration group, while no significant change in weight was observed in the Toc-HDO(scpBNA) administration group or the Toc-HDO(AmNA) administration 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 chain with scpBNA or AmNA, the toxicity of HDO can be reduced without diminishing its gene-repressing effect.

[0188] <Example 5: Gene suppression effect and hepatotoxicity reduction effect of multiple intravenous administrations of Chol-HDO (scpBNA)> (the purpose) This study will investigate the inhibitory effect of multiple intravenous administrations of a double-stranded nucleic acid complex consisting of scpBNA / DNA gapmer-type antisense nucleic acid and cholesterol-binding complementary strands, targeting SR-B1, on suppressing target gene expression and reducing toxicity.

[0189] (method) (1) Preparation of nucleic acids 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] [Table 6]

[0191] The first nucleic acid strand described above targets the SR-B1 gene and consists 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 consists of cholesterol-binding complementary RNA strands that have a sequence complementary to the first nucleic acid strand and have cholesterol bound to their 5' end.

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

[0194] (2) in vivo experiment The mice administered with the double-stranded nucleic acid complex were male C57BL / 6 mice weighing 20g and 6-7 weeks old. Experiments were conducted with n=4 for each condition. The double-stranded nucleic acid complex was administered intravenously to mice via the tail vein at a dose of 50 mg / kg in multiple doses (once a week, for a total of four doses). Furthermore, a negative control group of mice was created that received only PBS as a single dose. Blood samples were taken 72 hours after administration, and the mice were perfused with PBS. The mice were then dissected, and various parts of the brain and the entire body were removed.

[0195] (3) Expression analysis mRNA was extracted from each tissue according to the protocol using a high-throughput fully automated nucleic acid extraction system, MagNA Pure 96 (Roche Life Sciences). cDNA was synthesized according to the protocol of Transcriptor Universal cDNA Master (Roche Life Sciences). Quantitative RT-PCR was performed using TaqMan (Roche Life Sciences). The primers used in quantitative RT-PCR were products designed and manufactured by Thermo Fisher Scientific based on various gene counts. The PCR conditions (temperature and time) were 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second per cycle, repeated for 40 cycles. The resulting amplification products were quantified by quantitative RT-PCR, and based on the results, the expression levels of SR-B1 mRNA and Actin mRNA (internal standard gene) were calculated, and the relative expression levels were obtained from the ratio of the two. The mean and standard error of the relative expression levels were calculated. The differences between each group were analyzed by t-test.

[0196] (4) Analysis of serum The serum obtained from blood samples was analyzed by SRL Hachioji Lab Co., Ltd.

[0197] (result) The results of the gene expression analysis are shown in Figure 18. Chol-HDO (scpBNA) showed a strong gene suppression effect in the brain (Figure 18A) and in all organs (Figure 18B). The results of the serum analysis are shown in Figure 19. In the Chol-HDO(LNA) administration group, AST / ALT levels were significantly elevated 3 days after the first dose, and death occurred 5-6 days later. In contrast, in the Chol-HDO(scpBNA) administration group, patients survived even after 4 doses, and the elevation of AST / ALT levels was mild.

[0198] These results demonstrate that even with multiple doses, the toxicity of HDO is reduced by positioning scpBNA in the wing region of the first nucleic acid chain.

[0199] <Example 6: Effect of single intravenous administration of Toc-HDO (scpBNA) on reducing serum inflammatory cytokine / chemokine expression> (the purpose) This study investigates the expression of inflammatory cytokines / chemokines in the blood after intravenous administration of a double-stranded nucleic acid complex consisting of scpBNA / DNA gapmer-type antisense nucleic acid and a tocopherol-bound complementary strand.

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

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

[0202] (3) Quantification of inflammatory cytokines / chemokines in the blood Blood samples were analyzed using the MILLIPLEX MAP Mouse Cytokine / Chemokine Magnetic Bead Panel - Immunology Multiplex (Millipore) according to the instructions provided. To evaluate the toxicity of nucleic acid agents, the following protein groups were targeted: 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 after Toc-HDO(LNA) administration, were measured as indicators of inflammation.

[0203] (result) Figures 20-22 show the results of measuring the amount of inflammatory cytokines in the blood. As shown in Figures 20 to 22, the expression levels of TNFα (Figure 20), IP-10 (Figure 21), and RANTES (Figure 22) were lower in the Toc-HDO(scpBNA) and Toc-HDO(AmNA) administration groups compared to the Toc-HDO(LNA) administration group. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A double-stranded nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid strand can hybridize to at least a portion of the target gene or its transcript, and has an antisense effect on the target gene or its transcript. The second nucleic acid chain contains a base sequence complementary to the first nucleic acid chain. The first nucleic acid chain is, (1) A central region containing at least four consecutive deoxyribonucleosides, (2) A 5' wing region containing a non-natural nucleoside, located on the 5' end side of the central region, (3) The central region includes a 3' wing region located on the 3' end side, which contains a non-natural nucleoside, The 5' wing region and / or the 3' wing region in the first nucleic acid chain are given by the following formula (I) or formula (II): 【Chemistry 1】 (In the formula, R represents a hydrogen atom or a methyl group.) The double-stranded nucleic acid complex comprising at least one cross-linked non-natural nucleoside and an LNA nucleoside represented by .

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

3. The double-stranded nucleic acid complex according to claim 1, wherein the second nucleic acid chain further comprises at least two consecutive deoxyribonucleosides.

4. The double-stranded nucleic acid complex according to claim 1, wherein the second nucleic acid strand contains a cross-linked non-natural 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.

5. The double-stranded nucleic acid complex according to claim 1, wherein the first nucleic acid chain and / or the second nucleic acid chain comprises at least one ribose-2' 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.

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

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

8. The second nucleic acid chain is bound to tocopherol or a tocopherol analog, or cholesterol or a cholesterol analog. Tocopherol analogs are selected from the group consisting of α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. The double-stranded nucleic acid complex according to claim 1, wherein the cholesterol analog is selected from the group consisting of cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

9. A 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 linked via a cleavable or non-cleavable linker.

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

12. A pharmaceutical composition according to claim 11 for treating a central nervous system disorder in a subject.

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

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

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 a dose of 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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