BBB-crossing lipid ligands of heterogeneous nucleic acids

Abstract

To provide a nucleic acid agent that is efficiently delivered to the nervous system, particularly the central nervous system where drug delivery is hindered by the BBB mechanism, and that exerts an antisense effect on a target transcript at the delivery site; and to provide a composition containing the same.SOLUTION: The present invention provides a double-stranded nucleic acid complex formed by annealing a first nucleic acid strand that hybridizes to a portion of a target transcript and has an antisense effect on the target transcript, and a second nucleic acid strand that includes a base sequence complementary to the first nucleic acid strand and is bound to tocopherol or an analog thereof, cholesterol or an analog thereof, or a substituted or unsubstituted C1-30 alkyl group, a substituted or unsubstituted C2-30 alkenyl group, or a substituted or unsubstituted C1-30 alkoxy group.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to nucleic acid complexes capable of producing antisense effects in the nervous system, particularly the central nervous system. The present invention relates to a composition comprising the compound or a salt thereof. [Background technology]

[0002] In recent years, oligonucleotides have been used in the ongoing development of medicines called nucleic acid drugs. It has attracted a lot of attention, especially in terms of its high target gene selectivity and low toxicity. The development of nucleic acid medicines using the antisense method is being actively promoted. The partial sequence of mRNA or miRNA transcribed from the target gene is used as the target sense strand, and the complementary Antisense oligonucleotides (often referred to herein as "ASOs") By introducing the “AntiSense Oligonucleotide” (hereinafter “AntiSense Oligonucleotide”) into cells, the target gene is detected. Selectively modify or inhibit the expression of gene-encoded proteins or the activity of miRNAs The method includes:

[0003] In Patent Document 1, a first oligomer compound and a second oligomer compound containing a conjugated group such as cholesterol are used. The present invention relates to a method for the treatment of hepatitis, comprising administering to a patient a therapeutically effective amount of a hepatic enzyme comprising the steps of: A double-stranded nucleic acid molecule capable of regulating the amount or activity of a target nucleic acid, and a nucleic acid comprising the double-stranded nucleic acid molecule Antisense compounds that

[0004] In order to exert an antisense effect in the central nervous system including the brain, the AS Nucleic acid drugs such as O need to be delivered to the central nervous system. The blood-brain barrier (BLB) selects and restricts substances that are transferred. There is a mechanism called the BBB (often abbreviated as BBB) that blocks harmful substances. While protecting the brain from the effects of the immune system, it also acts as a barrier to drug delivery to the brain. There is a need for methods to deliver nucleic acid agents, such as ASOs, to the nervous system. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2017 / 053999 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a method for efficiently delivering drugs to the nervous system, particularly the central nervous system, where drug delivery is hindered by the BBB mechanism. and a nucleic acid agent that exerts an antisense effect on a target transcript at the site of delivery, The objective of the present invention is to provide a composition comprising the above. [Means for solving the problem]

[0007] In order to solve the above problems, the present inventors have conducted extensive research and have found that ASO and tocopherol The ASO is annealed to a complementary strand of the ASO bound to ethanol, cholesterol, or its analogues. The nucleic acid complex containing the nucleotide was efficiently delivered to the central nervous system, where it showed a high antisense effect. Based on these findings, the present inventors have completed the present invention. The present invention includes the following aspects.

[0008] [1] A nucleic acid complex or a salt thereof comprising a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid strand comprises a salt capable of hybridizing to at least a portion of a target transcript. It contains a base sequence and has an antisense effect on a target transcript, The second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, and (1) Tocopherol or its analogues, (2) Cholesterol or its analogues, or (3) Substituted or unsubstituted C1~ 30 The alkyl groups, substituted or unsubstituted is unsubstituted C2~ 30 or a substituted or unsubstituted alkenyl group of the formula: Not here C1~ 30 Alkoxy group It is combined with The nucleic acid complex or a salt thereof, wherein the first nucleic acid strand is annealed to the second nucleic acid strand. [2] (1) Tocopherol or its analogues, (2) Cholesterol or its analogues, or (3) Substituted or unsubstituted C1~ 30 The alkyl groups, substituted or unsubstituted is unsubstituted C2~ 30 or a substituted or unsubstituted alkenyl group of the formula: Not here C1~ 30 Alkoxy group a nucleic acid strand linked to the target transcript, said nucleic acid strand hybridizing to at least a portion of the target transcript; and having an antisense effect on a target transcript. The nucleic acid chain or a salt thereof. [3] The tocopherol or its analogue has the following formula (I): [ka] (In the formula, R 1 is a substituted or unsubstituted C1~ 30 Alkyl or substituted Substituted or unsubstituted C2~ 30 (represents an alkenyl group) The nucleic acid complex, nucleic acid strand, or salt thereof according to [1] or [2], [4] The cholesterol or its analogue is represented by the following formula (II), (V), (VI), or (VII): : [ka] (In the formula, Ring A is a substituted or unsubstituted cyclohexane or a substituted or unsubstituted cyclohexane. represents unsubstituted benzene, Ring B is a substituted or unsubstituted cyclohexene, a substituted or unsubstituted Unsubstituted cyclohexane or substituted or unsubstituted cyclohexadiene represents the Ring C is a substituted or unsubstituted cyclohexene or a substituted or unsubstituted cyclohexene. represents unsubstituted cyclohexane, R 2 is a hydrogen atom, a substituted or unsubstituted C 30 Alkyl groups, substituted Substituted or unsubstituted C2~ 30 alkenyl groups, substituted or unsubstituted C1-6 alkyl-carbonyl group, substituted or unsubstituted C1 ~6 alkyl-carbonyl-oxy group or oxo group, R 3 represents a hydrogen atom, R 2 and R 3 together form a substituted or unsubstituted 1,6-dioxa may form a spiro[4.5]decane ring, L 1 is -O-, -NH-, [ka] or [ka] ), [ka] (In the formula, R 4 is a substituted or unsubstituted C1~ 30 The alkyl group or the substituent Substituted or unsubstituted C2~ 30 represents an alkenyl group; [ka] (In the formula, R 5 and R 6 each independently represents a substituted or unsubstituted C1 ~ 30 or a substituted or unsubstituted C2- 30 Alkenyl represents a group), and

[0009] [ka] (In the formula, R 7 is a hydroxy group, a substituted or unsubstituted C 30 Alki a substituted or unsubstituted C1- 30 Arke represents the aryl-carbonyl-oxy group) The nucleic acid complex according to any one of [1] to [3], which is represented by a formula selected from the group consisting of: , a nucleic acid chain, or a salt thereof. [5] The cholesterol or its analogue has the following formula (IIa): [ka] (In the formula, Ring A is a substituted or unsubstituted cyclohexene, a substituted or unsubstituted Unsubstituted cyclohexane or substituted or unsubstituted cyclohexadiene represents the R 1 is a substituted or unsubstituted C1~ 30 The alkyl group, if substituted, Mostly unsubstituted C2 30 Alkenyl groups, substituted or unsubstituted, C1-6 alkyl-carbonyl group, substituted or unsubstituted C1-6 alkyl represents a aryl-carbonyl-oxy group or an oxo group; L 1 is -O-, -NH-, [ka] or [ka] (represents The nucleic acid complex, nucleic acid strand, or salt thereof according to [1] or [2], [6] The cholesterol or its analogue is represented by the following formula (IIa-1), (IIa-2), or (IIa-3): And (IIa-4): [ka] (IIa-1), TIFF2025063018000012.tif3265 (IIa-2), TIFF2025063018000013.tif3265 (IIa-3), TIFF2025063018000014.tif3865 (IIa-4) (In the formula, L 1 is -O-, -NH-, [ka] or [ka] ), The nucleic acid complex or the nucleic acid chain according to [1] or [2], Or its salt. [7] The second nucleic acid strand has the following formula (VIII): [ka] (In the formula, L 2 is a substituted or unsubstituted C1~ 12 alkylene groups, or unsubstituted C3-8 cycloalkylene group, -(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-, L 3 represents -NH- or a bond, L 4 is a substituted or unsubstituted C1~ 12 alkylene groups, or an unsubstituted C3-8 cycloalkylene group, -(CH2)2-[O-(CH 2)2] m - or a bond, where m is an integer of 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond. Through a linker represented by (1) Tocopherol or its analogues, (2) Cholesterol or its analogues, or (3) Substituted or unsubstituted C1~ 30 The alkyl groups, substituted or unsubstituted is unsubstituted C2~ 30 or a substituted or unsubstituted alkenyl group of the formula: Not here C1~ 30 Alkoxy group The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of [1] to [6], wherein the nucleic acid complex, nucleic acid strand, or salt thereof is bound to: [8] The first nucleic acid strand comprises at least four consecutive deoxyribonucleosides. The nucleic acid complex, the nucleic acid strand, or a salt thereof according to any one of [1] to [7]. [9] The nucleic acid according to any one of [1] to [8], wherein the first nucleic acid strand is a gapmer. A complex, a nucleic acid chain, or a salt thereof.

[10] The second nucleic acid strand is a nucleic acid sequence that is a sequence of at least four consecutive deoxyribonucleic acid residues in the first nucleic acid strand. [8] or The nucleic acid complex, the nucleic acid strand, or a salt thereof according to [9].

[11] The nucleic acid according to any one of [1] to [7], wherein the first nucleic acid strand is a mixmer. An acid complex, a nucleic acid strand, or a salt thereof.

[12] The method according to any one of [1] to

[11] , wherein the first nucleic acid strand has a length of 13 to 20 bases. The nucleic acid complex, nucleic acid strand, or salt thereof described above.

[13] Any of [1] to

[12] , wherein the second nucleic acid strand does not contain natural ribonucleosides. The nucleic acid complex, the nucleic acid strand, or a salt thereof according to any one of the above.

[14] The nucleic acid portions of the second nucleic acid strand are linked by modified or unmodified internucleoside bonds. The nucleic acid complex, nucleic acid chain, or salt thereof according to any one of [1] to

[0013] , which comprises a deoxyribonucleoside and / or a sugar-modified nucleoside linked thereto.

[15] A nucleic acid complex, a nucleic acid chain, or a salt thereof according to any one of [1] to

[14] . A composition for regulating the expression or editing of a target transcript in the central nervous system of a subject.

[16] The composition described in

[15] , which is for treating a central nervous system disease in a subject.

[17] The composition according to

[16] , wherein the central nervous system disease is an immune-mediated central nervous system disease. .

[18] A nucleic acid complex, a nucleic acid chain, or a salt thereof according to any one of [1] to

[14] . A composition for delivering an agent to the central nervous system of a subject.

[19] The central nervous system includes the cerebral cortex, the basal ganglia, the cerebral white matter, the diencephalon, the brain stem, the cerebellum, and The composition according to any one of

[15] to

[18] , selected from the group consisting of spinal cord.

[20] The central nervous system includes the frontal lobe, the temporal lobe, the hippocampus, the parahippocampal gyrus, the parietal lobe, the occipital lobe, and the striatum. , globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigra, pons, medulla oblongata, cerebellar cortex, cerebellar nuclei, cervical spinal cord, thoracic The composition according to any one of

[15] to

[18] , selected from the group consisting of spinal cord and lumbar spinal cord.

[21] The composition according to any one of

[15] to

[20] , which is for intravenous or subcutaneous administration. Composition.

[22] A nucleic acid complex, a nucleic acid strand, or a salt thereof, containing 5 mg / kg or more of the nucleic acid complex, the nucleic acid strand, or a salt thereof in a single dose. The composition according to any one of

[15] to

[21] .

[23] The composition described in any of

[0015] to

[22] , wherein the nucleic acid complex, nucleic acid strand, or salt thereof passes through the blood-brain barrier (BBB).

[24] The method according to

[17] , wherein the immune-mediated central nervous disease is a microglia-associated disease. Composition.

[25] The microglia-related disease is Alzheimer's disease, multiple sclerosis, ALS, or The composition described in

[24] , which is for neuropathic pain.

[26] To regulate the expression or editing of target transcripts in microglia,

[15] The composition described in any one of

[25] . This specification incorporates the disclosures of Japanese Patent Application No. 2018-055372, which is the priority basis of this application. Includes. Effect of the Invention

[0010] The present invention provides a method for efficiently delivering a gene to the central nervous system and exerting an antisense effect at the site of delivery. It is possible to provide a nucleic acid agent and a composition comprising the same. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing an example of a specific embodiment of a nucleic acid complex used in the present invention. In this diagram, four patterns are shown according to the binding position of tocopherol or cholesterol in the second nucleic acid strand. FIG. 1a shows a nucleic acid complex in which tocopherol is bound to the 5' end of the second nucleic acid strand. FIG. 1b shows a nucleic acid complex in which cholesterol is bound to the 5' end of the second nucleic acid strand. FIG. 1c shows a nucleic acid complex in which tocopherol is bound to the 3' end of the second nucleic acid strand. FIG. 1d shows a nucleic acid complex in which cholesterol is bound to the 3' end of the second nucleic acid strand. [Diagram 2] Figure 2 shows an example of the general mechanism of the antisense method. In the figure, "X" indicates the site of suppression or inhibition in the process from gene expression to translation. The figure within the dashed line is a schematic diagram showing how the heteroduplex portion is recognized by RNase H and the mRNA of the target gene is degraded. [Diagram 3] FIG. 3 shows the structures of various bridged nucleic acids. [Figure 4] FIG. 4 shows the structures of various natural and non-natural nucleotides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] A first aspect of the present invention is a nucleic acid complex, more preferably a blood-brain barrier penetrating nucleic acid complex. The nucleic acid complex includes a first nucleic acid strand and a second nucleic acid strand. The second nucleic acid strand is complementary to the first nucleic acid strand. In the nucleic acid complex, the first nucleic acid strand is a nucleotide chain that contains a base sequence similar to that of the second nucleic acid strand. In one embodiment, the second nucleic acid strand is annealed to (1) tocopherol or (2) cholesterol or its analogues; or (3) substituted or unsubstituted Unconverted C1~ 30 Alkyl groups of substituted or unsubstituted C2-3 0 alkenyl group, or a substituted or unsubstituted C 30 Alcoki (In this specification, the above (1) to (3) are often collectively referred to as “tocopherol”) (Also written as "cholesterol or cholesterol or its analogues, etc.")

[0013] A representative schematic diagram of the nucleic acid complex is shown in Figure 1. Figure 1a shows a tocopherol-containing nucleic acid complex having a tocopherol-containing 5' end at the second nucleic acid strand. Figure 1b shows a nucleic acid complex with cholesterol attached to the 5' end of the second nucleic acid strand. FIG. 1c shows a nucleic acid complex in which tocopherol is bound to the 3' end of the second nucleic acid strand. FIG. 1d shows a nucleic acid complex in which cholesterol is bound to the 3' end of the second nucleic acid strand. However, as described below, tocopherol or cholesterol or The analogues thereof may be bound to the 5' end, 3' end or both ends of the second nucleic acid strand. It may be attached to an internal nucleotide of the nucleic acid strand.

[0014] In one embodiment, the first nucleic acid strand hybridizes to at least a portion of the target transcript. In a particular embodiment, the first The nucleic acid strand is a nucleic acid having an antisense effect on the transcription product of a target gene or a target transcription product. It is a nucleotide chain.

[0015] In one embodiment, the present invention relates to a method for producing a tocopherol-based composition comprising: (1) tocopherol or an analog thereof; (3) substituted or unsubstituted C1 ~ 30 Alkyl groups of substituted or unsubstituted C2~ 30 an alkenyl group of or substituted or unsubstituted C1~ 30 is bonded to the alkoxy group of The nucleic acid strand corresponds to the first nucleic acid strand of the nucleic acid complex, and is a target nucleic acid strand. A nucleotide sequence capable of hybridizing to at least a portion of a target transcript is provided. In certain embodiments, the nucleic acid strand is a transcription product of a target gene or a target transcription product. It is a nucleotide chain that has an antisense effect on the transcript.

[0016] (Definition of terms) As used herein, a "target transcript" refers to a transcript that can be targeted by the nucleic acid complex of the present invention and is a DNA. Any RNA synthesized by A-dependent RNA polymerase. Generally, Specifically, the target gene is transcribed into mRNA (mature mRNA, pre-mRNA, etc.). non-coding RNA (including mRNA with no base modification), miRNA, etc. gRNA, ncRNA).

[0017] As used herein, the term "target gene" is not particularly limited, but may be, for example, the nucleic acid complex of the present invention. Genes from the organism into which the fusion is introduced, e.g. genes whose expression increases in various diseases Target transcripts include those derived from genomic DNA that encodes the target gene. mRNA that is transcribed, and also mRNA that has not undergone base modifications, processed "Target transcript" includes not only mRNA but also non-coding RNA such as miRNA. More generally, a "transcript" can refer to a DNA-dependent RNA or a non-coding RNA (ncRNA). In one embodiment, the target RNA may be any RNA synthesized by an endogenous RNA polymerase. The target transcript is, for example, scavenger receptor B1 (scavenger receptor B1 Scavenger receptor B1 (often referred to as "SR-B1 mRNA" in this specification) ) or metastasis associated lung adenocarcinoma transcript 1 pt 1: often referred to herein as "Malat1") may be a non-coding RNA. SEQ ID NO:3 is the nucleotide sequence of mouse Malat1 non-coding RNA, and SEQ ID NO:4 is the nucleotide sequence of human Mal The base sequence of at1 non-coding RNA is shown. SEQ ID NO:5 shows the base sequence of mouse SR-B1 mRNA. The base sequence of human SR-B1 mRNA is shown in SEQ ID NO:6, and the base sequence of mouse DM The base sequence of human DMPK mRNA is shown in SEQ ID NO: 8. In all of the above, the base sequence of mRNA is replaced with the base sequence of DNA. The base sequence information of the transcripts is available, for example, from the NCBI (National Center for Biotechnology Information) database. These can be obtained from known databases such as:

[0018] As used herein, the term "antisense oligonucleotide (ASO)" or "antisense nucleic acid An "acid" refers to at least a portion of a target transcript, e.g., a nucleic acid capable of hybridizing to any target region. It contains a complementary base sequence capable of suppressing the expression of the transcription product of the target gene by the antisense effect. Alternatively, it refers to a single-stranded oligonucleotide capable of suppressing and regulating the level of a target transcript. In the nucleic acid complex, the first nucleic acid strand functions as an ASO, and its target regions are the 3'UTR, 5'UTR, exon 1, and 2'UTR. A sequence, intron, coding region, translation initiation region, translation termination region, or any other nucleic acid region. The target region of the target transcript may include a region of at least 8 bases in length, for example, 10 to 3 May be 5 bases long, 12-25 bases long, 13-20 bases long, 14-19 bases long, or 15-18 bases long. can.

[0019] The "antisense effect" refers to the hybridization of an ASO to a target transcript (e.g., the sense strand of RNA). The effect of regulating expression or editing of the target transcript is measured by encoding the target transcript. "Modulating the expression or editing of a target transcript" means modulating the expression of a target gene or the editing of a target transcript. The expression level of a target transcript (as used herein, the "expression level of a target transcript" is often referred to as the "level of a target transcript"). suppression or reduction of the transcription factor, inhibition of translation, alteration of splicing function (e.g., For example, as shown in Figure 2, In the inhibition of , oligonucleotides (e.g., RNA) are introduced into cells as ASOs, and the ASOs bind to the target It binds to mRNA, a gene transcription product, to form a partial double strand. acts as a cover to prevent translation by ribosomes, thereby targeting Expression of the protein encoded by the gene is inhibited at the translational level (Figure 2, dashed cross mark). On the other hand, when DNA-containing oligonucleotides are introduced into cells as ASOs, they are transformed into partial DNA-RNA fragments. This heteroduplex is recognized by RNase H, resulting in the formation of a target The mRNA of the target gene is degraded, and expression of the protein encoded by the target gene is suppressed at the expression level. This is called the "RNase H-dependent pathway." In examples, the antisense effect can also be achieved by targeting an intron of a pre-mRNA. Antisense effects can also be achieved by targeting miRNAs. In this case, the function of the miRNA is inhibited and the gene whose expression the miRNA normally controls is suppressed. In one embodiment, the modulation of expression of the target transcript can be achieved by increasing the amount of the target transcript. It may be a decrease.

[0020] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a nucleic acid molecule that is a monomer. If it is an oligomer, it is an oligonucleotide. If it is a polymer, it is a If so, it means a polynucleotide.

[0021] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of the side is usually, but not limited to, composed of a pentofuranosyl sugar, specific examples of which include Examples of nucleosides include ribose and deoxyribose. The base portion of a nucleoside (nucleobase) is , usually a heterocyclic base moiety. Examples include, but are not limited to, adenine, cytosine, guanine, Examples include thymine or uracil, and other modified nucleic acid bases (modified bases).

[0022] "Nucleotide" refers to 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 2', 3', or 5' position of the sugar is usually A phosphate group is linked to the hydroxyl group.

[0023] An "oligonucleotide" is a compound in which the hydroxyl groups of the sugar moiety and the ribozyme are bonded between adjacent nucleotides. A linear oligomer formed by linking several to several tens of phosphate groups through covalent bonds. A "polynucleotide" refers to a nucleic acid having more nucleosides than an oligonucleotide. The peptide is formed by linking several tens or more, preferably several hundreds or more of peptides by the covalent bond. The term "internal" refers to a linear polymer formed from an oligonucleotide or polynucleotide structure. In, the phosphate groups are commonly considered to form the internucleoside linkages.

[0024] As used herein, the term "nucleic acid strand" or simply "strand" refers to an oligonucleotide or means polynucleotide. Nucleic acid chains can be synthesized chemically, for example, using an automated synthesizer. or by enzymatic steps using polymerases, ligases, or restriction reactions to produce full length chains or A nucleic acid strand can be made up of natural nucleotides and / or non-natural nucleotides. It may contain tides.

[0025] As used herein, the term "natural nucleoside" refers to a nucleoside that occurs in nature. For example, a nucleic acid consisting of ribose and the bases adenine, cytosine, guanine, or uracil Ribonucleosides, deoxyribose and the adenine, cytosine, guanine, or thymine Deoxyribonucleosides, which consist of bases such as ribonucleoside and ribonuclease, are also known. The ribonucleosides found in ribonucleosides and deoxyribonucleosides found in DNA are called "DNA nucleosides," respectively. Similarly, they are also called "natural nucleotides." " refers to a naturally occurring nucleotide in which a phosphate group is attached to the sugar moiety of the natural nucleoside. Covalently linked molecules. For example, ribonucleosides are linked to phosphate groups, forming the building blocks of RNA. The units known as ribonucleotides and deoxyribonucleosides are joined by phosphate groups. Examples of such nucleic acids include deoxyribonucleotides, which are known as the building blocks of DNA.

[0026] As used herein, the term "unnatural nucleoside" refers to any nucleoside other than a natural nucleoside. Nucleosides include, for example, modified nucleosides and nucleoside mimetics. In this context, "modified nucleoside" refers to a nucleoside having a modified sugar moiety and / or a modified nucleobase. Nucleic acid chains containing non-natural oligonucleotides are often used, for example, in cell cultures. enhanced binding, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or They are preferred over the native forms due to desirable properties such as increased inhibitory activity.

[0027] As used herein, a "mimetic" refers to a compound that replaces the sugar, nucleobase, and / or internucleoside linkage. In general, mimetics are functional groups that modify the sugar or sugar-internucleoside linkage combination. Instead, the nucleobase is used for hybridization to a selected target. A "nucleoside mimetic" is a nucleoside mimetic that is at least one position of an oligomeric compound. To replace sugars, or to replace sugars and bases, or to form oligomeric compounds "Oligo" includes structures used to replace the bonds between monomer subunits. A "mer compound" is a linked monoclonal antibody that is at least capable of hybridizing to a region of a nucleic acid molecule. A nucleoside mimetic refers to a polymer of mer subunits. Nucleoside mimetics include, for example, morpholino lino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar Mimetics include, for example, nucleoside mimetics that have non-furanose sugar units.

[0028] As used herein, a "bicyclic nucleoside" is a modified nucleoside that contains a bicyclic sugar moiety. Nucleic acids containing bicyclic sugar moieties are commonly called bridged nucleic acids (BNAs). As used herein, nucleosides that contain a bicyclic sugar moiety are referred to as "bridged nucleosides." Some examples of cross-linked nucleic acids are shown in Figure 3.

[0029] Bicyclic sugars are those in which the 2' and 4' carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. One subgroup is 4'-(CH2) p -O-2',4'-(CH2) p -CH2-2',4'-(CH2) p -S-2',4'-(CH2) p - OCH2O-2',4'-(CH2) n -N(R3)-O-(CH2) m -2' [wherein p, m and n are each an integer of 1 to 4, 0 R3 represents a hydrogen atom, an alkyl group, an alkenyl group, Cycloalkyl groups, aryl groups, aralkyl groups, acyl groups, sulfonyl groups, and units Substituents (fluorescent or chemiluminescent labeling molecules, functional groups with nucleic acid cleavage activity, intracellular or nuclear The 2'-position carbon atom and the 4'-position carbon atom are bridged by a peptide (representing a signal peptide for intracellular localization, etc.) Further, with respect to the BNA according to certain embodiments, the carbon atom at the 3' position can be described as having In the OR2 substituent on the atom and the OR1 substituent on the 5' carbon atom, R1 and R2 are typically are hydrogen atoms, but may be the same or different. Protective groups for the hydroxyl group, alkyl groups, alkenyl groups, cycloalkyl groups, aryl groups alkyl groups, aralkyl groups, acyl groups, sulfonyl groups, silyl groups, phosphate groups, for nucleic acid synthesis or P(R4)R5, where R4 and R5 are the same as each other; They may be the same or different, and serve as a protecting group for hydroxyl groups and nucleic acid synthesis, respectively. Hydroxyl groups, mercapto groups, and nucleic acid synthesis protecting groups are used to protect the A protected mercapto group, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 6 carbon atoms; a cyanoalkoxy group having 1 to 6 carbon atoms; or an amino group substituted with an alkyl group having 1 to 5 carbon atoms. Non-limiting examples of such BNAs include methyleneoxy (4'-CH2-O-2') BNAs (LNAs (Lo 2',4'-BNA), e.g., α-L-methyl 4'-CH2-O-2')BNA or β-D-methyleneoxy(4'-CH2-O-2')BNA, ethyleneoxy β-D-thio (4'-CH2-S-2') BNA, α-D-thio (4'-(CH2)2-O-2') BNA (also known as ENA), Aminooxy (4'-CH2-ON(R3)-2') BNA, oxyamino (4'-CH2-N(R3)-O-2') BNA (2',4'-BNA NC Also known as 2',4'-BNA coc , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(C (H3)-O-2') BNA (also known as cEt BNA), (4'-CH(CH2OCH3)-O-2') BNA (also known as cMOE BNA) Amide BNA (4'-C(O)-N(R)-2') BNA (R = H, Me) (also known as AmNA) ), 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (also known as scpBNA), and Other BNAs known to those of skill in the art include methyleneoxy (4'-CH2-O-2') Bridged bicyclic nucleosides are sometimes referred to as "LNA nucleosides."

[0030] As used herein, a "non-natural nucleotide" refers to any nucleotide other than a naturally occurring nucleotide. As used herein, the term "modified nucleotide" refers to a nucleotide that is a nucleotide or nucleotide mimic, and includes modified nucleotides and nucleotide mimetics. "Modified nucleotide" means any of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase. It means a nucleotide having one or more of the above.

[0031] A "nucleotide mimetic" refers to a compound that is a nucleoside at one or more positions of an oligomeric compound. Nucleotide mimetics include structures used to replace bonds and linkages. Nucleotide mimetics include, for example, For example, peptide nucleic acids or morpholino nucleic acids (-N(H)-C(=O)-O- or other non-phosphodiester bonds) Peptide Nucleic Acid (PNA) PNA) has a backbone in which N-(2-aminoethyl)glycine is linked by amide bonds instead of sugar. An example of the structure of a morpholino nucleic acid is shown in FIG. In this regard, nucleic acid chains containing non-natural oligonucleotides are often used, for example, to enhance cellular uptake. the ability to selectively bind to a nucleic acid target, increase affinity for the nucleic acid target, increase stability in the presence of nucleases, or decrease inhibitory activity They have desirable properties such as increased nucleic acid sequence and are therefore preferred over natural nucleotides.

[0032] As used herein, a "modified internucleoside bond" refers to a naturally occurring internucleoside bond. Nucleosides with substitutions or any changes from the phosphodiester bond (i.e., Modified internucleoside linkages include phosphorus-containing internucleoside linkages that contain a phosphorus atom. These include non-phosphorus-containing internucleoside linkages and non-phosphorus-containing internucleoside linkages that do not contain a phosphorus atom. Internucleotide bonds include phosphodiester bonds, phosphorothioate bonds, and phosphorodithioate bonds. Oate bond, phosphotriester bond, alkylphosphonate bond, alkylthiophos Examples of such bonds include phosphate bonds, boranophosphate bonds, and phosphoramidate bonds. Phosphorothioate bonds are non-bridging acids of phosphodiester bonds. The internucleoside bond is a bond in which a phosphorus atom is replaced by a sulfur atom. Methods for preparation are well known. Modified internucleoside linkages are naturally nuclease resistant. Higher linkages than internucleoside linkages are preferred.

[0033] As used herein, a "modified nucleic acid base" or a "modified base" refers to adenine, cytosine, guanine, "Nucleobases" refers to any nucleobase other than cytosine, thymine, or uracil. Examples of modified nucleobases include The following are available: 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, N6-methyladenine, 8-bromoadenine, N2-methylguanine, or 8 Preferred modified nucleobases include, but are not limited to, 5-bromoguanine. An "unmodified nucleobase" or "unmodified base" is a base that is the same as a natural nucleobase. The purine bases adenine (A) and guanine (G), as well as the pyrimidine base It stands for thymine (T), cytosine (C), and uracil (U).

[0034] As used herein, a "modified sugar" refers to a sugar that is a natural sugar moiety (i.e., DNA (2'-H) or RNA (2'-OH) As used herein, the term "sugar" refers to sugars that have substitutions and / or any changes from the sugar moieties found in In the present invention, the nucleic acid strand may contain one or more modified nucleosides, optionally containing modified sugars. Sugar-modified nucleosides may provide enhanced nuclease stability, increased binding affinity, or some other benefit. Nucleosides are chemically modified ribofuranoses that can confer beneficial biological properties to nucleic acid chains. Examples of chemically modified ribofuranose rings include, but are not limited to, However, addition of substituents (including 5' and 2' substituents) and bridging of non-geminal ring atoms may result in bifunctionalization. Formation of cyclic nucleic acids (bridged nucleic acids, BNAs), the ribosyl ring oxygen atom is S, N(R), or C(R1)(R2)(R, R1 and R2 are each independently H, C1-C 12 alkyl, or protecting groups), and As used herein, the term "nucleoside having a modified sugar moiety" includes any of the above combinations. Examples include, but are not limited to, 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F (2'- fluoro group), 2'-OCH3 (2'-OMe group or 2'-O-methyl group), and 2'-O(CH2)2OCH3 substituents. The 2'-position substituent may also be an allyl, amino, azido, thio, -O-Allyl, -O-C1-C 10 Alkyl, -OCF3, -O(CH2)2SCH3, -O(CH2)2-ON(Rm)(Rn), and OC H2-C(=O)-N(Rm)(Rn), each Rm and Rn is independently H or substituted or unsubstituted. or unsubstituted C1-C 10 As used herein, a "2'-modified sugar" is a sugar that is modified at the 2' position. It means a furanosyl sugar.

[0035] Methods for preparing modified sugars are well known to those of skill in the art. The nucleobase moiety (natural, modified, or a combination thereof) is capable of hybridizing with an appropriate nucleic acid target. The cells may be maintained for denaturation.

[0036] Generally, modifications are made such that nucleotides in the same strand can be independently modified differently. In addition, the same nucleic acid sequence can be used to confer resistance to enzymatic cleavage. wherein the nucleotides have modified internucleoside linkages (e.g., phosphorothioate linkages), Additionally, they can have modified sugars (e.g., 2'-O-methyl modified sugars or bicyclic sugars). The nucleotides may also have modified nucleobases (e.g., 5-methylcytosine) and may further include modified It can have sugars (eg, 2'-O-methyl modified sugars or bicyclic sugars).

[0037] The number, type and position of non-natural nucleotides in a nucleic acid strand are determined by the nucleic acid complex of the present invention. The choice of modification may affect the sequence of the target gene, etc. Although the sequence of the antisense molecule may vary depending on the sequence, those skilled in the art will be familiar with the literature related to antisense technology (e.g., WO 2014 / 023363). 2007 / 143315, WO 2008 / 043753, and WO 2008 / 049085). In addition, the antisense nucleic acid complex after modification can be determined. When the effect is measured, the measured value thus obtained is compared with that of the nucleic acid complex before modification. If the measured value obtained after modification is not significantly lower than that of the nucleic acid complex before modification (for example, ≥ 70%, ≥ 80%, or ≥ 90% of the measured values), the relevant modification can be evaluated. Cut.

[0038] The term "complementary" as used herein means that the nucleic acid bases are connected through hydrogen bonds, i.e. Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteiger base pairs) In the present invention, the first nucleic acid strand is a target nucleic acid strand capable of forming a base pair (e.g., a quinone base pair). Perfect complementarity to at least a portion of a transcript (e.g., a transcript of a target gene) Although not necessarily required, the base sequence is at least 70%, preferably at least 80%, and More preferably, at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementary. Similarly, the complementary region in the second nucleic acid strand is at least It is not necessary that the base sequence is completely complementary to any part of the sequence. , preferably at least 80%, even more preferably at least 90% (e.g., 95%, 96 %, 97%, 98%, or 99% or greater) is acceptable.

[0039] As used herein, the term "blood-brain barrier (BBB)" refers to the blood-brain barrier (BBB) ​​that provides access to the brain, as described above. It is a mechanism for selecting and restricting substances and plays a role in protecting the brain from harmful substances.

[0040] As used herein, the term "central nervous system" refers to the organs that comprise the brain and spinal cord, and which are connected to the peripheral nervous system. Both are tissues that make up the nervous system. The brain is made up of the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon, It includes the brainstem (midbrain, substantia nigra, pons, medulla oblongata), cerebellum (cerebellar cortex, cerebellar nuclei), and The spinal cord includes the cervical, thoracic, lumbar, sacral and coccygeal spinal cord. Any of these regions may be involved, but the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), The cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brain stem, cervical spinal cord, thoracic spinal cord, or lumbar spinal cord is preferred.

[0041] As used herein, the term "salt thereof" refers to a salt of the nucleic acid complex of the present invention. Physiologically and pharma- ceutically acceptable salts of the complexes, i.e., the desired nucleic acid complexes, A salt that retains biological activity and does not impart undesired toxicological effects. Examples of such salts include alkali salts such as sodium salts, potassium salts, and lithium salts. Metal salts; alkaline earth metal salts such as calcium salts, magnesium salts; aluminum salts, Metal salts such as iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; inorganic salts such as ammonium salts Organic salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, Phenylglycine alkyl ester salt, ethylenediamine salt, guanidine salt, diethylamine salt salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylene Diamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl -Phenethylamine salt, piperazine salt, tetramethylammonium salt, tris(hydroxy amine salts such as dimethylaminomethane salts, diolamine salts, and meglumine salts; Hydrohalides such as perchlorates, hydrochlorides, hydrobromides, hydroiodides; nitrates, Inorganic acid salts such as perchlorates, sulfates, and phosphates; methanesulfonates, trifluoromethanesulfonates Sulfonates, lower alkanesulfonates such as ethanesulfonates; benzenesulfonates phosphates, arylsulfonates such as p-toluenesulfonates; acetates, malates , fumarate, succinate, citrate, tartrate, oxalate, maleate, and other organic salts salts of glycine, lysine, arginine, ornithine, glutamate, Included are amino acid salts such as aspartate.

[0042] In certain embodiments, the nucleic acid complexes of the present invention are any pharma- ceutically acceptable form of the nucleic acid complex. Suitable pharma- ceutically acceptable salts include those of the nucleic acid complex, esters of the nucleic acid complex, or salts of the esters. Acceptable salts include sodium, potassium and meglumine salts. Not limited.

[0043] (Configuration of the first and second nucleic acid strands) In one aspect, the present invention relates to a nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand.

[0044] The first nucleic acid strand comprises a base pair capable of hybridizing to at least a portion of the target transcript. A single-stranded oligonucleotide that contains a sequence and exerts an antisense effect on a target transcript. It's a chain.

[0045] The second nucleic acid strand is a single-stranded oligonucleotide strand that contains a base sequence complementary to the first nucleic acid strand. The second nucleic acid strand is bound to tocopherol or cholesterol or its analogue. In the nucleic acid complex, the second nucleic acid strand binds to the first nucleic acid strand through hydrogen bonds of complementary base pairs. It's Neil.

[0046] In one embodiment, the present invention relates to a method for producing a tocopherol-based composition comprising: (1) tocopherol or an analog thereof; (3) substituted or unsubstituted C1 ~ 30 Alkyl groups of substituted or unsubstituted C2~ 30 an alkenyl group of or substituted or unsubstituted C1~ 30 is bonded to the alkoxy group of It relates to a single-stranded nucleic acid chain. The single-stranded nucleic acid is composed of tocopherol or cholesterol. or an analog thereof. The structure of the acetylcholine or cholesterol or its analogues and the form of its linkage with the nucleic acid chain are as follows: As described herein in the nucleic acid complex.

[0047] The base length of the first nucleic acid strand and the second nucleic acid strand is usually at least 8 bases long, at least 9 bases long, At least 10 bases in length, at least 11 bases in length, at least 12 bases in length, at least 13 bases in length, The length may be at least 14 bases or at least 15 bases, but is not particularly limited. The base length of the first nucleic acid strand and the second nucleic acid strand is 35 bases or less, 30 bases or less, 25 bases or less, 24 ≤ 23 bases, ≤ 22 bases, ≤ 21 bases, ≤ 20 bases, ≤ 19 bases , 18 bases or less, 17 bases or less, or 16 bases or less. The strand may be about 100 bases long, or may be 10-35 bases long, 12-25 bases long, 13-20 bases long, The first nucleic acid strand and the second nucleic acid strand may be 14 to 19 bases long, or 15 to 18 bases long. Even if they are the same length, they may be of different lengths (e.g., one of them is 1-3 bases shorter or longer). The double-stranded structure formed by the first nucleic acid strand and the second nucleic acid strand may include a bulge. The choice of length may depend on, among other factors, e.g., cost, synthesis yield, etc., the antisense oligonucleotide sequence. This can be determined by the balance between the strength of the ionizing effect and the specificity of the nucleic acid strand to the target. .

[0048] The internucleoside linkages in the first and second nucleic acid strands are naturally occurring nucleosides. The first nucleic acid strand and the second nucleic acid strand may be, but are not limited to, an internucleoside linkage and / or a modified internucleoside linkage. and / or at least one from the end (5' end, 3' end or both ends) of the second nucleic acid strand, At least two or at least three internucleoside linkages are modified internucleoside linkages. Here, for example, the two internucleoside bonds from the end of the nucleic acid chain are The internucleoside bond closest to the end and the adjacent internucleoside bond opposite the end The modified internucleoside bond in the terminal region of the nucleic acid chain is This is preferred because it can suppress or inhibit undesired degradation of the nucleic acid strand. All internucleoside linkages of the nucleic acid strand and / or the second nucleic acid strand are modified internucleoside linkages. The modified internucleoside linkage may be a phosphorothioate linkage.

[0049] At least one (e.g., three) internucleoside bond(s) from the 3'-terminus of the second nucleic acid strand are RNase resistant. The internucleoside linkage may be a modified internucleoside linkage, such as a phosphorothioate linkage, which has high affinity for the second nucleoside. When the 3' end of the nucleic acid chain contains a modified internucleoside bond such as a phosphorothioate modification, This is preferable because it improves the gene suppression activity of the strand nucleic acid complex.

[0050] At the 5' and 3' ends of the second nucleic acid strand, tocopherol or cholesterol or The internucleoside bonds of the 2 to 6 bases at the end that are not bound to the analogues are modified nucleosides. The bond may be an inter-mer bond (eg, a phosphorothioate bond).

[0051] At least one (e.g., three) nucleosides from the 3' end of the second nucleic acid strand are cleaved by, for example, RNase The second nucleic acid strand may be a modified nucleoside such as 2'F-RNA or 2'-OMe, which is highly resistant. Gene suppression activity of double-stranded nucleic acid complexes containing modified nucleosides such as 2'F-RNA and 2'-OMe This is preferable because it increases

[0052] At the 5' and 3' ends of the second nucleic acid strand, tocopherol or cholesterol or The terminal 1 to 5 nucleosides that are not bound to the analogues are, for example, 2'F, which is highly RNase resistant. - It may be a modified nucleoside such as RNA.

[0053] The nucleosides in the first and second nucleic acid strands are natural nucleosides (deoxyribonucleic acid). nucleosides, ribonucleosides, or both) and / or non-natural nucleosides good.

[0054] As used herein, the base sequence of the first nucleic acid strand corresponds to at least a part of the base sequence of the target transcript. Because it is complementary, it is capable of hybridizing (or annealing) to the target transcript. The complementarity of a base sequence can be determined by using a BLAST program or the like. Those skilled in the art can determine the conditions (temperature) under which the two strands can hybridize, taking into account the degree of complementarity between the strands. In addition, those skilled in the art can easily determine the concentration of the target gene, e.g. Based on the gene sequence information, antisense nucleic acids complementary to the target transcript can be easily designed. It can also be calculated.

[0055] Hybridization conditions can be, for example, low stringency conditions and high stringency conditions. The conditions may be of various stringency, such as low stringency conditions. The conditions are relatively low temperature and high salt concentration, for example, 30°C, 2xSSC, 0.1% SDS. High stringency conditions are relatively high temperature and low salt conditions, for example, 65°C. , 0.1×SSC, 0.1% SDS. By changing the conditions such as temperature and salt concentration, The stringency of hybridization can be adjusted. Here, 1×SSC contains 150 mM salt. Contains 15 mM sodium chloride and 15 mM sodium citrate.

[0056] The first nucleic acid strand is recognized by RNase H when hybridized to the target transcript. At least 4, at least 5, at least 6, or at least 7 consecutive nuclei It can contain nucleotides. Usually, it is a sequence of 4 to 20 bases, 5 to 16 bases, or 6 to 12 bases. Any region containing nucleosides is acceptable. Nucleosides recognized by RNase H are For example, natural deoxyribonucleosides can be used. Suitable nucleosides, including bonucleosides and other bases, are well known in the art. In addition, nucleosides having a hydroxy group at the 2' position, such as ribonucleosides, can be used as the nucleoside. It is also known that "at least four consecutive nucleotides are not suitable as nucleotides." The suitability of a nucleoside for use in this region, including "nucleosides," can be readily determined. In one embodiment, the first nucleic acid strand comprises at least four consecutive deoxyribonucleic acids. It may include

[0057] In one embodiment, the entire length of the first nucleic acid strand does not consist of only natural ribonucleosides. The first nucleic acid strand contains no or less than half of the natural ribonucleosides in its entirety. is preferred.

[0058] In one embodiment, the second nucleic acid strand is a sequence of at least four consecutive nucleotides as described above in the first nucleic acid strand. At least four consecutive ribonucleosides complementary to a ribonucleoside (e.g., a deoxyribonucleoside) The second nucleic acid strand may contain a nucleotide. The second nucleic acid strand may be a partial DNA-RNA heteroduplex with the first nucleic acid strand. This is to form a small nucleotide sequence in the second nucleic acid strand that can be recognized and cleaved by RNase H. At least four consecutive ribonucleosides are preferably naturally occurring nucleosides. They are linked by inter-linkages, i.e., phosphodiester bonds.

[0059] The second nucleic acid strand is a nucleic acid in which all nucleosides are ribonucleosides and / or modified nucleosides. All nucleosides of the second nucleic acid strand may be composed of deoxyribonucleosides. It may be composed of ribonucleosides and / or modified nucleosides and does not contain ribonucleosides. This is also fine.

[0060] The first and / or second nucleic acid strands constituting the nucleic acid complex of the present invention are gapmers. As used herein, the term "gapmer" refers to a region having a central region (a DNA gap region). The 5' wing region and the 3' wing region are arranged on both sides of the 5' end and the 3' end of the 5' wing region. The central region in a gapmer is a single-stranded nucleic acid consisting of at least four consecutive and the 5' wing region and the 3' wing region are non-naturally occurring nucleic acid. The non-natural nucleosides constituting the 5' wing region and the 3' wing region are When the gapmer comprises or consists of a bridged nucleoside, it is specifically referred to as a "BNA / DNA gapmer." The non-natural nucleosides that make up the 5' and 3' wing regions are called "champers." When the gapmer comprises or consists of a peptide nucleic acid, the gapmer is specifically referred to as a "peptide nucleic acid." The non-natural nucleotides constituting the 5' wing region and the 3' wing region are called "acid gapmers." When the peptide comprises or consists of a peptide nucleic acid, the gapmer is specifically referred to as a "morpholine gapmer." The 5' wing region and the 3' wing region contain bridged nucleic acids, which are called "ribonucleic acid gapmers." The number of nucleotides may be 2 or 3. The bridged nucleosides may be present contiguously or non-contiguously in the 5' wing region and the 3' wing region. The bridged nucleoside may further include a modified nucleobase (e.g., 5-methylcytosine). When the bridged nucleoside is an LNA nucleoside, the gap The LNA / DNA gapmer is called an "LNA / DNA gapmer." are each independently at least 2 bases long, for example, 2 to 10 bases long, 2 to 7 bases long, or 3 The 5' wing region and the 3' wing region may contain non-natural nucleosides. It is sufficient that at least one type is contained, and natural nucleosides may be further contained.

[0061] The first nucleic acid strand and / or the second nucleic acid strand constituting the gapmer are each composed of 2 to 7 3-5 base-long bridged nucleosides, 4-15 base-long or 8-12 base-long ribonucleosides Nucleosides or deoxyribonucleosides and bridges of 2-7 bases or 3-5 bases It may be composed of nucleosides.

[0062] The first and / or second nucleic acid strands constituting the nucleic acid complex of the present invention are mixmers. As used herein, a "mixmer" refers to a mixture of periodic or random segments. Contains alternating natural and non-natural nucleosides of different lengths, and has 4 or more consecutive Mixed nucleotides are nucleic acid chains that do not contain deoxyribonucleosides or ribonucleosides. In the mer, the non-natural nucleoside is a bridged nucleoside and the natural nucleoside is A mixmer in which the end is a deoxyribonucleoside is specifically called a "BNA / DNA mixmer." In the mixmer, the non-natural nucleoside is a peptide nucleic acid, and the natural Mixmers in which the nucleosides are deoxyribonucleosides are specifically called "peptide nucleic acids (DNs)." In a mixmer, the unnatural nucleoside is a morpholinoyl nucleoside. A mixmer which is a nucleic acid and in which the natural nucleosides are deoxyribonucleosides. In particular, we call it a "morpholino nucleic acid / DNA mixmer." A mixmer is a combination of two types of nucleosides. Mixmers are not limited to containing only natural or modified nucleosides. or any number of types of nucleosides, whether or not they are nucleoside mimetics. For example, one nucleotide separated by a bridge nucleoside (e.g., an LNA nucleoside) can be used. Or, two consecutive deoxyribonucleosides may be present. It may further comprise a modified nucleobase (eg, 5-methylcytosine).

[0063] At least one or at least two from the end (5' end, 3' end, or both ends) of the second nucleic acid strand At least three, or at least four, of the nucleosides may be modified nucleosides. The modified nucleoside may contain a modified sugar and / or a modified nucleobase. The modified nucleobase may be a 5-methyl, 2'-modified sugar (e.g., a sugar containing a 2'-O-methyl group). It can also be lucitsine.

[0064] The second nucleic acid strand is composed of modified nucleosides ( For example, modified nucleosides containing 2'-modified sugars), 4-15 bases or 8-12 bases in length (in the case ribonucleosides or deoxyribonucleosides linked by modified internucleoside bonds nucleosides, and modified nucleosides of 2-7 bases or 3-5 bases (e.g., 2'-modified sugar In this case, the first nucleic acid strand may be composed of a modified nucleoside containing a gap It may also be a mer.

[0065] The first and second nucleic acid strands may be composed entirely or in part of nucleoside or nucleotide mimetics. Nucleotide mimetics may include peptide nucleic acids and / or morpholino nucleic acids. The first nucleic acid strand may include at least one modified nucleoside. A nucleoside may contain a 2'-modified sugar, which may contain a 2'-O-methyl group. It may also be sugar.

[0066] The first and second nucleic acid strands may have any of the above-mentioned modified internucleoside bonds and modified nucleosides. It may include any combination.

[0067] The second nucleic acid strand is (1) tocopherol or an analogue thereof, (2) cholesterol or or its analogues, or (3) substituted or unsubstituted C1- 30 Alkyl Group, substituted or unsubstituted C2~ 30 or a substituted alkenyl group or unsubstituted C1 30 It is bound to the alkoxy group of tocopherol. Alternatively, cholesterol or its analogs can be prepared by a method known per se by those skilled in the art. It can be constructed.

[0068] As used herein, the term "analog" refers to a similar compound having the same or a similar basic structure. Analogs refer to compounds that have the same structure and properties. Examples of analogs include biosynthetic intermediates and metabolites. Whether a compound is an analog of another compound can be determined by one of ordinary skill in the art.

[0069] Tocopherols include α-tocopherol, β-tocopherol, γ-tocopherol, and and δ-tocopherol. The tocopherol analogues include: Various unsaturated analogues of tocopherol, e.g., α-tocotrienol, β-tocotrienol Tocotrienol, γ-tocotrienol, δ-tocotrienol, etc. are preferred. The tocopherol is α-tocopherol.

[0070] Cholesterol analogues are various cholesterol analogues that are alcohols with a sterol backbone. This refers to teratol metabolites and analogues, including but not limited to cholestanol, Lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol etc.

[0071] The second nucleic acid strand bound to tocopherol or its analogue is a group represented by the following general formula (I): may have the following structure:

[0072] [ka] [In the formula, R 1 is a substituted or unsubstituted C1~ 30 Alkyl groups (e.g., methyl 4,8,12-trimethyltridecyl) or substituted or unsubstituted C2-3 0 represents an alkenyl group (e.g., 4,8,12-trimethyl-3,7,11-tridecatrien-1-yl) (wherein wherein the substitution is preferably with a halogen atom.

[0073] The second nucleic acid strand bound to cholesterol or its analogue is represented by the following general formula (II), (V), or (VI): and (VII): [ka] (In the formula, Ring A is a substituted or unsubstituted cyclohexane or a substituted or unsubstituted cyclohexane. Ring B represents a substituted or unsubstituted cyclohexane. cyclohexane, substituted or unsubstituted cyclohexane or substituted or unsubstituted cyclohexane Ring C represents a substituted or unsubstituted cyclohexadiene. R represents cyclohexene or substituted or unsubstituted cyclohexane; 2 is a hydrogen atom, a substituted or unsubstituted C 30 Alkyl groups (e.g., ethyl isopropyl, 1-methylpropyl, 1,5-dimethylhexyl, 4-ethyl-1,5-dimethyl hexyl), substituted or unsubstituted C2~ 30 Alkenyl groups (e.g., 1,5- Dimethyl-4-hexen-1-yl, 1,4,5-trimethyl-2-hexen-1-yl, 4-ethyl-1,5-di methyl-2-hexen-1-yl), substituted or unsubstituted C1-6 alkyl- Carbonyl group (e.g., 1-oxoethyl), substituted or unsubstituted C1-6 alkyl represents an alkyl-carbonyl-oxy group (e.g., (1-oxoheptyl)oxy), or an oxo group; , R 3 represents a hydrogen atom, R 2 and R 3 taken together, are replaced or substituted (It may also form an unsubstituted 1,6-dioxaspiro[4.5]decane ring).

[0074] In the formula, L 1 is -O-, -NH-, [ka] or [ka] ), [ka] (In the formula, R 4is a substituted or unsubstituted C1~ 30 Alkyl groups (e.g., 1, 5-dimethylhexyl), or substituted or unsubstituted C2 30 Arken represents a aryl group), [ka] (In the formula, R 5 and R 6 each independently represents a substituted or unsubstituted C1 ~ 30 alkyl groups (e.g., butyl, 1,5-dimethylhexyl), or substituted or unsubstituted Unconverted C2~ 30 represents an alkenyl group, and [ka] (In the formula, R 7 is a hydroxy group, a substituted or unsubstituted C 30 Alki aryl-carbonyl-oxy groups (e.g., (1-oxododecyl)oxy), or substituted or Unsubstituted C1~ 30 Alkenyl-carbonyl-oxy groups (e.g., (1-oxo-9-oxo (tadekenyl)oxy). A compound selected from the group consisting of (II), (V), (VI), and (VII). In the groups, the substitutions are preferably halogen atoms (e.g., fluorine atoms), C1 to 3Alkyl groups (e.g., methyl), hydroxyl groups, C1-6 alkyl-carbonyl groups (e.g., 1- oxo-2-methylpropyl), 1-5 C1-3 alkoxy groups substituted with 1-3 halogen atoms Carbamoyl groups mono- or di-substituted with phenyl groups substituted with alkyl groups (e.g., [3,5- (trifluoromethyl)benzoyl]amino), or C1-6 alkyl-carbonyl group ( For example, 2-methyl-1-oxopropyl.

[0075] In one embodiment, the second nucleic acid strand comprises a substituted or unsubstituted C 30 Alkyl groups (e.g., heptadecyl, 16-methylheptadecyl, henicosyl), substituted or unsubstituted C2~ 30 Alkenyl groups (e.g., 2,6-dimethyl-1,3,5,7-octyl 8-Heptadecen-1-yl, 8,11-Heptadecadien-1-yl, 8,11,1 4-heptadecatrien-1-yl), or substituted or unsubstituted C1- 30 wherein the substitution is preferably or a cyclohexenyl group substituted with a halogen atom or 1 to 5 C1-3 alkyl groups ( For example, 2,6,6-trimethyl-1-cyclohexen-1-yl.

[0076] In one embodiment, the second nucleic acid strand bound to tocopherol or an analogue thereof is It may have a group represented by formula (IIa): [ka] (In the formula, Ring A is a substituted or unsubstituted cyclohexene, a substituted or unsubstituted Unsubstituted cyclohexane or substituted or unsubstituted cyclohexadiene represents the R 1 is a substituted or unsubstituted C1~ 30 Alkyl groups (e.g., C2- 20、 C4~ 16、 C6~ 12 or C8~10 alkyl group), substituted or unsubstituted Not yet C2~ 30 Alkenyl groups (e.g., C2- 20、 C4~ 16、 C6~ 12 or C8~ 10 alkenyl group), substituted or unsubstituted C1-6 alkenyl group alkyl-carbonyl group, substituted or unsubstituted C1-6 alkyl-carbonyl represents an oxy group or an oxo group; L 1 is -O-, -NH-, [ka] or [ka] (represents

[0077] In one embodiment, the second nucleic acid strand bound to tocopherol or an analogue thereof is It may have groups represented by formulae (IIa-1), (IIa-2), (IIa-3), and (IIa-4). [ka] (IIa-1) TIFF2025063018000029.tif3563 (IIa-2) TIFF2025063018000030.tif3263 (IIa-3) TIFF2025063018000031.tif3765 (IIa-4) (In the formula, L 1 is -O-, -NH-, [ka] or [ka] ), is selected from the group consisting of:

[0078] In one embodiment, the second nucleic acid strand bound to tocopherol or an analogue thereof is It does not have a group represented by formula (IIa-3). [ka]

[0079] Tocopherol or cholesterol or an analogue thereof is a 5'-terminal of the second nucleic acid strand. Alternatively, tocopherol or Cholesterol or its analogue is linked to an internal nucleotide of the second nucleic acid strand. For example, cholesterol can be attached to the 5' end of the second nucleic acid strand. In an embodiment, the second nucleic acid strand is tocopherol or cholesterol or a combination thereof. and / or Alternatively, they may be linked as a group to one position of the second nucleic acid strand. Cholesterol or its analogue is located at the 5' end and the 3' end of the second nucleic acid strand, respectively. Cholesterol or an analogue thereof may be linked to the 5' end and 3' end of the second nucleic acid strand. may be linked to each of the

[0080] The binding between the second nucleic acid strand and tocopherol, cholesterol, or an analog thereof is directly It does not matter whether the bond is direct or indirect. A direct bond means that two molecules are directly bonded. Indirect binding means that the two molecules to be bound are bound via another substance.

[0081] In one embodiment, the second nucleic acid strand and tocopherol or cholesterol and / or The binding with the analogue or the like may occur at the 5' end, 3' end, or an internal nucleoside of the second nucleic acid strand. The nucleotide sequence may be linked to the tide via a phosphate ester or phosphorothioate bond.

[0082] In one embodiment, the second nucleic acid strand has a phosphate bond or a phosphorothioate bond at the 5' end. They are connected via bonds.

[0083] In one embodiment, it is attached to the 5' end of the second nucleic acid strand via a phosphate bond.

[0084] The second nucleic acid strand and tocopherol, cholesterol, or an analog thereof are indirectly bound to each other. In this case, the linker is connected via a linking group (often referred to as a "linker" herein). The linker may be located at the 5' end, the 3' end, or at an internal nucleotide of the second nucleic acid strand. The nucleotide may be linked to the nucleotide sequence via a phosphate ester bond or a phosphorothioate bond.

[0085] In one embodiment, the linker comprises a phosphate ester bond or a phospho ester bond at the 5' end of the second nucleic acid strand. The linkage is via a thiolate bond.

[0086] In one embodiment, the linker is attached to the 5' end of the second nucleic acid strand via a phosphate bond. It fits together.

[0087] A specific example of the linker is a linker represented by the following general formula (VIII): R: [ka] (In the formula, L 2 is a substituted or unsubstituted C1~ 12 Alkylene groups (e.g., propylene hexylene, dodecylene), substituted or unsubstituted C3-8 cycloalkyl alkylene group (e.g., cyclohexylene), -(CH2)2-O-(CH2)2-O-(CH 2) 2-O-(CH2)3-, or CH(CH2-OH)-CH2-O-(CH2)2- O-(CH2)2-O-(CH2)2-O-(CH2)3-, L 3 -NH- or represents a bond, and L 4 is a substituted or unsubstituted C1~ 12 The alkylene group (e.g., ethylene, pentylene, heptylene, undecylene), substituted or unsubstituted Cycloalkylene groups of C3-8 not bound to the aryl group (e.g., cyclohexylene), -(CH2)2- [O-(CH2)2] m - or a bond, where m is an integer of 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond (wherein the substitution is preferred). or halogen atoms).

[0088] The second nucleic acid strand and tocopherol, cholesterol, or an analog thereof are indirectly bound to each other. In this case, the two may be linked via a cleavable linker. " can be cleaved under physiological conditions, e.g., within a cell or within an animal body (e.g., within the human body) Cleavable linkers are linking groups that can be cleaved by endogenous enzymes such as nucleases, peptidases, It may be selectively cleaved under acidic conditions, reductive conditions, etc. Specific examples of such cleavage include For example, one of an amide bond, an ester bond, a phosphate ester bond, a phosphodiester bond One or both of ester bonds, carbamate bonds, and disulfide bonds, as well as natural Examples of such linkers include nucleotide linkers such as DNA linkers.

[0089] On the other hand, the second nucleic acid strand and tocopherol or cholesterol or an analogue thereof are indirectly bonded to each other. In the case of binding, the two may be bound via an uncleavable linker. A "non-cleavable linker" refers to a linking group that is not cleaved under physiological conditions. Non-cleavable linkers include, for example, phosphorothioate bonds and Modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by a bond Examples of the linker include a linker composed of a nucleoside.

[0090] The length of the cleavable or non-cleavable linker can be determined by the length of the nucleic acid such as DNA or an oligonucleotide. In the case of a peptide, although there is no particular limitation, it is usually 1 to 20 bases long, 1 to 10 bases long, or 1 to 6 bases long. good.

[0091] In certain embodiments, the nucleic acid complexes of the present invention are optically isomeric, stereoisomeric, positional isomeric In the case where the nucleic acid complex contains isomers or rotational isomers, these are also included in the nucleic acid complex of the present invention. Both can be obtained as single products by known synthesis and separation methods. For example, when optical isomers exist in the nucleic acid complex of the present invention, the optical isomers separated from the compound may be used. Enantiomeric isomers are also encompassed within the nucleic acid complexes of the present invention.

[0092] In certain embodiments, the nucleic acid complex of the present invention comprises a prodrug and The present invention also includes a prodrug of the nucleic acid complex of the present invention and a pharma- ceutically acceptable salt thereof. Pharmaceutically acceptable salts of the compounds are formed by reactions with enzymes, gastric acid, etc. under physiological conditions in the body. The compound that converts the nucleic acid complex of the present invention into the nucleic acid complex by enzymatic oxidation, reduction, hydrolysis, etc. The compound which is converted into the nucleic acid complex of the present invention is hydrolyzed by gastric acid or the like to produce the nucleic acid complex of the present invention. In certain embodiments, the prodrug of the nucleic acid complex is a compound that converts the nucleic acid complex into a prodrug of the nucleic acid complex. The nucleic acid fragment is a tocopherol or cholesterol fragment bound to the first or second nucleic acid strand. The compound contains one or more of the following:

[0093] In the nucleic acid complex of the present invention, the first nucleic acid strand has an antisense nucleic acid for a target transcript. The effect can be measured by a method known in the art. For example, after introducing the nucleic acid complex into a cell, Using known techniques such as Northern blotting, quantitative PCR, or Western blotting, Specifically, the expression level of a target gene or the target transcript level in a cell can be measured. The level of a substance (e.g., mRNA amount or RNA amount such as microRNA, cDNA amount, protein amount, etc.) The reduction due to the antisense effect can be verified using the above-mentioned publicly known techniques.

[0094] Measurement of the antisense effect of the nucleic acid complex of the present invention in the central nervous system and The passage can be measured by a method known in the art. The acid complex is administered to a subject (e.g., a mouse), and several days to several months later (e.g., 2 to 7 days or 1 The expression level of the target gene or the level of the target transcript in the central nervous system was suppressed after 3 months. The determination can be made by measuring whether the expression of the target gene is increased or decreased. The amount or measured amount of the target transcript is less than that of a negative control (e.g., vehicle-administered). at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least The nucleic acid complex of the present invention is capable of crossing the blood-brain barrier when the amount of the nucleic acid complex that crosses the blood-brain barrier is reduced by at least 30% or at least 40%. It can be determined that the drug crossed the blood-brain barrier and exerted an antisense effect on the central nervous system. The passage of the nucleic acid complex of the present invention is judged by administering the complex to a subject (e.g., a mouse) and measuring the passage of the complex several days to several months later. The amount (concentration) of the nucleic acid complex of the present invention in the central nervous system is measured (e.g., 2 to 7 days or 1 month later). This can be determined by measuring the degree of

[0095] As described above, exemplary embodiments of the nucleic acid complex of the present invention have been described. The acid complex is not limited to the above exemplary embodiment. In addition, those skilled in the art can easily prepare the acid complex by known methods. By appropriately selecting the first nucleic acid complex constituting the nucleic acid complex according to various embodiments of the present invention, One nucleic acid strand and a second nucleic acid strand can be produced. For example, the nucleic acid molecules of the invention can be used to Designing each nucleic acid molecule based on information on the base sequence of the product (e.g., the base sequence of the target gene); For example, commercially available products from GE Healthcare, Thermo Fisher Scientific, Beckman Coulter, etc. Nucleic acids were synthesized using an automated nucleic acid synthesizer, and the resulting oligonucleotides were then reverse-phase It can be produced by purifying it using a column or the like.

[0096] The second nucleic acid strand further comprises at least one functional moiety attached to the polynucleotide. The functional moiety may be a desired component of the nucleic acid complex and / or the strand to which the functional moiety is attached. No particular limitation is imposed on the structure of the "functional moiety" according to a particular embodiment, so long as it provides the function of The desired functions include a labeling function and a purification function. Examples of such compounds include fluorescent proteins and luciferase. Examples of moieties that can be used include biotin, avidin, His-tag peptides, GST-tag peptides, and FLAG. The binding site and binding site of the functional moiety in the second nucleic acid strand are The type of the compound is tocopherol, cholesterol, or an analog thereof, and the second nucleic acid strand. As described above for binding to.

[0097] In one embodiment, the nucleic acid complex to which the functional moiety is attached is a nucleic acid complex to which the functional moiety has previously been attached. The nucleic acid species can be used to prepare the nucleic acid by carrying out the synthesis, purification and annealing steps described above. For example, tocopherol, cholesterol, or analogues thereof may be bound in advance. The combined nucleic acid species is used to prepare a second nucleic acid strand by carrying out the synthesis and purification steps described above. It is possible.

[0098] In one embodiment, the second nucleic acid strand produced by carrying out the synthesis and purification described above is Tocopherol, cholesterol or its analogues are bound to the compound by a method known per se. Methods for linking functional moieties to nucleic acids are well known in the art. The nucleic acid produced by this method is mixed in an appropriate buffer solution and heated to about 90°C to 98°C for several minutes. The nucleic acid is then denatured (e.g., for 5 minutes) and then annealed at about 30°C to 70°C for about 1 to 8 hours to form the nucleic acid of the present invention. It is possible to produce one of the above-mentioned nucleic acid complexes. In addition, the nucleic acid strand is a base sequence and a modification site. Specify the type and order from various manufacturers (e.g. Gene Design Co., Ltd.) and obtain the product. The annealing step may be performed by leaving the substrate at room temperature (about 10° C. to about 35° C.) for about 5 to 60 minutes. This can be done by:

[0099] The first nucleic acid strand and the second nucleic acid strand are each dissolved in a buffer solution (e.g., phosphate buffered saline) at about 70°C to 98°C. Salt water) or water, mix the two resulting solutions, and heat the mixture at about 70°C to 98°C for several minutes. (e.g., for 5 minutes), and then heat the mixture at about 30°C to 70°C (or 30°C to 50°C) for about 1 to 8 hours. The first nucleic acid strand and the second nucleic acid strand may be separated and then mixed to prepare a nucleic acid complex according to some embodiments of the present invention. The acid chains were each dissolved in a buffer solution (e.g., phosphate buffered saline) at room temperature (about 10°C to about 35°C) or It can also be dissolved in water.

[0100] However, the annealing conditions (time and temperature) for preparing the nucleic acid complex should be the same as above. In addition, suitable conditions for promoting annealing of nucleic acid strands are not limited to the above conditions. It is well known in the field.

[0101] (Effect of nucleic acid complex) The nucleic acid complex of the present invention inhibits the effect of a target miRNA in the central nervous system of a subject. For example, specifically, the first nucleic acid strand can hybridize to at least a portion of the target miRNA. It contains a base sequence capable of encoding a target miRNA and has an antisense effect on the target miRNA. the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, and or cholesterol or its analogues, and the first and second nucleic acid strands are bound to each other. The nucleic acid complex anneals to the target miRNA. By inhibiting the expression of genes that the target miRNA normally downregulates, can be upregulated.

[0102] The nucleic acid complex of the present invention is capable of regulating the expression or editing of a target RNA in the central nervous system of a subject. For example, the first nucleic acid strand can specifically hybridize to at least a portion of the target RNA. It contains a base sequence capable of recoding and exerts an antisense effect on the target RNA. The second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and is a tocopherol or or cholesterol or its analogue, and the first and second nucleic acid strands are mutually In this case, the term "regulation of expression of a target RNA" refers to, for example, For example, the expression level of a target RNA is up-regulated or down-regulated. Regulation of splicing by exon skipping and exon inclusion The target RNA may be a viral or bacterial RNA, or a toxic RNA. good.

[0103] The nucleic acid complex of the present invention is capable of inhibiting translation of a target mRNA in the central nervous system of a subject. For example, the first nucleic acid strand can be specifically hybridized to at least a portion of the target mRNA. and has an antisense effect on a target mRNA, The second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and is selected from the group consisting of tocopherol and The first nucleic acid strand and the second nucleic acid strand are bound to cholesterol or its analogues, and anneal with each other. The nucleic acid complex binds the first nucleic acid strand to the target mRNA. Binding creates a steric block, inhibiting translation of the mRNA.

[0104] The nucleic acid complex of the present invention is not limited to, but may be used in the microenvironment of the central nervous system. Effective use in regulating the expression or editing of target transcripts in glia (microglia) It is possible.

[0105] <Composition> The second aspect of the present invention is a composition. The composition of the present invention is a nucleic acid complex according to the first aspect or The first aspect includes a single-stranded nucleic acid chain as an active ingredient and / or as a drug delivery molecule. The nucleic acid complex or single-stranded nucleic acid strand crosses the BBB and increases the expression level of the target transcript in the central nervous system. can be regulated (e.g., reduced expression) by the antisense effect. Therefore, the composition of the present invention, when administered to a subject, delivers the nucleic acid complex and treats the subject. The composition of the present invention may be a composition for treating a disease or a pharmaceutical composition. including, but not limited to, targeting in microglia of the central nervous system These genes can be effectively used in regulating the expression or editing of target transcripts.

[0106] In one embodiment of the present invention, a composition containing a nucleic acid complex is administered to each central nervous system. The present invention relates to therapeutic methods for treating systemic diseases.

[0107] (Formulation) In the present specification, the composition can be formulated by a method known per se. For example, The composition may be administered in the form of a capsule, tablet, pill, liquid, powder, granule, microgranule, film-coated tablet, etc. tablets, pellets, lozenges, sublingual tablets, peptizers, buccal tablets, pastes , syrup, suspension, elixir, emulsion, coating, ointment, plaster , cataplasm, transdermal, lotion, inhalant, aerosol, eye drops, injection The composition can be used orally or parenterally in the form of tablets and suppositories.

[0108] Regarding the formulation of these preparations, pharma- ceutically acceptable carriers or solvents or food and beverage The composition may be suitably incorporated with a commercially acceptable carrier or solvent. As a solvent, specifically, sterilized water, physiological saline, vegetable oil, base, emulsifier, suspending agent, Surfactants, pH adjusters, stabilizers, flavors, fragrances, excipients, vehicles, preservatives, binders, Diluents, isotonicity agents, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, coloring agents , sweeteners, thickeners, flavoring agents, dissolution aids, and other additives.

[0109] (Dosage form and dosage) As used herein, there is no particular limitation on the preferred administration form of the composition. For example, oral administration Specific examples of parenteral administration include intravenous administration, intraarterial administration, and Intraperitoneal, subcutaneous, intradermal, tracheal / bronchial, rectal, and intramuscular administration, and Administration by intramuscular injection, intravenous drip, or implantable continuous It can also be administered subcutaneously. Subcutaneous administration allows patients to self-inject. In the case of intravenous administration, the nucleic acid complex contained in one dose of the composition is preferably The amount, i.e., the single dose of the nucleic acid complex, can be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0. 01mg / kg or more, 00.25mg / kg or more, 0.5mg / kg or more, 1mg / kg or more, 2.5mg / kg or more, 5mg / kg or more , 10mg / kg or more, 20mg / kg or more, 30mg / kg or more, 40mg / kg or more, 50mg / kg or more, 75mg / kg or more, 100mg / kg or more, 150mg / kg or more, 200mg / kg or more, 300mg / kg or more, 400mg / kg or more, or 50 For example, any concentration in the range of 0.001 mg / kg to 500 mg / kg can be used. Amount (e.g., 0.001mg / kg, 0.01mg / kg, 0.1mg / kg, 1mg / kg, 5mg / kg, 10mg / kg, 50mg / kg, 1 00mg / kg, or 200mg / kg) can be selected appropriately.

[0110] (Subject / Applicable Subject) As used herein, the term "subject" refers to a subject to which the composition of the present invention is applied. In addition, the subject may be an organ, tissue, or cell. When the subject is an individual, the compositions of the present invention may be used in combination with a human subject. The present invention can be applied to any animal. Non-human subjects include, for example, various domestic animals, Potential applications include poultry, pets, and laboratory animals. The subjects express target transcripts in the central nervous system. or an individual in need of treatment for a central nervous system disorder. Good too.

[0111] The composition of the present invention has the following features: By using the antisense effect, the expression level of the target transcript in the central nervous system is reduced. can be done.

[0112] When the composition of the present invention is applied to treat a central nervous system disease, the target disease is a genetic disease. Central nervous system disorders associated with increased or decreased gene expression, particularly target transcript or target gene expression. Diseases (tumors, etc.) associated with increased expression are preferred. Examples include, but are not limited to, brain tumors, Alzheimer's disease, and the like. Hymer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, etc. Some examples include:

[0113] In one embodiment, the composition of the present invention is applied for the treatment of immune-mediated central nervous system diseases. Examples of immune-mediated central nervous system diseases include microglia-related diseases. Examples of chromosome-related diseases include Alzheimer's disease, multiple sclerosis, ALS, and neurological disorders. Examples include sexual pain.

[0114] The delivery site of the composition of the present invention, more specifically, the delivery site of the active ingredient contained in the composition, is Although not particularly limited, more effective results can be achieved by delivering the drug to an appropriate site depending on each disease. For example, in the treatment of Alzheimer's disease, the hippocampus and / or parietal In addition, delivery of drugs to the lobes may be effective in treating frontotemporal dementia (FTD) (frontotemporal lobar degeneration) (including FTLD, semantic dementia (SD), progressive non-fluent aphasia (PNFA), etc.), and Pick's disease In the treatment of encephalopathy, drug delivery to the frontal lobe, temporal lobe, and / or substantia nigra may be effective. In the treatment of Parkinson's disease dementia, drug delivery to the occipital lobe, substantia nigra, and / or striatum is effective. In addition, drug delivery to the substantia nigra and / or striatum may be effective in treating Parkinson's disease. In the treatment of corticobasal degeneration (CBD), the frontal lobe, parietal lobe, basal ganglia and / or In the treatment of progressive supranuclear palsy (PSP), the frontal lobe, Drug delivery to the basal ganglia and / or substantia nigra may be effective in treating amyotrophic lateral sclerosis. In some cases, delivery of drugs to the frontal lobe, parietal lobe, basal ganglia, and / or substantia nigra may be effective. In the treatment of cerebral degeneration (SCA) types SCA1 to SCA34, drug delivery to the brainstem and / or cerebellum is effective. In the treatment of dentatorubral-pallidoluysian degeneration (DRPLA), and / or cerebellar drug delivery may be effective in the treatment of spinal and bulbar atrophy (SBMA). In Friedreich's ataxia (FA), drug delivery to the brain stem and / or spinal cord may be effective. In treatment, drug delivery to the brainstem and / or cerebellum may be effective. In this case, drug delivery to the striatum, frontal lobe, parietal lobe, and / or basal ganglia may be effective. In the treatment of On-diseases (including mad cow disease and GSS), the cerebral cortex, cerebral white matter, basal ganglia and / or substantia nigra Drug delivery to the cerebral white matter may be effective in the treatment of cerebral leukoencephalopathy. Encephalitis (including viral, bacterial, fungal and tuberculous), meningitis (viral Brain-wide drug delivery could be effective in treating inflammatory bowel disease (including inflammatory, bacterial, fungal, and tuberculous) In the treatment of metabolic, toxic, and nutritional encephalopathy, drug delivery to the entire brain is effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective. Drug delivery to the entire brain is effective in treating cerebral hemorrhage, subarachnoid hemorrhage, moyamoya disease, and anoxic encephalopathy. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective. Drug delivery to the cerebral white matter is effective in treating diffuse axonal injury. In the treatment of head trauma, drug delivery to the whole brain may be effective. ), and in the treatment of neuromyelitis optica (NMO), targeting of the cerebral white matter, cerebral cortex, optic nerve, and / or spinal cord Drug delivery can be effective. In the treatment of myotonic dystrophy (DM1, DM2), , delivery of drugs to the myocardium, cerebral cortex and / or cerebral white matter may be effective. Drug delivery to the parietal lobe and / or spinal cord may be effective in treating HSP. In the treatment of strophies, drug delivery to skeletal muscle, cerebral cortex and / or cerebral white matter is effective. In the treatment of dementia with Lewy bodies (DLB), the substantia nigra, striatum, occipital lobe, frontal lobe and / or In the treatment of multiple system atrophy (MSA), drug delivery to the striatum, Drug delivery to the basal ganglia, cerebellum, substantia nigra, frontal lobe and / or temporal lobe may be effective. Drug delivery to the cerebral white matter may be effective in the treatment of Kissander disease. In treatment, drug delivery to the cerebral white matter can be effective.

[0115] When the composition is applied by administration or ingestion, the amount administered or ingested depends on the age (months) of the subject. age, age in weeks, weight, symptoms and health condition, type of composition (medicine, food and beverage, etc.) The effective amount of the composition of the present invention to be ingested by a subject may be selected, for example, according to the following: , the nucleic acid complex contained therein is 0.00001 mg / kg / day to 10,000 mg / kg / day, or 0.001 mg / kg / day to 100 mg / kg The composition may be administered in a single dose or in multiple doses. In the case of multiple doses, the dose should be administered daily or at appropriate intervals (e.g., 1 day, 2 days, 3 days, 1 week, 2 weeks, etc.). The nucleic acid complex may be administered, for example, 2 to 20 times at intervals of 1 month. The dosage is, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 00.25 mg / kg or more. , 0.5mg / kg or more, 1mg / kg or more, 2.5mg / kg or more, 0.5mg / kg or more, 1.0mg / kg or more, 2.0mg / kg or more Above, 3.0mg / kg or more, 4.0mg / kg or more, 5mg / kg or more, 10mg / kg or more, 20mg / kg or more, 30mg / kg or more Above, 40mg / kg or more, 50mg / kg or more, 75mg / kg or more, 100mg / kg or more, 150mg / kg or more, 200mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, for example, Any amount in 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, 1mg / kg, 5mg / kg, 10mg / kg, 50mg / kg, 100mg / kg, or 200mg / kg) It is possible.

[0116] The nucleic acid complex of the present invention is administered twice a week at a dose of 0.01 to 10 mg / kg (for example, about 6.25 mg / kg) for 4 days. Alternatively, the nucleic acid complex may be administered once a week at a dose of 0.05 to 30 mg / kg (for example, about 25 mg / kg). The drug may be administered 2 to 4 times at a frequency of 1 to 2 times a week, for example, twice a week. The use of split doses reduces toxicity and improves patient outcomes compared to a single, higher dose. The load can be reduced.

[0117] Although there is a limit (upper limit) to the amount of nucleic acid complex that can pass through the BBB and BNB after a single administration, It is believed that the inhibitory effect is additive even when the amount of β-lactam is increased intracellularly. At doses higher than the upper limit (e.g., 25 mg / kg or more), increasing the dose at one time does not increase efficacy. However, the efficacy can be improved by repeated administration at a certain interval (e.g., half a day or more). It is believed that this can be improved.

[0118] (Drug Delivery) The composition of the present invention contains as an active ingredient a nucleic acid complex or a single-stranded nucleic acid chain according to the first aspect of the present invention. Taking advantage of the fact that a specific drug can be efficiently delivered to the central nervous system through the BB, and / or a second nucleic acid strand, thereby delivering the agent to the nervous system, particularly the central nervous system. The drug to be delivered to the nervous system can be, but is not limited to, a peptide, a tadpole, or the like. Protein or nucleic acid drugs, or other organic compounds, such as antitumor drugs, hormonal drugs, Antibiotics, antivirals, anti-inflammatory drugs, etc. The drug is preferably a small molecule drug. The term "small molecule drug" is well understood in the art. Typically, this refers to a drug that has a molecular weight of less than 1,000 daltons. The drug is a lipophilic drug. Examples of nucleic acid drugs include, but are not limited to, ASO, antagomir, splice inhibitors, and the like. Switching oligonucleotides, aptamers, single-stranded siRNAs, microRNAs, pre-my The binding position and type of the drug in the second nucleic acid strand may be tocopherol. The binding of cholesterol or cholesterol or its analogue to the second nucleic acid strand is described above. As stated above.

[0119] The compositions of the present invention are delivered to the central nervous system with high efficiency, as disclosed in the following examples. The expression of the target gene or the level of the target transcript can be effectively altered or suppressed. Thus, the present invention provides a method for reducing the expression level of a target transcript in the central nervous system of a subject. In accordance with another aspect of the present invention, a method is provided for administering to a subject a composition comprising the nucleic acid complex described above. The method may be a method for treating a central nervous system disorder in a subject. A method for delivering a drug to the central nervous system comprising administering to a subject a composition comprising the nucleic acid complex described above. Also provided is a method of drug delivery, comprising: EXAMPLES

[0120] The present invention is further illustrated by the following Reference Examples and Examples, which are merely illustrative of the present invention. are not intended to be limiting and may be varied without departing from the scope of the present invention.

[0121] In the following examples, "room temperature" generally refers to about 10°C to about 35°C. Ratios are by volume unless otherwise specified. % is by weight unless otherwise specified.

[0122] Unless otherwise specified, elution in column chromatography in the examples was performed by TLC (T Thin Layer Chromatography The TLC observation was performed under observation. Made in 60 F 254 As the developing solvent, The solvent used was the same as that used in the previous study. A UV detector was used for detection. In chromatography, when C18 is written, it means octadecyl bonded silica gel. The ratio of the dissolution solvents shown is a volume ratio unless otherwise specified. 1 For H NMR analysis, ACD / SpecManager (product name) software etc. The proton peaks of hydroxyl groups and amino groups are very gentle. There are some things that I haven't done.

[0123] The meanings of the abbreviations used in the examples are shown below. M: Molar concentration N: Normality CDCl3: deuterated chloroform DMSO-d6: deuterated dimethyl sulfoxide 1 H NMR: Proton nuclear magnetic resonance LC / MS: Liquid chromatograph mass spectrometer DIPEA: N,N-diisopropylethylamine DMAP: 4-dimethylaminopyridine DMF: Dimethylformamide DMSO: Dimethyl sulfoxide HATU:O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetrahydrofuran Methyluronium hexafluorophosphate NMP: N-methyl-2-pyrrolidone PBS: Phosphate-buffered saline TEA: Triethylamine TEAA: Triethylamine acetate THF: tetrahydrofuran

[0124] The structures of the oligonucleotides used in the following examples are summarized in Table 1. Among the oligonucleotides, ASO(Malat1), Y61-cRNA(Malat1), Y62-cRNA(Malat1), and Y63-cRNA(Malat1) were used. RNA (Malat1), Y64-cRNA (Malat1), Y59-cRNA (Malat1), Y60-cRNA (Malat1) and Chol-cRNA (M alat1) was synthesized by Gene Design Inc. (Osaka, Japan).

[0125] [Table 1] TIFF2025063018000037.tif209152 TIFF2025063018000038.tif209153 TIFF2025063018000039.tif209154 TIFF2025063018000040.tif228124

[0126] The 5'-terminal structure of the oligonucleotide Y61-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0127] [ka]

[0128] The 5'-terminal structure of the oligonucleotide Y62-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0129] [ka]

[0130] The 5'-terminal structure of the oligonucleotide Y63-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0131] [ka]

[0132] The 5'-terminal structure of the oligonucleotide Y64-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0133] [ka]

[0134] The 5'-terminal structure of the oligonucleotide Y1-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0135] [ka]

[0136] The 5'-terminal structure of the oligonucleotide Y3-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0137] [ka]

[0138] The 5'-terminal structure of the oligonucleotide Y4-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0139] [ka]

[0140] The 5'-terminal structure of the oligonucleotide Y5-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0141] [ka]

[0142] The 5'-terminal structure of the oligonucleotide Y6-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0143] [ka]

[0144] The 5'-terminal structure of the oligonucleotide Y7-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0145] [ka]

[0146] The 5'-terminal structure of the oligonucleotide Y8-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0147] [ka]

[0148] The 5'-terminal structure of the oligonucleotide Y9-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0149] [ka]

[0150] The 5'-terminal structure of the oligonucleotide Y10-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0151] [ka]

[0152] The 5'-terminal structure of the oligonucleotide Y11-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0153] [ka]

[0154] The 5'-terminal structure of the oligonucleotide Y12-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0155] [ka]

[0156] The 5'-terminal structure of the oligonucleotide Y13-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0157] [ka]

[0158] The 5'-terminal structure of the oligonucleotide Y14-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0159] [ka]

[0160] The 5'-terminal structure of the oligonucleotide Y15-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0161] [ka]

[0162] The 5'-terminal structure of the oligonucleotide Y16-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0163] [ka]

[0164] The 5'-terminal structure of the oligonucleotide Y17-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0165] [ka]

[0166] The 5'-terminal structure of the oligonucleotide Y18-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0167] [ka]

[0168] The 5'-terminal structure of the oligonucleotide Y19-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0169] [ka]

[0170] The 5'-terminal structure of the oligonucleotide Y20-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0171] [ka]

[0172] The 5'-terminal structure of the oligonucleotide Y59-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0173] [ka]

[0174] The 5'-terminal structure of the oligonucleotide Y60-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0175] [ka]

[0176] The 5'-terminal structure of the oligonucleotide Y21-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0177] [ka]

[0178] The 5'-terminal structure of the oligonucleotide Y22-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0179] [ka]

[0180] The 5'-terminal structure of the oligonucleotide Y23-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0181] [ka]

[0182] The 5'-terminal structure of the oligonucleotide Y24-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0183] [ka]

[0184] The 5'-terminal structure of the oligonucleotide Y25-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0185] [ka]

[0186] The 5'-terminal structure of the oligonucleotide Y26-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0187] [ka]

[0188] The 5'-terminal structure of the oligonucleotide Y27-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0189] [ka]

[0190] The 5'-terminal structure of the oligonucleotide Y28-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0191] [ka]

[0192] The 5'-terminal structure of the oligonucleotide Y29-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0193] [ka]

[0194] The 5'-terminal structure of the oligonucleotide Y30-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0195] [ka]

[0196] The 5'-terminal structure of the oligonucleotide Y31-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0197] [ka]

[0198] The 5'-terminal structure of the oligonucleotide Y32-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0199] [ka]

[0200] The 5'-terminal structure of the oligonucleotide Y33-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0201] [ka]

[0202] The 5'-terminal structure of the oligonucleotide Y34-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0203] [ka]

[0204] The 5'-terminal structure of the oligonucleotide Y35-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0205] [ka]

[0206] The 5'-terminal structure of the oligonucleotide Y36-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0207] [ka]

[0208] The 5'-terminal structure of the oligonucleotide Y37-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0209] [ka]

[0210] The 5'-terminal structure of the oligonucleotide Y38-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0211] [ka]

[0212] The 5'-terminal structure of the oligonucleotide Y39-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0213] [ka]

[0214] The 5'-terminal structure of the oligonucleotide Y40-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0215] [ka]

[0216] The 5'-terminal structure of the oligonucleotide Y41-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0217] [ka]

[0218] The 5'-terminal structure of the oligonucleotide Y42-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0219] [ka]

[0220] The 5'-terminal structure of the oligonucleotide Y43-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0221] [ka]

[0222] The 5'-terminal structure of the oligonucleotide Y44-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0223] [ka]

[0224] The 5'-terminal structure of the oligonucleotide Y45-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0225] [ka]

[0226] The 5'-terminal structure of the oligonucleotide Y46-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0227] [ka]

[0228] The 5'-terminal structure of the oligonucleotide Y47-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0229] [ka]

[0230] The 5'-terminal structure of the oligonucleotide Y48-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0231] [ka]

[0232] The 5'-terminal structure of the oligonucleotide Y49-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0233] [ka]

[0234] The 5'-terminal structure of the oligonucleotide Y50-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0235] [ka]

[0236] The 5'-terminal structure of the oligonucleotide Y51-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0237] [ka]

[0238] The 5'-terminal structure of the oligonucleotide Y52-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0239] [ka]

[0240] The 5'-terminal structure of the oligonucleotide Y53-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0241] [ka]

[0242] The 5'-terminal structure of the oligonucleotide Y54-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0243] [ka]

[0244] The 5'-terminal structure of the oligonucleotide Y55-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0245] [ka]

[0246] The 5'-terminal structure of the oligonucleotide Y57-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the designation "oligo" refers to an oligonucleotide.

[0247] [ka]

[0248] The 5'-terminal structure of the oligonucleotide Chol-cRNA (Malat1) shown in Table 1 is shown below. In the formulas below, the designation "oligo" refers to an oligonucleotide.

[0249] [ka]

[0250] Reference example 1 Synthesis of IY1 A solution of 6-aminohexanoic acid (146 mg) in pyridine (5567 μL) was added with trimethylsilyl chloride (5 69 μL) was added at 0° C. The reaction mixture was stirred at room temperature for 30 minutes under a nitrogen atmosphere. Cholesterol (500 mg) was added at 0°C, and the reaction mixture was stirred at room temperature for 30 min under a nitrogen atmosphere. The reaction mixture was neutralized with 1N aqueous hydrochloric acid at 0° C. and extracted with ethyl acetate. The layers were separated, washed with water, then saturated saline, dried over magnesium sulfate, and concentrated to dryness. The obtained solid was washed with diisopropyl ether to obtain 200 mg of the title compound.

[0251] In the same manner as in Reference Example 1, the compounds of Reference Examples 7, 8, 14 and 15 were prepared.

[0252] Table 2 below shows the chemical structures and NMR data of the compounds of Reference Examples 1, 7, 8, 14 and 15.

[0253] [Table 2] TIFF2025063018000104.tif24872

[0254] Reference example 9 Synthesis of IY9 A solution of estradiol enanthate (200 mg) and pyridine (535 μL) in THF (5 mL) was Phenyl carbonochloridate (210 mg) was added at 0° C. The reaction mixture was allowed to react at room temperature for 3 hours. The precipitate was removed by filtration through Celite, and the filtrate was washed with diisopropyl ether. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane The extract was purified by HPLC (HPLC) to obtain 80 mg of the title compound.

[0255] In the same manner as in Reference Example 9, the compounds of Reference Examples 10, 36, 39 and 40 were prepared. The chemical structures and NMR data of the compounds of Reference Examples 9, 10, 36, 39 and 40 are shown in Table 3 below. .

[0256] [Table 3]

[0257] Reference example 11 Synthesis of IY11 A) Synthesis of IY11-1 Cholesterol chloroformate (300 mg) and 3-(2-aminoethoxy)propanoic acid (89 mg) were used. The title compound was obtained by the same method as in Reference Example 1. This compound was used in the next step without purification. Ta.

[0258] B) Synthesis of IY11 A solution of IY11-1 obtained in step A in tetrahydrofuran (6700 μL) was added to bis(pentafluoro Phenyl) carbonate (528 mg) was added, and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. The reaction mixture was added to water at room temperature and extracted with ethyl acetate. The extract was washed with water and saturated saline. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography. Purification by chromatography (ethyl acetate / hexane) afforded 127 mg of the title compound.

[0259] IY16-1 of Reference Example 16 and IY17-1 of Reference Example 17 were produced in the same manner as in step A) of Reference Example 11. IY16 of Reference Example 16 and IY17 of Reference Example 17 were produced in the same manner as in step B) of Reference Example 11. did.

[0260] Table 4 below shows the chemical structures and NMR data of IY11-1, IY11, IY16-1, IY16, IY17-1, and IY17. This indicates the data.

[0261] [Table 4] TIFF2025063018000107.tif248126

[0262] Reference example 18 Synthesis of IY18

[0263] [ka]

[0264] A solution of cholesterol chloroformate (49.1 mg) in THF (3 mL) was added to the sodium hydroxide solution of IY18-1 (138 mg). Aqueous sodium solution (0.1N, 2.4 mL) was added under ice cooling, and the mixture was stirred at room temperature for 5 hours. The reaction solution was acidified and extracted with THF-ethyl acetate (1:1). After drying over sodium, the mixture was concentrated to dryness to give 151 mg of the title compound.

[0265] The NMR results of the obtained compound are as follows: 1H NMR (300 MHz, CDCl3) δ 0.68 (3H, s), 0.80-1.69 (37H, m), 1.73-2.07 (6H, m), 2.18-2.71 (11H, m), 3.27-3.46 (3H, m), 3.47-3.73 (80H, m), 3.78 (2H, t, J = 6.1 Hz), 4.39-4.66 (1H, m), 5.24 (1H, brs), 5.37 (1H, d, J = 5.4 Hz), 6.98 (1H, s).

[0266] Reference example 19 Synthesis of IY19 Diosgenin (445 mg), Bis(2,5-dioxopyrrolidin-1-yl) carbonate (1650 mg ), triethylamine (2.244 mL) and acetonitrile (10 mL) were sonicated under 3 The reaction mixture was diluted with saturated sodium bicarbonate solution, and then mixed with ethyl acetate and distilled off. The extract was washed with water and saturated saline, then extracted with magnesium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography. (ethyl acetate / hexane) to give 55 mg of the title compound.

[0267] In the same manner as in Reference Example 19, Reference Examples 20, 21, 22, 24, 25, 28, 29, 32, 33, 34, 35, Compounds 43, 44, 48, 51 and 52 were prepared.

[0268] Table 5 below shows Reference Examples 19, 20, 21, 22, 24, 25, 28, 29, 32, 33, 34, 35, 43, 44, and 48. The chemical structures and NMR data of compounds 51, 52 and 53 are shown in the table.

[0269] [Table 5] TIFF2025063018000110.tif201135 TIFF2025063018000111.tif202150 TIFF2025063018000112.tif199146 TIFF2025063018000113.tif203147

[0270] Reference example 23 Synthesis of IY-23 [ka] A) Synthesis of IY23-2 Imidazole (152 mg) and DMAP (91 mg) were added to a dichloromethane solution (10 mL) of IY23-1 (300 mg). The mixture was added at room temperature and stirred for 15 minutes. The reaction mixture was added with water and extracted with ethyl acetate. The organic layers were combined, washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (ethyl acetate / hexane). Purification gave 150 mg of the title compound.

[0271] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl3) δ -0.01 (6H, s), 0.67 (3H, s), 0.66 - 0.96 (15H, m), 0. 91 - 1.14 (9H, m), 1.11 - 1.40 (8H, m), 1.42 (2H, dd, J = 8.0, 4.6 Hz), 1.44 - 1 .53 (6H, m), 1.77 - 1.89 (3H, m), 1.91 - 2.05 (2H, m), 2.17 - 2.33 (2H, m), 3.34 (1H, dd, J = 9.7, 6.8 Hz), 3.42 (1H, dd, J = 9.6, 6.0 Hz), 3.47 - 3.57 (1H, m), 5.34 (1H, d, J = 4.8 Hz).

[0272] B) Synthesis of IY23 Using IY23-1 obtained in Step A, the title compound was obtained in the same manner as in Reference Example 19. The NMR results of the obtained compound are as follows: 1H NMR (300 MHz, CDCl3) δ 0.04 (s, 6 H) 0.68 (s, 3 H) 0.77 - 1.63 (m, 38 H) 1.6 7 - 2.18 (m, 6 H) 2.48 (d, J = 7.54 Hz, 2 H) 2.83 (s, 4 H) 3.28 - 3.51 (m, 2 H) 4.51 - 4.70 (m, 1 H) 5.42 (d, J = 4.14 Hz, 1 H)

[0273] Reference example 27 Synthesis of IY27 [ka] A) Synthesis of IY27-2 A solution of lauric acid (148 mg) in dichloromethane (15 mL) was added to dicyclohexylcarbodiimide ( 226 mg), IY27-1 (250 mg), and DMAP (26.7 mg) were added and stirred at room temperature for 16 hours. The reaction solution was diluted with ethanol (50 mL), and the organic layer was washed with water and saturated saline, and then with anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate). The resultant was purified using ethyl acetate / hexane to give 230 mg of the title compound.

[0274] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl3) δ0.68 (3H, s), 0.87 (3H, t, J = 5.5 Hz), 0.95 (3H, d, J = 6.1 Hz), 1.01(3H, s), 1.05 - 1.18 (3H, m), 1.25 (16H, s), 1.36-1.65 (14H, m), 1.80 - 1.89 (4H, m), 1.91 - 2.04 (2H, m), 2.21 - 2.33 (4H, m), 2.34 - 2.45 (1H, m), 2.46 - 2.59 (1H, m), 4.55 - 4.65 (1H, m), 5.27 (2H, s), 5.36 (1H, d, J = 2. 8 Hz), 7.62 (2H, d, J = 8.4 Hz), 7.77 (2H, d, J = 8.5 Hz).

[0275] B) Synthesis of IY27 A solution of IY27-2 (180 mg) obtained in step A in dichloromethane (10 mL) was added with acetic acid (10 mL) and zinc powder. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane). This gave 100 mg of the title compound.

[0276] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl3) δ 0.67 (3H, s), 0.86 (3H, d, J = 6.9 Hz), 0.93 (3H, d, J = 6.3 Hz), 1.01(3H, s), 1.06 - 1.19 (3H, m), 1.24 (14H, s), 1.36 - 1.65 (14H, m), 1.80 - 1.88 (5H, m), 1.91 - 2.02 (2H, m),2.27 (7H, dt, J = 15.1, 7.3 Hz), 4. 53 - 4.67 (1H, m), 5.36 (1H, d, J = 5.8 Hz).

[0277] Reference example 37 Synthesis of IY37 [ka] A) Synthesis of IY37-2 Imidazole (460 mg) and DMAP (275 mg) were added to a DMF solution (10 mL) of IY37-1 (1.0 g) at room temperature. The mixture was stirred for 15 minutes. tert-Butyldimethylsilyl trifluoromethanesulfonate (1.03 mL) was added. L) was added and the mixture was stirred at room temperature for 16 hours. Water was added to the reaction mixture, which was then extracted with ethyl acetate. The layers were combined, washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain the 1 g of the title compound was obtained.

[0278] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl3) δ 0.04 (6H, d, J = 4.6 Hz), δ 0.66 (3H, s), 0.81 - 0.9 3 (17H, m), 0.95 - 1.17 (12H, m), 1.22 - 1.39 (6H, m), 1.39 - 1.54 (4H, m), 1.71 - 1.91 (5H, m), 1.94 - 2.06 (4H, m), 2.31 (2H, q, J = 7.5 Hz), 3.93 (1H, d, J = 7.9 Hz), 4.54 - 4.66 (1H, m), 5.29 (1H, s).

[0279] B) Synthesis of IY37-3 Hydroxylated IY37-2 (1 g) obtained in step A was dissolved in ethanol (4 mL) and dichloromethane (2 mL). Aqueous sodium solution (10%, 2.86 mL) was added and the mixture was stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure. Ethyl acetate was added to the residue, which was washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give 800 mg of the title compound.

[0280] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl3) δ -0.04 - 0.09 (6H, m), 0.66 (3H, s), 0.82 - 0.93 (18H, m), 0.94 - 1.16 (12H, m), 1.24 (1H, d, J = 7.3 Hz), 1.28 - 1.41 (4H, m), 1.52 ( 6H, d, J = 32.7 Hz), 1.73 - 1.86 (4H, m), 1.95 - 2.05 (1H, m), 2.24 (2H, d, J = 10.9 Hz), 3.47 - 3.58 (1H, m), 3.93 (1H, d, J = 7.6 Hz), 5.26 (1H, s).

[0281] C) Synthesis of IY37-4 A solution of IY37-3 (800 mg) obtained in step B in acetonitrile (4 mL) and dichloromethane (4 mL) triethylamine (3.26 mL) and bis(2,5-dioxopyrrolidin-1-yl) carbonate (2. 38 g) was added and stirred at 45° C. for 16 hours. The reaction mixture was filtered through Celite and diluted with 5% citric acid solution. The organic layer was washed with water and saturated saline, and then anhydrous sodium sulfate was added. The residue was purified by silica gel column chromatography (ethyl acetate / hexane). As a result, 800 mg of the title compound was obtained.

[0282] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl3) δ 0.05 (6H, d, J = 4.6 Hz), 0.66 (3H, s), 0.84 - 0.92 ( 17H, m), 0.95 - 1.16 (12H, m), 1.21 - 1.37 (6H, m), 1.40 - 1.55 (3H, m), 1.70 - 1.82 (3H, m), 1.89 (1H, d, J = 13.8 Hz), 1.95 - 2.06 (3H, m), 2.47 (2H, d, J = 7 .8 Hz), 2.82 (4H, s), 3.92 (1H, d, J = 7.5 Hz), 4.53 - 4.67 (1H, m), 5.31 (1H, d , J = 15.7 Hz).

[0283] D) Synthesis of IY37 To a solution of IY37-4 (800 mg) obtained in step C in THF (8 mL), triethylamine trihydrofluoride was added. (2.64 mL) was added at 0°C and stirred at room temperature for 16 hours. Water was added to the reaction mixture and extracted with ethyl acetate. The organic layers were combined, washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain the title compound. 110 mg was obtained.

[0284] The NMR results of the obtained compound are as follows: 1H NMR (400 MHz, CDCl3) δ 0.68 (3H, s), 0.86 (6H, d, J = 5.0 Hz), 0.91 (3H, d, J = 6.2 Hz), 0.98 - 1.17 (11H, m), 1.22 - 1.43 (8H, m), 1.41 - 1.53 (3H, m), 1.7 0 - 1.83 (2H, m), 1.84 - 1.96 (2H, m), 2.04 (2H, s), 2.51 (2H, t, J = 9.1 Hz), 2 .83 (4H, s), 3.85 (1H, s), 4.55 - 4.69 (1H, m), 5.35 (1H, s).

[0285] Reference example 38 Synthesis of IY38 [ka] Using 5α-cholestane-3β,5α,6β-triol, the same procedure as in step C of Reference Example 37 was carried out. The title compound was synthesized from the above.

[0286] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 0.63 (3H, s), 0.86 (8H, dd, J = 16.0, 5.6 Hz), 0.94 - 1.38 (21H, m), 1.44 - 1.69 (6H, m), 1.71 - 1.81 (1H, m), 1.82 - 1.97 (2H, m), 2.15 (1H, t, J = 12.0 Hz), 2.80 (4H, s), 3.38 (1H, s), 4.13 (1H, s), 4.64 (1H, d, J = 4.0 Hz), 5.03 - 5.14 (1H, m)

[0287] Reference example 41 Synthesis of IY41 [ka]

[0288] A) Synthesis of IY41-2 8N sodium hydroxide solution (2.92 mL) and hydrogen peroxide solution (14.3 mL) were added to IY41-1 (1.8 g). The reaction mixture was stirred at room temperature for 1 hour at 4°C for 24 hours. The mixture was stirred overnight. Hydrogen peroxide solution (1.43 mL) was added. Urea·hydrogen peroxide (1.32 g) was added and the mixture was stirred for 1 hour. The reaction mixture was neutralized with 6N aqueous hydrochloric acid, and an aqueous solution of sodium thiosulfate was added. The organic layer was separated and washed with 1N aqueous hydrochloric acid, saturated sodium bicarbonate, and The extract was washed with an aqueous solution and then with saturated saline, dried over magnesium sulfate, and the solvent was removed by distillation. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound. As a result, 700 mg of the product was obtained as a stereoisomer mixture (4α,5α epoxy form and 4β,5β epoxy form).

[0289] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.64-0.73 (3H, m), 0.80-0.93 (9H, m), 0.94-2.47 (31H, m), 2.98 (1H, s, 4-H beta), 3.03 (1H, 2, 4-H alpha).

[0290] B) Synthesis of IY41-3 A mixture of IY41-2 (700 mg) obtained in step A in acetic acid (10 mL) and THF (10 mL) was added with p-toluenesulfonyl chloride. Ensulfonyl hydrazide (342 mg) was added in portions and the reaction mixture was stirred at room temperature for 4 hours. Saturated saline (30 mL) was added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate. The mixture was extracted, washed with saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, and then the solvent was removed. The residue was purified by silica gel column chromatography (ethyl acetate / hexane). This gave 560 mg of the title compound.

[0291] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.73 (3H, s), 0.82-0.95 (9H, m), 0.96-1.08 (3H, m), 1 .09 (3H, s), 1.10-2.39 (25H, m), 2.44-2.62 (1H, m).

[0292] C) Synthesis of IY41-4 To a methanol solution (10 mL) of IY41-3 (560 mg) obtained in step B, sodium borohydride was added. (110 mg) was added at room temperature. The reaction mixture was stirred at room temperature for 30 minutes and then diluted with ethyl acetate. The organic layer was separated, washed with water and saturated saline, dried over sodium sulfate, and then the solvent was removed by distillation. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound. Compound (5α and 5β hydroxyisomers) was obtained in an amount of 560 mg.

[0293] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.65 (9H, d, J = 3.4 Hz), 0.74-2.36 (35H, m), 3.43-3. 56 (1H, m, 5-H alpha), (1H, d, J = 2.4 Hz, 5-H alpha).

[0294] D) Synthesis of IY41-5 A solution (7 mL) of IY41-4 (540 mg) obtained in step C in ethanol was added to palladium / carbon (7.4 mg The reaction mixture was filtered through an aminosilica pad, and the solvent was removed. The residue was distilled off to obtain 540 mg of the title compound as a stereoisomer mixture (5α and 5β hydroxy forms). .

[0295] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.61-0.69 (3H, m), 0.80 (3H, s), 0.81-1.91 (42H, m), 1.92-2.03 (1H, m), 3.43-3.55 (1H, m, 5-H alpha), 3.63 (1H, brs, 5-H beta).

[0296] E) Synthesis of IY41 Using IY41-5 obtained in Step D, the title compound was obtained as a single isomer in the same manner as in Reference Example 9. The α-hydroxy compound of IY41-5, which remained unreacted under these reaction conditions, was obtained during the purification process. Removed.

[0297] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.66 (4H, s), 0.73-2.07 (47H, m), 4.72 (1H, dd, J = 1 1.2, 4.2 Hz), 7.36 (2H, d, J = 8.9 Hz), 8.28 (2H, d, J = 8.3 Hz).

[0298] Reference example 42 Synthesis of IY42 [ka] A) Synthesis of IY42-2 To a solution of IY42-1 (3.03 g) in pyridine (30 mL), p-toluenesulfonyl chloride (2.40 g) was added. The reaction mixture was stirred for 3 hours, and then p-toluenesulfonyl chloride (2.40 g) was added. To the reaction mixture was added 1N aqueous hydrochloric acid at room temperature, and the mixture was extracted with ethyl acetate. The organic layer was separated, washed with saturated saline, dried over magnesium sulfate, and then the solvent was distilled off. The residue was washed with diisopropyl ether to give 1.45 g of the title compound. This was used in the next step without any further purification.

[0299] B) Synthesis of IY42-3 Sodium azide (238 mg) was added to IY42-2 (377 mg) obtained in step A in DMF (3 mL) at room temperature. The mixture was stirred at 70°C for 4 hours. The reaction mixture was added to water at room temperature and extracted with ethyl acetate. The layers were separated, washed with saturated saline, dried over magnesium sulfate, and the solvent was removed by distillation. The residue was washed with diisopropyl ether to give 310 mg of the title compound. The crude product was used in the next step without purification. It was used in the process.

[0300] C) Synthesis of IY42-5 The polymer-supported IY42-3 (493 mg) obtained in step B was dissolved in a mixture of THF (10 mL) and water (1 mL). Triphenylphosphine (3 mmol / g) (1.32 g) was added and the mixture was stirred at 70° C. for 3 hours. Insoluble solid The mixture was filtered and washed with ethyl acetate. Saturated saline was added to the filtrate, the organic layer was separated, and the mixture was washed with sulfuric acid. After drying over magnesium, the solvent was removed by distillation. The residue was dissolved in pyridine (10 mL) and 3,5- Bis(trifluoromethyl)-benzoyl chloride (0.241 mL) was added at room temperature. The mixture was stirred overnight, and then 1N aqueous hydrochloric acid was added, followed by extraction with ethyl acetate. After washing with saturated saline and drying over magnesium sulfate, the solvent was removed by distillation. The title compound was purified by silica gel column chromatography (ethyl acetate / hexane) to give 370 mg was obtained.

[0301] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.81 (3H, s), 0.94-1.74 (22H, m), 1.74-2.12 (5H, m), 2.33 (2H, d, J = 7.3 Hz), 4.20-4.38 (1H, m), 4.62 (1H, brs), 5.39 (1H, brs), 5.9 6 (1H, d, J = 8.9 Hz), 8.00 (1H, s), 8.16 (2H, s).

[0302] D) Synthesis of IY42-6 IY49-5 (370 mg) obtained in step C was dissolved in methanol (3 mL)-THF (3 mL), and potassium carbonate was added. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed by distillation to give a residue. A 1N aqueous solution of hydrochloric acid was added to the mixture, and the mixture was extracted with ethyl acetate. The mixture was neutralized with aqueous ethanol and extracted with ethyl acetate. The organic layer was separated and washed with water and then saturated sodium chloride solution. After washing with water and drying over magnesium sulfate, the solvent was removed by distillation. The residue was purified by silica gel column chromatography. Purification by chromatography (ethyl acetate / hexane) afforded 183 mg of the title compound.

[0303] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.80 (3H, s), 0.90-2.12 (25H, m), 2.16-2.65 (2H, m), 3.53 (1H, brs), 4.25 (1H, d, J = 6.8 Hz), 5.36 (1H, brs), 6.08 (1H, d, J =8.7 Hz) ), 7.99 (1H, s), 8.16 (2H, s).

[0304] E) Synthesis of IY42 The title compound was synthesized in the same manner as in Reference Example 19 using IY42-6 obtained in Step D.

[0305] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.81 (3H, s), 0.90-1.24 (7H, m), 1.32 (3H, d, J = 6.4 Hz), 1.41-2.16 (14H, m), 2.49 (2H, d, J = 7.0 Hz), 2.84 (4H, s), 4.27 (1H, s),4 .62 (1H, s), 5.42 (1H, d, J = 4.7 Hz), 5.92 (1H, d, J = 9.4 Hz), 8.00 (1H, s), 8 .15 (2H, s).

[0306] Reference example 46 Synthesis of IY46 [ka]

[0307] IY42-3 (830 mg) obtained in Step B of Reference Example 42 and triphenylphosphine (577 mg) in THF( The mixture of 10 mL of 1N hydrochloric acid and water (1 mL) was stirred at 70° C. for 1 hour. The reaction mixture was added to 1 N aqueous hydrochloric acid solution, The insoluble solid was filtered off. The organic layer was separated, washed with saturated saline, and dried over magnesium sulfate. The solvent was then distilled off. The residue was diluted with THF (20 mL), triethylamine (0.562 mL), and Phosgene (598 mg) was added at 0° C. and the mixture was stirred at room temperature overnight. The mixture was neutralized with an aqueous sodium solution at 0°C and extracted with a mixed solvent of ethyl acetate and THF. The mixture was separated, washed with water and then with saturated saline, dried over magnesium sulfate, and the solvent was removed by distillation. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound. The product was obtained in an amount of 485 mg.

[0308] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.67 (3H, s), 0.78-2.12 (38H, m), 2.35 (2H, d, J = 8. 3 Hz), 3.24 (1H, d, J = 9.0 Hz), 5.37 (1H, d, J = 5.1 Hz).

[0309] Reference example 47 Synthesis of IY47 [ka]

[0310] IY42-3 (570 mg) obtained in Step B of Reference Example 42, hepta-6-ynoic acid (0.226 mL), copper sulfate (I I) Ascorbic acid pentahydrate (346 mg), ethanol (5 mL), water (5 mL) and THF (10 mL) were added to a mixture. Sodium acetate (1920 mg) was added at room temperature and stirred overnight. The insoluble solid was filtered off, and the solvent of the filtrate was The mixture was evaporated under reduced pressure, and 1N aqueous hydrochloric acid was added to the residue, followed by the addition of ethyl acetate-THF (4:1). The product was separated, washed with water and then with saturated saline, dried over magnesium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the target compound. 188 mg of the title compound was obtained.

[0311] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.69 (3H, s), 0.79-2.19 (43H, m), 2.27-2.89 (6H, m), 4.34 (1H, brs), 5.45 (1H, brs), 7.32 (1H, s).

[0312] Reference example 49 Synthesis of IY49 [ka]

[0313] IY42-3 (200 mg) obtained in Step B of Reference Example 42 and (1R,9S)-bicyclo[6.1.0]non-4-yne-9 A mixture of 2,5-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate (142 mg) and THF (15 mL) was The mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was The extract was separated, washed with saturated saline, dried over magnesium sulfate, and the solvent was removed by distillation. The title compound was purified by silica gel column chromatography (ethyl acetate / hexane) to give 134 mg was obtained.

[0314] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) - 0.70 (3H, s), 0.80-1.66 (36H, m), 1.70-2.12 (6H, m), 2 .17-2.51 (4H, m), 2.68 (1H, ddt, J = 16.0, 10.0, 3.2 Hz), 2.85 (4H, s),2.89-3.27 (4H, m), 4.01 (1H, tt, J = 12.2, 3.9 Hz), 4.29-4.44 (1H, m), 4.47-4.61 (1H, m), 5.42 (1H, d, J = 1.9 Hz).

[0315] Reference example 50 Synthesis of IY50 [ka]

[0316] A) Synthesis of IY50-2 Starting from IY50-1 (1.02 g), the title compound was obtained in 8 portions in the same manner as in Step A of Reference Example 42. Obtained 25 mg.

[0317] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.64 (3H, s), 0.81-2.39 (33H, m), 2.45 (3H, s), 3.50 (2H, brs), 3.67-4.06 (3H, m), 4.71 (1H, brs), 5.33 (1H, brs), 7.34 (2H, d, J = 7 .5 Hz), 7.78 (2H, d, J = 7.9 Hz).

[0318] B) Synthesis of IY50-3 Starting from IY50-2 (411 mg) obtained in Step A, a similar reaction was carried out in the same manner as in Step B of Reference Example 42. Thus, 258 mg of the title compound was obtained.

[0319] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ ppm 0.69 (3H, s), 0.82-2.43 (33H, m), 3.04 (1H, dd, J = 11.8, 7.4 Hz), 3.28-3.68 (3H, m), 3.81-4.03 (1H, m), 4.72 (1H, brs), 5.35(1H, brs).

[0320] C) Synthesis of IY50-5 Starting from IY50-3 (258 mg) obtained in Step B, the same procedure as in Step C of Reference Example 42 was carried out. The title compound was obtained in an amount of 190 mg.

[0321] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.73 (3H, s), 0.87-1.43 (22H, m), 1.63-2.14 (8H, m), 2.37 (1H, s), 3.03-4.03 (7H, m), 4.72 (1H, brs), 5.36 (1H, s), 6.13 (1H, s), 8.0 1 (1H, s), 8.20 (2H, s).

[0322] D) Synthesis of IY50-6 IY50-5 (190 mg) obtained in step C, pyridinium p-toluenesulfonate (101 mg) and methionine The mixture was stirred under reflux for 1 hour. The solvent was removed under reduced pressure, and the residue was The mixture was poured into water and extracted with ethyl acetate. The organic layer was separated, washed with saturated saline, and then extracted with magnesium sulfate. After drying over methanol, the solvent was distilled off to give 170 mg of the title compound. It was used in the process.

[0323] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.74 (3H, s), 0.86-2.45 (28H, m), 3.10-3.33 (1H, m), 3.41-3.69 (2H, m), 5.35 (1H, brs), 6.15 (1H, brs), 8.01 (1H, s), 8.20 (2H, s).

[0324] E) Synthesis of IY50 Using IY53-6 ​​obtained in Step D, the title compound was obtained in the same manner as in Reference Example 19.

[0325] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.74 (3H, s), 0.88-2.21 (25H, m), 2.49 (2H, d, J = 7. 9 Hz), 2.84 (4H, s), 3.09-3.32 (1H, m), 3.59 (1H, d, J = 12.8 Hz), 4.60 (1H, brs), 5.43 (1H, brs), 6.15 (1H, brs), 8.01 (1H, s), 8.20 (2H, s).

[0326] Other compounds used in the examples are shown in the table below. Commercially available products may be used as they are. Alternatively, it can be produced by a method known per se or a method analogous thereto.

[0327] [Table 6] TIFF2025063018000125.tif28163

[0328] Example 1 A) Synthesis of cRNA with Y1-linked to the 5' end (Y1-cRNA(Malat1)) 50 mM NMP solution of IY1 (800 μL), 75 mM NMP solution of HATU (800 μL), 150 mM NMP solution of DIPEA (800 μL) The solution (800 μL) was mixed in an Eppendorf flask, stirred, and then allowed to react at room temperature for 30 minutes. The RNA strand (Y61-cRNA(Malat1)) shown in Table 1 was added to the solution (2000 nmol), NMP (2100 μL), and distilled water ( After stirring and settling, the mixture was allowed to react at room temperature for 1 hour. The solution was then passed through an ODS column (column: Purif-Pack®-EX ODS-50 size 60, manufactured by Shoko Science, The mobile phase was purified using TEAA / acetonitrile and then ultrafiltered (Amicon ultra 3kDa, Merck Millipore) to obtain the eluate. The resulting solution was desalted using 1 M meglumine acetate (Pore Inc., distilled water). The mixture was added in 10-fold the amount of the ion exchange solution, stirred thoroughly, and left for 5 minutes to carry out ion exchange. The solution was desalted using a micron ultra 3 kDa filter (Merck Millipore, distilled water). After filtration through a .mu.m membrane filter and lyophilization, 690 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y1-HDO A 16mer single-stranded LN targeting metastasis-associated lung adenocarcinoma transcript (Malat1) non-coding RNA A / DNA gapmer (ASO(Malat1)) consists of three LNA nucleosides at the 5' end and three at the 3' end. , and 10 DNA nucleosides between them. This LNA / DNA gapmer is Malat1 non-coding RNA (GenBank accession number NR_002847, SEQ ID NO: 3) of The ASO (Malat1) (first strand) and the Y obtained in the previous step A have a complementary base sequence to 1316-1331. 1-cRNA(Malat1)(2nd strand) was mixed in equimolar amounts, and the mixture was heated at 70°C for 5 minutes. The mixture was cooled slowly to room temperature, and the double-stranded nucleic acid agent, Y1-linked heteroduplex oligonucleotide (Y1 -conjugated heteroduplex oligonucleotide, Y1-HDO) was prepared.

[0329] Example 3 A) Synthesis of cRNA with Y3-linked at the 5' end (Y3-cRNA(Malat1)) 50 mM DMSO solution of IY3 (240 μL), 75 mM NMP solution of HATU (240 μL), DIPEA (240 μL) The mixture was mixed with 150 mM NMP solution (240 μL) in an Eppendorf flask, stirred, and allowed to settle, then incubated at room temperature for 15 minutes. An aqueous solution (253 μL, 1200 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1, DM SO (1980 μL), distilled water (47.4 μL), and DIPEA (38 μL) were added, stirred, and allowed to settle. The reaction solution was diluted with distilled water to an NMP concentration of 10 v / v%, and then acetic acid was added. Extract with ethyl acetate, and the aqueous layer is ultrafiltered (Amicon ultra 3kDa, Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter and freeze-dried. The title compound was obtained quantitatively. B) Synthesis of double-stranded nucleic acid agent Y3-HDO Using ASO (Malat1) (first strand) and Y3-cRNA (Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y3-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y3-conjugated heteroduplex oligonucleotide (Y3-HDO) was prepared.

[0330] Example 4 A) Synthesis of cRNA with Y4-linked at the 5' end (Y4-cRNA(Malat1)) The RNA strand (Y61-cRNA(Malat1)) shown in Table 1 was dissolved in water (2000 nmol) and IY4 was used to prepare the RNA strand of Example 1. The title compound (590 nmol) was obtained in the same manner as in Step A. B) Synthesis of double-stranded nucleic acid agent Y4-HDO Using ASO (Malat1) (first strand) and Y4-cRNA (Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y4-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is A Y4-conjugated heteroduplex oligonucleotide (Y4-HDO) was prepared.

[0331] Example 5 A) Synthesis of cRNA with Y5-linked at the 5' end (Y5-cRNA(Malat1)) The same procedure as in Example 3 was carried out using an aqueous solution (1000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 and IY5. The title compound was obtained in an amount of 776 nmol in the same manner as in Step A. B) Synthesis of double-stranded nucleic acid agent Y5-HDO Using ASO (Malat1) (first strand) and Y5-cRNA (Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y5-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is A Y5-conjugated heteroduplex oligonucleotide (Y5-HDO) was prepared.

[0332] Example 6 A) Synthesis of cRNA with Y6-linked at the 5' end (Y6-cRNA(Malat1)) The same procedure as in Example 3 was carried out using an aqueous solution (1000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 and IY6. The title compound (880 nmol) was obtained in the same manner as in Step A. B) Synthesis of double-stranded nucleic acid agent Y6-HDO Using ASO (Malat1) (first strand) and Y6-cRNA (Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y6-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y6-conjugated heteroduplex oligonucleotide (Y6-HDO) was prepared.

[0333] Example 7 A) Synthesis of cRNA with Y7-linked at the 5' end (Y7-cRNA(Malat1)) 1200 μL of 50 mM NMP solution of IY7, 1200 μL of 75 mM NMP solution of HATU, and 150 mM NMP solution of DIPEA The P solution (1200 μL) was mixed in an Eppendorf flask, stirred, and then allowed to react at room temperature for 30 minutes. In addition, an aqueous solution (548 μL, 3000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 and NMP (3150 μL ), distilled water (202 μL), and DIPEA (94 μL) were added, stirred, and allowed to settle, then incubated at room temperature for 1 hour. The reaction solution was then loaded onto an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, Shoko The mixture was purified using a mobile phase of TEAA / acetonitrile (manufactured by Jensen Co., Ltd.) and ultrafiltered using an Amicon ultra 3kDa The solution was desalted using distilled water (Merck Millipore). The mixture was dissolved in distilled water to give 780 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y7-HDO A 16mer single-stranded LN targeting metastasis-associated lung adenocarcinoma transcript (Malat1) non-coding RNA A / DNA gapmer (ASO(Malat1)) consists of three LNA nucleosides at the 5' end and three at the 3' end. , and 10 DNA nucleosides between them. This LNA / DNA gapmer is Malat1 non-coding RNA (GenBank accession number NR_002847, SEQ ID NO: 3) of The ASO (Malat1) (first strand) and the Y1 obtained in the previous step A have a complementary base sequence to 1316-1331. -cRNA(Malat1)(2nd strand) was mixed in equimolar amounts, and the mixture was heated at 70°C for 5 minutes. The mixture was gradually cooled to room temperature. It was then ion-exchanged to a sodium salt using BT AG 50W-X8 Resin. After exchange, the mixture was lyophilized and distilled water was added to obtain the double-stranded nucleic acid drug Y7-linked heteroduplex oligonucleotide. The oligonucleotide (Y1-conjugated heteroduplex oligonucleotide, Y7-HDO) was prepared. .

[0334] Example 8 A) Synthesis of cRNA with Y8-linked at the 5' end (Y8-cRNA(Malat1)) The same procedure as in Example 7 was carried out using an aqueous solution (3,000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 and IY8. The title compound (630 nmol) was obtained in the same manner as in Step A. B) Synthesis of double-stranded nucleic acid agent Y8-HDO The ASO (Malat1) (first strand) and the Y8-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 7, a Y8-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y8-conjugated heteroduplex oligonucleotide (Y8-HDO) was prepared.

[0335] Example 9 A) Synthesis of cRNA with Y9-linked at the 5' end (Y9-cRNA(Malat1)). The RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was added to an aqueous solution (260 μL, 1500 nmol) of 50 mM NMP Solution (900 μL), distilled water (190 μL), NMP (2100 μL), Borate Buffer, pH 8.5+ / -0.2, 5X C After mixing in an Eppendorf flask, stirring, and settling, the mixture was incubated at room temperature for 2 hours. The reaction solution was then loaded onto an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, Showa The mixture was purified using a mobile phase of TEAA / acetonitrile (manufactured by Hikari Science Co., Ltd.) and ultrafiltered (Amicon ultra 3 The solution was desalted using 1000 mL of 1000 mL of 1000 kDa (Merck Millipore, distilled water). The resulting solution was freeze-dried. As a result, 578 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y9-HDO Using ASO (Malat1) (first strand) and Y9-cRNA (Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 7, a Y9-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y9-conjugated heteroduplex oligonucleotide (Y9-HDO) was prepared.

[0336] Example 10 A) Synthesis of cRNA with Y10-linked at the 5' end (Y10-cRNA(Malat1)). An aqueous solution (421 μL, 2000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was diluted with 50 mM NMP Solution (800 μL), distilled water (79 μL), NMP (3200 μL), Borate Buffer, pH 8.5+ / -0.2, 5X Co The mixture was mixed in an Eppendorf flask, stirred, precipitated, and then incubated at room temperature for 1 hour. The reaction solution was diluted with distilled water to an NMP concentration of 10 v / v%, and then filtered through an ultrafiltration (Amicon Ultra 3kDa (Merck Millipore, distilled water) was used for concentration and removal of low molecular weight reagents such as NMP. The final product was filtered through a 0.20 μm membrane filter, freeze-dried, and then labeled. The compound was obtained in an amount of 1185 nmol. B) Synthesis of double-stranded nucleic acid agent Y10-HDO Using ASO(Malat1)(1st strand) and Y10-cRNA(Malat1)(2nd strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y10-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y10-conjugated heteroduplex oligonucleotide (Y10-HDO) was prepared.

[0337] Example 11 A) Synthesis of cRNA with Y11-linked at the 5' end (Y11-cRNA(Malat1)) An aqueous solution (173.5 μL, 1000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was diluted with 50 mM N MP solution (400 μL), distilled water (76.5 μL), NMP (1600 μL), ×10 PBS (250 μL), DIPEA (31 The reaction mixture was mixed in this order in an Eppendorf flask, stirred, precipitated, and then reacted at 70°C for 1 hour. The column was transferred to an ODS column (Purif-Pack®-EX ODS-50 size 60, manufactured by Shoko Science Co., Ltd.). The mixture was purified by ultrafiltration (Amicon ultra 3kDa, Merck Millipore) and then purified by ultrafiltration (Amicon ultra 3kDa, Merck Millipore). The solution was desalted using 10 mL of 1 M meglumine acetate (manufactured by Ajinomoto Co., Inc., distilled water). The mixture was added in double the amount, stirred thoroughly, and left for 5 minutes to carry out ion exchange. The resulting solution was desalted using a 0.20% NaCl solution (Merck Millipore, distilled water). After filtration through a 1.0 μm membrane filter and lyophilization, 592 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y11-HDO Using ASO(Malat1)(1st strand) and Y11-cRNA(Malat1)(2nd strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y11-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y11-conjugated heteroduplex oligonucleotide (Y11-HDO) was prepared.

[0338] Example 12 A) Synthesis of cRNA with Y12-linked at the 5' end (Y12-cRNA(Malat1)). Example 7 was performed using an aqueous solution (3000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY12. The title compound (1770 nmol) was obtained in the same manner as in step A of above. B) Synthesis of double-stranded nucleic acid agent Y12-HDO Using ASO(Malat1) (first strand) and Y12-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 7, a Y12-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is A Y12-conjugated heteroduplex oligonucleotide (Y12-HDO) was prepared.

[0339] Example 13 A) Synthesis of cRNA with Y13-linked at the 5' end (Y13-cRNA(Malat1)). Example 7 was performed using an aqueous solution (3000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY13. The title compound was obtained in an amount of 1,230 nmol in the same manner as in Step A. B) Synthesis of double-stranded nucleic acid agent Y13-HDO Using ASO(Malat1) (first strand) and Y13-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 7, a Y13-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y13-conjugated heteroduplex oligonucleotide (Y13-HDO) was prepared.

[0340] Example 14 A) Synthesis of cRNA with Y14-linked at the 5' end (Y14-cRNA(Malat1)). The same procedure as in Example 1 was carried out using an aqueous solution (3,000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 and IY14. The title compound was obtained in an amount of 1650 nmol in the same manner as in Step A. B) Synthesis of double-stranded nucleic acid agent Y14-HDO Using ASO(Malat1) (first strand) and Y14-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y14-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y14-conjugated heteroduplex oligonucleotide (Y14-HDO) was prepared.

[0341] Example 15 A) Synthesis of cRNA with Y15-linked at the 5' end (Y15-cRNA(Malat1)). Aqueous solutions (210 μl nmol, 1000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY15 were Using this, 900 nmol of the title compound was obtained in the same manner as in Step A of Example 3. B) Synthesis of double-stranded nucleic acid agent Y15-HDO Using ASO(Malat1) (first strand) and Y15-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y15-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y15-conjugated heteroduplex oligonucleotide (Y15-HDO) was prepared.

[0342] Example 16 A) Synthesis of cRNA with Y16-linked at the 5' end (Y16-cRNA(Malat1)). Using an aqueous solution (176 μL, 1000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY16 The title compound (532 nmol) was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y16-HDO Using ASO(Malat1) (first strand) and Y16-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y16-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y16-conjugated heteroduplex oligonucleotide (Y16-HDO) was prepared.

[0343] Example 17 A) Synthesis of cRNA with Y17-linked at the 5' end (Y17-cRNA(Malat1)). Using an aqueous solution (176 μL, 1000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY17 The title compound (686 nmol) was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y17-HDO Using ASO(Malat1) (first strand) and Y17-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y17-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y17-conjugated heteroduplex oligonucleotide (Y17-HDO) was prepared.

[0344] Example 18 A) Synthesis of cRNA with Y18-linked at the 5' end (Y18-cRNA(Malat1)). Using an aqueous solution (274 μL, 1500 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY18 The title compound (1398 nmol) was obtained in the same manner as in Step A of Example 3. B) Synthesis of double-stranded nucleic acid agent Y18-HDO Using ASO(Malat1) (first strand) and Y18-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y18-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y18-conjugated heteroduplex oligonucleotide (Y18-HDO) was prepared.

[0345] Example 19 A) Synthesis of cRNA with Y19-linked at the 5' end (Y19-cRNA(Malat1)). An aqueous solution (210.5 μL, 1000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was diluted with 50 mM N MP solution (400 μL), distilled water (39.5 μL), NMP (1600 μL), ×10 PBS (250 μL), DIPEA (31 The reaction mixture was mixed in this order in an Eppendorf flask, stirred, allowed to settle, and then reacted at room temperature for 1 hour. The solution was diluted with distilled water to an NMP concentration of 10 v / v%, and then ultrafiltered (Amicon ultra 3 kDa, The solution was concentrated and low molecular weight reagents such as NMP were removed using distilled water (Merck Millipore). The mixture was filtered through a 0.20 μm membrane filter, freeze-dried, and the title compound was analyzed by spectrometry at 927 nm. I got ol. B) Synthesis of double-stranded nucleic acid agent Y19-HDO Using ASO(Malat1)(1st strand) and Y19-cRNA(Malat1)(2nd strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y19-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y19-conjugated heteroduplex oligonucleotide (Y19-HDO) was prepared.

[0346] Example 20 A) Synthesis of cRNA with Y20-attached to the 5' end (Y20-cRNA(Malat1)). Using an aqueous solution (260 μL, 1500 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY19 The title compound (686 nmol) was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y20-HDO Using ASO(Malat1) (first strand) and Y20-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y20-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y20-conjugated heteroduplex oligonucleotide (Y20-HDO) was prepared.

[0347] Example 21 A) Synthesis of cRNA with Y21-linked at the 5' end (Y21-cRNA(Malat1)). The RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was added to an aqueous solution (260 μL, 1500 nmol) of 50 mM NMP solution (300 μL), distilled water (115 μL), NMP (2400 μL), ×10 PBS (375 μL), 50 mM DMAP (3 The mixture was mixed in this order in an Eppendorf flask, stirred, precipitated, and then reacted at 70°C for 1 hour. The solution was then passed through an ODS column (column: Purif-Pack®-EX ODS-50 size 60, manufactured by Shoko Science, The mobile phase was purified using TEAA / acetonitrile and then ultrafiltered (Amicon ultra 3kDa, Merck Millipore) to obtain the eluate. The final product was filtered through a 0.20 μm membrane filter. After filtration and lyophilization, 917 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y21-HDO Using ASO(Malat1)(1st strand) and Y21-cRNA(Malat1)(2nd strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y21-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y21-conjugated heteroduplex oligonucleotide (Y21-HDO) was prepared.

[0348] Example 22 A) Synthesis of cRNA with Y22-linked at the 5' end (Y22-cRNA(Malat1)). Using an aqueous solution (365 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY22 The title compound (1253 nmol) was obtained in the same manner as in Step A of Example 21. B) Synthesis of double-stranded nucleic acid agent Y22-HDO Using ASO(Malat1) (first strand) and Y22-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y22-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y22-conjugated heteroduplex oligonucleotide (Y22-HDO) was prepared.

[0349] Example 23 A) Synthesis of cRNA with Y23-linked at the 5' end (Y23-cRNA(Malat1)). An aqueous solution (382 μL, 2200 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was diluted with 50 mM NMP solution (440 μL), distilled water (168 μL), NMP (3520 μL), ×10 PBS (550 μL), 50 mM DMAP (4 40 μL) were mixed in an Eppendorf flask, stirred, and allowed to settle, after which the reaction was allowed to proceed at 70°C for 1 hour. The solution was then passed through an ODS column (column: Purif-Pack®-EX ODS-50 size 60, manufactured by Shoko Science, The mobile phase was purified using TEAA / acetonitrile and then ultrafiltered (Amicon ultra 3kDa, Merck Millipore) to obtain the eluate. The resulting solution was diluted with distilled water to 1000 μL. Distilled water (200 μL), NMP (400 μL), methanol (4400 μL), 50 mM hydrogen fluoride in DMSO The liquids (2000 μL) were mixed in this order in an Eppendorf flask, stirred, and then allowed to settle, and then reacted at room temperature for 1 hour. A 300 mM trimethylethoxysilane solution in DMSO (2000 μL) was added to the reaction mixture. The mixture was separated using an ODS column (column: Purif-Pack®-EX ODS-50 size 60, manufactured by Shoko Science, The mobile phase was purified using TEAA / acetonitrile and then ultrafiltered (Amicon ultra 3kDa, Merck Millipore) to obtain the eluate. The final product was filtered through a 0.20 μm membrane filter. After filtration and lyophilization, 1114 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y23-HDO Using ASO(Malat1) (first strand) and Y23-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 7, a Y23-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y23-conjugated heteroduplex oligonucleotide (Y23-HDO) was prepared.

[0350] Example 24 A) Synthesis of cRNA with Y24-linked at the 5' end (Y24-cRNA(Malat1)). Using an aqueous solution (174 μL, 1000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY24 The title compound (870 nmol) was obtained in the same manner as in Step A of Example 21. B) Synthesis of double-stranded nucleic acid agent Y24-HDO Using ASO(Malat1) (first strand) and Y24-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 7, a Y24-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, was A Y24-conjugated heteroduplex oligonucleotide (Y24-HDO) was prepared.

[0351] Example 25 A) Synthesis of cRNA with Y25-linked at the 5' end (Y25-cRNA(Malat1)). Using an aqueous solution (274 μL, 1500 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY25 The title compound (760 nmol) was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y25-HDO Using ASO(Malat1) (first strand) and Y25-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y25-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y25-conjugated heteroduplex oligonucleotide (Y25-HDO) was prepared.

[0352] Example 26 A) Synthesis of cRNA with Y26-linked at the 5' end (Y26-cRNA(Malat1)). 400 μL of 50 mM THF solution of IY26, 400 μL of 75 mM NMP solution of HATU, and 150 mM NMP solution of DIPEA The solution (400 μL) was mixed in an Eppendorf flask, stirred, precipitated, and then reacted at room temperature for 30 minutes. The RNA strand (Y61-cRNA(Malat1)) shown in Table 1 was added to the solution (315 μL, 2000 nmol) and NMP (1450 μL). Add THF (1850 μL), distilled water (184 μL), and DIPEA (63 μL), stir, settle, and then store at room temperature. The reaction was allowed to proceed for 1 hour. In addition, a 50 mM THF solution of IY26 (400 μL), a 75 mM NMP solution of HATU (4 00 μL) and 150 mM NMP solution of DIPEA (400 μL) were mixed in a Falcon tube and left to stand for 15 minutes. After that, the reaction solution was combined with DIPEA (125 μL) and reacted at room temperature for 2 hours. Column (Column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, Mobile phase: The mixture was purified using TEAA / acetonitrile and ultrafiltered (Amicon ultra 3kDa, Merck Millipore) The solution was desalted using 10 volumes of 1 M meglumine acetate. The mixture was stirred thoroughly and left for 5 minutes to carry out ion exchange. The final product was desalted using a 0.20 μm mesh filter (Merck Millipore, distilled water). After filtration through a membrane filter and lyophilization, 237 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y26-HDO Using ASO(Malat1) (first strand) and Y26-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y26-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y26-conjugated heteroduplex oligonucleotide (Y26-HDO) was prepared.

[0353] Example 27 A) Synthesis of cRNA with Y27-linked at the 5' end (Y27-cRNA(Malat1)). Using an aqueous solution (312 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY27 The title compound (705 nmol) was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y27-HDO Using ASO(Malat1) (first strand) and Y27-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y27-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y27-conjugated heteroduplex oligonucleotide (Y27-HDO) was prepared.

[0354] Example 28 A) Synthesis of cRNA with Y28-linked at the 5' end (Y28-cRNA(Malat1)). Using an aqueous solution (346 μL, 2530 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY28 The title compound (1046 nmol) was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y28-HDO Using ASO(Malat1) (first strand) and Y28-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y28-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y28-conjugated heteroduplex oligonucleotide (Y28-HDO) was prepared.

[0355] Example 29 A) Synthesis of cRNA with Y29-linked at the 5' end (Y29-cRNA(Malat1)). Using an aqueous solution (369 μL, 1500 nmol) of the RNA strand (Y62-cRNA(Malat1)) shown in Table 1 and IY29 The title compound (903 nmol) was obtained in the same manner as in Step A of Example 21. B) Synthesis of double-stranded nucleic acid agent Y29-HDO Using ASO(Malat1) (first strand) and Y29-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 7, a Y29-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, was A Y29-conjugated heteroduplex oligonucleotide (Y29-HDO) was prepared.

[0356] Example 30 A) Synthesis of cRNA with Y30-linked at the 5' end (Y30-cRNA(Malat1)). An aqueous solution (358 μL, 1500 nmol) of the RNA strand (Y63-cRNA(Malat1)) shown in Table 1 and chloroformate were Using esterol, 1228 nmol of the title compound was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y30-HDO The ASO (Malat1) (first strand) and the Y30-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y30-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y30-conjugated heteroduplex oligonucleotide (Y30-HDO) was prepared.

[0357] Example 31 A) Synthesis of cRNA with Y31-linked at the 5' end (Y31-cRNA(Malat1)). Using an aqueous solution (355 μL, 1400 nmol) of the RNA strand (Y64-cRNA(Malat1)) shown in Table 1 and IY29 The title compound (657 nmol) was obtained in the same manner as in Step A of Example 21. B) Synthesis of double-stranded nucleic acid agent Y31-HDO Using ASO(Malat1) (first strand) and Y31-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 7, a Y31-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, was A Y31-conjugated heteroduplex oligonucleotide (Y31-HDO) was prepared.

[0358] Example 32 A) Synthesis of cRNA with Y32-linked at the 5' end (Y32-cRNA(Malat1)). Using an aqueous solution (315 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY32 The title compound (864 nmol) was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y32-HDO The ASO (Malat1) (first strand) and the Y32-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y32-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y32-conjugated heteroduplex oligonucleotide (Y32-HDO) was prepared.

[0359] Example 33 A) Synthesis of cRNA with Y33-linked at the 5' end (Y33-cRNA(Malat1)). The RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was added to an aqueous solution (365 μL, 2000 nmol) of 50 mM NMP solution (400 μL), distilled water (135 μL), NMP (3200 μL), ×10 PBS (500 μL), 50 mM DMAP (4 The mixture was mixed in this order in an Eppendorf flask, stirred, precipitated, and then reacted at 70°C for 1 hour. The solution was then passed through an ODS column (column: Purif-Pack®-EX ODS-50 size 60, manufactured by Shoko Science, The mobile phase was purified using TEAA / acetonitrile and then ultrafiltered (Amicon ultra 3kDa, Merck Millipore) to obtain the eluate. The resulting solution was desalted using 1 M meglumine acetate (Pore Inc., distilled water). The mixture was added in 10-fold the amount of the ion exchange solution, stirred thoroughly, and left for 5 minutes to carry out ion exchange. The solution was desalted using a micron ultra 3 kDa filter (Merck Millipore, distilled water). After filtration through a .mu.m membrane filter and lyophilization, 963 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y33-HDO Using ASO(Malat1) (first strand) and Y33-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y33-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y33-conjugated heteroduplex oligonucleotide (Y33-HDO) was prepared.

[0360] Example 34 A) Synthesis of cRNA with Y34-linked at the 5' end (Y34-cRNA(Malat1)). Using an aqueous solution (365 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY34 The title compound (1083 nmol) was obtained in the same manner as in Step A of Example 33. B) Synthesis of double-stranded nucleic acid agent Y34-HDO The ASO (Malat1) (first strand) and the Y34-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y34-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y34-conjugated heteroduplex oligonucleotide (Y34-HDO) was prepared.

[0361] Example 35 A) Synthesis of cRNA with Y35-linked at the 5' end (Y35-cRNA(Malat1)). Example 3 was performed using an aqueous solution (315 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1. The title compound (830 nmol) was obtained in the same manner as in Step A of 3. B) Synthesis of double-stranded nucleic acid agent Y35-HDO Using ASO(Malat1) (first strand) and Y35-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y35-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y35-conjugated heteroduplex oligonucleotide (Y35-HDO) was prepared.

[0362] Example 36 A) Synthesis of cRNA with Y36-linked at the 5' end (Y36-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY36 The title compound (830 nmol) was obtained in the same manner as in Step A of Example 33. B) Synthesis of double-stranded nucleic acid agent Y36-HDO The ASO (Malat1) (first strand) and the Y36-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y36-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, was A Y36-conjugated heteroduplex oligonucleotide (Y36-HDO) was prepared.

[0363] Example 37 A) Synthesis of cRNA with Y37-linked at the 5' end (Y37-cRNA(Malat1)). Using an aqueous solution (400 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY37 The title compound (1189 nmol) was obtained in a similar manner to Step A of Example 33. B) Synthesis of double-stranded nucleic acid agent Y37-HDO The ASO (Malat1) (first strand) and the Y37-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y37-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y37-conjugated heteroduplex oligonucleotide (Y37-HDO) was prepared.

[0364] Example 38 A) Synthesis of cRNA with Y38-linked at the 5' end (Y38-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY38 The title compound (1032 nmol) was obtained in the same manner as in Step A of Example 33. B) Synthesis of double-stranded nucleic acid agent Y38-HDO Using ASO(Malat1) (first strand) and Y38-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y38-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y38-conjugated heteroduplex oligonucleotide (Y38-HDO) was prepared.

[0365] Example 39 A) Synthesis of cRNA with Y39-linked at the 5' end (Y39-cRNA(Malat1)). An aqueous solution (329 μL, 2000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was diluted with 50 mM NMP Solution (800 μL), distilled water (270 μL), NMP (2800 μL), Borate Buffer, pH 8.5+ / -0.2, 5X C After mixing in an Eppendorf flask, stirring, and settling, the mixture was incubated at room temperature for 1 hour. Then, 50 mM NMP solution of IY39 (400 μL) was added and reacted at room temperature for 1 hour. In addition, 50 mM NMP solution of IY39 (400 μL), Borate Buffer, pH 8.5+ / -0.2, 5X Concentr The reaction mixture was then loaded onto an ODS column (column: Purif-Pac k(registered trademark)-EX ODS-50 size 60, Shoko Science, mobile phase: TEAA / acetonitrile The mixture was purified and desalted by ultrafiltration (Amicon ultra 3 kDa, Merck Millipore, distilled water). To the resulting solution, 10 volumes of 1 M meglumine acetate were added, mixed well, and left to stand for 5 minutes. Ion exchange was then performed by ultrafiltration (Amicon ultra 3kDa, Merck Millipore). The final product was filtered through a 0.20 μm membrane filter. After filtration and lyophilization, 1554 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y39-HDO The ASO (Malat1) (first strand) and the Y39-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y39-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y39-conjugated heteroduplex oligonucleotide (Y39-HDO) was prepared.

[0366] Example 40 A) Synthesis of cRNA with Y40-linked at the 5' end (Y40-cRNA(Malat1)). Using an aqueous solution (364 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY40 The title compound (607 nmol) was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y40-HDO Using ASO(Malat1) (first strand) and Y40-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y40-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y40-conjugated heteroduplex oligonucleotide (Y40-HDO) was prepared.

[0367] Example 41 A) Synthesis of cRNA with Y41-linked at the 5' end (Y41-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY41 The title compound (1400 nmol) was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y41-HDO Using ASO(Malat1) (first strand) and Y41-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y41-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y41-conjugated heteroduplex oligonucleotide (Y41-HDO) was prepared.

[0368] Example 42 A) Synthesis of cRNA with Y42-linked at the 5' end (Y42-cRNA(Malat1)). Using an aqueous solution (365 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY42 The title compound (1061 nmol) was obtained in a manner similar to that of Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y42-HDO Using ASO(Malat1) (first strand) and Y42-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y42-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y42-conjugated heteroduplex oligonucleotide (Y42-HDO) was prepared.

[0369] Example 43 A) Synthesis of cRNA with Y43-linked at the 5' end (Y43-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY43 The title compound (820 nmol) was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y43-HDO Using ASO(Malat1) (first strand) and Y43-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y43-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y43-conjugated heteroduplex oligonucleotide (Y43-HDO) was prepared.

[0370] Example 44 A) Synthesis of cRNA with Y44-linked at the 5' end (Y44-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY44 The title compound (969 nmol) was obtained in a manner similar to that of Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y44-HDO The ASO (Malat1) (first strand) and the Y44-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y44-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y44-conjugated heteroduplex oligonucleotide (Y44-HDO) was prepared.

[0371] Example 45 A) Synthesis of cRNA with Y45-linked at the 5' end (Y45-cRNA(Malat1)). Using an aqueous solution (164 μL, 1000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY45 The title compound (653 nmol) was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y45-HDO Using ASO(Malat1) (first strand) and Y45-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y45-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y45-conjugated heteroduplex oligonucleotide (Y45-HDO) was prepared.

[0372] Example 46 A) Synthesis of cRNA with Y46-linked at the 5' end (Y46-cRNA(Malat1)). The RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was added to an aqueous solution (312 μL, 2000 nmol) of 50 mM NMP solution (400 μL), distilled water (188 μL), NMP (3200 μL), ×10 PBS (500 μL), 50 mM DMAP (4 The mixture was mixed in this order in an Eppendorf flask, stirred, precipitated, and then reacted at 70°C for 1 hour. Further, 50 mM NMP solution of IY46 (400 μL) and 50 mM DMAP (400 μL) were added, and the mixture was incubated at 70° C. for 1 hour. The reaction was allowed to proceed at room temperature overnight. In addition, a 50 mM NMP solution of IY46 (400 μL) and 150 mM DIPEA (400 The reaction solution was then loaded onto an ODS column (column: Purif-Pack (registered trademark) Purified with TEAA / acetonitrile (Trademark)-EX ODS-50 size 60, Shoko Science Co., Ltd., mobile phase: TEAA / acetonitrile). The solution was desalted by ultrafiltration (Amicon ultra 3 kDa, Merck Millipore, distilled water). Add 10 times the amount of 1 M meglumine acetate to the resulting solution, mix well, and leave to stand for 5 minutes. Ion exchange was then performed using ultrafiltration (Amicon ultra 3kDa, Merck Millipore Co., Ltd.). The final product was filtered through a 0.20 μm membrane filter. After lyophilization, 980 nmol of the title compound was obtained. B) Synthesis of double-stranded nucleic acid agent Y46-HDO The ASO (Malat1) (first strand) and the Y46-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y46-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y46-conjugated heteroduplex oligonucleotide (Y46-HDO) was prepared.

[0373] Example 47 A) Synthesis of cRNA with Y47-linked at the 5' end (Y47-cRNA(Malat1)). Using an aqueous solution (315 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY47 The title compound (569 nmol) was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y47-HDO The ASO (Malat1) (first strand) and the Y47-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y47-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y47-conjugated heteroduplex oligonucleotide (Y47-HDO) was prepared.

[0374] Example 48 A) Synthesis of cRNA with Y48-linked at the 5' end (Y48-cRNA(Malat1)). IY48 in 50 mM NMP (800 μL), HATU in 75 mM NMP (800 μL), DIPEA in 150 mM NMP The solution (800 μL) was mixed in an Eppendorf flask, stirred, and then allowed to react at room temperature for 30 minutes. The RNA strand (Y61-cRNA(Malat1)) shown in Table 1 was added to the solution (2000 nmol), NMP (2100 μL), and distilled water ( After stirring and settling, the mixture was allowed to react at room temperature for 1 hour. The solution was diluted with distilled water to an NMP concentration of 10 v / v%, and then ultrafiltered (Amicon ultra 3 kDa The solution was concentrated and low molecular weight reagents such as NMP were removed using distilled water (Merck Millipore). The final product was filtered through a 0.20 μm membrane filter, freeze-dried, and the title compound was extracted at 1085°C. nmol was obtained. B) Synthesis of double-stranded nucleic acid agent Y48-HDO Using ASO(Malat1) (first strand) and Y48-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y48-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y48-conjugated heteroduplex oligonucleotide (Y48-HDO) was prepared.

[0375] Example 49 A) Synthesis of cRNA with Y49-linked at the 5' end (Y49-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY49 The title compound (1176 nmol) was obtained in a manner similar to that of Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y49-HDO The ASO (Malat1) (first strand) and the Y49-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y49-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y49-conjugated heteroduplex oligonucleotide (Y49-HDO) was prepared.

[0376] Example 50 A) Synthesis of cRNA with Y50-linked at the 5' end (Y50-cRNA(Malat1)). Using an aqueous solution (315 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY50 The title compound (674 nmol) was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y50-HDO Using ASO(Malat1) (first strand) and Y50-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y50-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y50-conjugated heteroduplex oligonucleotide (Y50-HDO) was prepared.

[0377] Example 51 A) Synthesis of cRNA with Y51-linked at the 5' end (Y51-cRNA(Malat1)). Using an aqueous solution (315 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY51 The title compound (827 nmol) was obtained in a manner similar to that of Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y51-HDO The ASO (Malat1) (first strand) and the Y51-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y51-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y51-conjugated heteroduplex oligonucleotide (Y51-HDO) was prepared.

[0378] Example 52 A) Synthesis of cRNA with Y52-linked at the 5' end (Y52-cRNA(Malat1)). Using an aqueous solution (164 μL, 1000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY52 The title compound (653 nmol) was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y52-HDO Using ASO(Malat1) (first strand) and Y52-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y52-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y52-conjugated heteroduplex oligonucleotide (Y52-HDO) was prepared.

[0379] Example 53 A) Synthesis of cRNA with Y53-linked at the 5' end (Y53-cRNA(Malat1)). Using an aqueous solution (313 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY53 The title compound (865 nmol) was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y53-HDO Using ASO(Malat1) (first strand) and Y53-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y53-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y53-conjugated heteroduplex oligonucleotide (Y53-HDO) was prepared.

[0380] Example 54 A) Synthesis of cRNA with Y54-linked at the 5' end (Y54-cRNA(Malat1)). Using an aqueous solution (313 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY54 The title compound (1165 nmol) was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y54-HDO The ASO (Malat1) (first strand) and the Y54-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y54-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y54-conjugated heteroduplex oligonucleotide (Y54-HDO) was prepared.

[0381] Example 55 A) Synthesis of cRNA with Y55-linked at the 5' end (Y55-cRNA(Malat1)). Using an aqueous solution (313 μL, 2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY55 The title compound (1401 nmol) was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y55-HDO Using ASO(Malat1) (first strand) and Y55-cRNA(Malat1) (second strand) obtained in the previous step A, In the same manner as in step B of Example 1, a Y55-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y55-conjugated heteroduplex oligonucleotide (Y55-HDO) was prepared.

[0382] Example 57 A) Synthesis of cRNA with Y57-linked at the 5' end (Y57-cRNA(Malat1)). Using an aqueous solution (394 μL, 2500 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY57 The title compound (641 nmol) was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y57-HDO The ASO (Malat1) (first strand) and the Y57-cRNA (Malat1) (second strand) obtained in the previous step A were used to carry out the experiment. In the same manner as in step B of Example 1, a Y57-linked heteroduplex oligonucleotide, which is a double-stranded nucleic acid agent, is prepared. A Y57-conjugated heteroduplex oligonucleotide (Y57-HDO) was prepared.

[0383] Example 58 Synthesis of double-stranded nucleic acid agent Y59-HDO Using ASO (Malat1) (first strand) and Y59-cRNA (Malat1) (second strand) shown in Table 1, In the same manner as in step B, a double-stranded nucleic acid agent, a Y59-linked heteroduplex oligonucleotide (Y5 9-conjugated heteroduplex oligonucleotide, Y59-HDO) was prepared.

[0384] Example 59 Synthesis of double-stranded nucleic acid agent Y60-HDO Using ASO (Malat1) (first strand) and Y60-cRNA (Malat1) (second strand) shown in Table 1, In the same manner as in step B, a double-stranded nucleic acid agent, a Y60-linked heteroduplex oligonucleotide (Y6 A 0-conjugated heteroduplex oligonucleotide (Y60-HDO) was prepared.

[0385] Example 60 Synthesis of double-stranded nucleic acid agent Chol-HDO Using ASO (Malat1) (first strand) and Chol-cRNA (Malat1) (second strand) shown in Table 1, In the same manner as in step B, a Chol-linked heteroduplex oligonucleotide (C A hol-conjugated heteroduplex oligonucleotide (Chol-HDO) was prepared.

[0386] Example 61 (A) In vivo experiment The experimental animals used were 7-week-old male C57BL / 6J mice (Charles River Japan). In the experimental group, 2-4 mice were used for the experiment. A solution containing nucleic acid was administered to the tail vein of the mice at a dose of 5 mL / kg. In the control group, the solvent used in preparing the nucleic acid solution was used. (5% glucose solution) was administered intravenously to the mice in the same manner as in the experimental group.

[0387] (B) Expression analysis 72 hours after administration of the nucleic acid solution, pentobarbital was intraperitoneally administered to the mice at 50 mg / kg. The animals were anesthetized with oral administration, blood was collected, and the animals were sacrificed. The brains (cerebral cortex) were then removed. Total RNA extraction from the brain tissue was performed. The RNA was extracted using ISOGEN (Nippon Gene Co., Ltd.) as an RNA extraction reagent. Brain tissue was crushed and then extracted with chloroform to obtain RNA. The cDNA synthesis from total RNA was performed using the QuickGene RNA tissue kit SII (Kuraray Industries, Inc.). everTra Ace qPCR RT Kit (Toyobo Co., Ltd.) was used, and PCR was performed using THUNDERBIRD qPCR Mix (Toyobo Co., Ltd.). Quantitative PCR was performed using a fluorescent probe. lat1 (Integrated DNA Technologies) and mouse Gapdh (Thermo Fisher Scientific) The gene fragment amplification reaction conditions for quantitative PCR were as follows: THUNDERBIRD qPCR Mix (Toyobo) The protocol of the manufacturer (Inc.) was followed. The expression levels of mouse Malat1 and Gapdh (internal control genes) were compared. The relative expression level was calculated as Malat1 / Gapdh using a standard curve. The average relative expression level was calculated from the results of each mouse. The relative expression level of the experimental group is calculated by dividing the mean value of the Malat1 expression level by the mean value of the experimental group. The ncRNA expression levels were calculated.

[0388] (C) Result The results of Example 61 are shown in Table 7. In the table, the dose indicates the amount of ASO (Malat1).

[0389] The double strand numbers 1, 3-55, 57, 59, and 60 are all Malat1 non-coding regions in the cerebral cortex. From this result, it was confirmed that the nucleic acid complex of the present invention is delivered to the brain and inhibits the expression of the IL-1 protein. It has been demonstrated that antisense effects can be achieved by

[0390] [Table 7] TIFF2025063018000127.tif7998

[0391] Example 62 (A) In vivo experiment The experimental animals used were 10-week-old male C57BL / 6J mice (Charles River Japan). Three mice were used per group for the experiment. In the experimental group, a solution containing nucleic acid was administered into the tail vein of the mice at a dose of 5 mL / kg. The nucleic acid solution was administered intravenously once at the prescribed dose. Saline) was administered intravenously to the mice in the same manner as in the experimental group.

[0392] (B) Microglia isolation 72 hours after administration of the nucleic acid solution, the mice were anesthetized with isoflurane and the diaphragm was incised. The heart was exposed, physiological saline was injected into the left ventricle with a syringe, and the right atrium was cut open to remove blood. After the mice were incubated for 1 h, the brains were removed. The brains were then dissociated using Neural Tissue Dissociation Kits (Miltenyi Biotec). The enzyme treatment was performed according to the protocol, and Debris Removal Solution (Miltenyi Biotec Debris was removed using a cytoplasmic antibody (Cell Signaling Technology, Inc.) to prepare a brain cell suspension. Using croglia Microbeads (Miltenyi Biotec), the following protocol was followed to extract the brain cell fluid. Microglia were isolated.

[0393] (C) Expression analysis Total RNA was extracted from isolated microglia using the RNeasy Micro Kit (QIAGEN). cDNA synthesis was performed using the High Capacity cDNA RT Kit (Thermo Fisher Scientific). Quantitative PCR was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). The quantitative PCR was performed using a fluorescent probe method, and the mouse Malat1 (Thermo Mouse Gapdh (Thermo Fisher Scientific) and mouse Gapdh (Thermo Fisher Scientific) were used. The PCR reaction conditions for amplifying gene fragments were as follows: TaqMan Fast Advanced Master Mix (Thermo Fisheye The protocol of the manufacturer (R Scientific) was followed. The expression level was calculated by the comparative Ct method, and the relative expression level was calculated as Malat1 / Gapdh. The average relative expression level was calculated from the results of three mice. The percentage of the mean relative expression level of the experimental group when the mean level is taken as 100% is called the relative Ma The expression level of lat1 ncRNA was calculated.

[0394] (D)Result The results of Example 62 are shown in Table 8. In the table, the dose indicates the amount of ASO (Malat1). Duplex number 58 suppressed the expression of Malat1 non-coding RNA in microglia These results indicate that the nucleic acid complex of the present invention is delivered to the brain and has an anti-inflammatory effect in microglia. It has been demonstrated that this can have a sense effect.

[0395] [Table 8] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. It shall be incorporated.

Claims

1. A nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid strand comprises a salt capable of hybridizing to at least a portion of a target transcript. It contains a base sequence and has an antisense effect on a target transcript, The second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, and (1) Tocopherol or its analogues, (2) cholesterol or its analogues, or (3) Substituted or unsubstituted C １ ~ ３０ or a substituted alkyl group or unsubstituted C ２ ~ ３０ or a substituted or unsubstituted alkenyl group. Unconverted C １ ~ ３０ Alkoxy group It is combined with The first nucleic acid strand is annealed to the second nucleic acid strand.

2. The tocopherol or its analogue has the following formula (I): 【Chemistry 1】 (In the formula, R １ is a substituted or unsubstituted C １ ~ ３０ Alkyl or substituted C that has not been replaced or substituted ２ ~ ３０ represents an alkenyl group) The nucleic acid complex according to claim 1 ,

3. The cholesterol or its analogue is represented by the following formulae (II), (V), (VI), and (VII): 【Chemistry 2】 (In the formula, Ring A is a substituted or unsubstituted cyclohexane or a substituted or unsubstituted cyclohexane. represents unsubstituted benzene, Ring B is a substituted or unsubstituted cyclohexene, a substituted or unsubstituted cyclohexene, Unsubstituted cyclohexane or substituted or unsubstituted cyclohexadiene represents the Ring C is a substituted or unsubstituted cyclohexene or a substituted or unsubstituted cyclohexene. represents unsubstituted cyclohexane, R ２ is a hydrogen atom, a substituted or unsubstituted C １ ~ ３０ Alkyl groups, substituted C that has not been replaced or substituted ２ ~ ３０ alkenyl groups, substituted or unsubstituted Not yet １ ~ ６ Alkyl-carbonyl group, substituted or unsubstituted C １ ~ ６ represents an alkyl-carbonyl-oxy group or an oxo group, R ３ represents a hydrogen atom, R ２ and R ３ together represent a substituted or unsubstituted 1,6-dioxa may form a spiro[4.5]decane ring, L １ Yes, -O-, -NH-, 【Chemistry 3】 or 【Chemistry 4】 (representing 【Chemistry 5】 (In the formula, R ４ is a substituted or unsubstituted C １ ~ ３０ The alkyl group or the substituent Substituted or unsubstituted C ２ ~ ３０ represents an alkenyl group; 【Chemistry 6】 (In the formula, R ５ and R ６ each independently represents a substituted or unsubstituted C １ ~ ３０ or a substituted or unsubstituted C ２ ~ ３０ Alkenyl represents a group), and 【Chemistry 7】 (In the formula, R ７ is a hydroxy group, a substituted or unsubstituted C １ ~ ３０ Alki a substituted or unsubstituted C １ ~ ３０ Arke represents a cycloalkyl-carbonyl-oxy group) The nucleic acid complex of claim 1, represented by a formula selected from the group consisting of:

4. The cholesterol or an analog thereof has the following formula (IIa): 【Chemistry 8】 (In the formula, Ring A is a substituted or unsubstituted cyclohexene, a substituted or unsubstituted Unsubstituted cyclohexane or substituted or unsubstituted cyclohexadiene represents the R １ is a substituted or unsubstituted C １ ~ ３０ The alkyl group, if substituted, or unsubstituted C ２ ~ ３０ Alkenyl groups, substituted or unsubstituted, C １ ~ ６ Alkyl-carbonyl group, substituted or unsubstituted C １ ~ ６ Alki represents a aryl-carbonyl-oxy group or an oxo group; L １ Yes, -O-, -NH-, 【Chemistry 9】 or 【Chemistry 10】 (represents The nucleic acid complex according to claim 1 ,

5. The cholesterol or its analogue is represented by the following formulae (IIa-1), (IIa-2), (IIa-3), and (IIa-4): 【Chemistry 11】 (IIa-1), 【change】 (IIa-2), 【change】 (IIa-3), 【change】 (IIa-4) (In the formula, L １ Yes, -O-, -NH-, 【Chemistry 12】 or 【Chemistry 13】 (representing The nucleic acid complex of claim 1, represented by a formula selected from the group consisting of:

6. The second nucleic acid strand has the following formula (VIII): 【Chemistry 14】 (In the formula, L ２ is a substituted or unsubstituted C １ ~ １２ alkylene groups, or unsubstituted C ３ ~ ８ Cycloalkylene group, -(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -、 or CH(CH ２ -OH)-CH ２ -O- (CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ - represents L ３ represents -NH- or a bond; L ４ is a substituted or unsubstituted C １ ~ １２ alkylene groups, or unsubstituted C ３ ~ ８ a cycloalkylene group of -(CH ２ ) ２ -[O-(CH ２ ) ２ ] ｍ - or a bond, where m is an integer from 1 to 25; L ５ represents -NH-(C=O)-, -(C=O)-, or a bond. Through a linker represented by (1) Tocopherol or its analogues, (2) cholesterol or its analogues, or (3) Substituted or unsubstituted C １ ~ ３０ or a substituted alkyl group or unsubstituted C ２ ~ ３０ Alkenyl group The nucleic acid complex according to any one of claims 1 to 5, which is bound to

7. The first nucleic acid strand comprises at least four consecutive deoxyribonucleosides. Item 7. The nucleic acid complex according to any one of Items 1 to 6.

8. The nucleic acid complex according to any one of claims 1 to 7, wherein the first nucleic acid strand is a gapmer. Merge.

9. The second nucleic acid strand is a nucleic acid sequence of at least four consecutive deoxyribonucleotides in the first nucleic acid strand.

9. The method according to claim 7 or 8, comprising at least four consecutive ribonucleosides complementary to a ribonucleoside. The nucleic acid complex according to claim 1.

10. The nucleic acid complex according to any one of claims 1 to 6, wherein the first nucleic acid strand is a mixmer. Merge.

11. The first nucleic acid strand according to any one of claims 1 to 10, wherein the first nucleic acid strand has a length of 13 to 20 bases. Nucleic acid complex.

12. 12. The nucleic acid of claim 1, wherein the second nucleic acid strand does not contain natural ribonucleosides. The nucleic acid complex described.

13. The nucleic acid portions of the second nucleic acid strand are linked by modified or unmodified internucleoside bonds. The nucleic acid according to any one of claims 1 to 12, which comprises deoxyribonucleosides and / or sugar-modified nucleosides. A nucleic acid complex according to any one of claims 1 to 4.

14. A method for treating a central nervous system disorder comprising administering to a subject a nucleic acid complex according to any one of claims 1 to 13. A composition for regulating the expression or editing of a target transcript by a method comprising:

15. The composition of claim 14 for treating a central nervous system disorder in a subject.

16. The composition of claim 15, wherein the central nervous system disease is an immune-mediated central nervous system disease.

17. A method for administering a drug to the central nervous system of a subject, comprising administering to the subject a drug comprising the nucleic acid complex according to any one of claims 1 to 13. A composition for delivering

18. The central nervous system is selected from the group consisting of the cerebral cortex, the basal ganglia, the cerebral white matter, the diencephalon, the brain stem, the cerebellum, and the spinal cord. The composition of any one of claims 14 to 17, wherein the composition is selected from the group consisting of:

19. The central nervous system includes the frontal lobe, the temporal lobe, the hippocampus, the parahippocampal gyrus, the parietal lobe, the occipital lobe, the striatum, the pallidum, bulb, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigra, pons, medulla oblongata, cerebellar cortex, cerebellar nuclei, cervical spinal cord, thoracic spinal cord and The composition according to any one of claims 14 to 17, wherein the composition is selected from the group consisting of lumbar spinal cord.

20. The composition according to any one of claims 14 to 19, which is for intravenous or subcutaneous administration. 。

21. Any one of claims 14 to 20, wherein the nucleic acid complex is contained in a single dose of 5 mg / kg or more. The composition according to any one of claims 1 to 4.

22. Any one of claims 14 to 21, wherein the nucleic acid complex crosses the blood-brain barrier (BBB). The composition described in

23. The composition according to claim 16, wherein the immune-mediated central nervous system disease is a microglia-associated disease. 。

24. The microglia-associated disease is Alzheimer's disease, multiple sclerosis, ALS, or neurodegenerative diseases. The composition of claim 23, which is for pathologic pain.

25. The method according to claims 14 to 2 for regulating the expression or editing of a target transcript in microglia.

5. The composition according to any one of claims 4 to 4.

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