Nucleic acid complex

Abstract

The present invention addresses the problem of providing a nucleic acid agent, which is delivered efficiently to a nervous system such as the central nervous system where drug delivery might be impeded by BBB, etc. and which exerts an antisense effect on a target transcript at the delivery site, and a composition comprising the nucleic acid agent.　In one embodiment, the present invention provides a double-stranded nucleic acid complex obtained by annealing a first nucleic acid strand, which hybridizes with a part of a target transcript and has an antisense effect on the target transcript, and a second nucleic acid strand which contains a base sequence complementary to the first nucleic acid strand and binds to a C22-35 alkyl group optionally substituted by a hydroxy group or an analog thereof.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid complex or a salt thereof that can produce an antisense effect in the nervous system, for example, the central nervous system, and a composition containing the same. [Background technology]

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

[0003] Patent Document 1 discloses a double-stranded nucleic acid molecule that is composed of a first oligomeric compound and a second oligomeric compound containing a conjugated group such as cholesterol and that can regulate the amount or activity of a target nucleic acid in extrahepatic tissues or extrahepatic cells, or in hepatic tissues or hepatic cells, and an antisense compound that is composed of the double-stranded nucleic acid molecule.

[0004] In order to exert an antisense effect in the central nervous system, including the brain, nucleic acid agents such as ASOs must be delivered to the central nervous system. However, the brain contains mechanisms known as the blood-brain barrier (hereinafter often referred to as "BBB"), blood-cerebrospinal fluid barrier (hereinafter often referred to as "BCSFB"), and cerebrospinal fluid barrier (hereinafter often referred to as "CSFBB"), which select and restrict substances that are transported to the brain via blood or cerebrospinal fluid. While the BBB, BCSFB, and / or CSFBB mechanisms protect the brain from harmful substances, they also act as barriers to drug delivery to the brain. Therefore, a method for delivering nucleic acid agents such as ASOs to the central nervous system, including the brain, is needed. [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] An objective of the present invention is to provide a nucleic acid agent that can be efficiently delivered to the central nervous system, where drug delivery is hindered by the nervous system, for example, the BBB mechanism, the BCSFB mechanism, and / or the CSFBB mechanism (which may sometimes be collectively abbreviated as "BBB, etc." in this specification), and that exerts an antisense effect on a target transcript at the delivery site, and a composition containing the same. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems, and as a result, have discovered a compound comprising ASO and C 22 ~ 35They found that a nucleic acid complex annealed with a complementary strand of an ASO bound to an alkyl group or its analog is efficiently delivered to the central nervous system, where it exhibits a high antisense effect. Based on these findings, the present inventors have completed the present invention. Specifically, the present invention encompasses the following aspects. (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 base sequence capable of hybridizing to at least a part of a target transcript and has an antisense effect on the target transcript; The second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, and a C 22 ~ 35 or its analogue, The nucleic acid complex or a salt thereof, in which the first nucleic acid strand is annealed to the second nucleic acid strand (these may sometimes be collectively referred to as the "nucleic acid complex of the present invention" in this specification). (2) C optionally substituted with a hydroxy group 22 ~ 35 or its analog, the nucleic acid strand or a salt thereof comprising a base sequence capable of hybridizing to at least a portion of a target transcription product and having an antisense effect on the target transcription product. (3) The second nucleic acid strand or the first nucleic acid strand may be substituted with a hydroxy group via a linker represented by general formula I. 22 ~ 35 The nucleic acid complex, nucleic acid chain, or salt thereof according to (1) or (2), wherein the nucleic acid complex, nucleic acid chain, or salt thereof is bound to an alkyl group or an analog thereof. [ka] (4) C optionally substituted with a hydroxy group 22 ~ 35 or its analogue is an unsubstituted straight chain C 22 ~ 24 or linear C 26 The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of (1) to (3), wherein the group is an alkyl group. (5) The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of (1) to (4), wherein the first nucleic acid strand contains at least four consecutive deoxyribonucleosides. (6) The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of (1) to (5), wherein the first nucleic acid strand is a gapmer. (7) The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of (1) to (6), wherein the second nucleic acid strand contains at least four consecutive ribonucleosides complementary to at least four consecutive deoxyribonucleosides in the first nucleic acid strand. (8) The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of (1) to (4), wherein the first nucleic acid strand is a mixmer. (9) The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of (1) to (8), wherein the first nucleic acid strand has a length of 13 to 20 bases. (10) The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of (1) to (9), wherein the second nucleic acid strand does not contain a natural ribonucleoside. (11) The nucleic acid complex, nucleic acid chain, or salt thereof according to any one of (1) to (10), wherein the nucleic acid portion of the second nucleic acid strand is composed of deoxyribonucleosides and / or sugar-modified nucleosides linked by modified or unmodified internucleoside bonds. (12) A composition for regulating the expression or editing of a target transcript in the central nervous system, comprising the nucleic acid complex, nucleic acid strand, or salt thereof according to any one of (1) to (11). (13) The composition according to (12), which is for treating a central nervous system disease. (14) A composition for delivering a drug to the central nervous system, comprising the nucleic acid complex, nucleic acid chain, or salt thereof according to any one of (1) to (11). (15) The composition according to any one of (12) to (14), wherein the central nervous system is selected from the group consisting of the cerebral cortex, the basal ganglia, the cerebral white matter, the diencephalon, the brainstem, the cerebellum, and the spinal cord. (16) The composition according to any one of (12) to (14), wherein the central nervous system is selected from the group consisting of the frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, parietal lobe, occipital lobe, striatum, globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigra, pons, medulla oblongata, cerebellar cortex, cerebellar nuclei, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. (17) The composition according to any one of (12) to (16), which is for intravenous or subcutaneous administration. (18) The composition according to any one of (12) to (17), wherein a single dose contains 5 mg / kg or more of the nucleic acid complex, nucleic acid chain, or salt thereof. (19) The composition according to any one of (12) to (18), wherein the nucleic acid complex, nucleic acid chain, or salt thereof is transferred from blood to the brain (brain parenchyma). This specification includes the disclosure of Japanese Patent Application No. 2019-168930, from which this application claims priority. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a nucleic acid agent that can be efficiently delivered to the central nervous system and that exerts an antisense effect at the delivery site, and a composition containing the same. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 is a schematic diagram showing an example of a specific embodiment of a nucleic acid complex used in the present invention. This diagram shows two modes depending on the binding position of a C22-35 alkyl group or its analogue, which may be optionally substituted with a hydroxyl group, in the second nucleic acid strand (simply referred to as "alkyl group" in the diagram). Figure 1a shows a nucleic acid complex in which a C22-35 alkyl group or its analogue, which may be optionally substituted with a hydroxyl group, is bound to the 5'-end of the second nucleic acid strand. Figure 1b shows a nucleic acid complex in which a C22-35 alkyl group or its analogue, which may be optionally substituted with a hydroxyl group, is bound to the 3'-end of the second nucleic acid strand. [Figure 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 diagram within the dashed line is a schematic diagram showing how the heteroduplex portion is recognized by RNase H, resulting in degradation of the mRNA of the target gene. [Figure 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 INVENTION

[0010] <Nucleic acid complex> A first aspect of the present invention is a nucleic acid complex. The nucleic acid complex may be capable of migrating from blood to the brain (brain parenchyma), for example, passing through the BBB. The nucleic acid complex comprises a first nucleic acid strand and a second nucleic acid strand. The second nucleic acid strand is a nucleotide strand containing a base sequence complementary to that of the first nucleic acid strand. In the nucleic acid complex, the first nucleic acid strand is annealed to the second nucleic acid strand. In one embodiment, the second nucleic acid strand comprises a C -type nucleotide sequence optionally substituted with a hydroxy group. 22 ~ 35 is bonded to an alkyl group or its analogue.

[0011] A typical schematic diagram of the nucleic acid complex is shown in Figure 1. Figure 1a shows a C 1 optionally substituted with a hydroxy group at the 5' end of the second nucleic acid strand. 22 ~ 35 1b shows a nucleic acid complex having an alkyl group or its analog attached to the 3' end of the second nucleic acid strand, which may be substituted with a hydroxy group. 22 ~ 35 However, as described below, C which may be substituted with a hydroxy group is also used. 22 ~ 35 The alkyl group or its analogue may be bound to the 5' end, 3' end or both ends of the second nucleic acid strand, or may be bound to an internal nucleotide of the second nucleic acid strand.

[0012] In one embodiment, the first nucleic acid strand is a nucleotide strand comprising a base sequence capable of hybridizing to at least a portion of a target transcript. In a specific embodiment, the first nucleic acid strand is a nucleotide strand having an antisense effect on a transcript of a target gene or a target transcript.

[0013] In one embodiment, the present invention provides a C 22 ~ 35 or its analog. The nucleic acid strand may be capable of migrating from the blood to the brain (brain parenchyma), for example, passing through the BBB. The nucleic acid strand corresponds to the first nucleic acid strand of the nucleic acid complex and is a nucleotide strand containing a base sequence capable of hybridizing to at least a portion of a target transcription product. In a specific embodiment, the nucleic acid strand is a nucleotide strand having an antisense effect on a transcription product of a target gene or a target transcription product.

[0014] (Definition of terms) As used herein, the term "target transcript" refers to any RNA that can be targeted by the nucleic acid complex of the present invention and is synthesized by a DNA-dependent RNA polymerase. Generally, this refers to a transcript of a target gene. Specifically, this term may include mRNA (including mature mRNA, mRNA precursor, and mRNA without base modifications) transcribed from the target gene, and non-coding RNA (ncRNA) such as miRNA.

[0015] As used herein, the term "target gene" is not particularly limited, but includes, for example, a gene derived from an organism into which the nucleic acid complex of the present invention is introduced, such as a gene whose expression increases in various diseases. Furthermore, the target transcript includes mRNA transcribed from genomic DNA encoding the target gene, as well as mRNA that has not undergone base modification and unprocessed mRNA precursors. The "target transcript" may include not only mRNA but also non-coding RNA (ncRNA) such as miRNA. More generally, the "transcript" may be any RNA synthesized by a DNA-dependent RNA polymerase. In one embodiment, the "target transcript" may be, for example, a gene encoding metastasis-associated lung adenocarcinoma transcript 1 (often referred to herein as "Malat1"), scavenger receptor B1 (often referred to herein as "SR-B1 mRNA"), myotonic dystrophy protein kinase (often referred to herein as "DMPK"), transthyretin (often referred to herein as "TTR"), or apolipoprotein B (often referred to herein as "ApoB"), such as a non-coding RNA or mRNA thereof. SEQ ID NO: 3 shows the nucleotide sequence of mouse Malat1 non-coding RNA, and SEQ ID NO: 4 shows the nucleotide sequence of human Malat1 non-coding RNA. SEQ ID NO: 5 shows the nucleotide sequence of mouse SR-B1 mRNA, and SEQ ID NO: 6 shows the nucleotide sequence of human SR-B1. The nucleotide sequence of mRNA is shown in SEQ ID NO: 7. The nucleotide sequence of mouse DMPK mRNA is shown in SEQ ID NO: 8. Note that in all of SEQ ID NOs: 3 to 8, the nucleotide sequence of mRNA is replaced with the nucleotide sequence of DNA.The nucleotide sequence information of these genes and transcripts can be obtained from publicly known databases such as the NCBI (National Center for Biotechnology Information) database.

[0016] As used herein, "antisense oligonucleotide (ASO)" or "antisense nucleic acid" refers to a single-stranded oligonucleotide that contains a complementary base sequence capable of hybridizing to at least a portion of a target transcript, e.g., any target region, and that can inhibit and control the expression of the transcript of the target gene or the level of the target transcript through its antisense effect. In the nucleic acid complex of the present invention, the first nucleic acid strand functions as an ASO, and the target region may include a 3' UTR, a 5' UTR, an exon, an intron, a coding region, a translation initiation region, a translation termination region, or any other nucleic acid region. The target region of the target transcript can be at least 8 bases long, e.g., 10 to 35 bases long, 12 to 25 bases long, 13 to 20 bases long, 14 to 19 bases long, or 15 to 18 bases long.

[0017] The term "antisense effect" refers to the effect of ASOs hybridizing to a target transcript (e.g., the RNA sense strand) to modulate the expression or editing of that target transcript. "Modulating the expression or editing of a target transcript" refers to suppressing or reducing the expression of a target gene or the expression level of the target transcript (herein, "expression level of a target transcript" is often referred to as "target transcript level"), inhibiting translation, altering splicing function (e.g., exon skipping), or degrading the transcript. For example, as shown in Figure 2, when an oligonucleotide (e.g., RNA) is introduced into a cell as an ASO, the ASO binds to the target gene's transcript, such as mRNA, to form a partial duplex. This partial duplex acts as a cover to prevent translation by ribosomes, thereby inhibiting the expression of the protein encoded by the target gene at the translational level (Figure 2, dashed line, x). On the other hand, when a DNA-containing oligonucleotide is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase H, resulting in degradation of the target gene's mRNA and inhibition of expression of the protein encoded by the target gene at the expression level (shown within the dashed line in Figure 2). This is referred to as the "RNase H-dependent pathway." Furthermore, in certain instances, an antisense effect can be achieved by targeting an intron of a pre-mRNA. An antisense effect can also be achieved by targeting an miRNA, in which case the function of the miRNA can be inhibited and the expression of the gene whose expression the miRNA normally controls can be increased. In one embodiment, modulation of the expression of a target transcript can be a reduction in the amount of the target transcript.

[0018] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a monomer, a nucleoside or nucleotide, an oligomer, an oligonucleotide, or a polymer, a polynucleotide.

[0019] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, specific examples of which include ribose and deoxyribose. The base portion (nucleobase) of a nucleoside is typically a heterocyclic base moiety. Examples include, but are not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).

[0020] A "nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of a nucleoside. In the case of nucleotides containing a pentofuranosyl sugar, the phosphate group is typically linked to the 2', 3', or 5' hydroxyl group of the sugar.

[0021] An "oligonucleotide" refers to a linear oligomer formed by covalently linking several to several tens of hydroxyl groups in the sugar moieties and phosphate groups between adjacent nucleotides. A "polynucleotide" refers to a linear polymer formed by linking several tens or more, preferably several hundred or more, of nucleotides, more numerous than an oligonucleotide, by such covalent bonds. Within an oligonucleotide or polynucleotide structure, the phosphate groups are generally considered to form internucleoside bonds.

[0022] As used herein, the term "nucleic acid strand" or simply "strand" refers to an oligonucleotide or polynucleotide. A nucleic acid strand can be made into a full-length strand or a partial strand by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction reactions. A nucleic acid strand can include natural and / or non-natural nucleotides.

[0023] As used herein, "natural nucleosides" refer to nucleosides found in nature. Examples include ribonucleosides composed of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides composed of deoxyribose and a base such as adenine, cytosine, guanine, or thymine. Ribonucleosides found in RNA and deoxyribonucleosides found in DNA are sometimes referred to as "DNA nucleosides" and "RNA nucleosides," respectively. Similarly, "natural nucleotides" refer to naturally occurring nucleotides in which a phosphate group is covalently bonded to the sugar moiety of the natural nucleoside. Examples include ribonucleotides, known as building blocks of RNA, in which a phosphate group is bonded to a ribonucleoside, and deoxyribonucleotides, known as building blocks of DNA, in which a phosphate group is bonded to a deoxyribonucleoside.

[0024] As used herein, the term "unnatural nucleoside" refers to any nucleoside other than a natural nucleoside. For example, this term includes modified nucleosides and nucleoside mimetics. As used herein, the term "modified nucleoside" refers to a nucleoside having a modified sugar moiety and / or a modified nucleobase. Nucleic acid chains, including unnatural oligonucleotides, are often preferred over natural forms due to desirable properties such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0025] As used herein, the term "mimetic" refers to functional groups that replace sugars, nucleobases, and / or internucleoside linkages. Generally, mimetics are used in place of sugars or sugar-internucleoside linkage combinations, while maintaining the nucleobases for hybridization to a selected target. "Nucleoside mimics" include structures used to replace sugars, or sugars and bases, or linkages between monomeric subunits that comprise an oligomeric compound at one or more positions. "Oligomeric compound" refers to a polymer of linked monomeric subunits that is at least hybridizable to a region of a nucleic acid molecule. Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimetics, e.g., nucleoside mimetics having non-furanose sugar units.

[0026] As used herein, the term "bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are commonly referred to as bridged nucleic acids (BNAs). Nucleosides containing a bicyclic sugar moiety are also sometimes referred to as "bridged nucleosides" herein. Some examples of bridged nucleic acids are shown in Figure 3.

[0027] A bicyclic sugar may be a sugar in which the 2' and 4' carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. One subgroup of nucleic acids (BNAs) containing bicyclic sugars is the 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 represent an integer of 1 to 4, an integer of 0 to 2, and an integer of 1 to 3, respectively; and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a unit substituent (a fluorescent or chemiluminescent labeled molecule, a functional group having nucleic acid cleavage activity, an intracellular or intranuclear localization signal peptide, etc.)]. Furthermore, with respect to BNAs according to certain embodiments, in the OR2 substituent on the 3' carbon atom and the OR1 substituent on the 5' carbon atom, R1 and R2 are typically hydrogen atoms, but may be the same or different from each other and may also be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R4)R5 (wherein R4 and R5 may be the same or different from each other and represent, respectively, a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms). Non-limiting examples of such BNAs include methyleneoxy (4'-CH2-O-2') BNA (also known as LNA (Locked Nucleic Acid®, 2',4'-BNA), e.g., α-L-methyleneoxy (4'-CH2-O-2') BNA or β-D-methyleneoxy (4'-CH2-O-2') BNA, ethyleneoxy (4'-(CH2)2-O-2') BNA (also known as ENA), β-D-thio (4'-CH2-S-2') BNA, aminooxy (4'-CH2-ON(R3)-2') BNA, oxyamino (4'-CH2-N(R)-O-2') BNA (e.g., R = H, Me) (2',4'-BNA NC (e.g., R=H is 2',4'-BNA NC[NH], R=Me is 2',4'-BNA NC [N-Me]), 2',4'-BNA coc , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH3)-O-2') BNA (also known as cEt BNA), (4'-CH(CHOCH3)-O-2') BNA (also known as cMOE BNA), amide-bridged nucleic acid (4'-C(O)-N(R)-2') BNA (e.g., R = H, Me), AmNA (e.g., R = H is AmNA[NH], R = Me is AmNA), Bicyclic nucleosides having a methyleneoxy (4'-CH2-O-2') bridge are sometimes referred to herein as "LNA nucleosides." Examples of such nucleosides include guanidine-bridged nucleic acids (GuNAs (e.g., R = H in Figure 3 is also known as GuNA[NH], and R = Me is also known as GuNA[N-Me]), 2'-O,4'-C-spirocyclopropylene-bridged nucleic acids (also known as scpBNAs), amine-bridged nucleic acids (also known as 2'-Amino-LNAs) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substitutions), and other BNAs known to those skilled in the art. Bicyclic nucleosides having a methyleneoxy (4'-CH2-O-2') bridge are sometimes referred to herein as "LNA nucleosides."

[0028] As used herein, a "cationic nucleoside" refers to a modified nucleoside that exists in a cationic form relative to a neutral form (e.g., the neutral form of a ribonucleoside) at a certain pH (e.g., human physiological pH (approximately 7.4), the pH of a delivery site (e.g., an organelle, cell, tissue, organ, organism, etc.)). A cationic nucleoside may contain one or more cationic modifying groups at any position of the nucleoside. In one embodiment, the cationic nucleoside is an amine-bridged nucleic acid (also known as 2'-Amino-LNA) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substitutions), an aminoalkyl-modified nucleic acid (e.g., 2'-O-methyl and 4'-CH2CH2CH2NH2 substitutions), a guanidine-bridged nucleic acid (GuNA (e.g., in Figure 3, R = H is GuNA[NH], and R = Me is GuNA[N-Me]), etc.).

[0029] As used herein, the term "non-natural nucleotide" refers to any nucleotide other than a naturally occurring nucleotide, including modified nucleotides and nucleotide mimics. As used herein, the term "modified nucleotide" refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase.

[0030] The term "nucleotide mimic" includes structures used to replace nucleosides and linkages at one or more positions in an oligomeric compound. Examples of nucleotide mimics include peptide nucleic acids or morpholino nucleic acids (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Peptide nucleic acids (PNAs) are nucleotide mimics with a backbone in which N-(2-aminoethyl)glycine is linked via amide bonds instead of sugars. An example of the structure of a morpholino nucleic acid is shown in Figure 4. Nucleic acid chains, including non-natural oligonucleotides, often possess desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Therefore, they are preferred over natural nucleotides.

[0031] As used herein, the term "modified internucleoside linkage" refers to an internucleoside linkage that has a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage). Modified internucleoside linkages include phosphorus-containing internucleoside linkages that contain a phosphorus atom and non-phosphorus-containing internucleoside linkages that do not contain a phosphorus atom. Representative phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages (e.g., methyl phosphotriester linkages and ethyl phosphotriester linkages as described in U.S. Patent Registration No. 5,955,599), alkyl phosphonate linkages (e.g., methyl phosphonate linkages as described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, and methoxypropyl phosphonate linkages as described in WO 2015 / 168172), alkylthiophosphonate linkages, boranophosphate linkages, and internucleoside linkages containing cyclic guanidine moieties (e.g., those having the following structure: [ka] Examples of such modified internucleoside linkages include, but are not limited to, the internucleoside linkages and phosphoramidate linkages used in the self-neutralizing nucleic acids (ZONs) described in International Publication No. 2016 / 081600. A phosphorothioate linkage is an internucleoside linkage in which the non-bridging oxygen atom of a phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known. The modified internucleoside linkage is preferably one that is more nuclease-resistant than naturally occurring internucleoside linkages.

[0032] When an internucleoside bond has a chiral center, the internucleoside bond may be chiral controlled. "Chiral controlled" refers to a single diastereomer present with respect to the chiral center, e.g., chiral phosphorus. A chiral controlled internucleoside bond may be completely chirally pure or may have a high chiral purity, e.g., 90% de, 95% de, 98% de, 99% de, 99.5% de, 99.8% de, 99.9% de, or higher. As used herein, "chiral purity" refers to the proportion of one diastereomer in a mixture of diastereomers, expressed as diastereomeric excess (% de), and defined as (target diastereomer - other diastereomers) / (total diastereomers) × 100 (%).

[0033] For example, the internucleoside linkages may be phosphorothioate linkages chiral controlled in the Rp or Sp configuration, internucleoside linkages containing cyclic guanidine moieties (e.g., those having the following structure: [ka] ) etc.) A method for preparing a chiral internucleoside bond is known, and for example, a phosphorothioate bond chiral controlled to an Rp configuration or an Sp configuration can be prepared by the methods described in Naoki Iwamoto et al., Angew. Chem. Int. Ed. Engl. 2009, 48(3), 496-9, Natsuhisa Oka et al., J. Am. Chem. Soc. 2003, 125, 8307-8317, Natsuhisa Oka et al., J. Am. Chem. Soc. 2008, 130, 16031-16037, Yohei Nukaga et al., J. Org. Chem. 2016, 81, 2753-2762, Yohei Nukaga et al., J. Org. Chem. 2012, 77, They can be synthesized according to the method described in [Publication No. 7913-7922]. Chiral phosphorothioate bonds controlled in the Rp or Sp configuration are also known, and are known to have the effects described in, for example, Naoki Iwamoto et al., Nat. Biotechnol., 2017, 35(9), 845-851 and Anastasia Khvorova et al., Nat. Biotechnol., 2017, 35(3), 238-248.

[0034] As used herein, "modified nucleobase" or "modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. Examples of modified nucleobases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, N6-methyladenine, 8-bromoadenine, N2-methylguanine, or 8-bromoguanine. A preferred modified nucleobase is 5-methylcytosine. "Unmodified nucleobase" or "unmodified base" is synonymous with natural nucleobases and refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0035] As used herein, the term "modified sugar" refers to a sugar having a substitution and / or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)). A nucleic acid strand herein may contain one or more modified nucleosides, optionally containing modified sugars. Sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to a nucleic acid strand. A nucleoside may contain a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), the formation of bridges between non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNA, LNA, ENA, AmNA (AmNA[NH], AmNA[N-Me]), GuNA (GuNA[NH], GuNA[N-Me]), scpBNA, 2',4'-BNA NC (2',4'-BNA NC [NH], 2',4'-BNA NC [N-Me]), 2',4'-BNA coc and the like), the formation of a ribosyl ring oxygen atom by S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently H, C1-C 12 Examples of nucleosides having modified sugar moieties, as used herein, include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F (2'-fluoro), 2'-OCH (2'-OMe or 2'-O-methyl), and 2'-O(CH)OCH substituents. The 2'-position substituent may also be an allyl, amino, azido, thio, -O-allyl, -O-C-C 10 alkyl, —OCF, —O(CH)SCH, —O(CH)—ON(R)(R), and O—CH—C(═O)—N(R)(R), where each R and R is independently H or a substituted or unsubstituted C—C 10 As used herein, the term "2'-modified sugar" refers to a furanosyl sugar modified at the 2' position.

[0036] Methods for preparing modified sugars are well known to those of skill in the art. In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) may be maintained for hybridization with an appropriate nucleic acid target.

[0037] Generally, modifications can be made so that nucleotides in the same chain can be independently modified. The same nucleotide can also have a modified internucleoside linkage (e.g., a phosphorothioate linkage) and a modified sugar (e.g., a 2'-O-methyl modified sugar or a bicyclic sugar) to confer resistance to enzymatic cleavage. The same nucleotide can also have a modified nucleobase (e.g., a 5-methylcytosine) and a modified sugar (e.g., a 2'-O-methyl modified sugar or a bicyclic sugar).

[0038] The number, type, and position of non-natural nucleotides in a nucleic acid strand can affect the antisense effect, etc., provided by the nucleic acid complex of the present invention. The choice of modification can vary depending on the sequence of the target gene, etc., but those skilled in the art can determine a suitable embodiment by referring to the descriptions in literature related to antisense methods (e.g., WO 2007 / 143315, WO 2008 / 043753, and WO 2008 / 049085). Furthermore, when the antisense effect of a modified nucleic acid complex is measured, if the measured value thus obtained is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of that of the nucleic acid complex before modification), the relevant modification can be evaluated.

[0039] As used herein, the term "complementary" refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) through hydrogen bonds. In the present invention, the first nucleic acid strand does not necessarily have to be completely complementary to at least a portion of a target transcript (e.g., a transcript of a target gene), but it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. Similarly, the complementary region in the second nucleic acid strand does not necessarily have to be completely complementary to at least a portion of the first nucleic acid strand, but it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity.

[0040] In the present specification, the term "alkyl group" refers to a linear or branched, acyclic saturated aliphatic hydrocarbon. For example, examples of linear or branched alkyl groups having 1 to 35 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, 2,6,10-trimethylundecyl, pentadecyl, 3,7,11-trimethyldodecyl, hexadecano ... hexadecanoyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, 2,6,10-trimethylundecyl, hexadecanoyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, 2,6,10-trimethylundecyl, hexadecanoyl, cyclohexyl, heptadecyl, 1-hexadecylheptadecyl, octadecyl, 6,10,14-trimethylpentadecan-2-yl, nonadecyl, 2,6,10,14-tetramethylpentadecyl, icosyl, 3,7,11,15-tetramethylhexadecyl, henicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl, hentriacontyl, dotriacontyl, tritriacontyl, tetratriacontyl, pentatriacontyl, and the like.

[0041] As used herein, the term "blood-brain barrier (BBB)" is, as described above, a mechanism that selects and restricts substances that enter the brain, and plays a role in protecting the brain from harmful substances.

[0042] The term "blood-brain-spinal cord barrier (BCSFB)" as used herein is a mechanism that exists in the choroid plexus and, like the BBB, selects and restricts substances that are transferred to the brain.

[0043] As used herein, the term "cerebrospinal fluid barrier (CSFBB)" is a mechanism that, like the BBB, selects and restricts substances that are transferred to the brain.

[0044] As used herein, the term "central nervous system" refers to tissues consisting of the brain and spinal cord, which, together with the peripheral nervous system, constitute the nervous system. The brain includes the cerebrum (cerebral cortex, cerebral white matter, and basal ganglia), diencephalon (thalamus and subthalamic nucleus), cerebellum (cerebellar cortex and cerebellar nuclei), and brainstem (midbrain, substantia nigra, pons, and medulla oblongata). The spinal cord includes the cervical, thoracic, lumbar, sacral, and coccygeal spinal cord. The central nervous system herein may refer to any of these regions, but is preferably the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, and occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brainstem, cervical, thoracic, or lumbar spinal cord.

[0045] As used herein, the term "brain parenchyma" refers to brain tissue composed of neurons and / or glial cells (e.g., astrocytes, oligodendrocytes, microglia), and includes brain tissue of the cerebrum, diencephalon, cerebellum, and brainstem (midbrain, hindbrain, medulla oblongata).

[0046] As used herein, "salts thereof" refers to salts of the nucleic acid complex of the present invention that are physiologically and pharmaceutically acceptable salts of the nucleic acid complex of the present invention, i.e., salts that retain the desired biological activity of the nucleic acid complex and do not impart undesired toxicological effects. Examples of such salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts such as ammonium salt; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris(hydroxymethyl)aminomethane salt, diolamine salt, and meglumine salt. hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate.

[0047] In certain embodiments, the nucleic acid complexes of the present invention include any pharmaceutically acceptable salt of the nucleic acid complex, an ester of the nucleic acid complex, or a salt of the ester. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts, potassium salts, and meglumine salts.

[0048] (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. The first nucleic acid strand is a single-stranded oligonucleotide strand that contains a base sequence capable of hybridizing to at least a portion of a target transcript and exerts an antisense effect on the target transcript.

[0049] The second nucleic acid strand is a single-stranded oligonucleotide strand containing a base sequence complementary to that of the first nucleic acid strand. The second nucleic acid strand is a C 22 ~ 35 In the nucleic acid complex, the second nucleic acid strand is annealed to the first nucleic acid strand through hydrogen bonding of complementary base pairs.

[0050] In one embodiment, the present invention provides a C 22 ~ 35 The single-stranded nucleic acid is bonded to an alkyl group or its analogue. The single-stranded nucleic acid has a structure of C 22 ~ 35 The first nucleic acid strand is identical to the first nucleic acid strand except that it is linked to an alkyl group or its analogue. 22 ~ 35 The structure of the alkyl group or its analog and the form of its connection to the nucleic acid chain are as described in the nucleic acid complex herein.

[0051] The base lengths of the first and second nucleic acid strands are generally at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases, but are not particularly limited thereto. The base lengths of the first and second nucleic acid strands may be 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. The first and second nucleic acid strands may be approximately 100 bases long, or 10 to 35 bases long, 12 to 25 bases long, 13 to 20 bases long, 14 to 19 bases long, or 15 to 18 bases long. The first and second nucleic acid strands may be the same length or different lengths (e.g., one may be 1 to 3 bases shorter or longer). The double-stranded structure formed by the first and second nucleic acid strands may contain a bulge. The length can be determined by balancing the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield.

[0052] The internucleoside linkages in the first and second nucleic acid strands may be naturally occurring internucleoside linkages and / or modified internucleoside linkages. Preferably, at least one, at least two, or at least three internucleoside linkages from the termini (5'-terminus, 3'-terminus, or both) of the first and / or second nucleic acid strands are modified internucleoside linkages. Here, for example, the two internucleoside linkages from the terminus of the nucleic acid strand refer to the internucleoside linkage closest to the terminus of the nucleic acid strand and the adjacent internucleoside linkage located opposite the terminus. Modified internucleoside linkages in the terminal region of the nucleic acid strand are preferred because they can suppress or inhibit undesired degradation of the nucleic acid strand. In one embodiment, all internucleoside linkages in the first and / or second nucleic acid strands may be modified internucleoside linkages. The modified internucleoside linkages may be phosphorothioate linkages.

[0053] In one embodiment, the modified internucleoside linkage of the first nucleic acid strand and / or the second nucleic acid strand is such that at a certain pH (e.g., human physiological pH (about 7.4), the pH of the delivery site (e.g., organelle, cell, tissue, organ, organism, etc.)), the modified internucleoside linkage is in an anionic form (e.g., —OP(O)(O - )-O- (anionic form of the natural phosphate bond), -OP(O)(S - In one embodiment, the modified internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand comprise neutral internucleoside linkages. In one embodiment, the modified internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand comprise cationic internucleoside linkages. In one embodiment, the non-negatively charged internucleoside linkages (e.g., neutral internucleoside linkages), when in their neutral form, do not have moieties with a pKa less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, or less than 14. In one embodiment, the non-negatively charged internucleoside linkage is, for example, a methyl phosphonate linkage as described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, a methyl phosphotriester linkage as described in U.S. Patent Registration No. 5,955,599, an ethyl phosphotriester linkage, a methoxypropyl phosphonate linkage as described in WO 2015 / 168172, or an internucleoside linkage used in self-neutralizing nucleic acids (ZON) as described in WO 2016 / 081600. In one embodiment, the non-negatively charged internucleoside linkage comprises a triazole moiety or an alkyne moiety. In one embodiment, the non-negatively charged internucleoside linkage comprises a cyclic guanidine moiety. In one embodiment, the modified internucleoside linkage comprising a cyclic guanidine moiety is [ka] In one embodiment, the neutral internucleoside linkage comprising a cyclic guanidine moiety is chiral controlled. In one embodiment, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleoside linkage and at least one phosphorothioate internucleoside linkage. Without wishing to be bound by any particular theory, in at least some cases, the neutral internucleoside linkage can improve properties and / or activity compared to a comparable nucleic acid that does not comprise a neutral internucleoside linkage, such as improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape, and / or improved nuclear uptake.

[0054] In one embodiment, the first nucleic acid strand and the second nucleic acid strand may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more modified internucleoside linkages. In one embodiment, the first nucleic acid strand and the second nucleic acid strand may each contain at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more modified internucleoside linkages.

[0055] In one embodiment, the first and second nucleic acid strands may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more chiral-controlled internucleoside linkages. In one embodiment, the first and second nucleic acid strands may each contain at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more chiral-controlled internucleoside linkages.

[0056] In one embodiment, the first nucleic acid strand and the second nucleic acid strand may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more non-negatively charged internucleoside linkages (preferably neutral internucleoside linkages). In one embodiment, the first nucleic acid strand and the second nucleic acid strand may each contain at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more non-negatively charged internucleoside linkages.

[0057] At least one (e.g., three) internucleoside linkages from the 3'-end of the second nucleic acid strand may be a modified internucleoside linkage such as a phosphorothioate linkage, which has high RNase resistance. Inclusion of a modified internucleoside linkage such as a phosphorothioate modification at the 3'-end of the second nucleic acid strand is preferred because it improves the gene silencing activity of the double-stranded nucleic acid complex.

[0058] C optionally substituted with a hydroxy group at the 5'-end and 3'-end of the second nucleic acid strand22 ~ 35 The internucleoside bonds of the terminal 2 to 6 bases not bound to the alkyl group or its analogue may be modified internucleoside bonds (for example, phosphorothioate bonds).

[0059] At least one (e.g., three) nucleosides from the 3'-end of the second nucleic acid strand may be, for example, a modified nucleoside with high RNase resistance, such as 2'F-RNA or 2'-OMe. Inclusion of a modified nucleoside, such as 2'F-RNA or 2'-OMe, at the 3'-end of the second nucleic acid strand is preferred because it enhances the gene suppression activity of the double-stranded nucleic acid complex.

[0060] C optionally substituted with a hydroxy group at the 5'-end and 3'-end of the second nucleic acid strand 22 ~ 35 The terminal 1 to 5 nucleosides not bound to the alkyl group or its analog may be modified nucleosides such as 2'F-RNA, which has high RNase resistance.

[0061] The nucleosides in the first and second nucleic acid strands can be natural nucleosides (deoxyribonucleosides, ribonucleosides, or both) and / or non-natural nucleosides.

[0062] In one embodiment, the first nucleic acid strand and the second nucleic acid strand comprise cationic nucleosides.

[0063] As used herein, the base sequence of the first nucleic acid strand is complementary to at least a portion of the base sequence of the target transcript, and therefore can hybridize (or anneal) to the target transcript. The complementarity of the base sequences can be determined using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design an antisense nucleic acid complementary to the target transcript, for example, based on information on the base sequence of the target gene.

[0064] Hybridization conditions may be of various stringent conditions, such as low stringency conditions and high stringency conditions. Low stringency conditions may be conditions of relatively low temperature and high salt concentration, for example, 30°C, 2xSSC, 0.1% SDS. High stringency conditions may be conditions of relatively high temperature and low salt concentration, for example, 65°C, 0.1xSSC, 0.1% SDS. Hybridization stringency can be adjusted by changing conditions such as temperature and salt concentration. Here, 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0065] The first nucleic acid strand, when hybridized to a target transcript, can contain at least 4, at least 5, at least 6, or at least 7 consecutive nucleosides recognized by RNase H. Typically, the region may contain 4 to 20, 5 to 16, or 6 to 12 consecutive nucleosides. Examples of nucleosides recognized by RNase H include natural deoxyribonucleosides. Modified deoxyribonucleosides and suitable nucleosides containing other bases are well known in the art. It is also known that nucleosides containing a hydroxy group at the 2' position, such as ribonucleosides, are unsuitable as such nucleosides. The suitability of nucleosides for use in this region containing "at least 4 consecutive nucleosides" can be easily determined. In one embodiment, the first nucleic acid strand can contain at least 4 consecutive deoxyribonucleosides.

[0066] In one embodiment, the entire length of the first nucleic acid strand and / or the second nucleic acid strand is not composed solely of natural ribonucleosides, and it is preferred that the natural ribonucleosides make up less than half of the entire length of the first nucleic acid strand and / or the second nucleic acid strand, or that they are not present at all.

[0067] In one embodiment, the second nucleic acid strand may contain at least four consecutive ribonucleosides complementary to the at least four consecutive nucleosides (e.g., deoxyribonucleosides) in the first nucleic acid strand, such that the second nucleic acid strand forms a partial DNA-RNA heteroduplex with the first nucleic acid strand and is recognized and cleaved by RNase H. The at least four consecutive ribonucleosides in the second nucleic acid strand are preferably linked by naturally occurring internucleoside linkages, i.e., phosphodiester bonds.

[0068] All nucleosides of the second nucleic acid strand may be composed of ribonucleosides and / or modified nucleosides. All nucleosides of the second nucleic acid strand may be composed of deoxyribonucleosides and / or modified nucleosides, or may not contain ribonucleosides.

[0069] The first nucleic acid strand and / or the second nucleic acid strand constituting the nucleic acid complex of the present invention may be a gapmer. As used herein, the term "gapmer" refers to a single-stranded nucleic acid comprising a central region (DNA gap region) and 5' and 3' wing regions located on either side of the central region (DNA gap region). In a gapmer, the central region contains at least four consecutive deoxyribonucleosides, and the 5' and 3' wing regions contain unnatural nucleosides. When the unnatural nucleosides constituting the 5' and 3' wing regions contain or consist of bridged nucleosides, the gapmer is specifically referred to as a "BNA / DNA gapmer." When the unnatural nucleosides constituting the 5' and 3' wing regions contain or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer." When the non-natural nucleosides constituting the 5' wing region and the 3' wing region comprise or consist of peptide nucleic acids, the gapmer is specifically referred to as a "morpholino nucleic acid gapmer." The number of bridged nucleosides contained in the 5' wing region and the 3' wing region may be two or three. The bridged nucleosides contained in the 5' wing region and the 3' wing region may be contiguous or discontinuous within the 5' wing region and the 3' wing region. The bridged nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine). When the bridged nucleoside is an LNA nucleoside, the gapmer is specifically referred to as an "LNA / DNA gapmer." The base lengths of the 5' wing region and the 3' wing region may each independently be at least two bases long, for example, 2 to 10 bases long, 2 to 7 bases long, or 3 to 5 bases long. The 5' wing region and the 3' wing region may contain at least one unnatural nucleoside, and may further contain natural nucleosides.

[0070] The first nucleic acid strand and / or the second nucleic acid strand constituting the gapmer may be composed of, in order from the 5' end, a bridged nucleoside having a length of 2 to 7 bases or 3 to 5 bases, a ribonucleoside or deoxyribonucleoside having a length of 4 to 15 bases or 8 to 12 bases, and a bridged nucleoside having a length of 2 to 7 bases or 3 to 5 bases.

[0071] The first nucleic acid strand and / or the second nucleic acid strand constituting the nucleic acid complex of the present invention may be a mixmer. As used herein, a "mixmer" refers to a nucleic acid strand that contains alternating natural and unnatural nucleosides of periodic or random segment lengths, but does not contain four or more consecutive deoxyribonucleosides and ribonucleosides. A mixmer in which the unnatural nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "BNA / DNA mixmer." A mixmer in which the unnatural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "peptide nucleic acid / DNA mixmer." In a mixmer, a mixmer in which the non-natural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "morpholino nucleic acid / DNA mixmer." A mixmer is not limited to containing only two types of nucleosides. A mixmer can contain any number of types of nucleosides, regardless of whether they are natural or modified nucleosides or nucleoside mimics. For example, a mixmer may have one or two consecutive deoxyribonucleosides separated by a bridged nucleoside (e.g., an LNA nucleoside). The bridged nucleoside may further contain a modified nucleobase (e.g., 5-methylcytosine).

[0072] At least one, at least two, at least three, or at least four nucleosides from the end (5'-end, 3'-end, or both ends) of the second nucleic acid strand may be modified nucleosides. The modified nucleosides may contain a modified sugar and / or a modified nucleobase. The modified sugar may be a 2'-modified sugar (e.g., a sugar containing a 2'-O-methyl group). The modified nucleobase may also be 5-methylcytosine.

[0073] The second nucleic acid strand may be composed of, from the 5'-terminus, modified nucleosides (e.g., modified nucleosides containing 2'-modified sugars) each having a length of 2 to 7 bases or 3 to 5 bases, ribonucleosides or deoxyribonucleosides (optionally linked via modified internucleoside linkages) each having a length of 4 to 15 bases or 8 to 12 bases, and modified nucleosides (e.g., modified nucleosides containing 2'-modified sugars). In this case, the first nucleic acid strand may be a gapmer.

[0074] The first nucleic acid strand and the second nucleic acid strand may comprise, in whole or in part, a nucleoside mimic or a nucleotide mimic. The nucleotide mimic may be a peptide nucleic acid and / or a morpholino nucleic acid. The first nucleic acid strand may comprise at least one modified nucleoside. The modified nucleoside may comprise a 2'-modified sugar. The 2'-modified sugar may comprise a 2'-O-methyl group.

[0075] The first nucleic acid strand and the second nucleic acid strand may comprise, in whole or in part, cationic nucleosides. In one embodiment, the cationic nucleosides include amine-bridged nucleic acids (also known as 2'-Amino-LNA) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substitutions), aminoalkyl-modified nucleic acids (e.g., 2'-O-methyl and 4'-CH2CH2CH2NH2 substitutions), GuNA (e.g., in Figure 3, R = H is GuNA[NH], and R = Me is GuNA[N-Me]), etc.

[0076] The first and second nucleic acid strands may contain any combination of the modified internucleoside linkages and modified nucleosides described above.

[0077] The second nucleic acid strand may be a C 22 ~ 35 or its analogue. C optionally substituted with a hydroxy group 22 ~ 35 The alkyl group or its analogue can be produced by a method known per se by a person skilled in the art.

[0078] As used herein, the term "analog" refers to a compound having a similar structure and properties, which has the same or a similar basic skeleton. Analogs include, for example, biosynthetic intermediates, metabolic products, etc. Whether a compound is an analog of another compound can be determined by one skilled in the art.

[0079] In one embodiment, C optionally substituted with a hydroxy group 22 ~ 35 The alkyl group (e.g., docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl, 1-hexadecylheptadecyl) may be, for example, C 22 ~ 26 or C 28 Alkyl groups (e.g., docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, octacosyl), such as C optionally substituted with a hydroxyl group 22 In one embodiment, C optionally substituted with a hydroxy group is an alkyl group (e.g., docosyl). 22 ~ 35 The alkyl group in 22 ~ 26 or C 28 Alkyl groups (e.g., docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, octacosyl), such as unsubstituted C 22 ~ 24 , or C 26Alkyl groups (e.g., docosyl, tricosyl, tetracosyl, hexacosyl), such as unsubstituted C 22 The alkyl group (e.g., docosyl) may be either a linear alkyl group or a branched alkyl group. In one embodiment, a C 22 ~ 35 The alkyl group of is an unsubstituted straight chain C 22 ~ 26 or linear C 28 Alkyl groups (e.g., docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, octacosyl), such as unsubstituted linear C 22 ~ 24 , or linear C 26 Alkyl groups (e.g., docosyl, tricosyl, tetracosyl, hexacosyl), such as unsubstituted linear C 22 In one embodiment, C optionally substituted with a hydroxy group is an alkyl group (e.g., docosyl). 22 ~ 35 The alkyl group of is an unsubstituted branched C 33 It is an alkyl group (e.g., 1-hexadecylheptadecyl).

[0080] C optionally substituted with a hydroxy group 22 ~ 35 The alkyl group or its analog may be linked to the 5'-end, 3'-end, or both ends of the second nucleic acid strand. 22 ~ 35 The alkyl group or its analog may be linked to an internal nucleotide of the second nucleic acid strand. For example, C 22 ~ 35 or its analog may be attached to the 5' end of the second nucleic acid strand. In another embodiment, the second nucleic acid strand is a C 22 ~ 35or its analogs, which may be linked to multiple positions on the second nucleic acid strand and / or may be linked as a group to one position on the second nucleic acid strand. 22 ~ 35 The alkyl group or its analog may be linked to the 5'-end and the 3'-end of the second nucleic acid strand, respectively. 22 ~ 35 The alkyl group or its analog may be linked to the 5' end and the 3' end of the second nucleic acid strand, respectively.

[0081] a second nucleic acid strand and C optionally substituted with a hydroxy group; 22 ~ 35 The bond to the alkyl group or its analogue may be a direct bond or an indirect bond. A direct bond means that two molecules are directly bonded. An indirect bond means that the two molecules to be bonded are bonded via another substance.

[0082] In one embodiment, the second nucleic acid strand is a C optionally substituted with a hydroxy group. 22 ~ 35 The bond to the alkyl group or its analogue may be via a phosphate bond or a phosphorothioate bond to the 5' end, 3' end, or an internal nucleotide of the second nucleic acid strand.

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

[0084] In one embodiment, it is attached to the 5' end of the second nucleic acid strand via a phosphate bond.

[0085] a second nucleic acid strand and C optionally substituted with a hydroxy group; 22 ~ 35When the alkyl group or its analog is indirectly bonded, it may be bonded via a linking group (often referred to as a "linker" herein). The linker may be bonded to the 5'-end, 3'-end, or an internal nucleotide of the second nucleic acid strand via a phosphate bond or a phosphorothioate bond.

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

[0087] In one embodiment, the linker is attached to the 5' end of the second nucleic acid strand via a phosphate bond.

[0088] a second nucleic acid strand and C optionally substituted with a hydroxy group; 22 ~ 35 When the alkyl group or its analog is indirectly bonded, the two may be linked via a cleavable linker. A "cleavable linker" refers to a linking group that can be cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within the human body). The cleavable linker may be selectively cleaved by endogenous enzymes such as nucleases and peptidases, under acidic conditions, under a reducing environment, or the like. Specific examples of such linkers include amide bonds, ester bonds, phosphate ester bonds, ester bonds at one or both ends of phosphodiester bonds, carbamate bonds, and disulfide bonds, as well as nucleotide linkers such as natural DNA linkers.

[0089] a second nucleic acid strand and C optionally substituted with a hydroxy group; 22 ~ 35 When the alkyl group or its analog is indirectly bonded, they may be bonded via a non-cleavable linker. A "non-cleavable linker" refers to a linking group that is not cleaved under physiological conditions. Examples of such non-cleavable linkers include a phosphorothioate bond, and a linker consisting of modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked via a phosphorothioate bond.

[0090] The chain length of the cleavable linker or non-cleavable linker is not particularly limited in the case of nucleic acids such as DNA or oligonucleotides, but may usually be 1 to 20 bases long, 1 to 10 bases long, or 1 to 6 bases long.

[0091] A specific example of the linker is a linker represented by the following general formula (II): [ka] (In the formula, L 2 is a substituted or unsubstituted C1~ 12 (e.g., propylene, hexylene, dodecylene), a substituted or unsubstituted C3-8 cycloalkylene group (e.g., cyclohexylene), —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, or CH(CH2-OH)—CH2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-; L 3 represents -NH- or a bond, and L 4 is a substituted or unsubstituted C1~ 12 alkylene groups (e.g., ethylene, pentylene, heptylene, undecylene), substituted or unsubstituted C3-8 cycloalkylene groups (e.g., cyclohexylene), -(CH2)2-[O-(CH2)2] m - or a bond, where m represents an integer of 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond (wherein the substitution is preferably made by a halogen atom).

[0092] In one embodiment, the linker of formula (II) is L 2 is an unsubstituted C3 to C6 alkylene group (e.g., propylene, hexylene), —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, or —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, and L3 is -NH-, and L 4 and L 5 is a bond.

[0093] A specific example of the linker is a linker represented by the following general formula (I): [ka]

[0094] In certain embodiments, the linker comprises one or more groups selected from an alkyl group, an amino group, an oxo group, an amide group, a disulfide group, a polyethylene glycol group, an ether group, a thioether group, and a hydroxylamino group. In certain such embodiments, the linker comprises a group selected from an alkyl group, an amino group, an oxo group, an amide group, and an ether group. In certain embodiments, the linker comprises a group selected from an alkyl group and an amide group. In certain embodiments, the linker comprises a group selected from an alkyl group and an ether group. In certain embodiments, the linker comprises at least one phosphorus moiety. In certain embodiments, the linker comprises at least one phosphate group. In certain embodiments, the linker comprises at least one neutral conjugate group.

[0095] In certain embodiments, the linker comprises a bifunctional linking moiety. Generally, the bifunctional linking moiety comprises at least two functional groups, one of which is selected to bind to a specific site on the second nucleic acid strand, and the other of which is a C, optionally substituted with a hydroxy group. 22 ~ 35 or analogs thereof. Examples of functional groups used in the bifunctional linking moiety include, but are not limited to, electrophiles that react with nucleophilic groups and nucleophiles that react with electrophilic groups. In certain embodiments, the bifunctional linking moiety comprises one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[0096] Examples of linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other linkers include, but are not limited to, optionally substituted C 1-10 Alkyl, optionally substituted C 2-10 Alkenyl, or optionally substituted C 2-10 Alkynyl is included, where a non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0097] In certain embodiments, the linker comprises 1 to 10 linker nucleosides. In certain embodiments, the conjugate linker comprises 2 to 5 linker nucleosides. In certain embodiments, the linker comprises exactly 3 linker nucleosides. In certain embodiments, the linker comprises a TCA motif. In certain embodiments, the linker nucleoside is a non-natural nucleoside. In certain embodiments, the linker nucleoside comprises a modified sugar moiety. In certain embodiments, the linker nucleoside is a natural nucleoside. In certain embodiments, the linker nucleoside comprises an optionally protected heterocyclic base selected from a purine base, a modified purine base, a pyrimidine base, or a modified pyrimidine base. Linker nucleosides are not considered part of the oligonucleotide herein. Thus, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and / or a specified percent complementarity to a reference nucleic acid, and in which the oligomeric compound comprises a linker that includes linker nucleosides, these linker nucleosides are not counted in the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide to the reference nucleic acid.

[0098] In certain embodiments, when the nucleic acid complex of the present invention contains optical isomers, stereoisomers, positional isomers, or rotational isomers, these are also contained in the nucleic acid complex of the present invention, and each can be obtained as a single product by a synthesis method or separation method known per se. For example, when an optical isomer is present in the nucleic acid complex of the present invention, the optical isomer separated from the compound is also included in the nucleic acid complex of the present invention.

[0099] In certain embodiments, the nucleic acid complex of the present invention includes a prodrug and a pharmaceutically acceptable salt of the prodrug. The prodrug of the nucleic acid complex of the present invention and a pharmaceutically acceptable salt of the prodrug refer to a compound that is converted into the nucleic acid complex of the present invention by a reaction with an enzyme, gastric acid, or the like under physiological conditions in vivo, i.e., a compound that is enzymatically oxidized, reduced, hydrolyzed, or the like to convert into the nucleic acid complex of the present invention, or a compound that is hydrolyzed by gastric acid or the like to convert into the nucleic acid complex of the present invention. In certain embodiments, the prodrug of the nucleic acid complex is a C 22 ~ 35 It contains one or more alkyl groups or their analogs.

[0100] In the nucleic acid complex of the present invention, the antisense effect of the first nucleic acid strand on a target transcript can be measured by methods known in the art. For example, after introducing the nucleic acid complex into cells, measurement can be performed using known techniques such as Northern blotting, quantitative PCR, or Western blotting. Specifically, the aforementioned known techniques can be used to verify that the antisense effect reduces the expression level of the target gene or the level of the target transcript (e.g., mRNA level, RNA level such as microRNA, cDNA level, protein level, etc.) in the cell.

[0101] The antisense effect of the nucleic acid complex of the present invention in the central nervous system and the passage of the BBB or the like can also be measured by methods known in the art. For example, and without limitation, this can be achieved by administering the nucleic acid complex of the present invention to a subject (e.g., a mouse) and measuring whether the expression level of a target gene or the level of a target transcript in the central nervous system is suppressed several days to several months later (e.g., 2 to 7 days or 1 month later). The criteria for this determination are that the nucleic acid complex of the present invention has crossed the BBB or the like and exerted an antisense effect on the central nervous system if the measured value of the expression level of the target gene or the target transcript is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 40% compared to the measured value of a negative control (e.g., vehicle administration). Furthermore, passage through the BBB or the like can be determined by administering the nucleic acid complex of the present invention to a subject (e.g., a mouse) and measuring the amount (concentration) of the nucleic acid complex of the present invention in the central nervous system several days to several months later (e.g., 2 to 7 days later or 1 month later).

[0102] Although exemplary embodiments of the nucleic acid complex of the present invention have been described above, the nucleic acid complex of the present invention is not limited to these exemplary embodiments. Furthermore, those skilled in the art can prepare the first and second nucleic acid strands constituting the nucleic acid complexes of various embodiments of the present invention by appropriately selecting known methods. For example, the nucleic acid molecules of the present invention can be prepared by designing each nucleic acid molecule based on information about the base sequence of the target transcript (e.g., the base sequence of the target gene), synthesizing the nucleic acid using a commercially available automated nucleic acid synthesizer, for example, from GE Healthcare, Thermo Fisher Scientific, Beckman Coulter, etc., and then purifying the resulting oligonucleotides using a reverse-phase column or the like.

[0103] The second nucleic acid strand may further include at least one functional moiety bound to the polynucleotide. There are no particular limitations on the structure of the "functional moiety" in certain embodiments, as long as the functional moiety confers a desired function to the nucleic acid complex and / or the strand to which the functional moiety is bound. Desired functions include labeling and purification functions. Examples of moieties that confer labeling function include compounds such as fluorescent proteins and luciferase. Examples of moieties that confer purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide. Furthermore, from the viewpoint of highly specific and efficient delivery of the first nucleic acid strand to a target site and highly effective suppression of target gene expression by the nucleic acid, it is preferable that the second nucleic acid strand be bound to a molecule having the activity of delivering the double-stranded nucleic acid complex of certain embodiments to a target site. Examples of moieties that confer target delivery function include lipids, antibodies, aptamers, and ligands for specific receptors. The binding position and type of the functional moiety on the second nucleic acid strand may be C, which may be substituted with a hydroxy group. 22 ~ 35 The bond between the alkyl group or its analog and the second nucleic acid strand is as described above.

[0104] In one embodiment, the second nucleic acid strand is bound to a lipid, including, but not limited to, tocopherol, cholesterol, fatty acids, phospholipids and their analogs, folic acid, vitamin C, vitamin B1, vitamin B2, estradiol, androstane and their analogs, steroids and their analogs, ligands for LDLR, SRBI, or LRP1 / 2, FK-506, and cyclosporine.

[0105] In one embodiment, a nucleic acid complex having a functional moiety bound thereto can be prepared by carrying out the synthesis, purification, and annealing described above using a nucleic acid species to which a functional moiety has already been bound. For example, a C optionally substituted with a hydroxy group can be prepared. 22 ~ 35The second nucleic acid strand can be produced by carrying out the synthesis and purification described above using a nucleic acid species to which an alkyl group or an analog thereof has been previously bound.

[0106] In one embodiment, the second nucleic acid strand produced by carrying out the synthesis and purification described above may be treated with C, which may be substituted with a hydroxy group by a method known per se. 22 ~ 35 The nucleic acid complex of the present invention can be produced by mixing the nucleic acid produced by this method in an appropriate buffer solution, denaturing it at about 90°C to 98°C for several minutes (e.g., 5 minutes), and then annealing the nucleic acid at about 30°C to 70°C for about 1 to 8 hours. Alternatively, nucleic acid chains can be ordered and purchased from various manufacturers (e.g., Gene Design, Inc.) by specifying the base sequence and the modification site and type. The annealing step can be carried out by leaving the mixture at room temperature (about 10°C to about 35°C) for about 5 to 60 minutes.

[0107] The nucleic acid complex of some embodiments of the present invention may be prepared by dissolving the first nucleic acid strand and the second nucleic acid strand in a buffer solution (e.g., phosphate-buffered saline) or water at about 70°C to 98°C, mixing the resulting two solutions, and maintaining the mixture at about 70°C to 98°C for several minutes (e.g., 5 minutes), and then maintaining 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 can also be dissolved in a buffer solution (e.g., phosphate-buffered saline) or water at room temperature (about 10°C to about 35°C).

[0108] However, the annealing conditions (time and temperature) when preparing the nucleic acid complex are not limited to the above conditions, and conditions suitable for promoting the annealing of nucleic acid strands are well known in the art.

[0109] (Effect of nucleic acid complexes) The nucleic acid complex of the present invention can inhibit the effect of a target miRNA in the central nervous system of a subject. Specifically, for example, the first nucleic acid strand comprises a base sequence capable of hybridizing to at least a portion of the target miRNA and has an antisense effect on the target miRNA, and the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and optionally substituted with a hydroxy group. 22 ~ 35 or its analog, and annealed to a first nucleic acid strand and a second nucleic acid strand. By inhibiting the effect of a target miRNA with this nucleic acid complex, it is possible to upregulate the expression of a gene that is normally downregulated by the target miRNA.

[0110] The nucleic acid complex of the present invention can regulate the expression or editing of a target RNA in the central nervous system of a subject. Specifically, for example, the first nucleic acid strand comprises a base sequence capable of hybridizing to at least a portion of the target RNA and has an antisense effect on the target RNA, and the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and optionally substituted with a hydroxy group. 22 ~ 35 Examples of such nucleic acid complexes include those in which the first nucleic acid strand and the second nucleic acid strand are bound to an alkyl group or its analog, and the first and second nucleic acid strands are annealed to each other. Here, "regulating expression of a target RNA" includes, for example, upregulation and downregulation of expression levels. Furthermore, "regulating editing of a target RNA" includes regulation of splicing by RNA editing, such as exon skipping and exon inclusion. The target RNA may be viral or bacterial RNA, or toxic RNA.

[0111] The nucleic acid complex of the present invention can inhibit the translation of a target mRNA in the central nervous system of a subject. Specifically, for example, the first nucleic acid strand comprises a base sequence capable of hybridizing to at least a portion of the target mRNA and has an antisense effect on the target mRNA, and the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and optionally substituted with a hydroxy group. 22 ~ 35 or its analog, and the first and second nucleic acid strands are annealed to each other. When the first nucleic acid strand binds to the target mRNA through this nucleic acid complex, a steric block occurs, inhibiting translation of the mRNA.

[0112] The nucleic acid complex of the present invention can be effectively used in, but is not limited to, regulating the expression or editing of target transcripts in microglia (microglial cells) of the central nervous system.

[0113] <Composition> A second aspect of the present invention is a composition. The composition of the present invention includes the nucleic acid complex or single-stranded nucleic acid strand of the first aspect as an active ingredient and / or a drug delivery molecule. The nucleic acid complex or single-stranded nucleic acid strand of the first aspect can pass through the BBB or the like and regulate (e.g., reduce) the expression level of a target transcript in the central nervous system by its antisense effect. In one embodiment, the nucleic acid complex or single-stranded nucleic acid strand of the first aspect can migrate from the blood to the brain (brain parenchyma) and regulate (e.g., reduce) the expression level of a target transcript in the brain parenchyma by its antisense effect. Therefore, the composition of the present invention may be a composition that delivers a nucleic acid complex to treat a subject by administration to the subject, or may be a pharmaceutical composition. In one embodiment, the composition of the present invention is a composition for regulating the expression or editing of a target transcript in the central nervous system and / or for delivering a drug to the central nervous system. The composition of the present invention can be effectively used, but is not limited to, for regulating the expression or editing of a target transcript in microglia (microglial cells) of the central nervous system.

[0114] Furthermore, one embodiment of the present invention relates to a method for treating each central nervous system disease by administering a composition containing a nucleic acid complex.

[0115] (Formulation) The composition herein can be formulated by a method known per se. For example, the composition can be used orally or parenterally in the form of capsules, tablets, pills, liquids, powders, granules, microgranules, film-coated formulations, pellets, troches, sublingual tablets, peptizers, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal formulations, lotions, inhalants, aerosols, eye drops, injections, and suppositories.

[0116] For the formulation of these preparations, a pharmaceutically acceptable carrier or solvent or a carrier or solvent acceptable for food and beverage products may be appropriately incorporated. Specific examples of such carriers or solvents include sterilized water, physiological saline, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavors, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonicity adjusters, soothing agents, bulking agents, disintegrating agents, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizing agents, and other additives.

[0117] (Dosage form and dosage) Herein, there is no particular limitation on the preferred administration form of the composition. For example, oral administration or parenteral administration may be used. Specific examples of parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, tracheal / bronchial administration, rectal administration, intrathecal administration, intraventricular administration, intranasal administration, and intramuscular administration, as well as administration by blood transfusion. Administration can also be by intramuscular injection, intravenous drip administration, or implanted continuous subcutaneous administration. Subcutaneous administration is preferred because it allows patients to self-inject. Furthermore, in the case of intravenous administration, the amount of nucleic acid complex contained in one dose of the composition, i.e., the single dose of the nucleic acid complex, can be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.025 mg / kg or more, 0.1 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more. For example, any amount within the range of 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg) can be appropriately selected.

[0118] (Subject / Applicable object) As used herein, the term "subject" refers to a target to which the composition of the present invention is applied. Subjects include individuals as well as organs, tissues, and cells. When the subject is an individual, the composition of the present invention can be applied to any animal, including humans. Non-human subjects may include, for example, various livestock, poultry, pets, laboratory animals, etc. The subject may be an individual in need of reducing the expression level of a target transcript in the central nervous system or an individual in need of treatment for a central nervous system disease.

[0119] The composition of the present invention can reduce the expression level of a target transcription product in the central nervous system by the migration of the nucleic acid complex or single-stranded nucleic acid strand of the first aspect it comprises from the blood to the brain (e.g., by passing through the BBB, etc.) and by its antisense effect.

[0120] When the composition of the present invention is used to treat central nervous system diseases, the target disease is preferably a central nervous system disease associated with increased or decreased gene expression, particularly a disease (such as a tumor) associated with increased expression of a target transcript or target gene, including, but not limited to, brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Huntington's disease.

[0121] In one embodiment, the composition of the present invention is used for the treatment of immune-mediated central nervous system diseases, such as microglia-associated diseases, including Alzheimer's disease, multiple sclerosis, ALS, and neuropathic pain.

[0122] The delivery site of the composition of the present invention, more specifically, the delivery site of the active ingredient contained in the composition, is not particularly limited, but more effective results can be obtained by delivering the composition to an appropriate site depending on the disease. Specifically, in the treatment of Alzheimer's disease, drug delivery to the hippocampus and / or parietal lobe can be effective. Furthermore, in the treatment of frontotemporal dementia (FTD) (including frontotemporal lobar degeneration (FTLD), semantic dementia (SD), progressive non-fluent aphasia (PNFA), etc.) and Pick's disease, drug delivery to the frontal lobe, temporal lobe, and / or substantia nigra can be effective. Furthermore, in the treatment of Parkinson's disease dementia, drug delivery to the occipital lobe, substantia nigra, and / or striatum can be effective. Furthermore, in the treatment of Parkinson's disease, drug delivery to the substantia nigra and / or striatum can be effective. In the treatment of corticobasal degeneration (CBD), drug delivery to the frontal lobe, parietal lobe, basal ganglia, and / or substantia nigra can be effective. In the treatment of progressive supranuclear palsy (PSP), drug delivery to the frontal lobe, basal ganglia, and / or substantia nigra may be effective. In the treatment of amyotrophic lateral sclerosis, drug delivery to the frontal lobe, parietal lobe, basal ganglia, and / or substantia nigra may be effective. In the treatment of spinocerebellar degeneration (SCD) SCA1 to SCA34, drug delivery to the brainstem and / or cerebellum may be effective. In the treatment of dentatorubral-pallidoluysian degeneration (DRPLA), drug delivery to the basal ganglia, brainstem, and / or cerebellum may be effective. In the treatment of spinal-bulbar atrophy (SBMA), drug delivery to the brainstem and / or spinal cord may be effective. In the treatment of Friedreich's ataxia (FA), drug delivery to the brainstem and / or cerebellum may be effective. In the treatment of Huntington's disease, drug delivery to the striatum, frontal lobe, parietal lobe, and / or basal ganglia may be effective. In the treatment of prion diseases (including mad cow disease and GSS), drug delivery to the cerebral cortex, cerebral white matter, basal ganglia, and / or substantia nigra may be effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective. In the treatment of encephalitis (including viral, bacterial, fungal, and tuberculous) and meningitis (including viral, bacterial, fungal, and tuberculous), drug delivery to the entire brain may be effective. In the treatment of metabolic encephalopathy, toxic encephalopathy, and nutritional encephalopathy, drug delivery to the cerebral white matter may be effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective.Drug delivery to the entire brain can be effective in treating cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, moyamoya disease, and anoxic encephalopathy. Drug delivery to the cerebral white matter can be effective in treating cerebral leukoencephalopathy. Drug delivery to the cerebral white matter can be effective in treating diffuse axonal injury. Drug delivery to the entire brain can be effective in treating head trauma. Drug delivery to the cerebral white matter, cerebral cortex, optic nerve, and / or spinal cord can be effective in treating multiple sclerosis (MS) and neuromyelitis optica (NMO). Drug delivery to skeletal muscle, cardiac muscle, cerebral cortex, and / or cerebral white matter can be effective in treating myotonic dystrophy (DM1, DM2). Drug delivery to the parietal lobe and / or spinal cord can be effective in treating familial spastic paraplegia (HSP). In the treatment of Fukuyama muscular dystrophy, drug delivery to skeletal muscle, cerebral cortex, and / or cerebral white matter may be effective. In the treatment of dementia with Lewy bodies (DLB), drug delivery to the substantia nigra, striatum, occipital lobe, frontal lobe, and / or parietal lobe may be effective. In the treatment of multiple system atrophy (MSA), drug delivery to the striatum, basal ganglia, cerebellum, substantia nigra, frontal lobe, and / or temporal lobe may be effective. In the treatment of Alexander disease, drug delivery to cerebral white matter may be effective. In the treatment of CADASIL and CARASIL, drug delivery to cerebral white matter may be effective.

[0123] When the composition is administered or ingested, the dosage or intake can be appropriately selected based on the subject's age (including age in months and weeks), body weight, symptoms and health condition, and type of composition (drug, food, beverage, etc.). The effective amount of the composition of the present invention ingested by a subject can be, for example, 0.00001 mg / kg / day to 10,000 mg / kg / day, or 0.001 mg / kg / day to 100 mg / kg / day of the nucleic acid complex contained therein. The composition can be administered in a single dose or multiple doses. In the case of multiple doses, the composition can be administered daily or at appropriate intervals (e.g., at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times. The single dose of the nucleic acid complex may be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.025 mg / kg or more, 0.1 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 3.0 mg / kg or more, 4.0 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more The dose can be 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, and can be appropriately selected from any amount within the range of, for example, 0.001 mg / kg to 500 mg / kg (for example, 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg).

[0124] The nucleic acid complex of the present invention may be administered at a dose of 0.01 to 10 mg / kg (e.g., about 6.25 mg / kg) twice a week for four doses. Alternatively, the nucleic acid complex may be administered at a dose of 0.05 to 30 mg / kg (e.g., about 25 mg / kg) once or twice a week for two to four doses, for example, twice a week for two doses. The use of such a dosing regimen (divided administration) can reduce toxicity and the burden on the subject compared to a single administration of a higher dose.

[0125] Although there are limits (upper limits) to the amount of nucleic acid complex that can cross the BBB, BCSFB, CSFBB, and blood-nerve barrier (BNB) after a single administration, it is believed that the inhibitory effect is additive within cells even with repeated administration. That is, at doses (e.g., 25 mg / kg or higher) that exceed the limits of BBB, BCSFB, CSFBB, and BNB passage, the enhancement of efficacy is reduced by increasing the single dose, but it is believed that efficacy can be improved by repeated administration with a certain interval between doses (e.g., half a day or more).

[0126] In certain embodiments, the nucleic acid complex of the present invention has excellent properties as a pharmaceutical, such as excellent solubility in water, the second fluid of the Japanese Pharmacopoeia dissolution test, or the second fluid of the Japanese Pharmacopoeia disintegration test, excellent pharmacokinetics (e.g., blood drug half-life, brain transferability, metabolic stability, CYP inhibition), low toxicity (e.g., superior as a pharmaceutical in terms of acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interactions, carcinogenicity, phototoxicity, etc.), and few side effects (e.g., suppression of excessive sedation, avoidance of lamellar necrosis).

[0127] (drug delivery) The composition of the present invention utilizes the fact that the nucleic acid complex or single-stranded nucleic acid strand of the first aspect contained as an active ingredient can cross the BBB and be efficiently delivered to the central nervous system. By binding a specific drug to the first nucleic acid strand and / or the second nucleic acid strand, the drug can be delivered to the nervous system, preferably the central nervous system, and more preferably the brain (brain parenchyma). Drugs delivered to the nervous system include, but are not limited to, peptides, proteins, nucleic acid drugs, or other organic compounds, such as antitumor drugs, hormone drugs, antibiotics, antiviral agents, and anti-inflammatory drugs. The drug is preferably a small molecule drug. The term "small molecule drug" is well understood in the art. Small molecule drugs typically refer to drugs with a molecular weight of less than 1,000 daltons. The drug may be a lipophilic drug. Nucleic acid drugs include, but are not limited to, ASOs, antagomir (miR), splice-switching oligonucleotides, aptamers, single-stranded siRNAs, microRNAs, and pre-microRNAs. The binding position and type of the drug in the second nucleic acid strand may be C, which may be substituted with a hydroxy group. 22 ~ 35 The bond between the alkyl group or its analog and the second nucleic acid strand is as described above.

[0128] As disclosed in the examples below, the compositions of the present invention can be delivered to the central nervous system (preferably the brain (brain parenchyma)) with high efficiency and effectively modify or suppress the expression of a target gene or the level of a target transcript. Therefore, a method for reducing the expression level of a target transcript in the central nervous system of a subject is provided, the method comprising administering to the subject a composition comprising the nucleic acid complex described above. The method may be a method for treating a central nervous system disease in a subject. Also provided is a drug delivery method for delivering a drug to the central nervous system of a subject, the drug delivery method comprising administering to the subject a composition comprising the nucleic acid complex described above. [Example]

[0129] The present invention will be further explained in detail by the following Reference Examples and Examples, but these do not limit the present invention and may be modified within the scope of the present invention.

[0130] In the following examples, "room temperature" generally refers to about 10°C to about 35°C. Ratios shown for mixed solvents are by volume unless otherwise specified. % means % by weight unless otherwise specified.

[0131] 1 H NMR analysis was performed using ACD / SpecManager (trade name) software, etc. Very gentle proton peaks such as those of hydroxyl groups and amino groups may not be recorded.

[0132] The meanings of the abbreviations used in the examples are shown below. M: Molar concentration N: Normality CDCl3: deuterated chloroform 1 H NMR: proton nuclear magnetic resonance DIPEA: N,N-diisopropylethylamine DMSO: dimethyl sulfoxide HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate NMP: N-methyl-2-pyrrolidone PBS: phosphate buffered saline TEAA: Triethylamine acetate THF: tetrahydrofuran ODS: Octadecyl-bonded silica gel

[0133] The structures of the oligonucleotides used in the following examples are summarized in Table 1. Among the oligonucleotides used in the examples, ASO (Malat1) and Y13-cRNA (Malat1) were synthesized by Gene Design Inc. (Osaka, Japan).

[0134] [Table 1]

[0135] The 5'-terminal structure of the oligonucleotide Y13-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0136] [ka]

[0137] The 5'-terminal structure of oligonucleotide Y1-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0138] [ka]

[0139] The 5'-terminal structure of oligonucleotide Y2-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0140] [ka]

[0141] The 5'-terminal structure of oligonucleotide Y3-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0142] [ka]

[0143] The 5'-terminal structure of the oligonucleotide Y4-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0144] [ka]

[0145] The 5'-terminal structure of the oligonucleotide Y5-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0146] [ka]

[0147] The 5'-terminal structure of the oligonucleotide Y6-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0148] [ka]

[0149] The 5'-terminal structure of the oligonucleotide Y7-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0150] [ka]

[0151] The 5'-terminal structure of the oligonucleotide Y8-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0152] [ka]

[0153] The 5'-terminal structure of the oligonucleotide Y9-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0154] [ka]

[0155] The 5'-terminal structure of the oligonucleotide Y10-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0156] [ka]

[0157] The 5'-terminal structure of the oligonucleotide Y11-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0158] [ka]

[0159] The 5'-terminal structure of the oligonucleotide Y12-cRNA (Malat1) shown in Table 1 is shown below. In the chemical formulas below, the term "oligo" refers to an oligonucleotide.

[0160] [ka]

[0161] Other compounds used in the examples are shown in the table below. Commercially available products may be used as they are, or they may be prepared by known methods or methods analogous thereto.

[0162] [Table 2]

[0163] Example 1 A) Synthesis of cRNA with Y1-attached to the 5' end (Y1-cRNA(Malat1)) A 50 mM NMP solution of IY1 (1200 μL), a 75 mM NMP solution of HATU (1200 μL), and a 150 mM NMP solution of DIPEA (1200 μL) were mixed in a microtube, stirred, and allowed to settle. The mixture was then incubated at room temperature for 15 minutes. An aqueous solution of the RNA strand (Y13-cRNA(Malat1)) shown in Table 1 (3000 nmol), NMP (3150 μL), distilled water (153 μL), and DIPEA (94 μL) were added. The mixture was stirred and allowed to settle, and then incubated at room temperature for 1 hour. The reaction mixture was purified using an ODS column (column: Purif-Pack®-EX ODS-50 size 60, Shoko Science, mobile phase: TEAA / acetonitrile) and desalted by ultrafiltration (Amicon Ultra 3 kDa, Merck Millipore, distilled water). The resulting solution was mixed with 10 volumes of 1 M meglumine acetate, stirred thoroughly, and left to stand for 5 minutes to allow for ion exchange. The mixture was then desalted by ultrafiltration (Amicon Ultra 3 kDa, Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter and lyophilized to obtain 1446 nmol of the title compound.

[0164] B) Synthesis of double-stranded nucleic acid agent Y1-HDO A 16-mer single-stranded LNA / DNA gapmer (ASO(Malat1)) targeting Malat1 non-coding RNA (NCR) contains three LNA nucleosides at the 5' end and three at the 3' end, with 10 DNA nucleosides between them. This LNA / DNA gapmer has a base sequence complementary to bases 1316-1331 of mouse Malat1 non-coding RNA (GenBank accession number NR_002847, SEQ ID NO: 3). Equimolar amounts of ASO(Malat1) (first nucleic acid strand) and Y1-cRNA(Malat1) (second nucleic acid strand) obtained in step A were mixed, and the mixture was heated at 70°C for 5 minutes. The mixture was then slowly cooled to room temperature to prepare the double-stranded nucleic acid agent, a Y1-conjugated heteroduplex oligonucleotide (Y1-HDO).

[0165] Example 2 A) Synthesis of cRNA with Y2-attached to the 5' end (Y2-cRNA(Malat1)) Using an aqueous solution (3000 nmol) of an RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and IY2, 1245 nmol of the title compound was obtained in the same manner as in Step A of Example 1.

[0166] B) Synthesis of double-stranded nucleic acid agent Y2-HDO A double-stranded nucleic acid agent, a Y2-conjugated heteroduplex oligonucleotide (Y2-HDO), was prepared in the same manner as in step B of Example 1 using ASO (Malat1) (first nucleic acid strand) and Y2-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step A.

[0167] Example 3 A) Synthesis of cRNA with Y3-attached to the 5' end (Y3-cRNA(Malat1)) Using an aqueous solution (3000 nmol) of an RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and IY3, 702 nmol of the title compound was obtained in the same manner as in Step A of Example 1.

[0168] B) Synthesis of double-stranded nucleic acid agent Y3-HDO Using ASO (Malat1) (first nucleic acid strand) and Y3-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y3-conjugated heteroduplex oligonucleotide (Y3-HDO), was prepared in the same manner as in step B of Example 1.

[0169] Example 4 A) Synthesis of cRNA with Y4-attached to the 5' end (Y4-cRNA(Malat1)) Using an aqueous solution (3000 nmol) of an RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and IY4, 1277 nmol of the title compound was obtained in the same manner as in Step A of Example 1.

[0170] B) Synthesis of double-stranded nucleic acid agent Y4-HDO A double-stranded nucleic acid agent, Y4-conjugated heteroduplex oligonucleotide (Y4-HDO), was prepared in the same manner as in step B of Example 1 using ASO (Malat1) (first nucleic acid strand) and Y4-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step A.

[0171] Example 5 A) Synthesis of cRNA with Y5-attached to the 5' end (Y5-cRNA(Malat1)) Using an aqueous solution (3000 nmol) of an RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and IY5, 1330 nmol of the title compound was obtained in the same manner as in Step A of Example 1.

[0172] B) Synthesis of double-stranded nucleic acid agent Y5-HDO A double-stranded nucleic acid agent, Y5-conjugated heteroduplex oligonucleotide (Y5-HDO), was prepared in the same manner as in step B of Example 1 using ASO (Malat1) (first nucleic acid strand) and Y5-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step A.

[0173] Example 6 A) Synthesis of cRNA with Y6-linked at the 5' end (Y6-cRNA(Malat1)) Using an aqueous solution (3,000 nmol) of the RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and IY6, 713 nmol of the title compound was obtained in the same manner as in Step A of Example 1. However, THF was used as the solvent for dissolving IY6.

[0174] B) Synthesis of double-stranded nucleic acid agent Y6-HDO Using ASO (Malat1) (first nucleic acid strand) and Y6-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y6-conjugated heteroduplex oligonucleotide (Y6-HDO), was prepared in the same manner as in step B of Example 1.

[0175] Example 7 A) Preparation of NHS-modified fatty acid IY7 A mixture of nonacosylic acid (256 mg) and thionyl chloride solution (1.49 mL) was stirred under reflux for 30 minutes. After cooling to room temperature, the solvent was removed under reduced pressure, and the residue was azeotroped twice with toluene. The resulting residue was dissolved in pyridine (2.0 mL), and 1-hydroxypyrrolidine-2,5-dione (77 mg) was added. The mixture was stirred under reflux for 30 minutes. After cooling to room temperature, 1 N hydrochloric acid, THF, and ethyl acetate were added, and the resulting solid was collected by filtration to give 232 mg of the title compound. The NMR results of the resulting compound are as follows: 1H NMR (300 MHz, CDCl3) δ 0.88 (3H, brs), 1.11-1.85 (52H, m), 2.54-2.67 (2H, m), 2.84 (4H, brs).

[0176] B) Synthesis of cRNA with Y7-linked at the 5' end (Y7-cRNA(Malat1)) An aqueous solution (672 μL, 4000 nmol) of the RNA strand (Y13-cRNA (Malat1)) shown in Table 1 was mixed in a microtube with 800 μL of 50 mM IY7 in NMP, 328 μL of distilled water, 7200 μL of NMP, 10× PBS (1000 μL), and 125 μL of DIPEA, in that order. The mixture was stirred and allowed to settle, then incubated at 70°C for 1 hour and 30 minutes. The reaction solution was purified using an ODS column (column: Purif-Pack®-EX ODS-50 size 60, Shoko Science, mobile phase: TEAA / acetonitrile) and desalted by ultrafiltration (Amicon Ultra 3 kDa, Merck Millipore, distilled water). A 10-fold volume of 1 M meglumine acetate was added to the resulting solution, stirred thoroughly, and allowed to stand for 5 minutes for ion exchange. Thereafter, desalting was carried out by ultrafiltration (Amicon Ultra 3 kDa, manufactured by Merck Millipore, distilled water), and the final product was filtered through a 0.20 μm membrane filter and lyophilized to obtain 1692 nmol of the title compound.

[0177] C) Synthesis of double-stranded nucleic acid agent Y7-HDO A double-stranded nucleic acid agent, Y7-conjugated heteroduplex oligonucleotide (Y7-HDO), was prepared in the same manner as in step B of Example 1 using ASO (Malat1) (first nucleic acid strand) and Y7-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step B.

[0178] Example 8 A) Preparation of NHS-modified fatty acid IY8 Using melissic acid (188 mg), 177 mg of the title compound was obtained in the same manner as in Step A of Example 7. The NMR results of the obtained compound are as follows. 1H NMR (300 MHz, CDCl3) δ 0.88 (3H, t, J = 6.4 Hz), 1.11-1.87 (54H, m), 2.60 (2H, t, J = 7.2 Hz), 2.84 (4H, s).

[0179] B) Synthesis of cRNA with Y8-linked at the 5' end (Y8-cRNA(Malat1)) Using an aqueous solution (4000 nmol) of an RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and IY8, 1089 nmol of the title compound was obtained in the same manner as in Step B of Example 7.

[0180] C) Synthesis of double-stranded nucleic acid agent Y8-HDO Using ASO (Malat1) (first nucleic acid strand) and Y8-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step B, a double-stranded nucleic acid agent, Y8-conjugated heteroduplex oligonucleotide (Y8-HDO), was prepared in the same manner as in step B of Example 1.

[0181] Example 9 A) Preparation of NHS-modified fatty acid IY9 Using hentriacontanoic acid (100 mg), 66 mg of the title compound was obtained in the same manner as in Step A of Example 7. The NMR results of the obtained compound are as follows. 1H NMR (300 MHz, CDCl3) δ 0.83-0.94 (3H, m), 1.20-1.80 (56H, m), 2.60 (2H, t, J = 7.5 Hz), 2.83 (4H, s).

[0182] B) Synthesis of cRNA with Y9-linked at the 5' end (Y9-cRNA(Malat1)) Using an aqueous solution (3500 nmol) of an RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and a THF solution of IY9, 955 nmol of the title compound was obtained in the same manner as in Step B of Example 7.

[0183] C) Synthesis of double-stranded nucleic acid agent Y9-HDO A double-stranded nucleic acid agent, Y9-conjugated heteroduplex oligonucleotide (Y9-HDO), was prepared in the same manner as in step B of Example 1 using ASO (Malat1) (first nucleic acid strand) and Y9-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step B.

[0184] Example 10 A) Synthesis of cRNA with Y10-attached to the 5' end (Y10-cRNA(Malat1)) Using an aqueous solution (3000 nmol) of the RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and IY10, 2159 nmol of the title compound was obtained in the same manner as in Step A of Example 1. However, a mixture of THF, DMSO, and water was used as the solvent.

[0185] B) Synthesis of double-stranded nucleic acid agent Y10-HDO Using ASO (Malat1) (first nucleic acid strand) and Y10-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y10-conjugated heteroduplex oligonucleotide (Y10-HDO), was prepared in the same manner as in step B of Example 1.

[0186] Example 11 A) Synthesis of cRNA with Y11-attached to the 5' end (Y11-cRNA(Malat1)) Using an aqueous solution (3,000 nmol) of the RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and IY11, 549 nmol of the title compound was obtained in the same manner as in Step A of Example 1. However, a mixture of THF, DMSO, and water was used as the solvent.

[0187] B) Synthesis of double-stranded nucleic acid agent Y11-HDO Using ASO (Malat1) (first nucleic acid strand) and Y11-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y11-conjugated heteroduplex oligonucleotide (Y11-HDO), was prepared in the same manner as in step B of Example 1.

[0188] Example 12 A) Synthesis of cRNA with Y12-attached to the 5' end (Y12-cRNA(Malat1)) Using an aqueous solution (3000 nmol) of an RNA strand (Y13-cRNA(Malat1)) shown in Table 1 and a THF solution of IY12, 977 nmol of the title compound was obtained in the same manner as in Step A of Example 1.

[0189] B) Synthesis of double-stranded nucleic acid agent Y12-HDO Using ASO (Malat1) (first nucleic acid strand) and Y12-cRNA (Malat1) (second nucleic acid strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y12-conjugated heteroduplex oligonucleotide (Y12-HDO), was prepared in the same manner as in step B of Example 1.

[0190] Example 13 (A) In vivo experiment Seven-week-old male C57BL / 6J mice (Charles River Japan) were used as experimental animals, with 2-4 mice per group. In the experimental group, a solution containing nucleic acids was administered intravenously via the tail vein of the mice at a dose of 5 mL / kg. In the control group, the solvent used to prepare the nucleic acid solution (5% glucose solution) was administered intravenously to mice using the same procedure as in the experimental group.

[0191] (B) Expression analysis Seventy-two hours after administration of the nucleic acid solution, the mice were anesthetized by intraperitoneal administration of 50 mg / kg pentobarbital, bled, sacrificed, and their brains (cerebral cortex) were removed. Total RNA was extracted from the brain tissue using the RNA extraction reagent ISOGEN (Nippon Gene Co., Ltd.). The extracted brain tissue was homogenized in ISOGEN solution and then isolated as an RNA fraction using chloroform. This was then performed using the QuickGene RNA tissue kit SII (Kurashiki Boseki Co., Ltd.) nucleic acid isolation system. cDNA was synthesized from the total RNA using the ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd.), and quantitative PCR was performed using THUNDERBIRD qPCR Mix (Toyobo Co., Ltd.). The fluorescent probe method was used for quantitative PCR, and the fluorescent probes used were mouse Malat1 (Integrated DNA Technologies) and mouse Gapdh (Thermo Fisher Scientific). The quantitative PCR gene fragment amplification reaction conditions followed the protocol for the THUNDERBIRD qPCR Mix (Toyobo Co., Ltd.) described above. The expression levels of mouse Malat1 and Gapdh (internal control genes) were calculated using a relative calibration curve, and the relative expression level was calculated as Malat1 / Gapdh. The average relative expression level was calculated from the results of two mice per group. The relative Malat1 ncRNA expression level was calculated as the ratio of the average relative expression level of the experimental group to the average relative expression level of the control group, which was set at 100%.

[0192] (C) Result The results of Example 13 are shown in Table 3. In the table, the dose indicates the amount of ASO (Malat1). All of duplexes Nos. 1 to 12 suppressed the expression of Malat1 non-coding RNA in the cerebral cortex. This result demonstrated that the nucleic acid complex of the present invention can be delivered to the brain and exert an antisense effect there.

[0193] [Table 3] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A composition for regulating expression or editing of a target transcript in the nervous system, comprising a nucleic acid complex or a salt thereof, the nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand comprises a base sequence capable of hybridizing to at least a part of a target transcript and has an antisense effect on the target transcript; the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and is linked to an unsubstituted linear C 22-24 or unsubstituted linear C 26 alkyl group; The composition wherein the first nucleic acid strand is annealed to the second nucleic acid strand.

2. The composition of claim 1, wherein the second nucleic acid strand is linked to an unsubstituted linear C 22-24 or linear C 26 alkyl group via a linker represented by general formula I: 【Chemical 1】

3. 3. The composition of claim 1 or 2, wherein the first nucleic acid strand comprises at least four consecutive deoxyribonucleosides.

4. The composition of any one of claims 1 to 3, wherein the first nucleic acid strand is a gapmer.

5. 5. The composition of claim 1, wherein the second nucleic acid strand comprises at least four consecutive ribonucleosides complementary to at least four consecutive deoxyribonucleosides in the first nucleic acid strand.

6. The composition of any one of claims 1 to 3, wherein the first nucleic acid strand is a mixmer.

7. The composition according to any one of claims 1 to 6, wherein the first nucleic acid strand is 13 to 20 bases in length.

8. The composition of any one of claims 1 to 7, wherein the second nucleic acid strand does not contain natural ribonucleosides.

9. The composition of any one of claims 1 to 8, wherein the nucleic acid portion of the second nucleic acid strand consists of deoxyribonucleosides and / or sugar-modified nucleosides linked by modified or unmodified internucleoside linkages.

10. The composition according to any one of claims 1 to 9, wherein the first nucleic acid strand contains no or less than half of natural ribonucleosides across its entire length.

11. A composition described in any one of claims 1 to 10, wherein the nervous system is the central nervous system.

12. The composition according to claim 11, which is for treating a central nervous system disease.

13. The composition of claim 11 for delivering a drug to the central nervous system.

14. 14. The composition of any one of claims 11 to 13, wherein the central nervous system is selected from the group consisting of the cerebral cortex, basal ganglia, cerebral white matter, diencephalon, brainstem, cerebellum, and spinal cord.

15. 14. The composition of any one of claims 11 to 13, wherein the central nervous system is selected from the group consisting of the frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, parietal lobe, occipital lobe, striatum, globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigra, pons, medulla oblongata, cerebellar cortex, cerebellar nuclei, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.

16. The composition according to any one of claims 1 to 15, which is for intravenous or subcutaneous administration.

17. The composition according to any one of claims 1 to 16, comprising 5 mg / kg or more of the nucleic acid complex or a salt thereof in a single dose.

18. The composition according to any one of claims 11 to 17, wherein the nucleic acid complex or a salt thereof is transferred from blood to the brain.

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