Heteronucleotides containing morpholino nucleic acids

Abstract

In one embodiment, the present invention addresses the problem of providing a double-stranded nucleic acid complex which can have excellent activity.　In one embodiment, the present invention pertains to a double-stranded nucleic acid complex comprising a first nucleic acid chain and a second nucleic acid chain, wherein: the first nucleic acid chain can hybridize to at least a part of a target gene or a transcript thereof, has an antisense effect on the target gene or the transcript thereof, and contains at least two morpholino nucleic acids; the second nucleic acid chain contains a base sequence complementary to that of the first nucleic acid chain; and the first nucleic acid chain is annealed to the second nucleic acid chain.

Description

[Technical Field]

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

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

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

[0004] The mechanism of action of the above double-stranded nucleic acid complex is not limited, but is thought to be partially as follows: When introduced into cells, a portion of the RNA region complementary to the antisense oligonucleotide in the complementary strand is cleaved by RNase H, releasing the antisense oligonucleotide. Subsequently, this antisense oligonucleotide can act, for example, to modify the activity or function of a transcript (see, for example, Patent Document 2). This is called the "RNase H-dependent pathway," and it is desirable that the nucleic acid portion is unmodified for cleavage by RNase H to occur. On the other hand, if the nucleic acid portion is unmodified, it may be degraded by nucleases in vivo and may not be able to exert sufficient activity. [Prior art documents] [Patent Documents]

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

[0006] [Non-Patent Document 1] Nishina K, et. al., "DNA / RNA heteroduplex oligonucleotide for highly efficient gene silencing", Nature Communication, 2015, 6:7969. [Non-Patent Document 2] Asami Y, et al., "Drug delivery system of therapeutic oligonucleotides", Drug Discoveries & Therapeutics. 2016; 10(5):256-262. [Overview of the project] [Problems that the invention aims to solve]

[0007] There is insufficient knowledge regarding which nucleic acids should be used to maintain the activity of double-stranded nucleic acid complexes while suppressing degradation by nucleolytic enzymes in vivo. In one embodiment, the present invention aims to provide a novel double-stranded nucleic acid complex that may have excellent activity. [Means for solving the problem]

[0008] The inventors have discovered that double-stranded nucleic acid complexes containing morpholino nucleic acids can exhibit excellent antisense effects. Furthermore, the inventors have unexpectedly discovered that double-stranded nucleic acid complexes containing antisense oligonucleotides composed solely of morpholino nucleic acids, which do not exhibit activity due to the RNase H-dependent pathway, also exhibit antisense effects.

[0009] The present invention is at least partially based on the above findings and provides the following embodiments. [1] A double-stranded nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid strand can hybridize to at least a portion of the target gene or its transcript, has an antisense effect on the target gene or its transcript, and contains at least two morpholino nucleic acids. The second nucleic acid chain contains a base sequence complementary to the first nucleic acid chain, and The double-stranded nucleic acid complex, wherein the first nucleic acid strand is annealed to the second nucleic acid strand. [2] A double-stranded nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, The first nucleic acid chain can specifically bind to a particular target molecule, has at least one aptamer, decoy, and bait effect on the target molecule, and comprises at least two morpholino nucleic acids. The second nucleic acid chain contains a base sequence complementary to the first nucleic acid chain, and The double-stranded nucleic acid complex, wherein the first nucleic acid strand is annealed to the second nucleic acid strand. [3] The double-stranded nucleic acid complex according to [1] or [2], which does not contain four consecutive natural ribonucleosides. [4] The double-stranded nucleic acid complex according to any one of [1] to [3], wherein 33% or more of the nucleic acids in the first nucleic acid strand are morpholino nucleic acids. [5] The double-stranded nucleic acid complex according to [4], wherein 100% of the nucleic acids in the first nucleic acid strand are morpholino nucleic acids. [6] The double-stranded nucleic acid complex according to any one of [1] to [5], wherein the second nucleic acid strand is bound to a functional moiety having a function selected from a labeling function, a purification function, and a delivery function to a target. [7] The double-stranded nucleic acid complex according to [6], wherein the functional moiety is a lipid. [8] The double-stranded nucleic acid complex according to [7], wherein the lipid is cholesterol or an analog thereof, or tocopherol or an analog thereof. [9] The double-stranded nucleic acid complex according to any one of [6] to [8], wherein the functional moiety is bound to the 5'-end and / or 3'-end of the second nucleic acid strand.

[10] The double-stranded nucleic acid complex according to any one of [1] to [9], wherein the first nucleic acid strand and the second nucleic acid strand are bound via a cleavable or uncleavable linker.

[11] In a subject, for performing at least one of the following functions: [[ID=. / 17]] Suppressing or enhancing the expression level of a transcription product or a translation product of a target gene; Inhibiting the function of a transcription product or a translation product of a target gene; Controlling RNA splicing; and Inhibiting the binding of a target gene to a protein. The double-stranded nucleic acid complex according to any one of [1] to

[10] .

[12] The double-stranded nucleic acid complex according to

[11] for exon skipping, exon inclusion, steric blocking, and enhancing RNA expression.

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

[12] for intrathecal administration or intracerebroventricular administration. A pharmaceutical composition containing a double-stranded nucleic acid complex as described in any of

[14] [1] to

[13] as an active ingredient. This specification includes the disclosures of Japanese Patent Application No. 2020-045137, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0010] The present invention provides a double-stranded nucleic acid complex having a novel structure. [Brief explanation of the drawing]

[0011] [Figure 1] Figures 1A and 1B are schematic diagrams illustrating specific embodiments of the nucleic acid complex according to the present invention, in which the second nucleic acid chain contains lipids. [Figure 2] Figures 2A to 2C are schematic diagrams illustrating specific embodiments of the nucleic acid complex according to the present invention, in which the second nucleic acid chain contains lipids and includes complementary and overhang regions. [Figure 3] Figure 3 shows an example of a general mechanism of the antisense method. [Figure 4] Figure 4 shows the structures of various natural and unnatural nucleotides. [Figure 5] Figure 5 shows the structures of various cross-linked nucleic acids. [Figure 6] Figure 6 shows the chemical modifications and structures of the oligonucleotides used in Examples 1 and 2. [Figure 7] Figure 7 shows the exon skipping effect in the cerebrum following systemic administration of heteronucleotide-type PMOs. PBS refers to the PBS-administered group, PMO refers to the single-stranded antisense oligonucleotide (ASO)-administered group, Toc HDO refers to the tocopherol-bound heterodouble-stranded oligonucleotide-administered group, and Chol HDO refers to the cholesterol-bound heterodouble-stranded oligonucleotide-administered group. [Figure 8]Figure 8 shows the exon skipping effect in the cerebral cortex (A), hippocampus (B), cerebellum (C), brainstem (D), and striatum (E) following intracerebroventricular administration of heteronucleotide-type PMOs. HDO refers to the group administered ligand-free heterodouble-stranded oligonucleotides. The meanings of PBS, PMO, Toc HDO, and Chol HDO are the same as in Figure 7. [Figure 9] Figure 9 shows the antisense effects in the frontal cortex, occipital cortex, striatum, hippocampus, brainstem, and cerebellum following intracerebroventricular administration of heteronucleotides that do not contain morpholino nucleic acids. ASO(mMalat1) refers to the group administered single-stranded oligonucleotides without ligands and without morpholino nucleic acids. PBS refers to the group administered PBS, and Chol HDO(control) refers to the group administered cholesterol-binding hetero-double-stranded oligonucleotides that do not contain morpholino nucleic acids. [Figure 10] Figure 10 shows the exon skipping effect in the liver (A) and kidney (B) following systemic administration of heteronucleotide-type PMOs. The meanings of PBS, PMO, Toc HDO, and Chol HDO are the same as in Figure 7. [Figure 11] Figure 11 shows the chemical modification and structure of the oligonucleotide used in Example 4. [Figure 12] Figure 12 shows the exon skipping effect in the cerebellum (A), brainstem (B), and striatum (C) upon intracerebroventricular administration of heteronucleic acid-type PMOs. CholHDO (DNA gap), CholHDO (full OMe), 3'Chol (default), and C3 (default) represent the administration groups of heteroduplex oligonucleotides containing ASO PMO (mDystrophin) as the first nucleic acid strand and Chol-cRNA (DNA gap), Chol-cRNA (full OMe), 3'Chol-cRNA (default), and C3-cRNA (default), respectively, as the second nucleic acid strands. [Figure 13]Figure 13 shows the exon skipping effect in the hippocampus (A), posterior cortex (B), and cervical spine (C) upon intracerebroventricular administration of heteronucleic acid-type PMOs. The meanings of PBS and PMO are the same as in Figure 7. HDO (default), CholHDO (default), CholHDO (DNA gap), CholHDO (full OMe), 3'Chol (default), and C3 (default) represent the administration groups of heteroduplex oligonucleotides containing ASO PMO (mDystrophin) as the first nucleic acid strand and cRNA (default), Chol-cRNA (default), Chol-cRNA (DNA gap), Chol-cRNA (full OMe), 3'Chol-cRNA (default), and C3-cRNA (default), respectively, as the second nucleic acid strands. [Modes for carrying out the invention]

[0012] 1. Double-stranded nucleic acid complex 1-1. Overview In one embodiment, the present invention relates to a double-stranded nucleic acid complex comprising a first nucleic acid chain and a second nucleic acid chain, wherein the first nucleic acid chain can hybridize to at least a portion of a target gene or its transcript, has an antisense effect on the target gene or its transcript, and contains at least two morpholino nucleic acids, the second nucleic acid chain contains a base sequence complementary to the first nucleic acid chain, and the first nucleic acid chain is annealed to the second nucleic acid chain.

[0013] In one embodiment, the present invention relates to a double-stranded nucleic acid complex comprising a first nucleic acid chain and a second nucleic acid chain, wherein the first nucleic acid chain is capable of specifically binding to a particular target molecule, has at least one effect of aptamer, decoy, and bait on the target molecule, and comprises at least two morpholino nucleic acids, the second nucleic acid chain comprises a base sequence complementary to the first nucleic acid chain, and the first nucleic acid chain is annealed to the second nucleic acid chain.

[0014] 1-2. Definitions of Terms In this specification, "target gene" refers to a gene to which the first nucleic acid strand of the double-stranded nucleic acid complex of the present invention can bind. "Target molecule" refers to a molecule (e.g., peptide, protein, nucleic acid, etc.) that can be targeted by the double-stranded nucleic acid complex of the present invention. "Target gene" or "target molecule" is, for example, a gene or molecule that can be targeted by the antisense effect, aptamer, decoy, or bait of the double-stranded nucleic acid complex of the present invention.

[0015] The type of target gene is not particularly limited as long as it is expressed in vivo, but examples include genes of organisms into which the double-stranded nucleic acid complex according to the present invention is introduced, such as genes whose expression is increased in various diseases. Examples include the dystrophin gene, the scavenger receptor B1 (often referred to as "SR-B1" herein) gene, the DMPK (dystrophia myotonica-protein kinase) gene, and the metastasis-associated lung adenocarcinoma transcript 1 (often referred to as "Malat1" herein) gene.

[0016] The dystrophin gene codes for the dystrophin protein, and in Duchenne muscular dystrophy, exon skipping, which involves skipping the abnormal exons, is known to be an effective treatment.

[0017] The DMPK gene encodes myotonin protein kinase and is known as the causative gene for myotonic dystrophy, the most common type of muscular dystrophy in adults. Abnormal elongation of the CTG repeat sequence in the 3' untranslated region of the DMPK gene is considered to be the cause of this disease.

[0018] Scavenger receptors are all receptor membrane proteins for denatured lipoproteins and are known to be involved in cholesterol and lipoprotein metabolism. SR-B1 is a two-pass transmembrane protein belonging to the evolutionarily conserved CD36 family, and has a long extracellular domain and two short intracellular domains, one containing an amino terminus and the other a carboxyl terminus.

[0019] Malat1 is a long non-coding RNA (lncRNA) that is highly expressed in malignant tumors, including lung cancer, and is known to reside in the nucleus of muscle cells.

[0020] In this specification, "target transcript" refers to any RNA that is a direct target of the nucleic acid complex of the present invention and is synthesized by RNA polymerase. Generally, this refers to the "transcript of a target gene." Specifically, it may include mRNA transcribed from a target gene (including mature mRNA, mRNA precursor, unmodified mRNA, etc.), non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA. Examples of target gene transcripts include pre-mRNA, which is the transcript of the dystrophin gene; DMPK mRNA, which is the transcript of the DMPK gene; SR-B1 mRNA, which is the transcript of the SR-B1 gene; and Malat1 non-coding RNA, which is the transcript of the Malat1 gene.

[0021] Specific examples of target transcripts include the exon 23 / intron 23 boundary region of Dystrophin pre-mRNA (GenBank accession number: NC_000086.7), for example, positions 83803482 to 83803566, and for example, positions 83803512 to 83803536. Other specific examples of target transcripts include the mouse SR-B1 mRNA sequence in SEQ ID NO: 5 and the human SR-B1 mRNA sequence in SEQ ID NO: 6. Furthermore, the mouse malat1 non-coding RNA sequence in SEQ ID NO: 7 and the human Malat1 non-coding RNA sequence in SEQ ID NO: 8. Additionally, the mouse DMPK mRNA sequence in SEQ ID NO: 9 and the human DMPK mRNA sequence in SEQ ID NO: 10. Note that in SEQ ID NOs: 5-10, the mRNA sequences have been replaced with DNA sequences. Sequence information for these genes and transcripts can be obtained from publicly known databases, such as the NCBI (National Center for Biotechnology Information) database.

[0022] Furthermore, the nucleotide sequences of known antisense drugs can also be used. For example, the nucleotide sequence shown in Sequence ID No. 11, which constitutes ISIS 598769 (IONIS), a drug used to treat myotonic dystrophy and targeting the DMPK gene, the causative gene for myotonic dystrophy; the nucleotide sequence shown in Sequence ID No. 12, which constitutes Eteplirsen (Sarepta; Exondys 51), a drug known to treat Dusenne muscular dystrophy and induce pre-mRNA exon skipping of the dystrophin gene; the nucleotide sequence shown in Sequence ID No. 13, which constitutes Golodirsen (Sarepta); the nucleotide sequence shown in Sequence ID No. 14, which constitutes NS-065 / NCNP-01 (Nippon Shinyaku); and the nucleotide sequence shown in Sequence ID No. 15, which constitutes Casimersen (Sarepta) may be used.

[0023] In this specification, “antisense oligonucleotide (ASO)” refers to a single-stranded oligonucleotide that contains a complementary nucleotide sequence that can hybridize to all or part of a target transcript, for example, to any target region, and which can control the expression of the target gene transcript or the level of the target transcript by an antisense effect. In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand functions as an ASO, and its target region may include a 3'UTR, 5'UTR, exon, intron, coding region, translation start region, translation termination region, or any other nucleic acid region. The target region of the target transcript can be at least 8 nucleotides long, for example, 8-40 nucleotides, 10-35 nucleotides, 12-25 nucleotides, 13-20 nucleotides, 14-19 nucleotides, or 15-18 nucleotides.

[0024] "Antisense effect" refers to the effect of an ASO (Antisense Organisation) hybridizing to a target transcript (e.g., an RNA sense strand) to regulate the expression or editing of that target transcript. "Regulating the expression or editing of a target transcript" includes suppression or reduction, enhancement, inhibition of translation, inhibition of the function of the translation product, regulation of RNA splicing (e.g., splicing switch, exon inclusion, exon skipping, etc.), degradation of the transcript, or inhibition of the binding of the target gene to a protein (see Figure 3). For example, in post-transcriptional inhibition of a target gene, when an RNA oligonucleotide is introduced into a cell as an ASO, the ASO forms a partial double helix by annealing with mRNA, the transcript of the target gene. This partial double helix acts as a cover to prevent ribosome translation, thereby inhibiting the expression of the target protein encoded by the target gene at the translational level (steric blocking, indicated by the dashed line outside the X in Figure 3). On the other hand, when an oligonucleotide containing DNA is introduced into cells as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase H, resulting in the degradation of the target gene's mRNA and the inhibition of the expression of the protein encoded by the target gene at the expression level (within the dashed line in Figure 3). Furthermore, the antisense effect can also be induced by targeting introns in the mRNA precursor. Moreover, the antisense effect can also be induced by targeting miRNA. In this case, inhibition of the miRNA's function can increase the expression of the gene whose expression is normally regulated by that miRNA. In one embodiment, the regulation of the target transcript's expression may be achieved by reducing the amount of the target transcript.

[0025] In this specification, "translation product of a target gene" means any polypeptide or protein that is a direct target of the nucleic acid complex of the present invention and is synthesized by the translation of the target transcript or the transcript of the target gene. Examples of translation products of target genes include DMPK protein, which is the translation product of the DMPK gene; SR-B1 protein, which is the translation product of the SR-B1 gene; and Malat1 protein, which is the translation product of the Malat1 gene.

[0026] In this specification, "aptamer" means a nucleic acid molecule that specifically binds to a particular target molecule inside, on, or outside the cell, for example, on or outside the cell membrane. Aptamers can be prepared by methods known in the art, such as in vitro selection using the SELEX (systematic evolution of ligands by exponential enrichment) method.

[0027] In this specification, "decoy" refers to a nucleic acid having the same or a similar sequence as the binding site of a transcription factor (e.g., NF-κB), and by introducing these into cells as a "decoy," the action of the transcription factor is suppressed (transcription is suppressed in the case of a transcription activator, and transcription is promoted in the case of a transcription repressor). Decoy nucleic acids can be easily designed based on information about the binding sequence of the target transcription factor.

[0028] In this specification, "bait" refers to a nucleic acid molecule that specifically binds to a particular target molecule within a cell and modifies the function of the target molecule. The target that interacts with the bait is also called a "prey."

[0029] As used herein, the terms "nucleic acid" or "nucleic acid molecule" refer to a nucleoside or nucleotide if it is a monomer, an oligonucleotide if it is an oligomer, and a polynucleotide if it is a polymer.

[0030] A "nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is not limited to but is usually composed of pentofuranosyl sugars, with specific examples including ribose and deoxyribose. The base portion (nucleic acid base) of a nucleoside is usually a heterocyclic base. While not limited to these, examples include adenine, cytosine, guanine, thymine, or uracil, or other modified nucleic acid bases (modified bases).

[0031] A "nucleotide" is a molecule in which a phosphate group is covalently bonded to the sugar portion of the nucleoside. In the case of nucleotides containing pentofuranosyl sugars, the phosphate group is usually linked to the hydroxyl group at the 2', 3', or 5' position of the sugar.

[0032] An "oligonucleotide" is a linear oligomer formed by the covalent linkage of several to tens of hydroxyl groups and phosphate groups of the sugar portion between adjacent nucleotides. A "polynucleotide" is a linear polymer formed by the covalent linkage of tens or more, preferably hundreds or more, nucleotides, which is more numerous than that of an oligonucleotide. Within the oligonucleotide or polynucleotide structure, the phosphate groups are generally considered to form internucleoside bonds.

[0033] In this specification, “nucleic acid chain” or simply “chain” means oligonucleotide or polynucleotide. Nucleic acid chains can be prepared as full-length or partial chains by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerase, ligase, or restriction reactions. Nucleic acid chains may contain native and / or non-native nucleotides.

[0034] In this specification, "natural nucleoside" refers to a nucleoside that exists in nature. Examples include ribonucleosides, which consist of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides, which consist of deoxyribose and a base such as adenine, cytosine, guanine, or thymine. In this specification, ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to as "DNA nucleosides" and "RNA nucleosides," respectively.

[0035] In this specification, "natural nucleotide" refers to a nucleotide that exists in nature, in which a phosphate group is covalently bonded to the sugar portion of the natural nucleoside. Examples include ribonucleotides, known as the building blocks of RNA, in which a phosphate group is bonded to a ribonucleoside, and deoxyribonucleotides, known as the building blocks of DNA, in which a phosphate group is bonded to a deoxyribonucleoside.

[0036] In this specification, "non-natural nucleoside" means any nucleoside other than a natural nucleoside. This includes, for example, modified nucleosides and nucleoside mimetic compounds. In this specification, "modified nucleoside" means a nucleoside having a modified sugar moiety and / or modified nucleic acid base. Nucleic acid chains containing non-natural oligonucleotides are often preferred over the natural type due to desirable properties such as enhanced cellular uptake, enhanced affinity to nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0037] In this specification, “mimetic” refers to a functional group that substitutes sugars, nucleic acid bases, and / or nucleoside bonds. Generally, mimetics are used in place of sugars or sugar-nucleoside bond combinations, and nucleic acid bases are retained for hybridization to a selected target. “Nucreoside mimetic” as used herein includes structures used to substitute sugars at one or more positions in an oligomeric compound, or to substitute sugars and bases, or to substitute bonds between monomer subunits constituting an oligomeric compound. “Oligomer compound” means a polymer of linked monomer subunits that can hybridize to at least one region of a nucleic acid molecule. Examples of nucleoside mimetics include morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar mimetics, such as nucleoside mimetics having non-furanose sugar units. Figure 4 shows the structures of various native and non-native nucleotides.

[0038] In this specification, "bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are generally called bridged nucleic acids (BNAs). In this specification, nucleosides containing a bicyclic sugar moiety may also be referred to as "bridged nucleosides." Figure 5 shows some examples of bridged nucleic acids.

[0039] A bicyclic sugar may be a sugar in which the carbon atoms at the 2' and 4' positions 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) that include bicyclic sugars is 4'-(CH2) p -O-2', 4'-(CH2) p -CH2-2', 4'-(CH2) p -S-2', 4'-(CH2) p -OCO-2', 4'-(CH2) n -N(R3)-O-(CH2) mIt can be described as having a carbon atom at the 2' position and a carbon atom at the 4' position that are cross-linked by -2'[wherein p, m, and n represent integers from 1 to 4, 0 to 2, and 1 to 3, respectively; and R3 represents a hydrogen atom, alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, sulfonyl group, and unit substituents (fluorescent or chemiluminescent labeled molecules, functional groups with nucleic acid cleavage activity, intracellular or nuclear localized signal peptides, etc.)]. Furthermore, with respect to BNA according to a particular embodiment, in the OR2 substituent on the 3' carbon atom and the OR1 substituent on the 5' carbon atom, R1 and R2 are typically hydrogen atoms, but may be the same or different from each other, and may also be a hydroxyl group protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R4)R5 [wherein R4 and R5 may be the same or different from each other, and each represents a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms]. Non-limiting examples of such BNA include methyleneoxy(4'-CH2-O-2')BNA (LNA (Locked Nucleic Acid®, also known as 2',4'-BNA)), for example, α-L-methyleneoxy(4'-CH2-O-2')BNA or β-D-methyleneoxy(4'-CH2-O-2')BNA, ethyleneoxy(4'-(CH2)2-O-2')BNA (also known as ENA), β-D-thio(4'-CH2-S-2')BNA, aminooxy(4'-CH2-ON(R3)-2')BNA, oxyamino(4'-CH2-N(R3)-O-2')BNA (2',4'-BNA) NC Also known as; R=H is 2',4'-BNA NC[N-H], R = Me is 2',4'-BNA NC [N-Me]), 2',4'-BNA coc , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH3)-O-2')BNA (also known as cEt BNA), (4'-CH(CH2OCH3)-O-2')BNA (also known as cMOE BNA), amide BNA (4'-C(O)-N(R)-2')BNA (R = H, Me) (also known as AmNA; R = H is AmNA[N-H], R = Me is AmNA[N-Me])), guanidine BNA (also known as GuNA (e.g., R = H in Figure 5 is GuNA[N-H], R = Me is GuNA[N-Me])), amine BNA (also known as 2'-Amino-LNA), 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (also known as scpBNA), and other BNAs known to those skilled in the art. Bicyclic nucleosides having a methyleneoxy (4'-CH2-O-2') bridge may also be referred to as LNA nucleosides.

[0040] In this specification, "non-natural nucleotide" refers to any nucleotide other than natural nucleotides, and includes modified nucleotides and nucleotide mimetic. In this specification, "modified nucleotide" means a nucleotide having one or more of the following: a modified sugar moiety, a modified nucleoside bond, and a modified nucleic acid base. In this specification, "nucleotide mimetic" refers to a structure used to substitute nucleosides and bonds at one or more positions in an oligomeric compound. Examples of nucleotide mimetics include peptide nucleic acids or morpholino nucleic acids (morpholino linked by -N(H)-C(=O)-O- or other non-phosphodiester bonds). Peptide nucleic acid (PNA) is a nucleotide mimetic having a main chain in which N-(2-aminoethyl)glycine is linked by an amide bond instead of sugar. In this specification, nucleic acid chains containing non-natural oligonucleotides often have desirable properties such as enhanced cellular uptake, enhanced affinity to nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Therefore, they are preferred over natural nucleotides.

[0041] In this specification, "modified nucleoside bond" refers to a nucleoside bond that has been substituted or modified from a naturally occurring nucleoside bond (i.e., a phosphodiester bond). Modified nucleoside bonds include phosphorus-containing nucleoside bonds containing a phosphorus atom and non-phosphorus-containing nucleoside bonds that do not contain a phosphorus atom. Typical phosphorus-containing nucleoside bonds include, but are not limited to, phosphodiester bonds, phosphorothioate bonds, phosphorodithioate bonds, phosphotriester bonds, alkylphosphonate bonds, alkylthiophosphonate bonds, boranophosphate bonds, and phosphorodiamidates. A phosphorothioate bond is a nucleoside bond in which the non-bridged oxygen atom of a phosphodiester bond is substituted with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known. It is preferable that the modified nucleoside bond has higher nuclease resistance than naturally occurring nucleoside bonds.

[0042] If the nucleoside bond has a chiral center, the nucleoside bond may be chiral-controlled. "Chiral-controlled" means that it exists as a single diastereomer with respect to the chiral center, for example, the chiral-bound phosphorus. A chiral-controlled nucleoside bond may be completely chiral pure, or it may have a high chiral purity, e.g., 90%de, 95%de, 98%de, 99%de, 99.5%de, 99.8%de, 99.9%de, or higher. In this specification, "chiral purity" refers to the proportion of one diastereomer in a mixture of diastereomers, expressed as diastereomer excess (%de), and defined as (diastereomer of interest - other diastereomers) / (total diastereomers) × 100 (%).

[0043] For example, the nucleoside bond may be a phosphorothioate bond that is chiralized to an Rp configuration or an Sp configuration. Methods for preparing chiralally controlled nucleoside bonds are known; for example, phosphorothioate bonds chiralized to Rp or Sp configurations are described in Naoki Iwamoto et al., Angew. Chem. Int. Ed. Engl. 2009, 48(3), 496-9, Natsuhisa Oka et al., J. Am. Chem. Soc. 2003, 125, 8307-8317, Natsuhisa Oka et al., J. Am. Chem. Soc. 2008, 130, 16031-16037, Yohei Nukaga et al., J. Org. Chem. 2016, 81, 2753-2762, Yohei Nukaga et al., J. Org. Chem. 2012, 77. It can be synthesized according to the methods described in 7913-7922. Chiral-controlled phosphorothioate bonds in Rp or Sp configurations are also known and are known to exert effects such as those described 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. For example, in one embodiment, a chiral-controlled phosphorothioate bond in the Sp configuration is more stable than one in the Rp configuration, and / or a chiral-controlled ASO in the Sp configuration promotes target RNA cleavage by RNase H1, resulting in a more sustained response in vivo.

[0044] In this specification, “modified nucleic acid base” or “modified base” means any nucleic acid base other than adenine, cytosine, guanine, thymine, or uracil. Examples of modified nucleic acid bases 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 nucleic acid base is 5-methylcytosine.

[0045] "Unmodified nucleic acid bases" or "unmodified bases" are synonymous with natural nucleic acid bases and refer to the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0046] In this specification, “modified sugar” means a sugar that has been substituted and / or altered from the natural sugar moiety (i.e., the sugar moiety found in DNA(2'-H) or RNA(2'-OH)). In this specification, a nucleic acid chain may optionally contain one or more modified nucleosides, including modified sugars. Sugar-modified nucleosides can confer to the nucleic acid chain enhanced nuclease stability, increased binding affinity, or any other beneficial biological properties. The nucleosides 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 bicyclic nucleic acids (bridged nucleic acids, BNAs) by crosslinking of non-geminal ring atoms, and the S, N(R), or C(R1)(R2)(R, where R1 and R2 are independently H, C1-C) of the ribosyl ring oxygen atom. 12Substitutions include alkyl groups (representing protecting groups) and combinations thereof. Examples of nucleosides having a modified sugar moiety as used herein include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F (2'-fluoro group), 2'-OCH3 (2'-OMe group or 2'-O-methyl group), and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position also include allyl, amino, azide, thio, -O-allyl, and -O-C1-C 10 The following can be selected: alkyl, -OCF3, -O(CH2)2SCH3, -O(CH2)2-ON(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn can independently be H or substituted or unsubstituted C1-C 10 It is alkyl. In this specification, "2'-modified sugar" means a furanosyl sugar modified at the 2' position.

[0047] Generally, modifications can be carried out in such a way that nucleotides within the same chain can undergo different modifications independently. Furthermore, to provide resistance to enzymatic cleavage, the same nucleotide may have modified nucleoside bonds (e.g., phosphorothioate bonds) and modified sugars (e.g., 2'-O-methyl-modified sugars or bicyclic sugars). The same nucleotide may also have modified nucleic acid bases (e.g., 5-methylcytosine) and modified sugars (e.g., 2'-O-methyl-modified sugars or bicyclic sugars).

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

[0049] As used herein, the term "complementary" means a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) via hydrogen bonding. In the present invention, the first nucleic acid chain does not necessarily have to be completely complementary to all or part of the target transcript (e.g., the transcript of the target gene), but is acceptable if its base sequence has at least 70%, preferably at least 80%, and more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. Similarly, the complementary region in the second nucleic acid chain does not necessarily have to be completely complementary to all or part of the first nucleic acid chain, but is acceptable if its base sequence has at least 70%, preferably at least 80%, and more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity.

[0050] In this specification, "tocopherol" is a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) having a cyclic structure called chroman. Tocorol has strong antioxidant properties, and therefore, in the body, it functions as an antioxidant to eliminate free radicals generated by metabolism and protect cells from damage.

[0051] Tocopherol is known to exist in several different forms, consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, based on the position of the methyl group bonded to chroman. In this specification, tocopherol may refer to any of these forms. Furthermore, analogs of tocopherol include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. Preferably, the tocopherol is α-tocopherol.

[0052] In this specification, "cholesterol" refers to a type of sterol, also known as a steroid alcohol, which is particularly abundant in animals. Cholesterol plays an important role in metabolic processes within the body and, in animal cells, is a major component of the cell membrane system along with phospholipids. Cholesterol analogs refer to, but are not limited to, various cholesterol metabolites and analogs, which are alcohols with a sterol skeleton, and include cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0053] In this specification, "analog" refers to a compound having the same or similar basic skeleton and similar structure and properties. Analogs include, for example, biosynthetic intermediates, metabolites, and substituted compounds. Whether a compound is an analog of another compound can be determined by common technical knowledge of the art.

[0054] In this specification, "subject" refers to the object to which the double-stranded nucleic acid complex or pharmaceutical composition of the present invention is applied. The subject includes individuals, organs, tissues, and cells. When the subject is an individual, it may include any animal, including humans. Examples of non-human animals include various livestock, poultry, pets, and laboratory animals. The subject may also be an individual requiring a reduction in the expression level of a target transcript or control of splicing (e.g., exon skipping).

[0055] 1-3. Structure The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand. The specific composition of each nucleic acid strand is shown below.

[0056] In one embodiment, the first nucleic acid chain is a single-stranded oligonucleotide chain comprising a base sequence capable of hybridizing to all or part of a target gene or its transcript, thereby exerting an antisense effect on the target gene or its transcript. In another embodiment, the first nucleic acid chain is a single-stranded oligonucleotide chain capable of specifically binding to a particular target molecule and having at least one effect of aptamer, decoy, and bait on the target molecule.

[0057] The second nucleic acid chain is a single-stranded oligonucleotide chain containing a base sequence complementary to the first nucleic acid chain. In the double-stranded nucleic acid complex, the second nucleic acid chain is annealed to the first nucleic acid chain via hydrogen bonds of complementary base pairs. An example of this embodiment is the heteroduplex oligonucleotide (HDO) disclosed in International Publication No. 2013 / 089283, Nishina K, et. al., Nature Communication, 2015, 6:7969, and Asami Y, et al., Drug Discoveries & Therapeutics. 2016; 10(5):256-262 (Figures 1A and B).

[0058] In further embodiments, the second nucleic acid chain may further include at least one overhang region located on either or both the 5' and 3' ends of the complementary region. An example of this embodiment is described in International Publication No. 2018 / 062510. An “overhang region” refers to a nucleotide region in the second nucleic acid chain that, when the first and second nucleic acid chains anneal to form a double-stranded structure, extends beyond the 3' end of the first nucleic acid chain and / or extends beyond the 5' end of the first nucleic acid chain; in other words, a nucleotide region in the second nucleic acid chain that protrudes from the double-stranded structure. The overhang region in the second nucleic acid chain may be located on the 5' end (Figure 2A) or the 3' end (Figure 2B) of the complementary region. The overhang region in the second nucleic acid chain may be located on both the 5' and 3' ends of the complementary region (Figure 2C).

[0059] In one embodiment, the base sequence of the first nucleic acid strand is complementary to all or part of the base sequence of the target transcript, allowing it to hybridize (or anneal) to the target transcript. Base sequence complementarity can be determined using a BLAST program or similar. 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 the base sequence information of the target gene.

[0060] Hybridization conditions may vary, including low-stringent and high-stringent conditions. Low-stringent conditions may be relatively low temperature and high salt concentration conditions, for example, 30°C, 2×SSC, 0.1%SDS. High-stringent conditions may be relatively high temperature and low salt concentration conditions, for example, 65°C, 0.1×SSC, 0.1%SDS. The stringency of hybridization can be adjusted by changing conditions such as temperature and salt concentration. Here, 1×SSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0061] The base lengths of the first and second nucleic acid chains are not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. Alternatively, the base lengths of the first and second nucleic acid chains may be 40 bases or less, 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. The first and second nucleic acid chains may be the same length or of different lengths (for example, one may be 1 to 3 bases shorter or longer than the other). The double-stranded structure formed by the first and second nucleic acid chains may include a bulge. The choice of length can be determined by balancing the strength of the antisense effect with the specificity of the nucleic acid chain to the target, among other factors such as cost and synthesis yield. If nucleic acids such as aptamers are bound to the first and / or second nucleic acid chains, the overall base length of the first and second nucleic acid chains may be the sum of the above base length and the base length of the bound nucleic acid. In this case, the base length of the bound nucleic acid is not limited, but may be, for example, at least 10 bases, at least 15 bases, or at least 20 bases, and may also be 100 bases or less, 80 bases or less, 60 bases or less, 40 bases or less, or 30 bases or less.

[0062] The nucleosides in the first and second nucleic acid strands may be natural nucleosides (deoxyribonucleosides, ribonucleosides, or both) and / or non-natural nucleosides.

[0063] The nucleoside bonds in the first and second nucleic acid chains may be naturally occurring nucleoside bonds and / or modified nucleoside bonds. Preferably, however limited, at least one, at least two, or at least three nucleoside bonds from the ends (5' end, 3' end, or both ends) of the first and / or second nucleic acid chains are modified nucleoside bonds. Here, for example, the two nucleoside bonds from the end of the nucleic acid chain refer to the nucleoside bond closest to the end of the nucleic acid chain and the adjacent nucleoside bond located on the opposite side of the end. Modified nucleoside bonds in the terminal region of the nucleic acid chain are preferred because they can suppress or inhibit undesirable degradation of the nucleic acid chain. In one embodiment, all nucleoside bonds in the first and / or second nucleic acid chains may be modified nucleoside bonds. The modified nucleoside bonds may be phosphorothioate bonds.

[0064] The first and second nucleic acid chains may contain, in whole or in part, nucleoside mimetic or nucleotide mimetic. The nucleotide mimetic may be peptide nucleic acid and / or morpholino nucleic acid.

[0065] The first nucleic acid chain contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 morpholino nucleic acids. In one embodiment, 25% or more, 33% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the nucleic acids in the first nucleic acid chain are morpholino nucleic acids. The nucleoside bonds between morpholino nucleic acids are not limited, but some or all of them may be phosphorodiamidate bonds.

[0066] In one embodiment, the first nucleic acid strand does not contain at least four consecutive nucleosides that are recognized by RNase H when hybridized to the target transcript. Typically, regions containing 4-20 bases, 5-16 bases, or 6-12 bases of consecutive nucleosides are recognized by RNase H. Examples of nucleosides recognized by RNase H include natural deoxyribonucleosides. Those skilled in the art can easily determine, for example, whether or not they are cleaved by RNase H. The fact that a double-stranded nucleic acid agent without containing "at least four consecutive nucleosides" recognized by RNase H has an antisense effect was unexpected, considering the common technical knowledge that the main part of the antisense effect of double-stranded nucleic acid agents is due to the RNase H-dependent pathway.

[0067] In one embodiment, the natural ribonucleosides of the first nucleic acid chain constitute less than half of the total length, or are not present at all.

[0068] The second nucleic acid chain may consist entirely of ribonucleosides and / or modified nucleosides. The second nucleic acid chain may consist entirely of deoxyribonucleosides and / or modified nucleosides, and may not contain any ribonucleosides. In one embodiment, the second nucleic acid chain may consist entirely of deoxyribonucleosides and / or modified nucleosides.

[0069] At least one, at least two, at least three, or at least four nucleosides from the ends of the second nucleic acid chain (5' end, 3' end, or both ends) may be modified nucleosides. Modified nucleosides may contain modified sugars and / or modified nucleic acid bases. Modified sugars may be 2'-modified sugars (e.g., sugars containing a 2'-O-methyl group). Modified nucleic acid bases may also be 5-methylcytosine.

[0070] The second nucleic acid chain may consist, in order from the 5' end, of a modified nucleoside of 2-7 nucleotides or 3-5 nucleotides in length (e.g., a modified nucleoside containing a 2'-modified sugar), a ribonucleoside or deoxyribonucleoside of 4-15 nucleotides or 8-12 nucleotides in length (optionally linked by intermodified nucleoside bonds), and a modified nucleoside of 2-7 nucleotides or 3-5 nucleotides in length (e.g., a modified nucleoside containing a 2'-modified sugar).

[0071] At least one (e.g., three) nucleoside bonds at the 3' end of the second nucleic acid strand may be modified nucleoside bonds, such as phosphorothioate bonds, which have high RNase resistance. It is preferable to include modified nucleoside bonds, such as phosphorothioate modifications, at the 3' end of the second nucleic acid strand because this improves the gene repression activity of the double-stranded nucleic acid complex.

[0072] At least one (e.g., three) nucleosides from the 3' end of the second nucleic acid strand may be modified nucleosides such as RNase-resistant 2'F-RNA or 2'-OMe. The presence of modified nucleosides such as 2'F-RNA or 2'-OMe at the 3' end of the second nucleic acid strand is preferable because it enhances the gene repression activity of the double-stranded nucleic acid complex.

[0073] The first and second nucleic acid strands may contain any combination of the modified nucleoside bonds and modified nucleosides described above.

[0074] The first and / or second nucleic acid strands constituting the double-stranded nucleic acid complex of the present invention may be a mixmer. In this specification, "mixmer" means a nucleic acid strand that contains alternating natural and non-natural nucleosides of periodic or random segment lengths, and does not contain four or more consecutive deoxyribonucleosides and ribonucleosides. In a mixmer, a mixmer in which the non-natural nucleosides are cross-linked nucleosides and the natural nucleosides are deoxyribonucleosides is specifically referred to as a "BNA / DNA mixmer". In a mixmer, a mixmer in which the non-natural nucleosides are peptide nucleic acids and the natural nucleosides are deoxyribonucleosides is specifically referred to as a "peptide nucleic acid / DNA mixmer". In a mixmer, a mixmer in which the non-natural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "morpholino nucleic acid / DNA mixmer." A mixmer is not limited to containing only two types of nucleosides. A mixmer may contain any number of types of nucleosides, whether natural or modified nucleosides or nucleoside mimetic compounds. For example, it may have one or two consecutive deoxyribonucleosides separated by a cross-linking nucleoside (e.g., an LNA nucleoside). The cross-linking nucleoside may further contain a modified nucleic acid base (e.g., 5-methylcytosine).

[0075] The first and second nucleic acid strands may be linked via a cleavable or uncleavable linker. In this case, the first and second nucleic acid strands may be linked via the linker to form a single strand. However, since the functional region has the same configuration as the double-stranded nucleic acid complex, this specification also includes such single-stranded nucleic acids as one embodiment of the double-stranded nucleic acid complex of the present invention. The linker may be any polymer. Examples include polynucleotides, polypeptides, alkylenes, etc. Specifically, it may be composed of natural nucleotides such as DNA and RNA, or unnatural nucleotides such as peptide nucleic acids or morpholino nucleic acids. When the linker is made of nucleic acid, the chain length of the linker may be at least one base, for example, 3 to 10 bases or 4 to 6 bases. Preferably, the chain length is 4 bases. In this case, the linker may take the form of a hinge (hairpin loop). The linker can be located on either the 5' or 3' end of the first nucleic acid strand. For example, in a configuration where the second nucleic acid strand is attached to the 5' end of the first nucleic acid strand, the 5' end of the first nucleic acid strand and the 3' end of the second nucleic acid strand will be linked via the linker. Further details regarding cleavable or non-cleavable linkers are described below in relation to the functional components.

[0076] In one embodiment, the functional moiety may be bound to the first nucleic acid chain and / or the second nucleic acid chain, for example, the second nucleic acid chain. The binding between the first nucleic acid chain and / or the second nucleic acid chain and the functional moiety may be direct or indirect, mediated by other substances. In one embodiment, it is preferable that the first nucleic acid chain and / or the second nucleic acid chain and the functional moiety are directly bound by a covalent bond, ionic bond, hydrogen bond, etc., and a covalent bond is more preferable from the viewpoint of obtaining a more stable bond.

[0077] In one embodiment, the structure of the "functional portion" is not particularly limited and confers a desired function to the double-stranded nucleic acid complex to which it is bound. Desired functions include labeling, purification, and target delivery. Examples of portions that confer labeling function include fluorescent proteins and luciferases. Examples of portions that confer purification function include biotin, avidin, His-tagged peptides, GST-tagged peptides, and FLAG-tagged peptides. Furthermore, from the viewpoint of delivering the first nucleic acid strand to the target site with high specificity and efficiency, and very effectively suppressing the expression of the target gene by said nucleic acid, it is preferable that a molecule having the activity to deliver the double-stranded nucleic acid complex in one embodiment to the target site is bound to the first and / or second nucleic acid strand as a functional portion. Examples of portions that provide target delivery function include lipids, antibodies, aptamers, and ligands for specific receptors.

[0078] In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain, for example, the second nucleic acid chain, is bound to a lipid. Examples of lipids include, but are not limited to, tocopherol, cholesterol, fatty acids, phospholipids and their analogues; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstan and their analogues; steroids and their analogues; ligands for LDLR, SRBI, or LRP1 / 2; FK-506, and cyclosporine; and lipids described in PCT / JP2019 / 12077 and PCT / JP2019 / 10392. The lipid is tocopherol or its analogues and / or cholesterol or its analogues, substituted or unsubstituted C1~ 30 alkyl groups, substituted or unsubstituted C2~ 30 The alkenyl group, or a substituted or unsubstituted C1~ 30 It may also be an alkoxy group.

[0079] Replaced or unreplaced C1~ 30The alkyl group may be, for example, a linear alkyl group having 3 to 15, 6 to 14, or 9 to 13 carbon atoms, where the substituent may be a hydroxyl group, a halogen atom, or an alkyl group having 1 to 3 carbon atoms.

[0080] The second nucleic acid chain bonded to a substituted or unsubstituted alkyl group may have a group represented by the following general formula (I). [ka] [In the formula, R x This is a linear alkylene group having 3 to 24 carbon atoms, preferably 6 to 14 or 9 to 13 carbon atoms.

[0081] The second nucleic acid chain bonded to a substituted or unsubstituted alkyl group may have a group represented by the following general formula (II). [ka] [In the formula, R y This is a linear alkylene group having 1 to 15 carbon atoms, preferably 3 to 15, 6 to 14, or 9 to 13 carbon atoms.

[0082] The functional moiety may be ligated to the 5' end, 3' end, or both ends of the first and / or second nucleic acid chain. Alternatively, the functional moiety may be ligated to nucleotides within the first and / or second nucleic acid chain. The first and / or second nucleic acid chain may contain two or more functional moieties, such as lipids, which may be ligated to multiple positions on the first and / or second nucleic acid chain, or / or ligated as a group at one position on the first and / or second nucleic acid chain. The functional moiety may be ligated one at each end of the 5' and 3' ends of the first and / or second nucleic acid chain.

[0083] The bond between the first nucleic acid chain and / or the second nucleic acid chain and the functional moiety may be a direct bond or an indirect bond mediated by another substance. However, in certain embodiments, it is preferable that the functional moiety is directly bonded to the first nucleic acid chain and / or the second nucleic acid chain via a covalent bond, ionic bond, hydrogen bond, etc., and a covalent bond is more preferable in that it allows for a more stable bond.

[0084] The functional portion may also be linked to the first and / or second nucleic acid strands via cleavable or uncleavable linkers. “Cleavable linker” means a linking group that is cleaved under physiological conditions, for example, intracellularly or within an animal body (e.g., in the human body). In certain embodiments, the cleavable linker is selectively cleaved by an endogenous enzyme such as a nuclease. Examples of cleavable linkers include amides, esters, one or both of phosphodiesters, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers.

[0085] A "non-cleavable linker" refers to a linker that is not cleaved under physiological conditions, for example, within cells or within animals (e.g., within humans). Non-cleavable linkers are not limited to those consisting of phosphorothioate bonds and modified or unmodified deoxyribonucleosides linked by phosphorothioate bonds, or modified or unmodified ribonucleosides. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not particularly limited, but is usually 2 to 20 nucleotides, 3 to 10 nucleotides, or 4 to 6 nucleotides.

[0086] One specific example of a linker is the linker represented by the following equation (I).

[0087] [ka] (In the formula, L 2C1~C, whether substituted or not. 12 The alkylene group (e.g., propylene, hexylene, dodecylene), substituted or unsubstituted C3-C8 cycloalkylene group (e.g., cyclohexylene), -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-, -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-, or CH(CH2-OH)-CH2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3- represents L 3 represents -NH- or a bond, L 4 C1~C, whether substituted or not. 12 Alkylene groups (e.g., ethylene, pentylene, heptylene, andesylene), substituted or unsubstituted C3-C8 cycloalkylene groups (e.g., cyclohexylene), -(CH2)2-[O-(CH2)2] m - represents a combination, where m is an integer from 1 to 25, and L 5 represents -NH-(C=O)-, -(C=O)-, or a bond (where the substitution is preferably made by a halogen atom).

[0088] In one embodiment, the linker represented by formula (III) is L 2 However, it is an unsubstituted C3-C6 alkylene group (e.g., propylene, hexylene), -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-, or -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-, L 3 However, it is -NH- and L 4 and L 5 However, it is a combination.

[0089] In the double-stranded nucleic acid complex of the present invention, the antisense effect of the first nucleic acid strand on the target transcript can be measured by methods known in the art. For example, after introducing the double-stranded nucleic acid complex into cells, it can be measured using known techniques such as Northern blotting, quantitative PCR, or Western blotting. By measuring the expression level of the target gene or the level of the target transcript (e.g., mRNA or microRNA levels, cDNA levels, protein levels, etc.) in a specific tissue, it is possible to determine whether or not the double-stranded nucleic acid complex suppresses target gene expression at those sites. Furthermore, in the case of exon skipping, for example, the effect can be determined by comparing the product produced by exon skipping with the product produced without exon skipping.

[0090] As described above, exemplary embodiments of the double-stranded nucleic acid complex of the present invention have been explained, but the double-stranded nucleic acid complex of the present invention is not limited to the exemplary embodiments described above.

[0091] 1-4. Method for producing double-stranded nucleic acid complexes The double-stranded nucleic acid complex of the present invention can be manufactured by those skilled in the art by appropriately selecting known methods. While not limited to these methods, the process typically begins with designing and manufacturing the first and second nucleic acid strands that constitute the double-stranded nucleic acid complex. For example, the first nucleic acid strand is designed based on the base sequence information of the target transcript (e.g., the base sequence of the target gene), and the second nucleic acid strand is designed as its complementary strand. Subsequently, each nucleic acid strand can be synthesized based on the designed base sequence information using commercially available automated nucleic acid synthesizers from companies such as GE Healthcare, Thermo Fisher Scientific, and Beckman Coulter. The resulting oligonucleotides can then be purified using a reverse-phase column or the like.

[0092] Furthermore, in the case of a double-stranded nucleic acid complex to which a functional moiety is attached, the first nucleic acid strand may be manufactured according to the method described above. On the other hand, the second nucleic acid strand to which the functional moiety is attached can be manufactured by performing the above synthesis and purification using a nucleic acid species to which the functional moiety is already attached. For example, the second nucleic acid strand may be manufactured by performing the above synthesis and purification using a nucleic acid species to which the functional moiety is already attached. Alternatively, the functional moiety may be attached to the second nucleic acid strand manufactured by performing the above synthesis and purification using a known method. After manufacturing each nucleic acid strand, a double-stranded nucleic acid complex to which the desired functional moiety is attached can be manufactured by performing annealing on the first and second nucleic acid strands, as described later.

[0093] Methods for linking functional portions to nucleic acids are well known in the art. A double-stranded nucleic acid complex of the present invention can be produced by mixing nucleic acids prepared by this method in a suitable buffer solution, denaturing them at approximately 90°C to 98°C for several minutes (e.g., 5 minutes), and then annealing the nucleic acids at approximately 30°C to 70°C for approximately 1 to 8 hours. Alternatively, nucleic acid strands can be ordered and obtained from various manufacturers (e.g., Gene Design Co., Ltd.) by specifying the base sequence, modification site, and type. The annealing process can be carried out by standing the mixture at room temperature (approximately 10°C to 35°C) for approximately 5 to 60 minutes. Alternatively, a double-stranded nucleic acid complex of a certain embodiment of the present invention may be prepared by dissolving the first and second nucleic acid strands, respectively, in a buffer solution (e.g., phosphate-buffered saline) or water at approximately 70°C to 98°C, mixing the two resulting solutions, holding the mixture at approximately 70°C to 98°C for several minutes (e.g., 5 minutes), and then holding the mixture at approximately 30°C to 70°C (or 30°C to 50°C) for approximately 1 to 8 hours. The first and second nucleic acid strands can each be dissolved in a buffer (e.g., phosphate-buffered saline) or water at room temperature (approximately 10°C to 35°C). The annealing conditions (time and temperature) during the preparation of the double-stranded nucleic acid complex are not limited to those described above. Furthermore, suitable conditions for promoting nucleic acid strand annealing are well known in the art.

[0094] 1-5. Applications of double-stranded nucleic acid complexes In one embodiment, the double-stranded nucleic acid complex of the present invention may be for at least one of the following actions: suppressing or increasing the expression level of the transcript or translation product of a target gene, inhibiting the function of the transcript or translation product of a target gene, regulating RNA splicing, and inhibiting the binding of a target gene to a protein, for example, for exon skipping. For example, the double-stranded nucleic acid complex of the present invention may be for at least one of the following actions: exon skipping, exon inclusion, steric blocking, and RNA expression enhancement. The double-stranded nucleic acid complex of the present invention may exert the above actions in specific tissues, such as the brain, spinal cord, kidney, liver, lung, intestine, spleen, adrenal gland, eye, retina, skin, peripheral nerves, for example, the brain. The brain may be the cerebrum, diencephalon, brainstem, or cerebellum, for example, one or more of the cerebrum (cerebral cortex, etc.), brainstem, cerebellum, hippocampus, and striatum. Furthermore, the double-stranded nucleic acid complex of the present invention may exert the above actions in tissues other than muscle tissue, including cardiac muscle and skeletal muscle.

[0095] In one embodiment, the double-stranded nucleic acid complex of the present invention is for disease or prevention. Diseases include, for example, muscular dystrophy (Duchenne muscular dystrophy, myotonic dystrophy type 1 (DM1), Fukuyama muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, etc.), congenital myopathy, primary age-related tauopathy (PART), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration / corticobasal syndrome (CBD), Pick's disease, frontotemporal dementia, neuroinclusion disease, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Huntington's disease, hereditary spinocerebellar degeneration (SCA), multiple system atrophy, hereditary spastic paraplegia, multiple sclerosis, cerebral infarction, epilepsy, and encephalitis.

[0096] 2. Pharmaceutical Compositions 2-1. Overview In one embodiment, the present invention relates to a pharmaceutical composition. The pharmaceutical composition of the present invention contains a double-stranded nucleic acid complex described herein as an active ingredient. In one embodiment, the pharmaceutical composition described herein may be used for the application of the double-stranded nucleic acid complex described herein. The pharmaceutical composition of the present invention may consist essentially of the double-stranded nucleic acid complex described herein. That is, the pharmaceutical composition of the present invention may further contain auxiliary components such as carriers in addition to the double-stranded nucleic acid complex described herein. Alternatively, the pharmaceutical composition of the present invention may consist solely of the double-stranded nucleic acid complex described herein.

[0097] 2-2. Composition The pharmaceutical composition of the present invention may include an active ingredient and a carrier. Each component will be described in detail below.

[0098] 2-2-1. Active Ingredients The pharmaceutical composition of the present invention comprises at least one double-stranded nucleic acid complex as described herein as an active ingredient. The pharmaceutical composition of the present invention may contain two or more of the double-stranded nucleic acid complexes.

[0099] The amount (content) of the double-stranded nucleic acid complex contained in the pharmaceutical composition varies depending on the type of double-stranded nucleic acid complex, the site to be delivered (e.g., the brain), the dosage form of the pharmaceutical composition, the dosage of the pharmaceutical composition, and the type of carrier described later. Therefore, it should be determined appropriately considering each of these conditions. Usually, the pharmaceutical composition is adjusted so that an effective amount of double-stranded nucleic acid complex is contained in a single dose. "Effective amount" refers to the amount necessary for the double-stranded nucleic acid complex to exert its function as an active ingredient. The "effective amount" may cause little to no harmful side effects to the organism to which it is applied. This effective amount may change depending on various conditions such as the subject's information, the route of administration, and the number of administrations. Ultimately, it is determined by the judgment of a physician, veterinarian, or pharmacist. "Subject's information" refers to various individual information of the organism to which the pharmaceutical composition is applied. For example, if the subject is human, it includes age, weight, sex, diet, health status, disease progression and severity, drug sensitivity, presence or absence of concomitant drugs, etc.

[0100] 2-2-2. Carrier The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers. "pharmaceutically acceptable carriers" refers to additives commonly used in the pharmaceutical technology field. Examples include solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavorings, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonic agents, sedatives, bulking agents, disintegrants, buffering agents, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizers, and other additives.

[0101] The solvent may be, for example, water or any other pharmaceutically acceptable aqueous solution, or any pharmaceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, and sodium acetate buffers. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0102] The above-mentioned carrier is used to avoid or suppress the degradation of the active ingredient, the double-stranded nucleic acid complex, by enzymes in the body, as well as to facilitate formulation and administration methods, and to maintain the dosage form and efficacy. It should be used as appropriate as needed.

[0103] 2-2-3. Dosage Form The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a form that can exert the pharmacological effect of the active ingredient in the body without inactivating the double-stranded nucleic acid complex described herein, which is the active ingredient, by degradation or the like.

[0104] The specific dosage form will vary depending on the method of administration and / or prescription conditions. Since the methods of administration can be broadly classified into parenteral administration and oral administration, the dosage form should be appropriate for each method of administration.

[0105] If the method of administration is parenteral, the preferred dosage form is a liquid preparation that can be administered directly to the target site or systemically via the circulatory system. An example of a liquid preparation is an injectable preparation. Injectable preparations can be formulated by mixing them with the aforementioned excipients, elixirs, emulsifiers, suspensions, surfactants, stabilizers, pH adjusters, etc., in a unit dose form generally accepted for pharmaceutical production. Other possible forms include ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, eye drops, and suppositories.

[0106] If the method of administration is oral, preferred dosage forms include solid preparations (including tablets, capsules, drops, and lozenges), granules, powders, and liquid preparations (including oral aqueous preparations, emulsions, and syrups). If a solid preparation is used, it may be a dosage form with a coating known in the art, such as a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multi-layer tablet, as needed.

[0107] The specific shapes and sizes of each of the above dosage forms are not particularly limited, as long as they fall within the range of dosage forms known in the relevant art. The pharmaceutical composition of the present invention may be manufactured according to conventional methods in the relevant art.

[0108] 2-3. Dosage Form and Dosage In this specification, there are no specific limitations on the preferred mode of administration of the pharmaceutical composition. Administration may be systemic or topical. The route of administration may be oral or parenteral. Specific examples of parenteral administration include intravenous administration, intra-arterial administration, transfusion, intraperitoneal administration, intracerebroventricular administration, intrathecal administration, intraocular administration, intramuscular administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, intravesical administration, vaginal administration, rectal administration, inhalation or nasal administration, and tracheal / bronchial administration. When the target site of the present invention is the brain, intracerebroventricular or intrathecal administration, which are the target sites, are preferred.

[0109] When a pharmaceutical composition is administered or ingested, the dosage or intake should be such that the amount of the double-stranded nucleic acid complex contained is, for example, 0.00001 mg / kg / day to 10000 mg / kg / day, or 0.001 mg / kg / day to 100 mg / kg / day. The pharmaceutical composition may be administered as a single dose or multiple doses. In the case of multiple doses, it may be administered daily or at appropriate time intervals (for example, at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times. The single dose of the above double-stranded nucleic acid complex is, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.25 mg / kg or more, 0.5 mg / kg or more, 1 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, 75 mg The amount can be 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 (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) can be appropriately selected.

[0110] The double-stranded nucleic acid complex of the present invention may be administered in doses of 0.01 to 10 mg / kg (e.g., about 6.25 mg / kg) twice a week for four doses. Alternatively, the double-stranded nucleic acid complex may be administered in doses of 0.05 to 30 mg / kg (e.g., about 25 mg / kg) once or twice a week for two to four doses, for example, twice a week for two doses. By adopting such a dosing regimen (divided dose), toxicity (e.g., avoiding platelet reduction) and the burden on the subject can be reduced compared to a single dose of a higher dose.

[0111] The inhibitory effects of pharmaceutical compositions are additive even with repeated administration. Furthermore, when administering repeatedly, allowing a certain interval between doses (for example, half a day or more) can improve efficacy.

[0112] 3. Method In one embodiment, the present invention relates to a method for inducing an antisense effect on a target gene or its transcript, or for inducing at least one of the effects of an aptamer, decoy, and bait on a target molecule, comprising administering a nucleic acid complex or composition described herein to a subject. The method may also be a method for treating or preventing a disease in the subject. [Examples]

[0113] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0114] [Example 1: Exon skipping effect in the brain by systemic administration of heteronucleotide-type PMOs] In Example 1, the exon-skipping effect in the cerebrum was evaluated by multiple administrations of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide (phosphodiamide morpholino oligomer (hereinafter also referred to as "PMO")) that targets the exon 23 / intron 23 boundary region (causing exon skipping) in mdx mice (Duchenne muscular dystrophy model mice) and a tocopherol or cholesterol-bound complementary chain.

[0115] (Materials and Methods) (1) Preparation of nucleic acid agents A double-stranded nucleic acid agent was compared to a single-stranded antisense oligonucleotide (ASO) control. The control (ASO) was a 25-mer single-stranded morpholino nucleic acid targeting exon 23 / intron 23 of the mouse dystrophin gene (dystrophin) pre-mRNA. This ASO consists entirely of 25 mers of morpholino nucleic acid, and all nucleoside bonds are phosphorodiamidate bonds. This morpholino nucleic acid has a nucleotide sequence complementary to positions 83803536~83803512 of mouse dystrophin pre-mRNA (GenBank accession number: NC_000086.7).

[0116] By annealing this morpholino nucleic acid (first nucleic acid chain) with tocopherol-conjugated Toc-cRNA (mDystrophin) or cholesterol-conjugated Chol-cRNA (mDystrophin), double-stranded nucleic acid agents, namely tocopherol-conjugated heteroduplex oligonucleotide (Toc HDO) or cholesterol-conjugated heteroduplex oligonucleotide (Chol HDO), were prepared. The first and second nucleic acid chains were mixed in equimolar amounts, the solution was heated at 95°C for 5 minutes, and then cooled to 37°C and held for 1 hour to anneal the nucleic acid chains and prepare the above-mentioned double-stranded nucleic acid agents. The annealed nucleic acids were stored at 4°C or on ice. The prepared double-stranded nucleic acid agents are referred to as Toc HDO and Chol HDO.

[0117] The sequences, chemical modifications, and structures of the oligonucleotides used in Example 1 are shown in Table 1 and Figure 6. All oligonucleotides were manufactured by GeneDesign Co., Ltd. (Osaka, Japan).

[0118] [Table 1]

[0119] (2) in vivo administration The mice used were 6-7 week old male mdx mice (CREA Japan) weighing 20g. All experiments using mice were performed with n=2. Nucleic acid agents were intravenously injected into the mice once a week for a total of 5 times at a dose of 100 mg / kg. In addition, mice were also prepared that were injected with PBS alone or ASO PMO (mDystrophin) (instead of the double-stranded nucleotide agent).

[0120] (3) RNA expression analysis Two weeks after the final administration of nucleic acid agents, mice were dissected and their brains were removed. mRNA was then extracted from each tissue using Isogen II (Nippon Gene Co., Ltd.). One-step RT-PCR was performed on 1 μg of the extracted total RNA using the Qiagen One Step RT-PCR Kit (Qiagen). The reaction mixture was prepared according to the protocol provided with the kit. A LifeECO thermal cycler (Bioer Technology) was used. The RT-PCR program used is as follows: 42°C for 30 minutes: Reverse transcription reaction 95°C for 15 minutes: Thermal denaturation [94°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds] x 35 cycles: PCR amplification 72°C, 7 minutes: Final extension reaction

[0121] The nucleotide sequences of the forward and reverse primers used in RT-PCR are as follows. Forward primer: 5'-ATCCAGCAGTCAGAAAGCAAA-3'(Sequence ID 3) Reverse primer: 5'-CAGCCATCCATTTCTGTAAGG-3'(Sequence ID 4)

[0122] One μl of the RT-PCR reaction product described above was analyzed using a Bioanalyzer 2100 (Agilent) with the Agilent DNA 1000 kit. The polynucleotide amount "A" of the band where exon 23 was skipped and the polynucleotide amount "B" of the band where exon 23 was not skipped were measured. Based on these measured values ​​of "A" and "B", the skipping efficiency was calculated according to the following formula. Skipping efficiency (%) = A / (A + B) × 100

[0123] (result) The results are shown in Figure 7. As shown in Figure 7, exon skipping was observed only in Chol HDO, a cholesterol-binding heterodouble-stranded oligonucleotide. This indicates that heteronucleation of ASO PMO (mDystrophin), which normally does not penetrate the brain, makes it possible for it to penetrate the brain even after systemic administration. Furthermore, the observation of exon skipping activity in Chol HDO, a morpholino nucleic acid in which the entire first nucleic acid strand is RNase H resistant, was unexpected considering the common technical understanding that the main part of the antisense effect of double-stranded nucleic acid agents is due to the RNase H-dependent pathway.

[0124] [Example 2: Exon skipping effect in the brain by intracerebroventricular administration of heteronucleotide-type PMOs] In Example 2, the exon-skipping effect in the brain of mdx mice (Duchenne muscular dystrophy model mice) was evaluated by a single intracerebroventricular administration of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide (PMO) that targets the exon 23 / intron 23 boundary region (causing exon skipping) and a tocopherol or cholesterol-bound complementary strand.

[0125] (Materials and Methods) (1) Preparation of nucleic acid agents The nucleic acid agent used in this example is the same as the nucleic acid agent prepared in Example 1. However, for comparison, a ligand-less cRNA was prepared and annealed with ASO PMO (mDystrophin) in the same manner as in Example 1. The prepared double-stranded nucleic acid agent is referred to as HDO.

[0126] The sequences, chemical modifications, and structures of the oligonucleotides used in Example 2 are shown in Table 2 and Figure 6. All oligonucleotides were manufactured by Gene Design Co., Ltd. (Osaka, Japan).

[0127] [Table 2]

[0128] (2) in vivo administration The mice used were 6-7 week old male mdx mice (CREA Japan) weighing 20g. All experiments using mice were performed with n=3. Nucleic acid agents were administered intracerebroventricularly to the left ventricular ventricle of the mice at a dose of 10 μL (10 nmol of nucleic acid). In addition, mice were prepared that were injected with PBS alone, PMO alone, or ligand-less HDO (instead of the double-stranded nucleotide agent).

[0129] (3) RNA expression analysis Two weeks after the final administration of nucleic acid agents, mice were dissected and the cerebrum, cerebellum, hippocampus, striatum, and brainstem were extracted. Subsequently, mRNA was extracted from each tissue using IsogenII (Nippon Gene Co., Ltd.). One-step RT-PCR was performed on 1 μg of the extracted total RNA using the Qiagen One Step RT-PCR Kit (Qiagen). The reaction mixture was prepared according to the protocol provided with the kit. A LifeECO thermal cycler (Bioer Technology Co., Ltd.) was used. The RT-PCR program used, as well as the forward and reverse primers used for RT-PCR, are as described in Example 1.

[0130] (result) The results are shown in Figure 8. As shown in Figure 8, the exon skipping effect was particularly pronounced with intracerebroventricular administration of TocHDO and CholHDO. The increased efficacy of intracerebroventricular administration of PMO by heteronucleation and ligand addition was unexpected, considering that, as shown in Comparative Example 1 below, no increase in efficacy was observed with intracerebroventricular administration of nucleic acids that do not contain morpholino nucleic acids by heteronucleation and ligand addition.

[0131] [Comparative Example 1: Antisense effect in the brain due to intracerebroventricular administration of nucleic acids that do not contain morpholino nucleic acids] In Comparative Example 1, the antisense effect in the brain was evaluated by a single intracerebroventricular administration of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide targeting malat 1 in C57BL / 6 mice and a tocopherol or cholesterol-bound complementary chain.

[0132] (Materials and Methods) (1) Preparation of nucleic acid agents A 16-mer single-stranded LNA / DNA gapmer (ASO(mMalat1)) (first nucleotide strand) targeting malat1 non-coding RNA was prepared. This LNA / DNA gapmer is a 16-nucleotide oligonucleotide containing three LNA nucleosides at the 5' end and three LNA nucleosides at the 3' end, and ten DNA nucleosides between them. This LNA / DNA gapmer has a nucleotide sequence complementary to positions 1316-1331 of mouse malat1 non-coding RNA (SEQ ID NO: 1).

[0133] A complementary RNA strand (Chol-cRNA(mMalat1)) (second nucleic acid strand) was prepared, having a base sequence complementary to this ASO and with cholesterol covalently bonded to its 5' end. The second strand is a 16-nucleotide oligonucleotide containing three 2'-O-methyl modified ribonucleosides at each end and ten ribonucleosides in between. The first and second strands were mixed in equimolar amounts, the solution was heated at 95°C for 5 minutes, then cooled to 37°C and held for 1 hour to anneal the nucleic acid strands and prepare a double-stranded nucleic acid agent. The annealed nucleic acid was stored at room temperature, 4°C, or on ice. The prepared double-stranded nucleic acid agent is called Chol HDO (control).

[0134] Table 3 shows the sequence, chemical modification, and structure of the oligonucleotides used in Comparative Example 1. All oligonucleotides were manufactured by Gene Design Co., Ltd. (Osaka, Japan).

[0135] [Table 3]

[0136] (2) in vivo administration The mice used were 6-7 week old male C57BL / 6 mice weighing 20g. All experiments using mice were performed with n=4. Nucleic acid agents (25 μg in ASO equivalent) were administered intracerebroventricularly to each mouse. In addition, mice were prepared that were injected with PBS alone (instead of the nucleic acid agent).

[0137] (3) RNA expression analysis Seven days after the final administration of nucleic acid agents, mice were perfused with PBS, and then dissected to extract various brain regions. mRNA was then extracted from each tissue using a high-throughput fully automated nucleic acid extraction system, MagNA Pure 96 (Roche Lifesciences), according to the protocol. cDNA was synthesized using Transcriptor Universal cDNA Master (Roche Lifesciences) according to the protocol. Quantitative RT-PCR was performed using TaqMan (Roche Lifesciences). The primers used in quantitative RT-PCR were products designed and manufactured by Thermo Fisher Scientific based on various gene counts. The amplification conditions (temperature and time) were as follows: [95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 1 second] × 45 cycles.

[0138] Based on the quantitative RT-PCR results obtained in this manner, the expression levels of mRNA(malat1) and mRNA(ACTB; internal standard gene) were calculated to obtain relative expression levels. The mean and standard error of the relative expression levels were calculated. The results of each group were also compared, and the results were further evaluated using a t-test.

[0139] (result) The results are shown in Figure 9. As shown in Figure 9, previously reported hetero-double-stranded oligonucleotides that do not contain morpholino nucleic acid did not show significantly superior gene suppression effects compared to single-stranded nucleic acids (ASOs) that do not contain morpholino nucleic acid, even when cholesterol was similarly bound to the complementary strand.

[0140] [Example 3: Exon skipping effect in the liver and kidneys by systemic administration of heteronucleotide-type PMOs] In Example 3, the exon-skipping effect in the liver and kidneys was evaluated by multiple administrations of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide (PMO) targeting the exon 23 / intron 23 boundary region (causing exon skipping) in mdx mice (Duchenne muscular dystrophy model mice) and a tocopherol or cholesterol-bound complementary strand.

[0141] The preparation of nucleic acid agents, in vivo administration, and RNA expression analysis were carried out according to Example 1. However, in this example, the liver or kidney was used instead of the brain for RNA expression analysis.

[0142] The results are shown in Figure 10. As shown in Figure 10, systemic administration of nucleic acid agents also produced exon skipping effects in the liver and kidneys, and the effect was particularly pronounced with TocHDO and CholHDO. [Example 4: Exon skipping effect by intracerebroventricular administration of heteronucleotide-type PMOs containing various complementary strands] In Example 4, the exon-skipping effect of a double-stranded nucleic acid complex consisting of an antisense oligonucleotide (PMO) targeting the exon 23 / intron 23 boundary region (causing exon skipping) in mdx mice (Duchenne muscular dystrophy model mice) and various complementary strands was evaluated by a single intracerebroventricular administration.

[0143] The sequences, chemical modifications, and structures of the oligonucleotides used in Example 4 are shown in Table 4 and Figure 11. All oligonucleotides were manufactured by Gene Design Co., Ltd. (Osaka, Japan).

[0144] [Table 4]

[0145] The 3' terminal structure of the oligonucleotide 3'Chol-cRNA (default) shown in Table 4 is represented by the following formula (IV). [ka]

[0146] The 5' terminal structure of the oligonucleotide C3-cRNA (default) shown in Table 4 is represented by the following formula (V). [ka]

[0147] Heteroduplex oligonucleotides were prepared containing ASO PMO (mDystrophin) as the first nucleic acid chain and various complementary strands as shown in Table 4 as the second nucleic acid chain. The nucleic acid agent was administered intracerebroventricularly to the left ventricular ventricle of mice in a volume of 10 μL (10 nmol of nucleic acid). Furthermore, mice were also prepared in which PBS alone or PMO alone was injected (instead of the double-stranded nucleic acid agent). One week after administration of the nucleic acid agent, the mice were dissected and the cerebellum, brainstem, striatum, hippocampus, posterior cortex, and cervical vertebrae were extracted, and RNA expression analysis was performed. Experiments using mice were performed with n=2-3. Other methods for preparing nucleic acid agents, in vivo administration, and RNA expression analysis followed those of Example 1.

[0148] The results are shown in Figures 12-13. In Figures 12-13, heterodouble-stranded oligonucleotides containing ASO PMO (mDystrophin) as shown in Table 4 as the first nucleic acid strand and cRNA (default), Chol-cRNA (default), Chol-cRNA (DNA gap), Chol-cRNA (full OMe), 3'Chol-cRNA (default), and C3-cRNA (default) as the second nucleic acid strand are shown as HDO (default), CholHDO (default), CholHDO (DNA gap), CholHDO (full OMe), 3'Chol (default), and C3 (default), respectively.

[0149] As shown in Figures 12-13, CholHDO (DNA gap), CholHDO (full OMe), 3'Chol (default), and C3 (default) exhibited exon skipping effects in the cerebellum, brainstem, striatum, hippocampus, posterior cortex, and cervical spine, with the effect being particularly pronounced with CholHDO (DNA gap). All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A pharmaceutical composition for intrathecal or intraventricular administration, It contains a double-stranded nucleic acid complex, which includes a first nucleic acid strand and a second nucleic acid strand, as an active ingredient. The first nucleic acid strand can hybridize to at least a portion of the target gene or its transcript, has an antisense effect on the target gene or its transcript, and 100% of the nucleic acids in the first nucleic acid strand are morpholino nucleic acids. The second nucleic acid chain contains a base sequence complementary to the first nucleic acid chain and is composed of ribonucleosides. The first nucleic acid strand is annealed to the second nucleic acid strand, and The pharmaceutical composition wherein the second nucleic acid chain is bound to cholesterol.

2. The pharmaceutical composition according to claim 1, wherein the ribonucleoside is a natural ribonucleoside and / or a modified ribonucleoside.

3. The pharmaceutical composition according to claim 1 or 2, wherein the cholesterol is bonded to the 5' and / or 3' ends of the second nucleic acid chain.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the first nucleic acid chain and the second nucleic acid chain are linked via a cleavable or uncleavable linker.

5. In the subject, at least one of the following functions: To suppress or enhance the expression level of the transcript or translation product of a target gene, Inhibits the function of the transcript or translation product of the target gene. Regulates RNA splicing, and Inhibits the binding of target genes to proteins. A pharmaceutical composition according to any one of claims 1 to 4, for the purpose of achieving the above.

6. The pharmaceutical composition according to claim 5 for exon skipping, exon inclusion, steric blocking, and RNA expression enhancement.

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