WO2024071362A1 - Nervous system delivery enhancer - Google Patents

Nervous system delivery enhancer Download PDF

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WO2024071362A1
WO2024071362A1 PCT/JP2023/035565 JP2023035565W WO2024071362A1 WO 2024071362 A1 WO2024071362 A1 WO 2024071362A1 JP 2023035565 W JP2023035565 W JP 2023035565W WO 2024071362 A1 WO2024071362 A1 WO 2024071362A1
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nucleic acid
pharmaceutical composition
delivery enhancer
delivery
group
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PCT/JP2023/035565
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French (fr)
Japanese (ja)
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隆徳 横田
耕太郎 吉岡
哲也 永田
倫太朗 原
博中 五十嵐
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国立大学法人東京医科歯科大学
国立大学法人新潟大学
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Publication of WO2024071362A1 publication Critical patent/WO2024071362A1/en

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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
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    • A61K9/127—Liposomes
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/76—Nitrogen atoms to which a second hetero atom is attached
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to a delivery enhancer for enhancing the delivery of a drug to the central nervous system or peripheral nervous system, and a pharmaceutical composition for treating a central nervous system disease or a peripheral nervous system disease.
  • oligonucleotides have attracted attention in the ongoing development of pharmaceuticals known as nucleic acid drugs, and in particular, the development of nucleic acid drugs using the antisense method is being actively pursued, given their high selectivity for target genes and low toxicity.
  • the antisense method involves selectively modifying or inhibiting the expression of proteins encoded by target genes or the activity of miRNA by introducing complementary oligonucleotides (antisense oligonucleotides: often referred to as "ASOs (Antisense Oligonucleotides)" in this specification) into cells, using a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand.
  • ASOs Antisense Oligonucleotides
  • the present inventors have developed a double-stranded nucleic acid complex (heteroduplex oligonucleotide, HDO) in which an antisense oligonucleotide is annealed to its complementary strand (Patent Documents 1 and 2, Non-Patent Documents 1 and 2).
  • the double-stranded nucleic acid complex is a technological technology with a strong antisense effect.
  • the goal is to provide a new method to improve the efficiency of delivery of drugs, such as nucleic acid medicines, to the nervous system.
  • Aquaporin 4 is a member of the aquaporin family with extremely high water selectivity, and is the main AQP involved in water transport in the nervous system. In the central nervous system, including the brain and spinal cord, AQP4 is found in large amounts, particularly in the endfeet membranes of astrocytes that contact the basement membrane.
  • the periarterial space/perivenous space that exists between the blood vessels and brain tissue in the brain is called the Virchow-Robin space.
  • CSF cerebrospinal fluid
  • This transport is similar to the lymphatic system, and the mechanism for excreting waste products from the brain through the perivascular space is called the Glymphatic system.
  • Patent Document 3 International Publication No. 2017/150704 describes that the accumulation of amyloid beta in the brain is suppressed by promoting water transport by AQP4 in the brain and thereby promoting the excretion function of the glymphatic system.
  • the inventors attempted to activate the glymphatic system by simultaneously administering an AQP4 function promoter when administering nucleic acid drugs such as antisense nucleic acids or siRNA into the ventricles of mice. As a result, they found that the amount of nucleic acid drugs detected in the nervous system after administration increased dramatically, and the inhibitory effect on the target gene was significantly improved. This result is a surprising effect that could not be predicted from the effect of promoting the excretion of amyloid ⁇ .
  • a delivery enhancer for promoting delivery of a drug to the central nervous system and/or peripheral nervous system the delivery enhancer consisting of an aquaporin 4 function enhancer or an aquaporin 4 function modifier.
  • the aquaporin 4 function promoter or aquaporin 4 function modifier is represented by the following formula (I): or a derivative thereof, or a salt thereof.
  • the derivative is represented by the following formula (III): The delivery enhancer according to (2), (4) The delivery enhancer according to (2) or (3), wherein the compound or the derivative is conjugated to a carrier molecule.
  • the protein is albumin, lipoprotein, or an antibody or antibody fragment.
  • the polymer comprises polyethylene glycol (PEG) or a PEG-grafted polymer.
  • PEG polyethylene glycol
  • a pharmaceutical composition for treating a central nervous system disease or a peripheral nervous system disease comprising a therapeutically effective amount of a drug and a delivery enhancer according to any one of (1) to (8).
  • the pharmaceutical composition according to (14) which is administered intrathecally, nasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly.
  • the pharmaceutical composition according to (15), wherein the intrathecal administration is intraventricular administration, posterior fossa puncture, or lumbar puncture.
  • the nucleic acid drug is selected from the group consisting of an antisense nucleic acid, a heteronucleic acid, an siRNA, an shRNA, an miRNA, an mRNA, an lncRNA, a plasmid DNA, an aptamer, a decoy, a bait nucleic acid, a ribozyme, and a nucleic acid vector.
  • nucleic acid drug comprises a nucleic acid molecule capable of hybridizing to at least a portion of a target gene or its transcription product and having an antisense effect on the target gene or its transcription product.
  • nucleic acid molecule is 12 to 30 bases in length.
  • nucleic acid molecule comprises one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides.
  • nucleic acid molecule is a mixmer.
  • nucleic acid molecule comprises a morpholino nucleic acid, or the entire nucleic acid of the nucleic acid molecule consists of a morpholino nucleic acid.
  • nucleic acid molecule is a gapmer.
  • the nucleic acid molecule (1) a central region containing at least three consecutive deoxyribonucleosides; (2) a 5' wing region comprising an unnatural nucleoside and disposed on the 5' end of the central region; and (3) a 3' wing region comprising an unnatural nucleoside and disposed on the 3' end of the central region.
  • the bridged nucleoside is selected from the group consisting of an LNA nucleoside, a 2',4'-BNA NC nucleoside, a cEt BNA nucleoside, an ENA nucleoside, an AmNA nucleoside, a GuNA nucleoside, a scpBNA nucleoside, a scpBNA2 nucleoside, and a BANA3 nucleoside.
  • nucleic acid drug comprises a double-stranded nucleic acid complex comprising a first nucleic acid strand consisting of the nucleic acid molecule and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand.
  • second nucleic acid strand is at least 8 bases in length.
  • the delivery enhancer or pharmaceutical composition according to (41) or (42), wherein the second nucleic acid strand comprises any one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides.
  • linker comprises a group represented by the following formula (IV):
  • L2 represents a substituted or unsubstituted C1-12 alkylene group, a substituted or unsubstituted C3- C8 cycloalkylene group, -( CH2 ) 2 -O-(CH2) 2 -O-( CH2 ) 2 -O-(CH2) 3- , or CH( CH2 - OH) -CH2 -O-( CH2 ) 2 -O-( CH2 ) 2 -O-( CH2 ) 2 -O-( CH2 ) 2- -O-( CH2 ) 3- ;
  • L3 represents -NH- or a bond;
  • L4 represents a substituted or unsubstituted C 1 -12 alkylene group, a substituted or unsubstituted C 3 -C 8 cycloalkylene group, -(CH 2 ) 2
  • the linker comprises a nucleic acid, a polyether group, and/or an alkylamino group.
  • the polyether group is a polyethylene glycol group or a triethylene glycol group.
  • the delivery enhancer or pharmaceutical composition according to (57), wherein the alkylamino group is a hexylamino group.
  • the delivery enhancer or pharmaceutical composition according to (66), wherein the lipid is selected from the group consisting of cholesterol or an analogue thereof, tocopherol or an analogue thereof, folic acid, phosphatidylethanolamine, and a substituted or unsubstituted alkyl group having 16 to 30 carbon atoms.
  • the linker comprises a nucleic acid, a polyether group, and/or an alkylamino group.
  • the central nervous system is selected from the group consisting of the cerebral cortex, the basal ganglia, the cerebral white matter, the diencephalon, the brainstem, the cerebellum, and the spinal cord.
  • the central nervous system is selected from the group consisting of the frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, parietal lobe, occipital lobe, striatum, globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain,
  • This specification includes the disclosure of Japanese Patent Application No. 2022-158781, which is the priority basis of this application.
  • the present invention provides a delivery enhancer that improves the efficiency of drug delivery to the nervous system.
  • FIG. 1 shows the structures of various natural and non-natural nucleotides.
  • FIG. 2 shows the structures of various bridged nucleic acids.
  • Figure 3 shows the amount of Malat1-targeting ASO delivered to the central nervous system and its target gene suppression effect.
  • Figure 3A shows the results of measuring ASO concentration in the hippocampus 3 hours or 7 days after intracerebroventricular administration.
  • Figure 3B shows Malat1 RNA expression levels in the hippocampus 7 days after intracerebroventricular administration.
  • ASO only indicates the group administered ASO alone, and "ASO + TGN-073" indicates the group administered ASO/TGN-073 together. Error bars indicate standard error.
  • Figure 4 shows the amount of ASO delivered to the central nervous system and the target gene suppression effect in the central nervous system when Malat1-targeting ASO was administered intracerebroventricularly at various concentrations.
  • ASO only indicates the group administered ASO alone, and
  • ASO + TGN-073 indicates the group administered ASO/TGN-073 together. Error bars indicate standard error.
  • Figure 5 shows Mapt mRNA expression levels in the hippocampus 7 days after intracerebroventricular administration of Mapt-targeting ASO.
  • “ASO only” indicates the group administered ASO alone, and "ASO + TGN-073” indicates the group administered ASO/TGN-073 together. Error bars indicate standard error.
  • FIG. 6 shows the scoring system used for behavioral assessment.
  • Figure 7 shows the results of central neurotoxicity evaluation of Malat1-targeting ASO.
  • the figure shows the acute tolerability scores 30 minutes, 1 hour, and 2 hours after intracerebroventricular administration.
  • TGN-073 only indicates the group administered TGN-073 alone
  • ASO only indicates the group administered ASO alone
  • ASO + TGN-073 indicates the group administered ASO/TGN-073 co-administration.
  • Error bars indicate standard error.
  • Figure 8 shows the results of evaluating motor function in an open field test 1 hour after intracerebroventricular administration.
  • Figure 8A shows the maximum movement speed.
  • Figure 8B shows the movement time.
  • TGN-073 only indicates the group administered TGN-073 alone
  • ASO only indicates the group administered ASO alone
  • ASO + TGN-073 indicates the group administered ASO/TGN-073 co-administration. Error bars indicate standard error.
  • Figure 9 shows Malat1 RNA expression levels in the hippocampus 7 days after intracerebroventricular administration of 2'-O-MOE-modified ASO targeting the Malat1 gene.
  • PBS indicates the negative control PBS-administered group
  • ASO only indicates the ASO-only administration group
  • ASO + TGN-073 indicates the ASO/TGN-073 co-administration group. Error bars indicate standard error.
  • FIG. 10 shows the structure of the nucleic acid used in Example 5.
  • Figure 11 shows the ASO concentration in the central nervous system (amount delivered to the central nervous system) and the target gene suppression effect when single-stranded or double-stranded nucleic acid agents targeting the Malat1 gene were administered intracerebroventricularly.
  • Figure 11A shows the results of measuring the ASO concentration in the hippocampus 7 days after intracerebroventricular administration.
  • Figure 11B shows the Malat1 RNA expression level in the hippocampus 7 days after intracerebroventricular administration.
  • +/- indicates the presence or absence of co-administration of TGN-073 when the nucleic acid agent was administered intracerebroventricularly. Error bars indicate standard error.
  • Figure 12 shows the target gene suppression effect when siRNA targeting the Sod1 gene was administered intracerebroventricularly.
  • PBS indicates the negative control PBS-administered group
  • siRNA only indicates the siRNA-only administered group
  • siRNA + TGN-073 indicates the siRNA/TGN-073 co-administered group. Error bars indicate standard error.
  • Figure 13 shows the ASO concentration in the central nervous system (amount delivered to the central nervous system) and target gene suppression effect when TGN-073-mes, a derivative of TGN-073, was administered intracerebroventricularly together with ASO.
  • Figure 13A shows the results of measuring the ASO concentration in the hippocampus 7 days after intracerebroventricular administration.
  • Figure 13B shows the Mapt mRNA expression level in the hippocampus 7 days after intracerebroventricular administration.
  • PBS indicates the negative control PBS-administered group
  • TGN-073-mes only indicates the TGN-073-mes alone-administered group
  • ASO only indicates the ASO alone-administered group
  • ASO + TGN-073-mes indicates the ASO/TGN-073-mes co-administered group.
  • Error bars indicate standard error.
  • Figure 14 shows the efficiency of exon 23 skipping in the brain 7 days after intracerebroventricular administration of phosphorodiamidate morpholino oligomers (PMOs) targeting exon 23/intron 23 of dystrophin pre-mRNA.
  • Figure 14A shows the efficiency of skipping in the hippocampus.
  • Figure 14B shows the efficiency of skipping in the striatum.
  • Figure 14C shows the efficiency of skipping in the cortex. Error bars show standard error.
  • FIG. 15 shows the structure of the nucleic acid used in Example 8.
  • Figure 16 shows the skipping efficiency of exon 23 in the brain 7 days after intracerebroventricular administration of a double-stranded nucleic acid complex containing a PMO targeting exon 23/intron 23 of dystrophin pre-mRNA.
  • Figure 16A shows the skipping efficiency in the hippocampus.
  • Figure 16B shows the skipping efficiency in the striatum.
  • Figure 16C shows the skipping efficiency in the cortex. Error bars show standard error.
  • Figure 17 shows the delivery amount of Malat1-targeting ASO to the lumbar spinal cord and the target gene suppression effect.
  • Figure 17A shows the results of measuring the ASO concentration in the lumbar spinal cord 7 days after intracerebroventricular administration.
  • Figure 17B shows the Malat1 RNA expression level in the lumbar spinal cord 7 days after intracerebroventricular administration.
  • PBS indicates the negative control PBS-administered group
  • ASO only indicates the ASO-only administered group
  • ASO + TGN-073 indicates the ASO/TGN-073 co-administered group. Error bars indicate standard error.
  • Figure 18 shows the amount of Malat1-targeting ASO delivered to the central nervous system after intrathecal administration.
  • Figure 18A shows the ASO concentration in spinal cord tissue.
  • Figure 18B shows the ASO concentration in the left hippocampus.
  • ASO only indicates the group administered ASO alone, and "ASO + TGN-073” indicates the group administered ASO/TGN-073 together. Error bars indicate standard error.
  • Figure 19 shows the amount of VHH antibody delivered to the central nervous system after intracerebroventricular administration.
  • Figure 19A shows the VHH antibody concentration in the left hippocampus.
  • Figure 19B shows the VHH antibody concentration in the left occipital cortex.
  • Figure 19C shows the VHH antibody concentration in the left basal ganglia.
  • VHH only indicates the group administered VHH antibody alone, and
  • VHH + TGN-073 indicates the group administered VHH antibody/TGN-073 together. Error bars indicate standard error.
  • Figure 20 shows the amount of IgG antibody delivered to the central nervous system after intracerebroventricular administration.
  • IgG only indicates the group administered IgG antibody alone
  • IgG + TGN-073 indicates the group administered IgG antibody/TGN-073 together. Error bars indicate standard error.
  • Figure 21 shows the results of observing the fluorescence of GFP protein expressed from the AAV9 vector administered intracerebroventricularly in coronal sections at the hippocampal level.
  • AAV only indicates the group administered AAV alone
  • AAV + TGN-073 indicates the group administered AAV/TGN-073 co-administration.
  • a first aspect of the present invention is a delivery enhancer.
  • the delivery enhancer of the present invention is an aquaporin 4 function enhancer or an aquaporin 4 function modifier.
  • a drug such as a nucleic acid drug, a peptide, or a low molecular weight compound, the efficiency of delivery of the drug to the central nervous system and/or the peripheral nervous system can be improved.
  • the "transcription product" of a target gene refers to any RNA synthesized by RNA polymerase. Specifically, it may include mRNA (including mature mRNA, mRNA precursor, and mRNA without base modification) transcribed from a target gene, non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA.
  • ncRNA non-coding RNA
  • miRNA miRNA
  • lncRNA long non-coding RNA
  • lncRNA long non-coding RNA
  • a "target gene” refers to a gene whose transcription or translation product expression level can be suppressed or enhanced, whose transcription or translation product function can be inhibited, or whose steric blocking, splicing control (e.g., splicing switch, exon skipping, exon inclusion), base editing, or RNA editing can be induced by the antisense effect of a nucleic acid molecule or double-stranded nucleic acid complex.
  • the type of target gene is not particularly limited.
  • Examples include genes derived from an organism into which a nucleic acid strand, nucleic acid molecule, or double-stranded nucleic acid complex is introduced, and include genes whose expression increases in various diseases (e.g., central nervous system diseases and peripheral nervous system diseases) and genes expressed in living organisms such as the nervous system.
  • diseases e.g., central nervous system diseases and peripheral nervous system diseases
  • SR-B1 scavenger receptor B1
  • Malat1 metastasis associated lung adenocarcinoma transcript 1
  • Mapt microtubule-associated protein tau
  • BACE1 beta-secretase 1
  • Hdac2 histone deacetylase 2
  • DMPK dystrophia myotonic-protein kinase
  • target transcript refers to a transcript that is the direct target of a nucleic acid molecule or a double-stranded nucleic acid complex
  • transcript of a target gene also falls under the category of target transcript.
  • Information on the base sequences of target transcripts and target genes can be obtained from publicly known databases, such as the NCBI (National Center for Biotechnology Information) database.
  • antisense nucleic acid refers to a single-stranded nucleic acid molecule that contains a base sequence capable of hybridizing (i.e., complementary) to at least a portion of a target transcript (mainly a transcript of a target gene) and can exert an antisense effect on the target transcript.
  • antisense oligonucleotide refers to an antisense nucleic acid composed of an oligonucleotide.
  • antisense nucleic acid or “antisense oligonucleotide” is often referred to as "ASO.”
  • ASO antisense nucleic acid
  • the nucleic acid molecule of the present invention or the first nucleic acid strand of the double-stranded nucleic acid complex functions as an ASO, and the target region may include the 3'UTR, 5'UTR, exon, intron, coding region, translation initiation region, translation termination region, or any other nucleic acid region.
  • the target region of the target transcript can be at least 8 bases long, for example, 10-35 bases long, 12-25 bases long, 13-20 bases long, 14-19 bases long, or 15-18 bases long, or 13-22 bases long, 16-22 bases long, or 16-20 bases long.
  • the term "antisense effect” refers to any effect that occurs when an ASO hybridizes to a target transcript (e.g., an RNA sense strand), such as the effect of regulating the expression or editing of a target transcript.
  • a target transcript e.g., an RNA sense strand
  • “Regulating the expression or editing of a target transcript” refers to suppression or reduction of the expression of a target gene or the expression level of a target transcript (herein, "the expression level of a target transcript” is often referred to as “the level of a target transcript”), inhibition of translation, RNA editing, base editing, splicing control or splicing function modification effects (e.g., splicing switch, exon inclusion, exon skipping, etc.), steric blocking, or degradation of a transcript.
  • RNA oligonucleotide when introduced into a cell as an ASO, the ASO forms a partial duplex by annealing with the mRNA, which is the transcription product of the target gene.
  • This partial duplex acts as a cover to prevent translation by ribosomes, thereby inhibiting the expression of a target protein encoded by the target gene at the translational level (steric blocking).
  • an oligonucleotide containing DNA is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed.
  • This heteroduplex structure is recognized by RNase H, resulting in degradation of the mRNA of the target gene and inhibition of expression of the protein encoded by the target gene at the expression level.
  • an antisense effect can also be achieved by targeting an intron in a pre-mRNA.
  • an antisense effect can also be achieved by targeting an miRNA.
  • inhibition of the function of the miRNA can increase the expression of a gene whose expression is normally controlled by the miRNA.
  • the modulation of expression of the target transcript can be a reduction in the amount of the target transcript.
  • the antisense effect can be measured, for example, by administering a test nucleic acid compound to a subject (e.g., a mouse) and measuring, for example, several days later (e.g., 2 to 7 days later), the expression level of a target gene or the level (amount) of a target transcript (e.g., the amount of mRNA or RNA such as microRNA, the amount of cDNA, the amount of protein, etc.) whose expression is regulated by the antisense effect provided by the test nucleic acid compound.
  • a test nucleic acid compound e.g., a mouse
  • the expression level of a target gene or the level (amount) of a target transcript e.g., the amount of mRNA or RNA such as microRNA, the amount of cDNA, the amount of protein, etc.
  • a decrease in the measured expression level of the target gene or the level of the target transcript by at least 10%, at least 20%, at least 25%, at least 30%, or at least 40% compared to a negative control (e.g., vehicle administration) indicates that the test nucleic acid compound can produce an antisense effect (e.g., a reduction in the amount of the target transcript).
  • nucleic acid strand may affect the antisense effect provided by the nucleic acid strand, nucleic acid molecule or nucleic acid complex.
  • the choice of modification may vary depending on the sequence of the target gene, etc., but a person skilled in the art can determine a suitable embodiment by referring to the descriptions in the literature related to antisense methods (e.g., WO 2007/143315, WO 2008/043753, and WO 2008/049085).
  • the relevant modification can be evaluated if the measured value thus obtained is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of the measured value of the nucleic acid complex before modification).
  • translation product of a target gene refers to any polypeptide or protein synthesized by translation of the target transcript or the transcription product of a target gene that is the direct target of a nucleic acid molecule or a double-stranded nucleic acid complex.
  • decoy refers to a nucleic acid that has a sequence of the binding site of a transcription factor (e.g., NF-kB) or a similar sequence, and is introduced into a cell as a “decoy” to suppress the action of the transcription factor (if it is a transcription activator, it suppresses transcription, and if it is a transcription repressor, it promotes transcription).
  • a transcription factor e.g., NF-kB
  • Decoy nucleic acids can be easily designed based on information on the binding sequence of the target transcription factor.
  • bait refers to a nucleic acid molecule that specifically binds to a specific target molecule within a cell and modifies the function of the target molecule.
  • a target that interacts with a bait is also called a "prey.”
  • nucleic acid or “nucleic acid molecule” as used herein may refer to a monomeric nucleotide or nucleoside, an oligonucleotide composed of multiple monomers, or multiple nucleosides linked by internucleoside bonds, and also includes polynucleotides if they are polymers.
  • Natural nucleic acid refers to a nucleic acid that exists in nature. Natural nucleic acids include natural nucleosides and natural nucleotides, etc., described below.
  • Non-natural nucleic acid or “artificial nucleic acid” refers to any nucleic acid other than natural nucleic acid. Non-natural nucleic acid or artificial nucleic acid includes non-natural nucleosides and non-natural nucleotides, etc., described below.
  • nucleic acid strand refers to two or more nucleosides linked by internucleoside bonds, and may be, for example, an oligonucleotide or a polynucleotide.
  • a nucleic acid strand may be made full length or partial by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction reactions.
  • a nucleic acid strand may contain natural and/or non-natural nucleotides.
  • Nucleoside generally refers to a molecule that is composed of a combination of a base and a sugar.
  • the sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, examples of which include ribose and deoxyribose.
  • the base portion of a nucleoside is typically a heterocyclic base moiety, including, but not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).
  • Nucleotide refers to a molecule in which a phosphate group is covalently linked to the sugar portion of the nucleoside.
  • the phosphate group is usually linked to the hydroxyl group at the 2', 3', or 5' position of the sugar.
  • Oligonucleotide refers to a linear oligomer formed by covalently linking several to several dozen hydroxyl groups and phosphate groups in the sugar moieties between adjacent nucleotides.
  • Polynucleotide refers to a linear polymer formed by linking several dozen or more, preferably several hundred or more, nucleotides that are more numerous than an oligonucleotide, by the covalent bonds.
  • the phosphate groups are generally considered to form internucleoside bonds.
  • natural nucleosides refer to nucleosides that exist in nature. Examples include ribonucleosides consisting of ribose and bases such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides consisting of deoxyribose and bases such as adenine, cytosine, guanine, or thymine.
  • ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to as “RNA nucleosides” and “DNA nucleosides,” respectively.
  • natural nucleotide refers to a nucleotide that exists in nature and is a molecule in which a phosphate group is covalently bonded to the sugar portion of the natural nucleoside.
  • examples include ribonucleotides, which are known as the building blocks of RNA and in which a phosphate group is bonded to a ribonucleoside, and deoxyribonucleotides, which are known as the building blocks of DNA and in which a phosphate group is bonded to a deoxyribonucleoside.
  • non-natural nucleotide refers to any nucleotide other than a natural nucleotide, and includes modified nucleotides and nucleotide mimetics.
  • modified nucleotide refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase.
  • nucleotide mimetics includes structures used to replace nucleosides and linkages at one or more positions of an oligomeric compound.
  • Peptide nucleic acids PNA are nucleotide mimetics with a backbone in which N-(2-aminoethyl)glycine is linked by amide bonds in place of sugars.
  • nucleic acid strands including non-natural oligonucleotides often have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Thus, they are preferred over natural nucleotides.
  • unnatural nucleoside refers to any nucleoside other than a natural nucleoside. For example, it includes modified nucleosides and nucleoside mimetics.
  • modified nucleoside refers to a nucleoside having a modified sugar moiety and/or a modified nucleobase.
  • mimetic refers to functional groups that replace the sugar, nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of a sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
  • nucleoside mimic includes structures that are used to replace the sugar, or the sugar and base, at one or more positions of an oligomeric compound, or to replace the linkage between the monomeric subunits that make up the oligomeric compound, etc.
  • oligomeric compound is meant a polymer of linked monomeric subunits that is at least capable of hybridizing to a region of a nucleic acid molecule.
  • Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar mimetics, e.g., nucleoside mimetics having non-furanose sugar units.
  • Modified sugar refers to a sugar having a substitution and/or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)), and "sugar modification” refers to a substitution and/or any change from a natural sugar moiety.
  • a nucleic acid strand may optionally include one or more modified nucleosides, including modified sugars.
  • “Sugar-modified nucleoside” refers to a nucleoside having a modified sugar moiety. Such sugar-modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to a nucleic acid strand.
  • the nucleoside includes a chemically modified ribofuranose ring moiety.
  • chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNAs), replacement of ribosyl ring oxygen atoms with S, N(R), or C(R1)(R2) (wherein R, R1, and R2 each independently represent H, C1 - C12 alkyl, or a protecting group), and combinations thereof.
  • sugar modified nucleosides include, but are not limited to, nucleosides containing 5'-vinyl, 5'-alkyl (e.g., 5'-methyl (R or S), 5'-ethyl (R or S)), 5'-allyl (R or S), 4'-S, 2'-F (2'-fluoro group), 2'- OCH3 (2'-O-Me group or 2'-O-methyl group), 2'-O-[2-(N-methylcarbamoyl)ethyl] (2'-O-MCE group), and 2'-O - methoxyethyl (2'-O-MOE or 2-O( CH2 ) 2OCH3 ) substituents.
  • nucleosides containing 5'-vinyl, 5'-alkyl (e.g., 5'-methyl (R or S), 5'-ethyl (R or S)), 5'-allyl (R or S), 4'-S, 2'-F (2
  • “2'-modified sugar” refers to a furanosyl sugar modified at the 2'-position.
  • a nucleoside containing a 2'-modified sugar may also be referred to as a "2'-modified nucleoside” or a "2'-sugar modified nucleoside.”
  • “5'-modified sugar” refers to a furanosyl sugar modified at the 5'-position.
  • Nucleosides containing a 5'-modified sugar are referred to as “5'-modified nucleosides" or “5'-sugar-modified nucleosides,” and are specifically distinguished as “5'-modified deoxyribonucleosides” and "5'-modified ribonucleosides” and “5'-modified ribonucleosides,” respectively.
  • BNA nucleoside refers to a modified nucleoside that contains a bicyclic sugar moiety. Nucleics that contain a bicyclic sugar moiety are commonly referred to as bridged nucleic acids (BNAs). Nucleosides that contain a bicyclic sugar moiety are also sometimes referred to as “bridged nucleosides,” “bridged non-natural nucleosides,” or “BNA nucleosides.” Some examples of bridged nucleic acids are shown in Figure 2.
  • a bicyclic sugar may be a sugar in which the 2' and 4' carbon atoms are bridged by two or more atoms.
  • bicyclic sugars are known to those of skill in the art.
  • One subgroup of bicyclic sugar-containing nucleic acids (BNAs) or BNA nucleosides can be described as having the 2' and 4 ' carbon atoms bridged by 4'-( CH2 ) p -O-2', 4'-( CH2 ) p - CH2-2 ', 4'-( CH2 ) p -S-2', 4'-( CH2 )p-OCO-2', 4'-(CH2) n -N( R3 )-O-( CH2 ) m -2', where p, m and n represent integers from 1 to 4, 0 to 2 and 1 to 3, respectively; and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an ary
  • R 1 and R 2 are typically hydrogen atoms, but may be the same as or different from each other, and may also be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R 4 )R 5 (wherein R 4 and R 5 may be the same as or different from each other and respectively represent a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto
  • amine BNAs also known as 2'-Amino-LNAs
  • 2'-Amino-LNAs e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substitutions
  • 2'-O,4'-C-spirocyclopropylene bridged nucleic acids also known as scpBNAs
  • BNA nucleosides include methyleneoxy (4'-CH 2 -O-2') BNA nucleosides (also known as LNA nucleosides, 2',4'-BNA nucleosides) (e.g., ⁇ -L-methyleneoxy (4'-CH 2 -O-2') BNA nucleosides, ⁇ -D-methyleneoxy (4'-CH 2 -O-2') BNA nucleosides), ethyleneoxy (4'-(CH 2 ) 2 -O-2') BNA nucleosides (also known as ENA nucleosides), ⁇ -D-thio (4'-CH 2 -S-2') BNA nucleosides, aminooxy (4'-CH 2 -ON(R 3 )-2') BNA nucleosides, oxyamino (4'-CH 2 -N(R 3 )-O-2') BNA nucleosides (2',4'-BNA Also known as NC nucle
  • a "cationic nucleoside” is a modified nucleoside that exists in a cationic form relative to a neutral form (such as the neutral form of a ribonucleoside) at a certain pH (e.g., human physiological pH (about 7.4), the pH of a delivery site (e.g., an organelle, cell, tissue, organ, organism, etc.) etc.).
  • a cationic nucleoside may contain one or more cationic modifying groups at any position of the nucleoside.
  • Bicyclic nucleosides with a methyleneoxy (4'- CH2 -O-2') bridge are sometimes referred to as LNA nucleosides.
  • modified internucleoside linkage refers to an internucleoside linkage having a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage).
  • Modified internucleoside linkages include internucleoside linkages that contain a phosphorus atom and internucleoside linkages that do not contain a phosphorus atom.
  • Representative phosphorus-containing internucleoside bonds include phosphodiester bonds, phosphorothioate bonds, phosphorodithioate bonds, phosphotriester bonds (e.g., methyl phosphotriester bonds and ethyl phosphotriester bonds as described in U.S. Patent Registration No.
  • alkyl phosphonate bonds e.g., methyl phosphonate bonds as described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, and methoxypropyl phosphonate bonds as described in WO 2015/168172
  • alkylthiophosphonate bonds e.g., methylthiophosphonate bonds
  • boranophosphate bonds e.g., a cyclic guanidine moiety
  • internucleoside linkage containing a guanidine moiety e.g., a tetramethylguanidine (TMG) moiety
  • TMG tetramethylguanidine
  • Phosphorothioate linkages refer to internucleoside linkages in which the non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.
  • the modified internucleoside linkage is preferably one that is more resistant to nucleases than naturally occurring internucleoside linkages.
  • internucleoside linkage When an internucleoside linkage has a chiral center, the internucleoside linkage may be chiral controlled.
  • chiral controlled it is intended that the internucleoside linkage exists as a single diastereomer about a chiral center, e.g., a chiral phosphorus linkage.
  • the internucleoside linkage may be a phosphorothioate linkage chirally controlled in the Rp or Sp configuration, an internucleoside linkage containing a guanidine moiety substituted with one to four C1-6 alkyl groups (e.g., a tetramethylguanidine (TMG) moiety; see, for example, Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811), and/or an internucleoside linkage containing a cyclic guanidine moiety.
  • TMG tetramethylguanidine
  • Chirally controlled phosphorothioate linkages in the Rp or Sp configuration are also known, and phosphorothioate linkages chirally controlled in the Sp configuration are known to be more stable than those in the Rp configuration, and ASOs chirally controlled in the Sp configuration are also known to promote target RNA cleavage by RNase H1 and result in a more sustained response in vivo.
  • nucleobase refers to the base component (heterocyclic moiety) that constitutes a nucleic acid, and the main known bases are adenine, guanine, cytosine, thymine, and uracil.
  • nucleobase or “base” includes both modified and unmodified nucleic acid bases (bases), unless otherwise specified.
  • a purine base may be either a modified or unmodified purine base.
  • a pyrimidine base may be either a modified or unmodified pyrimidine base.
  • Modified nucleobase or “modified base” means any nucleobase other than adenine, cytosine, guanine, thymine, or uracil.
  • Unmodified nucleobase or “unmodified base” (natural nucleobase) means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
  • modified nucleobases include, but are not limited to, hypoxanthine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, or N4-methylcytosine; N6-methyladenine, or 8-bromoadenine; 2-thio-thymine; and N2-methylguanine, or 8-bromoguanine.
  • a “mixmer” refers to a nucleic acid chain 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.
  • a mixmer in which the non-natural nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "BNA/DNA mixmer.”
  • a mixmer in which the non-natural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "peptide nucleic acid/DNA mixmer.”
  • a mixmer in which the non-natural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "morpholino nucleic acid/DNA mixmer.”
  • a mixmer is not limited to containing only two types of nucleosides.
  • a mixmer can include any number of types of nucleosides, whether natural or modified nucleosides or nucleoside mimetics. For example, it may have one or two consecutive deoxyribonucleosides separated by a bridged nucleoside (e.g., an LNA nucleoside).
  • the bridged nucleoside may further include a modified nucleobase (e.g., 5-methylcytosine).
  • the term "gapmer” refers, in principle, to a single-stranded nucleic acid that includes or consists of a "central region” (DNA gap region) and wing regions (referred to as the "5' wing region” and “3' wing region”, respectively) located directly at the 5' end and 3' end of the central region.
  • the central region in a gapmer contains at least three or at least four consecutive deoxyribonucleosides.
  • each of the 5' wing region and the 3' wing region contains at least one non-natural nucleoside.
  • the central region can be functionally defined as a region that can be recognized by RNase H (e.g., RNase H1).
  • RNase H e.g., RNase H1
  • "recognizable by RNase H” means that when the gapmer binds to a target RNA, the paired sequence in the target RNA can be cleaved by RNase H.
  • the boundary positions can be determined by defining the region in a gapmer that can be recognized by RNase H as the central region, and the regions that are not recognized by RNase H (more specifically, regions in which cleavage activity by RNase H is not substantially detectable under physiological conditions) as the wing regions (5' wing region and 3' wing region).
  • the terminal nucleosides adjacent to the central region in the 5' wing region and 3' wing region are non-natural nucleosides (e.g., 2'-modified nucleosides or bridged nucleosides), and the nucleosides adjacent to the 5' wing region or 3' wing region in the central region are deoxyribonucleosides or sugar-modified versions thereof.
  • both nucleosides adjacent to the 5' wing region and 3' wing region in the central region are deoxyribonucleosides.
  • the central region may contain a modified nucleic acid base recognized by RNase H, for example, 5-methylcytosine.
  • the central region may also contain non-natural nucleosides, such as 2'-modified nucleosides and 5'-modified nucleosides, except for the two terminal nucleosides adjacent to the 5' and 3' wing regions.
  • the central region may be configured such that all internucleoside bonds in the region that it pairs with in the target RNA are susceptible to cleavage by RNase H.
  • non-natural nucleosides contained in the wing regions typically have a stronger binding strength to RNA than natural nucleosides and are highly resistant to nucleic acid degrading enzymes (nucleases, etc.).
  • the non-natural nucleosides that make up the 5' and 3' wing regions may be, for example, bridged nucleosides and/or 2'-modified nucleosides.
  • the gapmer is specifically referred to as a "BNA/DNA gapmer.”
  • the number of bridged nucleosides in the 5' and 3' wing regions is at least one, and may be, for example, two or three.
  • the bridged nucleosides in the 5' and 3' wing regions may be contiguous or non-contiguous in the 5' and 3' wing regions.
  • the bridged nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine).
  • the bridged nucleoside may be an LNA nucleoside or an ENA nucleoside.
  • the gapmer When the bridged nucleoside is an LNA nucleoside, the gapmer is referred to as an "LNA/DNA gapmer.” When the bridged nucleoside is an ENA nucleoside, the gapmer is referred to as an "ENA/DNA gapmer.” When the non-natural nucleosides constituting the 5' and 3' wing regions comprise or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer.” When the non-natural nucleosides constituting the 5' wing region and the 3' wing region comprise or consist of morpholino nucleic acid, the gapmer is specifically referred to as a "morpholino nucleic acid gapmer”.
  • the 2'-modified group of the 2'-modified nucleoside may be a 2'-O-methyl group or a 2'-O-methoxyethyl group.
  • the number of 2'-modified nucleosides contained in the 5' wing region and the 3' wing region is at least one, and may be, for example, two or three.
  • the 2'-modified nucleosides contained in the 5' wing region and the 3' wing region may be contiguous or non-contiguous in the 5' wing region and the 3' wing region.
  • the 2'-modified nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine).
  • a modified nucleobase e.g., 5-methylcytosine
  • the nucleosides may be composed of a combination of two or more types of bridged nucleosides and/or 2'-modified nucleosides.
  • a nucleic acid strand having a wing region only on either the 5' end or the 3' end is called a "hemigapmer" in the art, and in this specification, hemi-gapmers are also included in the term "gapmer.”
  • nucleobases can form so-called Watson-Crick base pairs (natural base pairs) or Wobble base pairs (guanine-thymine or guanine-uracil) through hydrogen bonds, and a relationship in which similar base pairs can be formed between natural nucleobases and modified nucleobases or between modified nucleobases themselves.
  • the antisense region of the nucleic acid molecule does not necessarily have to be completely complementary to at least a portion of the target transcript (e.g., the transcript of the target gene), but is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity.
  • the antisense region of the nucleic acid molecule can hybridize to the target transcript when the base sequence is complementary (typically when the base sequence is complementary to at least a portion of the base sequence of the target transcript).
  • the complementary region in the second nucleic acid strand does not necessarily have to be completely complementary to at least a portion of the nucleic acid molecule that is the first nucleic acid strand, but is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity.
  • a complementary region in the second nucleic acid strand can anneal when the base sequence is complementary to at least a portion of the first nucleic acid strand.
  • the complementarity of the base sequence can be determined by using a BLAST program or the like.
  • Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can anneal or hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design an antisense nucleic acid complementary to a target transcription product, for example, based on information on the base sequence of the target gene.
  • Hybridization conditions may be various stringent conditions, such as low stringency conditions and high stringency conditions.
  • Low stringency conditions may be conditions of relatively low temperature and high salt concentration, for example, 30°C, 2xSSC, 0.1% SDS.
  • High stringency conditions may be conditions of relatively high temperature and low salt concentration, for example, 65°C, 0.1xSSC, 0.1% SDS.
  • Hybridization stringency can be adjusted by changing conditions such as temperature and salt concentration.
  • 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.
  • delivery to the central nervous system and/or peripheral nervous system means delivery to any site in the central nervous system and/or peripheral nervous system or to the entire central nervous system and/or peripheral nervous system.
  • the nervous system is divided into the central nervous system and the peripheral nervous system.
  • the central nervous system consists of the brain and spinal cord.
  • the brain includes the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon (thalamus, subthalamic nucleus), cerebellum (cerebellar cortex, cerebellar nuclei), and brainstem (midbrain, substantia nigra, pons, medulla oblongata).
  • the spinal cord includes the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, sacral spinal cord, and coccygeal spinal cord.
  • the "central nervous system” as used herein may be any of these regions, but may particularly be the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brainstem, cervical spinal cord, thoracic spinal cord, or lumbar spinal cord.
  • An example of the central nervous system is the brain.
  • the peripheral nerves consist of the cranial nerves and spinal nerves, including the ventral root of the spinal cord, the dorsal root, the cranial nerves 1 to 12, the cauda equina, and the dorsal root ganglion.
  • enhanced delivery to the central nervous system means an increase in the amount of drug delivered to the central nervous system and/or peripheral nervous system, or an increase in the efficiency of delivery to the central nervous system and/or peripheral nervous system.
  • the amount of drug delivered to the central nervous system and/or peripheral nervous system under co-administration conditions may be increased compared to the amount of drug delivered to the central nervous system and/or peripheral nervous system under conditions in which the delivery enhancer of the present invention is not co-administered.
  • the term "subject” refers to an object to which a drug or pharmaceutical composition is applied.
  • Subjects include individuals, as well as organs, tissues, and cells. When the subject is an individual, it may be any animal, including humans. Examples of subjects other than humans include various livestock, poultry, pets, and laboratory animals. Without being limited thereto, the subject may be an individual in which the expression level of a target transcript needs to be reduced, or an individual in which treatment or prevention of a disease, such as a central nervous system disease, is required.
  • multiple refers to, for example, 2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-10, 2-12, 2-14, 2-16, 2-18, 2-20, 2-25, 2-30, 2-35, 2-40, or more.
  • the drug delivery enhancer of this embodiment comprises an aquaporin 4 function enhancer or an aquaporin 4 function modifier.
  • aquaporin 4 function promoter is not limited to any substance that can activate aquaporin 4, and any known aquaporin 4 function promoter can be used.
  • aquaporin 4 function modifier is not limited to any substance that can modify the function of aquaporin 4, and any known aquaporin 4 function modifier can be used.
  • An example of the aquaporin 4 function promoter or aquaporin 4 function modifier is a compound represented by the following formula (I): or a derivative thereof, or a salt thereof.
  • the compound represented by the above formula (I) is disclosed in International Publication No. 2017/150704 and the inventors' previous publication (Huber VJ et al., NeuroReport, 2018, 29(9):697-703) as being capable of promoting the function of aquaporin 4, and is known under the generic name TGN-073, but can also be written as N-(3-benzyloxypyridin-2-yl)-benzene-sulfonamide or 2-(phenylsulfonamido)-3-benzyloxypyridine.
  • the derivative of the compound represented by the above formula (I) is any derivative capable of inducing a function-promoting activity or a function-modifying activity on aquaporin 4.
  • An example of the derivative is a compound represented by the following formula (III): Examples of the compound include those represented by the following formula:
  • the salt of the compound represented by formula (I) above is not particularly limited, and may be any pharma- ceutically acceptable salt.
  • pharma-ceutically acceptable salts include metal salts, salts of inorganic acids, and salts of organic acids.
  • the metal salt may be a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or a strontium salt.
  • the salt of an inorganic acid may be a salt of hydrochloric acid, bromic acid, phosphoric acid, sulfuric acid, or disulfuric acid.
  • the salt of an organic acid may be a salt of formic acid, acetic acid, propionic acid, lactic acid, oxalic acid, tartaric acid, malic acid, maleic acid, citric acid, fumaric acid, besylic acid, camsylic acid, edisylic acid, trichloroacetic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or aspartic acid.
  • the compound represented by formula (I) or a derivative thereof is bound to a carrier molecule.
  • the carrier molecule is any molecule bound to the delivery enhancer for the purpose of adjusting the delivery efficiency of the delivery enhancer to the target delivery site.
  • the carrier molecule can adjust the delivery efficiency to the target delivery site based on the control of its molecular size, hydrophilicity/hydrophobicity, etc.
  • the carrier molecule is not particularly limited, and examples thereof include proteins (e.g., proteins of 1 kDa or more), polymers, lipid molecules, and contrast agents. Examples of proteins include albumin, lipoproteins (e.g., HDL-like particles), and antibodies or antibody fragments (e.g., IgG, Fc, modified Fc, etc.). Examples of polymers include polyethylene glycol (PEG) or PEG-grafted polymers. Examples of lipids include tocopherol, cholesterol, fatty acids, phospholipids, and the lipid examples described below.
  • the compound of formula (I) or a derivative thereof can be bound or associated with a microbubble, micelle, or liposome.
  • the manner of binding or association is not limited, but can be, for example, via a carrier molecule, such as the above-mentioned lipid or polymer, bound to the compound of formula (I) or a derivative thereof.
  • the delivery enhancer of the present invention By controlling the molecular size and hydrophilicity/hydrophobicity of the delivery enhancer of the present invention, it is possible to prevent penetration into tissues near the administration site, which is advantageous because it is possible to prevent a decrease in the efficiency of delivery to the desired delivery site.
  • drug also referred to as a drug, medicine, or pharmaceutical product
  • drug is not particularly limited and includes nucleic acid medicines, peptides, low molecular weight compounds, viral vectors, cellular medicines, nanoparticles, liposomes, micelles, and exosomes.
  • a drug to be delivered to the central nervous system and/or peripheral nervous system in combination with the delivery enhancer of the present invention is often referred to as a "drug to be delivered to the nervous system.”
  • peptide refers to an amino acid polymer having one or more peptide bonds.
  • the term “peptide” is not limited by the number of amino acid residues contained in the peptide.
  • peptides include everything from oligopeptides containing several amino acid residues, such as dipeptides and tripeptides, to polypeptides containing many amino acid residues.
  • peptides include not only so-called proteins, but also fragmented peptides and peptides linked to other peptides by peptide bonds. Examples of peptides include antibodies or antibody fragments, and enzymes.
  • peptides include exogenous peptides such as protein pharmaceuticals (e.g., protein preparations), and do not include protein degradation products and other cellular waste products. Furthermore, peptides may be naturally derived or non-naturally derived. Furthermore, peptides may be either cyclic or non-cyclic.
  • the peptide is an antibody or an antibody fragment.
  • antibody refers to a protein that exhibits immune responsiveness to an antigen.
  • species of organism from which the antibody is derived There are no particular limitations on the species of organism from which the antibody is derived. Antibodies are preferably derived from birds and mammals. Examples include chicken, ostrich, mouse, rat, guinea pig, rabbit, goat, donkey, sheep, camel, horse, and human. The antibody may be a full-length antibody.
  • fragment of an antibody refers to an antibody fragment that is composed of a part of an antibody and exhibits immune responsiveness to an antigen like an antibody, and is an antigen-binding fragment.
  • fragments include Fab, Fab', F(ab') 2 , Fv fragment, Fv fragment stabilized by a disulfide bond (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), diabody stabilized by a disulfide bond (dsdiabody), single-chain antibody molecule (scFv), dimeric scFv (bivalent diabody), multispecific antibody, heavy chain antibody such as camelized single domain antibody (camelized antibody; VHH antibody), nanobody, domain antibody, and bivalent domain antibody.
  • Fab is an antibody fragment generated by cleavage of an IgG molecule at the N-terminal side of the disulfide bond in the hinge region with papain, and is composed of the C H 1 and V H adjacent to the V H of the three domains (C H 1, C H 2, C H 3) that constitute the H chain constant region (heavy chain constant region: hereinafter referred to as C H ) , and a full-length L chain.
  • C H H chain constant region
  • Fab' can be obtained by reducing the Fab' dimer (F(ab') 2 ) generated by cleavage of an IgG molecule at the C-terminal side of the disulfide bond in the hinge region with pepsin under mild conditions to cleave the disulfide bond in the hinge region. All of these antibody fragments contain an antigen-binding site and therefore have the ability to specifically bind to an antigen epitope.
  • the term "low molecular weight compound” is not particularly limited, and may be a therapeutic drug for a central nervous system disease or a peripheral nervous system disease, a psychiatric drug, an anticancer drug, or the like. Note that the above-mentioned delivery enhancer itself is excluded from the low molecular weight compound to be delivered to the nervous system in this specification.
  • viral vector is not particularly limited, and examples of usable viral vectors include retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotype vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Viral vectors that lack replication ability so as not to replicate autonomously within infected cells may also be used.
  • the viral vector can encode a gene for gene therapy.
  • cell medicine is not particularly limited, and may be, for example, a cell used in the treatment of any central nervous system disease or peripheral nervous system disease.
  • cells contained in cell medicines include nerve cells, T cells (e.g., CAR-T cells), NK cells, NKT cells, hematopoietic stem cells, peripheral blood mononuclear cells, mesenchymal stem cells, iPS cells, and ES cells.
  • nanoparticles refers to particles with a particle size on the order of nanometers (nm). In principle, nanoparticles refer to particles with a particle size of 1 nm to several hundred nm. Specific examples of nanoparticles include polymer nanoparticles, metal nanoparticles, and dendrimers.
  • liposome refers to a vesicle that contains a lipid membrane and an aqueous medium encapsulated in the lipid membrane.
  • the lipid membrane of a liposome is composed of one or more lipid layers. For example, it is composed of a lipid bilayer containing phospholipids, etc.
  • micelle refers to a vesicle formed by a single molecular membrane.
  • components of micelles include amphiphilic molecules such as surfactants.
  • nanoparticles, liposomes, and micelles can contain nucleic acid drugs, peptides, low molecular weight compounds, etc., making them useful for drug delivery.
  • Exosomes are small vesicles enclosed in a lipid bilayer membrane that are secreted from cells. Exosomes originate from multivesicular endosomes, and when released into the extracellular environment, they may contain biological materials such as nucleic acids (RNA, DNA, etc.) and proteins.
  • nucleic acid medicine refers to a drug containing any nucleic acid molecule.
  • nucleic acid medicine generally refers to a drug containing two or more nucleosides, and the nucleosides contained in the nucleic acid medicine may be natural or non-natural nucleosides.
  • nucleic acid molecule contained in the nucleic acid medicine may be a single-stranded or double-stranded or more nucleic acid.
  • nucleic acid medicines include, but are not limited to, antisense nucleic acids as nucleic acid molecules described below, heterogeneous nucleic acids as double-stranded nucleic acid complexes described below, siRNA, shRNA, miRNA, mRNA, lncRNA, aptamers, plasmid DNA, decoys, bait nucleic acids, ribozymes, and nucleic acid vectors such as plasmid vectors.
  • the nucleic acid medicine comprises a nucleic acid molecule capable of hybridizing to at least a part of a target gene or its transcription product and having an antisense effect on the target gene or its transcription product.
  • the base length of the nucleic acid molecule is not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases.
  • the base length of the nucleic acid molecule 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 base length of the nucleic acid molecule may be, for example, 10 to 40 bases, 12 to 30 bases, or 15 to 25 bases.
  • the length can be determined by the balance between the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield.
  • the base length of the nucleic acid molecule as a whole may be the above-mentioned base length plus the base length of the bound nucleic acid.
  • the nucleosides contained in the nucleic acid molecule may be natural nucleosides (deoxyribonucleosides, ribonucleosides, or both) and/or non-natural nucleosides.
  • the nucleic acid molecule may be a mixmer.
  • the nucleic acid molecule may be a gapmer.
  • the central region (gap region) of a gapmer may be, for example, 3-12 bases long, 4-11 bases long, 5-10 bases long, 6-9 bases long, or 7-8 bases long.
  • the base length of the 5' wing region and the 3' wing region of the gapmer may each independently be at least 2 bases long, for example, 2 to 10 bases long, 2 to 7 bases long, 3 to 5 bases long, 3 to 4 bases long, or 3 bases long.
  • the nucleic acid molecule can include 2'-modified nucleosides and/or bridged nucleosides in the 5' and 3' wing regions.
  • the 2'-modified nucleosides can be, for example, 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleosides, or 2'-fluoro modified nucleosides.
  • the bridged nucleosides can be, for example, LNA nucleosides, 2',4'-BNA NC nucleosides, cEt BNA nucleosides, ENA nucleosides, AmNA nucleosides, GuNA nucleosides, scpBNA nucleosides, scpBNA2 nucleosides, or BANA3 nucleosides.
  • 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-LNA or ENA may be combined, and the types of modifications may include 1 to 4 types, 2 to 3 types, for example 2 types, which may be the same or different in the 5' wing region and the 3' wing region.
  • the nucleic acid molecule comprises a 2'-modified nucleoside at the terminal base position adjacent to the central region in the 5' wing region, the second base position from the 5' side of the central region, and/or the eighth base position from the 5' side of the central region.
  • the "terminal base position adjacent to the central region in the 5' wing region” refers to the base position located at the 3' end in the 5' wing region.
  • the "Nth base position from the 5' side of the central region” refers to the Nth base in the direction from the terminal base adjacent to the 5' wing region in the central region.
  • the second base position from the 5' side of the central region refers to the second base in the 3' direction from the terminal base adjacent to the 5' wing region in the central region
  • the eighth base position from the 5' side of the central region refers to the eighth base in the 3' direction from the terminal base adjacent to the 5' wing region in the central region.
  • the 2'-modification in the 2'-modified nucleoside may be, for example, a 2'-O-methyl group, a 2'-O-[2-(N-methylcarbamoyl)ethyl] group, a 2'-O-methoxyethyl group, or a 2'-fluoro group, but a 2'-O-methyl group is preferred.
  • the 5' wing region and/or the 3' wing region are comprised of two or more 2'-modified nucleosides and/or bridged nucleosides linked by internucleoside linkages.
  • examples of base lengths of the 5' wing region, central region, and 3' wing region include 2-12-3, 3-12-2, 3-12-3, 4-12-3, 2-11-3, 3-11-2, 3-11-3, 4-11-3, 2-10-3, 3-10-2, 3-10-3, 4-10-3, 2-9-3, 3-9-2, 3-9-3, 4-9-3, 2-8-3, 3-8-2, 3-7-3, 4-6-3, 3-6-4, 4-5-4, 4-7-3, 3-7-4, 4-6-4, 5-6-3, 3-6-5, 3-7-5, 5-7-3, 4-7-4, 4-6-5, 5-6-4, 5-5-5, 5-6-5, etc.
  • A-B-C indicates the base length of the 5' wing region
  • B indicates the base length of the central region
  • C indicates the base length of the 3' wing region.
  • the internucleoside bond in the nucleic acid molecule may be a naturally occurring internucleoside bond and/or a modified internucleoside bond. Although not limited thereto, it is preferred that at least one, at least two, or at least three internucleoside bonds from the end (5' end, 3' end, or both ends) of the nucleic acid molecule are modified internucleoside bond.
  • two internucleoside bonds from the end of the nucleic acid chain refer to the internucleoside bond closest to the end of the nucleic acid chain and the internucleoside bond adjacent thereto and located on the opposite side to the end.
  • Modified internucleoside bonds in the terminal region of the nucleic acid chain are preferred because they can suppress or inhibit undesired degradation of the nucleic acid chain.
  • all or a portion of the internucleoside linkages of the nucleic acid molecule may be modified internucleoside linkages.
  • the modified internucleoside linkages may be phosphorothioate linkages.
  • the nucleic acid molecule may comprise, in whole or in part, a nucleoside mimic or a nucleotide mimic.
  • the nucleotide mimic may be a peptide nucleic acid and/or a morpholino nucleic acid.
  • 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% (all) of the nucleic acids in the nucleic acid molecule are morpholino nucleic acids.
  • the internucleoside linkages between the morpholino nucleic acids are not limited, and some or all of them may be phosphorodiamidate linkages.
  • the antisense effect of the nucleic acid molecule on the target transcript can be measured by a method known in the art. For example, after introducing the nucleic acid molecule into cells, the effect 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., the amount of RNA such as mRNA, the amount of cDNA, etc.) in a specific tissue, it can be determined whether the expression of the target gene is suppressed by the nucleic acid molecule at those sites.
  • the expression level of the target gene or the level of the target transcript e.g., the amount of RNA such as mRNA, the amount of cDNA, etc.
  • test nucleic acid compound can have an antisense effect.
  • the nucleic acid drug consists of a double-stranded nucleic acid complex including a first nucleic acid strand consisting of any of the above-mentioned nucleic acid molecules and a second nucleic acid strand including a base sequence complementary to the first nucleic acid strand.
  • the double-stranded nucleic acid complex contains a first nucleic acid strand and a second nucleic acid strand.
  • the first nucleic acid strand is any of the nucleic acid molecules described above, so a detailed explanation of it will be omitted here.
  • the second nucleic acid strand is a nucleic acid molecule that contains a base sequence complementary to the first nucleic acid strand.
  • the second nucleic acid strand is annealed to the first nucleic acid strand through hydrogen bonds of complementary base pairs.
  • the second nucleic acid strand may contain deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and/or bridged nucleosides.
  • the second nucleic acid strand may be such that all nucleosides in a region consisting of a base sequence complementary to the central region of the first nucleic acid strand are (a) deoxyribonucleosides; (b) deoxyribonucleosides and ribonucleosides; (c) deoxyribonucleosides and 2'-modified nucleosides; (d) ribonucleosides and 2'-modified nucleosides; or (e) deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides.
  • the second nucleic acid strand includes a region containing at least three or at least four consecutive ribonucleosides and/or deoxyribonucleosides that are complementary to at least three or at least four consecutive deoxyribonucleosides in a central region of the first nucleic acid strand.
  • the second nucleic acid strand may include a region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand.
  • the region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand may include at least one non-natural nucleoside, which may be a bridged nucleoside and/or a 2'-modified nucleoside.
  • the 2'-modified group of the 2'-modified nucleoside in the second nucleic acid strand may be a 2'-O-methyl group or a 2'-O-methoxyethyl group.
  • the bridged nucleoside and/or the 2'-modified nucleoside in the first nucleic acid strand and the second nucleic acid strand may be the same or different.
  • the internucleoside linkages in the second nucleic acid strand may be naturally occurring internucleoside linkages and/or modified internucleoside linkages. It is preferred, but not limited to, that at least one, at least two, or at least three internucleoside linkages from the ends (5' end, 3' end, or both ends) of the second nucleic acid strand are modified internucleoside linkages. In one embodiment, all or a portion of the internucleoside linkages in the second nucleic acid strand may be modified internucleoside linkages.
  • the second nucleic acid strand may include modified internucleoside linkages in a region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand.
  • the modified internucleoside linkages may be phosphorothioate linkages.
  • the second nucleic acid strand can include 2'-modified nucleosides (e.g., 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleosides, or 2'-fluoro modified nucleosides).
  • 2'-modified nucleosides e.g., 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleosides, or 2'-fluoro modified nucleosides.
  • the number of 2'-modified nucleosides in the second nucleic acid strand is not limited.
  • At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% of the total number of nucleosides in the second nucleic acid strand may be 2'-modified nucleosides. In one embodiment, all of the nucleosides in the second nucleic acid strand are 2'-modified nucleosides.
  • the second nucleic acid strand may include one or more consecutive 2'-modified nucleosides (e.g., 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleosides, or 2'-fluoro modified nucleosides) located at the 5' end and/or one or more consecutive 2'-modified nucleosides located at the 3' end.
  • the number of 2'-modified nucleosides located at the 5' end and/or the 3' end is not limited.
  • the second nucleic acid strand may include one or two, three, four, five, six, or seven consecutive 2'-modified nucleosides located at the 5' end and/or one or two, three, four, five, six, or seven consecutive 2'-modified nucleosides located at the 3' end.
  • the first nucleic acid strand and/or the second nucleic acid strand may comprise modified nucleobases.
  • the number of modified nucleobases is not limited and may be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6.
  • the second nucleic acid strand may include non-complementary bases and/or an insertion sequence and/or deletion of one or more bases relative to the first nucleic acid strand.
  • the number of non-complementary bases in the second nucleic acid strand is not limited, but may be, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 or 2.
  • the number of bases of the insertion sequence in the second nucleic acid strand is not limited, but may be, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 or 2.
  • the length of the deleted contiguous bases in the second nucleic acid strand is not limited, but may be, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 or 2.
  • the region composed of non-complementary bases or an inserted sequence may form a bulge.
  • the second nucleic acid strand may further include at least one overhang region located at one or both of the 5'-end and 3'-end of the complementary region.
  • overhang region refers to a region adjacent to a complementary region, in which, when the first and second nucleic acid strands anneal to form a double-stranded structure, the 5'-end of the second nucleic acid strand extends beyond the 3'-end of the first nucleic acid strand and/or the 3'-end of the second nucleic acid strand extends beyond the 5'-end of the first nucleic acid strand, that is, a nucleotide region in the second nucleic acid strand that protrudes from the double-stranded structure.
  • the overhang region in the second nucleic acid strand may be located at the 5'-end or 3'-end of the complementary region.
  • the overhang region in the second nucleic acid strand may be located at the 5'-end and 3'-end of the complementary region.
  • the base length of the overhang region is not particularly limited and may be 1 to 30 bases long or 1 to 20 bases long.
  • a functional moiety may be bound to the first nucleic acid strand and/or the second nucleic acid strand, for example, the second nucleic acid strand.
  • the bond between the first nucleic acid strand and/or the second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond via another substance, but in one embodiment, it is preferable that the first nucleic acid strand and/or the second nucleic acid strand and the functional moiety are directly bound to each other via a covalent bond, an ionic bond, a hydrogen bond, or the like, and a covalent bond is more preferable from the viewpoint of obtaining a more stable bond.
  • the structure of the "functional portion” is not particularly limited, and it confers a desired function to the double-stranded nucleic acid complex to which it is bound.
  • the desired function include a labeling function, a purification function, and a target delivery function.
  • the portion that confers the labeling function is, for example, a lipid or a peptide, and specific examples thereof include compounds such as fluorescent proteins and luciferase.
  • the portion that confers the purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.
  • a molecule having an activity of delivering the double-stranded nucleic acid complex in a certain embodiment to a target site is bound as a functional portion to the first nucleic acid strand and/or the second nucleic acid strand.
  • the portion that confers a target delivery function include lipids, antibodies, aptamers, and ligands for specific receptors.
  • the first nucleic acid strand and/or the second nucleic acid strand, e.g., the second nucleic acid strand, is bound to a lipid.
  • the lipid may include, but is not limited to, tocopherol, cholesterol, fatty acids, phospholipids (e.g., phosphatidylethanolamine) and analogs thereof; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstane and analogs thereof; steroids and analogs thereof; ligands of LDLR, SRBI, or LRP1/2; FK-506, and cyclosporine; lipids described in WO2019/182109 and WO2019/177061, etc.
  • the lipid may be tocopherol or an analog thereof and/or cholesterol or an analog thereof, a substituted or unsubstituted C 1-30 alkyl group, a substituted or unsubstituted C 2-30 alkenyl group, or a substituted or unsubstituted C 1-30 alkoxy group.
  • the second nucleic acid strand may be conjugated to tocopherol or cholesterol or an analogue thereof.
  • tocopherol is a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) with a ring structure called chroman.
  • Tocorol has a strong antioxidant effect, and therefore, as an antioxidant in the body, it has the function of eliminating free radicals generated by metabolism and protecting cells from damage.
  • Tocopherol is known in several different forms, consisting of ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -tocopherol, and ⁇ -tocopherol, based on the position of the methyl group bound to the chroman.
  • tocopherol may be any tocopherol.
  • examples of tocopherol analogs include various unsaturated analogs of tocopherol, such as ⁇ -tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol, and ⁇ -tocotrienol.
  • the tocopherol is ⁇ -tocopherol.
  • cholesterol refers to a type of sterol, also known as a steroid alcohol, and is found in large amounts in animals. Cholesterol plays an important role in metabolic processes in the body, and in animal cells, it is also a major component of the cell membrane system along with phospholipids. Furthermore, cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols with a sterol skeleton, and include, but are not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.
  • analog or “derivative” refers to a compound that has an identical or similar basic skeleton and similar structure and properties. Analogs include, for example, biosynthetic intermediates, metabolic products, and compounds with substituents. Those skilled in the art can determine whether a compound is an analog of another compound based on their common technical knowledge.
  • the functional moiety may be linked to the 5' end, or the 3' end, or both ends of the first and/or second nucleic acid strand.
  • the functional moiety may be linked to an internal nucleotide of the first and/or second nucleic acid strand.
  • the first and/or second nucleic acid strand may contain two or more functional moieties, such as lipids, which may be linked to multiple positions on the first and/or second nucleic acid strand and/or may be linked as a group to one position on the first and/or second nucleic acid strand.
  • the functional moieties may be linked to the 5' end and the 3' end of the first and/or second nucleic acid strand, one each.
  • the bond between the first and/or second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond mediated by another substance. However, in certain embodiments, it is preferred that the functional moiety is directly bonded to the first and/or second nucleic acid strand via a covalent bond, ionic bond, hydrogen bond, etc., and a covalent bond is more preferred in terms of obtaining a more stable bond.
  • the functional portion may also be linked to the first and/or second nucleic acid strands via a cleavable or uncleavable linker.
  • the first and second nucleic acid strands may be linked via a linker to form a single strand.
  • the functional region has the same structure as in the double-stranded nucleic acid complex, and therefore, in this specification, such a single-stranded nucleic acid is also included as an embodiment of the double-stranded nucleic acid complex of the present invention.
  • the linker may be any polymer. Examples include polynucleotides, polypeptides, and alkylenes.
  • the linker may be composed of natural nucleotides such as DNA and RNA, or non-natural nucleotides such as peptide nucleic acids and morpholino nucleic acids.
  • the chain length of the linker may be at least 1 base, for example, 3 to 10 bases or 4 to 6 bases. The chain length is preferably 4 bases.
  • the linker may take the form of a hinge (hairpin loop).
  • the linker can be located on either the 5' or 3' side of the first nucleic acid strand, but for example, in the case of a configuration in which the second nucleic acid strand is bound to the 5' side 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 are linked via a linker.
  • “Cleavable linker” refers to a linking group that is cleaved under physiological conditions, e.g., within a cell or within an animal (e.g., within the human body). In certain embodiments, the cleavable linker is selectively cleaved by an endogenous enzyme, such as a nuclease. Cleavable linkers include amides, esters, phosphodiesters or both esters, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers.
  • Non-cleavable linker means a linker that is not cleaved under physiological conditions, for example, within a cell or an animal body (for example, within the human body).
  • Non-cleavable linkers include, but are not limited to, linkers consisting of phosphorothioate bonds, and modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by phosphorothioate bonds.
  • the linker is a nucleic acid such as DNA or an oligonucleotide
  • the chain length is not particularly limited, but may be usually 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.
  • linker represented by the following formula (IV):
  • L 2 represents a substituted or unsubstituted C 1 -C 12 alkylene group (e.g., propylene, hexylene, dodecylene), a substituted or unsubstituted C 3 -C 8 cycloalkylene group (e.g., cyclohexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, or CH(CH 2 -OH)-CH 2 -O- (CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -;
  • L 3 represents -NH- or a bond;
  • L 4 represents a substituted or
  • L5 represents an alkylene group having 1 to 12 carbon atoms (e.g., ethylene, pentylene, heptylene, undecylene), a substituted or unsubstituted cycloalkylene group having 3 to 8 carbon atoms (e.g., cyclohexylene), -( CH2
  • the linker represented by formula (VI) is one in which L 2 is an unsubstituted C 3 to C 6 alkylene group (e.g., propylene, hexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, or -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, L 3 is -NH-, and L 4 and L 5 are bonds.
  • L 2 is an unsubstituted C 3 to C 6 alkylene group (e.g., propylene, hexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, or -(CH 2 ) 2 -O-(CH 2 )
  • the linker includes a nucleic acid, a polyether group, and/or an alkylamino group.
  • the nucleic acid may be, for example, one or two to ten nucleosides and/or non-natural nucleosides linked by internucleoside bonds.
  • Examples of polyether groups include polyethylene glycol groups, triethylene glycol groups, and tetraethylene glycol groups.
  • alkylamino groups include hexylamino groups.
  • the base length of the first nucleic acid strand and the second nucleic acid strand is not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases.
  • the base length of the first nucleic acid strand and the second nucleic acid strand 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 nucleic acid strand and the second nucleic acid strand may be the same length or different lengths (for example, one of them may be 1 to 3 bases shorter or longer).
  • the double-stranded structure formed by the first nucleic acid strand and the second nucleic acid strand may include a bulge.
  • the length can be selected based on the balance between the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield.
  • the overall base length of the first nucleic acid strand and the second nucleic acid strand may be the above-mentioned base length plus the base length of the bound nucleic acid.
  • 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, or may be 100 bases or less, 80 bases or less, 60 bases or less, 40 bases or less, or 30 bases or less.
  • the nucleic acid drug is an siRNA.
  • siRNA short-interfering RNA refers to a double-stranded nucleic acid having about 19 to 25 base pairs that can induce suppression of expression of a target gene by RNAi.
  • An siRNA is composed of two nucleic acid strands, a guide strand and a passenger strand, which will be described later.
  • the two nucleic acid strands that constitute the siRNA of the present invention can contain not only ribonucleosides but also deoxynucleosides and/or any modified nucleoside.
  • siRNAs that can induce RNAi activity are not limited to those consisting of ribonucleosides, and siRNAs containing deoxynucleosides or modified nucleosides can also be incorporated into the RISC described later to recognize target mRNA.
  • RNAi RNA interference
  • siRNA RNA interference
  • RNAi by siRNA can be explained as follows. First, one strand of the siRNA introduced into the cell is incorporated into a complex called RISC (RNA-induced Silencing Complex), which recognizes the mRNA of the target gene, which has a highly complementary sequence. The mRNA of the target gene is cleaved by RISC at the center of the highly complementary sequence. The cleaved mRNA can then be degraded.
  • RISC RNA-induced Silencing Complex
  • the siRNA is composed of a guide strand that can hybridize to at least a portion of a target gene or its transcription product, and a passenger strand that contains a base sequence complementary to the guide strand.
  • guide strand refers to a nucleic acid strand that contains a sequence complementary to the mRNA of a target gene.
  • passenger strand refers to a nucleic acid strand that contains a sequence complementary to the guide strand (i.e., contains a sequence homologous to the mRNA of a target gene).
  • the guide strand anneals with the passenger strand to generate siRNA.
  • the guide strand can bind to the mRNA of a target gene to induce RNAi.
  • the siRNA can include one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides. These modified nucleosides can be located in the guide strand and/or passenger strand of the siRNA.
  • the siRNA can also include the various configurations described above for the double-stranded nucleic acid complex. That is, the guide strand of the siRNA of the present invention can include each of the configurations described above for the first nucleic acid strand, and similarly, the passenger strand can include each of the configurations described above for the second nucleic acid strand.
  • the siRNA of the present invention may be bound to a functional moiety.
  • the functional moiety may be a lipid or a peptide, as in the case of the double-stranded nucleic acid complex.
  • the lipid may also be cholesterol or an analog thereof, tocopherol or an analog thereof, folic acid, phosphatidylethanolamine, or a substituted or unsubstituted alkyl group having 16 to 30 carbon atoms.
  • the nucleic acid drug of the present invention may contain two siRNAs (hereinafter, each siRNA is referred to as the first siRNA and the second siRNA).
  • each siRNA is referred to as the first siRNA and the second siRNA.
  • the guide strands contained in the two siRNAs may be linked to each other via a linker.
  • the linker binding positions in the first guide strand and the second guide strand are not particularly limited and may be the 5' end and/or the 3' end, respectively, but it is preferable that the 3' end of the first guide strand and the 3' end of the second guide strand are linked to the linker.
  • linkers are similar to those described for the double-stranded nucleic acid complex, and may include, for example, a nucleic acid, a polyether group, and/or an alkylamino group.
  • the structure in which two siRNAs are linked is known as a divalent siRNA (Alterman J.F. et al., Nature Biotechnology, 2019, 37:884-894).
  • the delivery enhancer of the present invention can increase the efficiency of delivery of a drug to the central nervous system by using the delivery enhancer of the present invention in combination with a drug to be delivered to the nervous system.
  • the administration route and/or administration timing of the drug and the delivery enhancer of the present invention may be the same or different.
  • administration form of the delivery enhancer of this embodiment and the drug to be delivered to the nervous system may be systemic or local.
  • the route of administration may be oral or parenteral.
  • parenteral administration include intrathecal administration (intracerebroventricular administration, posterior fossa puncture, or lumbar puncture), intranasal administration, intravenous administration, intraarterial administration, administration by blood transfusion, intraperitoneal administration, intraocular administration, intramuscular administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, intravesical administration, intravaginal administration, rectal administration, inhalation or nasal administration, and tracheal/bronchial administration.
  • Intrathecal administration may be administered using a shunt, an indwelling catheter, or a subcutaneous port.
  • the administration form of the delivery enhancer of this embodiment and the drug to be delivered to the nervous system may be independently selected from the administration methods described above.
  • the delivery enhancer of this aspect is administered intranasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly, and the agent to be delivered to the nervous system is administered intrathecally.
  • the delivery enhancer of this embodiment and the drug to be delivered to the nervous system may be administered simultaneously.
  • they may be administered as a composition, as described below, that includes the delivery enhancer of this embodiment and the drug to be delivered to the nervous system.
  • the delivery enhancer of this embodiment is administered before or after the drug to be delivered to the nervous system.
  • the delivery enhancer of this embodiment and the drug to be delivered to the nervous system may be administered within one week, three days, one day, 12 hours, six hours, three hours, or one hour.
  • the delivery enhancer of this embodiment is administered intravenously or subcutaneously before the drug, and the drug to be delivered to the nervous system is administered intrathecally after the delivery enhancer.
  • the dosage of the delivery enhancer of this embodiment can be appropriately selected as an effective amount depending on the type of drug used in combination and the target disease.
  • the delivery enhancer may be administered at 0.01 mg or more, 0.1 mg or more, or 1 mg or more, for example, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 75 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, or 500 mg or more, or 0.01 mg to 10 g, 0.1 mg to 1 g, or 1 mg to 100 mg, and in the case of mice, 1 ⁇ g or more may be administered.
  • the delivery enhancer may be administered in a single dose or multiple doses.
  • the dosage of the drug to be delivered to the nervous system can be appropriately selected as an effective amount depending on the type of drug and target disease.
  • the drug such as a nucleic acid drug may be administered at 0.01 mg or more, 0.1 mg or more, or 1 mg or more, for example, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 75 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, or 500 mg or more, or 0.01 mg to 1000 mg, 0.1 mg to 200 mg, or 1 mg to 20 mg, and in the case of mice, 1 ⁇ g or more may be administered.
  • the drug to be delivered to the nervous system may be administered in a single dose or multiple doses.
  • the dosage of a drug, such as a nucleic acid drug, to be delivered to the nervous system may be, for example, 0.00001 mg/kg/day to 10,000 mg/kg/day, or 0.001 mg/kg/day to 100 mg/kg/day.
  • the drug may be administered in a single dose or multiple doses. In the case of multiple doses, the drug 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 dosage of a single dose of a drug such as a nucleic acid drug may be, for example, 0.001 mg/kg or more, 0.005 mg/kg or more, 0.01 mg/kg or more, 0.25 mg/kg or more, 0.5 mg/kg or more, 1.0 mg/kg or more, 2.0 mg/kg or more, 2.5 mg/kg or more, 3.0 mg/kg or more, 4.0 mg/kg or more, 5 mg/kg or more, 10 mg/kg or more, 20 mg/kg or more, 30 mg/kg or more, 40 mg/kg or more, 50 mg/kg or more, 75 mg/kg or more, 100 mg/kg or more, g/kg or more, 150 mg/kg or more, 200 mg/kg or more, 300 mg/kg or more, 400 mg/kg or more, or 500 mg/kg or more, and can be appropriately selected from any amount within the range of, for example, 0.001 mg/kg to 500 mg/kg (e.g., 0.001 mg/kg, 0.01 mg/kg, 0.1 mg
  • the delivery enhancer of the present invention By using the delivery enhancer of the present invention in combination with a drug such as a nucleic acid drug, the delivery efficiency of the drug can be dramatically improved. Therefore, the delivery enhancer of the present invention can be called a delivery enhancer or a delivery auxiliary.
  • the present invention also provides a method for improving the efficiency of delivery of a drug to the central nervous system and/or peripheral nervous system, comprising administering a delivery enhancer of the present invention to a subject, as compared to a subject not administered the delivery enhancer of the present invention.
  • a second aspect of the present invention is a pharmaceutical composition for treating a central or peripheral nervous system disorder, comprising a therapeutically effective amount of a drug and a delivery enhancer as described in the first aspect.
  • the pharmaceutical composition of this embodiment contains a drug and a delivery enhancer as active ingredients.
  • the composition of the drug and the delivery enhancer are similar to those described in the first embodiment, and therefore the description thereof will be omitted here.
  • the amount (content) of the drug and delivery enhancer contained in the pharmaceutical composition of this embodiment varies depending on the type of drug and delivery enhancer, the delivery site, the dosage form of the composition, the dosage amount of the composition, and the type of carrier described below. Therefore, it may be determined appropriately taking into account each condition.
  • the composition is adjusted so that an effective amount of the drug is contained in a single dose.
  • An "effective amount” refers to an amount that is necessary for the drug to function as an active ingredient and that gives little or no harmful side effects to the living body to which it is applied. This effective amount may vary depending on various conditions such as information on the subject, the route of administration, and the number of administrations. It is ultimately determined by the judgment of a doctor, veterinarian, pharmacist, etc.
  • Subject information refers to various individual information of the living body to which the composition is applied. For example, if the subject is a human, it includes age, weight, sex, diet, health condition, progression and severity of the disease, drug sensitivity, and the presence or absence of concomitant drugs.
  • the pharmaceutical composition of this embodiment can include a pharma- ceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to an additive commonly used in the field of formulation technology.
  • solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, excipients, vehicles, preservatives, binders, diluents, isotonicity agents, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, thickeners, dissolution aids, and other additives can be mentioned.
  • the solvent may be, for example, water or any other pharma- ceutically acceptable aqueous solution, or a pharma-ceutically acceptable organic solvent.
  • aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer.
  • adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.
  • the above-mentioned carriers are used to avoid or suppress the decomposition of the drug and delivery enhancer by enzymes in the body, to facilitate formulation and administration, and to maintain the dosage form and efficacy, and may be used appropriately as needed.
  • the dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a form that can deliver the drug and delivery enhancer to the target site without inactivating them by decomposition or the like, and can exert the pharmacological effect of the active ingredients (antisense effect on the expression of the target gene) in the body.
  • the specific dosage form may be a dosage form suitable for intrathecal administration.
  • an example of a preferred dosage form is an injection.
  • the injection can be formulated by appropriately combining the above-mentioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH regulators, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.
  • each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art.
  • the pharmaceutical composition of this embodiment may be formulated according to standard methods in the art.
  • the dosage form and dosage amount of the pharmaceutical composition of this embodiment are not particularly limited, and should conform to the description of "(Dosage form of delivery enhancer/drug)" in the first embodiment.
  • Administration may be systemic or local.
  • the route of administration may be oral or parenteral.
  • parenteral administration include intrathecal administration (intraventricular administration, posterior fossa puncture, or lumbar puncture), nasal administration, intravenous administration, intraarterial administration, administration by blood transfusion, intraperitoneal administration, intraocular administration, intramuscular administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, intravesical administration, intravaginal administration, rectal administration, inhalation or nasal drop administration, and tracheal/bronchial administration.
  • intrathecal administration intraventricular administration, posterior fossa puncture, or lumbar puncture
  • nasal administration intravenous administration
  • intraarterial administration administration by blood transfusion
  • intraperitoneal administration intraocular administration
  • intramuscular administration subcutaneous administration (including implantable continuous subcutaneous administration)
  • intradermal administration intravesical administration
  • intravaginal administration rectal administration
  • inhalation or nasal drop administration and tracheal/bronchial administration.
  • intrathecal administration and nasal administration which are advantageous for delivery to the target site, are preferred, but delivery to the central nervous system is also possible by passing through the blood-brain barrier, for example, by intravenous administration, subcutaneous administration, intraperitoneal administration, oral administration, inhalation, or intramuscular administration.
  • the diseases to which the pharmaceutical composition is applied are, for example, central nervous system diseases or peripheral nervous system diseases.
  • the target diseases can be diseases that can be controlled by delivering the drug contained in the pharmaceutical composition of the present invention to the central nervous system and/or peripheral nervous system.
  • the target diseases can be diseases that involve genes that can suppress or enhance the expression level of a target gene or its transcription product or translation product, inhibit the function of the transcription product or translation product, or induce steric blocking, splicing switch, RNA editing, exon skipping, or exon inclusion due to its antisense effect.
  • the pharmaceutical composition may be used in animals, including humans, as subjects. However, there is no particular limitation on animals other than humans, and various livestock, poultry, pets, laboratory animals, etc. may be subjects in some embodiments.
  • the subject may be one in which it is necessary to reduce the expression level of a target transcript in the central and/or peripheral nervous system.
  • the subject may also be one in which it is necessary to treat a neurological disorder, such as a brain disorder.
  • the disease to be treated may be a central nervous system disease or peripheral nervous system disease associated with increased or decreased gene expression, particularly a disease (such as a tumor) associated with increased expression of a target transcript or target gene.
  • central nervous system diseases include, but are not limited to, brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, etc.
  • the pharmaceutical composition of this aspect can target central nervous system diseases other than Alzheimer's disease, cerebral infarction, brain tumor, demyelinating disease, epilepsy, and neuropathic pain.
  • the nervous system is divided into the central nervous system and the peripheral nervous system.
  • the central nervous system consists of the brain and spinal cord.
  • the brain includes the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon (thalamus, subthalamic nucleus), cerebellum (cerebellar cortex, cerebellar nuclei), and brainstem (midbrain, substantia nigra, pons, medulla oblongata).
  • the spinal cord includes the cervical, thoracic, lumbar, sacral, and coccygeal spinal cord.
  • the central nervous system in this specification may be any of these regions, but in particular may be the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brainstem, cervical spinal cord, thoracic spinal cord, or lumbar spinal cord.
  • the peripheral nerves consist of the cranial nerves and spinal nerves, and include the spinal cord anterior root, dorsal root, cranial nerves 1 to 12, cauda equina, and dorsal root ganglion.
  • central nervous system disease refers to a disease associated with any part of the central nervous system or the entire central nervous system.
  • the central nervous system includes the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon (thalamus, subthalamic nucleus), cerebellum (cerebellar cortex, cerebellar nuclei), and brain stem (midbrain, substantia nigra, pons, medulla oblongata).
  • the spinal cord includes the cervical, thoracic, lumbar, sacral, and coccygeal spinal cord.
  • the central nervous system disease may be a disease associated with any of these parts, but may particularly be a disease associated with the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brain stem, cervical spinal cord, thoracic spinal cord, or lumbar spinal cord.
  • cerebral cortex frontal lobe, temporal lobe, parietal lobe, occipital lobe
  • cerebellum cerebellum
  • striatum striatum
  • globus pallidus globus pallidus
  • claustrum claustrum
  • hippocampus hippocampus
  • parahippocampal gyrus brain stem
  • cervical spinal cord thoracic spinal cord
  • lumbar spinal cord lumbar spinal cord
  • FTD frontotemporal dementia
  • SD semantic dementia
  • PNFA progressive non-fluent aphasia
  • Pick's disease drug delivery to the frontal lobe, temporal lobe and/or substantia nigra may be effective.
  • Parkinson's disease dementia drug delivery to the occipital lobe, substantia nigra and/or striatum may be effective.
  • drug delivery to the substantia nigra and/or striatum may be effective.
  • corticobasal degeneration In the treatment of corticobasal degeneration (CBD), drug delivery to the frontal lobe, parietal lobe, basal ganglia and/or substantia nigra may be effective. In the treatment of progressive supranuclear palsy (PSP), drug delivery to the frontal lobe, basal ganglia and/or substantia nigra may be effective. In the treatment of amyotrophic lateral sclerosis, drug delivery to the frontal lobe, parietal lobe, basal ganglia, and/or substantia nigra may be effective.
  • CBD corticobasal degeneration
  • PPSP progressive supranuclear palsy
  • amyotrophic lateral sclerosis drug delivery to the frontal lobe, parietal lobe, basal ganglia, and/or substantia nigra may be effective.
  • SCD spinocerebellar degeneration
  • DPLA dentatorubral-pallidoluysian degeneration
  • SBMA spinal-bulbar atrophy
  • FA Friedreich's ataxia
  • striatum In the treatment of Huntington's disease, drug delivery to the striatum, frontal lobe, parietal lobe, and/or basal ganglia may be effective.
  • prion diseases mad cow disease, GSS
  • drug delivery to the cerebral cortex, cerebral white matter, basal ganglia and/or substantia nigra In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective.
  • encephalitis viral, bacterial, fungal, tuberculous
  • meningitis viral, bacterial, fungal, tuberculous
  • drug delivery to the entire brain may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • cerebral infarction cerebral hemorrhage, subarachnoid hemorrhage, moyamoya disease, and anoxic encephalopathy
  • drug delivery to the entire brain may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • head trauma drug delivery to the entire brain may be effective.
  • MM ⁇ sclerosis multiple sclerosis
  • NMO neuromyelitis optica
  • drug delivery to the cerebral white matter, cerebral cortex, optic nerve, and/or spinal cord may be effective.
  • myotonic dystrophy DM1, DM2
  • drug delivery to skeletal muscle, cardiac muscle, cerebral cortex, and/or cerebral white matter may be effective.
  • HSP familial spastic paraplegia
  • drug delivery to the parietal lobe and/or spinal cord may be effective.
  • Fukuyama muscular dystrophy drug delivery to skeletal muscle, cerebral cortex, and/or cerebral white matter may be effective.
  • ⁇ nigra In the treatment of dementia with Lewy bodies (DLB), drug delivery to the substantia nigra, striatum, occipital lobe, frontal lobe, and/or parietal lobe may be effective.
  • MSA multiple system atrophy
  • drug delivery to the striatum, basal ganglia, cerebellum, substantia nigra, frontal lobe, and/or temporal lobe In the treatment of Alexander disease, drug delivery to the cerebral white matter may be effective. In the treatment of CADASIL and CARASIL, drug delivery to the cerebral white matter may be effective.
  • peripheral nervous system disease refers to a disease associated with any part of the peripheral nervous system or the entire peripheral nervous system.
  • the peripheral nerves consist of the cranial nerves and spinal nerves, and include the ventral root of the spinal cord, the dorsal root, the first to twelfth cranial nerves, the cauda equina, and the dorsal root ganglion.
  • the peripheral nervous system disease may be a disease associated with any of these parts, but may particularly be a disease associated with the ventral root of the spinal cord, the dorsal root, the first to twelfth cranial nerves, the cauda equina, or the dorsal root ganglion.
  • the pharmaceutical composition of the present invention contains a delivery enhancer in addition to a therapeutically effective amount of a drug, thereby dramatically improving the efficiency of delivery of the drug to the central nervous system and/or peripheral nervous system, thereby increasing the drug efficacy.
  • the present invention also provides a method for treating and/or preventing a disease, such as a central nervous system disease or a peripheral nervous system disease, which comprises administering (e.g., intrathecally administering) the pharmaceutical composition of this embodiment to a subject, such as a human.
  • a disease such as a central nervous system disease or a peripheral nervous system disease
  • administering e.g., intrathecally administering
  • Example 1 Identification of novel compounds that enhance the delivery efficiency of antisense nucleic acids to the nervous system and the gene suppression effect in the nervous system (the purpose)
  • the candidate compound TGN-073 is administered intracerebroventricularly in mice together with ASO.
  • the delivery efficiency of antisense nucleic acid to the central nervous system and the effect of coadministration of TGN-073 on the target gene suppression effect in the central nervous system are verified by in vivo experiments.
  • Malat1 target ASO is used as ASO.
  • the compound represented by the above formula (I) can also be written as 2-(phenylsulfonamido)-3-benzyloxypyridine.
  • the compound represented by the above formula (I) is called “TGN-073” in the previous publication by the present inventors (Huber VJ et al., NeuroReport, 2018, 29(9):697-703), and is called “2A” in International Publication No. 2017/150704.
  • TGN-073 the compound represented by the above formula (I) and its salts are referred to as "TGN-073".
  • TGN-073 The synthesis of TGN-073 was carried out according to the method for preparing compound 2A described in WO 2017/150704. Specifically, 2-amino-3-benzyloxypyridine (1.50 g, 7.50 mmol) was added to a 50 mL round-bottom flask containing dry dichloromethane (35 mL). Aqueous 2,6-lutidine (2.62 mL, 22.5 mmol) was then added to the stirring solution using a syringe, and the reaction vessel was backfilled with argon. Benzenesulfonyl chloride (1.05 mL, 8.25 mmol) was then added to the stirring solution using a syringe.
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid targeting mouse metastasis associated lung adenocarcinoma transcript 1 (Malat1) non-coding RNA, and has a base sequence complementary to a portion of Malat1 RNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them linked by phosphorothioate bonds. All oligonucleotides were synthesized by Gene Design Co., Ltd. (Osaka, Japan).
  • ASO/TGN-073 co-administration group ASO mixed with TGN-073 was administered, and in the group administered only with ASO (hereinafter referred to as the "ASO alone administration group"), only ASO was administered without mixing with TGN-073.
  • the dose of TGN-073 in the ASO/TGN-073 co-administration group was 0.25 mg per mouse. After intraventricular administration, the skin was sutured with nylon thread.
  • mice administered only TGN-073 hereinafter referred to as the "TGN-073 administration group”
  • mice administered only PBS hereinafter referred to as the "PBS administration group” were also prepared.
  • Figure 3A shows the ASO concentration in the hippocampus 3 hours or 7 days after intracerebroventricular administration.
  • the ASO/TGN-073 co-administration group showed a dramatic increase in ASO concentration in the brain (amount of nucleic acid delivered to the brain) compared with the ASO alone administration group.
  • Figure 3B shows the levels of Malat1 RNA expression in the hippocampus 7 days after intracerebroventricular administration.
  • the ASO/TGN-073 co-administration group showed a significantly improved inhibitory effect on the Malat1 gene compared to the ASO alone administration group.
  • Example 2 Dose dependency of ASO administered intracerebroventricularly with TGN-073 (the purpose) In order to quantitatively evaluate the effects of TGN-073 found in Example 1, various concentrations of ASO will be administered intracerebroventricularly together with TGN-073, and the delivery efficiency of ASO to the central nervous system and its effect of suppressing target genes in the central nervous system will be verified through in vivo experiments.
  • Example 1 the ASO targeting Malat1 used in Example 1 (ASO listed in Table 1) was used. Preparation of nucleic acid, in vivo experiment, and evaluation of ASO concentration in the brain (amount of nucleic acid delivered to the brain) and target gene suppression effect were performed according to Example 1. However, in this example, the ASO dosage per mouse was 1.25 ⁇ g, 5 ⁇ g, and 20 ⁇ g in the ASO alone administration group, and 0.075 ⁇ g, 0.3 ⁇ g, and 1.25 ⁇ g in the ASO/TGN-073 co-administration group.
  • Figure 4A shows the ASO concentration in the hippocampus 7 days after intraventricular administration of the nucleic acid agent.
  • the ASO/TGN-073 co-administration group achieved the same amount of intracerebral delivery at a concentration approximately 16- to 20-fold lower than the ASO alone administration group.
  • Figure 4B shows the levels of Malat1 RNA expression in the hippocampus 7 days after intracerebroventricular administration of the nucleic acid agent.
  • the ASO/TGN-073 co-administration group achieved the same gene suppression effect at approximately 16- to 20-fold lower concentrations than the ASO alone administration group.
  • Example 3 Effect of coadministration of TGN-073 with intracerebroventricular administration of Mapt-targeting ASO (the purpose) ASO targeting the Mapt gene (hereinafter referred to as "Mapt-targeted ASO") will be co-administered intracerebroventricularly with TGN-073.
  • Mapt-targeted ASO ASO targeting the Mapt gene
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets mouse microtubule-associated protein tau (Mapt) mRNA, has a base sequence complementary to a portion of Mapt mRNA, and has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds.
  • Mapt mouse microtubule-associated protein tau
  • nucleic acid preparation for the ASOs listed in Table 2, nucleic acid preparation, in vivo experiments, and evaluation of the target gene suppression effect were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid agent administered per mouse was 9.4 nmol (50 ⁇ g).
  • each category includes two behavioral evaluation items. Each behavioral evaluation item is scored on a five-point scale from 0 to 4 points ( Figure 6, scores 0 to 4), with normal being scored as 0 and higher scores indicating higher toxicity. In each category, the higher score of the two behavioral evaluation items is adopted as the score for that category. The sum of the scores for the five categories represents the acute tolerability score (0 to 20 points).
  • Figure 5 shows the expression levels of Mapt RNA in the hippocampus 7 days after intracerebroventricular administration of the nucleic acid agent.
  • the ASO/TGN-073 co-administration group showed a significant increase in the target gene suppression effect compared to the ASO alone administration group.
  • Figure 7 shows the results of the central neurotoxicity evaluation.
  • the group administered TGN-073 alone had an acute tolerability score of 0 at 30 minutes, 1 hour, and 2 hours after intracerebroventricular administration, showing no toxicity. Furthermore, the acute tolerability scores of the ASO/TGN-073 co-administration group were almost equivalent to those of the ASO alone administration group.
  • Figure 8 shows the results of evaluating motor function in an open field test 1 hour after administration.
  • the ASO/TGN-073 co-administration group showed nearly equivalent maximum movement speed (Figure 8A) and movement time (Figure 8B) compared to the ASO alone administration group.
  • Example 4 Effect of coadministration of TGN-073 with intracerebroventricular administration of 2'-O-MOE-modified ASO (the purpose)
  • the Malat1 targeting ASO used in Example 1 is co-administered with TGN-073 when the ASO is intracerebroventricularly administered, in which 2'-MOE modification is introduced into the wing region as a different nucleic acid chemical modification.
  • the effect of TGN-073 co-administration on the target gene suppression effect in the central nervous system is verified by in vivo experiments.
  • the ASO used in this example is a 2'-O-MOE-RNA/DNA gapmer-type antisense nucleic acid that targets Malat1 non-coding RNA, has a base sequence complementary to a portion of Malat1 ncRNA, and has a structure in which five 2'-O-MOE-RNA nucleosides at the 5' end, five 2'-O-MOE-RNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds.
  • nucleic acid preparation for the ASOs listed in Table 3, nucleic acid preparation, in vivo experiments, and evaluation of the target gene suppression effect were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid agent administered per mouse was 1.7 nmol (12.5 ⁇ g).
  • Figure 9 shows the levels of Malat1 RNA expression in the hippocampus 7 days after intracerebroventricular administration.
  • the ASO/TGN-073 co-administration group showed enhanced target gene silencing effects compared to the ASO alone administration group.
  • Example 5 Effect of coadministration of TGN-073 with intracerebroventricular administration of heteroduplex nucleic acid (the purpose) TGN-073 is co-administered when a heteroduplex oligonucleotide (hereinafter referred to as "HDO") containing a first nucleic acid strand consisting of the single-stranded ASO used in Example 1 and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is intracerebroventricularly administered.
  • HDO heteroduplex oligonucleotide
  • the ASOs listed in Table 4 were prepared in a manner similar to that of Example 1.
  • the heteroduplex oligonucleotide (HDO) used in this example contains the above-mentioned ASO as a first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • Heteroduplex oligonucleotides with cholesterol bound to the 5' end contain the above ASO as the first nucleic acid strand, and cholesterol is linked to the 5' end of the second nucleic acid strand.
  • Figure 11A shows the ASO concentration in the hippocampus 7 days after intracerebroventricular administration. All groups co-administered with TGN-073 along with ASO, HDO, and Chol-HDO showed a dramatic increase in ASO concentration in the central nervous system compared to groups administered with the nucleic acid agent alone without co-administration of TGN-073.
  • Figure 11B shows the levels of Malat1 RNA expression in the hippocampus 7 days after intracerebroventricular administration.
  • the effect of target gene inhibition in the central nervous system was significantly improved compared to the groups in which each nucleic acid agent was administered alone without co-administration of TGN-073.
  • TGN-073 can dramatically improve the amount of nucleic acid delivered to the central nervous system and the effect of suppressing target genes in the central nervous system, not only for single-stranded nucleic acid agents but also for double-stranded nucleic acid agents.
  • Example 6 Effect of coadministration of TGN-073 with intracerebroventricular administration of siRNA (the purpose) siRNA will be co-administered intracerebroventricularly with TGN-073, and the effect of co-administration of TGN-073 on target gene silencing in the central nervous system will be examined in vivo.
  • the siRNA used in this example was an siRNA targeting copper/zinc superoxide dismutase 1 (Sod1) mRNA, and for the siRNAs listed in Table 5, in vivo experiments and evaluation of the target gene suppression effect were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid agent administered per mouse was 7.0 nmol (100 ⁇ g).
  • (result) 12 shows the Sod1 mRNA expression level in the hippocampus 7 days after intracerebroventricular administration.
  • the TGN-073 co-administration group showed a marked increase in the effect of suppressing the target gene in the brain compared to the siRNA alone administration group.
  • Example 7 TGN-073 derivatives (the purpose) To investigate the effect of TGN-073 derivatives, ASO targeting the Mapt gene will be co-administered with TGN-073-mes, a derivative of TGN-073, when administered intracerebroventricularly. The amount of ASO delivered to the central nervous system and its effect on target gene suppression in the central nervous system will be verified by in vivo experiments.
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets mouse Mapt mRNA, has a base sequence complementary to a portion of Mapt mRNA, and has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds.
  • TGN-073-mes which is co-administered with ASO in this embodiment, is a derivative of TGN-073X shown in formula (I) above, and its chemical structure is shown in formula (III) below.
  • TGN-073-mes the compound represented by the above formula (III) and its salts are referred to as "TGN-073-mes.”
  • TGN-073-mes the sodium salt of the compound represented by the above formula (III) was used.
  • the sodium salt of the compound represented by the above formula (III) is represented by the following formula (VII).
  • nucleic acid preparation for the ASOs listed in Table 6, nucleic acid preparation, in vivo experiments, and evaluation of ASO concentration and target gene suppression effect in the central nervous system were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid administered per mouse was 9.4 nmol (50 ⁇ g).
  • TGN-073-mes was mixed with ASO at a dose of 0.125 mg/mouse and administered intracerebroventricularly.
  • Figure 13 shows the expression levels of Mapt RNA in the hippocampus 7 days after intracerebroventricular administration.
  • the ASO/TGN-073 co-administration group showed a significant increase in ASO concentration in the central nervous system (Figure 13A) and in the target gene silencing effect (Figure 13B) compared with the ASO alone administration group.
  • Example 8 Effect of coadministration of TGN-073 on intracerebroventricular administration of single-chain PMO (the purpose) Phosphorodiamidate morpholino oligomers (hereinafter also referred to as "PMO") are administered intracerebroventricularly to mice as antisense oligonucleotides targeting the exon 23/intron 23 boundary region of dystrophin pre-mRNA for exon skipping.
  • PMO Phosphorodiamidate morpholino oligomers
  • TGN-073 is co-administered to evaluate the effect of TGN-073 co-administration on exon skipping in the brain.
  • the PMO described in Table 7 is a 25-mer single-stranded morpholino nucleic acid that targets exon 23/intron 23 of mouse dystrophin pre-mRNA.
  • the entire 25-mer of this PMO is composed of morpholino nucleic acid, and all internucleoside linkages are phosphorodiamidate bonds.
  • This morpholino nucleic acid has a base sequence complementary to positions 83803536 to 83803512 of mouse dystrophin pre-mRNA (GenBank accession number: NC000086.7).
  • the PMO was contracted for synthesis by Gene Design Co., Ltd. (Osaka, Japan).
  • mice used were 6-week-old male C57BL/6J mice weighing approximately 20 g.
  • 2.5 nmol of PMO (PMO alone administration group) or 2.5 nmol of PMO plus 250 ⁇ g of TGN-073 (PMO/TGN-073 co-administration group) were administered in a volume of 10 ⁇ L into the left ventricle of the mice.
  • mice were dissected and the hippocampus, striatum, and cortex (occipital lobe) were removed. Then, mRNA was extracted from each tissue using Isogen II. One-Step RT-PCR was performed on the extracted total RNA (900 ng) using the Qiagen One Step RT-PCR Kit (Qiagen). The reaction mixture was prepared according to the protocol attached to the kit. The thermal cycler used was LifeECO (Bioer Technology).
  • RT-PCR program used was reverse transcription reaction at 42°C for 30 minutes, followed by thermal denaturation at 95°C for 15 minutes, followed by 35 cycles of PCR amplification reaction (94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds) as one cycle, and a final extension reaction at 72°C for 7 minutes.
  • RT-PCR was performed using the forward (Fw) primer (5'-ATCCAGCAGTCAGAAAGCAAA-3', SEQ ID NO: 10) and reverse (Rv) primer base sequences (5'-CAGCCATCCATTTCTGTAAGG-3', SEQ ID NO: 11).
  • skipping efficiency A/(A+B) x 100.
  • Figure 14 shows the exon 23 skipping efficiency in the hippocampus ( Figure 14A), striatum ( Figure 14B), and cortex ( Figure 14C) 7 days after intracerebroventricular administration. In all brain regions, the PMO/TGN-073 co-administration group showed a significant increase in the skipping efficiency compared to the PMO alone administration group.
  • Example 9 Effect of coadministration of TGN-073 on intracerebroventricular administration of a double-stranded nucleic acid complex containing a PMO (the purpose)
  • a double-stranded nucleic acid complex consisting of a PMO targeting the exon 23/intron 23 boundary region of dystrophin pre-mRNA for exon skipping and its complementary sequence bound to cholesterol (hereinafter referred to as "Chol-HDO (PMO)”) is administered intracerebroventricularly to mice, and TGN-073 is co-administered.
  • PMO complementary sequence bound to cholesterol
  • Chol-HDO (PMO) described in Table 8 is composed of PMO and Chol-cRNA.
  • PMO is the same single-stranded morpholino nucleic acid as in Example 8.
  • Chol-cRNA has a base sequence complementary to the PMO, cholesterol is bound to the 5' side, and has a structure in which three 2'-O-methyl modified RNA nucleosides at the 5' end, three 2'-O-methyl modified RNA nucleosides at the 3' end, and 19 DNA nucleosides between them are linked by phosphorothioate bonds and phosphodiester bonds.
  • PMO and Chol-cRNA were synthesized by Gene Design Co., Ltd.
  • double-stranded Chol-HDO was prepared by annealing the two. Specifically, equimolar amounts of PMO and Chol-cRNA were mixed, and the solution was heated to 95°C for 5 minutes, then cooled to 37°C and held for 1 hour, thereby annealing the nucleic acid strands and preparing the above-mentioned double-stranded nucleic acid agent.
  • Example 10 Effect of coadministration of TGN-073 on spinal delivery of nucleic acid chains (the purpose)
  • the Malat1-targeting ASO used in Example 1 is administered intracerebroventricularly together with TGN-073.
  • the effect of coadministration of TGN-073 on the target gene suppression effect and the amount of nucleic acid delivered in the spinal cord is verified by in vivo experiments.
  • the ASO used in this example was the Malat1-targeting ASO described in Example 1. Specifically, for the ASOs listed in Table 1, nucleic acid preparation, in vivo experiments, and evaluation of the target gene suppression effect were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid agent administered per mouse was 1.7 nmol (12.5 ⁇ g), and the amount of nucleic acid delivered and the target gene suppression effect were evaluated in the lumbar spinal cord.
  • Figure 17A shows the ASO concentration in the lumbar spinal cord 7 days after intracerebroventricular administration.
  • the ASO/TGN-073 co-administration group showed a dramatic increase in ASO concentration in the lumbar spinal cord compared to the ASO alone administration group.
  • Figure 17B shows the Malat1 RNA expression levels in the lumbar spinal cord 7 days after intracerebroventricular administration.
  • the ASO/TGN-073 co-administration group showed significantly improved target gene suppression effects in the lumbar spinal cord compared to the ASO alone administration group.
  • Example 11 Effect of TGN-073 co-administration with intrathecal ASO administration (the purpose) The effect of TGN-073 co-administration will be verified by intrathecal administration, which is non-intracerebroventricular administration. Specifically, the Mapt-targeted ASO used in Example 3 will be administered intrathecally, and the effect of TGN-073 co-administration on the amount of nucleic acid delivered to the spinal cord tissue and hippocampus will be verified by in vivo experiments.
  • Intrathecal administration of ASO and measurement of ASO concentration were performed as follows. A catheter was inserted into the spinal subarachnoid space of 8-week-old Slc:SD rats (male), and 76 nmol (400 ⁇ g) of ASO and/or TGN-073 (1.5 g/rat) were administered intrathecally per rat at 9 weeks of age.
  • rats administered only ASO intrathecally are referred to as the "ASO alone administration group," and rats administered both ASO and TGN-073 intrathecally are referred to as the "ASO/TGN-073 co-administration group.”
  • ASO alone administration group rats administered only ASO and TGN-073 intrathecally are referred to as the "ASO/TGN-073 co-administration group.”
  • ASO/TGN-073 co-administration group Seven days after intrathecal administration, the spinal cord and left hippocampus were removed from the rats, and the ASO concentration in each tissue was evaluated using the same method as in Example 1.
  • Figure 18 shows ASO concentrations in the spinal cord tissue (Figure 18A) and left hippocampus ( Figure 18B) 7 days after intrathecal administration.
  • the ASO/TGN-073 co-administration group showed a dramatic increase in ASO concentrations in the spinal cord and left hippocampus compared to the ASO alone administration group.
  • Example 12 Effect of co-administration of TGN-073 with intracerebroventricular administration of VHH antibody (the purpose) To verify the effect of TGN-073 co-administration on drug modalities other than nucleic acid drugs, we will verify the effect of TGN-073 co-administration on intracerebroventricular administration of VHH antibodies through in vivo experiments in mice.
  • VHH antibody Alpaca anti-Rabbit IgG Nano (VHH) Recombinant Secondary Antibody, Alexa FluorTM 647, Invitrogen, #SA5-10327
  • VHH antibody Alpaca anti-Rabbit IgG Nano (VHH) Recombinant Secondary Antibody, Alexa FluorTM 647, Invitrogen, #SA5-10327
  • TGN-073 250 ⁇ g/mouse
  • mice that received only VHH antibody intracerebroventricularly are referred to as the "VHH antibody alone administration group," and mice that received both VHH antibody and TGN-073 intracerebroventricularly are referred to as the "VHH antibody/TGN-073 co-administration group.”
  • VHH antibody alone administration group mice that received only VHH antibody intracerebroventricularly
  • VHH antibody/TGN-073 co-administration group mice that received both VHH antibody and TGN-073 intracerebroventricularly.
  • Brain tissue samples from the left hippocampus, left occipital cortex, and left basal ganglia were collected from the mice 3 hours after intraventricular administration, and the VHH antibody concentration in each brain tissue was evaluated by measuring the Alexa647 fluorescence intensity per brain weight using a fluorescent plate reader (TECAN, M1000Pro).
  • Figure 19 shows the VHH antibody concentrations in the left hippocampus (Figure 19A), left occipital cortex ( Figure 19B), and left basal ganglia ( Figure 19C) after intraventricular administration.
  • the VHH antibody/TGN-073 co-administration group showed a dramatic increase in VHH antibody concentration in the central nervous system compared to the VHH antibody alone administration group.
  • Example 13 Effect of coadministration of TGN-073 with intracerebroventricular administration of IgG antibody (the purpose) The efficacy of co-administration of TGN-073 for delivery of IgG antibodies will also be verified through in vivo experiments in mice.
  • Alexa647 fluorescently labeled IgG antibody Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 (Invitrogen, #A-21245) was administered at a dose of 10 ⁇ g/mouse, either alone or in combination with TGN-073 (250 ⁇ g/mouse), into the left lateral ventricle of 8-week-old ICR mice (female).
  • mice that received only IgG antibody intracerebroventricular administration will be referred to as the “IgG antibody alone administration group”
  • mice that received both IgG antibody and TGN-073 intracerebroventricular administration will be referred to as the “IgG/TGN-073 co-administration group”.
  • Brain tissue samples from the left hippocampus were collected from the mice 3 hours after intracerebroventricular administration, and the IgG antibody concentration was evaluated by measuring the Alexa647 fluorescence intensity per brain weight using a fluorescent plate reader (TECAN, M1000Pro).
  • Figure 20 shows the IgG antibody concentration in the left hippocampus after intracerebroventricular administration.
  • the IgG/TGN-073 co-administration group showed a dramatic increase in IgG antibody concentration in the central nervous system compared to the IgG antibody alone administration group.
  • TGN-073 can also dramatically improve the efficiency of delivery of IgG antibodies into the central nervous system when administered intracerebroventricularly.
  • Example 14 Effect of coadministration of TGN-073 in intracerebroventricular administration of viral vectors (the purpose) The effect of coadministration of TGN-073 with intracerebroventricular administration of AAV gene therapy drugs will be verified through in vivo experiments in mice.
  • GFP-expressing AAV9 (AAV9-CMV-hrGFP) was administered as an adeno-associated virus (AAV) vector at a dose of 26 ⁇ 10 9 vg/mouse, either alone or in combination with TGN-073 (250 ⁇ g/mouse), into the left lateral ventricle of 7-week-old ICR mice (female).
  • AAV9-CMV-hrGFP adeno-associated virus
  • mice that received only the AAV vector intraventricularly will be referred to as the "AAV alone administration group,” and mice that received both the AAV vector and TGN-073 intraventricularly will be referred to as the "AAV/TGN-073 co-administration group.”
  • AAV alone administration group mice that received only the AAV vector intraventricularly
  • AAV/TGN-073 co-administration group mice that received both the AAV vector and TGN-073 intraventricularly
  • mice that received both the AAV vector and TGN-073 intraventricularly will be referred to as the "AAV/TGN-073 co-administration group.”
  • Figure 21 shows coronal sections at the hippocampal level 3 weeks after intraventricular injection.
  • the AAV/TGN-073 co-injection group showed significantly more extensive GFP expression in the central nervous system than the AAV mono-injection group, and also showed increased expression.

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Abstract

The present invention addresses the problem of providing a new method for improving the delivery efficiency of a drug such as a nucleic acid drug to the nervous system.　Provided is a delivery enhancer for enhancing the delivery of a drug to the central nervous system and/or peripheral nervous system, the delivery enhancer comprising an aquaporin-4 function enhancer or an aquaporin-4 function modifier.

Description

神経系送達促進剤Nervous System Delivery Enhancers
　本発明は、薬剤の中枢神経系又は末梢神経系への送達を促進するための送達促進剤及び中枢神経系疾患又は末梢神経系疾患の治療のための医薬組成物に関する。 The present invention relates to a delivery enhancer for enhancing the delivery of a drug to the central nervous system or peripheral nervous system, and a pharmaceutical composition for treating a central nervous system disease or a peripheral nervous system disease.
　近年、核酸医薬と呼ばれる医薬品の現在進行中の開発において、オリゴヌクレオチドが関心を集めており、また特に、標的遺伝子の高い選択性及び低毒性の点から考えて、アンチセンス法を利用する核酸医薬の開発が積極的に進められている。アンチセンス法とは、標的遺伝子より転写されたmRNAやmiRNAの部分配列を標的センス鎖として、それに相補的なオリゴヌクレオチド（アンチセンスオリゴヌクレオチド：本明細書ではしばしば「ASO（Antisense Oligonucleotide）」と表記する）を細胞に導入することによって、標的遺伝子によってコードされたタンパク質の発現やmiRNAの活性を選択的に改変又は阻害することを含む方法である。 In recent years, oligonucleotides have attracted attention in the ongoing development of pharmaceuticals known as nucleic acid drugs, and in particular, the development of nucleic acid drugs using the antisense method is being actively pursued, given their high selectivity for target genes and low toxicity. The antisense method involves selectively modifying or inhibiting the expression of proteins encoded by target genes or the activity of miRNA by introducing complementary oligonucleotides (antisense oligonucleotides: often referred to as "ASOs (Antisense Oligonucleotides)" in this specification) into cells, using a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand.
　アンチセンス法を利用した核酸として、本発明者らは、アンチセンスオリゴヌクレオチドとそれに対する相補鎖とをアニーリングさせた二本鎖核酸複合体(ヘテロ二本鎖オリゴヌクレオチド(heteroduplex oligonucleotide、HDO))を開発した(特許文献1及び2、非特許文献1及び2)。二本鎖核酸複合体は高いアンチセンス効果を有する画期的な技術である。 As a nucleic acid utilizing the antisense method, the present inventors have developed a double-stranded nucleic acid complex (heteroduplex oligonucleotide, HDO) in which an antisense oligonucleotide is annealed to its complementary strand ( Patent Documents 1 and 2, Non-Patent Documents 1 and 2). The double-stranded nucleic acid complex is a groundbreaking technology with a strong antisense effect.
　中枢神経系疾患又は末梢神経系疾患の投薬治療では、神経系への薬剤の送達効率がしばしば問題となる。治療効果を向上させるために薬剤の中枢神経系及び／又は末梢神経系への送達効率を向上させる方法が求められている。 In drug therapy for central or peripheral nervous system diseases, the efficiency of drug delivery to the nervous system is often an issue. To improve the therapeutic effect, there is a demand for a method to improve the efficiency of drug delivery to the central and/or peripheral nervous system.
国際公開第2013/089283号International Publication No. 2013/089283 国際公開第2014/192310号International Publication No. 2014/192310 国際公開第2017/150704号International Publication No. 2017/150704
　核酸医薬等の薬剤の神経系への送達効率を向上させる新たな方法を提供することである。 The goal is to provide a new method to improve the efficiency of delivery of drugs, such as nucleic acid medicines, to the nervous system.
　神経系における細胞内外の水の輸送は、アクアポリン（Aquaporin；AQP）等の膜貫通の水チャネルにより調整されている。アクアポリン4（AQP4）は、水選択性の極めて高いアクアポリンファミリーの一つであり、神経系における水の輸送に関与する主要なAQPである。また、AQP4は、脳や脊髄を含む中枢神経系においては、特に基底膜と接する星状膠細胞のエンドフィート（end-feet）膜に多く存在する。 　Water transport in and out of cells in the nervous system is regulated by transmembrane water channels such as aquaporins (AQPs). Aquaporin 4 (AQP4) is a member of the aquaporin family with extremely high water selectivity, and is the main AQP involved in water transport in the nervous system. In the central nervous system, including the brain and spinal cord, AQP4 is found in large amounts, particularly in the endfeet membranes of astrocytes that contact the basement membrane.
　脳内に分布する血管と脳組織との間に存在する血管周囲腔（periarterial space/perivenous space）は、Virchow-Robin腔と呼ばれている。近年、タンパク質の分解産物及びその他の細胞における老廃物は、大静脈内に再吸収され得るように、Virchow-Robin腔を介して輸送され、脳室中の脳脊髄液（cerebrospinal fluid；CSF）に流れ込むことが示唆された。この輸送はリンパ系に類似しており、血管周囲腔を介して脳から老廃物を排出する仕組みは、Glymphatic system（グリンパティックシステム）と呼ばれている。 The periarterial space/perivenous space that exists between the blood vessels and brain tissue in the brain is called the Virchow-Robin space. In recent years, it has been suggested that protein breakdown products and other cellular waste products are transported through the Virchow-Robin space and flow into the cerebrospinal fluid (CSF) in the ventricles so that they can be reabsorbed into the vena cava. This transport is similar to the lymphatic system, and the mechanism for excreting waste products from the brain through the perivascular space is called the Glymphatic system.
　上記の特許文献3（国際公開第2017/150704号）には、脳におけるAQP4による水の輸送を促進することにより、グリンパティックシステムによる排出機能を促進させることによって、脳におけるアミロイドβの蓄積が抑制されることが記載されている。 The above-mentioned Patent Document 3 (International Publication No. 2017/150704) describes that the accumulation of amyloid beta in the brain is suppressed by promoting water transport by AQP4 in the brain and thereby promoting the excretion function of the glymphatic system.
　本発明者らは、アンチセンス核酸やsiRNA等の核酸医薬をマウスの脳室内に投与する際にAQP4機能促進剤を同時に投与することによって、グリンパティックシステムの活性化を試みた。その結果、投与後の神経系における核酸医薬の検出量が飛躍的に増大し、標的遺伝子に対する抑制効果が著しく向上することを見出した。この結果は、アミロイドβに対する排出促進効果からは予想し得ない、驚くべき効果である。 The inventors attempted to activate the glymphatic system by simultaneously administering an AQP4 function promoter when administering nucleic acid drugs such as antisense nucleic acids or siRNA into the ventricles of mice. As a result, they found that the amount of nucleic acid drugs detected in the nervous system after administration increased dramatically, and the inhibitory effect on the target gene was significantly improved. This result is a surprising effect that could not be predicted from the effect of promoting the excretion of amyloid β.
　本発明は上記知見に基づくものであって、以下を提供する。
（１）薬剤の中枢神経系及び／又は末梢神経系への送達を促進するための送達促進剤であって、アクアポリン4機能促進剤又はアクアポリン4機能修飾剤からなる、前記送達促進剤。
（２）前記アクアポリン4機能促進剤又はアクアポリン4機能修飾剤が、下記式（I）：
Figure JPOXMLDOC01-appb-C000004
 で示される化合物若しくはその誘導体、又はそのいずれかの塩である、（１）に記載の送達促進剤。
（３）前記誘導体が、下記式(III)：
Figure JPOXMLDOC01-appb-C000005
 で示される、（２）に記載の送達促進剤。
（４）前記化合物又は前記誘導体がキャリア分子と結合している、（２）又は（３）に記載の送達促進剤。
（５）前記キャリア分子が、1kDa以上のタンパク質、ポリマー、脂質分子、又は造影剤である、（４）に記載の送達促進剤。
（６）前記タンパク質が、アルブミン、リポタンパク質、又は抗体若しくは抗体断片である、（５）に記載の送達促進剤。
（７）前記ポリマーが、ポリエチレングリコール(PEG)又はPEG graftedポリマーを含む、（５）に記載の送達促進剤。
（８）前記ポリマー又は前記脂質分子が、マイクロバブル、リポソーム、又はミセルを形成する、（５）又は（７）に記載の送達促進剤。
（９）前記薬剤が、髄腔内投与、経鼻投与、静脈内投与、皮下投与、腹腔内投与、経口投与、吸入投与、又は筋肉内投与される、（１）～（８）のいずれかに記載の送達促進剤。
（１０）前記髄腔内投与が脳室内投与、後頭窩穿刺、又は腰椎穿刺である、（９）に記載の送達促進剤。
（１１）前記薬剤が、シャント、留置カテーテル、又は皮下ポートを用いて投与される、（１）～（１０）のいずれかに記載の送達促進剤。
（１２）前記送達促進剤が、前記薬剤と同時に投与される、又は前記薬剤よりも先に若しくは後に投与される、（１）～（１１）のいずれかに記載の送達促進剤。
（１３）前記送達促進剤が、髄腔内投与、経鼻投与、静脈内投与、皮下投与、腹腔内投与、経口投与、吸入投与、又は筋肉内投与される、（１）～（１２）のいずれかに記載の送達促進剤。 The present invention is based on the above findings and provides the following.
(1) A delivery enhancer for promoting delivery of a drug to the central nervous system and/or peripheral nervous system, the delivery enhancer consisting of an aquaporin 4 function enhancer or an aquaporin 4 function modifier.
(2) The aquaporin 4 function promoter or aquaporin 4 function modifier is represented by the following formula (I):
Figure JPOXMLDOC01-appb-C000004
 or a derivative thereof, or a salt thereof.
(3) The derivative is represented by the following formula (III):
Figure JPOXMLDOC01-appb-C000005
 The delivery enhancer according to (2),
(4) The delivery enhancer according to (2) or (3), wherein the compound or the derivative is conjugated to a carrier molecule.
(5) The delivery enhancer according to (4), wherein the carrier molecule is a protein of 1 kDa or more, a polymer, a lipid molecule, or a contrast agent.
(6) The delivery enhancer according to (5), wherein the protein is albumin, lipoprotein, or an antibody or antibody fragment.
(7) The delivery enhancer according to (5), wherein the polymer comprises polyethylene glycol (PEG) or a PEG-grafted polymer.
(8) The delivery enhancer according to (5) or (7), wherein the polymer or lipid molecule forms a microbubble, a liposome, or a micelle.
(9) The delivery enhancer according to any one of (1) to (8), wherein the drug is administered intrathecally, nasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly.
(10) The delivery enhancer described in (9), wherein the intrathecal administration is intraventricular administration, posterior fossa puncture, or lumbar puncture.
(11) The delivery enhancer according to any one of (1) to (10), wherein the agent is administered using a shunt, an indwelling catheter, or a subcutaneous port.
(12) The delivery enhancer according to any one of (1) to (11), wherein the delivery enhancer is administered simultaneously with the drug, or administered prior to or after the drug.
(13) The delivery enhancer according to any one of (1) to (12), wherein the delivery enhancer is administered intrathecally, nasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly.
（１４）中枢神経系疾患又は末梢神経系疾患の治療のための医薬組成物であって、治療有効量の薬剤、及び（１）～（８）のいずれかに記載の送達促進剤を含む、前記医薬組成物。
（１５）髄腔内投与、経鼻投与、静脈内投与、皮下投与、腹腔内投与、経口投与、吸入投与、又は筋肉内投与される、（１４）に記載の医薬組成物。
（１６）前記髄腔内投与が脳室内投与、後頭窩穿刺、又は腰椎穿刺である、（１５）に記載の医薬組成物。
（１７）シャント、留置カテーテル、又は皮下ポートを用いて投与される、（１４）～（１６）のいずれかに記載の医薬組成物。
（１８）前記薬剤が、核酸医薬、ペプチド、低分子化合物、ウイルスベクター、細胞医薬、ナノ粒子、リポソーム、ミセル、又はエクソソームである、（１）～（１３）のいずれかに記載の送達促進剤、又は（１４）～（１７）のいずれかに記載の医薬組成物。
（１９）前記ペプチドが、天然ペプチド又は非天然ペプチドである、（１８）に記載の送達促進剤又は医薬組成物。
（２０）前記ペプチドが、環状ペプチドである、（１８）に記載の送達促進剤又は医薬組成物。
（２１）前記ペプチドが、タンパク製剤である、（１８）に記載の送達促進剤又は医薬組成物。
（２２）前記ペプチドが抗体又は抗体断片である、（１８）に記載の脳送達促進剤又は医薬組成物。
（２５）前記核酸医薬が、アンチセンス核酸、ヘテロ核酸、siRNA、shRNA、miRNA、mRNA、lncRNA、プラスミドDNA、アプタマー、デコイ、bait核酸、リボザイム、及び核酸ベクターからなる群から選択される、（１８）に記載の送達促進剤又は医薬組成物。
（２６）前記核酸医薬が、標的遺伝子又はその転写産物の少なくとも一部にハイブリダイズすることができ、かつ前記標的遺伝子又はその転写産物に対してアンチセンス効果を有する核酸分子を含む、（１８）に記載の送達促進剤又は医薬組成物。
（２７）前記核酸分子が、12～30塩基長である、（２６）に記載の送達促進剤又は医薬組成物。
（２８）前記核酸分子が、デオキシリボヌクレオシド、2'-修飾ヌクレオシド、5'-修飾ヌクレオシド、及び架橋ヌクレオシドからなる群から選択されるいずれか１以上を含む、（２６）又は（２７）に記載の送達促進剤又は医薬組成物。
（２９）前記核酸分子が、ミックスマーである、（２６）～（２８）のいずれかに記載の送達促進剤又は医薬組成物。
（３０）前記核酸分子が、モルホリノ核酸を含む、又は前記核酸分子の核酸の全部がモルホリノ核酸からなる、（２６）～（２９）のいずれかに記載の送達促進剤又は医薬組成物。
（３１）前記核酸分子が、ギャップマーである、（２６）～（３０）のいずれかに記載の送達促進剤又は医薬組成物。
（３２）前記核酸分子が、
　(1)少なくとも3個の連続するデオキシリボヌクレオシドを含む中央領域、
　(2)前記中央領域の5'末端側に配置された、非天然ヌクレオシドを含む5’ウイング領域、及び
　(3)前記中央領域の3'末端側に配置された、非天然ヌクレオシドを含む3’ウイング領域を含む、（３１）に記載の送達促進剤又は医薬組成物。
（３３）前記5'ウイング領域において前記中央領域に隣接する末端塩基位置、前記中央領域の5'側から2番目の塩基位置、及び／又は前記中央領域の5'側から8番目の塩基位置に、2'-修飾ヌクレオシドを含む、（３２）に記載の送達促進剤又は医薬組成物。
（３４）前記核酸分子において、前記5'ウイング領域及び前記3'ウイング領域が、架橋ヌクレオシド及び／又は2'-修飾ヌクレオシドを含む、（３２）に記載の送達促進剤又は医薬組成物。
（３５）前記架橋ヌクレオシドが、LNAヌクレオシド、2',4'-BNANCヌクレオシド、cEt BNAヌクレオシド、ENAヌクレオシド、AmNAヌクレオシド、GuNAヌクレオシド、scpBNAヌクレオシド、scpBNA2ヌクレオシド、及びBANA3ヌクレオシドからなる群から選択される、（３４）に記載の送達促進剤又は医薬組成物。
（３６）前記2'-修飾ヌクレオシドが、2'-O-メチル修飾ヌクレオシド、2'-O-メトキシエチル修飾ヌクレオシド、2'-O-[2-(N-メチルカルバモイル)エチル]修飾ヌクレオシド、又は2'-フルオロ修飾ヌクレオシドである、（３４）又は（３５）に記載の送達促進剤又は医薬組成物。
（３７）前記核酸分子のヌクレオシド間結合の全部又は一部が修飾ヌクレオシド間結合である、（２６）～（３６）のいずれかに記載の送達促進剤又は医薬組成物。
（３８）前記修飾ヌクレオシド間結合がホスホロチオエート結合である、（３７）に記載の送達促進剤又は医薬組成物。
（３９）前記核酸分子が、修飾核酸塩基を含む、（２６）～（３８）のいずれかに記載の送達促進剤又は医薬組成物。
（４０）前記核酸分子が、前記標的遺伝子又はその転写産物に対してステリックブロッキング、スプライシング制御、発現低下、発現上昇、及び／又は塩基編集を誘導することができる、（２６）～（３９）のいずれかに記載の送達促進剤又は医薬組成物。 (14) A pharmaceutical composition for treating a central nervous system disease or a peripheral nervous system disease, comprising a therapeutically effective amount of a drug and a delivery enhancer according to any one of (1) to (8).
(15) The pharmaceutical composition according to (14), which is administered intrathecally, nasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly.
(16) The pharmaceutical composition according to (15), wherein the intrathecal administration is intraventricular administration, posterior fossa puncture, or lumbar puncture.
(17) The pharmaceutical composition according to any one of (14) to (16), which is administered using a shunt, an indwelling catheter, or a subcutaneous port.
(18) The delivery enhancer according to any one of (1) to (13), or the pharmaceutical composition according to any one of (14) to (17), wherein the drug is a nucleic acid drug, a peptide, a low molecular weight compound, a viral vector, a cellular drug, a nanoparticle, a liposome, a micelle, or an exosome.
(19) The delivery enhancer or pharmaceutical composition according to (18), wherein the peptide is a natural peptide or a non-natural peptide.
(20) The delivery enhancer or pharmaceutical composition according to (18), wherein the peptide is a cyclic peptide.
(21) The delivery enhancer or pharmaceutical composition according to (18), wherein the peptide is a protein preparation.
(22) The brain delivery-enhancing agent or pharmaceutical composition described in (18), wherein the peptide is an antibody or an antibody fragment.
(25) The delivery enhancer or pharmaceutical composition according to (18), wherein the nucleic acid drug is selected from the group consisting of an antisense nucleic acid, a heteronucleic acid, an siRNA, an shRNA, an miRNA, an mRNA, an lncRNA, a plasmid DNA, an aptamer, a decoy, a bait nucleic acid, a ribozyme, and a nucleic acid vector.
(26) The delivery enhancer or pharmaceutical composition described in (18), wherein the nucleic acid drug comprises a nucleic acid molecule capable of hybridizing to at least a portion of a target gene or its transcription product and having an antisense effect on the target gene or its transcription product.
(27) The delivery enhancer or pharmaceutical composition according to (26), wherein the nucleic acid molecule is 12 to 30 bases in length.
(28) The delivery enhancer or pharmaceutical composition according to (26) or (27), wherein the nucleic acid molecule comprises one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides.
(29) The delivery enhancer or pharmaceutical composition according to any one of (26) to (28), wherein the nucleic acid molecule is a mixmer.
(30) The delivery enhancer or pharmaceutical composition according to any one of (26) to (29), wherein the nucleic acid molecule comprises a morpholino nucleic acid, or the entire nucleic acid of the nucleic acid molecule consists of a morpholino nucleic acid.
(31) The delivery enhancer or pharmaceutical composition according to any one of (26) to (30), wherein the nucleic acid molecule is a gapmer.
(32) The nucleic acid molecule,
(1) a central region containing at least three consecutive deoxyribonucleosides;
(2) a 5' wing region comprising an unnatural nucleoside and disposed on the 5' end of the central region; and (3) a 3' wing region comprising an unnatural nucleoside and disposed on the 3' end of the central region.
(33) The delivery enhancer or pharmaceutical composition described in (32), comprising a 2'-modified nucleoside at the terminal base position adjacent to the central region in the 5' wing region, the second base position from the 5' side of the central region, and/or the eighth base position from the 5' side of the central region.
(34) The delivery enhancer or pharmaceutical composition according to (32), wherein in the nucleic acid molecule, the 5' wing region and the 3' wing region comprise bridged nucleosides and/or 2'-modified nucleosides.
(35) The delivery enhancer or pharmaceutical composition according to (34), wherein the bridged nucleoside is selected from the group consisting of an LNA nucleoside, a 2',4'-BNA NC nucleoside, a cEt BNA nucleoside, an ENA nucleoside, an AmNA nucleoside, a GuNA nucleoside, a scpBNA nucleoside, a scpBNA2 nucleoside, and a BANA3 nucleoside.
(36) The delivery enhancer or pharmaceutical composition according to (34) or (35), wherein the 2'-modified nucleoside is a 2'-O-methyl modified nucleoside, a 2'-O-methoxyethyl modified nucleoside, a 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleoside, or a 2'-fluoro modified nucleoside.
(37) The delivery enhancer or pharmaceutical composition according to any one of (26) to (36), wherein all or a part of the internucleoside linkages of the nucleic acid molecule are modified internucleoside linkages.
(38) The delivery enhancer or pharmaceutical composition according to (37), wherein the modified internucleoside bond is a phosphorothioate bond.
(39) The delivery enhancer or pharmaceutical composition according to any one of (26) to (38), wherein the nucleic acid molecule comprises a modified nucleic acid base.
(40) The delivery enhancer or pharmaceutical composition according to any one of (26) to (39), wherein the nucleic acid molecule is capable of inducing steric blocking, splicing control, decreased expression, increased expression, and/or base editing of the target gene or its transcription product.
（４１）前記核酸医薬が、前記核酸分子からなる第1核酸鎖と、前記第1核酸鎖に相補的な塩基配列を含む第2核酸鎖とを含む二本鎖核酸複合体からなる、（２６）～（４０）のいずれかに記載の送達促進剤又は医薬組成物。
（４２）前記第2核酸鎖が少なくとも8塩基長である、（４１）に記載の送達促進剤又は医薬組成物。
（４３）前記第2核酸鎖が、デオキシリボヌクレオシド、2'-修飾ヌクレオシド、5'-修飾ヌクレオシド、及び架橋ヌクレオシドからなる群から選択されるいずれか１以上を含む、（４１）又は（４２）に記載の送達促進剤又は医薬組成物。
（４４）前記第2核酸鎖が、前記第1核酸鎖に対して、非相補的塩基、及び／又は1塩基以上の、挿入配列及び／又は欠失を含む、（４１）～（４３）のいずれかに記載の送達促進剤又は医薬組成物。
（４５）前記第2核酸鎖が、前記非相補的塩基を1～3個含む、（４４）に記載の送達促進剤又は医薬組成物。
（４６）前記挿入配列が1～8塩基からなる、（４４）又は（４５）に記載の送達促進剤又は医薬組成物。
（４７）前記欠失が連続する1～4塩基からなる、（４４）～（４６）のいずれかに記載の送達促進剤又は医薬組成物。
（４８）前記第2核酸鎖が、前記第1核酸鎖に相補的な塩基配列からなる領域の5'末端側及び/又は3'末端側に位置する少なくとも1つのオーバーハング領域を含む、（４１）～（４７）のいずれかに記載の送達促進剤又は医薬組成物。
（４９）前記オーバーハング領域が1～30塩基長である、（４８）に記載の送達促進剤又は医薬組成物。
（５０）前記第2核酸鎖が、機能性部分と結合している、（４１）～（４９）のいずれかに記載の送達促進剤又は医薬組成物。
（５１）前記機能性部分が、脂質又はペプチドである、（５０）に記載の送達促進剤又は医薬組成物。
（５２）前記ペプチドが抗体である、（５１）に記載の送達促進剤又は医薬組成物。
（５３）前記脂質が、コレステロール若しくはその類縁体、トコフェロール若しくはその類縁体、葉酸、ホスファチジルエタノールアミン、及び置換された若しくは置換されていない炭素数16～30のアルキル基からなる群から選択される、（５１）に記載の送達促進剤又は医薬組成物。
（５４）前記第1核酸鎖と前記第2核酸鎖とがリンカーを介して結合している、（４１）～（５３）のいずれかに記載の送達促進剤又は医薬組成物。
（５５）前記リンカーが、切断性（cleavable）又は非切断性（uncleavable）のリンカーである、（５４）に記載の送達促進剤又は医薬組成物。
（５６）前記リンカーが、以下の式（IV）で表される基を含む、（５４）又は（５５）に記載の送達促進剤又は医薬組成物。
Figure JPOXMLDOC01-appb-C000006
 （式中、
L2は、置換された若しくは置換されていないC1～12のアルキレン基、置換された若しくは置換されていないC3～C8シクロアルキレン基、-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-、又はCH(CH2-OH)-CH2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-を表し、
L3は、-NH-又は結合を表し、
L4は、置換された若しくは置換されていないC1～12のアルキレン基、置換された若しくは置換されていないC3～C8のシクロアルキレン基、-(CH2)2-[O-(CH2)2]m-、又は結合を表し、ここで、mは1～25の整数を表し、
L5は、-NH-(C=O)-、-(C=O)-、又は結合を表す）
（５７）前記リンカーが、核酸、ポリエーテル基、並びに／又はアルキルアミノ基を含む、（５４）～（５６）のいずれかに記載の送達促進剤又は医薬組成物。
（５８）前記核酸が、1個、又はヌクレオシド間結合により連結された2～10個のヌクレオシド及び／若しくは非天然ヌクレオシドからなる、（５７）に記載の送達促進剤又は医薬組成物。
（５９）前記ポリエーテル基が、ポリエチレングリコール基又はトリエチレングリコール基である、（５７）に記載の送達促進剤又は医薬組成物。
（６０）前記アルキルアミノ基が、ヘキシルアミノ基である、（５７）に記載の送達促進剤又は医薬組成物。
（６１）前記第2核酸鎖のヌクレオシド間結合の全部又は一部が修飾ヌクレオシド間結合である、（４１）～（６０）のいずれかに記載の送達促進剤又は医薬組成物。
（６２）前記修飾ヌクレオシド間結合がホスホロチオエート結合である、（６１）に記載の送達促進剤又は医薬組成物。
（６３）前記核酸医薬が、siRNAである、（１８）に記載の送達促進剤又は医薬組成物。
（６４）前記siRNAが、デオキシリボヌクレオシド、2'-修飾ヌクレオシド、5'-修飾ヌクレオシド、架橋ヌクレオシド、及び修飾ヌクレオシド間結合からなる群から選択されるいずれか１以上を含む、（６３）に記載の送達促進剤又は医薬組成物。
（６５）前記siRNAが、機能性部分と結合している、（６３）又は（６４）に記載の送達促進剤又は医薬組成物。
（６６）前記機能性部分が、脂質又はペプチドである、（６５）に記載の送達促進剤又は医薬組成物。
（６７）前記脂質が、コレステロール若しくはその類縁体、トコフェロール若しくはその類縁体、葉酸、ホスファチジルエタノールアミン、及び置換された若しくは置換されていない炭素数16～30のアルキル基からなる群から選択される、（６６）に記載の送達促進剤又は医薬組成物。
（６８）前記核酸医薬が2つのsiRNAを含み、前記2つのsiRNAに含まれるガイド鎖がリンカーを介して互いに結合している、（６３）に記載の送達促進剤又は医薬組成物。
（６９）前記リンカーが、核酸、ポリエーテル基、並びに／又はアルキルアミノ基を含む、（６８）に記載の送達促進剤又は医薬組成物。
（７０）前記中枢神経系が、大脳皮質、大脳基底核、大脳白質、間脳、脳幹、小脳、及び脊髄からなる群から選択される、（１）～（６９）のいずれかに記載の送達促進剤又は医薬組成物。
（７１）前記中枢神経系が、前頭葉、側頭葉、海馬、海馬傍回、頭頂葉、後頭葉、線条体、淡蒼球、前障、視床、視床下核、中脳、黒質、橋、延髄、小脳皮質、小脳核、頸髄、胸髄、及び腰髄からなる群から選択される、（７０）に記載の送達促進剤又は医薬組成物。
（７２）前記末梢神経系が、脊髄前根、後根、第１～第１２脳神経、馬尾、及び後根神経節からなる群から選択される、（１）～（６９）のいずれかに記載の送達促進剤又は医薬組成物。
（７３）前記送達促進剤が被験体に0.01mg～10g投与される、（１）～（７２）のいずれかに記載の送達促進剤又は医薬組成物。
　本明細書は本願の優先権の基礎となる日本国特許出願番号2022-158781号の開示内容を包含する。 (41) The delivery enhancer or pharmaceutical composition according to any one of (26) to (40), wherein the nucleic acid drug comprises a double-stranded nucleic acid complex comprising a first nucleic acid strand consisting of the nucleic acid molecule and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand.
(42) The delivery enhancer or pharmaceutical composition according to (41), wherein the second nucleic acid strand is at least 8 bases in length.
(43) The delivery enhancer or pharmaceutical composition according to (41) or (42), wherein the second nucleic acid strand comprises any one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides.
(44) The delivery enhancer or pharmaceutical composition according to any one of (41) to (43), wherein the second nucleic acid strand contains non-complementary bases and/or an insertion sequence and/or a deletion of one or more bases relative to the first nucleic acid strand.
(45) The delivery enhancer or pharmaceutical composition according to (44), wherein the second nucleic acid strand contains 1 to 3 non-complementary bases.
(46) The delivery enhancer or pharmaceutical composition according to (44) or (45), wherein the insertion sequence consists of 1 to 8 bases.
(47) The delivery enhancer or pharmaceutical composition according to any one of (44) to (46), wherein the deletion consists of 1 to 4 consecutive bases.
(48) The delivery enhancer or pharmaceutical composition according to any one of (41) to (47), wherein the second nucleic acid strand comprises at least one overhang region located on the 5'-end and/or 3'-end side of a region consisting of a base sequence complementary to the first nucleic acid strand.
(49) The delivery enhancer or pharmaceutical composition according to (48), wherein the overhang region is 1 to 30 bases in length.
(50) The delivery enhancer or pharmaceutical composition according to any one of (41) to (49), wherein the second nucleic acid strand is linked to a functional moiety.
(51) The delivery enhancer or pharmaceutical composition according to (50), wherein the functional moiety is a lipid or a peptide.
(52) The delivery enhancer or pharmaceutical composition according to (51), wherein the peptide is an antibody.
(53) The delivery enhancer or pharmaceutical composition according to (51), wherein the lipid is selected from the group consisting of cholesterol or an analogue thereof, tocopherol or an analogue thereof, folic acid, phosphatidylethanolamine, and a substituted or unsubstituted alkyl group having 16 to 30 carbon atoms.
(54) The delivery enhancer or pharmaceutical composition according to any one of (41) to (53), wherein the first nucleic acid strand and the second nucleic acid strand are linked via a linker.
(55) The delivery enhancer or pharmaceutical composition according to (54), wherein the linker is a cleavable or noncleavable linker.
(56) The delivery enhancer or pharmaceutical composition according to (54) or (55), wherein the linker comprises a group represented by the following formula (IV):
Figure JPOXMLDOC01-appb-C000006
 (Wherein,
L2 represents a substituted or unsubstituted C1-12 alkylene group, a substituted or unsubstituted C3- C8 cycloalkylene group, -( CH2 ) 2 -O-(CH2) 2 -O-( CH2 ) 2 -O-(CH2) 3- , or CH( CH2 - OH) -CH2 -O-( CH2 ) 2 -O-( CH2 ) 2 -O-( CH2 ) 2 -O-( CH2 ) 2 -O-( CH2 ) 3- ;
L3 represents -NH- or a bond;
L4 represents a substituted or unsubstituted C 1 -12 alkylene group, a substituted or unsubstituted C 3 -C 8 cycloalkylene group, -(CH 2 ) 2 -[O-(CH 2 ) 2 ] m -, or a bond, where m represents an integer of 1 to 25;
L5 represents -NH-(C=O)-, -(C=O)-, or a bond.
(57) The delivery enhancer or pharmaceutical composition according to any one of (54) to (56), wherein the linker comprises a nucleic acid, a polyether group, and/or an alkylamino group.
(58) The delivery enhancer or pharmaceutical composition according to (57), wherein the nucleic acid consists of one or 2 to 10 nucleosides and/or non-natural nucleosides linked by internucleoside bonds.
(59) The delivery enhancer or pharmaceutical composition according to (57), wherein the polyether group is a polyethylene glycol group or a triethylene glycol group.
(60) The delivery enhancer or pharmaceutical composition according to (57), wherein the alkylamino group is a hexylamino group.
(61) The delivery enhancer or pharmaceutical composition according to any one of (41) to (60), wherein all or a part of the internucleoside bonds of the second nucleic acid strand are modified internucleoside bonds.
(62) The delivery enhancer or pharmaceutical composition according to (61), wherein the modified internucleoside bond is a phosphorothioate bond.
(63) The delivery enhancer or pharmaceutical composition described in (18), wherein the nucleic acid drug is siRNA.
(64) The delivery enhancer or pharmaceutical composition described in (63), wherein the siRNA comprises any one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, bridged nucleosides, and modified internucleoside linkages.
(65) The delivery enhancer or pharmaceutical composition according to (63) or (64), wherein the siRNA is linked to a functional moiety.
(66) The delivery enhancer or pharmaceutical composition according to (65), wherein the functional moiety is a lipid or a peptide.
(67) The delivery enhancer or pharmaceutical composition according to (66), wherein the lipid is selected from the group consisting of cholesterol or an analogue thereof, tocopherol or an analogue thereof, folic acid, phosphatidylethanolamine, and a substituted or unsubstituted alkyl group having 16 to 30 carbon atoms.
(68) The delivery enhancer or pharmaceutical composition described in (63), wherein the nucleic acid drug contains two siRNAs, and the guide strands contained in the two siRNAs are linked to each other via a linker.
(69) The delivery enhancer or pharmaceutical composition according to (68), wherein the linker comprises a nucleic acid, a polyether group, and/or an alkylamino group.
(70) The delivery enhancer or pharmaceutical composition according to any one of (1) to (69), wherein the central nervous system is selected from the group consisting of the cerebral cortex, the basal ganglia, the cerebral white matter, the diencephalon, the brainstem, the cerebellum, and the spinal cord.
(71) The delivery enhancer or pharmaceutical composition according to (70), wherein the central nervous system is selected from the group consisting of the frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, parietal lobe, occipital lobe, striatum, globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigra, pons, medulla oblongata, cerebellar cortex, cerebellar nuclei, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.
(72) The delivery enhancer or pharmaceutical composition according to any one of (1) to (69), wherein the peripheral nervous system is selected from the group consisting of the ventral root of the spinal cord, the dorsal root, the first to twelfth cranial nerves, the cauda equina, and the dorsal root ganglion.
(73) The delivery enhancer or pharmaceutical composition according to any one of (1) to (72), wherein the delivery enhancer is administered to a subject in an amount of 0.01 mg to 10 g.
This specification includes the disclosure of Japanese Patent Application No. 2022-158781, which is the priority basis of this application.
　本発明によれば、薬剤の神経系への送達効率を向上させるための送達促進剤が提供される。 The present invention provides a delivery enhancer that improves the efficiency of drug delivery to the nervous system.
図1は、様々な天然ヌクレオチド又は非天然ヌクレオチドの構造を示す。FIG. 1 shows the structures of various natural and non-natural nucleotides. 図2は、様々な架橋核酸の構造を示す。FIG. 2 shows the structures of various bridged nucleic acids. 図3は、Malat1標的ASOの中枢神経系への送達量及び標的遺伝子抑制効果を示す。図3Aは、脳室内投与から3時間後又は7日後の海馬においてASO濃度を測定した結果を示す。図3Bは、脳室内投与から7日後の海馬におけるMalat1 RNA発現レベルを示す。「ASO only」はASO単独投与群を示し、「ASO + TGN-073」はASO/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 3 shows the amount of Malat1-targeting ASO delivered to the central nervous system and its target gene suppression effect. Figure 3A shows the results of measuring ASO concentration in the hippocampus 3 hours or 7 days after intracerebroventricular administration. Figure 3B shows Malat1 RNA expression levels in the hippocampus 7 days after intracerebroventricular administration. "ASO only" indicates the group administered ASO alone, and "ASO + TGN-073" indicates the group administered ASO/TGN-073 together. Error bars indicate standard error. 図4は、Malat1標的ASOを様々な濃度で脳室内投与した際のASOの中枢神経系への送達量及び中枢神経系における標的遺伝子抑制効果を示す。「ASO only」はASO単独投与群を示し、「ASO + TGN-073」はASO/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 4 shows the amount of ASO delivered to the central nervous system and the target gene suppression effect in the central nervous system when Malat1-targeting ASO was administered intracerebroventricularly at various concentrations. "ASO only" indicates the group administered ASO alone, and "ASO + TGN-073" indicates the group administered ASO/TGN-073 together. Error bars indicate standard error. 図5は、Mapt標的ASOの脳室内投与から7日後の海馬におけるMapt mRNA発現レベルを示す。「ASO only」はASO単独投与群を示し、「ASO + TGN-073」はASO/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 5 shows Mapt mRNA expression levels in the hippocampus 7 days after intracerebroventricular administration of Mapt-targeting ASO. "ASO only" indicates the group administered ASO alone, and "ASO + TGN-073" indicates the group administered ASO/TGN-073 together. Error bars indicate standard error. 図6は、行動評価に用いたスコアリングシステムを示す。FIG. 6 shows the scoring system used for behavioral assessment. 図7は、Malat1標的ASOの中枢神経毒性評価の結果を示す。図は、脳室内投与から30分後、1時間後、及び2時間後における急性期忍容性スコア（Acute tolerability score）を示す。「TGN-073 only」はTGN-073単独投与群を示し、「ASO only」はASO単独投与群を示し、「ASO + TGN-073」はASO/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 7 shows the results of central neurotoxicity evaluation of Malat1-targeting ASO. The figure shows the acute tolerability scores 30 minutes, 1 hour, and 2 hours after intracerebroventricular administration. "TGN-073 only" indicates the group administered TGN-073 alone, "ASO only" indicates the group administered ASO alone, and "ASO + TGN-073" indicates the group administered ASO/TGN-073 co-administration. Error bars indicate standard error. 図8は、脳室内投与から1時間後のオープンフィールドテストにおいて運動機能を評価した結果を示す。図8Aは、最大移動速度を示す。図8Bは、移動時間を示す。「TGN-073 only」はTGN-073単独投与群を示し、「ASO only」はASO単独投与群を示し、「ASO + TGN-073」はASO/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 8 shows the results of evaluating motor function in an open field test 1 hour after intracerebroventricular administration. Figure 8A shows the maximum movement speed. Figure 8B shows the movement time. "TGN-073 only" indicates the group administered TGN-073 alone, "ASO only" indicates the group administered ASO alone, and "ASO + TGN-073" indicates the group administered ASO/TGN-073 co-administration. Error bars indicate standard error. 図9は、Malat1遺伝子を標的とする2'-O-MOE修飾ASOの脳室内投与から7日後の海馬におけるMalat1 RNA発現レベルを示す。「PBS」は陰性対照のPBS投与群を示し、「ASO only」はASO単独投与群を示し、「ASO + TGN-073」はASO/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 9 shows Malat1 RNA expression levels in the hippocampus 7 days after intracerebroventricular administration of 2'-O-MOE-modified ASO targeting the Malat1 gene. "PBS" indicates the negative control PBS-administered group, "ASO only" indicates the ASO-only administration group, and "ASO + TGN-073" indicates the ASO/TGN-073 co-administration group. Error bars indicate standard error. 図10は、実施例5で用いた核酸の構造を示す。FIG. 10 shows the structure of the nucleic acid used in Example 5. 図11は、Malat1遺伝子を標的とする1本鎖又は2本鎖の核酸剤を脳室内投与した際の中枢神経系におけるASO濃度（中枢神経系への送達量）及び標的遺伝子抑制効果を示す。図11Aは、脳室内投与から7日後の海馬においてASO濃度を測定した結果を示す。図11Bは、脳室内投与から7日後の海馬におけるMalat1 RNA発現レベルを示す。図中、核酸剤の脳室内投与時にTGN-073共投与の有無を+/-で示す。エラーバーは標準誤差を示す。Figure 11 shows the ASO concentration in the central nervous system (amount delivered to the central nervous system) and the target gene suppression effect when single-stranded or double-stranded nucleic acid agents targeting the Malat1 gene were administered intracerebroventricularly. Figure 11A shows the results of measuring the ASO concentration in the hippocampus 7 days after intracerebroventricular administration. Figure 11B shows the Malat1 RNA expression level in the hippocampus 7 days after intracerebroventricular administration. In the figures, +/- indicates the presence or absence of co-administration of TGN-073 when the nucleic acid agent was administered intracerebroventricularly. Error bars indicate standard error. 図12は、Sod1遺伝子を標的とするsiRNAを脳室内投与した際の標的遺伝子抑制効果を示す。「PBS」は陰性対照のPBS投与群を示し、「siRNA only」はsiRNA単独投与群を示し、「siRNA + TGN-073」はsiRNA/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 12 shows the target gene suppression effect when siRNA targeting the Sod1 gene was administered intracerebroventricularly. "PBS" indicates the negative control PBS-administered group, "siRNA only" indicates the siRNA-only administered group, and "siRNA + TGN-073" indicates the siRNA/TGN-073 co-administered group. Error bars indicate standard error. 図13は、TGN-073の誘導体であるTGN-073-mesをASOと共に脳室内投与した際の中枢神経系におけるASO濃度（中枢神経系への送達量）及び標的遺伝子抑制効果を示す。図13Aは、脳室内投与から7日後の海馬においてASO濃度を測定した結果を示す。図13Bは、脳室内投与から7日後の海馬におけるMapt mRNA発現レベルを示す。「PBS」は陰性対照のPBS投与群を示し、「TGN-073-mes only」はTGN-073-mes単独投与群を示し、「ASO only」はASO単独投与群を示し、「ASO + TGN-073-mes」はASO/TGN-073-mes共投与群を示す。エラーバーは標準誤差を示す。Figure 13 shows the ASO concentration in the central nervous system (amount delivered to the central nervous system) and target gene suppression effect when TGN-073-mes, a derivative of TGN-073, was administered intracerebroventricularly together with ASO. Figure 13A shows the results of measuring the ASO concentration in the hippocampus 7 days after intracerebroventricular administration. Figure 13B shows the Mapt mRNA expression level in the hippocampus 7 days after intracerebroventricular administration. "PBS" indicates the negative control PBS-administered group, "TGN-073-mes only" indicates the TGN-073-mes alone-administered group, "ASO only" indicates the ASO alone-administered group, and "ASO + TGN-073-mes" indicates the ASO/TGN-073-mes co-administered group. Error bars indicate standard error. 図14は、ジストロフィンpre-mRNAのエキソン23/イントロン23を標的とするホスホロジアミデートモルホリノオリゴマー(PMO)の脳室内投与から7日後の脳におけるエキソン23のスキッピング効率を示す。図14Aは、海馬におけるスキッピング効率を示す。図14Bは、線条体におけるスキッピング効率を示す。図14Cは、皮質におけるスキッピング効率を示す。エラーバーは標準誤差を示す。Figure 14 shows the efficiency of exon 23 skipping in the brain 7 days after intracerebroventricular administration of phosphorodiamidate morpholino oligomers (PMOs) targeting exon 23/intron 23 of dystrophin pre-mRNA. Figure 14A shows the efficiency of skipping in the hippocampus. Figure 14B shows the efficiency of skipping in the striatum. Figure 14C shows the efficiency of skipping in the cortex. Error bars show standard error. 図15は、実施例8で用いた核酸の構造を示す。FIG. 15 shows the structure of the nucleic acid used in Example 8. 図16は、ジストロフィンpre-mRNAのエキソン23/イントロン23を標的とするPMOを含む二本鎖核酸複合体の脳室内投与から7日後の脳におけるエキソン23のスキッピング効率を示す。図16Aは、海馬におけるスキッピング効率を示す。図16Bは、線条体におけるスキッピング効率を示す。図16Cは、皮質におけるスキッピング効率を示す。エラーバーは標準誤差を示す。Figure 16 shows the skipping efficiency of exon 23 in the brain 7 days after intracerebroventricular administration of a double-stranded nucleic acid complex containing a PMO targeting exon 23/intron 23 of dystrophin pre-mRNA. Figure 16A shows the skipping efficiency in the hippocampus. Figure 16B shows the skipping efficiency in the striatum. Figure 16C shows the skipping efficiency in the cortex. Error bars show standard error. 図17は、Malat1標的ASOの腰髄部への送達量及び標的遺伝子抑制効果を示す。図17Aは、脳室内投与から7日後の腰髄部においてASO濃度を測定した結果を示す。図17Bは、脳室内投与から7日後の腰髄部におけるMalat1 RNA発現レベルを示す。「PBS」は陰性対照であるPBS投与群を示し、「ASO only」はASO単独投与群を示し、「ASO + TGN-073」はASO/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 17 shows the delivery amount of Malat1-targeting ASO to the lumbar spinal cord and the target gene suppression effect. Figure 17A shows the results of measuring the ASO concentration in the lumbar spinal cord 7 days after intracerebroventricular administration. Figure 17B shows the Malat1 RNA expression level in the lumbar spinal cord 7 days after intracerebroventricular administration. "PBS" indicates the negative control PBS-administered group, "ASO only" indicates the ASO-only administered group, and "ASO + TGN-073" indicates the ASO/TGN-073 co-administered group. Error bars indicate standard error. 図18は、髄腔内投与したMalat1標的ASOの中枢神経系への送達量を示す。図18Aは、脊髄組織におけるASO濃度を示す。図18Bは、左海馬におけるASO濃度を示す。「ASO only」はASO単独投与群を示し、「ASO + TGN-073」はASO/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 18 shows the amount of Malat1-targeting ASO delivered to the central nervous system after intrathecal administration. Figure 18A shows the ASO concentration in spinal cord tissue. Figure 18B shows the ASO concentration in the left hippocampus. "ASO only" indicates the group administered ASO alone, and "ASO + TGN-073" indicates the group administered ASO/TGN-073 together. Error bars indicate standard error. 図19は、脳室内投与したVHH抗体の中枢神経系への送達量を示す。図19Aは、左海馬におけるVHH抗体濃度を示す。図19Bは、左後頭葉皮質におけるVHH抗体濃度を示す。図19Cは、左基底核におけるVHH抗体濃度を示す。「VHH only」はVHH抗体単独投与群を示し、「VHH + TGN-073」はVHH抗体/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 19 shows the amount of VHH antibody delivered to the central nervous system after intracerebroventricular administration. Figure 19A shows the VHH antibody concentration in the left hippocampus. Figure 19B shows the VHH antibody concentration in the left occipital cortex. Figure 19C shows the VHH antibody concentration in the left basal ganglia. "VHH only" indicates the group administered VHH antibody alone, and "VHH + TGN-073" indicates the group administered VHH antibody/TGN-073 together. Error bars indicate standard error. 図20は、脳室内投与したIgG抗体の中枢神経系への送達量を示す。「IgG only」はIgG抗体単独投与群を示し、「IgG + TGN-073」はIgG抗体/TGN-073共投与群を示す。エラーバーは標準誤差を示す。Figure 20 shows the amount of IgG antibody delivered to the central nervous system after intracerebroventricular administration. "IgG only" indicates the group administered IgG antibody alone, and "IgG + TGN-073" indicates the group administered IgG antibody/TGN-073 together. Error bars indicate standard error. 図21は、脳室内投与したAAV9ベクターから発現したGFPタンパク質の蛍光を海馬レベル冠状断像において観察した結果を示す。「AAV only」はAAV単独投与群を示し、「AAV + TGN-073」はAAV/TGN-073共投与群を示す。Figure 21 shows the results of observing the fluorescence of GFP protein expressed from the AAV9 vector administered intracerebroventricularly in coronal sections at the hippocampal level. "AAV only" indicates the group administered AAV alone, and "AAV + TGN-073" indicates the group administered AAV/TGN-073 co-administration.
＜１．送達促進剤＞
　本発明の第1態様は送達促進剤である。本発明の送達促進剤は、アクアポリン4機能促進剤又はアクアポリン4機能修飾剤からなる。本発明の送達促進剤を核酸医薬、ペプチド、又は低分子化合物等の薬剤と併用することによって、薬剤の中枢神経系及び／又は末梢神経系への送達効率が向上させることができる。 1. Delivery enhancers
A first aspect of the present invention is a delivery enhancer. The delivery enhancer of the present invention is an aquaporin 4 function enhancer or an aquaporin 4 function modifier. By using the delivery enhancer of the present invention in combination with a drug such as a nucleic acid drug, a peptide, or a low molecular weight compound, the efficiency of delivery of the drug to the central nervous system and/or the peripheral nervous system can be improved.
（用語の定義）
　本明細書において、標的遺伝子の「転写産物」とは、RNAポリメラーゼによって合成される任意のRNAをいう。具体的には、標的遺伝子から転写されるmRNA（成熟mRNA、mRNA前駆体、塩基修飾を受けていないmRNA等を含む）、miRNA等のノンコーディングRNA(non-coding RNA、ncRNA)、ロングノンコーディングRNA（lncRNA）、ナチュラルアンチセンスRNAを含み得る。 (Definition of terms)
As used herein, the "transcription product" of a target gene refers to any RNA synthesized by RNA polymerase. Specifically, it may include mRNA (including mature mRNA, mRNA precursor, and mRNA without base modification) transcribed from a target gene, non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA.
　本明細書において「標的遺伝子」とは、核酸分子又は二本鎖核酸複合体のアンチセンス効果により、その転写産物又は翻訳産物の発現量が抑制若しくは亢進され得る、その転写産物又は翻訳産物の機能が阻害され得る、又はステリックブロッキング、スプライシング制御（例えばスプライシングスイッチ、エキソンスキッピング、エキソンインクルージョン）、塩基編集、若しくはRNA編集が誘導され得る遺伝子である。標的遺伝子の種類は、特に限定しない。例えば、核酸鎖、核酸分子、又は二本鎖核酸複合体を導入する生物由来の遺伝子が挙げられ、様々な疾患（例えば中枢神経系疾患や末梢神経系疾患）においてその発現が増加する遺伝子や神経系等の生体内で発現する遺伝子が挙げられる。具体例としては、スカベンジャー受容体B1(scavenger receptor B1：本明細書では、しばしば「SR-B1」と表記する）遺伝子、転移関連肺腺癌転写産物1（metastasis associated lung adenocarcinoma transcript 1：本明細書では、しばしば「Malat1」と表記する）遺伝子、微小管結合タンパク質タウ（microtubule-associated protein tau：本明細書では、しばしば「Mapt」と表記する）遺伝子、β-セクレターゼ1(beta-secretase 1：本明細書では、しばしば「BACE1」と表記する)遺伝子、ヒストンデアセチラーゼ2（histone deacetylase 2：本明細書では、しばしば「Hdac2」と表記する）遺伝子、DMPK(dystrophia myotonica-protein kinase)遺伝子、及びジストロフィン遺伝子等が挙げられる。 As used herein, a "target gene" refers to a gene whose transcription or translation product expression level can be suppressed or enhanced, whose transcription or translation product function can be inhibited, or whose steric blocking, splicing control (e.g., splicing switch, exon skipping, exon inclusion), base editing, or RNA editing can be induced by the antisense effect of a nucleic acid molecule or double-stranded nucleic acid complex. The type of target gene is not particularly limited. Examples include genes derived from an organism into which a nucleic acid strand, nucleic acid molecule, or double-stranded nucleic acid complex is introduced, and include genes whose expression increases in various diseases (e.g., central nervous system diseases and peripheral nervous system diseases) and genes expressed in living organisms such as the nervous system. Specific examples include the scavenger receptor B1 (often referred to herein as "SR-B1") gene, the metastasis associated lung adenocarcinoma transcript 1 (often referred to herein as "Malat1") gene, the microtubule-associated protein tau (often referred to herein as "Mapt") gene, the beta-secretase 1 (often referred to herein as "BACE1") gene, the histone deacetylase 2 (often referred to herein as "Hdac2") gene, the dystrophia myotonic-protein kinase (DMPK) gene, and the dystrophin gene.
　本明細書において「標的転写産物」とは、核酸分子又は二本鎖核酸複合体の直接的な標的となる転写産物を意味し、「標的遺伝子の転写産物」も標的転写産物に該当する。標的転写産物や標的遺伝子の塩基配列情報は、例えばNCBI(米国国立生物工学情報センター)データベース等の公知のデータベースから入手できる。 In this specification, the term "target transcript" refers to a transcript that is the direct target of a nucleic acid molecule or a double-stranded nucleic acid complex, and "transcript of a target gene" also falls under the category of target transcript. Information on the base sequences of target transcripts and target genes can be obtained from publicly known databases, such as the NCBI (National Center for Biotechnology Information) database.
　本明細書において「アンチセンス核酸」とは、標的転写産物(主として標的遺伝子の転写産物)の少なくとも一部にハイブリダイズすることが可能な(すなわち、相補的な)塩基配列を含み、標的転写産物にアンチセンス効果をもたらすことができる、一本鎖の核酸分子を指す。また、本明細書において「アンチセンスオリゴヌクレオチド」とは、オリゴヌクレオチドで構成されるアンチセンス核酸を意味する。本明細書では、「アンチセンス核酸」又は「アンチセンスオリゴヌクレオチド」をしばしば「ASO」と表記する。本発明における核酸分子、又は二本鎖核酸複合体の第1核酸鎖は、ASOとして機能し、その標的領域は、3'UTR、5'UTR、エキソン、イントロン、コード領域、翻訳開始領域、翻訳終結領域、又は他のいずれの核酸領域を含んでいてもよい。標的転写産物の標的領域は、少なくとも8塩基長、例えば、10～35塩基長、12～25塩基長、13～20塩基長、14～19塩基長、若しくは15～18塩基長、又は13～22塩基長、16～22塩基長、若しくは16～20塩基長とすることができる。 As used herein, "antisense nucleic acid" refers to a single-stranded nucleic acid molecule that contains a base sequence capable of hybridizing (i.e., complementary) to at least a portion of a target transcript (mainly a transcript of a target gene) and can exert an antisense effect on the target transcript. Also, as used herein, "antisense oligonucleotide" refers to an antisense nucleic acid composed of an oligonucleotide. In this specification, "antisense nucleic acid" or "antisense oligonucleotide" is often referred to as "ASO." The nucleic acid molecule of the present invention or the first nucleic acid strand of the double-stranded nucleic acid complex functions as an ASO, and the target region may include the 3'UTR, 5'UTR, exon, intron, coding region, translation initiation region, translation termination region, or any other nucleic acid region. The target region of the target transcript can be at least 8 bases long, for example, 10-35 bases long, 12-25 bases long, 13-20 bases long, 14-19 bases long, or 15-18 bases long, or 13-22 bases long, 16-22 bases long, or 16-20 bases long.
　本明細書において「アンチセンス効果」とは、ASOが標的転写産物（例えばRNAセンス鎖）にハイブリダイズすることによって生じる任意の効果、例えば標的転写産物にもたらされる発現又は編集を調節する効果をいう。「標的転写産物の発現又は編集を調節する」とは、標的遺伝子の発現又は標的転写産物の発現量（本明細書では、「標的転写産物の発現量」をしばしば「標的転写産物のレベル」と表記する）の抑制又は低下、翻訳の阻害、RNA編集、塩基編集、スプライシング制御若しくはスプライシング機能改変効果（例えばスプライシングスイッチ、エキソンインクルージョン、エキソンスキッピング等）、ステリックブロッキング、又は転写産物の分解をいう。例えば、標的遺伝子の転写後阻害では、ASOとしてRNAオリゴヌクレオチドが細胞に導入されると、ASOは標的遺伝子の転写産物であるmRNAとアニーリングによって部分的二本鎖を形成する。この部分的二本鎖はリボソームによる翻訳を妨げるためのカバーとしての役割を果たし、それによって標的遺伝子にコードされた標的タンパク質の発現が翻訳レベルで阻害される（ステリックブロッキング）。一方、ASOとしてDNAを含むオリゴヌクレオチドが細胞に導入されると、部分的DNA-RNAヘテロ二本鎖が形成される。このヘテロ二本鎖構造がRNase Hによって認識される結果、標的遺伝子のmRNAが分解され、標的遺伝子にコードされたタンパク質の発現が発現レベルで阻害される。さらに、アンチセンス効果は、mRNA前駆体におけるイントロンを標的としてももたらされ得る。さらに、アンチセンス効果は、miRNAを標的としてももたらされ得る。この場合、そのmiRNAの機能阻害により、当該miRNAが通常発現を制御している遺伝子の発現が増加し得る。一実施形態で、標的転写産物の発現調節は、標的転写産物量の低下であってもよい。 As used herein, the term "antisense effect" refers to any effect that occurs when an ASO hybridizes to a target transcript (e.g., an RNA sense strand), such as the effect of regulating the expression or editing of a target transcript. "Regulating the expression or editing of a target transcript" refers to suppression or reduction of the expression of a target gene or the expression level of a target transcript (herein, "the expression level of a target transcript" is often referred to as "the level of a target transcript"), inhibition of translation, RNA editing, base editing, splicing control or splicing function modification effects (e.g., splicing switch, exon inclusion, exon skipping, etc.), steric blocking, or degradation of a transcript. For example, in post-transcriptional inhibition of a target gene, when an RNA oligonucleotide is introduced into a cell as an ASO, the ASO forms a partial duplex by annealing with the mRNA, which is the transcription product of the target gene. This partial duplex acts as a cover to prevent translation by ribosomes, thereby inhibiting the expression of a target protein encoded by the target gene at the translational level (steric blocking). On the other hand, when an oligonucleotide containing DNA is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase H, resulting in degradation of the mRNA of the target gene and inhibition of expression of the protein encoded by the target gene at the expression level. Furthermore, an antisense effect can also be achieved by targeting an intron in a pre-mRNA. Furthermore, an antisense effect can also be achieved by targeting an miRNA. In this case, inhibition of the function of the miRNA can increase the expression of a gene whose expression is normally controlled by the miRNA. In one embodiment, the modulation of expression of the target transcript can be a reduction in the amount of the target transcript.
　アンチセンス効果の測定は、例えば、被験核酸化合物を被験体(例えばマウス)に投与し、例えば数日後(例えば2～7日後)に、被験核酸化合物によって提供されるアンチセンス効果により発現が調節される標的遺伝子の発現量又は標的転写産物のレベル(量)(例えば、mRNA量若しくはマイクロRNA等のRNA量、cDNA量、タンパク質量等)を測定することによって、実施することができる。 The antisense effect can be measured, for example, by administering a test nucleic acid compound to a subject (e.g., a mouse) and measuring, for example, several days later (e.g., 2 to 7 days later), the expression level of a target gene or the level (amount) of a target transcript (e.g., the amount of mRNA or RNA such as microRNA, the amount of cDNA, the amount of protein, etc.) whose expression is regulated by the antisense effect provided by the test nucleic acid compound.
　例えば、測定された標的遺伝子の発現量又は標的転写産物のレベルが、陰性対照(例えばビヒクル投与)と比較して、少なくとも10％、少なくとも20％、少なくとも25％、少なくとも30％、又は少なくとも40％減少している場合に、被験核酸化合物がアンチセンス効果(例えば、標的転写産物量の低下)をもたらし得ることが示される。 For example, a decrease in the measured expression level of the target gene or the level of the target transcript by at least 10%, at least 20%, at least 25%, at least 30%, or at least 40% compared to a negative control (e.g., vehicle administration) indicates that the test nucleic acid compound can produce an antisense effect (e.g., a reduction in the amount of the target transcript).
　核酸鎖における非天然ヌクレオチドの数、種類及び位置は、核酸鎖、核酸分子、又は核酸複合体によって提供されるアンチセンス効果等に影響を及ぼし得る。修飾の選択は、標的遺伝子等の配列によって異なり得るが、当業者であれば、アンチセンス法に関連する文献(例えば、WO 2007/143315、WO 2008/043753、及びWO 2008/049085)の説明を参照することによって好適な実施形態を決定することができる。さらに、修飾後の核酸複合体が有するアンチセンス効果が測定される場合、このようにして得られた測定値が修飾前の核酸複合体の測定値と比較して有意に低くない場合(例えば、修飾後に得られた測定値が、修飾前の核酸複合体の測定値の70％以上、80％以上又は90％以上である場合)、関連修飾を評価することができる。 The number, type and position of non-natural nucleotides in a nucleic acid strand may affect the antisense effect provided by the nucleic acid strand, nucleic acid molecule or nucleic acid complex. The choice of modification may vary depending on the sequence of the target gene, etc., but a person skilled in the art can determine a suitable embodiment by referring to the descriptions in 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 thus obtained is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of the measured value of the nucleic acid complex before modification).
　本明細書において「標的遺伝子の翻訳産物」とは、核酸分子又は二本鎖核酸複合体の直接的な標的となる前記標的転写産物又は標的遺伝子の転写産物の翻訳によって合成される任意のポリペプチド又はタンパク質をいう。 As used herein, the term "translation product of a target gene" refers to any polypeptide or protein synthesized by translation of the target transcript or the transcription product of a target gene that is the direct target of a nucleic acid molecule or a double-stranded nucleic acid complex.
　本明細書において、「デコイ」とは、転写因子（例えばNF-kB）の結合部位の配列又は類似の配列を有する核酸を指し、これらを「おとり」として細胞内に導入することによって転写因子の作用を抑制（転写活性化因子であれば転写を抑制、転写抑制因子であれば転写を促進）するものをいう。デコイ核酸は、標的となる転写因子の結合配列の情報に基づいて容易に設計することができる。 In this specification, the term "decoy" refers to a nucleic acid that has a sequence of the binding site of a transcription factor (e.g., NF-kB) or a similar sequence, and is introduced into a cell as a "decoy" to suppress the action of the transcription factor (if it is a transcription activator, it suppresses transcription, and if it is a transcription repressor, it promotes transcription). Decoy nucleic acids can be easily designed based on information on the binding sequence of the target transcription factor.
　本明細書において、「bait(ベイト)」とは、細胞内で特定の標的分子と特異的に結合する核酸分子であって、標的分子の機能を修飾するものをいう。baitと相互作用する標的を、「prey(プレイ)」ともいう。 As used herein, "bait" refers to a nucleic acid molecule that specifically binds to a specific target molecule within a cell and modifies the function of the target molecule. A target that interacts with a bait is also called a "prey."
　本明細書中で使用される用語「核酸」又は「核酸分子」は、モノマーのヌクレオチド又はヌクレオシドを指してもよいし、複数のモノマーで構成されるオリゴヌクレオチドやヌクレオシド間結合によって連結された複数のヌクレオシドを意味してもよく、またポリマーであればポリヌクレオチドが含まれる。「天然核酸」とは、自然界に存在する核酸をいう。天然核酸には後述の天然ヌクレオシドや天然ヌクレオチド等が含まれる。「非天然核酸」又は「人工核酸」とは、天然核酸以外の任意の核酸を指す。非天然核酸又は人工核酸には、後述の非天然ヌクレオシドや非天然ヌクレオチド等が含まれる。 The term "nucleic acid" or "nucleic acid molecule" as used herein may refer to a monomeric nucleotide or nucleoside, an oligonucleotide composed of multiple monomers, or multiple nucleosides linked by internucleoside bonds, and also includes polynucleotides if they are polymers. "Natural nucleic acid" refers to a nucleic acid that exists in nature. Natural nucleic acids include natural nucleosides and natural nucleotides, etc., described below. "Non-natural nucleic acid" or "artificial nucleic acid" refers to any nucleic acid other than natural nucleic acid. Non-natural nucleic acid or artificial nucleic acid includes non-natural nucleosides and non-natural nucleotides, etc., described below.
　本明細書において「核酸鎖」又は単なる「鎖」とは、ヌクレオシド間結合によって連結された2以上のヌクレオシドを意味し、例えばオリゴヌクレオチド又はポリヌクレオチドであってもよい。核酸鎖は、例えば自動合成装置を使用した化学的合成法により、又はポリメラーゼ、リガーゼ、又は制限反応による酵素的工程により全長鎖又は部分鎖を作製することができる。核酸鎖は、天然ヌクレオチド及び／又は非天然ヌクレオチドを含み得る。 As used herein, a "nucleic acid strand" or simply a "strand" refers to two or more nucleosides linked by internucleoside bonds, and may be, for example, an oligonucleotide or a polynucleotide. A nucleic acid strand may be made full length or partial by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction reactions. A nucleic acid strand may contain natural and/or non-natural nucleotides.
　「ヌクレオシド」とは、一般に塩基及び糖の組み合わせからなる分子をいう。ヌクレオシドの糖部分は、限定はしないが、通常、ペントフラノシル糖で構成され、その具体例としてリボースやデオキシリボースが挙げられる。ヌクレオシドの塩基部分（核酸塩基）は、通常は、複素環式塩基部分である。限定はしないが、アデニン、シトシン、グアニン、チミン、又はウラシルや、それ以外の修飾核酸塩基（修飾塩基）が挙げられる。 "Nucleoside" generally refers to a molecule that is composed of a combination of a base and a sugar. The sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, examples of which include ribose and deoxyribose. The base portion of a nucleoside (nucleobase) is typically a heterocyclic base moiety, including, but not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).
　「ヌクレオチド」とは、前記ヌクレオシドの糖部分にリン酸基が共有結合した分子をいう。ペントフラノシル糖を含むヌクレオチドの場合、通常、糖の2'位、3'位、又は5'位のヒドロキシル基にリン酸基が連結されている。 "Nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of the nucleoside. In the case of nucleotides containing a pentofuranosyl sugar, the phosphate group is usually linked to the hydroxyl group at the 2', 3', or 5' position of the sugar.
　「オリゴヌクレオチド」とは、隣接するヌクレオチド間で糖部分のヒドロキシル基とリン酸基が共有結合によって数個～数十個連結することによって形成される直鎖状のオリゴマーをいう。また「ポリヌクレオチド」とは、オリゴヌクレオチドよりも多数のヌクレオチドが前記共有結合によって数十個以上、好ましくは数百個以上連結することによって形成される直鎖状のポリマーをいう。オリゴヌクレオチド又はポリヌクレオチド構造の内部で、リン酸基は、一般にヌクレオシド間結合を形成するとみなされる。 "Oligonucleotide" refers to a linear oligomer formed by covalently linking several to several dozen hydroxyl groups and phosphate groups in the sugar moieties between adjacent nucleotides. "Polynucleotide" refers to a linear polymer formed by linking several dozen or more, preferably several hundred or more, nucleotides that are more numerous than an oligonucleotide, by the covalent bonds. Within an oligonucleotide or polynucleotide structure, the phosphate groups are generally considered to form internucleoside bonds.
　本明細書において「天然ヌクレオシド」とは、自然界に存在するヌクレオシドをいう。例えば、リボースと前記アデニン、シトシン、グアニン、又はウラシル等の塩基からなるリボヌクレオシドや、デオキシリボースと前記アデニン、シトシン、グアニン、又はチミン等の塩基からなるデオキシリボヌクレオシドが挙げられる。なお、RNA中に見られるリボヌクレオシド、及びDNA中に見られるデオキシリボヌクレオシドを、本明細書では、しばしば、それぞれ「RNAヌクレオシド」及び「DNAヌクレオシド」と称する。 In this specification, "natural nucleosides" refer to nucleosides that exist in nature. Examples include ribonucleosides consisting of ribose and bases such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides consisting of deoxyribose and bases such as adenine, cytosine, guanine, or thymine. In this specification, ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to as "RNA nucleosides" and "DNA nucleosides," respectively.
　本明細書において「天然ヌクレオチド」とは、自然界に存在するヌクレオチドで、前記天然ヌクレオシドの糖部分にリン酸基が共有結合した分子をいう。例えば、リボヌクレオシドにリン酸基が結合した、RNAの構成単位として知られるリボヌクレオチド、及びデオキシリボヌクレオシドにリン酸基が結合した、DNAの構成単位として知られるデオキシリボヌクレオチドが挙げられる。 In this specification, the term "natural nucleotide" refers to a nucleotide that exists in nature and is a molecule in which a phosphate group is covalently bonded to the sugar portion of the natural nucleoside. Examples include ribonucleotides, which are known as the building blocks of RNA and in which a phosphate group is bonded to a ribonucleoside, and deoxyribonucleotides, which are known as the building blocks of DNA and in which a phosphate group is bonded to a deoxyribonucleoside.
　本明細書において「非天然ヌクレオチド」とは、天然ヌクレオチド以外の任意のヌクレオチドを指し、修飾ヌクレオチド及びヌクレオチド模倣体を含む。本明細書において「修飾ヌクレオチド」とは、修飾糖部分、修飾ヌクレオシド間結合、及び修飾核酸塩基のいずれか1つ以上を有するヌクレオチドを意味する。ここでいう「ヌクレオチド模倣体」とは、オリゴマー化合物の一以上の位置において、ヌクレオシド及び結合を置換するために使用される構造体を含む。ヌクレオチド模倣体としては、例えば、ペプチド核酸又はモルホリノ核酸(-N(H)-C(=O)-O-又は他の非ホスホジエステル結合によって結合されるモルホリノ)が挙げられる。ペプチド核酸(Peptide Nucleic Acid、PNA）は、糖の代わりにN-(2-アミノエチル)グリシンがアミド結合で結合した主鎖を有するヌクレオチド模倣体である。本明細書において非天然オリゴヌクレオチドを含む核酸鎖は、多くの場合、例えば、細胞取り込みの強化、核酸標的への親和性の強化、ヌクレアーゼ存在下での安定性の増加、又は阻害活性の増加等の望ましい特性を有する。したがって、天然ヌクレオチドよりも好ましい。 As used herein, the term "non-natural nucleotide" refers to any nucleotide other than a natural nucleotide, and includes modified nucleotides and nucleotide mimetics. As used herein, the term "modified nucleotide" refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase. As used herein, the term "nucleotide mimetics" includes structures used to replace nucleosides and linkages at one or more positions of an oligomeric compound. Nucleotide mimetics include, for example, peptide nucleic acids or morpholino nucleic acids (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Peptide nucleic acids (PNA) are nucleotide mimetics with a backbone in which N-(2-aminoethyl)glycine is linked by amide bonds in place of sugars. As used herein, nucleic acid strands including non-natural oligonucleotides often have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Thus, they are preferred over natural nucleotides.
　本明細書において「非天然ヌクレオシド」とは、天然ヌクレオシド以外の任意のヌクレオシドをいう。例えば、修飾ヌクレオシド及びヌクレオシド模倣体を含む。本明細書において「修飾ヌクレオシド」とは、修飾糖部分及び／又は修飾核酸塩基を有するヌクレオシドを意味する。 As used herein, "unnatural nucleoside" refers to any nucleoside other than a natural nucleoside. For example, it includes modified nucleosides and nucleoside mimetics. As used herein, "modified nucleoside" refers to a nucleoside having a modified sugar moiety and/or a modified nucleobase.
　本明細書において「模倣体」とは、糖、核酸塩基、及び／又はヌクレオシド間結合を置換する官能基を指す。一般に、模倣体は、糖又は糖－ヌクレオシド間結合の組み合わせの代わりに使用され、核酸塩基は、選択される標的に対するハイブリダイゼーションのために維持される。ここでいう「ヌクレオシド模倣体」とは、オリゴマー化合物の一以上の位置において糖を置換するために、又は糖及び塩基を置換するために、又はオリゴマー化合物を構成するモノマーサブユニット間の結合等を置換するために使用される構造体を含む。「オリゴマー化合物」とは、核酸分子のある領域に少なくともハイブリダイズ可能な連結したモノマーサブユニットのポリマーを意味する。ヌクレオシド模倣体としては、例えば、モルホリノ、シクロヘキセニル、シクロヘキシル、テトラヒドロピラニル、二環式又は三環式糖模倣体、例えば、非フラノース糖単位を有するヌクレオシド模倣体が挙げられる。 As used herein, the term "mimetic" refers to functional groups that replace the sugar, nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of a sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target. As used herein, "nucleoside mimic" includes structures that are used to replace the sugar, or the sugar and base, at one or more positions of an oligomeric compound, or to replace the linkage between the monomeric subunits that make up the oligomeric compound, etc. By "oligomeric compound" is meant a polymer of linked monomeric subunits that is at least capable of hybridizing to a region of a nucleic acid molecule. Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar mimetics, e.g., nucleoside mimetics having non-furanose sugar units.
　「修飾糖」とは、天然糖部分(すなわち、DNA(2'-H)又はRNA(2'-OH)中に認められる糖部分)からの置換及び／又は任意の変化を有する糖を指し、「糖修飾」とは、天然糖部分からの置換及び／又は任意の変化をいう。核酸鎖は、場合により、修飾糖を含む1つ以上の修飾ヌクレオシドを含んでもよい。「糖修飾ヌクレオシド」とは、修飾糖部分を有するヌクレオシドをいう。かかる糖修飾ヌクレオシドは、ヌクレアーゼ安定性の強化、結合親和性の増加、又は他の何らかの有益な生物学的特性を核酸鎖に付与し得る。特定の実施形態では、ヌクレオシドは、化学修飾リボフラノース環部分を含む。化学修飾リボフラノース環の例としては、限定するものではないが、置換基(5'及び2'置換基を含む)の付加、非ジェミナル環原子の架橋形成による二環式核酸(架橋核酸、BNA)の形成、リボシル環酸素原子のS、N(R)、又はC(R1)(R2)(R、R1及びR2は、それぞれ独立して、H、C1-C12アルキル、又は保護基を表す)での置換、及びそれらの組み合わせが挙げられる。 "Modified sugar" refers to a sugar having a substitution and/or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)), and "sugar modification" refers to a substitution and/or any change from a natural sugar moiety. A nucleic acid strand may optionally include one or more modified nucleosides, including modified sugars. "Sugar-modified nucleoside" refers to a nucleoside having a modified sugar moiety. Such sugar-modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to a nucleic acid strand. In certain embodiments, the nucleoside includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNAs), replacement of ribosyl ring oxygen atoms with S, N(R), or C(R1)(R2) (wherein R, R1, and R2 each independently represent H, C1 - C12 alkyl, or a protecting group), and combinations thereof.
　糖修飾ヌクレオシドの例としては、限定するものではないが、5'-ビニル、5'-アルキル（例：5'-メチル(R又はS)、5'-エチル(R又はS)）、5'-アリル(R又はS)、4'-S、2'-F(2'-フルオロ基)、2'-OCH3(2'-O-Me基若しくは2'-O-メチル基)、2'-O-[2-(N-メチルカルバモイル)エチル]（2'-O-MCE基）、及び2'-O-メトキシエチル（2'-O-MOE若しくは2-O(CH2)2OCH3）置換基を含むヌクレオシドが挙げられる。2'位の置換基はまた、アリル、アミノ、アジド、チオ、-O-アリル、-O-C1-C10アルキル、-OCF3、-O(CH2)2SCH3、-O(CH2)2-O-N(Rm)(Rn)、及びO-CH2-C(=O)-N(Rm)(Rn)から選択することができ、各Rm及びRnは、独立して、H又は置換若しくは非置換C1-C10アルキルである。「2'-修飾糖」は、2'位で修飾されたフラノシル糖を意味する。2'-修飾糖を含むヌクレオシドを「2'-修飾ヌクレオシド」又は「2'-糖修飾ヌクレオシド」と称することもある。また、「5'-修飾糖」は、5'位で修飾されたフラノシル糖を意味する。5'-修飾糖を含むヌクレオシドを「5'-修飾ヌクレオシド」又は「5'-糖修飾ヌクレオシド」と称し、特に5'-修飾糖を含むデオキシリボヌクレオシドを「5'-修飾デオキシリボヌクレオシド」、5'-修飾糖を含むリボヌクレオシドを「5'-修飾リボヌクレオシド」等として区別する。 Examples of sugar modified nucleosides include, but are not limited to, nucleosides containing 5'-vinyl, 5'-alkyl (e.g., 5'-methyl (R or S), 5'-ethyl (R or S)), 5'-allyl (R or S), 4'-S, 2'-F (2'-fluoro group), 2'- OCH3 (2'-O-Me group or 2'-O-methyl group), 2'-O-[2-(N-methylcarbamoyl)ethyl] (2'-O-MCE group), and 2'-O - methoxyethyl (2'-O-MOE or 2-O( CH2 ) 2OCH3 ) substituents. The substituent at the 2'-position can also be selected from allyl, amino, azido, thio, -O-allyl, -OC1 - C10 alkyl, -OCF3 , -O( CH2 ) 2SCH3 , -O( CH2 ) 2 - ON (Rm)(Rn), and O- CH2 -C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1 - C10 alkyl. "2'-modified sugar" refers to a furanosyl sugar modified at the 2'-position. A nucleoside containing a 2'-modified sugar may also be referred to as a "2'-modified nucleoside" or a "2'-sugar modified nucleoside." Additionally, "5'-modified sugar" refers to a furanosyl sugar modified at the 5'-position. Nucleosides containing a 5'-modified sugar are referred to as "5'-modified nucleosides" or "5'-sugar-modified nucleosides," and are specifically distinguished as "5'-modified deoxyribonucleosides" and "5'-modified ribonucleosides" and "5'-modified ribonucleosides," respectively.
　「二環式ヌクレオシド」は、二環式糖部分を含む修飾ヌクレオシドを指す。二環式糖部分を含む核酸は、一般に架橋核酸(bridged nucleic acid、BNA)と称される。二環式糖部分を含むヌクレオシドは、「架橋ヌクレオシド」、「架橋型の非天然ヌクレオシド」、又は「BNAヌクレオシド」と称することもある。図2に架橋核酸を一部例示する。 "Bicyclic nucleoside" refers to a modified nucleoside that contains a bicyclic sugar moiety. Nucleics that contain a bicyclic sugar moiety are commonly referred to as bridged nucleic acids (BNAs). Nucleosides that contain a bicyclic sugar moiety are also sometimes referred to as "bridged nucleosides," "bridged non-natural nucleosides," or "BNA nucleosides." Some examples of bridged nucleic acids are shown in Figure 2.
　二環式糖は、2'位の炭素原子及び4'位の炭素原子が2つ以上の原子によって架橋されている糖であってよい。二環式糖の例は当業者に公知である。二環式糖を含む核酸(BNA)又はBNAヌクレオシドの1つのサブグループは、4'-(CH2)p-O-2'、4'-(CH2)p-CH2-2'、4'-(CH2)p-S-2'、4'-(CH2)p-OCO-2'、4'-(CH2)n-N(R3)-O-(CH2)m-2'[式中、p、m及びnは、それぞれ1～4の整数、0～2の整数、及び1～3の整数を表し；またR3は、水素原子、アルキル基、アルケニル基、シクロアルキル基、アリール基、アラルキル基、アシル基、スルホニル基、及びユニット置換基(蛍光若しくは化学発光標識分子、核酸切断活性を有する機能性基、細胞内又は核内局在化シグナルペプチド等)を表す]により架橋された2'位の炭素原子と4'位の炭素原子を有すると説明することができる。さらに、特定の実施形態によるBNA又はBNAヌクレオシドに関し、3'位の炭素原子上のOR2置換基及び5'位の炭素原子上のOR1置換基において、R1及びR2は、典型的には水素原子であるが、互いに同一であっても異なっていてもよく、さらにまた、核酸合成のためのヒドロキシル基の保護基、アルキル基、アルケニル基、シクロアルキル基、アリール基、アラルキル基、アシル基、スルホニル基、シリル基、リン酸基、核酸合成のための保護基によって保護されているリン酸基、又はP(R4)R5[ここで、R4及びR5は、互いに同一であっても異なっていてもよく、それぞれヒドロキシル基、核酸合成のための保護基によって保護されているヒドロキシル基、メルカプト基、核酸合成のための保護基によって保護されているメルカプト基、アミノ基、1～5個の炭素原子を有するアルコキシ基、1～5個の炭素原子を有するアルキルチオ基、1～6個の炭素原子を有するシアノアルコキシ基、又は1～5個の炭素原子を有するアルキル基で置換されているアミノ基を表す]であってもよい。このようなBNAの非限定的な例としては、メチレンオキシ(4'-CH2-O-2')BNA(LNA(Locked Nucleic Acid(登録商標)、2',4'-BNAとしても知られている)（例えば、α-L-メチレンオキシ(4'-CH2-O-2')BNA若しくはβ-D-メチレンオキシ(4'-CH2-O-2')BNA）、エチレンオキシ(4'-(CH2)2-O-2')BNA(ENAとしても知られている)、β-D-チオ(4'-CH2-S-2')BNA、アミノオキシ(4'-CH2-O-N(R3)-2')BNA、オキシアミノ(4'-CH2-N(R3)-O-2')BNA(2',4'-BNANCとしても知られている；R=Hは2',4'-BNANC[N-H]、R=Meは2',4'-BNANC[N-Me])、2',4'-BNAcoc、3'-アミノ-2',4'-BNA、5'-メチルBNA、(4'-CH(CH3)-O-2')BNA(cEt BNAとしても知られている)、(4'-CH(CH2OCH3)-O-2')BNA(cMOE BNAとしても知られている)、アミドBNA（アミド架橋型核酸）若しくは(4'-C(O)-N(R)-2')BNA(R=H、Me)(AmNAとしても知られている；図2のR=HはAmNA[N-H]、R=MeはAmNA[N-Me])、グアニジンBNA(GuNA（例、図2のR=HはGuNA[N-H]、R=MeはGuNA[N-Me]）としても知られる)、アミンBNA(2'-Amino-LNAとしても知られている)(例、3-(Bis(3-アミノプロピル)アミノ)プロパノイル置換体)、2'-O,4'-C-スピロシクロプロピレン架橋型核酸(scpBNAとしても知られている)及び当業者に公知の他のBNAが挙げられる。このようなBNAヌクレオシドの非限定的な例としては、メチレンオキシ(4'-CH2-O-2')BNAヌクレオシド(LNAヌクレオシド、2',4'-BNAヌクレオシドとしても知られている)（例、α-L-メチレンオキシ(4'-CH2-O-2')BNAヌクレオシド、β-D-メチレンオキシ(4'-CH2-O-2')BNAヌクレオシド)、エチレンオキシ(4'-(CH2)2-O-2')BNAヌクレオシド(ENAヌクレオシドとしても知られている)、β-D-チオ(4'-CH2-S-2')BNAヌクレオシド、アミノオキシ(4'-CH2-O-N(R3)-2')BNAヌクレオシド、オキシアミノ(4'-CH2-N(R3)-O-2')BNAヌクレオシド(2',4'-BNANCヌクレオシドとしても知られている；R=Hは2',4'-BNANC[N-H]ヌクレオシド、R=Meは2',4'-BNANC[N-Me]ヌクレオシド)、2',4'-BNAcocヌクレオシド、3'-アミノ-2',4'-BNAヌクレオシド、5'-メチルBNAヌクレオシド、(4'-CH(CH3)-O-2')BNAヌクレオシド(cEtヌクレオシドとしても知られている)、(4'-CH(CH2OCH3)-O-2')BNAヌクレオシド(cMOEヌクレオシドとしても知られている)、アミドBNAヌクレオシド若しくは(4'-C(O)-N(R)-2')BNAヌクレオシド(R=H、Me)(AmNAヌクレオシドとしても知られている；図2のR=HはAmNA[N-H]ヌクレオシド、R=MeはAmNA[N-Me]ヌクレオシド)、グアニジンBNAヌクレオシド(GuNAヌクレオシド（例、図2のR=HはGuNA[N-H]ヌクレオシド、R=MeはGuNA[N-Me]ヌクレオシド）としても知られる)、アミンBNAヌクレオシド(2'-Amino-LNAヌクレオシドとしても知られている)(例、3-(Bis(3-アミノプロピル)アミノ)プロパノイル置換ヌクレオシド)、2'-O,4'-C-スピロシクロプロピレン架橋ヌクレオシド(scpBNAヌクレオシドとしても知られている)及び当業者に公知の他のBNAヌクレオシドが挙げられる。 A bicyclic sugar may be a sugar in which the 2' and 4' carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those of skill in the art. One subgroup of bicyclic sugar-containing nucleic acids (BNAs) or BNA nucleosides can be described as having the 2' and 4 ' carbon atoms bridged by 4'-( CH2 ) p -O-2', 4'-( CH2 ) p - CH2-2 ', 4'-( CH2 ) p -S-2', 4'-( CH2 )p-OCO-2', 4'-(CH2) n -N( R3 )-O-( CH2 ) m -2', where p, m and n represent integers from 1 to 4, 0 to 2 and 1 to 3, respectively; and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, and a unit substituent (such as a fluorescent or chemiluminescent labeling molecule, a functional group having nucleic acid cleavage activity, or an intracellular or nuclear localization signal peptide). Furthermore, with respect to BNAs or BNA nucleosides according to certain embodiments, in the OR 2 substituent on the 3' carbon atom and the OR 1 substituent on the 5' carbon atom, R 1 and R 2 are typically hydrogen atoms, but may be the same as or different from each other, and may also be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R 4 )R 5 (wherein R 4 and R 5 may be the same as or different from each other and respectively represent a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms). Non-limiting examples of such BNAs include methyleneoxy (4'- CH2 -O-2') BNAs (also known as LNAs (Locked Nucleic Acids®), 2',4'-BNAs) (e.g., α-L-methyleneoxy (4'- CH2 -O-2') BNAs or β-D-methyleneoxy (4'- CH2 -O-2') BNAs), ethyleneoxy (4'-( CH2 ) 2 -O-2') BNAs (also known as ENAs), β-D-thio (4'- CH2 -S-2') BNAs, aminooxy (4'- CH2 -ON( R3 )-2') BNAs, oxyamino (4'- CH2 -N( R3 )-O-2') BNAs (also known as 2',4'-BNA NC ; where R=H is 2',4'-BNA NC [NH] and R=Me is 2',4'-BNA NC) . [N-Me]), 2',4'-BNA coc , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH 3 )-O-2')BNA (also known as cEt BNA), (4'-CH(CH 2 OCH 3 )-O-2')BNA (cMOE BNAs), amide BNAs (amide bridged nucleic acids) or (4'-C(O)-N(R)-2')BNAs (R=H, Me) (also known as AmNAs; R=H in FIG. 2 is AmNA[NH] and R=Me is AmNA[N-Me]), guanidine BNAs (also known as GuNAs (e.g., R=H in FIG. 2 is GuNA[NH] and R=Me is GuNA[N-Me]), amine BNAs (also known as 2'-Amino-LNAs) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substitutions), 2'-O,4'-C-spirocyclopropylene bridged nucleic acids (also known as scpBNAs), and other BNAs known to those of skill in the art. Non-limiting examples of such BNA nucleosides include methyleneoxy (4'-CH 2 -O-2') BNA nucleosides (also known as LNA nucleosides, 2',4'-BNA nucleosides) (e.g., α-L-methyleneoxy (4'-CH 2 -O-2') BNA nucleosides, β-D-methyleneoxy (4'-CH 2 -O-2') BNA nucleosides), ethyleneoxy (4'-(CH 2 ) 2 -O-2') BNA nucleosides (also known as ENA nucleosides), β-D-thio (4'-CH 2 -S-2') BNA nucleosides, aminooxy (4'-CH 2 -ON(R 3 )-2') BNA nucleosides, oxyamino (4'-CH 2 -N(R 3 )-O-2') BNA nucleosides (2',4'-BNA Also known as NC nucleosides; R=H is 2',4'-BNA NC [NH] nucleosides, R=Me is 2',4'-BNA NC [N-Me] nucleosides), 2',4'-BNA coc nucleosides, 3'-amino-2',4'-BNA nucleosides, 5'-methyl BNA nucleosides, (4'-CH(CH 3 )-O-2')BNA nucleosides (also known as cEt nucleosides), (4'-CH(CH 2 OCH 3 )-O-2') BNA nucleosides (also known as cMOE nucleosides), amide BNA nucleosides or (4'-C(O)-N(R)-2') BNA nucleosides (R=H, Me) (also known as AmNA nucleosides; R=H in Figure 2 is AmNA[NH] nucleosides and R=Me is AmNA[N-Me] nucleosides), guanidine BNA nucleosides (GuNA nucleosides (e.g., R=H in Figure 2 is GuNA[NH] nucleosides), nucleosides, where R=Me is a GuNA[N-Me] nucleoside), amine BNA nucleosides (also known as 2'-Amino-LNA nucleosides) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substituted nucleosides), 2'-O,4'-C-spirocyclopropylene bridged nucleosides (also known as scpBNA nucleosides) and other BNA nucleosides known to those of skill in the art.
　本明細書において、「カチオン性ヌクレオシド」は、あるpH（例えば、ヒトの生理学的pH（約7.4）、送達部位（例えば、オルガネラ、細胞、組織、臓器、生物体等）のpH等）において、中性形態（リボヌクレオシドの中性形態等）と比較して、カチオン形態として存在する修飾ヌクレオシドである。カチオン性ヌクレオシドはヌクレオシドの任意の位置に１つ以上のカチオン性修飾基を含んでもよい。一実施形態では、カチオン性ヌクレオシドは、2'-Amino-LNAヌクレオシド(例、3-(Bis(3-アミノプロピル)アミノ)プロパノイル置換ヌクレオシド)、アミノアルキル修飾ヌクレオシド（例、2'-O-メチル及び4'-CH2CH2CH2NH2置換ヌクレオシド)、GuNAヌクレオシド(例、図2のR=HはGuNA[N-H]ヌクレオシド、R=MeはGuNA[N-Me]ヌクレオシド)等である。メチレンオキシ(4'-CH2-O-2')架橋を有する二環式ヌクレオシドを、LNAヌクレオシドと称することもある。 As used herein, a "cationic nucleoside" is a modified nucleoside that exists in a cationic form relative to a neutral form (such as the neutral form of a ribonucleoside) at a certain pH (e.g., human physiological pH (about 7.4), the pH of a delivery site (e.g., an organelle, cell, tissue, organ, organism, etc.) etc.). A cationic nucleoside may contain one or more cationic modifying groups at any position of the nucleoside. In one embodiment, the cationic nucleoside is a 2'-Amino-LNA nucleoside (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substituted nucleoside ) , an aminoalkyl modified nucleoside (e.g., 2'-O-methyl and 4'- CH2CH2CH2NH disubstituted nucleosides ), a GuNA nucleoside (e.g., R=H in Figure 2 is a GuNA[NH] nucleoside, and R=Me is a GuNA[N-Me] nucleoside), etc. Bicyclic nucleosides with a methyleneoxy (4'- CH2 -O-2') bridge are sometimes referred to as LNA nucleosides.
　本明細書において「修飾ヌクレオシド間結合」とは、天然に存在するヌクレオシド間結合(すなわち、ホスホジエステル結合)からの置換又は任意の変化を有するヌクレオシド間結合を指す。修飾ヌクレオシド間結合には、リン原子を含むヌクレオシド間結合、及びリン原子を含まないヌクレオシド間結合が含まれる。代表的なリン含有ヌクレオシド間結合としては、ホスホジエステル結合、ホスホロチオエート結合、ホスホロジチオエート結合、ホスホトリエステル結合(米国特許登録番号5,955,599記載のメチルホスホトリエステル結合、エチルホスホトリエステル結合)、アルキルホスホネート結合(例、米国特許登録番号5,264,423及び5,286,717記載のメチルホスホネート結合、国際公開第2015/168172号記載のメトキシプロピルホスホネート結合)、アルキルチオホスホネート結合、メチルチオホスホネート結合、ボラノホスフェート結合、環状グアニジン部分を含むヌクレオシド間結合（例、以下の式(V)で表される部分構造：
Figure JPOXMLDOC01-appb-C000007
 ）、１～４個のＣ１～６のアルキル基で置換されたグアニジン部分（例えば、テトラメチルグアニジン（TMG）部分）を含むヌクレオシド間結合（例、以下の式(VI)で表される部分構造：
Figure JPOXMLDOC01-appb-C000008
 ）、国際公開第2016/081600号記載の自己中和核酸(ZON)に用いられるヌクレオシド間結合及びホスホロアミデート結合が挙げられるが、これらに限定されない。ホスホロチオエート結合は、ホスホジエステル結合の非架橋酸素原子を硫黄原子に置換したヌクレオシド間結合を指す。リン含有及び非リン含有結合の調製方法は周知である。修飾ヌクレオシド間結合は、ヌクレアーゼ耐性が天然に存在するヌクレオシド間結合よりも高い結合であることが好ましい。 As used herein, "modified internucleoside linkage" refers to an internucleoside linkage having a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage). Modified internucleoside linkages include internucleoside linkages that contain a phosphorus atom and internucleoside linkages that do not contain a phosphorus atom. Representative phosphorus-containing internucleoside bonds include phosphodiester bonds, phosphorothioate bonds, phosphorodithioate bonds, phosphotriester bonds (e.g., methyl phosphotriester bonds and ethyl phosphotriester bonds as described in U.S. Patent Registration No. 5,955,599), alkyl phosphonate bonds (e.g., methyl phosphonate bonds as described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, and methoxypropyl phosphonate bonds as described in WO 2015/168172), alkylthiophosphonate bonds, methylthiophosphonate bonds, boranophosphate bonds, and internucleoside bonds containing a cyclic guanidine moiety (e.g., a partial structure represented by the following formula (V):
Figure JPOXMLDOC01-appb-C000007
 ), an internucleoside linkage containing a guanidine moiety (e.g., a tetramethylguanidine (TMG) moiety) substituted with one to four C 1-6 alkyl groups (e.g., a moiety represented by the following formula (VI):
Figure JPOXMLDOC01-appb-C000008
 ), and phosphoramidate linkages used in self-neutralizing nucleic acids (ZONs) described in WO 2016/081600. Phosphorothioate linkages refer to internucleoside linkages in which the non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known. The modified internucleoside linkage is preferably one that is more resistant to nucleases than naturally occurring internucleoside linkages.
　ヌクレオシド間結合がキラル中心を有する場合、ヌクレオシド間結合はキラル制御されたものであってもよい。「キラル制御された」とは、キラル中心、例えばキラル結合リンに関して単一のジアステレオマーで存在することを意図する。 When an internucleoside linkage has a chiral center, the internucleoside linkage may be chiral controlled. By "chiral controlled" it is intended that the internucleoside linkage exists as a single diastereomer about a chiral center, e.g., a chiral phosphorus linkage.
　例えば、ヌクレオシド間結合は、Rp配置又はSp配置にキラル制御されたホスホロチオエート結合、１～４個のＣ１～６のアルキル基で置換されたグアニジン部分（例えば、テトラメチルグアニジン（TMG）部分；例えばAlexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811を参照のこと）を含むヌクレオシド間結合、及び／又は環状グアニジン部分を含むヌクレオシド間結合であってよい。Rp配置又はSp配置にキラル制御されたホスホロチオエート結合も公知であり、Sp配置にキラル制御されたホスホロチオエート結合は、Rp配置のものよりも安定であることが知られており、Sp配置にキラル制御されたASOは、RNase H1による標的RNA切断を促進し、生体内でより持続的な応答をもたらすことも知られている。 For example, the internucleoside linkage may be a phosphorothioate linkage chirally controlled in the Rp or Sp configuration, an internucleoside linkage containing a guanidine moiety substituted with one to four C1-6 alkyl groups (e.g., a tetramethylguanidine (TMG) moiety; see, for example, Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811), and/or an internucleoside linkage containing a cyclic guanidine moiety. Chirally controlled phosphorothioate linkages in the Rp or Sp configuration are also known, and phosphorothioate linkages chirally controlled in the Sp configuration are known to be more stable than those in the Rp configuration, and ASOs chirally controlled in the Sp configuration are also known to promote target RNA cleavage by RNase H1 and result in a more sustained response in vivo.
　本明細書中で使用される用語「核酸塩基」又は「塩基」とは、核酸を構成する塩基成分(複素環部分)であって、主としてアデニン、グアニン、シトシン、チミン、及びウラシルが知られる。本明細書において「核酸塩基」又は「塩基」は、特に断りのない場合、修飾又は非修飾の核酸塩基（塩基）のいずれをも包含する。したがって、特に断りのない場合、プリン塩基は修飾又は非修飾のプリン塩基のいずれであってもよい。また、特に断りのない場合、ピリミジン塩基は修飾又は非修飾のピリミジン塩基のいずれであってもよい。 The term "nucleobase" or "base" as used herein refers to the base component (heterocyclic moiety) that constitutes a nucleic acid, and the main known bases are adenine, guanine, cytosine, thymine, and uracil. In this specification, "nucleobase" or "base" includes both modified and unmodified nucleic acid bases (bases), unless otherwise specified. Thus, unless otherwise specified, a purine base may be either a modified or unmodified purine base. Furthermore, unless otherwise specified, a pyrimidine base may be either a modified or unmodified pyrimidine base.
　「修飾核酸塩基」又は「修飾塩基」とは、アデニン、シトシン、グアニン、チミン、又はウラシル以外のあらゆる核酸塩基を意味する。「非修飾核酸塩基」又は「非修飾塩基」(天然核酸塩基)とは、プリン塩基であるアデニン(A)及びグアニン(G)、並びにピリミジン塩基であるチミン(T)、シトシン(C)、及びウラシル(U)を意味する。修飾核酸塩基の例としては、ヒポキサンチン、5-メチルシトシン、5-フルオロシトシン、5-ブロモシトシン、5-ヨードシトシン又はN4-メチルシトシン；N6-メチルアデニン又は8-ブロモアデニン；2-チオ-チミン；並びにN2-メチルグアニン又は8-ブロモグアニンが挙げられるが、これらに限定されない。 "Modified nucleobase" or "modified base" means any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. "Unmodified nucleobase" or "unmodified base" (natural nucleobase) means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Examples of modified nucleobases include, but are not limited to, hypoxanthine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, or N4-methylcytosine; N6-methyladenine, or 8-bromoadenine; 2-thio-thymine; and N2-methylguanine, or 8-bromoguanine.
　本明細書において「ミックスマー」とは、周期的又は無作為セグメント長の交互型の天然ヌクレオシド及び非天然ヌクレオシドを含み、かつ4個以上の連続するデオキシリボヌクレオシド及びリボヌクレオシドを含まない核酸鎖をいう。ミックスマーにおいて、前記非天然ヌクレオシドが架橋ヌクレオシドであり、かつ天然ヌクレオシドがデオキシリボヌクレオシドであるミックスマーを特に「BNA／DNAミックスマー」と称する。ミックスマーにおいて、前記非天然ヌクレオシドがペプチド核酸であり、かつ天然ヌクレオシドがデオキシリボヌクレオシドであるミックスマーを特に「ペプチド核酸／DNAミックスマー」と称する。ミックスマーにおいて、前記非天然ヌクレオシドがモルホリノ核酸であり、かつ天然ヌクレオシドがデオキシリボヌクレオシドであるミックスマーを特に「モルホリノ核酸／DNAミックスマー」と称する。ミックスマーは、2種のヌクレオシドのみを含むようには制限されない。ミックスマーは、天然若しくは修飾のヌクレオシド又はヌクレオシド模倣体であるか否かに関わらず、任意の数の種類のヌクレオシドを含むことができる。例えば、架橋ヌクレオシド(例えば、LNAヌクレオシド)により分離された1又は2個の連続するデオキシリボヌクレオシドを有してもよい。架橋ヌクレオシドは、修飾核酸塩基(例えば、5-メチルシトシン)をさらに含んでもよい。 As used herein, a "mixmer" refers to a nucleic acid chain 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 nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "BNA/DNA mixmer." In a mixmer, a mixmer in which the non-natural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "peptide nucleic acid/DNA mixmer." In a mixmer, a mixmer in which the non-natural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "morpholino nucleic acid/DNA mixmer." A mixmer is not limited to containing only two types of nucleosides. A mixmer can include any number of types of nucleosides, whether natural or modified nucleosides or nucleoside mimetics. For example, it may have one or two consecutive deoxyribonucleosides separated by a bridged nucleoside (e.g., an LNA nucleoside). The bridged nucleoside may further include a modified nucleobase (e.g., 5-methylcytosine).
　本明細書において「ギャップマー」とは、原則として、「中央領域」（DNAギャップ領域）と、その5'末端及び3'末端に直接配置されたウイング領域（それぞれ、「5'ウイング領域」及び「3'ウイング領域」と称する）を含む、又はからなる一本鎖核酸をいう。ギャップマーにおける中央領域は少なくとも3個又は少なくとも4個の連続するデオキシリボヌクレオシドを含む。また、5'ウイング領域及び3'ウイング領域の各々は、少なくとも１つの非天然ヌクレオシドを含む。ギャップマーにおいて中央領域（DNAギャップ領域）と5'ウイング領域及び3'ウイング領域との境界位置は、当業者であればヌクレオシドの配列から容易に判定することができる。例えば中央領域は、RNase H（例えばRNase H1）によって認識され得る領域として機能的に定義することができる。ここで「RNase Hによって認識され得る」とは、ギャップマーが標的RNAと結合したときに標的RNA中で対合した配列がRNase Hによって切断され得ることをいう。したがって、ギャップマーにおいてRNase Hによって認識され得る領域を中央領域、RNase Hによって認識されない領域（より具体的には、生理学的条件下でRNase Hによる切断活性が実質的に検出されない領域）をウイング領域（5'ウイング領域及び3'ウイング領域）として、境界位置を決定することができる。なお、本明細書において、5'ウイング領域及び3'ウイング領域において中央領域と隣接した末端ヌクレオシドは非天然ヌクレオシド（例えば2'-修飾ヌクレオシド又は架橋型ヌクレオシド）とし、中央領域において5'ウイング領域又は3'ウイング領域と隣接したヌクレオシドはデオキシリボヌクレオシド又はその糖修飾体とする。一実施形態では、中央領域において5'ウイング領域及び3'ウイング領域と隣接したヌクレオシドの両方がデオキシリボヌクレオシドである。ギャップマーにおいて中央領域はRNase Hによって認識される修飾核酸塩基を含んでいてもよく、例えば、5-メチルシトシンを含んでもよい。また中央領域は、5'ウイング領域及び3'ウイング領域と隣接した2つの末端ヌクレオシドを除いて、2'-修飾ヌクレオシドや5'-修飾ヌクレオシド等の非天然ヌクレオシドを含むこともできる。一実施形態において、中央領域は、その標的RNA中で対合する領域における全てのヌクレオシド間結合がRNase Hによる切断を受け得るように構成してもよい。また、限定はしないが、ウイング領域に含まれる非天然ヌクレオシドは、通常、天然ヌクレオシドよりもRNAとの結合力が高く、核酸分解酵素(ヌクレアーゼ等)に対する耐性の高い性質を有する。5'ウイング領域及び3'ウイング領域を構成する非天然ヌクレオシドは、例えば、架橋ヌクレオシド及び／又は2'-修飾ヌクレオシドであってもよい。ウイング領域を構成する非天然ヌクレオシドが架橋ヌクレオシドを含む、又はそれからなる場合、前記ギャップマーを特に「BNA／DNAギャップマー」と称する。5'ウイング領域及び3'ウイング領域に含まれる架橋ヌクレオシドの数は、少なくとも1個であり、例えば、2又は3個であってもよい。5'ウイング領域及び3'ウイング領域に含まれる架橋ヌクレオシドは、5'ウイング領域及び3'ウイング領域内に連続又は非連続に存在していてもよい。架橋ヌクレオシドは、修飾核酸塩基(例えば、5-メチルシトシン）をさらに含むことができる。架橋ヌクレオシドはLNAヌクレオシド又はENAヌクレオシドであってもよい。架橋ヌクレオシドがLNAヌクレオシドである場合、前記ギャップマーを「LNA／DNAギャップマー」と称する。架橋ヌクレオシドがENAヌクレオシドである場合、前記ギャップマーを「ENA／DNAギャップマー」と称する。5'ウイング領域及び3'ウイング領域を構成する非天然ヌクレオシドがペプチド核酸を含む、又はそれからなる場合、前記ギャップマーを特に「ペプチド核酸ギャップマー」と称する。5'ウイング領域及び3'ウイング領域を構成する非天然ヌクレオシドがモルホリノ核酸を含む、又はそれからなる場合、前記ギャップマーを特に「モルホリノ核酸ギャップマー」と称する。また、5'ウイング領域及び3'ウイング領域を構成する非天然ヌクレオシドが2'-修飾ヌクレオシドを含む、又はそれからなる場合、2'-修飾ヌクレオシドの2'-修飾基は2'-O-メチル基又は2'-O-メトキシエチル基であってもよい。5'ウイング領域及び3'ウイング領域に含まれる2'-修飾ヌクレオシドの数は、少なくとも1個であり、例えば、2又は3個であってもよい。5'ウイング領域及び3'ウイング領域に含まれる2'-修飾ヌクレオシドは、5'ウイング領域及び3'ウイング領域内に連続又は非連続に存在していてもよい。2'-修飾ヌクレオシドは、修飾核酸塩基(例えば、5-メチルシトシン）をさらに含むことができる。5'ウイング領域及び3'ウイング領域を構成する非天然ヌクレオシドは、架橋ヌクレオシド、2'-修飾ヌクレオシドを含む、又はそれからなる場合、2種類以上の架橋ヌクレオシド及び／又は2'-修飾ヌクレオシドが組み合わされて構成されていてもよい。なお、ウイング領域を5'末端側又は3'末端側のいずれか一方にのみ有する核酸鎖は、当該分野では「ヘミギャップマー」と呼ばれるが、本明細書においては、ヘミギャップマーもギャップマーに包含されるものとする。 As used herein, the term "gapmer" refers, in principle, to a single-stranded nucleic acid that includes or consists of a "central region" (DNA gap region) and wing regions (referred to as the "5' wing region" and "3' wing region", respectively) located directly at the 5' end and 3' end of the central region. The central region in a gapmer contains at least three or at least four consecutive deoxyribonucleosides. Furthermore, each of the 5' wing region and the 3' wing region contains at least one non-natural nucleoside. The boundaries between the central region (DNA gap region) and the 5' wing region and the 3' wing region in a gapmer can be easily determined by a person skilled in the art from the sequence of nucleosides. For example, the central region can be functionally defined as a region that can be recognized by RNase H (e.g., RNase H1). Here, "recognizable by RNase H" means that when the gapmer binds to a target RNA, the paired sequence in the target RNA can be cleaved by RNase H. Therefore, the boundary positions can be determined by defining the region in a gapmer that can be recognized by RNase H as the central region, and the regions that are not recognized by RNase H (more specifically, regions in which cleavage activity by RNase H is not substantially detectable under physiological conditions) as the wing regions (5' wing region and 3' wing region). In this specification, the terminal nucleosides adjacent to the central region in the 5' wing region and 3' wing region are non-natural nucleosides (e.g., 2'-modified nucleosides or bridged nucleosides), and the nucleosides adjacent to the 5' wing region or 3' wing region in the central region are deoxyribonucleosides or sugar-modified versions thereof. In one embodiment, both nucleosides adjacent to the 5' wing region and 3' wing region in the central region are deoxyribonucleosides. In a gapmer, the central region may contain a modified nucleic acid base recognized by RNase H, for example, 5-methylcytosine. The central region may also contain non-natural nucleosides, such as 2'-modified nucleosides and 5'-modified nucleosides, except for the two terminal nucleosides adjacent to the 5' and 3' wing regions. In one embodiment, the central region may be configured such that all internucleoside bonds in the region that it pairs with in the target RNA are susceptible to cleavage by RNase H. Additionally, and without being limited thereto, non-natural nucleosides contained in the wing regions typically have a stronger binding strength to RNA than natural nucleosides and are highly resistant to nucleic acid degrading enzymes (nucleases, etc.). The non-natural nucleosides that make up the 5' and 3' wing regions may be, for example, bridged nucleosides and/or 2'-modified nucleosides. When the non-natural nucleosides that make up the wing regions include or consist of bridged nucleosides, the gapmer is specifically referred to as a "BNA/DNA gapmer." The number of bridged nucleosides in the 5' and 3' wing regions is at least one, and may be, for example, two or three. The bridged nucleosides in the 5' and 3' wing regions may be contiguous or non-contiguous in the 5' and 3' wing regions. The bridged nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine). The bridged nucleoside may be an LNA nucleoside or an ENA nucleoside. When the bridged nucleoside is an LNA nucleoside, the gapmer is referred to as an "LNA/DNA gapmer." When the bridged nucleoside is an ENA nucleoside, the gapmer is referred to as an "ENA/DNA gapmer." When the non-natural nucleosides constituting the 5' and 3' wing regions comprise or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer." When the non-natural nucleosides constituting the 5' wing region and the 3' wing region comprise or consist of morpholino nucleic acid, the gapmer is specifically referred to as a "morpholino nucleic acid gapmer". When the non-natural nucleosides constituting the 5' wing region and the 3' wing region comprise or consist of 2'-modified nucleosides, the 2'-modified group of the 2'-modified nucleoside may be a 2'-O-methyl group or a 2'-O-methoxyethyl group. The number of 2'-modified nucleosides contained in the 5' wing region and the 3' wing region is at least one, and may be, for example, two or three. The 2'-modified nucleosides contained in the 5' wing region and the 3' wing region may be contiguous or non-contiguous in the 5' wing region and the 3' wing region. The 2'-modified nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine). When the non-natural nucleosides constituting the 5' wing region and the 3' wing region include or consist of a bridged nucleoside or a 2'-modified nucleoside, the nucleosides may be composed of a combination of two or more types of bridged nucleosides and/or 2'-modified nucleosides. Note that a nucleic acid strand having a wing region only on either the 5' end or the 3' end is called a "hemigapmer" in the art, and in this specification, hemi-gapmers are also included in the term "gapmer."
　本明細書中で使用される用語「相補的」とは、核酸塩基が水素結合を介して、いわゆるワトソン-クリック塩基対（天然型塩基対）又はWobble塩基対（グアニン-チミン、又はグアニン-ウラシル）を形成し得る関係や、天然核酸塩基と修飾核酸塩基との間又は修飾核酸塩基同士で類似する塩基対を形成し得る関係が挙げられる。 The term "complementary" as used herein includes a relationship in which nucleobases can form so-called Watson-Crick base pairs (natural base pairs) or Wobble base pairs (guanine-thymine or guanine-uracil) through hydrogen bonds, and a relationship in which similar base pairs can be formed between natural nucleobases and modified nucleobases or between modified nucleobases themselves.
　本発明において、核酸分子のアンチセンス領域は、標的転写産物(例えば、標的遺伝子の転写産物)の少なくとも一部と完全に相補的であることは必ずしも必要ではなく、塩基配列が少なくとも70％、好ましくは少なくとも80％、さらにより好ましくは少なくとも90％(例えば、95％、96％、97％、98％、又は99％以上)の相補性を有していれば許容される。核酸分子のアンチセンス領域は、塩基配列が相補的である場合に(典型的には、塩基配列が標的転写産物の少なくとも一部の塩基配列に相補的である場合に)、標的転写産物にハイブリダイズすることができる。同様に、二本鎖核酸複合体において第2核酸鎖中の相補的領域は、第1核酸鎖である核酸分子の少なくとも一部と完全に相補的であることは必ずしも必要ではなく、塩基配列が少なくとも70％、好ましくは少なくとも80％、さらにより好ましくは少なくとも90％(例えば、95％、96％、97％、98％、又は99％以上)の相補性を有していれば許容される。第2核酸鎖中の相補的領域は、第1核酸鎖の少なくとも一部と塩基配列が相補的である場合に、アニールすることができる。塩基配列の相補性は、BLASTプログラム等を使用することによって決定することができる。当業者であれば、鎖間の相補度を考慮して、2本の鎖がアニール又はハイブリダイズし得る条件(温度、塩濃度等)を容易に決定することができる。またさらに、当業者であれば、例えば標的遺伝子の塩基配列の情報に基づいて、標的転写産物に相補的なアンチセンス核酸を容易に設計することができる。 In the present invention, the antisense region of the nucleic acid molecule does not necessarily have to be completely complementary to at least a portion of the target transcript (e.g., the transcript of the target gene), but is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. The antisense region of the nucleic acid molecule can hybridize to the target transcript when the base sequence is complementary (typically when the base sequence is complementary to at least a portion of the base sequence of the target transcript). Similarly, in a double-stranded nucleic acid complex, the complementary region in the second nucleic acid strand does not necessarily have to be completely complementary to at least a portion of the nucleic acid molecule that is the first nucleic acid strand, but is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. A complementary region in the second nucleic acid strand can anneal when the base sequence is complementary to at least a portion of the first nucleic acid strand. The complementarity of the base sequence can be determined by using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can anneal or hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design an antisense nucleic acid complementary to a target transcription product, for example, based on information on the base sequence of the target gene.
　ハイブリダイゼーション条件は、例えば、低ストリンジェントな条件及び高ストリンジェントな条件等の様々なストリンジェントな条件であってもよい。低ストリンジェントな条件は、比較的低温で、かつ高塩濃度の条件、例えば、30℃、2×SSC、0.1％SDSであってよい。高ストリンジェントな条件は、比較的高温で、かつ低塩濃度の条件、例えば、65℃、0.1×SSC、0.1％SDSであってよい。温度及び塩濃度等の条件を変えることによって、ハイブリダイゼーションのストリンジェンシーを調整できる。ここで、1×SSCは、150mM塩化ナトリウム及び15mMクエン酸ナトリウムを含む。 Hybridization conditions may be various stringent conditions, such as low stringency conditions and high stringency conditions. Low stringency conditions may be conditions of relatively low temperature and high salt concentration, for example, 30°C, 2xSSC, 0.1% SDS. High stringency conditions may be conditions of relatively high temperature and low salt concentration, for example, 65°C, 0.1xSSC, 0.1% SDS. Hybridization stringency can be adjusted by changing conditions such as temperature and salt concentration. Here, 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.
　本明細書において「中枢神経系及び／又は末梢神経系への送達」とは、中枢神経系及び／又は末梢神経系における任意の部位又は中枢神経系全体及び／又は末梢神経系全体への送達を意味する。神経系は、中枢神経系及び末梢神経系に分けられる。中枢神経系は、脳及び脊髄からなる。脳は大脳(大脳皮質、大脳白質、大脳基底核)、間脳(視床、視床下核)、小脳(小脳皮質、小脳核)及び脳幹(中脳、黒質、橋、延髄)を含む。脊髄は、頸髄、胸髄、腰髄、仙髄及び尾髄を含む。本明細書における「中枢神経系」は、これらのいずれの領域であってもよいが、特に、大脳皮質(前頭葉、側頭葉、頭頂葉、後頭葉)、小脳、線条体、淡蒼球、前障、海馬、海馬傍回、脳幹、頸髄、胸髄又は腰髄であり得る。中枢神経系は例えば脳である。末梢神経は、脳神経及び脊髄神経からなり、脊髄前根、後根、第１～第１２脳神経、馬尾、及び後根神経節を含む。 As used herein, "delivery to the central nervous system and/or peripheral nervous system" means delivery to any site in the central nervous system and/or peripheral nervous system or to the entire central nervous system and/or peripheral nervous system. The nervous system is divided into the central nervous system and the peripheral nervous system. The central nervous system consists of the brain and spinal cord. The brain includes the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon (thalamus, subthalamic nucleus), cerebellum (cerebellar cortex, cerebellar nuclei), and brainstem (midbrain, substantia nigra, pons, medulla oblongata). The spinal cord includes the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, sacral spinal cord, and coccygeal spinal cord. The "central nervous system" as used herein may be any of these regions, but may particularly be the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brainstem, cervical spinal cord, thoracic spinal cord, or lumbar spinal cord. An example of the central nervous system is the brain. The peripheral nerves consist of the cranial nerves and spinal nerves, including the ventral root of the spinal cord, the dorsal root, the cranial nerves 1 to 12, the cauda equina, and the dorsal root ganglion.
　本明細書において中枢神経系への「送達促進」とは、上述の中枢神経系及び／又は末梢神経系への送達量や、中枢神経系及び／又は末梢神経系への送達効率が増大することを意味する。例えば、本発明の送達促進剤を共投与しない条件下における薬剤の中枢神経系及び／又は末梢神経系への送達量と比較して、共投与条件下で当該薬剤の送達量が増大すればよい。 In this specification, "enhanced delivery" to the central nervous system means an increase in the amount of drug delivered to the central nervous system and/or peripheral nervous system, or an increase in the efficiency of delivery to the central nervous system and/or peripheral nervous system. For example, the amount of drug delivered to the central nervous system and/or peripheral nervous system under co-administration conditions may be increased compared to the amount of drug delivered to the central nervous system and/or peripheral nervous system under conditions in which the delivery enhancer of the present invention is not co-administered.
　本明細書で「被験体」とは、薬物又は医薬組成物を適用する対象をいう。被験体は、個体の他、器官、組織、及び細胞を含む。被験体が個体の場合、ヒトを含むあらゆる動物が該当し得る。例えば、ヒト以外では、様々な家畜、家禽、ペット、実験動物等が挙げられる。限定はしないが、被験体は、標的転写産物の発現量を減少させる必要がある個体や、中枢神経系疾患等の疾患の治療又は予防が必要な個体であってもよい。 As used herein, the term "subject" refers to an object to which a drug or pharmaceutical composition is applied. Subjects include individuals, as well as organs, tissues, and cells. When the subject is an individual, it may be any animal, including humans. Examples of subjects other than humans include various livestock, poultry, pets, and laboratory animals. Without being limited thereto, the subject may be an individual in which the expression level of a target transcript needs to be reduced, or an individual in which treatment or prevention of a disease, such as a central nervous system disease, is required.
　本明細書において「複数個」とは、例えば、2個、2～3個、2～4個、2～5個、2～6個、2～7個、2～8個、2～10個、2～12個、2～14個、2～16個、2～18個、2～20個、2～25個、2～30個、2～35個、2～40個、若しくはそれ以上をいう。 In this specification, "multiple" refers to, for example, 2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-10, 2-12, 2-14, 2-16, 2-18, 2-20, 2-25, 2-30, 2-35, 2-40, or more.
（送達促進剤）
　本態様の薬剤送達促進剤は、アクアポリン4機能促進剤又はアクアポリン4機能修飾剤からなる。 Delivery Enhancers
The drug delivery enhancer of this embodiment comprises an aquaporin 4 function enhancer or an aquaporin 4 function modifier.
　本明細書において「アクアポリン4機能促進剤」は、アクアポリン4を活性化し得る物質であれば制限されず、任意の公知のアクアポリン4機能促進剤を使用することができる。また、本明細書において「アクアポリン4機能修飾剤」は、アクアポリン4の機能を修飾し得る物質であれば制限されず、任意の公知のアクアポリン4機能修飾剤を使用することができる。 In this specification, the term "aquaporin 4 function promoter" is not limited to any substance that can activate aquaporin 4, and any known aquaporin 4 function promoter can be used. In addition, in this specification, the term "aquaporin 4 function modifier" is not limited to any substance that can modify the function of aquaporin 4, and any known aquaporin 4 function modifier can be used.
　アクアポリン4機能促進剤又はアクアポリン4機能修飾剤の一例としては、下記式（I）：
Figure JPOXMLDOC01-appb-C000009
 で示される化合物若しくはその誘導体、又はそのいずれかの塩が挙げられる。なお、上記式(I)で示される化合物は、国際公開第2017/150704号及び本発明者らの過去の文献（Huber V.J. et al., NeuroReport, 2018, 29(9):697-703）においてアクアポリン4の機能を促進し得ることが開示されており、一般名TGN-073として公知であるが、N-(3-benzyloxypyridin-2-yl)-benzene-sulfonamide又は2-(phenylsulfonamido)-3-benzyloxypyridineと表記することもできる。 An example of the aquaporin 4 function promoter or aquaporin 4 function modifier is a compound represented by the following formula (I):
Figure JPOXMLDOC01-appb-C000009
 or a derivative thereof, or a salt thereof. The compound represented by the above formula (I) is disclosed in International Publication No. 2017/150704 and the inventors' previous publication (Huber VJ et al., NeuroReport, 2018, 29(9):697-703) as being capable of promoting the function of aquaporin 4, and is known under the generic name TGN-073, but can also be written as N-(3-benzyloxypyridin-2-yl)-benzene-sulfonamide or 2-(phenylsulfonamido)-3-benzyloxypyridine.
　上記(I)で示される化合物の誘導体は、アクアポリン4に対する機能促進活性又は機能修飾活性を誘導し得る任意の誘導体である。誘導体の一例として、下記式(III)：
Figure JPOXMLDOC01-appb-C000010
 で示される化合物を挙げることができる。 The derivative of the compound represented by the above formula (I) is any derivative capable of inducing a function-promoting activity or a function-modifying activity on aquaporin 4. An example of the derivative is a compound represented by the following formula (III):
Figure JPOXMLDOC01-appb-C000010
 Examples of the compound include those represented by the following formula:
　また、上記式(I)で示される化合物の塩は特に制限されず、薬学的に許容される塩であればよい。薬学的に許容される塩の例として、金属塩、無機酸の塩、及び有機酸の塩が挙げられる。金属塩は、ナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩、又はストロンチウム塩であってもよい。無機酸の塩は、塩酸、臭素酸、リン酸、硫酸、又は二硫酸の塩であってもよい。有機酸の塩は、ギ酸、酢酸、プロピオン酸、乳酸、シュウ酸、酒石酸、リンゴ酸、マレイン酸、クエン酸、フマル酸、ベシル酸、カンシル酸、エジシル酸、トリクロロ酢酸、トリフルオロ酢酸、安息香酸、グルコン酸、メタンスルホン酸、グリコール酸、コハク酸、4-トルエンスルホン酸、ガラクツロン酸、エンボン酸、グルタミン酸、エタンスルホン酸、ベンゼンスルホン酸、p-トルエンスルホン酸、又はアスパラギン酸の塩であってもよい。 　The salt of the compound represented by formula (I) above is not particularly limited, and may be any pharma- ceutically acceptable salt. Examples of pharma-ceutically acceptable salts include metal salts, salts of inorganic acids, and salts of organic acids. The metal salt may be a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or a strontium salt. The salt of an inorganic acid may be a salt of hydrochloric acid, bromic acid, phosphoric acid, sulfuric acid, or disulfuric acid. The salt of an organic acid may be a salt of formic acid, acetic acid, propionic acid, lactic acid, oxalic acid, tartaric acid, malic acid, maleic acid, citric acid, fumaric acid, besylic acid, camsylic acid, edisylic acid, trichloroacetic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or aspartic acid.
　一実施形態において、式（I）で示す化合物又はその誘導体は、キャリア分子と結合している。キャリア分子は、送達促進剤の目的の送達部位への送達効率を調節する目的で送達促進剤に結合される任意の分子である。キャリア分子は、その分子サイズや親水性／疎水性等の制御に基づいて目的の送達部位への送達効率を調節することができる。キャリア分子は特に制限されず、例えばタンパク質（例えば1kDa以上のタンパク質）、ポリマー、脂質分子、又は造影剤を挙げることができる。タンパク質としては、アルブミン、リポタンパク質（例：HDL like particle）、又は抗体若しくは抗体断片（例：IgG、Fc、修飾Fc等）が例示される。ポリマーとしては、ポリエチレングリコール(PEG)又はPEG graftedポリマーが例示される。脂質としては、トコフェロール、コレステロール、脂肪酸、リン脂質の他、後述する脂質の例も挙げられる。 In one embodiment, the compound represented by formula (I) or a derivative thereof is bound to a carrier molecule. The carrier molecule is any molecule bound to the delivery enhancer for the purpose of adjusting the delivery efficiency of the delivery enhancer to the target delivery site. The carrier molecule can adjust the delivery efficiency to the target delivery site based on the control of its molecular size, hydrophilicity/hydrophobicity, etc. The carrier molecule is not particularly limited, and examples thereof include proteins (e.g., proteins of 1 kDa or more), polymers, lipid molecules, and contrast agents. Examples of proteins include albumin, lipoproteins (e.g., HDL-like particles), and antibodies or antibody fragments (e.g., IgG, Fc, modified Fc, etc.). Examples of polymers include polyethylene glycol (PEG) or PEG-grafted polymers. Examples of lipids include tocopherol, cholesterol, fatty acids, phospholipids, and the lipid examples described below.
　さらなる実施形態において、式（I）で示す化合物又はその誘導体は、マイクロバブル、ミセル、又はリポソームと結合又は会合することができる。結合又は会合の様式は限定しないが、例えば式（I）で示す化合物又はその誘導体と結合した上述の脂質やポリマー等のキャリア分子を介することができる。 In a further embodiment, the compound of formula (I) or a derivative thereof can be bound or associated with a microbubble, micelle, or liposome. The manner of binding or association is not limited, but can be, for example, via a carrier molecule, such as the above-mentioned lipid or polymer, bound to the compound of formula (I) or a derivative thereof.
　本発明の送達促進剤の分子サイズや親水性／疎水性の制御によって、投与部位の付近で組織への浸透を防ぎ、それにより目的の送達部位への送達効率の低下を防ぐことができるため好適である。 By controlling the molecular size and hydrophilicity/hydrophobicity of the delivery enhancer of the present invention, it is possible to prevent penetration into tissues near the administration site, which is advantageous because it is possible to prevent a decrease in the efficiency of delivery to the desired delivery site.
（薬剤）
　本明細書において「薬剤」（薬物、医薬、又は医薬品ともいう）は特に限定されず、核酸医薬、ペプチド、低分子化合物、ウイルスベクター、細胞医薬、ナノ粒子、リポソーム、ミセル、又はエクソソームを包含する。なお、本明細書において本発明の送達促進剤と併用され、中枢神経系及び／又は末梢神経系に送達されるべき薬剤をしばしば「神経系に送達されるべき薬剤」と称する。 (Drugs)
As used herein, the term "drug" (also referred to as a drug, medicine, or pharmaceutical product) is not particularly limited and includes nucleic acid medicines, peptides, low molecular weight compounds, viral vectors, cellular medicines, nanoparticles, liposomes, micelles, and exosomes. Note that, as used herein, a drug to be delivered to the central nervous system and/or peripheral nervous system in combination with the delivery enhancer of the present invention is often referred to as a "drug to be delivered to the nervous system."
　本明細書において「ペプチド」とは、1つ以上のペプチド結合を有するアミノ酸ポリマーをいう。ペプチドは、ペプチドに含まれるアミノ酸残基の数によって限定されない。したがって、ペプチドには、ジペプチドやトリペプチド等の数個のアミノ酸残基を含むオリゴペプチドから、多数のアミノ酸残基を含むポリペプチドまでが包含される。したがって、いわゆるタンパク質のみならず、断片化されたものや、ペプチド結合によって他のペプチドが連結されたものも包含される。ペプチドの例として、抗体又は抗体断片、及び酵素が挙げられる。なお、ペプチドは、原則としてタンパク質医薬（例：タンパク製剤）等の外因性のペプチドが該当し、タンパク質の分解産物及びその他の細胞における老廃物等を含まない。また、ペプチドは天然由来のものであっても、非天然であってもよい。さらに、ペプチドは環状又は非環状のいずれであってもよい。 In this specification, the term "peptide" refers to an amino acid polymer having one or more peptide bonds. The term "peptide" is not limited by the number of amino acid residues contained in the peptide. Thus, peptides include everything from oligopeptides containing several amino acid residues, such as dipeptides and tripeptides, to polypeptides containing many amino acid residues. Thus, peptides include not only so-called proteins, but also fragmented peptides and peptides linked to other peptides by peptide bonds. Examples of peptides include antibodies or antibody fragments, and enzymes. In principle, peptides include exogenous peptides such as protein pharmaceuticals (e.g., protein preparations), and do not include protein degradation products and other cellular waste products. Furthermore, peptides may be naturally derived or non-naturally derived. Furthermore, peptides may be either cyclic or non-cyclic.
　一実施形態において、ペプチドは、抗体又は抗体断片である。 In one embodiment, the peptide is an antibody or an antibody fragment.
　本明細書において、「抗体」は、抗原に対して免疫応答性を示すタンパク質を意味する。抗体の由来生物種は、特に限定しない。好ましくは鳥類及び哺乳動物由来の抗体である。例えば、ニワトリ、ダチョウ、マウス、ラット、モルモット、ウサギ、ヤギ、ロバ、ヒツジ、ラクダ、ウマ、又はヒト等が挙げられる。抗体は全長抗体であってもよい。 In this specification, "antibody" refers to a protein that exhibits immune responsiveness to an antigen. There are no particular limitations on the species of organism from which the antibody is derived. Antibodies are preferably derived from birds and mammals. Examples include chicken, ostrich, mouse, rat, guinea pig, rabbit, goat, donkey, sheep, camel, horse, and human. The antibody may be a full-length antibody.
　本明細書において抗体の「断片」とは、抗体の一部からなり、かつ抗体と同様に抗原に対して免疫応答性を示す抗体断片であり、抗原結合性断片である。例えば、Fab、Fab'、F(ab')2、Fvフラグメント、ジスルフィド結合により安定化したFvフラグメント（dsFv）、(dsFv)2、二重特異性dsFv（dsFv-dsFv'）、ジスルフィド結合により安定化したディアボディ（dsディアボディ）、単鎖抗体分子（scFv）、二量体scFv（2価ディアボディ）、多重特異性抗体、ラクダ化シングルドメイン抗体（ラクダ化抗体；VHH抗体）等の重鎖抗体、ナノボディ、ドメイン抗体、及び2価ドメイン抗体等が該当する。Fabは、IgG分子がパパインによってヒンジ部のジスルフィド結合よりもN末端側で切断されて生じる抗体断片であって、H鎖定常領域（重鎖定常領域：以下CHと表記する）を構成する3つのドメイン（CH1、CH2、CH3）のうちVHに隣接するCH1とVH、及び完全長のL鎖から構成される。Fab'は、Fabよりもヒンジ部を含む分だけH鎖が若干長いが実質的にはFabと同等の構造を有する。Fab'は、IgG分子がペプシンによってヒンジ部のジスルフィド結合よりもC末端側で切断されて生じるFab'の二量体（F(ab')2）をマイルドな条件下で還元し、ヒンジ領域のジスルフィド連結を切断することによって得ることができる。これらの抗体断片は、いずれも抗原結合部位を包含していることから、抗原エピトープと特異的に結合する能力を有している。 As used herein, the term "fragment" of an antibody refers to an antibody fragment that is composed of a part of an antibody and exhibits immune responsiveness to an antigen like an antibody, and is an antigen-binding fragment. Examples of such fragments include Fab, Fab', F(ab') 2 , Fv fragment, Fv fragment stabilized by a disulfide bond (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), diabody stabilized by a disulfide bond (dsdiabody), single-chain antibody molecule (scFv), dimeric scFv (bivalent diabody), multispecific antibody, heavy chain antibody such as camelized single domain antibody (camelized antibody; VHH antibody), nanobody, domain antibody, and bivalent domain antibody. Fab is an antibody fragment generated by cleavage of an IgG molecule at the N-terminal side of the disulfide bond in the hinge region with papain, and is composed of the C H 1 and V H adjacent to the V H of the three domains (C H 1, C H 2, C H 3) that constitute the H chain constant region (heavy chain constant region: hereinafter referred to as C H ) , and a full-length L chain. Fab' has a slightly longer H chain than Fab because it contains the hinge region, but has a substantially similar structure to Fab. Fab' can be obtained by reducing the Fab' dimer (F(ab') 2 ) generated by cleavage of an IgG molecule at the C-terminal side of the disulfide bond in the hinge region with pepsin under mild conditions to cleave the disulfide bond in the hinge region. All of these antibody fragments contain an antigen-binding site and therefore have the ability to specifically bind to an antigen epitope.
　本明細書において「低分子化合物」は特に制限されず、中枢神経系疾患又は末梢神経系疾患の治療薬、精神薬、又は抗癌剤等であってもよい。なお、上述の送達促進剤自体は、本明細書において神経系に送達されるべき低分子化合物からは除外されるものとする。 In this specification, the term "low molecular weight compound" is not particularly limited, and may be a therapeutic drug for a central nervous system disease or a peripheral nervous system disease, a psychiatric drug, an anticancer drug, or the like. Note that the above-mentioned delivery enhancer itself is excluded from the low molecular weight compound to be delivered to the nervous system in this specification.
　本明細書において「ウイルスベクター」は特に制限されず、例えばレトロウイルスベクター（オンコレトロウイルスベクター、レンチウイルスベクター、及び偽型ベクターを含む）、アデノウイルスベクター、アデノ随伴ウイルス（AAV）ベクター、シミアンウイルスベクター、ワクシニアウイルスベクター、センダイウイルスベクター、エプスタイン－バーウイルス（EBV）ベクター、及びHSVベクター等のウイルスベクターが使用可能である。感染細胞内で自己複製しないように複製能を欠くウイルスベクターを使用してもよい。ウイルスベクターは遺伝子治療用の遺伝子をコードすることができる。 In this specification, the term "viral vector" is not particularly limited, and examples of usable viral vectors include retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotype vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Viral vectors that lack replication ability so as not to replicate autonomously within infected cells may also be used. The viral vector can encode a gene for gene therapy.
　本明細書において「細胞医薬」は特に制限されず、例えば任意の中枢神経系疾患又は末梢神経系疾患の治療に用いられる細胞を使用することができる。細胞医薬に含まれる細胞として、神経細胞、T細胞（例：CAR-T細胞）、NK細胞、NKT細胞、造血幹細胞、末梢血単核球、間葉系幹細胞、iPS細胞、及びES細胞等が挙げられる。 In this specification, the term "cell medicine" is not particularly limited, and may be, for example, a cell used in the treatment of any central nervous system disease or peripheral nervous system disease. Examples of cells contained in cell medicines include nerve cells, T cells (e.g., CAR-T cells), NK cells, NKT cells, hematopoietic stem cells, peripheral blood mononuclear cells, mesenchymal stem cells, iPS cells, and ES cells.
　本明細書において「ナノ粒子」とは、ナノメートル（nm）オーダーの粒子径を有する粒子をいう。ナノ粒子は、原則として1nm～数百nmの粒子径を有する粒子を意味する。ナノ粒子の具体例としては、ポリマーナノ粒子、金属ナノ粒子、及びデンドリマー等が挙げられる。 In this specification, "nanoparticles" refers to particles with a particle size on the order of nanometers (nm). In principle, nanoparticles refer to particles with a particle size of 1 nm to several hundred nm. Specific examples of nanoparticles include polymer nanoparticles, metal nanoparticles, and dendrimers.
　本明細書において「リポソーム」とは、脂質膜、及び脂質膜に内包された水性媒体を含む小胞を意味する。リポソームの脂質膜は、1又は2以上の脂質層で構成される。例えばリン脂質等を含む脂質二重層で構成される。 In this specification, "liposome" refers to a vesicle that contains a lipid membrane and an aqueous medium encapsulated in the lipid membrane. The lipid membrane of a liposome is composed of one or more lipid layers. For example, it is composed of a lipid bilayer containing phospholipids, etc.
　本明細書において「ミセル」とは、一層の分子膜により形成される小胞を意味する。ミセルの構成成分の例として、界面活性剤等の両親媒性分子が挙げられる。 In this specification, "micelle" refers to a vesicle formed by a single molecular membrane. Examples of components of micelles include amphiphilic molecules such as surfactants.
　上述のナノ粒子、リポソーム、及びミセルは、核酸医薬、ペプチド、低分子化合物等を含むことが可能であり、薬物の送達に便利である。 The above-mentioned nanoparticles, liposomes, and micelles can contain nucleic acid drugs, peptides, low molecular weight compounds, etc., making them useful for drug delivery.
　本明細書において「エクソソーム」とは、細胞から分泌される脂質二重膜に包まれた小胞である。エクソソームは多胞エンドソームに由来し、細胞外環境に放出される際にRNA、DNA等の核酸やタンパク質等の生体物質を内部に含むことがある。 As used herein, "exosomes" are small vesicles enclosed in a lipid bilayer membrane that are secreted from cells. Exosomes originate from multivesicular endosomes, and when released into the extracellular environment, they may contain biological materials such as nucleic acids (RNA, DNA, etc.) and proteins.
（核酸医薬）
　本明細書において「核酸医薬（nucleic acid medicine）」は、任意の核酸分子を含む薬剤である。本明細書において、核酸医薬は原則として2以上のヌクレオシドを含むものを意味し、核酸医薬に含まれるヌクレオシドは天然ヌクレオシド又は非天然ヌクレオシドを問わない。また、核酸医薬に含まれる核酸分子は、一本鎖又は二本鎖以上の核酸であってもよい。核酸医薬の具体例としては、限定するものではないが、核酸分子として後述するアンチセンス核酸、二本鎖核酸複合体として後述するヘテロ核酸、siRNA、shRNA、miRNA、mRNA、lncRNA、アプタマー、プラスミドDNA、デコイ、bait核酸、リボザイム、及びプラスミドベクター等の核酸ベクターを挙げることできる。 (Nucleic acid medicine)
In this specification, the term "nucleic acid medicine" refers to a drug containing any nucleic acid molecule. In this specification, the term "nucleic acid medicine" generally refers to a drug containing two or more nucleosides, and the nucleosides contained in the nucleic acid medicine may be natural or non-natural nucleosides. In addition, the nucleic acid molecule contained in the nucleic acid medicine may be a single-stranded or double-stranded or more nucleic acid. Specific examples of nucleic acid medicines include, but are not limited to, antisense nucleic acids as nucleic acid molecules described below, heterogeneous nucleic acids as double-stranded nucleic acid complexes described below, siRNA, shRNA, miRNA, mRNA, lncRNA, aptamers, plasmid DNA, decoys, bait nucleic acids, ribozymes, and nucleic acid vectors such as plasmid vectors.
（核酸分子）
　一実施形態において、核酸医薬は、標的遺伝子又はその転写産物の少なくとも一部にハイブリダイズすることができ、かつ前記標的遺伝子又はその転写産物に対してアンチセンス効果を有する核酸分子を含む。 (Nucleic Acid Molecules)
In one embodiment, the nucleic acid medicine comprises a nucleic acid molecule capable of hybridizing to at least a part of a target gene or its transcription product and having an antisense effect on the target gene or its transcription product.
　核酸分子の塩基長は、特に限定されないが、少なくとも8塩基長、少なくとも9塩基長、少なくとも10塩基長、少なくとも11塩基長、少なくとも12塩基長、少なくとも13塩基長、少なくとも14塩基長、又は少なくとも15塩基長であればよい。また、核酸分子の塩基長は、40塩基長以下、35塩基長以下、30塩基長以下、25塩基長以下、24塩基長以下、23塩基長以下、22塩基長以下、21塩基長以下、20塩基長以下、19塩基長以下、18塩基長以下、17塩基長以下、又は16塩基長以下であればよい。核酸分子の塩基長は、例えば、10～40塩基長、12～30塩基長、又は15～25塩基長であってもよい。長さの選択は、例えば費用、合成収率等の他の因子の中でも特に、アンチセンス効果の強度と標的に対する核酸鎖の特異性とのバランスによって決定することができる。なお、核酸分子にアプタマー等の核酸が結合している場合、核酸分子の全体としての塩基長は、上記塩基長に結合した核酸の塩基長を加えたものであってよい。 The base length of the nucleic acid molecule is not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. The base length of the nucleic acid molecule 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 base length of the nucleic acid molecule may be, for example, 10 to 40 bases, 12 to 30 bases, or 15 to 25 bases. The length can be determined by the balance between the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield. In addition, when a nucleic acid such as an aptamer is bound to the nucleic acid molecule, the base length of the nucleic acid molecule as a whole may be the above-mentioned base length plus the base length of the bound nucleic acid.
　核酸分子に含まれるヌクレオシドは、天然ヌクレオシド(デオキシリボヌクレオシド、リボヌクレオシド、若しくは両者)及び／又は非天然ヌクレオシドであってよい。 The nucleosides contained in the nucleic acid molecule may be natural nucleosides (deoxyribonucleosides, ribonucleosides, or both) and/or non-natural nucleosides.
　一実施形態において、核酸分子は、ミックスマー（mixmer）であってもよい。 In one embodiment, the nucleic acid molecule may be a mixmer.
　別の実施形態において、核酸分子はギャップマーであってもよい。 In another embodiment, the nucleic acid molecule may be a gapmer.
　ギャップマーの中央領域（ギャップ領域）は、例えば3～12塩基長、4～11塩基長、5～10塩基長、6～9塩基長、又は7～8塩基長であってもよい。 The central region (gap region) of a gapmer may be, for example, 3-12 bases long, 4-11 bases long, 5-10 bases long, 6-9 bases long, or 7-8 bases long.
　また、ギャップマーの5'ウイング領域及び3'ウイング領域の塩基長は、それぞれ独立して、少なくとも2塩基長、例えば、2～10塩基長、2～7塩基長、3～5塩基長、3～4塩基長、又は3塩基長であってもよい。 Furthermore, the base length of the 5' wing region and the 3' wing region of the gapmer may each independently be at least 2 bases long, for example, 2 to 10 bases long, 2 to 7 bases long, 3 to 5 bases long, 3 to 4 bases long, or 3 bases long.
　核酸分子は、5'ウイング領域及び3'ウイング領域に2'-修飾ヌクレオシド及び／又は架橋型ヌクレオシドを含むことができる。2'-修飾ヌクレオシドは、例えば2'-O-メチル修飾ヌクレオシド、2'-O-メトキシエチル修飾ヌクレオシド、2'-O-[2-(N-メチルカルバモイル)エチル]修飾ヌクレオシド、又は2'-フルオロ修飾ヌクレオシドであってもよい。また架橋型ヌクレオシドは、例えばLNAヌクレオシド、2',4'-BNANCヌクレオシド、cEt BNAヌクレオシド、ENAヌクレオシド、AmNAヌクレオシド、GuNAヌクレオシド、scpBNAヌクレオシド、scpBNA2ヌクレオシド、又はBANA3ヌクレオシドであってもよい。好ましくは、2'-O-メチル修飾ヌクレオシド、2'-O-メトキシエチル修飾ヌクレオシド、2'-LNA又はENAを組みあわせてもよく、その修飾の種類は、1～4種類、2～3種類、例えば2種類を含んでもよく、それらの種類は、5'ウイング領域及び3'ウイング領域で同じであっても又は異なっていてもよい。 The nucleic acid molecule can include 2'-modified nucleosides and/or bridged nucleosides in the 5' and 3' wing regions. The 2'-modified nucleosides can be, for example, 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleosides, or 2'-fluoro modified nucleosides. The bridged nucleosides can be, for example, LNA nucleosides, 2',4'-BNA NC nucleosides, cEt BNA nucleosides, ENA nucleosides, AmNA nucleosides, GuNA nucleosides, scpBNA nucleosides, scpBNA2 nucleosides, or BANA3 nucleosides. Preferably, 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-LNA or ENA may be combined, and the types of modifications may include 1 to 4 types, 2 to 3 types, for example 2 types, which may be the same or different in the 5' wing region and the 3' wing region.
　一実施形態において、核酸分子は、5'ウイング領域において中央領域に隣接する末端塩基位置、中央領域の5'側から2番目の塩基位置、及び／又は中央領域の5'側から8番目の塩基位置に2'-修飾ヌクレオシドを含む。「5'ウイング領域において前記中央領域に隣接する末端塩基位置」とは、5'ウイング領域において最も3'側の末端に位置する塩基位置を意味する。また、本明細書において「中央領域の5'側からN番目の塩基位置」とは、中央領域において5'ウイング領域に隣接する末端塩基を起点として5'側から3'側への方向にN番目に位置する塩基を意味する。したがって、中央領域の5'側から2番目の塩基位置とは、中央領域において5'ウイング領域に隣接する末端塩基を起点として3'方向に数えて2番目に位置する塩基であり、中央領域の5'側から8番目の塩基位置とは、中央領域において5'ウイング領域に隣接する末端塩基を起点として3'方向に数えて8番目に位置する塩基を意味する。2'-修飾ヌクレオシドにおける2'-修飾に制限はなく、例えば2'-O-メチル基、2'-O-[2-(N-メチルカルバモイル)エチル]基、2'-O-メトキシエチル基、又は2'-フルオロ基であってもよいが、好ましくは2'-O-メチル基である。 In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleoside at the terminal base position adjacent to the central region in the 5' wing region, the second base position from the 5' side of the central region, and/or the eighth base position from the 5' side of the central region. The "terminal base position adjacent to the central region in the 5' wing region" refers to the base position located at the 3' end in the 5' wing region. In addition, in this specification, the "Nth base position from the 5' side of the central region" refers to the Nth base in the direction from the terminal base adjacent to the 5' wing region in the central region. Therefore, the second base position from the 5' side of the central region refers to the second base in the 3' direction from the terminal base adjacent to the 5' wing region in the central region, and the eighth base position from the 5' side of the central region refers to the eighth base in the 3' direction from the terminal base adjacent to the 5' wing region in the central region. There are no limitations on the 2'-modification in the 2'-modified nucleoside, and it may be, for example, a 2'-O-methyl group, a 2'-O-[2-(N-methylcarbamoyl)ethyl] group, a 2'-O-methoxyethyl group, or a 2'-fluoro group, but a 2'-O-methyl group is preferred.
　さらなる実施形態において、5'ウイング領域及び／又は3'ウイング領域は、ヌクレオシド間結合で連結された2以上の2'-修飾ヌクレオシド及び／又は架橋型ヌクレオシドからなる。 In further embodiments, the 5' wing region and/or the 3' wing region are comprised of two or more 2'-modified nucleosides and/or bridged nucleosides linked by internucleoside linkages.
　ギャップマーにおいて、5'ウイング領域、中央領域、及び3'ウイング領域の各領域の塩基長の例としては、2-12-3、3-12-2、3-12-3、4-12-3、2-11-3、3-11-2、3-11-3、4-11-3、2-10-3、3-10-2、3-10-3、4-10-3、2-9-3、3-9-2、3-9-3、4-9-3、2-8-3、3-8-2、3-7-3、4-6-3、3-6-4、4-5-4、4-7-3、3-7-4、4-6-4、5-6-3、3-6-5、3-7-5、5-7-3、4-7-4、4-6-5、5-6-4、5-5-5、5-6-5等が挙げられる。ここで、「A-B-C」の表記において、「A」は5'ウイング領域の塩基長を示し、「B」は中央領域の塩基長を示し、「C」は3'ウイング領域の塩基長を示す。 In gapmers, examples of base lengths of the 5' wing region, central region, and 3' wing region include 2-12-3, 3-12-2, 3-12-3, 4-12-3, 2-11-3, 3-11-2, 3-11-3, 4-11-3, 2-10-3, 3-10-2, 3-10-3, 4-10-3, 2-9-3, 3-9-2, 3-9-3, 4-9-3, 2-8-3, 3-8-2, 3-7-3, 4-6-3, 3-6-4, 4-5-4, 4-7-3, 3-7-4, 4-6-4, 5-6-3, 3-6-5, 3-7-5, 5-7-3, 4-7-4, 4-6-5, 5-6-4, 5-5-5, 5-6-5, etc. Here, in the notation "A-B-C," "A" indicates the base length of the 5' wing region, "B" indicates the base length of the central region, and "C" indicates the base length of the 3' wing region.
　核酸分子におけるヌクレオシド間結合は、天然に存在するヌクレオシド間結合及び／又は修飾ヌクレオシド間結合であってよい。限定はしないが、核酸分子の末端（5'末端、3'末端若しくは両端）から少なくとも1個、少なくとも2個、又は少なくとも3個のヌクレオシド間結合が修飾ヌクレオシド間結合であることが好ましい。ここで、例えば核酸鎖の末端から2つのヌクレオシド間結合とは、核酸鎖の末端に最も近接するヌクレオシド間結合と、それに隣接し、かつ末端とは反対側に位置するヌクレオシド間結合を意味する。核酸鎖の末端領域における修飾ヌクレオシド間結合は、核酸鎖の望ましくない分解を抑制又は阻害できるために好ましい。 The internucleoside bond in the nucleic acid molecule may be a naturally occurring internucleoside bond and/or a modified internucleoside bond. Although not limited thereto, it is preferred that at least one, at least two, or at least three internucleoside bonds from the end (5' end, 3' end, or both ends) of the nucleic acid molecule are modified internucleoside bond. Here, for example, two internucleoside bonds from the end of the nucleic acid chain refer to the internucleoside bond closest to the end of the nucleic acid chain and the internucleoside bond adjacent thereto and located on the opposite side to the end. Modified internucleoside bonds in the terminal region of the nucleic acid chain are preferred because they can suppress or inhibit undesired degradation of the nucleic acid chain.
　一実施形態では、核酸分子のヌクレオシド間結合の全部又は一部が修飾ヌクレオシド間結合であってもよい。修飾ヌクレオシド間結合は、ホスホロチオエート結合であってよい。 In one embodiment, all or a portion of the internucleoside linkages of the nucleic acid molecule may be modified internucleoside linkages. The modified internucleoside linkages may be phosphorothioate linkages.
　核酸分子は、全部又は一部にヌクレオシド模倣体又はヌクレオチド模倣体を含んでもよい。ヌクレオチド模倣体は、ペプチド核酸及び／又はモルホリノ核酸であってもよい。さらなる実施形態において、核酸分子中の核酸の25％以上、33％以上、40%以上、50％以上、60％以上、70％以上、80％以上、90%以上、95%以上又は100%（全部）がモルホリノ核酸である。モルホリノ核酸間のヌクレオシド間結合は限定しないが、その一部又は全てがホスホロジアミデート結合であってよい。 The nucleic acid molecule may comprise, in whole or in part, a nucleoside mimic or a nucleotide mimic. The nucleotide mimic may be a peptide nucleic acid and/or a morpholino nucleic acid. In further embodiments, 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% (all) of the nucleic acids in the nucleic acid molecule are morpholino nucleic acids. The internucleoside linkages between the morpholino nucleic acids are not limited, and some or all of them may be phosphorodiamidate linkages.
　核酸分子が有する標的転写産物に対するアンチセンス効果は、当該分野で公知の方法で測定できる。例えば、核酸分子を細胞等に導入した後、ノザンブロッティング、定量PCR、又はウェスタンブロッティング等の公知技術を使用することにより測定すればよい。特定の組織における標的遺伝子の発現量又は標的転写産物のレベル(例えば、mRNA量等のRNA量、cDNA量等)を測定することで、それらの部位において核酸分子によって標的遺伝子発現が抑制されるか否かを判定できる。測定された標的遺伝子の発現量又は標的転写産物のレベルが、陰性対照(例えばビヒクル投与又は無処置)と比較して、少なくとも20％、少なくとも25％、少なくとも30％、少なくとも40％又は少なくとも50％減少している場合に、被験核酸化合物がアンチセンス効果をもたらし得ることが示される。 The antisense effect of the nucleic acid molecule on the target transcript can be measured by a method known in the art. For example, after introducing the nucleic acid molecule into cells, the effect 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., the amount of RNA such as mRNA, the amount of cDNA, etc.) in a specific tissue, it can be determined whether the expression of the target gene is suppressed by the nucleic acid molecule at those sites. If the measured expression level of the target gene or the level of the target transcript is reduced by at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% compared to a negative control (e.g., vehicle administration or no treatment), it is indicated that the test nucleic acid compound can have an antisense effect.
（二本鎖核酸複合体）
　一実施形態において、核酸医薬は、上記のいずれかの核酸分子からなる第1核酸鎖と、前記第1核酸鎖に相補的な塩基配列を含む第2核酸鎖とを含む二本鎖核酸複合体からなる。 (Double-stranded nucleic acid complex)
In one embodiment, the nucleic acid drug consists of a double-stranded nucleic acid complex including a first nucleic acid strand consisting of any of the above-mentioned nucleic acid molecules and a second nucleic acid strand including a base sequence complementary to the first nucleic acid strand.
　二本鎖核酸複合体は、第1核酸鎖及び第2核酸鎖を含む。第1核酸鎖は、上述のいずれかの核酸分子であるため、ここでの説明は省略する。 The double-stranded nucleic acid complex contains a first nucleic acid strand and a second nucleic acid strand. The first nucleic acid strand is any of the nucleic acid molecules described above, so a detailed explanation of it will be omitted here.
　二本鎖核酸複合体において、第2核酸鎖は、第1核酸鎖に相補的な塩基配列を含む核酸分子である。二本鎖核酸複合体において、第2核酸鎖は、相補的塩基対の水素結合を介して第1核酸鎖にアニールしている。 In a double-stranded nucleic acid complex, the second nucleic acid strand is a nucleic acid molecule that contains a base sequence complementary to the first nucleic acid strand. In a double-stranded nucleic acid complex, the second nucleic acid strand is annealed to the first nucleic acid strand through hydrogen bonds of complementary base pairs.
　二本鎖核酸複合体において、第2核酸鎖は、デオキシリボヌクレオシド、2'-修飾ヌクレオシド、5'-修飾ヌクレオシド、及び／又は架橋ヌクレオシドを含むことができる。例えば、第2核酸鎖は、第1核酸鎖の中央領域に相補的な塩基配列からなる領域の全てのヌクレオシドが、(a)デオキシリボヌクレオシド；(b)デオキシリボヌクレオシド及びリボヌクレオシド；(c)デオキシリボヌクレオシド及び2'-修飾ヌクレオシド；(d)リボヌクレオシド及び2'-修飾ヌクレオシド；又は(e)デオキシリボヌクレオシド、リボヌクレオシド、及び2'-修飾ヌクレオシドであってもよい。 In the double-stranded nucleic acid complex, the second nucleic acid strand may contain deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and/or bridged nucleosides. For example, the second nucleic acid strand may be such that all nucleosides in a region consisting of a base sequence complementary to the central region of the first nucleic acid strand are (a) deoxyribonucleosides; (b) deoxyribonucleosides and ribonucleosides; (c) deoxyribonucleosides and 2'-modified nucleosides; (d) ribonucleosides and 2'-modified nucleosides; or (e) deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides.
　一実施形態では、第2核酸鎖は、第1核酸鎖の中央領域における少なくとも3個又は少なくとも4個の連続するデオキシリボヌクレオシドに相補的な、少なくとも3個又は少なくとも4個の連続するリボヌクレオシド及び／又はデオキシリボヌクレオシドを含む領域を含む。 In one embodiment, the second nucleic acid strand includes a region containing at least three or at least four consecutive ribonucleosides and/or deoxyribonucleosides that are complementary to at least three or at least four consecutive deoxyribonucleosides in a central region of the first nucleic acid strand.
　さらなる実施形態では、第2核酸鎖は、第1核酸鎖の5'ウイング領域及び／又は3'ウイング領域に相補的な塩基配列からなる領域を含んでもよい。第2核酸鎖において、第1核酸鎖の5'ウイング領域及び／又は3'ウイング領域に相補的な塩基配列からなる領域は、少なくとも１つの非天然ヌクレオシドを含んでもよく、当該非天然ヌクレオシドは、架橋ヌクレオシド及び／又は2'-修飾ヌクレオシドであってもよい。また、第2核酸鎖における2'-修飾ヌクレオシドの2'-修飾基は、2'-O-メチル基又は2'-O-メトキシエチル基であってもよい。なお、第1核酸鎖と第2核酸鎖が共に架橋ヌクレオシド及び／又は2'-修飾ヌクレオシドを含む場合、第1核酸鎖及び第2核酸鎖における架橋ヌクレオシド及び／又は2'-修飾ヌクレオシドは、同一であっても異なっていてもよい。 In a further embodiment, the second nucleic acid strand may include a region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand. In the second nucleic acid strand, the region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand may include at least one non-natural nucleoside, which may be a bridged nucleoside and/or a 2'-modified nucleoside. In addition, the 2'-modified group of the 2'-modified nucleoside in the second nucleic acid strand may be a 2'-O-methyl group or a 2'-O-methoxyethyl group. Note that when both the first nucleic acid strand and the second nucleic acid strand include a bridged nucleoside and/or a 2'-modified nucleoside, the bridged nucleoside and/or the 2'-modified nucleoside in the first nucleic acid strand and the second nucleic acid strand may be the same or different.
　第2核酸鎖におけるヌクレオシド間結合は、天然に存在するヌクレオシド間結合及び／又は修飾ヌクレオシド間結合であってよい。限定はしないが、第2核酸鎖の末端（5'末端、3'末端若しくは両端）から少なくとも1個、少なくとも2個、又は少なくとも3個のヌクレオシド間結合が修飾ヌクレオシド間結合であることが好ましい。一実施形態では、第2核酸鎖のヌクレオシド間結合の全部又は一部が修飾ヌクレオシド間結合であってもよい。一実施形態では、第2核酸鎖は、第1核酸鎖の5'ウイング領域及び／又は3'ウイング領域に相補的な塩基配列からなる領域に修飾ヌクレオシド間結合を含んでもよい。修飾ヌクレオシド間結合は、ホスホロチオエート結合であってよい。 The internucleoside linkages in the second nucleic acid strand may be naturally occurring internucleoside linkages and/or modified internucleoside linkages. It is preferred, but not limited to, that at least one, at least two, or at least three internucleoside linkages from the ends (5' end, 3' end, or both ends) of the second nucleic acid strand are modified internucleoside linkages. In one embodiment, all or a portion of the internucleoside linkages in the second nucleic acid strand may be modified internucleoside linkages. In one embodiment, the second nucleic acid strand may include modified internucleoside linkages in a region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand. The modified internucleoside linkages may be phosphorothioate linkages.
　さらなる実施形態において、第2核酸鎖は、2'-修飾ヌクレオシド（例えば2'-O-メチル修飾ヌクレオシド、2'-O-メトキシエチル修飾ヌクレオシド、2'-O-[2-(N-メチルカルバモイル)エチル]修飾ヌクレオシド、又は2'-フルオロ修飾ヌクレオシド）を含むことができる。第2核酸鎖において、2'-修飾ヌクレオシドの数は限定しない。例えば、前記第2核酸鎖におけるヌクレオシドの総数の少なくとも20％、少なくとも30％、少なくとも40％、少なくとも50％、少なくとも60％、少なくとも70％、少なくとも75％、少なくとも80％、少なくとも85％、少なくとも90％、若しくは少なくとも95％、又は100％が、2'-修飾ヌクレオシドであってもよい。一実施形態では、第2核酸鎖において、ヌクレオシドの全てが2'-修飾ヌクレオシドである。 In a further embodiment, the second nucleic acid strand can include 2'-modified nucleosides (e.g., 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleosides, or 2'-fluoro modified nucleosides). The number of 2'-modified nucleosides in the second nucleic acid strand is not limited. For example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% of the total number of nucleosides in the second nucleic acid strand may be 2'-modified nucleosides. In one embodiment, all of the nucleosides in the second nucleic acid strand are 2'-modified nucleosides.
　さらなる実施形態において、第2核酸鎖は、5'末端に位置する1個又は連続する2個以上の2'-修飾ヌクレオシド（例えば2'-O-メチル修飾ヌクレオシド、2'-O-メトキシエチル修飾ヌクレオシド、2'-O-[2-(N-メチルカルバモイル)エチル]修飾ヌクレオシド、又は2'-フルオロ修飾ヌクレオシド）、及び／又は3'末端に位置する1個又は連続する2個以上の2'-修飾ヌクレオシドを含むことができる。5'末端及び／又は3'末端に位置する2'-修飾ヌクレオシドの数は限定しない。例えば、第2核酸鎖は、5'末端に位置する1個又は連続する2個、3個、4個、5個、6個、若しくは7個の2'-修飾ヌクレオシド、及び／又は3'末端に位置する1個又は連続する2個、3個、4個、5個、6個、若しくは7個の2'-修飾ヌクレオシドを含んでもよい。 In a further embodiment, the second nucleic acid strand may include one or more consecutive 2'-modified nucleosides (e.g., 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleosides, or 2'-fluoro modified nucleosides) located at the 5' end and/or one or more consecutive 2'-modified nucleosides located at the 3' end. The number of 2'-modified nucleosides located at the 5' end and/or the 3' end is not limited. For example, the second nucleic acid strand may include one or two, three, four, five, six, or seven consecutive 2'-modified nucleosides located at the 5' end and/or one or two, three, four, five, six, or seven consecutive 2'-modified nucleosides located at the 3' end.
　一実施形態において、第1核酸鎖及び／又は第2核酸鎖は修飾核酸塩基を含んでもよい。修飾核酸塩基の数は限定せず、例えば少なくとも1個、少なくとも2個、少なくとも3個、少なくとも4個、少なくとも5個、又は少なくとも6個であってもよい。 In one embodiment, the first nucleic acid strand and/or the second nucleic acid strand may comprise modified nucleobases. The number of modified nucleobases is not limited and may be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6.
　一実施形態において、第2核酸鎖は、第1核酸鎖に対して、非相補的塩基、及び／又は1塩基以上の、挿入配列及び／又は欠失を含み得る。第2核酸鎖における非相補的塩基の数は、限定しないが、例えば1～10個、1～9個、1～8個、1～7個、1～6個、1～5個、1～4個、1～3個、1～2個、又は1個又は2個であってもよい。第2核酸鎖における挿入配列の塩基長数は、限定しないが、例えば1～10個、1～9個、1～8個、1～7個、1～6個、1～5個、1～4個、1～3個、1～2個、又は1個又は2個であってもよい。第2核酸鎖における欠失の連続する塩基長は、限定しないが、例えば1～10個、1～9個、1～8個、1～7個、1～6個、1～5個、1～4個、1～3個、1～2個、又は1個又は2個であってもよい。非相補塩基又は挿入配列から構成される領域はバルジを形成していてもよい。 In one embodiment, the second nucleic acid strand may include non-complementary bases and/or an insertion sequence and/or deletion of one or more bases relative to the first nucleic acid strand. The number of non-complementary bases in the second nucleic acid strand is not limited, but may be, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 or 2. The number of bases of the insertion sequence in the second nucleic acid strand is not limited, but may be, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 or 2. The length of the deleted contiguous bases in the second nucleic acid strand is not limited, but may be, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 or 2. The region composed of non-complementary bases or an inserted sequence may form a bulge.
　一実施形態において、第2核酸鎖は、相補的領域の5'末端側及び3'末端側の一方又は両方に位置する少なくとも1つのオーバーハング領域をさらに含み得る。本実施形態の一例は、国際公開第2018/062510に記載される。「オーバーハング領域」とは、相補的領域に隣接する領域で、第1核酸鎖と第2核酸鎖がアニールして二本鎖構造を形成した場合、第2核酸鎖の5'末端が第1核酸鎖の3'末端を超えて伸長する、及び／又は第2核酸鎖の3'末端が第1核酸鎖の5'末端を超えて伸長する、つまり、二本鎖構造から突出した第2核酸鎖中のヌクレオチド領域を指す。第2核酸鎖中のオーバーハング領域は、相補的領域の5'末端側に位置してもよく、3'末端側に位置してもよい。第2核酸鎖中のオーバーハング領域は、相補的領域の5'末端側及び3'末端側に位置してもよい。オーバーハング領域の塩基長は特に制限されず、1～30塩基長又は1～20塩基長であってもよい。 In one embodiment, the second nucleic acid strand may further include at least one overhang region located at one or both of the 5'-end and 3'-end of the complementary region. An example of this embodiment is described in WO 2018/062510. The term "overhang region" refers to a region adjacent to a complementary region, in which, when the first and second nucleic acid strands anneal to form a double-stranded structure, the 5'-end of the second nucleic acid strand extends beyond the 3'-end of the first nucleic acid strand and/or the 3'-end of the second nucleic acid strand extends beyond the 5'-end of the first nucleic acid strand, that is, a nucleotide region in the second nucleic acid strand that protrudes from the double-stranded structure. The overhang region in the second nucleic acid strand may be located at the 5'-end or 3'-end of the complementary region. The overhang region in the second nucleic acid strand may be located at the 5'-end and 3'-end of the complementary region. The base length of the overhang region is not particularly limited and may be 1 to 30 bases long or 1 to 20 bases long.
　一実施形態において、第1核酸鎖及び／又は第2核酸鎖、例えば第2核酸鎖に、機能性部分が結合していてもよい。第1核酸鎖及び／又は第2核酸鎖と機能性部分との結合は、直接的な結合であってもよく、他の物質を介した間接的な結合であってもよいが、ある実施形態において、共有結合、イオン結合、水素結合等で第1核酸鎖及び／又は第2核酸鎖と機能性部分とが直接的に結合していることが好ましく、より安定した結合が得られるという観点から、共有結合がより好ましい。 In one embodiment, a functional moiety may be bound to the first nucleic acid strand and/or the second nucleic acid strand, for example, the second nucleic acid strand. The bond between the first nucleic acid strand and/or the second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond via another substance, but in one embodiment, it is preferable that the first nucleic acid strand and/or the second nucleic acid strand and the functional moiety are directly bound to each other via a covalent bond, an ionic bond, a hydrogen bond, or the like, and a covalent bond is more preferable from the viewpoint of obtaining a more stable bond.
　一実施形態において、「機能性部分」の構造は、特に制限はなく、それを結合する二本鎖核酸複合体に所望の機能を付与する。所望の機能としては、標識機能、精製機能及び標的への送達機能が挙げられる。標識機能を付与する部分は例えば脂質やペプチドであり、その具体例としては、蛍光タンパク質、ルシフェラーゼ等の化合物が挙げられる。精製機能を付与する部分の例としては、ビオチン、アビジン、Hisタグペプチド、GSTタグペプチド、FLAGタグペプチド等の化合物が挙げられる。また、第1核酸鎖を特異性高く効率的に標的部位に送達し、かつ当該核酸によって標的遺伝子の発現を非常に効果的に抑制するという観点から、第1核酸鎖及び／又は第2核酸鎖に機能性部分として、ある実施形態における二本鎖核酸複合体を標的部位に送達させる活性を有する分子が結合していることが好ましい。標的への送達機能を与える部分の例としては、脂質、抗体、アプタマー、特定のレセプターに対するリガンド等が挙げられる。 In one embodiment, the structure of the "functional portion" is not particularly limited, and it confers a desired function to the double-stranded nucleic acid complex to which it is bound. Examples of the desired function include a labeling function, a purification function, and a target delivery function. The portion that confers the labeling function is, for example, a lipid or a peptide, and specific examples thereof include compounds such as fluorescent proteins and luciferase. Examples of the portion that confers the purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide. In addition, from the viewpoint of delivering the first nucleic acid strand to a target site with high specificity and efficiency, and suppressing the expression of a target gene very effectively by the nucleic acid, it is preferable that a molecule having an activity of delivering the double-stranded nucleic acid complex in a certain embodiment to a target site is bound as a functional portion to the first nucleic acid strand and/or the second nucleic acid strand. Examples of the portion that confers a target delivery function include lipids, antibodies, aptamers, and ligands for specific receptors.
　一実施形態において、第1核酸鎖及び／又は第2核酸鎖、例えば第2核酸鎖は、脂質と結合している。脂質としては、トコフェロール、コレステロール、脂肪酸、リン脂質（例：ホスファチジルエタノールアミン）及びそれらの類縁体；葉酸、ビタミンC、ビタミンB1、ビタミンB2；エストラジオール、アンドロスタン及びそれらの類縁体；ステロイド及びその類縁体；LDLR、SRBI又はLRP1/2のリガンド；FK-506、及びシクロスポリン；WO2019/182109及びWO2019/177061記載の脂質等が挙げられるが、これらに限定されない。脂質は、トコフェロール又はその類縁体及び／又はコレステロール又はその類縁体、置換された若しくは置換されていないＣ1～３０のアルキル基、置換された若しくは置換されていないＣ２～３０のアルケニル基、若しくは置換された若しくは置換されていないＣ1～３０のアルコキシ基であってもよい。一実施形態では、第2核酸鎖はトコフェロール若しくはコレステロール又はそれらの類縁体と結合していてもよい。 In one embodiment, the first nucleic acid strand and/or the second nucleic acid strand, e.g., the second nucleic acid strand, is bound to a lipid. The lipid may include, but is not limited to, tocopherol, cholesterol, fatty acids, phospholipids (e.g., phosphatidylethanolamine) and analogs thereof; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstane and analogs thereof; steroids and analogs thereof; ligands of LDLR, SRBI, or LRP1/2; FK-506, and cyclosporine; lipids described in WO2019/182109 and WO2019/177061, etc. The lipid may be tocopherol or an analog thereof and/or cholesterol or an analog thereof, a substituted or unsubstituted C 1-30 alkyl group, a substituted or unsubstituted C 2-30 alkenyl group, or a substituted or unsubstituted C 1-30 alkoxy group. In one embodiment, the second nucleic acid strand may be conjugated to tocopherol or cholesterol or an analogue thereof.
　本明細書において「トコフェロール」は、トコロールのメチル化誘導体で、クロマンと呼ばれる環状構造を有する脂溶性ビタミン（ビタミンE）である。トコロールは、強い抗酸化作用を有しており、それ故に、生体内では、抗酸化物質として、代謝によって生じるフリーラジカルを消失させ、細胞を傷害から保護する機能を有する。 In this specification, "tocopherol" is a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) with a ring structure called chroman. Tocorol has a strong antioxidant effect, and therefore, as an antioxidant in the body, it has the function of eliminating free radicals generated by metabolism and protecting cells from damage.
　トコフェロールは、クロマンに結合するメチル基の位置に基づき、α-トコフェロール、β-トコフェロール、γ-トコフェロール、及びδ-トコフェロールからなる複数の異なる型が知られている。本明細書におけるトコフェロールは、いずれのトコフェロールであってもよい。また、トコフェロールの類縁体としては、トコフェロールの種々の不飽和類縁体、例えば、α-トコトリエノール、β-トコトリエノール、γ-トコトリエノール、δ-トコトリエノール等が挙げられる。好ましくは、トコフェロールは、α-トコフェロールである。 Tocopherol is known in several different forms, consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, based on the position of the methyl group bound to the chroman. In this specification, tocopherol may be any tocopherol. In addition, examples of tocopherol analogs include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. Preferably, the tocopherol is α-tocopherol.
　本明細書において「コレステロール」とは、ステロイドアルコールとも呼ばれるステロールの1種であり、特に動物において多く存在する。コレステロールは、生体内における代謝過程で重要な機能を果たしている他、動物細胞では、リン脂質と共に細胞の膜系を構成する主要な構成成分でもある。また、コレステロールの類縁体は、ステロール骨格を有するアルコールである、種々のコレステロール代謝産物及び類縁体等を指し、限定されるものではないが、コレスタノール、ラノステロール、セレブロステロール、デヒドロコレステロール、及びコプロスタノール等を含む。 In this specification, "cholesterol" refers to a type of sterol, also known as a steroid alcohol, and is found in large amounts in animals. Cholesterol plays an important role in metabolic processes in the body, and in animal cells, it is also a major component of the cell membrane system along with phospholipids. Furthermore, cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols with a sterol skeleton, and include, but are not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.
　本明細書において「類縁体(analog)」又は「誘導体(derivative)」とは、同一又は類似の基本骨格を有する類似した構造及び性質を有する化合物を指す。類縁体は、例えば、生合成中間体、代謝産物、置換基を有する化合物等を含む。ある化合物が他の化合物の類縁体であるか否かは、当業者であれば技術常識に基づき判定できる。 As used herein, "analog" or "derivative" refers to a compound that has an identical or similar basic skeleton and similar structure and properties. Analogs include, for example, biosynthetic intermediates, metabolic products, and compounds with substituents. Those skilled in the art can determine whether a compound is an analog of another compound based on their common technical knowledge.
　機能性部分は、第1核酸鎖及び／又は第2核酸鎖の5'末端、又は3'末端、或いは両端に連結されていてもよい。或いは、機能性部分は、第1核酸鎖及び／又は第2核酸鎖の内部のヌクレオチドに連結されていてもよい。第1核酸鎖及び／又は第2核酸鎖は、脂質等の機能性部分を2つ以上含み、これらは第1核酸鎖及び／又は第2核酸鎖の複数の位置に連結されていてもよく、及び／又は第1核酸鎖及び／又は第2核酸鎖の1つの位置に一群として連結されていてもよい。機能性部分は、第1核酸鎖及び／又は第2核酸鎖の5'末端と3'末端にそれぞれ1つずつ連結されていてもよい。 The functional moiety may be linked to the 5' end, or the 3' end, or both ends of the first and/or second nucleic acid strand. Alternatively, the functional moiety may be linked to an internal nucleotide of the first and/or second nucleic acid strand. The first and/or second nucleic acid strand may contain two or more functional moieties, such as lipids, which may be linked to multiple positions on the first and/or second nucleic acid strand and/or may be linked as a group to one position on the first and/or second nucleic acid strand. The functional moieties may be linked to the 5' end and the 3' end of the first and/or second nucleic acid strand, one each.
　第1核酸鎖及び／又は第2核酸鎖と機能性部分との間の結合は、直接結合であってもよいし、別の物質によって介在される間接結合であってもよい。しかし、特定の実施形態においては、機能性部分は、共有結合、イオン性結合、水素結合等を介して第1核酸鎖及び／又は第2核酸鎖に直接結合されていることが好ましく、またより安定した結合を得ることができるという点から考えると、共有結合がより好ましい。 The bond between the first and/or second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond mediated by another substance. However, in certain embodiments, it is preferred that the functional moiety is directly bonded to the first and/or second nucleic acid strand via a covalent bond, ionic bond, hydrogen bond, etc., and a covalent bond is more preferred in terms of obtaining a more stable bond.
　機能性部分はまた、切断性(cleavable)又は非切断性(uncleavable)リンカーを介して第1核酸鎖及び／又は第2核酸鎖に結合されていてもよい。この場合、第1核酸鎖と第2核酸鎖は、リンカーを介して連結され、一本鎖を形成し得る。しかし、その場合も機能領域は二本鎖核酸複合体と同じ構成であることから、本明細書では、このような一本鎖核酸も本発明における二本鎖核酸複合体の一実施形態として包含する。リンカーは、任意のポリマーでありうる。例えば、ポリヌクレオチド、ポリペプチド、アルキレン等が挙げられる。具体的には、例えば、DNA、RNAといった天然のヌクレオチド又はペプチド核酸、モルホリノ核酸といった非天然のヌクレオチドから構成されうる。リンカーが核酸からなる場合、リンカーの鎖長は少なくとも1塩基、例えば、3～10塩基又は4～6塩基の鎖長をとりうる。好ましくは4塩基の鎖長である。この場合、リンカーはヒンジ(ヘアピンループ)の形態をとり得る。リンカーの位置は、第1核酸鎖の5'側でも3'側のいずれでも可能であるが、例えば、第1核酸鎖の5'側に第2核酸鎖を結合させた構成の場合には、第1核酸鎖の5'末端と第2核酸鎖の3'末端とがリンカーを介して連結されることになる。 The functional portion may also be linked to the first and/or second nucleic acid strands via a cleavable or uncleavable linker. In this case, the first and second nucleic acid strands may be linked via a linker to form a single strand. However, even in this case, the functional region has the same structure as in the double-stranded nucleic acid complex, and therefore, in this specification, such a single-stranded nucleic acid is also included as an embodiment of the double-stranded nucleic acid complex of the present invention. The linker may be any polymer. Examples include polynucleotides, polypeptides, and alkylenes. Specifically, the linker may be composed of natural nucleotides such as DNA and RNA, or non-natural nucleotides such as peptide nucleic acids and morpholino nucleic acids. When the linker is composed of a nucleic acid, the chain length of the linker may be at least 1 base, for example, 3 to 10 bases or 4 to 6 bases. The chain length is preferably 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' side of the first nucleic acid strand, but for example, in the case of a configuration in which the second nucleic acid strand is bound to the 5' side 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 are linked via a linker.
　「切断性リンカー」とは、生理学的条件下で、例えば細胞内又は動物体内(例えば、ヒト体内)で、切断される連結基を意味する。特定の実施形態では、切断性リンカーは、ヌクレアーゼ等の内在性酵素によって選択的に切断される。切断性リンカーとしては、アミド、エステル、ホスホジエステルの一方若しくは両方のエステル、リン酸エステル、カルバメート、及びジスルフィド結合、並びに天然DNAリンカーが挙げられる。 "Cleavable linker" refers to a linking group that is cleaved under physiological conditions, e.g., within a cell or within an animal (e.g., within the human body). In certain embodiments, the cleavable linker is selectively cleaved by an endogenous enzyme, such as a nuclease. Cleavable linkers include amides, esters, phosphodiesters or both esters, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers.
　「非切断性リンカー」とは、生理学的条件下、例えば、細胞内又は動物体内(例えば、ヒト体内)で切断されないリンカーを意味する。非切断性リンカーは、限定はしないが、ホスホロチオエート結合、及びホスホロチオエート結合で連結された修飾若しくは非修飾のデオキシリボヌクレオシド又は修飾若しくは非修飾のリボヌクレオシドからなるリンカー等が挙げられる。リンカーがDNA等の核酸又はオリゴヌクレオチドの場合、鎖長は、特に限定はされないが、通常は2～20塩基長、3～10塩基長又は4～6塩基長であればよい。 "Non-cleavable linker" means a linker that is not cleaved under physiological conditions, for example, within a cell or an animal body (for example, within the human body). Non-cleavable linkers include, but are not limited to, linkers consisting of phosphorothioate bonds, and modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by phosphorothioate bonds. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not particularly limited, but may be usually 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.
　リンカーの一具体例として、以下の式(IV)で表されるリンカーが挙げられる。 One specific example of a linker is the linker represented by the following formula (IV):
Figure JPOXMLDOC01-appb-C000011
 （式中、
L2は、置換された若しくは置換されていないC1～C12のアルキレン基（例、プロピレン、ヘキシレン、ドデシレン）、置換された若しくは置換されていないC3～C8シクロアルキレン基（例、シクロヘキシレン）、-(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-、又はCH(CH2-OH)-CH2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-を表し、L3は、-NH-又は結合を表し、L4は、置換された若しくは置換されていないC1～C12のアルキレン基（例、エチレン、ペンチレン、へプチレン、アンデシレン）、置換された若しくは置換されていないC3～C8のシクロアルキレン基（例、シクロヘキシレン）、-(CH2)2-[O-(CH2)2]m-、又は結合を表し、ここで、mは1～25の整数を表し、L5は、-NH-(C=O)-、-(C=O)-、又は結合を表す(ここで、該置換は、好ましくはハロゲン原子によりなされる）。
Figure JPOXMLDOC01-appb-C000011
 (Wherein,
L 2 represents a substituted or unsubstituted C 1 -C 12 alkylene group (e.g., propylene, hexylene, dodecylene), a substituted or unsubstituted C 3 -C 8 cycloalkylene group (e.g., cyclohexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, or CH(CH 2 -OH)-CH 2 -O- (CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -; L 3 represents -NH- or a bond; L 4 represents a substituted or unsubstituted C 3 -C 8 cycloalkylene group (e.g. , cyclohexylene); L5 represents an alkylene group having 1 to 12 carbon atoms (e.g., ethylene, pentylene, heptylene, undecylene), a substituted or unsubstituted cycloalkylene group having 3 to 8 carbon atoms (e.g., cyclohexylene), -( CH2 ) 2- [O-( CH2 ) 2 ] m- , or a bond, where m represents an integer of 1 to 25, and L5 represents -NH-(C=O)-, -(C=O)-, or a bond (wherein the substitution is preferably by a halogen atom).
　一実施形態において、式(VI)で表されるリンカーは、L2が、置換されていないC3～C6のアルキレン基（例、プロピレン、ヘキシレン）、-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-、又は-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-であり、L3が、-NH-であり、L4及びL5が、結合である。 In one embodiment, the linker represented by formula (VI) is one in which L 2 is an unsubstituted C 3 to C 6 alkylene group (e.g., propylene, hexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, or -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, L 3 is -NH-, and L 4 and L 5 are bonds.
　一実施形態において、リンカーは、核酸、ポリエーテル基、及び／又はアルキルアミノ基を含む。核酸は、例えば、1個、又はヌクレオシド間結合により連結された2～10個のヌクレオシド及び／若しくは非天然ヌクレオシドからなるものであってもよい。また、ポリエーテル基の例として、ポリエチレングリコール基、トリエチレングリコール基、又はテトラエチレングリコール基が挙げられる。アルキルアミノ基の例として、ヘキシルアミノ基が挙げられる。 In one embodiment, the linker includes a nucleic acid, a polyether group, and/or an alkylamino group. The nucleic acid may be, for example, one or two to ten nucleosides and/or non-natural nucleosides linked by internucleoside bonds. Examples of polyether groups include polyethylene glycol groups, triethylene glycol groups, and tetraethylene glycol groups. Examples of alkylamino groups include hexylamino groups.
　第1核酸鎖及び第2核酸鎖の塩基長は、特に限定されないが、少なくとも8塩基長、少なくとも9塩基長、少なくとも10塩基長、少なくとも11塩基長、少なくとも12塩基長、少なくとも13塩基長、少なくとも14塩基長、又は少なくとも15塩基長であればよい。また、第1核酸鎖及び第2核酸鎖の塩基長は、40塩基長以下、35塩基長以下、30塩基長以下、25塩基長以下、24塩基長以下、23塩基長以下、22塩基長以下、21塩基長以下、20塩基長以下、19塩基長以下、18塩基長以下、17塩基長以下、又は16塩基長以下であればよい。第1核酸鎖及び第2核酸鎖は、同じ長さであっても、異なる長さ（例えば、いずれか一方が1～3塩基短い又は長い長さ）であってもよい。第1核酸鎖及び第2核酸鎖が形成する二本鎖構造は、バルジを含んでいてもよい。長さの選択は、例えば費用、合成収率等の他の因子の中でも特に、アンチセンス効果の強度と標的に対する核酸鎖の特異性とのバランスによって決定することができる。なお、第1核酸鎖及び／又は第2核酸鎖にアプタマー等の核酸が結合している場合、第1核酸鎖及び第2核酸鎖の全体としての塩基長は、上記塩基長に結合した核酸の塩基長を加えたものであってよい。この場合、結合する核酸の塩基長は限定しないが、例えば少なくとも10塩基長、少なくとも15塩基長、又は少なくとも20塩基長であってよく、また100塩基長以下、80塩基長以下、60塩基長以下、40塩基長以下、又は30塩基長以下であってよい。 The base length of the first nucleic acid strand and the second nucleic acid strand is not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. The base length of the first nucleic acid strand and the second nucleic acid strand 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 nucleic acid strand and the second nucleic acid strand may be the same length or different lengths (for example, one of them may be 1 to 3 bases shorter or longer). The double-stranded structure formed by the first nucleic acid strand and the second nucleic acid strand may include a bulge. The length can be selected based on the balance between the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield. When a nucleic acid such as an aptamer is bound to the first nucleic acid strand and/or the second nucleic acid strand, the overall base length of the first nucleic acid strand and the second nucleic acid strand may be the above-mentioned base length plus 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, or may be 100 bases or less, 80 bases or less, 60 bases or less, 40 bases or less, or 30 bases or less.
（siRNA）
　一実施形態において、核酸医薬は、siRNAである。本明細書において「siRNA」(short-interfering RNA)とは、RNAiにより標的遺伝子の発現抑制を誘導し得る約19～25塩基対を有する2本鎖の核酸を意味する。siRNAは、後述のガイド鎖及びパッセンジャー鎖の2本の核酸鎖から構成される。本発明におけるsiRNAを構成する2本の核酸鎖は、リボヌクレオシドのみならず、デオキシヌクレオシド及び／又は任意の修飾ヌクレオシドを含むことができる。RNAi活性を誘導し得るsiRNAは、リボヌクレオシドからなるものに限定されず、デオキシヌクレオシドや修飾ヌクレオシドを含むsiRNAも後述のRISCに取り込まれて標的mRNAを認識できるためである。 (siRNA)
In one embodiment, the nucleic acid drug is an siRNA. As used herein, "siRNA" (short-interfering RNA) refers to a double-stranded nucleic acid having about 19 to 25 base pairs that can induce suppression of expression of a target gene by RNAi. An siRNA is composed of two nucleic acid strands, a guide strand and a passenger strand, which will be described later. The two nucleic acid strands that constitute the siRNA of the present invention can contain not only ribonucleosides but also deoxynucleosides and/or any modified nucleoside. This is because siRNAs that can induce RNAi activity are not limited to those consisting of ribonucleosides, and siRNAs containing deoxynucleosides or modified nucleosides can also be incorporated into the RISC described later to recognize target mRNA.
　本明細書において「RNAi」(RNA interference)とは、標的遺伝子配列と相補的な配列を含むsiRNA等の2本鎖の核酸鎖を導入した細胞内で、標的遺伝子の発現が特異的に抑制される現象を指す。siRNAによるRNAiは以下のように説明することができる。まず、細胞内に導入されたsiRNAの一方の鎖がRISC(RNA-induced Silencing Complex)と呼ばれる複合体に取り込まれ、相補性の高い配列を有する標的遺伝子のmRNAを認識する。標的遺伝子のmRNAは、RISCによって相補性の高い配列の中心部分で切断される。その後、切断されたmRNAは分解され得る。 As used herein, "RNAi" (RNA interference) refers to the phenomenon in which the expression of a target gene is specifically suppressed in a cell into which a double-stranded nucleic acid strand, such as siRNA, containing a sequence complementary to the target gene sequence, has been introduced. RNAi by siRNA can be explained as follows. First, one strand of the siRNA introduced into the cell is incorporated into a complex called RISC (RNA-induced Silencing Complex), which recognizes the mRNA of the target gene, which has a highly complementary sequence. The mRNA of the target gene is cleaved by RISC at the center of the highly complementary sequence. The cleaved mRNA can then be degraded.
　具体的には、本発明においてsiRNAは、標的遺伝子又はその転写産物の少なくとも一部にハイブリダイズすることができるガイド鎖、及び前記ガイド鎖に相補的な塩基配列を含むパッセンジャー鎖から構成される。 Specifically, in the present invention, the siRNA is composed of a guide strand that can hybridize to at least a portion of a target gene or its transcription product, and a passenger strand that contains a base sequence complementary to the guide strand.
　本明細書において「ガイド鎖」とは、標的遺伝子のmRNAに相補的な配列を含む核酸鎖を意味する。また、「パッセンジャー鎖」とは、ガイド鎖に相補的な配列を含む(すなわち、標的遺伝子のmRNAと相同な配列を含む)核酸鎖を意味する。ガイド鎖は、パッセンジャー鎖とアニーリングしてsiRNAを生成する。ガイド鎖は、標的遺伝子のmRNAと結合してRNAiを誘導し得る。 As used herein, "guide strand" refers to a nucleic acid strand that contains a sequence complementary to the mRNA of a target gene. Additionally, "passenger strand" refers to a nucleic acid strand that contains a sequence complementary to the guide strand (i.e., contains a sequence homologous to the mRNA of a target gene). The guide strand anneals with the passenger strand to generate siRNA. The guide strand can bind to the mRNA of a target gene to induce RNAi.
　一実施形態においてsiRNAは、デオキシリボヌクレオシド、2'-修飾ヌクレオシド、5'-修飾ヌクレオシド、及び架橋ヌクレオシドからなる群から選択されるいずれか１以上を含むことができる。これらの修飾型ヌクレオシドは、siRNAのガイド鎖及び／又はパッセンジャー鎖に配置することができる。 In one embodiment, the siRNA can include one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides. These modified nucleosides can be located in the guide strand and/or passenger strand of the siRNA.
　またsiRNAは、二本鎖核酸複合体について上述した様々な構成を包含することもできる。すなわち、本発明におけるsiRNAのガイド鎖は上述の第1核酸鎖について説明した各構成を包含することができ、同様にパッセンジャー鎖は上述の第2核酸鎖について説明した各構成を包含することができる。例えば、本発明におけるsiRNAは機能性部分と結合していてもよい。機能性部分は、二本鎖核酸複合体の場合と同様に脂質又はペプチドであってもよい。また脂質は、コレステロール若しくはその類縁体、トコフェロール若しくはその類縁体、葉酸、ホスファチジルエタノールアミン、又は置換された若しくは置換されていない炭素数16～30のアルキル基であってもよい。 The siRNA can also include the various configurations described above for the double-stranded nucleic acid complex. That is, the guide strand of the siRNA of the present invention can include each of the configurations described above for the first nucleic acid strand, and similarly, the passenger strand can include each of the configurations described above for the second nucleic acid strand. For example, the siRNA of the present invention may be bound to a functional moiety. The functional moiety may be a lipid or a peptide, as in the case of the double-stranded nucleic acid complex. The lipid may also be cholesterol or an analog thereof, tocopherol or an analog thereof, folic acid, phosphatidylethanolamine, or a substituted or unsubstituted alkyl group having 16 to 30 carbon atoms.
　一実施形態において、本発明における核酸医薬は、2つのsiRNA（以下、各siRNAを第1のsiRNA、及び第2のsiRNAと称する）を含むことができる。例えば、2つのsiRNAに含まれるガイド鎖（第1のsiRNAに含まれる第1のガイド鎖、及び第2のsiRNAに含まれる第2のガイド鎖）がリンカーを介して互いに結合していてもよい。第1のガイド鎖及び第2のガイド鎖においてリンカーの結合位置は特に制限されず、それぞれ5'末端及び／又は3'末端であってもよいが、第1のガイド鎖の3’末端と第2のガイド鎖の3'末端とがリンカーに結合していることが好ましい。リンカーの例は、二本鎖核酸複合体について述べた例に準じ、例えば核酸、ポリエーテル基、並びに／又はアルキルアミノ基を含むことができる。なお、2つのsiRNAが連結された構造は、divalent siRNAとして公知である（Alterman J.F. et al., Nature Biotechnology, 2019, 37:884-894）。 In one embodiment, the nucleic acid drug of the present invention may contain two siRNAs (hereinafter, each siRNA is referred to as the first siRNA and the second siRNA). For example, the guide strands contained in the two siRNAs (the first guide strand contained in the first siRNA and the second guide strand contained in the second siRNA) may be linked to each other via a linker. The linker binding positions in the first guide strand and the second guide strand are not particularly limited and may be the 5' end and/or the 3' end, respectively, but it is preferable that the 3' end of the first guide strand and the 3' end of the second guide strand are linked to the linker. Examples of linkers are similar to those described for the double-stranded nucleic acid complex, and may include, for example, a nucleic acid, a polyether group, and/or an alkylamino group. The structure in which two siRNAs are linked is known as a divalent siRNA (Alterman J.F. et al., Nature Biotechnology, 2019, 37:884-894).
（送達促進剤／薬剤の投与形態）
　本発明の送達促進剤は、神経系に送達されるべき薬剤と併用することによって、薬剤の中枢神経系への送達効率を増大させることができる。当該薬剤及び本発明の送達促進剤の投与経路及び／又は投与時期は、同一であってもよく、或いは異なっていてもよい。 Delivery Enhancers/Drug Administration Forms
The delivery enhancer of the present invention can increase the efficiency of delivery of a drug to the central nervous system by using the delivery enhancer of the present invention in combination with a drug to be delivered to the nervous system. The administration route and/or administration timing of the drug and the delivery enhancer of the present invention may be the same or different.
　本態様の送達促進剤、及び神経系に送達されるべき薬剤の投与形態には特定の限定はなく、投与は全身投与であっても局所投与であってもよい。投与経路は、経口投与又は非経口投与であってよい。非経口投与の具体例として、髄腔内投与（脳室内投与、後頭窩穿刺、又は腰椎穿刺）、経鼻投与、静脈内投与、動脈内投与、輸血による投与、腹腔内投与、眼内投与、筋肉内投与、皮下投与（埋め込み型持続皮下投与を含む）、皮内投与、膀胱内投与、経膣内投与、直腸投与、吸入又は点鼻投与、並びに気管／気管支投与が挙げられる。髄腔内投与は、シャント、留置カテーテル、又は皮下ポートを用いて投与してもよい。本態様の送達促進剤及び神経系に送達されるべき薬剤の投与形態は、上述の投与方法から独立に選択することができる。 There are no particular limitations on the administration form of the delivery enhancer of this embodiment and the drug to be delivered to the nervous system, and administration may be systemic or local. The route of administration may be oral or parenteral. Specific examples of parenteral administration include intrathecal administration (intracerebroventricular administration, posterior fossa puncture, or lumbar puncture), intranasal administration, intravenous administration, intraarterial administration, administration by blood transfusion, intraperitoneal administration, intraocular administration, intramuscular administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, intravesical administration, intravaginal administration, rectal administration, inhalation or nasal administration, and tracheal/bronchial administration. Intrathecal administration may be administered using a shunt, an indwelling catheter, or a subcutaneous port. The administration form of the delivery enhancer of this embodiment and the drug to be delivered to the nervous system may be independently selected from the administration methods described above.
　一実施形態において、本態様の送達促進剤は、経鼻投与、静脈内投与、皮下投与、腹腔内投与、経口投与、吸入投与、又は筋肉内投与され、神経系に送達されるべき薬剤は髄腔内投与される。 In one embodiment, the delivery enhancer of this aspect is administered intranasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly, and the agent to be delivered to the nervous system is administered intrathecally.
　一実施形態において、本態様の送達促進剤、及び神経系に送達されるべき薬剤は同時に投与してもよい。例えば、本態様の送達促進剤及び神経系に送達されるべき薬剤を含む後述のような組成物として、投与してもよい。 In one embodiment, the delivery enhancer of this embodiment and the drug to be delivered to the nervous system may be administered simultaneously. For example, they may be administered as a composition, as described below, that includes the delivery enhancer of this embodiment and the drug to be delivered to the nervous system.
　一実施形態において、本態様の送達促進剤は、神経系に送達されるべき薬剤よりも先に又は後に投与される。例えば1週間以内、3日以内、1日以内、12時間以内、6時間以内、3時間以内、又は1時間以内に、本態様の送達促進剤及び神経系に送達されるべき薬剤を投与してもよい。例えば、本態様の送達促進剤は薬剤よりも先に静脈内投与や皮下投与により投与され、神経系に送達されるべき薬剤は送達促進剤よりも後に髄腔内投与される。 In one embodiment, the delivery enhancer of this embodiment is administered before or after the drug to be delivered to the nervous system. For example, the delivery enhancer of this embodiment and the drug to be delivered to the nervous system may be administered within one week, three days, one day, 12 hours, six hours, three hours, or one hour. For example, the delivery enhancer of this embodiment is administered intravenously or subcutaneously before the drug, and the drug to be delivered to the nervous system is administered intrathecally after the delivery enhancer.
　本態様の送達促進剤の投与量は、併用する薬剤や対象疾患の種類に応じて有効量を適宜選択することができる。サルやヒトの場合、送達促進剤の薬剤を0.01mg以上、0.1mg以上、又は1mg以上、例えば、2mg以上、3mg以上、4mg以上、5mg以上、10mg以上、20mg以上、30mg以上、40mg以上、50mg以上、75mg以上、100mg以上、200mg以上、300mg以上、400mg以上、又は500mg以上投与してもよく、0.01mg～10g、0.1mg～1g、又は1mg～100mg投与してもよく、マウスの場合には1μg以上投与してもよい。送達促進剤の投与は、単回投与でも、複数回投与であってもよい。 The dosage of the delivery enhancer of this embodiment can be appropriately selected as an effective amount depending on the type of drug used in combination and the target disease. In the case of monkeys and humans, the delivery enhancer may be administered at 0.01 mg or more, 0.1 mg or more, or 1 mg or more, for example, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 75 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, or 500 mg or more, or 0.01 mg to 10 g, 0.1 mg to 1 g, or 1 mg to 100 mg, and in the case of mice, 1 μg or more may be administered. The delivery enhancer may be administered in a single dose or multiple doses.
　神経系に送達されるべき薬剤の投与量は、薬剤や対象疾患の種類に応じて有効量を適宜選択することができる。サルやヒトの場合、核酸医薬等の薬剤を0.01mg以上、0.1mg以上、又は1mg以上、例えば、2mg以上、3mg以上、4mg以上、5mg以上、10mg以上、20mg以上、30mg以上、40mg以上、50mg以上、75mg以上、100mg以上、200mg以上、300mg以上、400mg以上、又は500mg以上投与してもよく、0.01mg～1000mg、0.1mg～200mg、又は1mg～20mg投与してもよく、マウスの場合には1μg以上投与してもよい。神経系に送達されるべき薬剤の投与は、単回投与でも、複数回投与であってもよい。 The dosage of the drug to be delivered to the nervous system can be appropriately selected as an effective amount depending on the type of drug and target disease. In the case of monkeys and humans, the drug such as a nucleic acid drug may be administered at 0.01 mg or more, 0.1 mg or more, or 1 mg or more, for example, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 75 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, or 500 mg or more, or 0.01 mg to 1000 mg, 0.1 mg to 200 mg, or 1 mg to 20 mg, and in the case of mice, 1 μg or more may be administered. The drug to be delivered to the nervous system may be administered in a single dose or multiple doses.
　神経系に送達されるべき核酸医薬等の薬剤の投与量は、例えば0.00001mg/kg/日～10000mg/kg/日、又は0.001mg/kg/日～100mg/kg/日となるようにすればよい。薬剤の投与は、単回投与でも、複数回投与であってもよい。複数回投与の場合、毎日若しくは適当な時間間隔で（例えば1日、2日、3日、1週間、2週間、1ヶ月の間隔で）、例えば2～20回投与することもできる。核酸医薬等の薬剤の1回の投与量は、例えば、0.001mg/kg以上、0.005mg/kg以上、0.01mg/kg以上、0.25mg/kg以上、0.5mg/kg以上、1.0mg/kg以上、2.0mg/kg以上、2.5mg/kg以上、3.0mg/kg以上、4.0mg/kg以上、5mg/kg以上、10mg/kg以上、20mg/kg以上、30mg/kg以上、40mg/kg以上、50mg/kg以上、75mg/kg以上、100mg/kg以上、150mg/kg以上、200mg/kg以上、300mg/kg以上、400mg/kg以上、若しくは500mg/kg以上とすることができ、例えば、0.001mg/kg～500mg/kgの範囲に含まれる任意の量（例えば、0.001mg/kg、0.01mg/kg、0.1mg/kg、1mg/kg、5mg/kg、10mg/kg、50mg/kg、100mg/kg、若しくは200mg/kg）を適宜選択することができる。 The dosage of a drug, such as a nucleic acid drug, to be delivered to the nervous system may be, for example, 0.00001 mg/kg/day to 10,000 mg/kg/day, or 0.001 mg/kg/day to 100 mg/kg/day. The drug may be administered in a single dose or multiple doses. In the case of multiple doses, the drug 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 dosage of a single dose of a drug such as a nucleic acid drug may be, for example, 0.001 mg/kg or more, 0.005 mg/kg or more, 0.01 mg/kg or more, 0.25 mg/kg or more, 0.5 mg/kg or more, 1.0 mg/kg or more, 2.0 mg/kg or more, 2.5 mg/kg or more, 3.0 mg/kg or more, 4.0 mg/kg or more, 5 mg/kg or more, 10 mg/kg or more, 20 mg/kg or more, 30 mg/kg or more, 40 mg/kg or more, 50 mg/kg or more, 75 mg/kg or more, 100 mg/kg or more, g/kg or more, 150 mg/kg or more, 200 mg/kg or more, 300 mg/kg or more, 400 mg/kg or more, or 500 mg/kg or more, and can be appropriately selected from any amount within the range of, for example, 0.001 mg/kg to 500 mg/kg (e.g., 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, 100 mg/kg, or 200 mg/kg).
（効果）
　本発明の送達促進剤を核酸医薬等の薬剤と併用することによって、薬剤の送達効率を飛躍的に向上させることができる。したがって、本発明の送達促進剤は、送達増強剤又は送達補助剤等と言い換えることができる。 (effect)
By using the delivery enhancer of the present invention in combination with a drug such as a nucleic acid drug, the delivery efficiency of the drug can be dramatically improved. Therefore, the delivery enhancer of the present invention can be called a delivery enhancer or a delivery auxiliary.
　また、本発明によれば、本発明の送達促進剤を対象に投与することを含む、本発明の送達促進剤を投与しない対象と比較して、薬剤の中枢神経系及び／又は末梢神経系への送達効率を向上させる方法も提供される。 The present invention also provides a method for improving the efficiency of delivery of a drug to the central nervous system and/or peripheral nervous system, comprising administering a delivery enhancer of the present invention to a subject, as compared to a subject not administered the delivery enhancer of the present invention.
　国際公開第2017/150704号には、AQP4機能促進剤の投与によって、アミロイドβの脳外への排出が促進されることが示唆されていた。したがって、本願実施例において核酸医薬の脳内送達量が飛躍的に増大させる効果は、従来技術から予想外の効果である。 　International Publication No. 2017/150704 suggests that administration of an AQP4 function promoter promotes the excretion of amyloid beta from the brain. Therefore, the effect of dramatically increasing the amount of nucleic acid drug delivered to the brain in the examples of this application is an unexpected effect based on conventional technology.
＜２．医薬組成物＞
　本発明の第2の態様は中枢神経系疾患又は末梢神経系疾患を治療するための医薬組成物である。本態様の医薬組成物は、治療有効量の薬剤、及び第1態様に記載の送達促進剤を包含する。 2. Pharmaceutical Compositions
A second aspect of the present invention is a pharmaceutical composition for treating a central or peripheral nervous system disorder, comprising a therapeutically effective amount of a drug and a delivery enhancer as described in the first aspect.
（有効成分）
　本態様の医薬組成物は、有効成分として薬剤及び送達促進剤を含む。薬剤及び送達促進剤の構成は第1態様の記載に準じるものとし、ここでの説明を省略する。 (Active ingredient)
The pharmaceutical composition of this embodiment contains a drug and a delivery enhancer as active ingredients. The composition of the drug and the delivery enhancer are similar to those described in the first embodiment, and therefore the description thereof will be omitted here.
　本態様の医薬組成物に含まれる薬剤及び送達促進剤の量（含有量）は、薬剤及び送達促進剤の種類、送達部位、組成物の剤形、組成物の投与量、並びに後述する担体の種類によって異なる。したがって、それぞれの条件を勘案して適宜定めればよい。通常は、単回投与量の組成物に有効量の薬剤が包含されるように調整する。「有効量」とは、薬剤が有効成分としての機能を発揮する上で必要な量であって、かつそれを適用する生体に対して有害な副作用をほとんど又は全く付与しない量をいう。この有効量は、被験体の情報、投与経路、及び投与回数等の様々な条件によって変化し得る。最終的には医師、獣医師又は薬剤師等の判断によって決定される。「被験体の情報」とは、組成物を適用する生体の様々な個体情報である。例えば、被験体がヒトであれば、年齢、体重、性別、食生活、健康状態、疾患の進行度や重症度、薬剤感受性、併用薬剤の有無等を含む。 The amount (content) of the drug and delivery enhancer contained in the pharmaceutical composition of this embodiment varies depending on the type of drug and delivery enhancer, the delivery site, the dosage form of the composition, the dosage amount of the composition, and the type of carrier described below. Therefore, it may be determined appropriately taking into account each condition. Usually, the composition is adjusted so that an effective amount of the drug is contained in a single dose. An "effective amount" refers to an amount that is necessary for the drug to function as an active ingredient and that gives little or no harmful side effects to the living body to which it is applied. This effective amount may vary depending on various conditions such as information on the subject, the route of administration, and the number of administrations. It is ultimately determined by the judgment of a doctor, veterinarian, pharmacist, etc. "Subject information" refers to various individual information of the living body to which the composition is applied. For example, if the subject is a human, it includes age, weight, sex, diet, health condition, progression and severity of the disease, drug sensitivity, and the presence or absence of concomitant drugs.
（担体）
　本態様の医薬組成物は、薬学的に許容可能な担体を含むことができる。「薬学的に許容可能な担体」とは、製剤技術分野において通常使用する添加剤をいう。例えば、溶媒、植物性油、基剤、乳化剤、懸濁化剤、界面活性剤、pH調整剤、安定化剤、賦形剤、ビヒクル、防腐剤、結合剤、希釈剤、等張化剤、鎮静剤、増量剤、崩壊剤、緩衝剤、コーティング剤、滑沢剤、増粘剤、溶解助剤、及び他の添加剤が挙げられる。 (Carrier)
The pharmaceutical composition of this embodiment can include a pharma- ceutically acceptable carrier. The term "pharma-ceutically acceptable carrier" refers to an additive commonly used in the field of formulation technology. For example, solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, excipients, vehicles, preservatives, binders, diluents, isotonicity agents, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, thickeners, dissolution aids, and other additives can be mentioned.
　溶媒には、例えば、水若しくはそれ以外の薬学的に許容し得る水溶液、又は薬学的に許容される有機溶剤のいずれであってもよい。水溶液としては、例えば、生理食塩水、ブドウ糖やその他の補助剤を含む等張液、リン酸塩緩衝液、酢酸ナトリウム緩衝液が挙げられる。補助剤としては、例えば、D-ソルビトール、D-マンノース、D-マンニトール、塩化ナトリウム、その他にも低濃度の非イオン性界面活性剤、ポリオキシエチレンソルビタン脂肪酸エステル類等が挙げられる。 The solvent may be, for example, water or any other pharma- ceutically acceptable aqueous solution, or a pharma-ceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.
　上記担体は、薬剤及び送達促進剤の生体内での酵素等による分解を回避又は抑制する他、製剤化や投与方法を容易にし、剤形及び薬効を維持するために用いられるものであり、必要に応じて適宜使用すればよい。 The above-mentioned carriers are used to avoid or suppress the decomposition of the drug and delivery enhancer by enzymes in the body, to facilitate formulation and administration, and to maintain the dosage form and efficacy, and may be used appropriately as needed.
（剤形）
　本発明の医薬組成物の剤形は、薬剤及び送達促進剤を分解等により不活化させることなく、標的部位まで送達し、生体内でその有効成分の薬理効果（標的遺伝子の発現に対するアンチセンス効果）を発揮し得る形態であれば特に限定しない。 (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 deliver the drug and delivery enhancer to the target site without inactivating them by decomposition or the like, and can exert the pharmacological effect of the active ingredients (antisense effect on the expression of the target gene) in the body.
　本発明の医薬組成物が髄腔内投与される場合には、具体的な剤形は髄腔内投与に適した剤形にすればよい。この場合、好ましい剤形の例としては、注射剤が挙げられる。注射剤は、前記賦形剤、エリキシル剤、乳化剤、懸濁剤、界面活性剤、安定剤、pH調節剤等と適宜組み合わせて、一般に認められた製薬実施に要求される単位用量形態で混和することによって製剤化することができる。 When the pharmaceutical composition of the present invention is administered intrathecally, the specific dosage form may be a dosage form suitable for intrathecal administration. In this case, an example of a preferred dosage form is an injection. The injection can be formulated by appropriately combining the above-mentioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH regulators, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.
　なお、上記各剤形の具体的な形状、大きさについては、いずれもそれぞれの剤形において当該分野で公知の剤形の範囲内にあればよく、特に限定はしない。本態様の医薬組成物の製造方法については、当該技術分野の常法に従って製剤化すればよい。 The specific shape and size of each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art. The pharmaceutical composition of this embodiment may be formulated according to standard methods in the art.
（投与形態及び投与量）
　本態様の医薬組成物の投与形態及び投与量には特定の限定はなく、第1態様における「（送達促進剤／薬剤の投与形態）」の記載に準じるものとする。 (Dosage form and dosage)
The dosage form and dosage amount of the pharmaceutical composition of this embodiment are not particularly limited, and should conform to the description of "(Dosage form of delivery enhancer/drug)" in the first embodiment.
　投与は全身投与であっても局所投与であってもよい。投与経路は、経口投与又は非経口投与であってよい。非経口投与の具体例として、髄腔内投与（脳室内投与、後頭窩穿刺、又は腰椎穿刺）、経鼻投与、静脈内投与、動脈内投与、輸血による投与、腹腔内投与、眼内投与、筋肉内投与、皮下投与（埋め込み型持続皮下投与を含む）、皮内投与、膀胱内投与、経膣内投与、直腸投与、吸入又は点鼻投与、並びに気管／気管支投与が挙げられる。本発明の適用対象部位は中枢神経系及び／又は末梢神経系であるため、対象部位への送達に有利な髄腔内投与や経鼻投与は好適であるが、例えば静脈内投与、皮下投与、腹腔内投与、経口投与、吸入投与、又は筋肉内投与によって脳血液関門を通過させることにより中枢神経系に送達することもできる。 Administration may be systemic or local. The route of administration may be oral or parenteral. Specific examples of parenteral administration include intrathecal administration (intraventricular administration, posterior fossa puncture, or lumbar puncture), nasal administration, intravenous administration, intraarterial administration, administration by blood transfusion, intraperitoneal administration, intraocular administration, intramuscular administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, intravesical administration, intravaginal administration, rectal administration, inhalation or nasal drop administration, and tracheal/bronchial administration. Since the target site of application of the present invention is the central nervous system and/or peripheral nervous system, intrathecal administration and nasal administration, which are advantageous for delivery to the target site, are preferred, but delivery to the central nervous system is also possible by passing through the blood-brain barrier, for example, by intravenous administration, subcutaneous administration, intraperitoneal administration, oral administration, inhalation, or intramuscular administration.
（適用対象疾患）
　医薬組成物の適用対象となる疾患は、例えば中枢神経系疾患又は末梢神経系疾患である。本発明の医薬組成物に含まれる薬剤が中枢神経系及び／又は末梢神経系に送達されることにより制御され得る疾患を対象とすることができる。例えば、薬剤が核酸医薬である場合、そのアンチセンス効果により、標的遺伝子又はその転写産物又は翻訳産物の発現量が抑制若しくは亢進され得る、その転写産物又は翻訳産物の機能が阻害され得る、又はステリックブロッキング、スプライシングスイッチ、RNA編集、エキソンスキッピング若しくはエキソンインクルージョンが誘導され得る遺伝子が関係し得る疾患を対象とすることができる。 (Applicable diseases)
The diseases to which the pharmaceutical composition is applied are, for example, central nervous system diseases or peripheral nervous system diseases. The target diseases can be diseases that can be controlled by delivering the drug contained in the pharmaceutical composition of the present invention to the central nervous system and/or peripheral nervous system. For example, when the drug is a nucleic acid drug, the target diseases can be diseases that involve genes that can suppress or enhance the expression level of a target gene or its transcription product or translation product, inhibit the function of the transcription product or translation product, or induce steric blocking, splicing switch, RNA editing, exon skipping, or exon inclusion due to its antisense effect.
　医薬組成物は、被験体としてヒトを含む動物に使用することができる。しかし、ヒトを除く動物には特定の限定はなく、様々な家畜、家禽、ペット、実験動物などが一部の実施形態の被験体となり得る。被験体は、中枢神経系及び／又は末梢神経系において標的転写産物の発現量を減少させることが必要な被験体であってもよい。また被験体は、脳疾患等の神経疾患の治療が必要な被験体であってもよい。 The pharmaceutical composition may be used in animals, including humans, as subjects. However, there is no particular limitation on animals other than humans, and various livestock, poultry, pets, laboratory animals, etc. may be subjects in some embodiments. The subject may be one in which it is necessary to reduce the expression level of a target transcript in the central and/or peripheral nervous system. The subject may also be one in which it is necessary to treat a neurological disorder, such as a brain disorder.
　薬剤が核酸医薬である場合、治療対象の疾患は、遺伝子発現の増加又は減少に関連する中枢神経系疾患又は末梢神経系疾患、特に標的転写産物又は標的遺伝子の発現の増加に関連する疾患(腫瘍等)であり得る。中枢神経系疾患としては、特に限定されないが、例えば、脳腫瘍、アルツハイマー病、パーキンソン病、筋萎縮性側索硬化症、多発性硬化症、ハンチントン病などが挙げられる。 When the drug is a nucleic acid drug, the disease to be treated may be a central nervous system disease or peripheral nervous system disease associated with increased or decreased gene expression, particularly a disease (such as a tumor) associated with increased expression of a target transcript or target gene. Examples of central nervous system diseases include, but are not limited to, brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, etc.
　一実施形態において、本態様の医薬組成物では、アルツハイマー病、脳梗塞、脳腫瘍、脱髄疾患、てんかん、及び神経因性疼痛以外の中枢神経系疾患を対象とすることができる。 In one embodiment, the pharmaceutical composition of this aspect can target central nervous system diseases other than Alzheimer's disease, cerebral infarction, brain tumor, demyelinating disease, epilepsy, and neuropathic pain.
　神経系は、中枢神経系及び末梢神経系に分けられる。中枢神経系は、脳及び脊髄からなる。脳は大脳(大脳皮質、大脳白質、大脳基底核)、間脳(視床、視床下核)、小脳(小脳皮質、小脳核)及び脳幹(中脳、黒質、橋、延髄)を含む。脊髄は、頸髄、胸髄、腰髄、仙髄及び尾髄を含む。本明細書における中枢神経系は、これらのいずれの領域であってもよいが、特に、大脳皮質(前頭葉、側頭葉、頭頂葉、後頭葉)、小脳、線条体、淡蒼球、前障、海馬、海馬傍回、脳幹、頸髄、胸髄又は腰髄であり得る。末梢神経は、脳神経及び脊髄神経からなり、脊髄前根、後根、第１～第１２脳神経、馬尾、及び後根神経節を含む。 The nervous system is divided into the central nervous system and the peripheral nervous system. The central nervous system consists of the brain and spinal cord. The brain includes the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon (thalamus, subthalamic nucleus), cerebellum (cerebellar cortex, cerebellar nuclei), and brainstem (midbrain, substantia nigra, pons, medulla oblongata). The spinal cord includes the cervical, thoracic, lumbar, sacral, and coccygeal spinal cord. The central nervous system in this specification may be any of these regions, but in particular may be the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brainstem, cervical spinal cord, thoracic spinal cord, or lumbar spinal cord. The peripheral nerves consist of the cranial nerves and spinal nerves, and include the spinal cord anterior root, dorsal root, cranial nerves 1 to 12, cauda equina, and dorsal root ganglion.
　本明細書において「中枢神経系疾患」とは、中枢神経系における任意の部位又は中枢神経系全体に関連した疾患を意味する。中枢神経系は大脳(大脳皮質、大脳白質、大脳基底核)、間脳(視床、視床下核)、小脳(小脳皮質、小脳核)及び脳幹(中脳、黒質、橋、延髄)を含む。脊髄は、頸髄、胸髄、腰髄、仙髄及び尾髄を含む。中枢神経系疾患は、これらのいずれの部位に関連する疾患であってもよいが、特に、大脳皮質(前頭葉、側頭葉、頭頂葉、後頭葉)、小脳、線条体、淡蒼球、前障、海馬、海馬傍回、脳幹、頸髄、胸髄又は腰髄に関連する疾患であり得る。 As used herein, the term "central nervous system disease" refers to a disease associated with any part of the central nervous system or the entire central nervous system. The central nervous system includes the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon (thalamus, subthalamic nucleus), cerebellum (cerebellar cortex, cerebellar nuclei), and brain stem (midbrain, substantia nigra, pons, medulla oblongata). The spinal cord includes the cervical, thoracic, lumbar, sacral, and coccygeal spinal cord. The central nervous system disease may be a disease associated with any of these parts, but may particularly be a disease associated with the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brain stem, cervical spinal cord, thoracic spinal cord, or lumbar spinal cord.
　例えば、アルツハイマー病の治療においては、海馬及び/又は頭頂葉への薬剤送達が有効となり得る。前頭側頭型認知症(FTD)(前頭側頭葉変性症(FTLD)、意味性認知症(SD)、進行性非流暢性失語(PNFA))、ピック病の治療においては、前頭葉、側頭葉及び/又は黒質への薬剤送達が有効となり得る。パーキンソン病認知症の治療においては、後頭葉、黒質及び/又は線条体への薬剤送達が有効となり得る。パーキンソン病の治療においては、黒質及び/又は線条体への薬剤送達が有効となり得る。皮質基底核変性症(CBD)の治療においては、前頭葉、頭頂葉、大脳基底核及び/又は黒質への薬剤送達が有効となり得る。進行性核上性麻痺(PSP)の治療においては、前頭葉、大脳基底核及び/又は黒質への薬剤送達が有効となり得る。筋萎縮性側索硬化症の治療においては、前頭葉、頭頂葉、大脳基底核及び/又は黒質への薬剤送達が有効となり得る。脊髄小脳変性症(SCD)SCA1型～SCA34型までの治療においては、脳幹及び/又は小脳への薬剤送達が有効となり得る。歯状核赤核淡蒼球ルイ体変性症(DRPLA)の治療においては、大脳基底核、脳幹及び/又は小脳への薬剤送達が有効となり得る。球脊髄性萎縮症(SBMA)の治療においては、脳幹及び/又は脊髄への薬剤送達が有効となり得る。フリードライヒ失調症(FA)の治療においては、脳幹及び/又は小脳への薬剤送達が有効となり得る。ハンチントン病の治療においては、線条体、前頭葉、頭頂葉及び/又は大脳基底核への薬剤送達が有効となり得る。プリオン病(狂牛病、GSS)の治療においては、大脳皮質、大脳白質、大脳基底核及び/又は黒質への薬剤送達が有効となり得る。大脳白質性脳症の治療においては、大脳白質への薬剤送達が有効となり得る。脳炎(ウイルス性、細菌性、真菌性、結核性)、髄膜炎(ウイルス性、細菌性、真菌性、結核性)の治療においては、脳全体への薬剤送達が有効となり得る。代謝性脳症、中毒性脳症、栄養障害性脳症の治療においては、脳全体への薬剤送達が有効となり得る。大脳白質性脳症の治療においては、大脳白質への薬剤送達が有効となり得る。脳梗塞、脳出血、くも膜下出血、もやもや病、無酸素脳症の治療においては、脳全体への薬剤送達が有効となり得る。大脳白質性脳症の治療においては、大脳白質への薬剤送達が有効となり得る。びまん性軸索損傷(Diffuse axonal injury)の治療においては、大脳白質への薬剤送達が有効となり得る。頭部外傷の治療においては、脳全体への薬剤送達が有効となり得る。多発性硬化症(MS)、視神経脊髄炎(NMO)の治療においては、大脳白質、大脳皮質、視神経及び/又は脊髄への薬剤送達が有効となり得る。筋緊張型ジストロフィー症(DM1, DM2)の治療においては、骨格筋、心筋、大脳皮質及び/又は大脳白質への薬剤送達が有効となり得る。家族性痙性対麻痺(HSP)の治療においては、頭頂葉及び/又は脊髄への薬剤送達が有効となり得る。福山型筋ジストロフィーの治療においては、骨格筋、大脳皮質及び/又は大脳白質への薬剤送達が有効となり得る。レビー小体型認知症(DLB)の治療においては、黒質、線条体、後頭葉、前頭葉及び/又は頭頂葉への薬剤送達が有効となり得る。多系統萎縮症(MSA)の治療においては、線条体、大脳基底核、小脳、黒質、前頭葉及び/又は側頭葉への薬剤送達が有効となり得る。アレキサンダー病の治療においては、大脳白質への薬剤送達が有効となり得る。CADASIL、CARASILの治療においては、大脳白質への薬剤送達が有効となり得る。 For example, in the treatment of Alzheimer's disease, drug delivery to the hippocampus and/or parietal lobe may be effective. In the treatment of frontotemporal dementia (FTD) (frontotemporal lobar degeneration (FTLD), semantic dementia (SD), progressive non-fluent aphasia (PNFA)) and Pick's disease, drug delivery to the frontal lobe, temporal lobe and/or substantia nigra may be effective. In the treatment of Parkinson's disease dementia, drug delivery to the occipital lobe, substantia nigra and/or striatum may be effective. In the treatment of Parkinson's disease, drug delivery to the substantia nigra and/or striatum may be effective. In the treatment of corticobasal degeneration (CBD), drug delivery to the frontal lobe, parietal lobe, basal ganglia and/or substantia nigra may be effective. In the treatment of progressive supranuclear palsy (PSP), drug delivery to the frontal lobe, basal ganglia and/or substantia nigra may be effective. In the treatment of amyotrophic lateral sclerosis, drug delivery to the frontal lobe, parietal lobe, basal ganglia, and/or substantia nigra may be effective. In the treatment of spinocerebellar degeneration (SCD) SCA1 to SCA34, drug delivery to the brainstem and/or cerebellum may be effective. In the treatment of dentatorubral-pallidoluysian degeneration (DRPLA), drug delivery to the basal ganglia, brainstem, and/or cerebellum may be effective. In the treatment of spinal-bulbar atrophy (SBMA), drug delivery to the brainstem and/or spinal cord may be effective. In the treatment of Friedreich's ataxia (FA), drug delivery to the brainstem and/or cerebellum may be effective. In the treatment of Huntington's disease, drug delivery to the striatum, frontal lobe, parietal lobe, and/or basal ganglia may be effective. In the treatment of prion diseases (mad cow disease, GSS), drug delivery to the cerebral cortex, cerebral white matter, basal ganglia and/or substantia nigra may be effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective. In the treatment of encephalitis (viral, bacterial, fungal, tuberculous) and meningitis (viral, bacterial, fungal, tuberculous), drug delivery to the entire brain may be effective. In the treatment of metabolic encephalopathy, toxic encephalopathy, and nutritional encephalopathy, drug delivery to the entire brain may be effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective. In the treatment of cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, moyamoya disease, and anoxic encephalopathy, drug delivery to the entire brain may be effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective. In the treatment of diffuse axonal injury, drug delivery to the cerebral white matter may be effective. In the treatment of head trauma, drug delivery to the entire brain may be effective. In the treatment of multiple sclerosis (MS) and neuromyelitis optica (NMO), drug delivery to the cerebral white matter, cerebral cortex, optic nerve, and/or spinal cord may be effective. In the treatment of myotonic dystrophy (DM1, DM2), drug delivery to skeletal muscle, cardiac muscle, cerebral cortex, and/or cerebral white matter may be effective. In the treatment of familial spastic paraplegia (HSP), drug delivery to the parietal lobe and/or spinal cord may be effective. In the treatment of Fukuyama muscular dystrophy, drug delivery to skeletal muscle, cerebral cortex, and/or cerebral white matter may be effective. In the treatment of dementia with Lewy bodies (DLB), drug delivery to the substantia nigra, striatum, occipital lobe, frontal lobe, and/or parietal lobe may be effective. In the treatment of multiple system atrophy (MSA), drug delivery to the striatum, basal ganglia, cerebellum, substantia nigra, frontal lobe, and/or temporal lobe may be effective. In the treatment of Alexander disease, drug delivery to the cerebral white matter may be effective. In the treatment of CADASIL and CARASIL, drug delivery to the cerebral white matter may be effective.
　本明細書において「末梢神経系疾患」とは、末梢神経系における任意の部位又は末梢神経系全体に関連した疾患を意味する。末梢神経は脳神経及び脊髄神経からなり、脊髄前根、後根、第１～第１２脳神経、馬尾、及び後根神経節を含む。末梢神経系疾患は、これらのいずれの部位に関連する疾患であってもよいが、特に、脊髄前根、後根、第１～第１２脳神経、馬尾、又は後根神経節に関連する疾患であり得る。 In this specification, the term "peripheral nervous system disease" refers to a disease associated with any part of the peripheral nervous system or the entire peripheral nervous system. The peripheral nerves consist of the cranial nerves and spinal nerves, and include the ventral root of the spinal cord, the dorsal root, the first to twelfth cranial nerves, the cauda equina, and the dorsal root ganglion. The peripheral nervous system disease may be a disease associated with any of these parts, but may particularly be a disease associated with the ventral root of the spinal cord, the dorsal root, the first to twelfth cranial nerves, the cauda equina, or the dorsal root ganglion.
（効果）
　本発明の医薬組成物は、治療有効量の薬剤に加えて送達促進剤を含むことによって、薬剤の中枢神経系及び／又は末梢神経系への送達効率を飛躍的に向上させることに基づいて薬効を増大させることができる。 (effect)
The pharmaceutical composition of the present invention contains a delivery enhancer in addition to a therapeutically effective amount of a drug, thereby dramatically improving the efficiency of delivery of the drug to the central nervous system and/or peripheral nervous system, thereby increasing the drug efficacy.
　また、本発明によれば、本態様の医薬組成物をヒト等の被験体に投与（例えば、髄腔内投与）することを含む、中枢神経系疾患又は末梢神経系疾患等の疾患を治療及び／又は予防する方法もまた提供される。 The present invention also provides a method for treating and/or preventing a disease, such as a central nervous system disease or a peripheral nervous system disease, which comprises administering (e.g., intrathecally administering) the pharmaceutical composition of this embodiment to a subject, such as a human.
　医薬の製造における、第1態様の送達促進剤、又は第2態様の医薬組成物の使用もまた提供される。 There is also provided the use of a delivery enhancer of the first aspect, or a pharmaceutical composition of the second aspect, in the manufacture of a medicament.
　以下、実施例を用いて本発明をさらに具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。 The present invention will be explained in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.
＜実施例1：アンチセンス核酸の神経系への送達効率及び神経系における遺伝子抑制効果を増強する新規化合物の同定＞
（目的）
　Malat1遺伝子を標的とするアンチセンス核酸（以下、「Malat1標的ASO」と称する）に基づく神経系における遺伝子抑制効果を増強し得る新規化合物を同定するために、候補化合物であるTGN-073をASOと共にマウス脳室内に投与する。中枢神経系へのアンチセンス核酸の送達効率、及び中枢神経系における標的遺伝子抑制効果に対するTGN-073共投与の影響をin vivo実験により検証する。本実施例では、ASOとしてMalat1標的ASOを使用する。 Example 1: Identification of novel compounds that enhance the delivery efficiency of antisense nucleic acids to the nervous system and the gene suppression effect in the nervous system
(the purpose)
In order to identify a novel compound that can enhance the gene suppression effect in the nervous system based on antisense nucleic acid targeting Malat1 gene (hereinafter referred to as "Malat1 target ASO"), the candidate compound TGN-073 is administered intracerebroventricularly in mice together with ASO. The delivery efficiency of antisense nucleic acid to the central nervous system and the effect of coadministration of TGN-073 on the target gene suppression effect in the central nervous system are verified by in vivo experiments. In this example, Malat1 target ASO is used as ASO.
（方法）
（１）TGN-073の合成
　候補化合物として以下の式(I)で示すN-(3-benzyloxypyridin-2-yl)-benzene-sulfonamideを合成した。 (Method)
(1) Synthesis of TGN-073 As a candidate compound, N-(3-benzyloxypyridin-2-yl)-benzene-sulfonamide shown in the following formula (I) was synthesized.
Figure JPOXMLDOC01-appb-C000012
 　上記式(I)で示す化合物は、2-(phenylsulfonamido)-3-benzyloxypyridineとも表記することもできる。また、上記式(I)で示す化合物は、本発明者らの過去の文献（Huber V.J. et al., NeuroReport, 2018, 29(9):697-703）では「TGN-073」と呼ばれ、国際公開第2017/150704号では「2A」と呼ばれている。本明細書では、上記式(I)で示す化合物及びその塩を「TGN-073」と称することとする。
Figure JPOXMLDOC01-appb-C000012
 The compound represented by the above formula (I) can also be written as 2-(phenylsulfonamido)-3-benzyloxypyridine. The compound represented by the above formula (I) is called "TGN-073" in the previous publication by the present inventors (Huber VJ et al., NeuroReport, 2018, 29(9):697-703), and is called "2A" in International Publication No. 2017/150704. In this specification, the compound represented by the above formula (I) and its salts are referred to as "TGN-073".
　TGN-073の合成は、国際公開第2017/150704号に記載の化合物2Aの製造方法に準じて行った。具体的には、2-アミノ-3-ベンジルオキシピリジン（1.50ｇ、7.50mmol）を乾燥したジクロロメタン（35mL）を添加した50mL容量の丸底フラスコに加えた。続いて、2,6-ルチジン（2.62mL、22.5mmol）水溶液を、シリンジを用いて撹拌中の溶液に加え、反応容器にアルゴンを充填した。続いて、ベンゼンスルホニルクロリド（1.05mL、8.25mmol）を、シリンジを用いて撹拌中の溶液に加えた。続いて、72時間アルゴン雰囲気下、室温で撹拌しながら反応を行った。続いて、得られた黄色の溶液を10％クエン酸溶液（2×25mL）及び飽和炭酸ナトリウム溶液（2×25mL）で洗浄した。続いて、硫酸マグネシウムを用いて乾燥させ、濾過し、水分を蒸発させることにより、暗赤色の半固体の結晶を得た。続いて、得られた暗赤色の結晶をヘキサンで洗浄した。続いて、シリカゲル（ワコーゲル300）を用いたフラッシュクロマトグラフィーカラムにより、33％酢酸エチル／ヘキサン用出来で溶出し、白色の結晶（化合物TGN-073）を得た（収量0.309g、収率13.5％）。TGN-073の合成経路を以下に示す。 The synthesis of TGN-073 was carried out according to the method for preparing compound 2A described in WO 2017/150704. Specifically, 2-amino-3-benzyloxypyridine (1.50 g, 7.50 mmol) was added to a 50 mL round-bottom flask containing dry dichloromethane (35 mL). Aqueous 2,6-lutidine (2.62 mL, 22.5 mmol) was then added to the stirring solution using a syringe, and the reaction vessel was backfilled with argon. Benzenesulfonyl chloride (1.05 mL, 8.25 mmol) was then added to the stirring solution using a syringe. The reaction was then carried out under argon atmosphere at room temperature with stirring for 72 hours. The resulting yellow solution was then washed with 10% citric acid solution (2 × 25 mL) and saturated sodium carbonate solution (2 × 25 mL). The product was then dried over magnesium sulfate, filtered, and evaporated to give dark red semi-solid crystals. The resulting dark red crystals were then washed with hexane. They were then eluted with 33% ethyl acetate/hexane in a flash chromatography column using silica gel (Wakogel 300) to obtain white crystals (compound TGN-073) (yield: 0.309 g, 13.5%). The synthetic route for TGN-073 is shown below.
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000013
　なお、以下の実施例においてTGN-073を投与する場合には、上記式(I)で示す化合物のナトリウム塩を使用した。上記式(I)で示す化合物のナトリウム塩を以下の式(II)で示す。 In the following examples, when TGN-073 was administered, the sodium salt of the compound represented by the above formula (I) was used. The sodium salt of the compound represented by the above formula (I) is represented by the following formula (II).
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000014
（２）核酸の調製
　本実施例で用いたASOの塩基配列と化学修飾を以下の表1に示す。 (2) Preparation of Nucleic Acid The base sequence and chemical modification of the ASO used in this example are shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
　本実施例で用いたASOは、マウスの転移関連肺腺癌転写産物1(metastasis associated lung adenocarcinoma transcript 1、Malat1)ノンコーディングRNAを標的とするLNA/DNAギャップマー型アンチセンス核酸であり、Malat1 RNAの一部に相補的な塩基配列を有し、5’末端の3個のLNAヌクレオシド、3’末端の3個のLNAヌクレオシド、及びそれらの間の10個のDNAヌクレオシドがホスホロチオエート結合で連結された構造を有する。全てのオリゴヌクレオチドは株式会社ジーンデザイン(Gene Design)(大阪、日本)によって委託合成された。 The ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid targeting mouse metastasis associated lung adenocarcinoma transcript 1 (Malat1) non-coding RNA, and has a base sequence complementary to a portion of Malat1 RNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them linked by phosphorothioate bonds. All oligonucleotides were synthesized by Gene Design Co., Ltd. (Osaka, Japan).
（３）in vivo実験
　7週齢雌のICRマウスを、2.5～4％イソフルレン麻酔下にて脳定位固定装置に固定した。その後、耳間に前後2～3cmで皮膚を切開し、ブレグマ(bregma)の1mm左方かつ0.2mm後方に1mm径ドリルで穿孔した。ハミルトン(Hamilton)シリンジ内に核酸剤を充填した。穿孔部より針を3mm程度刺入し、マウス1匹当たり0.95nmol(5μg)のASOを左側脳室内に投与した(n=4～7／群)。ASO及びTGN-073を共投与した群（以下、「ASO/TGN-073共投与群」という）では、TGN-073と混合したASOを投与し、ASOのみを投与した群（以下、「ASO単独投与群」という）ではTGN-073を混合することなくASOのみを投与した。ASO/TGN-073共投与群におけるTGN-073の用量は、マウス1匹当たり0.25mgとした。脳室内投与後、ナイロン糸で皮膚縫合した。また、対照として、TGN-073のみを投与したマウス（以下、「TGN-073投与群」という）、及びPBSのみを投与したマウス（以下、「PBS投与群」という）も作製した。 (3) In vivo experiment Seven-week-old female ICR mice were fixed in a stereotaxic apparatus under 2.5-4% isoflurane anesthesia. Then, the skin was incised 2-3 cm in front and behind the ears, and a hole was made with a 1 mm diameter drill 1 mm left and 0.2 mm behind the bregma. The nucleic acid agent was loaded into a Hamilton syringe. The needle was inserted about 3 mm into the hole, and 0.95 nmol (5 μg) of ASO per mouse was administered into the left lateral ventricle (n = 4-7/group). In the group co-administered with ASO and TGN-073 (hereinafter referred to as the "ASO/TGN-073 co-administration group"), ASO mixed with TGN-073 was administered, and in the group administered only with ASO (hereinafter referred to as the "ASO alone administration group"), only ASO was administered without mixing with TGN-073. The dose of TGN-073 in the ASO/TGN-073 co-administration group was 0.25 mg per mouse. After intraventricular administration, the skin was sutured with nylon thread. As controls, mice administered only TGN-073 (hereinafter referred to as the "TGN-073 administration group") and mice administered only PBS (hereinafter referred to as the "PBS administration group") were also prepared.
（４）遺伝子抑制効果の評価
　脳室内投与から3時間後又は7日後のマウスから左海馬を摘出した。IsogenIキット(株式会社ジーンデザイン)を使用して、摘出した左海馬からRNAを抽出した。cDNAは、Transcriptor Universal cDNA Master, DNase (ロシュ・ダイアグノスティックス社(Roche Diagnostics))を使用してプロトコルに従って合成した。 (4) Evaluation of gene suppression effect The left hippocampus was excised from the mice 3 hours or 7 days after intracerebroventricular administration. RNA was extracted from the excised left hippocampus using an IsogenI kit (Gene Design Co., Ltd.). cDNA was synthesized using Transcriptor Universal cDNA Master, DNase (Roche Diagnostics) according to the protocol.
　次に、定量RT-PCRは、TaqMan(ロシュ・アプライドサイエンス社(Roche Applied Science))により実施した。定量RT-PCRにおいて使用したプライマーは、様々な遺伝子数に基づいて、サーモ・フィッシャー・サイエンティフィック社(Thermo Fisher Scientific、旧ライフ・テクノロジーズ社(Life Technologies Corp))によって設計および製造された製品であった。増幅条件(温度および時間)は以下のとおりであった：95℃で15秒、60℃で30秒、及び72℃で1秒(1サイクル)を40サイクル繰り返した。Actb mRNA(内部標準遺伝子)の発現量に対するMalat1 RNAの発現量の比率を計算し、PBS投与群の値に対して標準化した値を相対的Malat1 RNAレベルとした。 Next, quantitative RT-PCR was performed using TaqMan (Roche Applied Science). The primers used in quantitative RT-PCR were products designed and manufactured by Thermo Fisher Scientific (formerly Life Technologies Corp) based on various gene numbers. The amplification conditions (temperature and time) were as follows: 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second (1 cycle), repeated for 40 cycles. The ratio of Malat1 RNA expression to Actb mRNA (internal control gene) expression was calculated, and the value normalized to the value of the PBS-treated group was used as the relative Malat1 RNA level.
　脳内ASO濃度（脳内核酸送達量）の測定は、上記と同様に摘出された左海馬からRNAを抽出し、ASOを増幅するためのプライマー対、及びTaqMan Small RNA Assay(ロシュ・アプライドサイエンス社)を使用する定量RT-PCRにより行った。U6 snRNA（内部標準遺伝子）に対する相対的なASO量を計算することによって脳内ASO濃度を得た。 To measure ASO concentration in the brain (amount of nucleic acid delivered to the brain), RNA was extracted from the left hippocampus excised as described above, and quantitative RT-PCR was performed using a primer pair for amplifying ASO and TaqMan Small RNA Assay (Roche Applied Science). The ASO concentration in the brain was obtained by calculating the relative amount of ASO to U6 snRNA (internal standard gene).
（結果）
　図3Aは、脳室内投与から3時間後又は7日後の海馬におけるASO濃度を示す。ASO/TGN-073共投与群では、ASO単独投与群と比較して、脳内ASO濃度（脳内核酸送達量）の飛躍的な増大を示した。 (result)
Figure 3A shows the ASO concentration in the hippocampus 3 hours or 7 days after intracerebroventricular administration. The ASO/TGN-073 co-administration group showed a dramatic increase in ASO concentration in the brain (amount of nucleic acid delivered to the brain) compared with the ASO alone administration group.
　図3Bは、脳室内投与から7日後の海馬におけるMalat1 RNA発現レベルを示す。ASO/TGN-073共投与群では、ASO単独投与群と比較して、Malat1遺伝子に対する抑制効果が著しく向上した。 Figure 3B shows the levels of Malat1 RNA expression in the hippocampus 7 days after intracerebroventricular administration. The ASO/TGN-073 co-administration group showed a significantly improved inhibitory effect on the Malat1 gene compared to the ASO alone administration group.
　以上の結果から、TGN-073の共投与によって、ASOの中枢神経系への送達効率が著しく改善され、中枢神経系における標的遺伝子抑制効果が飛躍的に増強される驚くべき効果が明らかになった。 These results reveal the surprising effect of co-administration of TGN-073, which significantly improves the efficiency of ASO delivery to the central nervous system and dramatically enhances the effect of suppressing target genes in the central nervous system.
＜実施例2：TGN-073と共に脳室内投与されるASOの用量依存性＞
（目的）
　実施例1で見出されたTGN-073の効果を定量的に評価する目的で、様々な濃度のASOをTGN-073と共に脳室内投与し、ASOの中枢神経系への送達効率及び中枢神経系における標的遺伝子抑制効果をin vivo実験により検証する。 Example 2: Dose dependency of ASO administered intracerebroventricularly with TGN-073
(the purpose)
In order to quantitatively evaluate the effects of TGN-073 found in Example 1, various concentrations of ASO will be administered intracerebroventricularly together with TGN-073, and the delivery efficiency of ASO to the central nervous system and its effect of suppressing target genes in the central nervous system will be verified through in vivo experiments.
（方法と結果）
　本実施例では実施例1で使用したMalat1を標的とするASO（表1に記載のASO）を使用した。核酸の調製、in vivo実験、並びに脳内ASO濃度（脳内核酸送達量）及び標的遺伝子抑制効果の評価は、実施例1に準じて行った。但し、本実施例ではマウス1匹当たりのASO投与量は、ASO単独投与群では1.25μg、5μg、及び20μg、ASO/TGN-073共投与群では0.075μg、0.3μg、及び1.25μgとした。 (Methods and Results)
In this example, the ASO targeting Malat1 used in Example 1 (ASO listed in Table 1) was used. Preparation of nucleic acid, in vivo experiment, and evaluation of ASO concentration in the brain (amount of nucleic acid delivered to the brain) and target gene suppression effect were performed according to Example 1. However, in this example, the ASO dosage per mouse was 1.25 μg, 5 μg, and 20 μg in the ASO alone administration group, and 0.075 μg, 0.3 μg, and 1.25 μg in the ASO/TGN-073 co-administration group.
　図4Aは、核酸剤の脳室内投与から7日後の海馬におけるASO濃度を示す。ASO/TGN-073共投与群では、ASO単独投与群と比較して、約16倍～20倍低い濃度で同等の脳内送達量を達成した。 Figure 4A shows the ASO concentration in the hippocampus 7 days after intraventricular administration of the nucleic acid agent. The ASO/TGN-073 co-administration group achieved the same amount of intracerebral delivery at a concentration approximately 16- to 20-fold lower than the ASO alone administration group.
　図4Bは、核酸剤の脳室内投与から7日後の海馬におけるMalat1 RNA発現レベルを示す。ASO/TGN-073共投与群では、ASO単独投与群と比較して、約16倍～20倍低い濃度で同等の遺伝子抑制効果を達成した。 Figure 4B shows the levels of Malat1 RNA expression in the hippocampus 7 days after intracerebroventricular administration of the nucleic acid agent. The ASO/TGN-073 co-administration group achieved the same gene suppression effect at approximately 16- to 20-fold lower concentrations than the ASO alone administration group.
　以上の結果から、ASOの中枢神経系への送達量及びASOに基づく中枢神経系ｍにおける標的遺伝子抑制効果は、TGN-073の共投与によって約16倍～20倍に向上することが示された。 These results show that the amount of ASO delivered to the central nervous system and the ASO-based effect of suppressing target genes in the central nervous system are increased by approximately 16- to 20-fold by coadministration of TGN-073.
＜実施例3：Mapt標的ASOの脳室内投与におけるTGN-073共投与の効果＞
（目的）
　Mapt遺伝子を標的とするASO（以下、「Mapt標的ASO」と表記する）をTGN-073と共に脳室内に投与する。中枢神経系における標的遺伝子抑制効果に対するTGN-073共投与の影響，及び中枢神経毒性をin vivo実験により検証する。 Example 3: Effect of coadministration of TGN-073 with intracerebroventricular administration of Mapt-targeting ASO
(the purpose)
ASO targeting the Mapt gene (hereinafter referred to as "Mapt-targeted ASO") will be co-administered intracerebroventricularly with TGN-073. The effects of co-administration of TGN-073 on the target gene suppression effect in the central nervous system and central nervous toxicity will be verified by in vivo experiments.
（方法）
　本実施例で用いたASOの塩基配列と化学修飾を以下の表2に示す。 (Method)
The base sequence and chemical modifications of the ASO used in this example are shown in Table 2 below.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　本実施例で用いたASOは、マウスのmicrotubule-associated protein tau (Mapt) mRNAを標的とするLNA/DNAギャップマー型アンチセンス核酸であり、Mapt mRNAの一部に相補的な塩基配列を有し、5’末端の3個のLNAヌクレオシド、3’末端の3個のLNAヌクレオシド、及びそれらの間の10個のDNAヌクレオシドがホスホロチオエート結合で連結された構造を有する。 The ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets mouse microtubule-associated protein tau (Mapt) mRNA, has a base sequence complementary to a portion of Mapt mRNA, and has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds.
　表2に記載されるASOについて、実施例1と同様の方法により核酸の調製、in vivo実験、及び標的遺伝子抑制効果の評価を行った。但し、本実施例ではマウス1匹当たりの核酸剤投与量は9.4nmol（50μg）とした。 For the ASOs listed in Table 2, nucleic acid preparation, in vivo experiments, and evaluation of the target gene suppression effect were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid agent administered per mouse was 9.4 nmol (50 μg).
　脳室内投与後のマウスにおける中枢神経毒性評価及び運動機能評価は以下の方法で行った。 Central neurotoxicity and motor function evaluation in mice after intracerebroventricular administration were performed using the following methods.
　中枢神経毒性評価では、脳室内投与から30分後、1時間後、及び2時間後に、図6に示すスコアリングシステムを用いて行動評価を行った。図6に示すスコアリングシステムでは、5つのカテゴリーに属する行動が評価対象となる（図6、カテゴリー1～5）。各カテゴリーには各々2つの行動評価項目が含まれている。各行動評価項目は0～4点（図6、スコア0～4）の5段階で評価され、正常であれば0点となり、高毒性になるほどスコアが高くなる。各カテゴリーでは、2つの行動評価項目のうち高い方のスコアをそのカテゴリーのスコアとして採用する。5つのカテゴリーのスコアを合計した値を急性期忍容性スコア（0～20点）とする。 In the central neurotoxicity evaluation, behavioral assessment was performed 30 minutes, 1 hour, and 2 hours after intracerebroventricular administration using the scoring system shown in Figure 6. In the scoring system shown in Figure 6, behaviors belonging to five categories are evaluated (Figure 6, categories 1 to 5). Each category includes two behavioral evaluation items. Each behavioral evaluation item is scored on a five-point scale from 0 to 4 points (Figure 6, scores 0 to 4), with normal being scored as 0 and higher scores indicating higher toxicity. In each category, the higher score of the two behavioral evaluation items is adopted as the score for that category. The sum of the scores for the five categories represents the acute tolerability score (0 to 20 points).
　運動機能評価では、脳室内投与後の各時点でオープンフィールドテストを行った。具体的には、マウスをケージ（幅50cm×直径50cm×高さ40cm）の中央に配置し、マウスの軌跡を5分間記録した。記録データに基づき、総移動最大移動速度(m/s)及び移動時間(s)をビデオトラッキングソフト（ANY-maze）により計測した。 To evaluate motor function, an open field test was performed at each time point after intracerebroventricular administration. Specifically, the mouse was placed in the center of a cage (50 cm wide x 50 cm diameter x 40 cm high), and the mouse's trajectory was recorded for 5 minutes. Based on the recorded data, the total maximum movement speed (m/s) and movement time (s) were measured using video tracking software (ANY-maze).
（結果）
　図5は、核酸剤の脳室内投与から7日後の海馬におけるMapt RNA発現レベルを示す。ASO/TGN-073共投与群では、ASO単独投与群と比較して、標的遺伝子抑制効果の著しい増強を示した。 (result)
Figure 5 shows the expression levels of Mapt RNA in the hippocampus 7 days after intracerebroventricular administration of the nucleic acid agent. The ASO/TGN-073 co-administration group showed a significant increase in the target gene suppression effect compared to the ASO alone administration group.
　図7は、中枢神経毒性評価の結果を示す。TGN-073単独投与群は、脳室内投与から30分後、1時間後、及び2時間後のいずれの時点においても急性期忍容性スコアは0であり、毒性を示さなかった。また、ASO/TGN-073共投与群では、ASO単独投与群と比較して、急性忍容性スコアはほぼ同等であった。 Figure 7 shows the results of the central neurotoxicity evaluation. The group administered TGN-073 alone had an acute tolerability score of 0 at 30 minutes, 1 hour, and 2 hours after intracerebroventricular administration, showing no toxicity. Furthermore, the acute tolerability scores of the ASO/TGN-073 co-administration group were almost equivalent to those of the ASO alone administration group.
　図8は、投与1時間後のオープンフィールドテストにおいて運動機能を評価した結果を示す。ASO/TGN-073共投与群は、ASO単独投与群と比較して、ほぼ同等の最大移動速度（図8A）及び移動時間（図8B）を示した。 Figure 8 shows the results of evaluating motor function in an open field test 1 hour after administration. The ASO/TGN-073 co-administration group showed nearly equivalent maximum movement speed (Figure 8A) and movement time (Figure 8B) compared to the ASO alone administration group.
　以上の結果から、TGN-073共投与によってASOに基づく中枢神経系における標的遺伝子抑制効果が飛躍的に増大する一方で、神経毒性の増強は全く認められないことが示された。 These results demonstrate that coadministration of TGN-073 dramatically increases the effect of ASO-based target gene suppression in the central nervous system, without any increase in neurotoxicity.
＜実施例4：2'-O-MOE修飾ASOの脳室内投与におけるTGN-073共投与の効果＞
（目的）
　実施例1で用いたMalat1標的ASOにおいて異なる核酸化学修飾として2'-MOE修飾をウイング領域に導入したASOを脳室内投与する際にTGN-073を共投与する。中枢神経系における標的遺伝子抑制効果に対するTGN-073共投与の影響をin vivo実験により検証する。 Example 4: Effect of coadministration of TGN-073 with intracerebroventricular administration of 2'-O-MOE-modified ASO
(the purpose)
In Example 1, the Malat1 targeting ASO used in Example 1 is co-administered with TGN-073 when the ASO is intracerebroventricularly administered, in which 2'-MOE modification is introduced into the wing region as a different nucleic acid chemical modification. The effect of TGN-073 co-administration on the target gene suppression effect in the central nervous system is verified by in vivo experiments.
（方法）
　本実施例で用いたASOの塩基配列と化学修飾を以下の表3に示す。 (Method)
The base sequence and chemical modifications of the ASO used in this example are shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
　本実施例で用いたASOは、Malat1ノンコーディングRNAを標的とする2'-O-MOE-RNA/DNAギャップマー型アンチセンス核酸であり、Malat1 ncRNAの一部に相補的な塩基配列を有し、5’末端の5個の2'-O-MOE-RNAヌクレオシド、3’末端の5個の2'-O-MOE-RNAヌクレオシド、及びそれらの間の10個のDNAヌクレオシドがホスホロチオエート結合で連結された構造を有する。 The ASO used in this example is a 2'-O-MOE-RNA/DNA gapmer-type antisense nucleic acid that targets Malat1 non-coding RNA, has a base sequence complementary to a portion of Malat1 ncRNA, and has a structure in which five 2'-O-MOE-RNA nucleosides at the 5' end, five 2'-O-MOE-RNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds.
　表3に記載されるASOについて、実施例1と同様の方法により核酸の調製、in vivo実験、並びに標的遺伝子抑制効果の評価を行った。但し、本実施例ではマウス1匹当たりの核酸剤投与量は、1.7nmol(12.5μg)とした。 For the ASOs listed in Table 3, nucleic acid preparation, in vivo experiments, and evaluation of the target gene suppression effect were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid agent administered per mouse was 1.7 nmol (12.5 μg).
（結果）
　図9は、脳室内投与から7日後の海馬におけるMalat1 RNA発現レベルを示す。ASO/TGN-073共投与群では、ASO単独投与群と比較して、標的遺伝子抑制効果の増強を示した。 (result)
Figure 9 shows the levels of Malat1 RNA expression in the hippocampus 7 days after intracerebroventricular administration. The ASO/TGN-073 co-administration group showed enhanced target gene silencing effects compared to the ASO alone administration group.
　以上の結果からアンチセンス核酸における化学修飾の種類によらず、脳内標的遺伝子抑制効果がTGN-073共投与によって飛躍的に向上され得ることが示された。 These results demonstrate that the effect of suppressing target genes in the brain can be dramatically improved by coadministration of TGN-073, regardless of the type of chemical modification in the antisense nucleic acid.
＜実施例5：ヘテロ2本鎖核酸の脳室内投与におけるTGN-073共投与の効果＞
（目的）
　実施例１で用いた1本鎖ASOからなる第1核酸鎖、及び第1核酸鎖に相補的な塩基配列を有する第2核酸鎖を含むヘテロ二重鎖オリゴヌクレオチド（以下、「HDO」と称する）を脳室内投与する際にTGN-073を共投与する。中枢神経系における核酸送達量及び標的遺伝子抑制効果に対するTGN-073共投与の影響をin vivo実験により検証する。 Example 5: Effect of coadministration of TGN-073 with intracerebroventricular administration of heteroduplex nucleic acid
(the purpose)
TGN-073 is co-administered when a heteroduplex oligonucleotide (hereinafter referred to as "HDO") containing a first nucleic acid strand consisting of the single-stranded ASO used in Example 1 and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is intracerebroventricularly administered. The effect of TGN-073 co-administration on the amount of nucleic acid delivered in the central nervous system and the target gene suppression effect is verified by an in vivo experiment.
（方法）
　本実施例で用いた1本鎖及び2本鎖の核酸の塩基配列と化学修飾を図10及び以下の表4に示す。 (Method)
The base sequences and chemical modifications of the single-stranded and double-stranded nucleic acids used in this example are shown in FIG. 10 and Table 4 below.
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
　表4に記載されるASOは、実施例1と同様の方法により調製した。
　本実施例で用いたヘテロ二重鎖オリゴヌクレオチド（HDO）は、上記ASOを第1核酸鎖として含み、第2核酸鎖は第1核酸鎖に相補的な配列を有する。 The ASOs listed in Table 4 were prepared in a manner similar to that of Example 1.
The heteroduplex oligonucleotide (HDO) used in this example contains the above-mentioned ASO as a first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
　5'側にコレステロールが結合したヘテロ二重鎖オリゴヌクレオチド（Chol-HDO）は、上記ASOを第1核酸鎖として含み、第2核酸鎖の5’末端にはコレステロールが連結されている。 Heteroduplex oligonucleotides with cholesterol bound to the 5' end (Chol-HDO) contain the above ASO as the first nucleic acid strand, and cholesterol is linked to the 5' end of the second nucleic acid strand.
　表4に記載されるASO、HDO、及びChol-HDOについて、実施例1と同様の方法によりin vivo実験、中枢神経系におけるASO濃度の測定、及び標的遺伝子抑制効果の評価を行った。但し、本実施例ではマウス1匹当たりの核酸剤投与量は、0.95nmolとした。 For the ASO, HDO, and Chol-HDO listed in Table 4, in vivo experiments, measurement of ASO concentration in the central nervous system, and evaluation of the target gene suppression effect were performed using methods similar to those used in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 0.95 nmol.
（結果）
　図11Aは、脳室内投与から7日後の海馬におけるASO濃度を示す。ASO、HDO、及びChol-HDOと共にTGN-073を共投与した群はいずれも、TGN-073を共投与せず核酸剤を単独投与した群と比較して、中枢神経系におけるASO濃度の飛躍的な増大を示した。 (result)
Figure 11A shows the ASO concentration in the hippocampus 7 days after intracerebroventricular administration. All groups co-administered with TGN-073 along with ASO, HDO, and Chol-HDO showed a dramatic increase in ASO concentration in the central nervous system compared to groups administered with the nucleic acid agent alone without co-administration of TGN-073.
　図11Bは、脳室内投与から7日後の海馬におけるMalat1 RNA発現レベルを示す。いずれのTGN-073共投与群においても、TGN-073を共投与せず各核酸剤を単独投与した群と比較して、中枢神経系における標的遺伝子抑制効果が著しく向上した。 Figure 11B shows the levels of Malat1 RNA expression in the hippocampus 7 days after intracerebroventricular administration. In all TGN-073 co-administration groups, the effect of target gene inhibition in the central nervous system was significantly improved compared to the groups in which each nucleic acid agent was administered alone without co-administration of TGN-073.
　以上の結果から1本鎖の核酸剤のみならず、2本鎖の核酸剤に対しても、TGN-073共投与によって中枢神経系への核酸送達量及び中枢神経系における標的遺伝子抑制効果を飛躍的に向上し得ることが示された。 These results show that co-administration of TGN-073 can dramatically improve the amount of nucleic acid delivered to the central nervous system and the effect of suppressing target genes in the central nervous system, not only for single-stranded nucleic acid agents but also for double-stranded nucleic acid agents.
＜実施例6：siRNA脳室内投与におけるTGN-073共投与の効果＞
（目的）
　siRNAをTGN-073と共に脳室内に投与する。中枢神経系における標的遺伝子抑制効果に対するTGN-073共投与の影響をin vivo実験により検証する。 Example 6: Effect of coadministration of TGN-073 with intracerebroventricular administration of siRNA
(the purpose)
siRNA will be co-administered intracerebroventricularly with TGN-073, and the effect of co-administration of TGN-073 on target gene silencing in the central nervous system will be examined in vivo.
（方法）
　本実施例で用いたsiRNAの塩基配列と化学修飾を以下の表5に示す。 (Method)
The base sequences and chemical modifications of the siRNAs used in this example are shown in Table 5 below.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
　本実施例で用いたsiRNAは、銅／亜鉛スーパーオキシドディスムターゼ1(Sod1) mRNAを標的とするsiRNAであり，表5に記載されるsiRNAについて、実施例1と同様の方法によりin vivo実験、及び標的遺伝子抑制効果の評価を行った。但し、本実施例ではマウス1匹当たりの核酸剤投与量は、7.0nmol(100μg)とした。 The siRNA used in this example was an siRNA targeting copper/zinc superoxide dismutase 1 (Sod1) mRNA, and for the siRNAs listed in Table 5, in vivo experiments and evaluation of the target gene suppression effect were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid agent administered per mouse was 7.0 nmol (100 μg).
（結果）
　図12は、脳室内投与から7日後の海馬におけるSod1 mRNA発現レベルを示す。TGN-073共投与群は、siRNA単独投与群と比較して、脳内標的遺伝子抑制効果の顕著な増強を示した。 (result)
12 shows the Sod1 mRNA expression level in the hippocampus 7 days after intracerebroventricular administration. The TGN-073 co-administration group showed a marked increase in the effect of suppressing the target gene in the brain compared to the siRNA alone administration group.
　以上の結果から、siRNAの脳室内投与において、TGN-073共投与によって中枢神経系における標的遺伝子抑制効果を飛躍的に向上し得ることが示された。 These results demonstrate that co-administration of TGN-073 with intracerebroventricular administration of siRNA can dramatically improve the effect of target gene suppression in the central nervous system.
＜実施例7：TGN-073誘導体＞
（目的）
　TGN-073の誘導体について効果を検討するために、Mapt遺伝子を標的とするASOを脳室内投与する際に、TGN-073の誘導体であるTGN-073-mesを共投与する。中枢神経系へのASO送達量及び中枢神経系における標的遺伝子抑制効果に対する影響をin vivo実験により検証する。 Example 7: TGN-073 derivatives
(the purpose)
To investigate the effect of TGN-073 derivatives, ASO targeting the Mapt gene will be co-administered with TGN-073-mes, a derivative of TGN-073, when administered intracerebroventricularly. The amount of ASO delivered to the central nervous system and its effect on target gene suppression in the central nervous system will be verified by in vivo experiments.
（方法）
　本実施例で用いたASOの塩基配列と化学修飾を以下の表6に示す。 (Method)
The base sequence and chemical modifications of the ASO used in this example are shown in Table 6 below.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
　本実施例で用いたASOは、マウスのMapt mRNAを標的とするLNA/DNAギャップマー型アンチセンス核酸であり、Mapt mRNAの一部に相補的な塩基配列を有し、5’末端の3個のLNAヌクレオシド、3’末端の3個のLNAヌクレオシド、及びそれらの間の10個のDNAヌクレオシドがホスホロチオエート結合で連結された構造を有する。 The ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets mouse Mapt mRNA, has a base sequence complementary to a portion of Mapt mRNA, and has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds.
　本実施例においてASOと共投与するTGN-073-mesは、上記式(I)で示すTGN-073Xの誘導体であり、その化学構造を以下の式(III)で示す。 TGN-073-mes, which is co-administered with ASO in this embodiment, is a derivative of TGN-073X shown in formula (I) above, and its chemical structure is shown in formula (III) below.
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000021
　本明細書では、上記式(III)で示す化合物及びその塩を「TGN-073-mes」と称することとする。なお、以下の実施例においてTGN-073-mesを投与する場合には、上記式(III)で示す化合物のナトリウム塩を使用した。上記式(III)で示す化合物のナトリウム塩を以下の式(VII)で示す。 In this specification, the compound represented by the above formula (III) and its salts are referred to as "TGN-073-mes." In the following examples, when TGN-073-mes was administered, the sodium salt of the compound represented by the above formula (III) was used. The sodium salt of the compound represented by the above formula (III) is represented by the following formula (VII).
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
　表6に記載されるASOについて、実施例1と同様の方法により核酸の調製、in vivo実験、並びに中枢神経系におけるASO濃度及び標的遺伝子抑制効果の評価を行った。但し、本実施例ではマウス1匹当たりの核酸剤投与量は、9.4nmol(50μg)とした。また、ASO/TGN-073-mes共投与群では、TGN-073-mesを0.125mg/匹の用量でASOと混合して脳室内投与を行った。 For the ASOs listed in Table 6, nucleic acid preparation, in vivo experiments, and evaluation of ASO concentration and target gene suppression effect in the central nervous system were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid administered per mouse was 9.4 nmol (50 μg). In addition, in the ASO/TGN-073-mes co-administration group, TGN-073-mes was mixed with ASO at a dose of 0.125 mg/mouse and administered intracerebroventricularly.
（結果）
　図13は、脳室内投与から7日後の海馬におけるMapt RNA発現レベルを示す。ASO/TGN-073共投与群は、ASO単独投与群と比較して、中枢神経系におけるASO濃度（図13A）及び標的遺伝子抑制効果（図13B）の著しい向上を示した。 (result)
Figure 13 shows the expression levels of Mapt RNA in the hippocampus 7 days after intracerebroventricular administration. The ASO/TGN-073 co-administration group showed a significant increase in ASO concentration in the central nervous system (Figure 13A) and in the target gene silencing effect (Figure 13B) compared with the ASO alone administration group.
＜実施例8：一本鎖PMOの脳室内投与に対するTGN-073共投与の効果＞
（目的）
　ジストロフィンpre-mRNAのエキソン23/イントロン23境界領域をエキソンスキッピングの標的とするアンチセンスオリゴヌクレオチドとして、ホスホロジアミデートモルホリノオリゴマー(以下、「PMO」とも記載する)をマウスの脳室内に投与する際に、TGN-073を共投与する。脳内でのエキソンスキッピング効果に対するTGN-073共投与の効果を評価する。 Example 8: Effect of coadministration of TGN-073 on intracerebroventricular administration of single-chain PMO
(the purpose)
Phosphorodiamidate morpholino oligomers (hereinafter also referred to as "PMO") are administered intracerebroventricularly to mice as antisense oligonucleotides targeting the exon 23/intron 23 boundary region of dystrophin pre-mRNA for exon skipping. TGN-073 is co-administered to evaluate the effect of TGN-073 co-administration on exon skipping in the brain.
（方法）
　本実施例で用いたPMOの塩基配列を以下の表7に示す。 (Method)
The base sequences of the PMOs used in this example are shown in Table 7 below.
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000023
　表7に記載されるPMOは、マウス・ジストロフィン遺伝子(dystrophin)のpre-mRNAのエキソン23/イントロン23を標的とする25merの一本鎖モルホリノ核酸である。このPMOは25merの全てがモルホリノ核酸で構成され、ヌクレオシド間結合は全てホスホロジアミデート結合である。このモルホリノ核酸は、マウスのDystrophin pre-mRNA(GenBankアクセッション番号:NC000086.7)の83803536～83803512位に相補的な塩基配列を有する。PMOは株式会社ジーンデザイン(Gene Design)(大阪、日本)によって委託合成された。 The PMO described in Table 7 is a 25-mer single-stranded morpholino nucleic acid that targets exon 23/intron 23 of mouse dystrophin pre-mRNA. The entire 25-mer of this PMO is composed of morpholino nucleic acid, and all internucleoside linkages are phosphorodiamidate bonds. This morpholino nucleic acid has a base sequence complementary to positions 83803536 to 83803512 of mouse dystrophin pre-mRNA (GenBank accession number: NC000086.7). The PMO was contracted for synthesis by Gene Design Co., Ltd. (Osaka, Japan).
　マウスは、体重20g前後の6週齢の雄のC57BL/6Jマウスを使用した。以降の実施例では、マウスを使用する実験は全てn=3で実施した。PMO2.5nmol（PMO単独投与群）又はPMO 2.5nmolにTGN-073 250μgを加えたもの（PMO/TGN-073共投与群）を、マウスの左脳室内に10μL量で投与した。 The mice used were 6-week-old male C57BL/6J mice weighing approximately 20 g. In the following examples, all experiments using mice were performed with n=3. 2.5 nmol of PMO (PMO alone administration group) or 2.5 nmol of PMO plus 250 μg of TGN-073 (PMO/TGN-073 co-administration group) were administered in a volume of 10 μL into the left ventricle of the mice.
　脳室内投与から1週間後に、マウスを解剖して海馬、線条体、及び皮質(後頭葉)を摘出した。続いて、mRNAを各組織からIsogen IIを使用して抽出した。抽出したtotal RNA（900ng）に対し、Qiagen One Step RT-PCR Kit（Qiagen社製）を用いてOne-Step RT-PCRを行った。キットに添付のプロトコルに従って、反応液を調製した。サーマルサイクラーはLifeECO（Bioer Technology社製）を用いた。用いたRT-PCRのプログラムは、42℃にて30分間で逆転写反応を行った後、95℃にて15分間、熱変性を行い、続いて、[94℃で30秒間；60℃で30秒間；72 ℃で60秒間]を1サイクルとして35サイクルPCR増幅反応を行い、72℃にて7分間、最後の伸長反応を行った。RT-PCRは、フォワード（Fw）プライマー（5'-ATCCAGCAGTCAGAAAGCAAA-3'、配列番号10）及びリバース（Rv）プライマーの塩基配列（5'-CAGCCATCCATTTCTGTAAGG-3'、配列番号11）を用いて実施した。 One week after intraventricular administration, the mice were dissected and the hippocampus, striatum, and cortex (occipital lobe) were removed. Then, mRNA was extracted from each tissue using Isogen II. One-Step RT-PCR was performed on the extracted total RNA (900 ng) using the Qiagen One Step RT-PCR Kit (Qiagen). The reaction mixture was prepared according to the protocol attached to the kit. The thermal cycler used was LifeECO (Bioer Technology). The RT-PCR program used was reverse transcription reaction at 42°C for 30 minutes, followed by thermal denaturation at 95°C for 15 minutes, followed by 35 cycles of PCR amplification reaction (94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds) as one cycle, and a final extension reaction at 72°C for 7 minutes. RT-PCR was performed using the forward (Fw) primer (5'-ATCCAGCAGTCAGAAAGCAAA-3', SEQ ID NO: 10) and reverse (Rv) primer base sequences (5'-CAGCCATCCATTTCTGTAAGG-3', SEQ ID NO: 11).
　上記RT-PCRの反応産物1μLをBioanalyzer2100（Agilent社製）を使用して、Agilent DNA1000キットを用いて解析した。エキソン23がスキップしたバンドのヌクレオチドモル数「A」と、エキソン23がスキップしなかったバンドのヌクレオチドモル数「B」を測定した。これら「A」及び「B」の測定値に基づき、以下の式：スキッピング効率（％）＝A/(A＋B)×100に従って、スキッピング効率を求めた。 1μL of the reaction product from the above RT-PCR was analyzed using a Bioanalyzer2100 (Agilent) with the Agilent DNA1000 kit. The number of nucleotide moles "A" of the band in which exon 23 was skipped and the number of nucleotide moles "B" of the band in which exon 23 was not skipped were measured. Based on the measured values of "A" and "B", the skipping efficiency was calculated according to the following formula: skipping efficiency (%) = A/(A+B) x 100.
（結果）
　図14は、脳室内投与から7日後の海馬（図14A）、線条体（図14B）、及び皮質（図14C）におけるExon 23スキッピング効率を示す。いずれの脳部位においても、PMO/TGN-073共投与群では、PMO単独投与群と比較して、スキッピング効率が顕著に上昇した。 (result)
Figure 14 shows the exon 23 skipping efficiency in the hippocampus (Figure 14A), striatum (Figure 14B), and cortex (Figure 14C) 7 days after intracerebroventricular administration. In all brain regions, the PMO/TGN-073 co-administration group showed a significant increase in the skipping efficiency compared to the PMO alone administration group.
＜実施例9：PMOを含む二本鎖核酸複合体の脳室内投与に対するTGN-073共投与の効果＞
（目的）
　ジストロフィンpre-mRNAのエキソン23/イントロン23境界領域をエキソンスキッピングの標的とするPMOとその相補配列を含みコレステロールを結合した相補鎖とからなる二本鎖核酸複合体（以下、「Chol-HDO(PMO)」という）をマウスの脳室内に投与する際に、TGN-073を共投与する。脳内でのエキソンスキッピング効果に対するTGN-073共投与の効果を評価する。 Example 9: Effect of coadministration of TGN-073 on intracerebroventricular administration of a double-stranded nucleic acid complex containing a PMO
(the purpose)
A double-stranded nucleic acid complex consisting of a PMO targeting the exon 23/intron 23 boundary region of dystrophin pre-mRNA for exon skipping and its complementary sequence bound to cholesterol (hereinafter referred to as "Chol-HDO (PMO)") is administered intracerebroventricularly to mice, and TGN-073 is co-administered. The effect of TGN-073 co-administration on exon skipping in the brain is evaluated.
（方法）
　本実施例で用いたPMO及び相補鎖の塩基配列を以下の表8及び図15に示す。 (Method)
The base sequences of the PMO and complementary strand used in this example are shown in Table 8 below and FIG.
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-T000024
　表8に記載されるChol-HDO(PMO)は、PMO及びChol-cRNAにより構成される。PMOは実施例8と同一の一本鎖モルホリノ核酸である。Chol-cRNAは、上記PMOに相補的な塩基配列を有し、5'側にコレステロールが結合し、5'末端の3個の2'-O-メチル修飾RNAヌクレオシド、3'末端の3個の2'-O-メチル修飾RNAヌクレオシド、及びそれらの間の19個のDNAヌクレオシドがホスホロチオエート結合及びホスホジエステル結合で連結された構造を有する。PMO及びChol-cRNAは株式会社ジーンデザイン(Gene Design)(大阪、日本)によって委託合成され、両者をアニールさせることにより、二本鎖のChol-HDOを調製した。具体的には、PMOとChol-cRNAとを等モル量で混合し、溶液を95℃で5分間加熱し、その後37℃に冷却して1時間保持し、これにより核酸鎖をアニールして上記の二本鎖核酸剤を調製した。 Chol-HDO (PMO) described in Table 8 is composed of PMO and Chol-cRNA. PMO is the same single-stranded morpholino nucleic acid as in Example 8. Chol-cRNA has a base sequence complementary to the PMO, cholesterol is bound to the 5' side, and has a structure in which three 2'-O-methyl modified RNA nucleosides at the 5' end, three 2'-O-methyl modified RNA nucleosides at the 3' end, and 19 DNA nucleosides between them are linked by phosphorothioate bonds and phosphodiester bonds. PMO and Chol-cRNA were synthesized by Gene Design Co., Ltd. (Osaka, Japan), and double-stranded Chol-HDO was prepared by annealing the two. Specifically, equimolar amounts of PMO and Chol-cRNA were mixed, and the solution was heated to 95°C for 5 minutes, then cooled to 37°C and held for 1 hour, thereby annealing the nucleic acid strands and preparing the above-mentioned double-stranded nucleic acid agent.
　実施例8の方法に準じて、Chol-HDO(PMO)2.5nmol（Chol-HDO(PMO)単独投与群）又はChol-HDO(PMO)2.5nmolにTGN-073 250μgを加えたもの（Chol-HDO(PMO)/TGN-073共投与群）を、マウスの左脳室内に10μL量で投与して、スキッピング効率を評価した。
（結果）
　図16は、脳室内投与から7日後の海馬（図16A）、線条体（図16B）、及び皮質（図16C）におけるExon 23スキッピング効率を示す。いずれの脳部位においても、Chol-HDO(PMO)/TGN-073共投与群では、Cho-HDO(PMO)単独投与群と比較して、スキッピング効率が顕著に上昇した。 According to the method of Example 8, 2.5 nmol of Chol-HDO(PMO) (Chol-HDO(PMO) alone administration group) or 2.5 nmol of Chol-HDO(PMO) plus 250 μg of TGN-073 (Chol-HDO(PMO)/TGN-073 co-administration group) was administered into the left ventricle of mice in an amount of 10 μL to evaluate skipping efficiency.
(result)
Figure 16 shows the Exon 23 skipping efficiency in the hippocampus (Figure 16A), striatum (Figure 16B), and cortex (Figure 16C) 7 days after intracerebroventricular administration. In all brain regions, the Chol-HDO(PMO)/TGN-073 co-administration group showed a significant increase in the skipping efficiency compared to the Cho-HDO(PMO) alone administration group.
＜実施例10：核酸鎖の脊髄送達に対するTGN-073共投与の効果＞
（目的）
　実施例1で用いたMalat1標的ASOをTGN-073と共に脳室内に投与する。脊髄における標的遺伝子抑制効果及び核酸送達量に対するTGN-073共投与の影響をin vivo実験により検証する。 Example 10: Effect of coadministration of TGN-073 on spinal delivery of nucleic acid chains
(the purpose)
The Malat1-targeting ASO used in Example 1 is administered intracerebroventricularly together with TGN-073. The effect of coadministration of TGN-073 on the target gene suppression effect and the amount of nucleic acid delivered in the spinal cord is verified by in vivo experiments.
（方法）
　本実施例で用いたASOは、実施例1に記載のMalat1標的ASOとした。具体的には、表1に記載されるASOについて、実施例1と同様の方法により核酸の調製、in vivo実験、並びに標的遺伝子抑制効果の評価を行った。但し、本実施例ではマウス1匹当たりの核酸剤投与量は1.7nmol(12.5μg)とし、核酸送達量及び標的遺伝子抑制効果を腰髄部において評価した。 (Method)
The ASO used in this example was the Malat1-targeting ASO described in Example 1. Specifically, for the ASOs listed in Table 1, nucleic acid preparation, in vivo experiments, and evaluation of the target gene suppression effect were performed in the same manner as in Example 1. However, in this example, the amount of nucleic acid agent administered per mouse was 1.7 nmol (12.5 μg), and the amount of nucleic acid delivered and the target gene suppression effect were evaluated in the lumbar spinal cord.
（結果）
　図17Aは、脳室内投与から7日後の腰髄部におけるASO濃度を示す。ASO/TGN-073共投与群は、ASO単独投与群と比較して、腰髄部におけるASO濃度の飛躍的な増大を示した。 (result)
Figure 17A shows the ASO concentration in the lumbar spinal cord 7 days after intracerebroventricular administration. The ASO/TGN-073 co-administration group showed a dramatic increase in ASO concentration in the lumbar spinal cord compared to the ASO alone administration group.
　図17Bは、脳室内投与から7日後の腰髄部におけるMalat1 RNA発現レベルを示す。ASO/TGN-073共投与群では、ASO単独投与群と比較して、腰髄部における標的遺伝子抑制効果が著しく向上した。 Figure 17B shows the Malat1 RNA expression levels in the lumbar spinal cord 7 days after intracerebroventricular administration. The ASO/TGN-073 co-administration group showed significantly improved target gene suppression effects in the lumbar spinal cord compared to the ASO alone administration group.
　以上の結果から脳組織及び脊髄を含む中枢神経系への核酸送達効率、並びに脳組織及び脊髄を含む中枢神経系における標的遺伝子抑制効果が、TGN-073共投与によって飛躍的に向上され得ることが示された。 These results demonstrate that the efficiency of nucleic acid delivery to the central nervous system, including brain tissue and the spinal cord, as well as the effect of target gene suppression in the central nervous system, including brain tissue and the spinal cord, can be dramatically improved by coadministration of TGN-073.
＜実施例11：ASO髄腔内投与におけるTGN-073共投与の効果＞
（目的）
　TGN-073共投与の効果を、非脳室内投与である髄腔内投与について検証する。具体的には、実施例3で用いたMapt標的ASOを髄腔内投与し、脊髄組織及び海馬への核酸送達量に対するTGN-073共投与の影響をin vivo実験により検証する。 Example 11: Effect of TGN-073 co-administration with intrathecal ASO administration
(the purpose)
The effect of TGN-073 co-administration will be verified by intrathecal administration, which is non-intracerebroventricular administration. Specifically, the Mapt-targeted ASO used in Example 3 will be administered intrathecally, and the effect of TGN-073 co-administration on the amount of nucleic acid delivered to the spinal cord tissue and hippocampus will be verified by in vivo experiments.
（方法）
　本実施例では、実施例3で用いたASO（表２に記載のASO）と同一の構成を有するASOを使用し、実施例1と同様の方法により核酸の調製を行った。 (Method)
In this example, an ASO having the same configuration as the ASO used in Example 3 (ASO listed in Table 2) was used, and nucleic acid was prepared in the same manner as in Example 1.
　ASOの髄腔内投与及びASO濃度の測定は、以下の方法で行った。8週齢のSlc:SDラット（雄）の脊髄くも膜下にカテーテルを挿入し、9週齢時にラット1匹当たり76nmol（400μg）のASO及び／又はTGN-073（1.5g/匹）を髄腔内投与した。以下、ASOのみを髄腔内投与したラットを「ASO単独投与群」と呼び、ASO及びTGN-073を共に髄腔内投与したラットを「ASO/TGN-073共投与群」と呼ぶ。髄腔内投与から7日後のラットから脊髄部及び左海馬を摘出し、実施例1と同様の方法により各組織内のASO濃度を評価した。 　Intrathecal administration of ASO and measurement of ASO concentration were performed as follows. A catheter was inserted into the spinal subarachnoid space of 8-week-old Slc:SD rats (male), and 76 nmol (400 μg) of ASO and/or TGN-073 (1.5 g/rat) were administered intrathecally per rat at 9 weeks of age. Hereinafter, rats administered only ASO intrathecally are referred to as the "ASO alone administration group," and rats administered both ASO and TGN-073 intrathecally are referred to as the "ASO/TGN-073 co-administration group." Seven days after intrathecal administration, the spinal cord and left hippocampus were removed from the rats, and the ASO concentration in each tissue was evaluated using the same method as in Example 1.
(結果）
　図18は、髄腔内投与から7日後の脊髄組織（図18A）及び左海馬（図18B）におけるASO濃度を示す。ASO/TGN-073共投与群では、ASO単独投与群と比較して、脊髄部及び左海馬におけるASO濃度の飛躍的な増大を示した。 (result)
Figure 18 shows ASO concentrations in the spinal cord tissue (Figure 18A) and left hippocampus (Figure 18B) 7 days after intrathecal administration. The ASO/TGN-073 co-administration group showed a dramatic increase in ASO concentrations in the spinal cord and left hippocampus compared to the ASO alone administration group.
　以上の結果から脳室内投与の場合だけでなく髄腔内投与の場合でも、中枢神経系への核酸送達効率がTGN-073共投与によって飛躍的に向上され得ることが示された。 These results demonstrate that the efficiency of nucleic acid delivery to the central nervous system can be dramatically improved by coadministration of TGN-073, not only in the case of intracerebroventricular administration but also in the case of intrathecal administration.
＜実施例12：VHH抗体の脳室内投与におけるTGN-073共投与の効果＞
（目的）
　核酸医薬以外の薬物モダリティに対するTGN-073共投与の効果を検証するために、VHH抗体の脳室内投与におけるTGN-073共投与の影響をマウスin vivo実験により検証する。 Example 12: Effect of co-administration of TGN-073 with intracerebroventricular administration of VHH antibody
(the purpose)
To verify the effect of TGN-073 co-administration on drug modalities other than nucleic acid drugs, we will verify the effect of TGN-073 co-administration on intracerebroventricular administration of VHH antibodies through in vivo experiments in mice.
（方法）
　本実施例では、蛍光標識ラベルされたラクダ化シングルドメイン抗体（VHH抗体；Alpaca anti-Rabbit IgG Nano (VHH) Recombinant Secondary Antibody, Alexa Fluor（商標）647, invitrogen, #SA5-10327)を29pmol/匹の投与量にて、単独又はTGN-073（250μg/匹）と組み合わせて、8週齢のICRマウス（雌）の脳室内に投与した。以下、VHH抗体のみを脳室内投与したマウスを「VHH抗体単独投与群」と呼び、VHH抗体及びTGN-073を共に脳室内投与したマウスを「VHH抗体/TGN-073共投与群」と呼ぶ。脳室内投与から3時間後のマウスから左海馬、左後頭葉皮質、及び左基底核の脳組織サンプルを採取し、蛍光プレートリーダー(TECAN社, M1000Pro)を用いて脳重量当たりのAlexa647蛍光強度を測定することにより、各脳組織におけるVHH抗体の濃度を評価した。 (Method)
In this example, a fluorescently labeled camelized single domain antibody (VHH antibody; Alpaca anti-Rabbit IgG Nano (VHH) Recombinant Secondary Antibody, Alexa Fluor™ 647, Invitrogen, #SA5-10327) was administered intracerebroventricularly at a dose of 29 pmol/mouse, either alone or in combination with TGN-073 (250 μg/mouse) to 8-week-old ICR mice (female). Hereinafter, mice that received only VHH antibody intracerebroventricularly are referred to as the "VHH antibody alone administration group," and mice that received both VHH antibody and TGN-073 intracerebroventricularly are referred to as the "VHH antibody/TGN-073 co-administration group." Brain tissue samples from the left hippocampus, left occipital cortex, and left basal ganglia were collected from the mice 3 hours after intraventricular administration, and the VHH antibody concentration in each brain tissue was evaluated by measuring the Alexa647 fluorescence intensity per brain weight using a fluorescent plate reader (TECAN, M1000Pro).
(結果）
　図19は、脳室内投与後の左海馬（図19A）、左後頭葉皮質（図19B）、及び左基底核（図19C）の各部におけるVHH抗体濃度を示す。VHH抗体/TGN-073共投与群は、VHH抗体単独投与群と比較して、中枢神経系におけるVHH抗体濃度の飛躍的な増大を示した。 (result)
Figure 19 shows the VHH antibody concentrations in the left hippocampus (Figure 19A), left occipital cortex (Figure 19B), and left basal ganglia (Figure 19C) after intraventricular administration. The VHH antibody/TGN-073 co-administration group showed a dramatic increase in VHH antibody concentration in the central nervous system compared to the VHH antibody alone administration group.
　以上の結果から、核酸医薬のみならずVHH抗体についても、脳室内投与時の中枢神経内への送達効率がTGN-073共投与によって飛躍的に向上され得ることが示された。 These results show that the efficiency of delivery to the central nervous system when administered intracerebroventricularly, not only for nucleic acid drugs but also for VHH antibodies, can be dramatically improved by coadministration with TGN-073.
＜実施例13：IgG抗体の脳室内投与におけるTGN-073共投与の効果＞
（目的）
　IgG抗体の送達についてもTGN-073共投与の有効性をマウスin vivo実験により検証する。 Example 13: Effect of coadministration of TGN-073 with intracerebroventricular administration of IgG antibody
(the purpose)
The efficacy of co-administration of TGN-073 for delivery of IgG antibodies will also be verified through in vivo experiments in mice.
（方法）
　本実施例ではAlexa647蛍光標識ラベルされたIgG抗体（Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor（商標） 647 (Invitrogen, #A-21245)を10μg/匹の投与量にて、単独又はTGN-073（250μg/匹）と組み合わせて、8週齢のICRマウス（雌）の左側脳室内に投与した。以下、IgG抗体のみを脳室内投与したマウスを「IgG抗体単独投与群」と呼び、IgG抗体及びTGN-073を共に脳室内投与したマウスを「IgG/TGN-073共投与群」と呼ぶ。脳室内投与から3時間後のマウスから左海馬の脳組織サンプルを採取し、蛍光プレートリーダー(TECAN社, M1000Pro)を用いて脳重量当たりのAlexa647蛍光強度を測定することにより、IgG抗体濃度を評価した。 (Method)
In this example, Alexa647 fluorescently labeled IgG antibody (Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Invitrogen, #A-21245) was administered at a dose of 10 μg/mouse, either alone or in combination with TGN-073 (250 μg/mouse), into the left lateral ventricle of 8-week-old ICR mice (female). Hereinafter, mice that received only IgG antibody intracerebroventricular administration will be referred to as the “IgG antibody alone administration group”, and mice that received both IgG antibody and TGN-073 intracerebroventricular administration will be referred to as the “IgG/TGN-073 co-administration group”. Brain tissue samples from the left hippocampus were collected from the mice 3 hours after intracerebroventricular administration, and the IgG antibody concentration was evaluated by measuring the Alexa647 fluorescence intensity per brain weight using a fluorescent plate reader (TECAN, M1000Pro).
(結果）
　図20は、脳室内投与後の左海馬におけるIgG抗体濃度を示す。IgG/TGN-073共投与群は、IgG抗体単独投与群と比較して、中枢神経系におけるIgG抗体濃度の飛躍的な増大を示した。 (result)
Figure 20 shows the IgG antibody concentration in the left hippocampus after intracerebroventricular administration. The IgG/TGN-073 co-administration group showed a dramatic increase in IgG antibody concentration in the central nervous system compared to the IgG antibody alone administration group.
　以上の結果から、IgG抗体についてもTGN-073共投与によって脳室内投与時の中枢神経内への送達効率が飛躍的に向上され得ることが示された。 These results show that co-administration of TGN-073 can also dramatically improve the efficiency of delivery of IgG antibodies into the central nervous system when administered intracerebroventricularly.
＜実施例14：ウイルスベクターの脳室内投与におけるTGN-073共投与の効果＞
（目的）
　AAV遺伝子治療薬の脳室内投与に対するTGN-073共投与の影響をマウスin vivo実験により検証する。 Example 14: Effect of coadministration of TGN-073 in intracerebroventricular administration of viral vectors
(the purpose)
The effect of coadministration of TGN-073 with intracerebroventricular administration of AAV gene therapy drugs will be verified through in vivo experiments in mice.
（方法）
　本実施例ではアデノ随伴ウイルス（AAV）ベクターとしてGFP発現AAV9(AAV9-CMV-hrGFP)を26×109vg/匹の投与量にて、単独又はTGN-073（250μg/匹）と組み合わせて、7週齢のICRマウス（雌）の左側脳室内に投与した。以下、AAVベクターのみを脳室内投与したマウスを「AAV単独投与群」と呼び、AAVベクター及びTGN-073を共に脳室内投与したマウスを「AAV/TGN-073共投与群」と呼ぶ。脳室内投与から3週間後、マウスを4％パラホルムアルデヒドで灌流固定した。PBSに溶解した10％～30％のショ糖溶液で抗凍結処理をした後、ミクロトームを用いて厚さ40μmの切片を作製した。蛍光顕微鏡（VHS-7000、キーエンス）を用いてGFP蛍光（海馬レベル冠状断像）を撮像した。 (Method)
In this example, GFP-expressing AAV9 (AAV9-CMV-hrGFP) was administered as an adeno-associated virus (AAV) vector at a dose of 26×10 9 vg/mouse, either alone or in combination with TGN-073 (250 μg/mouse), into the left lateral ventricle of 7-week-old ICR mice (female). Hereinafter, mice that received only the AAV vector intraventricularly will be referred to as the "AAV alone administration group," and mice that received both the AAV vector and TGN-073 intraventricularly will be referred to as the "AAV/TGN-073 co-administration group." Three weeks after intraventricular administration, the mice were perfused and fixed with 4% paraformaldehyde. After cryoprotection with a 10% to 30% sucrose solution dissolved in PBS, 40 μm-thick sections were prepared using a microtome. GFP fluorescence (hippocampal level coronal section) was captured using a fluorescence microscope (VHS-7000, Keyence).
(結果）
　図21は、脳室内投与から3週間後の海馬レベルでの冠状断像を示す。AAV/TGN-073共投与群は、AAV単独投与群と比較して、中枢神経系において大幅に広い範囲でGFP発現が観察され、発現量の増大をも示した。 (result)
Figure 21 shows coronal sections at the hippocampal level 3 weeks after intraventricular injection. The AAV/TGN-073 co-injection group showed significantly more extensive GFP expression in the central nervous system than the AAV mono-injection group, and also showed increased expression.
　以上の結果から、ウイルスベクターの脳室内投与についても、TGN-073共投与によって中枢神経内への送達効率が飛躍的に向上され得ることが示された。
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 These results indicate that co-administration of TGN-073 can dramatically improve the efficiency of intraventricular administration of viral vectors into the central nervous system.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (73)

  1. 　薬剤の中枢神経系及び／又は末梢神経系への送達を促進するための送達促進剤であって、アクアポリン4機能促進剤又はアクアポリン4機能修飾剤からなる、前記送達促進剤。 A delivery enhancer for enhancing the delivery of a drug to the central nervous system and/or peripheral nervous system, the delivery enhancer being composed of an aquaporin 4 function enhancer or an aquaporin 4 function modifier.
  2. 　前記アクアポリン4機能促進剤又はアクアポリン4機能修飾剤が、下記式（I）：
    Figure JPOXMLDOC01-appb-C000001
     で示される化合物若しくはその誘導体、又はそのいずれかの塩である、請求項１に記載の送達促進剤。     The aquaporin 4 function promoter or aquaporin 4 function modifier is represented by the following formula (I):
    Figure JPOXMLDOC01-appb-C000001
     The delivery enhancer of claim 1 , which is a compound represented by the following formula (I):
  3. 　前記誘導体が、下記式(III)：
    Figure JPOXMLDOC01-appb-C000002
     で示される、請求項２に記載の送達促進剤。     The derivative has the following formula (III):
    Figure JPOXMLDOC01-appb-C000002
     The delivery enhancer of claim 2 , wherein
  4. 　前記化合物又は前記誘導体がキャリア分子と結合している、請求項２に記載の送達促進剤。 The delivery enhancer of claim 2, wherein the compound or the derivative is conjugated to a carrier molecule.
  5. 　前記キャリア分子が、1kDa以上のタンパク質、ポリマー、脂質分子、又は造影剤である、請求項４に記載の送達促進剤。 The delivery enhancer according to claim 4, wherein the carrier molecule is a protein of 1 kDa or more, a polymer, a lipid molecule, or a contrast agent.
  6. 　前記タンパク質が、アルブミン、リポタンパク質、又は抗体若しくは抗体断片である、請求項５に記載の送達促進剤。 The delivery enhancer of claim 5, wherein the protein is albumin, a lipoprotein, or an antibody or antibody fragment.
  7. 　前記ポリマーが、ポリエチレングリコール(PEG)又はPEG graftedポリマーを含む、請求項５に記載の送達促進剤。 The delivery enhancer of claim 5, wherein the polymer comprises polyethylene glycol (PEG) or a PEG-grafted polymer.
  8. 　前記ポリマー又は前記脂質分子が、マイクロバブル、リポソーム、又はミセルを形成する、請求項５に記載の送達促進剤。 The delivery enhancer of claim 5, wherein the polymer or lipid molecules form microbubbles, liposomes, or micelles.
  9. 　前記薬剤が、髄腔内投与、経鼻投与、静脈内投与、皮下投与、腹腔内投与、経口投与、吸入投与、又は筋肉内投与される、請求項１に記載の送達促進剤。 The delivery enhancer of claim 1, wherein the agent is administered intrathecally, intranasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly.
  10. 　前記髄腔内投与が脳室内投与、後頭窩穿刺、又は腰椎穿刺である、請求項９に記載の送達促進剤。 The delivery enhancer according to claim 9, wherein the intrathecal administration is intraventricular administration, posterior fossa puncture, or lumbar puncture.
  11. 　前記薬剤が、シャント、留置カテーテル、又は皮下ポートを用いて投与される、請求項１に記載の送達促進剤。 The delivery enhancer of claim 1, wherein the agent is administered using a shunt, an indwelling catheter, or a subcutaneous port.
  12. 　前記送達促進剤が、前記薬剤と同時に投与される、又は前記薬剤よりも先に若しくは後に投与される、請求項１に記載の送達促進剤。 The delivery enhancer of claim 1, wherein the delivery enhancer is administered simultaneously with the drug, or administered prior to or after the drug.
  13. 　前記送達促進剤が、髄腔内投与、経鼻投与、静脈内投与、皮下投与、腹腔内投与、経口投与、吸入投与、又は筋肉内投与される、請求項１に記載の送達促進剤。 The delivery enhancer of claim 1, wherein the delivery enhancer is administered intrathecally, nasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly.
  14. 　中枢神経系疾患又は末梢神経系疾患の治療のための医薬組成物であって、治療有効量の薬剤、及び請求項１に記載の送達促進剤を含む、前記医薬組成物。 A pharmaceutical composition for treating a central nervous system disease or a peripheral nervous system disease, comprising a therapeutically effective amount of a drug and a delivery enhancer according to claim 1.
  15. 　髄腔内投与、経鼻投与、静脈内投与、皮下投与、腹腔内投与、経口投与、吸入投与、又は筋肉内投与される、請求項１４に記載の医薬組成物。 The pharmaceutical composition of claim 14, which is administered intrathecally, nasally, intravenously, subcutaneously, intraperitoneally, orally, by inhalation, or intramuscularly.
  16. 　前記髄腔内投与が脳室内投与、後頭窩穿刺、又は腰椎穿刺である、請求項１５に記載の医薬組成物。 The pharmaceutical composition according to claim 15, wherein the intrathecal administration is intraventricular administration, posterior fossa puncture, or lumbar puncture.
  17. 　シャント、留置カテーテル、又は皮下ポートを用いて投与される、請求項１４に記載の医薬組成物。 The pharmaceutical composition of claim 14, which is administered using a shunt, an indwelling catheter, or a subcutaneous port.
  18. 　前記薬剤が、核酸医薬、ペプチド、低分子化合物、ウイルスベクター、細胞医薬、ナノ粒子、リポソーム、ミセル、又はエクソソームである、請求項１～１３のいずれか一項に記載の送達促進剤、又は請求項１４～１７のいずれか一項に記載の医薬組成物。 The delivery enhancer according to any one of claims 1 to 13, or the pharmaceutical composition according to any one of claims 14 to 17, wherein the drug is a nucleic acid drug, a peptide, a low molecular weight compound, a viral vector, a cell drug, a nanoparticle, a liposome, a micelle, or an exosome.
  19. 　前記ペプチドが、天然ペプチド又は非天然ペプチドである、請求項１８に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 18, wherein the peptide is a natural peptide or a non-natural peptide.
  20. 　前記ペプチドが、環状ペプチドである、請求項１８に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 18, wherein the peptide is a cyclic peptide.
  21. 　前記ペプチドが、タンパク製剤である、請求項１８に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 18, wherein the peptide is a protein preparation.
  22. 　前記ペプチドが抗体又は抗体断片である、請求項１８に記載の脳送達促進剤又は医薬組成物。 The brain delivery enhancer or pharmaceutical composition according to claim 18, wherein the peptide is an antibody or an antibody fragment.
  23. 　前記抗体が、全長抗体である、請求項２２に記載の脳送達促進剤又は医薬組成物。 The brain delivery enhancer or pharmaceutical composition according to claim 22, wherein the antibody is a full-length antibody.
  24. 　前記抗体断片が、Fab、Fab'、scFv、又はVHHである、請求項２２に記載の脳送達促進剤又は医薬組成物。 The brain delivery enhancer or pharmaceutical composition according to claim 22, wherein the antibody fragment is a Fab, a Fab', a scFv, or a VHH.
  25. 　前記核酸医薬が、アンチセンス核酸、ヘテロ核酸、siRNA、shRNA、miRNA、mRNA、lncRNA、プラスミドDNA、アプタマー、デコイ、bait核酸、リボザイム、及び核酸ベクターからなる群から選択される、請求項１８に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 18, wherein the nucleic acid drug is selected from the group consisting of antisense nucleic acid, heteronucleic acid, siRNA, shRNA, miRNA, mRNA, lncRNA, plasmid DNA, aptamer, decoy, bait nucleic acid, ribozyme, and nucleic acid vector.
  26. 　前記核酸医薬が、標的遺伝子又はその転写産物の少なくとも一部にハイブリダイズすることができ、かつ前記標的遺伝子又はその転写産物に対してアンチセンス効果を有する核酸分子を含む、請求項１８に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 18, wherein the nucleic acid drug comprises a nucleic acid molecule capable of hybridizing to at least a portion of a target gene or its transcription product and having an antisense effect on the target gene or its transcription product.
  27. 　前記核酸分子が、12～30塩基長である、請求項２６に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 26, wherein the nucleic acid molecule is 12 to 30 bases in length.
  28. 　前記核酸分子が、デオキシリボヌクレオシド、2'-修飾ヌクレオシド、5'-修飾ヌクレオシド、及び架橋ヌクレオシドからなる群から選択されるいずれか１以上を含む、請求項２６に記載の送達促進剤又は医薬組成物。 27. The delivery enhancer or pharmaceutical composition of claim 26, wherein the nucleic acid molecule comprises one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides.
  29. 　前記核酸分子が、ミックスマーである、請求項２６に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 26, wherein the nucleic acid molecule is a mixmer.
  30. 　前記核酸分子がモルホリノ核酸を含む、又は前記核酸分子の核酸の全部がモルホリノ核酸からなる、請求項２６に記載の送達促進剤又は医薬組成物。 27. The delivery enhancer or pharmaceutical composition of claim 26, wherein the nucleic acid molecule comprises a morpholino nucleic acid, or the entire nucleic acid of the nucleic acid molecule consists of a morpholino nucleic acid.
  31. 　前記核酸分子が、ギャップマーである、請求項２６に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 26, wherein the nucleic acid molecule is a gapmer.
  32. 　前記核酸分子が、
    　(1)少なくとも3個の連続するデオキシリボヌクレオシドを含む中央領域、
    　(2)前記中央領域の5'末端側に配置された、非天然ヌクレオシドを含む5’ウイング領域、及び
    　(3)前記中央領域の3'末端側に配置された、非天然ヌクレオシドを含む3’ウイング領域を含む、請求項３１に記載の送達促進剤又は医薬組成物。     The nucleic acid molecule comprises:
    (1) a central region containing at least three consecutive deoxyribonucleosides;
    The delivery enhancer or pharmaceutical composition of claim 31 , comprising: (2) a 5' wing region comprising an unnatural nucleoside located on the 5' end of the central region; and (3) a 3' wing region comprising an unnatural nucleoside located on the 3' end of the central region.
  33. 　前記5'ウイング領域において前記中央領域に隣接する末端塩基位置、前記中央領域の5'側から2番目の塩基位置、及び／又は前記中央領域の5'側から8番目の塩基位置に、2'-修飾ヌクレオシドを含む、請求項３２に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 32, comprising a 2'-modified nucleoside at the terminal base position adjacent to the central region in the 5' wing region, the second base position from the 5' side of the central region, and/or the eighth base position from the 5' side of the central region.
  34. 　前記核酸分子において、前記5'ウイング領域及び前記3'ウイング領域が、架橋ヌクレオシド及び／又は2'-修飾ヌクレオシドを含む、請求項３２に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 32, wherein the 5' wing region and the 3' wing region of the nucleic acid molecule comprise bridged nucleosides and/or 2'-modified nucleosides.
  35. 　前記架橋ヌクレオシドが、LNAヌクレオシド、2',4'-BNANCヌクレオシド、cEt BNAヌクレオシド、ENAヌクレオシド、AmNAヌクレオシド、GuNAヌクレオシド、scpBNAヌクレオシド、scpBNA2ヌクレオシド、及びBANA3ヌクレオシドからなる群から選択される、請求項３４に記載の送達促進剤又は医薬組成物。 35. The delivery-enhancing agent or pharmaceutical composition of claim 34, wherein the bridged nucleoside is selected from the group consisting of an LNA nucleoside, a 2',4'-BNA NC nucleoside, a cEt BNA nucleoside, an ENA nucleoside, an AmNA nucleoside, a GuNA nucleoside, a scpBNA nucleoside, a scpBNA2 nucleoside, and a BANA3 nucleoside.
  36. 　前記2'-修飾ヌクレオシドが、2'-O-メチル修飾ヌクレオシド、2'-O-メトキシエチル修飾ヌクレオシド、2'-O-[2-(N-メチルカルバモイル)エチル]修飾ヌクレオシド、又は2'-フルオロ修飾ヌクレオシドである、請求項３４に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 34, wherein the 2'-modified nucleoside is a 2'-O-methyl modified nucleoside, a 2'-O-methoxyethyl modified nucleoside, a 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleoside, or a 2'-fluoro modified nucleoside.
  37. 　前記核酸分子のヌクレオシド間結合の全部又は一部が修飾ヌクレオシド間結合である、請求項２６に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 26, wherein all or a portion of the internucleoside linkages of the nucleic acid molecule are modified internucleoside linkages.
  38. 　前記修飾ヌクレオシド間結合がホスホロチオエート結合である、請求項３７に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 37, wherein the modified internucleoside linkage is a phosphorothioate linkage.
  39. 　前記核酸分子が、修飾核酸塩基を含む、請求項２６に記載の送達促進剤又は医薬組成物。 27. The delivery enhancer or pharmaceutical composition of claim 26, wherein the nucleic acid molecule comprises a modified nucleic acid base.
  40. 　前記核酸分子が、前記標的遺伝子又はその転写産物に対してステリックブロッキング、スプライシング制御、発現低下、発現上昇、及び／又は塩基編集を誘導することができる、請求項２６に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 26, wherein the nucleic acid molecule is capable of inducing steric blocking, splicing control, expression downregulation, expression upregulation, and/or base editing of the target gene or its transcription product.
  41. 　前記核酸医薬が、前記核酸分子からなる第1核酸鎖と、前記第1核酸鎖に相補的な塩基配列を含む第2核酸鎖とを含む二本鎖核酸複合体からなる、請求項２６に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 26, wherein the nucleic acid drug is a double-stranded nucleic acid complex comprising a first nucleic acid strand comprising the nucleic acid molecule and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand.
  42. 　前記第2核酸鎖が少なくとも8塩基長である、請求項４１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 41, wherein the second nucleic acid strand is at least 8 bases in length.
  43. 　前記第2核酸鎖が、デオキシリボヌクレオシド、2'-修飾ヌクレオシド、5'-修飾ヌクレオシド、及び架橋ヌクレオシドからなる群から選択されるいずれか１以上を含む、請求項４１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 41, wherein the second nucleic acid strand comprises one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides.
  44. 　前記第2核酸鎖が、前記第1核酸鎖に対して、非相補的塩基、及び／又は1塩基以上の、挿入配列及び／又は欠失を含む、請求項４１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 41, wherein the second nucleic acid strand contains non-complementary bases and/or one or more inserted sequences and/or deletions relative to the first nucleic acid strand.
  45. 　前記第2核酸鎖が、前記非相補的塩基を1～3個含む、請求項４４に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 44, wherein the second nucleic acid strand contains 1 to 3 non-complementary bases.
  46. 　前記挿入配列が1～8塩基からなる、請求項４４に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 44, wherein the insertion sequence consists of 1 to 8 bases.
  47. 　前記欠失が連続する1～4塩基からなる、請求項４４に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 44, wherein the deletion consists of 1 to 4 consecutive bases.
  48. 　前記第2核酸鎖が、前記第1核酸鎖に相補的な塩基配列からなる領域の5'末端側及び/又は3'末端側に位置する少なくとも1つのオーバーハング領域を含む、請求項４１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 41, wherein the second nucleic acid strand includes at least one overhang region located on the 5'-end and/or 3'-end of a region consisting of a base sequence complementary to the first nucleic acid strand.
  49. 　前記オーバーハング領域が1～30塩基長である、請求項４８に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 48, wherein the overhang region is 1 to 30 bases in length.
  50. 　前記第2核酸鎖が、機能性部分と結合している、請求項４１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 41, wherein the second nucleic acid strand is linked to a functional moiety.
  51. 　前記機能性部分が、脂質又はペプチドである、請求項５０に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 50, wherein the functional moiety is a lipid or a peptide.
  52. 　前記ペプチドが抗体である、請求項５１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 51, wherein the peptide is an antibody.
  53. 　前記脂質が、コレステロール若しくはその類縁体、トコフェロール若しくはその類縁体、葉酸、ホスファチジルエタノールアミン、及び置換された若しくは置換されていない炭素数16～30のアルキル基からなる群から選択される、請求項５１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 51, wherein the lipid is selected from the group consisting of cholesterol or an analogue thereof, tocopherol or an analogue thereof, folic acid, phosphatidylethanolamine, and a substituted or unsubstituted alkyl group having 16 to 30 carbon atoms.
  54. 　前記第1核酸鎖と前記第2核酸鎖とがリンカーを介して結合している、請求項４１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 41, wherein the first nucleic acid strand and the second nucleic acid strand are linked via a linker.
  55. 　前記リンカーが、切断性（cleavable）又は非切断性（uncleavable）のリンカーである、請求項５４に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 54, wherein the linker is a cleavable or uncleavable linker.
  56. 　前記リンカーが、以下の式（IV）で表される基を含む、請求項５４に記載の送達促進剤又は医薬組成物。
    Figure JPOXMLDOC01-appb-C000003
     （式中、
    L2は、置換された若しくは置換されていないC1～12のアルキレン基、置換された若しくは置換されていないC3～C8シクロアルキレン基、-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-、又はCH(CH2-OH)-CH2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)3-を表し、
    L3は、-NH-又は結合を表し、
    L4は、置換された若しくは置換されていないC1～12のアルキレン基、置換された若しくは置換されていないC3～C8のシクロアルキレン基、-(CH2)2-[O-(CH2)2]m-、又は結合を表し、ここで、mは1～25の整数を表し、
    L5は、-NH-(C=O)-、-(C=O)-、又は結合を表す）     55. The delivery enhancer or pharmaceutical composition of claim 54, wherein the linker comprises a group represented by formula (IV):
    Figure JPOXMLDOC01-appb-C000003
     (Wherein,
    L2 represents a substituted or unsubstituted C1-12 alkylene group, a substituted or unsubstituted C3- C8 cycloalkylene group, -( CH2 ) 2 -O-(CH2) 2 -O-( CH2 ) 2 -O-(CH2) 3- , or CH( CH2 - OH) -CH2 -O-( CH2 ) 2 -O-( CH2 ) 2 -O-( CH2 ) 2 -O-( CH2 ) 2 -O-( CH2 ) 3- ;
    L3 represents -NH- or a bond;
    L4 represents a substituted or unsubstituted C 1 -12 alkylene group, a substituted or unsubstituted C 3 -C 8 cycloalkylene group, -(CH 2 ) 2 -[O-(CH 2 ) 2 ] m -, or a bond, where m represents an integer of 1 to 25;
    L5 represents -NH-(C=O)-, -(C=O)-, or a bond.
  57. 　前記リンカーが、核酸、ポリエーテル基、並びに／又はアルキルアミノ基を含む、請求項５４に記載の送達促進剤又は医薬組成物。 55. The delivery enhancer or pharmaceutical composition of claim 54, wherein the linker comprises a nucleic acid, a polyether group, and/or an alkylamino group.
  58. 　前記核酸が、1個、又はヌクレオシド間結合により連結された2～10個のヌクレオシド及び／若しくは非天然ヌクレオシドからなる、請求項５７に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 57, wherein the nucleic acid consists of one or two to ten nucleosides and/or non-natural nucleosides linked by internucleoside bonds.
  59. 　前記ポリエーテル基が、ポリエチレングリコール基又はトリエチレングリコール基である、請求項５７に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 57, wherein the polyether group is a polyethylene glycol group or a triethylene glycol group.
  60. 　前記アルキルアミノ基が、ヘキシルアミノ基である、請求項５７に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 57, wherein the alkylamino group is a hexylamino group.
  61. 　前記第2核酸鎖のヌクレオシド間結合の全部又は一部が修飾ヌクレオシド間結合である、請求項４１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 41, wherein all or a portion of the internucleoside bonds of the second nucleic acid strand are modified internucleoside bonds.
  62. 　前記修飾ヌクレオシド間結合がホスホロチオエート結合である、請求項６１に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 61, wherein the modified internucleoside linkage is a phosphorothioate linkage.
  63. 　前記核酸医薬が、siRNAである、請求項１８に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 18, wherein the nucleic acid drug is siRNA.
  64. 　前記siRNAが、デオキシリボヌクレオシド、2'-修飾ヌクレオシド、5'-修飾ヌクレオシド、架橋ヌクレオシド、及び修飾ヌクレオシド間結合からなる群から選択されるいずれか１以上を含む、請求項６３に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 63, wherein the siRNA comprises one or more selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, bridged nucleosides, and modified internucleoside linkages.
  65. 　前記siRNAが、機能性部分と結合している、請求項６３に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 63, wherein the siRNA is conjugated to a functional moiety.
  66. 　前記機能性部分が、脂質又はペプチドである、請求項６５に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 65, wherein the functional moiety is a lipid or a peptide.
  67. 　前記脂質が、コレステロール若しくはその類縁体、トコフェロール若しくはその類縁体、葉酸、ホスファチジルエタノールアミン、及び置換された若しくは置換されていない炭素数16～30のアルキル基からなる群から選択される、請求項６６に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 66, wherein the lipid is selected from the group consisting of cholesterol or an analogue thereof, tocopherol or an analogue thereof, folic acid, phosphatidylethanolamine, and a substituted or unsubstituted alkyl group having 16 to 30 carbon atoms.
  68. 　前記核酸医薬が2つのsiRNAを含み、前記2つのsiRNAに含まれるガイド鎖がリンカーを介して互いに結合している、請求項６３に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition according to claim 63, wherein the nucleic acid drug comprises two siRNAs, and the guide strands contained in the two siRNAs are linked to each other via a linker.
  69. 　前記リンカーが、核酸、ポリエーテル基、並びに／又はアルキルアミノ基を含む、請求項６８に記載の送達促進剤又は医薬組成物。 The delivery enhancer or pharmaceutical composition of claim 68, wherein the linker comprises a nucleic acid, a polyether group, and/or an alkylamino group.
  70. 　前記中枢神経系が、大脳皮質、大脳基底核、大脳白質、間脳、脳幹、小脳、及び脊髄からなる群から選択される、請求項１に記載の送達促進剤又は請求項１４に記載の医薬組成物。 The delivery enhancer of claim 1 or the pharmaceutical composition of claim 14, wherein the central nervous system is selected from the group consisting of the cerebral cortex, the basal ganglia, the cerebral white matter, the diencephalon, the brainstem, the cerebellum, and the spinal cord.
  71. 　前記中枢神経系が、前頭葉、側頭葉、海馬、海馬傍回、頭頂葉、後頭葉、線条体、淡蒼球、前障、視床、視床下核、中脳、黒質、橋、延髄、小脳皮質、小脳核、頸髄、胸髄、及び腰髄からなる群から選択される、請求項７０に記載の送達促進剤又は医薬組成物。 71. The delivery enhancer or pharmaceutical composition according to claim 70, wherein the central nervous system is selected from the group consisting of the frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, parietal lobe, occipital lobe, striatum, globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigra, pons, medulla oblongata, cerebellar cortex, cerebellar nuclei, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.
  72. 　前記末梢神経系が、脊髄前根、後根、第１～第１２脳神経、馬尾、及び後根神経節からなる群から選択される、請求項１に記載の送達促進剤又は請求項１４に記載の医薬組成物。 The delivery enhancer of claim 1 or the pharmaceutical composition of claim 14, wherein the peripheral nervous system is selected from the group consisting of the ventral spinal root, dorsal root, cranial nerves 1 to 12, cauda equina, and dorsal root ganglion.
  73. 　前記送達促進剤が被験体に0.01mg～10g投与される、請求項１に記載の送達促進剤又は請求項１４に記載の医薬組成物。 The delivery enhancer of claim 1 or the pharmaceutical composition of claim 14, wherein the delivery enhancer is administered to a subject in an amount of 0.01 mg to 10 g.
PCT/JP2023/035565 2022-09-30 2023-09-29 Nervous system delivery enhancer WO2024071362A1 (en)

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