Preventive and / or therapeutic agent for cystic kidney disease

Abstract

This invention provides a preventive and / or therapeutic agent for cystic kidney disease. The preventive and / or therapeutic agent for cystic kidney disease comprises a single-stranded oligonucleotide containing a base sequence complementary to at least a portion of the base sequence of miR-21.

Description

Technical Field

[0001] This invention relates to preventive and / or therapeutic agents for cystic kidney disease, etc. More specifically, it relates to preventive and / or therapeutic agents for cystic kidney disease, etc., containing a single-stranded oligonucleotide comprising a base sequence complementary to at least a portion of the base sequence of miR-21. Background Technology

[0002] Autosomal dominant polycystic kidney disease (PCK), a type of cystic kidney disease, is a congenital disorder caused by a single gene mutation (PKD1 / 2). It is reported to occur in approximately 1 in 3,000 people, making it one of the most common single-gene mutation diseases. Cysts form in the liver and kidneys, leading to kidney enlargement, and about half of patients require maintenance dialysis in the second half of their lives after age 60.

[0003] miR-21 is a miRNA associated with cell proliferation, apoptosis inhibition, and fibrosis. Anti-miRNA oligosaccharides (AMOs) targeting miR-21 are also under research and development.

[0004] In addition, it has been reported that knocking out miR-21 inhibits cyst growth in an orthologous mouse model of ADPKD (Non-Patent Literature 1).

[0005] Existing technical documents Patent documents Patent Document 1: International Publication No. 2021 / 153762 Patent Document 2: International Publication No. 2023 / 140040 Non-patent literature Non-patent literature 1: J Am Soc Nephrol. 2016 Aug; 27(8): 2319-30. doi: 10.1681 / ASN.2015060634. Epub 2015 Dec 17. Summary of the Invention

[0006] The problem that the invention aims to solve The present invention aims to provide a preventive and / or therapeutic agent for cystic kidney disease.

[0007] Technical solutions for solving the problem The inventors of this invention discovered that miR-21 expression is upregulated in the kidney tissue of mice with cystic disease model, and that cyst growth is inhibited by antisense oligonucleotides of miR-21, and further improved upon this.

[0008] This invention includes, for example, the subject matter described in the following items.

[0009] Item 1. A preventive and / or therapeutic agent for cystic kidney disease, comprising: a single-stranded oligonucleotide containing a base sequence complementary to at least a portion of the base sequence of miR21.

[0010] Item 1A. A method for the prevention and / or treatment of cystic kidney disease, comprising: administering to a subject requiring prevention and / or treatment of cystic kidney disease (preferably a subject with cystic kidney disease) a single-stranded oligonucleotide comprising a base sequence complementary to at least a portion of the base sequence of miR21.

[0011] Item 1B. A single-stranded oligonucleotide containing a base sequence complementary to at least a portion of the base sequence of miR21 for the purpose of prevention and / or treatment of cystic kidney disease.

[0012] Item 1C. Use of single-stranded oligonucleotides containing a base sequence complementary to at least a portion of the base sequence of miR21 in the manufacture of preventive and / or therapeutic agents for cystic kidney disease.

[0013] Item 1D. Use of single-stranded oligonucleotides containing a base sequence complementary to at least a portion of the base sequence of miR21 for the prevention and / or treatment of cystic kidney disease.

[0014] Item 2. The formulation described in item 1, wherein the single-chain oligonucleotide comprises modified nucleoside structural units and / or modified nucleoside inter-bonds.

[0015] Item 3. The formulation described in item 2, wherein the modified nucleoside structural unit is a non-cyclic nucleoside structural unit and / or a locked artificial nucleic acid (LNA) structural unit; or The modified nucleoside internucleotide bonds are thiophosphate bonds.

[0016] Item 4. The formulation described in item 3, wherein the non-cyclic nucleoside structural unit is the structural unit shown in the following general formula (1).

[0017] [Chemistry 1] [In the formula: R] 1 and R 2 Same or different, indicating hydrogen atoms or organic groups (wherein, excluding R) 1 and R 2 (This applies when both parties are organic groups). [Base represents a nucleic acid base.] Item 5. The formulation described in item 4, wherein... In general formula (1), R 1 It is a hydrogen atom or a methyl group, and R 2 It is a hydrogen atom.

[0018] Item 6. The formulation as described in any one of items 1 to 5, wherein, Among the above single-stranded oligonucleotides, The bases complementary to at least one uracil at positions 5, 6, 8, 14, 17, 19, and 20 of the base sequence shown in Serial No. 1 are 2,6-diaminopurine, and / or The bases that are complementary to at least one adenine from positions 7, 10, and 16 of the base sequence shown in Serial No. 1 are 2-thiouracil.

[0019] Item 7. The formulation described in item 3, wherein... (1) The above single-stranded oligonucleotide contains a base sequence that is complementary to the base sequence of miR-21 by at least 16 bases, preferably 18 bases; (2) The nucleoside structural units constituting the above single-stranded oligonucleotides are the modified nucleoside structural units shown in the following general formula (1): [Chemistry 2] [In the formula: R] 1 and R 2 Same or different, indicating hydrogen atoms or organic groups (wherein, excluding R) 1 and R 2 (This applies when both parties are organic groups). [Base represents a nucleic acid base.] (3) In the above single-stranded oligonucleotide, the base that is complementary to at least one uracil selected from the base sequence shown in sequence number 1 at positions 5, 6, 8, 14, 17, 19 and 20 is 2,6-diaminopurine. (4) The bases complementary to at least one adenine at positions 7, 10, and 16 of the sequence shown in Serial No. 1 are 2-thiouracil; and (5) The number of internucleotide bonds in the single-chain oligonucleotides is 100%, and the number of thiophosphate bonds is more than 20%.

[0020] Item 8. The formulation described in item 7, wherein... The above single-stranded oligonucleotides are: (iii-a): Oligonucleotides in which all nucleoside structural units are SNA structural units and / or L-aTNA structural units, and the base sequence is composed of the base sequence shown in sequence number 6; (iii-b): Oligonucleotides containing the base sequence of (iii-a) above, in which one or more bases are missing, substituted or added.

[0021] Item 9. The formulation as described in any one of items 1 to 6, wherein, The above single-stranded oligonucleotides are: (ia): an oligonucleotide consisting of the base sequence shown in sequence number 2; (ib): an oligonucleotide in which one or more bases are missing, substituted or added in the base sequence of the oligonucleotide consisting of the base sequence shown in Serial No. 2; (ii-a): The base sequence is GATAAG m CT, an oligonucleotide whose entire nucleoside structural unit is an LNA structural unit and whose entire internucleotide bond is a phosphate thioester bond, in the sequence, m C represents 5-methylcytosine; or (ii-b): Oligonucleotides contained in (ii-a) whose base sequences are missing, substituted, or have one or more base sequences added.

[0022] Invention Effects A preventive and / or therapeutic agent for cystic kidney disease is provided. Attached Figure Description

[0023] Figure 1 The results of miR-21 expression in mouse kidney tissue are presented (n=3 per group, *P < 0.05; t-test).

[0024] Figure 2 This indicates the results of miR-21 detection in mouse kidney tissue.

[0025] Figure 3 This indicates the results of miR-21 detection in human kidney tissue.

[0026] Figure 4 This represents the in vivo dynamics evaluation results of disulfoCy5-antimiR-21 (SNA).

[0027] Figure 5 This indicates the staining results of mouse kidney tissue.

[0028] Figure 6The results represent the measurement of kidney weight / body weight (KW / BW) and blood urea nitrogen (BUN) concentration in mice.

[0029] Figure 7 This indicates the results of the measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood of mice.

[0030] Figure 8 The results of miR-21 detection in the kidney tissue of mice after oligonucleotide administration are shown (n=5, *P <0.05; one-way ANOVA).

[0031] Figure 9 The results represent the expression of PPARα and BCL2 in mouse kidney tissue.

[0032] Figure 10 The results show the expression of TGF-β, SMAD7, and αSMA in mouse kidney tissue.

[0033] Figure 11 This indicates the measurement of cyst size in a three-dimensional culture of primary tubular cells from a kidney removed from a patient with cystic kidney disease.

[0034] Figure 12 This indicates the measurement results of creatinine (Cre) concentration in mouse serum.

[0035] Figure 13 The results represent the kidney weight / body weight (KW / BW) and serum creatinine (Cre) concentration in mice. Detailed Implementation

[0036] The embodiments included in this invention will now be described in more detail.

[0037] The preventive and / or therapeutic agents for cystic kidney disease included in this invention contain a single-stranded oligonucleotide comprising a base sequence complementary to at least a portion of the base sequence of miR-21. In this specification, the agent is sometimes referred to as "the formulation of this invention," etc. Additionally, the single-stranded oligonucleotide is sometimes referred to as "the single-stranded oligonucleotide of this invention," etc.

[0038] The base sequence shown in sequence number 1 is the base sequence (22 bases in length) that constitutes miR-21.

[0039] "At least a portion of the base sequence of miR-21" is not particularly limited as long as the single-stranded oligonucleotide of the present invention can form a complementary strand with miR-21, for example, a length of 6 to 22 bases. The lower limit of this range is not particularly limited, but is preferably 7 bases long, more preferably 8 bases long, further preferably 10 bases long, even more preferably 14 bases long, particularly preferably 18 bases long, and even more particularly preferably 21 bases long.

[0040] In this specification, "complementarity" includes not only relationships of complete base complementarity (complete complementarity: e.g., A hybridization with T or U, and G hybridization with C without mismatches), but also complementarity to the degree to which hybridization can occur under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of nucleic acids, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). For example, wash conditions after hybridization typically include "1×SSC, 0.1% SDS, 37°C". It is preferable that washing under such conditions also maintains the hybridization state. While not particularly restrictive, wash conditions of "0.5×SSC, 0.1% SDS, 42°C" can be listed as more stringent hybridization conditions, and wash conditions of "0.1×SSC, 0.1% SDS, 65°C" can be listed as even more stringent hybridization conditions.

[0041] Furthermore, as mentioned above, complementarity is based on a one-to-one correspondence of bases. Therefore, a certain base sequence X and a base sequence Y that is complementary to base sequence X have the same base length.

[0042] The number of bases constituting the single-stranded oligonucleotide of the present invention is preferably about 6 to 25. The lower limit of this range is not particularly limited, but is preferably 7, more preferably 8, further preferably 10, even more preferably 14, and particularly preferably 18. The upper limit of this range is not particularly limited, but is preferably 24, more preferably 23, further preferably 22, and particularly preferably 21.

[0043] The single-stranded oligonucleotide of the present invention is an oligonucleotide comprising a base sequence having, for example, 85% or more identity with respect to at least a portion of the base sequence of miR-21, preferably 90% or more identity, more preferably 95% or more identity, further preferably 98% or more identity, even more preferably 99% or more identity, and particularly preferably 100% identity.

[0044] In this specification, the “identity” of a base sequence refers to the degree of consistency between two or more comparable base sequences. Therefore, the higher the consistency between two base sequences, the higher the identity or similarity of these sequences. The level of identity of a base sequence can be determined, for example, using the FASTA sequence analysis procedure with default parameters. Alternatively, it can be determined using the BLAST algorithm of Karlin and Altschul (Karlin S, Altschul SF, “Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF, “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on such a BLAST algorithm. The specific methods used in these analyses are well-known and can be found on the website of the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0045] The single-stranded oligonucleotides of the present invention are preferably oligonucleotides that do not form their own double strands. Examples of self-double strands include intramolecular (hairpin) double strands and intermolecular (dimer) double strands. Oligonucleotides that do not form their own double strands can be obtained, for example, by omitting base sequence portions that would form their own double strands, introducing substitutions and / or arbitrary changes to base sequence portions that would form their own double strands.

[0046] The single-stranded oligonucleotides of the present invention can be DNA, RNA, etc., or oligonucleotides based on them and containing modified nucleoside structural units and / or modified nucleoside inter-bonds.

[0047] Modified nucleoside structural units are structural units corresponding to the nucleosides that constitute oligonucleotides; there are no particular restrictions as long as they fall within this range. Examples of modified nucleoside structural units include molecules that have undergone chemical modifications to ribonucleosides, deoxyribonucleosides, DNA, RNA, etc. For example, examples include nucleosides modified with 2'-OMe, nucleosides modified with 2'-MOE, LNA (registered trademark), ENA (registered trademark), AmNA (registered trademark), GuNA, scpBNA, and other glycoside modified nucleoside structural units.

[0048] Modified sugars are sugars that have been substituted and / or arbitrarily altered in their natural sugar moieties (i.e., the sugar moieties found in DNA (2'-H) or RNA (2'-OH)). Sugar-modified nucleosides are modified nucleosides that contain modified sugars. Glycoside-modified nucleosides refer to any form of nucleoside in which any chemical structure is added to or substituted for part or all of the sugar's chemical structure. Examples include: morpholino nucleic acids (PMOs) such as those with a 2'-O-methyl substituted sugar, a 2'-O-propyl substituted sugar, a 2'-methoxyethoxy substituted sugar, a 2'-O-methoxyethyl substituted sugar, a 2'-O-[2-(guanidino)ethyl] substituted sugar, a 2'-O-fluorine substituted sugar, and a sugar with a morpholino ring substituted; bridged nucleic acids (BNAs) with two ring structures obtained by introducing a cross-linking structure into the sugar; more specifically, locked nucleic acids (LNAs) obtained by cross-linking the oxygen atom at the 2' position and the carbon atom at the 4' position via a methylene group; and ethylene-bridged nucleic acids. Nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)], etc., and also peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)] and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)], etc. In addition, as described later, non-cyclic nucleoside structural units can also be used as the modifying nucleoside structural units.

[0049] As a modified nucleoside, other possible alternatives include nucleosides in which the base portion of nucleic acid atoms (e.g., hydrogen atoms, oxygen atoms) or functional groups (e.g., hydroxyl, amino groups) are replaced by other atoms (e.g., hydrogen atoms, sulfur atoms), functional groups (e.g., amino groups), or alkyl groups having 1 to 6 carbon atoms, or nucleosides protected with protecting groups (e.g., methyl or acyl groups), or molecules obtained by adding other chemical substances such as lipids, phospholipids, phenazines, folic acid, phenanthridine, anthraquinones, acridine, fluorescein, rhodamine, coumarin, pigments, etc.

[0050] The modified nucleoside structural unit is preferably an LNA structural unit and / or an acyclic nucleoside structural unit, more preferably an acyclic nucleoside structural unit.

[0051] One type of nucleoside structural unit can be used, or a combination of two or more types can be used.

[0052] The proportion of modified nucleoside structural units relative to 100% of the nucleoside structural units constituting the single-chain oligonucleotide of the present invention is, for example, 50% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, even more preferably 95%, and particularly preferably 100%.

[0053] In one embodiment, the single-stranded oligonucleotide of the present invention preferably comprises acyclic nucleoside structural units. The proportion of acyclic nucleoside structural units is, for example, 50% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, even more preferably 95%, and particularly preferably 100%, relative to 100% of the nucleoside structural units (number of nucleosides) constituting the single-stranded oligonucleotide of the present invention.

[0054] In this specification, "acyclic nucleoside structural unit" refers to a structural unit corresponding to a nucleoside that constitutes an oligonucleotide, which only needs to have an acyclic backbone without a sugar backbone, and there are no particular restrictions. A representative example of an acyclic nucleoside structural unit is the structural unit shown in general formula (1): [Chemical 3] [In the formula: R] 1 and R 2 Same or different, indicating hydrogen atoms or organic groups (wherein, excluding R) 1 and R 2 (This applies when both parties are organic groups). [Base represents a nucleic acid base.] There are no particular restrictions on organic groups; for example, hydrocarbon groups can be listed.

[0055] As a hydrocarbon group, chain-like hydrocarbon groups are preferred. Examples of chain-like hydrocarbon groups include alkyl, alkenyl, and alkynyl groups, with alkyl groups being preferred. Specific examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl, and 3-methylpentyl. Methyl is preferred. The number of carbon atoms in the hydrocarbon group is not particularly limited. The number of carbon atoms is preferably 1 to 8, more preferably 1 to 6, further preferably 1 to 4, even more preferably 1 to 2, and particularly preferably 1. Furthermore, alkyl groups that contain an alkynyl group (-C≡C-, -C≡CH) at the terminal or internally, which can introduce various functional groups through click reactions or the like, are more preferred.

[0056] In addition to the groups mentioned above, monovalent groups formed by removing one hydrogen atom or functional group from various molecules, such as those used for modifying nucleosides, can also be used as organic groups. Examples of such molecules include polyethylene glycol chains, pigment molecules, polycations (spermine), groove binders, amino groups, hydroxyl groups, thiol groups, metal ligands, photolytic functional groups, and sugar chains. These groups can be directly or indirectly linked to the backbone of the aforementioned structural units. For example, they can be linked using click reactions (such as the reaction between alkynes and azides described above).

[0057] In a preferred embodiment of the invention, from the viewpoint of the bonding affinity to miR-21, R is preferred. 1 It is a hydrogen atom or a chain hydrocarbon group (preferably methyl) and R 2 It is a hydrogen atom. In other words, as a non-cyclic nucleoside structural unit, it is preferably an SNA (Serinol Nucleic acid) structural unit (R... 1 and R 2 (for hydrogen atoms), and / or L-aTNA (acyclic L-threoninol nucleic acid) structural units (R 1 It is methyl, and R 2 (For hydrogen atoms).

[0058] As nucleic acid bases, they can be used without restriction from the bases that make up nucleic acids. The bases that make up nucleic acids include not only the typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also bases other than these, such as hypoxanthine (I) and modified bases. Examples of modified bases include 2,6-diaminopurine, 2-thiouracil, 2-thiothymine, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1- Methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, etc.

[0059] In general formula (1), *1 and *2 indicate the orientation when forming oligonucleotides. R 1 It is an organic group and R 2 When the atom is hydrogen, *1 is on the 3' side and *2 is on the 1' side. R 1 It is a hydrogen atom and R 2 When the radical is an organic group, *1 is on the 1' side and *2 is on the 3' side. R 1 It is a hydrogen atom and R 2 When the atom is hydrogen, *1 is on the (S) side and *2 is on the (R) side.

[0060] The (S) end and (R) end (SNA in Chemical 4), the 3' side and 1' side mentioned above correspond to the 5' end and 3' end of DNA (LNA in Chemical 5), respectively. Here, for convenience, the (S) end and (R) end, the 3' end and 1' end are sometimes referred to as the 5' end and 3' end, respectively.

[0061] Furthermore, when the modified nucleoside structural unit shown in general formula (1) is located at the end, its *1 and *2 are added with hydrogen atoms, sulfur atoms, and phosphate groups as described above.

[0062] Modified nucleoside bonds refer to nucleoside bonds that have undergone substitution or arbitrary alteration from naturally occurring nucleoside bonds (i.e., phosphodiester bonds). Modified nucleoside bonds include those containing phosphorus atoms and those without. As modified nucleoside bonds, any chemical substance can be added to or substituted for part or all of the chemical structure of the phosphodiester bond of the nucleotide. Examples of modified nucleoside bonds include, for instance, thiophosphate bonds (also called thiophosphate bonds), dithiophosphate bonds, phosphotriester bonds, methylphosphonate bonds, methylthiophosphonate bonds, borane phosphate bonds, and phosphoramide ester bonds. This is intended to prevent degradation by hydrolases such as phosphatases.

[0063] As a modification of internucleotide bonds, thiophosphate bonds are particularly preferred.

[0064] As a modifier of nucleoside internucleotide bonds, one type can be used alone, or a combination of two or more types can be used.

[0065] The number of modified nucleoside internucleotides relative to 100% of the number of nucleoside internucleotides in the single-stranded oligonucleotide of the present invention is not particularly limited, but is, for example, 20% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more. In one embodiment, all nucleoside internucleotides can be modified nucleoside internucleotides (e.g., phosphate thioester bonds).

[0066] In one embodiment, the single-chain oligonucleotide of the present invention may be 2,6-diaminopurine, whose bases are complementary to at least one uracil in the base sequence shown in Serial No. 1, and / or 2-thiouracil, whose bases are complementary to at least one adenine in the base sequence shown in Serial No. 1. Complementarity refers to the relationship between miR21 and the single-chain oligonucleotide of the present invention when they are arranged in a complementary manner. By positioning 2,6-diaminopurine and 2-thiouracil opposite to each other, it is expected to inhibit the formation of self-double strands of the single-chain oligonucleotide and further improve its binding affinity to miR-21.

[0067] Uracil, as represented by the base sequence shown in Serial No. 1, can be uracil at positions 1, 5, 6, 8, 14, 17, 19, and 20. In the single-stranded oligonucleotide of the present invention, the base complementary to at least one uracil selected from positions 5, 6, 8, 14, 17, 19, and 20 is preferably 2,6-diaminopurine. For example, the base complementary to the uracil at positions 5, 6, 8, 14, 17, 19, and 20 can be 2,6-diaminopurine. The proportion of 2,6-diaminopurine in the single-stranded oligonucleotide of the present invention is, for example, 40–100%, preferably 50–100%, and more preferably 60–100%, relative to 100% of the amount of uracil contained in at least a portion of the base sequence of miR-21.

[0068] Adenine, as represented by the base sequence shown in Serial No. 1, can be adenine at positions 2, 7, 10, 12, 16, and 22. In the single-chain oligonucleotide of the present invention, the base complementary to at least one adenine selected from positions 7, 10, and 16 is preferably 2-thiouracil. For example, the base complementary to the adenine at positions 7, 10, and 16 can be 2-thiouracil.

[0069] The proportion of 2-thiouracil contained in the single-chain oligonucleotide of the present invention is, for example, 20-100%, preferably 30-100%, and more preferably 40-100%, relative to 100% of the adenine content in at least a portion of the base sequence of miR21.

[0070] In one embodiment, the single-chain oligonucleotide of the present invention preferably has one or more pairs of 2,6-diaminopurine and 2-thiouracil bases that are opposite to each other. More preferably, it has two or more, and even more preferably, it has three. The relative relationships can be the relationships within the single-chain oligonucleotide of the present invention or the relationships between the single-chain oligonucleotides of the present invention. Preferably, it is the relationship between the single-chain oligonucleotides of the present invention.

[0071] As a preferred embodiment of the single-chain oligonucleotide of the present invention, examples include oligonucleotides with the base sequence (S)-C DDC DsUCDG T CsUGDsU DDG CTA- (R) (where D represents 2,6-diaminopurine and sU represents 2-thiouracil) (Sequence No. 6).

[0072] As a preferred embodiment of the single-chain oligonucleotide of the present invention, at least one, two, three, four, or all (five) oligonucleotides satisfying (1) to (5) below can be listed.

[0073] (1) The above single-stranded oligonucleotide contains a base sequence that is complementary to the base sequence of miR-21 by at least 18 bases; (2) The nucleoside structural units constituting the above single-stranded oligonucleotides are the modified nucleoside structural units shown in the following general formula (1): [Chemical 4] [In the formula: R] 1 and R 2 Same or different, indicating hydrogen atoms or organic groups (wherein, excluding R) 1 and R 2 (This applies when both parties are organic groups). [Base represents a nucleic acid base.] (3) In the above single-stranded oligonucleotide, the base that is complementary to at least one uracil selected from the base sequence shown in sequence number 1 at positions 5, 6, 8, 14, 17, 19 and 20 is 2,6-diaminopurine. (4) The bases complementary to at least one adenine at positions 7, 10, and 16 of the sequence shown in Serial No. 1 are 2-thiouracil; and (5) The number of internucleotide bonds in the single-chain oligonucleotides is 100%, and the number of thiophosphate bonds is more than 20%.

[0074] As a preferred embodiment of the single-chain oligonucleotide of the present invention, examples include: an oligonucleotide with the base sequence (S)-C DDC DsUCDG T CsUGDsU DDG CTA- (R) (where D represents 2,6-diaminopurine and sU represents 2-thiouracil), all nucleoside structural units being SNA structural units, and all nucleoside internucleotide bonds being thiophosphate bonds (Sequence No. 2); an oligonucleotide with the base sequence (S)-C DDC DsUCDG T CsUGDsU DDG CTA- (R) (where D represents 2,6-diaminopurine and sU represents 2-thiouracil), all nucleoside structural units being L-aTNA structural units, and all nucleoside internucleotide bonds being thiophosphate bonds (Sequence No. 3); and an oligonucleotide with the base sequence (S)-C* D*D*C* DsUCDG T*C*sU*GDsU DDG Oligonucleotides such as CT*A-(R) (Sequence No. 5) (in the sequence, D represents 2,6-diaminopurine, sU represents 2-thiouracil, and the * between the bases indicates that the bond between the nucleosides is a thiophosphate bond.) and all of their nucleoside structural units are SNA structural units (Sequence No. 5), etc. In this specification, these oligonucleotides are sometimes referred to as oligonucleotides composed of the base sequence shown in Sequence No. 2, oligonucleotides composed of the base sequence shown in Sequence No. 3, etc.

[0075] Furthermore, as a preferred embodiment of the single-stranded oligonucleotide of the present invention, for example, oligonucleotides listed in sequence numbers 2, 3, 5, or 6 may contain a base sequence with one or more bases missing, substituted, or added. The upper limit for the number of missing, substituted, or added bases is not particularly limited, as long as the single-stranded oligonucleotide of the present invention contains a base sequence complementary to at least a portion of the base sequence of miR-21; for example, it can be 10, 9, 8, 7, 6, 5, 4, or 3.

[0076] As a preferred embodiment of the single-chain oligonucleotide of the present invention, for example, the base sequence 5'-GAT AAG can be listed. m CT-3' (in the sequence,m C represents 5-methylcytosine. Oligonucleotides, etc., whose entire nucleoside structural unit is an LNA structural unit and whose entire inter-nucleoside bond is a thiophosphate bond.

[0077] Furthermore, as a preferred embodiment of the single-chain oligonucleotide of the present invention, for example, in the form of a base sequence of 5'-GAT AAG... m CT-3' (in the sequence, m C represents 5-methylcytosine. The oligonucleotide whose base sequence comprises all LNA structural units and all internucleotide bonds are phosphate thioester bonds contains a sequence of one or more missing, substituted, or added bases. The upper limit for the number of missing, substituted, or added bases is as long as the single-stranded oligonucleotide of the present invention contains at least a portion of a base sequence complementary to the base sequence of miR-21; there is no particular limitation, for example, it can be 4 or 3.

[0078] The single-stranded oligonucleotides of this invention can be linked to other molecules. There are no particular limitations on these other molecules; examples include fluorescent markers, biotin, azide groups, ethynyl groups, etc. Examples of fluorescent markers include fluorescein, rhodamine, Texas red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM (trademark), Cy (registered trademark) 3, Cy (registered trademark) 5, and the Alexa Fluor (registered trademark) series, etc.

[0079] The single-stranded oligonucleotides of the present invention can be easily produced using known genetic engineering methods, artificial nucleic acid manufacturing methods, etc. For example, they can be produced using PCR, restriction enzyme digestion, nucleic acid ligation technology, etc.

[0080] In the formulations of the present invention, the single-chain oligonucleotides of the present invention are preferably contained as active ingredients.

[0081] In the formulation of the present invention, the content of the single-chain oligonucleotide of the present invention is not particularly limited, for example, it can be about 0.0001 to 100% by weight.

[0082] The formulations of the present invention contain the single-chain oligonucleotides of the present invention, and may also contain other components. Examples of such other components include, for instance, pharmacologically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc.

[0083] The formulation of the present invention can be used for the prevention and / or treatment of cystic kidney disease.

[0084] Examples of cystic kidney diseases include, for example, polycystic kidney disease. Furthermore, examples of polycystic kidney diseases include, for example, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). Among these, ADPKD is preferred.

[0085] As examples of the objects to which the formulations of the present invention are applied, humans and other mammals (e.g., rats, mice, rabbits, cattle, pigs, dogs, cats, sheep, monkeys, etc.) can be listed.

[0086] The formulations of this invention can be in any dosage form, such as oral preparations like tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including beverages, suspensions, syrups), gels, etc., or injectable preparations (e.g., intravenous infusion preparations, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), topical preparations (e.g., ointments, plasters, lotions), suppositories, inhalers, eye drops, eye ointments, nasal drops, ear drops, etc.). Furthermore, the active ingredient can be administered either in a state co-located with particles (e.g., lipid particles, exosomes) or encapsulated within the particles.

[0087] As for the administration route of the formulation of the present invention, it is not particularly limited as long as the desired effect can be obtained. Examples include: oral administration; enteral administration such as enteral nutrition and enema; non-oral administration such as intravenous administration, arterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and nasal administration.

[0088] The dosage of the formulation of the present invention is not particularly limited as long as it is an effective amount to exert the therapeutic effect. Generally, the weight of the active ingredient is 0.1 to 1000 mg / kg body weight per day for oral administration, preferably 0.5 to 500 mg / kg body weight per day, and 0.01 to 100 mg / kg body weight per day for non-oral administration, preferably 0.05 to 50 mg / kg body weight per day. The above dosage and dosing interval can also be appropriately increased or decreased according to age, condition, symptoms, etc.

[0089] While not wishing to be limited by theory, it can be considered that the single-chain oligonucleotides of the present invention can inhibit the enlargement of renal cysts and / or hepatic cysts by promoting the expression of PPARα, inhibiting the expression of fibroblasts (e.g., promoting the expression of SMAD7, inhibiting the expression of TGF-β, and / or inhibiting the expression of αSMA, etc.) and / or inhibiting the expression of BCL2 as an anti-apoptotic protein, thereby preventing and / or treating cystic kidney disease.

[0090] Furthermore, in this specification, the term "comprising" includes both "consisting essentially of" and "consisting of." Additionally, this invention encompasses all arbitrary combinations of the constituent elements described in this specification.

[0091] Furthermore, the various characteristics (properties, structures, functions, etc.) described in the above embodiments of the present invention can be arbitrarily combined to determine the subject matter encompassed by the present invention. That is, the present invention encompasses all subject matter constituted by all combinations of the combinable characteristics described in this specification.

[0092] Example The invention is illustrated by the following experimental examples. However, the invention is not limited to these examples. Unless otherwise specified, all experiments were conducted at atmospheric pressure and room temperature. And, unless otherwise specified, "%" refers to "mass %".

[0093] Animal models of cystic diseases Male DBA / 2 mice (SLC Corporation, Japan) and male DBA / 2FG-pcy mice (Kyudo Co., Ltd.) were used as a mouse model of cystic disease.

[0094] This study was approved by the Animal Care and Use Committee of Nagoya University School of Medicine and was conducted in accordance with the Animal Experimentation Guidelines of Nagoya University School of Medicine.

[0095] Experimental Design Male DBA / 2 and male DBA / 2FG-pcy mice were administered the drug subcutaneously at a dose of 10 mg / kg three times weekly for 6 weeks, from 4 to 10 weeks of age. The injection was administered subcutaneously via the back under isoflurane anesthesia, into the left scapula. After 18 injections over 6 weeks, the mice were sacrificed, and kidney weight / body weight (KW / BW), blood tests, and kidney tissue analysis were performed.

[0096] Human Organization Kidney tissue from healthy individuals and ADPKD patients, removed from Nagoya University Hospital, was used as the test samples. Consent from patients regarding the use of these samples was obtained in accordance with the regulations of Nagoya University Hospital.

[0097] qRT-PCR analysis Total RNA was extracted from tissue samples using the TRIzol Reagent (Thermo Fisher Scientific). For miRNA detection, total RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific), and analyzed using TaqMan MicroRNA Assays (Thermo Fisher Scientific).

[0098] Targets were amplified using real-time PCR with the Step One Plus Real-Time PCR System (Thermo Fisher Scientific) and Fast Advanced Master Mix (Thermo Fisher Scientific). All samples were measured in duplicate. Quantitative evaluation of target expression was performed using the ΔΔCT method, and miRNA expression levels were normalized to U6 snRNA expression levels.

[0099] miRNA scope detection The miRNAscope HD Reagent Kit - RED (Advanced Cell Diagnostics) was used for the detection of miR-21 in tissues. The miRNAscope Positive Control Probe - SR-RNU6-S1 (Advanced Cell Diagnostics) was used as a positive control probe, and the miRNAscope Negative Control Probe - SR-Scramble-S1 (Advanced Cell Diagnostics) was used as a negative control probe. The miRNAscope Probe - SR-mmu-miR-21a-5q-S1 (Advanced Cell Diagnostics) was used for the detection of miR-21 in mouse kidney tissue. The miRNAscope Probe - SR-hsa-miR-21-5p-S1 (Advanced Cell Diagnostics) was used for the detection of miR-21 in human kidney tissue. Tissue specimens stained with the kit were observed using a BZ-X810 (KEYENCE).

[0100] Anti-miR-21 (SNA), Anti-miR-21 (LNA), Scramble In the following discussion Figures 4-13 The antisense oligonucleotides of miR-21 (anti-miR-21 (SNA) and anti-miR-21 (LNA)) used as the target drugs in the experiments: Figures 4-11 Scramble: Figures 4-13 As described below.

[0101] The structure of the SNA used is shown below.

[0102] [Chemical 5] The base sequences of anti-miR-21 (SNA; serine nucleic acid) and Scramble are shown below. All constituent nucleotides are SNA.

[0103] Anti-miR-21 (SNA) (S-D7sU3): (S) -C DDC DsUCDG T CsUGDsU DDG CTA- (R) (Serial No. 2) Scramble: (S) -TAT GAT GTC CAT GTC GTA CGC- (R) (Serial Number 4) In the base sequence of antimiR-21 (SNA), D represents 2,6-diaminopurine and sU represents 2-thiouracil. AntimiR-21 (SNA) and Scramble were synthesized by the method described in Patent Document 2 (using a protecting group that can be removed by bases) and were provided by the Asanuma Laboratory, Faculty of Engineering, Nagoya University.

[0104] The structure of the LNA used is shown below.

[0105] [Chemical 6] The base sequence of anti-miR-21 (LNA; Locked Nucleic acid) is shown below. All its structural nucleotides are LNA.

[0106] Anti-miR-21 (LNA): 5'-GAT AAG m CT-3' (in the sequence, m C represents 5-methylcytosine. Anti-miR-21 (LNA) was purchased from Gene Design.

[0107] In all anti-miR-21 (SNA), Scramble, and anti-miR-21 (LNA) formulations, all internucleotide bonds were modified with thiosulfate (PS) as shown in the above structural formulas.

[0108] S-45371 The following Figure 12 The antisense oligonucleotide (S-45371) of miR-21 used as the target drug in the experiment is shown below. Its entire nucleotide structure is SNA.

[0109] S-45371: (S)-C* D*D*C* DsUCDG T*C*sU*GDsU DDG CT*A-(R) (Serial Number 5) In the S-45371 base sequence, D and sU are as described above. An asterisk (*) between the bases indicates that the bond between the nucleosides is a thiophosphate bond. If no asterisk is marked between the bases, it indicates that the bond between the nucleosides is a phosphodiester bond. The synthesis of S-45371 is carried out in the same manner as that of anti-miR-21 (SNA) and Scramble.

[0110] T-D7sU3 (PS) The following Figure 13 The antisense oligonucleotide (T-D7sU3(PS)) of miR-21 used as the target drug in the experiment is shown below. Its complete nucleotide structure is L-aTNA.

[0111] T-D7sU3 (PS): (S)-C DDC DsUCDG T CsUGDsU DDG CTA-(R) (Serial Number 3) In the T-D7sU3 (PS) nucleotide sequence, D and sU are as described above. Furthermore, all nucleoside bonds are phosphate thioester bonds. The synthesis of T-D7sU3 (PS) is performed in the same manner as that of anti-miR-21 (SNA) and Scramble.

[0112] Ex vivo imaging Anti-miR-21 (SNA) was indicated using a disulfoCy5-tagged sequence (provided by Asanuma Laboratory). Mice were sacrificed 1 and 12 hours after SNA administration. Fresh organs were harvested and placed in an IVIS Spectrum in vivo imaging system (PerkinElmer, Waltham, MA, USA). Cy5 was excited at 643 nm and detected at 667 nm. Data were expressed as photons per second. -1 cm -2 Measurements. Fluorescent tissue specimens were observed using BZ-X810 (KEYENCE) and SpinSR10 (Olympus LifeScience).

[0113] Histological analysis The organs were fixed with 10% paraformaldehyde, embedded in paraffin, cut into 4μm thick sections, and stained with hematoxylin-eosin.

[0114] Blood biochemistry analysis The concentrations of blood urea nitrogen (BUN), serum creatinine (Cre), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in serum were measured using an automated chemical analysis device (SRL Co., Ltd., Tokyo, Japan).

[0115] Western blot analysis β-actin antibody (4917; cell Signaling), PPARα antibody (PA1-822A; invitrogen), BCL2 antibody (ab182858; abcam), TGF-β antibody (3711; cell Signaling), SMAD7 antibody (42-0400; invitrogen), and αSMA antibody (19245; cell Signaling) were used as primary antibodies.

[0116] Evaluation of the effect of three-dimensional culture of renal tubular cells on inhibiting cyst formation via antisense introduction. Madin-Darby canine kidney (MDCK) cells were purchased from ATCC (CCL-34; American Type Culture Collection) and cultured at 37°C and 5% CO2 in Eagle's Minimum Essential Medium (051-07615; FUJIFILM WAKO) medium supplemented with 10% heat-inactivated fetal bovine serum. Primary cultured human ADPKD renal tubule cells were prepared from human ADPKD kidneys obtained from patients admitted to Nagoya University Hospital. They were cultured in Dulbecco's modified Eagle medium / nutrient mixture F-12 (048-29785; FUJIFILM WAKO) supplemented with L-glutamine and 15 mM HEPES (D8437; Sigma-Aldrich), 1% penicillin-streptomycin (15140122; Thermo Fisher Scientific), and 1% insulin-transferrin-selenium (41400045; Thermo Fisher Scientific) at 37°C and 5% CO2. These MDCK2 cells and the primary cultured human ADPKD renal tubule cells were cultured in six-well plates at a density of 0.3 × 10⁻⁶ cells / well. 6 Cells were seeded at a density of [number] cells / mL. Two days later, at 80% confluence, cells were transfected with Scramble-SNA and Anti-miR-21-SNA using Lipofectamine 3000 Transfection Reagent (L3000008; Invitrogen). Forty-eight hours later, cells were recovered and seeded at 1.0 × 10⁶ cells / mL on Matrigel (356234; CORNING) in 24-well plates. 4 Cells were seeded at a density of [number] cells / mL and treated with 10 μM Forskolin (067-2191; FUJIFILMWAKO) for 48 h. Cells were then observed using a BZ-X810 microscope (KEYENCE). Images were taken randomly, and cyst size and number were determined. Cyst and cell area were analyzed using Image J (13.0.6).

[0117] The expression of miR-21 in the kidney tissues of DBA / 2 and DBA / 2FG-pcy mice was analyzed by qRT-PCR. Figure 1 As shown, miR-21 expression was upregulated in the kidney tissue of mice with cystic kidney model.

[0118] The expression of miR-21 in the kidney tissues of DBA / 2 and DBA / 2FG-pcy mice was analyzed using miRNA scope. Figure 2 As shown, miR-21 expression was upregulated in the kidney tissue of mice with cystic disease compared to normal mouse kidney tissue.

[0119] In addition, the expression of miR-21 in the kidney tissues of normal individuals and ADPKD patients was analyzed using miRNA scope. Figure 3 As shown, compared with normal human kidney tissue, upregulated expression of miR-21 was also confirmed in the kidney tissue of ADPKD patients.

[0120] As mentioned above, in cystic kidney disease, upregulated expression of miR-21 has been identified in kidney tissue, which is involved in the growth of kidney cysts.

[0121] In mice treated with disulfoCy5-antimiR-21 (SNA), the in vivo dynamics of SNA were evaluated, and the results were as follows: Figure 4 As shown, strong aggregation was confirmed in the kidneys, and it was taken up by renal tubular cells.

[0122] Kidney tissues from the untreated group, the Scramble-treated group, and the anti-miR-21 (SNA)-treated group were stained, such as... Figure 5 As shown, cyst growth was inhibited in the SNA-treated group. Furthermore, in the anti-miR-21 (LNA)-treated group, although there were significant individual differences compared to the SNA-treated group, similar effects were observed in some individuals.

[0123] Kidney weight / body weight (KW / BW) and blood urea nitrogen (BUN) concentrations were compared in the untreated group, the scramble-treated group, and the anti-miR-21 (SNA)-treated group. Figure 6 As shown, in the SNA-treated group, a decrease in KW / BW and BUN concentrations was confirmed, along with cyst growth inhibition and improved renal function. However, no liver dysfunction was observed. Figure 7 Furthermore, in the anti-miR-21 (LNA) group, although there were significant individual differences compared to the SNA group, individuals with the same effects were also observed.

[0124] The expression of miR-21 in kidney tissues of the untreated group, the Scramble-treated group, and the anti-miR-21 (SNA)-treated group was analyzed using miRNA scope. Figure 8 As shown, reduced miR-21 expression was confirmed in the SNA-treated group.

[0125] The expression of PPARα and BCL2 in kidney tissues was evaluated in the untreated group, the Scramble-treated group, and the anti-miR-21 (SNA)-treated group. PPARα expression is known to be inhibited by miR-21 and participates in cyst enlargement through mitochondrial metabolism. Additionally, BCL2, an anti-apoptotic protein, is known to be upregulated by miR-21 and participates in cyst enlargement. Figure 9 As shown, the SNA-treated group was confirmed to promote PPARα expression and inhibit BCL2 expression. Furthermore, in the anti-miR-21 (LNA)-treated group, although there were significant individual differences compared to the SNA-treated group, individuals with similar effects were also observed.

[0126] The expression of TGF-β, SMAD7, and αSMA in kidney tissues of the untreated group, the Scramble-treated group, and the anti-miR-21 (SNA)-treated group was evaluated. It is known that SMAD7 expression is inhibited by miR-21, while the expression of TGF-β and αSMA is upregulated by miR-21, both of which are involved in fibrosis. Figure 10 As shown, in the SNA-treated group, promotion of SMAD7 expression and inhibition of TGF-β and αSMA expression were confirmed. Furthermore, in the anti-miR-21 (LNA)-treated group, although there were significant individual differences compared to the SNA-treated group, similar effects were observed in some individuals.

[0127] Primary renal tubular cells were cultured from kidneys removed from patients with cystic kidney disease, and then subjected to three-dimensional culture. The inhibitory effect of Scramble-added and anti-miR-21 (SNA)-added groups on cyst formation was evaluated in this culture. Figure 11 As shown, cyst formation inhibition was confirmed in the SNA-treated group.

[0128] Serum creatinine (Cre) concentrations were compared between the Scramble and S-45371 dosing groups. Figure 12 As shown, in the SNA (S-45371) administration group, Cre concentration decreased, confirming an improvement in renal function.

[0129] In the Scramble-treated group and the T-D7sU3 (PS)-treated group, kidney weight / body weight (KW / BW) and serum creatinine (Cre) concentration were compared. Figure 13 As shown, in the SNA(T-D7sU3(PS)) administration group, a decrease in KW / BW and Cre concentration, inhibition of cyst growth, and improvement in renal function were confirmed.

Claims

1. A preventive and / or therapeutic agent for cystic kidney disease, characterized in that, contain: A single-stranded oligonucleotide containing a base sequence that is complementary to at least a portion of the base sequence of miR-21.

2. The formulation according to claim 1, characterized in that: The single-stranded oligonucleotide contains modified nucleoside structural units and / or modified nucleoside inter-units.

3. The formulation according to claim 2, characterized in that: The modified nucleoside structural unit is a non-cyclic nucleoside structural unit, or The modified nucleoside inter-bond is a thiophosphate bond.

4. The formulation according to claim 3, characterized in that: The acyclic nucleoside structural unit is the structural unit shown in the following general formula (1). In the formula: R 1 and R 2 "Same" or "different" indicates a hydrogen atom or an organic group, excluding R. 1 and R 2 When both are organic groups, Base represents a nucleic acid base.

5. The formulation according to claim 4, characterized in that: In general formula (1), R 1 It is a hydrogen atom or a methyl group, and R 2 It is a hydrogen atom.

6. The formulation according to claim 1 or 2, characterized in that: In the single-stranded oligonucleotide, The bases complementary to at least one uracil at positions 5, 6, 8, 14, 17, 19, and 20 of the base sequence shown in Serial No. 1 are 2,6-diaminopurine, and / or The bases that are complementary to at least one adenine from positions 7, 10, and 16 of the base sequence shown in Serial No. 1 are 2-thiouracil.

7. The formulation according to claim 3, characterized in that: (1) The single-stranded oligonucleotide contains a base sequence that is complementary to the base sequence of miR-21 by at least 18 bases; (2) The nucleoside structural unit constituting the single-stranded oligonucleotide is the modified nucleoside structural unit shown in the following general formula (1): In the formula: R 1 and R 2 "Same" or "different" indicates a hydrogen atom or an organic group, excluding R. 1 and R 2 When both components are organic groups, Base represents a nucleic acid base. (3) In the single-stranded oligonucleotide, the base that is complementary to at least one uracil selected from the base sequence shown in Serial No. 1 at positions 5, 6, 8, 14, 17, 19 and 20 is 2,6-diaminopurine; (4) The bases complementary to at least one adenine at positions 7, 10, and 16 of the sequence shown in Serial No. 1 are 2-thiouracil; and (5) The number of nucleotide inter-bonds in the single-chain oligonucleotide is 100%, and the number of thiophosphate bonds is more than 20%.

8. The formulation as described in claim 7, characterized in that: The single-stranded oligonucleotide is: (iii-a): Oligonucleotides in which all nucleoside structural units are SNA structural units and / or L-aTNA structural units, and the base sequence is composed of the base sequence shown in sequence number 6; (iii-b): Oligonucleotides containing the oligonucleotides of (iii-a) whose base sequences are missing, substituted or added to one or more bases.

9. The formulation according to claim 1 or 2, characterized in that: The single-stranded oligonucleotide is: (ia): an oligonucleotide consisting of the base sequence shown in sequence number 2; (ib): an oligonucleotide in which one or more bases are missing, substituted or added in the base sequence of the oligonucleotide consisting of the base sequence shown in Serial No. 2; (ii-a): The base sequence is GATAAG m CT, an oligonucleotide whose entire nucleoside structural unit is an LNA structural unit and whose entire internucleotide bond is a phosphate thioester bond, in the sequence, m C represents 5-methylcytosine; or (ii-b): Oligonucleotides contained in (ii-a) whose base sequences are missing, substituted, or have one or more base sequences added.

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