Prophylactic and / or therapeutic agent for cystic kidney diseases

Abstract

The present invention provides a prophylactic and / or therapeutic agent for cystic kidney diseases. Provided is a prophylactic and / or therapeutic agent for cystic kidney diseases comprising a single-strand oligonucleotide that includes a base sequence which is complementary to at least a portion of a base sequence of miR-21.

Description

Preventive and / or therapeutic agent for cystic kidney disease

[0001] The present disclosure relates to a preventive and / or therapeutic agent for cystic kidney disease, etc. More specifically, the present disclosure relates to a preventive and / or therapeutic agent for cystic kidney disease, etc., comprising a single-stranded oligonucleotide comprising a base sequence complementary to at least a portion of the base sequence of miR-21.

[0002] Autosomal dominant polycystic kidney disease (PCKD), a type of cystic kidney disease, is a congenital disorder caused by a single gene mutation in PKD1 / 2. Its incidence rate is estimated at 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 renal enlargement, and approximately half of patients require maintenance dialysis by the time they reach their late 60s.

[0003] miR-21 is a miRNA involved in cell proliferation, apoptosis suppression, and fibrosis promotion, and antisense oligonucleotides (anti-miRNA oligos: AMOs) targeting miR-21 as a therapeutic target are also being researched and developed.

[0004] It has also been reported that knocking out miR-21 suppressed cyst growth in an orthologous mouse model of ADPKD (Non-Patent Document 1).

[0005] International Publication No. 2021 / 153762 International Publication No. 2023 / 140040

[0006] J Am Soc Nephrol. 2016 Aug;27(8):2319-30. doi: 10.1681 / ASN.2015060634. Epub 2015 Dec 17.

[0007] An objective of the present disclosure is to provide an agent for preventing and / or treating cystic kidney disease.

[0008] The inventors discovered that miR-21 expression was elevated in the kidney tissue of cystic disease model mice, and that antisense oligonucleotides to miR-21 inhibited cyst growth, and further improved the method.

[0009] The present disclosure includes, for example, the subject matter described in the following sections: Item 1. A preventive and / or therapeutic agent for cystic kidney disease, comprising a single-stranded oligonucleotide comprising a nucleotide sequence complementary to at least a portion of the nucleotide sequence of miR21. Item 1A. A method for preventing and / or treating cystic kidney disease, comprising administering to a subject in need thereof (preferably a subject with cystic kidney disease) a single-stranded oligonucleotide comprising a nucleotide sequence complementary to at least a portion of the nucleotide sequence of miR21. Item 1B. A single-stranded oligonucleotide comprising a nucleotide sequence complementary to at least a portion of the nucleotide sequence of miR21, for use in preventing and / or treating cystic kidney disease. Item 1C. Use of a single-stranded oligonucleotide comprising a nucleotide sequence complementary to at least a portion of the nucleotide sequence of miR21 for the manufacture of a preventive and / or therapeutic agent for cystic kidney disease. Item 1D. Use of a single-stranded oligonucleotide comprising a nucleotide sequence complementary to at least a portion of the nucleotide sequence of miR21 for the prevention and / or treatment of cystic kidney disease. Item 2. Item 1. The agent according to Item 1, wherein the single-stranded oligonucleotide comprises a modified nucleoside building block and / or a modified internucleoside bond. Item 3. The agent according to Item 2, wherein the modified nucleoside building block is an acyclic nucleoside building block and / or a locked artificial nucleic acid (LNA) building block, or the modified internucleoside bond is a phosphorothioate bond. Item 4. The agent according to Item 3, wherein the acyclic nucleoside building block is a building block represented by the following general formula (1): [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups.) Base represents a nucleic acid base.] Item 5. In general formula (1), R 1 is a hydrogen atom or a methyl group, and R 2Item 6. The agent according to any one of Items 1 to 5, wherein in the single-stranded oligonucleotide, the base complementary to at least one uracil selected from the group consisting of positions 5, 6, 8, 14, 17, 19, and 20 of the base sequence shown in SEQ ID NO: 1 is 2,6-diaminopurine, and / or the base complementary to at least one adenine selected from the group consisting of positions 7, 10, and 16 of the base sequence shown in SEQ ID NO: 1 is 2-thiouracil. Item 7. (1) The single-stranded oligonucleotide comprises a base sequence complementary to a base sequence of at least 16 bases, preferably at least 18 bases, in length of miR-21, and (2) the nucleoside building blocks constituting the single-stranded oligonucleotide are modified nucleoside building blocks represented by the following general formula (1): [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2(Except when both are organic groups). Base represents a nucleic acid base.], (3) in the single-stranded oligonucleotide, the base complementary to at least one uracil selected from the group consisting of positions 5, 6, 8, 14, 17, 19, and 20 of the base sequence shown in SEQ ID NO: 1 is 2,6-diaminopurine, (4) the base complementary to at least one adenine selected from the group consisting of positions 7, 10, and 16 of the base sequence shown in SEQ ID NO: 1 is 2-thiouracil, and (5) the number of phosphorothioate bonds relative to the number of internucleoside bonds in the single-stranded oligonucleotide is 20% or more. Item 7. Item 7. The agent according to Item 7, wherein the single-stranded oligonucleotide is: (iii-a): an oligonucleotide in which all nucleoside building blocks are SNA building blocks and / or L-aTNA building blocks and the base sequence consists of the base sequence shown in SEQ ID NO: 6; (iii-b): an oligonucleotide containing a base sequence in which one or more bases have been deleted, substituted or added in the base sequence of the oligonucleotide of (iii-a). Item 9. The agent according to Item 7, wherein the single-stranded oligonucleotide is: (ia): an oligonucleotide containing the base sequence shown in SEQ ID NO: 2; (ib): an oligonucleotide containing a base sequence in which one or more bases have been deleted, substituted or added in the base sequence of the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2; (ii-a): an oligonucleotide containing the base sequence GATAAG m CT (in the sequence, m (ii-b): an oligonucleotide comprising the base sequence of the oligonucleotide (ii-a) in which one or more bases are deleted, substituted, or added.

[0010] An agent for the prevention and / or treatment of cystic kidney disease is provided.

[0011] Figure 1 shows the results of measuring miR-21 expression levels in mouse kidney tissue (n = 3 in each group. *P < 0.05; t-test). Figure 2 shows the results of detecting miR-21 in mouse kidney tissue. Figure 3 shows the results of detecting miR-21 in human kidney tissue. Figure 4 shows the results of evaluating the pharmacokinetics of disulfoCy5-anti-miR-21 (SNA). Figure 5 shows the results of staining mouse kidney tissue. Figure 6 shows the results of measuring mouse kidney weight / body weight (KW / BW) and blood urea nitrogen (BUN) concentrations. Figure 7 shows the results of measuring mouse blood aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Figure 8 shows the results of detecting miR-21 in mouse kidney tissue after oligonucleotide administration (n = 5, *P < 0.05; one-way ANOVA). Figure 9 shows the results of measuring PPARα and BCL2 expression in mouse kidney tissue. Figure 10 shows the results of measuring TGF-β, SMAD7, and αSMA expression in mouse kidney tissue. This shows the results of measuring cyst size in 3D culture of primary cultured tubular cells from kidneys removed from human patients with cystic kidney disease. This shows the results of measuring creatinine (Cre) concentrations in mouse serum. This shows the results of measuring mouse kidney weight / body weight (KW / BW) and creatinine (Cre) concentrations in serum.

[0012] Each embodiment of the present disclosure will be described in more detail below. The preventive and / or therapeutic agent for cystic kidney disease included in the present disclosure includes 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 may be referred to as "the agent of the present disclosure" or the like. The single-stranded oligonucleotide may also be referred to as "the single-stranded oligonucleotide of the present disclosure" or the like.

[0013] The base sequence shown in SEQ ID NO: 1 is the base sequence (22 bases long) that constitutes miR-21.

[0014] The term "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 disclosure can form a complementary strand with miR-21, and is, for example, 6 to 22 bases in length. The lower limit of this range is not particularly limited, but is preferably 7 bases in length, more preferably 8 bases in length, even more preferably 10 bases in length, even more preferably 14 bases in length, particularly preferably 18 bases in length, and even more particularly preferably 21 bases in length.

[0015] As used herein, "complementary" refers not only to a perfect base complementarity (perfectly complementary: for example, A and T or U, G and C, etc., hybridize without mismatches), but also to a degree of complementarity that allows hybridization under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, typical post-hybridization washing conditions include 1×SSC, 0.1% SDS, and 37°C. It is preferable that the hybridized state is maintained even after washing under such conditions. Although not particularly limited, examples of more stringent hybridization conditions include approximately "0.5xSSC, 0.1% SDS, 42°C," and examples of even more stringent hybridization conditions include washing conditions of approximately "0.1xSSC, 0.1% SDS, 65°C."

[0016] As mentioned above, the complementary relationship is based on a one-to-one relationship between bases, so a certain base sequence X and a base sequence Y complementary to the base sequence X have the same base length.

[0017] The number of bases constituting the single-stranded oligonucleotide of the present disclosure is preferably, for example, about 6 to 25. The lower limit of this range is not particularly limited, but is preferably 7, more preferably 8, even more preferably 10, still more preferably 14, and particularly preferably 18. The upper limit of this range is not particularly limited, but is preferably 24, more preferably 23, even more preferably 22, and particularly preferably 21.

[0018] The single-stranded oligonucleotide of the present disclosure is an oligonucleotide containing a base sequence that has, for example, 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity, even more preferably 98% or more identity, still more preferably 99% or more identity, and particularly preferably 100% identity to a base sequence that is completely complementary to at least a portion of the base sequence of miR-21.

[0019] As used herein, the "identity" of a base sequence refers to the degree of match between the base sequences of two or more comparable base sequences. Therefore, the greater the match between two base sequences, the greater the identity or similarity between those sequences. The level of identity between base sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by 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, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known, and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0020] The single-stranded oligonucleotide of the present disclosure is preferably an oligonucleotide that does not form a self-duplex.Self-duplexes include, for example, intramolecular (hairpin) duplexes, intermolecular (dimer) duplexes, etc.The oligonucleotide that does not form a self-duplex does not, for example, contain a base sequence portion that can form a self-duplex; it can be obtained by introducing substitution and / or any change into the base sequence portion that can form a self-duplex.

[0021] The single-stranded oligonucleotides of the present disclosure can be DNA, RNA, etc., and can be based on these and contain modified nucleoside building blocks and / or modified internucleoside linkages.

[0022] The modified nucleoside building block is a building block corresponding to the nucleoside constituting the oligonucleotide, and is not particularly limited thereto. Examples of the modified nucleoside building block include molecules obtained by chemically modifying ribonucleosides, deoxyribonucleosides, DNA, RNA, etc., and examples thereof include nucleosides modified with 2'-OMe, nucleosides modified with 2'-MOE, and sugar-modified nucleoside building blocks such as LNA (registered trademark), ENA (registered trademark), AmNA (registered trademark), GuNA, and scpBNA.

[0023] Modified sugar refers to a sugar having a substitution and / or any change from a natural sugar moiety (i.e., the sugar moiety found in DNA (2'-H) or RNA (2'-OH)), and sugar-modified nucleoside refers to a modified nucleoside containing a modified sugar. The sugar-modified nucleoside may be any nucleoside in which any chemical structural substance has been added or substituted to a part or all of the chemical structure of the sugar of the nucleoside, and examples thereof include modified nucleosides substituted with 2'-O-methyl in the sugar moiety, modified nucleosides substituted with 2'-O-propyl, modified nucleosides substituted with 2'-methoxyethoxy, modified nucleosides substituted with 2'-O-methoxyethyl, modified nucleosides substituted with 2'-O-[2-(guanidium)ethyl], modified nucleosides substituted with 2'-O-fluoro, morpholino nucleic acids (commonly known as PMOs) which are modified nucleosides in which the sugar moiety is substituted with a morpholine ring, bridged nucleic acids (BNAs) which have two ring structures by introducing a bridged structure into the sugar moiety, and more specifically, locked artificial nucleic acids in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via a methylene. Examples of the modified nucleoside building block include cyclic nucleoside building blocks, such as ethylene-bridged artificial nucleic acids (LNA) and ethylene-bridged artificial nucleic acids (ENA) [Nucleic Acid Research, 32, e175 (2004)], as well as peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and peptide ribonucleic acids (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)]. As described below, acyclic nucleoside building blocks can also be used as modified nucleoside building blocks.

[0024] Other examples of modified nucleosides include those in which an atom (e.g., hydrogen atom, oxygen atom) or functional group (e.g., hydroxyl group, amino group) in the base portion of a nucleic acid is substituted with another atom (e.g., hydrogen atom, sulfur atom), functional group (e.g., amino group), or alkyl group having 1 to 6 carbon atoms, or those protected with a protecting group (e.g., methyl group or acyl group), and molecules in which another chemical substance, such as a lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, or dye, is attached to a nucleoside.

[0025] The modified nucleoside building block is preferably an LNA building block and / or an acyclic nucleoside building block, more preferably an acyclic nucleoside building block.

[0026] The modified nucleoside building blocks may be used alone or in combination of two or more.

[0027] The proportion of modified nucleoside constituent units relative to 100% of the nucleoside constituent units (number of nucleosides) constituting the single-stranded oligonucleotide of the present disclosure is, for example, 50% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, still more preferably 95%, and particularly preferably 100%.

[0028] In one embodiment, the single-stranded oligonucleotide of the present disclosure preferably comprises acyclic nucleoside structural units.The ratio of acyclic nucleoside structural units to 100% of the nucleoside structural units (nucleoside number) constituting the single-stranded oligonucleotide of the present disclosure is, for example, 50% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95%, particularly preferably 100%.

[0029] In the present specification, the term "acyclic nucleoside structural unit" refers to a structural unit corresponding to a nucleoside constituting an oligonucleotide, and is not particularly limited as long as it does not contain a sugar skeleton and has an acyclic skeleton. Representative examples of the acyclic nucleoside structural unit include those represented by the general formula (1):

[0030] [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 and the like are both organic groups.) Base represents a nucleic acid base.]

[0031] The organic group is not particularly limited, and examples thereof include hydrocarbon groups.

[0032] The hydrocarbon group is preferably a chain-like hydrocarbon group. Examples of the chain-like hydrocarbon group include alkyl groups, alkenyl groups, and alkynyl groups, with alkyl groups being preferred. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl, and 3-methylpentyl groups. Of these, a methyl group 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, even more preferably 1 to 4, still more preferably 1 to 2, and particularly preferably 1. Furthermore, alkyl groups containing an alkynyl group (—C≡C—, —C≡CH) at the end or inside thereof, which allows for the introduction of various functional groups by click reactions and the like, are even more preferred.

[0033] In addition to the above, the organic group can also be a monovalent group formed by removing one hydrogen atom or functional group from various molecules, such as molecules used to modify nucleosides. Examples of such molecules include polyethylene glycol chains, dye molecules, polycations (spermine), groove binders, amino groups, hydroxyl groups, thiol groups, metal ligands, photocleavable functional groups, and sugar chains. These can be linked directly or indirectly to the skeleton of the above-mentioned structural units. For example, they can be linked using a click reaction (e.g., the reaction between an alkyne and an azide, as described above).

[0034] In a preferred embodiment of the present disclosure, R 1is a hydrogen atom or a chain hydrocarbon group (preferably a methyl group), and R 2 is preferably a hydrogen atom. In other words, the acyclic nucleoside structural unit is preferably an SNA (Serinol Nucleic acid) structural unit (R 1 and R 2 is a hydrogen atom), and / or an L-aTNA (acyclic L-threoninol nucleic acid) building block (R 1 is a methyl group, and R 2 is a hydrogen atom).

[0035] As the nucleic acid base, any base constituting a nucleic acid can be used without any particular limitation. The base constituting a nucleic acid includes not only typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also other bases such as hypoxanthine (I), modified bases, etc. Modified bases include, for example, 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- ... Examples of amino acids that can be used include thiouracil, 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, and xanthine.

[0036] In general formula (1), *1 and *2 indicate the direction in which the oligonucleotide is constructed. 1 is an organic group and R 2 When R is a hydrogen atom, *1 is the 3' side and *2 is the 1' side. 1 is a hydrogen atom and R 2 When R is an organic group, *1 is the 1' side and *2 is the 3' side. 1 is a hydrogen atom and R 2 When is a hydrogen atom, *1 is the (S) side and *2 is the (R) side. The (S) end and (R) end (SNA in Chemical Formula 4) and the 3' end and 1' end correspond to the 5' end and 3' end of DNA (LNA in Chemical Formula 5), ​​respectively. For convenience, the (S) end and (R) end, the 3' end and 1' end may be referred to as the 5' end and the 3' end, respectively. When the modified nucleoside building block represented by general formula (1) is at the terminal, a hydrogen atom, a sulfur atom, or a phosphate group is added to *1 and *2, as described above.

[0037] Modified internucleoside linkages refer to internucleoside linkages that have been substituted or have any change from naturally occurring internucleoside linkages (i.e., phosphodiester linkages), and include internucleoside linkages that contain a phosphorus atom and internucleoside linkages that do not contain a phosphorus atom. Modified internucleoside linkages may be those in which any chemical substance has been added to or substituted for part or all of the chemical structure of the phosphodiester bond of a nucleotide. Examples of modified internucleoside linkages include phosphorothioate linkages (also called thiophosphate linkages), phosphorodithioate linkages, phosphotriester linkages, methylphosphonate linkages, methylthiophosphonate linkages, boranophosphate linkages, and phosphoramidate linkages. This is expected to prevent degradation by hydrolases such as nucleases.

[0038] A particularly preferred example of the modified internucleoside bond is a phosphorothioate bond.

[0039] The modified internucleoside bond may be employed singly or in combination of two or more types.

[0040] The number of modified internucleoside bonds relative to the number of internucleoside bonds (100%) of the single-stranded oligonucleotide of the present disclosure is not particularly limited, but is, for example, 20% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more. In one embodiment, all internucleoside bonds can be modified internucleoside bonds (for example, phosphorothioate bonds).

[0041] In one embodiment, in the single-stranded oligonucleotide of the present disclosure, the base complementary to at least one uracil in the base sequence set forth in SEQ ID NO: 1 may be 2,6-diaminopurine, and / or the base complementary to at least one adenine in the base sequence set forth in SEQ ID NO: 1 may be 2-thiouracil. The complementary relationship refers to a relationship in which miR21 and the single-stranded oligonucleotide of the present disclosure are aligned in a complementary manner, resulting in opposing pairs. The opposing pairs of 2,6-diaminopurine and 2-thiouracil are expected to suppress the formation of a self-duplex of the single-stranded oligonucleotide while further enhancing its binding affinity to miR-21.

[0042] Examples of uracils in the base sequence shown in SEQ ID NO: 1 include uracils at positions 1, 5, 6, 8, 14, 17, 19, and 20. In the single-stranded oligonucleotide of the present disclosure, the base complementary to at least one uracil selected from the group consisting of 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 may be 2,6-diaminopurine. The ratio of 2,6-diaminopurine contained in the single-stranded oligonucleotide of the present disclosure to the number of uracils (100%) contained in at least a portion of the base sequence of miR-21 is, for example, 40 to 100%, preferably 50 to 100%, and more preferably 60 to 100%.

[0043] Examples of adenines in the base sequence shown in SEQ ID NO: 1 include adenines at positions 2, 7, 10, 12, 16, and 22. In the single-stranded oligonucleotide of the present disclosure, it is preferable that the base complementary to at least one adenine selected from the group consisting of positions 7, 10, and 16 is 2-thiouracil. For example, the bases complementary to the adenines at positions 7, 10, and 16 may be 2-thiouracil. The ratio of 2-thiouracil contained in the single-stranded oligonucleotide of the present disclosure to the number of adenines contained in at least a portion of the base sequence of miR21 (100%) is, for example, 20 to 100%, preferably 30 to 100%, and more preferably 40 to 100%.

[0044] In one embodiment, the single-stranded oligonucleotide of the present disclosure preferably has one or more pairs of opposing 2,6-diaminopurine and 2-thiouracil. More preferably, two pairs, and even more preferably, three pairs. The opposing relationship may be within the single-stranded oligonucleotide of the present disclosure, or may be between single-stranded oligonucleotides of the present disclosure. Of these, the opposing relationship within the single-stranded oligonucleotide of the present disclosure is preferred.

[0045] A preferred embodiment of the single-stranded oligonucleotide of the present disclosure includes, for example, an oligonucleotide (SEQ ID NO: 6) having the base sequence (S)-C DDC DsUCDG T CsUGDsU DDG CTA- (R) (in the sequence, D represents 2,6-diaminopurine, and sU represents 2-thiouracil).

[0046] A preferred embodiment of the single-stranded oligonucleotide of the present disclosure is an oligonucleotide that satisfies at least one, two, three, or four, or all (five) of the following (1) to (5): (1) the single-stranded oligonucleotide contains a base sequence that is complementary to a base sequence of at least 18 bases in length of miR-21, (2) the nucleoside building blocks that constitute the single-stranded oligonucleotide are modified nucleoside building blocks represented by the following general formula (1): [In the formula: R 1 and R 2are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups). Base represents a nucleic acid base.] (3) in the single-stranded oligonucleotide, the base complementary to at least one uracil selected from the group consisting of positions 5, 6, 8, 14, 17, 19, and 20 of the base sequence shown in SEQ ID NO: 1 is 2,6-diaminopurine, (4) the base complementary to at least one adenine selected from the group consisting of positions 7, 10, and 16 of the base sequence shown in SEQ ID NO: 1 is 2-thiouracil, (5) the number of phosphorothioate bonds relative to 100% of the number of internucleoside bonds in the single-stranded oligonucleotide is 20% or more.

[0047] Preferred embodiments of the single-stranded oligonucleotide of the present disclosure include, for example, an oligonucleotide (SEQ ID NO: 2) having the base sequence (S)-C DDC DsUCDG T CsUGDsU DDG CTA- (R) (in the sequence, D represents 2,6-diaminopurine and sU represents 2-thiouracil), in which all nucleoside building blocks are SNA building blocks and all internucleoside linkages are phosphorothioate linkages; an oligonucleotide (SEQ ID NO: 3) having the base sequence (S)-C DDC DsUCDG T CsUGDsU DDG CTA- (R) (in the sequence, D represents 2,6-diaminopurine and sU represents 2-thiouracil), in which all nucleoside building blocks are L-aTNA building blocks and all internucleoside linkages are phosphorothioate linkages; DDG CT*A-(R) (SEQ ID NO: 5) (in the sequence, D represents 2,6-diaminopurine, sU represents 2-thiouracil, and the * between bases indicates that the bond between nucleosides is a phosphorothioate bond), and all nucleoside building blocks are SNA building blocks (SEQ ID NO: 5). In this specification, these oligonucleotides may be referred to as oligonucleotides consisting of the base sequence shown in SEQ ID NO: 2, oligonucleotides consisting of the base sequence shown in SEQ ID NO: 3, etc.

[0048] Furthermore, a preferred embodiment of the single-stranded oligonucleotide of the present disclosure includes, for example, an oligonucleotide comprising a base sequence in which one or more bases have been deleted, substituted, or added in the base sequence of the oligonucleotide shown in SEQ ID NO: 2, 3, 5, or 6. The upper limit of the number of deleted, substituted, or added bases is not particularly limited as long as the single-stranded oligonucleotide of the present disclosure comprises a base sequence complementary to at least a portion of the base sequence of miR-21, and may be, for example, 10, 9, 8, 7, 6, 5, 4, or 3 bases.

[0049] A preferred embodiment of the single-stranded oligonucleotide of the present disclosure is, for example, a single-stranded oligonucleotide having a base sequence of 5'-GAT AAG m CT-3' (in the sequence m C represents 5-methylcytosine), and oligonucleotides in which all nucleoside building blocks are LNA building blocks and all internucleoside linkages are phosphorothioate linkages.

[0050] In addition, a preferred embodiment of the single-stranded oligonucleotide of the present disclosure is, for example, a single-stranded oligonucleotide having a base sequence of 5'-GAT AAG m CT-3' (in the sequence m (C represents 5-methylcytosine.) Examples of oligonucleotides include oligonucleotides containing a base sequence in which one or more bases have been deleted, substituted, or added in an oligonucleotide in which all nucleoside building blocks are LNA building blocks and all internucleoside linkages are phosphorothioate bonds. The upper limit of the number of deleted, substituted, or added bases is not particularly limited as long as the single-stranded oligonucleotide of the present disclosure contains a base sequence complementary to at least a portion of the base sequence of miR-21, and may be, for example, 4 or 3.

[0051] The single-stranded oligonucleotide of the present disclosure may be linked to another molecule. Examples of other molecules include, but are not limited to, fluorescent labels, biotin, azide, and ethynyl groups. Examples of fluorescent labels include fluorescein, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM™, Cy®3, Cy®5, and the Alexa Fluor® series.

[0052] The single-stranded oligonucleotide of the present disclosure can be easily prepared according to known genetic engineering techniques, techniques for producing artificial nucleic acids, etc. For example, it can be prepared using PCR, restriction enzyme cleavage, nucleic acid ligation techniques, etc.

[0053] The agent of the present disclosure preferably contains the single-stranded oligonucleotide of the present disclosure as an active ingredient.

[0054] The content of the single-stranded oligonucleotide of the present disclosure in the agent of the present disclosure is not particularly limited, but can be, for example, about 0.0001 to 100% by weight.

[0055] The agent of the present disclosure contains the single-stranded oligonucleotide of the present disclosure and may further contain other ingredients, such as pharmacologically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0056] The agent of the present disclosure can be used for the prevention and / or treatment of cystic kidney disease.

[0057] Cystic kidney diseases include, for example, polycystic kidney disease. Examples of polycystic kidney disease include autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). Among these, ADPKD is preferred.

[0058] The agent of the present disclosure can be applied to, for example, humans and non-human mammals (for example, rats, mice, rabbits, cows, pigs, dogs, cats, sheep, monkeys, etc.).

[0059] The agent of the present disclosure can be in any dosage form, for example, oral formulations such as 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 drinks, suspensions, syrups), and jellies; or parenteral formulations such as injectable formulations (e.g., drip injections (e.g., intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye drops, eye ointments, nasal drops, and ear drops. Furthermore, the active ingredient can be administered in a complexed state with particles (e.g., lipid particles or exosomes) or encapsulated in the particles.

[0060] The route of administration of the agent of the present disclosure is not particularly limited as long as the desired effect can be obtained, and examples include oral administration; enteral administration such as tube feeding and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and nasal administration.

[0061] The dosage of the agent of the present disclosure is not particularly limited as long as it is an effective amount that exerts a medicinal effect, and is typically 0.1 to 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day in terms of the weight of the active ingredient, when administered orally, and 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day in terms of the weight of the active ingredient, when administered parenterally. The dosage and administration interval can be increased or decreased as appropriate depending on the age, pathological condition, symptoms, etc.

[0062] Without wishing to be bound by theory, it is believed that the single-stranded oligonucleotides of the present disclosure can prevent and / or treat cystic kidney disease by inhibiting the growth of kidney and / or liver cysts through promoting the expression of PPARα, suppressing fibroblasts (e.g., promoting the expression of SMAD7, suppressing the expression of TGF-β, and / or suppressing the expression of αSMA), and / or suppressing the expression of the anti-apoptotic protein BCL2.

[0063] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure encompasses any and all combinations of the constituent elements described in this specification.

[0064] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0065] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0066] Male DBA / 2 mice (SLC Japan) and male DBA / 2FG-pcy mice (Kyudo Co., Ltd.) were used as animal models of cystic disease. This study was approved by the Nagoya University School of Medicine Animal Care and Use Committee and conducted in accordance with the Nagoya University School of Medicine Animal Experiment Guidelines.

[0067] Experimental Design: Male DBA / 2 and male DBA / 2FG-pcy mice were injected subcutaneously with the target drug at a dose of 10 mg / kg three times a week for 6 weeks from 4 to 10 weeks of age. Under isoflurane anesthesia, the drug was injected dorsally into the subcutaneous tissue under the left scapula. After a total of 18 injections over 6 weeks, the mice were sacrificed and kidney weight / body weight (KW / BW), blood tests, and renal tissue were evaluated.

[0068] Human tissues: Normal human kidney tissues and kidney tissues from ADPKD patients were collected at Nagoya University Hospital. Patients gave informed consent for research use in accordance with the hospital's regulations.

[0069] qRT-PCR analysis. Total RNA was extracted from tissue samples using 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). Targets were amplified by real-time PCR using the Step One Plus Real-Time PCR System (Thermo Fisher Scientific) and Fast Advanced Master Mix (Thermo Fisher Scientific). All samples were run in duplicate. Quantitative evaluation of target expression was performed using the ΔΔCT method, and miRNA expression levels were normalized to those of U6 snRNA.

[0070] miRNAscope Assay: The miRNAscope HD Reagent Kit-RED (Advanced Cell Diagnostics) was used to detect miR-21 in tissues. The miRNAscope Positive Control Probe-SR-RNU6-S1 (Advanced Cell Diagnostics) was used as the positive control probe, and the miRNAscope Negative Control Probe-SR-Scramble-S1 (Advanced Cell Diagnostics) was used as the negative control probe. The miRNAscope Probe-SR-mmu-miR-21a-5q-S1 (Advanced Cell Diagnostics) was used to detect miR-21 in mouse kidney tissue. The miRNAscope Probe-SR-hsa-miR-21-5p-S1 (Advanced Cell Diagnostics) was used to detect miR-21 in human kidney tissue. Tissue specimens stained with the kit were observed using a BZ-X810 (KEYENCE).

[0071] Anti-miR-21 (SNA), anti-miR-21 (LNA), Scramble The miR-21 antisense oligonucleotides used as target drugs in the tests shown in Figures 4 to 13 below (anti-miR-21 (SNA) and anti-miR-21 (LNA): Figures 4 to 11, Scramble: Figures 4 to 13) are as follows. The structural formula of the SNA used is shown below. The base sequences of anti-miR-21 (SNA; Serinol Nucleic acid) and Scramble are as follows. In both cases, all constituent nucleotides are SNA. Anti-miR-21 (SNA) (S-D7sU3): (S) -C DDC DsUCDG T CsUGDsU DDG CTA- (R) (SEQ ID NO: 2) Scramble: (S) -TAT GAT GTC CAT GTC GTA CGC- (R) (SEQ ID NO: 4) In the base sequence of anti-miR-21 (SNA), D represents 2,6-diaminopurine and sU represents 2-thiouracil. Anti-miR-21 (SNA) and Scramble were synthesized by the method described in Patent Document 2 (a method using a base-removable protecting group) and provided by the Asanuma Laboratory, School of Engineering, Nagoya University.

[0072] The structural formula of the LNA used is shown below. The base sequence of anti-miR-21 (LNA; Locked Nucleic Acid) is as follows. All constituent nucleotides are LNA. Anti-miR-21 (LNA): 5'-GAT AAG m CT- 3' (in the sequence m (C indicates 5-methylcytosine.) Anti-miR-21 (LNA) was purchased from Gene Design. All internucleoside bonds in anti-miR-21 (SNA), Scramble, and anti-miR-21 (LNA) were phosphorothioated (PS) as shown in the structural formulas above.

[0073] S-45371 The miR-21 antisense oligonucleotide (S-45371) used as the target drug in the test shown in Figure 12 below is as follows. All constituent nucleotides are SNA. S-45371: (S)-C* D*D*C* DsUCDG T*C*sU*GDsU DDG CT*A-(R) (SEQ ID NO: 5). In the base sequence of S-45371, D and sU are as described above. An * between bases indicates a phosphorothioate bond between nucleosides, and the absence of an * between bases indicates a phosphodiester bond between nucleosides. S-45371 was synthesized in the same manner as anti-miR-21 (SNA) and Scramble.

[0074] T-D7sU3(PS) The miR-21 antisense oligonucleotide (T-D7sU3(PS)) used as the target drug in the test shown in Figure 13 below is as follows. All constituent nucleotides are L-aTNA. T-D7sU3(PS): (S)-C DDC DsUCDG T CsUGDsU DDG CTA-(R) (SEQ ID NO: 3). In the base sequence of T-D7sU3(PS), D and sU are as described above. All internucleoside bonds are phosphorothioate bonds. T-D7sU3(PS) was synthesized in the same manner as anti-miR-21(SNA) and Scramble.

[0075] Ex vivo imaging: Anti-miR-21 (SNA) was labeled with disulfoCy5 (provided by the 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, and data were collected in photons per second. -1 cm -2 The fluorescent tissue specimens were observed using a BZ-X810 (KEYENCE) and a SpinSR10 (Olympus Life Science).

[0076] Histological Analysis Organs were fixed in 10% paraformaldehyde, embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin.

[0077] Blood biochemistry analysis Serum blood urea nitrogen (BUN), serum creatinine (Cre), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) concentrations were measured using an automatic chemistry analyzer (SRL Corporation, Tokyo, Japan).

[0078] 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.

[0079] Three-dimensional culture of renal tubular cells and evaluation of the effect of antisense transfection on cyst formation suppression Madin-Darby canine kidney (MDCK) cells were purchased from ATCC (CCL-34; American Type Culture Collection) and cultured in Eagle's Minimum Essential Medium (051-07615; FUJIFILM WAKO) supplemented with 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. Primary human ADPKD tubule cells were prepared from human ADPKD kidneys obtained from patients at Nagoya University Hospital. MDCK2 cells and primary human ADPKD tubule cells were cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle medium / nutrient mixture F-12 (048-29785; FUJIFILM WAKO) supplemented with L-glutamine, 15 mM HEPES (D8437; Sigma-Aldrich), 1% penicillin-streptomycin (15140122; Thermo Fisher Scientific), and 1% insulin-transferrin-selenium (41400045; Thermo Fisher Scientific). MDCK2 cells and primary human ADPKD tubule cells were plated at 0.3 × 10 cells / well in a 6-well plate. 6 Cells were seeded at a density of 1.0 × 10 cells / mL. Two days later, when the cells reached 80% confluence, they were transfected with Scramble-SNA and Anti-miR-21-SNA using Lipofectamine 3000 Transfection Reagent (L3000008; Invitrogen). After 48 hours, the cells were harvested and plated at 1.0 × 10 cells / mL on Matrigel (356234; CORNING) in a 24-well plate. 4 Cells were seeded at a density of 1000 cells / mL and treated with 10 μM forskolin (067-2191; FUJIFILM WAKO) for 48 hours. Cells were then observed under a BZ-X810 microscope (KEYENCE). Images were taken randomly, and the size and number of cysts were measured. Cyst and cell area were analyzed using Image J (13.0.6).

[0080] The expression of miR-21 in the kidney tissues of DBA / 2 and DBA / 2FG-pcy mice was analyzed by qRT-PCR. As shown in Figure 1, elevated miR-21 expression was confirmed in the kidney tissues of the polycystic kidney disease model mice.

[0081] The expression of miR-21 in kidney tissue from DBA / 2 and DBA / 2FG-pcy mice was analyzed using miRNAscope. As shown in Figure 2, miR-21 expression was confirmed to be elevated in kidney tissue from cystic disease model mice compared to normal mouse kidney tissue. Furthermore, the expression of miR-21 in normal human kidney tissue and kidney tissue from ADPKD patients was analyzed using miRNAscope. As shown in Figure 3, miR-21 expression was also confirmed to be elevated in kidney tissue from ADPKD patients compared to normal human kidney tissue. These results confirm that miR-21 expression is elevated in kidney tissue in cystic kidney disease and is involved in the growth of renal cysts.

[0082] When the pharmacokinetics of SNA was evaluated in mice administered disulfoCy5-anti-miR-21 (SNA), strong accumulation in the kidney was observed, and uptake into renal tubular cells was confirmed, as shown in Figure 4.

[0083] Staining of kidney tissue from the non-treated, Scramble-treated, and anti-miR-21 (SNA)-treated groups revealed that cyst growth was suppressed in the SNA-treated group, as shown in Figure 5. In addition, although there was greater individual variation compared to the SNA-treated group, some individuals in the anti-miR-21 (LNA)-treated group showed a similar effect.

[0084] We compared kidney weight / body weight (KW / BW) and blood urea nitrogen (BUN) concentrations in the non-treated, scramble-treated, and anti-miR-21 (SNA)-treated groups. As shown in Figure 6, the SNA-treated group showed a decrease in KW / BW and BUN concentrations, as well as suppression of cyst growth and improvement of renal function. No liver dysfunction was observed (Figure 7). Although there was greater individual variation in the anti-miR-21 (LNA)-treated group compared to the SNA-treated group, some individuals showed similar effects.

[0085] The expression of miR-21 in kidney tissues of the non-treated group, the Scramble group, and the anti-miR-21 (SNA) group was analyzed using miRNAscope. As shown in Figure 8, the expression of miR-21 was confirmed to be reduced in the SNA group.

[0086] The expression of PPARα and BCL2 was evaluated in kidney tissue from the non-treated, scrambled, and anti-miR-21 (SNA) groups. PPARα expression is known to be downregulated by miR-21 and contribute to cyst growth via mitochondrial metabolism. The expression of the anti-apoptotic protein BCL2 is also known to be upregulated by miR-21 and contribute to cyst growth. As shown in Figure 9, the SNA group demonstrated enhanced PPARα expression and suppressed BCL2 expression. The anti-miR-21 (LNA) group also demonstrated similar effects, although there was greater individual variation compared to the SNA group.

[0087] The expression of TGF-β, SMAD7, and αSMA was evaluated in kidney tissue from the non-treated group, the Scramble group, and the anti-miR-21 (SNA) group. It is known that SMAD7 expression is suppressed by miR-21, while TGF-β and αSMA expression are increased by miR-21, contributing to fibrosis. As shown in Figure 10, the SNA group showed enhanced SMAD7 expression and suppressed TGF-β and αSMA expression. The anti-miR-21 (LNA) group also showed similar effects, although there was greater individual variation compared to the SNA group.

[0088] Renal tubule cells were cultured in primary culture from kidneys removed from patients with human cystic kidney disease, and then cultured in 3D. The effects of adding Scramble and anti-miR-21 (SNA) on the suppression of cyst formation in these cultures were evaluated. As shown in Figure 11, the SNA-administered group demonstrated suppression of cyst formation.

[0089] Serum creatinine (Cre) levels were compared between the Scramble and S-45371 groups. As shown in Figure 12, Cre levels were reduced in the SNA (S-45371) group, indicating improvement in renal function.

[0090] The kidney weight / body weight (KW / BW) and serum creatinine (Cre) concentrations were compared between the Scramble and T-D7sU3(PS) groups. As shown in Figure 13, the KW / BW and Cre concentrations were reduced in the SNA(T-D7sU3(PS)) group, indicating suppression of cyst growth and improvement of renal function.

Claims

1. A preventive and / or therapeutic agent for cystic kidney disease, comprising a single-stranded oligonucleotide comprising a base sequence complementary to at least a portion of the base sequence of miR-21.

2. The agent of claim 1, wherein the single-stranded oligonucleotide comprises modified nucleoside building blocks and / or modified internucleoside linkages.

3. The agent according to claim 2, wherein the modified nucleoside building block is an acyclic nucleoside building block, or the modified internucleoside bond is a phosphorothioate bond.

4. The agent according to claim 3, wherein the acyclic nucleoside constituent unit is a constituent unit represented by the following general formula (1): [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups.) Base represents a nucleic acid base.) 5. In general formula (1), R 1 is a hydrogen atom or a methyl group, and R 2 The agent according to claim 4, wherein is a hydrogen atom.

6. The agent according to claim 1 or 2, wherein in the single-stranded oligonucleotide, a base complementary to at least one uracil selected from the group consisting of positions 5, 6, 8, 14, 17, 19, and 20 of the base sequence shown in SEQ ID NO: 1 is 2,6-diaminopurine, and / or a base complementary to at least one adenine selected from the group consisting of positions 7, 10, and 16 of the base sequence shown in SEQ ID NO: 1 is 2-thiouracil.

7. (1) The single-stranded oligonucleotide comprises a base sequence complementary to at least 18 bases of the base sequence of miR-21; (2) the nucleoside building block constituting the single-stranded oligonucleotide is a modified nucleoside building block represented by the following general formula (1): [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (excluding the case where both are organic groups), and Base represents a nucleic acid base.], (3) in the single-stranded oligonucleotide, a base complementary to at least one uracil selected from the group consisting of positions 5, 6, 8, 14, 17, 19, and 20 of the base sequence shown in SEQ ID NO: 1 is 2,6-diaminopurine, (4) a base complementary to at least one adenine selected from the group consisting of positions 7, 10, and 16 of the base sequence shown in SEQ ID NO: 1 is 2-thiouracil, and (5) the number of phosphorothioate bonds relative to 100% of the number of internucleoside bonds in the single-stranded oligonucleotide is 20% or more.

8. The agent according to claim 7, wherein the single-stranded oligonucleotide is: (iii-a): an oligonucleotide in which all nucleoside building blocks are SNA building blocks and / or L-aTNA building blocks and the base sequence consists of the base sequence shown in SEQ ID NO: 6; (iii-b): an oligonucleotide containing a base sequence in which one or more bases have been deleted, substituted or added in the base sequence of the oligonucleotide (iii-a).

9. The single-stranded oligonucleotide is: (ia): an oligonucleotide having the base sequence shown in SEQ ID NO: 2; (ib): an oligonucleotide having a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of the oligonucleotide having the base sequence shown in SEQ ID NO: 2; (ii-a): an oligonucleotide having the base sequence GATAAG m CT (in the sequence, m The agent according to claim 1 or 2, which is an oligonucleotide in which all nucleoside building blocks are LNA building blocks and all internucleoside bonds are phosphorothioate bonds, or (ii-b): an oligonucleotide comprising a base sequence in which one or more bases have been deleted, substituted or added in the base sequence of the oligonucleotide (ii-a).

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