Oligonucleotide for mir-204, and composition for alleviating, preventing or treating osteoarthritis, comprising same

Abstract

The present specification provides an oligonucleotide specifically hybridized with miR-204, and a composition for alleviating, preventing or treating osteoarthritis, comprising same. The oligonucleotide according to one aspect of the present invention is designed to have a structure in which miR-204 degradation efficacy and formulation stability are optimized, and effectively blocks pathophysiological processes of osteoarthritis such as decreased cartilage matrix synthesis and increased inflammatory response, and thus is useful for preventing or treating osteoarthritis, and exhibits excellent delivery ability to chondrocytes and stable retention characteristics in tissues, thereby providing a structural advantage of implementing a long-lasting therapeutic effect and safety suitable for use as a pharmaceutical composition since the immunogenicity-inducing potential and toxicity are low, and enabling miR-204 in canine-derived chondrocytes to be effectively inhibited such that the oligonucleotide can be used as an animal therapeutic agent and / or a feed composition.

Description

Oligonucleotide for miR-204, and a composition for improving, preventing, or treating osteoarthritis comprising the same

[0001] This application claims priority to Korean Patent Application No. 10-2024-0198889, filed on December 27, 2025, the entire contents of which are incorporated by reference into this application. Additionally, this application claims priority to Korean Patent Application No. 10-2025-0194238, filed on December 9, 2025, the entire contents of which are incorporated by reference into this application.

[0002] The present specification discloses an oligonucleotide that specifically hybridizes with miR-204, and a composition for improving, preventing, or treating osteoarthritis comprising the same.

[0003] Description of government-funded research projects

[0004] [National R&D projects that supported this invention]

[0005] [Project ID] 2420016229

[0006] [Project No.] RS-2024-00437503

[0007] [Ministry Name] Ministry of SMEs and Startups

[0008] [Name of Project Management (Specialized) Agency] Korea Technology Information Promotion Agency for SMEs

[0009] [Research Project Name] Startup Growth Technology Development

[0010] [Project Title] Development of a Next-Generation Osteoarthritis Treatment Based on MicroRNA Degrader

[0011] [Project Performing Organization Name] Lifeflex Science Co., Ltd.

[0012] [Research Period] 2024.07.01 ~ 2027.06.30

[0013] Osteoarthritis (OA) is a disease in which the number of patients increased by 48% globally between 1990 and 2019, affecting 4.18 million people in Korea alone as of 2022. The demand for osteoarthritis treatment is surging due to the increase in patients resulting from an aging population and growing interest in quality of life. Cartilage is a tissue that undergoes progressive degeneration when damaged by various etiological risk factors such as aging, mechanical overload, obesity, and metabolic diseases; osteoarthritis is known to develop as a result of cartilage degeneration. It is characterized by pathological changes such as cartilage degeneration, subchondral bone progression, osteophyte formation, and synovial inflammation.

[0014] Existing research on osteoarthritis treatment has been limited to slowing cartilage degeneration by blocking the "degradation" mechanism of the extracellular matrix, and treatment strategies rely on prescribing anti-inflammatory drugs or artificial joint replacement surgery due to the lack of treatments that fundamentally improve the disease.

[0015] miR-204 is a type of microRNA, specifically referring to miR-204-5p. miR-204 is a microRNA whose expression levels significantly increase in the cartilage of various osteoarthritis species. It exacerbates osteoarthritis by simultaneously inhibiting multiple sGAG synthetases that constitute the cartilage matrix and contributing to the formation of SASPs. Since the therapeutic effect of inhibiting miR-204 on osteoarthritis is known, it is now time to develop therapeutic drugs that effectively "degrade" miR-204 with minimal side effects. However, despite the importance of sGAG synthesis reduction and SASPs induced by miR-204 in the pathogenesis of osteoarthritis, no effective therapeutic agent has been developed to date that specifically binds to miR-204 to restore cartilage matrix synthesis or inhibit inflammation-related factors.

[0016] The inventors identified miR-204 as a biomarker for improving, preventing, or treating osteoarthritis and conducted research on nucleic acid therapeutics targeting it. As a result, they confirmed that an oligonucleotide specifically hybridizes with miR-204, and that a nucleotide at a specific position among the nucleotides constituting the oligonucleotide contains a fixed sugar modification or a non-fixed 2'-substituted sugar modification, thereby providing excellent effects in improving, preventing, or treating osteoarthritis by degrading miR-204.

[0017] Accordingly, in one aspect, the object of the present invention is to provide an oligonucleotide that specifically hybridizes with miR-204 and has an excellent degradation effect on miR-204.

[0018] In another aspect, the object of the present invention is to provide a composition for improving, preventing, or treating osteoarthritis comprising the oligonucleotide.

[0019] In one aspect, the present invention relates to an oligonucleotide that specifically hybridizes with miR-204, wherein the oligonucleotide comprises 20 to 22 nucleotides linked together and is composed of a first region, a second region, and a third region in the order of 5' end to 3' end, wherein the first region comprises three nucleotides linked together that are all complementary to the tail region of miR-204 and include a fixed sugar modification, wherein the second region comprises three nucleotides linked together, wherein the nucleotides constituting the second region each independently include a fixed sugar modification or a non-fixed 2'-substituted sugar modification, wherein the third region comprises a plurality of nucleotides linked together, wherein the nucleotides constituting the third region each independently include a non-fixed 2'-substituted sugar modification, and wherein the oligonucleotide comprises 30% or less of nucleotides including a fixed sugar modification based on the total number of nucleotides. Provides oligonucleotides.

[0020] In another aspect, the present invention provides a composition for improving, preventing, or treating osteoarthritis, comprising the oligonucleotide.

[0021] An oligonucleotide according to one aspect of the present invention is an antisense oligonucleotide that reduces the expression of miR-204 by inducing its degradation, and is designed to have a structure with optimized miR-204 inhibitory efficacy and formulation stability by comparing and verifying various chemical modifications, such as sugar modification, length adjustment, and the number and positioning of fixed sugar modifications.

[0022] Accordingly, the antisense oligonucleotide of the present invention effectively blocks pathophysiological processes of osteoarthritis, such as reduced cartilage matrix synthesis and increased inflammatory response, and is useful for pain relief, prevention, or treatment of osteoarthritis.

[0023] In addition, the oligonucleotide according to one aspect of the present invention exhibits excellent delivery capacity to chondrocytes and stable retention characteristics within the tissue, thereby providing structural advantages that enable long-term sustained therapeutic effects.

[0024] An oligonucleotide according to one aspect of the present invention provides safety suitable for use in pharmaceutical compositions due to its low immunogenicity potential and toxicity.

[0025] An oligonucleotide according to one aspect of the present invention has been confirmed to effectively inhibit miR-204 not only in human chondrocytes but also in canine-derived chondrocytes. Furthermore, since the nucleotide sequence of miR-204 is conserved among various species such as humans, dogs, and monkeys, the oligonucleotide can be utilized as a therapeutic agent and / or a feed additive composition in various animal species.

[0026] Since the sequence of the target miR-204 is conserved across species, the oligonucleotide according to one aspect of the present invention can be utilized in human-derived chondrocytes or in chondrocytes of other species (e.g., dogs, monkeys, etc.) as well as in humans or other species.

[0027] Figure 1 is an experimental example of the present invention, and shows the results of measuring the miR-204 degradation effect in a C28 / I2 chondrocyte cell line by treatment of Example 1 according to Experimental Example 1 using qRT-PCR.

[0028] Figure 2 shows the results of confirming the delivery efficiency of Example 1 in mouse-derived chondrocytes according to one experimental example of the present invention. Figure 2a is a fluorescence microscope image of the fluorescently labeled Example 1 accumulated in mouse-derived chondrocytes, and Figures 2b to 2c are the results of quantifying the ratio of Cy3 fluorescently labeled Example 1 positive cells confirmed through flow cytometry.

[0029] Figure 3 is a fluorescence microscope image confirming the delivery efficiency of Example 1 in cartilage tissue derived from an osteoarthritis patient using fluorescent label Example 1 according to one experimental example of the present invention.

[0030] Figure 4 is a graph showing the results of measuring the expression level of miR-204 by qRT-PCR when treated with Full DNA or ASO containing sequence mismatch (Examples 3-1 to 3-3) according to Experimental Example 4, which is an experimental example of the present invention.

[0031] Figure 5 is a graph showing the results of measuring the expression level of miR-204 by qRT-PCR when treated with ASO (Examples 4-1 and 4-2) in which one or two bases are deleted from the 5' end of Full DNA or microRNA, respectively, according to Experimental Example 5, which is an experimental example of the present invention.

[0032] Figures 6 and 7 are graphs showing experimental results of measuring the expression level of miR-204 by qRT-PCR to determine whether there is a difference in miR-204 resolution depending on whether each nucleotide included in the first and second regions of an ASO specific to miR-204 according to one embodiment of the present invention includes a fixed sugar modification (Full DNA, Examples 5-1 to 5-7, and Comparative Examples 1-1 and 1-2).

[0033] FIG. 8 is a graph showing the experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution in the case where an ASO specific to miR-204 includes an LNA at one or more of the 1st to 6th positions in the direction from the 3' end to the 5' end (Example 1, Comparative Examples 2-1 to 2-4) as a comparative example of the present invention.

[0034] FIGS. 9 and 10 are graphs showing experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution when, compared to Example 1, which is Full DNA or an ASO according to an embodiment of the present invention, an ASO specific to miR-204 as a comparative example of the present invention randomly contains LNA and / or has a shorter length than the ASO of an embodiment of the present invention (Comparative Examples 3 and 4).

[0035] FIGS. 11a and 11b are a graph (Fig. 11a) showing experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm miR-204 degradation ability when an ASO specific to miR-204 contains 15 or more LNAs as a comparative example of the present invention (Comparative Examples 5-1 to 5-3), and a photograph (Fig. 11b) taken to confirm formulation stability when the ASO is dissolved.

[0036] FIGS. 12 and 13 are graphs showing experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm miR-204 resolution when an ASO specific to miR-204 according to one embodiment of the present invention contains one or more nucleotides including MOE or OME instead of LNA in the second region (Examples 6-1 to 6-14).

[0037] FIG. 14 is a graph showing the experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm miR-204 resolution when an ASO specific to miR-204 according to one embodiment of the present invention is composed of a PO bond (backbone) instead of a PS bond (backbone) (Example 2).

[0038] FIG. 15 is a graph showing pathway enrichment results in the form of a bubble plot based on transcriptome analysis results between the Example 1 treatment group and the vehicle control group according to one embodiment of the present invention. In FIG. 15, the size of the dot represents the number of genes included in the corresponding pathway, and the color represents the fold enrichment value.

[0039] FIGS. 16a to 16c illustrate changes in gene and protein expression related to ECM composition or inflammation induction in mouse-derived chondrocytes following treatment with Example 1 as an embodiment of the present invention. FIG. 16a is a graph showing the results of measuring mRNA expression of enzymes related to the sGAG biosynthesis pathway by qRT-PCR in chondrocytes damaged by bleomycin. FIG. 16b is a band image showing the results of analyzing protein expression related to sGAG synthesis by Western blot. FIG. 16c is a graph showing the results of measuring changes in ECM biosynthesis enzymes and inflammation-related gene expression by qRT-PCR after treatment with Example 1 and SM04690 under conditions of treatment with the inflammatory cytokine IL-1β.

[0040] FIGS. 17a to 17c illustrate changes in sGAG content within chondrocytes according to the treatment of Example 1 as an embodiment of the present invention. FIG. 17a is a graph showing changes in sGAG content according to the treatment concentration of Example 1 in chondrocytes damaged by bleomycin. FIG. 17b is a graph re-plotted on a logarithmic scale in the form of a concentration-response curve of the results of Example 1 in FIG. 17a, showing the maximum effect (E max This is a graph showing the results of calculating the values ​​for ) and half-maximum effective concentration (EC50). Figure 17c is a graph showing the change in sGAG content according to Example 1 and SM04690 treatment under the inflammatory cytokine IL-1β treatment conditions.

[0041] FIGS. 18a to 18f illustrate the therapeutic effect of a single intra-articular administration of Example 1 as an embodiment of the present invention in a mouse model of osteoarthritis induced by DMM surgery. FIG. 18a is a figure showing histological images of Safranin O staining after administration of Example 1 and a vehicle control group. FIG. 18b is a graph quantifying the pathological progression stage by quantifying the intensity of cartilage damage based on the image in FIG. 18a. FIG. 18c is an image showing microscopic changes in the cartilage tissue structure through microcomputed tomography (μCT). FIG. 18d is an image confirming the distribution of Example 1 within the articular cartilage through in situ hybridization. FIG. 18e is a graph showing the mitigating effect of Example 1 on weight-bearing imbalance in a mouse model of osteoarthritis induced by DMM surgery. FIG. 18f is an image showing the results of immunohistochemistry analysis indicating changes in the expression of proteins related to cartilage matrix synthesis and degradation.

[0042] FIGS. 19a to 19d illustrate the therapeutic effect of two repeated intra-articular administrations of Example 1 as an embodiment of the present invention in an osteoarthritis model induced by DMM surgery. FIGS. 19a and 19b are images showing histological analysis of the cartilage of the Example 1 treatment group and the vehicle control group via Safranin O staining, and graphs quantifying the pathological progression stages. FIG. 19c is a graph showing the alleviating effect of Example 1 on weight-bearing imbalance. FIG. 19d is an image showing microscopic changes in the cartilage tissue structure via micro-computed tomography (μCT).

[0043] FIGS. 20a to 20e show experimental results for comparing the therapeutic effects of Example 1, SM04690, and Dexamethasone as an embodiment of the present invention in a mouse osteoarthritis model induced by DMM surgery. FIG. 20a is an image schematically illustrating the treatment period and treatment conditions for each drug. FIGS. 20b and 20c are graphs quantifying the Safranin O staining histological images and pathological progression stages of each drug treatment group. FIG. 20d is a graph showing the results of the analysis of weight-bearing imbalance in each drug treatment group through behavioral experiments. FIG. 20e is an image illustrating microscopic changes in the cartilage tissue structure through microcomputed tomography (μCT).

[0044] FIGS. 21a to 21d show the results of evaluating the therapeutic effect of Example 1 as an embodiment of the present invention in the late stage of osteoarthritis. FIGS. 21a and 21b are Safranin O stained histological images and graphs quantifying the pathological progression stages of each drug treatment group. FIG. 21c is an image showing microscopic changes in the cartilage tissue structure in each drug treatment group through microcomputed tomography (μCT). FIG. 21d is a graph showing the results of the analysis of weight-bearing imbalance in each drug treatment group through behavioral experiments.

[0045] FIGS. 22a to 22c show the analysis results for evaluating the off-target effect of Example 1 as an embodiment of the present invention. FIGS. 22a to 22c are bubble plot graphs showing the results of Gene Ontology (GO) term analysis using transcriptome data obtained after treating C28 / I2 cell lines overexpressing miR-204 with Example 1. The size of the dot indicates the number of genes included in the corresponding annotation, and the color indicates fold enrichment.

[0046] FIGS. 23a to 23c are graphs showing the results of the immunogenicity evaluation of Example 1 by performing a Toll-like receptor (TLR) activity analysis using HEK-Blue TLR7, TLR8, and TLR9 cell lines.

[0047] FIGS. 24a to 24c show the results of the cytotoxicity evaluation of Example 1 as an embodiment of the present invention. FIGS. 24a to 24c are graphs showing the cell viability in mouse primary cultured chondrocytes, hepatocytes, and kidneys, respectively, when Example 1 was treated at various concentrations.

[0048] FIGS. 25a and 25b show the results of a toxicity evaluation after administering Example 1 as a single intra-articular dose to a mouse as an embodiment of the present invention. FIG. 25a is a graph showing the organ-to-body ratio of major organs (liver, kidney, spleen) measured 14 days after administration. The top and middle sections of FIG. 25b are images showing the results of Safranin O staining of knee joint tissue, and the bottom section is an image showing the results of TUNEL staining analysis of the same tissue.

[0049] FIGS. 26a to 26d show the results of confirming the intracellular delivery efficiency and functional efficacy of Example 1 as an embodiment of the present invention in canine-derived chondrocytes. FIG. 26a is an image of canine-derived chondrocytes treated with Cy3 fluorescent labeling Example 1 observed under a fluorescence microscope. FIG. 26b is a graph showing the results of quantifying the ratio of fluorescence-positive cells through flow cytometry after treatment with Cy3 fluorescent labeling Example 1. FIG. 26c is a graph showing the results of measuring miR-204 expression levels by qRT-PCR when Example 1 was treated under cellular aging conditions. FIG. 26d is a graph showing the results of quantifying sGAG content under cellular aging conditions.

[0050] The present invention will be described in detail below.

[0051] In one aspect of the present invention, "microRNA-204," "miR-204," or "microRNA-204" is a collective concept for any variant, isoform, and species homolog of miR-204 that is naturally expressed by cells. Specifically, it may refer to human miR-204, but is not limited thereto, and may include miR-204 of other animals.

[0052] In one aspect of the present invention, the “tail region of miR-204” may be a nucleic acid region from the 5’ end of microRNA-204 (miR-204) to the 17th and beyond.

[0053] In one aspect of the present invention, the "supplementary region of miR-204" may be a nucleic acid region from the 5' end of microRNA-204 (miR-204) to the 13th to 16th.

[0054] In one aspect of the present invention, the “central region of miR-204” may be the 9th to 12th nucleic acid region from the 5’ end of microRNA-204 (miR-204).

[0055] In one aspect of the present invention, the “seed region of miR-204” may be a nucleic acid region from the 2nd to 8th end of microRNA-204 (miR-204).

[0056] In one aspect of the present invention, "oligonucleotide" means a short nucleic acid polymer that binds complementarily to a target nucleic acid, specifically a target and microRNA, to exhibit function, and said polymer may include natural or modified nucleotides, for example, sugar modifications such as 2'-O-methoxyethyl (MOE), 2'-O-methyl (OME), 2'-fluoro(2'-F), LNA, cEt, etc., and phosphorothioate (PS) bonds. In one aspect, "oligonucleotide" may be an antisense oligonucleotide.

[0057] In one aspect of the present invention, "a nucleotide containing a fixed sugar modification" may mean that the sugar included in the nucleotide has undergone a modification that fixes the conformational configuration of the sugar. In one aspect, the nucleotide containing the fixed sugar modification may be a nucleotide containing a bicyclic sugar (e.g., LNA, ENA, cEt, BNA, BNANC / BNANC, etc.).

[0058] In one aspect of the present invention, "a nucleotide comprising a non-constrained 2'-substituted sugar modification" may mean that the sugar included in the nucleotide is modified based on a 2'-position substitution so that the stereochemistry of the sugar is non-constrained (non-fixed). In one aspect, the non-constrained 2'-substituted sugar modification may include a sugar modification that is not a dicyclic sugar (e.g., 2'-deoxy, 2'-O-methyl(2'-O-Me), 2'-O-methoxyethyl(2'-O-MOE), 2'-O-alkyl, 2'-fluoro(2'-F), 2'-amino(2'-NH2), 2'-O-allyl, 2'-O-benzyl, ribose, arabinose (ANA), etc.).

[0059] In one aspect of the present invention, "osteoarthritis" is a group of diseases encompassing degenerative damage or dysfunction of various joint tissues such as articular cartilage, subchondral bone, ligaments, and synovial membranes, and may include primary or secondary osteoarthritis, and osteoarthritis of all parts such as the knee, hip, hand, and spine.

[0060] Oligonucleotides that specifically hybridize with miR-204

[0061] In one aspect, the present invention relates to an oligonucleotide that specifically hybridizes with miR-204, wherein the oligonucleotide comprises 20 to 22 nucleotides linked together and is composed of a first region, a second region, and a third region in the order of 5' end to 3' end, wherein the first region is complementary to the tail region of miR-204 and comprises three nucleotides including a fixed sugar modification linked together, wherein the second region comprises three nucleotides linked together, and the nucleotides constituting the second region each independently comprise a nucleotide including a fixed sugar modification or a non-fixed 2'-substituted sugar modification, wherein the third region comprises a plurality of nucleotides linked together, and the nucleotides constituting the third region each independently comprise a nucleotide including a non-fixed 2'-substituted sugar modification, and wherein the oligonucleotide comprises 30% or less of nucleotides including a fixed sugar modification based on the total number of nucleotides. Provides oligonucleotides.

[0062] An oligonucleotide according to one aspect of the present invention may specifically hybridize with miR-204.

[0063] An oligonucleotide according to one aspect of the present invention may have 20 to 22 nucleotides linked together. Specifically, the oligonucleotide may have 20, 21, or 22 nucleotides linked together.

[0064] An oligonucleotide according to one aspect of the present invention may be composed of a first region, a second region, and a third region in the order from the 5' end to the 3' end. Specifically, the first region may be a region containing the 1st to 3rd nucleotide in the order from the 5' end to the 3' end of the oligonucleotide. Additionally, the second region may be a region containing the 4th to 6th nucleotide in the order from the 5' end to the 3' end of the oligonucleotide. Additionally, the third region may be a region containing the 7th to the last (e.g., the 20th, 21st, or 22nd) nucleotide in the order from the 5' end to the 3' end of the oligonucleotide. More specifically, the first region may be a region composed of the 1st to 3rd nucleotide in the order from the 5' end to the 3' end of the oligonucleotide. Additionally, the second region may be a region composed of the 4th to 6th nucleotides in the order from the 5' end to the 3' end of the oligonucleotide. Additionally, the third region may be a region composed of the 7th to the last (e.g., the 20th, 21st, or 22nd) nucleotides in the order from the 5' end to the 3' end of the oligonucleotide.

[0065] An oligonucleotide according to one aspect of the present invention can be designed to improve binding affinity, in vivo stability, and functional inhibitory effect by selectively introducing LNA (lock nucleic acid), 5-methyl-deoxycytosine (iMe-dC), phosphorothioate (PS) bonds, phosphodiester (PO) bonds, etc.

[0066] Since LNA bases possess a bicyclic structure with a fixed equivalent structure, they can contribute to enhancing the ligand binding strength and structural rigidity of oligonucleotides. Additionally, 5-methyl-deoxycytosine (iMe-dC) is a modified base with increased resistance to oxidation and deamination compared to wild-type cytosine; as the most standardized synthetic base modification for stably and reproducibly mimicking actual biological phenomena, it can contribute to increasing the structural stability and functional persistence of the entire ASO. Furthermore, phosphorothioate (PS) binding can increase nuclease resistance and contribute to extending the in vivo half-life due to increased protein binding strength.

[0067] Meanwhile, the nucleotides included in or constituting the oligonucleotide according to one aspect of the present invention may be phosphorothioate (PS) bonds and / or phosphodiester (PO) bonds.

[0068] According to Experimental Example 1, in the ASO-treated group of Example 1, which is an embodiment of the present invention, the expression of miR-204 decreased in a concentration-dependent manner, and quantitative analysis results showed a maximum degradation efficacy of approximately 99.6%, and a DC that degrades miR-204 expression by 50% 50 It was calculated to be approximately 0.0055 μM, showing high resolution (Fig. 1). In addition, excellent miR-204 resolution was confirmed in the ASO-treated group of Example 2, which is an embodiment of the present invention, confirming that the oligonucleotide according to one aspect of the present invention can effectively degrade the target miR-204 regardless of the nucleotide-nucleotide binding method or the components of the backbone (Fig. 14).

[0069] MicroRNA is generally composed of 22-24 mers, and the 2nd to 8th nucleic acids from the 5' end are classified as the seed region, the 9th to 12th nucleic acids as the central region, the 13th to 16th nucleic acids as the supplementary region, and thereafter as the tail region. The first region of the ASO of Example 1, which is an embodiment of the present invention, has a sequence that is entirely complementary to the tail region.

[0070] According to Experimental Example 2, the ASO of Cy5.5-labeled Example 1 was identified as a strong fluorescent signal within the cytoplasm of chondrocytes, and in flow cytometry analysis, the intracellular fluorescence intensity in the Cy3-labeled Example 1 treatment group showed a concentration-dependent increase. Therefore, this demonstrates that the structural design of the ASO according to one embodiment of the present invention can contribute to effective delivery and absorption even in cells with high penetration difficulty, such as cartilage tissue (Fig. 2).

[0071] An oligonucleotide according to one aspect of the present invention can be efficiently delivered into chondrocytes or cartilage tissue. Specifically, an oligonucleotide according to one aspect of the present invention can effectively induce intracellular uptake into chondrocytes or retention within cartilage tissue.

[0072] According to Experimental Example 3, the ASO of Example 1 according to one embodiment of the present invention was well delivered into cells even in human-derived cartilage tissue (Fig. 3), which suggests that the ASO according to one embodiment of the present invention has physical and chemical properties that allow it to be stably accumulated in human cartilage cells.

[0073] According to Experimental Example 4, variants (Examples 3-1 to 3-3) containing a single or multiple mismatches in the central region or supplementary region of miR-204 were found to maintain excellent resolution without significantly reducing binding efficiency compared to a fully complementary ASO (Fig. 4). This suggests that the sequence design of the ASO according to one embodiment of the present invention is highly advantageous for securing high resolution efficiency, binding stability, and biological functionality.

[0074] In this specification, "mismatch" refers to a state in which a single base is substituted non-complementarily when compared to the complementary sequence of the target microRNA. That is, the expected Watson-Crick complementary base pair is not formed at a specific position of the oligonucleotide, and the position may take the form of a G·U wobble pair or non-canonical pairing, but is not limited thereto.

[0075] An oligonucleotide according to one aspect of the present invention may include one or more mismatch nucleotides with the miR-204. Specifically, the mismatch may be located in a region complementary to the central region or supplementary region of the miR-204. Additionally, specifically, the mismatch nucleotides may be included in the oligonucleotide as one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, and may be included as ten or fewer, nine or fewer, eight or fewer, seven or fewer, six or fewer, five or fewer, four or fewer, three or fewer, or two or fewer.

[0076] A second or third region of an oligonucleotide according to one aspect of the present invention may include one or more mismatch nucleotides with the miR-204. Specifically, the oligonucleotide may include a mismatch nucleotide at one or more selected positions among the 1st to 22nd nucleotides. More specifically, the oligonucleotide may include a mismatch nucleotide at one or more selected positions among the 7th to 22nd, 7th to 20th, 8th to 18th, 8th to 16th, or 8th to 14th nucleotide positions. More specifically, the oligonucleotide may include a mismatch nucleotide in one or more selected from the 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, or 22nd nucleotides, and preferably, may include a mismatch nucleotide in one or more selected from the 8th, 9th, 10th, 11th, 12th, 13th, or 14th nucleotides.

[0077] Additionally, the mismatched nucleotide may be present in the sequence complementary to one or more of the 1st to 22nd nucleotides from the 5' end of the miR-204. Specifically, the oligonucleotide may include a mismatched nucleotide at one or more selected nucleotide positions among the 1st to 22nd, 1st to 16th, 3rd to 16th, 6th to 16th, or 9th to 15th nucleotide positions of the miR-204. More specifically, the oligonucleotide may include a mismatch nucleotide in one or more selected from the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, or 16th nucleotides of miR-204, and even more specifically, may include a mismatch nucleotide in one or more selected from the 9th, 10th, 11th, 12th, 13th, 14th, or 15th nucleotides.

[0078] According to Experimental Example 5, ASOs with 1 to 2 3' terminal bases deleted compared to fully complementary ASOs (Examples 4-1 and 4-2) significantly degrade miR-204, and it was confirmed that binding affinity and degradation ability for miR-204 are maintained even at shortened lengths (Fig. 5).

[0079] This suggests that when designing a miR-204 targeted ASO, some truncation at the 3' end does not impair functional efficacy and can maintain miR-204 resolution.

[0080] A nucleotide comprising a fixed sugar modification according to one aspect of the present invention may be at least one selected from the group consisting of LNA (locked nucleic acid), ENA (2',4'-ethylene-bridged nucleic acid), cEt (constrained ethyl) nucleotide, BNA (bridged nucleic acid), and BNANC / BNANC (acyclic amino-bridged BNA). Specifically, the nucleotide comprising the fixed sugar modification may be at least one of LNA and ENA, and more specifically, may be LNA.

[0081] A non-fixed 2'-substituted sugar modification according to one aspect of the present invention may be at least one selected from the group consisting of non-fixed 2'-substituted sugar modifications of 2'-deoxy, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-alkyl, 2'-fluoro (2'-F), 2'-amino (2'-NH2), 2'-O-allyl, 2'-O-benzyl, ribose, and arabinose (ANA). More specifically, the non-fixed 2'-substituted sugar modification may be at least one selected from the group consisting of 2'-deoxy, 2'-O-methyl, and 2'-O-methoxyethyl.

[0082] An oligonucleotide according to one aspect of the present invention may include a first region complementary to the tail region of miR-204, and the first region may consist of three nucleotides containing a fixed sugar modification connected in succession. Specifically, the first region may include the first to third nucleotides in the direction from the 5' end to the 3' end of the oligonucleotide, and the three nucleotides may consist of three nucleotides containing a fixed sugar modification connected in succession. More specifically, the first region may consist of the first to third nucleotides in the direction from the 5' end to the 3' end of the oligonucleotide, and the three nucleotides may consist of three nucleotides consisting of a fixed sugar modification connected in succession.

[0083] An oligonucleotide according to one aspect of the present invention may include a second region complementary to the tail region of miR-204, and the nucleotides included in the second region may each independently be nucleotides including a fixed sugar modification or a non-fixed 2'-substituted sugar modification. Specifically, the second region may be located after the first region in the direction from the 5' end to the 3' end of the oligonucleotide. Additionally, the second region may include nucleotides from the 4th to the 6th in the direction from the 5' end to the 3' end of the oligonucleotide, and the three nucleotides may be three nucleotides including a fixed sugar modification or a non-fixed 2'-substituted sugar modification connected in succession. More specifically, the second region may consist of the 4th to 6th nucleotides in the direction from the 5' end to the 3' end of the oligonucleotide, and the three nucleotides may be three nucleotides connected in succession, consisting of a fixed sugar modification or a non-fixed 2'-substituted sugar modification.

[0084] According to one embodiment of the present invention, when an oligonucleotide according to one aspect of the present invention contains a fixed sugar modification at the 1st to 3rd positions (first region) in the direction from the 5' end to the 3' end (Examples 5-1 to 5-7), it has stable and efficient degradation efficacy against miR-204, and this same miR-204 degradation efficacy is achieved even when a non-fixed 2'-substituted sugar modification and a fixed sugar modification are mixed at the adjacent 4th to 6th positions (second region). However, an oligonucleotide containing only one or two fixed sugar modifications in the first region (Comparative Examples 1-1 and 1-2) showed a miR-204 expression level similar to the control group at a concentration of 10 μM, indicating almost no miR-204 degradation efficacy (see Experimental Example 6-1, and Figs. 6 and 7).

[0085] In addition, according to one embodiment of the present invention, the overall structural stability of the oligonucleotide and miR-204 degradation efficacy were maintained even when the oligonucleotide according to one aspect of the present invention included MOE or OME as a non-fixed 2'-substituted sugar modification at one or more of the 4th to 6th positions (second region) in the direction from the 5' end to the 3' end (see Examples 6-1 to 6-14, Experimental Example 8, and Figs. 12 and 13).

[0086] An oligonucleotide according to one aspect of the present invention may include a third region complementary to the supplementary region, central region, and seed region of miR-204, and the nucleotides included in the third region may each independently be nucleotides containing a non-fixed 2'-substituted sugar modification. Specifically, the third region may be located after the second region in the direction from the 5' end to the 3' end of the oligonucleotide. Additionally, the third region may include nucleotides from the 7th to the last in the direction from the 5' end to the 3' end of the oligonucleotide, and the plurality of nucleotides may be nucleotides containing a non-fixed 2'-substituted sugar modification connected in succession. More specifically, the third region may consist of nucleotides from the seventh to the last in the direction from the 5' end to the 3' end of the oligonucleotide, and the plurality of nucleotides may be a continuous connection of nucleotides consisting of non-fixed 2'-substituted sugar modifications. More specifically, all nucleotides constituting the third region may include 2'-deoxy.

[0087] An oligonucleotide according to one aspect of the present invention may include a region complementary to the seed region of miR-204, and the region complementary to the seed region of miR-204 may be a part of a third region of the oligonucleotide according to one aspect of the present invention, specifically a part adjacent to the 3' end. The sugars of the nucleotides included in the region complementary to the seed region may include non-fixed 2'-substituted sugar modifications.

[0088] In one aspect of the present invention, the sugar of one or more nucleotides at positions 1 to 16 from the 3' end of the oligonucleotide may be 2'-deoxy. Specifically, the sugar of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more nucleotides at positions 1 to 16 from the 3' end of the oligonucleotide may be 2'-deoxy, and the sugar of 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer nucleotides may be 2'-deoxy. More specifically, the sugar of the nucleotide at positions 1 and 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, or 1 to 16 from the 3' end of the oligonucleotide may be 2'-deoxy.

[0089] According to one embodiment of the present invention, when the oligonucleotide according to one aspect of the present invention does not include a fixed sugar modification in the first region and includes a fixed sugar modification only in the region near the 3' end (third region) (Comparative Examples 2-1 to 2-4) or randomly includes a fixed sugar modification in the second and third regions (Comparative Examples 3 and 4), the binding strength of ASO and the RNase H-mediated degradation efficiency are reduced, and the overall miR-204 degradation effect is reduced (see Experimental Examples 6-2 and 6-3, and FIGS. 8 to 10). Thus, it was found that the location of the fixed sugar modification in the oligonucleotide according to one aspect of the present invention has a decisive influence on the miR-204 degradation activity, and the best miR-204 degradation efficacy is exhibited when the fixed sugar modification is included centered on the 5' end, that is, in the first region.

[0090] An oligonucleotide according to one aspect of the present invention may contain 30% or less of a nucleotide containing a fixed sugar modification based on the total number of nucleotides. Specifically, the oligonucleotide may contain nucleotides containing fixed sugar modifications based on the total number of nucleotides in an amount of 30% or less, 29.5% or less, 29% or less, 28.5% or less, 28% or less, 27.5% or less, 27.3% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22.7% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18.2% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13.6% or less, 13% or less, 12% or less, 11% or less, or 10% or less. More specifically, the oligonucleotide may contain 13 to 30% of nucleotides containing fixed sugar modifications based on the total number of nucleotides.

[0091] Alternatively, an oligonucleotide according to one aspect of the present invention may comprise 3 to 14 nucleotides containing fixed sugar modifications. Specifically, the oligonucleotide may comprise 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 13 or more nucleotides containing fixed sugar modifications, and may comprise 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, or 4 or fewer. More specifically, the oligonucleotide may comprise 3 to 6 nucleotides containing fixed sugar modifications. According to one aspect of the present invention, the oligonucleotide exhibits excellent miR-204 degradation efficacy when all three nucleotides constituting the first region contain fixed sugar modifications, so the nucleotides containing fixed sugar modifications may be 13% or more or three or more.

[0092] According to one embodiment of the present invention, in one aspect of the present invention, oligonucleotides improve the miR-204 degradation effect as they contain more nucleotides containing fixed sugar modifications, but at the same time, this may lead to physical and formulation limitations as a drug due to increased viscosity and decreased solubility of the formulation. Therefore, considering both efficacy and formulation stability, a level of nucleotides containing fixed sugar modifications that is not excessive (e.g., 13 to 30% or 3 to 6) was determined to be the optimal structure that maintains a balance between miR-204 degradation efficacy and formulation suitability (see Experimental Example 7 and Fig. 11b).

[0093] Each nucleotide included in an oligonucleotide according to one aspect of the present invention may be connected by a phosphorohioate bond or a phosphodiester bond. The bonds between nucleotides included in an oligonucleotide according to one aspect of the present invention may each be independently a phosphorohioate bond or a phosphodiester bond. Specifically, in an oligonucleotide according to one aspect of the present invention, the phosphate bonds between nucleotides may each be independently a phosphorohioate bond and / or a phosphodiester bond. For example, some of the bonds between nucleotides may be phosphorohioate bonds and others may be phosphodiester bonds, and the number and location of these bonds are not particularly limited. As another example, all of the bonds between nucleotides may be phosphorohioate bonds or phosphodiester bonds.

[0094] An oligonucleotide according to one aspect of the present invention can effectively degrade miR-204 in chondrocytes. An oligonucleotide according to one aspect of the present invention can exhibit a preventive or therapeutic effect against osteoarthritis. Specifically, an oligonucleotide according to one aspect of the present invention can exhibit a preventive or therapeutic effect against osteoarthritis by effectively degrading miR-204 in chondrocytes.

[0095] In one embodiment, osteoarthritis may be at least one selected from the group consisting of degenerative (primary) osteoarthritis, post-traumatic osteoarthritis, post-surgical osteoarthritis, inflammatory osteoarthritis, and secondary osteoarthritis.

[0096] Oligonucleotides according to one aspect of the present invention are genes related to extracellular matrix (ECM) remodeling (e.g., ECM proteoglycan synthesis factor) and / or genes related to inflammation induction (e.g., IL6, PTGS2, NOS2, IL family, substrate degradation products) and / or genes related to chondrolytic enzymes (e.g., MMP family (e.g., MMP3), ADAMTS family, not limited thereto).

[0097] An oligonucleotide according to one aspect of the present invention can exhibit a preventive or therapeutic effect against osteoarthritis even upon repeated administration. Specifically, an oligonucleotide according to one aspect of the present invention can exhibit a stable and reproducible effect even upon repeated administration.

[0098] An oligonucleotide according to one aspect of the present invention can alleviate symptoms of osteoarthritis (e.g., cartilage damage, subcutaneous bone sclerosis, subchondral bone sclerosis) upon repeated administration. According to one embodiment of the present invention, when an oligonucleotide according to one aspect of the present invention is administered twice, both cartilage damage and subcutaneous bone sclerosis can be significantly reduced (see Experimental Example 10, and FIGS. 19a, 19b, and 19d).

[0099] An oligonucleotide according to one aspect of the present invention may exhibit a pain-relieving effect even upon repeated administration. According to one embodiment of the present invention, when an oligonucleotide according to one aspect of the present invention is administered twice, the pain-relieving effect may be sustained (see Experimental Example 10 and FIG. 19c).

[0100] In one embodiment, repeated administration means administering an oligonucleotide according to one aspect of the present invention two or more times. Specifically, repeated administration may mean administering an oligonucleotide of the same or different dosage two or more times at regular intervals. For example, repeated administration may be administered two, three, four, five or more times, but is not limited thereto.

[0101] An oligonucleotide according to one aspect of the present invention can exhibit reproducible efficacy when administered once or repeatedly.

[0102] An oligonucleotide according to one aspect of the present invention may have a superior effect in preventing or treating osteoarthritis compared to an oligonucleotide that does not follow the sugar modification pattern of the oligonucleotide according to one aspect of the present invention. According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention may exhibit significantly superior effects in terms of reducing cartilage damage, reducing subcutaneous bone sclerosis, and / or alleviating pain compared to other oligonucleotides that do not follow the sugar modification pattern of the oligonucleotide according to one aspect of the present invention under the same conditions (see Experimental Example 10 and FIGS. 19a to 19d).

[0103] An oligonucleotide according to one aspect of the present invention may exhibit superior preventive or therapeutic effects compared to existing osteoarthritis treatments (e.g., commercially available osteoarthritis treatments, osteoarthritis treatments currently in clinical use or under development). Specifically, an oligonucleotide according to one aspect of the present invention may exhibit superior cartilage protective effects, subcutaneous bone sclerosis improvement effects, pain relief effects, and / or cartilage regeneration effects compared to osteoarthritis treatments currently in clinical use or under development. For example, existing osteoarthritis treatments may be SM04690 or steroidal anti-inflammatory analgesics (e.g., dexamethasone), but are not limited thereto.

[0104] According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention may exhibit a superior cartilage protective effect compared to SM04690 (see Experimental Example 10 and FIGS. 20a to 20e). According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention may have superior cartilage regenerative ability compared to Dexamethasone (see Experimental Example 10 and FIGS. 20a to 20e).

[0105] An oligonucleotide according to one aspect of the present invention can protect and / or restore the cartilage structure. Specifically, an oligonucleotide according to one aspect of the present invention can improve osteoarthritis by inducing not only the regulation of inflammation but also the protection and / or restoration of the cartilage structure itself.

[0106] An oligonucleotide according to one aspect of the present invention can exhibit a preventive or therapeutic effect even in the late stages of osteoarthritis. Specifically, an oligonucleotide according to one aspect of the present invention can restore cartilage even when administered in the late stages of advanced osteoarthritis. An oligonucleotide according to one aspect of the present invention can exhibit a superior cartilage restoration effect compared to steric blockers and / or SM04690 in the late stages of osteoarthritis. According to one embodiment of the present invention, when administered 6 weeks after osteoarthritis induction, an oligonucleotide according to one aspect of the present invention can exhibit a statistically significant cartilage restoration effect compared to steric blockers (SEQ No. 22) and SM04690 (see Experimental Example 10 and FIGS. 21a to 21c).

[0107] An oligonucleotide according to one aspect of the present invention can exhibit a pain-improving effect even in the late stages of osteoarthritis. According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention can exhibit a statistically significant pain-improving effect when administered 6 weeks after osteoarthritis induction (see Experimental Example 10 and FIG. 21d).

[0108] In one embodiment, the late stage of osteoarthritis refers to a stage in which osteoarthritis has progressed for a certain period. Specifically, the late stage of osteoarthritis may refer to a stage in which pathological changes of osteoarthritis have progressed and cartilage damage, subcutaneous bone sclerosis, osteophyte formation, and / or synovitis are observed. For example, the late stage of osteoarthritis may be a point in time 4, 5, 6 weeks, or longer after osteoarthritis induction in an animal model, but is not limited thereto. For example, the late stage of osteoarthritis may be a point in time 4, 5, 6 weeks, or longer after osteoarthritis diagnosis, but is not limited thereto. In the case of humans, it may be a point in time 3, 4, or longer after KL grade (Kellgren-Lawrence grade). The late stage of osteoarthritis refers to a state in which pathological changes have progressed further and become entrenched compared to the initial stage, and may include a stage that is more difficult to treat.

[0109] An oligonucleotide according to one aspect of the present invention has high sequence specificity for miR-204 and may have extremely low off-target effects.

[0110] An oligonucleotide according to one aspect of the present invention may have high specificity for miR-204. Specifically, an oligonucleotide according to one aspect of the present invention may not non-specifically bind to targets other than miR-204 and / or its host gene, TRPM3 (e.g., human mRNA, microRNA, or pre-mRNA). According to one embodiment of the present invention, NCBI-BLAST analysis of the nucleotide sequence of the oligonucleotide according to one aspect of the present invention revealed no sequences expected to bind other than miR-204 and TRPM3 (see Experimental Example 11 and Table 14).

[0111] Oligonucleotides according to one aspect of the present invention may have low off-target effects. Specifically, oligonucleotides according to one aspect of the present invention may not have a significant effect on other genes or biological pathways other than miR-204 degradation. According to one embodiment of the present invention, as a result of Gene Ontology (GO) analysis using transcriptome data of cells treated with oligonucleotides according to one aspect of the present invention, no significant ontology terms were derived in any of the Biological Process, Molecular Function, and Cellular Component items (see Experimental Example 11 and FIGS. 22a to 22c).

[0112] An oligonucleotide according to one aspect of the present invention may have low immunogenicity. Specifically, an oligonucleotide according to one aspect of the present invention may not induce an immune response through Toll-like receptors (TLRs). For example, the TLR may be TLR7, TLR8, and / or TLR9, but is not limited thereto.

[0113] An oligonucleotide according to one aspect of the present invention may not increase TLR activity. According to one embodiment of the present invention, when treated with an oligonucleotide according to one aspect of the present invention, no significant increase in TLR7, TLR8, and TLR9 activity was observed (see Experimental Example 12 and FIGS. 23a to 23c).

[0114] An oligonucleotide according to one aspect of the present invention may have non-immunostimulatory properties toward innate immune receptor pathways. An oligonucleotide according to one aspect of the present invention may be an antisense oligonucleotide that does not induce immunogenicity.

[0115] An oligonucleotide according to one aspect of the present invention may not exhibit cytotoxicity or may exhibit low cytotoxicity. Specifically, an oligonucleotide according to one aspect of the present invention may not exhibit cytotoxicity or may exhibit low cytotoxicity in chondrocytes, hepatocytes, and / or kidney cells. According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention did not reduce cell viability to 80% or less in mouse cartilage-derived primary culture cells, a hepatocyte line (HepG2 cell line), and a kidney cell line (RPTEC cell line) (see Experimental Example 13 and FIGS. 24a to 24c).

[0116] An oligonucleotide according to one aspect of the present invention may not exhibit systemic toxicity. An oligonucleotide according to one aspect of the present invention may not exhibit systemic toxicity when administered intra-articularly. An oligonucleotide according to one aspect of the present invention may not exhibit toxicity to the liver, kidney, and / or spleen. An oligonucleotide according to one aspect of the present invention may not exhibit toxicity to the liver, kidney, and / or spleen when administered intra-articularly. According to one embodiment of the present invention, after a single intra-articular administration of an oligonucleotide according to one aspect of the present invention, the organ-to-body weight ratio of the liver, kidney, and spleen did not show a significant difference from the control group (see Experimental Example 13 and FIG. 25a).

[0117] An oligonucleotide according to one aspect of the present invention may not exhibit local toxicity. An oligonucleotide according to one aspect of the present invention may not exhibit local toxicity when administered into the joint cavity. Specifically, an oligonucleotide according to one aspect of the present invention may not induce cartilage damage, proteoglycan loss, and / or chondrocyte apoptosis. Specifically, an oligonucleotide according to one aspect of the present invention may not induce cartilage damage, proteoglycan loss, and / or chondrocyte apoptosis when administered into the joint cavity. According to one embodiment of the present invention, when an oligonucleotide according to one aspect of the present invention was administered into the joint cavity, no cartilage damage or proteoglycan loss was observed in the knee joint tissue, the matrix components within the cartilage were maintained normally, and apoptosis was not induced in the chondrocytes (see Experimental Example 13 and FIG. 25b).

[0118] An oligonucleotide according to one aspect of the present invention may have excellent local safety and / or systemic safety. Specifically, an oligonucleotide according to one aspect of the present invention may be a safe antisense oligonucleotide with no or low local toxicity and / or systemic toxicity at the site of administration.

[0119] An oligonucleotide according to one aspect of the present invention can be efficiently delivered into chondrocytes. Additionally, an oligonucleotide according to one aspect of the present invention can be efficiently delivered into chondrocytes derived from mammals (e.g., dogs). Specifically, an oligonucleotide according to one aspect of the present invention can be efficiently delivered into chondrocytes derived from mammals (e.g., dogs) and can be stably accumulated within the chondrocytes. According to one embodiment of the present invention, a fluorescently labeled oligonucleotide according to one aspect of the present invention was distinctly accumulated within dog-derived chondrocytes, and a fluorescence positive signal of more than 50% was observed under conditions treated at concentrations of 10 nM and 50 nM (see Experimental Example 14, and FIGS. 26a and 26b).

[0120] An oligonucleotide according to one aspect of the present invention can reduce miR-204 in canine-derived chondrocytes. According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention can statistically significantly reduce miR-204 in canine-derived chondrocytes with induced cellular senescence (see Experimental Example 14 and FIG. 26c).

[0121] An oligonucleotide according to one aspect of the present invention can restore the cartilage matrix synthesis function in canine-derived chondrocytes. Specifically, an oligonucleotide according to one aspect of the present invention can increase or restore the synthesis of sulfated glycosaminoglycans (sGAG) in canine-derived chondrocytes. According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention can statistically significantly increase the synthesis of sGAG, which is reduced by aging, in canine-derived chondrocytes with induced cellular aging (see Experimental Example 14 and FIG. 26d).

[0122] An oligonucleotide according to one aspect of the present invention may have cross-species efficacy. Specifically, an oligonucleotide according to one aspect of the present invention can effectively inhibit miR-204 and restore cartilage matrix synthesis function in mouse-derived chondrocytes as well as dog-derived chondrocytes. An oligonucleotide according to one aspect of the present invention may exhibit an inhibitory effect on miR-204 regardless of species specificity.

[0123] In addition, oligonucleotides according to one aspect of the present invention can be efficiently delivered without a separate nucleic acid carrier. Specifically, oligonucleotides according to one aspect of the present invention can be efficiently delivered to chondrocytes, cartilage, and / or joint tissue without a separate nucleic acid carrier. In this specification, the expressions “carrier-free” or “carrier-independent” mean that the oligonucleotides of the present invention can be introduced into cells without a separate nucleic acid carrier, such as cationic lipids, lipid nanoparticles, liposomes, cationic polymers (e.g., polyethyleneimide, poly-L-lysine, etc.), dendrimers, cell-penetrating peptides, or virus / virus-like particle-based vectors. The oligonucleotides according to one aspect of the present invention can be administered in a form dissolved alone in an aqueous solution (e.g., nuclease-free material, buffer solution, isotonic solution, etc.) that does not substantially contain such a carrier.

[0124] An oligonucleotide according to one aspect of the present invention may be prepared in the form of an intra-articular injection and may not contain a separate nucleic acid delivery vehicle, except for a pharmaceutically acceptable solvent (e.g., nuclease-free water, physiological saline, buffered saline, etc.) and a selective stabilizer, buffer, or isotonic agent. Despite being a delivery vehicle-free composition, the oligonucleotide of the present invention can remain stably within the articular cartilage for a long period (e.g., for several weeks after administration), degrade miR-204, and exhibit an effect of significantly improving pathological changes in osteoarthritis (see Experimental Examples). Therefore, the oligonucleotide of the present invention has the advantage of exhibiting excellent tissue penetration and sustained efficacy without requiring an expensive delivery vehicle.

[0125] An oligonucleotide according to one aspect of the present invention can be efficiently absorbed into chondrocytes (e.g., mouse chondrocytes, dog-derived chondrocytes, etc.) simply by being directly added to the culture medium without the need for separate transduction reagents (e.g., lipofectamine, cationic lipid complex, etc.) in a cell culture system. As such, since the oligonucleotide of the present invention exhibits excellent intracellular absorption and miR-204 degradation capabilities even under carrier-free conditions, it can provide the advantage of minimizing additional toxicity, immunogenicity, or manufacturing process complexity caused by the carrier.

[0126] Of course, if necessary, the oligonucleotide according to one aspect of the present invention may be used in combination with other pharmaceutical delivery systems selected by those skilled in the art, in addition to the carrier-free form as described above. For example, the oligonucleotide according to one aspect of the present invention may be delivered to chondrocytes, cartilage, and / or joint tissue via a carrier.

[0127] Composition for the prevention or treatment of osteoarthritis

[0128] In addition, one aspect of the present invention provides a composition for the prevention or treatment of osteoarthritis comprising an oligonucleotide according to the aforementioned aspect.

[0129] Oligonucleotides have been mentioned above, so a detailed explanation is omitted.

[0130] In this specification, the term "osteoarthritis (OA)" refers to a degenerative joint disease characterized by degeneration of articular cartilage, sclerosis of subchondral bone, formation of osteophytes, inflammation of the synovium, etc. Risk factors such as aging, obesity, and mechanical overload may contribute to the development and progression of osteoarthritis.

[0131] In this specification, the term "treatment" refers to any act of improving or beneficially altering the symptoms of osteoarthritis or diseases caused thereby by administering an oligonucleotide and / or composition according to one aspect of the present invention. A person skilled in the art to which this application pertains will be able to determine the precise criteria for the disease and assess the degree of improvement, enhancement, and treatment by referring to materials presented in the art.

[0132] In this specification, the term "prevention" refers to any act of suppressing or delaying the onset of osteoarthritis or a disease caused thereby by administering an oligonucleotide and / or a composition according to one aspect of the present invention. Additionally, "prevention" may include acts of reducing the risk of osteoarthritis or a disease caused thereby, slowing the progression of the disease, or suppressing the appearance of symptoms of the disease.

[0133] In one embodiment, osteoarthritis may be a degenerative disease in which articular cartilage normally formed at birth gradually degenerates due to acquired factors such as aging, trauma, mechanical overload, or inflammation. In one embodiment, osteoarthritis may include degenerative (primary) osteoarthritis, post-traumatic osteoarthritis, post-surgical osteoarthritis, inflammatory osteoarthritis, and secondary osteoarthritis. For example, degenerative osteoarthritis may be the most common form of osteoarthritis caused by age-related, natural degenerative changes occurring without a specific cause such as clear trauma, structural disease, or inflammatory disease. For example, post-traumatic osteoarthritis may be osteoarthritis in which degenerative changes are accelerated following mechanical trauma or damage to the joint structure, such as joint injury or fracture, ligament injury or rupture, or meniscus injury or rupture. For example, inflammatory osteoarthritis may be osteoarthritis characterized by synovitis or an increase in inflammatory cells in the joint fluid, in addition to structural degenerative changes, where inflammation of the synovium and surrounding tissues within the joint is prominent. Postoperative osteoarthritis may be osteoarthritis secondarily induced by structural changes, instability, or biomechanical changes following surgical treatment of the joint or surrounding tissues. Secondary osteoarthritis may be osteoarthritis resulting from changes in the joint structure or biological environment caused by clear underlying causes (trauma, congenital deformities, metabolic diseases, endocrine disorders, infections, crystal deposition diseases, etc.). In this case, congenital deformities may include hip dysplasia and varus / valgus deformities, while metabolic and endocrine disorders may include diabetes, thyroid disease, avascular necrosis, and obesity.

[0134] A composition according to one aspect of the present invention may be for the prevention or treatment of osteoarthritis in mammals. The mammal may be at least one selected from the group consisting of humans, dogs, cats, rabbits, cattle, horses, pigs, sheep, mice, rats, monkeys, and chimpanzees, but is not limited thereto. Specifically, the mammal may be a human, a dog, or a cat.

[0135] A composition according to one aspect of the present invention may be used to alleviate various pathological changes of osteoarthritis. Specifically, the composition may be used to alleviate at least one selected from the group consisting of cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, loss of cartilage matrix, and / or osteoarthritis-related pain, but is not limited thereto. In this regard, one aspect of the present invention may provide a composition for alleviating cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, loss of cartilage matrix, and / or osteoarthritis-related pain, comprising an oligonucleotide according to one aspect of the present invention.

[0136] A composition according to one aspect of the present invention may be used to restore the extracellular matrix (ECM) synthesis function of chondrocytes. Specifically, the composition may be used to increase or restore the expression of proteoglycan biosynthetic enzymes. Additionally, the composition may be used to increase or restore the content of sulfated glycosaminoglycans (sGAG). In this regard, one aspect of the present invention may provide a composition for increasing proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans (sGAG), comprising an oligonucleotide according to one aspect of the present invention.

[0137] A composition according to one aspect of the present invention may be used to reduce or restore the expression of a cartilage matrix degrading enzyme, or to suppress or alleviate inflammation. Specifically, the composition may be used to reduce the expression of a cartilage matrix degrading enzyme, and said cartilage matrix degrading enzyme may include, but is not limited to, Mmp3. In addition, said composition may be used to reduce the expression of inflammatory cytokines. said inflammatory cytokines may be IL-6, IL-1β, TNF-α, or a combination thereof, but are not limited thereto. In this regard, one aspect of the present invention may provide a composition for reducing a cartilage matrix degrading enzyme and inflammatory cytokines, comprising an oligonucleotide according to one aspect of the present invention.

[0138] A composition according to one aspect of the present invention may be for the prevention or treatment of the early and / or late stages of osteoarthritis. The composition may restore cartilage even in the late stages where osteoarthritis has already progressed. For example, the early stage of osteoarthritis may be a stage within 1, 2, 3, or 4 weeks after the induction or diagnosis of osteoarthritis, in which minor cartilage damage is observed but subcutaneous bone sclerosis, osteophyte formation, etc., have not progressed, but is not limited thereto. For example, the late stage of osteoarthritis may be a stage after 4 weeks after the induction or diagnosis of osteoarthritis, in which severe cartilage damage is observed and subcutaneous bone sclerosis, osteophyte formation, etc., have progressed, but is not limited thereto.

[0139] A composition according to one aspect of the present invention may exhibit superior effects compared to existing osteoarthritis treatments. Specifically, the composition may exhibit superior cartilage protective effects, cartilage recovery effects, cartilage regeneration effects, subcutaneous bone sclerosis improvement effects, inflammation relief effects, sGAG synthesis increase effects, and / or pain relief effects compared to existing osteoarthritis treatments. Existing osteoarthritis treatments may be SM04690 or Dexamethasone, but are not limited thereto.

[0140] A composition according to one aspect of the present invention may be for animals (e.g., mammals), but is not limited thereto. For example, the animal may be at least one selected from the group consisting of humans, dogs, cats, rabbits, cattle, horses, pigs, sheep, mice, rats, monkeys, and chimpanzees, but is not limited thereto. Specifically, the animal may be a human or a dog. An oligonucleotide according to one aspect of the present invention may be applied to animals.

[0141] A composition according to one aspect of the present invention may be a pharmaceutical composition, a food composition, a health functional food composition, or an oral composition, but is not limited thereto.

[0142] In the case where a composition according to one aspect of the present invention is a pharmaceutical composition, the composition may further include a pharmaceutically acceptable carrier, a diluent, an excipient, a stabilizer, a buffer, an isotonic agent, etc. The pharmaceutically acceptable carrier, diluent, excipient, stabilizer, buffer, or isotonic agent may be any known in the art without limitation and may be, for example, at least one selected from the group consisting of saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof, but is not limited thereto. Additionally, other conventional additives such as antioxidants and bacteriostatic agents may be further included as needed.

[0143] A composition according to one aspect of the present invention may not include a carrier. A composition according to one aspect of the present invention can effectively deliver an oligonucleotide according to one aspect of the present invention into a cell even under carrier-free conditions, thereby exhibiting miR-204 degradation ability and demonstrating an effect of preventing or treating osteoarthritis. Meanwhile, a composition according to one aspect of the present invention may include a carrier if necessary. The carrier may be, but is not limited to, a cationic lipid, a lipid nanoparticle, a liposome, a cationic polymer (e.g., polyethyleneimide, poly-L-lysine, etc.), a dendrimer, a cell-penetrating peptide, a virus particle-based vector, and / or a virus-like particle-based vector.

[0144] A composition according to one aspect of the present invention may be prepared in various formulations. For example, the composition may be formulated in the form of an injectable, liquid, suspension, emulsion, syrup, powder, granule, tablet, capsule, gel, patch, emulsion, syrup, aerosol, or a combination thereof, but is not limited thereto. Specifically, the composition may be formulated as an intra-articular injectable. The composition may be for intra-articular injection. For example, intra-articular injection can deliver a drug locally at a high concentration, thereby maximizing the therapeutic effect while minimizing systemic side effects.

[0145] An oligonucleotide or composition according to one aspect of the present invention may be administered by various routes. For example, the composition may be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, intranasally, pulmonaryly, rectally, locally (e.g., intra-articular injection, other local injections, topical application), or intra-articularly, but is not limited thereto. Specifically, the composition may be administered by intra-articular administration. Although the embodiment according to one aspect of the present invention was verified through intra-articular administration, those skilled in the art will understand that it can be appropriately applied to other routes of administration considering pharmacokinetics / mechanisms of delivery.

[0146] An oligonucleotide or composition according to one aspect of the present invention may be administered in a pharmaceutically effective amount. In one aspect of this specification, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0147] The dosage of an oligonucleotide or composition according to one aspect of the present invention may be appropriately selected by a person skilled in the art, taking into account the patient's age, gender, weight, severity of the disease, route of administration, and frequency of administration. For example, the oligonucleotide may be 0.1 mg to 50 mg per single administration based on an adult (e.g., an adult weighing 60 kg). For example, based on an adult (e.g., an adult weighing 60 kg), the oligonucleotide may be administered in a single dose of 0.1 mg or more, 1 mg or more, 5 mg or more, 9 mg or more, 10 mg or more, 11 mg or more, 15 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 0.1 mg or less, 1 mg or less, 5 mg or less, 9 mg or less, 10 mg or less, 11 mg or less, 15 mg or less, 20 mg or less, 30 mg or less, 40 mg or less, 50 mg or less, or a combination thereof (e.g., 10 to 30 mg), but is not limited thereto. The above composition may be administered once or several times a day, or may be administered repeatedly at intervals of several days, weeks, or months.

[0148] An oligonucleotide or composition according to one aspect of the present invention may be administered once or repeatedly. Specifically, the oligonucleotide or composition may be administered once, twice, three times, four times, five times or more.

[0149] A composition according to one aspect of the present invention may be administered simultaneously, separately, or sequentially in combination with an oligonucleotide according to one aspect of the present invention and another osteoarthritis treatment (e.g., a commercially available osteoarthritis treatment or a treatment currently in clinical trials).

[0150] In the case where a composition according to one aspect of the present invention is a food composition or a health functional food composition, said composition may be prepared in a form that can be consumed like ordinary food. There are no particular restrictions on the types of said food composition, and they may be, for example, meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, or health functional foods, but are not limited thereto.

[0151] A composition according to one aspect of the present invention can prevent or treat osteoarthritis by effectively degrading miR-204. For example, the composition can prevent or treat osteoarthritis by degrading or reducing miR-204, regulating genes related to ECM remodeling and biological pathways related to maintaining cartilage structure, and reducing the expression of proteins related to cartilage matrix destruction.

[0152] In addition, one aspect of the present invention provides a method for preventing or treating osteoarthritis, comprising the step of administering the aforementioned oligonucleotide or composition to an individual.

[0153] In one embodiment, the individual may be a subject requiring treatment or prevention of osteoarthritis.

[0154] In one embodiment, the individual may include, but is not limited to, mammals. For example, the mammal may be at least one selected from the group consisting of humans, dogs, cats, rabbits, cattle, horses, pigs, sheep, mice, rats, monkeys, and chimpanzees, but is not limited thereto. Specifically, the mammal may be a human or a dog.

[0155] In a method for preventing or treating osteoarthritis according to one aspect of the present invention, the oligonucleotide or composition may be administered to an individual in an amount effective for the prevention or treatment of osteoarthritis.

[0156] Additionally, one aspect of the present invention provides a method for alleviating cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, loss of cartilage matrix, and / or pain associated with osteoarthritis, comprising the step of administering the aforementioned oligonucleotide or composition to an individual. Additionally, one aspect of the present invention provides a method for increasing proteoglycan biosynthesizing enzymes and / or sulfated glycosaminoglycans (sGAG), comprising the step of administering the aforementioned oligonucleotide or composition to an individual. Additionally, one aspect of the present invention provides a method for decreasing cartilage matrix degrading enzymes and / or inflammatory cytokines, comprising the step of administering the aforementioned oligonucleotide or composition to an individual.

[0157] In one embodiment, the subject may be a subject requiring relief of cartilage destruction, subcutaneous bone sclerosis, osteophyte formation, synovitis, loss of cartilage matrix, and / or pain associated with osteoarthritis. In one embodiment, the subject may be a subject requiring an increase in proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans. In one embodiment, the subject may be a subject requiring a decrease in cartilage matrix degrading enzymes and / or inflammatory cytokines.

[0158] In one embodiment, the oligonucleotide or composition may be administered to an individual in an amount effective for the relief of cartilage destruction, subcutaneous bone sclerosis, osteophyte formation, synovitis, loss of cartilage matrix, and / or pain associated with osteoarthritis. In one embodiment, the oligonucleotide or composition may be administered to an individual in an amount effective for increasing proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans. In one embodiment, the oligonucleotide or composition may be administered to an individual in an amount effective for decreasing cartilage matrix degrading enzymes and / or inflammatory cytokines.

[0159] As the route of administration, dosage, frequency of administration, concomitant administration, etc. have been previously described, a detailed explanation is omitted.

[0160] In addition, one aspect of the present invention provides a use for preparing a composition for the prevention or treatment of osteoarthritis of the aforementioned oligonucleotide.

[0161] In addition, one aspect of the present specification provides the use of the aforementioned oligonucleotide for the prevention or treatment of osteoarthritis.

[0162] As oligonucleotides, treatment, prevention, and osteoarthritis have been previously discussed, a detailed explanation is omitted.

[0163] In addition, one aspect of the present invention provides a use for preparing a composition for the relief of pain associated with cartilage destruction, subcutaneous bone sclerosis, osteophyte formation, synovitis, loss of cartilage matrix, and / or osteoarthritis using the aforementioned oligonucleotide. In addition, one aspect of the present invention provides a use for preparing a composition for increasing proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans using the aforementioned oligonucleotide. In addition, one aspect of the present invention provides a use for preparing a composition for reducing cartilage matrix degrading enzymes and / or inflammatory cytokines using the aforementioned oligonucleotide.

[0164] Additionally, one aspect of the present specification provides the use of the aforementioned oligonucleotide for the relief of pain associated with chondrosis, subcutaneous bone sclerosis, osteophyte formation, synovitis, loss of cartilage matrix, and / or osteoarthritis. Additionally, one aspect of the present specification provides the use of the aforementioned oligonucleotide for increasing proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans. Additionally, one aspect of the present specification provides the use of the aforementioned oligonucleotide for reducing cartilage matrix degrading enzymes and / or inflammatory cytokines.

[0165] The structure and effects of the present invention will be explained in more detail below through examples and experimental examples. However, the following examples are provided for illustrative purposes only to aid in understanding the present invention, and the scope and range of the present invention are not limited by them.

[0166] [Preparation Example] Preparation of ASO specific to miR-204

[0167] Oligonucleotides specific to miR-204, specifically antisense oligonucleotides (ASOs), were prepared in the following manner (Examples 1 and 2).

[0168] Example 1 is a 22-mer antisense oligonucleotide (ASO) composed of the sequence 5'-+A+G+G+C+A+TAGGATGA / iMe-dC / AAAGGGAA-3' (Sequence No. 1). Example 2 is a 22-mer ASO composed of the sequence 5'-+A*+G*+G*+C*+A*+T*A*G*G*A*T*G*A* / iMe-dC / *A*A*A*G*G*G*A*A-3' (Sequence No. 2). In this specification, uppercase letters denote DNA bases, "+X" denotes locked nucleic acid (LNA) bases, and "iMe-dC" denotes 5-methyl-deoxycytosine bases. Also, the "*" mark indicates that the bases are connected by phosphodiester (PO) bonds, and the absence of the "*" mark means that the bases are connected by phosphorothioate (PS) bonds.

[0169] ASO was synthesized and purified by a contract manufacturing organization (CMO) and supplied in powder form. The supplied material was dissolved in nuclease-free water (NFW; AM9932, Thermo Fisher Scientific) to prepare a concentration of 5 mM and stored at -80 ℃.

[0170] For cell experiments, the solution was thawed at room temperature immediately before use, diluted in culture medium to the target concentration, and treated with cells. For animal experiments, the solution was thawed at room temperature immediately before use, diluted in NFW to the target concentration, and injected into the joint cavity in a final volume of 5 μL.

[0171] [Experimental Example 1] Confirmation of miR-204 resolution of an ASO specific to miR-204

[0172] [Experimental Example 1-1] Confirmation of miR-204 resolution of ASO of Example 1 (1) Experimental method

[0173] C28 / I2 cell lines (chondrocyte cell lines) overexpressing microRNA-204 (miR-204) were 3 x 10 5 Cells were inoculated into 12-well plates at a density of cells / well and cultured for 24 hours. Cells were maintained at 37°C under conditions of 5% CO2 and atmospheric oxygen concentration, and DMEM / F-12 medium supplemented with 10% FBS and 1% antibiotic (penicillin-streptomycin) was used as the culture medium.

[0174] After 24 hours of culture, the antisense oligonucleotide (ASO) of Example 1 was diluted to the culture medium at concentrations of 0.001, 0.01, 0.1, 0.316, 13.16, and 10 μM, respectively, and treated. To do this, the existing culture medium was removed and replaced with fresh medium containing ASO, and the cells were treated.

[0175] Forty-eight hours after the ASO treatment of Example 1, cells were harvested and total RNA was extracted using TRI Reagent (Molecular Research Center, Inc.). The extracted RNA was synthesized into DNA using the miRCURY LNA RT kit (Qiagen). The expression level of miR-204 was analyzed using the synthesized cDNA via quantitative reverse transcription polymerase chain reaction (qRT-PCR) with the miRCURY LNA microRNA PCR Kit (Qiagen). Let-7e-5p (miRCURY LNA TM The miRNA PCR assay (Qiagen) was used as an internal control. The maximum degradation efficacy of Example 1 (D max ) and reaction concentration that decomposes miR-204 by 50% (DC 50 ) was calculated using GraphPad prism (Dotmatics).

[0176] (2) Experimental results

[0177] Since miR-204 exists at a very low basal level in normal chondrocytes, a chondrogenic cell line (C28 / I2) overexpressing miR-204 was used to sensitively evaluate the miR-204 degradation efficacy of Example 1. As a result of treating the cell line with Example 1 at various concentrations, it was confirmed that miR-204 expression decreased in a dose-dependent manner in the Example 1 treatment group (Fig. 1a). Quantitative analysis results showed that the maximum degradation efficacy of Example 1 (D max The level was approximately 99.6%, and the reaction concentration that degrades miR-204 by 50% (DC 50 ) was calculated to be approximately 0.0055 μM (Fig. 1b).

[0178] [Experimental Example 1-2] Confirmation of miR-204 resolution of the ASO of Example 2

[0179] (1) Experimental method

[0180] C28 / I2 chondrocytes overexpressing miR-204 were cultured in 12-well plates in DMEM / F-12 medium supplemented with 10% FBS and 1% penicillin-streptomycin under conditions of 5% CO2 atmospheric oxygen and 37°C. At this time, some of the cells were 3 x 10 5After inoculating cells / well, the cells were cultured for 24 hours. After removing the culture medium, 1 μM of Example 2 and the transfection reagent messengerMAX reagent, opti-mem, were diluted and cultured at room temperature for 10 minutes. Subsequently, the cells were treated with Example 2 diluted with the transfection reagent, opti-mem, after being replaced with 0.5 ml of fresh medium, and the medium was replaced with fresh medium after 6 hours. Cells were harvested 48 hours later (Fig. 14). Total RNA was extracted from the harvested cells using TRI Reagent (Molecular Research Center, Inc.), cDNA was synthesized using the miRCURY LNA RT Kit (Qiagen), and miR-204 expression was analyzed by qRT-PCR using the miRCURY LNA microRNA PCR Kit (Qiagen). Let-7e-5p was used as an endogenous control.

[0181] For statistical analysis, all quantitative data were expressed as Mean ± Standard Error (Mean ± SEM). Statistical significance testing was performed using GraphPad Prism 10 software (version 10.4.2, GraphPad Software), and Student's t-test was applied. Statistical significance is indicated within the graph as follows: *, **, ***, ****: P < 0.05, 0.01, 0.001, and 0.0001, respectively.

[0182] (2) Experimental results

[0183] As a result of qRT-PCR analysis, the mean expression level of miR-204 in vehicle trf was 1.000 (SEM 0.000), whereas the mean expression level of miR-204 in Example 2, specifically transfected (carried on a carrier) (0.1 μM), was 0.323 (SEM 0.030), confirming that the ASO composed of a PO backbone also induces the degradation of miR-204 (see Fig. 14).

[0184] This degradation effect of miR-204 suggests that when an oligonucleotide (ASO) according to one aspect of the present invention is delivered into a cell regardless of the backbone components, the target miR-204 can be effectively degraded.

[0185] [Experimental Example 2] Confirmation of delivery ability of miR-204-specific ASO (Example 1) to mouse chondrocytes

[0186] (1) Experimental method

[0187] 1) Mouse chondrocyte primary culture

[0188] Mouse articular chondrocytes were isolated from the femoral condyle and tibial plateau of 5–6 day old ICR mice. The isolated tissues were enzymatically digested using 0.2% collagenase solution (ref), after which single-cell suspensions were obtained. The acquired cells were subjected to flow cytometry in 2 x 10⁻³ of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). 5Cells were seeded into 12-well plates at a cell density of cells / well and cultured. Cells were maintained under conditions of 37°C, 5% CO2, and 21% O2. After 72 hours from seeding, antisense oligonucleotide (ASO) treatment groups and vehicle control groups were established according to each experimental objective. For fluorescence imaging, 1 x 10 5 Cells were inoculated into 12-well plates at a cell density of cells / well and cultured. Cells were maintained under conditions of 37°C, 5% CO2, and 21% O2. After 96 hours from cell inoculation, antisense oligonucleotide (ASO) treatment groups and vehicle control groups were established according to the experimental purpose.

[0189] 2) Verification of the delivery efficiency of the ASO in Example 1

[0190] To visualize the intracellular uptake pattern of Example 1 in chondrocytes, cultured cells were incubated with 1 μM of the fluorescence-labeled Cy5.5 of Example 1 for 6 hours. Subsequently, 1 μg / ml of Hoechst was added to the culture medium, and co-incubation was performed for an additional 18 hours. For fluorescence microscopy analysis, the cells were washed three times with phosphate buffer (PBS), and fluorescence images were acquired using an EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0191] In addition, the degree of intracellular uptake of Example 1 was quantitatively evaluated through flow cytometry. For this purpose, mouse articular chondrocytes were cultured with Cy3 fluorescent label Example 1 (10 nM or 50 nM) for 24 hours. After treatment, the cells were detached from the attachment surface with trypsin, washed with PBS, and recovered by centrifugation. The recovered cells were resuspended twice in a PBS solution containing 5% FBS.

[0192] Flow cytometry analysis was performed using the FACSAria™ III Cell Sorter (BD Biosciences) instrument, and analysis data were collected and quantitatively analyzed using FACSDiva 9.7 software.

[0193] (2) Experimental results

[0194] To confirm the delivery efficiency of Example 1 to mouse-derived chondrocytes, intracellular uptake was evaluated using the Cy5.5 or Cy3 fluorescently labeled Example 1. Fluorescence microscopy analysis confirmed that fluorescent signals were significantly accumulated intracellularly in the mouse-derived chondrocytes of the Example 1 treatment group compared to the vehicle treatment group (Fig. 2a). In addition, flow cytometry results showed that more than 50% of fluorescence-positive signals were observed under all conditions treated with Example 1 at concentrations of 10 nM and 50 nM, quantitatively confirming the excellent intracellular uptake of Example 1 (Figs. 2b, 2c). These results suggest that Example 1 possesses physical and chemical properties that allow it to be efficiently delivered into chondrocytes and stably accumulated.

[0195] [Experimental Example 3] Confirmation of delivery power of Example 1 in osteoarthritis patient tissue

[0196] (1) Experimental method

[0197] 1) In vitro culture of human cartilage tissue

[0198] Osteoarthritis patient tissues obtained from total joint replacement surgery were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). The tissues were maintained under conditions of 37°C, 5% CO2, and 21% O2. Antisense oligonucleotide (ASO) treatment groups and vehicle control groups were established according to each experimental objective.

[0199] 2) Verification of delivery of Example 1

[0200] To visualize the intracellular uptake pattern of Example 1 in chondrocytes, cultured cells were incubated with 1 μM of the fluorescence-labeled Cy5.5 of Example 1 for 24 hours. Subsequently, 1 μg / ml of Hoechst was added to the culture medium, and co-incubation was performed for an additional 2 hours. For fluorescence microscopy analysis, the cells were washed three times with phosphate buffer (PBS), and fluorescence images were acquired using an EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0201] (2) Experimental results

[0202] To confirm the delivery of Example 1 to cartilage tissue derived from osteoarthritis patients, intracellular uptake was evaluated using the Cy5.5 fluorescently labeled Example 1. Fluorescence microscopy analysis confirmed that the fluorescent signal accumulated significantly within the patient-derived chondrocytes of the Example 1 treatment group compared to the vehicle treatment group (Fig. 3). These results suggest that Example 1 possesses physical and chemical properties that allow it to be efficiently delivered and stably accumulated not only in mice but also in human chondrocytes.

[0203] [Experimental Example 4] Confirmation of miR-204 resolution of ASO specific to miR-204

[0204] To confirm the miR-204 resolution of Full DNA, which has the same base sequence as the ASO of Example 1 prepared in the above preparation example but is composed entirely of DNA, and ASOs with a mismatch (indicated in bold) which have a different base sequence from it (Examples 3-1 to 3-3, Table 1 below), the following experiment was performed.

[0205] Here, Full DNA refers to an ASO having a base sequence that is completely complementary to the target miR204, and the sugars of the nucleotides constituting Full DNA do not include LNA sugars or modified sugars and are all 2'-deoxy.

[0206] Separation Sequence (5'-3') Sequence Number Full DNA AGGCATAGGATGA / iMe-dC / AAAGGGAA40 Example 3-1+A+G+G+C+A+TAGGATGAGAAAGGGAA3 Example 3-2+A+G+G+C+A+TAGGAAGA / iMe-dC / AAAGGGAA4 Example 3-3+A+G+G+C+A+TACGAAGA / iMe-dC / AAAGGGAA5

[0207] (1) Experimental method

[0208] An experiment was performed to analyze the expression level of miR-204 via qRT-PCR in the same manner as in Experimental Example 1 above (however, the treatment concentrations of Examples 1 and 3-1 to 3-3 above are 1 μM and 10 μM).

[0209] For statistical analysis, all quantitative data were expressed as mean ± standard error (Mean ± SEM). Statistical significance testing was performed using GraphPad Prism 10 software (version 10.4.2, GraphPad Software); one-way ANOVA was conducted followed by the application of Tukey's post-hoc test. The statistical significance indicators within the graph are as follows.

[0210] *, **, ***, ****: Comparison relative to Vehicle (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0211] #, ##, ###, ####: Comparison of ASO (1 μM concentration group) against Full DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0212] $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) against Full DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0213] (2) Experimental results

[0214] As a result of qRT-PCR analysis, it was observed that miR-204 expression was statistically significantly reduced in the treatment groups of Examples 3-1 to 3-3 compared to the control group without LNA (Full DNA) (see Table 2, Figure 4).

[0215] [Table 2]

[0216]

[0217] Under the 1 μM treatment condition, Examples 3-1 to 3-3 all showed a significant decrease in expression compared to Full DNA, and the same trend was maintained under the 10 μM treatment condition.

[0218] In particular, despite having one mismatch each in the central region (nucleotide 9-12 positions) of miR-204 and two mismatches in the supplementary region (nucleotide 13-16 positions) of miR-204, miR-204 degradation activity was maintained. This suggests that a single or two or more mismatches within the central or supplementary regions of miR-204 do not significantly affect the binding efficiency of the ASO, and that complete complementarity at those sites is not essential.

[0219] Accordingly, ASO variants (Examples 3-1 to 3-3) containing one or more mismatches within the central or supplementary region of miR-204 are determined to maintain excellent stability, binding affinity, and degradation-inducing efficacy for miR-204.

[0220] [Experimental Example 5] Confirmation of miR-204 resolution according to ASO length

[0221] To determine the resolution of miR-204 according to the length of the ASO, experiments were performed on shorter length ASOs (Examples 4-1 and 4-2, Table 3 below) in which some nucleotide sequences in the region near the 3' end of the ASO prepared in the above preparation example were deleted, using the same method as in Experimental Example 1.

[0222] Example sequence (5'-3') Sequence No. 4-1+A+G+G+C+A+TAGGATGA / iMe-dC / AAAGGGA6 4-2+A+G+G+C+A+TAGGATGA / iMe-dC / AAAGGG7

[0223] (1) Experimental method

[0224] An experiment was conducted to analyze the expression levels of miR-204 according to the treatments of Examples 4-1 and 4-2 using qRT-PCR in the same manner as in Experimental Example 4. At this time, the statistical significance in the graph is indicated as follows.

[0225] *, **, ***, ****: Comparison relative to Vehicle (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0226] #, ##, ###, ####: Comparison of ASO (1 μM concentration group) against Full DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0227] $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) against Full DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0228] (2) Experimental results

[0229] As a result of qRT-PCR analysis, it was observed that miR-204 expression was statistically significantly reduced in the antisense oligonucleotide treatment groups (Examples 4-1 and 4-2) in which one (n-1) and two (n-2) bases, respectively, from the 5' end of the miR-204 sequence were deleted, compared to the fully complementary sequence (Full DNA) of miR-204 without LNA (see Table 4, Figure 5).

[0230] [Table 4]

[0231]

[0232] In both concentration conditions of 1 μM and 10 μM, Examples 4-1 and 4-2 showed a tendency for low miR-204 expression relative to Full DNA to be maintained or even enhanced. In particular, Example 4-2 (n-2) exhibited the lowest miR-204 expression level in both concentration conditions, confirming that miR-204 target binding and degradation induction were efficiently maintained even when the number of bases was shortened by two.

[0233] These results suggest that when designing the antisense for miR-204, effective binding and degradation induction are possible even in truncated forms with partial deletions at the n-1 or n-2 level, even if the sequence is not fully complementary.

[0234] Therefore, antisense oligonucleotide variants with 1 to 2 bases missing relative to the complete complementary sequence of miR-204 (Examples 4-1 and 4-2) are considered candidates capable of significantly degrading miR-204.

[0235] [Experimental Example 6] Confirmation of the effect of LNA position on the miR-204 resolution of ASO

[0236] In Experimental Examples 1 to 4 above, it was confirmed that the miR-204 resolution of the ASO of Example 1 was excellent. Since the ASO of Example 1 consists of six LNAs connected in succession within a first region, the following experiments were performed to determine whether the miR-204 resolution of the ASO is affected by the position of the LNAs.

[0237] [Experimental Example 6-1] Effect of LNA position when LNA is included at the 5' end of the ASO

[0238] First, ASOs containing LNAs at the 5' end of the ASO, but with different numbers and positions of LNAs (Examples 5-1 to 5-7, and Comparative Examples 1-1 and 1-2, Table 5 below) were prepared in the same manner as the above preparation example, and their miR-204 resolution was evaluated.

[0239] Classification No. Sequence (5'→3') Sequence Number Example 5-1+A+G+G / iMe-dC / +A+TAGGATGA / iMe-dC / AAAGGGAA85-2+A+G+G+CA+TAGGATGA / iMe-dC / AAAGGGAA95-3+A+G+G+C+ATAGGATGA / iMe-dC / AAAGGGAA105-4+A+G+G / iMe-dC / A+TAGGATGA / iMe-dC / AAAGGGAA115-5+A+G+G+CATAGGA TGA / iMe-dC / AAAGGGAA125-6+A+G+G / iMe-dC / +ATAGGATGA / iMe-dC / AAAGGGAA135-7+A+G+G / iMe-dC / ATAGGATGA / iMe-dC / AAAGGGAA14Comparative Example 1-1+AGG / iMe-dC / ATAGGATGA / iMe-dC / AAAGGGAA151-2+A+GG / iMe-dC / ATAGGATGA / iMe-dC / +AAAGGGAA16

[0240] (1) Experimental method

[0241] Experiments were conducted to analyze the expression levels of miR-204 according to the treatments of Examples 5-1 to 5-7 and Comparative Examples 1-1 and 1-2 using qRT-PCR in the same manner as in Experimental Example 4. At this time, the statistical significance in the graph is indicated as follows.

[0242] The statistical significance indicators within the graph are as follows.

[0243] *, **, ***, ****: Comparison relative to Vehicle (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0244] #, ##, ###, ####: Comparison of ASO (1 μM concentration group) against Full DNA in Examples 5-1 to 5-7 / Comparison of ASO (1 μM concentration group) against Example 1 in Comparative Examples 1-1 to 1-2 (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0245] $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) against Full DNA in Examples 5-1 to 5-7 / Comparison of ASO (10 μM concentration group) against Example 1 in Comparative Examples 1-1 to 1-2 (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0246] (2) Interpretation of experimental results

[0247] As a result of qRT-PCR analysis, it was observed that miR-204 expression was statistically significantly reduced in both ASOs with three consecutive LNAs at the 5' end (where all three nucleotides in the first region were LNAs) (Examples 5-7) and ASOs with one or two LNAs at the 4th-6th base positions from the 5' end (where at least one of the three nucleotides in the second region was LNAs) (Examples 5-1 to 5-6) compared to the control group without LNAs (Full DNA) (see Table 6 below, and Figures 6 and 7).

[0248] [Table 6]

[0249]

[0250] Under two treatment concentration conditions of 1 μM and 10 μM, the ASOs of all Examples 5-1 to 5-7 showed a significant miR-204 degradation effect compared to Full DNA, and in particular, Examples 5-1 to 5-7 showed the same trend.

[0251] These results suggest that for miR-204 target binding, LNA can be included in the 5' end (first region, nucleotide 1-3) while LNA or DNA can be placed in the second region (nucleotide 4-6 from the 5' end), and even in that case, excellent miR-204 degradation activity can be maintained.

[0252] Therefore, if the placement of LNA is concentrated near the 5' end (first region), it is determined that the ASO with DNA or LNA mixed in the adjacent second region has stable and efficient degradation efficacy against miR-204.

[0253] [Experimental Example 6-2] Effect of LNA position when LNA is included at the 3' end of the ASO

[0254] In Experimental Example 6-1 above, it was confirmed that ASOs in which at least three of the 1st to 6th nucleotides of the 5' end are LNAs, specifically in which at least all of the 1st to 3rd nucleotides are LNAs, have excellent miR-204 resolution. Accordingly, to determine whether there is a difference in miR-204 resolution when the ASO contains LNAs at the 3' end rather than the 5' end (Comparative Examples 2-1 to 2-4, Table 7 below), miR-204 resolution was evaluated using the same method as in Experimental Example 1 above.

[0255] Classification No. Sequence (5'→3') Sequence Number Comparison Example 2-1 AGG / iMe-dC / ATAGGATGA / iMe-dC / AAAGGGA+A172-2 AGG / iMe-dC / ATAGGATGA / iMe-dC / AAAGGG+A+A182-3 AGG / iMe-dC / ATAGGATGA / iMe-dC / AAAG+G+G+A+A192-4 AGG / iMe-dC / ATAGGATGA / iMe-dC / AA+A+G+G+G+A+A20

[0256] (1) Experimental method

[0257] An experiment was conducted to analyze the expression levels of miR-204 according to the treatments of Comparative Examples 2-1 to 2-4 using qRT-PCR in the same manner as in Experimental Example 4. At this time, the statistical significance in the graph is indicated as follows.

[0258] *, **, ***, ****: Comparison relative to Vehicle (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0259] #, ##, ###, ####: Comparison of ASO (1 μM concentration group) against Example 1 (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0260] $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) against Example 1 (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0261] (2) Interpretation of experimental results

[0262] The effect of the terminal position of the LNA on the degradation of miR-204 was evaluated through qRT-PCR analysis. Example 1, in which the LNA was positioned at the 5' end, was compared with ASO variants in which the LNA was positioned at the 3' end (Comparative Examples 2-1 to 2-4). As a result, the ASOs with the LNA located at the 3' end (Comparative Examples 2-1 to 2-4) showed reduced degradation of miR-204 at both concentrations (1 μM and 10 μM) compared to LA001, in which the LNA was positioned at the 5' end (see Table 8 and Figure 8 below).

[0263] [Table 8]

[0264]

[0265] Under the 1 μM treatment condition, Comparative Examples 2-1 and 2-2 showed a statistically significant reduction in the miR-204 degradation effect compared to Example 1, while Comparative Examples 2-3 and 2-4 showed a decreasing trend. Under the 10 μM treatment condition, a statistically significant reduction in the degradation effect was confirmed in Comparative Examples 2-1, 2-3, and 2-4, and Comparative Example 2-2 also showed a similar decreasing trend.

[0266] These results suggest that the position of the LNA has a decisive influence on the miR-204 degradation activity of ASO, and that the most superior efficacy is exhibited, particularly when the LNA is located at the 5' end. In other words, it is determined that when the LNA is moved to the 3' end, the binding affinity of ASO and the RNase H-mediated degradation efficiency are reduced, thereby decreasing the overall miR-204 degradation effect.

[0267] [Experimental Example 6-3] Effect of LNA Location When LNAs Are Randomly Included in ASO

[0268] Through Experimental Example 6-2 above, it was found that even if three or more LNAs are included in the ASO, the miR-204 degradation effect is not significant if they are included only at the 3' end. Accordingly, to evaluate the miR-204 degradation when LNAs are randomly included in the ASO (Comparative Examples 3 and 4, Table 9 below), the following experiment was performed.

[0269] Classification No. Sequence (5'→3') Sequence Number Comparison Example 3+AG+GAT+G+ACA+AA+GGG+A214+CAT+AGGA+TGA+CAAA+GGG+A22

[0270] (1) Experimental method

[0271] C28 / I2 chondrocytes overexpressing miR-204 were cultured in 12-well plates in DMEM / F-12 medium supplemented with 10% FBS and 1% penicillin-streptomycin under conditions of 5% CO2 atmospheric oxygen and 37°C. At this time, some of the cells were 3 x 10 5 After inoculating cells / well, the cells were cultured for 24 hours. After removing the culture medium, the medium was replaced with fresh medium diluted to 10 μM of Comparative Example 3 for a single treatment, and the cells were harvested 48 hours after treatment (Group 1, Fig. 9). Meanwhile, the remaining cells were 2 × 10 4After inoculating cells / well and culturing for 24 hours, Comparative Example 4 was treated at 256 nM, 1.6 μM, and 10 μM while changing the culture medium. Then, a second treatment at the same concentration was performed 72 hours after the first treatment, and cells were harvested 48 hours after the second treatment (Group 2, Fig. 10). Total RNA was extracted from the cells harvested from each group using TRI Reagent (Molecular Research Center, Inc.), cDNA was synthesized using the miRCURY LNA RT Kit (Qiagen), and miR-204 expression was analyzed by qRT-PCR using the miRCURY LNA microRNA PCR Kit (Qiagen), and Let-7e-5p was used as an endogenous control.

[0272] For statistical analysis, all quantitative data were expressed as mean ± standard error (Mean ± SEM). Statistical significance testing was performed using GraphPad Prism 10 software (version 10.4.2, GraphPad Software); one-way ANOVA was conducted followed by the application of Tukey's post-hoc test. The statistical significance indicators within the graph are as follows.

[0273] *, **, ***, ****: P < 0.05, 0.01, 0.001, 0.0001 respectively

[0274] (2) Interpretation of experimental results

[0275] As a result of qRT-PCR analysis, Comparative Example 3, in which LNA was randomly included in Group 1, showed a statistically significantly lower miR-204 degradation effect compared to Example 1 (see Table 10 and Fig. 9 below).

[0276] [Table 10]

[0277]

[0278] This low miR-204 degradation effect implies that the position and arrangement regularity of LNAs act as more important factors in miR-204 degradation activity than the inclusion of LNAs within the ASO. Similarly, Comparative Example 4, another form in which LNAs are randomly included, also showed lower miR-204 degradation activity than Example 1 in all concentration groups (see Fig. 10). In other words, in structures where LNAs are randomly arranged as in Comparative Examples 3 and 4, efficient binding to and degradation induction of miR-204 is difficult, suggesting that an ASO according to one aspect of the present invention, such as Example 1, which is arranged around the 5' end (around the first and second regions), is more effective for miR-204 degradation.

[0279] [Experimental Example 7] Confirmation of the effect of the number of LNAs on the miR-204 resolution of ASO

[0280] In Experimental Example 6 above, it was confirmed that the miR-204 resolution varied depending on the position of the LNA within the ASO. To confirm the effect of the number of LNAs within the ASO, ASOs were prepared in which the LNAs were positioned from the 5' end of the ASO of Example 1 and there were 15 or more LNAs (Comparative Examples 5-1 to 5-3, Table 11 below), and their formulation stability was evaluated.

[0281] Category No. Sequence (5'→3') Sequence number comparison example 5-1+A+G+G+C+A+T+A+G+G+A+T+G+A+C+AAAGGGAA235-2+A+G+G+C+A+T+A+G +G+A+T+G+A+C+A+A+A+G+GGAA245-3+A+G+G+C+A+T+A+G+G+A+T+G+A+C+A+A+A+G+G+G+A+A25

[0282] (1) Experimental method

[0283] An experiment was conducted to analyze the expression levels of miR-204 according to the treatments of Comparative Examples 5-1 to 5-3 using qRT-PCR in the same manner as in Experimental Example 4. At this time, the statistical significance in the graph is indicated as follows.

[0284] *, **, ***, ****: Comparison relative to Vehicle (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0285] In addition, to confirm the physical properties (viscosity) of the dissolved ASOs of Comparative Examples 5-1 to 5-3, the tube containing each solution was photographed from below at an angle, and after collecting the solution using a 1ml pipette tip, the solution inside the tip was photographed.

[0286] (2) Interpretation of experimental results

[0287] The effect of the number of LNAs in antisense oligonucleotides (ASOs) on the degradation of miR-204 was evaluated through qRT-PCR analysis. Experiments were conducted on variants with progressively increased numbers of LNAs (Comparative Examples 5-1 to 5-3), and as a result, the degradation effect of miR-204 remained similar even as the number of LNAs increased (see Fig. 11a).

[0288] However, in the variants with an excessively increased number of LNAs (Comparative Examples 5-2 and 5-3), a physical phenomenon was observed in which the viscosity increased rapidly during the dissolution process and the solution aggregated into a jelly-like form (see Fig. 11b), and even in the variant with a relatively small number of LNAs (Comparative Example 5-1), higher viscosity was observed than in Example 1. This is considered to be a phenomenon in which water solubility decreases as the interaction between ASOs is strengthened with increasing LNA content.

[0289] Synthesizing these results, it was found that while an increase in the number of LNAs enhances the miR-204 degradation effect, it can simultaneously lead to physical and formulation limitations as a drug due to increased viscosity and decreased solubility of the formulation. Therefore, considering both efficacy and formulation stability, a level of LNAs that is not excessive (e.g., the level of Example 1) is considered the optimal structure that maintains a balance between miR-204 inhibitory efficacy and formulation suitability.

[0290] [Experimental Example 8] Effect of modifications other than LNA on miR-204 resolution

[0291] In Experimental Example 6-1 above, it was confirmed that the miR-204 resolution of ASOs is excellent for which at least three of the 1st to 6th nucleotides at the 5' end are LNAs (ASOs in which at least three of the nucleotides in the first and second regions are LNAs), specifically, for which at least all of the 1st to 3rd nucleotides are LNAs (ASOs in which all three nucleotides in the first region are LNAs). In cases where at least three of the 1st to 6th nucleotides at the 5' end of the ASO are LNAs as in Experimental Example 6-1, specifically where at least all of the 1st to 3rd nucleotides are LNAs, but additionally other modification sugars (e.g., non-fixed 2'-substituted sugar modifications) other than LNA modification sugars are present at other positions (Examples 6-1 to 6-14, Table 12 below; MOE modification is X M , the OME variant is X O Indicated as (X is a base)) miR-204 resolution was evaluated to determine if there was a difference in miR-204 resolution.

[0292] [Table 12]

[0293]

[0294] (1) Experimental method

[0295] An experiment was conducted to analyze the expression levels of miR-204 according to the treatments of Examples 6-1 to 6-14 using qRT-PCR in the same manner as in Experimental Example 4. At this time, the statistical significance indicated in the graph is as follows.

[0296] *, **, ***, ****: Comparison relative to Vehicle (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0297] #, ##, ###, ####: Comparison of ASO (1 μM concentration group) against Full DNA in Examples 6-1 to 6-14 (P < 0.05, 0.01, 0.001, 0.0001, respectively);

[0298] $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) against Full DNA in Examples 6-1 to 6-14 (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0299] (2) Interpretation of experimental results

[0300] In order to determine the effect of introducing a different modified sugar instead of LNA in the second region (the 4th to 7th nucleotide positions relative to the ASO) on the efficacy of the ASO against miR-204, experiments were conducted. To this end, ASOs containing 1 to 3 2'-O-methoxyethyl (MOE) at the corresponding positions (Examples 6-1 to 6-7) and ASOs containing 2'-O-Methyl (OME) at the same positions (Examples 6-8 to 6-14) were designed, and experiments were conducted under the same conditions as in Example 1.

[0301] As a result of qRT-PCR analysis, the miR-204 degradation effect was maintained even when MOE or OME was introduced into the second region, and statistically significantly higher miR-204 degradation ability was confirmed compared to the control group (Full DNA) in which LNA was completely excluded (see Figures 12 and 13), confirming that the efficacy of ASO is not significantly reduced even when modified sugars other than LNA are partially introduced into the second region.

[0302] These results suggest that even if MOE or OME is introduced instead of LNA in the second region (position 4-7), the overall structural stability of the ASO and miR-204 degradation activity can be maintained, and demonstrate that it is possible to design modified ASOs with chemical diversity.

[0303] [Experimental Example 9] Effects of miR-204-specific ASO in a cell model

[0304] In Experimental Example 1 above, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, to confirm whether the ASO of Example 1 above exhibits an effect of preventing or treating osteoarthritis by degrading miR-204 in a cell model, the following experiment was performed.

[0305] (1) Experimental method

[0306] 1) Mouse chondrocyte primary culture

[0307] Mouse articular chondrocytes were isolated from the femoral condyle and tibial plateau of 5–6 day old ICR mice. The isolated tissues were enzymatically digested using a 0.2% collagenase solution to obtain single-cell suspensions. The acquired cells were inoculated and cultured (primary culture) at different cell densities according to each experimental objective in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). The cells were maintained under hypoxic conditions of 37°C, 5% CO2, and 3% O2. After 72 hours from cell inoculation, antisense oligonucleotide (ASO) treatment groups and vehicle control groups were established according to each experimental objective. To induce senescence in chondrocytes, primary cultured chondrocytes were treated with 40 μg / ml of bleomycin (Cayman) for 24 hours. Subsequently, the medium was removed and replaced with a growth medium containing nuclease-free water (NFW) as the vehicle or a growth medium containing Example 1 (SEQ No. 1) (final concentration 2 μM). Cells were harvested for mRNA analysis at 48 hours after treatment, with an inoculation density of 5 × 10⁻¹⁰ 4 It was cells / well. In addition, cells were harvested for protein analysis 72 hours after treatment with Example 1 (final concentrations 0.1, 1, and 10 μM), and the inoculation density at this time was 2 × 10 based on a 6-well plate. 5The cell count was cells / well. To induce an inflammatory response in chondrocytes, primary cultured chondrocytes were treated with 1 ng / ml of IL-1β (Genscript) and simultaneously cultured for 72 hours with the co-administration of Example 1 (10 μM) or SM04690 (30 nM). Subsequently, cells were harvested and mRNA analysis was performed, with an inoculation density of 0.83 x 10⁻¹⁰ based on a 12-well plate. 5 It was cells / well.

[0308] 2) Analysis of changes in gene expression according to Example 1 via qRT-PCR and Western blot

[0309] Cultured cells were harvested, and total RNA was extracted using TRI Reagent (Molecular Research Center, Inc.). The extracted RNA was synthesized into cDNA using SuperScript IV Reverse Transcriptase (Invitrogen). The synthesized cDNA was used in quantitative reverse transcription polymerase chain reaction (qRT-PCR) with SYBR Green qPCR Master Mix (ThermoFisher) to analyze the expression levels of genes related to chondrocyte ECM composition and inflammation. The Hprt gene was used as an internal control, and the relative expression level of each gene was calculated as relative to the vehicle control using the ΔΔCt method. For statistical analysis, all quantitative data were expressed as mean ± standard error (Mean ± SEM). Statistical significance was tested using GraphPad Prism 10 software (version 10.4.2); Tukey's post-hoc test was applied after conducting one-way ANOVA. The primers used for qRT-PCR are shown in Table 13 below.

[0310] [Table 13]

[0311]

[0312] For protein expression analysis, cultured cells were harvested and total protein was extracted using RIPA buffer. Quantified protein samples were separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to an NC membrane. Subsequently, primary and secondary antibody reactions were performed against cartilage matrix synthesis-related proteins (HPLN1, CHSY1) and endogenous control protein (Vinculin). Protein signals were detected using SuperSignal West Dura Extended Duration Substrate ECL solution (ThermoFisher) and confirmed using a chemiluminescence signal detection system (iBright FL1500, ThermoFisher). The HPLN1 antibody (ab98038) was purchased from Abcam, the CHSY1 antibody (NBP3-47604) from Novus Biologicals, and the Vinculin antibody (13901) from Cell Signaling Technology.

[0313] 3) sGAG assay

[0314] Quantitative analysis of sGAG was performed using primary cultured mouse chondrocytes according to the method described above. To induce cellular senescence, cells were treated with bleomycin at a concentration of 40 μg / mL for 24 hours. After treatment, the cells were cultured for an additional 3 days in fresh complete medium containing or excluding Example 1. Subsequently, the medium was replaced and cultured again for 3 days, after which the culture supernatant was collected and used for sGAG analysis. The concentration of sGAG in the collected culture medium was measured using the 1,9-dimethylmethylene blue (DMMB) staining method. Absorbance was measured at 525 nm, and the sGAG content was quantified based on a standard curve prepared using chondroitin sulfate (CO335; Tokyo Chemical Industry). To compensate for differences in cell viability and cell density, an MTT assay was performed on the same cells. For this purpose, a PBS-based MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, T-030-1; Goldbio) was added at a concentration of 100 μg / mL and reacted for 2 hours. After the reaction, the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm. All sGAG measurements were normalized based on the MTT results for comparison and analysis. The maximum effect of Example 1 (E max ) and semi-maximum effective concentration (EC 50 ) was calculated using GraphPad prism (Dotmatics).

[0315] 4) Pathway Analysis

[0316] A list of differentially expressed genes (DEG list) was derived from transcriptome data obtained after treating C28 / I2 cell lines overexpressing miR-204 with Example 1 at a concentration of 1 μM. Reactome pathway analysis was performed using the EnrichR platform with the derived DEG list. The analysis results selected the top 20 annotations based on adjusted p-values ​​and visualized them in the form of a bubble plot.

[0317] (2) Experimental results

[0318] Since miR-204 exists at a very low basal level in normal chondrocytes, a chondrogenic cell line (C28 / I2) overexpressing miR-204 was used to sensitively evaluate the miR-204 degradation efficacy of Example 1. Transcriptome analysis confirmed that gene annotations related to ECM remodeling, including ECM proteoglycans, and biological pathways related to maintaining cartilage structure were significantly altered (Fig. 15). This suggests that the inhibition of miR-204 by Example 1 is not limited to simple microRNA-level regulation but is also involved in structural recovery mechanisms such as ECM composition and homeostasis maintenance within chondrocytes.

[0319] Based on the above results, the protective mechanism of action of Example 1 was verified at the molecular level in primary cultured mouse chondrocytes. To this end, changes in the expression of key enzymes of the sulfated proteoglycan biosynthesis pathway were analyzed, consistent with the results confirmed in the transcriptome analysis. When Example 1 was applied to chondrocytes damaged by bleomycin, the expression of key enzymes of the sGAG biosynthesis pathway increased statistically significantly, while the expression of substrate-degrading enzymes decreased significantly (Fig. 16a). This recovery of expression indicates that the cartilage matrix synthesis pathway, which had been inhibited by miR-204, was restored to normal levels by Example 1, and the same increasing trend was confirmed at the protein level following treatment with Example 1 (Fig. 16b). These results molecularly and functionally demonstrate the mechanism by which Example 1 effectively inhibits the pathological action of miR-204 while simultaneously restoring the matrix synthesis ability of chondrocytes.

[0320] Next, to evaluate the effect of Example 1 under inflammatory stimulation conditions, experiments were conducted using primary cultured chondrocytes treated with the inflammatory cytokine IL-1β. As a result, similar to the results observed when damage was induced with bleomycin, the expression of proteoglycan biosynthetic enzymes was significantly restored in the Example 1 treatment group, while the expression of the inflammatory cytokine IL-6 was statistically significantly reduced (Fig. 16c). In particular, Example 1 showed a better effect compared to SM04690, which has currently completed Phase 3 clinical trials.

[0321] These results show that Example 1 effectively inhibits the miR-204-mediated pathway in both aging and inflammatory environments, thereby performing a dual action of promoting cartilage matrix synthesis and alleviating inflammatory responses.

[0322] Next, additional experiments were conducted to verify whether the action of Example 1 was limited to changes at the gene and protein levels, or whether it also influenced the functional phenotype of actual cells. As a result of treating aging-induced chondrocytes with Example 1, it was confirmed that the sulfated glycosaminoglycan (sGAG) content recovered in a concentration-dependent manner (Fig. 17a). Quantitative analysis results showed the maximum effect of Example 1 (E max ) is 114.3%, semi-maximum effective concentration (EC 50 ) was calculated to be 273.5 nM (Fig. 17b). In addition, an increase in sGAG content by Example 1 was confirmed even in an inflammatory environment, and the effect was superior to that of SM04690 (Fig. 17c). This phenomenon of substrate content recovery means that Example 1 does not merely stop at inhibiting miR-204 expression or regulating downstream protein expression, but actually functionally restores the ability to synthesize cartilage matrix at the cellular level.

[0323] That is, Example 1 was proven to be a therapeutic candidate that structurally and functionally restores the ECM synthesis function of chondrocytes by effectively blocking the pathological signaling pathway of miR-204 in both aging and inflammatory environments.

[0324] [Experimental Example 10] Confirmation of the effect of an ASO specific to miR-204 in an animal model

[0325] In Experimental Example 1 above, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, to confirm whether the ASO of Example 1 exhibits an effect of preventing or treating osteoarthritis by degrading miR-204 in an animal model, the following experiment was performed.

[0326] (1) Experimental method

[0327] 1) Construction of an osteoarthritis mouse model

[0328] The male C57BL / 6 mice (12 weeks old, WT) used in the experiment were purchased from Daehan Biolink Co., Eumsung, Korea. All animal experiments were approved (SNU-240710-5-1) by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. Experimental animals were randomly assigned and housed in a specific pathogen-free facility at Seoul National University. Housing conditions were controlled to a temperature of 23–25°C, humidity of 45–65%, and a 12-hour light / dark cycle. All mice were allowed to freely consume standard experimental solid feed. Animal experiment reporting followed the ARRIVE guidelines. To induce post-traumatic osteoarthritis (OA), destabilization of the medial meniscus (DMM) was performed on the right knee of 12-week-old male mice, while sham surgery was performed as a control group under the same conditions.

[0329] To evaluate the therapeutic efficacy of Example 1 for osteoarthritis, Example 1 and SM04690 were administered intra-articularly into the knee joint at 4 weeks post-surgery, and dexamethasone was administered once a week to the same site starting from 2 weeks post-surgery. Mice were sacrificed at 6 weeks post-administration for histological analysis. In addition, to evaluate the therapeutic effect on the late stage of osteoarthritis, Example 1 was injected intra-articularly a total of 2 times at 2-week intervals starting from 4 weeks post-surgery, and mice were sacrificed at 4 weeks post-surgery for histological analysis. A static weight-bearing analysis was performed one day prior to sacrifice.

[0330] 2) Histological analysis, immunohistochemistry, and in situ hybridization

[0331] The legs of mice sacrificed for histological analysis were excised, placed in a 4% paraformaldehyde solution, and fixed at 4°C. The fixed leg tissues were decalcified in a 0.5 M EDTA (pH 7.4) solution at 4°C for 4 weeks, followed by stepwise dehydration with ethanol, xylene treatment, and paraffin embedding. The prepared paraffin blocks were sliced ​​to a thickness of 5 μm to prepare slides.

[0332] Slides were deparaffinized with xylene and then rehydrated with ethanol of progressively lower concentrations. Subsequently, Safranin O staining was performed to observe changes in cartilage tissue, and the degree of medial tibial cartilage damage was evaluated in a double-blind manner according to OARSI criteria (grade 0-6).

[0333] Immunohistochemistry staining was performed using SLC35D1 (ab113717, Abcam), IL6 (sc-130326, Stanta Cruz Biotechnology), and CTXII (NBP2-59386, Novus Biologicals) antibodies diluted 1:100, and CHSY1 (NBP3-47604, Novus Biologicals), HPLN1 (ab98038, Abcam), and HAS2 antibodies (NBP3-03787, Novus Biologicals) diluted 1:50.

[0334] For in situ hybridization, the probe for miR-204 detection and the probe for Example 1 (manufactured by BIONEER) were used, and the probe for U6 snRNA detection was purchased from Exiqon. Tissues were treated with 5 μg / ml proteinase K at 37°C for 30 minutes, followed by refixation with a 4% paraformaldehyde solution at room temperature for 20 minutes. The hybridization reaction was performed under the following conditions: the U6 snRNA probe (1 nM) was reacted at 50°C for 1 hour, the probe for Example 1 (50 nM) at 42°C for 2 hours, and the miR-204 probe (50 nM) at 36°C for 2 hours. Afterward, the anti-DIG-AP antibody (1:800 dilution, Roche, 11093274910) was reacted at room temperature for 1 hour, and then the signal was developed using NBT / BCIP solution (Thermo Fisher, 34070).

[0335] All histological, immunohistochemical, and in situ hybridization images were acquired by connecting a DS-Ri2 camera (Nikon) to a Nikon Eclipse Ni-U optical microscope.

[0336] 3) Micro-computed tomography (μCT) analysis

[0337] To evaluate the therapeutic effect of Example 1 and potential side effects regarding bone remodeling, the knee joints of mice were resected and fixed using the method described above. The therapeutic effect was evaluated using a Bruker SKYSCAN 1276 in vivo μCT system (70 kV, 57 μA, 0.5 mm Al filter, scanning time 13 min), and the obtained images were reconstructed using NRecon software (Bruker, version 1.7.3.2). 3D images were generated using CTvox (Bruker, version 3.3.0).

[0338] (2) Interpretation of experimental results

[0339] The therapeutic efficacy of Example 1 was evaluated in a surgically induced osteoarthritis mouse model. In the DMM surgical group injected with Vehicle (administration control group), typical pathological changes of osteoarthritis, such as cartilage destruction, subchondral bone sclerosis, osteophyte development, and synovial inflammation, were severe. In contrast, in the group administered Example 1, cartilage damage induced by DMM was statistically significantly inhibited, and the degree of subchondral bone sclerosis, osteophyte maturation, and synovial inflammation was also significantly alleviated (Figs. 18a to 18c). In situ hybridization analysis confirmed that Example 1 remained continuously present in the articular cartilage at a dose of 10 μg or more even after 6 weeks of administration (Fig. 18d), suggesting that Example 1 can remain stably within the cartilage for a long period and exert a sustained therapeutic effect. In behavioral analysis as well, intra-articular administration of Example 1 statistically significantly restored weight-bearing imbalance caused by DMM surgery, which means that the drug can substantially alleviate osteoarthritis-associated pain behavior (Fig. 18e). As a result of molecular analysis, the expression of enzymes involved in chondroitin sulfate synthesis was restored in the treatment group of Example 1, and the expression of proteins related to cartilage matrix destruction was significantly reduced (Fig. 18f), which shows that it contributes to the structural recovery and functional restoration of cartilage tissue.

[0340] The efficacy of Example 1 was consistently maintained even in a two-dose repeated administration experiment. Even when the ASO was injected twice, both cartilage damage and subcutaneous bone sclerosis were significantly reduced, and the pain relief effect also persisted. In particular, the efficacy of Example 1 was generally superior compared to a steric blocker (Comparative Example 4) (Sequence No. 22) compared under the same conditions, showing a stable and reproducible effect even after repeated administration (Figs. 19a to 19d).

[0341] To verify the therapeutic efficacy of Example 1, a comparative experiment was conducted with drugs currently in clinical use or under development. The experiment was conducted using SM04690, an osteoarthritis treatment that has completed Phase 3 clinical trials, and Dexamethasone, a steroidal anti-inflammatory analgesic, as control groups. As a result, SM04690 showed some pathological improvement but had limited cartilage protective effects compared to Example 1, and while Dexamethasone also showed effects of alleviating inflammation and pain, its cartilage regenerative capacity was low. On the other hand, the group administered Example 1 showed the most significant reduction in cartilage destruction, as well as the best improvement in subcutaneous bone sclerosis and pain relief (Figs. 20a to 20e). These results suggest that, unlike existing inflammation-modifying drugs, Example 1 improves the pathophysiological root cause by inducing the protection and restoration of the cartilage structure itself.

[0342] In addition, to evaluate the potential for treatment in the late stage of osteoarthritis, drugs were administered 6 weeks after OA induction. As a result, statistically significant cartilage recovery was observed only in the group administered in Example 1, while no significant effect was confirmed in the groups administered with Steric blocker (Comparative Example 4) (Sequence No. 22) and SM04690 (Figs. 21a to 21c). On the other hand, the pain improvement effect was confirmed to be statistically significant in all three groups: Example 1, Steric blocker (Comparative Example 4) (Sequence No. 22), and SM04690 (Fig. 21d). Micro-CT analysis also showed that Example 1 most effectively alleviated subcutaneous bone sclerosis, which provides a mechanistic basis for restoring the structural stability of the subchondral bone.

[0343] Synthesizing the above results, it was confirmed that Example 1 is an excellent therapeutic candidate that inhibits the pathological progression of osteoarthritis at multiple levels compared to other drugs. The substance demonstrated excellent therapeutic effects in structural, functional, and behavioral aspects through a complex mechanism of action comprising (1) inhibition of cartilage destruction and promotion of regeneration, (2) reduction of subcutaneous bone sclerosis and osteophyte formation, (3) alleviation of synovitis and inhibition of inflammatory response, (4) improvement of pain and recovery of function, and (5) maintenance of long-term efficacy through continuous retention within the cartilage. In particular, Example 1 showed superior cartilage protection and pain relief effects compared to existing clinical-stage drugs or steroids, and maintained reproducible efficacy in both single and repeated administrations. These results strongly suggest that Example 1 is not merely a symptom reliever, but a novel ASO drug candidate with a mechanism capable of improving the fundamental pathological condition of osteoarthritis.

[0344] [Experimental Example 11] Confirmation of off-target effect of Example 1

[0345] In Experimental Example 1 above, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, to evaluate the off-target effect of the ASO of Example 1 above, the following experiment was performed.

[0346] (1) Experimental method

[0347] 1) Predicted target analysis of Example 1

[0348] Expected target genes (human mRNA, microRNA, and pre-mRNA) were analyzed for sequence variants including the nucleotide sequence (n) of Example 1, a form with one nucleotide deleted at the 3' end (n-1), and a form with one nucleotide repeated and added at the 3' end (n+1). Sequence-based homology analysis was performed using NCBI-BLAST (Basic Local Alignment Search Tool), and the human gene databases provided by NCBI (human RefSeq_RNA and human RefSeq_Gene) were used for the analysis. When evaluating sequence alignment, a case where there were two or fewer mismatches was set as the criterion for a significant match.

[0349] 2) Gene ontology (GO) term analysis

[0350] A list of differentially expressed genes (DEG list) was derived from transcriptome data obtained after treating C28 / I2 cell lines overexpressing miR-204 with Example 1 at a concentration of 1 μM. Gene Ontology (GO) term analysis was performed using the EnrichR platform based on the derived DEG list. The analysis results selected the top 10 annotations based on adjusted p-values ​​and visualized them in the form of a bubble plot.

[0351] (2) Interpretation of experimental results

[0352] Based on the nucleotide sequence of Example 1, expected target genes (human mRNA, microRNA, and pre-mRNA) capable of binding were analyzed. As a result of evaluating homology using NCBI-BLAST, no sequences expected to bind were identified other than TRPM3, the host gene of miR-204, and miR-204, the on-target (see Table 14). Table 14 shows the results of sequence homology analysis performed using NCBI-BLAST on the nucleotide sequence and sequence variants of Example 1.

[0353] Number of bases in the sequence Human mRNA or microRNA Human pre-mRNA nNATRPM3n-1NATRPM3n+1NATRPM3

[0354] *TRPM3 is the host gene of miR-204. Additionally, Gene Ontology (GO) analysis was performed using transcriptome data obtained after treating C28 / I2 cell lines overexpressing miR-204 with Example 1. The analysis was performed using EnrichR, and the top 10 annotations were selected based on adjusted p-values. As a result, no significant ontology terms were derived for Biological Process, Molecular Function, or Cellular Component (see Figs. 22a to 22c). These results suggest that Example 1 is a highly specific ASO with high sequence specificity for miR-204 and extremely low off-target effects.

[0355] [Experimental Example 12] Confirmation of Immunogenicity of Example 1

[0356] In Experimental Example 1 above, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, to confirm the immunogenicity of Example 1, the following experiment was performed.

[0357] (1) Experimental method - Toll-like receptor (TLR) activity assay

[0358] To evaluate the effect of Example 1 on Toll-like receptor (TLR) activity, a TLR activity assay was performed using HEK-Blue cell lines (InvivoGen) overexpressing TLR7, TLR8, and TLR9, respectively. The HEK-Blue TLR7 cell line was 5 x 10 4 Cells were seeded into a 96-well plate at a cell / well density and cultured for 24 hours. Subsequently, Example 1 (10 μM) or ORN6S (positive control) was treated with 0.1 mM Guanosine (Sigma, G6264). Six hours after drug treatment, the cell culture supernatant was collected, and Secreted Alkaline Phosphatase (SEAP) activity in the culture supernatant was measured. HEK-Blue TLR8 cell lines were seeded at the same density and cultured for 24 hours, and then treated with Example 1 (10 μM) or ORN6S along with 1 mM Uridine (Sigma, U3003). Six hours after treatment, the culture supernatant was collected, and SEAP activity was measured. HEK-Blue TLR9 cell lines were cultured under the same conditions and then treated with Example 1 (10 μM) or ODN2006 (positive control). Six hours after treatment, the cell culture supernatant was collected, and SEAP activity was measured.

[0359] (2) Interpretation of experimental results

[0360] To evaluate the immunogenicity of Example 1, changes in the activity of Toll-like receptors (TLR) 7, 8, and 9 were measured. As a result of analyzing the ligand-dependent SEAP expression using HEK-Blue TLR7, TLR8, and TLR9 cell lines, ORN6S (for TLR7 and TLR8 assays) and ODN2006 (for TLR9 assays), which were used as positive controls, both induced receptor activity, and SEAP activity significantly increased.

[0361] On the other hand, no significant increase in TLR7, TLR8, and TLR9 activity was observed under any conditions treated with Example 1 (10 μM) (see Figs. 23a to 23c). These results indicate that Example 1 does not induce an immune response through TLR7, TLR8, and TLR9 and has non-immunostimulatory characteristics toward innate immune receptor pathways. Therefore, Example 1 suggests that it is a safe antisense oligonucleotide with a low potential for inducing immunogenicity.

[0362] [Experimental Example 13] Confirmation of toxicity of Example 1

[0363] In Experimental Example 1 above, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, as it is necessary to examine safety when utilizing a composition containing Example 1 as a pharmaceutical composition, the following experiment was performed to confirm the toxicity of Example 1.

[0364] (1) Experimental method

[0365] 1) Cytotoxicity assay

[0366] To evaluate the cytotoxicity of Example 1, mouse primary chondrocytes, a hepatocyte line (HepG2 cell line), and a kidney cell line (RPTEC cell line) were used. The HepG2 and RPTEC cell lines were purchased from the American Cell Line Bank (ATCC).

[0367] Each cell line was seeded into a 96-well plate under the following conditions: Mouse primary culture chondrocytes: 1 x 10 4 cells / well; HepG2 cells: 1 x 10 3 cells / well; RPTEC cells: 4 x 10 4 cells / well. Each cell line was cultured for 72 hours (chondrocytes) and 24 hours (HepG2 and RPTEC cells) under each condition, after which Example 1 was treated at concentrations of 2 μM, 5 μM, and 10 μM.

[0368] After 72 hours of treatment, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega).

[0369] 2) Single-dose toxicity studies in mice

[0370] To evaluate the toxicological characteristics of Example 1, two single-dose toxicity studies were performed. First, a 2-week single-dose toxicity study was conducted using 9-week-old female CD-1 mice (Daehan Biolink Co., Eumsung, Korea). This study was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (Approval No.: SNU-230531-6-1). The animals were housed in the Specific Pathogen-Free (SPF) Animal Laboratory at Seoul National University and were given a standard diet free of charge under conditions of 23-25°C, 45-65% relative humidity, and a 12-hour light / dark cycle. Mice were randomly assigned to three groups, and each group received a single injection of 5 μL of nuclease-free water containing Example 1 into the joint cavity (20 μg, 40 μg, and 80 μg, respectively). Body weight was measured on the day of administration (Day 0), Day 4, Day 11, and Day 14. On Day 14, after a one-day fast, blood was collected via the retro-orbital sinus, and the blood was centrifuged (1,500 x g, 15 min, 4°C) to separate the plasma. Clinical chemistry analysis was commissioned to an external analysis agency (Cellpurics, Korea) and performed using an Adivia 1800 automated analyzer (Siemens). Subsequently, the animals were sacrificed, and major organs (liver, spleen, kidney) were excised, weighed, and visually inspected for abnormalities. The tissue of the right leg was excised and fixed in a 4% paraformaldehyde solution at 4°C for 1 night and 2 days, and the cartilage and bone tissues were analyzed for abnormalities through histological analysis using the method described above.Chondrocyte apoptosis was performed using the TUNEL assay (Takara, MK500) according to the manufacturer's instructions, and the nuclei were stained with DAPI at room temperature for 10 minutes. Fluorescence signals were observed using the EVOS M7000 Imaging System (Thermo Fisher, AMF7000).

[0371] (2) Interpretation of experimental results

[0372] The cytotoxicity of Example 1 was verified in primary cultured cells derived from mouse cartilage, a hepatocyte cell line (HepG2 cell line), and a kidney cell line (RPTEC cell line). The experimental results showed that no significant level of cytotoxicity was observed in any of the three cell types, with cell viability decreasing to 80% or less (see Figs. 24a, 24b, and 24c). Next, to evaluate the local and systemic safety of Example 1, a 14-day toxicity study was conducted on mice following a single intra-articular (IA) administration. Example 1 was administered at doses of 20, 40, and 80 μg / animal, respectively. As a result, the organ-to-body weight ratios of the liver, kidney, and spleen in all administration groups did not show a significant difference compared to the control group (see Fig. 25a). This suggests that no systemic or organ-specific toxicity occurred following the administration of Example 1. Histological analysis of the administration site revealed no cartilage damage or loss of proteoglycans within the knee joint tissue, and Safranin-O staining confirmed that the matrix components within the cartilage remained normal (Fig. 25b, top and middle). Additionally, TUNEL staining analysis confirmed that no chondrocyte apoptosis was induced, thereby demonstrating the excellent local tissue tolerability of Example 1 (Fig. 25b, bottom). The above results serve as evidence that Example 1 is a safe antisense oligonucleotide that simultaneously exhibits excellent local safety and the absence of systemic toxicity when administered into the joint cavity.

[0373] [Experimental Example 14] Confirmation of the effect of miR-204-specific ASO on dog-derived chondrocytes

[0374] In Experimental Example 1 above, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, to confirm whether the ASO of Example 1 above can effectively inhibit miR-204 in canine-derived chondrocytes, the following experiment was performed.

[0375] (1) Experimental method

[0376] 1) Canine chondrocytes cell line culture method

[0377] Canine chondrocytes (Canine chondrocytes cell line; Cell Applications, Inc.) 2 x 10 5 Cells were seeded into 12-well plates at a density of cells / well and cultured for 24 hours. Cells were cultured under 5% CO₂. 2 The temperature was maintained at 37°C under atmospheric oxygen concentration conditions, and the culture medium used was Canine chondrocytes growth medium (Cell Applications, Inc., Cn411-500) supplemented with Growth supplement (Cell Applications, Inc., Cn411-GS). After 24 hours of culture, cells were used according to each experiment.

[0378] 2) Verification of the transfer efficiency of Example 1

[0379] To visualize the intracellular uptake pattern of Example 1 in canine-derived chondrocytes, cultured cells were incubated with 1 μM of the fluorescence-labeled Cy3 from Example 1 for 24 hours. Subsequently, 1 μg / ml of Hoechst was added to the culture medium and co-incubated for an additional 20 minutes. For fluorescence microscopy analysis, the cells were washed three times with phosphate buffer (PBS), and fluorescence images were acquired using an EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0380] In addition, the degree of intracellular uptake of Example 1 was quantitatively evaluated through flow cytometry. For this purpose, canine-derived chondrocytes were cultured with Cy3 fluorescent label Example 1 (10 nM or 50 nM) for 24 hours. After treatment, the cells were detached from the attachment surface with trypsin, washed with PBS, and recovered by centrifugation. The recovered cells were resuspended twice in a PBS solution containing 5% FBS.

[0381] Flow cytometry analysis was performed using the FACSAria™ III Cell Sorter (BD Biosciences) instrument, and analysis data were collected and quantitatively analyzed using FACSDiva 9.7 software.

[0382] 3) qRT-PCR

[0383] After culturing canine-derived chondrocytes for 24 hours, they were treated with 0.25 μM doxorubicin (Sigma-Aldrich) to induce cellular senescence. The cells were maintained under hypoxic conditions of 37°C, 5% CO2, and 3% O2. After 72 hours, the cells were treated again with the same concentration of doxorubicin; however, half of the existing culture medium was removed, and the remaining half was diluted and treated by mixing it with an equal volume of fresh culture medium. After an additional 72 hours had elapsed, the culture medium was replaced with fresh medium while treating with Example 1 at a concentration of 1 μM, and the cells were harvested 48 hours after treatment.

[0384] Cells were harvested, and total RNA was extracted using TRI Reagent (Molecular Research Center, Inc.). The extracted RNA was synthesized into DNA using the miRCURY LNA RT kit (Qiagen). The expression level of miR-204 was analyzed using the synthesized cDNA via quantitative reverse transcription polymerase chain reaction (qRT-PCR) with the miRCURY LNA microRNA PCR Kit (Qiagen). Let-7e-5p was used as an internal control.

[0385] 4) sGAG assay

[0386] Quantitative analysis of sGAG was performed using primary cultured canine-derived chondrocytes according to the method described above. To induce cellular senescence, doxorubicin 0.25 μM (Sigma-Aldrich) was administered. Cells were maintained under hypoxic conditions at 37°C, 5% CO2, and 3% O2. After 72 hours, the cells were treated again with the same concentration of doxorubicin; however, half of the existing culture medium was removed, and the remaining half was diluted and treated by mixing it with an equal volume of fresh culture medium. After an additional 72 hours had elapsed, the cells were treated with Example 1 at concentrations of 0.1, 1, and 10 μM while replacing the culture medium with fresh medium, and the medium was replaced with fresh medium 48 hours after treatment. After culturing for 2 days, the culture supernatant was collected and used for sGAG analysis. The concentration of sGAG in the recovered culture medium was measured using the 1,9-dimethylmethylene blue (DMMB) staining method. Absorbance was measured at 525 nm, and the sGAG content was quantified based on a standard curve prepared using chondroitin sulfate (CO335; Tokyo Chemical Industry). To compensate for differences in cell viability and cell density, an MTT assay was performed on the same cells. For this purpose, a PBS-based MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, T-030-1; Goldbio) was added at a concentration of 100 μg / mL and reacted for 2 hours. After the reaction, the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm. All sGAG measurements were normalized based on MTT results and compared and analyzed.Statistical significance testing was performed using GraphPad Prism 10 software (GraphPad Software), and Tukey's post-hoc test was applied after conducting one-way ANOVA.

[0387] (2) Interpretation of experimental results

[0388] To confirm the delivery efficiency of Example 1 to canine-derived chondrocytes, intracellular uptake was evaluated using Cy3 fluorescently labeled Example 1. Fluorescence microscopy analysis confirmed that fluorescent signals were significantly accumulated within the canine-derived chondrocytes of the Example 1 treatment group compared to the vehicle treatment group (Fig. 26a). In addition, flow cytometry results showed that more than 50% of fluorescence-positive signals were observed under all conditions treated with Example 1 at concentrations of 10 nM and 50 nM, quantitatively confirming the excellent intracellular uptake of Example 1 (Fig. 26b). These results suggest that Example 1 possesses physical and chemical properties that allow it to be efficiently delivered into chondrocytes and stably accumulated.

[0389] Subsequently, the decrease in miR-204 by Example 1 was confirmed in canine-derived chondrocytes in which cellular senescence was induced. When Example 1 was delivered after treatment with Doxorubicin to induce cellular senescence, a statistically significant decrease in miR-204 expression was confirmed (Fig. 26c).

[0390] Next, the effect of Example 1 on the recovery of the synthesis function of cartilage matrix components was evaluated. When Example 1 was applied to dog-derived chondrocytes with induced cellular aging, it was confirmed that the synthesis of sulfated glycosaminoglycans (sGAG), which had decreased due to aging, significantly increased (Fig. 26d). These results indicate that Example 1 possesses functional efficacy in restoring ECM synthesis function in both mouse chondrocytes and dog-derived chondrocytes through the degradation of miR-204, supporting matrix recovery and anti-aging effects at the cellular level.

[0391] These results suggest that Example 1 has cross-species efficacy in mammals such as dogs and cats, capable of effectively inhibiting miR-204 in mouse-derived chondrocytes as well as dog-derived chondrocytes, and support the preservation of the miR-204 target inhibition mechanism independent of the species specificity of the substance.

Claims

1. As an oligonucleotide that specifically hybridizes with miR-204, The above oligonucleotide consists of 20 to 22 nucleotides linked together and is composed of a first region, a second region, and a third region in order from the 5' end to the 3' end. The above first region is complementary to the tail region of miR-204 and consists of three nucleotides connected in succession, including a fixed sugar modification. The second region is composed of three connected nucleotides, and the nucleotides constituting the second region are each nucleotides that independently include a fixed sugar modification or a non-fixed 2'-substituted sugar modification, and The above third region is composed of a plurality of connected nucleotides, and the nucleotides constituting the above third region are each nucleotides independently comprising a non-fixed 2'-substituted sugar modification, and The above oligonucleotide is an oligonucleotide comprising 30% or less of nucleotides containing fixed sugar modifications based on the total number of nucleotides.

2. In Paragraph 1, The nucleotide comprising the above-mentioned fixed sugar modification is an oligonucleotide selected from the group consisting of at least one of LNA (locked nucleic acid), ENA (2',4'-ethylene-bridged nucleic acid), cEt (constrained ethyl) nucleotide, BNA (bridged nucleic acid), and BNANC / BNANC (acyclic amino-bridged BNA).

3. In Paragraph 1, The above-mentioned non-fixed 2'-substituted sugar modification is at least one selected from the group consisting of non-fixed 2'-substituted sugar modifications of 2'-deoxy, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-alkyl, 2'-fluoro (2'-F), 2'-amino (2'-NH2), 2'-O-allyl, 2'-O-benzyl, ribose, and arabinose (ANA), an oligonucleotide.

4. In Paragraph 1, The nucleotides constituting the third region above are all oligonucleotides containing a 2'-deoxy sugar.

5. In Paragraph 1, The sugars of the nucleotides at positions 1 and 2 from the 3' end of the above oligonucleotide are 2'-deoxygen, oligonucleotide.

6. In Paragraph 1, The above oligonucleotide comprises a region complementary to the seed region of the miR-204, and the sugars of the nucleotides included in the region complementary to the seed region are non-fixed sugar modifications.

7. In Paragraph 1, An oligonucleotide in which the inter-nucleotide bonds included in the above oligonucleotide are each independently a phosphorothioate bond or a phosphodiester bond.

8. In Paragraph 1, The above oligonucleotide is an oligonucleotide that is one or more of the oligonucleotides represented by SEQ ID NOs. 1 to 14 and 26 to 39.

9. In Paragraph 1, The second or third region is an oligonucleotide comprising one or more mismatch nucleotides with the miR-204.

10. In Paragraph 1, The above oligonucleotide is an oligonucleotide that is an oligonucleotide for the degradation of miR-204.

11. A composition for improving, preventing, or treating osteoarthritis, comprising an oligonucleotide according to any one of claims 1 to 10.

12. A composition according to claim 11, wherein the osteoarthritis is at least one selected from the group consisting of degenerative osteoarthritis, post-traumatic osteoarthritis, post-surgical osteoarthritis, inflammatory osteoarthritis, and secondary osteoarthritis.

13. In claim 11, the composition is for improving, preventing, or treating osteoarthritis in mammals.

14. In paragraph 11, the composition is a pharmaceutical composition, a food composition, a health functional food composition, or an oral composition.

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