Oligonucleotides targeting miR-204 and compositions comprising the same for improving, preventing or treating osteoarthritis

Abstract

This specification discloses an oligonucleotide capable of specifically hybridizing with miR-204, and compositions comprising the oligonucleotide for improving, preventing, or treating osteoarthritis. In one aspect, the oligonucleotide is designed with an optimized structure for miR-204 degradation and formulation stability, effectively blocking pathophysiological processes of osteoarthritis such as decreased cartilage matrix synthesis and increased inflammatory response, thereby preventing or treating osteoarthritis. Furthermore, it exhibits excellent delivery capability to chondrocytes and stable residence within tissues, thus possessing structural advantages for achieving long-term, sustained therapeutic effects. It also has low immunogenicity and toxicity, possessing safety suitable for use in pharmaceutical compositions. In addition, it effectively inhibits miR-204 in canine chondrocytes, therefore it can be used in animal therapeutics and / or feed compositions, etc.

Description

Technical Field

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

[0002] This specification discloses an oligonucleotide that specifically hybridizes with miR-204, and compositions comprising the oligonucleotide for improving, preventing or treating osteoarthritis.

[0003] Description of government-funded research projects [National R&D projects supporting this invention] [Project Number] 2420016229 [Project Number] RS-2024-00437503 [Department Name] Small and Medium Enterprises and Startups Department [Project Management (Professional) Organization Name] Small and Medium Enterprise Technology Information Promotion Institute [Research Project Title] Enterprise Growth Technology Development [Research Topic Title] Development of Next-Generation Osteoarthritis Treatments Based on microRNA Degraders [Project Implementing Agency Name] Liflex Science Inc. [Research Period] 2024.07.01 ~ 2027.06.30. Background Technology

[0004] The number of patients with osteoarthritis (OA) increased by 48% globally between 1990 and 2019, reaching 4.18 million in South Korea alone by 2022. The demand for OA treatment is rapidly increasing due to population aging and growing focus on quality of life. Cartilage is a tissue that is damaged and progressively degenerates due to various etiological risk factors such as aging, mechanical overload, obesity, and metabolic diseases; osteoarthritis is known to result from cartilage degeneration. It is characterized by pathological changes such as cartilage degeneration, subchondral bone sclerosis, osteophyte formation, and synovial inflammation.

[0005] Current research on osteoarthritis treatment is limited to slowing cartilage degeneration by blocking the "degradation" mechanism of the extracellular matrix of chondrocytes. Due to the lack of drugs that can fundamentally improve the disease, treatment strategies for osteoarthritis rely on prescription anti-inflammatory drugs or artificial joint replacement surgery.

[0006] miR-204 is a type of microRNA, specifically miR-204-5p. miR-204 is a microRNA whose expression is significantly elevated in cartilage across various types of osteoarthritis. It inhibits multiple sGAG synthases that constitute the cartilage matrix and promotes the formation of inflammatory inflammatory factors (SASPs), thus exacerbating osteoarthritis. The therapeutic effects of miR-204 inhibition on osteoarthritis have been confirmed, and the current stage involves developing therapeutic drugs that can effectively "degrade" miR-204 with fewer side effects. However, although the importance of miR-204-mediated reduction in sGAG synthesis and SASPs in the pathogenesis of osteoarthritis has been established, to date, no effective therapeutic drugs have been developed that specifically bind to miR-204 to restore cartilage matrix synthesis or inhibit inflammatory factors. Summary of the Invention

[0007] Technical problems to be solved The inventors of this application have identified miR-204 as a biomarker for improving, preventing, or treating osteoarthritis, and have conducted research on nucleic acid therapeutic agents targeting it. The results show that among oligonucleotides that can specifically hybridize with miR-204, when the nucleotides at a specific position in the nucleotides constituting the oligonucleotide contain fixed sugar modifications or non-fixed 2'-substituted sugar modifications, degradation of miR-204 can have excellent effects in improving, preventing, or treating osteoarthritis.

[0008] Therefore, on the one hand, the purpose of this invention is to provide an oligonucleotide that can specifically hybridize with miR-204 and has excellent degradation effect on miR-204.

[0009] On the other hand, the object of the present invention is to provide a composition comprising the oligonucleotides for improving, preventing or treating osteoarthritis.

[0010] Problem Solving Methods On one hand, the present invention provides an oligonucleotide capable of specifically hybridizing with miR-204, wherein the oligonucleotide is composed of 20 to 22 nucleotides linked together and is composed of a first region, a second region, and a third region sequentially from the 5' end to the 3' end; the first region is complementary to the tail region of miR-204 and consists of three consecutive nucleotides containing immobilized sugar modifications; the second region consists of three nucleotides linked together, and the nucleotides constituting the second region are each independently nucleotides containing immobilized sugar modifications or non-immobilized 2'-substituted sugar modifications; the third region consists of multiple nucleotides linked together, and the nucleotides constituting the third region are each independently nucleotides containing non-immobilized 2'-substituted sugar modifications; and, based on the total number of nucleotides, the oligonucleotide contains less than 30% nucleotides containing immobilized sugar modifications.

[0011] In another aspect, the present invention provides a composition comprising the oligonucleotide for improving, preventing or treating osteoarthritis.

[0012] The effects of the invention The oligonucleotide described in one aspect of this invention is an antisense oligonucleotide that reduces miR-204 expression by inducing miR-204 degradation. It is designed to have optimized miR-204 inhibitory efficacy and formulation stability by comparing and verifying various chemical modifications such as sugar modification, length regulation, and the selection of the number and position of immobilized sugar modifications.

[0013] Therefore, the antisense oligonucleotides of the present invention can effectively block the pathophysiological processes of osteoarthritis, such as decreased cartilage matrix synthesis and increased inflammatory response, and can be used to relieve pain, prevent or treat osteoarthritis.

[0014] Furthermore, the oligonucleotides described in this invention exhibit excellent delivery capabilities to chondrocytes and stable retention characteristics within tissues, thereby providing structural advantages that enable long-term, sustained therapeutic effects.

[0015] The oligonucleotides described in one aspect of the present invention have a low likelihood of inducing immunogenicity and low toxicity, providing a safety profile suitable for use as pharmaceutical compositions.

[0016] The oligonucleotides described in this invention have been shown to effectively inhibit miR-204 not only in human chondrocytes but also in canine chondrocytes. Furthermore, since the base sequence of miR-204 is conserved among multiple species such as humans, dogs, and monkeys, the oligonucleotides can be used as therapeutic agents and / or feed additive compositions in various animal species.

[0017] Because the sequence of the target miR-204 is conserved across species, the oligonucleotide described in one aspect of this invention can be used not only in human chondrocytes or humans, but also in chondrocytes of other species (e.g., dogs, monkeys) or other species. Attached Figure Description

[0018] Figure 1 is an experimental example of the present invention, which shows the degradation effect of miR-204 in the C28 / I2 chondrocyte cell line by treatment according to Example 1 and measured by qRT-PCR.

[0019] Figure 2 shows the results of confirming the delivery efficiency of Example 1 in mouse chondrocytes according to an experimental example of the present invention. Figure 2a This is a fluorescence micrograph of Example 1, showing the accumulation of fluorescently labeled cells in mouse-derived chondrocytes; Figures 2b to 2c The results are the proportion of Cy3 fluorescently labeled positive cells in Example 1, which were quantitatively determined by flow cytometry.

[0020] Figure 3 This is a fluorescence micrograph of Example 1, which confirms the delivery efficiency of Example 1 in osteoarthritis patient-derived cartilage tissue using fluorescent labeling, according to an experimental example of the present invention.

[0021] Figure 4 This is a graph showing the results of measuring miR-204 expression levels by qRT-PCR when processing Full DNA or ASO containing mismatches in the sequence (Examples 3-1 to 3-3) according to an experimental example (Experiment 4) of the present invention.

[0022] Figure 5 This is a graph showing the results of measuring miR-204 expression levels by qRT-PCR when treating full DNA or ASO with microRNA as a reference, which has one or two bases deleted from the 5' end (Examples 4-1 and 4-2).

[0023] Figure 6 and Figure 7 According to an embodiment of the present invention, the experimental results of determining the miR-204 expression level by qRT-PCR were obtained by confirming whether the nucleotides contained in the first and second regions of the miR-204-specific ASO contained fixed sugar modifications (Full DNA, Examples 5-1 to 5-7, and Comparative Examples 1-1 and 1-2).

[0024] Figure 8This is a comparative example of the present invention. To confirm the miR-204 degradation ability when one or more of the 1st to 6th positions in the 3' to 5' direction of the miR-204-specific ASO contain LNA (Example 1, Comparative Examples 2-1 to 2-4), the experimental results of measuring the miR-204 expression level by qRT-PCR are presented in the figure.

[0025] Figure 9 and Figure 10 This is a graph showing the experimental results of measuring miR-204 expression levels by qRT-PCR when compared with Full DNA or Example 1 of the ASO described in one embodiment of the present invention. The miR-204 degradation ability of the miR-204-specific ASO in one comparative example of the present invention is confirmed when it randomly contains LNA and / or is shorter than the ASO length of one embodiment of the present invention (Comparative Examples 3 and 4).

[0026] Figure 11a and 11b This is a graph showing the experimental results of measuring miR-204 expression levels by qRT-PCR to confirm the miR-204 degradation ability in a comparative example of the present invention when the miR-204-specific ASO contains 15 or more LNAs (Comparative Examples 5-1 to 5-3). Figure 11a ) and photographs taken to confirm formulation stability during the dissolution of the ASO ( Figure 11b ).

[0027] Figure 12 and 13 This is a graph showing the experimental results of determining the expression level of miR-204 by qRT-PCR to confirm the miR-204 degradation ability when the miR-204-specific ASO contains one or more nucleotides containing MOE or OME (instead of LNA) in the second region (Examples 6-1 to 6-14) in one embodiment of the present invention.

[0028] Figure 14 This is a graph showing the experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 degradation ability when the miR-204-specific ASO is constructed by replacing the PS bond with a PO bond (backbone) in one embodiment of the present invention (Example 2).

[0029] Figure 15 This is a graph illustrating the pathway enrichment results in the form of a bubble plot, based on the transcriptomic analysis results between the treatment group and the vehicle control group of Example 1, according to one embodiment of the present invention. Figure 15In the diagram, the size of the dot represents the number of genes contained in the pathway, and the color represents the fold enrichment.

[0030] Figures 16a to 16c This is a diagram showing the changes in ECM composition or inflammation-induced gene and protein expression in mouse chondrocytes caused by the treatment in Example 1, which is an embodiment of the present invention. Figure 16a This is a graph showing the results of measuring the mRNA expression of enzymes related to the sGAG biosynthesis pathway by qRT-PCR in bleomycin-induced chondrocytes. Figure 16b This image shows the bands representing the results of Western blot analysis of protein expression associated with sGAG synthesis. Figure 16c The chart shows the results of changes in the expression of ECM biosynthetic enzymes and inflammation-related genes by qRT-PCR after treatment with the inflammatory cytokine IL-1β in Example 1 and SM04690.

[0031] Figures 17a to 17c The changes in intracellular sGAG content in chondrocytes caused by the treatment in Example 1, which is an embodiment of the present invention, are shown. Figure 17a A graph showing the changes in sGAG content at different treatment concentrations in Example 1 in bleomycin-induced chondrocytes. Figure 17b To be Figure 17a The results of Example 1 were plotted as a concentration-response curve on a logarithmic scale, showing the maximum effect (E). max The calculation results of the concentration and half maximum effective concentration (EC50) values. Figure 17c The graph shows the changes in sGAG content caused by treatment with the inflammatory cytokine IL-1β in Example 1 and SM04690.

[0032] Figures 18a to 18f The therapeutic effect of a single intra-articular application in Example 1, as an embodiment of the present invention, is shown in a mouse model of osteoarthritis induced by DMM surgery. Figure 18a The illustration shows a schematic diagram of histological images stained with Safranin O after application to Example 1 and the vehicle control group. Figure 18b Based on Figure 18a Images, quantifying the intensity of cartilage damage, and charts that quantify the stages of pathological progression. Figure 18c Images illustrating the microscopic changes in cartilage tissue structure using micro-computed tomography (μCT). Figure 18d Images were taken to confirm the distribution of Example 1 within the articular cartilage using in situ hybridization. Figure 18e The graph shows the effect of Example 1 on alleviating weight-bearing imbalance in a DMM-induced mouse model of osteoarthritis. Figure 18f Images show the results of immunohistochemical analysis of changes in protein expression related to cartilage matrix synthesis and degradation.

[0033] Figures 19a to 19d The therapeutic effect of repeated intra-articular administration in Example 1, an embodiment of the present invention, is shown in a DMM surgery-induced osteoarthritis model. Figure 19a and Figure 19b Images and graphs showing the pathological progression stages of cartilage from the Example 1 treatment group and the vehicle control group, stained with Safranin O. Figure 19c A graph illustrating the effect of Example 1 on alleviating weight imbalance is shown. Figure 19d Images illustrating the microscopic changes in cartilage tissue structure using micro-computed tomography (μCT).

[0034] Figures 20a to 20e The experimental results are shown in a DMM surgery-induced mouse model of osteoarthritis, comparing the therapeutic effects of Example 1, SM04690, and dexamethasone as embodiments of the present invention. Figure 20a This is a schematic diagram showing the treatment cycle and conditions for each drug. Figure 20b and Figure 20c Histological images of each drug treatment group stained with Safranin O and charts that quantify the pathological progression stages. Figure 20d A graph showing the results of the weight imbalance analysis in each drug treatment group was used to illustrate the behavioral experiment. Figure 20e Images illustrating the microscopic changes in cartilage tissue structure using micro-computed tomography (μCT).

[0035] Figures 21a to 21d The results of evaluating the therapeutic effect of Example 1, which is an embodiment of the present invention, in the late stage of osteoarthritis are shown. Figure 21a and Figure 21b Histological images of each drug treatment group stained with Safranin O and charts that quantify the pathological progression stages. Figure 21c Images illustrating the microscopic changes in cartilage tissue structure in each drug treatment group were obtained using microcomputed tomography (μCT). Figure 21d A graph showing the results of the weight imbalance analysis in each drug treatment group was used to illustrate the behavioral experiment.

[0036] Figures 22a to 22cThe analysis results used to evaluate the off-target effect of Example 1, which is an embodiment of the present invention, are shown. Figures 22a to 22c The bubble plot shows the results of gene ontology (GO) term analysis performed on the obtained transcriptome data in the C28 / I2 cell line overexpressing miR-204 after treatment in Example 1. The size of the dot represents the number of genes included in the annotation, and the color represents the fold enrichment.

[0037] Figures 23a to 23c The results of Toll-like receptor (TLR) activity analysis using HEK-Blue TLR7, TLR8, and TLR9 cell lines to evaluate the immunogenicity of Example 1 are shown in the figure.

[0038] Figures 24a to 24c The results of the cytotoxicity assessment for Example 1, which is an embodiment of the present invention, are shown. Figures 24a to 24c In Example 1, graphs showing cell viability in primary cultured mouse chondrocytes, hepatocytes, and kidney cells were presented when treated with different concentrations.

[0039] Figures 25a to 25b The results of a single intra-articular toxicity assessment in mice are shown in Example 1, which is an embodiment of the present invention. Figure 25a A chart showing the organ weight ratios of major organs (liver, kidney, spleen) measured 14 days after administration is presented. Figure 25b The top and middle images show the results of Safranin O staining of the knee joint tissue, while the bottom image shows the results of TUNEL staining analysis of the same tissue.

[0040] Figures 26a to 26d The results show the results of confirming the intracellular delivery efficiency and functional efficacy of Example 1, which is an embodiment of the present invention, in canine chondrocytes. Figure 26a Images of canine chondrocytes treated with Cy3 fluorescent labeling in Example 1 were observed using a fluorescence microscope. Figure 26b The chart shows the results of quantitative analysis of the proportion of fluorescently positive cells by flow cytometry after treatment with Cy3 fluorescent labeling in Example 1. Figure 26c The graph shows the results of measuring miR-204 expression levels by qRT-PCR under cell senescence conditions treated with Example 1. Figure 26d This is a graph showing the results of quantitative analysis of sGAG content under cellular senescence conditions. Detailed Implementation

[0041] The present invention will now be described in detail.

[0042] In one aspect of the invention, "microRNA-204", "miR-204", or "microRNA-204" is a collective term for any variant, isoform, and species homolog of miR-204 naturally expressed by cells. Specifically, it may refer to, but is not limited to, human miR-204, and may also include miR-204 from other animals.

[0043] In one aspect of the invention, the "tail region of miR-204" can be the nucleic acid region of microRNA-204 (miR-204) starting from the 17th position from the 5' end.

[0044] In one aspect of the invention, the "supplementary region of miR-204" may be the nucleic acid region of microRNA-204 (miR-204) from position 13 to 16 starting from the 5' end.

[0045] In one aspect of the invention, the "central region of miR-204" may be the nucleic acid region of microRNA-204 (miR-204) from position 9 to position 12 starting from the 5' end.

[0046] In one aspect of the invention, the “seed region of miR-204” can be the nucleic acid region of microRNA-204 (miR-204) from position 2 to position 8 starting from the 5' end.

[0047] In one aspect of the invention, "oligonucleotide" refers to a targeted nucleic acid, specifically a short nucleic acid polymer that functions by complementary binding to a target microRNA, said polymer may contain native or modified nucleotides (e.g., sugar modifications such as 2'-O-methoxyethyl (MOE), 2'-O-methyl (OME), 2'-fluoro (2'-F), LNA, cEt, and phosphate thioester (PS) bonds). In another aspect, "oligonucleotide" may be an antisense oligonucleotide.

[0048] In one aspect of the invention, "nucleotide containing a fixed sugar modification" can refer to a nucleotide in which the sugar is conformationally constrained. In another aspect, the nucleotide containing a fixed sugar modification can be a nucleotide containing a bridging sugar (e.g., LNA, ENA, cEt, BNA, BNANC, etc.).

[0049] In one aspect of the invention, "nucleotide comprising a non-fixed 2'-substituted sugar" can refer to the sugar contained in the nucleotide, whose stereoconformation is not fixed based on 2'-position substitution. In one aspect, the non-fixed sugar includes 2'-deoxy, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-alkyl, 2'-fluorine (2'-F), 2'-amino (2'-NH2), 2'-O-allyl, 2'-O-benzyl, ribose, arabinose, etc.

[0050] 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 synovium. It may include primary or secondary osteoarthritis, as well as osteoarthritis in all parts of the body such as the knee, hip, hand, and spine.

[0051] Oligonucleotides that specifically hybridize with miR-204 In one aspect, the present invention provides an oligonucleotide capable of specifically hybridizing with miR-204, the oligonucleotide being composed of 20 to 22 nucleotides linked together and consisting of a first region, a second region, and a third region sequentially from the 5' end to the 3' end; the first region is complementary to the tail region of miR-204 and consists of three consecutive nucleotides containing immobilized sugar modifications; the second region consists of three nucleotides linked together, and the nucleotides constituting the second region are each independently nucleotides containing immobilized sugar modifications or non-immobilized 2'-substituted sugar modifications; the third region consists of multiple nucleotides linked together, and the nucleotides constituting the third region are each independently nucleotides containing non-immobilized 2'-substituted sugar modifications; and, based on the total number of nucleotides, the oligonucleotide contains less than 30% nucleotides containing immobilized sugar modifications.

[0052] The oligonucleotides described in one aspect of this invention can specifically hybridize with miR-204.

[0053] The oligonucleotide described in one aspect of the present invention can be composed of 20 to 22 nucleotides linked together. Specifically, the oligonucleotide can be composed of 20, 21, or 22 nucleotides linked together.

[0054] The oligonucleotide described in one aspect of the present invention may be composed of a first region, a second region, and a third region in a direction from the 5' end to the 3' end. Specifically, the first region may be a region containing the 1st to 3rd nucleotides of the oligonucleotide in the direction from the 5' end to the 3' end. Furthermore, the second region may be a region containing the 4th to 6th nucleotides of the oligonucleotide in the direction from the 5' end to the 3' end. Furthermore, the third region may be a region containing the 7th to the last (e.g., the 20th, 21st, or 22nd) nucleotides of the oligonucleotide in the direction from the 5' end to the 3' end. More specifically, the first region may be a region composed of the 1st to 3rd nucleotides of the oligonucleotide in the direction from the 5' end to the 3' end. Furthermore, the second region may be a region composed of the 4th to 6th nucleotides of the oligonucleotide in the direction from the 5' end to the 3' end. Furthermore, the third region may be a region composed of the 7th to the last (e.g., the 20th, 21st, or 22nd) nucleotides of the oligonucleotide in the direction from the 5' end to the 3' end.

[0055] The oligonucleotides described in this invention can be selectively introduced with lock nucleic acid (LNA), 5-methyl-deoxycytosine (iMe-dC), phosphorothioate (PS) bonds, phosphodiester (PO) bonds, etc., to improve binding affinity, in vivo stability and functional inhibitory effect.

[0056] The LNA base gene possesses a bicyclic structure with a fixed sugar ring, which helps improve the ligand binding strength and structural rigidity of oligonucleotides. Furthermore, 5-methyl-deoxycytosine (iMe-dC), a modified base with higher resistance to oxidation and deamination compared to wild-type cytosine, is the most standardized synthetic base modification that stably and reproducibly mimics real biological phenomena, contributing to improved structural stability and functional persistence of the entire ASO. In addition, the phosphorothioate (PS) bond enhances resistance to nucleases and prolongs the in vivo half-life by improving protein binding capacity.

[0057] On the other hand, the nucleotides contained in or constituting the oligonucleotide described in one aspect of the present invention may be phosphate thioate (PS) bonds and / or phosphate diester (PO) bonds.

[0058] According to Experiment 1, in the ASO treatment group of Example 1 of this invention, the expression of miR-204 decreased in a concentration-dependent manner. Quantitative analysis showed that its maximum degradation efficiency was approximately 99.6%, and the DC that reduced miR-204 expression by 50% was... 50 The value was approximately 0.0055 μM, indicating high degradation ability (Figure 1). Furthermore, excellent miR-204 degradation ability was also confirmed in the ASO-treated group of another embodiment of the present invention (Example 2), demonstrating that regardless of the internucleotide binding mode or backbone composition of the oligonucleotides, the oligonucleotides described in one aspect of the present invention can effectively degrade the target miR-204. Figure 14 ).

[0059] MicroRNAs typically consist of 22-24 megohms, with the seed region consisting of positions 2 to 8 from the 5' end, the central region consisting of positions 9 to 12, the supplementary region consisting of positions 13 to 16, and the tail region. In Example 1 of this invention, the first region of the ASO has a sequence completely complementary to the tail region.

[0060] According to Experimental Example 2, the Cy5.5-labeled ASO of Example 1 exhibited a strong fluorescent signal in the cytoplasm of chondrocytes, and in flow cytometry analysis, the intracellular fluorescence intensity of the Cy3-labeled Example 1 treatment group also showed a concentration-dependent increasing trend. Therefore, this indicates that the structural design of the ASO described in one embodiment of the present invention facilitates its effective delivery and absorption in cells with high permeability, such as cartilage tissue (Figure 2).

[0061] The oligonucleotides described in one aspect of this invention can be effectively delivered to chondrocytes or cartilage tissue. Specifically, the oligonucleotides described in one aspect of this invention can effectively induce intracellular uptake in chondrocytes or retention within cartilage tissue.

[0062] According to Experimental Example 3, the ASO of Example 1 in one embodiment of the present invention also showed good intracellular delivery in human cartilage tissue. Figure 3 This indicates that the ASO described in one embodiment of the present invention has physicochemical properties that allow it to accumulate stably in human chondrocytes.

[0063] According to Experimental Example 4, variants containing one or more mismatches in the central region or supplementary region of miR-204 (Examples 3-1 to 3-3) did not show a significant decrease in binding efficiency compared to fully complementary ASOs and maintained excellent degradation capabilities. Figure 4This indicates that the sequence design of ASO described in one embodiment of the present invention is highly advantageous in ensuring high degradation efficiency, binding stability, and biological functionality.

[0064] In this specification, "mismatch" refers to a state in which a single base is substituted without complementarity 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. This position can be in the form of G·U wobble pairing or non-canonical pairing, but is not limited to these.

[0065] The oligonucleotide described in one aspect of the present invention may contain one or more mismatched nucleotides with miR-204. Specifically, the mismatch may be located in a region complementary to the central region or complementary region of miR-204. Furthermore, the mismatched nucleotides may comprise 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 comprise fewer than ten, fewer than nine, fewer than eight, fewer than seven, fewer than six, fewer than five, fewer than four, fewer than three, or fewer than two.

[0066] The second or third region of the oligonucleotide described in one aspect of the present invention may contain one or more mismatched nucleotides with miR-204. Specifically, the oligonucleotide may contain mismatched nucleotides at one or more positions selected from nucleotides 1 to 22. More specifically, the oligonucleotide may contain mismatched nucleotides at one or more positions selected from nucleotides 7 to 22, 7 to 20, 8 to 18, 8 to 16, or 8 to 14. More specifically, the oligonucleotide may contain mismatched nucleotides at one or more positions selected from the 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, or 22nd nucleotides (preferably mismatched nucleotides may be contained at one or more positions selected from the 8th, 9th, 10th, 11th, 12th, 13th, or 14th nucleotides).

[0067] Furthermore, the mismatched nucleotide may be present at one or more complementary positions of the nucleotides 1 to 22 from the 5' end of the miR-204. Specifically, the oligonucleotide may contain the mismatched nucleotide at one or more selected positions from the nucleotide positions 1 to 22, 1 to 16, 3 to 16, 6 to 16, or 9 to 15 of the miR-204. More specifically, the oligonucleotide may contain mismatched nucleotides at one or more positions selected from the nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of miR-204; and even more specifically, it may contain mismatched nucleotides at one or more positions selected from the nucleotides at positions 9, 10, 11, 12, 13, 14, or 15.

[0068] According to Experiment 5, compared to fully complementary ASOs, ASOs lacking 1-2 bases at the 3' end (Examples 4-1 and 4-2) can still significantly degrade miR-204, confirming that even with shortened length, their binding affinity and degradation ability for miR-204 are maintained. Figure 5 ).

[0069] This indicates that when designing ASOs targeting miR-204, partial deletion (truncation) at the 3' end does not impair their functional efficacy and can maintain the ability to degrade miR-204.

[0070] The nucleotide containing immobilized sugar modification described in one aspect of the present invention can be at least one selected from the group consisting of: locked nucleic acid (LNA), 2',4'-ethylene-bridged nucleic acid (ENA), constrained ethyl (cEt) nucleotide, bridged nucleic acid (BNA), and acyclic amino-bridged BNA (BNANC). Specifically, the nucleotide containing immobilized sugar modification can be at least one of LNA and ENA, more specifically, it can be LNA.

[0071] The non-fixed 2'-substituted sugar modification described in one aspect of the present invention can be at least one selected from the group consisting 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 can be at least one selected from the group consisting of: 2'-deoxy, 2'-O-methyl, and 2'-O-methoxyethyl.

[0072] In one aspect, the oligonucleotide of this invention may include a first region complementary to the tail region of miR-204, wherein the first region may be three consecutive nucleotides containing immobilized sugar modifications. Specifically, the first region may include the nucleotides at positions 1 to 3 from the 5' end to the 3' end of the oligonucleotide, and the three nucleotides may be three consecutive nucleotides containing immobilized sugar modifications. More specifically, the first region may be composed of the nucleotides at positions 1 to 3 from the 5' end to the 3' end of the oligonucleotide, and the three nucleotides may be three consecutive nucleotides composed of immobilized sugar modifications.

[0073] In one aspect, the oligonucleotide of this invention may include a second region complementary to the tail region of miR-204, wherein the nucleotides included in the second region may be independently nucleotides containing fixed sugar modifications or non-fixed 2'-substituted sugar modifications. Specifically, the second region may be located immediately following the first region in the direction from the 5' end to the 3' end of the oligonucleotide. Furthermore, the second region may include nucleotides at positions 4 to 6 in the direction from the 5' end to the 3' end of the oligonucleotide, and these three nucleotides may be three consecutive nucleotides containing fixed sugar modifications or non-fixed 2'-substituted sugar modifications. More specifically, the second region may be composed of nucleotides at positions 4 to 6 in the direction from the 5' end to the 3' end of the oligonucleotide, and these three nucleotides may be three consecutive nucleotides composed of fixed sugar modifications or non-fixed 2'-substituted sugar modifications.

[0074] According to one embodiment of the present invention, when the oligonucleotide described in one aspect of the present invention contains a fixed sugar modification at positions 1 to 3 (first region) in the direction from the 5' end to the 3' end (Examples 5-1 to 5-7), it exhibits stable and efficient degradation efficacy against miR-204, even when non-fixed 2'-substituted sugar modifications and fixed sugar modifications are mixed at adjacent positions 4 to 6 (second region), it still exhibits the same miR-204 degradation efficacy. However, oligonucleotides containing only one or two fixed sugar modifications in the first region (Comparative Examples 1-1 and 1-2) exhibit similar miR-204 expression levels to the control group at a concentration of 10 μM, with almost no miR-204 degradation efficacy (see Experimental Example 6-1). Figure 6 and Figure 7 ).

[0075] Furthermore, according to one embodiment of the present invention, even if the non-fixed 2'-substituted sugar modification comprises MOE or OME at one or more positions (second region) of the oligonucleotide in the direction from the 5' end to the 3' end, the overall structural stability of the oligonucleotide and the miR-204 degradation efficiency are still maintained (see Examples 6-1 to 6-14, Experimental Example 8). Figure 12 and Figure 13 ).

[0076] In one aspect, the oligonucleotide 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 be independently a nucleotide modified with a non-fixed 2'-substituted sugar. Specifically, the third region may be located immediately after the second region in the direction from the 5' end to the 3' end of the oligonucleotide. Furthermore, the third region may include the 7th to the last nucleotide in the direction from the 5' end to the 3' end of the oligonucleotide, and the plurality of nucleotides may be consecutively linked nucleotides modified with a non-fixed 2'-substituted sugar. More specifically, the third region may be composed of the 7th to the last nucleotide in the direction from the 5' end to the 3' end of the oligonucleotide, and the plurality of nucleotides may be consecutively linked nucleotides formed by non-fixed 2'-substituted sugar modification. Even more specifically, the nucleotides constituting the third region may all contain 2'-deoxy.

[0077] The oligonucleotide described in 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 part of a third region of the oligonucleotide described in one aspect of the present invention, specifically, a portion adjacent to the 3' end. The sugar of the nucleotide contained in the region complementary to the seed region may include a non-fixed 2'-substituted sugar modification.

[0078] In one aspect of the present invention, the oligonucleotide may have a sugar of one or more nucleotides at positions 1 to 16 from its 3' end that is 2'-deoxy. Specifically, the oligonucleotide may have a 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 its 3' end that is 2'-deoxy; while 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 nucleotides may have a sugar of 2'-deoxy. More specifically, the sugar in the oligonucleotide 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 may be 2'-deoxy.

[0079] According to one embodiment of the present invention, when the oligonucleotide described in one aspect of the present invention does not contain immobilized sugar modification in the first region, but only in the region near the 3' end (the third region) (Comparative Examples 2-1 to 2-4), or when it randomly contains immobilized sugar modification in the second and third regions (Comparative Examples 3 and 4), the binding force of ASO and the RNase H-mediated degradation efficiency are reduced, thereby reducing the overall degradation effect of miR-204 (see Experimental Examples 6-2 and 6-3 and...). Figures 8 to 10 Therefore, the study found that the position of the fixed sugar modification of the oligonucleotide described in one aspect of the present invention has a key influence on the miR-204 degradation activity. When it contains the fixed sugar modification at the 5' end center (i.e., the first region), it exhibits the best miR-204 degradation efficiency.

[0080] The oligonucleotides described in one aspect of the present invention may contain less than 30% of nucleotides with fixed-type sugar modifications, based on the total number of nucleotides. Specifically, the oligonucleotides, based on the total number of nucleotides, contain less than 30%, less than 29.5%, less than 29%, less than 28.5%, less than 28%, less than 27.5%, less than 27.3%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22.7%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18.2%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13.6%, less than 13%, less than 12%, less than 11%, or less than 10% of nucleotides with fixed-type sugar modifications. More specifically, the oligonucleotides, based on the total number of nucleotides, contain between 13% and 30% of nucleotides with fixed-type sugar modifications.

[0081] Alternatively, the oligonucleotide described in one aspect of the present invention may comprise 3 to 14 nucleotides with 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 with fixed sugar modifications; or it may comprise fewer than 14, 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 nucleotides with fixed sugar modifications. More specifically, the oligonucleotide may comprise 3 to 6 nucleotides with fixed sugar modifications. Since the oligonucleotide described in one aspect of the present invention exhibits excellent miR-204 degradation efficiency when all three nucleotides constituting the first region contain fixed sugar modifications, the nucleotides containing fixed sugar modifications may be 13% or more or 3 or more.

[0082] According to one embodiment of the present invention, the more nucleotides with immobilized sugar modifications a oligonucleotide contains, the more it enhances the degradation effect of miR-204. However, this also leads to limitations in terms of physical properties and formulation as a drug due to increased formulation viscosity and decreased solubility. Therefore, when considering both efficacy and formulation stability, a moderate level of the number of nucleotides with immobilized sugar modifications (e.g., 13% to 30% or 3 to 6) is considered the optimal structure that can balance the degradation efficacy of miR-204 with formulation suitability (see Experimental Example 7 and...). Figure 11b ).

[0083] In one aspect of the present invention, the oligonucleotides contained therein are linked by either phosphorothioate bonds or phosphodiester bonds. Specifically, in the oligonucleotides described in one aspect of the present invention, the phosphate bonds between the nucleotides can be independently phosphorothioate bonds and / or phosphodiester bonds. For example, some nucleotide links can be phosphorothioate bonds, and others can be phosphodiester bonds; the number and position of these links are not particularly limited. Furthermore, the nucleotide links can be entirely phosphorothioate bonds or entirely phosphodiester bonds.

[0084] The oligonucleotides described in one aspect of this invention can effectively degrade miR-204 in chondrocytes. These oligonucleotides can exhibit preventative or therapeutic effects against osteoarthritis. Specifically, the oligonucleotides described in one aspect of this invention can exhibit preventative or therapeutic effects against osteoarthritis by effectively degrading miR-204 in chondrocytes.

[0085] In one embodiment, osteoarthritis can be at least one selected from the group consisting of: degenerative (primary) osteoarthritis, post-traumatic osteoarthritis, postoperative osteoarthritis, inflammatory osteoarthritis, and secondary osteoarthritis.

[0086] The oligonucleotides described in one aspect of the present invention can regulate extracellular matrix (ECM) remodeling-related genes (e.g., ECM proteoglycan synthesis factors), and / or inflammation-induced genes (e.g., IL6, PTGS2, NOS2, IL family, matrix degradation products), and / or cartilage matrix degradation enzyme-related genes (e.g., but not limited to MMP family (e.g., MMP3, ADAMTS family)).

[0087] The oligonucleotides described in one aspect of the present invention exhibit preventive or therapeutic effects against osteoarthritis even with repeated administration. Specifically, the oligonucleotides described in one aspect of the present invention exhibit stable and reproducible effects even with repeated administration.

[0088] The oligonucleotides described in one aspect of this invention can alleviate osteoarthritis symptoms (e.g., cartilage damage, subcutaneous bone sclerosis, subchondral bone sclerosis) upon repeated administration. According to one embodiment of this invention, when the oligonucleotides described in one aspect of this invention are repeatedly administered twice, both cartilage damage and subchondral bone sclerosis can be significantly reduced (see Experimental Example 10). Figure 19a , 19b and Figure 19d ).

[0089] The oligonucleotide described in one aspect of the present invention exhibits an analgesic effect even upon repeated administration. According to one embodiment of the present invention, when the oligonucleotide described in one aspect of the present invention is administered twice, its analgesic effect can be sustained (see Experimental Example 10 and...). Figure 19c ).

[0090] In one embodiment, repeated application means applying the oligonucleotide described in one aspect of the present invention two or more times. Specifically, repeated application may refer to applying the same or different doses of the oligonucleotide two or more times at certain time intervals. For example, repeated application may be applying 2, 3, 4, 5 or more times, but is not limited thereto.

[0091] The oligonucleotides described in one aspect of the present invention exhibit reproducible efficacy whether applied once or repeatedly.

[0092] The oligonucleotides described in one aspect of this invention, compared with oligonucleotides that do not follow the sugar modification pattern of the oligonucleotides described in one aspect of this invention, can have superior preventive or therapeutic effects on osteoarthritis. According to one embodiment of this invention, under the same conditions, the oligonucleotides described in one aspect of this invention, compared with other oligonucleotides that do not follow the sugar modification pattern of the oligonucleotides described in one aspect of this invention, show significantly superior effects in reducing cartilage damage, reducing subchondral bone sclerosis, and / or relieving pain (see Experimental Example 10 and...). Figures 19a to 19d ).

[0093] The oligonucleotides described in one aspect of the present invention can exhibit superior preventive or therapeutic effects compared to existing osteoarthritis treatments (e.g., commercially available osteoarthritis treatments, those currently in clinical use, or those under development). Specifically, the oligonucleotides described in one aspect of the present invention can have superior cartilage protection, subchondral bone sclerosis improvement, pain relief, and / or cartilage regeneration effects compared to currently used or under development osteoarthritis treatments. For example, existing osteoarthritis treatments may include SM04690, or steroidal anti-inflammatory analgesics (e.g., dexamethasone), but are not limited thereto.

[0094] According to one embodiment of the present invention, the oligonucleotide described in one aspect of the present invention can exhibit a superior chondroprotective effect compared to SM04690 (see Experimental Example 10 and...). Figures 20a to 20e According to one embodiment of the present invention, the oligonucleotide described in one aspect of the present invention can have superior cartilage regeneration capacity compared to dexamethasone (see Experimental Example 10 and...). Figures 20a to 20e).

[0095] The oligonucleotides described in one aspect of this invention can protect and / or restore cartilage structure. Specifically, the oligonucleotides described in one aspect of this invention can not only regulate inflammation, but also improve osteoarthritis by inducing the protection and / or restoration of cartilage structure itself.

[0096] The oligonucleotides described in one aspect of this invention exhibit preventative or therapeutic effects even in the late stages of osteoarthritis. Specifically, the oligonucleotides described in one aspect of this invention can restore cartilage even when administered in the late stages of osteoarthritis. In the late stages of osteoarthritis, the oligonucleotides described in one aspect of this invention show superior cartilage recovery compared to steric blockers and / or SM04690. According to one embodiment of this invention, when administered 6 weeks after osteoarthritis induction, the oligonucleotides described in one aspect of this invention show statistically significant cartilage recovery compared to steric blockers (serial number 22) and SM04690 (see Experimental Example 10 and...). Figures 21a to 21c ).

[0097] The oligonucleotides described in one aspect of this invention exhibit pain-relieving effects even in the late stages of osteoarthritis. According to one embodiment of the invention, when administered 6 weeks after osteoarthritis induction, the oligonucleotides described in one aspect of this invention showed statistically significant pain-relieving effects (see Experimental Example 10 and...). Figure 21d ).

[0098] In one implementation, the late stage of osteoarthritis refers to a stage where osteoarthritis has progressed to a certain point. Specifically, the late stage of osteoarthritis can refer to a stage where the pathological changes in osteoarthritis have progressed, with observed cartilage damage, subchondral bone sclerosis, osteophyte formation, and / or synovitis. For example, in animal models, the late stage of osteoarthritis can be a time point of 4, 5, 6 weeks or longer after the induction of osteoarthritis, but is not limited to these. For example, the late stage of osteoarthritis can be a time point of 4, 5, 6 weeks or longer after the diagnosis of osteoarthritis, but is not limited to these. In humans, this can occur when the disease progresses to Kellgren-Lawrence grade 3, 4 or higher, but is not limited to these. The late stage of osteoarthritis means that, compared to the early stages, the pathological changes have further progressed and tended to solidify, and may include stages that are more difficult to treat.

[0099] The oligonucleotides described in one aspect of this invention have high sequence specificity for miR-204 and extremely low off-target effects.

[0100] The oligonucleotides described in one aspect of this invention can exhibit high specificity for miR-204. Specifically, the oligonucleotides described in one aspect of this invention do not nonspecifically 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 this invention, NCBI-BLAST analysis of the base sequence of the oligonucleotides described in one aspect of this invention showed that no other expected binding sequences were found besides miR-204 and TRPM3 (see Experimental Example 11 and Table 14).

[0101] The oligonucleotides described in one aspect of this invention can have a low off-target effect. Specifically, the oligonucleotides described in one aspect of this invention may not significantly affect other genes or biological pathways besides miR-204 degradation. According to one embodiment of this invention, gene ontology (GO) analysis of transcriptome data from cells treated with the oligonucleotides described in one aspect of this invention showed that no significant ontology terms were found in biological processes, molecular functions, and cellular components (see Experimental Example 11 and...). Figures 22a to 22c ).

[0102] The oligonucleotides described in one aspect of this invention may have low immunogenicity. Specifically, the oligonucleotides described in one aspect of this invention may not induce an immune response through Toll-like receptors (TLRs). For example, the TLR may be TLR7, TLR8, and / or TLR9, but are not limited thereto.

[0103] The oligonucleotides described in one aspect of this invention may not increase TLR activity. According to one embodiment of this invention, no significant increase in TLR7, TLR8, and TLR9 activity was observed after treatment with the oligonucleotides described in one aspect of this invention (see Experimental Example 12 and...). Figures 23a to 23c ).

[0104] The oligonucleotides described in one aspect of this invention can have non-immunostimulatory properties for the innate immune receptor pathway. The oligonucleotides described in one aspect of this invention can be antisense oligonucleotides that do not induce immunogenicity.

[0105] The oligonucleotides described in one aspect of this invention may exhibit no cytotoxicity or low cytotoxicity. Specifically, the oligonucleotides described in one aspect of this invention may exhibit no cytotoxicity or low cytotoxicity in chondrocytes, hepatocytes, and / or kidney cells. According to one embodiment of this invention, the oligonucleotides described in one aspect of this invention did not reduce the cell viability of primary cultured cells of mouse cartilage, the HepG2 cell line, and the RPTEC cell line to below 80% (see Experimental Example 13 and...). Figures 24a to 24c ).

[0106] The oligonucleotides described in one aspect of this invention may not exhibit systemic toxicity. The oligonucleotides described in one aspect of this invention may not exhibit systemic toxicity when administered intra-articularly. The oligonucleotides described in one aspect of this invention may not exhibit toxicity to the liver, kidneys, and / or spleen. The oligonucleotides described in one aspect of this invention may not exhibit toxicity to the liver, kidneys, and / or spleen when administered intra-articularly. According to one embodiment of this invention, after a single intra-articular administration of the oligonucleotides described in one aspect of this invention, the organ-to-body weight ratio of the liver, kidneys, and spleen did not show a significant difference compared to the control group (see Experimental Example 13 and...). Figure 25a ).

[0107] The oligonucleotides described in one aspect of this invention may not exhibit local toxicity. Specifically, the oligonucleotides described in one aspect of this invention may not induce cartilage damage, proteoglycan loss, and / or chondrocyte apoptosis when administered intra-articularly. In particular, the oligonucleotides described in one aspect of this invention may not induce cartilage damage, proteoglycan loss, and / or chondrocyte apoptosis when administered intra-articularly. According to one embodiment of this invention, when the oligonucleotides described in one aspect of this invention are administered intra-articularly, no cartilage damage or proteoglycan loss is observed in the knee joint tissue, the cartilage matrix components remain normal, and chondrocyte apoptosis is not induced (see Experimental Example 13 and...). Figure 25b ).

[0108] The oligonucleotides described in one aspect of this invention can have excellent local and / or systemic safety. Specifically, the oligonucleotides described in one aspect of this invention can be safe antisense oligonucleotides with no or low local and / or systemic toxicity at the site of application.

[0109] The oligonucleotides described in one aspect of this invention can be efficiently delivered into chondrocytes. Furthermore, the oligonucleotides described in one aspect of this invention can be efficiently delivered into mammalian (e.g., canine) chondrocytes. Specifically, the oligonucleotides described in one aspect of this invention can be efficiently delivered into mammalian (e.g., canine) chondrocytes and stably accumulate within said chondrocytes. According to one embodiment of this invention, fluorescently labeled oligonucleotides described in one aspect of this invention significantly accumulate in canine chondrocytes, and more than 50% of the fluorescence positive signal was observed under treatment conditions of 10 nM and 50 nM concentrations (see Experimental Example 14 and...). Figure 26a and 26b ).

[0110] The oligonucleotides described in one aspect of this invention can reduce miR-204 in canine chondrocytes. According to one embodiment of this invention, the oligonucleotides described in one aspect of this invention can statistically significantly reduce miR-204 in senescence-induced canine chondrocytes (see Experimental Example 14 and...). Figure 26c ).

[0111] The oligonucleotides described in one aspect of this invention can restore the cartilage matrix synthesis function in canine chondrocytes. Specifically, the oligonucleotides described in one aspect of this invention can increase or restore the synthesis of sulfated glycosaminoglycans (sGAG) in canine chondrocytes. According to one embodiment of this invention, the oligonucleotides described in one aspect of this invention can statistically significantly increase the senescence-induced decrease in sGAG synthesis in canine chondrocytes (see Experimental Example 14 and...). Figure 26d ).

[0112] The oligonucleotides described in one aspect of this invention can exhibit cross-species efficacy. Specifically, the oligonucleotides described in one aspect of this invention can effectively inhibit miR-204 not only in mouse chondrocytes but also in canine chondrocytes, and can restore cartilage matrix synthesis function. The oligonucleotides described in one aspect of this invention can exhibit miR-204 inhibitory effects without species specificity.

[0113] Furthermore, the oligonucleotides described in one aspect of the present invention can be delivered efficiently without additional nucleic acid delivery carriers. Specifically, the oligonucleotides described in one aspect of the present invention can be delivered efficiently to chondrocytes, cartilage, and / or joint tissues without additional nucleic acid delivery carriers. In this specification, the expression "carrier-free" or "carrier-independent" means that the oligonucleotides of the present invention can be introduced into cells without additional nucleic acid delivery carriers (e.g., cationic lipids, lipid nanoparticles, liposomes, cationic polymers (e.g., polyethyleneimine, poly-L-lysine, etc.), dendrimers, cell-penetrating peptides, virus / virus-like particle-based delivery carriers, etc.). The oligonucleotides described in one aspect of the present invention can be administered alone in the form of an aqueous solution that substantially does not contain such delivery carriers (e.g., nuclease-free water, buffer, isotonic solution, etc.).

[0114] The oligonucleotides described in one aspect of this invention can be prepared as intra-articular injections, and may not contain any additional nucleic acid delivery carriers, except for pharmaceutically acceptable solvents (e.g., nuclease-free water, physiological saline, buffered saline, etc.) and optional stabilizers, buffers, and isotonic agents. Despite this carrier-free composition, the oligonucleotides of this invention can remain stably stationary in articular cartilage for a long period (e.g., several weeks after administration), degrade miR-204, and exhibit significant effects in improving the pathological changes of osteoarthritis (see various experimental examples). Therefore, the oligonucleotides of this invention have the advantage of exhibiting excellent tissue penetration and sustained efficacy without the need for expensive delivery carriers.

[0115] The oligonucleotides described in one aspect of the present invention can be efficiently absorbed into chondrocytes (e.g., mouse chondrocytes, canine chondrocytes, etc.) even in cell culture systems without the need for additional transduction reagents; they can be added directly to the culture medium (e.g., liposome transfection reagents, cationic lipid complexes, etc.). Thus, the oligonucleotides of the present invention exhibit excellent intracellular uptake and miR-204 degradation capabilities even without delivery carriers, providing the advantage of minimizing additional toxicity, immunogenicity, or manufacturing complexity caused by delivery carriers.

[0116] Of course, as needed, the oligonucleotides described in one aspect of the present invention, in addition to the carrier-free form described above, can also be used in combination with other pharmaceutical delivery systems selected by those skilled in the art. For example, the oligonucleotides described in one aspect of the present invention can be delivered to chondrocytes, cartilage, and / or joint tissues via a delivery carrier.

[0117] Compositions for the prevention or treatment of osteoarthritis In addition, one aspect of the present invention provides a composition comprising the oligonucleotides described in the foregoing aspect for the prevention or treatment of osteoarthritis.

[0118] Oligonucleotides, as described above, are omitted here for further details.

[0119] In this specification, the term "osteoarthritis (OA)" refers to a degenerative joint disease characterized by degeneration of articular cartilage, sclerosis of subchondral bone, osteophyte formation, and synovitis. Risk factors such as aging, obesity, and mechanical overload can promote the occurrence and progression of osteoarthritis.

[0120] In this specification, the term "treatment" refers to any action that improves or beneficially alters the symptoms of osteoarthritis or the disease caused therefrom by applying the oligonucleotides and / or compositions described in one aspect of this invention. Those skilled in the art, upon reference to the information available in the field, are able to determine the precise criteria for the disease and the extent of improvement, enhancement, and treatment.

[0121] In this specification, the term "prevention" refers to any action that inhibits or delays the onset of osteoarthritis or the disease caused therefrom by applying the oligonucleotides and / or compositions described in one aspect of this invention. Furthermore, "prevention" may include actions that reduce the risk of osteoarthritis or the disease caused therefrom, slow the progression of the disease, or suppress the appearance of disease symptoms.

[0122] In one embodiment, osteoarthritis can be a degenerative disease in which articular cartilage, normally formed at birth, gradually degenerates due to acquired factors such as aging, trauma, mechanical overload, and inflammation. In another embodiment, osteoarthritis includes degenerative (primary) osteoarthritis, post-traumatic osteoarthritis, postoperative osteoarthritis, inflammatory osteoarthritis, and secondary osteoarthritis. For example, degenerative osteoarthritis can be the most common form of osteoarthritis, caused by age-related, natural degenerative changes without a specific cause such as trauma, structural disease, or inflammatory disease. For example, post-traumatic osteoarthritis can be osteoarthritis that occurs after mechanical trauma or joint structural damage, such as joint injury or fracture, ligament injury or tear, or meniscus injury or tear, due to accelerated degenerative changes. For example, inflammatory osteoarthritis can be osteoarthritis with prominent inflammation of the synovium and surrounding tissues in addition to structural degenerative changes; it can be osteoarthritis characterized by synovitis or an increase in inflammatory cells in the synovial fluid. Postoperative osteoarthritis can be osteoarthritis that occurs secondary to structural changes or instability and biomechanical changes following surgical treatment of the intra-articular or surrounding tissues. Secondary osteoarthritis can be osteoarthritis caused by changes in joint structure or biological environment due to a clear underlying cause (trauma, congenital malformation, metabolic disease, endocrine abnormality, infection, crystal deposition disease, etc.). In this case, congenital malformation may include hip dysplasia, varus / valgus deformity, etc.; metabolic and endocrine abnormalities may include diabetes, thyroid disease, ischemic necrosis, obesity, etc.

[0123] The composition described in one aspect of this invention can be used to prevent or treat 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.

[0124] The compositions described in one aspect of this invention can be used to alleviate various pathological changes in osteoarthritis. Specifically, the compositions can be used to alleviate at least one selected from the group consisting of cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, cartilage matrix loss, and / or osteoarthritis-related pain, but are not limited thereto. Relatedly, one aspect of this invention can provide compositions comprising the oligonucleotides described in one aspect of this invention for alleviating cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, cartilage matrix loss, and / or osteoarthritis-related pain.

[0125] The composition described in one aspect of the present invention can be used to restore the extracellular matrix (ECM) synthesis function of chondrocytes. Specifically, the composition can be used to increase or restore the expression of proteoglycan biosynthetic enzymes. Furthermore, the composition can be used to increase or restore the content of sulfated glycosaminoglycans (sGAGs). Relatedly, one aspect of the present invention can provide a composition comprising the oligonucleotides described in one aspect of the present invention for increasing proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans (sGAGs).

[0126] The composition described in one aspect of the present invention can be used to reduce or restore the expression of cartilage matrix degrading enzymes, or to inhibit or alleviate inflammation. Specifically, the composition can be used to reduce the expression of cartilage matrix degrading enzymes, which may include, but are not limited to, Mmp3. Furthermore, the composition can be used to reduce the expression of inflammatory cytokines. The inflammatory cytokines may be IL-6, IL-1β, TNF-α, or combinations thereof, but are not limited thereto. Relatedly, one aspect of the present invention can provide a composition comprising the oligonucleotides described in one aspect of the present invention for reducing cartilage matrix degrading enzymes and inflammatory cytokines.

[0127] The compositions described in one aspect of this invention can be used to prevent or treat early and / or late stages of osteoarthritis. The compositions can restore cartilage even when osteoarthritis has progressed to a late stage. For example, in the early stage of osteoarthritis, within 1, 2, 3, or 4 weeks after induction or diagnosis, mild cartilage damage may be observed, but subchondral bone sclerosis, osteophyte formation, etc., have not yet progressed, but are not limited thereto. Similarly, in the late stage of osteoarthritis, beyond 4 weeks after induction or diagnosis, severe cartilage damage may be observed, and subchondral bone sclerosis, osteophyte formation, etc., have progressed, but are not limited thereto.

[0128] The composition described in one aspect of this invention can exhibit superior effects compared to existing osteoarthritis treatments. Specifically, compared to existing osteoarthritis treatments, the composition can demonstrate superior effects in cartilage protection, cartilage recovery, cartilage regeneration, improvement of subchondral bone sclerosis, inflammation relief, increased sGAG synthesis, and / or pain relief. Existing osteoarthritis treatments may include SM04690 or dexamethasone, but are not limited to these.

[0129] The compositions described in one aspect of this invention can be used for animals (e.g., mammals), but are not limited thereto. For example, the animal can 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 can be a human or a dog. The oligonucleotides described in one aspect of this invention can be applied to animals.

[0130] The compositions described in one aspect of this invention may be pharmaceutical compositions, food compositions, health functional food compositions, or oral compositions, but are not limited thereto.

[0131] When the composition described in one aspect of this invention is a pharmaceutical composition, the composition may further include pharmaceutically acceptable carriers, diluents, excipients, stabilizers, buffers, isotonic agents, etc. The pharmaceutically acceptable carriers, diluents, excipients, stabilizers, buffers, or isotonic agents may be of any kind known in the art without limitation, for example, at least one selected from the group consisting of physiological saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof, but not limited thereto. Furthermore, other conventional additives, such as antioxidants and antibacterial agents, may be added as needed.

[0132] The compositions described in one aspect of the present invention may not contain a delivery carrier. Even without a delivery carrier, the compositions described in one aspect of the present invention can effectively deliver the oligonucleotides described in one aspect of the present invention into cells, thereby exhibiting miR-204 degradation capabilities and demonstrating preventative or therapeutic effects against osteoarthritis. Alternatively, the compositions described in one aspect of the present invention may contain a delivery carrier as needed. The delivery carrier may be, but is not limited to, cationic lipids, lipid nanoparticles, liposomes, cationic polymers (e.g., polyethyleneimine, poly-L-lysine, etc.), dendrimers, cell-penetrating peptides, viral particle delivery carriers, and / or virus-like particle delivery carriers.

[0133] The compositions described in one aspect of this invention can be formulated into various preparations. For example, the compositions can be formulated as injections, liquids, suspensions, emulsions, syrups, powders, granules, tablets, capsules, gels, patches, emulsions, syrups, aerosols, or combinations thereof, but are not limited thereto. Specifically, the compositions can be formulated as intra-articular injections. The compositions can be used for intra-articular injection. For example, intra-articular injection can deliver drugs locally at high concentrations, thereby maximizing therapeutic effects while minimizing systemic side effects.

[0134] The oligonucleotides or compositions described in one aspect of this invention can be administered via a variety of routes. For example, the compositions can be administered orally, via enteral administration, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, nasally, pulmonaryly, rectally, or topically (e.g., intra-articular injection, other local injections, topical application), but are not limited thereto. Specifically, the compositions can be administered intra-articularly. The embodiments described in one aspect of this invention are validated through intra-articular administration, but those skilled in the art will understand that, considering pharmacokinetic / delivery mechanisms, other routes of administration can also be appropriately applied.

[0135] The oligonucleotides or compositions described in one aspect of this invention can be administered in pharmaceutically effective amounts. In one aspect of this specification, "pharmaceutically effective amount" means an amount suitable for medical treatment, sufficient to treat a disease with a reasonable benefit / risk ratio. The effective dose level can be determined based on the patient's disease type, severity, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors known in the medical field.

[0136] The dosage of the oligonucleotide or composition described in one aspect of this invention can be appropriately selected by those skilled in the art, taking into account factors such as the patient's age, sex, weight, severity of disease, route of administration, and frequency of administration. For example, based on an adult (e.g., a 60 kg adult), a single dose of oligonucleotide can be 0.1 mg to 50 mg. For example, based on an adult (e.g., a 60 kg adult), a single dose of oligonucleotide can be 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, or 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 composition can be applied once or several times daily, or repeatedly at intervals of several days, weeks or months.

[0137] The oligonucleotides or compositions described in one aspect of the present invention can be applied once or repeatedly. Specifically, the oligonucleotides or compositions can be applied repeatedly once, twice, three times, four times, five times or more.

[0138] The composition described in one aspect of the present invention can be administered simultaneously, separately, or sequentially in combination with the oligonucleotide described in one aspect of the present invention and another osteoarthritis treatment agent (e.g., a commercially available osteoarthritis treatment agent or a treatment agent in clinical trials).

[0139] When the composition described in one aspect of this invention is a food composition or a health functional food composition, the composition can be prepared in a form that can be consumed like a conventional food. There are no particular limitations on the type of food composition; for example, it can be meat, sausage, bread, chocolate, candy, snacks, pastries, pizza, instant noodles, other noodle products, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinking preparations, alcoholic beverages, vitamin complexes, or health functional foods, but it is not limited thereto.

[0140] The composition described in 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 ECM remodeling-related genes and biological pathways for maintaining cartilage structure, and reducing the expression of proteins related to cartilage matrix destruction.

[0141] In addition, the present invention provides a method for preventing or treating osteoarthritis, comprising administering the aforementioned oligonucleotides or compositions to an individual.

[0142] In one implementation, the individual may be someone who needs treatment or prevention for osteoarthritis.

[0143] In one implementation, the individual may include, but is not limited to, mammals. For example, a 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.

[0144] In one aspect of the osteoarthritis prevention or treatment method described in this invention, the oligonucleotide or composition may be applied to an individual in an effective amount for preventing or treating osteoarthritis.

[0145] Furthermore, this invention provides a method for alleviating pain associated with cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, cartilage matrix loss, and / or osteoarthritis, comprising administering the aforementioned oligonucleotides or compositions to an individual. Additionally, this invention provides a method for increasing proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans (sGAGs), comprising administering the aforementioned oligonucleotides or compositions to an individual. Furthermore, this invention provides a method for reducing cartilage matrix degrading enzymes and / or inflammatory cytokines, comprising administering the aforementioned oligonucleotides or compositions to an individual.

[0146] In one embodiment, the individual may be a subject requiring relief from pain associated with cartilage destruction, subchondral bone sclerosis, osteophyte formation, synovitis, cartilage matrix loss, and / or osteoarthritis. In one embodiment, the individual may be a subject requiring increased proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans. In one embodiment, the individual may be a subject requiring decreased cartilage matrix degrading enzymes and / or inflammatory cytokines.

[0147] In one embodiment, the oligonucleotide or composition may be applied to an individual in an effective amount to alleviate cartilage destruction, subchondral bone sclerosis, osteophyte formation, synovitis, cartilage matrix loss, and / or osteoarthritis-related pain. In one embodiment, the oligonucleotide or composition may be applied to an individual in an effective amount to increase proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans. In one embodiment, the oligonucleotide or composition may be applied to an individual in an effective amount to reduce cartilage matrix degrading enzymes and / or inflammatory cytokines.

[0148] The route of administration, dosage, frequency of administration, and combined administration are as described above, and specific details are omitted here.

[0149] Furthermore, the present invention provides, in one aspect, the use of the aforementioned oligonucleotide preparations for the prevention or treatment of osteoarthritis.

[0150] In addition, one aspect of this specification provides the use of the aforementioned oligonucleotides for the prevention or treatment of osteoarthritis.

[0151] Regarding oligonucleotides, treatment, prevention, and osteoarthritis, as mentioned above, specific details are omitted here.

[0152] Furthermore, this invention provides, in one aspect, the use of the aforementioned oligonucleotides in preparing compositions for relieving pain associated with cartilage destruction, subchondral bone sclerosis, osteophyte formation, synovitis, cartilage matrix loss, and / or osteoarthritis. Furthermore, this invention provides, in another aspect, the use of the aforementioned oligonucleotides in preparing compositions that increase proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans. Furthermore, this invention provides, in yet another aspect, the use of the aforementioned oligonucleotides in preparing compositions that reduce cartilage matrix degrading enzymes and / or inflammatory cytokines.

[0153] Furthermore, one aspect of this specification provides the use of the aforementioned oligonucleotides for relieving pain associated with cartilage destruction, subchondral bone sclerosis, osteophyte formation, synovitis, cartilage matrix loss, and / or osteoarthritis. Additionally, one aspect of this specification provides the use of the aforementioned oligonucleotides for increasing proteoglycan biosynthetic enzymes and / or sulfated glycosaminoglycans. Furthermore, one aspect of this specification provides the use of the aforementioned oligonucleotides for reducing cartilage matrix degrading enzymes and / or inflammatory cytokines.

[0154] The following examples and experimental cases provide a more detailed description of the structure and effects of the present invention. However, these examples are provided for illustrative purposes only to aid in understanding the present invention, and the scope and effect of the invention are not limited thereto.

[0155] [Preparation Example] Preparation of ASO specifically targeting miR-204 The following methods were used to prepare miR-204-specific oligonucleotides, specifically antisense oligonucleotides (ASO) (Examples 1 and 2).

[0156] 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 represent DNA bases, "+X" represents locked nucleic acid (LNA) bases, and "iMe-dC" represents 5-methyl-deoxycytosine bases. In addition, the "*" mark indicates that the bases are linked by a phosphodiester (PO) bond, while the absence of the "*" mark indicates that the bases are linked by a phosphothioate (PS) bond.

[0157] ASO is synthesized and purified by a contract manufacturing organization (CMO) and supplied in powder form. The supplied substance is dissolved in nuclease-free water (NFW: AM9932, Thermo Fisher Scientific) to a concentration of 5 mM and stored at -80 °C.

[0158] For cell experiments, thaw at room temperature before use, dilute in culture medium to the target concentration, and then treat the cells. For animal experiments, thaw at room temperature before use, dilute in NFW to the target concentration, and finally inject into the joint cavity in a 5 μL volume.

[0159] [Experimental Example 1] Confirmation of miR-204 degradation ability against miR-204-specific ASO [Experimental Example 1-1] Confirmation of the miR-204 degradation ability of ASO in Example 1 (1) Experimental Method C28 / I2 cell lines (chondrocytes) overexpressing microRNA-204 (miR-204) were used at 3 × 10⁻⁶. 5Cells were seeded at a density of 12-well plates and cultured for 24 hours. Cells were maintained at 37 °C under conditions of 5% CO2 and atmospheric oxygen concentration, and the culture medium used was DMEM / F-12 medium supplemented with 10% FBS and 1% antibiotic (penicillin-streptomycin).

[0160] After culturing for 24 hours, the antisense oligonucleotide (ASO) from Example 1 was diluted in the culture medium at concentrations of 0.001, 0.01, 0.1, 0.316, 1, 3.16, and 10 μM for treatment. For this purpose, the original culture medium was removed and replaced with fresh culture medium containing ASO for cell treatment.

[0161] After 48 hours of ASO treatment in Example 1, cells were recovered, 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 efficiency (D) in Example 1 was measured. max ) and the reaction concentration (DC) required to degrade miR-204 by 50%. 50 The computation is performed using GraphPad Prism (Dotmatics).

[0162] (2) Experimental results Because miR-204 exists at a very low basal level in normal chondrogenic cells, a chondrogenic cell line overexpressing miR-204 (C28 / I2) was used to sensitively assess the miR-204 degradation efficacy of Example 1. Treatment of this cell line with different concentrations of the miR-204 described in Example 1 showed that, in the Example 1 treatment group, miR-204 expression was confirmed to decrease in a concentration-dependent manner. Figure 1a Quantitative analysis results show that the maximum degradation efficiency (D) of Example 1 is [missing information]. maxThe concentration (DC) is approximately 99.6%, which causes a 50% degradation of miR-204. 50 The calculated value is approximately 0.0055 μM. Figure 1b ).

[0163] [Experimental Examples 1-2] Confirmation of miR-204 degradation ability of ASO in Example 2 (1) Experimental methods 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 point, a portion of the cells were cultured at 3 × 10⁻⁶ cells / well. 5 After inoculating cells / wells for 24 hours, the culture medium was removed, and 1 μM of the reagent from Example 2 was diluted with MessengerMAX reagent and Opti-MEM for transfection. The cells were then incubated at room temperature for 10 minutes. Subsequently, the cells were replaced with 0.5 mL of fresh culture medium, and the solution of Example 2 diluted with transfection reagent and Opti-MEM was added. After 6 hours, the culture medium was replaced with fresh medium. Cells were recovered 48 hours after this point. Figure 14 Total RNA was extracted from the recovered cells using TRI Reagent (Molecular Research Center, Inc.), and cDNA was synthesized using the miRCURY LNA RT Kit (Qiagen). miR-204 expression was then analyzed by qRT-PCR using the miRCURY LNA microRNA PCR Kit (Qiagen), with Let-7e-5p used as an internal control.

[0164] For statistical analysis, all quantitative data are expressed as mean ± standard error (Mean ± SEM). Statistical significance was tested using GraphPad Prism 10 software (version 10.4.2, GraphPad Software), and Student's t-test was applied. Statistical significance markers in the figures are as follows: *, **, ***, ****: corresponding to P < 0.05, 0.01, 0.001, and 0.0001, respectively. (2) Experimental results qRT-PCR analysis showed that the mean expression level (Mean) of miR-204 in the vector control (trf) was 1.000 (0.000 on SEM), while in Example 2, specifically the transfected (vector-loaded) (0.1 μM) miR-204 (Mean) was 0.323 (0.030 on SEM), confirming that ASO composed of PO bonds (backbone) can also induce miR-204 degradation (see [link to documentation]). Figure 14 ).

[0165] This miR-204 degradation effect demonstrates that, regardless of the backbone components, the oligonucleotide (ASO) described in one aspect of the present invention can effectively degrade the target miR-204 when delivered into cells.

[0166] [Experimental Example 2] Confirmation of the delivery capability of miR-204-specific ASO (Example 1) to mouse chondrocytes. (1) Experimental methods 1) Primary culture of mouse chondrocytes Articular chondrocytes were isolated from the femoral condyle and tibial plateau of 5-6 day old ICR mice. The isolated tissues were digested with 0.2% collagenase solution (ref) to obtain single-cell suspensions. For flow cytometry analysis, the obtained cells were cultured at 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 × 10⁶ cells / well in 12-well plates and cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). Cells were maintained at 37 °C, 5% CO₂, and 21% O₂. 72 hours after seeding, antisense oligonucleotide (ASO) treatment groups and vehicle control groups were established according to the experimental objectives. For fluorescence imaging, cells were seeded at a density of 1 × 10⁶ cells / well. 5Cells were seeded at a density of cells / well in 12-well plates and cultured. Cells were maintained at 37 °C, 5% CO2, and 21% O2. Ninety-six hours after seeding, antisense oligonucleotide (ASO) treatment groups and vehicle control groups were established according to the experimental objectives.

[0167] 2) Confirm the delivery efficiency of ASO in Example 1 To visualize the intracellular uptake trend of Example 1 in chondrocytes, cultured cells were co-cultured with 1 μM Cy5.5 fluorescently labeled Example 1 for 6 hours. Subsequently, 1 μg / mL Hoechst was added to the culture medium, and the cells were co-incubated for another 18 hours. For fluorescence microscopy analysis, cells were washed three times with phosphate-buffered saline (PBS), and fluorescence images were acquired using an EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0168] Furthermore, the intracellular uptake of Example 1 was quantitatively assessed by flow cytometry. For this purpose, mouse articular chondrocytes were co-cultured with Cy3-labeled Example 1 cells (10 nM or 50 nM) for 24 hours. After treatment, cells were separated from the attachment surface using trypsin, washed with PBS, and centrifuged. The recovered cells were resuspended twice in PBS containing 5% FBS.

[0169] Flow cytometry analysis was performed using a FACSAria™ III cell sorter (BD Biosciences), and data were acquired and quantified using FACSDiva 9.7 software.

[0170] (2) Experimental results To confirm the delivery efficiency of mouse-derived chondrocytes from Example 1, their intracellular uptake capacity was assessed using Cy5.5 or Cy3 fluorescent labeling. Fluorescence microscopy analysis showed that, compared to the vehicle-treated group, significant accumulation of fluorescent signals was observed intracellularly in the mouse-derived chondrocytes of the Example 1-treated group. Figure 2a Furthermore, flow cytometry analysis revealed over 50% fluorescence positive signals under all conditions of treatment with 10 nM and 50 nM concentrations in Example 1, thus quantitatively confirming the excellent intracellular uptake capacity of Example 1. Figure 2b , Figure 2cThese results indicate that Example 1 possesses physicochemical properties that enable efficient delivery and stable accumulation within chondrocytes.

[0171] [Experimental Example 3] Confirmation of the delivery capability of Example 1 in osteoarthritis patient tissues (1) Experimental methods 1) In vitro culture of human cartilage tissue Osteoarthritis tissue obtained from total joint replacement surgery was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). The tissues were maintained at 37 °C, 5% CO2, and 21% O2. Antisense oligonucleotide (ASO) treatment groups and vehicle control groups were set up according to the experimental objectives.

[0172] 2) Confirm the delivery status of Example 1 To visualize the intracellular uptake trend of Example 1 in chondrocytes, cultured cells were co-cultured with 1 μM Cy5.5 fluorescently labeled Example 1 for 24 hours. Subsequently, 1 μg / mL Hoechst was added to the culture medium for further co-incubation for 2 hours. For fluorescence microscopy analysis, cells were washed three times with phosphate-buffered saline (PBS), and fluorescence images were acquired using an EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0173] (2) Experimental results To confirm the delivery of Example 1 to osteoarthritis patient-derived cartilage tissue, its intracellular uptake capacity was assessed using Cy5.5 fluorescent labeling. Fluorescence microscopy analysis showed that, compared to the vehicle-treated group, the fluorescence signal significantly accumulated intracellularly in patient-derived chondrocytes of the Example 1-treated group. Figure 3 These results demonstrate that Example 1 possesses physicochemical properties that enable efficient delivery and stable accumulation not only in mice but also in human chondrocytes.

[0174] [Experimental Example 4] Confirmation of miR-204 degradation ability against miR-204-specific ASO To confirm the degradation ability of complete DNA (Full DNA) consisting entirely of DNA, which has the same base sequence as the ASO prepared in Example 1 described above, and ASO (Examples 3-1 to 3-3, Table 1 below) that differs from it in some base sequences, i.e. has mismatches (in bold), on miR-204, the following experiments were conducted.

[0175] Among them, full DNA refers to an ASO with a base sequence that is completely complementary to the target miR-204. The sugars in the nucleotides that make up full DNA do not contain LNA sugars or modifying sugars, and are all 2'-deoxy ASOs.

[0176] Table 1

[0177] (1) Experimental methods The expression level of miR-204 was analyzed by qRT-PCR using the same method as in Example 1 (however, the treatment concentrations in Examples 1 and 3-1 to 3-3 were 1 μM and 10 μM, respectively).

[0178] For statistical analysis, all quantitative data are expressed as mean ± standard error (Mean ± SEM). Statistical significance was tested using GraphPad Prism 10 software (version 10.4.2), employing one-way ANOVA followed by a Tukey post-hoc test. Statistical significance markers in the figures are shown below.

[0179] *, **, ***, ****: Comparison with solvent (Vehicle) (corresponding to P<0.05, 0.01, 0.001, 0.0001 respectively); #, ##, ###, ####: Comparison of ASO (1 μM concentration group) with intact DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively); $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) with intact DNA (P<0.05, 0.01, 0.001, 0.0001, respectively). (2) Experimental results qRT-PCR analysis showed that, compared with the control group (Full DNA) which did not contain LNA, miR-204 expression was statistically significantly decreased in the treatment groups of Examples 3-1 to 3-3 (see Table 2). Figure 4 ).

[0180] Table 2

[0181] Under 1 μM treatment, Examples 3-1 to 3-3 all showed a significant decrease in expression compared to full DNA, and the same trend was maintained under 10 μM treatment.

[0182] In particular, Examples 3-1 and 3-2 each contained one mismatch in the central region of miR-204 (nucleotide positions 9-12), and Example 3-3 contained two mismatches in the supplementary region (nucleotide positions 13-16). Despite this, the degradation activity of miR-204 was maintained. This indicates that a single or two or more mismatches in the central or supplementary region of miR-204 do not significantly affect the binding efficiency of ASO, and that perfect complementarity within this region is not required.

[0183] Therefore, ASO variants containing one or more mismatches within the central or supplementary region of miR-204 (Examples 3-1 to 3-3) were deemed to maintain excellent stability, binding affinity, and degradation-inducing efficacy against miR-204.

[0184] [Experimental Example 5] Confirmation of miR-204 degradation capacity based on ASO length To confirm the miR-204 degradation ability based on ASO length, experiments were conducted on shorter ASOs (Examples 4-1 and 4-2, Table 3 below) with partial base sequence deletions near the 3' end of the ASO prepared in Example 1, using the same method as in Experiment 1.

[0185] Table 3

[0186] (1) Experimental methods The expression level of miR-204 treated according to Examples 4-1 and 4-2 was analyzed by qRT-PCR using the same method as in Example 4. The statistical significance markers in the graphs are as follows.

[0187] *, **, ***, ****: Comparison with solvent (Vehicle) (corresponding to P<0.05, 0.01, 0.001, 0.0001 respectively); #, ##, ###, ####: Comparison of ASO (1 μM concentration group) with intact DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively); $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) with complete DNA (P<0.05, 0.01, 0.001, 0.0001, respectively).

[0188] (2) Experimental results qRT-PCR analysis showed that, compared with the LNA-free and fully complementary miR-204 sequence (Full DNA), the antisense oligonucleotides (Examples 4-1 and 4-2) with a deletion of 1 (n-1) and 2 (n-2) bases from the 5' end of the miR-204 sequence, respectively, resulted in statistically significant reductions in miR-204 expression (see Table 4). Figure 5 ).

[0189] Table 4

[0190] At both 1 μM and 10 μM concentrations, Examples 4-1 and 4-2 maintained lower miR-204 expression compared to full DNA, or even showed an enhanced trend. In particular, Example 4-2 (n-2) showed the lowest miR-204 expression level at both concentrations, confirming that even with a reduction of 2 bases, the targeted binding and degradation induction of miR-204 were still efficiently maintained.

[0191] These results demonstrate that, when designing antisense miR-204, even in the case of incomplete sequence complementarity, partial deletion (truncated) at the n-1 or n-2 level can still effectively bind and induce degradation.

[0192] Therefore, compared to the fully complementary sequence of miR-204, antisense oligonucleotide variants missing 1-2 bases (Examples 4-1 and 4-2) were identified as candidates that could significantly degrade miR-204.

[0193] [Experimental Example 6] Confirming the effect of LNA location on the miR-204 degradation ability of ASO In Experiments 1 to 4, the excellent miR-204 degradation ability of the ASO of Example 1 was confirmed. The ASO of Example 1 had 6 LNAs continuously connected in the first region. To confirm whether the miR-204 degradation ability of the ASO was affected by the LNA position, the following experiment was conducted.

[0194] [Experimental Example 6-1] The Influence of LNA Position When the 5' End of the ASO Contains an LNA First, ASOs containing locked nucleic acids at their 5' ends, but with different numbers and positions of LNAs, were prepared using the same method as in the preparation examples described above (Examples 5-1 to 5-7, and Comparative Examples 1-1 and 1-2, Table 5 below), and their miR-204 degradation ability was evaluated.

[0195] Table 5

[0196] (1) Experimental methods The expression levels of miR-204 treated according to Examples 5-1 to 5-7 and Comparative Examples 1-1 and 1-2 were analyzed by qRT-PCR using the same method as in Experimental Example 4. The statistical significance markers in the graphs are as follows.

[0197] The statistical significance markers in the chart are as follows.

[0198] *, **, ***, ****: Comparison with solvent (Vehicle) (corresponding to P<0.05, 0.01, 0.001, 0.0001 respectively); #, ##, ###, ####: Comparison of ASO (1 μM concentration group) with complete DNA in Examples 5-1 to 5-7 / Comparison of ASO (1 μM concentration group) with Example 1 in Comparative Examples 1-1 to 1-2 (corresponding to P<0.05, 0.01, 0.001, 0.0001, respectively). $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) with complete DNA in Examples 5-1 to 5-7 / Comparison of ASO (10 μM concentration group) with Example 1 in Comparative Examples 1-1 to 1-2 (corresponding to P<0.05, 0.01, 0.001, 0.0001, respectively).

[0199] (2) Interpretation of experimental results qRT-PCR analysis showed that, compared with the control group (Full DNA) without LNA, ASOs with three consecutive LNAs at the 5' end (all three nucleotides in the first region are LNAs) (Examples 5-7) and ASOs with one or two LNAs at the 4th-6th base positions at the 5' end (nucleotides 4-6) (at least one or more of the three nucleotides in the second region are LNAs) (Examples 5-1 to 5-6) all exhibited statistically significant reductions in miR-204 expression (see Table 6 below). Figure 6 and Figure 7 ).

[0200] Table 6

[0201] Under two treatment concentrations of 1 μM and 10 μM, all ASOs in Examples 5-1 to 5-7 showed a significant miR-204 degradation effect compared to intact DNA (Full DNA), with Examples 5-1 to 5-7 showing the same trend.

[0202] These results demonstrate that even when miR-204 targets and binds, excellent miR-204 degradation activity can be maintained even if the 5' end (first region, nucleotides 1–3) contains LNA, while LNA or DNA can also be arranged in the second region (nucleotides 4–6 from 5').

[0203] Therefore, if the LNA is concentrated near the 5' end (first region), the ASO of DNA or LNA mixed in the adjacent second region has a stable and efficient degradation effect on miR-204.

[0204] [Experimental Example 6-2] The Influence of LNA Position When the LNA is Included at the 3' End in the ASO In Experiment 6-1, it was confirmed that ASO containing three or more LNAs at positions 1 to 6 of the 5' end, specifically, ASO with all nucleotides at positions 1 to 3 being LNAs exhibited excellent miR-204 degradation ability. Therefore, to confirm whether there is a difference in miR-204 degradation ability when ASO contains LNAs at the 3' end rather than the 5' end (Comparative Examples 2-1 to 2-4, Table 7 below), the miR-204 degradation ability was evaluated using the same method as in Experiment 1.

[0205] Table 7

[0206] (1) Experimental methods The expression level of miR-204 treated according to Comparative Examples 2-1 to 2-4 was analyzed by qRT-PCR using the same method as in Experimental Example 4. The statistical significance markers in the graphs are as follows.

[0207] *, **, ***, ****: Comparison with solvent (Vehicle) (corresponding to P<0.05, 0.01, 0.001, 0.0001 respectively); #, ##, ###, ####: Comparison of ASO (1 μM concentration group) with Example 1 (corresponding to P<0.05, 0.01, 0.001, 0.0001, respectively); $, $$, $$$, $$$: Comparison of ASO (10 μM concentration group) with Example 1 (corresponding to P<0.05, 0.01, 0.001, 0.0001, respectively).

[0208] (2) Interpretation of experimental results The effect of the LNA terminal position on miR-204 degradation was evaluated using qRT-PCR analysis. Example 1 (LNA at the 5' end) and ASO variants (Comparative Examples 2-1 to 2-4) (LNA at the 3' end) were compared. Results showed that, compared to Example 1 (LNA at the 5' end), ASO variants (Comparative Examples 2-1 to 2-4) with LNA at the 3' end exhibited reduced miR-204 degradation at both concentrations (1 μM and 10 μM) (see Table 8 below). Figure 8 ).

[0209] Table 8

[0210] Under 1 μM treatment, Comparative Examples 2-1 and 2-2 showed a statistically significant reduction in miR-204 degradation compared to Example 1, while Comparative Examples 2-3 and 2-4 showed a decreasing trend. Under 10 μM treatment, a statistically significant reduction in degradation was confirmed in Comparative Examples 2-1, 2-3, and 2-4, and Comparative Example 2-2 also showed a similar decreasing trend.

[0211] These results indicate that the position of the LNA has a decisive influence on the miR-204 degradation activity of ASO, especially when the LNA is located at the 5' end, it exhibits the best efficacy. That is, when the LNA moves to the 3' end, the binding force of ASO and the RNase H-mediated degradation efficiency decrease, resulting in a decrease in the overall miR-204 degradation effect.

[0212] [Experimental Example 6-3] The Influence of LNA Position When LNAs are Randomly Included in the ASO As demonstrated in Experiment 6-2, even if the ASO contains three or more LNAs, the degradation effect of miR-204 is not significant if they are only contained on the 3' end side. Therefore, to evaluate the miR-204 degradation capacity when the ASO contains LNAs randomly (Comparative Examples 3 and 4, Table 9 below), the following experiments were conducted.

[0213] Table 9

[0214] (1) Experimental methods 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 point, a portion of the cells were cultured at 3 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of cells / well and cultured for 24 hours. The culture medium was then removed and replaced with fresh medium diluted 10 μM (Comparative Example 3) for a single treatment. Cells were recovered 48 hours after treatment (Group 1). Figure 9 On the other hand, the remaining cells were divided into 2 × 10⁻⁶ cells. 4 Cells were seeded at a density of cells / well and cultured for 24 hours. While changing the culture medium, Comparative Example 4 was treated with 256 nM, 1.6 μM, and 10 μM concentrations. 72 hours after the first treatment, a second treatment was performed at the same concentrations. Cells were recovered 48 hours after the second treatment (Group 2). Figure 10 Total RNA was extracted from the recovered cells in each group using TRI Reagent (Molecular Research Center, Inc.), and cDNA was synthesized using the miRCURYLNA RT Kit (Qiagen). The expression of miR-204 was then analyzed by qRT-PCR using the miRCURY LNA microRNA PCR Kit (Qiagen), with Let-7e-5p used as an internal control.

[0215] For statistical analysis, all quantitative data are expressed as mean ± standard error (Mean ± SEM). Statistical significance was tested using GraphPad Prism 10 software (version 10.4.2), employing one-way ANOVA followed by a Tukey post-hoc test. Statistical significance markers in the figures are shown below.

[0216] *, **, ***, ****: represent P < 0.05, 0.01, 0.001, and 0.0001, respectively. (2) Interpretation of experimental results qRT-PCR analysis showed that in Group 1, the degradation effect of miR-204 in Comparative Example 3, which randomly contained LNA, was statistically significantly lower than that in Example 1 (see Table 10 below). Figure 9 ).

[0217] Table 10

[0218] Such a low miR-204 degradation effect indicates that the position and arrangement regularity of LNAs play a more significant role in miR-204 degradation activity compared to the presence of LNAs in ASO. Similarly, Comparative Example 4, with another form containing random LNAs, also exhibited lower miR-204 degradation ability than Example 1 across all concentration groups (see Example 1). Figure 10 That is, in the structures of randomly arranged LNAs in Comparative Examples 3 and 4, it is difficult to efficiently bind and induce degradation of miR-204. This indicates that the ASOs described in one aspect of the present invention, for example, the structure of Example 1 with the 5' end as the center (with the first and second regions as the center), are more effective in degrading miR-204.

[0219] [Experimental Example 7] Confirming the effect of LNA quantity on the miR-204 degradation ability of ASO. In Experiment 6, it was confirmed that the miR-204 degradation ability in ASO varied depending on the position of LNA. To confirm the effect of the number of LNAs in ASO, ASOs with 15 or more LNAs arranged from the 5' end were prepared (Comparative Examples 5-1 to 5-3, Table 11 below) and their formulation stability was evaluated.

[0220] Table 11

[0221] (1) Experimental methods The expression level of miR-204 treated according to Comparative Examples 5-1 to 5-3 was analyzed by qRT-PCR using the same method as in Experimental Example 4. The statistical significance markers in the graphs are as follows.

[0222] *, **, ***, ****: Comparison with solvent (Vehicle) (corresponding to P<0.05, 0.01, 0.001, 0.0001 respectively).

[0223] In addition, to confirm the physical properties (viscosity) of the dissolved ASO in Comparative Examples 5-1 to 5-3, test tubes containing their respective solutions were photographed from below at an angle. After the solution was drawn into a 1 mL pipette tip using a 1 mL pipette, the solution remaining in the tip was photographed.

[0224] (2) Interpretation of experimental results The effect of the amount of LNA within antisense oligonucleotides (ASOs) on the degradation efficiency of miR-204 was evaluated using qRT-PCR analysis. Variants with progressively increased LNA amounts (Comparative Examples 5-1 to 5-3) were used in the experiments. The results showed that even with increased LNA amounts, the degradation efficiency of miR-204 remained at a similar level (see [link to qRT-PCR analysis]). Figure 11a ).

[0225] However, variants with an excessive increase in LNA (Comparative Examples 5-2 and 5-3) exhibited a sharp increase in viscosity during dissolution, and the physical phenomenon of the solution coagulating into a gel-like form was observed (see [link]). Figure 11b In variants with lower LNA content (Comparative Example 5-1), higher viscosity was also observed compared to Example 1. This was attributed to the increased interaction between ASO groups as LNA content increased, leading to decreased water solubility.

[0226] In summary, these results indicate that increasing the number of LNAs improves the degradation effect of miR-204, but it also leads to physical and formulation limitations due to increased formulation viscosity and decreased solubility. Therefore, when considering both efficacy and formulation stability, the optimal structure for maintaining a balance between miR-204 inhibitory efficacy and formulation suitability is determined to be at a level where the number of LNAs is not excessive (e.g., the level in Example 1).

[0227] [Experimental Example 8] Effects of modified sugars other than LNA on miR-204 degradation ability In Experiment 6-1, it was confirmed that ASOs with three or more LNAs at positions 1 to 6 of the 5' end (ASOs with three or more LNAs in both the first and second regions), specifically ASOs with all three LNAs at positions 1 to 3 (all three LNAs in the first region), exhibited excellent miR-204 degradation ability. To confirm that, as in Experiment 6-1, the ASO has three or more LNAs at positions 1 to 6 of the 5' end, specifically with all three LNAs at positions 1 to 3, but with additional non-LNA modified sugars present at other positions (e.g., non-fixed 2'-substituted sugar modifications) (Examples 6-1 to 6-14, Table 12 below; MOE modification X), M This indicates that OME is modified by X. O (X represents a base)) Whether there are differences in the degradation ability of miR-204, and to evaluate the degradation ability of miR-204.

[0228] Table 12

[0229] (1) Experimental methods The expression level of miR-204 treated according to Examples 6-1 to 6-14 was analyzed by qRT-PCR using the same method as in Example 4. The statistical significance markers in the graphs are as follows.

[0230] *, **, ***, ****: Comparison with solvent (Vehicle) (corresponding to P<0.05, 0.01, 0.001, 0.0001 respectively); #, ##, ###, ####: Comparison of ASO (1 μM concentration group) with intact DNA (Full DNA) in Examples 6-1 to 6-14 (corresponding to P<0.05, 0.01, 0.001, 0.0001, respectively). $, $$, $$$, $$$$: In Examples 6-1 to 6-14, ASO (10 μM concentration group) compared with complete DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0231] (2) Interpretation of experimental results To confirm the effect of introducing other modified sugars to replace LNA in the second region (nucleotide positions 4-7 based on ASO) on the efficacy of ASO for miR-204, experiments were conducted. For this purpose, ASOs containing 1 to 3 2'-O-methoxyethyl (MOE) nucleotides (Examples 6-1 to 6-7) and ASOs containing 2'-O-Methyl (OME) nucleotides at the same position were designed and tested under the same conditions as in Example 1.

[0232] qRT-PCR analysis showed that the degradation effect of miR-204 was maintained even when MOE or OME was introduced into the second region, confirming a statistically significant increase in miR-204 degradation ability compared to the control group (Full DNA) with complete LNA exclusion (see [link to qRT-PCR analysis]). Figure 12 and Figure 13 This confirms that even with the introduction of modifying sugars other than LNA in the second region, the efficacy of ASO was not significantly reduced.

[0233] These results indicate that even when MOE or OME is introduced into the second region (positions 4-7) to replace LNA, the overall structural stability of ASO and the degradation activity of miR-204 can be maintained, demonstrating that it is feasible to design modified ASOs with chemical diversity.

[0234] [Experimental Example 9] Effect of miR-204-specific ASO in a cell model In Experiment 1, the degradation effect of miR-204 in Example 1 was confirmed. Furthermore, to confirm whether the ASO of Example 1 also exhibited a preventative or therapeutic effect on osteoarthritis in a cell model by degrading miR-204, the following experiment was conducted.

[0235] (1) Experimental methods 1) Primary culture of mouse chondrocytes Articular chondrocytes were isolated from the femoral condyle and tibial plateau of 5-6 day old ICR mice. The isolated tissues were digested with 0.2% collagenase solution to obtain single-cell suspensions. Depending on the experimental objectives, the obtained cells were seeded at different cell densities in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich) for primary culture. Cells were maintained under hypoxic conditions at 37 °C, 5% CO2, and 3% O2. 72 hours after seeding, antisense oligonucleotide (ASO) treatment groups and vehicle control groups were established according to the experimental objectives. To induce chondrocyte senescence, primary cultured chondrocytes were treated with bleomycin (Cayman) at 40 μg / mL for 24 hours. Subsequently, the culture medium was removed and replaced with growth medium using nuclease-free water (NFW) or growth medium containing the solution from Example 1 (Sequence No. 1) (final concentration 2 μM). Forty-eight hours post-treatment, cells were recovered for mRNA analysis at a seeding density of 5 × 10⁶ cells per well in a 12-well plate. 4 Cells / well. Furthermore, 72 hours after treatment in Example 1 (final concentrations of 0.1, 1, and 10 μM), cells were recovered for protein analysis at a seeding density of 2 × 10⁶ cells / well in a 6-well plate. 5 Cells / well. To induce an inflammatory response in chondrocytes, primary cultured chondrocytes were treated with IL-1β (Genscript) 1 ng / mL, in combination with either Example 1 (10 μM) or SM04690 (30 nM), and cultured for 72 hours. Cells were then recovered for mRNA analysis at a seeding density of 0.83 × 10⁶ cells / well in a 12-well plate. 5 cells / well.

[0236] 2) Gene expression changes induced in Example 1 were analyzed by qRT-PCR and Western blot. Cells in culture were recovered, 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 for 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 levels of each gene were calculated relative to the vehicle control group using the ΔΔCt method. For statistical analysis, all quantitative data are expressed as mean ± standard error (Mean ± SEM). Statistical significance tests were performed using GraphPad Prism 10 software (version 10.4.2, GraphPad Software), first using one-way ANOVA, then applying Tukey's post-hoc test. The primers used for qRT-PCR are shown in Table 13 below. Table 13

[0237] To perform protein expression analysis, cultured cells were recovered, and total protein was extracted using RIPA buffer. Quantified protein samples were separated by SDS-PAGE and then transferred to an NC membrane. Primary and secondary antibody reactions were then performed against cartilage matrix synthesis-related proteins (HPLN1, CHSY1) and the internal 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). HPLN1 antibody (ab98038) was purchased from Abcam, CHSY1 antibody (NBP3-47604) from Novus Biologicals, and Vinculin antibody (13901) from Cell Signaling Technology.

[0238] 3) sGAG analysis sGAG quantification was performed using primary cultured mouse chondrocytes according to the described method. To induce cellular senescence, cells were treated with bleomycin at a concentration of 40 μg / mL for 24 hours. After treatment, cells were cultured for 3 days in fresh complete medium, with or without the medium from Example 1. The medium was then replaced, and cells were cultured for another 3 days. The culture supernatant was then recovered for sGAG analysis. The sGAG concentration in the recovered culture medium was determined using 1,9-dimethylmethylene blue (DMMB) staining. Absorbance was measured at 525 nm, and sGAG content was quantified based on a standard curve plotted using chondroitin sulfate (C0335; Tokyo Chemical Industry). MTT assays were performed on the same cells to correct for differences in cell viability and density. For this purpose, a PBS-based MTT solution (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide, T-030-1; Goldbio) at a concentration of 100 μg / mL was added, and the reaction was carried out for 2 hours. After the reaction, the generated formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at a wavelength of 570 nm. All sGAG measurements were normalized according to the MTT results and then compared and analyzed. The maximum effect (E) of Example 1 was... max ) and half-maximum effective concentration (EC) 50 The computation is performed using GraphPad Prism (Dotmatics).

[0239] 4) Pathway analysis In the C28 / I2 cell line overexpressing miR-204, after treatment with 1 μM of the drug from Example 1, a list of differentially expressed genes (DEG list) was derived from the obtained transcriptome data. Reactome pathway analysis was performed using the derived DEG list on the EnrichR platform. The top 20 annotations were selected based on adjusted p-values ​​and visualized as a bubble chart.

[0240] (2) Experimental results Because miR-204 exists at a very low basal level in normal chondrogenic cells, a chondrogenic cell line overexpressing miR-204 (C28 / I2) was used to sensitively assess the miR-204 degradation efficacy in Example 1. Transcriptome analysis revealed significant changes in ECM remodeling-related gene populations containing ECM proteoglycans and in gene annotations related to cartilage structure maintenance. Figure 15 This indicates that the inhibition of miR-204 in Example 1 is not limited to the regulation of microRNA levels, but also participates in structural repair mechanisms such as ECM formation and homeostasis maintenance in chondrocytes.

[0241] Based on these results, the protective mechanism of action of Example 1 was validated at the molecular level in actual primary cultured mouse chondrocytes. To this end, the expression changes of key enzymes in the sulfated proteoglycan biosynthesis pathway were analyzed, consistent with the results confirmed in the transcriptomic analysis. Treatment of bleomycin-induced damaged chondrocytes with Example 1 showed a statistically significant increase in the expression of key enzymes in the sGAG biosynthesis pathway, while a significant decrease in the expression of matrix-degrading enzymes. Figure 16a This restoration of expression refers to the recovery of the cartilage matrix synthesis pathway, which was inhibited by miR-204, to normal levels as described in Example 1, and the same increasing trend observed at the protein level after treatment with Example 1 was also confirmed. Figure 16b These results confirm at the molecular and functional levels that Example 1 can effectively inhibit the pathological effects of miR-204 while restoring the matrix synthesis capacity of chondrocytes.

[0242] Then, to evaluate the effect of Example 1 under inflammatory stimulation, experiments were conducted using primary cultured chondrocytes treated with the inflammatory cytokine IL-1β. The results showed that, similar to those observed with bleomycin-induced injury, the expression of proteoglycan biosynthesizers was significantly restored in the Example 1 treatment group, while the expression of the inflammatory cytokine IL-6 was statistically significantly reduced (…). Figure 16c In particular, Example 1 showed superior performance compared to SM04690, which has completed Phase III clinical trials.

[0243] These results indicate that Example 1 effectively inhibits the miR-204-mediated pathway not only in aging environments but also in inflammatory environments, exerting a dual action of promoting cartilage matrix synthesis and alleviating inflammatory responses.

[0244] Then, to verify whether the effects of Example 1 extended beyond changes at the gene and protein levels, and also influenced the actual functional phenotype of cells, supplementary experiments were conducted. The results of treatment with Example 1 in senescent chondrocytes showed that the content of sulfated glycosaminoglycans (sGAG) recovered in a concentration-dependent manner. Figure 17a Quantitative analysis results showed that the maximum effect of Example 1 (E) was calculated. max The half-maximum effective concentration (EC50) was 114.3%, and the half-maximum effective concentration (EC50) was 114.3%. 50 The value is 273.5 nM. Figure 17b Furthermore, the increase in sGAG content induced by Example 1 was also confirmed in an inflammatory environment, and its effect was superior to SM04690 ( ). Figure 17c This restoration of matrix content means that Example 1 is not limited to inhibiting miR-204 expression or regulating downstream protein expression, but actually functionally restores the cartilage matrix synthesis capacity at the cellular level.

[0245] That is, Example 1 was demonstrated to be a therapeutic candidate that restores the ECM synthesis function of chondrocytes structurally and functionally by effectively blocking the pathological signaling pathway of miR-204 in both aging and inflammatory environments.

[0246] [Experimental Example 10] Validating the effect of miR-204-specific ASO in an animal model In Experiment 1, the degradation effect of miR-204 in Example 1 was confirmed. Furthermore, to confirm whether the ASO of Example 1 also exhibited a preventative or therapeutic effect on osteoarthritis in animal models by degrading miR-204, the following experiment was conducted.

[0247] (1) Experimental methods 1) Establish a mouse model of osteoarthritis C57BL / 6 male mice (12 weeks old, wild type) used in the experiments were purchased from Daehan Biolink Co., Eumsung, Korea. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (approval number: SNU-240710-5-1). Experimental animals were randomly assigned to groups and housed in a specific pathogen-free facility at Seoul National University. Housing conditions were maintained at a temperature of 23–25 °C, humidity of 45–65%, and a 12-hour (light / dark) cycle. All mice had free access to standard laboratory solid feed. Animal experiment reports followed the ARRIVE guidelines. To induce post-traumatic osteoarthritis (OA), medial meniscus destabilization (DMM) surgery was performed on the right knee of 12-week-old male mice, and a control group underwent sham surgery under the same conditions.

[0248] To evaluate the therapeutic effect of Example 1 on osteoarthritis, Example 1 and SM04690 were administered intra-articularly to the knee joint at week 4 post-surgery, while dexamethasone was administered weekly at the same site starting week 2 post-surgery. Mice were sacrificed at week 6 post-administration for histological analysis. Furthermore, to evaluate the therapeutic effect on late-stage osteoarthritis, Example 1 was injected intra-articularly twice at 2-week intervals, starting week 4 post-surgery. Mice were sacrificed at week 4 after the last injection for histological analysis. Static weight-bearing analysis was performed the day before sacrifice.

[0249] 2) Histological analysis, immunohistochemistry, and in situ hybridization For histological analysis, the legs of mice were excised after euthanasia and fixed in 4% paraformaldehyde solution at 4 °C. The fixed leg tissue was then decalcified in 0.5 M EDTA (pH 7.4) solution at 4 °C for 4 weeks, followed by gradient dehydration with ethanol, xylene treatment, and paraffin embedding. The prepared paraffin blocks were cut into 5 μm thick sections.

[0250] After dewaxing with xylene, the sections were rehydrated with ethanol at decreasing concentrations. Safranin O staining was then performed to observe changes in cartilage tissue, and the degree of damage to the medial tibial cartilage was assessed using a double-blind method according to the OARSI scoring scale (0-6).

[0251] Immunohistochemistry staining was performed using SLC35D1 (ab113717, Abcam), IL6 (sc-130326, Stata Cruz Biotechnology), and CTXII (NBP2-59386, Novus Biologicals) antibodies diluted 1:100; and CHSY1 (NBP3-47604, Novus Biologicals), HPLN1 (ab98038, Abcam), and HAS2 (NBP3-03787, Novus Biologicals) antibodies diluted 1:50.

[0252] In situ hybridization was performed using probes for detecting miR-204 and the probe from Example 1 (synthesized by BIONEER). The probe for detecting U6 snRNA was purchased from Exiqon. Tissues were treated with 5 μg / mL proteinase K at 37 °C for 30 min, followed by fixation with 4% paraformaldehyde solution at room temperature for 20 min. Hybridization reactions were performed under the following conditions: U6 snRNA probe (1 nM) reacted at 50 °C for 1 h, the Example 1 probe (50 nM) reacted at 42 °C for 2 h, and the miR-204 probe (50 nM) reacted at 36 °C for 2 h. Subsequently, after reacting with anti-DIG-AP antibody (1:800 dilution, Roche, 11093274910) at room temperature for 1 hour, color development was performed using NBT / BCIP solution (Thermo Fisher, 34070).

[0253] All histological, immunohistochemical, and in situ hybridization images were acquired using a Nikon Eclipse Ni-U optical microscope connected to a DS-Ri2 camera (Nikon).

[0254] 3) Micro-computed tomography (μCT) analysis To evaluate the therapeutic effect of Example 1 and its potential side effects on bone remodeling, the knee joints of mice were removed and fixed as 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, 13-minute imaging time), and the resulting images were reconstructed using NRecon software (Bruker, version 1.7.3.2). Three-dimensional images were generated using CTvox (Bruker, version 3.3.0).

[0255] (2) Interpretation of experimental results The therapeutic effect of Example 1 was evaluated in a surgically induced osteoarthritis mouse model. In the DMM-treated group (administered as a Vehicle, control group), severe typical pathological changes characteristic of osteoarthritis were observed, including cartilage destruction, subchondral bone sclerosis, osteophyte development, and synovial inflammation. In contrast, in the group treated with Example 1, DMM-induced cartilage damage was statistically significantly inhibited, and the maturation of subchondral bone sclerosis and osteophytes, as well as the severity of synovial inflammation, were significantly alleviated. Figures 18a to 18c In situ hybridization analysis confirmed that, 6 weeks after administration, the material from Example 1 persisted in the articular cartilage at doses of 10 μg or higher. Figure 18d This indicates that Example 1 can remain stably in the cartilage for a long period, exerting a sustained therapeutic effect. In behavioral analysis, intra-articular administration of Example 1 also statistically significantly restored the weight-bearing imbalance induced by DMM surgery, meaning that this drug can substantially alleviate osteoarthritis-associated pain behavior. Figure 18e Molecular-level analysis showed that in the Example 1 treatment group, the expression of enzymes involved in chondroitin sulfate synthesis was restored, while the expression of proteins related to cartilage matrix destruction was significantly decreased. Figure 18f This indicates that it helps in the structural repair and functional recovery of cartilage tissue.

[0256] The effects of Example 1 remained consistent in two repeated administration experiments. Even with two ASO injections, cartilage damage and subchondral bone sclerosis were significantly reduced, and the pain relief effect persisted. In particular, under the same conditions, Example 1 demonstrated superior overall efficacy compared to the steric blocker (Comparative Example 4, Serial No. 22), showing stable and reproducible effects even after repeated administration. Figures 19a to 19d ).

[0257] To verify the therapeutic effect of Example 1, we conducted a comparative experiment with drugs currently in clinical use or under development. SM04690, a drug for treating osteoarthritis that has completed Phase III clinical trials, and dexamethasone, a steroidal anti-inflammatory analgesic, were used as control groups. The results showed that while SM04690 demonstrated some pathological improvement, its cartilage-protective effect was relatively limited compared to Example 1. Dexamethasone also showed confirmed anti-inflammatory and analgesic effects, but its cartilage regeneration capacity was low. In contrast, the group treated with Example 1 showed the most significant reduction in cartilage damage, and the best improvement in subchondral bone sclerosis and pain relief. Figures 20a to 20e These results indicate that, unlike traditional drugs that focus on inflammation control, Example 1 improves the pathophysiological root of the disease by inducing the protection and restoration of the cartilage structure itself.

[0258] Furthermore, to assess its therapeutic potential in the late stage of osteoarthritis, the drug was administered 6 weeks after OA induction. The results showed that statistically significant cartilage recovery was observed only in the Example 1 administration group, while no significant effect was confirmed in the steric blocker (Comparative Example 4, Serial No. 22) and SM04690 administration groups. Figures 21a to 21c In contrast, in terms of pain improvement, all three groups—Example 1, the steric blocker (Comparative Example 4, Serial No. 22), and SM04690—showed statistically significant levels. Figure 21d Micro-CT analysis also showed that Example 1 was the most effective in alleviating subchondral bone sclerosis, which provides a mechanistic basis for restoring the stability of subchondral bone structure.

[0259] In summary, these results demonstrate that Example 1 is a superior candidate for treatment that, compared to other drugs, can inhibit the pathological progression of osteoarthritis at multiple levels. This substance exhibits superior therapeutic effects at the structural, functional, and behavioral levels through the following combined mechanisms of action: (1) inhibiting cartilage destruction and promoting regeneration; (2) reducing subchondral bone sclerosis and osteophyte formation; (3) relieving synovitis and inhibiting the inflammatory response; (4) improving pain and restoring function; and (5) maintaining long-term efficacy through sustained intracartilage residence. Of particular note is that Example 1 demonstrates superior cartilage protection and pain relief compared to existing clinical-stage drugs or steroids, and maintains reproducible efficacy in both single and repeated administrations. These results strongly suggest that Example 1 is not merely a symptom reliever, but a mechanism-based ASO drug candidate capable of improving the underlying pathology of osteoarthritis.

[0260] [Experimental Example 11] Verify the off-target effect of Example 1 In Experiment 1, the degradation effect of miR-204 in Example 1 was confirmed. Furthermore, to evaluate off-target effects, the ASO of Example 1 underwent the following experiment.

[0261] (1) Experimental methods 1) Analysis of the predicted target in Example 1 Sequence variants containing the base sequence of Example 1 (n), a variant of Example 1 with a 3' end missing a base (n-1), and a variant with a 3' end repeat added a base (n+1) were analyzed against their expected target genes (human mRNA, microRNA, and pre-mRNA). Sequence-based homology analysis was performed using NCBI-BLAST (Basic Local Alignment Search Tool), utilizing the human gene databases (humanRefSeq_RNA and humanRefSeq_Gene) provided by NCBI. When assessing sequence matching, a mismatch of two or fewer bases was defined as a significant match.

[0262] 2) Analysis of Gene Ontology (GO) entries In the C28 / I2 cell line overexpressing miR-204, after treatment with 1 μM of the drug from Example 1, a list of differentially expressed genes (DEG list) was derived from the obtained transcriptome data. Using the derived DEG list, Gene Ontology (GO) term analysis was performed on the EnrichR platform. The analysis results were used to select the top 10 annotations based on adjusted p-values ​​and visualized as a bubble chart.

[0263] (2) Interpretation of experimental results The expected target genes (human mRNA, microRNA, and pre-mRNA) that might bind to the base sequences based on Example 1 were analyzed. Homology assessment using NCBI-BLAST showed that, apart from the host gene TRPM3 of miR-204 and the target miR-204, no other expected binding sequences were found (see Table 14). Table 14 shows the results of sequence homology analysis using NCBI-BLAST for the base sequences and sequence variants of Example 1.

[0264] Table 14

[0265] *TRPM3 is the host gene of miR-204. Furthermore, in the C28 / I2 cell line overexpressing miR-204, after treatment in Example 1, gene ontology (GO) analysis was performed using the obtained transcriptome data. The analysis was performed using EnrichR, and the top 10 annotations were selected based on adjusted p-values. The results showed that no significant ontology entries were found in any category, including biological process, molecular function, and cellular component (see [link to example]). Figures 22a to 22c These results indicate that Example 1 has high sequence specificity for miR-204 and is a highly specific ASO with extremely low off-target effects.

[0266] [Experimental Example 12] Verification of the immunogenicity of Example 1 In Experiment 1, the degradation effect of miR-204 in Example 1 was confirmed. Furthermore, to confirm the immunogenicity of Example 1, the following experiment was conducted.

[0267] (1) Experimental method—Detection of Toll-like receptor (TLR) activity To evaluate the effect of Example 1 on Toll-like receptor (TLR) activity, TLR activity was measured using HEK-Blue cell lines (InvivoGen) overexpressing TLR7, TLR8, and TLR9, respectively. HEK-Blue TLR7 cell lines were used at 5 × 10⁻⁶ cells / cells. 4 Cells / well were seeded at a density of 1 / 2 well in 96-well plates and cultured for 24 hours. Subsequently, they were treated with Example 1 (10 μM) or ORN6S (positive control) and 0.1 mM guanosine (Sigma, G6264). Six hours after treatment, the cell culture medium was collected, and the activity of secreted alkaline phosphatase (SEAP) in the culture supernatant was measured. HEK-Blue TLR8 cells were seeded at the same density and cultured for 24 hours, then treated with Example 1 (10 μM) or ORN6S and 1 mM uridine (Sigma, U3003). Six hours after treatment, the culture supernatant was collected, and SEAP activity was measured. HEK-Blue TLR9 cells 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 medium was collected, and SEAP activity was measured.

[0268] (2) Interpretation of experimental results To assess the immunogenicity of Example 1, changes in the activity of Toll-like receptors (TLRs) 7, 8, and 9 were measured. Analysis of ligand-dependent SEAP expression using HEK-Blue TLR7, TLR8, and TLR9 cell lines showed that both ORN6S (used for TLR7 and TLR8 detection) and ODN2006 (used for TLR9 detection), as positive controls, induced receptor activity, leading to a significant increase in SEAP activity.

[0269] In contrast, no significant increase in TLR7, TLR8, or TLR9 activity was observed under all conditions of the treatment in Example 1 (10 μM) (see Example 1). Figures 23a to 23cThese results indicate that Example 1 does not induce an immune response via TLR7, TLR8, or TLR9, implying a non-immunostimulatory property to innate immune receptor pathways. Therefore, Example 1 demonstrates to be a safe antisense oligonucleotide with low immunogenicity induction potential.

[0270] [Experimental Example 13] Toxicity verification of Example 1 In Experiment 1, the degradation effect of miR-204 in Example 1 was confirmed. Furthermore, considering the need to review the safety of the composition comprising Example 1 when using it as a pharmaceutical composition, the following experiment was conducted to verify the toxicity of Example 1.

[0271] (1) Experimental methods 1) Cytotoxicity assay To evaluate the cytotoxicity of Example 1, mouse primary chondrocytes, the HepG2 cell line, and the RPTEC cell line were used. The HepG2 and RPTEC cell lines were purchased from the American Type Culture Collection (ATCC).

[0272] Each cell line was seeded in a 96-well plate under the following conditions: Primary mouse chondrocytes: 1 × 10⁻⁶ 4 cells / well; HepG2 cells: 1 × 10³ cells / well; RPTEC cells: 4 × 10³ cells / well 4 Cells / wells. Each cell line was cultured for 72 hours (chondrocytes) and 24 hours (HepG2 and RPTEC cells) under the specified conditions, and then treated with 2 μM, 5 μM, and 10 μM concentrations of the solution from Example 1.

[0273] After 72 hours of treatment, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega).

[0274] 2) Single-dose toxicity studies in mice To evaluate the toxicological properties of Example 1, two single-dose toxicity studies were conducted. The first, a 2-week study, was performed using 9-week-old female CD-1 mice (Daehan Biolink Co., Eumsung, Korea). This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (Approval No.: SNU-230531-6-1). Animals were housed in the specific pathogen-free (SPF) animal laboratory at Seoul National University under environmental conditions of 23-25 ​​°C, 45-65% relative humidity, and a 12-hour light / dark cycle, with free access to standard feed. Mice were randomly assigned to three groups, each receiving a single intra-articular injection of 5 μL of nuclease-free water containing Example 1 (20 μg, 40 μg, and 80 μg, respectively). Body weight was measured on days 0, 4, 11, and 14 after administration. On day 14 post-administration, after a one-day fast, blood was collected via the retro-orbital venous plexus, and plasma was separated by centrifugation (1,500 × g, 15 min, 4 °C). Clinical chemistry analysis was performed using an external analytical institution (Cellpurics, Korea) using an Advia 1800 automated analyzer (Siemens). Animals were subsequently euthanized, and major organs (liver, spleen, kidney) were harvested, weighed, and visually inspected for abnormalities. After excision of the right leg tissue, it was fixed in 4% paraformaldehyde solution at 4 °C for two days and one night, and histological analysis of cartilage and bone tissue was performed using the aforementioned method to assess for abnormalities. Chondrocyte apoptosis was detected using the TUNEL assay (Takara, MK500) according to the manufacturer's instructions, and cell nuclei were stained with DAPI at room temperature for 10 minutes. Fluorescence signals were observed using the EVOS M7000 imaging system (Thermo Fisher, AMF7000).

[0275] (2) Interpretation of experimental results The cytotoxicity of Example 1 was confirmed in primary cultured cells derived from mouse cartilage, the HepG2 cell line, and the RPTEC cell line. The results showed that no significant level of cytotoxicity resulting in cell viability below 80% was observed in any of the three cell lines (see [link to example 1]). Figure 24a , 24bNext, to assess the local and systemic safety of Example 1, a 14-day intra-articular (IA) toxicity study was conducted in mice following a single-dose administration. Example 1 was administered at doses of 20, 40, and 80 μg / mouse, respectively. Results showed that in all administration groups, the organ-to-body weight ratios of the liver, kidney, and spleen were not significantly different from those in the control group (see [reference]). Figure 25a This indicates that the application of Example 1 did not cause systemic or organ-specific toxicity. Histological analysis of the application site showed no cartilage damage or proteoglycan loss in the knee joint tissue, and Safranin O staining also confirmed that the intracartilaginous matrix components remained normal. Figure 25b (See top and middle images). Furthermore, TUNEL staining analysis confirmed that no chondrocyte apoptosis was induced, demonstrating the excellent tolerability of Example 1 in local tissues. Figure 25b (See figure below). In summary, these results demonstrate that Example 1, when administered intra-articularly, exhibits excellent local safety and no systemic toxicity, making it a safe antisense oligonucleotide.

[0276] [Experimental Example 14] Validation of the effect of miR-204-specific ASO in canine chondrocytes In Experiment 1, the degradation effect of miR-204 in Example 1 was confirmed. Furthermore, to confirm whether the ASO in Example 1 could also effectively inhibit miR-204 in canine chondrocytes, the following experiment was conducted.

[0277] (1) Experimental methods 1) Canine chondrocyte cell line culture method Canine chondrocytes (Cell Applications, Inc.) were used at a rate of 2 × 10⁻⁶. 5Cells were seeded at a density of 12-well plates and cultured for 24 hours. Cells were cultured at 37 °C under 5% CO2 and atmospheric oxygen conditions using canine chondrocyte growth medium (Cell Applications, Inc., Cn411-500) supplemented with growth enhancer (Cell Applications, Inc., Cn411-GS). After 24 hours of culture, cells were used according to the specific requirements of each experiment.

[0278] 2) Confirm the delivery efficiency of Example 1 To visualize intracellular uptake in canine chondrocytes of Example 1, cultured cells were co-cultured with 1 μM Cy3-labeled cells of Example 1 for 24 hours. Subsequently, 1 μg / mL Hoechst was added to the culture medium, and co-incubation continued for 20 minutes. For fluorescence microscopy analysis, cells were washed three times with phosphate-buffered saline (PBS), and fluorescence images were acquired using an EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0279] Furthermore, the intracellular uptake of Example 1 was quantitatively assessed by flow cytometry. For this purpose, canine chondrocytes were co-cultured with Cy3-labeled Example 1 cells (10 nM or 50 nM) for 24 hours. After treatment, cells were separated from the attachment surface using trypsin, washed with PBS, and centrifuged. The recovered cells were resuspended twice in PBS containing 5% FBS.

[0280] Flow cytometry analysis was performed using a FACSAria™ III cell sorter (BD Biosciences), and data were acquired and quantified using FACSDiva 9.7 software.

[0281] 3) qRT-PCR After culturing canine chondrocytes for 24 hours, they were treated with 0.25 μM doxorubicin (Sigma-Aldrich) to induce cell senescence. Cells were maintained under hypoxic conditions at 37 °C, 5% CO2, and 3% O2. After 72 hours, they were treated again with the same concentration of doxorubicin by removing half of the original culture medium and adding an equal volume of fresh culture medium to the remaining half for dilution. Subsequently, at another 72 hours, they were treated with 1 μM doxorubicin (Example 1) while replacing the medium with fresh culture medium. Cells were recovered 48 hours after treatment.

[0282] Cells were recovered, 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 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.

[0283] 4) sGAG detection sGAG quantitative analysis was performed using primary cultured canine chondrocytes according to the described method. To induce cell senescence, cells were treated with 0.25 μM doxorubicin (Sigma-Aldrich). Cells were maintained under hypoxic conditions at 37 °C, 5% CO2, and 3% O2. After 72 hours, cells were treated again with the same concentration of doxorubicin by removing half of the original culture medium and diluting the remaining half with an equal volume of fresh culture medium. Subsequently, at 72 hours later, cells were treated with 0.1, 1, and 10 μM doxorubicin (Example 1) while replacing the medium with fresh medium. Fresh medium was replaced after 48 hours of treatment. Cells were then cultured for 2 days, and the culture supernatant was recovered for sGAG analysis. The sGAG concentration in the recovered culture medium was determined using 1,9-dimethylmethylene blue (DMMB) staining. Absorbance was measured at 525 nm, and sGAG content was quantified based on a standard curve plotted using chondroitin sulfate (C0335; Tokyo Chemical Industry). To correct for differences in cell viability and density, MTT assays were performed on the same cells. For this, 100 μg / mL of PBS-based MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, T-030-1; Goldbio) was added, and the reaction was allowed to proceed for 2 hours. After the reaction, the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm. All sGAG measurements were standardized according to the MTT results before comparative analysis. Statistical significance tests were performed using GraphPad Prism 10 software. One-way ANOVA was performed first, followed by Tukey's post-hoc test.

[0284] (2) Interpretation of experimental results To confirm the delivery efficiency of Example 1 to canine chondrocytes, intracellular uptake was assessed using Cy3 fluorescent labeling. Fluorescence microscopy analysis showed that, compared to the vehicle-treated group, a significant accumulation of fluorescent signal was observed intracellularly in the canine chondrocytes of the Example 1-treated group. Figure 26aFurthermore, flow cytometry analysis showed that under all conditions of treatment with 10 nM and 50 nM concentrations in Example 1, more than 50% of the fluorescence was positive, quantitatively confirming the excellent intracellular uptake capacity of Example 1. Figure 26b These results indicate that Example 1 possesses physicochemical properties that enable efficient delivery and stable accumulation within chondrocytes.

[0285] Subsequently, the reducing effect of Example 1 on miR-204 was confirmed in canine chondrocytes induced with cellular senescence. A statistically significant decrease in miR-204 expression was observed upon delivery of Example 1 after treatment with doxorubicin to induce cellular senescence. Figure 26c ).

[0286] Next, the effect of Example 1 on restoring the synthetic function of cartilage matrix components was evaluated. Treatment with Example 1 in senescent canine chondrocytes showed a significant increase in the synthesis of sulfated glycosaminoglycans (sGAGs), which is reduced due to senescence. Figure 26d These results suggest that Example 1, by degrading miR-204, not only restored ECM synthesis function in mouse chondrocytes but also in canine chondrocytes, supporting matrix restoration and anti-aging effects at the cellular level.

[0287] These results indicate that Example 1 effectively inhibits miR-204 not only in mouse chondrocytes but also in canine chondrocytes, suggesting that it has cross-species efficacy in mammals such as dogs and cats, and supporting the conservation of the species-independent mechanism by which this substance targets and inhibits miR-204.

Claims

1. An oligonucleotide capable of specifically hybridizing with miR-204, The oligonucleotide is composed of 20 to 22 nucleotides linked together, and consists of a first region, a second region, and a third region sequentially from the 5' end to the 3' end; The first region is complementary to the tail region of miR-204 and consists of three consecutive nucleotides containing immobilized sugar modifications. The second region is composed of three nucleotides linked together, and the nucleotides constituting the second region are each independently a nucleotide containing either a fixed sugar modification or a non-fixed 2'-substituted sugar modification; The third region is composed of multiple nucleotides linked together, and each nucleotide constituting the third region is independently a nucleotide modified with a non-fixed 2'-substituted sugar; and The oligonucleotides, based on the total number of nucleotides, contain less than 30% of nucleotides modified with fixed sugars.

2. The oligonucleotide according to claim 1, The nucleotide containing the fixed sugar modification is selected from at least one of the following groups: locked nucleic acid (LNA), 2',4'-ethylene-bridged nucleic acid (ENA), constrained ethyl (cEt) nucleotide, bridged nucleic acid (BNA), and acyclic amino-bridged BNA (BNANC).

3. The oligonucleotide according to claim 1, The non-fixed 2'-substituted sugar modification is a non-fixed 2'-substituted sugar modification selected from the group consisting of at least one of the following: 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).

4. The oligonucleotide according to claim 1, The nucleotides constituting the third region all contain 2'-deoxy sugars.

5. The oligonucleotide according to claim 1, The sugars in the first and second nucleotides from the 3' end of the oligonucleotide are 2'-deoxy.

6. The oligonucleotide according to claim 1, The oligonucleotide contains a region complementary to the seed region of miR-204, and the sugars of the nucleotides contained in the region complementary to the seed region are non-fixed sugar modifications.

7. The oligonucleotide according to claim 1, The oligonucleotides contain nucleotides linked by either a phosphorothioate bond or a phosphodiester bond.

8. The oligonucleotide according to claim 1, The oligonucleotide is one or more oligonucleotides shown in sequence numbers 1 to 14 and 26 to 39.

9. The oligonucleotide according to claim 1, The second or third region contains one or more nucleotides that are mismatched with miR-204.

10. The oligonucleotide according to claim 1, The oligonucleotide is an oligonucleotide used to reduce and / or degrade the miR-204.

11. Use of the oligonucleotide of claim 1 in the preparation of compositions for improving, preventing or treating osteoarthritis.

12. The use according to claim 11, wherein the osteoarthritis is at least one selected from the group consisting of: degenerative osteoarthritis, post-traumatic osteoarthritis, postoperative osteoarthritis, inflammatory osteoarthritis, and secondary osteoarthritis.

13. The use according to claim 11, wherein the composition is used to improve, prevent or treat osteoarthritis in mammals.

14. The use according to claim 11, wherein the composition is a pharmaceutical composition, a food composition, a health functional food composition, or an oral composition.

Images