Oligonucleotides for miR-204, and compositions containing the same for the improvement, prevention, or treatment of osteoarthritis.

Abstract

This specification discloses oligonucleotides that specifically hybridize with miR-204, and compositions comprising the same for the improvement, prevention, or treatment of osteoarthritis. [Solution] An oligonucleotide according to one aspect of the present invention is designed to have a structure optimized for miR-204 degradation and formulation stability, effectively inhibiting pathophysiological processes of osteoarthritis such as decreased cartilage matrix synthesis and increased inflammatory response, and is useful for the prevention or treatment of osteoarthritis. It exhibits excellent delivery to chondrocytes and stable retention characteristics within tissues, thus providing structural advantages that enable long-term sustained therapeutic effects, as well as safety suitable for use as a pharmaceutical composition due to its low potential for immunogenicity induction and toxicity. Furthermore, it can effectively inhibit miR-204 even in canine chondrocytes, making it suitable for use as an animal therapeutic agent and / or feed composition.

Description

[Technical Field]

[0001] This application claims priority based on Republic of Korea Patent Application No. 10-2024-0198889, filed on 27 December 2025, with its entire specification incorporated as a reference to this application. Furthermore, this application claims priority based on Republic of Korea Patent Application No. 10-2025-0198889, filed on 9 December 2025, with its entire specification incorporated as a reference to this application.

[0002] This specification discloses oligonucleotides that specifically hybridize with miR-204, and compositions comprising the same for the improvement, prevention, or treatment of osteoarthritis. Explanation of government-supported research projects [National research and development project that supported this invention] [Project-specific number] 2420003227 [Issue Number] RS-2024-00437503 [Ministry / Agency Name] Small and Medium Enterprises and Startups Department [Name of the specialized organization for issue management] Small and Medium Enterprise Technology Information Promotion Agency [Research Project Name] Startup Growth Technology Development [Graduate School Name] Development of next-generation osteoarthritis treatments based on microRNA derivatives [Project Implementation Organization Name] Lyplex Science Co., Ltd. [Research Period] July 1, 2024 - June 30, 2027 [Background technology]

[0003] Osteoarthritis (OA) is a disease in which the number of patients worldwide increased by 48% between 1990 and 2019, with 4.18 million people affected in South Korea alone as of 2022. Due to the increasing number of patients associated with aging and growing concern for quality of life, the demand for treatment of osteoarthritis is rapidly increasing. Cartilage is a tissue that is damaged and undergoes progressive degeneration due to various etiological risk factors such as aging, mechanical overload, obesity, and metabolic diseases, and osteoarthritis is known to develop as a result of cartilage degeneration. It is characterized by pathological changes such as cartilage degeneration, subchondral bone progression, osteophyte formation, and synovial inflammation.

[0004] Conventional research on the treatment of osteoarthritis has been limited to slowing cartilage degeneration by inhibiting the "degradation" mechanism of the extracellular matrix of chondrocytes. Since there are no therapeutic agents that fundamentally improve the disease, treatment strategies for osteoarthritis rely on prescribing anti-inflammatory drugs or performing artificial joint replacement surgery.

[0005] miR-204 is a type of microRNA, specifically miR-204-5p. miR-204 is significantly increased in expression in various types of osteoarthritis cartilage, simultaneously inhibiting multiple sGAG synthases that constitute the cartilage matrix, and contributing to the formation of SASP, thus exacerbating osteoarthritis. Because the therapeutic effect of miR-204 inhibition on osteoarthritis is known, there is now a need for the development of therapeutic agents that effectively "degrade" miR-204 with minimal side effects. However, despite the importance of miR-204-mediated sGAG synthesis reduction and SASP in the pathogenesis of osteoarthritis, no effective therapeutic agent has yet been developed that specifically binds to miR-204 to restore cartilage matrix synthesis or inhibits inflammation-related factors. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The inventors identified miR-204 as a biomarker for improving, preventing, or treating osteoarthritis, and conducted research on nucleic acid therapeutic agents targeting it. As a result, they confirmed that oligonucleotides that specifically hybridize with miR-204, and in which nucleotides at specific positions among the nucleotides constituting the oligonucleotide include fixed-type glycosylation or unfixed-type 2'-substituted glycosylation, degrade miR-204, thereby exhibiting excellent effects in improving, preventing, or treating osteoarthritis.

[0007] Therefore, in one embodiment, the present invention aims to provide an oligonucleotide that specifically hybridizes with miR-204 and exhibits excellent degradation effects on miR-204.

[0008] In another embodiment, the present invention aims to provide a composition for improving, preventing, or treating osteoarthritis, comprising the oligonucleotide. [Means for solving the problem]

[0009] In one embodiment, the present invention provides an oligonucleotide that specifically hybridizes with miR-204, wherein the oligonucleotide is composed of 20 to 22 linked nucleotides, and is composed of a first region, a second region, and a third region in order from the 5' end to the 3' end, the first region is entirely complementary to the tail region of miR-204 and consists of three linked nucleotides containing fixed glycosylation, the second region consists of three linked nucleotides, each of which independently contains either fixed glycosylation or non-fixed 2'-substituted glycosylation, the third region consists of multiple linked nucleotides, each of which independently contains non-fixed 2'-substituted glycosylation, and the oligonucleotide contains 30% or less of nucleotides containing fixed glycosylation relative to the total number of nucleotides.

[0010] In another aspect, the present invention provides a composition for improving, preventing or treating osteoarthritis, which contains the oligonucleotide.

Advantages of the Invention

[0011] The oligonucleotide according to one aspect of the present invention is an antisense oligonucleotide that induces the degradation of miR-204 and thereby reduces its expression. By comparing and verifying various chemical modifications such as sugar modification, length adjustment, selection of the number and position of fixed sugar modifications, etc., it is designed to have a structure in which the miR-204 inhibitory ability and formulation stability are optimized.

[0012] Thereby, the antisense oligonucleotide of the present invention effectively inhibits the pathophysiological processes of osteoarthritis such as reduced synthesis of cartilage matrix and increased inflammatory response, and is useful for pain relief, prevention or treatment of osteoarthritis.

[0013] In addition, the oligonucleotide according to one aspect of the present invention exhibits excellent delivery ability to chondrocytes and stable retention characteristics in tissues, thus providing a structural advantage capable of realizing a long-term continuous therapeutic effect.

[0014] The oligonucleotide according to one aspect of the present invention has low potential for inducing immunogenicity and toxicity, and provides safety suitable for use as a pharmaceutical composition.

[0015] The oligonucleotide according to one aspect of the present invention has been confirmed to effectively inhibit miR-204 not only in human chondrocytes but also in chondrocytes derived from dogs. Furthermore, since the base sequence of miR-204 is conserved in multiple species such as humans, dogs, monkeys, etc., the oligonucleotide can be used as a therapeutic agent and / or a composition for feed addition in various animal species.

[0016] Since the target sequence miR-204 is conserved across species, the oligonucleotide according to one aspect of the present invention can be used in human chondrocytes or in chondrocytes of other species (e.g., dogs, monkeys, etc.) as well as in humans. [Brief explanation of the drawing]

[0017] [Figure 1] This is an experimental example of the present invention, and the figure shows the results of measuring the miR-204 degradation effect in the C28 / I2 chondrocyte line by qRT-PCR following the treatment in Example 1, according to Experimental Example 1. [Figure 2A] This figure shows the results of confirming the delivery efficiency of Example 1 in mouse-derived chondrocytes according to one experimental example of the present invention, and is a fluorescence micrograph of fluorescently labeled Example 1 accumulated in mouse-derived chondrocytes. [Figure 2B] This figure shows the results of confirming the delivery efficiency of Example 1 in mouse-derived chondrocytes according to an experimental example of the present invention, and quantifies the percentage of Cy3-fluorescently labeled Example 1-positive cells as confirmed by flow cytometry. [Figure 2C] This figure shows the results of confirming the delivery efficiency of Example 1 in mouse-derived chondrocytes according to an experimental example of the present invention, and quantifies the percentage of Cy3-fluorescently labeled Example 1-positive cells as confirmed by flow cytometry. [Figure 3] This is a fluorescence micrograph showing the delivery efficiency of Example 1 in cartilage tissue derived from a patient with osteoarthritis, using fluorescently labeled Example 1 according to an experimental example of the present invention. [Figure 4] This graph shows the results of measuring the expression level of miR-204 by qRT-PCR when ASOs (Examples 3-1 to 3-3) containing full DNA or intra-sequence mismatches were treated according to Experimental Example 4, which is an experimental example of the present invention. [Figure 5]This graph shows the results of measuring the expression level of miR-204 by qRT-PCR when ASOs (Examples 4-1 and 4-2) with one or two bases deleted from the 5' end, respectively, based on Full DNA or microRNA, were treated according to Experimental Example 5, which is an experimental example of the present invention. [Figure 6] This graph shows experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm whether there is a difference in miR-204 resolution depending on whether each of the nucleotides contained in the first and second regions of the miR-204-specific ASO according to one embodiment of the present invention contains fixed glycosylation (Full DNA, Examples 5-1 to 5-7, and Comparative Examples 1-1 and 1-2). [Figure 7] This graph shows experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm whether there is a difference in miR-204 resolution depending on whether each of the nucleotides contained in the first and second regions of the miR-204-specific ASO according to one embodiment of the present invention contains fixed glycosylation (Full DNA, Examples 5-1 to 5-7, and Comparative Examples 1-1 and 1-2). [Figure 8] As a comparative example of the present invention, this graph shows the experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution when the miR-204-specific ASO contains one or more LNAs at positions 1 to 6 from the 3' end to the 5' end (Example 1, Comparative Examples 2-1 to 2-4). [Figure 9] This graph shows experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution in comparative examples of the present invention, where the miR-204-specific ASO randomly contains LNA or is shorter in length than the ASO of one example of the present invention (Comparative Examples 3 and 4), compared with Example 1, which is full DNA or an ASO according to one embodiment of the present invention. [Figure 10]This graph shows experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution in comparative examples of the present invention, where the miR-204-specific ASO randomly contains LNA or is shorter in length than the ASO of one example of the present invention (Comparative Examples 3 and 4), compared with Example 1, which is full DNA or an ASO according to one embodiment of the present invention. [Figure 11A] As a comparative example of the present invention, this graph shows the experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution when the miR-204-specific ASO contains 15 or more LNAs (Comparative Examples 5-1 to 5-3). [Figure 11B] As a comparative example of the present invention, this is a photograph taken to confirm the stability of the formulation when the miR-204-specific ASO containing 15 or more LNAs (Comparative Examples 5-1 to 5-3) was dissolved in order to confirm the miR-204 resolution. [Figure 12] This graph shows experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution when the miR-204-specific ASO according to one embodiment of the present invention contains at least one nucleotide containing MOE or OME instead of LNA in the second region (Examples 6-1 to 6-14). [Figure 13] This graph shows experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution when the miR-204-specific ASO according to one embodiment of the present invention contains at least one nucleotide containing MOE or OME instead of LNA in the second region (Examples 6-1 to 6-14). [Figure 14] This graph shows the experimental results of measuring the expression level of miR-204 by qRT-PCR to confirm the miR-204 resolution when the miR-204-specific ASO according to one embodiment of the present invention is composed of PO-binding (backbone) instead of PS-binding (backbone) (Example 2). [Figure 15]This graph shows pathway enrichment results in the form of a bubble plot, based on the analysis results of transcripts between the treatment group of Example 1 according to one embodiment of the present invention and the vehicle control group. In Figure 15, the size of the points indicates the number of genes included in the corresponding pathway, and the hue indicates the Fold enrichment value. [Figure 16A] This figure shows, as one embodiment of the present invention, changes in the expression of genes and proteins related to ECM composition or inflammation induction in mouse-derived chondrocytes following treatment in Example 1. The graph shows the results of measuring the mRNA expression of sGAG biosynthesis pathway-related enzymes in chondrocytes damaged by bleomycin using qRT-PCR. [Figure 16B] This figure shows changes in the expression of genes and proteins related to ECM composition or inflammation induction in mouse-derived chondrocytes following treatment in Example 1, as one embodiment of the present invention. The figure is a band image showing the results of analyzing protein expression related to sGAG synthesis using Western blot. [Figure 16C] This figure shows the changes in the expression of genes and proteins related to ECM composition or inflammation induction in mouse-derived chondrocytes as one embodiment of the present invention, following treatment with Example 1. The graph shows the results of measuring changes in the expression of ECM biosynthesis enzymes and inflammation-related genes by qRT-PCR after treatment with Example 1 and SM04690 under inflammatory cytokine IL-1β treatment conditions. [Figure 17A] As an embodiment of the present invention, this figure shows the change in sGAG content in chondrocytes following treatment in Example 1, and is a graph showing the change in sGAG content in chondrocytes damaged by bleomycin according to the treatment concentration in Example 1. [Figure 17B] As an embodiment of the present invention, the figure shows the change in sGAG content in chondrocytes after treatment in Example 1. This graph is a replot of the results of Example 1 in Figure 17A as a logarithmic concentration-response curve, and is a graph showing the calculated maximum effect (Emax) and maximum half-effect concentration (EC50) values. [Figure 17C]As an embodiment of the present invention, this figure shows the change in sGAG content in chondrocytes after treatment in Example 1, and is a graph showing the change in sGAG content after treatment in Example 1 and SM04690 under inflammatory cytokine IL-1β treatment conditions. [Figure 18A] In a mouse model of DMM surgery-induced osteoarthritis, this figure shows the therapeutic effect of a single intra-articular administration of Example 1 as one embodiment of the present invention, and displays histological images stained with safranin O after administration to Example 1 and the vehicle control group. [Figure 18B] In a mouse model of DMM surgery-induced osteoarthritis, this figure shows the therapeutic effect of a single intra-articular administration of Example 1 as one embodiment of the present invention. The graph quantifies the intensity of cartilage damage and numerically represents the pathological progression stage based on the image in Figure 18A. [Figure 18C] In a mouse model of DMM surgery-induced osteoarthritis, this figure shows the therapeutic effect of a single intra-articular administration of Example 1 as one embodiment of the present invention, and is an image showing subtle changes in the cartilage tissue structure as measured by microclaving (μCT). [Figure 18D] In a mouse model of DMM surgery-induced osteoarthritis, this figure shows the therapeutic effect of a single intra-articular administration of Example 1 as one embodiment of the present invention, and is an image confirming the distribution of Example 1 within the articular cartilage by in situ hybridization. [Figure 18E] In a mouse model of DMM surgery-induced osteoarthritis, this figure shows the therapeutic effect of a single intra-articular administration of Example 1 as one embodiment of the present invention, and is a graph showing the mitigating effect of Example 1 on weight-bearing imbalance in a DMM surgery-induced mouse osteoarthritis model. [Figure 18F] In a mouse model of DMM surgery-induced osteoarthritis, this figure shows the therapeutic effect of a single intra-articular administration of Example 1 as one embodiment of the present invention, and is an image showing the results of immunohistochemistry analysis indicating changes in the expression of proteins related to cartilage matrix synthesis and degradation. [Figure 19A] In a DMM surgery-induced osteoarthritis model, this figure shows the therapeutic effect of repeating intra-articular administration of Example 1 twice as one embodiment of the present invention. The figure includes images of histological analysis of cartilage from the treatment group and vehicle control group of Example 1, stained with safranin O, and a graph quantifying the pathological progression stage. [Figure 19B] In a DMM surgery-induced osteoarthritis model, this figure shows the therapeutic effect of repeating intra-articular administration of Example 1 twice as one embodiment of the present invention. The figure includes images of histological analysis of cartilage from the treatment group and vehicle control group of Example 1, stained with safranin O, and a graph quantifying the pathological progression stage. [Figure 19C] In a DMM surgery-induced osteoarthritis model, this figure shows the therapeutic effect of repeating intra-articular administration of Example 1 twice as one embodiment of the present invention, and is a graph showing the mitigating effect of Example 1 on weight-bearing imbalance. [Figure 19D] In a DMM surgery-induced osteoarthritis model, this figure shows the therapeutic effect of repeating intra-articular administration of Example 1 twice as one embodiment of the present invention, and is an image showing subtle changes in the cartilage tissue structure as measured by microclaving (μCT). [Figure 20A] This is a schematic diagram showing experimental results for comparing the therapeutic effects of Example 1, SM04690, and dexamethasone in a DMM surgery-induced mouse osteoarthritis model, as one embodiment of the present invention, and illustrating the treatment period and treatment conditions for each drug. [Figure 20B] In a DMM surgery-induced mouse osteoarthritis model, as one embodiment of the present invention, this is an experimental results diagram comparing the therapeutic effects of Example 1, SM04690, and dexamethasone, showing safranin O stained histological images and a graph quantifying the pathological progression stage for each drug treatment group. [Figure 20C] In a DMM surgery-induced mouse osteoarthritis model, as one embodiment of the present invention, this is an experimental results diagram comparing the therapeutic effects of Example 1, SM04690, and dexamethasone, showing safranin O stained histological images and a graph quantifying the pathological progression stage for each drug treatment group. [Figure 20D] In a DMM surgery-induced mouse osteoarthritis model, this is an experimental result diagram comparing the therapeutic effects of Example 1, SM04690, and dexamethasone as one embodiment of the present invention, and is a graph showing the results of an analysis of the imbalance in body weight load in each drug treatment group based on behavioral experiments. [Figure 20E] In a DMM surgery-induced mouse osteoarthritis model, this is an experimental result diagram comparing the therapeutic effects of Example 1, SM04690, and dexamethasone as one embodiment of the present invention, and is an image showing subtle changes in cartilage tissue structure by microccopy (μCT). [Figure 21A] This figure shows the results of evaluating the therapeutic effect of Example 1 as one embodiment of the present invention in the late stage of osteoarthritis, and is a graph that quantifies the safranin O stained histological images and pathological progression stages of each drug treatment group. [Figure 21B] This figure shows the results of evaluating the therapeutic effect of Example 1 as one embodiment of the present invention in the late stage of osteoarthritis, and is a graph that quantifies the safranin O stained histological images and pathological progression stages of each drug treatment group. [Figure 21C] This figure shows the results of evaluating the therapeutic effect of Example 1 as one embodiment of the present invention in the late stage of osteoarthritis, and is an image showing the subtle changes in the cartilage tissue structure in each drug treatment group by micro-tomography (μCT). [Figure 21D] This figure shows the results of evaluating the therapeutic effect of Example 1 as one embodiment of the present invention in the late stage of osteoarthritis, and is a graph showing the results of the analysis of the imbalance in body weight load in each drug treatment group based on behavioral experiments. [Figure 22A] As an embodiment of the present invention, this is an analysis result diagram for evaluating the off-target effect of Example 1. The diagram is a bubble plot graph showing the Gene Ontology (GO) term analysis results using transcript data obtained after treating a C28 / I2 cell line overexpressing miR-204 with Example 1. The size of the points indicates the number of genes included in the corresponding item (annotation), and the hue indicates fold enrichment. [Figure 22B] As an embodiment of the present invention, this is an analytical result diagram for evaluating the non-target effect of Example 1. It is a bubble plot graph showing the Gene Ontology (GO) term analysis results using transcript data obtained after treating a C28 / I2 cell line overexpressing miR-204 with Example 1. The size of the points indicates the number of genes included in the corresponding item (annotation), and the hue indicates fold enrichment. [Figure 22C] As an embodiment of the present invention, this is an analytical result diagram for evaluating the non-target effect of Example 1. It is a bubble plot graph showing the Gene Ontology (GO) term analysis results using transcript data obtained after treating a C28 / I2 cell line overexpressing miR-204 with Example 1. The size of the points indicates the number of genes included in the corresponding item (annotation), and the hue indicates fold enrichment. [Figure 23A] This graph shows the results of the immunogenicity evaluation of Example 1, performed by analyzing Toll-like receptor (TLR) activity using HEK-Blue TLR7, TLR8, and TLR9 cell lines. [Figure 23B] This graph shows the results of the immunogenicity evaluation of Example 1, performed by analyzing Toll-like receptor (TLR) activity using HEK-Blue TLR7, TLR8, and TLR9 cell lines. [Figure 23C] This graph shows the results of the immunogenicity evaluation of Example 1, performed by analyzing Toll-like receptor (TLR) activity using HEK-Blue TLR7, TLR8, and TLR9 cell lines. [Figure 24A] As one embodiment of the present invention, this is a graph showing the evaluation results of cytotoxicity in Example 1, which shows the cell viability in mouse primary cultured chondrocytes when Example 1 was treated at various concentrations. [Figure 24B] As one embodiment of the present invention, this is a graph showing the evaluation results of cytotoxicity in Example 1, which shows the cell viability in hepatocyte lines when Example 1 was treated at various concentrations. [Figure 24C] As one embodiment of the present invention, this is a graph showing the evaluation results of the cytotoxicity of Example 1, which shows the cell viability in a kidney cell line when Example 1 was treated at various concentrations. [Figure 25A] As one embodiment of the present invention, this graph shows the results of toxicity evaluation after a single intra-articular administration of Example 1 to mice, and illustrates the ratio of organ weight to body weight of major organs (liver, kidney, spleen) measured 14 days after administration. [Figure 25B] As one embodiment of the present invention, the image shows the results of toxicity evaluation after a single intra-articular administration of Example 1 to mice. The upper and middle panels show images of safranin O staining results of knee joint tissue, and the lower panel shows images of TUNEL staining analysis results of the same tissue. [Figure 26A] This figure shows the results of confirming the intracellular delivery efficiency and functional effect of Example 1 as one embodiment of the present invention in canine chondrocytes, and is an image of canine chondrocytes treated with Cy3 fluorescently labeled Example 1 observed with a fluorescence microscope. [Figure 26B] This figure shows the results of confirming the intracellular delivery efficiency and functional effect of Example 1 as one embodiment of the present invention in canine chondrocytes. The graph shows the results of quantifying the percentage of fluorescently positive cells by flow cytometry after processing with Cy3-fluorescently labeled Example 1. [Figure 26C] This figure shows the results of confirming the intracellular delivery efficiency and functional effect of Example 1 as one embodiment of the present invention in canine chondrocytes. The graph shows the results of measuring the miR-204 expression level by qRT-PCR when Example 1 was treated under cellular senescence conditions. [Figure 26D] This figure shows the results of confirming the intracellular delivery efficiency and functional effects of Example 1 in canine chondrocytes, as one embodiment of the present invention, and is a graph showing the results of quantitative analysis of sGAG content under cellular senescence conditions. [Modes for carrying out the invention]

[0018] The present invention will be described in detail below.

[0019] In one aspect of the present invention, "microRNA-204," "miR-204," or "microRNA-204" is a general term encompassing any variant, isoform, and species homolog of miR-204 that is 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.

[0020] In one embodiment of the present invention, the "tail region of miR-204" may refer to the nucleic acid region from the 5' end of microRNA-204 (miR-204) starting from the 17th position.

[0021] In one embodiment of the present invention, the "supplementary region of miR-204" may be the nucleic acid region from the 5' end to the 13th to 16th positions of microRNA-204 (miR-204).

[0022] In one embodiment of the present invention, the "central region of miR-204" may refer to the nucleic acid region from the 9th to 12th position from the 5' end of microRNA-204 (miR-204).

[0023] In one embodiment of the present invention, the "seed region of miR-204" may be the nucleic acid region from the 2nd to the 8th position from the 5' end of microRNA-204 (miR-204).

[0024] In one embodiment of the present invention, "oligonucleotide" means a target nucleic acid, specifically a short nucleic acid polymer that binds complementaryally to a target and microRNA to exhibit function, wherein the polymer may include native or modified nucleotides, such as sugar modifications and phosphorothioate (PS) bonds, including 2'-O-methoxyethyl (MOE), 2'-O-methyl (OME), 2'-fluoro (2'-F), LNA, cEt, etc. In one embodiment, "oligonucleotide" may also be an antisense oligonucleotide.

[0025] In one embodiment of the present invention, "nucleotide containing fixed sugar modification" may mean that the sugar contained in the nucleotide has undergone a modification that fixes the conformation of the sugar (conformationally constrained). In one embodiment, the nucleotide containing fixed sugar modification may be a nucleotide containing a bicyclic sugar (for example, LNA, ENA, cEt, BNA, BNANC / BNANC, etc.).

[0026] In one embodiment of the present invention, "nucleotides containing non-constrained 2'-substituted sugar modifications" may mean that the sugars contained in the nucleotides are modified based on 2' positional substitution so that the stereochemistry of the sugars is not fixed (non-constrained, non-fixed). In one embodiment, the non-constrained 2'-substituted sugar modifications may include sugar modifications that are not bicyclic sugars (for example, 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 (ribose), arabinose (ANA), etc.).

[0027] In one embodiment of the present invention, "osteoarthritis" is a group of diseases encompassing degenerative damage or dysfunction of various joint tissues such as articular cartilage, subchondral bone, ligaments, and synovial membrane, and may include primary or secondary osteoarthritis, and osteoarthritis of any part of the body such as the knee, hip, hand, and spine.

[0028] Oligonucleotides that specifically hybridize with miR-204 In one embodiment, the present invention provides an oligonucleotide that specifically hybridizes 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 in order from the 5' end to the 3' end, the first region is entirely complementary to the tail region of miR-204 and consists of three nucleotides linked together, each of which independently contains either fixed or unfixed 2'-substituted nucleotides, the third region is composed of multiple nucleotides linked together, each of which independently contains unfixed 2'-substituted nucleotides, and the oligonucleotide contains 30% or less of nucleotides with fixed nucleotide modifications relative to the total number of nucleotides.

[0029] An oligonucleotide according to one aspect of the present invention may specifically hybridize with miR-204.

[0030] An oligonucleotide according to one aspect of the present invention may have 20 to 22 nucleotides linked together. Specifically, the oligonucleotide may have 20, 21, or 22 nucleotides linked together.

[0031] An oligonucleotide according to one aspect of the present invention may consist of a first region, a second region, and a third region, in order from the 5' end to the 3' end. Specifically, the first region may be a region containing the 1st to 3rd nucleotides in order from the 5' end to the 3' end of the oligonucleotide. The second region may be a region containing the 4th to 6th nucleotides in order from the 5' end to the 3' end of the oligonucleotide. The third region may be a region containing the 7th to the last (e.g., the 20th, 21st, or 22nd) nucleotide in order from the 5' end to the 3' end of the oligonucleotide. More specifically, the first region may consist of the 1st to 3rd nucleotides in order from the 5' end to the 3' end of the oligonucleotide. The second region may consist of the 4th to 6th nucleotides in order from the 5' end to the 3' end of the oligonucleotide. Furthermore, the third region may consist of the 7th to the last (for example, the 20th, 21st, or 22nd) nucleotides, sequentially from the 5' end to the 3' end of the oligonucleotide.

[0032] Oligonucleotides according to one aspect of the present invention may be designed to improve binding affinity, in vivo stability, and functional inhibitory effect by selectively incorporating LNA (lock nucleic acid), 5-methyl-deoxycytosine (iMe-dC), phosphorothioate (PS) bonds, phosphodiester (PO) bonds, and the like.

[0033] LNA bases, possessing a fixed bicyclic structure, can contribute to improving the ligand binding strength and structural rigidity of oligonucleotides. Furthermore, 5-methyldeoxycytosine (iMe-dC) is a modified base with improved resistance to oxidation and deamination compared to wild-type cytosine. In fact, it is the most standardized synthetic base modification for stably and reproducibly mimicking biological phenomena, and can contribute to improving the overall structural stability and functional persistence of ASOs. Additionally, phosphorothioate (PS) binding can increase nuclease resistance and contribute to extending the half-life in the body by increasing protein binding affinity.

[0034] On the other hand, the nucleotides contained in or constituting an oligonucleotide according to one aspect of the present invention may be linked by phosphorothioate (PS) bonds and / or phosphodiester (PO) bonds.

[0035] According to Experimental Example 1, in the ASO-treated group of Example 1, which is one embodiment of the present invention, miR-204 expression decreased in a concentration-dependent manner. Quantitative analysis revealed that the maximum resolution was approximately 99.6%, and DC degraded miR-204 expression by 50%. 50 The resolution was calculated to be approximately 0.0055 μM, demonstrating high resolution (Figure 1). Furthermore, excellent miR-204 resolution was also confirmed in the ASO treatment group of Example 2, which is one embodiment of the present invention. Therefore, it was confirmed that oligonucleotides according to one aspect of the present invention can effectively degrade the target miR-204 regardless of the method of bonding between nucleotides in the oligonucleotide or the components of the backbone (Figure 14).

[0036] microRNAs are generally composed of 22-24 mers, with the 2nd to 8th nucleic acids from the 5' end being the seed region, the 9th to 12th nucleic acids being the central region, the next 13th to 16th nucleic acids being the supplementary region, and the remainder being the tail region. In Example 1, one embodiment of the present invention, the first region of the ASO has sequences that are entirely complementary to the tail region.

[0037] According to Experimental Example 2, the Cy5.5-labeled ASO of Example 1 was confirmed to exhibit a strong fluorescence signal in the cytoplasm of chondrocytes, and in flow cytometry, the intracellular fluorescence intensity in the treatment group of Cy3-labeled Example 1 tended to increase in a concentration-dependent manner. Therefore, the structural design of the ASO according to one embodiment of the present invention can contribute to effective delivery and uptake even in cells that are difficult to penetrate, such as cartilage tissue (Figure 2).

[0038] Oligonucleotides according to one aspect of the present invention may be efficiently delivered into chondrocytes or cartilage tissue. Specifically, oligonucleotides according to one aspect of the present invention can effectively induce intracellular uptake into chondrocytes or retention within cartilage tissue.

[0039] According to Experimental Example 3, the ASO of Example 1, according to one embodiment of the present invention, was successfully delivered into cells even in human-derived cartilage tissue (Figure 3). This suggests that the ASO according to one embodiment of the present invention has physical and chemical properties that allow it to accumulate stably in human chondrocytes.

[0040] According to Experimental Example 4, mutants containing one or more mismatches in the central or supplemental region of miR-204 (Examples 3-1 to 3-3) were found to maintain excellent degradation without a significant decrease in binding efficiency compared to fully complementary ASOs (Figure 4). This suggests that the sequence design of ASOs according to one embodiment of the present invention is highly advantageous in ensuring high degradation efficiency, binding stability, and biological functionality.

[0041] In this specification, "mismatch" refers to a state in which a single base is non-complementarily substituted when compared to the complementary sequence of the target microRNA. That is, the expected Watson-Crick complementary base pair may not be formed at a specific position of the oligonucleotide, and the position may take the form of a G·U wobble bond or a non-canonical pairing, but is not limited to these.

[0042] An oligonucleotide according to 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 supplementary region of miR-204. Furthermore, specifically, the oligonucleotide may contain 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 mismatched nucleotides, and may contain 10 or fewer, nine or fewer, eight or fewer, seven or fewer, six or fewer, five or fewer, four or fewer, three or fewer, or two or fewer.

[0043] A second or third region of an oligonucleotide according to one aspect of the present invention may contain one or more mismatched nucleotides with miR-204. Specifically, the oligonucleotide may contain a mismatched nucleotide at one or more selected positions among the 1st to 22nd nucleotides. More specifically, the oligonucleotide may contain a mismatched nucleotide at one or more selected positions among the 7th to 22nd, 7th to 20th, 8th to 18th, 8th to 16th, or 8th to 14th nucleotides. Even more specifically, the oligonucleotide may contain a mismatched nucleotide at one or more selected positions among the 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, or 22nd nucleotides, and preferably at one or more selected positions among the 8th, 9th, 10th, 11th, 12th, 13th, or 14th nucleotides.

[0044] Furthermore, the mismatched nucleotide may be located in a complementary sequence at one or more positions among the 1st to 22nd nucleotides from the 5' end of miR-204. Specifically, the oligonucleotide may contain a mismatched nucleotide at one or more selected nucleotide positions from the 1st to 22nd, 1st to 16th, 3rd to 16th, 6th to 16th, or 9th to 15th nucleotide positions of miR-204. More specifically, the oligonucleotide may contain a mismatched nucleotide at one or more selected nucleotide positions from the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, or 16th nucleotides of miR-204, and more specifically, it may contain a mismatched nucleotide at one or more selected nucleotide positions from the 9th, 10th, 11th, 12th, 13th, 14th, or 15th nucleotides.

[0045] According to Experimental Example 5, compared to fully complementary ASOs, ASOs with 1-2 3' terminal base deletions (Examples 4-1 and 4-2) significantly degraded miR-204, confirming that they maintained binding affinity and degrading ability for miR-204 even at the shortened length (Figure 5).

[0046] This suggests that when designing miR-204-targeted ASOs, a partial deletion (truncation) at the 3' end can maintain miR-204 resolution without hindering functional effectiveness.

[0047] A nucleotide containing fixed sugar modification according to one aspect of the present invention may be at least one selected from the group consisting of LNA (locked nucleic acid), ENA (2',4'-ethylene-bridged nucleic acid), cEt (constrained ethyl) nucleotide, BNA (bridged nucleic acid), and BNANC / BNANC (acyclic amino-bridged BNA). Specifically, the nucleotide containing fixed sugar modification may be at least one of LNA and ENA, and more specifically, it may be LNA.

[0048] The non-fixed 2'-substituted sugar modification according to one aspect of the present invention may be at least one selected from the group consisting of non-fixed 2'-substituted sugar modifications of 2'-deoxy, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-alkyl, 2'-fluoro (2'-F), 2'-amino (2'-NH2), 2'-O-allyl, 2'-O-benzyl, ribose, and arabinose (ANA). More specifically, the non-fixed 2'-substituted sugar modification may be at least one selected from the group consisting of 2'-deoxy, 2'-O-methyl, and 2'-O-methoxyethyl.

[0049] An oligonucleotide according to one aspect of the present invention may include a first region complementary to the tail region of miR-204, and the first region may consist of three nucleotides linked in succession, including fixed-type sugar modifications. Specifically, the first region may include the 1st to 3rd nucleotides from the 5' end to the 3' end of the oligonucleotide, and the three nucleotides may consist of three nucleotides linked in succession, including fixed-type sugar modifications. More specifically, the first region may consist of the 1st to 3rd nucleotides from the 5' end to the 3' end of the oligonucleotide, and the three nucleotides may consist of three nucleotides linked in succession, including fixed-type sugar modifications.

[0050] An oligonucleotide according to one aspect of the present invention may include a second region complementary to the tail region of miR-204, and the nucleotides included in the second region may each independently contain fixed or unfixed 2'-substituted nucleotides. Specifically, the second region may be located next to the first region, moving from the 5' end to the 3' end of the oligonucleotide. Furthermore, the second region may include the 4th to 6th nucleotides, moving from the 5' end to the 3' end of the oligonucleotide, and these three nucleotides may be three nucleotides linked together in a sequence, containing either fixed or unfixed 2'-substituted nucleotides. More specifically, the second region may consist of the 4th to 6th nucleotides, moving from the 5' end to the 3' end of the oligonucleotide, and these three nucleotides may be three nucleotides linked together in a sequence, consisting of either fixed or unfixed 2'-substituted nucleotides.

[0051] According to one embodiment of the present invention, when an oligonucleotide according to one aspect of the present invention contains fixed glycosylation at the 1st to 3rd positions (first region) from the 5' end to the 3' end (Examples 5-1 to 5-7), it exhibits stable and efficient resolution of miR-204. This same miR-204 resolution is also observed when unfixed 2'-substituted glycosylation and fixed glycosylation are mixed in the adjacent 4th to 6th positions (second region). However, oligonucleotides containing only one or two fixed glycosylation in the first region (Comparative Examples 1-1 and 1-2) showed miR-204 expression levels at a concentration of 10 μM similar to the control group and exhibited almost no resolution of miR-204 (see Experimental Example 6-1 and Figures 6 and 7).

[0052] Furthermore, according to one embodiment of the present invention, even when an oligonucleotide according to one aspect of the present invention contains MOE or OME at one or more positions 4 to 6 (second region) from the 5' end to the 3' end by non-fixed 2'-substituted sugar modification, the overall structural stability of the oligonucleotide and its miR-204 decomposition ability were maintained (see Examples 6-1 to 6-14, Experimental Example 8, and Figures 12 and 13).

[0053] An oligonucleotide according to one aspect of the present invention may include a third region complementary to the supplementary region, central region, and seed region of miR-204, and each nucleotide in the third region may independently contain a non-fixed 2'-substituted sugar modification. Specifically, the third region may be located next to the second region, moving from the 5' end to the 3' end of the oligonucleotide. Furthermore, the third region may include nucleotides from the 7th to the last nucleotide, moving from the 5' end to the 3' end of the oligonucleotide, and the plurality of nucleotides may be a series of linked nucleotides containing a non-fixed 2'-substituted sugar modification. More specifically, the third region may consist of nucleotides from the 7th to the last nucleotide, moving from the 5' end to the 3' end of the oligonucleotide, and the plurality of nucleotides may be a series of linked nucleotides consisting of a non-fixed 2'-substituted sugar modification. Even more specifically, all nucleotides constituting the third region may contain 2'-deoxy.

[0054] An oligonucleotide according to one aspect of the present invention may include a region complementary to the seed region of miR-204, and the region complementary to the seed region of miR-204 may be a part of a third region of the oligonucleotide according to one aspect of the present invention, specifically a part adjacent to the 3' end. The sugar of the nucleotide included in the region complementary to the seed region may include an unfixed 2'-substituted sugar modification.

[0055] In an oligonucleotide according to one aspect of the present invention, the sugar of one or more nucleotides from position 1 to 16 from the 3' end of the oligonucleotide may be 2'-deoxy. Specifically, in the oligonucleotide, the sugar of 1 or more nucleotides from position 1 to 16 from the 3' end of the oligonucleotide may be 2'-deoxy, and the sugar of nucleotides 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less may be 2'-deoxy. More specifically, the oligonucleotide may have a 2'-deoxy sugar at the nucleotides at positions 1 and 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, or 1 to 16 from the 3' end of the oligonucleotide.

[0056] According to one embodiment of the present invention, when an oligonucleotide according to one aspect of the present invention does not contain fixed glycosylation in the first region and contains fixed glycosylation only in the region near the 3' end (third region) (Comparative Examples 2-1 to 2-4), or when fixed glycosylation is randomly contained in the second and third regions (Comparative Examples 3 and 4), the binding affinity to ASO and the efficiency of RNase H-mediated degradation are reduced, and the overall miR-204 degradation effect is reduced (see Experimental Examples 6-2 and 6-3, and Figures 8 to 10). This shows that the position of fixed glycosylation has a decisive effect on the miR-204 degradation activity of the oligonucleotide according to one aspect of the present invention, and that the best miR-204 degradation is observed when fixed glycosylation is centered around the 5' end, i.e., in the first region.

[0057] An oligonucleotide according to one aspect of the present invention may contain 30% or less of nucleotides containing fixed-type sugar modifications relative to the total number of nucleotides. Specifically, the oligonucleotide may contain 30% or less, 29.5% or less, 29% or less, 28.5% or less, 28% or less, 27.5% or less, 27.3% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22.7% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18.2% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13.6% or less, 13% or less, 12% or less, 11% or less, or 10% or less of nucleotides containing fixed-type sugar modifications relative to the total number of nucleotides. More specifically, the oligonucleotide may contain 13 to 30% of nucleotides containing fixed-type sugar modifications relative to the total number of nucleotides.

[0058] Alternatively, an oligonucleotide according to one aspect of the present invention may contain 3 to 14 nucleotides containing fixed-type sugar modifications. Specifically, the oligonucleotide may contain 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 13 or more nucleotides containing fixed-type sugar modifications, and may contain 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, or 4 or fewer nucleotides. More specifically, the oligonucleotide may contain 3 to 6 nucleotides containing fixed-type sugar modifications. The oligonucleotide according to one aspect of the present invention exhibits excellent miR-204 resolution when all three nucleotides constituting the first region contain fixed-type sugar modifications; therefore, the nucleotides containing fixed-type sugar modifications may be 13% or more, or 3 or more.

[0059] According to one embodiment of the present invention, the oligonucleotide according to one aspect of the present invention exhibits improved miR-204 degradation as the number of nucleotides containing fixed glycosylation increases. However, this simultaneously increases the viscosity and decreases the solubility of the formulation, potentially leading to physical and pharmaceutical limitations as a drug. Therefore, considering both efficacy and formulation stability, a moderate number of nucleotides containing fixed glycosylation (e.g., 13-30% or 3-6 nucleotides) was determined to be the optimal structure for maintaining a good balance between miR-204 degradation and formulation compatibility (see Experimental Example 7 and Figure 11B).

[0060] Each nucleotide in an oligonucleotide according to one aspect of the present invention may be linked by a phosphorothioate bond or a phosphodiester bond. The bonds between nucleotides in an oligonucleotide according to one aspect of the present invention may each be independently a phosphorothioate bond or a phosphodiester bond. Specifically, in an oligonucleotide according to one aspect of the present invention, the phosphate bonds between nucleotides may each be independently a phosphorothioate bond and / or a phosphodiester bond. For example, some of the bonds between nucleotides may be phosphorothioate bonds and others may be phosphodiester bonds, and the number and position of these bonds are not particularly limited. As another example, all of the bonds between nucleotides may be phosphorothioate bonds or phosphodiester bonds.

[0061] An oligonucleotide according to one aspect of the present invention can effectively degrade miR-204 in chondrocytes. An oligonucleotide according to one aspect of the present invention can exert a preventive or therapeutic effect on osteoarthritis. Specifically, an oligonucleotide according to one aspect of the present invention can exert a preventive or therapeutic effect on osteoarthritis by effectively degrading miR-204 in chondrocytes.

[0062] In one example, the osteoarthritis may be at least one selected from the group consisting of degenerative (primary) osteoarthritis, post-traumatic osteoarthritis, post-surgical osteoarthritis, inflammatory osteoarthritis, and secondary osteoarthritis.

[0063] Oligonucleotides according to one aspect of the present invention include, but are not limited to, extracellular matrix (ECM) remodeling-related genes (e.g., ECM proteoglycan synthesis factors) and / or inflammation-inducing-related genes (e.g., IL6, PTGS2, NOS2, IL family, matrix degradation products) and / or cartilage matrix-degrading enzyme-related genes (e.g., MMP family (e.g., MMP3), ADAMTS family).

[0064] Oligonucleotides according to one aspect of the present invention can exert preventive or therapeutic effects on osteoarthritis even when administered repeatedly. Specifically, oligonucleotides according to one aspect of the present invention can exert stable and reproducible effects even when administered repeatedly.

[0065] An oligonucleotide according to one aspect of the present invention can alleviate osteoarthritis symptoms (e.g., cartilage damage, subcutaneous bone sclerosis, subchondral bone sclerosis) when administered repeatedly. According to one embodiment of the present invention, when the oligonucleotide according to one aspect of the present invention is administered twice repeatedly, both cartilage damage and subcutaneous bone sclerosis are significantly reduced (see Experimental Example 10, and Figures 19A, 19B, and 19D).

[0066] An oligonucleotide according to one aspect of the present invention can exert a pain-relieving effect even when administered repeatedly. According to one embodiment of the present invention, when an oligonucleotide according to one aspect of the present invention is administered twice repeatedly, the pain-relieving effect is sustained (see Experimental Example 10 and Figure 19C).

[0067] In one embodiment, repeated administration means administering an oligonucleotide according to one aspect of the present invention two or more times. Specifically, repeated administration may mean administering the same or different doses of the oligonucleotide two or more times at regular intervals. For example, repeated administration may be two, three, four, five or more times, but is not limited to these.

[0068] Oligonucleotides according to one aspect of the present invention can exert reproducible effects in both single-dose and repeated-dose administrations.

[0069] Oligonucleotides according to one aspect of the present invention may exhibit superior preventive or therapeutic effects on osteoarthritis compared to oligonucleotides that do not follow the glycosylation pattern of oligonucleotides according to one aspect of the present invention. According to one embodiment of the present invention, oligonucleotides according to one aspect of the present invention can exhibit remarkably superior effects in reducing cartilage damage, reducing subcutaneous bone sclerosis, and / or pain relief compared to other oligonucleotides that do not follow the glycosylation pattern of oligonucleotides according to one aspect of the present invention under the same conditions (see Experimental Example 10 and Figures 19A to 19D).

[0070] Oligonucleotides according to one aspect of the present invention can exhibit superior preventive or therapeutic effects compared to conventional osteoarthritis treatments (e.g., commercially available osteoarthritis treatments, or osteoarthritis treatments currently in clinical use or under development). Specifically, oligonucleotides according to one aspect of the present invention may exhibit superior cartilage protection, subcutaneous bone sclerosis improvement, pain relief, and / or cartilage regeneration effects compared to osteoarthritis treatments currently in clinical use or under development. For example, conventional osteoarthritis treatments may be SM04690 or steroidal anti-inflammatory analgesics (e.g., dexamethasone), but are not limited to these.

[0071] According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention can exhibit a superior cartilage protective effect compared to SM04690 (see Experimental Example 10 and Figures 20A to 20E). According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention may have superior cartilage regeneration ability compared to dexamethasone (see Experimental Example 10 and Figures 20A to 20E).

[0072] Oligonucleotides according to one aspect of the present invention can protect and / or repair cartilage structures. Specifically, oligonucleotides according to one aspect of the present invention can improve osteoarthritis not only by controlling inflammation but also by inducing the protection and / or restoration of the cartilage structure itself.

[0073] An oligonucleotide according to one aspect of the present invention can exert preventive or therapeutic effects even in the later stages of osteoarthritis. Specifically, an oligonucleotide according to one aspect of the present invention can restore cartilage even when administered in the later stages of advanced osteoarthritis. An oligonucleotide according to one aspect of the present invention can exhibit a superior cartilage restoration effect compared to Steric blocker and / or SM04690 in the later stages of osteoarthritis. According to one embodiment of the present invention, when administered 6 weeks after the induction of osteoarthritis, an oligonucleotide according to one aspect of the present invention can exhibit a statistically significant level of cartilage restoration effect compared to Steric blocker (SEQ ID NO: 22) and SM04690 (see Experimental Example 10 and Figures 21A-21C).

[0074] An oligonucleotide according to one aspect of the present invention can exert a pain-relieving effect even in the later stages of osteoarthritis. According to one embodiment of the present invention, when administered 6 weeks after the induction of osteoarthritis, the oligonucleotide according to one aspect of the present invention can exert a statistically significant level of pain relief (see Experimental Example 10 and Figure 21D).

[0075] In one case, the late stage of osteoarthritis refers to the stage in which osteoarthritis has progressed for a certain period of time. Specifically, the late stage of osteoarthritis may refer to the stage in which pathological changes in osteoarthritis have progressed and cartilage damage, subcutaneous bone sclerosis, osteophyte formation, and / or synovitis are observed. For example, the late stage of osteoarthritis may be, but is not limited to, four, five, six weeks, or more after induction of osteoarthritis in an animal model. For example, the late stage of osteoarthritis may be, but is not limited to, four, five, six weeks, or more after diagnosis of osteoarthritis. In humans, it may be, but is not limited to, the stage in which KL grade (Kellgren-Lawrence grade) stages 3, 4, or higher have progressed. The late stage of osteoarthritis refers to a state in which pathological changes have progressed further and become fixed compared to the initial stage, and may include a stage in which treatment is more difficult.

[0076] Oligonucleotides according to one aspect of the present invention may have high sequence specificity for miR-204 and extremely low non-targeting effects.

[0077] An oligonucleotide according to one aspect of the present invention may have high specificity for miR-204. Specifically, an oligonucleotide according to one aspect of the present invention does not need to bind nonspecifically to other targets (e.g., human mRNA, microRNA, or pre-mRNA) other than miR-204 and / or its host gene, TRPM3. According to one embodiment of the present invention, NCBI-BLAST analysis of the nucleotide sequence of an oligonucleotide according to one aspect of the present invention revealed no sequences other than miR-204 and TRPM3 that were expected to bind (see Experimental Example 11 and Table 14).

[0078] Oligonucleotides according to one aspect of the present invention may have a low off-target effect. Specifically, oligonucleotides according to one aspect of the present invention do not significantly affect any other genes or biological pathways other than miR-204 degradation. According to one embodiment of the present invention, Gene Ontology (GO) analysis using transcriptome data from cells treated with oligonucleotides according to one aspect of the present invention showed no significant ontological terms detected in any of the items of Biological Process, Molecular Function, and Cellular Component (see Experimental Example 11 and Figures 22A-22C).

[0079] Oligonucleotides according to one aspect of the present invention may have low immunogenicity. Specifically, oligonucleotides according to one aspect of the present invention do not induce an immune response mediated by Toll-like receptors (TLRs). For example, the TLR may be TLR7, TLR8, and / or TLR9, but is not limited to these.

[0080] Oligonucleotides according to one aspect of the present invention do not necessarily have to increase TLR activity. According to one embodiment of the present invention, when oligonucleotides according to one aspect of the present invention were treated, no significant increase in TLR7, TLR8, and TLR9 activity was observed (see Experimental Example 12 and Figures 23A to 23C).

[0081] An oligonucleotide according to one aspect of the present invention may have non-immunostimulatory properties with respect to the innate immune receptor pathway. An oligonucleotide according to one aspect of the present invention may be an antisense oligonucleotide that does not induce immunogenicity.

[0082] Oligonucleotides according to one aspect of the present invention may be non-cytotoxic or have low cytotoxicity. Specifically, oligonucleotides according to one aspect of the present invention may be non-cytotoxic or have low cytotoxicity in chondrocytes, hepatocytes, and / or kidney cells. According to one embodiment of the present invention, oligonucleotides according to one aspect of the present invention did not reduce the cell viability to 80% or less in primary cultured cells derived from mouse cartilage, hepatocyte line (HepG2 cell line), and kidney cell line (RPTEC cell line) (see Experimental Example 13 and Figures 24A to 24C).

[0083] Oligonucleotides according to one aspect of the present invention do not have to exhibit systemic toxicity. Oligonucleotides according to one aspect of the present invention do not have to exhibit systemic toxicity when administered intra-articular. Oligonucleotides according to one aspect of the present invention do not have to exhibit toxicity to the liver, kidney, and / or spleen. Oligonucleotides according to one aspect of the present invention do not have to exhibit toxicity to the liver, kidney, and / or spleen when administered intra-articular. According to one embodiment of the present invention, after a single intra-articular administration of oligonucleotides according to one aspect of the present invention, the organ-to-body weight ratios of the liver, kidney, and spleen did not show a significant difference compared to the control group (see Experimental Example 13 and Figure 25A).

[0084] Oligonucleotides according to one aspect of the present invention do not have to exhibit local toxicity. Oligonucleotides according to one aspect of the present invention do not have to exhibit local toxicity when administered intra-articularly. Specifically, oligonucleotides according to one aspect of the present invention do not induce cartilage damage, proteoglycan loss, and / or chondrocyte apoptosis. Specifically, oligonucleotides according to one aspect of the present invention do not have to induce cartilage damage, proteoglycan loss, and / or chondrocyte apoptosis when administered intra-articularly. According to one embodiment of the present invention, when oligonucleotides according to one aspect of the present invention were administered intra-articularly, no cartilage damage or proteoglycan loss was observed in the knee joint tissue, the intracartilage matrix components were maintained normally, and no apoptosis was induced in chondrocytes (see Experimental Example 13 and Figure 25B).

[0085] Oligonucleotides according to one aspect of the present invention may have excellent local and / or systemic safety. Specifically, oligonucleotides according to one aspect of the present invention may be safe antisense oligonucleotides that have no or low local toxicity and / or systemic toxicity at the administration site.

[0086] Oligonucleotides according to one aspect of the present invention may be efficiently delivered within chondrocytes. Furthermore, oligonucleotides according to one aspect of the present invention may be efficiently delivered into chondrocytes derived from mammals (e.g., dogs). Specifically, oligonucleotides according to one aspect of the present invention may be efficiently delivered into chondrocytes derived from mammals (e.g., dogs) and stably accumulated within said chondrocytes. According to one embodiment of the present invention, fluorescently labeled oligonucleotides according to one aspect of the present invention were significantly accumulated in dog-derived chondrocytes, and a fluorescence-positive signal of 50% or more was observed under conditions of treatment at concentrations of 10 nM and 50 nM (see Experimental Example 14, and Figures 26A and 26B).

[0087] An oligonucleotide according to one aspect of the present invention may reduce miR-204 in canine chondrocytes. According to one embodiment of the present invention, an oligonucleotide according to one aspect of the present invention can statistically significantly reduce miR-204 in canine chondrocytes in which cellular senescence has been induced (see Experimental Example 14 and Figure 26C).

[0088] Oligonucleotides according to one aspect of the present invention may restore cartilage matrix synthesis function in canine chondrocytes. Specifically, oligonucleotides according to one aspect of the present invention can increase or restore sulfated glycosaminoglycan (sGAG) synthesis in canine chondrocytes. According to one embodiment of the present invention, oligonucleotides according to one aspect of the present invention can statistically significantly increase sGAG synthesis, which has decreased due to aging, in canine chondrocytes in which cellular senescence has been induced (see Experimental Example 14 and Figure 26D).

[0089] Oligonucleotides according to one aspect of the present invention may have cross-species effects. Specifically, oligonucleotides according to one aspect of the present invention can effectively inhibit miR-204 not only in mouse-derived chondrocytes but also in canine-derived chondrocytes, thereby restoring cartilage matrix synthesis function. Oligonucleotides according to one aspect of the present invention can exert miR-204 inhibitory effects regardless of species specificity.

[0090] Furthermore, oligonucleotides according to one aspect of the present invention may be efficiently delivered without the use of a separate nucleic acid carrier. Specifically, oligonucleotides according to one aspect of the present invention can be efficiently delivered to chondrocytes, cartilage, and / or joint tissue without the use of a separate nucleic acid carrier. In this specification, the terms "carrier-free" or "carrier-independent" mean that the oligonucleotides of the present invention can be introduced into cells without the use of a separate nucleic acid carrier such as cationic lipids, lipid nanoparticles, liposomes, cationic polymers (e.g., polyethyleneimide, poly-L-lysine, etc.), dendrimers, cell-penetrating peptides, or virus / virus-like particle-based vectors. Oligonucleotides according to one aspect of the present invention may be administered in a form dissolved alone in an aqueous solution substantially free of such carriers (e.g., nuclease-free substances, buffer solutions, isotonic solutions, etc.).

[0091] An oligonucleotide according to one aspect of the present invention may be manufactured in the form of an intra-articular injection and may not contain a separate nucleic acid carrier, except for a pharmaceutically acceptable solvent (e.g., nuclease-free water, physiological saline, buffered saline, etc.) and a selective stabilizer, buffer, or isotonic agent. Despite being such a carrier-free composition, the oligonucleotide of the present invention can remain stably in the articular cartilage for a long period (e.g., several weeks after administration) and exert an effect of significantly improving the pathological changes of osteoarthritis by degrading miR-204 (see experimental examples). Therefore, the oligonucleotide of the present invention has the advantage of not requiring expensive carriers and exhibiting excellent tissue penetration and sustained efficacy.

[0092] Oligonucleotides according to one aspect of the present invention may be efficiently taken up into chondrocytes (e.g., mouse chondrocytes, canine chondrocytes, etc.) simply by being added directly to the culture medium in a cell culture system, without requiring a separate transduction reagent (e.g., lipofectamine, cationic lipid complex, etc.). Thus, since the oligonucleotides of the present invention exhibit excellent intracellular uptake and miR-204 degradation even under carrier-free conditions, they offer advantages such as minimizing additional toxicity, immunogenicity, or complexity of the manufacturing process caused by carriers.

[0093] Of course, if necessary, oligonucleotides according to one aspect of the present invention may be used in combination with other pharmaceutical delivery systems selected by those skilled in the art, in addition to the carrier-free form described above. For example, oligonucleotides according to one aspect of the present invention may be delivered to chondrocytes, cartilage, and / or joint tissue via a carrier.

[0094] Composition for the prevention or treatment of osteoarthritis Furthermore, one aspect of the present invention provides a composition for the prevention or treatment of osteoarthritis, comprising an oligonucleotide according to the aforementioned aspect.

[0095] Since oligonucleotides have already been discussed, a detailed explanation will be omitted.

[0096] In this specification, the term "osteoarthritis (OA)" refers to a degenerative joint disease characterized by degeneration of articular cartilage, sclerosis of subchondral bone, formation of osteophytes, and inflammation of the synovium. Risk factors such as aging, obesity, and mechanical overload contribute to the development and progression of osteoarthritis.

[0097] In this specification, the term "treatment" means any action that improves or beneficially alters the symptoms of osteoarthritis or a disease caused thereby by administering oligonucleotides and / or compositions according to one aspect of the present invention. A person with ordinary skill in the art to which the present invention belongs will be able to understand the precise criteria of the disease and determine the degree of improvement, enhancement, and treatment by referring to the materials presented in the art.

[0098] In this specification, the term “prevention” means any action that inhibits or delays the onset of osteoarthritis or related diseases by administering oligonucleotides and / or compositions according to one aspect of the present invention. “Prevention” may also include actions that reduce the risk of osteoarthritis or related diseases, slow the progression of the disease, or inhibit the appearance of symptoms of the disease.

[0099] In one real-world example, osteoarthritis may be a degenerative disease in which articular cartilage, normally formed at birth, gradually degenerates due to acquired factors such as aging, trauma, mechanical overload, and inflammation. In one real-world example, osteoarthritis may include degenerative (primary) osteoarthritis, post-traumatic osteoarthritis, post-surgical osteoarthritis, inflammatory osteoarthritis, and secondary osteoarthritis. For example, degenerative osteoarthritis may be the most common form of osteoarthritis, triggered by natural degenerative changes associated with aging, occurring without a specific cause such as clear trauma, structural disease, or inflammatory disease. For example, post-traumatic osteoarthritis may be osteoarthritis that occurs after mechanical trauma or damage to joint structures, such as joint injury or fracture, ligament injury or rupture, or meniscal injury or rupture, with accelerated degenerative changes. For example, inflammatory osteoarthritis is osteoarthritis in which inflammation of the intra-articular synovial membrane and surrounding tissues is prominent in addition to structural degenerative changes, and may be characterized by synovitis or an increase in synovial fluid inflammatory cells. Postoperative osteoarthritis may be osteoarthritis that is secondarily induced by structural changes, instability, or biomechanical changes following surgical procedures on intra-articular or surrounding tissues. Secondary osteoarthritis may be osteoarthritis that arises from changes in the joint structure or biological environment due to an apparent underlying cause (such as trauma, congenital malformation, metabolic disorder, endocrine abnormality, infection, or crystal deposition disorder). In this case, congenital malformations may include hip dysplasia, varus / valgus deformity, etc., and metabolic and endocrine abnormalities may include diabetes, thyroid disease, avascular necrosis, obesity, etc.

[0100] A composition according to one aspect of the present invention may be used for the prevention or treatment of osteoarthritis in mammals. The mammal may be, but is not limited to, at least one selected from the group consisting of humans, dogs, cats, rabbits, cattle, horses, pigs, sheep, mice, rats, monkeys, and chimpanzees. Specifically, the mammal may be a human, a dog, or a cat.

[0101] A composition according to one aspect of the present invention may be used to alleviate various pathological changes in osteoarthritis. Specifically, the composition may be used to alleviate at least one selected from the group consisting of cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, loss of cartilage matrix, and / or osteoarthritis-related pain, but is not limited to these. In this regard, one aspect of the present invention can provide a composition for alleviating cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, loss of cartilage matrix, and / or osteoarthritis-related pain, comprising an oligonucleotide according to one aspect of the present invention.

[0102] A composition according to one aspect of the present invention may be used to restore the extracellular matrix (ECM) synthesis function of chondrocytes. Specifically, the composition may be used to increase or restore the expression of proteoglycan biosynthesis enzymes. The composition may also be used to increase or restore the sulfated glycosaminoglycan (sGAG) content. In this regard, one aspect of the present invention can provide a composition for increasing proteoglycan biosynthesis enzymes and / or sulfated glycosaminoglycans (sGAGs), comprising an oligonucleotide according to one aspect of the present invention.

[0103] A composition according to one aspect of the present invention may be used to reduce or restore the expression of cartilage matrix-degrading enzymes, or to inhibit or alleviate inflammation. Specifically, the composition may be used to reduce the expression of cartilage matrix-degrading enzymes, and the cartilage matrix-degrading enzymes may include, but are not limited to, Mmp3. The composition may also be used to reduce the expression of inflammatory cytokines. The inflammatory cytokines may be, but are not limited to, IL-6, IL-1β, TNF-α, or a combination thereof. In this regard, one aspect of the present invention can provide a composition for reducing cartilage matrix-degrading enzymes and inflammatory cytokines, comprising an oligonucleotide according to one aspect of the present invention.

[0104] A composition according to one aspect of the present invention may be used for the prevention or treatment of the early and / or late stages of osteoarthritis. The composition can also restore cartilage even in the late stages when osteoarthritis has already progressed. For example, the early stage of osteoarthritis may be the stage within one, two, three, or four weeks after induction or diagnosis of osteoarthritis, in which minor cartilage damage is observed, but subcutaneous bone sclerosis, osteophyte formation, etc. have not yet occurred, but are not limited to these. For example, the late stage of osteoarthritis may be the stage four weeks or more after induction or diagnosis of osteoarthritis, in which serious cartilage damage is observed, and subcutaneous bone sclerosis, osteophyte formation, etc. have occurred, but are not limited to these.

[0105] A composition according to one aspect of the present invention can exhibit superior effects compared to conventional osteoarthritis treatments. Specifically, the composition is superior to conventional osteoarthritis treatments in terms of cartilage protection, cartilage restoration, cartilage regeneration, improvement of subcutaneous bone sclerosis, inflammation reduction, sGAG synthesis increase, and / or pain relief. Conventional osteoarthritis treatments may be SM04690 or dexamethasone, but are not limited to these.

[0106] A composition according to one aspect of the present invention may, but is not limited to, be used for animals (e.g., mammals). For example, the animal may, but is not limited to, at least one selected from the group consisting of humans, dogs, cats, rabbits, cattle, horses, pigs, sheep, mice, rats, monkeys, and chimpanzees. Specifically, the animal may be a human or a dog. Oligonucleotides according to one aspect of the present invention may be applied to animals.

[0107] A composition according to one aspect of the present invention may be, but is not limited to, a pharmaceutical composition, a food composition, a functional food composition, or an oral composition.

[0108] If a composition according to one aspect of the present invention is a pharmaceutical composition, the composition may further contain pharmaceutically acceptable carriers, diluents, excipients, stabilizers, buffers, isotonic agents, etc. The pharmaceutically acceptable carriers, diluents, excipients, stabilizers, buffers, or isotonic agents may be any known in the art without limitation, and may be, for example, at least one selected from the group consisting of saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof. In addition, other common additives such as antioxidants and bacteriostatic agents may be further included as needed.

[0109] A composition according to one aspect of the present invention may not contain a carrier. Even under carrier-free conditions, a composition according to one aspect of the present invention can effectively deliver oligonucleotides according to one aspect of the present invention into cells, exhibit miR-204 degradation, and exert preventive or therapeutic effects on osteoarthritis. On the other hand, a composition according to one aspect of the present invention may contain a carrier as needed. The carrier may be, but is not limited to, a cationic lipid, lipid nanoparticle, liposome, cationic polymer (e.g., polyethyleneimide, poly-L-lysine, etc.), dendrimer, cell-penetrating peptide, virus particle-based vector, and / or virus-like particle-based vector.

[0110] A composition according to one aspect of the present invention may be manufactured in various formulation forms. For example, the composition may be formulated in the form of an injection, a liquid, a suspension, an emulsion, a syrup, a powder, a granule, a tablet, a capsule, a gel, a patch, an emulsion, a syrup, an aerosol, or a combination thereof, but is not limited thereto. Specifically, the composition may be formulated as an intra-articular injection. The composition may be for intra-articular injection. For example, intra-articular injection allows for the localized delivery of a high concentration of the drug, maximizing the therapeutic effect while minimizing systemic side effects.

[0111] Oligonucleotides or compositions according to one aspect of the present invention may be administered by various routes. For example, the compositions may be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, nasally, transpulmonaryly, rectally, topically (e.g., intra-articular injection, other local injections, topical application), or intra-articularly. Specifically, the compositions may be administered by intra-articularly. While the examples according to one aspect of the present invention are verified by intra-articular administration, those skilled in the art will understand that other administration routes are also appropriately applicable, taking into account the pharmacokinetic / delivery mechanisms.

[0112] Oligonucleotides or compositions according to one aspect of the present invention may be administered in a pharmaceutically effective amount. In one aspect of this specification, “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit-to-risk ratio applicable to medical treatment, and the effective dose level may be determined by the type and severity of the patient’s disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and elimination rate, the duration of treatment, factors including drugs used concurrently, and other factors known in the medical field.

[0113] The dosage of an oligonucleotide or composition according to one aspect of the present invention can be appropriately selected by a person skilled in the art, taking into consideration the patient's age, sex, weight, disease severity, route of administration, and number of administrations. For example, based on an adult (e.g., an adult weighing 60 kg), the oligonucleotide per single dose may be 0.1 to 50 mg. For example, based on an adult (e.g., an adult weighing 60 kg), the oligonucleotide per single dose may 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, or 50 mg or more, and may be 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 of these (e.g., 10 to 30 mg), but is not limited to these. The composition may be administered once to several times a day, or repeatedly at intervals of several days, several weeks, or several months.

[0114] An oligonucleotide or composition according to one aspect of the present invention may be administered as a single dose or repeatedly. Specifically, the oligonucleotide or composition may be administered once, twice, three times, four times, five times, or more times.

[0115] A composition according to one aspect of the present invention may be administered simultaneously, separately, or sequentially in combination with an oligonucleotide according to one aspect of the present invention and other osteoarthritis treatment agents (for example, commercially available osteoarthritis treatment agents or treatment agents in clinical trials).

[0116] If a composition according to one aspect of the present invention is a food composition or a functional food composition, the composition may be manufactured in a form that can be consumed in the same way as ordinary food. There are no special restrictions on the type of food composition, and it may be, but is not limited to, meats, sausages, bread, chocolate, candies, snacks, sweets, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, or functional foods.

[0117] A composition according to one aspect of the present invention can prevent or treat osteoarthritis by effectively degrading miR-204. For example, the composition can prevent or treat osteoarthritis by degrading or reducing miR-204, modulating ECM reconstruction-related genes and cartilage structure maintenance-related biological pathways, and reducing the expression of cartilage matrix disruption-related proteins.

[0118] Furthermore, one aspect of the present invention provides a method for preventing or treating osteoarthritis, comprising the step of administering the aforementioned oligonucleotide or composition to an individual.

[0119] In one implementation example, the individual may be a subject requiring treatment or prevention of osteoarthritis.

[0120] In one implementation example, the individual may include, but is not limited to, a mammal. For example, the mammal may be, but is not limited to, at least one selected from the group consisting of humans, dogs, cats, rabbits, cattle, horses, pigs, sheep, mice, rats, monkeys, and chimpanzees. Specifically, the mammal may be a human or a dog.

[0121] In a method for preventing or treating osteoarthritis according to one aspect of the present invention, the oligonucleotide or composition may be administered to an individual in an amount effective for preventing or treating osteoarthritis.

[0122] Furthermore, one aspect of the present invention provides a method for alleviating cartilage destruction, subchondral bone sclerosis, osteophyte development, synovial inflammation, cartilage matrix loss, and / or osteoarthritis-related pain, comprising the step of administering the aforementioned oligonucleotide or composition to an individual. Furthermore, one aspect of the present invention provides a method for increasing proteoglycan biosynthesis enzymes and / or sulfated glycosaminoglycans (sGAGs), comprising the step of administering the aforementioned oligonucleotide or composition to an individual. Furthermore, one aspect of the present invention provides a method for decreasing cartilage matrix-degrading enzymes and / or inflammatory cytokines, comprising the step of administering the aforementioned oligonucleotide or composition to an individual.

[0123] In one implementation example, the individual may require relief of cartilage destruction, subcutaneous osteosclerosis, osteophyte formation, synovitis, loss of cartilage matrix, and / or osteoarthritis-related pain. In one implementation example, the individual may require an increase in proteoglycan biosynthesis enzymes and / or sulfated glycosaminoglycans. In one implementation example, the individual may require a decrease in cartilage matrix-degrading enzymes and / or inflammatory cytokines.

[0124] In one implementation example, the oligonucleotide or composition may be administered to an individual in an amount effective for alleviating cartilage destruction, subcutaneous osteosclerosis, osteophyte formation, synovitis, loss of cartilage matrix, and / or osteoarthritis-related pain. In one implementation example, the oligonucleotide or composition may be administered to an individual in an amount effective for increasing proteoglycan biosynthesis enzymes and / or sulfated glycosaminoglycans. In one implementation example, the oligonucleotide or composition may be administered to an individual in an amount effective for decreasing cartilage matrix-degrading enzymes and / or inflammatory cytokines.

[0125] Since the route of administration, dosage, frequency of administration, and concomitant administration have been described above, a detailed explanation will be omitted.

[0126] Furthermore, one aspect of the present invention provides an application for producing the aforementioned oligonucleotide compositions for the prevention or treatment of osteoarthritis.

[0127] Furthermore, one aspect of this specification provides the aforementioned oligonucleotides for the prevention or treatment of osteoarthritis.

[0128] Since oligonucleotides, treatment, prevention, and their relationship to osteoarthritis have been discussed previously, I will omit further explanation.

[0129] Furthermore, one aspect of the present invention provides a use for producing compositions for alleviating cartilage destruction, subcutaneous osteosclerosis, osteophyte formation, synovitis, cartilage matrix loss, and / or osteoarthritis-related pain using the aforementioned oligonucleotides. Furthermore, one aspect of the present invention provides a use for producing compositions for increasing proteoglycan biosynthesis enzymes and / or sulfated glycosaminoglycans using the aforementioned oligonucleotides. Furthermore, one aspect of the present invention provides a use for producing compositions for reducing cartilage matrix-degrading enzymes and / or inflammatory cytokines using the aforementioned oligonucleotides.

[0130] Furthermore, one aspect of this specification provides the use of the aforementioned oligonucleotides for alleviating cartilage destruction, subcutaneous osteosclerosis, osteophyte formation, synovitis, loss of cartilage matrix, and / or osteoarthritis-related pain. Furthermore, one aspect of this specification provides the use of the aforementioned oligonucleotides for increasing proteoglycan biosynthesis 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.

[0131] The configuration and effects of the present invention will be described in more detail below with reference to examples and experimental cases. However, the following examples are provided to aid in understanding the present invention and do not limit the scope and scope of the present invention.

[0132] [Manufacturing example] Production of miR-204-specific ASO Oligonucleotides specific to miR-204, specifically ASOs (antisense oligonucleotides), were produced by the following method (Examples 1 and 2).

[0133] 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 ID 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 ID 2). In this specification, uppercase letters represent DNA bases, "+X" represents LNA (locked nucleic acid) bases, and "iMe-dC" represents 5-methyldeoxycytosine bases. Also, "*" indicates that the bases are linked by phosphodiester (PO) bonds, and if there is no "*", it means that the bases are linked by phosphorothioate (PS) bonds.

[0134] ASO was synthesized and purified by a contract manufacturing organization (CMO) and supplied in powder form. The supplied material was dissolved in nuclear-free water (NFW; AM9932, Thermo Fisher Scientific) to a concentration of 5 mM and stored at -80°C.

[0135] In cell experiments, the solution was thawed at room temperature immediately before use, diluted in culture medium to the desired concentration, and then administered to the cells. In animal experiments, the solution was thawed at room temperature immediately before use, diluted in NFW to the desired concentration, and injected into the joint cavity at a final volume of 5 μL.

[0136] [Experimental Example 1] Confirmation of miR-204 resolution of ASO specific to miR-204. [Experimental Example 1-1] Confirmation of the miR-204 resolution of ASO in Example 1 (1) Experimental method 3 × 10⁶ C28 / I2 cell lines (chondrocyte line) overexpressing microRNA-204 (miR-204) 5 Cells were seeded in 12-well plates at a cell / well density and cultured for 24 hours. The 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% antibiotics (penicillin-streptomycin).

[0137] After 24 hours of incubation, the ASO (antisense oligonucleotide) 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, respectively, and used for treatment. Therefore, the conventional culture medium was removed and replaced with fresh medium containing the ASO, and the cells were treated.

[0138] After 48 hours of ASO treatment in Example 1, cells were harvested and total RNA was extracted using TRI Reagent (Molecular Research Center, Inc.). The extracted RNA was synthesized into DNA using the miRCURY LNA RT kit (Qiagen). The expression level of miR-204 in the synthesized cDNA was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the miRCURY LNA microRNA PCR Kit (Qiagen). TM The miRNA PCR assay (Qiagen) was used as the internal control. The maximum resolution (D) for Example 1 was used. max ) and the reaction concentration (DC) that decomposes miR-204 by 50% 50The result was calculated using GraphPad prism (Dotmatics).

[0139] (2) Experimental results In normal chondrocytes, miR-204 is present at a very low basal level. Therefore, to evaluate the miR-204 resolution of Example 1 with high sensitivity, a chondrogenic cell line (C28 / I2) overexpressing miR-204 was used. When this cell line was treated with Example 1 at different concentrations, it was confirmed that miR-204 expression decreased in a dose-dependent manner in the group treated with Example 1 (Figure 1A). Quantitative analysis revealed that the maximum resolution of Example 1 (D max ) is approximately 99.6%, and the reaction concentration (DC) that decomposes miR-204 by 50% is approximately 99.6%. 50 The concentration was calculated to be approximately 0.0055 μM (Figure 1B).

[0140] [Experimental Example 1-2] Confirmation of the miR-204 resolution of ASO in Example 2 (1) Experimental method 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 that time, a portion of the cells were divided into 3 × 10⁶ cells. 5After seeding cells in wells, they were cultured for 24 hours. The culture medium was then removed, and 1 μM of Example 2 was diluted with the transfection reagent messengerMAX reagent and opti-mem, and the cells were cultured at room temperature for 10 minutes. Subsequently, the cells were treated with Example 2 diluted with transfection reagent and opti-mem in 0.5 mL of fresh medium, and after 6 hours, the medium was changed back to fresh medium. The cells were then harvested after 48 hours (Figure 14). Total RNA was extracted from the harvested cells using TRI Reagent (Molecular Research Center, Inc.), cDNA was synthesized using the miRCURY LNA RT Kit (Qiagen), and miR-204 expression was analyzed by qRT-PCR using the miRCURY LNA microRNA PCR Kit (Qiagen). Let-7e-5p was used as the endogenous control group.

[0141] During statistical analysis, all quantitative data were expressed as mean ± standard error (Mean ± SEM). Statistical significance was verified using GraphPad Prism 10 software (version 10.4.2, GraphPad Software) and Student's t-test. Statistical significance in the graphs is indicated as follows: *, **, ***, ****: P < 0.05, 0.01, 0.001, 0.0001, respectively. (2) Experimental results qRT-PCR analysis revealed that the mean expression level of miR-204 in vehicle trf was 1.000 (0.000 in SEM), while the mean expression level of transfected (carrier-supported) (0.1 μM) miR-204 in Example 2 was 0.323 (0.030 in SEM), confirming that ASO composed of a PO backbone also induces the degradation of miR-204 (see Figure 14).

[0142] Such a degradation effect of miR-204 suggests that regardless of the backbone components, when the oligonucleotide (ASO) according to one aspect of the present invention is delivered into cells, it can effectively degrade the target miR-204.

[0143] [Experimental Example 2] Confirmation of the delivery ability of ASO (Example 1) specific to miR-204 to mouse chondrocytes (1) Experimental method 1) Primary culture of mouse chondrocytes Mouse articular chondrocytes were isolated from the femoral condyles and tibial plateaus of ICR mice at 5 - 6 days old. The isolated tissue was enzymatically digested using a 0.2% collagenase solution (ref), and then a single cell suspension was obtained. The obtained cells were seeded and cultured in a 12-well plate at a cell density of 2×10 5 cells / well for flow cytometry in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). The cells were maintained under conditions of 37°C, 5% CO2 and 21% O2. At the time point when 72 hours had elapsed after cell seeding, an ASO (antisense oligonucleotide) treatment group and a vehicle control group were set according to each experimental purpose. For fluorescence imaging, the cells were seeded and cultured in a 12-well plate at a cell density of 1×10 5 cells / well. The cells were maintained under conditions of 37°C, 5% CO2 and 21% O2. At the time point when 96 hours had elapsed after cell seeding, an ASO (antisense oligonucleotide) treatment group and a vehicle control group were set according to each experimental purpose.

[0144] 2) Confirmation of the delivery efficiency of the ASO of Example 1 To visualize the intracellular uptake of Example 1 in chondrocytes, cultured cells were incubated with 1 μM of Cy5.5-labeled Example 1 for 6 hours. Subsequently, 1 μg / mL of Hoechst was added to the culture medium, and co-incubation was performed for a further 18 hours. For fluorescence microscopy analysis, the cells were washed three times with phosphate-buffered solution (PBS), and fluorescence images were acquired using the EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0145] Furthermore, the degree of intracellular uptake of Example 1 was quantitatively evaluated by flow cytometry. For this purpose, mouse articular chondrocytes were cultured for 24 hours together with Cy3 fluorescently labeled Example 1 (10 nM or 50 nM). After treatment, the cells were separated from the adhesion surface with trypsin, washed with PBS, and then recovered by centrifugation. The recovered cells were resuspended twice in a PBS solution containing 5% FBS.

[0146] Flow cytometry is performed at FACSAria. TM The analysis was performed using a III Cell Sorter (BD Biosciences) system, and the data was collected and quantitatively analyzed using FACSDiva 9.7 software.

[0147] (2) Experimental results To confirm the delivery efficiency of Example 1 to mouse-derived chondrocytes, intracellular uptake was evaluated using Example 1 labeled with Cy5.5 or Cy3 fluorescence. Fluorescence microscopy analysis confirmed that, compared to the vehicle-treated group, the mouse-derived chondrocytes treated with Example 1 showed significant accumulation of fluorescent signals within the cells (Figure 2A). Furthermore, flow cytometry results showed that over 50% of samples were fluorescently positive under all conditions when Example 1 was treated at concentrations of 10 nM and 50 nM, quantitatively confirming the excellent intracellular uptake ability of Example 1 (Figures 2B and 2C). These results suggest that Example 1 possesses physical and chemical properties that allow it to be efficiently delivered to and stably accumulated in chondrocytes.

[0148] [Experimental Example 3] Confirmation of the delivery power of Example 1 in the tissue of a patient with osteoarthritis. (1) Experimental method 1) In vitro culture of human cartilage tissue Tissue from osteoarthritis patients obtained from artificial joint replacement surgery was cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). The tissue was maintained under conditions of 37°C, 5% CO2, and 21% O2. Depending on the experimental objective, an ASO (antisense oligonucleotide) treatment group and a vehicle control group were established.

[0149] 2) Confirmation of delivery in Example 1 To visualize the intracellular uptake of Example 1 in chondrocytes, cultured cells were incubated with 1 μM of Cy5.5-labeled Example 1 for 24 hours. Subsequently, 1 μg / mL of Hoechst was added to the culture medium, and co-incubation was performed for an additional 2 hours. For fluorescence microscopy analysis, the cells were washed three times with phosphate-buffered solution (PBS), and fluorescence images were acquired using the EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0150] (2) Experimental results To confirm the delivery of Example 1 to cartilage tissue derived from osteoarthritis patients, intracellular uptake was evaluated using Cy5.5-labeled Example 1. Fluorescence microscopy analysis revealed that, compared to the vehicle-treated group, the Example 1-treated group showed significant accumulation of fluorescent signals within patient-derived chondrocytes (Figure 3). These results suggest that Example 1 possesses physical and chemical properties that allow it to be efficiently delivered and stably accumulated not only in mouse cells but also in human chondrocytes.

[0151] [Experimental Example 4] Confirmation of miR-204 resolution of ASO specific to miR-204. In order to confirm the miR-204 resolution of ASOs (Examples 3-1 to 3-3, Table 1 below) that have the same base sequence as the ASO of Example 1 produced in the above manufacturing example, but are composed entirely of DNA (Full DNA), and ASOs that have a mismatch (shown in bold) in some base sequences, the following experiments were conducted.

[0152] Here, "Full DNA" refers to an ASO that has a base sequence completely complementary to the target miR204, and in which the sugars of the nucleotides constituting the Full DNA are all 2'-deoxy, without containing LNA sugars or modified sugars.

[0153] [Table 1]

[0154] (1) Experimental method Experiments were conducted to analyze the expression level of miR-204 using qRT-PCR in the same manner as in Experimental Example 1 (however, the treatment concentrations in Examples 1 and 3-1 to 3-3 were 1 μM and 10 μM, respectively).

[0155] All quantitative data in the statistical analysis were expressed as mean ± standard error (Mean ± SEM). Statistical significance was verified using GraphPad Prism 10 software (version 10.4.2, GraphPad Software), performing a one-way ANOVA followed by Tukey's post-hoc test. The statistical significance indicators in the graphs are as follows: *, **, ***, ****: Comparison with the vehicle (P<0.05, 0.01, 0.001, 0.0001 respectively) #, ##, ###, ####: Comparison of ASO (1 μM concentration group) relative to Full DNA (P<0.05, 0.01, 0.001, 0.0001 respectively) $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) relative to Full DNA (P<0.05, 0.01, 0.001, 0.0001, respectively). (2) Experimental results qRT-PCR analysis revealed a statistically significant decrease in miR-204 expression in the treatment groups of Examples 3-1 to 3-3 compared to the control group (Full DNA) which did not contain LNA (see Table 2 and Figure 4).

[0156] [Table 2]

[0157] Under a 1 μM treatment condition, all of Examples 3-1 to 3-3 showed a significant decrease in expression compared to Full DNA, and a similar trend was maintained even under a 10 μM treatment condition.

[0158] In particular, Examples 3-1 and 3-2 maintained miR-204 degradation activity despite each containing one mismatch in the central region (nucleotide positions 9-12) of miR-204, and Example 3-3 contained two mismatches in the supplementary region (nucleotide positions 13-16). This suggests that one or more mismatches in the central or supplementary region of miR-204 do not significantly affect the ASO binding efficiency, and that complete complementarity at the relevant site is not essential.

[0159] Therefore, ASO variants containing one or more mismatches within the central or supplemental region of miR-204 (Examples 3-1 to 3-3) are judged to maintain excellent stability, binding affinity, and degradation-inducing effect on miR-204.

[0160] [Experimental Example 5] Verification of miR-204 resolution based on ASO length To confirm the resolution of miR-204 according to the length of the ASO, experiments were conducted using the same method as in Experimental Example 1 on shorter ASOs (Examples 4-1 and 4-2, Table 3 below) in which a portion of the base sequence near the 3' end region of the ASO of Example 1 produced in the above manufacturing example was deleted.

[0161] [Table 3]

[0162] (1) Experimental method Using the same method as in Experimental Example 4, an experiment was conducted to analyze the expression levels of miR-204 after treatment in Examples 4-1 and 4-2 using qRT-PCR. The statistical significance indicators in the graph are as follows: *, **, ***, ****: Comparison with the vehicle (P<0.05, 0.01, 0.001, 0.0001 respectively) #, ##, ###, ####: Comparison of ASO (1 μM concentration group) relative to Full DNA (P<0.05, 0.01, 0.001, 0.0001 respectively) $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) relative to Full DNA (P<0.05, 0.01, 0.001, 0.0001, respectively).

[0163] (2) Experimental results qRT-PCR analysis revealed that miR-204 expression was statistically significantly reduced in all ASO (Examples 4-1 and 4-2) treatment groups, which had one (n-1) and two (n-2) bases deleted from the 5' end of the miR-204 sequence compared to a fully complementary sequence (Full DNA) that lacked a LNA (see Table 4 and Figure 5).

[0164] [Table 4]

[0165] In both concentration conditions, 1 μM and 10 μM, Examples 4-1 and 4-2 showed a tendency to maintain or even increase low miR-204 expression compared to Full DNA. In particular, Example 4-2(n-2) showed the lowest miR-204 expression level in both concentration conditions, confirming that miR-204 target binding and degradation induction are efficiently maintained even when the number of base pairs is shortened by two.

[0166] These results suggest that when designing antisense for miR-204, effective binding and degradation induction are possible even with truncated forms at the n-1 or n-2 level, rather than fully complementary sequences.

[0167] Therefore, ASO mutants (Examples 4-1 and 4-2) that have 1-2 bases deleted compared to the fully complementary sequence of miR-204 are judged to be candidates that can significantly degrade miR-204.

[0168] [Experimental Example 6] Confirmation of the effect of LNA position on the miR-204 resolution of ASO In Experimental Examples 1-4, it was confirmed that the miR-204 resolution of the ASO in Example 1 was superior. Since the ASO in Example 1 has six LNAs connected in a continuous manner within a first region, the following experiment was conducted to confirm whether the miR-204 resolution of the ASO is affected by the position of the LNAs.

[0169] [Experimental Example 6-1] Influence of LNA position when ASO contains LNA at its 5' end First, ASOs containing LNAs at the 5' end, but with varying numbers and positions of LNAs (Examples 5-1 to 5-7, and Comparative Examples 1-1 and 1-2, Table 5 below), were manufactured using the same method as described above, and their miR-204 resolution was evaluated.

[0170] [Table 5]

[0171] (1) Experimental method Using the same method as in Experimental Example 4, experiments were conducted to analyze the expression levels of miR-204 after treatment in Examples 5-1 to 5-7 and Comparative Examples 1-1 and 1-2 using qRT-PCR. The statistical significance indicators in the graphs are as follows: *, **, ***, ****: Comparison with the vehicle (P<0.05, 0.01, 0.001, 0.0001 respectively) #, ##, ###, ####: In Examples 5-1 to 5-7, ASO (1 μM concentration group) was compared to Full DNA / In Comparative Examples 1-1 to 1-2, ASO (1 μM concentration group) was compared to Example 1 (P < 0.05, 0.01, 0.001, 0.0001 respectively). $, $$, $$$, $$$$: In Examples 5-1 to 5-7, ASO (10 μM concentration group) was compared to Full DNA / In Comparative Examples 1-1 to 1-2, ASO (10 μM concentration group) was compared to Example 1 (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0172] (2) Analysis of experimental results qRT-PCR analysis revealed that, compared to the control group (Full DNA) without LNA, miR-204 expression was statistically significantly reduced in all ASOs (Examples 5-7) with three consecutive LNAs at the 5' end (all three nucleotides in the first region were LNAs) and in all ASOs (Examples 5-1 to 5-6) with one or two LNAs at the 4-6 base positions from the 5' end (one or more of the three nucleotides in the second region were LNAs) (see Table 6 and Figures 6 and 7 below).

[0173] [Table 6]

[0174] Under two treatment concentration conditions of 1 μM and 10 μM, all ASOs from Examples 5-1 to 5-7 showed a significant miR-204 degradation effect compared to Full DNA, and Examples 5-1 to 5-7 showed a particularly similar trend.

[0175] These results suggest that, during miR-204 target binding, it is possible to include LNA in the 5' end (first region, nucleotides 1-3) while positioning LNA or DNA in the second region (from 5' to nucleotides 4-6), and that even in this case, excellent miR-204 degradation activity is maintained.

[0176] Therefore, when the arrangement of LNA is concentrated near the 5' end (first region), ASOs in which DNA or LNA is mixed in the adjacent second region are judged to have stable and efficient resolution for miR-204.

[0177] [Experimental Example 6-2] Influence of LNA position when the ASO contains an LNA at its 3' end In Experimental Example 6-1, it was confirmed that ASOs in which three or more of the 1st to 6th nucleotides at the 5' end of the ASO were LNA, specifically all of at least the 1st to 3rd nucleotides, exhibited superior miR-204 resolution. Therefore, to determine whether there was a difference in miR-204 resolution when the ASO contained LNA at the 3' end instead of the 5' end (Comparative Examples 2-1 to 2-4, Table 7 below), the miR-204 resolution was evaluated using the same method as in Experimental Example 1.

[0178] [Table 7]

[0179] (1) Experimental method Using the same method as in Experimental Example 4, experiments were conducted to analyze the expression levels of miR-204 after treatment in Comparative Examples 2-1 to 2-4 using qRT-PCR. The statistical significance indicators in the graphs are as follows: *, **, ***, ****: Comparison with the vehicle (P<0.05, 0.01, 0.001, 0.0001 respectively) #, ##, ###, ####: Comparison of ASO (1 μM concentration group) with Example 1 (P < 0.05, 0.01, 0.001, 0.0001 respectively) $, $$, $$$, $$$$: Comparison of ASO (10 μM concentration group) for Example 1 (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0180] (2) Analysis of experimental results The effect of the terminal position of LNA on miR-204 degradation was evaluated by qRT-PCR analysis. Example 1, in which the LNA was located at the 5' end, was compared with ASO variants (Comparative Examples 2-1 to 2-4) in which the LNA was located at the 3' end. As a result, the ASO variants with the LNA located at the 3' end (Comparative Examples 2-1 to 2-4) showed reduced miR-204 degradation compared to LA001 with the LNA located at the 5' end at both concentrations (1 μM and 10 μM) (see Table 8 and Figure 8 below).

[0181] [Table 8]

[0182] Under a 1 μM treatment condition, Comparative Examples 2-1 and 2-2 showed a statistically significant decrease in the miR-204 degradation effect compared to Example 1, while Comparative Examples 2-3 and 2-4 showed a decreasing trend. Under a 10 μM treatment condition, a statistically significant decrease in the degradation effect was confirmed in Comparative Examples 2-1, 2-3, and 2-4, and Comparative Example 2-2 also showed a similar decreasing trend.

[0183] These results suggest that the position of the LNA has a decisive effect on the miR-204 degradation activity of ASO, and that the best effect is observed when the LNA is located at the 5' end. In other words, when the LNA is moved to the 3' end, the binding affinity to ASO and the efficiency of RNase H-mediated degradation are reduced, and the overall miR-204 degradation effect is thought to be diminished.

[0184] [Experimental Example 6-3] The effect of LNA location when LNAs are randomly included in ASO Experimental Example 6-2 showed that even if the ASO contains three or more LNAs, the miR-204 resolution effect is not significant if they are only present at the 3' end. Therefore, to evaluate the miR-204 resolution when LNAs are randomly included in the ASO (Comparative Examples 3 and 4, Table 9 below), the following experiment was conducted.

[0185] [Table 9]

[0186] (1) Experimental method 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 that time, a portion of the cells were divided into 3 × 10⁶ cells. 5 After seeding the cells in each well, they were cultured for 24 hours. The culture medium was then removed, and the cells were treated once with fresh medium diluted with 10 μM of Comparative Example 3. After 48 hours of treatment, the cells were harvested (Group 1, Figure 9). Meanwhile, the remaining cells were divided into 2 × 10⁶ cells. 4After seeding cells in wells, they were cultured for 24 hours. The culture medium was then changed and the cells were treated with Comparative Example 4 at concentrations of 256 nM, 1.6 μM, and 10 μM. A second treatment was administered at the same concentration 72 hours after the first treatment, and the cells were harvested 48 hours after the second treatment (Group 2, Figure 10). Total RNA was extracted from the cells harvested in each group using TRI Reagent (Molecular Research Center, Inc.), cDNA was synthesized using the miRCURY LNA RT Kit (Qiagen), and miR-204 expression was analyzed by qRT-PCR using the miRCURY LNA microRNA PCR Kit (Qiagen). Let-7e-5p was used as the endogenous control group.

[0187] During statistical analysis, all quantitative data were expressed as mean ± standard error (Mean ± SEM). Statistical significance was verified using GraphPad Prism 10 software (version 10.4.2, GraphPad Software), performing a one-way ANOVA followed by Tukey's post-hoc test. The statistical significance indicators in the graphs are as follows: *, **, ***, ****: P < 0.05, 0.01, 0.001, 0.0001 respectively (2) Analysis of experimental results qRT-PCR analysis revealed that Comparative Example 3, in which LNA was randomly included in the first group, showed a statistically significantly lower miR-204 degradation effect compared to Example 1 (see Table 10 and Figure 9 below).

[0188] [Table 10]

[0189] Such low miR-204 degradation effects suggest that the position and sequence regularity of LNA, rather than the LNA content within the ASO, are more important factors in the miR-204 degradation activity. Similarly, Comparative Example 4, another form in which LNA was randomly distributed, also showed lower miR-204 degradation than Example 1 in all concentration groups (see Figure 10). In other words, in structures where LNA is randomly arranged, as in Comparative Examples 3 and 4, efficient binding to and degradation induction of miR-204 is difficult, suggesting that an ASO according to one aspect of the present invention, such as a structure arranged around the 5' end (centered around the first and second regions) as in Example 1, is more effective in miR-204 degradation.

[0190] [Experimental Example 7] Confirmation of the effect of LNA number on the miR-204 resolution of ASO In Experimental Example 6, it was confirmed that the miR-204 resolution differed depending on the position of the LNAs in the ASO. To confirm the effect of the number of LNAs in the ASO, ASOs were manufactured in the same way as in Example 1, but with 15 or more LNAs, although the LNAs were located from the 5' end (Comparative Examples 5-1 to 5-3, Table 11 below). The stability of these formulations was then evaluated.

[0191] [Table 11]

[0192] (1) Experimental method Using the same method as in Experimental Example 4, experiments were conducted to analyze the expression levels of miR-204 after treatment in Comparative Examples 5-1 to 5-3 using qRT-PCR. The statistical significance indicators in the graphs are as follows: *, **, ***, ****: Comparison with the vehicle (P<0.05, 0.01, 0.001, 0.0001, respectively).

[0193] Furthermore, to confirm the physical properties (viscosity) of the dissolved ASOs of Comparative Examples 5-1 to 5-3, the tubes containing each solution were photographed from below at an angle, and after collecting the solution using a 1 mL pipette with a 1 mL pipette tip, the solution present in the tip was photographed.

[0194] (2) Analysis of experimental results The effect of the number of LNAs in an antisense oligonucleotide (ASO) on the miR-204 degradation effect was evaluated using qRT-PCR analysis. Experiments were conducted on mutants with progressively increased LNA counts (Comparative Examples 5-1 to 5-3), and the results showed that the effect of miR-204 degradation remained at a similar level even with an increased number of LNAs (see Figure 11A).

[0195] However, in mutants with an excessively increased number of LNAs (Comparative Examples 5-2 and 5-3), a physical phenomenon was observed in which the viscosity rapidly increased during the dissolution process, causing the solution to solidify into a jelly-like state (see Figure 11B). Even in the mutant with a relatively small number of LNAs (Comparative Example 5-1), higher viscosity was observed than in Example 1. This is judged to be a phenomenon in which the interaction between ASOs is strengthened as the LNA content increases, leading to a decrease in water solubility.

[0196] In summary, these results indicate that while increasing the number of LNAs improves the miR-204 degradation effect, it can also lead to increased viscosity and decreased solubility of the formulation, potentially limiting its physical and pharmaceutical properties. Therefore, considering both efficacy and formulation stability, a moderate number of LNAs (e.g., the level in Example 1) is considered the optimal structure for maintaining a good balance between miR-204 inhibitory activity and formulation compatibility.

[0197] [Experimental Example 8] Influence of other sugar modifiers besides LNA on miR-204 resolution In Experimental Example 6-1, it was confirmed that ASOs in which three or more of the 1st to 6th nucleotides at the 5' end of the ASO are LNA (ASOs in which three or more of the nucleotides at the 1st and 2nd regions are LNA), specifically ASOs in which at least all of the 1st to 3rd nucleotides are LNA (all three nucleotides in the 1st region are LNA), exhibit superior miR-204 resolution. As in Experimental Example 6-1, when three or more of the 1st to 6th nucleotides at the 5' end of the ASO are LNA, specifically when at least all of the 1st to 3rd nucleotides are LNA, but other modified sugars that are not LNA-modified sugars (e.g., non-fixed 2'-substituted sugar modifications) are present at other positions (Examples 6-1 to 6-14, Table 12 below, MOE modification is X). M OME modification is X O To determine whether there was a difference in miR-204 resolution (X represents a base), the miR-204 resolution was evaluated.

[0198] [Table 12]

[0199] (1) Experimental method Using the same method as in Experimental Example 4, experiments were conducted to analyze the expression levels of miR-204 after treatment in Examples 6-1 to 6-14 using qRT-PCR. The statistical significance indicators in the graph are as follows: *, **, ***, ****: Comparison with the vehicle (P<0.05, 0.01, 0.001, 0.0001 respectively) #, ##, ###, ####: In Examples 6-1 to 6-14, ASO (1 μM concentration group) was compared to Full DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively). $, $$, $$$, $$$$: In Examples 6-1 to 6-14, ASO (10 μM concentration group) was compared to Full DNA (P < 0.05, 0.01, 0.001, 0.0001, respectively).

[0200] (2) Analysis of experimental results In the effect of ASO on miR-204, experiments were conducted to confirm the influence when other modified sugars were introduced instead of LNA in the second region (the 4th to 7th nucleotide positions based on ASO). For this purpose, ASOs containing 1 to 3 2'-O-methoxyethyl (MOE) at the corresponding positions (Examples 6-1 to 6-7) and ASOs containing 2'-O-Methyl (OME) at the same positions (Examples 6-8 to 6-14) were designed and experiments were carried out under the same conditions as in Example 1.

[0201] As a result of qRT-PCR analysis, even when MOE or OME was introduced into the second region, the miR-204 degradation effect was maintained, and a statistically significantly higher miR-204 degradation ability was confirmed compared to the control group (Full DNA) in which LNA was completely excluded (see Figures 12 and 13). It was confirmed that even when modified sugars other than LNA were partially introduced into the second region, the effect of ASO did not significantly decrease.

[0202] Such results suggest that even when MOE or OME is introduced instead of LNA in the second region (positions 4-7), the overall structural stability of ASO and the miR-204 degradation activity are maintained, indicating that a modified ASO design ensuring chemical diversity is possible.

[0203] [Experimental Example 9] Effect of ASO specific to miR-204 in a cell model In Experimental Example 1, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, in order to confirm whether the ASO of Example 1 also shows the effect of degrading miR-204 to prevent or treat osteoarthritis in a cell model, the following experiments were conducted.

[0204] (1) Experimental method 1) Primary culture of mouse chondrocytes Mouse articular chondrocytes were isolated from the femoral condyle and tibial plateau of ICR mice aged 5-6 days. The isolated tissues were enzymatically digested with 0.2% collagenase solution to obtain single-cell suspensions. The obtained cells were seeded and cultured (primary culture) in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich) at different cell densities according to the experimental objective. The cells were maintained under hypoxic conditions of 37°C, 5% CO2, and 3% O2. 72 hours after cell seeding, an ASO (antisense oligonucleotide) treatment group and a vehicle control group were established according to the experimental objective. To induce chondrocyte senescence, primary cultured chondrocytes were treated with bleomycin (Cayman) 40 μg / mL for 24 hours. The culture medium was then removed and replaced with growth medium using nuclease-free water (NFW) as a vehicle or with growth medium containing Example 1 (SEQ ID NO: 1) (final concentration 2 μM). 48 hours after treatment, cells were harvested for mRNA analysis, with a seeding density of 5 × 10⁶ based on a 12-well plate. 4 The cell density was 1 / well. Additionally, 72 hours after treatment with Example 1 (final concentrations 0.1, 1, and 10 μM), cells were collected for protein analysis, with a seeding density of 2 × 10⁶ based on a 6-well plate. 5 The cell density was 1 / well. To induce an inflammatory response in chondrocytes, primary cultured chondrocytes were treated with 1 ng / mL of IL-1β (Genscript) and simultaneously cultured for 72 hours with either Example 1 (10 μM) or SM04690 (30 nM). Subsequently, the cells were harvested and mRNA analysis was performed. The seeding density at this time was 0.83 × 10⁶ based on a 12-well plate. 5 It was a cell / well.

[0205] 2) Analysis of gene expression changes according to Example 1 using qRT-PCR and Western blot Cells were harvested from culture 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) using SYBR Green qPCR Master Mix (ThermoFisher) to analyze the expression levels of chondrocyte ECM-related genes and inflammation-related genes. The Hprt gene was used as the internal control, and the relative expression levels of each gene were calculated as relative expression levels compared to the vehicle control group using the ΔΔCt method. All quantitative data in the statistical analysis were expressed as mean ± standard error (Mean ± SEM). Statistical significance was verified using GraphPad Prism 10 software (version 10.4.2), performing one-way ANOVA followed by Tukey's post-hoc test. The primers used during qRT-PCR are shown in Table 13 below.

[0206] [Table 13]

[0207] For protein expression analysis, cultured cells were harvested and total protein was extracted using RIPA buffer. The quantified protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to an NC membrane. Subsequently, primary and secondary antibody reactions were performed against cartilage matrix synthesis-related proteins (HPLN1, CHSY1) and endogenous control protein (Vinculin). Protein signals were detected using SuperSignal West Dura Extended Duration Substrate ECL solution (ThermoFisher) and confirmed using a chemiluminescent signal detection system (iBright FL1500, ThermoFisher). The HPLN1 antibody (ab98038) was purchased from Abcam, the CHSY1 antibody (NBP3-47604) from Novus Biologicals, and the Vinculin antibody (13901) from Cell Signaling Technology.

[0208] 3) sGAG assay Quantitative analysis of sGAG was performed using primary cultured mouse chondrocytes cultured by the method described above. To induce cellular senescence, cells were treated with bleomycin at a concentration of 40 μg / mL for 24 hours. After treatment, the cells were cultured for a further 3 days in fresh complete medium, with or without Example 1. Subsequently, the medium was changed and the cells were cultured again for 3 days, after which the culture supernatant was collected and used for sGAG analysis. The concentration of sGAG in the collected culture medium was measured using 1,9-dimethylmethylene blue (DMMB) staining. Absorbance was measured at 525 nM, and the sGAG content was quantified based on a standard curve created using chondroitin sulfate (C0335; Tokyo Chemical Industry). To correct for differences in cell viability and cell density, an MTT assay was performed on the same cells. Therefore, a PBS-based MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, T-030-1; Goldbio) was added at a concentration of 100 μg / mL and reacted for 2 hours. After the reaction, the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and the absorbance was measured at 570 nM. All sGAG measurements were normalized against the MTT results for comparison and analysis. The maximum effect (E) of Example 1 was measured. max ) and semi-maximal effective concentration (EC 50 The result was calculated using GraphPad prism (Dotmatics).

[0209] 4) Pathway analysis After treating C28 / I2 cell lines overexpressing miR-204 with a 1 μM dose of Example 1, a set of differentially expressed genes (DEG list) was derived from the obtained transcriptome data. Reactome pathway analysis was performed using the EnrichR platform with the derived DEG list. The analysis results were selected based on the adjusted p-value, with the top 20 annotations being visualized and represented in bubble plot format.

[0210] (2) Experimental results Since miR-204 is present at a very low basal level in normal chondrocytes, a chondrogenic cell line (C28 / I2) overexpressing miR-204 was used to highly sensitively evaluate the miR-204 resolution of Example 1. Transcriptome analysis revealed significant changes in ECM remodeling-related genes, including ECM proteoglycans, and gene annotations involved in maintaining cartilage structure (Figure 15). This suggests that the inhibition of miR-204 by Example 1 is not merely a microRNA-level regulation, but also involves structural recovery mechanisms such as ECM composition and homeostasis maintenance within chondrocytes.

[0211] Based on the above results, the protective mechanism of action of Example 1 was verified at the molecular level in primary cultured mouse chondrocytes. Therefore, similar to the results confirmed by transcription analysis, changes in the expression of major enzymes in the sulfated proteoglycan biosynthesis pathway were analyzed. When chondrocytes damaged by Bleomycin were treated with Example 1, the expression of core enzymes in the sGAG biosynthesis pathway increased statistically significantly, while the expression of matrix-degrading enzymes decreased significantly (Figure 16A). This restoration of expression means that the cartilage matrix synthesis pathway inhibited by miR-204 is restored to normal levels by Example 1, and the same increasing trend was confirmed at the protein level after treatment with Example 1 (Figure 16B). These results molecularly and functionally demonstrate the mechanism by which Example 1 effectively inhibits the pathological effects of miR-204 while simultaneously restoring the matrix synthesis ability of chondrocytes.

[0212] Next, to evaluate the effect of Example 1 under inflammatory stimulation conditions, experiments were conducted using primary cultured chondrocytes treated with the inflammatory cytokine IL-1β. As a result, similar to the results observed when damage was induced by Bleomycin, the expression of proteoglycan biosynthesis enzymes was significantly restored in the group treated with Example 1, while the expression of the inflammatory cytokine IL-6 was statistically significantly reduced (Figure 16C). In particular, Example 1 showed even better efficacy compared to SM04690, which has now completed Phase 3 clinical trials.

[0213] These results demonstrate that Example 1 effectively inhibits the miR-204-mediated pathway not only in aging environments but also in inflammatory environments, promoting cartilage matrix synthesis and mitigating inflammatory responses through a dual action.

[0214] Next, additional experiments were conducted to verify whether the effects of Example 1 were limited to changes at the gene and protein levels, or whether they actually affected the functional phenotype of cells. When senescent chondrocytes were treated with Example 1, it was confirmed that the sulfated glycosaminoglycan (sGAG) content was restored in a concentration-dependent manner (Figure 17A). Quantitative analysis revealed that the maximum effect of Example 1 was (E max ) is 114.3%, semi-maximal effective concentration (EC 50 The sGAG content was calculated to be 273.5 nM (Figure 17B). Furthermore, an increase in sGAG content by Example 1 was confirmed even in an inflammatory environment, and its effect was superior to that of SM04690 (Figure 17C). This matrix content recovery phenomenon indicates that Example 1 does not merely inhibit miR-204 expression or regulate lower-level protein expression, but actually functionally repairs cartilage matrix synthesis ability at the cellular level.

[0215] In other words, Example 1 was proven to be a candidate therapeutic agent that structurally and functionally restores the ECM synthesis function of chondrocytes by effectively inhibiting the pathological signaling pathway of miR-204 in all aging and inflammatory environments.

[0216] [Experimental Example 10] Confirmation of the effects of miR-204-specific ASO in animal models. In Experimental Example 1, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, in order to confirm whether the ASO of Example 1 also degrades miR-204 and has the effect of preventing or treating osteoarthritis in an animal model, the following experiment was conducted.

[0217] (1) Experimental method 1) Construction of a mouse model of osteoarthritis The C57BL / 6 male mice (12 weeks old, WT) used in the experiment were purchased from Daehan Biolink Co., Eumsung, Korea. All animal experiments were approved by the Seoul National University Institutional Animal Care and Use Committee (IACUC) (SNU-240710-5-1). Experimental animals were randomly assigned and housed in the Seoul National University specific pathogen-free facility. Housing conditions were adjusted to a temperature of 23-25°C, humidity of 45-65%, and a 12-hour light-dark cycle (day / night). All mice were given free access to standard laboratory solid feed. Animal experiment reports followed ARRIVE guidelines. To induce post-traumatic osteoarthritis (OA), 12-week-old male mice underwent DMM (destabilization of the medial meniscus) surgery on the right knee, while a control group underwent sham surgery under the same conditions.

[0218] To evaluate the therapeutic effect of Example 1 on osteoarthritis, Example 1 and SM04690 were administered intra-articularly at 4 weeks post-surgery, and dexamethasone was administered once a week at the same site starting at 2 weeks post-surgery. Mice were sacrificed at 6 weeks post-administration for histological analysis. Furthermore, to evaluate the therapeutic effect on the later stages of osteoarthritis, Example 1 was injected intra-articularly twice at 2-week intervals starting at 4 weeks post-surgery, and mice were sacrificed at 4 weeks after the final administration for histological analysis. Static weight-bearing analysis was performed one day prior to sacrifice.

[0219] 2) Histological analysis, immunohistochemistry, and in situ hybridization. The feet of the sacrificed mice for histological analysis were excised and fixed in a 4% paraformaldehyde solution at 4°C. The fixed foot tissues were decalcified in a 0.5 M EDTA (pH 7.4) solution at 4°C for 4 weeks, dehydrated stepwise with ethanol, treated with xylene, and then embedded in paraffin. The prepared paraffin blocks were cut at a thickness of 5 μm to produce slides.

[0220] The slides were deparaffinized with xylene and then rehydrated with ethanol of gradually decreasing concentration. Thereafter, safranin O staining was performed to observe changes in cartilage tissue, and the degree of damage to the medial subchondral bone was evaluated in a double-blind manner based on the OARSI criteria (grade 0 - 6).

[0221] Immunohistochemistry staining was performed by diluting the SLC35D1 (ab113717, Abcam), IL6 (sc - 130326, Stanta Cruz Biotechnology), and CTXII (NBP2 - 59386, Novus Biologicals) antibodies to 1:100, and the CHSY1 (NBP3 - 47604, Novus Biologicals), HPLN1 (ab98038, Abcam), and HAS2 antibodies (NBP3 - 03787, Novus Biologicals) to 1:50.

[0222] In situ hybridization was performed using the miR-204 detection probe and the detection probe from Example 1 (manufactured by BIONEER), while the U6 snRNA detection probe was purchased from Exiqon. The tissue was treated with 5 μg / mL proteinase K at 37°C for 30 minutes, and then re-fixed in 4% paraformaldehyde solution at room temperature for 20 minutes. The hybridization reaction was carried out under the following conditions: the U6 snRNA probe (1 nM) was reacted at 50°C for 1 hour, the probe from Example 1 (50 nM) at 42°C for 2 hours, and the miR-204 probe (50 nM) at 36°C for 2 hours. Subsequently, anti-DIG-AP antibody (1:800 dilution, Roche, 11093274910) was reacted at room temperature for 1 hour, and the signal was developed using NBT / BCIP solution (Thermo Fisher, 34070).

[0223] All histological, immunohistochemical, and in situ hybridization images were acquired by connecting a DS-Ri2 camera (Nikon) to a Nikon Eclipse Ni-U optical microscope.

[0224] 3) Analysis of micro-computed tomography (μCT) To evaluate the therapeutic effect and potential side effects of bone remodeling in Example 1, the knee joints of mice were resected and fixed using the method described above. The therapeutic effect was evaluated using a Bruker SKYSCAN 1276 in vivo μCT system (70kV, 57μA, 0.5mm Al filter, scanning time 13 minutes), and the obtained images were reconstructed using NRecon software (Bruker, version 1.7.3.2). Three-dimensional images were generated using CTvox (Bruker, version 3.3.0).

[0225] (2) Analysis of experimental results The therapeutic effect of Example 1 was evaluated in a surgically induced osteoarthritis mouse model. In the DMM surgery group injected with the vehicle (control group), typical pathological changes of osteoarthritis, such as cartilage destruction, subchondral bone sclerosis, osteophyte development, and synovial inflammation, were significantly observed. On the other hand, in the group administered Example 1, DMM-induced cartilage damage was statistically significantly inhibited, and the degree of subchondral bone sclerosis, osteophyte maturation, and synovial inflammation was also significantly reduced (Figures 18A-18C). In situ hybridization analysis confirmed that Example 1 remained persistently present in the articular cartilage at doses of 10 μg or more even 6 weeks after administration (Figure 18D), suggesting that Example 1 can remain stably in the cartilage for a long period and exert a sustained therapeutic effect. Behavioral analysis also showed that intra-articular administration in Example 1 statistically significantly restored weight-bearing imbalance caused by DMM surgery, indicating that this drug can substantially alleviate osteoarthritis-associated pain behavior (Figure 18E). Molecular analysis revealed that the treatment group in Example 1 showed restored expression of enzymes involved in chondroitin sulfate synthesis and a significant decrease in the expression of proteins associated with cartilage matrix destruction (Figure 18F), indicating that this contributes to the structural recovery and functional restoration of cartilage tissue.

[0226] The effect of Example 1 was consistently maintained even in the experiment involving two repeated administrations. Even after two injections of ASO, cartilage damage and subcutaneous bone sclerosis were clearly reduced, and the pain relief effect was also sustained. In particular, the effect of Example 1 was generally superior to that of Steric blocker (Comparative Example 4) (SEQ ID NO: 22) compared under the same conditions, and it showed a stable and reproducible effect even after repeated administrations (Figures 19A to 19D).

[0227] To verify the therapeutic effect of Example 1, comparative experiments were conducted with drugs currently in clinical use or under development. The osteoarthritis treatment SM04690, which has completed Phase 3 clinical trials, and the steroidal anti-inflammatory analgesic dexamethasone were used as control groups. The results showed that while SM04690 showed some pathological improvement, its cartilage protective effect was limited compared to Example 1. Dexamethasone also showed anti-inflammatory and pain-relieving effects, but its cartilage regeneration capacity was low. On the other hand, the group administered with Example 1 showed the most significant reduction in cartilage destruction, and demonstrated the best improvement in subcutaneous bone sclerosis and pain relief (Figures 20A-20E). These results suggest that Example 1, unlike conventional inflammation-controlling drugs, improves the pathophysiological root cause by inducing the protection and restoration of the cartilage structure itself.

[0228] Furthermore, to evaluate the potential for treatment in the later stages of osteoarthritis, drugs were administered 6 weeks after OA induction. Statistically significant levels of cartilage recovery were observed only in the group administered in Example 1, while no significant effects were confirmed in the groups administered with Steric blocker (Comparative Example 4) (SEQ ID NO: 22) and SM04690 (Figures 21A-21C). On the other hand, pain relief was confirmed to be statistically significant in all three groups: Example 1, Steric blocker (Comparative Example 4) (SEQ ID NO: 22), and SM04690 (Figure 21D). Micro-CT analysis also showed that Example 1 most effectively alleviated subcutaneous bone sclerosis, providing a mechanistic basis for restoring the structural stability of the subchondral bone.

[0229] In summary, the results of Example 1 confirm that it is an excellent therapeutic agent candidate that inhibits the pathological progression of osteoarthritis at multiple levels compared to other drugs. This substance exhibited excellent therapeutic effects in structural, functional, and behavioral aspects through a complex mechanism of action that includes (1) inhibition of cartilage destruction and promotion of regeneration, (2) reduction of subcutaneous bone sclerosis and osteophyte formation, (3) alleviation of synovitis and inhibition of inflammatory responses, (4) pain improvement and functional recovery, and (5) maintenance of long-term effects through sustained retention within the cartilage. In particular, Example 1 showed outstanding cartilage protection and pain relief effects compared to conventional clinical drugs and steroids, and maintained reproducible effects in all cases, whether single or repeated administration. These results strongly suggest that Example 1 is not merely a symptomatic agent, but a mechanism-based ASO drug candidate that can improve the underlying pathology of osteoarthritis.

[0230] [Experimental Example 11] Confirmation of off-target effects in Example 1 In Experimental Example 1, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, in order to evaluate the non-target effect of ASO in Example 1, the following experiment was conducted.

[0231] (1) Experimental method 1) Analysis of the predictive target in Example 1 We analyzed predicted target genes (human mRNA, microRNA, and pre-mRNA) using sequence variants that included the nucleotide sequence of Example 1 (n), a variant of Example 1 with one base deleted at the 3' end (n-1), and a variant with one repeating base added at the 3' end (n+1). Sequence-based homology analysis was performed using NCBI-BLAST (Basic Local Alignment Search Tool), and the human gene databases provided by NCBI (human RefSeq_RNA and human RefSeq_Gene) were used for the analysis. When evaluating sequence consistency, a criterion of two or fewer mismatched nucleotides was set as significant homology.

[0232] 2) Analysis of Gene Ontology (GO) term After treating C28 / I2 cell lines overexpressing miR-204 with a 1 μM dose of Example 1, a set of genes with significantly altered expression (differentially expressed genes, DEG list) was derived from the transcriptome data obtained. Gene Ontology (GO) term analysis was performed using the derived DEG list based on the EnrichR platform. The analysis results were selected based on the adjusted p-value, with the top 10 annotations being visualized and represented in the form of a bubble plot.

[0233] (2) Analysis of experimental results Based on the nucleotide sequence of Example 1, we analyzed the expected target genes (human mRNA, microRNA, and pre-mRNA) that could bind. Homology evaluation using NCBI-BLAST revealed that, other than the host gene TRPM3 and the on-target miR-204, no other sequences were expected to bind (see Table 14). Table 14 shows the results of sequence homology analysis performed using NCBI-BLAST on the nucleotide sequence and sequence variants of Example 1.

[0234] [Table 14] *TRPM3 is the host gene for miR-204.

[0235] Furthermore, Gene Ontology (GO) analysis was performed using transcriptome data obtained after treating C28 / I2 cell lines overexpressing miR-204 with Example 1. The analysis was performed using EnrichR, and the top 10 annotations were selected based on the adjusted p-value. As a result, no significant ontological terms were derived for any of the Biological Process, Molecular Function, or Cellular Component items (see Figures 22A-22C). These results suggest that Example 1 has high sequence specificity for miR-204 and is a highly specific ASO with extremely low non-targeting effects.

[0236] [Experimental Example 12] Confirmation of immunogenicity of Example 1 In Experimental Example 1, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, the following experiment was conducted to confirm the immunogenicity of Example 1.

[0237] (1) Experimental method - Toll-like receptor (TLR) activity assay To evaluate the effect of Example 1 on Toll-like receptor (TLR) activity, a TLR activity assay was performed using HEK-Blue cell lines (InvivoGen) overexpressing TLR7, TLR8, and TLR9, respectively. The HEK-Blue TLR7 cell line was 5 × 10⁶ 4Cells were seeded at a density of cells / well in a 96-well plate and cultured for 24 hours. Then, either Example 1 (10 μM) or ORN6S (positive control) was treated with 0.1 mM guanosine (Sigma, G6264). Six hours after drug treatment, the cell culture medium was collected and the Secreted Alkaline Phosphatase (SEAP) activity in the culture supernatant was measured. HEK-Blue TLR8 cell lines were seeded at the same density and cultured for 24 hours, then treated with either 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 cell lines were cultured under the same conditions and then treated with either Example 1 (10 μM) or ODN2006 (positive control). Six hours after treatment, the cell culture medium was collected and SEAP activity was measured.

[0238] (2) Analysis of experimental results To evaluate the immunogenicity of Example 1, changes in the activity of Toll-like receptors (TLRs) 7, 8, and 9 were measured. Using HEK-Blue TLR7, TLR8, and TLR9 cell lines, ligand-dependent SEAP expression was analyzed. The results showed that ORN6S (for TLR7 and TLR8 assays) and ODN2006 (for TLR9 assay), used as positive control groups, both induced receptor activity, and SEAP activity significantly increased.

[0239] On the other hand, no significant increase in TLR7, TLR8, and TLR9 activity was observed under any of the conditions in which Example 1 (10 μM) was treated (see Figures 23A to 23C). These results indicate that Example 1 does not induce an immune response via TLR7, TLR8, and TLR9, and has non-immunostimulatory properties toward the innate immune receptor pathway. Therefore, Example 1 is suggested to be a safe antisense oligonucleotide with a low potential for immunogenicity induction.

[0240] [Experimental Example 13] Confirmation of toxicity in Example 1 In Experimental Example 1, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, since it is necessary to consider the safety when using the composition containing Example 1 as a pharmaceutical composition, the following experiment was conducted to confirm the toxicity of Example 1.

[0241] (1) Experimental method 1)Cytotoxicity assay To evaluate the cytotoxicity of Example 1, mouse primary chondrocytes, a hepatocyte line (HepG2 cell line), and a renal cell line (RPTEC cell line) were used. The HepG2 and RPTEC cell lines were purchased from the American Cell Line Bank (ATCC).

[0242] Each cell line was seeded in a 96-well plate under the following conditions: Mouse primary cultured chondrocytes: 1 × 10⁶ 4 Cells / well, HepG2 cells: 1 × 10 3 Cells / well, RPTEC cells: 4 × 10 4 Cells / well. Each cell line was cultured under different conditions for 72 hours (chondrocytes) and 24 hours (HepG2 and RPTEC cells), and then treated with Example 1 at concentrations of 2 μM, 5 μM, and 10 μM.

[0243] After 72 hours of treatment, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega).

[0244] 2) Single-dose toxicity studies in mice To evaluate the toxicological characteristics of Example 1, two single-dose toxicity studies were conducted. First, a two-week single-dose toxicity study (2-week study) was performed using 9-week-old female CD-1 mice (Daehan Biolink Co., Eumsung, Korea). This study was conducted with the approval of the Seoul National University Institutional Animal Care and Use Committee (IACUC) (Approval Number: SNU-230531-6-1). The animals were housed in a specific pathogen-free (SPF) animal laboratory at Seoul National University and were given ad libitum access to standard feed in an environment with a temperature of 23-25°C, relative humidity of 45-65%, and a 12-hour light / dark cycle. Mice were randomly assigned to three groups. Each group received 5 μL of nuclease-free water containing Example 1 intra-articular injection (20 μg, 40 μg, and 80 μg, respectively). Body weight was measured on day 0, day 4, day 11, and day 14. On day 14, after a 1-day fast, blood was collected via the retro-orbital sinus, and the blood was centrifuged (1,500 × g, 15 minutes, 4°C) to separate the plasma. Clinical chemistry analysis was performed by an external analytical laboratory (Cellpurics, Korea) using an Adivia 1800 automated analyzer (Siemens). After the animals were sacrificial, the major organs (liver, spleen, and kidney) were removed, weighed, and observed for abnormalities with the naked eye. The tissue from the right foot was excised and fixed in a 4% paraformaldehyde solution at 4°C for 1 night and 2 days. The presence or absence of abnormalities in the cartilage and bone tissue was analyzed by histological analysis using the method described above. Chondrocyte apoptosis was performed using the TUNEL assay (Takara, MK500) according to the manufacturer's guidelines, and the nuclei were stained with DAPI at room temperature for 10 minutes. Fluorescence signals were observed using the EVOS M7000 Imaging System (Thermo Fisher, AMF7000).

[0245] (2) Analysis of experimental results The presence or absence of cytotoxicity in Example 1 was confirmed in primary cultured cells derived from mouse cartilage, hepatocyte cell line (HepG2 cell line), and kidney cell line (RPTEC cell line). The experimental results showed no significant level of cytotoxicity, such as a reduction in cell viability to below 80%, in any of the three cell types (see Figures 24A, 24B, and 24C). Next, to evaluate the local and systemic safety of Example 1, a 14-day toxicity study was conducted in mice after a single-dose intra-articular (IA) administration. Example 1 was administered at doses of 20, 40, and 80 μg / animal, respectively. The results showed no significant difference in organ-to-body weight ratios for the liver, kidney, and spleen compared to the control group in any of the administration groups (see Figure 25A). This suggests that no systemic or organ-specific toxicity occurred due to the administration of Example 1. Histological analysis of the administration site revealed no cartilage damage or proteoglycan loss within the knee joint tissue, and Safranin-O staining confirmed that the intrachondrocyte matrix components were normally maintained (Figure 25B, top and middle panels). Furthermore, TUNEL staining analysis confirmed that chondrocyte apoptosis was not induced, demonstrating the excellent local tissue tolerance of Example 1 (Figure 25B, bottom panel). These results support the conclusion that Example 1 is a safe antisense oligonucleotide that simultaneously exhibits excellent local safety and the absence of systemic toxicity when administered intra-articularly.

[0246] [Experimental Example 14] Confirmation of the effect of miR-204-specific ASO on canine chondrocytes. In Experimental Example 1, the miR-204 degradation effect of Example 1 was confirmed. Furthermore, in order to confirm whether the ASO of Example 1 can effectively inhibit miR-204 in canine chondrocytes, the following experiment was conducted.

[0247] (1) Experimental method 1)Canine chondrocyte cell line culture method Canine chondrocytes cell line (Cell Applications, Inc.) 2 × 10 5 Cells were seeded in 12-well plates at a cell / well density and cultured for 24 hours. The cells were maintained at 37°C under conditions of 5% CO2 and atmospheric oxygen concentration. The culture medium used was Canine chondrocyte growth medium (Cell Applications, Inc., Cn411-500) with a growth supplement (Cell Applications, Inc., Cn411-GS). After 24 hours of culture, the cells were used according to each experiment.

[0248] 2) Confirmation of delivery efficiency in Example 1 To visualize the intracellular uptake of Example 1 in canine chondrocytes, cultured cells were incubated with 1 μM Cy3-labeled cells from Example 1 for 24 hours. Subsequently, 1 μg / mL of Hoechst was added to the culture medium, and co-incubation was performed for an additional 20 minutes. After washing the cells three times with phosphate-buffered solution (PBS) for fluorescence microscopy analysis, fluorescence images were acquired using the EVOS M7000 Imaging System (Thermo-Fisher, AMF7000).

[0249] Furthermore, the degree of intracellular uptake of Example 1 was quantitatively evaluated by flow cytometry. For this purpose, canine chondrocytes were cultured for 24 hours together with Cy3 fluorescently labeled Example 1 (10 nM or 50 nM). After the treatment was complete, the cells were separated from the adhesion surface with trypsin, washed with PBS, and then recovered by centrifugation. The recovered cells were resuspended twice in a PBS solution containing 5% FBS.

[0250] Flow cytometry is performed at FACSAria.TM The analysis was performed using a III Cell Sorter (BD Biosciences) system, and the analytical data was collected and quantitatively analyzed using FACSDiva 9.7 software.

[0251] 3) qRT-PCR Canine chondrocytes were cultured for 24 hours and then treated with 0.25 μM doxorubicin (Sigma-aldrich) at a concentration of 0.25 μM to induce cellular senescence. The cells were maintained under hypoxic conditions of 37°C, 5% CO2, and 3% O2. After 72 hours, the cells were treated again with the same concentration of doxorubicin, but half of the original culture medium was removed and the remaining half was diluted with the same volume of fresh culture medium before treatment. After another 72 hours, the culture medium was replaced with fresh culture medium while treating with 1 μM doxorubicin, and the cells were harvested 48 hours after treatment.

[0252] Cells were harvested and total RNA was extracted using TRI Reagent (Molecular Research Center, Inc.). The extracted RNA was synthesized into DNA using the miRCURY LNA RT kit (Qiagen). The expression level of miR-204 in the synthesized cDNA was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the miRCURY LNA microRNA PCR Kit (Qiagen). Let-7e-5p was used as the internal control.

[0253] 4) sGAG assay Quantitative sGAG analysis was performed using canine chondrocytes cultured using the method described above. To induce cellular senescence, cells were treated with 0.25 μM doxorubicin (Sigma-aldrich) at a concentration of 0.25 μM. Cells were maintained under hypoxic conditions of 37°C, 5% CO2, and 3% O2. After 72 hours, the cells were treated again with the same concentration of doxorubicin, but half of the original culture medium was removed and the remaining half was diluted with the same volume of fresh culture medium before treatment. After another 72 hours, the culture medium was replaced with fresh medium while treating with concentrations of 0.1, 1, and 10 μM of Example 1, and the medium was replaced with fresh medium 48 hours after treatment. After culturing for 2 days, the culture medium was collected and used for sGAG analysis. The concentration of sGAG in the collected culture medium was measured using 1,9-dimethylmethylene blue (DMMB) staining. Absorbance was measured at 525 nM, and sGAG content was quantified based on a standard curve created using chondroitin sulfate (C0335; Tokyo Chemical Industry). To correct for differences in cell viability and cell density, an MTT assay was performed on the same cells. Therefore, a PBS-based MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, T-030-1; Goldbio) was added at a concentration of 100 μg / mL and reacted for 2 hours. After the reaction, the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm. All sGAG measurements were normalized against the MTT results for comparison and analysis. Statistical significance was verified using GraphPad Prism 10 software (GraphPad Software), with one-way ANOVA followed by Tukey's post-hoc test.

[0254] (2) Analysis of experimental results To confirm the delivery efficiency of Example 1 to canine chondrocytes, intracellular uptake was evaluated using Cy3-labeled Example 1. Fluorescence microscopy analysis confirmed that, compared to the vehicle-treated group, the canine chondrocytes treated with Example 1 showed a significant accumulation of fluorescent signals within the cells (Figure 26A). Furthermore, flow cytometry revealed that over 50% of samples were fluorescently positive under all conditions, including treatment with 10 nM and 50 nM concentrations of Example 1, quantitatively confirming the superior intracellular uptake ability of Example 1 (Figure 26B). These results suggest that Example 1 possesses physical and chemical properties that allow for efficient delivery and stable accumulation within chondrocytes.

[0255] Subsequently, a decrease in miR-204 by Example 1 was confirmed in canine chondrocytes in which cellular senescence had been induced. When Example 1 was delivered after treatment with Doxorubicin to induce cellular senescence, a statistically significant decrease in miR-204 expression was observed (Figure 26C).

[0256] Next, we evaluated the effect of Example 1 on restoring the synthesis function of cartilage matrix components. When canine chondrocytes with induced cellular senescence were treated with Example 1, we confirmed a significant increase in the amount of sulfated glycosaminoglycan (sGAG) synthesized, which had decreased due to aging (Figure 26D). These results indicate that Example 1 has a functional effect of restoring ECM synthesis function not only in mouse chondrocytes but also in canine chondrocytes by miR-204 degradation, supporting matrix restoration and anti-aging effects at the cellular level.

[0257] These results suggest that Example 1 has cross-species effects in mammals such as dogs and cats, as it can effectively inhibit miR-204 not only in mouse-derived chondrocytes but also in canine-derived chondrocytes, thus supporting the conservation of the miR-204 target inhibition mechanism of this substance, which is independent of species specificity.

Claims

1. An oligonucleotide that specifically hybridizes with miR-204, The oligonucleotide is composed of 20 to 22 nucleotides linked together, and is comprised of a first region, a second region, and a third region, in order from the 5' end to the 3' end. The first region is entirely complementary to the tail region of miR-204 and consists of three nucleotides linked together in sequence, including fixed glycosylation. The second region is a chain of three nucleotides, and each nucleotide constituting the second region independently contains either fixed-type or unfixed-type 2'-substituted-type sugar modification. The third region is formed by linking multiple nucleotides, and each nucleotide constituting the third region is independently a nucleotide containing an unfixed 2'-substituted sugar modification. The oligonucleotide is an oligonucleotide in which 30% or less of the total number of nucleotides are nucleotides containing fixed-type sugar modifications.

2. The oligonucleotide according to claim 1, wherein the nucleotide containing the fixed glycosylation is at least one selected from the group consisting of LNA (locked nucleotide), ENA (2',4'-ethylene-bridged nucleotide), cEt (contrained ethyl) nucleotide, BNA (bridged nucleotide), and BNANC / BNANC (acycloamino-bridged BNA).

3. The aforementioned non-immobilized 2'-substituted sugar modifications include 2'-deoxy, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-alkyl, 2'-fluoro (2'-F), and 2'-amino (2'-NH). ２ The oligonucleotide according to claim 1, which is at least one selected from the group consisting of non-fixed 2'-substituted glycosphagia modifications of ), 2'-O-allyl, 2'-O-benzyl, ribose, and arabinose (ANA).

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

5. The oligonucleotide according to claim 1, wherein the sugars of the nucleotides at the 1st and 2nd positions from the 3' end of the oligonucleotide are 2'-deoxy.

6. The oligonucleotide according to claim 1, wherein the oligonucleotide includes a region complementary to the seed region of miR-204, and the sugar of the nucleotide included in the region complementary to the seed region is an unfixed sugar modification.

7. The oligonucleotide according to claim 1, wherein the bonds between nucleotides contained in the oligonucleotide are, independently, phosphorothioate (phosphorothioate) bonds or phosphodiester (phosphodiester) bonds.

8. The oligonucleotide according to claim 1, wherein the oligonucleotide is at least one of the oligonucleotides represented by SEQ ID NOs: 1 to 14 and 26 to 39.

9. The oligonucleotide according to claim 1, wherein the second or third region comprises one or more mismatched nucleotides with miR-204.

10. The oligonucleotide according to claim 1, wherein the oligonucleotide is the miR-204 decomposition oligonucleotide.

11. A method for improving, preventing, or treating osteoarthritis, comprising administering the oligonucleotide described in claim 1 to an individual.

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

13. The method according to claim 11, wherein the individual is a mammal.

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