An antisense oligonucleotide jag-i9 aso and applications thereof

Abstract

The application provides an antisense oligonucleotide Jag-i9 ASO and application thereof, relates to the technical field of biological medicine, and the antisense oligonucleotide Jag-i9 ASO has the sequence of 5'-ACTGGGCCCTGCACCTGA-3'. By providing the antisense oligonucleotide of a specific sequence, the binding site of target heterogeneous ribonucleoprotein K and the Jag2 gene is targeted, the expression of the pro-inflammatory Jag2 subtype is inhibited, the Jag2 gene splicing site can be accurately targeted, the heterogeneous ribonucleoprotein K binding function is specifically blocked, a targeted intervention means is provided for the treatment of myocardial ischemia-reperfusion injury, the generation of the pro-inflammatory subtype is inhibited by efficiently and accurately regulating the alternative splicing of a specific gene, myocardial cell apoptosis and inflammatory response are reduced, myocardial function is improved, and a new approach is provided for the prevention and treatment of myocardial ischemia-reperfusion injury, the treatment effect is improved, and the advantages of reducing side effects are achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an antisense oligonucleotide Jag-i9 ASO and its applications. Background Technology

[0002] Myocardial ischemia-reperfusion injury (MIRI), a serious complication following myocardial infarction revascularization, involves a vicious cycle of cardiomyocyte apoptosis, inflammatory cascades, and oxidative stress. During reperfusion, myocardial tissue releases a large number of pro-inflammatory factors, activating neutrophil infiltration and NLRP3 inflammasomes, forming a continuously deteriorating inflammatory microenvironment that ultimately leads to irreversible cardiac damage. Current clinical treatments face significant limitations. Conventional antioxidants and anti-inflammatory drugs have broad targets and lack the ability to precisely intervene in myocardial-specific pathogenic processes, making efficacy difficult to guarantee and easily causing systemic side effects. Summary of the Invention

[0003] This invention aims to solve the problem that conventional drugs have broad targets, which makes it difficult to guarantee efficacy and easily causes systemic side effects.

[0004] To address the above problems, this invention provides an antisense oligonucleotide Jag-i9 ASO and its applications.

[0005] In a first aspect, the present invention provides an antisense oligonucleotide Jag-i9 ASO with the sequence 5'-ACTGGGCCCTGCACCTGA-3' (SEQ ID NO. 1).

[0006] Optionally, the antisense oligonucleotide Jag-i9 ASO specifically targets the binding site of heterologous ribonucleoprotein K to the Jag2 gene.

[0007] Optionally, the binding site of heterogeneous ribonucleoprotein K to the Jag2 gene is GCCCAG (SEQ ID NO.2).

[0008] Optionally, the antisense oligonucleotide Jag-i9 ASO has a main chain thiophosphate ester and a 2'-O-methoxyethyl modified structure.

[0009] In a second aspect, the present invention provides the use of the antisense oligonucleotide Jag-i9 ASO as described above in the preparation of medicaments for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0010] Alternatively, treatment of myocardial ischemia-reperfusion injury can be achieved by reducing the activation of the NF-κB signaling pathway in myocardial tissue.

[0011] Alternatively, myocardial ischemia-reperfusion injury can be treated by reducing the release of the pro-inflammatory cytokine IL-1β.

[0012] Alternatively, myocardial ischemia-reperfusion injury can be treated by reducing the release of the pro-inflammatory cytokine IL-6.

[0013] Alternatively, treatment of myocardial ischemia-reperfusion injury can be achieved by reducing the release of the pro-inflammatory cytokine TNF-α.

[0014] Alternatively, myocardial ischemia-reperfusion injury can be treated by reducing cardiomyocyte apoptosis.

[0015] The beneficial effects of the antisense oligonucleotide Jag-i9 ASO and its application of the present invention are as follows: By providing an antisense oligonucleotide with a specific sequence, it targets the binding site of heterologous ribonucleoprotein K and the Jag2 gene, inhibiting the expression of the pro-inflammatory Jag2 subtype. It has the ability to precisely target the Jag2 gene splicing site and specifically block the binding function of heterologous ribonucleoprotein K, providing a targeted intervention for the treatment of myocardial ischemia-reperfusion injury. By efficiently and precisely regulating the alternative splicing of specific genes, it inhibits the generation of pro-inflammatory subtypes, which helps to reduce cardiomyocyte apoptosis and inflammatory response, improve myocardial function, and thus provide a new approach for the prevention and treatment of myocardial ischemia-reperfusion injury, improving the therapeutic effect and reducing side effects. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the CLIP-qPCR experimental procedure and results according to an embodiment of the present invention;

[0017] Figure 2 This is a schematic diagram illustrating the protein expression verification of the Jag2 gene regulatory sequence by the HNRNPK protein in an embodiment of the present invention.

[0018] Figure 3 This is a schematic diagram of agarose gel electrophoresis of the RT-PCR products of the long transcript Jag2-L (SEQ ID NO. 9) (containing exon 10) and the short transcript Jag2-S in Example 1.

[0019] Figure 4 This is a schematic diagram of the experimental results of Jag-i9 ASO intervention for ischemia-reperfusion (IR) injury in Example 1;

[0020] Figure 5 This is a schematic diagram of the M-mode echocardiography and the quantitative left ventricular ejection fraction (LVEF, %) and shortening rate (FS, %) of Example 1.

[0021] Figure 6 This is a schematic diagram of DAPI staining and TUNEL staining of cardiomyocytes in Example 1;

[0022] Figure 7 This is a schematic diagram illustrating the quantitative analysis of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in myocardial tissue of Example 1.

[0023] Figure 8 This is a schematic diagram of the experimental results of key protein expression and NF-κB signaling pathway activation in myocardial tissue in Example 1. Detailed Implementation

[0024] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention's description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0026] The term "comprising" and its variations as used herein are open-ended inclusion, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the description below.

[0027] An embodiment of the present invention provides an antisense oligonucleotide Jag-i9 ASO with the sequence 5'-ACTGGGCCCTGCACCTGA-3' (SEQ ID NO. 1).

[0028] Specifically, antisense oligonucleotides (ASOs) are short-chain synthetic nucleotides that regulate gene expression by specifically binding to target RNA through Watson-Crick base pairing. ASOs can be designed, through chemical modifications, to guide RNA degradation in the cytoplasm or regulate the splicing of precursor mRNA in the nucleus.

[0029] Jag-i9 ASO is a specific antisense oligonucleotide proposed in this application, designed to specifically target genes associated with myocardial ischemia-reperfusion injury. The sequence 5'-ACTGGGCCCTGCACCTGA-3' (SEQ ID NO. 1) is the nucleotide sequence constituting Jag-i9 ASO, which determines its binding specificity to the target RNA.

[0030] The antisense oligonucleotide Jag-i9 ASO can be prepared in several ways. For example, it can be obtained by solid-phase synthesis, in which nucleotide monomers are linked together by phosphodiester bonds to form an oligonucleotide chain. Conventional DNA or RNA nucleotides can be used as building blocks during the synthesis process.

[0031] The sequence of the antisense oligonucleotide Jag-i9 ASO was determined to be 5'-ACTGGGCCCTGCACCTGA-3' (SEQ ID NO. 1). This sequence determination can be achieved by initially screening the target gene to identify potential binding regions. For example, the mRNA sequence of the Jag2 gene can be analyzed to find regions complementary to the antisense oligonucleotide. In this way, oligonucleotide sequences capable of binding to the target mRNA can be obtained.

[0032] In this embodiment, the antisense oligonucleotide Jag-i9 ASO, by providing a specific sequence of antisense oligonucleotides, targets the binding site of heterologous ribonucleoprotein K and the Jag2 gene, inhibiting the expression of the pro-inflammatory Jag2 subtype. It has the ability to precisely target the Jag2 gene splicing site and specifically block the binding of heterologous ribonucleoprotein K, providing a targeted intervention for the treatment of myocardial ischemia-reperfusion injury. By efficiently and precisely regulating the alternative splicing of specific genes and inhibiting the generation of pro-inflammatory subtypes, it helps to reduce cardiomyocyte apoptosis and inflammatory response, improve myocardial function, and thus provide a new approach for the prevention and treatment of myocardial ischemia-reperfusion injury, improving treatment efficacy and reducing side effects.

[0033] Optionally, the antisense oligonucleotide Jag-i9 ASO specifically targets the binding site of heterologous ribonucleoprotein K to the Jag2 gene.

[0034] Specifically, specific targeting refers to the ability of the antisense oligonucleotide Jag-i9 ASO to precisely recognize and bind to its pre-defined target nucleic acid sequence, thereby avoiding non-specific binding and off-target effects. Methods to achieve this specific targeting may include, but are not limited to: designing the nucleotide sequence of the antisense oligonucleotide Jag-i9 ASO (e.g., SEQ ID NO.1) to be highly complementary to the target sequence, ensuring stable binding via the Watson-Crick base pairing principle; or introducing specific chemical modifications (e.g., phosphate thioester backbone or 2'-O-methoxyethyl modification) to enhance the binding affinity, nuclease stability, and cellular uptake efficiency of the antisense oligonucleotide Jag-i9 ASO, while maintaining its specificity to the target.

[0035] The binding site of heteroribonucleoprotein K (HNRNPK) to the Jag2 gene refers to the specific nucleotide region on the Jag2 gene precursor mRNA where HNRNPK plays a splicing regulatory role. This binding site is a key node in regulating alternative splicing of the Jag2 gene, and its accurate identification is crucial for intervening in aberrant splicing of the Jag2 gene. The determination of this binding site can be achieved through various methods. For example, bioinformatics tools can be used to predict the binding motif of HNRNPK and match it within the Jag2 gene sequence; alternatively, in vitro or intracellular experimental methods, such as RNA immunoprecipitation (RIP) or cross-linked immunoprecipitation-sequencing (CLIP-seq), can be used to directly identify the interaction region between HNRNPK and Jag2 precursor mRNA.

[0036] In this optional embodiment, the antisense oligonucleotide Jag-i9 ASO can act on the binding site of heteroribonucleoprotein K and the Jag2 gene in a highly precise manner. This specific targeting ensures that the antisense oligonucleotide Jag-i9 ASO can directly interfere with the regulation of alternative splicing of the Jag2 gene by heteroribonucleoprotein K, thereby effectively reducing the generation of pro-inflammatory Jag2 isoforms. Given that Jag-i9 ASO (SEQ ID NO.1) itself has the ability to bind to target RNA, combined with the specific targeting of key binding sites, this application can significantly improve the efficiency and precision of intervention on the inflammatory cascade response in myocardial ischemia-reperfusion injury, avoid the potential side effects caused by non-specific effects, and thus more effectively reduce cardiomyocyte apoptosis and improve myocardial function, providing a more precise and effective strategy for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0037] Optionally, the binding site of heterogeneous ribonucleoprotein K to the Jag2 gene is GCCCAG (SEQ ID NO.2).

[0038] Specifically, the GCCCAG (SEQ ID NO.2) sequence precisely defines the region of interaction between heteroribonucleoprotein K and the Jag2 gene. Heteronucleoprotein K, as a key regulator of splicing decisions, is crucial for the alternative splicing process of the Jag2 gene. By specifically defining the binding site as the GCCCAG sequence, this application provides a highly specific target for the antisense oligonucleotide Jag-i9 ASO (sequence 5'-ACTGGGCCCTGCACCTGA-3' (SEQ ID NO.1)). Specifically, the antisense oligonucleotide Jag-i9 ASO can be designed to bind complementary to this GCCCAG sequence or its adjacent regions, thereby directly preventing the binding of heteroribonucleoprotein K to the Jag2 gene through steric hindrance, or indirectly affecting the recognition and binding efficiency of heteroribonucleoprotein K by altering the local RNA secondary structure.

[0039] In this optional embodiment, the binding site between heterologous ribonucleoprotein K and the Jag2 gene is identified as GCCCAG (SEQ ID NO. 2), enabling the antisense oligonucleotide Jag-i9 ASO to target this key site with extremely high specificity and precision. This precise targeting capability ensures that the antisense oligonucleotide Jag-i9 ASO can efficiently intervene in the heterologous ribonucleoprotein K-mediated alternative splicing process of the Jag2 gene, thereby effectively inhibiting the generation of pro-inflammatory subtypes. Therefore, this application can significantly improve the intervention effect on myocardial ischemia-reperfusion injury, providing a more precise and effective strategy for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0040] Optionally, the antisense oligonucleotide Jag-i9 ASO has a main chain thiophosphate ester and a 2'-O-methoxyethyl modified structure.

[0041] Specifically, the main-chain thiophosphate modification structure refers to replacing non-bridging oxygen atoms with sulfur atoms in the phosphodiester backbone of antisense oligonucleotides. This modification significantly enhances the resistance of antisense oligonucleotides to various nucleases in vivo (including endonucleases and exonucleases), effectively preventing their rapid degradation in complex biological environments and ensuring the long-term maintenance of their structural integrity and biological activity. In practical preparation, a solid-phase synthesis method can be used, employing a sulfidation reagent (such as Beaucage reagent or DDTT) instead of a traditional oxidizing reagent during oligonucleotide synthesis to achieve the conversion of phosphodiester bonds to thiophosphate bonds. Alternatively, the main-chain thiophosphate modification structure can also be introduced through a liquid-phase synthesis method using a sulfidation reaction in solution.

[0042] The 2'-O-methoxyethyl modification refers to the introduction of a methoxyethyl group at the 2' site of the ribosome loop of the antisense oligonucleotide. This modification significantly improves the binding affinity of the antisense oligonucleotide to the target RNA and enhances the thermal stability of its double strand formation, thereby ensuring that the antisense oligonucleotide Jag-i9 ASO can bind more stably and effectively to the binding sites of heterologous ribonucleoprotein K and the Jag2 gene. Simultaneously, the 2'-O-methoxyethyl modification further enhances the antinuclease degradation resistance of the antisense oligonucleotide and improves its intracellular uptake efficiency and pharmacokinetic properties. In the preparation process, a solid-phase synthesis method can be used, employing nucleotide monomers pre-contained with a 2'-O-methoxyethyl protecting group for sequential coupling to introduce this modification onto the synthetic strand.

[0043] In this optional embodiment, the antisense oligonucleotide Jag-i9 ASO effectively addresses the issues of easy degradation and low binding efficiency of unmodified oligonucleotides in vivo by introducing main-chain phosphate thioester and 2'-O-methoxyethyl modifications. Main-chain phosphate thioester modification enhances the antinuclease resistance of the antisense oligonucleotide, preventing its rapid degradation in the biological environment, thereby ensuring structural stability and durable effects. 2'-O-methoxyethyl modification improves the binding affinity and specificity of the oligonucleotide to target RNA, reducing off-target binding and ensuring precise intervention in the alternative splicing process of the Jag2 gene. These modifications synergistically enhance the reliability and efficacy of the antisense oligonucleotide in the treatment of myocardial ischemia-reperfusion injury, providing a solid foundation for drug application. In particular, when the antisense oligonucleotide Jag-i9 ASO has the sequence 5'-ACTGGGCCCTGCACCTGA-3' (SEQ ID NO.1) and specifically targets the binding site GCCCAG (SEQ ID NO.2) of heterogeneous ribonucleoprotein K and the Jag2 gene, these modifications ensure its stable presence and efficient function in vivo, thereby achieving effective prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0044] Another embodiment of the present invention provides the use of the antisense oligonucleotide Jag-i9 ASO as described above in the preparation of medicaments for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0045] Specifically, the application refers to using the antisense oligonucleotide Jag-i9 ASO as the active ingredient, through a specific preparation process, to make it a drug capable of preventing and / or treating myocardial ischemia-reperfusion injury. Specifically, this preparation process may include formulating the antisense oligonucleotide Jag-i9 ASO with pharmaceutically acceptable carriers, excipients, etc., to form various drug dosage forms. For example, it can be prepared as an intravenous injection solution for rapid administration before or after myocardial ischemia-reperfusion to achieve a systemic therapeutic effect. Furthermore, to improve its enrichment efficiency in target tissues (such as myocardium) and intracellular uptake, while protecting it from nuclease degradation, thereby enhancing efficacy and reducing off-target effects, the antisense oligonucleotide Jag-i9 ASO can also be encapsulated using drug delivery systems such as nanoparticles and liposomes. The prevention and / or treatment of myocardial ischemia-reperfusion injury refers to reducing the risk of injury or mitigating its severity through drug intervention, or mitigating pathological progression, promoting repair, and improving prognosis after injury has occurred. For example, prophylactic administration can be given before revascularization procedures such as percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) to preemptively suppress inflammatory responses and apoptosis pathways, thereby reducing postoperative myocardial reperfusion injury. Therapeutic administration can be given immediately after thrombolytic therapy or revascularization in patients with myocardial infarction to rapidly intervene in the inflammatory cascade, oxidative stress, and apoptosis induced by reperfusion, thereby limiting the extent of myocardial damage and protecting cardiac function.

[0046] This embodiment clarifies the specific use of the antisense oligonucleotide Jag-i9 ASO with the sequence 5'-ACTGGGCCCTGCACCTGA-3' (SEQ ID NO.1) in the treatment of cardiovascular diseases, namely, its use in the preparation of drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury. Given that myocardial ischemia-reperfusion injury (MIRI) is a serious complication following revascularization after diseases such as myocardial infarction, characterized by exacerbated inflammatory responses and cardiomyocyte apoptosis, and that existing treatments lack specificity, the application proposed in this application enables Jag-i9 ASO to specifically target and intervene in key pathogenic mechanisms of MIRI. Specifically, this antisense oligonucleotide Jag-i9 ASO specifically binds to the binding site of heterologous ribonucleoprotein K and the Jag2 gene, such as GCCCAG (SEQ ID NO.2), through a base pairing mechanism, thereby regulating the alternative splicing process of the Jag2 gene and inhibiting the generation of pro-inflammatory subtypes. This precise molecular intervention can effectively reduce the inflammatory cascade and apoptosis in myocardial tissue, thereby limiting the extent of myocardial damage, protecting cardiac function, and significantly improving patient prognosis. Compared to broad-spectrum drugs, this application provides a treatment strategy that is highly targeted, effective, and has few side effects, effectively solving the problem of the lack of precise targeted intervention for myocardial ischemia-reperfusion injury in existing technologies.

[0047] Alternatively, treatment of myocardial ischemia-reperfusion injury can be achieved by reducing the activation of the NF-κB signaling pathway in myocardial tissue.

[0048] Specifically, methods to reduce NF-κB signaling pathway activation in myocardial tissue can include any or a combination of the following: The NF-κB signaling pathway is a key intracellular pathway in response to inflammatory stimuli. Its activation typically involves a series of protein phosphorylation and nuclear translocation processes, ultimately leading to the expression of pro-inflammatory genes. Reducing its activation can be understood as decreasing NF-κB activity by intervening in any key step of this pathway. For example, this can be achieved by inhibiting the activity of key kinases in the NF-κB signaling pathway, such as by targeting and inhibiting the phosphorylation of IκB kinase (IKK), thereby preventing the degradation of its downstream repressor protein IκB, causing the NF-κB complex to remain in the cytoplasm and preventing its translocation to the nucleus to initiate the transcription of inflammatory genes. Alternatively, this can be achieved by enhancing the expression level or stability of endogenous NF-κB repressor proteins (such as IκBα), enabling them to more effectively bind to and isolate NF-κB, thus preventing its nuclear translocation. Or, this can be achieved by directly interfering with the DNA binding of NF-κB to the promoter region of target genes, for example by introducing competitive binding molecules or modifying the DNA-binding domain of the NF-κB protein, thereby blocking its transcriptional activity.

[0049] In this optional embodiment, the application of the antisense oligonucleotide Jag-i9 ASO in the preparation of drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury can directly target the core inflammatory problem in myocardial ischemia-reperfusion injury by reducing the activation of the NF-κB signaling pathway in myocardial tissue. Given that the NF-κB signaling pathway is a key regulator of the inflammatory response, and its activation triggers the release of downstream pro-inflammatory cytokines and amplifies the inflammatory cascade, precise intervention in this pathway can effectively inhibit the vicious cycle of the inflammatory cascade, thereby reducing cardiomyocyte damage and functional deterioration. This well-defined therapeutic strategy enhances the specificity and efficacy of the drug, solves the problem of lack of targeting in existing treatments, avoids the potential side effects of broad-spectrum drugs, and provides more precise and effective treatment options for clinical practice.

[0050] Alternatively, myocardial ischemia-reperfusion injury can be treated by reducing the release of the pro-inflammatory cytokine IL-1β.

[0051] Specifically, the pro-inflammatory cytokine IL-1β plays a central role in the inflammatory response. It is released in large quantities during myocardial ischemia-reperfusion injury (MIRI) and is a key mediator in the initiation and amplification of the inflammatory cascade. Reducing IL-1β release aims to directly intervene in the inflammatory pathological process of MIRI, thereby alleviating myocardial damage. Specifically, reducing IL-1β release can be achieved, including but not limited to: inhibiting the synthesis of IL-1β precursors by regulating the expression or splicing of related genes; or blocking the cleavage and release of IL-1β precursors into mature IL-1β by inhibiting the activation of inflammasomes (such as the NLRP3 inflammasome). Furthermore, its biological activity or secretion level can be indirectly reduced by regulating other key molecules in the IL-1β signaling pathway.

[0052] In this optional embodiment, the application of the antisense oligonucleotide Jag-i9 ASO in the preparation of drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury has been further optimized. This antisense oligonucleotide Jag-i9 ASO can specifically bind to the binding site of heterologous ribonucleoprotein K and the Jag2 gene, regulating alternative splicing of the Jag2 gene and inhibiting the generation of pro-inflammatory subtypes. Based on this, by reducing the release of the pro-inflammatory cytokine IL-1β, this application can precisely intervene in the inflammatory cascade response in myocardial ischemia-reperfusion injury. As a core initiator of the inflammatory response, the reduction of IL-1β release can effectively inhibit the transmission of downstream inflammatory signals, block the activation of the NLRP3 inflammasome and neutrophil infiltration, thereby significantly reducing the inflammatory response and cell damage in myocardial tissue. This mechanism of action avoids the non-specific side effects that may be caused by traditional broad-spectrum anti-inflammatory drugs, improves the targeting and effectiveness of treatment, and provides a more precise and efficient strategy for the treatment of myocardial ischemia-reperfusion injury.

[0053] Alternatively, myocardial ischemia-reperfusion injury can be treated by reducing the release of the pro-inflammatory cytokine IL-6.

[0054] Specifically, the pro-inflammatory cytokine IL-6 (interleukin-6) is a pleiotropic cytokine that plays a crucial role in inflammatory responses, immune responses, and hematopoiesis. In myocardial ischemia-reperfusion injury (MIRI), IL-6 is considered an important pro-inflammatory mediator; increased release significantly exacerbates the inflammatory cascade, leading to cardiomyocyte damage and functional deterioration. Reducing IL-6 release means inhibiting its production, secretion, or accelerating its degradation. This can be achieved through various mechanisms, such as inhibiting IL-6 gene transcription or mRNA stability, reducing IL-6 synthesis by targeting key transcription factors that regulate IL-6 gene expression or affecting IL-6 mRNA stability. Furthermore, it can also indirectly reduce IL-6 production by modulating upstream signaling pathways, blocking signaling pathways that activate IL-6 expression, such as NF-κB and MAPK.

[0055] Treatment of myocardial ischemia-reperfusion injury (MIRI) is achieved by reducing the release of the pro-inflammatory cytokine IL-6, aiming to directly intervene in the inflammatory cascade response within MIRI and alleviate myocardial damage by reducing the level of the key pro-inflammatory mediator IL-6. This approach can be achieved by directly inhibiting the production or secretion of IL-6, for example, by directly targeting the synthesis or release pathway of IL-6 through small molecule inhibitors, antibodies, or gene therapy. More preferably, as described in this application, by targeting the binding site of heterologous ribonucleoprotein K to the Jag2 gene using the antisense oligonucleotide Jag-i9 ASO, the alternative splicing of the Jag2 gene is regulated, inhibiting the generation of the pro-inflammatory Jag2 isoform. This regulatory effect can effectively block or weaken the activation of downstream inflammatory signaling pathways, thereby indirectly and effectively reducing the release of IL-6 and thus alleviating MIRI.

[0056] In this optional embodiment, the specific mechanism of action of the antisense oligonucleotide Jag-i9 ASO in the prevention and / or treatment of myocardial ischemia-reperfusion injury is clarified, namely, by reducing the release of the pro-inflammatory cytokine IL-6. Given that IL-6 plays a crucial "amplifier" role in the inflammatory cascade of myocardial ischemia-reperfusion injury (MIRI), its excessive release can significantly exacerbate cardiomyocyte damage. Therefore, by targeting the binding site of heterologous ribonucleoprotein K to the Jag2 gene with the antisense oligonucleotide Jag-i9 ASO, regulating the alternative splicing of the Jag2 gene, and inhibiting the generation of the pro-inflammatory isoform, the activation of downstream inflammatory signaling pathways can be effectively blocked or weakened, thereby directly and precisely reducing the release of IL-6. This mechanism can effectively alleviate the inflammatory microenvironment, inhibit cardiomyocyte apoptosis, and thus significantly improve myocardial function, providing a more targeted and effective strategy for the treatment of myocardial ischemia-reperfusion injury (MIRI), avoiding the limitations of traditional broad-spectrum anti-inflammatory drugs with poor targeting and uncertain efficacy.

[0057] Alternatively, treatment of myocardial ischemia-reperfusion injury can be achieved by reducing the release of the pro-inflammatory cytokine TNF-α.

[0058] Specifically, the pro-inflammatory cytokine TNF-α (tumor necrosis factor-α) is an important inflammatory mediator in myocardial ischemia-reperfusion injury. Its excessive release exacerbates the inflammatory response, induces apoptosis, and leads to tissue damage. Reducing TNF-α release is one of the key strategies for intervening in the inflammatory cascade response of myocardial ischemia-reperfusion injury. Reducing the release of the pro-inflammatory cytokine TNF-α can be achieved through multiple pathways. For example, the transcription or translation of the TNF-α gene can be inhibited, thereby reducing the production of TNF-α mRNA or protein at its source. This can be achieved by targeting transcription factors or signaling pathways that regulate TNF-α gene expression. Furthermore, the production and release of TNF-α can be indirectly reduced by regulating intracellular signal transduction pathways, such as inhibiting the activation of the NF-κB signaling pathway. NF-κB is a key regulator of the expression of various inflammatory factors, including TNF-α. Moreover, the degradation or decreased stability of TNF-α mRNA can be promoted, accelerating its clearance within cells, thereby reducing the amount of translatable TNF-α mRNA and consequently decreasing the synthesis and release of TNF-α protein.

[0059] In this optional embodiment, the strategy for treating myocardial ischemia-reperfusion injury focuses on reducing the release of the pro-inflammatory cytokine TNF-α, thereby achieving precise intervention in the inflammatory cascade. Given the central role of TNF-α in the inflammatory response of myocardial ischemia-reperfusion injury, this approach effectively blocks TNF-α-mediated cell damage and apoptosis, avoiding the non-specific side effects and uncertain efficacy issues that may arise from existing broad-spectrum treatments. Combined with the application of the aforementioned antisense oligonucleotide Jag-i9 ASO in the preparation of drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury, this ASO can specifically regulate gene expression or splicing associated with myocardial ischemia-reperfusion injury, thereby inhibiting the production or release of TNF-α. This targeted intervention mechanism makes the treatment more targeted, more effectively reducing inflammatory damage to myocardial tissue, improving myocardial function, and thus enhancing the precision and effectiveness of treatment for myocardial ischemia-reperfusion injury.

[0060] Alternatively, myocardial ischemia-reperfusion injury can be treated by reducing cardiomyocyte apoptosis.

[0061] Specifically, cardiomyocyte apoptosis refers to programmed cell death of cardiomyocytes under specific pathological conditions, such as myocardial ischemia-reperfusion injury. This apoptosis leads to a reduction in the number of cardiomyocytes and impaired myocardial function, and is one of the key links in the pathological progression of myocardial ischemia-reperfusion injury. Reducing cardiomyocyte apoptosis is an important strategy for directly intervening in myocardial injury and protecting myocardial function. By inhibiting programmed cell death of cardiomyocytes, the structural and functional integrity of myocardial tissue can be effectively preserved, thereby reducing the severity of myocardial ischemia-reperfusion injury. One way to achieve this is by regulating intracellular signaling pathways, such as inhibiting the expression or activity of pro-apoptotic proteins while promoting the expression or activity of anti-apoptotic proteins, thereby blocking the transmission of apoptotic signals. Another way is to intervene in mitochondrial function, such as stabilizing mitochondrial membrane potential, reducing cytochrome c release, or inhibiting the initiation of caspase cascade reactions to prevent the activation of mitochondrial-mediated apoptosis pathways. In addition, scavenging reactive oxygen free radicals can reduce the damage of oxidative stress to cardiomyocytes, thereby indirectly inhibiting apoptosis.

[0062] In this optional embodiment, the antisense oligonucleotide Jag-i9 ASO can treat myocardial ischemia-reperfusion injury by reducing cardiomyocyte apoptosis. Specifically, the antisense oligonucleotide Jag-i9 ASO specifically targets the binding site of heterologous ribonucleoprotein K to the Jag2 gene, regulating alternative splicing of the Jag2 gene and thereby inhibiting the generation of pro-inflammatory subtypes. Based on this, by reducing cardiomyocyte apoptosis, it directly intervenes in the core pathological process of myocardial ischemia-reperfusion injury, namely, programmed cell death of cardiomyocytes. This mechanism of action can effectively protect the survival of cardiomyocytes and maintain the integrity of myocardial tissue, thereby significantly alleviating myocardial dysfunction caused by myocardial ischemia-reperfusion injury. Compared with indirect means such as anti-inflammatory or antioxidant methods, the strategy of directly reducing cardiomyocyte apoptosis is more targeted and fundamental, and can more effectively improve patient prognosis and enhance treatment efficacy.

[0063] The present invention will be further described below with reference to specific embodiments.

[0064] Example 1

[0065] (1) Core target of antisense oligonucleotide Jag-i9 ASO: The binding site of HNRNPK to Jag2 pre-mRNA was selected as the intervention target. HNRNPK is a key splicing factor that regulates alternative splicing. Under normal conditions, HNRNPK binds less to Jag2 and tends to skip exon 10 (SEQ ID NO. 3) to form a short isoform. In MIRI, the binding of HNRNPK to Jag2 is enhanced, promoting the retention of exon 10 of Jag2. By promoting the Notch–NF-κB axis, it initiates a series of downstream inflammatory responses, leading to myocardial damage, decreased cardiac function, and myocardial remodeling.

[0066] (2) Jag-i9 ASO sequence design and synthesis: The binding site between HNRNPK and Jag2 precursor mRNA (SEQ ID NO. 4) was identified using CLIP-qPCR (Cross-Linking Immunoprecipitation Quantitative PCR) targeting the mouse Jag2 gene mRNA sequence (GenBank accession number: NM_010588.3). Cells were subjected to 0.15 J / cm². 2 Exposure to 365nm UV light for 15 minutes at the specified dose was used to achieve complete cross-linking between RNA and protein. Following the BersinBio CLIP-qPCR kit instructions, 10µg of anti-HNRNPK antibody or control IgG was incubated with 50µl of protein and magnetic beads for immunoprecipitation and enzyme digestion. RNA was then isolated from the complex, and RT-PCR was performed using primers targeting one exon upstream and downstream of the skipped exon 10 and its intron sequence (i.e., exon 9 (SEQ ID NO. 5), intron 9 (SEQ ID NO. 6), exon 10 (SEQ ID NO. 3), intron 10 (SEQ ID NO. 7), and exon 11 (SEQ ID NO. 8). This confirmed that HNRNPK primarily binds to intron 9 of Jag2. Figure 1 As shown. The binding site between HNRNPK and Jag2 was identified as GCCCAG using RIP-seq (RNA Immunoprecipitation Sequencing) and CLIP-qPCR (Cross-Linking Immunoprecipitation Quantitative PCR). Based on this binding site, an 18-base antisense oligonucleotide was designed. Its nucleotide sequence is: 5'-ACTGGGCCCTGCACCTGA-3' (SEQ ID NO. 1). To improve stability and research efficiency, the Jag-i9 ASO sequence was modified with a phosphate thioester backbone and a 2'-O-methoxyethyl (MOE) ribose group.

[0067] Prediction was performed using the online bioinformatics tool SpliceAid 2 (http: / / 193.206.120.249 / splicing_tissue.html). The Jag2 sequence containing intron 9 was entered in the dialog box. The predicted potential binding motif for HNRNPK was GCCCAG.

[0068] Furthermore, mutating GCCCAG to GTTTCA can inhibit the binding of HNRNPK to Jag2, such as... Figure 2 As shown, the HNRNPK protein plays a role in the regulatory sequence of the Jag2 gene, where... Figure 2 Four sets of experiments were presented (columns: HNRNPK-WT to HNRNPK-K405R), involving two core variables: reporter gene vectors: Jag2-reporter-WT (wild-type Jag2 regulatory sequence) and Jag2-reporter-mut (mutant Jag2 regulatory sequence); and HNRNPK protein forms: HNRNPK-WT (wild-type HNRNPK) and HNRNPK-K405R (HNRNPK K405 site mutant).

[0069] (3) In vitro cell experiments:

[0070] Model construction: Using HL-1 cardiomyocytes, an H / R model was established, consisting of 12 hours of hypoxia (94% N2, 5% CO2, 1% O2) followed by 4 hours of reoxygenation (normal oxygen concentration).

[0071] Intervention: 24 hours before the onset of hypoxia, the Jag2-i9 ASO (experimental group) and ASO-Ctrl (negative control group) of this invention were transfected into cells using lipo 3000.

[0072] The only difference between ASO-Ctrl (negative control group) and Jag2-i9 ASO (experimental group) was that Jag2-i9 ASO was not added.

[0073] Jag2-i9 ASO or ASO-Ctrl was transfected using a concentration gradient of adiponectin RNAiMAX. The transfection process was as follows: transfection was performed when cell confluence reached 30% to 50%, using serum-free medium (such as Opti-MEM) for dilution throughout the process.

[0074] Preparation of Jag2-i9 ASO (experimental group)

[0075] A. Dilute RNAiMAX reagent

[0076] Take a sterile 1.5 mL centrifuge tube and add 50 µL of Opti-MEM medium. Gently mix RNAiMAX reagent (do not vortex), and add 1.5 µL of the RNAiMAX reagent to the Opti-MEM medium. Gently pipette or tap the tube wall to mix, and let stand at room temperature for 5 minutes.

[0077] B. Dilute Jag2-i9 ASO

[0078] Take another sterile 1.5 mL centrifuge tube and add 50 µL of Opti-MEM medium. Calculate the volume of Jag2-i9 ASO stock solution needed based on the desired final concentration. Add it to the Opti-MEM medium.

[0079] C. Formation of transfection complex

[0080] Add the diluted RNAiMAX (tube A) completely to the diluted Jag2-i9 ASO (tube B). Note the order: add the liposomes to the nucleic acid. Gently pipette 3 to 5 times to mix, or tap the tube wall lightly to mix. Do not vortex. Let stand at room temperature for 10 to 15 minutes to allow the liposomes and Jag2-i9 ASO to fully form a stable complex. The solution may become slightly cloudy.

[0081] Add 100 µL of the prepared transfection complex dropwise and evenly to the wells containing cells. Gently shake the culture plate back and forth and side to side to distribute the complex evenly in the culture medium. Return to a 37°C, 5% CO2 incubator for further incubation.

[0082] Dosage screening: RT-PCR analysis was performed on Jag2 splicing in cardiomyocytes transfected with increasing concentrations (0 to 80 nM) of Jag2-i9 ASO or ASO-Ctrl.

[0083] Alternative splicing is a key regulatory mechanism in gene expression, producing diverse mRNA isoforms with different functional properties and promoting various biological processes. Some isoforms generated by alternative splicing are thought to be associated with the occurrence and progression of various diseases, including cancer, neurodegenerative diseases, and heart failure. We detected two transcripts of Jag2: the long transcript Jag2-L (SEQ ID NO. 9) (enclosed in exon 10) and the short transcript Jag2-S (SEQ ID NO. 10) (skipped from exon 10).

[0084] RNA was reverse transcribed into cDNA using a qPCR kit (TaKaRa, Japan). Semi-quantitative RT-PCR was performed using 2× Hieff® PCR master mix (Yeasen, Shanghai). RT-PCR products were validated by 1%–2% agarose gel electrophoresis. The grayscale values ​​of long and short transcripts were quantified using ImageJ software, and the splicing ratio was assessed by calculating the percentage of splicing in (PSI) value (PSI = splicing in / (splicing in + splicing out), e.g. Figure 3 As shown. Figure 3 It is evident that 20 nM Jag2-i9 ASO has the most significant inhibitory effect on long transcripts.

[0085] (4) In vivo animal experiments:

[0086] Model construction: Male C57BL / 6J mice were used. The left anterior descending coronary artery was surgically ligated for 45 minutes, and then released to achieve reperfusion for 6 hours to establish the MIRI model.

[0087] Dosage regimen: Before reperfusion, mice were injected via tail vein with Jag-i9 ASO and ASO-Ctrl (negative control group) using a 0.3 mL U-100 insulin injector. The dose was 3 mg / kg per injection, three times a week for three consecutive weeks.

[0088] Infarct area: 24 hours after reperfusion, the heart was harvested for TTC staining, and the ratio of myocardial infarction area to risk area was calculated.

[0089] like Figure 4 The figure shows the experimental results of Jag-i9 ASO intervention in ischemia-reperfusion (IR) injury. IR (ischemia-reperfusion) represents the experimental model of myocardial / tissue ischemia-reperfusion injury. Figure 4 The images show the quantification of risk area (AAR / LV) and infarct area relative to risk area (AAR) in the heart region stained with Evans Blue / TTC. It is evident that Jag-i9 ASO treatment can reduce the myocardial infarct area after ischemia-reperfusion injury compared to the negative control ASO (ASO-Ctrl) intervention.

[0090] Cardiac function: Left ventricular ejection fraction and fractional shortening were measured by echocardiography before reperfusion and 72 hours after reperfusion.

[0091] like Figure 5 As shown, the left image is a representative M-mode echocardiogram, with the sham group representing the sham surgery group. The right image shows the quantitative data of left ventricular ejection fraction (LVEF, %) and shortening rate (FS, %). It is evident that Jag-i9 ASO treatment can restore cardiac function.

[0092] Histology and Apoptosis: DAPI staining was used to observe the infiltration of inflammatory cells in the tissue, and TUNEL staining was used to quantify cardiomyocyte apoptosis.

[0093] like Figure 6 As shown, Merge is an overlay of DAPI and TUNEL, showing positive apoptotic cells and their quantification. It is evident that Jag-i9 ASO treatment can reduce cardiomyocyte apoptosis.

[0094] Molecular mechanism verification: Myocardial tissue was collected, and the quantification (pg / mg) of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in myocardial tissue was verified by Western Blot and ELISA.

[0095] like Figure 7 As shown, Jag-i9 ASO treatment can reduce the release of inflammatory factors.

[0096] To verify the molecular mechanism, myocardial tissue was collected, and the activation of the NF-κB signaling pathway in the heart was verified by Western blotting and ELISA.

[0097] like Figure 8 As shown, the levels of p-IKK, IKKα, p-p65, p65, p-IκBα, and IκBα are displayed. Quantification of phosphorylation ratios (p-IKK / Tubulin, p-p65 / p65, and p-IκBα / IκBα) is presented. It is evident that Jag-i9 ASO treatment can reduce the activation of the NF-κB pathway, an inflammatory pathway.

[0098] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. An antisense oligonucleotide Jag-i9 ASO, characterized in that, The sequence is 5'-ACTGGGCCCTGCACCTGA-3', as shown in SEQ ID NO.

1.

2. The antisense oligonucleotide Jag-i9 ASO according to claim 1, characterized in that, The antisense oligonucleotide Jag-i9 ASO specifically targets the binding site of Heterogeneous Ribonucleoprotein K and Jag2 gene.

3. The antisense oligonucleotide Jag-i9 ASO according to claim 2, characterized in that, The binding site of Heterogeneous Ribonucleoprotein K and Jag2 gene is GCCCAG, as shown in SEQ ID NO.

2.

4. The antisense oligonucleotide Jag-i9 ASO of claim 1, wherein, The antisense oligonucleotide Jag-i9 ASO has a main chain of phosphorothioate and a 2'-O-methoxyethyl modification structure.

5. The antisense oligonucleotide Jag-i9 ASO of claim 1 for use in the preparation of a medicament for preventing and / or treating myocardial ischemia-reperfusion injury.

6. The use of the antisense oligonucleotide Jag-i9 ASO according to claim 5 for the preparation of a medicament for the prevention and / or treatment of myocardial ischemia reperfusion injury, characterized in that, The treatment of myocardial ischemia-reperfusion injury is achieved by reducing the activation of NF-κB signaling pathway in myocardial tissue.

7. The use of the antisense oligonucleotide Jag-i9 ASO according to claim 5 for the preparation of a medicament for the prevention and / or treatment of myocardial ischemia reperfusion injury, characterized in that, The treatment of myocardial ischemia-reperfusion injury is achieved by reducing the release of pro-inflammatory cytokine IL-1β.

8. The use of the antisense oligonucleotide Jag-i9 ASO according to claim 5 for the preparation of a medicament for the prevention and / or treatment of myocardial ischemia reperfusion injury, characterized in that, The treatment of myocardial ischemia-reperfusion injury is achieved by reducing the release of pro-inflammatory cytokine IL-6.

9. The use of the antisense oligonucleotide Jag-i9 ASO according to claim 5 for the preparation of a medicament for the prevention and / or treatment of myocardial ischemia reperfusion injury, characterized in that, The treatment of myocardial ischemia-reperfusion injury is achieved by reducing the release of pro-inflammatory cytokine TNF-α.

10. The use of the antisense oligonucleotide Jag-i9 ASO according to claim 5 for the preparation of a medicament for the prevention and / or treatment of myocardial ischemia reperfusion injury, characterized in that, The treatment of myocardial ischemia-reperfusion injury is achieved by reducing the apoptosis of myocardial cells.

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